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<title>Microbiological Methods for Monitoring the Environment Water and Wastes</title>

<provider>NEPIS</provider>

<access>online</access>

<type>single page tiff</type>

<keyword>agar water broth sample laboratory colonies test media fecal tubes sterile microbiological dilution mpn medium filter membrane plate count part</keyword>

<author>Bordner, Robert.; Winter, John A.; Scarpino, Pasquale. Bordner, Robert. ; Winter, John A. ; Scarpino, Pasquale. Environmental Monitoring and Support Laboratory, Cincinnati, Ohio. Environmental Monitoring and Support Laboratory (Cincinnati, Ohio)</author>

<publisher>Environmental Protection Agency, Office of Research and Development, Environmental Monitoring and Support Laboratory ;</publisher>

<subject>Water--Microbiology; Water--Waste; Sewage--Microbiology; Aquatic microbiology; Water analysis; Water quality; Manuals; Potable water; Law enforcement; Monitoring; Surface waters; Coliform bacteria; Feces; Streptococcus; Salmonella; Actinomycetales; Culture media; Counting; Indicator organisms; Laboratory equipment; Enterobacteriaceae; Safety </subject>

<abstract>This first EPA manual contains uniform laboratory and field methods for microbiological analyses of waters and wastewaters, and is recommended in enforcement, monitoring and research activities. The procedures are prepared in detailed, stepwise form for the bench worker. The manual covers coliform, fecal coliform, fecal streptococci, Salmonella, actinomycetes, and Standard Plate Count organisms with the necessary support sections on sampling, equipment, media, basic techniques, safety, and quality assurance. </abstract>

EPA-600/8-78-017

December 1978

MICROBIOLOGICAL METHODS FOR MONITORING

THE ENVIRONMENT

Wafer and Wastes

Edited by

Robert Bordner and John Winter

Environmental Monitoring and Support Laboratory-Cincinnati

Cincinnati, Ohio 45268

and Pasquale Scarpino, University of Cincinnati

Cincinnati, Ohio 45219

Prepared in part under EPA Contract No, 68-03-0431

Project Officer

John Winter

Environmental Monitoring and Support Laboratory

Cincinnati, Ohio 45268

ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY

OFFICE OF RESEARCH AND DEVELOPMENT

U.S. ENVIRONMENTAL PROTECTION AGENCY

CINCINNATI, OHIO 45268

s/Q Printed on Recycled Paper

image:

DISCLAIMER

This report has been reviewed by the Environmental Monitoring and Support Laboratory-

Cincinnati, U.S. Environmental Protection Agency and approved for publication. Mention of trade

names or commercial products does not constitute endorsement or recommendation for use.

MICROBIOLOGICAL MANUAL 1978

image:

FOREWORD

Environmental measurements are required to determine the quality of ambient waters and the

Character of waste effluents. The Environmental Monitoring and Support Laboratory (EMSL^—

Cincinnati conducts research to: • • ••'*•'•

* Develop and evaluate techniques to measure the presence and concentration of

physical, chemical, and radiological pollutants in water, wastewater, bottom

sediments, and solid waste.

* Investigate methods for the concentration, recovery, and identification of viruses,

bacteria and other microorganisms in water.

* Conduct studies to determine the responses of aquatic organisms to water quality.

• Conduct an Agency-wide quality assurance program to assure standardization and

quality control of systems for monitoring water and wastewater.

This publication of EMSL-Cincinnati, contains the methods selected by consensus of EPA

senior microbiologists for parameters of interest to the Agency. Federal agencies, states,

municipalities, universities, private laboratories, and industry should find this manual of assistanpe

in monitoring and controlling microbiological pollution in the environment.

DwightG. Ballinger

Director, EMSL-Cincinnati

image:

PREFACE

The Federal Water Pollution Control Act Amendments of 1972, the Marine Protection,

Research, and Sanctuaries Act of 1972, and the Safe Drinking Water Act of 1974, require that EPA

develop and select methods for environmental monitoring and research on public and private

water supplies, rivers, lakes, ground waters, wastewaters and the marine environment for the

purposes of setting and enforcing environmental standards and ultimately enhancing the quality of

the environment. This manual of methodology supports these needs.

Under the direction of a Steering Committee formed for the development of an Agency

microbiology manual, a seminar was held among representative Agency microbiologists in San

Francisco, January, 1973. Assignments were made to committee members for the preparation of

first draft material. The basic design, format and content of the manual, were established and the

first drafts presented and reviewed at the second meeting of the Committee in January, 1974 at

Cincinnati.

The drafts submitted by the Steering Committee members were formatted and developed into

the initial version under EPA Contract No. 68-03-0431 by Dr. Pasquale Scarpino, Professor of

Environmental Engineering, Department of Civil and Environmental Engineering, University of

Cincinnati, working with the two EPA editors: Robert Bordner, Chief Microbiology Section,

Biological Methods Branch and John Winter, Chief, Quality Assurance Branch, both of EMSL-

Cincinnati. Subsquently, these editors added technical detail and the necessary information

reflecting Agency policies. Valuable source documents for This Manual were Current Practices in

Water Microbiology, National Training and Operational Technology Center and Handbook for

Evaluating Water Bacteriological Laboratories, Municipal Environmental Research Center, both of

U.S. EPA, Cincinnati, Ohio. The refined product is presented here.

Comments or questions concerning the manual should be directed to:

Robert Bordner or John Winter

U.S. Environmental Protection Agency

EMSC-Cincinnati

Cincinnati, OH 45268

<SER*\ MICROBIOLOGICAL MANUAL 1978

image:

TABLE OF CONTENTS

Page

Foreword iii

Preface iv

Figures vii

Tables x

Acknowledgements xv

PART I INTRODUCTION 1

PART II GENERAL OPERATIONS

A. Sample Collection, Preservation and Storage 5

B. Laboratory Equipment, Techniques and Media 32

C. Isolation and Enumeration of Bacteria 59

D. Selection of Analytical Methods 91

PART III ANALYTICAL METHODOLOGY

A. Standard Plate Count , 101

B. Total Coliforms 108

C. Fecal Coliforms 124

D. Fecal Streptococci 135

E. Salmonella. 154

F. Actinomycetes 186

PART IV QUALITY CONTROL

A. Laboratory Operations 194

B. Statistics for Microbiology 225

C. Analytical Quality Control Procedures 231

PART V LABORATORY MANAGEMENT

A. Development of a Quality Control Program 244

B. Manpower and Analytical Costs 246

C. Safety 259

D. Legal Considerations 277

image:

APPENDICES

A. Microbiological Activities under the Water Laws 289

B. Certification of Water Supply Laboratories 297

C. Bibliography 324

INDEX 325

Vi oV MICROBIOLOGICAL MANUAL 1978

image:

FIGURES

Number Page

Il-A-1 Suggested Sample Containers 7

II-A-2 Demonstration of Technique Used in Grab Sampling of Surface

Waters 9

II-A-3 Weighted Bottle Frame and Sample Bottle for Grab Sampling 10

II-A-4 Zobell J-Z Sampler,.,,.. , ,. 11

ll-A-5 Niskin Depth Sampler 12

II-A-6 New York State Dept of Health Depth Sampler 13

ll-A-7 Kemmerer Depth Sampler 15

II-A-8 Van Donsel-Geldreich Sediment Sampler 16

ll-A-9 Example of a Sample Label 18

M-A-10 Field Data Record 18

II-A-11 Sample Log Sheet 20

II-A-12 Chain of Custody Record..., 21

II-A-13 Sampling a Water Supply Reservoir 23

II-A-14 Sampling a Lake or Impoundment 26

II-A-15 Sampling a Large Stream 27

II-B-1 Finger-mounted Pipetting Device.. 35

ll-B-2 Enlargement of Pipetting Device Tip 35

ll-C-1 Microbiological Bench Cards for MF Analyses 60

ll-C-2 Bench Cards for MPN Analyses 60

II-C-3 Combined Microbiological Bench Card 61

vii

image:

II-C-4 Preparation of Decimal Dilution 63

ll-C-5 Suggested Pattern'for Preparing a Streak Plate , 67

1I-C-6 Membrane Filtration Units Made by Various Manufacturers for

Detection of Bacteria in Aqueous Suspensions ,..„ 72

II-C-7 Exploded View of a Stainless Steel Membrane Filtration Unit 73

ll-C-8 Colony Counting Pathway 76

ll-C-9 Enlarged Portion of Grid-Marked Square of Filter , 76

II-C-10 Packaging and Labelling of Microbiological Cultures for

Shipment „ , ..,.. 89

III-A-1 Typical Dilution Series for Standard Plate Count , 103

HI-B-1 Verification of Total Coliform Colonies on the Membrane Filter 115

III-B-2 Flow Chart for the Total Coliform MPN Test...., , 116

lll-C-1 Verification of Fecal Coliform Colonies on the Membrane Filter.... 131

lll-C-2 Flow Chart for the Fecal Coliform MPN Test , 133

lll-D-1 Verification Procedure for Fecal Streptococci ; , ,,..,...,. 140

lll-D-2 Isolation and Identification of Fecal Streptococcci, General

Scheme , 141

III-D-3 Identification of Fecal Streptococci, Separation of Enterococcus

Group by Species and by Original Source of Culture ,.,... 148

Ill-D-4 Identification of Fecal Streptococci, Separation of Enterococci

from Vegetation, Insect and Animal Sources.. 151

lll-E-1 Scheme for the Concentration, Isolation and Identification of

Salmonella ,..,.,.»...„ 156

lll-E-2 Simplified Scheme for Concentration, Isolation and Identification

of Salmonella..., 157

lll-E-3 Dimensions of the Gauze Swabs 158

IH-E-4 The Gauze Swab in Position , , 159

III-F-1 A Plate Containing Bacterial and Actinornycete Colonies., ,,.. 189

Vlii ©EPA MICROBIOLOGICAL MANUAL 1978

image:

lll-F-2 An Actinomycete Colony Showing the Branching Filaments that

Cause the Fuzzy Appearance of its Border 190

lll-F-3 A Bacterial Colony with its Relatively-Distinct, Smooth Border 190

IV-A-1 Equipment Operation Temperature Record 217

IV-B-1 Normal Distribution Curve 228

IV-B-2 Positively-Skewed Distribution Curve 228

V-C-1 Laminar Flow Cabinet 270

V-C-2 Example of Biohazard Sign... ..„ 271

image:

TABLES

Number ' Page

ll-A-1 Sampling Frequency Based on Population 25

H-B-1 Relationship of Steam Pressure to Temperature in the Autoclave. 37

II-C-1 Recommended Filtration Volumes of Samples in MF Analyses....... 64

ll-C-2 Number of Significant Figures (S.F.) Reported 70

H-C-3 Acceptable Limits '. . '. 77

lf-C-4 Most Probable Number Index and 95% Confidence Limits for

Five Tube, Three Dilution Series 82

H-C-5 Most Probable Number Index and 95% Confidence Limits for

Testing Potable Waters 83

II-C-6 Selection of Code Results, Five Tube Series 85

1I-D-1 Approved Test Procedures for the Analyses of Pollutants (40

CFR 136) .". , 93

II-D-2 Water Quality Standards.,! '. ".. 94

H-D-3 Water Quality Criteria 95

II-D-4 Selection of Methods for Problem Samples „.,...... i8

lll-B-1 Differentiation of the Conform and Related Organisms Based on

Biochemical Reactions 120

lll-C-1 Suggested Range of Sample Volumes for Fecal Coliform Tests

Using the Membrane Filter Methods^, 127

lll-E-1 Colonial Appearance of Salmonella and Other Enterics on

Isolation Media 166

III-E-2 Production Rate and Time Requirements of Multitest Systems 174

X &EFA MICROBIOLOGICAL MANUAL 1978

image:

III-E-3 Reported Shelf-Life of Multitest Systems With or Without

Refrigeration 174

HI-E-4 Cost and Source of Multitest Systems 175

lll-E-5 Biochemical Characteristics of the Enterobacteriaceae , 176

IV-A-1 Monitoring Laboratory Equipment.;.......' 212

IV-A-2 Glassware Maintenance , 218

IV-A-3 Laboratory Pure Water for Bacteriological Testing 219

IV-A-4 Quality Control of Media . 220

IVTA-5 Quality Control of Biochemical Tests 222

IV-B-1 Microbiological Results, count/100 ml 225

IV-B-2 Colifdrm Counts and Their. Logarithms 229

IV-B-3 Comparison of Frequency of MPN Data 230

IV-B-4 Comparison of Frequency of Log MPN Data 230

IV-C-1 Raw Sample Data from the Analysis of Chlorinated Sewage

Treatment Plant Effluents.^ '....... ':.......... ...:.. 237

IV-C-2 Logarithmic Transformation of the Data in Table IV-C-1 238

IV-C-3 Analysis of Difference Between Means 241

V-B-1 Estimated Time Required for Twenty MPN Analyses 248

V-B-2 General Equipment and Supplies Minimum Program, Yearly Basis 249

V-B-3 Equipment and Supplies for MF Analyses Minimum Program.... 251

V-B-4 Equipment and Supplies for MPN Analyses Minimum Program 253

V-B-5 General Equipment and Supplies Full Program in Microbiology

Weekly Basis .....I .....; 254

V-B-6 Equipment and Supplies for MF Analyses Full Program in

Microbiology, Weekly Basis...; 256

V-B-7 Equipment and Supplies for MPN Analyses Full Program in

Microbiology, Weekly Basis 257

V-B-8 Media for Full Program in Microbiology Laboratory Usage for

each Week/100 Samples : 258

image:

V-C-1 Laboratory-acquired Infections Related to Personnel and Work 260

V-C-2 Sources of Laboratory-acquired Infections 261

V-C-3 Normal Use Concentration of Disinfectants 267

Xii vyEFVX MICROBIOLOGICAL MANUAL 1978

image:

THE MICROBIOLOGY METHODS STEERING COMMITTEE

OF EPA

Cochairpersons: Robert Bordner and John Winter

Environmental Monitoring and Support Laboratory-Cincinnati

Members:

William Stang Harold Jeter (retired)

Edwin Geldreich Francis Brezenski

Kathleen Shimmin

xiii

image:

CONTRIBUTORS BY SECTION

Sampling '*•

William Stang

NEIC-Denver

General Laboratory Equipment/Media

Robert Bordner and John Winter

EMSL-Cincinnati

Pasquale Scarpino

UC Dept. of Environ. Engineering

Isolation and Enumeration of Bacteria

Robert Bordner and John Winter

EMSL-Cincinnati

Pasquale Scarpino

UC Dept. of Environ. Engineering

Selection of Analytical Methods

Robert Bordner and John Winter

EMSL-Cincinnati

Standard Plate Count

Raymond Taylor

MERL-Cincinnati

Total Coliforms

Harold Jeter (retired)

National Training Center

ERC-Cincinnati

Fecal Coliforms

Edwin Geldreich

MERL-Cincinnati

Fecal Streptococci

Francis Brezenski

Region II

Salmonella

Kathleen Shimmin

Alameda Laboratory

Region IX

Donald Spino

MERL-Cincinnati

Actinomycetes

Robert Safferman

EMSL-Cincinnati

Quality Control

Robert Bordner

EMSL-Cincinnati

Development of a Quality Control Program

John Winter

EMSL-Cincinnati

Manpower and Analytical Costs

Robert Bordner and John Winter

EMSL-Cincinnati

Legal Considerations

Dave Shedroff

Office of Enforcement

Washington, DC

Carroll Wills

NEIC-Denver

Safety

Robert Bordner and John Winter

EMSL-Cincinnati

Pasquale Scarpino

UC Dept of Environ. Engineering

MICROBIOLOGICAL MANUAL 1978

image:

ACKNOWLEDGEMENTS

The Committee wishes to acknowledge the many EPA microbiologists and others who

participated in the development or review of the manual. These include, in regional and program

order: .

Region I

Howard Davis and Edward Gritsavage •

Regional Laboratory

Needham Heights, MA

Victor Cabelli, Alfred Dufour and Morris Levin

Environmental Research Laboratory

Narragansett, RI •••...-,..

Region II

Isidore Seidenberg (retired)

Edison Water Laboratory

Edison, NJ

Region III .

Leonard Guarraia Don Lear' . '

Office of Water & Hazardous Materials Annapolis Field Station

Washington, DC Annapolis, MD

Region IV

Bobby Joe Carroll and Ralph Gentry A! Bourquin

S & A Division, SERL Pensacola Station

Athens, GA Pensacola, FL

Region V

James Adams

Central Regional Laboratory

Chicago, 1L

Region VI ,

Harold Cumiford

Houston Facility

Houston, TX

Region VII

Carl Bailey

Regional Laboratory

Kansas City, MO

- xv

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Region VIII

John Manhart

Regional Laboratory, Denver Federal Center

Denver, CO

Region IX

Harold Scotten

Alameda Laboratory

Alameda, CA

Region X

George J. Vasconcelos and Richard Bauer

Regional Laboratory

Seattle, WA

Martin Knittel

Environmental Research Laboratory

Corvallis, OR

Ronald Gordon

Alaska Water Laboratory

College, AK

Cincinnati Environmental Research Center

Joseph Santner and Rocco Russomanno

National Training Center

Martin Allen, Harry Nash and Don Reasoner

Municipal Environmental Research Laboratory

Louis Resi

Division of Technical Support

Bernard Kenner (retired)

Municipal Environmental Reserach Laboratory (AWTRL)

Paul Britton, Terry Covert and Herbert Manning

Environmental Monitoring and Support Laboratory

Elmer Akin and Walter Jakubowski

Health Effects Research Laboratory

PREPARATION OF THIS VOLUME

The editors acknowledge gratefully the excellent technical skills of organization, proofreading,

typing and computerized text editing performed by M. Mary Sullivan, Her contribution of hard work

and sacrifice of personal time to this manual cannot be overstated.

XVi V>EPA MICROBIOLOGICAL MANUAL 1978

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PART I. INTRODUCTION

As the only direct measures of pollution by man and other warm-blooded animals,

microbiological parameters contribute unique information on water and wastewater quality and

public health risk from waterborne disease. Microbiological analyses are conducted to:

Monitor ambient water quality for recreational, industrial, agricultural and water supply uses,

Assure the safety of potable water

Monitor municipal and industrial discharges.

Identify the sources of bacterial pollutants,

and evaluate water resources.

Role of the Aquatic Microbiologist

Although their primary role is to produce valid data for management decisions,

microbiologists should also participate in survey planning and evaluation, develop new microbjal

parameters and methodology, consult on microbiological problems, establish and monitor criteria

and standards, testify in administrative hearings and court cases, train laboratory staffs and

research special problems. Microbiologists should also go beyond sanitary microbiology to solve

taste and odor problems, to study microbiological transformations, and to apply other

measurements to the aquatic ecosystem.

Scope of the Microbiology Manual Series

This EPA manual provides uniform laboratory and field methods for microbiological analyses

of the environment. The analytical methods are standardized procedures recommended for use in

enforcement, monitoring and research. However, they are not intended to inhrbit or prevent

methods research and development. Exploratory and developmental methods are compiled sepa-

rately for evaluation but are part of the EPA Microbiology Manual Series.

The environmental areas covered will include:

* All waters — fresh, estuarine, marine, shellfish-growing, agricultural, ground, surface,

finished, recreational and industrial processing.

INTRODUCTION

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* All wastewaters of microbiological concern - domestic waste effluents, industrial

wastes such as food, dairy, meat, tanning, sugar, textile, pulp and paper, shellfish

processing and agricultural wastes such as feedlot and irrigation runoff.

• Other areas of the environment - air, sediments, soils, sludges, oils, leachates,

vegetation, etc.

Coverage of the First Edition of the Manual

Although the scope of the Manual Series is broad and inclusive of many parameters and

sample types, the first edition describes primarily the analytical methods that meet the immediate

needs of the Agency. These are the key parameters that are accepted and used for water quality,

compliance monitoring and enforcement under Federal Water Pollution Control Act, PL 92-500,

Marine Protection, Research, and Sanctuaries Act, PL 92-532 and the Safe Drinking Water Act, PL

93-523. The necessary supportive sections include: sample collection, equipment and techniques,

cultural media, glassware preparation, quality control, data handling, safety, legal considerations

and selection of analytical methods.

Focus of the Manual

This Manual is intended for use by the supervisor or analyst who may be a professional

microbiologist, a technician, chemist, engineer or plant operator. Regardless of other skills, the

supervisor and analyst should have received at least two weeks training in each parameter from a

federal or state agency or from a university.

To assist the newanalyst, Part II has been prepared as a basic discussion on laboratory operations

and for general guidance to permit use of the manual by those required to do microbiological

analyses. The trained analyst will be familiar and knowledgeable of most of these techniques.

The analytical procedures in Part HI are written in a stepwise manner so that the manual can be

used both at bench level and as a reference book. Part IV emphasizes the important, but often neglect-

ed need for quality control in microbiological analyses, while Part V describes general considerations

for laboratory management.

Objectives

The objectives of This Manual are to:

* Select the best method currently available for use in the environmental monitoring,

compliance monitoring, enforcement and research activities of the Agency,

» Establish uniform application of microbiological methods so that only the best methods

are used and perpetuated, data from different laboratories or surveys can be fairly

compared and/or results can be stored in a common data bank, e.g., STORET, for later

use.

» Provide guidance on the use of these methods, their" advantages, limitations and

application to various types of water and wastes.

• Establish recognized procedures for method selection and evaluation that will form the

baseline against which othertests forthe same or new parameters can be measured.

* Emphasize the analytical quality control and management practices that should be

performed in the laboratory to assure valid data.

2 4>EPA MICROBIOLOGICAL MANUAL 1978

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Criteria

The first edition of This Manual describes the parameters of health and sanitary significance.

In the future, the criteria for addition of a method to the Manual are:

• The method is required to satisfy new or changing needs of the Agency.

» The method is practical for field and laboratory use. Equipment, supplies and media are

available and the procedure provides results within reasonable time limits.

* The method offers significant advantages over current methods.

• The method has been validated by the developer or by others according to the criteria

for Comparative Testing of Methodology and Method Characterization. (See IV-C-1).

• The method criteria and characterization have been reviewed and accepted by the EPA

Steering Committee for Microbiology.

INTRODUCTION

image:

PART II. GENERAL OPERATIONS

This Part describes the general procedures which are applicable to the methods of analysis for

all parameters. The Sections provide the basic background information that must be understood

whan the analytical procedures are carried out. The procedures are divided here into broad areas

of function:

Section A Sample Collection, Preservation and Storage

Section B General Laboratory Equipment, Techniques and Media

Section C Isolation and Enumeration of Bacteria

Section D Selection of Analytical Methodology

MICROBIOLOGICAL MANUAL 1978

image:

PART II. GENERAL OPERATIONS

Section A Sample Collection, Preservation and Storage

Collection, preservation and storage of

water samples are critical to the results of

water quality analyses. The data are only as

valid as the water sample,

A sampling program must be planned to

satisfy the objectives of the study yet remain

within the limitations of available manpower,

time and money. The survey should use the

minimum number of samples that adequately

represent the effluent or body of water from

which they are taken. The number of samples

and location of sampling sites should be deter-

mined prior to the survey and must satisfy the

requirements needed to establish water qual-

ity standard or effluent permit violations.

The microbiologist should participate in

the planning which specifies the microbiologi-

cal tests needed, the number of analyses to be

performed, and the equipment required. Con-

sideration should be given to the weather and

other local conditions prior to the formulation

of a final plan. For example, seasonal varia-

tions in water temperature and flows would be

important factors in deciding when to study

the effects of thermal pollution on bacteria.

Sample collectors must know the exact loca-

tion of the sampling sites and be fully trained

in the aseptic technique of sample collection

as well as the use of any specialized sampling

equipment. The sample collector is responsi-

ble for the recording of all pertinent informa-

tion about the sample that might be significant

in the evaluation and interpretation of the

laboratory data or that might be necessary in

potential enforcement action.

This Section is organized as follows:

1. Sample Containers

2. Sampling Techniques

Composite Sampling

Surface Sampling by Hand

Surface Sampling by

Weighted Bottle Frame

Depth Sampling

Soil Sampling

Sediment Sampling

Water Tap Sampling

3. Sample Identification and

Handling

4, Chain of Custody Procedures

5. Selection of Sampling Sites and

Frequency

Potable Water Supplies

Lakes and Impoundments

Stream Sampling

Marine and Estuarine

Sampling

Domestic and Industrial

Waste Discharges

Recreational Waters

Shellfish-Harvesting Waters

Frequency of Sampling

6. Preservation and Transit of

Samples

SAMPLING TECHNIQUES

image:

1. Sample Containers

1.1 Sample Bottles: Bottles must be resis-

tant to sterilizing conditions and the solvent

action of water. Wide-mouth borosilicate glass

bottles with screw-cap or ground-glass stopper

or heat-resistant plastic bottles may be used

if they can be sterilized without producing toxic

materials {see suggested sample containers in

Figure II-A-1). Screw-caps must not produce

bacteriostatic or nutritive compounds upon

sterilization.

1.2 Selection and Cleansing of Bottles:

Sample bottles should be at least 125 ml vol-

ume for adequate sampling and for good mixing.

Bottles of 250 ml, 500 ml and 1000 ml volume

are often used for multiple analyses. Discard

bottles which have chips, cracks, and etched

surfaces. Bottle closures must be water-tight.

Before use, thoroughly cleanse bottles and

closures with detergent and hot water, fol-

lowed by a hot water rinse to remove all trace

of detergent. Then rinse them three times with

laboratory-pure water {II-B.6). A test for the

biological examination of glassware where

bacteriostatic or inhibitory residues may

be present, is described in Part 1V-A, 5.1.

1.5 Wrapping Bottles: Protect the tops

and necks of glass stoppered bottles from

contamination by covering them before sterili-

zation with aluminum foil or kraft paper.

1.6 Sterilization of Bottles: Autoclave

glass or heat-resistant plastic bottles at 121 C

for 15 minutes. Alternatively, dry glassware

may be sterilized in a hot air oven at 170 C for

not less than two hours. Ethylene oxide gas

sterilization is acceptable for plastic containers

that are not heat-resistant Sample bottles

sterilized by gas should be stored overnight

before being used to allow the last traces of

gas to dissipate. See Part il-B, 3 for steriliza-

tion procedures.

1.7 Plastic Bags: The commercially-

available bags (Whirl-pak) are a practical sub-

stitute for plastic or glass sample bottles in

sampling soil or sediment. See Figure ll-A-1.

The bags are sealed in manufacture and opened

only at time of sampling. The manufacturer

states that such bags are sterilized.

2. Sampling Techniques

1.3 Dechlorinating Agent: The agent

must be placed in the bottle when water and

wastewater samples containing residual chlo-

rine are anticipated. Add sodium thiosulfate to

the bottle before sterilization at a concentration

of 0.1 ml of a 10 percent solution for each

125 ml {4 oz.J sample volume (1). This concen-

tration will neutralize approximately 15 mg/l

of residue chlorine.

1,4 Chelating Agent: A chelating agent

should be added to sample bottles used to

collect samples suspected of containing

>0.01 mg/liter concentrations of heavy metals

such as copper, nickel or zinc, etc. Add 0.3

ml of a 15 percent solution of ethylenedia-

minetetraacetic acid (EDTA) tetrasodium salt,

for each 125 ml (4 oz.) sample volume prior to

sterilization {2, 3).

Samples are collected by hand or with a

sampling device if (1) depth samples are re-

quired or (2) the sampling site has difficult

access such as a manhole, dock, bridge or

bank adjacent to a surface water.

2,1 Chlorinated Samples: When samples,

such as treated waters, chlorinated wastewa-

ters or recreational waters, are collected, the

sample bottle must contain a dechlorinating

agent (see this Section, 1.3).

2.2 Composite Sampling: |_n no case

should a composite sample be collected for

bacteriological examination. Data from indi-

vidual samples show a range of values. A com-

posite sample will not display this range. Indi-

vidual results will give information about in-

dustrial process variations in flow and compo-

«»EPA MICROBIOLOGICAL MANUAL 1978

image:

FIGURE ll-A-1. Suggested Sample Containers

A Screw-cap Glass or Plastic Bottle.

B Plastic Bag (Whiri-pak).

C Glass Stoppered Bottle.

SAMPLING TECHNIQUES

image:

sition. Also, one or more portions that make up

a composite sample may contain toxic or nutri-

tive materials and cause erroneous results.

2.3 Surface Sampling by Hand: A grab

sample is obtained using a sample bottle pre-

pared as described in 1. above. Identify the

sampling site on a chain of custody tag if

required, or on the bottle label and on a field

log sheet (see 3J, Remove the bottle covering

and closure and protect from contamination.

Grasp the bottle at the base with one hand and

plunge the bottle mouth down into the water to

avoid introducing surface scum. Position the

mouth of the bottle into the current away from

the hand of the collector and away from the

side of the sampling platform or boat (see

Figure ll-A-2). The sampling depth should be

15 to 30 cm (6 to 12 inches) below the water

surface. If the water body is static, an artificial

current can be created, by moving the bottle

horizontally in the direction it is pointed and

away from the sampler. Tip the bottle slightly

upwards to allow air to exit and the bottle to

fill. After removal of the bottle from the stream,

pour out a small portion of the sample to allow

an air space of 2.5 to 5 cm (1 to 2 inches)

above each sample for proper mixing of the

sample before analyses. Tightly stopper and

label the bottle.

2A Surface Sampling by Weighted Bot-

tle Frame: When sampling from a bridge or

other structure above a stream or body of

water, the sample collector places the bottle in

a weighted frame (see Figure II-A-3) that holds

the bottle securely. Remove cover and lower

the device to the water. It is preferable to use

nylon rope which does not absorb water and

will not rot. Face the bottle mouth upstream by

swinging the sampling device first down-

stream, and then allow it to drop into the water,

without slack in the rope. Pull the sample de-

vice rapidly upstream and out of the water,

thus simulating the scooping motion of grab

sampling described in 2.3. Take care not to

dislodge dirt or other material that might fall

into the open bottle from the sampling

platform.

2.5 Depth Sampling: Several additional

devices are needed for collection of depth

samples from lakes, reservoirs, estuaries and

the oceans. These depth samplers require low-

ering the sampling device and/or container to

the desired depth, then opening, filling, and

closing the container and returning the device

to the surface. Although depth measurements

are best made with a pre-marked steel cable,

the sample depths can be determined by pre-

measuring and marking the nylon rope at inter-

vals with a non-smearing ink, paint, or finger-

nail polish. The following list of depth sam-

plers is not inclusive but can serve as a guide:

2.5.1 ZoBell J-Z Sampler: This sampler

described by ZoBell in 1941 (4) was designed

for deep sea sampling but is also used in fresh

waters. Figure ll-A-4 shows its general ap-

pearance. It has a metal frame (A), a heavy

metal messenger (B), a sealed glass tube (C)

attached to a rubber tube (D), and a sterile

350 ml glass bottle (E) or a collapsible neo-

prene rubber bulb for shallow waters. The

messenger (B) is released at the surface when

the sampler reaches the 'desired depth, and

breaks the glass tubing (C) at a file mark. The

bent rubber tubing (D) then straightens out and

the water is drawn in several inches from the

sampler. A partial vacuum created by auto-

claving of the sealed unit draws the water into

the bottle.

2.5.2 Niskin Sampler: This is sometimes

called a sterile-bag or "Book" sampler (see

Figure ll-A-5) (5). A messenger triggers the

opening of two plates (A) in V-fashion by spring

power, and causes the sterile plastic bag (B) to

inflate. At the same time a plastic filler tube (C)

leading to the plastic container is cut by a

guillotine knife (D) and the bag fills with water.

The bag is then automatically sealed with a

clamp (E) and the apparatus is brought to the

surface. Samplers are available that will hold

1,2,3, or 5 liters of water.

2.5.3 New York Dept. of Health Depth

Sampler: This device (see Figure ll-A-6) de-

pends upon a vane (A) and lever (B) mechanism

to lift the glass stopper (C) as water inertia is

applied by a sharp upward tug on the line (D)

attached to the apparatus. As the stopper is

lifted, the bottle fills before the detachment of

the stopper from the vane occurs and closes

the sample bottle (6).

MICROBIOLOGICAL MANUAL 1978

image:

FIGURE II-A-2. Demonstration of Technique Used in Grab Sampling of Surface Waters.

SAMPLING TECHNIQUES

image:

FIGURE II-A-3. Weighted Bottle Frame and Sample Bottle for Grab Sampling.

4>EPA MICROBIOLOGICAL MANUAL 1978

image:

FIGURE ll-A-4. Zobell J-Z Sampler. (A) metal frame, (B) messenger, (C) glass tube,

(D) rubber tube and (E) sterile sample bottle.

SAMPLING TECHNIQUES

image:

FIGURE II-A-5. Niskin Depth Sampler. (A) hinged plates, (B) plastic bag,

closure clamp.

<&EPA MICROBIOLOGICAL MANUAL 1978

image:

FIGURE ll-A-6. New York State Dept. of Health Depth Sampler. (A) vane, (B1) lever

in closed position, (B2) lever in open position, (C1) glass stopper in

closed position, (C ) glass stopper in open position, (D) suspension

line, and (E) metal frame.

SAMPLING TECHNIQUES

image:

2«5-4 Kemmerer Sampler (7): This depth

sampler (see Figure Il-A-7) has been used with-

out sterilization to collect bacteriological

water samples in high pollution areas. The

sampler consists of a cylindrical brass or plas-

tic tube (F) that contains a rubber stopper or

valve at either end (D and G). The valves are

connected to a rod (E) that passes through the

center of the cylinder. The device is lowered

into the water in the open position, and a water

sample is trapped in the cylinder when the

valves are closed by a dropped messenger (B).

The Kemmerer sampler should not be used for

collecting bacteriological samples without ob-

taining data that support its use without

sterilization.

2.6 Sediment Sampling with Van

Donsel-Geldreich Sampler (8): This device

(see Figure I1-A-8J collects sediment or mud in

sterile "Whlrl-Pak" plastic bags (A) down to 60

foot depth. The bag mouth is wrapped over

a nosepiece (B), and the bag is kept closed

during descent to the bottom by a bag clamp

bar (H). As the mud plate (D) contacts the bot-

tom, the nosepiece (B) is driven into the sedi-

ment by the weight (C) of the sampler. As the

nosepiece (B) moves downward, the bag (A)

slides through the bag clamp bar (H), opens,

and fills with sediment. The bag is sealed when

the double noose (F) tied to the bottom of the

bag is pulled, before the apparatus is returned

to the surface.

2.7 Water Tap Sampling: Make certain

that samples are not collected from spigots

that leak around their stems, or from spigots

that contain aeration devices or screens within

the faucet. For samples taken from direct water

main connections, the spigot should be flushed

for 2-3 minutes to clear the service line. For

wells equipped with hand or mechanical

pumps, pump the water to waste for five min-

utes before the sample is collected. Remove

the cap aseptically from the sample bottle. Hold

the sample bottle upright near the base while it

is being filled. Avoid splashing. Do not rinse the

bottle with the sample; fill it directly to within

2.5 cm (1 inch) from the top. Replace bottle

closure and hood covering. Caution must be

used to prevent contaminating the sample with

finger, gloves or other materials. If the well

does not have pumping machinery, collect the

sample using a weighted sterilized sample

bottle, such as described in 2.4 above, and

shown in Figure ll-A-3. Care must be taken to

avoid contaminating the sample with the sur-

face scum from the water surface.

2.8 Soil Sampling

2.8.1 Selection of the sampling site is

based on knowledge of the area and the pur-

poses of the analyses, i.e., surface sampling for

natural background, surface contamination, or

below surface sampling to monitor treatment

effect such as irrigation, or stormwater runoff.

The actual sites for sampling and the num-

ber of points to be sampled must be predeter-

mined by the survey objectives. Soil sampling

has the advantage of permitting the survey

planners to lay out a stable grid network for

sampling and resampling over a given time

period.

2.8.2 If a surface sample is desired, scrape

the top one inch of soil from a square foot area

using a sterile scoop or spoon.

If a subsurface sample is desired, use a

sterile scoop or spatula to remove the top

surface of one inch or more from a one foot

square area. Use a second sterile scoop or

spoon to take the sample.

Place samplings in a sterile one quart

screw-cap bottle until it is full. Depending on

the amount of moisture, a one quart bottle

holds 300-800 grams of soil. Label and tag

the bottle carefully and store at 4 C until

analyzed.

3. Sample Identification and Handling

3.1 Specific details on sample identifica-

tion are entered on a permanent label. Take

care in transcribing sampling information to

the label, because the enforcement action may

depend upon evidence of primary labeling.

See 4. in This Section. Labels must be clean.

&EPA MICROBIOLOGICAL MANUAL 1978

image:

42 em

FIGURE ll-A-7. Kemmerer Depth Sampler. (A) nylon line, (B) messenger, (C) catch set so

that the sampler is open, (D)top rubber valve, (E) connecting rod between

the valves,(F)tube body,(G) bottom rubbervalve, (H) knot atthe bottom of

the suspension line and (I) rubber tubing attached to the spring loaded

check valve.

SAMPLING TECHNIQUES

image:

FIGURE II-A-8. Van bonsel-Geidreich Sediment Sampler. (A) sterile "Whiri-Pak" plastic

bag, (B) nose piece, (C) weight, (D) mud plate, (E) slide bar, (F) part of the

double noose, (G) attachment for the suspension line and (H) bag clamp

bar.

MICROBIOLOGICAL MANUAL 1978

image:

waterproof, non-smearing and of sufficient

size for the necessary information. Label must

be securely attached to the sample bottle, but

removable when necessary. Do not accept in-

sufficiently or improperly labeled samples for

examination. A sample label showing the mini-

mum information required is pictured in Figure

ll-A-9.

3.2 Field Data Record: A field record

should be completed on each sample to record

the full details on sampling and other pertinent

remarks such as flooding, rain or extreme tem-

perature which are relevant to interpretation of

results. This record also provides a back-up

record of sample identification. One example

is shown in Figure ll-A-10.

3.3 Marking Device: A marking pen or

other device must be non-smearing if wetted,

and maintain a permanent legible mark.

3.4 Transport Container: Insulated ice

containers in which the sample can be held, are

recommended.

3.5 Storage of Samples: A refrigerator is

necessary for storage of samples at the labora-

tory. The temperature range of the refrigerator

is 1-4C.

4. Chain of Custody Procedures

4.1 General: An agency must demon-

strate the reliability of its evidence in pollution

cases by proving the.chain of possession and

custody of samples which are offered for evi-

dence or which form the basis of analytical

results introduced into evidence. It is impera-

tive that the office and laboratory prepare writ-

ten procedures to be followed whenever evi-

dence samples are collected, transferred,

stored, analyzed, or destroyed.

4.1.1 The primary objective of these pro-

cedures is to create an accurate written record

which can be used to trace the possession of

the sample from the moment of its collection

through its introduction into evidence. A sam-

ple is in custody if it is:

(a) in actual physical possession, or

(b) in view after being in physical posses-

sion, or

that no one could tamper with it.

4.1.2 Personnel should receive copies of

study plans and know the contents prior to the

study. A pre-study briefing shall be held to

appraise participants of the objectives, sample

locations and chain of custody procedures.

After chain of custody samples are collected, a

de-briefing is held in the field to determine

adherence to chain of custody procedures and

whether additional samples are required.

4.2 Rules for Sample Collection

4.2.1 Handle the samples as little as

possible.

4.2.2 Obtain stream and effluent samples

using standard microbiological sampling

techniques.

4.2.3 Attach sample tag or;label (Figure

II-A- 9 i to the sample container. The tag or

label should contain as a minimum: serial

number of label, location, date andtime taken,

type of sample, sequence number (first sample

of the day - sequence No. 1, second sample,

sequence No. 2, etc), analyses required and

sample collector. The tags must be filled out

legibly in waterproof ink.

4.2.4 Use a bound notebook to record

field measurements and other pertinent

information necessary to refresh the sampler's

memory if the person later becomes a witness

in an enforcment proceeding. A separate set of

field notebooks should be maintained for each

study and stored in a safe place where it can

be protected and accounted for. A sample log

sheet with a standard format should be

established to minimize field entries and

include the date, time, survey.type of samples,

volume of each sample, type of analyses,

label and sample numbers, sample location,

field measurements such as temperature,

conductivity, DO, pH, and any other pertinent

SAMPLING TECHNIQUES

image:

EPA, NATIONAL ENFORCEMENT

Station No. Date

Station Location

BOD Metals

Solids Oil and Grease

COD D.O.

.Nutrients Bact.

Other

Samplers!

INVESTIGATIONS CENTER

Time Sequence No.

Grab

Comp.

Remarks/Preservativet

FIGURE ll-A-9. Example of a Sample Label.

ERIAL SHEET NO.

STATION

(

San

FIELD DATA RECORD

SAMPLE

NUMBER

DATE OF

COUJCTION

TIME (MRS)

SAMPLE

TAXIN

SAMPLE

XlCtiVtO ff IM

(*«

TEMP

•c

OTHER

REMARKS

US EPA. NEIC-Donvar

FIGURE ll-A-10. Field Data Record.

MICROBIOLOGICAL MANUAL 1978

image:

information or observation (Figure ll-A-1 1). The

entries should be signed by the sample

collector. The responsibility for preparing and

storing sample notebooks during and after a

study should be assigned to a study

coordinator, or his designated representative.

4.2.5 A field collector is responsible for

the samples collected until properly dis-

patched to the receiving laboratory or turned

over to an assigned custodian. He must assure

that each container is in his physical posses-

sion or in his view at all times, or stored in a

locked place where no one can tamper with it.

4.2.6 Color slides or photographs should

be taken of the sample location and any visible

water pollution. The signature of the photo-

grapher, time, date, and site location must be

written on the back of the photo. Such photo-

graphs should be handled according to the

established chain of custody procedures to

prevent alteration.

4.3 Transfer of Custody and Shipment

In transfer of custody procedures, each

custodian of samples must sign, record and

date the transfer. Most regulatory agencies

develop chain of custody procedures tailored

to their needs. These procedures may vary in

format and language but contain the same

essential elements. Historically, sample trans-

fer under chain of custody has been on a

sample by sample basis which is awkward and

time-consuming. However, EPA's National En-

forcement Investigation Center (NEIC), Denver

has set a precedent with its bulk transfer of

samples. Bulk transfer is speedier, reduces

paperwork and the number of sample custodi-

ans. The following description of chain of cus-

tody is essentially that of NEIC-Denver (9).

4.3.1 Samples must be accompanied by a

Chain of Custody Record which includes the

name of the study, collector's signature, sta-

tion number, station location, date, time, type

of sample, sequence number, number of con-

tainers and analyses required (Figure ll-A-12).

When turning over the possession of samples,

the transferor and transferee sign, date and

note time on the sheet. This record sheet al-

lows transfer of custody of a group of samples

in the field, to the mobile laboratory or to the

NEIC-Denver laboratory.: When a custodian

transfers a portion of the samples identified on

the sheet to the field mobile laboratory, the

individual samples must be noted in the col-

umn with the signature of the person relin-

quishing the samples. The field laboratory per-

son receiving the samples acknowledges re-

ceipt by signing in the appropriate column.

4.3.2 If a custodian has not been

assigned, the field custodian or field sampler

has the responsibility for packaging and

dispatching samples to the laboratory for

analysis. The "Dispatch" portion of the Chain

of Custody Record must be filled out, dated,

and signed.

4.3.3 Samples must be carefully packed in

shipment containers such as ice chests, to

avoid breakage. The shipping containers are

padlocked for shipment to the receiving

laboratory.

4.3.4 Packages must be accompanied by

the Chain of Custody Record showing

identification of the contents. The original

must accompany the shipment. A copy is

retained by the survey coordinator.

4.3.5 If samples are delivered to the

laboratory when appropriate personnel are not

there to receive them, the samples must be

locked in a designated area within the

laboratory so that no one can tamper with

them. This same person must return to the

laboratory, unlock the samples and deliver

custody to the appropriate custodian.

4.4 Laboratory Custody Procedures

4.4.1 The laboratory shall designate a

"sample custodian" and an alternate to act in

his absence. In addition, the laboratory shall

set aside as a "sample storage security area",

an isolated room with sufficient refrigerator

space, which can be locked or just a locked

refrigerator in smaller laboratories.

4.4.2 Samples should be handled by the

minimum possible number of persons.

SAMPLING TECHNIQUES

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MICROBIOLOGICAL MANUAL 1979

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SERIAL SHEE

T IMC

ENVIRONMENTAL PROTECTION AGENCY

Office Of Enforcement

NATIONAL ENFORCEMENT INVESTIGATIONS CENTER

Building 53, Box 25227, Denver Federal Center

Denver, Colorado 60225

CHAIN OF CUSTODY RECORD

SURVEY

SIA1ION

NUMBER

STATION LOCATION

DATE

Relinquished by: fsignarurij

Relinquished by: is:aiaiut*i

Relinquished by: isigratuni

Relinquished by: isig"°><"»)

Dispatched by: (si8norur.) Date/

TIME

SAMPLERS: (sig™tu,*i

SAMPL8 TVpE

Water

Comp.

Crab.

Air

SEO.

NO.

NO. OF

CONTAINERS

ANALYSIS

REQUIRED

Received by: (Signoiur.j Date/Time

Received by: fsignaturej Date/Time

Received by: (s/onarure; Date/Time

Received by Mobile Laboratory for field Date/Time

analysis: tsignatu,,)

Time

Received for Laboratory by: Date/Time

Method of Shipment:

Diltribution: Orig.— Accompany Shipment

1 Copy— Survey Coordinator Field Filet

FIGURE ll-A-12. Chain of Custody Record.

SAMPLING TECHNIQUES

image:

4.4.3 incoming samples shall be received

only by the custodian, who will indicate receipt

by signing the Chain of Custody Record Sheet

accompanying the samples and retaining the

sheet as a permanent record. Couriers picking

up samples at the airport, post office, etc. shall

sign jointly with the laboratory custodian.

4,4.4 Immediately upon receipt, the custo-

dian places samples in the sample room,

which will be locked at all times except when

samples are removed or replaced by the custo-

dian. To the maximum extent possible, only the

custodian should be permitted in the sample

room.

4.4.5 The custodian shall ensure that

microbiological samples are properly stored

and maintained at 1-4 C.

4.4.6 Only the custodian will distribute

samples to personnel who are to perform tests.

4.4.7 The analyst records information in

his laboratory notebook or analytical work-

sheet, describing the sample, the procedures

performed and the results of the testing. The

notes shall be dated and indicate who per-

formed the tests. The notes shall be retained

as a permanent record in the laboratory and

should include any abnormalities which oc-

curred during the testing procedure. In the

event that the person who performed the tests

is not available as a witness at time of trial,

the government may be able to introduce the

notes in evidence under the Federal Business

Records Act.

4.4.8 Standard methods of laboratory

analyses shall be used as described in the

"Guidelines Establishing Test Procedures for

Analysis of Pollutants," (10) and amendments.

If laboratory personnel deviate from standard

procedures, they should be prepared to justify

their decision during cross-examination.

4.4.9 Laboratory personnel are responsi-

ble for the care and custody of a sample once it

is handed over to them and should be pre-

pared to testify that the sample was in their

possession and view or secured in the labora-

tory at all times from the moment it was

received from the custodian until the tests

were run.

4.4.10 Once the sample testing is com-

pleted, microbiological samples can be dis-

carded but identifying tags and laboratory

record should be returned to the custodian.

Other documentation of work will also be

given to the custodian.

4.4.11 Tags and laboratory records of

tests may be destroyed only upon the order of

the Laboratory Director, who will first confer

with the Chief, Enforcement Specialist Office,

to make certain that the information is no

longer required.

5. Selection of Sampling Sites and

Frequency

These will be described for potable and

recreational waters, streams, lakes, reservoirs,

estuarine, and marine waters as well as do-

mestic and industrial wastewaters.

5.1 Potable Water Supplies

An expanded program to maintain the san-

itary quality of potable water supplies has

been recently established by the National jn-

terim Primary Drinking Water Regulations (11).

The sampling program includes examination

of water as it enters and flows throughout the

distribution system. For application of the EPA

Drinking Water Standards, the frequency of

sampling and the location of sampling points

are established jointly by the utility, the

Reporting Agency, and the Certifying

Authority. Additionally, the laboratory, the

methods of analyses, and the technical

competence of personnel must be inspected

and approved by the Reporting Agency and

the Certifying Authority.

5-1-1 Sampling Water Supplies: Figure II-

A-13 shows how reservoirs and lakes used as

water supplies are sampled: (A) at inlets, (B) at

other possible sources of pollution, (C) at the

draw-off point, (D) at quarter point intervals

around the draw-off point at about the same

depth and (E) at the reservoir outlet.

&EFW MICROBIOLOGICAL MANUAL 1978

image:

•£»

'riteS^'

FIGURE ll-A-13. Sampling a Water Supply Reservoir. (A) influent stream, (B) possible

agricultural contamination, (C) water plant intake, (D) multi-point

sampling around intake and (E) reservoir outlet.

SAMPLING TECHNIQUES

image:

5.1.2 Sampling Treatment Systems: Sam-

pling should be representative of the distribu-

tion system and include sites such as munici-

pal buildings, public schools, airports and

parks, hydrants, restaurants, theaters, gas sta-

tions, industrial plants and private residences.

A systematic coverage of such points in the

distribution system should insure the detec-

tion of contamination from breaks in water-

lines, loss of pressure or crossconnections.

The sampling program should also include

special sampling locations such as dead-end

distribution lines that are sources of bacterial

contamination (12),

5.1.3 Sample Frequency: The minimum

number of samples which must be collected

and examined each month is based upon the

population density served by the distribution

system (Table II-A-1). Samples should be col-

lected at evenly spaced time intervals through-

out the month. In the event of an unsatisfactory

sample, repetitive samples must be collected

until two consecutive samples yield satisfac-

tory quality water. Repetitive samples from any

single point or special purpose samples must

not be counted in the overall total of monthly

samples.

5.1.4 Standard Sample: The standards for

microbiological quality are based upon the

number of organisms allowable in a standard

sample. A standard sample for the membrane

filter technique is at least 100 ml. For the MPN

test, a standard sample consists of five stan-

dard portions of either 10 ml or 100 ml.

5.2 Lakes and Impoundments

Figure II-A-14 shows the range of sam-

pling points in a recreational impoundment or

Jake: (A) inlets, (B) source of pollution, (C) grid or

transect across the long axis of the water body,

(D)bathing beach and (E) outlet.

5.3 Stream Sampling

The objectives of the initial survey dictate

the location, frequency and number of sam-

ples to be collected.

5.3.1 Selection of Sampling Sites: A

typical stream sampling program includes

sampling locations upstream of the area of

concern, upstream and downstream of waste

discharges, upstream and downstream from

tributary entrances to the river and upstream

of the mouth of the tributary. For more

complex situations, where several waste

discharges are involved, sampling includes

sites upstream and downstream from the

combined discharge area and samples taken

directly from each industrial or municipal

waste discharge. Using available

bacteriological, chemical and discharge rate

data, the contribution of each pollution source

can be determined. See Figure ll-A-14 and

II-A-15.

5.3.2 Small Streams: Small streams

should be sampled at background stations

upstream of the pollution sources and at

stations downstream from pollution sources.

Additional sampling sites should be located

downstream to delineate the zones of

pollution. Avoid sampling areas where

stagnation may occur (backwater of a

tributary) and areas located near the inside

bank of a curve in the stream which may not be

representative of the main channel.

5.3.3 Large Streams and Rivers: Large

streams are usually not well mixed laterally for

long distances downstream from the pollution

sources. Sampling sites below point source

pollution should be established to provide

desired downstream travel time and dispersal

as determined by flow rate measurements.

Particular care must be taken to establish the

proper sampling points as shown in Figure II-A-

15: Sampling point (A) is the upper reach control

station, (B) monitors a non-point source of

pollution, (C) samples the waste discharge as it

enters the stream, (D) shows quarter-point

sampling below the pollution to detect chan-

neling, |D) also serves as an upstream monitor

on the tributary measured as (E), and (F) moni-

tors the downstream effect of the tributary

after mixing. Occasionally, depth samples

are necessary to determine vertical mixing

patterns.

MICROBIOLOGICAL MANUAL 1978

image:

TABLE 1I-A-1

Sampling Frequency for Drinking Waters Based on Population

Population served:

25 to 1,000.

1,001 to 2,500

2,501 to 3,300

3,301 to 4,100

4,101 to 4,900

4,901 to 5,800

5,801 to 6,700

6,701 to 7,600

7,601 to 8,500

8,501 to 9,400

9,401 to 10,300

10,301 to 11,100

11,101 to 12,000

12,001 to 12,900

12,901 to 13,700

13,701 to 14,600

14,601 to 15,500

15,501 to 16,300

16,301 to 17,200

17,201 to 18,100

18,1O1 to 18.9OO

18,901 to 19,800

19,801 to 20,700

20,701 to 21.50O

21,501 to 22,300

22,301 to 23,200

23,201 to 24,000

24,001 to 24,900

24,901 to 25,000

25,001 to 28,000

28,001 to 33,000

33,001 to 37,000

37,000 to 41,000

41,001 to 46,000

46,001 to 50,000

50,001 to 54,000

54,001 to 59,000

59,001 to 64,000

64,001 to 70,000

70,001 to 76,000

76,001 to 83,000

83,001 to 90,000

Minimum number of

samples per month

1 1

1 3

1 4

Population served:

90,001 to 96,000

96,001 to 111,000

111,001 to 130,000

130,001 to 160,000

160,001 to 190,000

190,001 to 220,000

220,001 to 250,000

250,001 to 290,000

290,001 to 320,000

320,001 to 360,000

360,001 to 410,000

410,001 to 450,000

450,001 to 500,000

500,001 to 550,000

550,001 to 600,000

600,001 to 660,000

660,001 to 720,000

720,001 to 780,000

780,001 to 840,000

840,001 to 910,000

9 1O.OO1 to 97O.OOO

970,001 to 1,050,000

1,050,001 to 1,140,000...,

1,140,001 to 1,230,000....

1,230,001 to 1,320,000...,

1,320,001 to 1,420,000....

1,420,001 to 1,520,000....

1,520,001 to 1,630,000....

1,630,001 to 1,730,000...,

1,730,001 to 1,850,000....

1,850,001 to 1,970,000....

1,970,001 to 2,060,000....

2,060,001 to 2,270,000,.,.

2,270,001 to 2,510,000,,..

2,510,001 to 2,750,000.,..

2,750,001 to 3,020,000.,..

3,020,001 to 3,320,000....

3,320,001 to 3,620,000....

3,620,001 to 3,960,000....

3,960,001 to 4,310,000....

4,310,001 to 4,690,000...,

4,690,001 or more

Minimum number of

samples per month

100

110

120

130

140

150

160

170

180

190

200

210

220

230

240

250

260

270

280

29O

300

310

320

330

340

350

360

370

380

390

400

410

420

430

440

450

460

470

480

490

500

SAMPLING TECHNIQUES

image:

FIGURE ll-A-14. Sampling a Lake or Impoundment. (A) inlets, (B) potential source

of pollution, (B1) village, (B2) agricultural run-off, (B3) home septic

tank, (C) multi-point transect, (D) bathing beach and (E) outlet above

and below dam.

x>EFV\ MICROBIOLOGICAL MANUAL 1978

image:

FIGURE ll-A-15. Sampling a Large Stream. (A) control station, (B) agricultural pollution,

(F) downstream monitoring.

SAMPLING TECHNIQUES

image:

5.4 Marine and Estuarine Sampling

Sampling marine and estuarine waters

requires the consideration of other factors in

addition to those usually recognized in fresh

water sampling. They include tidal cycles,

current patterns, bottom currents and counter-

currents, stratification, seasonal fluctuations,

dispersion of discharges and multi-depth

samplings.

The frequency of sampling varies with the

objectives. When a sampling program is

started, it may be necessary to sample every

hour around the clock to establish pollutional

loads and dispersion patterns. The sewage

discharges may occur continuously or

Intermittently.

When the sampling strategy for a survey is

planned, data may be available from previous

hydrological studies done by Coast Guard,

Corps of Engineers, National Oceanic and

Atmospheric Administration (NOAA), U.S.

Geological Survey, or university and private

research investigations. In a survey, float

studies and dye studies are often carried out to

determine surface and undercurrents. Initially

depth samples are taken on the bottom and at

five feet increments between surface and

bottom. A random grid pattern for selecting

sampling sites is established statistically.

5.4.1 Marine Sampling: In ocean studies,

the environmental conditions are most diverse

along the coast where shore, atmosphere and

the surf are strong influences. The shallow

coastal waters are particularly susceptible to

daily fluctuations in temperature and seasonal

changes.

Sampling during the entire tidal cycle or

during a half cycle may be required. Many

ocean studies such as sampling over the conti-

nental shelf involve huge areas and no two

areas of water are the same.

Selection of sampling sites and depths are

most critical in marine waters. In winter, cool-

ing of coastal waters can result in water layers

which approach 0 C. In summer, the shallow

waters warm much faster than the deeper

waters. Despite the higher temperature, oxy-

gen concentrations are higher in shallow than

in deeper waters due to greater water move-

ment, surf action and photosynthetic activity

from macrophytes and the plankton.

Moving from the shallow waters to the

intermediate depths, one observes a modera-

tion of these shallow water characteristics. In

the deeper waters, there is a marked stabliza-

tion of conditions. Water temperatures are

lower and more stable. There is limited turbu-

lence, little penetration of light, sparse vegeta-

tion and the ocean floor is covered with a layer

of silts and sediments.

5.4.2 Estuarine Sampling: When a survey

is made on an estuary, samples are often taken

from a boat, usually making an end to end

traverse of the estuary. Another method in-

volves taking samples throughout a tidal cycle,

every hour or two hours from a bridge or from

an anchored boat at a number of fixed points.

In a large bay or estuary where many

square miles of area are involved, a grid or

series of stations may be necessary. Two sets

of samples are usually taken from an area on a

given day, one at ebb or flood slack water, and

the other three hours earlier, or later, at the half

tidal interval. Sampling is scheduled so that

the mid-sampling time of each run coincides

with the calculated occurrence of the tidal

condition.

In locating sampling sites, one must con-

sider points at which tributary waters enter the

main stream or estuary, location of shellfish

beds and bathing beaches. The sampling sta-

tions can be adjusted as data accumulate. For

example, if a series of stations half mile apart

consistently show similar values, some of

these stations may be dropped and other sta-

tions added in areas where data shows more

variability.

Considerable stratification can occur be-

tween the salt water from the sea and the fresh

water supplied by a river. It is essential when

starting a survey of an unknown estuary to find

out whether there is any marked stratification.

This can be done by chloride determinations at

different locations and depths. It is possible for

MICROBIOLOGICAL MANUAL 1978

image:

stratification to occur in one part of an estuary

and not in another.

On a.flood tide, the more dense salt water

pushing up into the less dense fresh river

water will cause an overlapping with the fresh

water flowing on top. A phenomenon called a

salt water wedge can form. As a result, stratifi-

cation occurs. If the discharge of pollution is in

the salt water layer, the contamination will be

concentrated near the bottom at the flood tide.

The flow or velocity of the fresh water will

influence the degree of stratification which

occurs. If one is sampling only at the surface, it

is possible that the data will not show the

polluted underflowing water which was con-

taminated at a point below the fresh water

river. Therefore, where stratification is sus-

pected, samples at different depths will be

needed to measure vertical distribution.

5.5 Domestic and Industrial Waste

Discharges

It is often necessary to sample secondary

and tertiary wastes from municipal waste

treatment plants and various industrial waste

treatment operations. In situations where the

plant treatment efficiency varies considerably,

grab samples are collected around the clock at

selected intervals for a three to five day period.

If it is known that the process displays little

variation, fewer samples are needed. In no

case should a composite sample be collected

for bacteriological examination. The NPDES

has established wastewater treatment plant

effluent limits for all dischargers. These are

often based on maximum and mean values. A

sufficient number of samples must be col-

lected to satisfy the permit and/or to provide

statistically sound data and give a fair repre-

sentation of the bacteriological quality of the

discharge.

5.6 Recreational Waters

5-6-1 Bathing Beaches: Sampling sites at

bathing beaches or other recreational areas

should include upstream or peripheral areas

and locations adjacent to natural drains that

would discharge stormwater, or run-off areas

draining septic wastes from restaurants, boat

marinas, or garbage collection areas (12).

Samples of bathing beach water should be

collected at locations and times of heaviest

use. Daily sampling, preferably in the after-

noon, is the optimum frequency during the

season. Weekends and holidays which are pe-

riods of highest use must be included in the

sampling program. Samples of estuarine bath-

ing waters should be obtained at high tide, ebb

tide and low tide in order to determine the

cyclic water quality and deterioration that

must be monitored during the swimming

season.

5-6.2 Swimming Pools: Swimming pool

water should be monitored at least daily dur-

ing maximum use periods, preferably at the

overflow. It is important to test swimming pool

samples for neutralization of residual chlorine

at pool side to assure that the dechlorinating

agent was effective.

6.7 Shellfish-Harvesting Waters

Water overlying shellfish-harvesting areas

should be sampled during periods of most

unfavorable hydrographic conditions, usually

at low tide after heavy precipitation. However,

shellfish beds are sometimes exposed during

low tide and must be sampled during other

tidal conditions. Procedures for sampling of

shellfish and water in shellfish growing areas

are governed by the National Shellfish Sanita-

tion Program's Manual of Operations (13).

5.8 Frequency of Sampling

The frequency of sampling depends upon

the type of pollution that is to be measured.

Cyclic pollution and its duration are measured

as frequently as practical immediately down-

stream from the source. Uniform pollution

loads are measured at greater distances down-

stream from the source and at less frequent

time intervals than cyclic pollution. Climatic

and tidal conditions must be considered. Jn

marine and estuarine sampling. A common

approach for short-term studies is to collect

samples from each site daily and advance the

sampling intervals one hour during each 24-

hour period to obtain data for a 7-10 day

study.

SAMPLING TECHNIQUES

image:

Often the numbers of samples to be

collected are specified by NPDES permits,

drinking water regulations, or by state

requirements. Some standards require a

minimum number of samples to be collected

each month. Other standards are less explicit

and simply indicate that the geometric mean

coliform density shall not exceed a certain

level each month, with no more than 10%,

20%, etc. of samples exceeding a certain

value. Where the number of samples required

is undetermined, a sufficient number should

be collected to measure the variations in

stream conditions.

6. Preservation and Transit of Samples

The adherence to sample preservation and

holding time limits is critical to the production

of valid data. Samples exceeding these limits

should not be analyzed. The following rules

must be observed.

6.1 Storage Temperature and Handling

Conditions

Bacteriological samples should be iced or

refrigerated at a temperature of 1-4 C during

transit to the laboratory. Insulated containers

are preferable to assure proper maintenance

of storage temperature. Care should be taken

that sample bottles are not totally immersed in

water during transit or storage.

6.2 Holding Time Limitations

Although samples should be examined as

soon as possible afte'r collection, they should

not be held longer than six hours between

collection and initiation of analyses (14). This

limit is applied to fresh waters, seawaters and

shellfish-bed waters. The exception is water

supply samples mailed in from water treat-

ment systems. Current regulations permit

these samples to be held up to 30 hours.

6.2.1 Despite the establishment of a six

hour limit, sewage samples, organically-rich

wastes and marine waters are particularly

susceptible to rapid increases or die-away and

hence should be held for the shortest time

possible to minimize change.

6.2.2 Temporary Field Laboratories: In sit-

uations where it is impossible to meet the six

hour maximum holding time between collec-

tion and processing of samples, the use of

temporary field laboratories located near the

collection site should be considered.

6.2.3 Delayed Incubation Procedure: If

sampling and transit conditions require more

than six hours, and the use of field laboratories

is impossible, the delayed incubation proce-

dure for total and fecal coliforms and fecal

streptococci should be considered.

6.2.4 Public Transportation: Occasionally,

commercial forms of transit such as airlines,

buslines or couriers can be used to transport

samples contained in ice chests to the labora-

tory. These should be considered only when

storage time and temperature requirements

and the proper disposition of the samples can

be assured.

&EFA MICROBIOLOGICAL MANUAL 1978

image:

REFERENCES

1, Public Health Laboratory Service Water Subcommittee, 1953. The effect of sodium thiosulfate

on the coliform and Bacterium co//counts of non-chlorinated water samples. J. Hyg. 51:572.

2. Shipe, E. L and A. Fields, 1954. Comparison of the molecular filter technique with agar plate

counts for enumeration of Escherichia cof/'m various aqueous concentrations of zinc and copper

sulfate. Appl. Microbiol. 2:382.

3. Shipe, E. L. and A. Fields, 1956. Chelation as a method for maintaining the coliform index in water

supplies. Public Health Rep. 71:974.

4. Zobell, C. E., 1941. Apparatus for collecting water samples from different depths for bacteriologi-

cal analysis. J. Marine Research 4:173.

5. Niskin, S., 1962. Water sampler for microbiological study. Deep Sea Research 9:501.

6. Funs, G.Wolfgang, 1977. Personal Communication; Director of the Environmental Health Center,

Division of Lab and Research, New York State Health Department, Albany, New York.

7. Welch, P.S. 1948. Limnological Methods. Blakiston Company, Philadelphia, PA.

8. Van Donsel, D. J. and E. E. Geldreich, 1972. Relationships of Salmonella to fecal coliforms in

bottom sediments. Water Research 5:1079.

9. Wills, Carroll, 1975 (June). Chain of Custody Procedures, National Enforcement Investigation

Center-Denver, Colorado, U.S. EPA.

10. Guidelines Establishing Test Procedures for the Analysis of Pollutants. 40 CFR Part 136, 52780,

as amended, December 1,197 6.

11. National Interim Primary Drinking Water Regulations, 40 Code of Federal Regulations, Amend-

ments to Part 141, December 24,1975.

12. Geldreich, E. E., 1975. Handbook for Evaluating Bacteriological Water Laboratories, (2nd ed.)

U.S. Environmental Protection Agency, Municipal Environmental Research Laboratory, Cincin-

nati, Ohio. EPA-670/9-75-006.

13. Mauser, L. S. (ed.), 1965. National Shellfish Sanitation Program. Manual of Operations, Part h_

Sanitation of shellfish growing areas. U.S. Public Health Service, Washington, D.C.

14. Public Health Laboratory Service Water Subcommittee, 1953. The effect of storage on the

coliform and Bacterium coli counts of water samples. Storage for six hours at room and

refrigerator temperatures. J. Hyg. S|:559.

SAMPLING TECHNIQUES 31

image:

PART II. GENERAL OPERATIONS

Section B Laboratory Equipment, Techniques and Media

Most equipment and supplies described in

This Manual are available in weH-equipped

bacteriology laboratories. Other items specific

for the membrane filter or multiple-tube dilu-

tion methods are used only in water laborator-

ies. This Section describes the required equip-

ment, media and preparation techniques used

in the laboratory or in the field. The contents

include:

1. Equipment and Supplies

2. Cleaning Glassware

3. Sterilization

4. Preparation and Use of

Culture Media

5. Media Composition

General Use Media

MF Media for Coliforms

MPN Media for Coliforms

Media for Fecal Streptococci

Media for Salmonella and

Other Enterics

Medium for Actinomycetes

6. Laboratory Pure Water

7. Dilution Water

1. Equipment and Supplies

1.1 Inoculating Needles and Loops:

Needles and loops of nichrome, platinum or

platinum-indium wire are used to transfer

microbes aseptically from one growth medium

to another. A 24-26 gauge nichrome wire is

recommended. The loop diameter should be at

least 3 mm. They are sterilized by heating to

redness in a gas flame or in an electric

incinerator. Resterilization is not required for

replicate transfers of the same bacteria or

bacteria-containing materials to a sterile

medium. Sterile, disposable hardwood

applicator sticks or plastic loops can be used

to inoculate fermentation tubes.

1.2 Plastic petri dishes (50 x 12mm) with

tight-fitting lids are preferred for MF proce-

dures because they retain humidity and are

more practical for use in plastic bags sub-

mersed in a water bath incubator. Petri dishes

(60 x 15 mm) with loose-fitting lids can be used

in incubators with controlled humidity or in

plastic boxes with tight covers, containing

moist towels.

1.3 Incubators: Incubators are constant

temperature air chambers or water baths

which provide controlled temperature environ-

ment for microbiological tests. The incubator

must control temperature within specified

tolerance limits of the tests. The air type

incubator can be water-jacketed, a dry air unit

or aluminum block equipped with thermostat-

controlled heating units that maintain 35 C ±

0.5 C. Overcrowding of incubators with plates

arid tubes must be avoided, because this will

interfere with the constancy of the desired

<&ERA MICROBIOLOGICAL MANUAL 1978

image:

temperature. Water bath or aluminum block

type incubators used in fecal coliform tests

must control the incubation temperature of

44.5 C±0.2 C. Covers are required. An accurate

thermometer in the water bath monitors tem-

perature. Recording thermometers are recom-

mended for use in the water baths.

1,4 Colony Counter: A standard counting

device equivalent to the Quebec Colony Counter

that provides good visibility and magnification

of at least 1.5 diameters on a non-glare ruled

guide plate is recommended.

1.5 pH Equipment; ElectrometricpH meter

must be accurate to at least 0.1 pH unit.

1.6 Balances: For routine weighing of

media and reagents, use a single pan top loader

balance having a sensitivity of 0.1 gram at a

load of 150 grams. For weighings of less than 2

grams, use a four place analytical balance

having a sensitivity of 1 mg at a load of 10

grams.

1.7 Media Preparation Utensils: Use suit-

able non-corrosive utensils of plastic, glass,

stainless steel, or non-chipped enamel. Utensils

must be chemically clean before use to prevent

contamination of media with chemicals such as

chlorine, copper, zinc, chromium and de-

tergents.

1.8 Pipets and Graduated Cylinders

1.8.1 Pipets: Because transfer pipets are an

important element in any microbiological

method, they must be properly used. If mouth-

pipetting is practiced for non-polluted waters,

pipets should be cotton-plugged for safety.

Blow-out pipets are not to be used.

The major types of transfer pipets (non-

volumetric) used in the laboratory are based

on the method of draining:

'a) l£ Contain (TC) Pipets: Pipets calibrated

to hold or contain the exact amount specified

by the calibration. Pipet must be completely

emptied to provide the stated volume. Ex-

amples: Transfer Micro and Dual Purpose

Pipets.

fa) Jjo Deliver (TD) Pipets: Pipets designed

to release the exact calibrated amount when

the pipet tip is held vertically against the re-

ceiving vessel wall until draining stops. Exam-

ples: bacteriological, Mohr, serological, and

volumetric pipets.

To Contain and To Deliver pipets are indic-

ated by the letters TC or TD marked respec-

tively on the neck with other calibration

information.

(G) Blow-Out Pipets: These pipets are in-

tended for rapid use in serology and are emp-

tied by forceful blow-out. Because the blow-

out action always produces an aerosol such

pipets should never be used in the microbiol-

ogy laboratory. The pipets do not deliver the

calibrated amount until they are completely

emptied.

Blow-Out pipets are marked with a double

band etching or fired-in marking on the neck.

(d) Dual Purpose Pipets: A new pipet which

combines three calibrations. The pipet has an

upper graduation mark which is the To Deliver

and Blow-Out line, a lower graduation which is

the To Contain line, and carries the double

band for Blow-Out

For microbiological analyses, only bacteri-

ological, serological or Mohr pipets are recom-

mended for use i£ the To Deliver (TD) mode.

Dual purpose or serological pipets which must

be used as blow-out pipets, are not recom-

mended for microbiology because of the dan-

ger of infection through aerosol and the possi-

ble mix-up of TD, TC and blow-out type pipets

with subsequent misuse and improper

delivery.

(e) Pipet Standards/Specifications: There

are several tolerance specifications used to

characterize measuring pipets. The analyst

should be aware of these limits. When accu-

racy of measurements is critical, use only pi-

pets within the following Class A limits:

NBS Specification for Mohr Measuring Pi-

pets, Class A Volume Tolerances Circular 602

and Federal Specification NNN-P-350

EQUIPMENT, TECHNIQUES AND MEDIA

image:

Capacity/Grad (ml)

1/10 in 1/100

2/10 in 1/100

1 in 1/100

1 in 1/10

2 In 1/10

5 In 1/10

10 in 1/10

26 In 1/10

Tolerance (ml)

±0.0025

±0.004

±0.01

±0.02

±0.03

±0.05

For routine use, the following tolerances

are acceptable:

APHA Specification, Bacteriological Pipets

Capacity/Grad (ml)

Tolerance (ml)

1 in 1

1,1 in 1.1, 1.0

2.2 in 2.2, 2.1

2.0, 1.0

11 in 11

±0.025

+0.040

±0.10

USPHS Specification, Bacteriological Pipe'ts

for Water Analyses

Capacity/Grad (ml)

11 in 11, 10, 1

Tolerance (ml)

±0.06

1-8.2 Pipetting Devices: Mechanical pipet-

ting devices are recommended for all purposes.

Although several devices have recently be-

come available, some are not practical for

water analyses because they are too slow and

cumbersome. A finger-mounted safety pipet-

tor which can be fabricated in the laboratory

(1, 2) is recommended as the most efficient It

can be fitted with a mouthpiece, hand-held

bulb or attached to a vacuum pump. Figure II-B-

1 shows the finger-held model and Figure II-B-

2 shows an enlargement of the plastic ring

suction device.

1.8.3 Graduates: For normal laboratory

operations, graduates are used for measuring

volumes greater than 10 ml. When extreme

accuracy is required above 10 ml, volumetric

pipets up to 200 ml and volumetric flasks from

10 ml to 6 liters are available. The tolerances

given below are acceptable for graduates.

1.9 Pipet Container: Only aluminum,

stainless steel, pyrex glass or other non-

corrosive heat-resistant containers, either cyl-

indrical or rectangular in shape, should be

used. Pipets may be also wrapped in kraft

paper. Copper or copper alloy containers must

not be used.

1.10 Dilution (Milk Dilution) Bottles or

Tubes: Borosilicate or other non-corrosive

glass bottles with screw-caps and inert liners.

1.11 Fermentation Tubes and Vials: The

fermentation tubes should be large enough to

contain the media and inocula in no more than

half of the tube depth. There should be suffi-

cient media in the outer fermentation tube to

fill the enclosed inverted vial after sterilization

and partially submerge it

NBS Specification, Tolerance of Graduates, in ml

Volume

Demarcations

NBS Class A

NNN-C-940

Type 1 Style 1

2000

1000

500

250

100

+ 1.3

±0.8

±0.4

±0.26

±10.0

±5,0

±2.6

+ 1.4

±0.6

V>EFV\ MICROBIOLOGICAL MANUAL 1978

image:

EQUJPMENT'

image:

1.12 Sample Bottles: Sample bottles

should be borosilicate glass or plastic, resis-

tant to sterilizing conditions and the solvent

action of water and which do not produce toxic

substances upon sterilization. Wide-mouth

ground-glass stopper or screw-cap bottles are

acceptable. Bottles equipped with screw-caps

are acceptable provided that bacteriostatic or

nutritive compounds are not produced from

caps or liners. Bottles should be at least 125

ml volume.

2. Cleaning Glassware

In microbiology, clean glassware is crucial

to insure valid results. Previously used or new

glassware must be thoroughly cleaned with a

phosphate-free laboratory detergent and hot

water, then rinsed repeatedly with hot water,

followed by at least three rinses with labora-

tory pure water. To determine whether the

detergent used contains inhibitory residues,

the test procedure for detergent suitability

should be performed each time a new type

detergent is purchased. See Part IV-A, 5.1.

3. Sterilization

Sterilization is the process that eliminates

living organisms from treated substances or

objects. Disinfection is the destruction or

removal of the infectious agents by chemical

or physical means. Usually chemical agents

are used as disinfectants (germicide and bac-

tericide are synonymous with the term, disin-

fectant). Pasteurization is a form of disinfection

used for materials which may be altered or

damaged by excessive heat. Low heat is applied

once or repeatedly to sensitive liquids to destroy

vegetative cells. Sterilization can be accom-

plished by moist heat, dry heat, incineration,

filtration, radiation or by the use of the chemical

agent, ethylene oxide. Bottles should have loos-

ened caps for penetration of steam or gas.

3.1 Moist Heat: The autoclave is used in

the laboratory for moist heat sterilization. It is

normally operated at 15 Ibs. per sq. in. steam

pressure for 15 minutes, producing a tempera-

ture inside the autoclave of 121.6 C (250F) at

sea level. Steam under pressure provides

effective sterilization since it has good penetra-

tion power and coagulates microbial proto-

plasm. The temperature in the autoclave should

be monitored. The relationship between the

pressure of steam and the temperature is

shown in Table II-B-1. The autoclave is used to

sterilize solid and liquid media, contaminated

materials, discarded cultures, glassware of all

types, filtering units, etc. Pressure cookers and

vertical autoclaves are not recommended.

3.2 Dry Heat; The hot-air oven is used to

sterilize glassware such as petri dishes, pipets,

sample bottles and flasks, hardwood applicator

sticks, and other articles, but not liquids or

materials which will evaporate or deteriorate.

Since moisture is not present in the oven, a

temperature of 165-170 C (329-338 F) is re-

quired for a 2 hour period.

3.3 Incineration: Contaminated materials

that are combustible may be disposed of by

burning. This method is also used for steriliz-

ing inoculating needles and loops, and flaming

the lips of test tubes and flasks.

3.4 Filtration. Filtration is used to sterilize

liquids that are heat-sensitive. Filters include

those composed of: asbestos-cellulose (Seitz

filter), cellulose esters (0.22/tm MF or molecu-

lar filter), unglazed porcelain (Chamberland-

Pasteur filter), or diatomaceous earth (Berkfeld

filter). Although filters are normally used

to remove bacteria, porosities small enough

.for the removal of viruses are available.

3.5 Ultraviolet Radiation: Ultraviolet light

includes radiations between 150 and 4000

Angstrom units (A.U.), but radiations less than

1800 A.U. are absorbed by atmospheric oxy-

gen. The greatest killing effect on microorga-

nisms occurs at 2600 A.U. Commercial germi-

cidal ultraviolet lamps emit primarily 2537

A.U. which has 85 percent of the germicidal

ability of 2600 A.U. Ultraviolet radiation at the

germicidal wavelength is used to sterilize labo-

ratory equipment such as membrane filter

units, inoculating rooms, bacteriological

hoods and glove boxes. See Part IV-A, 4.2, for

monitoring procedures.

3.6 Ethylene Oxide Chemical

Sterilization: Low temperature ethylene oxide

&ERA MICROBIOLOGICAL MANUAL 1978

image:

TABLE li-B-1

Relationship of Steam Pressure to Temperature in the Autoclave

Pounds of pressure of steam

per square inch

Corresponding Temperature

In Degrees*

Celsius

Fahrenheit

1OO.O

108.3

115.5

121.6

126.6

130.5

134.4

212

227

240

250

260

267

274

•Correct at sea level and only if all air is evacuated from the sterilizing chamber since a mixture

of steam and air at a given pressure gives a temperature that is less than that of pure steam only.

EQUIPMENT, TECHNIQUES AND MEDIA

image:

gas sterilization is used to sterilize plastics,

rubber goods, delicate instruments and other

materials that would be damaged by the high

temperature of the steam pressure autoclave.

Vent sterilized materials according to operating

instructions.

4, Preparation and Use of Culture Media

The preparation of culture media and solu-

tions is a critical aspect of water quality testing.

In many laboratories media are prepared by

nonprofessional personnel. If such personnel

are properly trained and guided, they can

perform the required tasks efficiently and

reliably. However, the supervisor should

monitor media preparation to maintain quality

control. Use commercial dehydrated media

which require only weighing and dissolving of

the powder in laboratory pure water for prepa-

ration and are much more likely to have uniform

high quality than media compounded in the

laboratory. See IV-A, 7.3-7.5 for quality control

on preparation of media.

4.1 Supplies of Dehydrated Media: The

laboratory worker should keep a record of the

lot numbers of commercial media in use. The

data of receipt of media and the date of open-

ing should be recorded in the quality control

log. It is suggested that supplies of dehydrated

media be purchased for anticipated use over

the next year. Whenever practical, one-quarter

pound bottles should be purchased to insure

minimal exposure of contents to atmosphere.

When a new lot number of medium is used, the

contents should be tested for expected per-

formance characteristics {see Part IV-A for

details). Stocks of dehydrated media should be

stored in a cool, dry place away from sunlight.

4.2 Rehydration of Media: In this Manual,

dry ingredients are added to 1 liter (1000 ml)

of laboratory pure water. However, liquids

such as ethanol are added to graduate and

bought up to volume with laboratory pure

water. Care must be taken to completely dis-

solve and mix the ingredients before dispensing

the medium into bottles, tubes or flasks. If

heat is necessary, it must be applied with cau-

tion and for the shortest possible time. Direct

heat, boiling water bath and flowing steam are

used selectively according to the type and

volume of medium as described below:

4,2.1 MF Broths and Agars: Small volumes

of broth are rehydrated in a boiling water bath

for 5 minutes, Agars and larger volumes of

broth require direct heating to the first bubble

of boil. Such heating must be applied with

stirring and constant attention until agar is

dissolved.

4.2.2 Other Broths: Some heat may be

required to dissolve the ingredients prior to

autoclaving. Heat can be applied by flame or

waterbath.

4.2.3 Other Agars: Heat is required for

complete solution. For large volumes, direct

heat will effect solution more rapidly but must

be applied with stirring and constant attention

to prevent scorching.

4.3 Sterilization: Media must be dissolved

before autoclaving, to insure timing for com-

plete sterilization. The specific recommenda-

tions for sterilization are described in subsec-

tion 5, Composition of Media, in This Section.

The following general recommendations

can be made:

Group

Sterilization

MF and Salmonella

Media

Heat to boiling only

Media Containing

Blood

Autoclave, cool,

then add blood

Litmus Milk and

Other Milk-con-

taining Media

115 C for 20

minutes

{10 Ibs. pressure)

Most Carbohydrate-

Containing Media

for Fermentation

Reactions such as

Phenol Red Broth,

Triple Sugar Iron

Agar

118 C for 15

minutes

(12 Ibs. pressure)

121 C for 12 minutes

MICROBIOLOGICAL MANUAL 1978

image:

MPN, some Salmonella

Media and General

Use Media such as

Trypticase Soy Broth

and Agar, Nutrient

Broth and Agar

121 Cfor

12-15 minutes

(15 Ibs. pressure)

As soon as the autoclave pressure has

fallen to zero, the sterilized media should be

removed from the autoclave for cooling before

use or storage. Refrigerated media should be

allowed to come to room temperature before

use. Incubate MPN tubed media overnight and

discard tubes showing bubbles. Media that

have been poured into petri dishes should be

used on the day of preparation or refrigerated.

Water loss from evaporation can be prevented

by storing plates in plastic bags. Plates should

be stored inverted. See Part IV-A, 7.9 for stor-

age limits.

Autoclaves should be inspected routinely

to insure proper functioning of pressure

gauges and .thermometers. The use of com-

mercially available temperature indicator de-

vices within the sterilizer (heat-resistant spore

preparations, chemically-impregnated tapes,

vials containing chemicals, etc.) is recom-

mended to insure sterility (see Part IV-A for

details).

5. Composition of Media

The formulas for media used in this Man-

ual and for other more commonly-used media

are given in detail in this Section. Normally,

these media should not be prepared from basic

ingredients when suitable commercial media

are available.

The catalogue numbers cited are those for

y* Ib. size if available, from the two U.S. manu-

facturers whose media were used in most of

the method development work. This listing is

not restrictive; other sources can be utilized if

the user confirms that the formulas are the

same as those cited here and that the media

produce comparable results.

Culture media used for the examination of

water and wastewaters are described sepa-

rately for:

5.1 General Use Media

5.2 MF Media for Coliforms

5.3 MPN Media for Coliforms

5.4 Media for Fecal Streptococci

5.5 Media for Sa/moneffaand

Other Enterics

5.6 Medium for Actinomycetes

5.1 General Use Media

Nutrient Agar (Difco 0001-02, BBL

11471)

Use: This medium is used to cultivate pure

culture isolates for subsequent gram stain and

other examinations and for general cultivation

of microorganisms that are not fastidious.

Composition:

Peptone

Beef Extract

Agar

Final pH: 6.8 ±.1

5.0

3.0

15.O

Preparation: Add 23 grams of nutrient

agar per liter of laboratory pure water and

mix well. Heat in boiling water bath to dissolve

the agar completely. Dispense in screw-cap

tubes, bottles or flasks and sterilize for 15

minutes at 121 C (15 Ibs. pressure). Remove

tubes and slant.

5-1 -2 Nutrient Broth (Difco 0003-02, BBL

11478)

Use: General laboratory use for the cultiva-

tion of non-fastidious microorganisms.

Composition:

Peptone

Beef Extract

Final pH: 6.8±.1

5.0 g

3.0 g

EQUIPMENT, TECHNIQUES AND MEDIA

image:

Preparation: Add 8 grams of nutrient broth

per liter of laboratory pure water and warm to

dissolve the medium completely. Dispense in

containers and sterilize for 15 minutes at 121

C(15 Ibs. pressure).

5.1.3 Trypticase Soy Agar (BBL 11042)

Tryptic Soy Agar(Difco 036902)

Use: A general purpose medium for the

cultivation of fastidious microorganisms. An

excellent blood agar is prepared by adding

sheep blood.

Composition:

Trypticase Peptone or

Tryptone

Phytone Peptone or

Soytone

Sodium Chloride

Agar

FinalpH:7.3 ±.2

15.0 g

5.0

15.0

Preparation: Add 40 grams of TS agar per

liter of laboratory pure water and mix well.

Heat in a boiling water bath to dissolve agar

completely. Dispense into tubes, bottles or

flasks and sterilize for 15 minutes at 121 C (15

Ibs. pressure).

For blood agar, cool the sterile, melted

agar to 45-46 C and add aseptically 5 ml of

sterile defibrinated sheep blood for each 100

ml of agar. Mix flask of agar by swirling and

dispense into petri dishes. Blood from other

species may be used for particular purposes.

5.1.4 Tryptic Soy Broth (Difco 0370-02

Trypticase Soy Broth (BBL 11767)

Use: A general purpose medium for the

cultivation of fastidious microorganisms.

Composition:

Tryptone or Trypticase

Peptone

Soytone or Phytone

Peptone

Sodium Chloride

17.0 g

3.0 g

5.0 g

Dextrose 2.5 g

Dipotassium Phosphate 2.5 g

Final pH: 7.3 ±,2

Preparation: Add 30 grams of TS broth per

liter of laboratory pure water. Warm the broth

and mix gently to dissolve the medium com-

pletely. Dispense and sterilize for 15 minutes

to 121 C (15 Ibs. pressure).

5-'1-5 Standard Methods Agar (BBL

11637) Plate Count Agar (Difco 0479-02)

(Tryptone Glucose Yeast Agar)

Use: Standard Plate Counts in water and in

general pour plate procedures.

Composition

Tryptone or Trypticase

Peptone 5.0 g

Yeast Extract 2.5 g

Dextrose 1.0 g

Agar 15.0 g

Final pH: 7.0 ±.2

Preparation: Add 23.5 grams of tryptone

glucose yeast agar per liter of laboratory pure

water. Mix well and heat in boiling water bath

to dissolve agar completely. Dispense into

screw-cap tubes, flasks or bottles and sterilize

for 15 minutes at 121 C (15 Ibs. pressure).

5-1-6 Phenol Red Broth Base (Difco

0092-02, BBL 11505)

Use: Phenol red broth base with the addi-

tion of carbohydrates is used in fermentation

studies of microorganisms because its pH

range of 6.9-8.5 indicates slight changes

toward acidity. Although 0.5-1.0%

carbohydrates have been used, the 1.0 per-

cent level is recommended to prevent reversal

of the reactions.

«»EPA MICROBIOLOGICAL MANUAL 1978

image:

Composition:

Dif co (0092-02)

Beef Extract

Proteose Peptone No. 3

Sodium Chloride

Phenol Red

BBL (11505)

Trypticase Peptone

Sodium Chloride

Phenol Red

Final pH: 7.4+ .2

1.0

10.0

5.0

0.018

10.0

5.0

0.018

Preparation: Add 15-16 grams (de-

pending on manufacturer) of phenol red broth

base per liter of laboratory pure water. Mix

well to dissolve. To this solution, add 10 grams

of test carbohydrate if heat-stable. Mix to com-

plete solution. Distribute the medium in fer-

mentation tubes and sterilize not more than 15

minutes at 118 C (12 Ibs. pressure). Heat-

sensitive carbohydrates or alcohols are filter-

sterilized and added to the sterile medium

tubes. Check pH and adjust if necessary with

0.1 N NaOH after addition and solution of the

carbohydrate.

5-1-7 purPle Broth Base (Difco 0227-02,

BBL 11558)

Use: For preparation of carbohydrate broths

in fermentation studies, for the cultural identi-

fication of pure cultures of microorganisms,

particularly the fecal streptococci. Although

0.5-1.0 percent carbohydrate has been used,

1.0 percent is recommended rather than 0.5

percent to prevent reversal of the reaction.

BBL product does not contain beef extract.

Composition:

Proteose Peptone No. 3

or Peptone

Beef Extract

Sodium Chloride

Brom Cresol Purple

Final pH: 6.8 ±.2

10.0

1.0

5.0

0.015

Preparation: Add 15-16 grams (depending

on manufacturer) of purple broth base per liter

of laboratory pure water and mix to dissolve.

Add 10 grams of the test carbohydrate and dis-

solve. Dispense in screw-cap fermentation

tubes and sterilize for not more than 15 minutes

§! Hi C (12 Ibs. pressure).

Ten percent solutions of the following car-

bohydrates are prepared for differentiation of

fecal streptococci.

L-Arabinose (Difco 0159, BBL 11960}

Raffinose (Difco 0174, BBL 12060)

D-SorbitoI (Difco 0179)

Glycerol (Difco 0282)

Lactose (Difco 0156,BBL11881)

Inositol(Difco0164)

L-Sorbose (Merck 7760)

Sterilize heat-labile carbohydrates and

alcohols by passage through a sterile 0.22 ,um

membrane filtration unit. The carbohydrates

made up as 10% solutions are sterilized and

added to the sterile base medium at a 1%

concentration, wt/unit volume. Check pH and

adjust if necessary with 0.1 N NaOH after

addition of carbohydrate. Careful use of asep-

tic techniques is necessary to prevent contami-

nation. As a QC check for contamination, incu-

bate the prepared tubes for 24 hours at 35 C.

5.1.8 Purple Broth Base with Sorbose, pH

(Medium may not be commercially

available).

Use: To determine the presence of Group

Q Streptococci.

Composition:

Same as purple broth base but add 1 %

sorbose and adjust pH to 10.0.

Final pH: 10.0+ .2

Preparation: Prepare and sterilize purple

broth base as in 5.1.7. Add sufficient volume

of a 10% filter-sterilized solution of sorbose to

produce a 1 % final concentration of sorbose.

Adjust pH to 10.0 with sterile 38% sodium

phosphate solution (Na3P04 • 12 H20).

EQUIPMENT, TECHNIQUES AND MEDIA

image:

5.1.9 IMViC Test Media

Use: Differentiation of the coliform group

based on indole production from tryptophane

broth, acid production in a glucose broth indic-

ated by methyl red color change, formation of

acetoin (actylmethylcarbinol) in salt peptone

glucose broth and use of citrate as the sole

carbon source.

(a) Tryptone 1% (Difco 0123-02) Trypto-

phane Broth (BBL 11920)

Use: For the detection of indole as a by-

product of the metabolism of tryptophane and

for the identification of bacteria.

Composition:

Tryptone or Trypticase

Peptone

Final pH: 7.2 ±.2

10.0 g

Preparation: Add 10 grams of Tryptone or

Trypticase to 900 ml of laboratory pure water

and heat with mixing until dissolved. Bring

solution to 1000 ml in a graduate or flask.

Dispense in five ml volume tubes and sterilize

for 15 minutes at 121 C (15 Ibs. pressure).

(b) MR-VP Broth (Buffered Glucose) (BBL

11382, Difco 0016-02)

Use: For the performance of the Methyl

Red and Voges-Proskauer Tests in differentia-

tion of the coliform group.

Composition:

Buffered Peptone or

Polypeptone

Dextrose

Dipotassium Phosphate

Final pH: 6.9 ± .2

7.0

5.0

Preparation: Add 17 grams of MR-VP me-

dium to 1 liter of laboratory pure water. Mix to

dissolve. Dispense 10 ml volumes into tubes

and sterilize for 15 minutes at 121 C (15 Ibs.

pressure).

Difco 0091-02)

Use: Differentiation of gram-negative en-

teric bacteria on the basis of citrate utilization.

Composition:

Magnesium Sulfate 0.2 g

Monoammonium Phosphate 1.0 g

Dipotassium Phosphate 1.0 g

Sodium Citrate 2.0 g

Sodium Chloride 5.0 g

Brom Thymol Blue 0.08 g

Agar 15.0 g

Final pH: 6.8 ±.2

Preparation: Add 24.2 grams of Simmons

Citrate agar per liter of laboratory pure water.

Heat in boiling water bath with mixing for

complete solution. Dispense into screw-cap

tubes and sterilize for 15 minutes at 121 C

(15 Ibs. pressure). Cool tubes as slants.

5.1.10 Motility Test Medium (Edwards and

Ewing)(BBL 11435)

Use: Detection of motility of gram-negative

enteric bacteria.

Composition:

Beef Extract

Peptone

Sodium Chloride

Agar

Final pH: 7.3 ±,2

3.0 g

10.0 g

5.0 g

4.0 g

Preparation: Add 22 grams of dry medium

to 1 liter of laboratory pure water. Add 0.05

grams of triphenyl tetrazolium chloride/liter.

Heat with mixing to boil for 1 minute. Dispense

10 ml volumes into tubes and sterilize for 15

minutes at 121 C (15 Ibs. pressure).

5.2 MF Media for Coliforms

Prepare heat-sensitive broths in sterile

flasks.

MICROBIOLOGICAL MANUAL 1978

image:

5.2.1 M-FC Broth Base (Difco 0883-02)

M-FC Broth (BBL 11364)

Use: Detection and enumeration of fecal

coliform microorganisms by the membrane fil-

ter procedure.

Composition:

Tryptose or Biosate

Peptone 10.0 g

Proteose Peptone No. 3

or Polypeptone 5.0 g

Yeast Extract 3.0 g

Sodium Chloride 5.0 g

Lactose 12.5 g

Bile Salts No. 3 or

Bile Salts Mixture 1.5 g

Aniline Blue 0.1 g

Final pH: 7,4 ± .2

Preparation:

Rosolic Acid

Dissolve 1 gram of rosolic acid in 1OO ml

of 0.2 N sodium hydroxide to make a rosolic

acid solution. Note: The quality of present sup-

plies is quite variable. Performance of new

batches should be compared against previous

batch before it is exhausted.

Autoclaving will decompose rosolic acid

reagent. Stock solutions should be stored in

the dark at 4 C. Discard after 2 weeks or sooner

if the color changes from dark red to muddy

brown. The rosolic acid may be omitted from

testing samples with stressed organisms and

low background count.

M-FC Broth

Add 37 grams of M-FC medium to 1 liter of

laboratory pure water containing 10 ml of the

rosolic acid solution. Heat in a boiling water

bath to dissolve before use. Store the prepared

medium at 4 C in a refrigerator. Discard unused

medium after 96 hours.

M-FC Agar

Prepare by adding 15 grams of agar per

liter of M-FC broth. Heat in boiling water bath

to solution then cool to about 45 C and add to

50 mm diameter glass or plastic petri dishes to

a minimal agar depth of 2-3 mm. Allow to

solidify. Protect the prepared medium from

light. It can be stored at 4 C for up to 2 weeks.

Caution: Do not autoclave M-FC Broth or

Agar.

5-2-2 M-Coliform Broth (BBL 11119) M-

Endo Broth MF (Difco 0749-02)

Use: A selective and differential medium

for enumeration of members of the coliform

group by the membranefiltertechnique.

Composition:

Tryptose or Polypeptone

Thiopeptone or Thiotone

Casitone or Trypticase

Yeast Extract

Lactose

Sodium Chloride

Dipotassium Hydrogen

Phosphate

Potassium Dihydrogen

Phosphate

Sodium Lauryl Sulfate

Sodium Desoxycholate

Sodium Sulfite

Basic Fuchsin

Final pH: 7.2+ .2

10.0

5.0

1.5

12.5

5.0

4.375 g

1.375

0.050

0.1

2.1

1.05

Preparation: Add 48 grams of M-Endo

medium to 1 liter of laboratory pure water

containing 20 ml of 95% ethanol. Denatured

alcohol should not be used. Heat in boiling

water bath for solution. Store prepared medium

in the dark at 4 C. Discard the unused medium

after 96 hours.

Prepare M-Endo agar by adding 15 grams

of agar per liter of M-Endo medium. Heat to

boiling, cool to about 45 C and dispense into

glass or plastic petri dishes to provide minimal

agar depth of 2-3 mm and allow to solidify.

Protect prepared medium from light. It can be

stored at 4 C for up to 2 weeks.

EQUIPMENT, TECHNIQUES AND MEDIA

image:

5.2,3 M-Endo Holding Medium

Composition:

Use: Holding Medium in the delayed incu-

bation total eoliform procedure.

Composition:

Same as M-Endo Broth but add 0.384

grams of sodium benzoate per 100 ml.

Preparation:

Prepare M-Endo Broth as described in

5,2.2 and add 3.2 ml of a 12% solution of

sodium benzoate per 100 ml of medium. Add

cycloheximide If needed.

Sodium Benzoate Solution

Final pH: 7.2 ± .2

Dissolve 12 grams of sodium benzoate in

about 85 ml of laboratory pure water, then

bring to 100 ml final volume. Sterilize by auto-

claving or filtration. Discard the solution after

6 months.

Cycloheximide Solution (Optional)

Cycloheximide is used for samples that

have shown problems of overgrowth with

fungi. Prepare an aqueous solution containing

1.25 grams of cycloheximide/100 ml of labora-

tory pure water. Store solution in refrigerator.

Discard and remake after 6 months. Add 4 ml

of the aqueous solution of cycloheximide per

100 ml of M-Endo Holding Medium.

Caution: Cycloheximide is a powerful skin

irritant that should be handled with care. See

Manufacturer's Directions (Actidione, Upjohn,

Kalamazoo, Ml).

5.2.4 M-Endo Agar LES (Difco 0736-02,

BBL11203)

Use: Determination of total conforms us-

ing a two-step membrane filter technique with

lauryl tryptose broth as the preliminary

enrichment.

Yeast Extract 1.2 g

Casitone or Trypticase Peptone 3.7 g

Thiopeptone or Thiotone 3.7 g

Tryptose or Biosate Peptone 7.5 g

Lactose 9.4 g

Dipotassium Hydrogen

Phosphate 3.3 g

Potassium Dihydrogen

Phosphate 1.0

Sodium Chloride 3.7

Sodium Desoxycholate 0.1

Sodium Lauryl Sulfate 0.05

Sodium Sulfite 1.6

Basic Fuchsin 0.8

Agar 14-15.0

Final pH: 7.2 ±.2

Preparation: Add 50 or 51 grams, depend-

ing on manufacturer, of agar per liter of labora-

tory pure water to which has been added 20 ml

of 95% ethanol. Heat in boiling water bath

to dissolve completely. Cool to about 45 C

and dispense into 60 mm glass or plastic petri

dishes to provide a minimal agar depth of 2-3

mm. Allow to solidify. If larger dishes are used,

dispense sufficient agar to give equivalent

depth. Protect prepared medium from light. It

can be stored at 4 C for up to 2 weeks.

Caution: Do not autoclave.

5.2.5 M-Coliform Holding Broth (Difco

0842-02) (LES Holding Medium)

Use: Holding medium in the delayed-

incubation total eoliform procedure.

Composition:

Tryptone or Trypticase

Peptone

M-Endo Broth MF

Dipotassium Hydrogen

Phosphate

Sodium Benzoate

Sulfanilamide

Paraminobenzoic Acid

Cycloheximide

3.0

3.0 g

1.0 g

1.2 g

0.5 g

«-EPA MICROBIOLOGICAL MANUAL 1978

image:

Final pH: 7.1 ±.2

Preparation: Add 12.7 grams per liter of

laboratory pure water and mix to dissolve. Do

not heat to dissolve medium.

5.2.6 M-VFC Holding Medium (3)

Use: Holding medium in the delayed incu-

bation test for fecal coliform microorganisms.

Composition:

Casitone, Vitamin Free

Sodium Benzoate

Sulfanilamide

Final pH: 6.7 ±.2

0.2

4.0

0.5

Preparation: Add 4.7 grams of medium

per liter of laboratory pure water containing

10 ml of 95% ethanol. Denatured alcohol

should not be used. Heat slightly to dissolve

ingredients, then sterilize by membrane filtra-

tion (0.22 urn). Store prepared medium at 4 C.

Discard after 1 month.

5.3 MPN Media for Coliforms

5.3.1 Lauryl Sulfate Broth (BBL 11338)

Lauryl Tryptose Broth (Difco 0241 -02)

Use: Primary medium for the Presumptive

Test for the tola I coliform group.

Composition:

Tryptose or Trypticase

Peptone

Lactose

Dipotassium Hydrogen

Phosphate

Potassium Dihydrogen

Phosphate

Sodium Chloride

Sodium Lauryl Sulfate

Final pH: 6.8 ±.2

20.0

5.0

2.75 g

2.75

5.0

0.1

Preparation: Add 35.6 grams of the me-

dium to 1 liter of laboratory pure water and mix

to dissolve. Dispense 10 ml volumes in fer-

mentation tubes (150 x 20 mm tubes contain-

ing 75 x 10 mm tubes) for testing 1 ml or less

of samples. For testing 10ml volumes of sam-

ples, add 71.2 grams of the medium per liter of

laboratory pure water and mix to dissolve. Dis-

pense in 10 ml amounts in fermentation tubes

(1 50 x 25 mm tubes containing 75x10 mm

tubes). Sterilize for 15 minutes at 121 C (15

Ibs. pressure). The concentration of the me-

dium should vary with the size of the sample

according to the table below.

Compensation in Lauryl Tryptose Broth

for Diluting Effects of Samples

LIB Medium

/Tube in ml

Sample Size

/Dilution

in ml

0.1 to 1.0

Medium

Concen-

tration

1.5x

Dehydrated

LIB in

grams/liter

35.6

71.2

53.4

5.3.2 Brilliant Green Bile 2% (Difco

0007-02) Brilliant Green Bile Broth 2% (BBL

11079)

Use: Recommended for the confirmation

of MPN Presumptive Tests for members of the

coliform group.

Composition:

Peptone

Lactose

Oxgall or Bile

Brilliant Green

Final Ph: 7.2+ .2

10.0

20.0

0.33

Preparation: Add 40 grams of brilliant

green bile broth to 1 liter of laboratory pure

water. Dispense 10 ml volumes of the broth in

fermentation tubes (1 50 x 20 mm tubes con-

taining 75 x 10 mm tubes). Sterilize for 15

minutes at 121 C(15 Ibs. pressure).

EQUIPMENT, TECHNIQUES AND MEDIA

image:

5.3.3 Levine's Eosin Methylene Blue Agar

(Difco 0005-02, BBL 11220)

Use: Isolation of coliform-like colonies as a

preliminary to total coliform Completed Test

Procedure.

Composition:

Peptone

Lactose

Dlpotassium Phosphate

Agar

Eosin Y

Methylene Blue

Final pH: 7.1 ±.2

10.0

2.0

15.0

0.4

0.065

Preparation: Add 37.5 grams of Levine's

E.M.B. agar to 1 liter of laboratory pure water

and heat in a boiling water bath until dissolved

completely. Dispense into tubes, flasks or bot-

tles and sterilize for 15 minutes at 121 C (15

Ibs. pressure). A flocculant precipitate may

form after autoclaving. Resuspend the precipi-

tate by gently shaking the flask prior to pour-

ing the medium into sterile petri dishes.

5.3.4 EC Medium (Difco 0314-02) EC Broth

(BBL 11187)

Use: Detection and enumeration of fecal

coliform bacteria.

Composition:

Tryptose or Trypticase

Peptone 20.0 g

Lactose 5.0 g

Bile Salts No. 3 or

Bile Salts Mixture 1.5 g

Dlpotassium Phosphate 4.0 g

Monopotassium Phosphate 1.5 g

Sodium Chloride 5.0 g

Final pH: 6.9 ±.2

Preparation: Add 37 grams of EC medium

to 1 liter of laboratory pure water. Dispense

into fermentation tubes (150 X 20 mm tubes

containing 75X10 mm tubes). Sterilize for 15

minutes at 121 C (15 Ibs. pressure).

10.0

5.0

10.0

20.0

1.0

0.4

0.015

20.0

5.4 Media for Fecal Streptococci

5.4.1 KF Streptococcus Agar (Difco

0496-02) KF Streptococcal Agar (BBL 11313)

Use: Selective cultivation and enumera-

tion of fecal streptococci by direct plating or

the membranefiltertechnique.

Composition:

Proteose Peptone No. 3

or Polypeptone

Yeast Extract

Sodium Chloride

Sodium Glycerophosphate

Maltose

Lactose

Sodium Azide

Brom Cresol Purple

(in Difco medium only)

Agar

Final pH: 7.2 ±.2

Preparation: Add 76.4 grams of the me-

dium per liter of laboratory pure water. Dis-

solve by heating in a boiling water bath with

agitation. Heat in boiling water bath for 5

minutes after solution is complete. Caution: Do

not autoclave. Cool to 60 C and add 1 ml of a

filter-sterilized 1 % aqueous solution of 2, 3, 5-

triphenyl tetrazolium chloride per 100 ml of

agar. If necessary, adjust pH to 7.2 with 10%

Na2CO3. Do not hold the completed medium

(with indicator) at 44-46 C for more than 4

hours before use. Store prepared medium

(without indicator) in the dark for up to 30 days

at 4 C. TTC solution is light-sensitive. It should

be stored in the refrigerator and protected

from light.

5.4.2 Azide Dextrose Broth (Difco 0837-02,

BBL 11000)

Use: Primary inoculation medium for Fecal

Streptococci Presumptive Test.

Composition:

Beef Extract

Tryptose or Polypeptone

4.5 g

15.0 g

S»EB\ MICROBIOLOGICAL MANUAL 1978

image:

7.5

0.2

Dextrose

Sodium Chloride

Sodium Azide

Final pH: 7.2+ .2

Preparation; Add 34.7 grams of azide dex-

trose broth to 1 liter of laboratory pure water.

Dissolve and dispense into tubes without inner

vials. Note: Azide dextrose broth should be

Ibs.

sterilized at 1 18 C for

Prepare the

minutes (1 2

multiple

pressure). Prepare the medium in

strength for larger inocula to preserve the cor-

rect concentration of ingredients. For exam-

ple, if 10 ml of inoculum is to be added to 10

ml of medium, the medium should be prepared

double strength.

5.4.3 Ethyl Violet Azide Broth (BBL 11 226)

EVA Broth (Difco 0606-02)

Use: Confirmed

streptococci.

Composition:

Tryptose or Biosate Peptone

Dextrose

Dipotassium Phosphate

Monopotassium Phosphate

Sodium Chloride

Sodium Azide

Ethyl Violet

Final pH: 7.0+ .2

Test for fecal

20.0

5.0

2.7

5.0

0.4

0.83 mg

Preparation: Add 35.8 grams of the me-

dium to 1 liter of laboratory pure water. Dis-

solve and dispense in 10 ml amounts into

tubes. Sterilize for 15 minutes at 121 C(15 Ibs.

pressure).

5.4.4 PSE Agar (Pfizer Selective Entero-

coccus Agar) 224B, formerly from Pfizer Diag-

nostics Division. Now available from Grand

Island Biological Company (GIBCO), 3175 Sta-

ley Road, Grand Island, NY 14072.

Use: Isolation of fecal streptococci.

Composition:

Pfizer Peptone C

Pfizer Peptone B

Pfizer Yeast Extract

Pfizer Bile

Sodium Chloride

Esculin

Sodium Citrate

Ferric Ammonium Citrate

Sodium Azide

Agar

Final pH: 7.1 +.2

17.0

3.0

5.0

10.0

5.2

1.0

0.5

0.25

15.0

Preparation: Add 58 grams of PSE agar to

1 liter of laboratory pure water. Heat in a boil-

ing water bath to complete solution. Dispense

into tubes or flasks and sterilize for 15 minutes

at 121 C (15 Ibs. pressure).

5.4.5 Brain Heart Infusion Broth (BHI)

(Difco 0037-02, BBL 1 1058)

Use: For separation of enterococcus group

organisms from S. bovis and S. equinus by

testing for growth at 10 C and 45 C. For

general cultivation of fastidious micro^

organisms.

Composition:

Calf Brain Infusion

Beef Heart Infusion

Peptone

Sodium Chloride

Disodium Phosphate

Dextrose

Final pH: 7.4+ .2

200.0

250.0

10.0

5.0

2.5

2.0

Preparation: Dissolve 37 grams of brain

heart infusion broth in 1 liter of laboratory pure

water. Dispense in 8-10 ml volumes in screw-

cap tubes and sterilize for 1 5 minutes at 1 21 C

(15 Ibs. pressure). If the medium is not used the

same day as prepared and sterilized, heat at

100 C for several minutes to remove absorbed

oxygen, and cool quickly without agitation,

justpriorto inoculation.

5.4.6 Brain Heart Infusion Agar (Difco

0418-02, BBL 11064)

EQUIPMENT, TECHNIQUES AND MEDIA

image:

Use: Cultivation of streptococci isolates or

other fastidious bacteria.

Composition: Brain heart infusion agar

contains the same components as BHI broth

(see 5.4.5 above) with the addition of 15.0

grams agar.

Preparation: Heat in boiling water bath

until dissolved. Dispense 10-12 ml of medium

in screw-cap test tubes and slant after steriliza-

tion. Sterilize for 15 minutes at 121 C{15 Ibs.

pressure).

Final pH:7.4 ±.2

5.4.7 Brain Heart Infusion (BHI) Broth with

6.5% NaCI

Use: Identification of fecal streptococci.

Composition: Brain heart infusion broth

with 6.5% NaCI is the same as BHI broth in

5.4.5 above with addition of 60.0 grams NaCI

per liter of medium.

Since most commercially available dehy-

drated media already contain sodium chloride,

this amount is taken into consideration for

determining the final NaCI percentage above.

5.4.8 Brain Heart Infusion Broth (BHI), pH

9.6

Use: Identification of fecal streptococci.

Composition: Same as for BHI broth above

in 5.4.5 with addition of sterile 38% sodium

phosphate solution (Na3P04«12 H20) to

produce a final pH of 9.6.

5.4.9 Brain Heart Infusion Broth with 40%

Bile

Use: Verification of fecal streptococci.

Composition: Same as for BHI broth above

in 5.4.5 with addition of 40 ml of sterile 10%

oxgall to 60 ml of basic medium or 668 ml to

each liter of medium.

Final pH: 7.4 ± ,2

Preparation of Medium: Add 37 grams of

BHI broth to 1 liter of laboratory pure water

and heat gently with agitation to dissolve. Dis-

pense 60 ml amounts of the medium into

screw-cap flasks. Sterilize for 15 minutes at

121 C (15 Ibs. pressure).

Preparation of 10% oxgall: Add 10 grams

of oxgall per 100 ml of laboratory pure water.

After dissolving and mixing, filter-sterilize the

solution.

Preparation of Final Medium: Cool the BHI

broth and add 40 ml of the sterile 10% oxgall

solution to each 60 ml of sterile, cool BHI

broth, resulting in a 40% bile concentration.

Dispense as needed aseptically in 10 ml vol-

umes into sterile culture tubes.

5.4.10 Starch Agar

(Medium may not be commercially

available).

Use: Starch hydrolysis tests for separation

and confirmation of fecal streptoccal species.

Composition:

Peptone

Yeast Extract

Sodium Chloride

Starch (Soluble)

Agar

Final pH: 6.8 ±.2

10.0

5.0

2.0

15.0

Preparation: Add 37 grams of starch agar

in 1 liter of cold laboratory pure water. Heat to

boiling to dissolve and dispense into tubes,

flasks or bottles. Sterilize for 15 minutes at

121 C (15 Ibs. pressure). Cool medium after

sterilization and pour into petri dishes. Allow to

solidify.

5.4.11 Starch Liquid Medium

(Medium may not be commercially

available).

Use: Starch hydrolysis for speciation of

fecal streptococci.

MICROBIOLOGICAL MANUAL 1978

image:

Composition:

Tryptone or Trypticase

Peptone

Yeast Extract

Dipotassium Phosphate

Glucose

Starch (Soluble)

Final pH: 6.8 ±.2

10.0

3.0

2.0

0.5

5.0

Preparation: Add 30.5 grams of dry ingre-

dients to 1 liter of laboratory pure water and

heat to boiling. Dispense into tubes and steril-

ize for 15 minutes at 121 C (15 Ibs. pressure).

5.4.12 Nutrient Gelatin (BBL 11481, Difco

0011 -02)

Use: Detection of gelatin liquefaction for

identification of the fecal streptococci and

other microorganisms.

Composition:

Peptone

Beef Extract

Gelatin

Final pH: 6.8 + .2

5.0 g

3.0 g

120.0 g

Preparation: Add 128 grams of nutrient

gelatin to 1 liter of cold laboratory pure water

and warm to about 50 C to dissolve the me-

dium. Dispense 5 ml in screw-cap test tubes

and sterilize for 15 minutes at 121 C (15 Ibs.

pressure). Store tubes in refrigerator until use.

5-4.13 L'tmus Milk (Difco 0107-02, BBL

11343}

Use: To separate and identify fecal strep-

tococci and generally to determine the action

of bacteria on milk.

Composition:

Skim Milk

Litmus

Final pH: 6.8 ± .2

100.0

0.75

Preparation: Add 100 grams of litmus milk

to 1 liter of laboratory pure water and warm to

about 50 C to dissolve the medium. Dispense

10 ml volumes into screw-cap tubes and steril-

ize for 20 minutes at 115 C (10 Ibs. pressure).

Do not overheat. Control pressure and time

carefully since overheating or prolonged heat-

ing during sterilization can caramelize the milk

sugar.

5.4.14 Skim Milk with 0.1% Methylene

Blue

Use: Identification of fecal streptococci by

reduction of methylene blue.

Composition:

Skim Milk Powder

Methylene Blue Powder

Final pH: 6.4+ .2

100.0g

1.0g

Preparation: Add 100 grams of skim milk

powder and 1 gram of methylene blue to 1 liter

of distilled water, and warm to 50 C to dissolve

the medium. Dispense 10 ml volumes into

screw-cap tubes and sterilize for 20 minutes

at 115 C (10 Ibs. pressure). Do not overheat.

Prolonged heating or overheating during sterili-

zation results in caramelization of the milk

sugar.

5.4.15 Brain Heart Infusion Agar with

0.04% Potassium Tellurite

Use: Identification of fecal streptococci by

tellurite reduction.

Composition:

Calf Brain Infusion

Beef Heart Infusion

Proteose Peptone

Dextrose

Sodium Chloride

Disodium Phosphate

Agar

Final pH: 7,4 ±.2

200.0

250.0

10.0

2.0

5.0

2.5

15.0

EQUIPMENT, TECHNIQUES AND MEDIA

image:

Preparation: Add 52 grams of brain heart

infusion agar to 1 liter of cold laboratory pure

water. Heat in a boiling water bath to dissolve

the agar and dispense 100 ml volumes in

screw-cap flasks and sterilize for 15 minutes at

121 C{15 Ibs. pressure). Cool to 50 C and add

1 ml of sterile warm (50 C) 4% potassium

tellurite to each 100 ml flask of brain heart

infusion agar. This should produce a final po-

tassium tellurite concentration of 0.04%. Dis-

pense melted sterile medium into sterile petri

dishes.

5.4.16 Blood Agar with 0.04% Potassium

Tellurite

Use: Identification of fecal streptococci by

tellurite reduction.

Composition:

Evans Peptone 10.0 g

Sodium Chloride 5.0 g

Meat Extract (Lab Lemco) 10.0 g

Yeast Extract 3.0 g

Agar 15.0 g

Final pH: 7.2 ±.2

Preparation: Add 43 grams of blood agar

base to 1 liter of laboratory pure water. Heat

in a boiling water bath to dissolve the agar and

dispense 100 ml volumes in screw-cap flasks,

sterilize for 15 minutes at 121 C (15 Ibs. pres-

sure) and cool to 44-46 C. Aseptically add 10%

sterile defibrinated horse blood to the medium.

The mixture is heated at 70 C for 10 minutes,

then cooled to 45 C, A filter-sterilized 4.0%

solution of potassium tellurite is added asep-

tically to give a final concentration of 0.04%.

Dispense the completed medium into petri

dishes,

5-4.17 Tetrazolium Glucose (TG) Agar or

2, 3, Sjrriphenyl Tetrazolium Chloride (TTC

Agar) (Medium may not be commercially

available).

Use: Identification of fecal streptococci by

tetrazolium reduction.

Composition:

Peptone

Beef Extract

Sodium Chloride

Agar

Final pH: 7.0-7.3

5.0 g

14.0 g

Preparation of 50% Glucose Solution:

Weigh out 50 grams of reagent grade glucose.

Add to 50 ml laboratory pure water in a 100 ml

volumetric flask. Dissolve glucose and bring

up to volume. Filter-sterilize solution and store

in a screw-cap flask.

Preparation of 1% TTC Solution: Weigh

out 1 gram of 2, 3, 5 triphenyl tetrazolium

chloride. Add to 50 ml laboratory pure water in

a 100 ml volumetric flask. Dissolve and bring

up to volume. Filter-sterilize solution and store

in a screw-cap flask.

Preparation of Final Medium: Add 39

grams of TG agar to 1 liter of cold laboratory

pure water and heat in a boiling water bath to

dissolve the agar. Sterilize for 15 minutes at

121 C (15 Ibs. pressure) and cool to about 50

C. To 970 ml of liquid TG agar, aseptically add

20 ml of 50% glucose solution and 10 ml of

1 % TTC solution. Mix well and pour into sterile

petri dishes.

5.4.18 Blood Agar Base (Optional - 10%

• Blood) (Difco 0045-02, BBL 11036)

Use: Identification of hemolytic properties

of fecal streptococci.

Composition:

BBL 11036

10.0

Beef Heart Infusion

Tryptose or Thiotone

(Peptone)

Sodium Chloride

Agar

Difco 0045-02

Beef Heart Infusion

Tryptose or Thiotone

(Peptone)

Sodium Chloride

Agar

Final pH: 7.4 ± .2

375.0 g

10.0

5.0

15.0

500.0 g

10.0

5.0

15.0

•SERA MICROBIOLOGICAL MANUAL 1978

image:

Preparation: Add 40 grams of blood agar

base to 1 liter of cold laboratory pure water.

Heat in a boiling water bath to dissolve the

agar. Dispense in 90 ml volumes into screw-

cap flask. Sterilize for 15 minutes at 121 C (15

Ibs. pressure). Store at 4 C for later use. When

ready to use, heat/cool the blood agar base to

45-50 C and add 10% by volume of fresh

sterile defibrinated horse blood. Mix and pour

into sterile petri dishes.

5.5 Media for Salmonella

5.5.1 Selenite F Broth (BBL 11607, Difco

0275-02)

Use: Primary enrichment of salmonellae.

Composition:

Tryptone or Polypeptone 5.0 g

Lactose 4.0 g

Disodium Hydrogen Phosphate 10.0 g

Sodium Selenite 4.0 g

Final pH:7.0±.2

Preparation: Add 23 grams of selenite

broth to 1 liter of laboratory pure water. Mix

and warm gently until dissolved. Dispense in

tubes to a depth of 6 cm and expose to flowing

steam for 15 minutes. Avoid excessive heat-

ing. Do not autoclave. Sterilization is unneces-

sary if broth is used immediately.

5.5.2 Tetrathionate Broth Base (Difco

0104-02, BBL 11706)

Use: Primary enrichment of salmonellae.

Composition:

Proteose Peptone or

Polypeptone

Bile Salts

Calcium Carbonate

Sodium Thiosulfate

Final pH: 7.8 ± .2

5.0 g

1.0 g

10.0 g

30.0 g

Preparation: Add 46 grams of tetrathion-

ate broth base to 1 liter of laboratory pure

water and heat to boiling. Cool to less than 45

C and add 20 ml of iodine solution." Mix and

dispense in 10 ml volumes into screw-cap

tubes. Do not heat after the addition of the

iodine. Do not autoclave. The tetrathionate

broth base without iodine may be stored for

later use. The complete medium (with iodine)

should be used on the day it is prepared.

"The iodine-iodide solution is prepared by dis-

solving 6 grams iodine crystals and 5 grams

potassium iodide in 20 ml of distilled water.

5.5.3 Dulcitol Selenite Broth

(Medium may not be commercially

available).

Use: Primary enrichment of salmonellae.

Composition:

Proteose Peptone

Yeast Extract

Dulcitol

Sodium Selenite

Disodium Hydrogen

Phosphate

Potassium Dihydrogen

Phosphate

Final pH: 6.9 ±.2

4.0

1.5

4.0

5.0

1.25 g

Preparation: Add 16.5 grams of dulcitol

selenite broth to 1 liter of laboratory pure

water and heat carefully to dissolve ingredi-

ents. Do not boH. The prepared medium should

be buff-colored. Dispense into screw-cap

tubes to a depth of 6 cm. Do not autoclave.

5.5.4 Tetrathionate Brilliant Green Broth

Use: Primary enrichment for salmonellae.

Composition: Same as tetrathionate broth

base (5.5.2) with addition of 0.01 gram of

brilliant green per liter.

EQUIPMENT, TECHNIQUES AND MEDIA

image:

5.5.5 Brilliant Green Agar (BBL 11072,

Difco 0285-02)

Use: As a primary plating medium for the

isolation of salmonellae.

Phenol Red

Agar

Final pH: 7.4 ±.2

0.08

15.0

Composition:

Yeast Extract

Polypeptone or Proteose

Peptone

Sodium Chloride

Lactose

Saccharose (Sucrose)

Phenol Red

Brilliant Green

Agar

Final pH: 6.9 ±.2

3.0

10.0

5.0

10.0

0.08

0.0125

20.0

Preparation: Add 58 grams of brilliant

green agar to 1 liter of cold laboratory pure

water and heat to boiling. Dispense in screw-

cap flasks and sterilize for 15 minutes at 121 C

(15 Ibs. pressure). Pour into sterile petri dishes.

Warning: A longer period of sterilization

will reduce the selectivity of the medium.

5.5.6 Xylose Lysine Brilliant Green (XLBG)

Agar

Use: Salmonella Differentiation.

Composition of XL Agar Base:

BBL11836

Xylose

L-Lysine

Lactose

Saccharose (Sucrose)

Sodium Chloride

Yeast Extract

Phenol Red

Agar

Difco 0555-02

Xylose

L-Lysine

Lactose

Saccharose (Sucrose)

Sodium Chloride

Yeast Extract

3.5

5.0

7.5

5.0

3.0

0.08

13.5

3.75

5.0

7.5

5.0

3.0

Preparation: Add 45 or 47 grams of XL

agar base to 1 liter of cold laboratory pure

water. Heat in a boiling water bath to dissolve

the agar. Prior to sterilization, add 1.25 ml of

1% aqueous brilliant green. Sterilize for 15

minutes at 121 C (15 Ibs. pressure). Cool the

sterilized medium to about 45-50 C and add

20 ml of a solution containing 34% sodium

thiosulfate and 4% ferric ammonium citrate.

Pour into sterile petri dishes.

5.5.7 Xylose Lysine Desoxycholate (XLD)

Agar

Use: Differentiation of Salmonella.

Composition:

BBL 11837

Xylose

L-Lysine

Lactose

Saccharose (Sucrose)

Sodium Chloride

Yeast Extract

Phenol Red

Agar

Sodium Desoxycholate

Sodium Thiosulfate

Ferric Ammonium Citrate

Difco 0788-02

Xylose

L-Lysine

Lactose

Saccharose (Sucrose)

Sodium Chloride

Yeast Extract

Phenol Red

Agar

Sodium Desoxycholate

Sodium Thiosulfate

Ferric Ammonium Citrate

Final pH: 7.4 ±.2

3.5

5.0

7.5

5.0

3.0

0.08

13.5

2.5

6.8

0.8

3.75

5.0

7.5

5.0

3.0

0.08

15.0

2.5

6.8

0.8

O-ERA MICROBIOLOGICAL MANUAL 1978

image:

Preparation: Add 55 or 57 grams of XLD

agar in 1 liter of cold laboratory pure water,

heat to boiling with mixing. Do not overheat

and do not autoclave. Pour into sterile petri

dishes.

Note: Taylor and Schelhart report better

recoveries by using XL Agar Base (BBL 11835

or Difco 9555), see 5.5.6, and adding sepa-

rately, sterile solutions of the last three ingredi-

ents (4).

5.5.8 Bismuth Sulfite Agar (Difco

0073-02, BBL 11030)

Use: Differentiation of salmonellae, espe-

cially S. typhosa.

Composition:

Polypeptone or Proteose

Peptone

Beef Extract

Dextrose

Disodium Hydrogen

Phosphate

Ferrous Sulfate

Bismuth Sulfite Indicator

Brilliant Green

Agar

Final pH: 7.6+ .2

Preparation: Add 52 grams of bismuth sul-

fite agar to 1 liter of cold laboratory pure water

and heat to boiling. Do not autoclave or over-

heat. Twirl the flask prior to pouring plates to

evenly dispense the characteristic precipitate.

Use the plated medium on the day prepared.

5.5.9 Triple Sugar Iron (TSI) Agar

Use: Differentiation of gram negative en-

terics by their differing ability to ferment dex-

trose, lactose and sucrose and ability to

produce hydrogen sulfide.

Composition:

Difco 0265-02

10.0

5.0

4.0

0.3

8.0

0.025

20.0

Yeast Extract

Peptone

Proteose Peptone

Lactose

Saccharose (Sucrose)

Dextrose

Ferrous Sulfate

Sodium Chloride

Sodium Thiosulfate

Agar

Phenol Red

BBL 11748

Peptone

Lactose

Saccharose (Sucrose)

Dextrose

Ferrous Sulfate

Sodium Chloride

Sodium Thiosulfate

Agar

Phenol Red

Final pH: 7.3+ .2

3.0

15.0

5.0

10.0

1.0

0.2

5.0

0.3

12.0

0.024

20.0

10.0

1.0

0.2

5.0

0.2

13.0

0.025

Preparation: Add 65 grams or 59.4 grams,

depending on manufacturer, of triple sugar

iron agar to 1 liter of cold laboratory pure

water and heat in a boiling water bath to dis-

solve the agar. Dispense into screw-cap tubes

and sterilize for 1 5 minutes at 1 1 8 C (1 2 Ibs.

pressure). Slant tubes for a generous butt.

Inoculated TSI slants must be incubated with

loosened caps to prevent complete blackening

of the medium from I-^S.

5.5.10 Lysine Iron Agar

Use: Differentiation of Proteus, Citrobacter

and Shigella from Salmonella based on deami-

nation of lysine and hydrogen sulfide produc-

tion. Salmonella cultures produce large

amounts of hydrogen sulfide and lysine decar-

boxylase.

Composition:

Difco 0849-02

Beef Extract

3.0

g Peptone

5.0 g

EQUIPMENT, TECHNIQUES AND MEDIA

image:

Yeast Extract

Dextrose

L-Lysine

Ferric Ammonium Citrate

Sodium Thiosulfate

Brom Cresol Purple

Agar

BBL 11362

Peptone

Yeast Extract

Dextrose

L-Lysine

Ferric Ammonium Citrate

Sodium Thiosulfate

Brom Cresol Purple

Agar

FinalpH: 6.7 ±.2

3.0

1.0

10.0

0.5

0.04

0.02

15.0

5.0

3.0

1.0

10.0

0.5

0.04

0.02

13.5

Preparation: Add 34.5 or 33 grams, de-

pending on manufacturer, of lysine iron agarto

1 liter of laboratory pure water. Heat in a

boiling water bath to dissolve the agar. Dis-

pense in 4 ml amounts in screw-cap tubes and

sterilize for 12 minutes at 121 C (15 Ibs.

pressure). Cool to give a deep butt and short

slant. Inoculated LIA slants must be incubated

with loosened caps.

5.5.11 Urea Agar Base (BBL 11794, Difco

0283-02)

Use: To differentiate enteric microorga-

nisms, especially Proteus sp. on basis of

urease activity.

Composition:

Peptone 1.0 g

Dextrose 1.0 g

Sodium Chloride 5.0 g

Monopotassium Phosphate 2.0 g

Urea 20.0 g

Phenol Red 0.012 g

Final pH: 6.8 ± .2

Preparation: Add 29 grams of urea agar

base to 100 ml of laboratory pure water. Dis-

solve and filter-sterilize. Add 15 grams of agar

to 900 ml laboratory pure water and boil to

dissolve. Sterilize for 15 minutes at 121 C (15

Ibs.pressure). Cool to 50-55 C and add asepti-

cally 100 ml of filter-sterilized urea agar base.

Mix and dispense in sterile tubes. Slant tubes

to form a 2 cm butt and 3 cm slant and cool.

Urea Agar Base 10X Concentrate (Difco

0284-60)

Use: Same as urea agar base, for prepara-

tion of small volumes of urea agar.

Composition: A filter-sterilized 10X solu-

tion of urea agar base, 10 ml volumes in tubes.

Refrigerate to store.

Preparation: Add 1.5 grams of agar to 90

ml of laboratory pure water and dissolve by

boiling. Sterilize for 1 5 minutes at 121 C (1 5

Ibs. pressure). Cool the agar to 50-55 C and

aseptically add a 10 ml tube urea agar base

concentrate. Mix agar and concentrate. Dis-

pense aseptically into sterile tubes and slant.

5.5.12 Phenylalanine Agar (BBL 11536,

Difco 0745-02)

Use: Differential tube medium for the sep-

aration of members of the Proteus and Provi-

dencia genera from other members of the En-

terobacteriaceae based on deaminase activity.

Composition:

Yeast Extract

DL-Phenylalanine

Disodium Phosphate

Sodium Chloride

Agar

Final pH: 7.3+ .2

3.0

2.0

1.0

5.0

12.0

Preparation: Add 23 grams of phenylala-

nine agar to 1 liter of cold laboratory pure

water. Heat in a boiling water bath to dissolve

the agar. Dispense in screw-cap tubes and

sterilize in the autoclave for 15 minutes at 121

C (15 Jbs. pressure). Slant and cool tubes.

MICROBIOLOGICAL MANUAL 1978

image:

5.5.13 Malonate Broth (Modified) (BBL

11398, Difco 0569-02)

Use: Differentiation of enteric organisms

based on utilization of malonate. Described by

Leifson and modified by Ewing, the medium is

used in differentiation of Salmonella.

Composition:

Yeast Extract

Ammonium Sulfate

Dipotassium Phosphate

Monopotassium Phosphate

Sodium Chloride

Sodium Malonate

Dextrose

Brom Thymol Blue

Final pH: 6.7+ .2

1.0

2.0

0.6

0.4

2.0

3.0

0.25

0.025

Preparation: Dissolve 9.3 grams in 1 liter

of laboratory pure water. Dispense into tubes

and sterilize for 15 minutes at 121 C (15 Ibs.

pressure).

5.5.14 Decarboxylase Medium Base (Difco

0872-02, BBL 11429)

Use: Differentiation of microorganisms

based on decarboxylase activity in presence of

L-lysine HCI, L-arginine HCI, L-ornithine HCI,

glutamic acid or other amino acids.

Composition:

Peptone

Yeast Extract

Dextrose

Brom Cresol Purple

Final pH: 6.5+ .2

5.0 g

3.0 g

1.0 g

0.02 g

Preparation: Add 9 grams of base to 1 liter

of cold laboratory pure water and warm to

dissolve completely. Add 5 grams L-lysine,

L-ornithine, L-arginine or other L-amino acids

as desired per liter of medium and warm to

dissolve completely. When D/L amino acids are

used add 10 g rather than 5 g. If ornithine HCI

is used, adjust pH with 10 N NaOH. (About 2.1

ml required for 1 liter of medium containing 5

grams of ornithine HCI). Lysine or arginine do

not require pH adjustment. Dispense in 5 ml

volumes into screw-cap tubes and sterilize for

15 minutes at 121 C (15 Ibs. pressure). The

proper pH for the complete medium (6.5) is

indicated by purple color of broth.

5.5.15 MotilityTest Medium (BBL 11435)

Use. Recommended for detection of motil-

ity of gram-negative enteric bacilli.

Composition:

Beef Extract

Peptone

Sodium Chloride

Agar

Final pH: 7.3+ .2

3.0 g

10.0 g

5.0 g

4.0 g

Preparation: Add 22 grams of motility test

medium to 1 liter of cold laboratory pure water

and heat in a boiling water bath to dissolve the

agar. Dispense in tubes and sterilize for 15

minutes at 121 C (15 Ibs. pressure).

To aid in recognizing motility, add 0.05

grams of triphenyl tetrazolium chloride/liter

after sterilization.

5.5.16 Motility Sulfide Medium (Difco

0450-17)

Use: Determination of motility and the

production of hydrogen sulfide from l-cystine.

Composition:

Beef Extract 3.0 g

Peptone No. 3 10.0 g

L-Cystine 0.2 g

Ferrous Ammonium Citrate 0.2 g

Sodium Citrate 2.0 g

Sodium Chloride 5.0 g

Gelatin 80.0 g

Agar 4.0 g

Final pH: 7.3 ±.2

Preparation: Add 104 grams of Motility

Sulfide Medium to 1 liter of cold laboratory

pure water. After wetting powder, heat care-

fully to boiling on a hot plate to dissolve com-

pletely. Dispense in 4 ml amounts in tubes, cap

EQUIPMENT, TECHNIQUES AND MEDIA

image:

loosely and sterilize for 15 minutes at 117 C

(10 Ibs. pressure).

5.5.1 7 H Broth (Difco 0451 -02)

Use: Preparation of H agglutinating anti-

gens of members of genus, Salmonella.

Composition:

BBL 11288

Thiotone (peptone)

Beef Extract

Dextrose

Sodium Chloride

Dipotassium Phosphate

Difco 0451-02

Thiotone (Peptone)

Tryptone

Beef Extract

Dextrose

Sodium Chloride

Dipotassium Phosphate

Final pH:7.2 ±.2

10.0 g

3.0 g

1.0 g

5.0 g

2.5 g

5.0

3.0

1.0

5.0

2.5

Preparation: Add 21.5 grams of H broth to

1 liter of laboratory pure water, mix well and

dissolve by warming. Dispense 5 ml amounts

In screw-cap test tubes. Sterilize for 15

minutes at 121 C (15 Ibs. pressure).

5.6 Medium for Actinomycetes

Starch-Casein Agar

(Medium may not be commercially

available).

Use: Isolation of actinomycetes from

water or soil.

Composition:

Soluble Starch (or

Glycerol)

Casein (Vitamin-free)

Sodium Nitrate

10.0

0.3

2.0

Sodium Chloride 2.0 g

Dipotassium Phosphate 2.0 g

Magnesium Sulfate»7 H20 0.05 g

Calcium Carbonate 0.02 g

Iron Sulfate»7 H20 0.01 g

Agar 15.0 g

Final pH: 7.0-7.2

Preparation: Weigh out ingredients and

add in turn to 1 liter of laboratory pure water in

a two liter flask. Dissolve ingredients using

gentle heat. Add the agar last and place in a

boiling water bath. Heat and stir occasionally

until dissolved. Dispense 250 ml volumes in

500 ml screw-cap flasks and 17 ml volumes in

screw-cap tubes. Sterilize for 15 minutes at

121 C (15 Ibs. pressure).

6. Laboratory Pure Water

.Distilled or deionized water free of nutri-

tive and toxic materials is required for prepa-

ration of media and dilution/rinse water.

6.1 Distilled Water Systems: Water dis-

tillation units can produce good grades of pure

water. Stills are dependable and long-lived if

maintained and cleaned properly. Use of sof-

tened water as the source water increases the

interval between cleanings of the still. Stills

characteristically produce a good grade of

water which gradually deteriorates as corro-

sion, leaching and fouling set in. There is no

sudden loss of water quality unless a structural

failure occurs. Stills are efficient in removing

dissolved chemicals but not dissolved gases.

Fresh laboratory pure water may contain chlo-

rine and ammonia. On storage, ammonia will

increase and CO2 will appear from air contami-

nation. Distilled water systems should be

monitored continuously for conductance and

analyzed monthly for chlorine, ammonia and

standard plate count and at least annually for

trace metals. See Table IV-A-3.

6.2 Deionized Water Systems: Deioniza-

tion systems produce a good grade of pure

water and can produce an ultrapure water

when combined with filtration and activated

carbon in a recirculating system.

MICROBIOLOGICAL MANUAL 1978

image:

In contrast to distilled water systems,

deionization systems do not gradually deterio-

rate. Rather, they continue to produce the

same quality water for a long period of time

until the resins and/or carbon are exhausted,

whereupon the quality deteriorates quickly.

Deioriized water systems should be monitored

continually with a conductance meter and

analyzed monthly for ammonia/amines, total

organic carbon, specific organic pollutants and

Standard Plate Count and at least annually for

metals. Amines may elute from the resin.

Organic carbon results from organic chemicals

in the water or from bacterial growth in the

columns. Use of a 0.22 ^urn final filter is recom-

mended to remove bacterial contamination.

See Table IV-A-3.

Avoid the sudden loss of good quality

water by continuously monitoring perfor-

mance of the system, anticipating the remain-

ing life of cartridges and replacing them be-

fore failure occurs.

6.3 Quality Control

Pure water systems should be monitored

carefully as a part of the intralaboratory QC

program. The water quality should meet the

standards set in this Manual, Part IV-A, 5.2-5.4,

7. Dilution Water

Dilution water is used to reduce the num-

ber of microbial cells/unit volume of sample so

thatthe density of cells is low enough to permit

enumeration or manipulation by the technique

of choice: pour-plate, spread plate, MF or MPN.

The ideal dilution water is neutral in effect.

It maintains bacterial populations without

stimulating cell growth and reproduction,

damaging cells or reducing their ability to sur-

vive, grow or reproduce. Its basic purpose is to

simulate the chemical conditions of the natural

environment which are favorable to cell

stability.

Microbiologists have tried different ap-

proaches to obtain an ideal diluent Some

workers have copied the natural environment

by use of sterile fresh or marine waters as

diluents, but these are non-standard. Other

workers have used tap waters with the same

lack of uniformity and an added potential for

toxicity. Certainly for comparability of microbi-

ological data the dilution water must be uni-

form between laboratories. The chemical ele-

ments and compounds required in natural con-

ditions to insure a balance of cell solutes and

maintain cell turgidity must be reproduced in

the laboratory.

Inorganic constituents such as sodium,

potassium, magnesium, phosphate, chloride

and sulfate, and soluble organics such as pep-

tone are used in synthetic dilution waters. pH

is usually held to a near-neutral reaction. Two

standard dilution waters are:

7.1 Phosphate Buffered Dilution Water

7.1.1 Stock Phosphate Buffer Solution

Potassium Dihydrogen

Phosphate (KH2P04)

Laboratory Pure Water

34.0 g

500 ml

Adjust the pH of the solution to 7.2 with 1

N NaOH and bring volume to 10OO ml with

laboratory pure water. Sterilize by filtration or

autoclave for 15 minutes at 121 C (15 Ibs.

pressure).

7.1.2 Storage of Stock Solution: After

sterilization of the stock solution store in the

refrigerator until used. Handle aseptically. If

evidence of mold or other contamination ap-

pears, the stock solution should be discarded

and a fresh solution prepared.

7.1.3 Working Solution

Stock Phosphate Buffer 1.25ml

MgCl2 Solution (38 g/liter) 5 ml

Laboratory Pure Water 1000 ml

Final pH: 7.2 10.1

7.2 Peptone Dilution Water

Composition:

Peptone 1.0 g

EQUIPMENT, TECHNIQUES AND MEDIA

image:

Laboratory Pure Water

Final pH: 7.0 ±0.1

1000 ml

7.3 Preparation of Dilution and Rinse

Water

Dispense 102 ml volumes of phosphate

buffer or peptone dilution water into borosili-

cate glass, screw-cap dilution bottles scribed

at 99 ml. Loosen screw-caps on bottles and

sterilize at 121 C for 15 minutes (15 Ibs.

pressure). Final volumes after sterilization

should be 99 ±2 ml. Cool and separate bottles

outside of the 99 ml ±2 ml limit. Tighten screw-

caps after sterilization and store in a cool

place.

Prepare dilution water for rinsing in iOO

ml or larger volumes and autoclave for 30

minutes or more. Bottles or flasks must be

separated sufficiently in the autoclave to per-

mit easy access for steam.

REFERENCES

1. Songer, J. R., J. F. Sullivan and J. W. Monroe, 1971. Safe, convenient pipetting device. Appl.

Microbiol. 21:1097.

2. Songer, J. R., D. T. Braymen and R. G. Mathis, 1975. Safe, convenient pipetting station. Appl.

Microbiol. 30:887.

3. Taylor, R., R. Bordner and P. Scarpino, 1973. Delayed incubation membrane-filter test for fecal

coliforrns. Appl. Microbiol. 25:363.

4. Taylor, W. I., 1965. Isolation of Shigellae. I. Xylose lysine agars; new media for isolation of enteric

pathogens. Ann. J. Clin. Path. 14:471.

GENERAL REFERENCES

Difco Laboratories. General Conditions Pertaining to the Cultivation of Microorganisms; and. Preparation

of Media from Dehydrated Culture Media. Difco Manual. 9th Edition. Difco Laboratories, Detroit, Ml. p. 16

(1953).

Baltimore Biological Laboratories. General Suggestions for Use of Media: Dehydrated-Prepared. BBL

Manual of Products and Laboratory Procedures. 5th Edition, BBL, Division of Becton-Dickinson and Co.,

Cockeysville, MD. p. 88 (1968).

Geldreich, E. E., 1975. Handbook for Evaluating Water Laboratories (2nd Edition). EPA-670/9-75-006,

U.S. Environmental Protection Agency, Cincinnati, OH p. 83.

American Public Health Association, 1976. Standard Methods for the Examination of Water and

Wastewater{14th Edition). American Public Health Association, Inc. p. 892.

£EPA MICROBIOLOGICAL MANUAL 1978

image:

PART II. GENERAL OPERATIONS

Section C Techniques for Isolation and Enumeration of Bacteria of

Sanitary Significance

This Section describes the fundamental

laboratory procedures needed for

microbiological analyses of water and

wastewater. Although experienced

microbiologists and technicians may not

require the depth of information and the

degree of detail given in this Section, it is

provided to serve the technical personnel who

are new to environmental microbiology. The

procedures included are:

1. Preparation for Analyses

2. Streak Plate, Pour Plate and

Spread Plate Methods

3. Membrane Filtration Method

4. Most Probable Number Method

5. Staining Procedures

6. Shipment of Cultures

The specific details unique to tests are

described in separate Sections of Part III:

Standard Plate Count, Total Coliform, Fecal

Coliform, Fecal Streptococci, Salmonella and

Actinomycetes. Refer to Part IV, A & C for de-

tails of quality control on these analyses.

1. Preparation for Analyses

1.1 Preparation of Data Sheets

Select a standard format for bench sheet

or card and use to record pertinent data:

sample identification, sampling conditions,

chlorine residual, temperature, pH, sampling

site, station number, date and time of

collection, sample collector, required chain of

custody information, data and time received

and analyzed, time elapsed from sample

collection, analyses performed, sample

volume(s), analyst, and laboratory

identification numbers. (See Figures ll-C-1, 2

and 3).

1.2 Disinfection of Work Area

Wipe the work area before and after use

with laboratory-strength disinfectant and allow

surface to dry before use. See Table V-C-3.

Keep a covered container of iodophor or

quaternary ammonium disinfectant available

for emergency use. Phenolics are acceptable if

analyses for these compounds are not per-

formed as part of laboratory work.

1.3 Pretreatment of Samples

Prior to dilution of samples for analyses,

the analyst should examine the sample for free

chlorine residual and possible uneven distribu-

tion of microorganisms.

ISOLATION AND ENUMERATION

image:

EPA-209 (Clnl

(R*v- 4-78)

PHOJ

SAMP

COLL

VOL.

Rirnir

MICROBIOLOGY LABORATORY RECORD

UEUBRANE FILTER ANALYSIS

fr.t' NO-

LE SOURCI

UNO nATFr TIME: FILTRATION DATE: TIME:

ECTOR:

TEMP: nH:

COUNT

COUNT/100 ml

k«!

VOL.

R,ma

COUNT

COUNT/100 ml

rk«:

ANALYST:

VOL.

Ramai

COUNT

COUNT/100 ml

bn:

FIGURE ll-C-1. Microbiological Bench Card for MF Analyses.

MICROBIOLOGY I

COLIFORM K

Sa.ppit • nn*»

.ABOI

iPN /

Time

1ATORY RE

WALYSIS

CORD

ST River

Tid« Rain pai» 24 Hr>. D«p»h Wa»«rT»mp. 'C Secchi Disc.

(XVition

Cdifwrn Pr«». LST 24Hra.

48 Mrs.

CoKform Conf. BGLB 48 Hr..

Fical Cdlfortn EC 24Hrj.

MPN/IOOml

AFO, Region III, USEPA

FIGURE ll-C-2. Bench Card for MPN Analyses.

MICROBIOLOGICAL MANUAL 1978

image:

BACTERIAL INDICATOR ORGANISMS OF POLLUTION

STATION.

.BENCH NO

LOCATION.

DATE

HOURS SAD NO.

COLLECTED .

EXAMINED .

ANALYST

M P N

DiL.

ml.

1 fl-

ic-

i fl-

ic-

CONF1RME

FECAL CO

PRESUMPTIVE

LTB

35'C

24 hrs. •

... ,„

AS hrs.

CONFIRMED

B.G.L.B.

35'C

24 hn.

D MPN ... . . . . .

LIFORM MPN . . .

48 hrs.

44.5'C

24 hrs.

PER 100 ML.

MEMBRANE FILTER

VOLUME

FILTERED

COLONY

COUNT

TOTAL

COLIFORMS

FECAL

COLIFORMS

ORGANISMS PER 100 ML.

cprai s

SALMONS

ISOLATIO

SEROTYPE

tsi

REMARKS:

SERL, Region IV. US EPA

FIGURE II-C-3. Combined Microbiological Bench Card

ISOLATION AND ENUMERATION

image:

1.3.1 Water Samples with High Solids

1.4 Dilution of Samples

(a) Blending of sediments, primary efflu-

ents, sludge and highly turbid waters is essen-

tial for representative subsampling.

(b) Blend the entire water sample contain-

ing particulates in a Waring-type blender. Use

only autoclavable pyrex glass, stainless steel

or plastic blender containers with safety screw

covers to prevent release of aerosols.

onds at about 5000 RPM to avoid overheating

or shearing damage.

(d) Dilute sediments or soils containing

limited amounts of water at a 1:1, 1:2 ratio or

more with dilution water to ensure good blend-

ing action and to reduce heat generation. Use

of a large blender container rather than smaller

units also reduces heat,

1.3.2 Dry Solid Samples

(a) Mix sample thoroughly and weigh 50

grams aliquot in a tared weighing pan. Dry at

105-110 C to constant weight. The final weight

is used in calculating numbers of organisms/

gram dry solids.

(b) Prepare the initial dilution by weighing

out a second aliquot of 11 grams of original

sample. Add to a 99 ml volume of buffered dilu-

tion water for a 1:10 dilution and blend sample

aseptically in a Waring-type blender at 5000

rpm for 30 seconds. Use only a pyrex glass,

stainless steel or plastic blender container with

safety screw lid to prevent release of aerosols.

dilution to a second dilution bottle containing

99 ml buffered dilution water and shake vigor-

ously about 25 times. Repeat this process until

the desired dilution is reached.

1.3.3 Analytical Method

Although high solids samples with low

microbial densities may require MPN or pour

plate procedures, high density samples such as

polluted soils, sludges and feces are diluted so

that the MF method is applicable.

1.4.1 Necessity for Dilutions: Dilutions of

the original sample of water, wastewater or

other material are often necessary to reduce

the number of bacterial cells to measureable

levels or to isolate single cells for purification

and identification (see Part II-B, 7 for details on

dilution water).

1.4.2 Serial Dilutions: A known quantity of

the sample (usually 1.0 ml, but other volumes

can be used) is transferred through a series of

known volumes (e.g., 9 or 99 ml) of dilution

water. This procedure is repeated until the

desired bacterial density is reached. After dilu-

tion of the sample, the bacteria are enumer-

ated using the membrane filtration, pour plate,

streak plate, or the most probable number

technique.

For ease of calculation and preparation,

serial dilutions are usually prepared in suc-

ceeding ten-fold volumes called decimal dilu-

tions. The decimal dilution procedure is shown

in Figure ll-C-4.

1.4.3 Special Dilutions for Membrane Fil-

tration Procedures: The normally accepted lim-

its for colonies per plate in membrane filtration

methods (20-60, 20-80 or 20-100) require

that decimal dilution series be modified to

assume an MF plate count within the accepted

limit.

The recommended method for obtaining

counts within these limits is to filter dilution

volumes of the decimal series which have a

factor of 3, 4 or 5 among them (see Table ll-C-1

for details).

1.4.4 Filtration Volumes for Membrane

Filter Analyses: For sample volumes of 1 -10 ml,

add 20 ml of dilution water to the funnel before

adding sample, to evenly disperse cells.

1.4.5 Preparation of Dilutions: Shake

sample bottle vigorously (about 25 times in 7

seconds) to evenly distribute the bacteria.

Take care to secure the screw-cap and prevent

leakage during shaking.

&ERA MICROBIOLOGICAL MANUAL 1978

image:

WATER

SAMPLE

-1 ml-

DEUVERY VOLUME: 10 ml 1 ml 0.1 ml

DILUTION A

10-2

DILUTION B

-4

99 ml

BLANK

99 ml

BLANK

1 ml 0.1 ml

VOLUME OF

ORIGINAL SAMPLE

IN TUBE/PLATE 10 ml

10-5 ml

FIGURE ll-C-4. Preparation of Decimal Dilution.

image:

TABLE II-C-1

Recommended Filtration Volumes of Samples in MF Analyses

MF Count Range 20-60

MF Count Range 20-80

MF Count Range 20-100

Sample vol., ml added as: Sample vol., ml added as: Sample vol., ml added as:

.01

.03

1.0

1 ml of 10"2

3 ml of 10~2

1 ml of 10"1

3 ml of 10~1

1 ml sample

3 ml sample

10 ml sample

30 ml sample

.04

.16

2.0

8.0

4 ml of 10~2

1.5 ml of 10~1

5 ml of 10~1

2 ml sample

8 ml sample

30 ml sample

.01

.05

.25

1.25

1 ml of 10~2

5 ml of 10~2

2.5 of 10"1

1.25 ml sample

6 ml sample

30 ml sample

-SEPA MICROBIOLOGICAL MANUAL 1978

image:

(a) Withdraw 1.0 ml or 0.1 ml of original

sample to test samples directly for a 1 x 10°

ml and 1 x 10~1 ml volumes, respectively.

(b) Transfer a second 1.0 ml volume to a

99 ml dilution water bottle (Dilution A). Shake

sample vigorously about 25 times and with-

draw 1.0 ml or 0.1 ml of diluted sample for

testing of 1 x 10~2 andil X'10~3iml sample

volumes. The.resultant dilution is calculated as

follows:

Volume of Sample

Volume of Dilution Blank

+ Volume of Sample

Dilution

Ratio

or.

1.0 mi

99.0 + 1.0 ml

or 10~2 (Dilution A)

100

tion bottle A to a second dilution bottle (B), the

dilution ratio for bottle B dilution shown in

Figure ll-C-4 is the product of the individual

dilutions as follows:

A x B = Final or Total Dilution Ratio

100

10,000

Volumes of 0.1 ml can be tested directly from

each serial dilution to provide intermediate

dilutions.

(d) Alternatively, if an initial sample vol-

ume of 1 1 ml is transferred into the first 99 ml

volume dilution blank, an intermediate dilution

can be obtained with the added precision re-

sulting from measurements of 1 ml volumes

serially as opposed to the measurement of 0.1

ml volumes in(c).

(e) Different dilutions can be obtained by

varying sample and dilution preparations. For

example, if 2.0 ml of the sample is transferred

to a 98.0 ml dilution blank, the final dilution

ratio is calculated as follows:

2.0 ml

98.0 + 2.0 ml

100

(f) If 0.5 ml is added to a dilution blank

containing 99.5 ml of buffer, the dilution ratio

is calculated as follows:

0.5 ml

99.5 + 0.5 ml

0.5

100

200

(g) Varying the final volume tested will also

permit modification of dilutions without

increasing the number of dilution bottles as

follows:

A 1:200 dilution can be obtained by test-

ing 0.5 ml of a 1:100 dilution.

A 1:500 dilution can be obtained by test-

ing 2 ml of a 1:1 000 dilution.

1.4.6 Prompt Use of Dilutions: The poten-

tial toxicity of phosphate dilution water and

the stimulatory effect of peptone dilution

water increase rapidly with time. Therefore,

dilutions of samples should be tested as soon

as possible after make-up and should be held

no longerthan 30 minutes after preparation.

2. Streak, Pour and Spread Plate Methods

2.1 Summary: There are three plate dilu-

tion techniques commonly used for isolating

and/or enumerating single colonies of bacteria:

the streak plate, pour plate and spread plate

procedures. These techniques described herein

use solid or melted agar plating media to dilute

out the microorganisms so that individual

species or cells can be selected or counted from

mixed cultures. Because colonies can originate

from more than one cell, results may be report-

ed as colony-forming-units (CPUs).

2.1.1 Streak Plate Method: To obtain a

pure culture the analyst dilutes and isolates

bacteria from a mixed culture by drawing a

small amount of the bacterial growth lightly

across the surface of an agar plate in a pattern

with an inoculating needle or loop.

ISOLATION AND ENUMERATION

image:

In one suggested pattern, the plate is

streaked in parallel lines over half of the sur-

face, rotated a quarter turn, streaked again,

rotated another quarter turn and streaked

once more. The inoculum is progressively di-

luted with each successive streak, and eventu-

ally single cells are deposited on the agar

surface. After suitable incubation, single iso-

lated colonies develop in the path of the

streak, (see Figure ll-C-5).

2.1.2 Pour Plate Method: The analyst di-

lutes apd isolates cells in a bacterial suspen-

sion by consecutively transferring a portion of

the original sample through a series of dilution

water blanks. After an appropriate series of

dilutions, the original bacterial population is

diluted out to a countable level, as described in

1.4. Aliquots of the diluted sample are added

to sterile petri dishes and mixed with melted

agar. After the agar solidifies, the plates are

inverted and incubated for a predetermined

time. Surface or subsurface colonies will de-

velop in some of the agar plates. These colo-

nies can be counted to provide a quantitative

value for the bacterial density of the original

sample, or they can be picked for further quali-

tative study.

2.1.3 Spread Plate Method: The analyst

isolates bacterial cells by delivering a small

volume of a diluted sample onto a solid agar

plate and spreading the inoculum by use of a

sterile glass rod bent at an angle of about 120°.

The Inoculum is spread uniformly by holding

the stick at a set angle on the agar and rotating

the agar plate or rotating the stick until the

Inoculum is distributed evenly.

2.2 Scope and Application

2.2.1 Streak and pour plate methods pro-

vide the means to separate individual bacteria

so that each cell will develop into an isolated

colony in or on a solid medium. The methods

can isolate specific bacteria by the use of se-

lective or differential media.

The streak plate is only qualitative but the

pour plate procedure can be used to quantitate

bacteria present in a sample as in the Standard

Plate Count Method (see Part III-A), The spread

plate method provides a quantitative method

for aerobic surface growth of cultures against

which other surface growth methods such as

the MF technique can be compared.

2.2.2 Because the volumes tested with the

spread plate technique are limited to

0.1-0.5 ml, the sample must be diluted to con-

tain at least 40-2000 cells/ml in order to have

a counting range of 20-200.

2.2.3 In the pour plate technique, test vol-

umes are limited to 0.1-2 ml per 100 ml petri

dish so the sample must be diluted to contain

60-3000 cells/ml to have a 30-300 counting

range.

2.3 Apparatus and Materials

2.3.1 Incubator set at 35 ± 0.5 C.

2.3.2 Water bath set at 44-46 C for tem-

pering agar.

2.3.3 Colony Counter, Quebec darkfield

model or equivalent.

2.3.4 Hand tally or electronic counting

device.

2.3.5 Thermometer which has been

checked against a National Bureau of

Standards-Certified Thermometer or one of

equivalent accuracy.

2.3.6 Inoculating needle and loop.

2.3.7 Pipet containers of stainless steel,

aluminum or pyrex for glass pipets.

2.3.8 Petri dish containers of stainless

steel, aluminum or pyrex glass for glass petri

dishes.

2.3.9 Glass spreader rods.

2.3.10 Sterile T.D. bacteriological or Mohr

pipets, glass or plastic of appropriate volume.

2.3.11 Sterile 100 mm x 15 mm petri

dishes, glass or plastic.

MICROBIOLOGICAL MANUAL 1978

image:

STEP 1

STEP 2

STEP 3

FIGURE II-C-5. Suggested Pattern for Preparing a Streak Plate,

ISOLATION AND ENUMERATION

image:

2.3.12 Dilution (milk dilution) bottles, py-

rex, marked at 99 ml volume, screw-cap with

neoprene rubber liner.

2.3.13 Bunsen/Fishertype burner or elec-

tric incinerator.

2.4 Media

2.4.1 Sterile agar medium dispensed in

bulk quantities in screwcapped bottles or

flasks.

2.4.2 Sterile dilution water in bottles con-

taining 99 ± 2 ml volumes.

2.5 Streak Plate Procedures

2.5.1 Melt the nonselective agar, such as

nutrient agar or Trypticase soy agar, temper at

44-46 C, and add about 15 ml agar to each

sterile petri dish. Allow to harden and dry for

bast results in streaking.

2.5.2 Bend an inoculation needle or loop

at an angle about 1 cm from the needle tip to

prevent cutting of the agar during streaking.

Sterilize the needle by heating it to redness in

a flame, and air-cool.

2.5.3 Remove screw cap and pick a small

amount of growth from an isolated colony or

from a mass of growth.

2.5.4 Draw the needle containing the

bacteria back and forth across the surface of a

previously-poured and hardened agar plate in

a specific pattern, such as shown in Figure II-C-

5. Streak 1/3-1/2 of the agar surface. Flame

and cool needle after each step and inoculate

plate further by drawing the needle across the

area previously streaked.

2.5.5 Rotate the plate one quarter turn

clockwise as each step is completed to make

the streaking easier. Streaking patterns other

than the model shown in Figure ll-C-5 can be

used; the objective is simply to deposit fewer

and fewer cells along the streak until single

cells are deposited on the agar surface. After

incubation, these cells will develop into well-

separated pure colonies of bacteria, each theo-

retically arising from a single bacterium.

2.5.6 After streaking, incubate the petri

dishes at 35 C for 24 hours (or other appropri-

ate conditions) in an inverted position to pre-

vent condensation of water on the agar sur-

face. Moisture interferes with development of

isolated colonies by spreading bacterial

growth over the agar surface.

2.5.7 For further purification, examine

plates after incubation for single, well-isolated

colonies. Pick typical colonies using a sterile

inoculating needle, suspend cells in dilution

water, and restreak on an agar plate, repeating

steps 2.5.2-2.5.6. Isolated, single colonies

from a plate containing like colonies may be

considered to be pure.

2.5.8 Streaking may also be done on se-

lective media, such as Endo or EMB agars or on

selective/differential media e.g.} in Salmonella

testing.

2.6 Pour Plate Procedure

2.6.1 Shake the sample bottle vigorously

about 25 times to disperse the bacteria. Dur-

ing shaking, close cap tightly to prevent leak-

age of sample and the danger of

contamination.

2.6.2 Using a 1.1 ml bacteriological pipet,

prepare the initial dilution by pipeting 1 ml of

the sample into a 99 ml dilution water blank.

This initial dilution represents a 10~2 dilution

(see this Section, 1.4, Dilution of Samples).

2.6.3 Using the same pipet transfer 0.1

and 1.0 ml from the undiluted sample to two

separate petri dishes.

2.6.4 Shake the 1:100 dilution bottle

vigorously again and pipet 0.1 and 1.0 ml of

the 1:100 dilution into two petri dishes using

another sterile 1.1 ml pipet.

2.6.5 Pour aseptically 12-15 ml of the

melted agar medium cooled to 44-46 C into

each petri dish. Mix agar and inoculum by

rotation, being careful to prevent spillover of

MICROBIOLOGICAL MANUAL 1978

image:

agar. One recommended technique uses a se-

quence of five rotations to the left, five to the

right and five forward and backward. Allow the

agar to solidify on a level surface.

2.6.6 Invert the dishes and incubate at the

specific temperature and time. After incuba-

tion, well-isolated surface and subsurface colo-

nies should develop in some of the plates.

2.6.7 When the pour plate technique is

used quantitatively, count plates containing

between 30 and 300 colonies. This is the

technique used in the Standard Plate Count

Method described in Part III-A. Pour plate

counts are reported as the count per ml.

Count/ml =

Sum of Plate Counts

Total Volume of Sample in ml

2.7 Spread Plate Procedure

2.7.1 Prepare the appropriate melted

agar. Pour about 15 ml of the melted agar into

each 100 mm petri dish. Keep covers opened

slightly until agars have hardened and mois-

ture or condensation have evaporated. Close

dishes and store in refrigerator. Warm at room

temperature before use.

2.7.2 Prepare a series of dilutions based

upon the estimated concentration of bacteria

so that 0.1-0.5 ml of inoculum will give a

20-200 count (equivalent to a 40-2000

count/ml in the diluted sample). The dilutions

should bracket the estimated density of

bacteria. The analyst must remember that if

only 0.1 ml volume is tested, it must be plated

on the agar plate marked with the next higher

dilution; for example, 0.1 ml of the 10~~1

dilution onto the surface of the plate marked

10~2. Inoculate agar plates.

2.7.3 Remove the glass spreader from al-

cohol and flame. Cool for 15 seconds. Test

glass rod on edge of agar to verify safe temper-

ature before use. This step can be simplified by

making and sterilizing a number of glass

spreaders.

2.7,4 Place cool glass spreader on agar

surface next to inoculum. Position spreader so

that the tip forms a radius from the center to

the plate edge. Holding spreader motionless,

rotate plate several revolutions, or hold plate

and move the spreader in a series of sweeping

arcs. The purpose is to spread the inoculum

uniformly over the entire surf ace of the agar.

2.7.5 Lift the glass spreader from the agar

and place in alcohol solution. Cover plate par-

tially, leaving open slightly to evaporate excess

moisture for 15-30 minutes.

2.7.6 When agar surfaces are dry, close

dishes, invert them and incubate as required

for the specific test.

2.7.7 After incubation at the proper time

and temperature, isolated surface colonies

should develop in one or more dilutions within

the acceptable counting range of 20-200 col-

lonies. The maximum recommended number of

colonies/spread plate is fewer than for other

plate techniques because surface colonies are

larger than subsurface colonies and crowding

can result at lower count levels.

2.7.8 Count the colonies by normal tech-

niques and report on a count/ml or count/100

ml basis dependent on the use of the data.

2.8 Reporting Results

^•8- 1 Significant Figures: To prevent false

precision in the reporting of counts, the plate

counts must be limited to the digit(s) known

definitely plus one digit which is in doubt.

These combined digits are termed the Signifi-

cant Figures (S.F.).

(a) For example, if an analyst reports a

plate count of 124 to three significant figures

he is indicating that he is certain of the first

two digits, 1 and 2, but is uncertain whether

the last digit is 3, 4 or 5. If the analyst were

reporting that same number to two significant

figures, he would report the first figures, 1, as

certain, the second figure, 2, as uncertain, and

the third figure, 4, as unknown. Hence he

would report it as 120, inserting the zero only

as a spacer. Large counts of 1200, 12,000 and

ISOLATION AND ENUMERATION

image:

12,000,000 only contain two significant fig-

ures. Of course, zeros can be significant in

counts of 10,60,105, etc.

(b) In plate count and MF methods, the

number of significant digits which can be re-

ported are dictated by the' method itself as

follows: within the acceptable counting range

of the method itself, i.e., 20-60, 20-80,

20-100 or 30-300 the actual number of colo-

nies observed is the best estimate of the true

density. The number of significant figures are

equal to the number of colonies.

TABLE ll-C-2

Number of Significant •

Figures (S.F.) Reported

Actual

Colony

Count

Pour Plate/

Spread Plate

Method

Membrane

Filtration

Method

1-9 1 S.F.

10-99 2 S.F.

100 - 300 3 S.F.

1 S.F.

2 S.F.

2.8.2 Rounding Off Counts: Since plate

counts must be limited to the number of signifi-

cant figures obtainable by the method, the

non-zero number which is not significant

should be treated by the standard scientific

convention:

(a) If the insignificant digit is less than five,

replace it with a zero, e.g., 3530 becomes

3500.

(b) If the insignificant digit is five, round

the preceding significant digit to the nearest

even number, e.g., with two S.F., 3450 be-

comes 3400, and 3550 becomes 3600.

five, drop the digit and increase the preceding

significant number by one, e.g., 3480 be-

comes 3500.

3. Membrane Filtration Method

3.1 Summary: The membrane filter

method provides a direct count of bacterial

colonies on the surface of the filter. The

sample is filtered as soon as possible after

collection. After the sample is filtered, the

membrane filter is placed on a nutrient

medium formulated to encourage growth of

the bacteria for which the.test is designed and

toouppressthegrowthofothermicroorganisms.

After incubation under the specified

conditions, the bacteria retained on the

surface of the membrane develop into visible

colonies. The medium and the temperature of

incubation influence the kinds and

appearance of bacteria that develop. Two-step

enrichment and delayed incubation MF

procedures can also be used. The two-step

procedure involves an acclimation period on

another medium before the selective growth

step.

3.2 Scope and Application

Membrane filter methods are preferred

over MPN or other techniques, where applica-

ble because of the following advantages.

3.2.1 Advantages

(a) One of the primary advantages of this

method is its speed. Definitive results for total

and fecal coliforms can be obtained in 22-26

hours, whereas 48-96 hours are required for

the multiple-tube fermentation method.

(b) Considerably larger, more representa-

tive water samples can be examined than with

the. MPN. With waters of low bacterial densi-

ties such as finished waters, larger sample

aliquots can be used to enhance the reliability

of the results.

the multiple-tube technique becausetheformer

makes a direct count of colonies/unit volume.

(d)The method represents savings in time,

labor, space, supplies and equipment.

(e) Because of its portability, this proce-

dure is also very practical for field studies.

SERA MICROBIOLOGICAL MANUAL 1978

image:

3.2.2 Limitations: Although the majority of

water samples can be tested by membrane

filtration, there are limitations with certain

samples and some problems with membrane

filters themselves.

(a) Some samples contain large quantities

of colloidal materials or suspended solids such

as iron, manganese or alum floes or clay (1).

Other samples may contain algae. These

substances can clog the filter pores and

prevent filtration or can cause the

development of spreading bacterial colonies.

When the bacterial counts of such samples are

high, a smaller volume or a higher sample

dilution can be used to minimize the effect of

sample turbidity. The membrane filter method

may be used with samples containing turbidity

by filtration of several smaller replicate sample

volumes and compositing the results.

However, with waters of high turbidity and low

bacterial count, the membrane filter method

may not be applicable. In the latter situation

the multiple-tube procedure should be used.

(b) Large non-specific populations may

mask the appearance of indicators on selective

media such as M-Endo MF medium (4).

zinc, copper, or other heavy metallic com-

pounds (2) which adsorb onto the membrane

surface and interfere with subsequent bacterial

development (1, 2, 3).

(d) MF analyses require preparation of

MPN tubed media for verification.

(e) Inhibition may result in seawater or

from toxic materials such as chlorine or

phenols.

(f) Indicator organisms stressed in the en-

vironment may be poorly recovered (5).

3.3 Apparatus and Materials

3.3.1 Incubator set at 35 ± O.5 C for total

coliform-test (Part III-B) and fecal streptococci

test (Part III-D).

3.3.2 Water bath or other type of incuba-

tors such as the aluminum heat sink incubator,

or equivalent, set at 44.5 ± 0.2 C for fecal

coliform test (Part ill-C).

3.3.3 Stereoscopic microscope, with mag-

nification of 10-15 X, wide-field type.

3.3.4 A microscope lamp producing dif-

fuse light from cool, white fluorescent lamps

adjusted to give maximum color or sheen

appearance.

3.3.5 Hand tally.

3.3.6 Pipet container of stainless steel,

aluminum or pyrex glass for glass pipets.

3.3.7 Graduated cylinders covered with

aluminum foil or kraft paper and sterilized.

3.3.8 Membrane filtration units (filter base

and funnel), glass, plastic or stainless steel, see

Figure ll-C-6. These are wrapped with alumi-

num foil or kraft paper and sterilized. See Fig-

ure ll-C-7 for an exploded view of a stainless

steel MF assembly and filter.

3.3.9 Ultraviolet sterilizer for the filter fun-

nel is optional (6).

3.3.10 Line vacuum, electric vacuum

pump or aspirator is used as a vacuum source.

in an emergency or in the field, a hand pump or

a syringe can be used. Such vacuum-

producing devices should be equipped with a

check valve to prevent the return flow of air.

3.3.11 Vacuum filter flask, usually 1 liter,

with appropriate tubing. Filter manifolds to

hold a number of filter bases are optional.

3.3.12 Safety trap flask placed between

the filter flask and the vacuum source.

3.3.13 Forceps, straight or curved, with

smooth tips to permit easy handling of filters

without damage.

3.3.14 Alcohol, ethanol or methanol, in

small wide mouthed vials, for sterilizing

forceps.

ISOLATION AND ENUMERATION

image:

FIGURE ll-C-6. Membrane Filtration Units Made by Various Manufacturers

for Detection of Bacteria in Aqueous Suspensions.

•&EFA MICROBIOLOGICAL MANUAL 1978

image:

Funnel

Locking King

Membrane Pilfer

Membrane Filter

Support Screen

Filter Base with

Bayonet Joint

Stopper

FIGURE ll-C-7. Exploded View of a Stainless Steel Membrane Filtration Unit.

ISOLATION AND ENUMERATION

image:

3.3.15 Bunsen or Fisher type burner or

electric incinerator unit.

3.3,16 Sterile T.D. bacteriological or Mohr

pipets, glass or plastic, of appropriate volume.

3.3.17 Sterile petri dishes, 50 x 12 mm,

with tight-fitting lids and 60 x 15 mm, with

loose-fitting lids glass or plastic.

3.3.18 Dilution bottles (milk dilution)

pyrex, marked at 99 ml, screw-cap with

neoprene liners.

3.3.19 Membrane filters, white, grid

marked, 47 mm diameter, with 0.45 um ± .02

um pore size or other pore size recommended

by the manufacturer for water analyses.

3.3.20 Absorbent pads of cellulosic

paper, 47 mm diameter. The paper should be

of high quality and free of sulfites or other

substances that could inhibit bacterial growth.

3.3.21 Waterproof plastic bags.

3.3.22 Inoculation loops, at least 3 mm

diameter, or needles, nichrome or platinum

wire, 26 B&S gauge, in suitable holder. Dispos-

able applicator sticks or plastic loops as alter-

natives to inoculation loops.

3.3.23 Media: Media required for a spe-

cific test should be prepared in pre-sterilized

erlenmeyer flasks with metal caps, aluminum

foil covers, or screw caps.

3.3.24 Dilution Water

(a) Sterile buffered or peptone dilution

water dispensed in 99 j^ 2 ml amounts in

screw-capped dilution bottles.

(b) Sterile dilution water prepared in larger

volumes for wetting membranes before addi-

tion of the sample and for rinsing the funnel

after sample filtration.

3.4 Procedure

3.4.1 Prepare the required media as out-

lined in Part II-B. If the medium is an agar, cool

to room temperature. Use sterile forceps for

manipulation of absorbent pads and mem-

brane filters, contacting the outer edges only,

to avoid touching the filtering area or damag-

ing the membrane filter surface. Sterilize for-

ceps by immersing the tips in ethanol and

flaming. Place absorbent pad in bottom of 50

or 6O mm petri dish. Add 1.8-2.0 ml broth to

the sterile absorbent pad. Saturate but do not

flood the pad. Tip the petri dish to drain off

excess, if agar medium is used, add about 5-6

ml (to a depth of 2-3 mm) in the petri dish.

3.4.2 Arrange petri dishes in rows accord-

ing to the dilution series. Mark each dish to

identify the sample, volume or dilution to be

filtered.

3.4.3 Using sterile forceps, place a mem-

brane filter, grid-side up, on the porous plate of

the filter base.

3.4.4 Attach the funnel to the base of the

filter unit, taking care not to damage or dis-

lodge the filter. The membrane filter is now

fitted between the funnel and the base.

3.4.5 Shake the sample container vigor-

ously about 25 times.

3.4.6 Prepare at least three sample incre-

ments according to 1,4.3, in this Section.

Measure the desired volume of sample into the

funnel with the vacuum turned off. To measure

the sample accurately and to obtain good distri-

bution of colonies on the filter surface, the

following methods are recommended:

(a) Sample volumes of 20 ml or more:

Measure the sample in a sterile graduated cyl-

inder and pour it into the funnel. Rinse the

graduate twice with sterile dilution water, and

add the rinse water to the funnel. For potable

waters, 100 ml volumes may be measured

directly in a precalibrated funnel.

(b) Sample volumes of 10-20 ml: Measure

the sample with a sterile 10 ml or 20 ml pipet

into the funnel.

10 ml of sterile dilution water into the funnel

and add the sample to the sterile water using

appropriate sterile pipet.

MICROBIOLOGICAL MANUAL 1978

image:

(d) Sample volumes of less than 0.1 ml:

Prepare appropriate dilutions in sterile dilution

water and proceed as applicable in steps (b) or

(e) The time elapsing between preparation

of sample dilutions and filtration should be

minimal and never more than 30 minutes.

3.4.7 Turn on the vacuum to filter the

sample. Leave the vacuum on and rinse down

the funnel walls at least twice with 20-30 ml of

sterile dilution water. Turn off vacuum.

3.4.8 Remove the funnel from the base of

the filter unit. An ultraviolet sterilizer unit can

be used to hold and sterilize the funnel be-

tween filtrations. At least 2 minutes exposure

time is required for funnel decontamination

(6). Protect eyes from UV irradiation with

glasses, goggles, or an enclosed UV chamber

(7).

3.4.9 Holding the membrane filter at its

edge with a sterilized forceps gently lift and

place the filter grid-side up in the culture dish.

Slide the filter onto the absorbent pad or agar,

using a rolling action to avoid trapping air

bubbles between the membrane filter and the

underlying pad or agar. Reseat the membrane

if non-wetted areas occur due to air bubbles.

3.4.10 Invert the petri dishes and incubate

at the appropriate temperature in an atmo-

sphere with close to 100% relative humidity

for the required time.

3.5 Counting Colonies: The grid lines are

used in counting the colonies.

3.5.1 Count the colonies for the parameter

of interest following a preset plan such as

shown in Figure ll-C-8. Some colonies will be in

contact with grid lines. A suggested procedure

to reduce error in counting these colonies is

shown in Figure ll-C-9. Count the colonies in

the squares indicated by the arrows.

3.5.2 The fluorescent lamp should be

nearly perpendicular to the membrane filter.

Count colonies individually, even if they are in

contact with each other. The technician must

learn to recognize the difference between two

or more colonies which have grown into con-

tact with each other and single, irregularly

shaped colonies which sometimes develop on

membrane filters. The latter colonies are usu-

ally associated with a fiber or paniculate ma-

terial and the colonies conform to the shape

and size of the fiber or particulates. Colonies

which have grown together almost invariably

show a very fine line of contact.

3.6 Calculation of Results: Select the

membrane filter with the number of colonies in

the acceptable range and calculate count per

100 ml according to the general formula:

count per 100 ml =

No. colonies counted

Volume of sample filtered, in ml

X 100

3.6.1 Counts Within the Acceptable Limits

(a) The acceptable range of colonies which

is countable on a membrane is a function of

the parameter as shown in Table ll-C-3.

(b) Assume that filtration of volumes of 50,

15, 5, 1.5, and 0.5 ml produced colony counts

of 200, 110,40, 10, 5, respectively.

colonies on all filters. By inspection he would

select the membrane filter(s) with 20-80 coli-

form colonies and then limit his actual count-

ing to such membranes.

(d) After selecting the best membrane

filter for counting, in this case the MF with a 40

colony count, the analyst counts colonies

according to the counting procedures in 3.6

and applies the general formula as follows:

Colonies per 100 ml =

X 100 = 800

ISOLATION AND ENUMERATION

image:

FIGURE ll-C-8, Colony-Counting Pathway, (The inner circle indicates the

effective filtering area, dashed line indicates the pathway.)

FIGURE ll-C-9. Enlarged Portion of Grid-Marked Square of Filter.

(Colonies are counted in squares indicated by the arrow.)

MICROBIOLOGICAL MANUAL 1978

image:

TABLE II-C-3

Acceptable Limits*

Parameters

Lower

Upper

Remarks

Total coliform bacteria

Fecal coliform bacteria

Fecal streptococci

20 (0 for potable waters)

100

Limit, 200 colonies

of all types

Colony counts < or > the limits cited above must be identified as outside of this range.

3.6.2 More Than One Acceptable Count

(a) If there are acceptable counts on repli-

cate plates, carry counts independently to final

reporting units, then calculate the arithmetic

mean of these counts to obtain the final re-

ported value.

For example, 1 ml volumes produce coli-

form counts of 26 and 36 or counts of 2600

and 3600/100 mi:

2600 + 3600

= 3100

and value

3100/100 ml

(b) If more than one dilution, indepen-

dently carry counts to final reporting units,

then average for final reported value.

For example, assume that volumes of 0.3,

0.1, 0.03 and 0.01 ml produced coliform colony

counts of TNTC (Too Numerous To Count), 75,

30 and 8, respectively. In this example, two

volumes, 0.1 and 0.03 produce colonies in

the acceptable counting range.

Independently carry each MF count to a

count per 100 ml:

— x 100 = 75,000/100 ml

0.1

0.03

Then calculate the arithmetic mean of these

counts to obtain the final reported value:

75,000 + 100.000

Report as: 88,000/100 ml.

3.6.3 If All MF Counts are Below the Lower

Limit, Select the Most Nea

Acceptable

Count (for non-potable waters)

For example, assume a count in which

sample volumes of 1,0.3 and .01 ml produced

colony counts of 14,3, and 0, respectively.

Here, no colony count falls within recom-

mended limits. Calculate on the basis of the

most nearly acceptable plate count, 14, and

report with a qualifying remark:

— x 100 = 1400

Report as: Estimated Count, 1400 per 100

X 100 =100,000/100 ml

ml.

3.6.4 If Counts from All Membranes are

Zero, Calculate Using Count from Largest Fil-

tration Volume

For example, sample volumes of 25, 10,

and 2 ml produced colony counts of 0, 0, and

0, respectively, and no actual calculation is

possible, even as an estimated report. Calcu-

late the number of colonies per 100 ml that

would have been reported if there had been

one colony on the filter representing the larg-

est filtration volume, thus:

J_ x 100 = 4

ISOLATION AND ENUMERATION

image:

Report as: < (Less than) 4 colonies per

100 ml.

3.6.5 if All Membrane Counts are Above

the Upper Limit, Calculate Count with Smallest

Volume Filtered

For example, assume that the volumes 1,

0.3, and 0.01 m! produced colony counts of

TNTC, 150, and 110 colonies. Since all colony

counts are above the recommended limit, use

the colony count from the smallest sample

volume filtered and estimate the count as:

X 100 = 1,100,000

Estimated Count

1,100,000 per 100 ml.

3.6.6 If Colonies are Too Numerous To

Count, Use Upper Limit Count with Smallest

Filtration Volume

Assume in Example 3.6.5 that the volumes

1.0, 0.3, and 0.01 ml, al[ produced too many

colonies to show separated colonies, and that

the laboratory bench record showed TNTC (Too

Numerous to Count).

Use 80 colonies (Upper Limit Count for

Total Coliform) as the basis of calculation with

the smallest filtration volume, thus:

0.01

X 100 = 800,000

Report as: > (Greater Than) 8000,000 per

100 mj.

3.6.7 If there is no result because of con-

fluency, lab accident, etc. Report as: No Result

and specify reason.

3.6.8 Reporting Results: Report bacterial

densities per 100 ml of sample. See Figure II-

C-1 and ll-C-3 for examples of forms for

reporting results,

3.7 Verification: A verified membrane fil-

ter test establishes the validity of colony differ-

entiation on a selective medium and provides

support evidence of colony interpretation.

3.7,1 A percent verification can be deter-

mined for any colony validation test:

No. of colonies meeting verification test

No. of colonies subjected to verification

X 100 = Percent verification

3.7.2 Verification is required for all posi-

tive samples from potable waters,

3.7.3 Verification is also recommended

for establishing quality control in research for

use with new test waters, new procedures or

new technicians, for identifying unusual col-

ony types and as support for data used in legal

actions.

3.7.4 The worker is cautioned not to apply

the percentage of verification determined for

one sample to other samples.

3.7.5 The careful worker may also pick

non-typical colonies and follow the verification

procedure to determine that false negative col-

onies do not occur.

3.8 Significant Figures: See 2.8.1.

3.9 Repeatability and Reproducibility of

Counts: Analysts should be able to duplicate

their own colony counts on the same mem-

brane within 5% and the counts of other ana-

lysts within 10%. Failure to agree within these

limits should trigger a review of procedures.

4. Most Probable Number (MPN) Method

4.1 Summary: The Most Probable Num-

ber procedure estimates the number of spe-

cific organisms in water and wastewater by

the use of probability tables.

Decimal dilutions of samples are inocu-

lated in series into liquid tube media. Positive

tests are indicated by growth and/or fermenta-

tive gas production. Bacterial densities are

based on combinations of positive and nega^

MICROBIOLOGICAL MANUAL 1978

image:

tive tube results read from the MPN table. The

MPN procedure may be carried to three stages

of completion:

4-1-1 The Presumptive Test provides a

preliminary estimate of bacterial density

based on enrichment in minimally-restrictive

tube media. The results of this test are never

used without further analyses,

4.1.2 The Confirmed Test, the second

stage of the MPN, is the usual extent of testing.

Growth from each positive Presumptive Test

tube is inoculated into a more selective inhibi-

tory medium. The tubes are incubated at the

prescribed temperature and time, the positive

reactions noted and counts calculated from

the MPN table.

4.1.3 The Completed Test is the third

stage of the MPN used for total coliform

analyses only. Positive tubes from the

Confirmed Test are submitted to additional

tests to verify the identification of the isolated

microorganisms. Although the Completed Test

provides the greatest reliability, the amount of

time and the workload restrict its use to

periodic substantiation of Confirmed Test

results, to other QC checks on methodology

and analysts, and to research.

4.2 Scope and Application

4.2.1 Advantages: The MPN procedure

has the advantages inherent in liquid nutrient

media.

(a) The Presumptive and Confirmed Tests

require only observing and recording of

gas/no gas for conforms and growth/no

growth for fecal streptococci. The tests require

minimal experience, training or interpretation

by the analyst.

(b) Water samples with high turbidity or

large numbers of algae have no apparent dele-

terious effect on the tube reactions.

sample, the resultant 1:10 or 1:100 dilution of

that sample in the liquid broth may reduce the

toxicity to the point of no effect.

(d) The MPN may be the only method appli-

cable to problem sample materials such as

bottom sludges, muds, soils and sediments

(with blending).

4.2.2 Limitations: The MPN procedure has

disadvantages:

(a) This method is ordinarily limited to a

maximum sample volume of 10 mi per tube, but

100 ml portions are used in shellfish waters.

(b) The time required for the test may be as

long as 96 hours for a Confirmed Test result.

tions and inherently have poor precision and

contain a 23% bias at the 5 tube, three dilution

levels normally used.

(d) The man-hour requirements to prepare

glassware and media and to perform the tests

are significant.

(e) Relatively large amounts of bench space,

incubator space and tube/rack storage space

are required.

(f) The procedure does not lend itself to

field work. As compliance monitoring of water

quality and effluent standards becomes a major

legal requirement, the time, precision and

equipment limitations cited in (b), {c), (d) and(e)

above are more serious for the large number of

field analyses which will be required.

(g) Background organisms or toxic con-

stituents in 10 ml volumes of marine water can

interfere and be undetected.

4.2.3 The minimum MPN test that is ac-

ceptable for water and wastewater analyses is_.

the Confirmed Test because of the high proba-

bility of false positive reactions in the Pre-

sumptive Test.

ISOLATION AND ENUMERATION

image:

4.3 Apparatus and Materials

4.3.1 Water bath or air incubator set at 35

j; 0.5 C for total coliform and fecal strepto-

cocci tests. Water bath at 44.5 ± 0.2 for fecal

coliform test

4.3.2 Pipet containers of stainless steel,

aluminum or pyrex glass for glass pipets.

4.3.3 Inoculation loops, 3 mm diameter,

and needle of nichrome or platinum wire, 26

B&S gauge, in suitable holder.

4.3.4 Disposable applicator sticks or plas-

tic loops as alternatives to inoculation loops in

4.3.3 above.

4.3.6 Compound microscope, oil immer-

sion, 1000X-

4.3.6 Culture tube racks, 10x5 open-

ings; each opening to accept 25 mm diameter

tubes.

4.3.7 Gas burner, Bunsen/Fisher types or

electric incinerator unit.

4.3.8 Sterile T.D. bacteriological or Mohr

pipets, glass or plastic, of appropriate sizes.

4.3.9 Dilution bottles (milk dilution), pyrex

glass, marked at 99 ml volume, screw cap with

neoprene liner.

4.3.10 Pyrex culture test tubes 150 x 25

or 150 X 20 mm containing inverted fermen-

tation vials, 75 x 10 mm and proper closures.

4.3.11 Gram stain solutions (optional).

See This Section 5.3.

(a) Hucker's Crystal Violet Solution (stain).

(b) Lugol's Iodine Solution (mordant).

(d)Safranin Solution (counterstain).

4.3.12 Glass microscope slides, 2.5 x 7.6

cm(1 X 3 inches).

4.4 Media: Appropriate media dispensed

in test tubes or in fermentation test tubes. See

Part IIB. for specific media.

4.5 Dilution Water: Sterile 99 ± 2 ml

volumes in screw-cap pyrex glass bottles. See

Partll-B,7.

4.6 Presumptive Test

4.6.1 Shake the sample or dilution con-

tainer vigorously about 25 times.

4.6.2 To perform the Presumptive Test,

arrange a series of three or more rows of

culture tubes containing the test medium in a

rack, providing for five replicates in each row.

Use five rows for samples of unknown density.

Inoculate each successive row with decreas-

ing decimal dilutions of the sample. For exam-

ple, in testing polluted waters for total coli-

forms, the initial sample inoculations might be

0.1,0.01,0.001,0.0001,0.00001 ml of origi-

nal sample into successive rows each contain-

ing five replicate volumes. This series of sam-

ple volumes would yield determinate results

from test waters containing up to 16,000,000

organisms per 100 ml by use of the MPN

tables.

When removing sample aliquots or dilu-

tions for further inoculations, do not insert the

pipet tip more than 2.5 cm (1 inch) below the

surface of the sample.

4.6.3 Incubate tubes for 24±2 hours at 35 C.

A positive presumptive test is gas production

for the coliforms or growth for fecal strepto-

cocci. After 24 hours incubation, examine the

tubes for gas formation and/or growth. Inocu-

late positive tubes into Confirmed Test media.

If there is no gas or growth reincubate these

negative tubes for an additional 24 hours.

4.6.4 If the Presumptive tubes are negative

after 48 ± 3 hours, discard tubes. If the Pre-

sumptive tubes are positive, the cultures are

verified in the Confirmed Test. Record the

negative and positive results.

&ERA MICROBIOLOGICAL MANUAL 1978

image:

4.7 Confirmed Test

4.7.1 The Confirmed Test is performed by

verifying positive tubes from the Presumptive

Test at 24 and 48 hours. If Presumptive tubes

are positive at 24 hours, confirm them at that

time.

4.7.2 A positive test is indicated by gas

production for the coliform bacteria or growth

for fecal streptococci. After 24 ± 2 hours incu-

bation, examine the tubes for gas formation

and/or growth. If there is no gas/growth, rein-

cubate these negative tubes for a second 24

hours.

4.7.3 After 48 hours ± 3 hours examine

tubes for gas and/or growth, record positive

and negative results. Discard negative tubes.

Retain positive tubes if the test is to be carried

to completion for total coliform tests.

4.7.4 The fecal coliform MPN test is per-

formed by inoculating EC Broth tubes with

growth from all positive Presumptive tubes and

incubating them at the elevated temperature of

44.5 C for 24 hours. Gas production is the posi-

tive reaction.

4.7.5 Passage of positive Presumptive cul-

tures through the ConfirmedTestcompletesthe

MPN series for fecal streptococci and fecal

coliform bacteria.

4.7.6 In routine practice, most sample ex-

aminations for total coliform are terminated at

the end of the Confirmed Test. However, for

quality control, at least five percent of the Con-

firmed Test samples (and a minimum of one

sample per test run) should be carried through

the Completed Test.

4.8 Completed Test for Total Coliform

MPN

Positjve Confirmed Test cultures may be

subjected to final Completed Test identification

through application of further culture tests,

as follows:

,4.8.1 Streak Levine'sEMB agar plates from

each positive confirmatory tube and incubate at

35 C for 24 hours. Pick typical coliform colonies

(or atypical colonies if no typical colonies are

present), inoculate into lauryl tryptose fermen-

tation tubes and incubate at 35 C for 24-48

hours. The formation of gas in any amount in

the fermentation tubes constitutes a positive

Completed Test for total conforms.

Typical colonies show a golden green

metallic sheen or reddish purple color with

nucleation.

Atypical colonies are red, pink or colorless,

unnucleated and mucoid.

4.8.2 Optional Gram Stain Procedure

The gram stain test has been used in the

Completed MPN Test for demonstrating gram

negative, nonsporeforming rods from isolated

colonies. Although the gram stain procedure is

proposed for revision of the 15th edition of

Standard Methods, it provides a final check on

results and remains useful for evaluating ques-

tionable colony types.

After incubation of the EMB agar plates for

24 hours at 35 C (in 4.8.1) pick at least two

typical colonies (or atypicals if no typical colo-

nies are present) and inoculate onto nutrient

agar slants. Incubate for 24 hours at 35 C, and

proceed as in 5, this Section.

4.9 Calculation of MPN Value

The calculated density of the Confirmed or

Completed Test may be obtained from the MPN

table based on the number of positive tubes

and reactions in each dilution.

4.9.1 Table ll-C-4 illustrates the MPN indi-

ces and 95% Confidence Limits for general use.

4.9.2 Table ll-C-5 shows the MPN indices

and limits for potable water testing.

4.9.3 Three dilutions are necessary to

formulate the MPN code. For example in Table

ll-C-4 if five 10 ml, five 1.0 ml, and five 0.1 ml

portions are used as inocula and positive results

are observed in five of the 10 ml inocula,

three of the 1.0 inocula, and none of the 0.1

ISOLATION AND ENUMERATION

image:

TABLE II4M

Most Probable Number Index and 95% Confidence Limits for Five Tube, Three Dilution Series (8,9)

No. of Tubes Giving

Positive Reaction out of

5 of 10

ml Each

5 of 1

ml Each

' 2

5 of 0. 1

ml Each

MPN

Index

per

100ml

95% Confidence

Limits

Lower

<0.5

<0,5

<0.5

<0,5

<0.5

Upper

4§

No. of Tubes Giving

Positive Reaction out of

5 of 10

ml Each

5 of 1

ml Each

6 Of 0. 1

ml Each

MPN

Index

per

100ml

110

140

180

130

170

220

280

350

240

350

540

920

1600

>_2400

95% Confidence

Limits

Lower

120

180

300

640

Upper

70;

110

120

150

130

170

220

190

250

340

500

300

490

700

850

1,000

750

1,000

1,400

3,200

5,800

image:

TABLE li-C-S

Most Probable Number Index and 95% Confidence Limits for Testing Potable Waters

Number of Positive Tubes

from five 10 ml Portions

MPN

Index/100 ml

95% Confidence Limits

Lower

Upper

<2.2

2.2

5.1

9.2

16.

0.1

0.5

1.6

3.3

8.0

6.0

12.6

19.2

29.4

52.9

Infinite

ISOLATION AND ENUMERATION

image:

ml inocula, the coded results of the test are

5-3-0. The code is located in the MPN Table,

and the MPN index of 79 per 100 ml is

recorded.

4,9.4 When the series of decimal dilutions

is other than 10, 1.0 and 0.1 ml, select the

MPN value from Table ll-C-4 and calculate

according to the following formula:

MPN (From Table) x

Largest Quantity Tested

MPN/100 ml

As an example, five out of five 0.01 ml

portions, two out of five 0.001 ml portions,

and zero out of five 0.0001 ml portions from a

sample of water, gave positive reactions. From

the code 5-2-0 in MPN Table (Table ll-C-4), the

MPN index 49 is adjusted for dilutions:

49 {From Table) X

0.01

= 49,000

The final corrected MPN Value =

49,000/100 ml.

4.9.5 If more than the above three sample

volumes are inoculated, the three significant

dilutions must be determined. The significant

dilutions are selected using the following

rules:

(a) Only three dilutions are used in the

code for calculating an MPN value.

(b) To obtain the proper three dilutions,

select the smallest sample volume giving all

positive results and the two succeeding lesser

sample volumes. See Table II-C-6, Test land 2.

tive tubes, select the three highest sample

volumes which will include the dilutions with

the positive tubes. See Table II-C-6, Test 3.

(d) If there are positive tubes in the dilu-

tions higher than these dilutions selected, pos-

itive results are moved up from these dilutions

sample volume to increase the positive tubes

in the highest dilution selected. See Table II-C-

6, Test 4.

(e) There should be no negative results in

higher sample volumes than those chosen.

However, if negative tubes are present, e.g.,

4/5, 5/5, 3/5 and 0/5 the highest sample vol-

ume with all positive tubes must be used along

with the next two lower sample volumes. See

Table II-C-6, Test 5.

(f) If all tubes are positive, choose the three

highest dilutions. See Table II-C-6, Test 6.

(g) If all tubes are negative, choose the

three lowest dilutions. See Table II-C-6, Test 7.

(h) If positive tubes skip a dilution, select

the highest dilution with positive tubes and the

two lower dilutions. See Table II-C-6, Test 8.

(i)lf only the middle dilution is positive,

select this dilution and one higher and

lower dilution. See Table II-C-6, Test 9.

4.9.6 A number of theoretically possible

combinations of positive tube results are omit-

ted in Table ll-C-4 because the probability of

their occurrence is less than 1 %. If such un-

likely tube combinations occur in more than

1 % of samples, review the laboratory proce-

dures for errors and note sample types. Collect

fresh samples for analyses.

4.9.7 The MPN can also be computed for

each sample based upon the number of

positive and negative Presumptive, Confirmed

or Completed Tests, and the total number of

milliliters tested (10). MPN/100 ml =

No. of Positive Tubes

100

(No. of ml in Negative Tubes) X

(No, of rnl in All Tubes)

Example: From a sample of water, five out

of five 10 ml portions, two out of five 1.0 rnl

portions, and zero out of five 0.1 ml portions

MICROBIOLOGICAL MANUAL 1378

image:

TABLE Il-C-fi

Selection of Coded Results, Five Tube Series

Test

Positive

10 1.0

5' 3

5 5

4 5_

Tubes/ml

0.1 0

Sample

.01

Volume

0.001

0.0001

Code

5-3-0

5-4-0

4-1-0

5-4-2

5-3-0

5-5-5

0-0-0

4-0-2

0-1-0

'Underlines indicate positive tube series selected for code.

ISOLATION AND ENUMERATION

image:

gave positive results. Therefore, MPN/100 ml

7 X 100

(55.5)

= 50.22

MPN/100 ml = 50

4.10 Reporting Results: Report the MPN

value for water samples on the basis of 100 ml

of sample. Report the MPN values of solid type

samples on the basis of 1 gram of dry weight

sample.

Examples of bench forms are shown in

Figuresll-C-1,2and3.

4.11 Precision and Accuracy

4.11.1 The precision of the MPN value

increases proportionately with the number of

replicates tested.

4.11.2 Multiple-tube values are generally

high because MPN tables include a 23% posi-

tive bias.

4.11.3 MPN numbers represent only a sta-

tistical estimate of the true bacterial density in

the sample. The 95% Confidence Limits for

each MPN value included in the Tables ll-C-4

and 5 show the limited precision of these

estimates.

5.1.3 Air-dry the smear and fix by quickly

passing the slide several times through a por-

tion of the flame.

5.2 Gram Stain

Gram staining is a general test for charac-

terization of bacteria and for examination of

culture purity.

5.2.1 Prepare and fix a bacterial smear as

in 5.1. For quality control, prepare a separate

smear of known gram positive cocci and gram

negative rods.

5.2.2 Flood the smear with ammonium

oxalate-crystal violet stain for one minute.

5.2.3 Wash the slide in a gentle stream of

tap or pure water and flood with Lugol's iodine

solution (mordant). Allow it to remain for one

minute.

5.2.4 Wash the slide in water and blot dry.

5. Staining Procedures

5.1 Preparation of Bacterial Smears

5.1.1 Place a small drop of laboratory pure

water on a clean slide.

5.1.2 Using a sterilized inoculating nee-

dle, pick a small amount of growth from the

agar slant. Mix the bacteria with the drop of

water on the slide and spread evenly over an

area the size of a quarter. Use loop for broth

cultures.

5.2.5 Decolorize with acetone alcohol,

either by adding it dropwise on the tilted slide

until the blue color stops flowing from the

smear, or by gently agitating the slide up and

down in a beaker containing the alcohol wash

for about 30 seconds.

5.2.6 Flood the smear with the safranin

counterstain for 10 seconds. Wash and air-dry.

MICROBIOLOGICAL MANUAL 1978

image:

5.2.7 Examine under the oil immersion

objective. Gram positive cells retain the crystal

violet stain and are blue in color. Gram negative

cells are decolorized by the acetone alcohol so

that they accept the safranin counterstain and

appear pink to red.

5.3 Stain Solutions

Only those stains and dyes which are certi-

fied by the National Biological Stain Commis-

sion should be used.

5.3.1 Loeffler's Methylene Blue

(a) Methylene Blue-ethyl alcohol solution.

(1) Dissolve 0.3 grams of methylene blue

(90% dye content) in 30 ml of 95% ethyl alcohol

(Solution A).

(2) Dissolve 0.01 grams of potassium

hydroxide in 100 ml of laboratory pure water

(Solution B).

(3) Mix solution A and B.

5.3.2 Solutions for Gram Staining

(a) Ammonium oxalate-crystal violet

solution:

(1) Dissolve 2 grams crystal violet (approxi-

mately 85% dye content) in 20 ml of 95%

ethyl alcohol (Solution A).

(2) Dissolve 0.8 grams ammonium oxalate

in 80 ml laboratory pure water (Solution B).

(3) Mix Solutions A and B.

(4) Filter through cheesecloth or coarse

filter paper.

(5) Problems with the gram stain tech-

nique are frequently traceable to the

ammonium oxalate-crystal violet solution. In

the event that decolorization is insufficient, the

amount of crystal violet in the solution can be

reduced to as little as 10% of the

recommended amount.

(b) Lugol's iodine: Dissolve 1 gram iodine

crystals and 2 grams potassium iodide in

about 5 ml of pure water. After crystals are in

solution, add sufficient laboratory pure water

to bring the final solution to a volume of 300

ml.

volumes of acetone and 95% ethyl alcohol.

(d) Safranin: Dissolve 2.5 grams of safra-

nin in 100 ml of 95% ethyl alcohol. Store as a

stock solution. For working solution, add 10 ml

of stock to 100 ml of laboratory pure water,

mix and store.

6. Shipment of Cultures

6.1 Confirmation or further identification

to serotype may be required if the bacteriologi-

cal data are to be used for specific needs such

as enforcement cases, epidemiological stud-

ies, tracing sources of pollution or scientific

publication. The selected cultures should be

sent to an official typing center or state health

laboratory with pertinent information for the

confirmatory identification. This service is usu-

ally available if the cultures are of public health

significance, but permission should be ob-

tained from the reference laboratory before

sending cultures.

Observe the following instructions:

6.1.1 Send only pure cultures.

6.1.2 Provide a culture with discernable

growth on brain heart infusion agar, blood

agar base or nutrient agar stab in a screw-cap

tube or vial sealed with a cork soaked in hot

paraffin. Triple sugar iron agar or other sugar-

containing agars should not be used.

6.1.3 Complete the reference laboratory

form which requires information on the source

of the culture, tests completed, results

obtained and identification of the originating

laboratory. Include form with cultures, but on

outside of secondary container.

ISOLATION AND ENUMERATION

image:

6.2 Shipping Regulations for Cultures:

Transportation (DOT) regulations apply to sur-

face and air transportation, but shipment of

cultures beyond 100-200 miles is only practi-

cal by air. Air freight service is limited in many

areas, hence passenger-carrying aircraft must

be used for safe and quick service. Strict ship-

ping regulations are imposed on such passen-

ger service shipments (11), Packaging and la-

beling of the cultures must conform with cur-

rent federal shipping regulations for etiologi-

cai agents described in: 49 CFR 173.387 (12)

and 42 CFR 72.25 (c) (13). The requirements in

6.2.1-6.2.8 that follow are also shown in Fig-

ure li-C-10.

6.2.1 Place each culture in a securely

closed, watertight PRIMARY CONTAINER

(screw-cap test tube or vial) and seal the cap

with tape.

6.2.2 Wrap the PRIMARY CONTAINER

with sufficient absorbent material (paper

towel, tissue, etc.) to absorb the entire con-

tents should breakage or leakage occur.

6.2.3 Place the wrapped, sealed,

PRIMARY CONTAINER in a durable, watertight

SECONDARY CONTAINER (screw-cap metal

mailing tube or sealed metal can). Screw-cap

metal mailing tubes should be sealed with

tape. Several PRIMARY CONTAINERS of

cultures, each individually wrapped in

absorbent material, may be placed in the

SECONDARY CONTAINER provided that the

total aggregate volume does not exceed 50

ml. (NOTE: Multiple secondary containers of

cultures, which individually meet the

packaging requirements for shipment of 50 ml

or less, can be overpacked in a single outer

shipping container, provided that the total

aggregate volume does not exceed 4000 ml).

6.2.4 Data forms, letters and other infor-

mation identifying or describing the cultures

should be placed around the outside of the

SECONDARY CONTAINER. DO NOT ENCLOSE

WITHIN THE SECONDARY CONTAINER.

6.2.5 Place the SECONDARY CONTAINER

and information form in an OUTER MAILING

TUBE OR BOX.

6.2.6 Place an address label and

ETIOLOGIC AGENT/MICROBIOLOGICAL CUL-

TURES label on the outer mailing tube or box.

6.2.7 Individual primary containers of

greater than 50 ml of culture material require

special packaging and cannot be transported

on passenger-carrying aircraft.

6.2.8 International shipments must also

conform to the added regulations: US Post

Office Publication 51, Air Cargo Restricted Art-

icles Tariff 6-D and DOT Regulations 49 CFR,

Section 173.

&EPA MICROBIOLOGICAL MANUAL 1978

image:

PART 1

PRIMARY

CONTAINER

CULTURE

(SCREW-CAP)

ABSORBENT

PACKING

MATERIAL

CAP

SECONDARY

CONTAINER

CAP

SHIPPING

CONTAINER

LABEL

ADDRESS

LABEL

CULTURE

INFORMATION

PART 3

PART 2

ET10LOGICAL AGENTS

MICROBIOLOGICAL

MATERIALS C

IN CASE OF DAMAGE

OR LEAKAGE

NOTIFY DIRECTOR, CDC

ATLANTA. GA

404-633-5313

WATERPROOF

TAPE

ABSORBENT

PACKING

MATERIAL

MICROBIOLOGICAL MATERIALS

PACKAGING CONFORMS

WITH STANDARDS IN 49

CFR 173.387, AND

42CFR72.25

CROSS SECTION

OF PROPER PACKING

FIGURE ll-C-10. Packaging and Labelling of Microbiological Cultures for Shipment.

image:

REFERENCES

1. Clark, H. F., P. W. Kablerand E. E. Geldreich, 1957. Advantages and limitations of the membrane

filter procedures. Water Sewage Works 104:385.

2. Ships, E. L and A. Fields, 1954. A comparison of the molecular filter technique with agar plate

count for enumeration of Escherichia coli. Appl. Microbiol. 2^382.

3. Snipe, E. L. and G. M. Cameron, 1954. A comparison of the membrane filter with the most

probable number method for coliform determinations from several waters. Appj. Microbiol. 2:85.

4. Geldreich, E. E., H. L. Jeter and J. A. Winter, 1967, Technical considerations in applying the

membrane filter procedure. Health Laboratory Science 4:113.

5. Proceedings of the Symposium on the Recovery of Indicator Organisms Employing Membrane

Filters. Jan 20-21,1975. Environmental Monitoring and Support Laboratory, U.S. Environmental

Protection Agency, Cincinnati, OH 45268. (1977).

6. Rhines, C. E. and W. P. Cheevers, 1965. Decontamination of membrane filter holders by

ultraviolet light. J. Am. Waterworks Association 57:500.

7. Manning, H., 1975. AQC Newsletter #26, July 1975. U.S. Environmental Protection Agency,

Environmental Monitoring and Support Laboratory, Cincinnati, OH 45268. p. 15.

8. Swaroop, S., 1938. Numerical estimation of B. coli by dilution method. Indian J. Med. Research

26:353. • ~

9, Swaroop, S., 1951. The range of variation of the most probable number of organisms estimated

by the dilution method. Indian J. Med. Research 39:107.

10. Thomas, H. A., Jr., 1942. Bacterial densities from fermentation tubes. J. Am. Water Works

Association 34:572.

11. Morbidity and Mortality Report, 1975. Center for Disease Control, Public Health Service,

USDHEW, Atlanta, GA 24:49.

12, Title 49, Code of Federal Regulations <CFR), Part 173.

13, Interstate Quarantine, regulations of the shipment of etiologic agents. Title 42, Code of Federal

Regulations, (CFR), Part 72, Section 25.

90 <8>Efik MICROBIOLOGICAL MANUAL 1978

image:

PART II. GENERAL OPERATIONS

Section D Selection of Analytical Methods

This Section discusses the selection of

methods for monitoring water and wastewater

in response to the Laws, the microbiological

standards that have been established, and the

criteria that have been recommended to en-

force the laws. The major problems that have

developed in.the application of the methods

9re identified and solutions are given where

they are available.

1. Methodology

1.1 National Interim Primary

Drinking Water Regu-

lations '

1.2 NPDES Guidelines

1.3 Marine Sanitation Regu-

lations

1.4 Water Quality Standards

1.5 Water Quality Criteria

1.6 Alternate Test Procedures

2. Problems in Application

2.1 Stressed Microorganisms

2.2 Incomplete Recovery/

Suppression

2.3 Interference by Turbidity

2.4 Analysis of Ground Water

2.5 Field Problems

2.6 Method Modifications

and Kits

2.7 Changes in Membrane

Filters and Methodology

2.8 Klebsiella'm Industrial

Wastes

3. Recommendations for Methods

in Waters and Wastewaters

1. Methodology

Test procedures have .been specified and

published in Federal Register for drinking

water, wastewater discharges (NPDES) and

vessel discharges.

1.1 National Interim Primary Drinking

Water Regulations

Although the National Interim Primary

Drinking Water Regulations (Title 40 CFR Part

141) state that the total coliform analyses can

be performed by the membrane filter or MPN

procedures, the MF procedure is preferred

because large volumes of samples can be

analyzed in a much shorter time, a critical

factor for potable water. Samples containing

excessive noncoliform populations or turbidity

must be analyzed by the MPN technique.

These regulations specify the testing of

sample sizes of 100 ml for the MF technique

and the testing of five replicate 10 or 100 ml

volumes for the MPN procedure. The law

directs that the samples be taken at points

representative of the distribution system.

The minimal schedules for the frequency

of sampling are based on population and

the required response is given for positive

test results. A detailed description of the

proposed criteria for interim certifi-

cation of microbiology laboratories under

METHOD SELECTION

image:

the Safe Drinking Water Act is given in

Appendix B,

1.2 National Pollution Discharge

Elimination System (NPDES) Guidelines

The NPDES established guidelines for

analysis of pollutants under PL 92-500,

Section 304 (g). The parameters and methods

are described in 40 CFR Part 136, as amended

(40 Code of Federal Regulations, Protection of

the Environment ch. 1 - Environmental

Protection Agency, Part 136, Guidelines

Establishing Test Procedures for the Analysis

of Pollutants). The method must be specified

and MPNs must be five tube, five dilution. See

Table H-D-1.

1.3 Marine Sanitation Regulations

The regulations for marine sanitation

devices (40 CFR Part 140) established

performance standards and specified the

analytical methods as those promulgated in

40 CFR Part 136, cited in 1.2 above.

1.4 Water Quality Standards

Water quality standards (limits) have been

established by law for drinking water and

certain sewage and industrial effluents. These

standards and the reference sources are listed

in Table ll-D-2. A standard must be specified in

the NPDES permitto be enforceable.

1.5 Water Quality Criteria

Water quality criteria have been

recommended by the EPA for certain types of

water classified according to use. These

criteria are listed in Table ll-D-3.

1.6 Alternate Test Procedures

The amendments to 304 (g) also provide

procedures for approval of alternate methods.

National approval for test methods is obtained

by application to EPA through EMSL-

Cincinnati while case by case approval is

obtained by application through the EPA

Regional Offices (40 CFR 136.4).

2. Problems in Application

Although the methods described in this

Manual are judged the best available, there are

difficulties in the application of methods in

different geographical areas, in certain wastes

and in some potable and surface waters. Addi-

tional problems can stem from the indiscrimi-

nate use of new and simplified equipment,

supplies or media that have been proposed for

use in these procedures.

2.1 Stressed Microorganisms

Some water and wastewater samples con-

tain microorganisms which should reproduce

but do not under the conditions of test. These

organisms have been described as injured or

stressed cells. The stress may be caused by

temperature changes or chemical treatment

such as chlorine or toxic wastes (1).

Stressed organisms are particularly

important in environmental measurements

because tests for bacterial indicators or

pathogens can give negative responses, then

recover later and multiply to produce

dangerous conditions. Subsections 2.1.1 and

2.1.2 describe efforts to recover stressed

microorganisms.

2.1-1 Ambient Temperature Effects

Extreme ambient temperatures stress

microorganisms and reduce recovery of

microbiological indicators. For example, in

Alaska and other extremely cold areas, the

severe change from cold stream temperature

to 44.5 C temperature of incubation reduces

recovery of fecal conforms. The two-step MF

test for fecal coliforms increases recoveries by

use of a 2-hour acclimation on an enrichment

medium at 35 C before normal incubation at

44.5 C.

In contrast, water samples from natural

waters at high temperatures may include large

numbers of non-coliform organisms which in-

terfere with sheen production on MF's and

with positive gas production in MPN analyses.

An improved MF medium that provides greater

selectivity is desirable but may not be possible

without sacrificing recovery.

MICROBIOLOGICAL MANUAL 1978

image:

TABLE H-D-1

Approved Test Procedures for the Analysis of Pollutants (40 CFR 136)

Parameter per 100 ml

Fecal

Coliforms

Fecal Conforms in

presence of chlorine

Total

Coliforms

Coliforms in

presence of Chlorine

Fecal

Streptococci

Method

MPN

Plate Count

Reference and Page No.

SM1 USGS2 This Manual

922

937

922

928, 937

916

928

916

933

943

944

947

Part III

45 Part III

Part III

35 Part III

Part III

50 Part III

Part III

C, 5

C, 2

C, 5

C, 2

B, 4

B, 2

B, 4

B, 2

D, 4

D, 2

D, 5

Standard Methods for the Examination of Water and Wastewater, 14th Edition, (1975).

Slack, K. V., etal. Methods for Collection and Analysis of Aquatic Biological and

Microbiological Samples. USGS Techniques of Water Resources Inv., Book 5, eh. A4 (1973).

Since the MF technique usually yields low and variable recovery from chlorinated

wastewaters, the MPN method will be required to resolve any controversies.

METHOD SELECTION

image:

Vegetables

Textiles

Effluents from Marine

Sanitation Devices with

TABLE ll-D-2

Water Quality Standards

Water or

Wastawater

Potable Water

Chlorinated Effluents

2* Treatment Wastes

Selected Industrial Wastes

Leather and Tanning

Feed Lots

Meat Products

Beet Sugar

Canned Fruits and

Microbiological

Standards

Coliforms/100 ml

Total Fecal

<5 —

— 200-400

— 400

Reference

Source

PL 93-523

PL 92-500

40 CFR Part 133

PL 92-500

40 CFR Part 425

40 CFR Part 412

40 CFR Part 432

40 CFR Part 409

40 CFR Part 407

400

40 CFR Part 410

Discharges

Type I

Type II

— 1000

— . 200

40 CFR Part 140

and Amendments

40 CFR Part 140

and Amendments

<&ER& MICROBIOLOGICAL MANUAL 1978

image:

TABLE ll-D-3

Water Quality Criteria

Water or

Wastewater

Public Water Supply

Recreational Water:

Primary Contact

General Contact

Agricultural Water

Shellfish-Raising Waters

.Statistical

Measure

log X

log X/30 days

maximum/30 days,

in 10% of Samples

log X/30 days

maximum/30 days,

in 10% of Samples

monthly X

Daily Median

Highest 10% of

Daily Values

Microbiological Criteria

Coliforms/ 100 mi

Total Fecal

20000 2666""

200

400

1000

,., 2000

5000 1000

70 14

230 43

Reference

Source

C & D ..

A Water Quality Criteria, EPA. March, 1973. Superintendent of Documents, U.S. Government Printing

Office, Washington, DC 20402.

B Water Quality Criteria, FWPCA, April 1,1968. Superintendent of Documents, U.S. Government Printing

Office, Washington, DC 20402.

C National Shellfish Sanitation Program Manual of Operation. U.S. Dept. of. HEW; 1965. Public Health

Service Publ. No. 33, Superintendent of Documents, U.S. Government Printing Office, Washington, DC

20402. .

D Quality Criteria for Water, July 1976, O.W.H.M., US EPA.

METHOD SELECTION

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2,1.2 Chlorinated Effluents and Toxic

Wastes

Although thiosulfate is added to all

samples suspected of containing chlorine, to

neutralize its toxic effects, the membrane filter

procedure yields poor recovery of conforms

from chlorinated effluents as compared to

MPN recovery (1-6). A recent amendment to

40 CFR 136 added Coliform bacteria (Fecal) in

the presence of chlorine, as a specific

parameter and recommended analysis by the

MF or MPN techniques (7). A qualifying state-

ment appended to the method in 40 CFR Part

136 requires the five tube, five dilution MPN

and states: "Since the membrane filter

technique usually yields low and variable

recovery from chlorinated wastewaters, the

MPN method will be required to resolve any

controversies." Therefore, the MPN procedure

should be used in analysis of chlorinated

effluents where the data may be challenged by

legal or enforcement actions. The MF may be

used currently for self-monitoring situations.

{See Table ll-D-1).

Proposed changes in MF materials and

procedures include new membrane filter for-

mulations, an agar overlay technique, modified

media and twostep methods (1), Present modi-

fications of the MF method have not produced

recoveries of fecal conforms from chlorinated

effluents equivalent to MPN recoveries. Thor-

ough evaluation and approval of proposed pro-

cedures by EPA are required before changes

will be acceptable.

Certain types of wastes show recovery

problems for total and fecal coliforms:

1. Primary and Chlorinated-Primary

Waste Effluents.

2. Chlorinated-Secondary and Chlor-

inated-Tertiary Waste Effluents.

3. Industrial wastes containing toxic

metals or phenols.

When turbidity and low recovery prevent

the application of the MF technique to coliform

analyses of primary and secondary effluents or

industrial wastes containing toxic materials,

the MPN procedure is required. However, the

two-step MF procedure for total coliforms

described in this Manual and in Standard

Methods is acceptable for toxic wastes.

If the MF procedure is applied to

chlorinated or toxic samples, the laboratory

should require data from at least 10 samples

collected over 1 week of plant processing (but,

not less than 5 calendar days) to show

comparability of the MFtothe MPN technique.

See Part IV-C, 3 for details.

2.2 Incomplete Recovery/Suppression

When coliforms are present in low num-

bers in drinking -water, high levels of non^

coliforms can suppress growth or mask detec-

tion. This problem may appear as a mass of

confluent growth on a membrane filter or as

spots of sheen in this confluent growth. In the

MPN procedure, presumptive tubes may show

heavy growth with no gas bubbles, dilution

skips or unusual tube combinations. When

these negative presumptive tubes are trans-

ferred to BGLB, they confirm in this more re-

strictive medium, indicating that the coliform

gas production in the Presumptive Test was

suppressed by non-coliforms.

2.3 Interference by Turbidity

The tendency of bacteria to clump and

adhere to particles can produce inaccurate

results in the analysis of water samples. The

National Interim Primary Drinking Water Regu-

lations (NIPDWR) specify one turbidity unit as

the primary maximum allowable level but per-

mit up to five turbidity units if this level does

not interfere with disinfection or microbiologi-

cal analyses. Turbidity can interfere with filtra-

tion by causing a clumping of indicators or

clogging of pores. The turbidity as organic

solids can also provide nutrients for bacterial

growth and subsequently produce higher

counts. The type of particles variably affects

the filtration rate; for example, clay, silt or

organic debris clog more easily than sand.

Background organisms may also be imbedded

V>EPA MICROBIOLOGICAL MANUAL 1978

image:

in the particles and interfere with the coliform

detection.

2.4 Analysis of Ground Water

Although total coliforms are a valid mea-

sure of pollution, their use as indicators in

analyzing ground waters and rural community

supplies may not sufficiently describe the

water quality. For example, ground waters fre-

quently contain high total counts of bacteria

with no coliforms. Such waters pass Interim

Drinking Water Regulations buttechnical judg-

ment must conclude these are not acceptable

as potable waters.

2.5 Field Problems

Assurance of data validity demands sam-

ple analyses within the shortest time interval

after collection. This need requires field ana-

lyses using either a mobile laboratory or field

kit equipment. Since a mobile laboratory may

not be available for a survey, it is likely that at

least a part of the analyses will need to be

completed in an onsite facility. If the analyses

can be done using membrane filtration tech-

niques, field kits such as Millipore's Water

Laboratory and MF Portable Incubator (heat

sink) are particularly helpful for rapid set-up

and analyses of limited samples. However, if

large numbers of samples are tested per day or

the survey covers more than a few days, the

heat-sink incubator is Impractical because of

limited capacity and high cost. In such surveys,

a mobile laboratory utilizing water-jacketed in-

cubators is more practical.

2.6 Method Modifications and Kits

Commercial manufacturers continue to of-

fer proprietary kits and method modifications

to speed or simplify the procedures used in

coliform and fecal coliform analyses, primarily

for field use. Most of these units have not been

demonstrated to produce results comparable

to the official procedures. If not tested to the

satisfaction of EPA, such method modifica-

tions and kits cannot be used for establishing

total or fecal coliform numbers for permits

under NPDES or for total coliform numbers

under the Safe Drinking Water Act. The proce-

dure required for acceptance of an alternative

procedure is described in 40 CFR Parts 136.4

and 136.5, as amended.

2.7 Changes in Membrane Filters and

Methodology

There is an expected pattern of changes

in materials and methodology used in the

manufacture of membrane filters. The changes

may or may not be announced by the manu-

facturer. Therefore, it is important for the

laboratory to monitor membrane performance

as described in Section A of Quality Control

in this Manual.

These changes include modification of

formulations and the replacement of the

0.45 Mm pore MF by a 0.7 Mm retention pore

MF for improved recovery. Tests by independ-

ent investigators show that several MF's give

comparable recovery (5, 6, 8, 9), however,

enrichment or two-temperature incubations

are needed before recoveries approach the

MPN values (See 2.1.2 in this Section).

This discussion of problems with new

methodology and membrane materials should

not be interpreted as indicating that EPA dis-

courages new developments. Rather EPA en-

courages the MF supply industry to test and

examine procedures, to innovate and to re-

search. The membrane filter manufacturers

should be commended and encouraged to

continue their efforts toward solving problems

and improving materials and techniques in

water microbiology.

2.8 Klebsiella\n Industrial Wastes

Ktebsiella bacteria (part of the coliform

group) multiply in certain industrial wastes, are

not differentiated from fecal coliforms by MF

and MPN procedures and consequently are

included in the results. These recoveries have

been reported in textile, paper and pulp mills

and other wastes. Objections have been raised

to the application of fecal coliform standards

METHOD SELECTION

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TABLE ll-D-4

Selection of Methods for Problem Samples

Problem Area

Treated Industrial Wastes

(non-chlorinated, non-toxic)

Low Solids Wastes

Parameter Chosen

Fecal Coliform

Method of Choice

Shellfish-harvesting waters

Marine & Estuarine Waters

Total boliform

Fecal Coliform

Total Coliform

Fecal Coliform

MPN' "

MPN'

MF/MPN

Toxic Industrial Wastes

(metals, phenolics) and

High Solids Wastes

Fecal Coliform

MPN or alternate

procedure, tested

and approved"

Primary and Chlorinated-

Primary Municipal/Industrial

Effluent

Total Coliform

Fecal Coliform

MPN or alternate

procedure, tested

,and approved"

MPN or alternate

procedure, tested

and approved"

Chlorinated-Secondary Effluent

Total Coliform

Fecal Coliform

Two-Step MF

MPN or alternate

procedure, tested

and approved"

*MPN recommended to conform with the MPN method specified for examination of

shellfish. . '.."'

"Requires proof of comparability under. EPA's specified test regime that the alternate

procedure (MF, streak plate, etc.) is valid. See This Manual, IV-C, 3.

&EPA MICROBIOLOGICAL MANUAL 1978

image:

to these wastes because Klebsiella originate

from other than sanitary sources. However,

EPA does consider large numbers of Kleb-

siella, Aeromonas and other noncoliforms as

indicators of organic pollution. Further, these

organisms do occur in low densities in human

and animal wastes.

3. Recommendations for Methods in Waters

and Wastewaters

The amended Federal Water Pollution

Control Act, the Marine Protection, Research

and Sanctuaries Act and the Safe Drinking

Water Act require recommendations on analyt-

ical methodology. Generally, the membrane

filter methods are preferred over MPN and

other techniques, where proven applicable.

In Table ll-D-4, problem samples are

identified and the analytical method recom-

mended for parameters of choice.

REFERENCES

1. Bordner, R. H,, C, F. Frith and J. A. Winter, eds., 1977. Proceedings of the Symposium on

Recovery of Indicator Organisms Employing Membrane Filters, U.S. Environmental Protection

Agency, EPA-600.19-77-024, EMSL-Cincinnati, Cincinnati, OH 45268.

2. Lin, S. D., 1973. Evaluation of coliform tests for chlorinated secondary effluents. JWPCF, 45:3:498.

3. Greene, R. A., R. H. Bordner and P. V. Scarpino, 1974. Applicability of the membrane filter and

most probable number coliform procedures to chlorinated wastewaters. Paper G87 given at

74th Annual Meeting of the American Society for Microbiology, May 12-17, 1974, Chicago, IL.

4. Rose, R; E., E. E. Geldreich and W. Litsky, 1975. Improved membrane filter method for fecal

coliform analysis, Appl. Microbiol. 29:4:532.

5. Lin, S. D., 1976. Evaluation of Millipore HA and HC membrane filters for the enumeration of

indicator bacteria. Appl. Environ. Microbiol. 32:300.

6. Green, 8. L., E. Clausen and W. Litsky, 1975. Comparison of the new Millipore HC with

conventional membrane filters for the enumeration of fecal coliform bacteria. Appl. Microbiol.

30:697.

7. Guidelines for Establishing Test Procedures, 40 Code of Federal Regulations (CFR) Part 136,

Published in Federal Register, 40, 52780, Dec. 1, 1976.

8. Tobin, R. S. and 8. J. Dutka, 1977. Comparison of the surface structure, metal binding, and fecal

coliform recoveries of nine membrane filters. Appl. Environ. Microbiol. 34:69.

9. Lin, S. D., 1977. Comparison of membranes for fecal coliform recovery in chlorinated effluents.

JWPCF, 49:2255.

METHOD SELECTION

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PART III. ANALYTICAL METHODOLOGY

Part 111 of the manual describes the specific analytical procedures selected in response to the

parameters required under PL 92-500 and 93-523 (see Part V-D, Legal Considerations) and to

related parameters for indicators and pathogens which.supplementthe required information. New

parameters and new methodology will be added as proven in actual usage. The methods are

presented in Sections as follows:

Section A Standard Plate Count

Section B Total Coliforms

Section C Fecal Coliforms

Section D Fecal Streptococci

Section E Salmonella

Section F Actinomycetes

100 <&ER& MICROBIOLOGICAL MANUAL 1978

image:

PART III. ANALYTICAL METHODOLOGY

Section A Standard Plate Count

1. Summary of Method

The Standard Plate Count (SPC) Method is

a direct quantitative measurement of the via-

ble aerobic and facultative anaerobic bacteria

in a water environment, capable of growth on

the selected plating medium. An aliquot of the

water sample or its dilution is pipetted into a

sterile glass or plastic petri dish and a liquified,

tempered agar medium added. The plate is

rotated to evenly distribute the bacteria. Each

colony that develops on or in the agar medium

originates theoretically from one bacterial cell.

Although no one set of plate count conditions

can enumerate all organisms present, the

Standard Plate Count Method provides the uni-

form technique required for comparative test-

ing and for monitoring water quality in se-

lected situations.

2. Scope and Application (1 -6)

This simple technique is a useful tool for

determining the bacterial density of potable

waters and for quality control studies of water

treatment processes. The Standard Plate

Count provides a method for monitoring

changes in the bacteriological quality of fin-

ished water throughout a distribution system,

thus giving an indication of the effectiveness

of chlorine in the system as well as the possi-

ble existence of cross-connections, sediment

accumulations and other problems within the

distribution lines. Jots.', bacterial densities

greater than 500-1000 organisms per ml may

indicate coliform suppression or desensitiza-

tion of quantitative tests for coliforms (1-3).

The procedure may also be used to monitor

quality changes in bottled water or emergency

water supplies.

2.1 Theoretically, each bacterium present

in a sample multiplies into a visible colony of

millions of bacteria. However, no standard

plate count or any other total count procedure

yields the true number because not all viable

bacterial cells in the water sample can repro-

duce under a single set of cultural conditions

imposed in the test. The number and types of

bacteria that develop are influenced by the

time and temperature of incubation, the pH of

the medium, the level of oxygen, the presence

of specific nutrients in the growth medium,

competition among cells for nutrients, anti-

biosis, predation, etc.

2.2 This procedure does not allow the

more fastidious aerobes or obligate anaerobes

to develop. Also, ba'cteria of possible impor-

tance in water such as Crenothrix, Sphaeroti-

lus, and the actinomycetes will not develop

within the incubation period specified for pota-

ble water analysis.

2.3 Clumps of organisms in the water sam-

ple which are not broken up by shaking result

in underestimates of bacterial density, since

an aggregate of cells will appear as one colony

on the growth medium.

STANDARD PLATE COUNT

101

image:

3. Apparatus and Materials

5. Procedure

3.1 Incubator that maintains a stable 35

±0.5 C. Temperature is checked against an

NBS certified thermometer or one of equivalent

accuracy.

3.2 Water bath for tempering agar set at

44-4 6 C.

3.3 Colony Counter, Quebec darkfield

model or equivalent.

3.4 Hand tally or electronic counting de-

vice {optional).

3.5 Pipet containers of stainless steel, alu-

minum or pyrex glass for glass pipets.

3.6 Petrl dish containers of stainless steel

or aluminum for glass petri dishes.

3.7 Thermometer certified by National

Bureau of Standards or one of equivalent

accuracy, with calibration chart.

3.8 Sterile TD (To Deliver) bacteriological

or Mohr pipets, glass or plastic of appropriate

volumes, see Part ll-B, 1.8.1.

3.9 Sterile 100 mm x 15 mm petri dishes,

glass or plastic.

3.10 Dilution bottles (milk dilution), pyrex

glass, marked at 99 ml volume, screw cap with

neoprene rubber liner.

3.11 Bunsen/Fisher gas burner or electric

Incinerator.

4. Media

4.1 Sterile Plate Count Agar (Tryptone Glu-

cose Yeast Agar) dispensed in tubes (15 to 20

ml per tube) or in bulk quantities in screw cap

flasks or dilution bottles. See Part II-B, 5.1.5.

4.2 Sterile buffered dilution water, 99 ± 2

ml volumes, in screwcapped dilution bottles.

See Part II-B, 7.

5.1 Dilution of Sample (See Part il-C, 1.4

for details)

5.1.1 The sample is diluted to obtain final

plate counts of 30-300 colonies. In this range,

the plate counts are *he most accurate and

precise possible. Sir^v the microbiai popula-

tion in the original water sample is not known

beforehand, a series of dilutions must be pre-

pared and plated to obtain a plate count within

this range.

•5.1.2 For most potable water samples,

countable plates can be obtained by plating 1

and 0.1 ml of the undiluted sample, and 1 ml of

the 1:100 sample dilution (see Figure lll-A-1).

Higher dilutions may be necessary with some

potable waters.

5.1.3 Shake the sample vigorously about

25 times.

5.1:4 Prepare an initial 1:100 dilution by

pipetting 1 ml of the sample into a 99 ml

dilution water blank using a sterile 1 ml pipet

(see Figure lll-A-1).

5.1.5 The 1:100 dilution bottle is vigor-

ously shaken and further dilutions made by

pipetting aliquots (usually 1 ml) into additional

dilution blanks. A new sterile pipet must be

used for each transfer and each dilution must

be thoroughly shaken before removing an ali-

quot for subsequent dilution.

5.1.6 When an aliquot is removed, the

pipet tip should not be inserted more than 2.5

cm (1 inch) below the surface of the liquid.

5.2 Preparation of Agar

5.2.1 Melt prepared plate count agar (tryp-

tone glucose yeast agar) by heating in boiling

water or by flowing steam in an autoclave at

100 C. Do not allow the medium to remain at

these high temperatures beyond the time nec-

essary to melt it. Prepared agar should be

melted once only.

5.2.2 Place melted agar in a tempering

water bath maintained at a temperature of

44-46 C. Do not hold agar at this temperature

102

MICROBIOLOGICAL MANUAL 1978

image:

FIGURE lll-A-1. Typical Dilution Series for Standard Plate Count

STANDARD PLATE COUNT

103

image:

longer than three hours because precipitates

may form which confuse the counting of colo-

nies. Maintain a thermometer immersed in a

separate bottle or flask in the water bath to

monitor the temperature.

5.3 Preparation for Plating

5.3.1 Prepare at least duplicate plates for

each sample or dilution tested. Mark and ar-

range plates in a reasonable order for use.

Prepare a bench sheet or card, including sam-

ple identity, dilutions, date and other relevant

information.

5.3.2 Aseptically pipet an aliquot from the

appropriate dilution into the bottom of each

petri dish. Use a separate sterile pipet to trans-

fer an aliquot to each set of petri dishes for

each sample or sample dilution used. Vigor-

ously shake the undiluted sample and dilution

containers before each transfer is made.

5.3.3 Pipet sample or sample dilution into

marked petri dish. After delivery, touch the tip

once to a dry spot in the dish.

5.3.4 To minimize bacterial density

changes in the samples, do not prepare any

more samples than can be diluted and plated

within 20-25 minutes.

5.4 Pouring Agar Plates

5.4.1 Use the thermometer in the control

bottle in the tempering bath to check the tem-

perature of the plating medium before

pouring.

5.4.2 Add not less than 12 ml (usually

12-15"ml) of the melted and cooled (44-46 C)

agar medium to each petri dish containing an

aliquot of the sample or its dilution. Mix the

inoculated medium carefully to prevent spill-

ing. Avoid splashing the inside of the cover.

One recommended technique rotates plate

five times to left, five times to the right and five

times in a back and forth motion.

5.4.3 Pipet a one m! volume of sterile

dilution water into a petri dish, add agar, mix

and incubate with test plates. This control

plate will check the sterility of pipets, agar,

dilution water and petri dishes. See Part IV-C,

1.3.

5.5 Incubation of Plated Samples

5.5.1 After agar plates have hardened on a

level surface (usually within 10 minutes), invert

the plates and immediately incubate at 35 C.

5.5.2 Incubate tests on all water samples

except bottled water at 35 ± 0.5 C for 48 ± 3

hours. Incubate the tests on bottled water at

35 .± 0.5 C for 72 ± 4 hours. The longer

incubation is required to recover organisms in

bottled water with longer generation times.

5.5.3 Stacks of plates should be at least

2.5 cm from adjacent stacks, the top or sides

of the incubator. Do not stack plates more than

four high. These precautions allow proper

circulation of air to maintain uniform tempera-

ture throughout the incubator and speed

equilibration.

5.6 Counting and Recording Colonies:

After the required incubation period, examine

plates and select those with 30-300 colonies.

Count these plates immediately. A Quebec-

type colony counter equipped with a guide

plate, appropriate magnification and light is

recommended for use with a hand tally.

5.6.1 Electronic-assist devices are avail-

able which register colony counts with a sens-

ing probe and automatically tabulate the total

plate count.

Fully-automatic colony counters are avail-

able which count all colonies (particles) larger

than a preset threshold-size. These counters

scan and provide digital register and a visual

image of the plate for further examination and

recounting with different threshold if so

desired.

Because the accuracy of automatic cen-

ters varies with the size and number of colo-

nies per plate, the analyst should periodically

compare its results with manual counts.

104

oEPA MICROBIOLOGICAL MANUAL 1978

image:

5.6.2 The following rules should be used

to report the Standard Plate Count:

^ p|ates with 30 to 300 Colonies: Count

all colonies and divide by the volume tested (in

ml). If replicate plates from one dilution are

countable (30-300), sum the counts of colo-

nies on all plates and divide by the volumes

tested (in mi) as follows:

Sum of Colonies

Sum of Volumes Tested, ml

= S.P. Count/ml

Record the dilutions used, the number of

colonies on each plate and report as the Stan-

dard Plate Count per milliliter.

If two or more consecutive dilutions are

countable, independently carry each calcula-

tion of plate count to a final count per ml, then

calculate the mean of these counts/ml for the

reported value.

For example, if 280 and 34 colonies are

counted in the 1:100 and 1:1000 dilutions of a

water sample, the calculation is:

= 28,000/ml

= 34,000/ml

28000 + 34000

Reporting Value =

= 31000 SPC/mi

(b) All Plates with Fewer than 30 Colonies:

If there are less than 30 colonies on all plates,

record the actual number of colonies on the

lowest dilution plated and report the count as:

Estimated Standard Plate Count per milliliter.

For example, if volumes of 0.1,0.01 and 0.001

ml were plated and produced counts of 22, 2

and 0 colonies respectively, the colony count

of 22 from the largest sample volume (0.1 ml)

would be selected, calculated and reported as

follows:

Plate Count

Volume Plate

0.1

Count reported: Estimated Standard Plate

Count, 220/ml.

produce counts < 30, actual counts are

reported.

(d) Plate with No Colonies: If all plates from

dilutions tested show no colonies, report the

count as < 1 times the lowest dilution plated.

For example, if 0.1, 0.01 and 0.001 ml vol-

umes of sample were tested with no visible

colonies developing, the lowest dilution, 0,1

ml would be used to calculate a less than (<)

count as follows:

Volume Tested

"o~T

= < 10

Count reported: Standard Plate Count,

^ Al1 Plates Greater than 300 Colonies:

When counts per plate in the highest dilution

exceed 300 colonies, compute the count by

multiplying the mean count by the dilution

used and report as a greater than ( > ), Standard

Plate Count per milliliter. For example, if

duplicate 1.0, 0.1 and 0.01 volumes of sample

were tested with average counts of > 500,

> 500 and 340 developing in the dilutions, the

count would be calculated as follows:

Plate Count

Volume Tested

= 34,000

220

or count reported as: Standard Plate Count,

> 34,000/ml.

5.6.3 Count Estimations on Crowded

Plates: The square divisions of the grid on the

Quebec or similar colony counter can be used

to estimate the numbers of bacteria per plate.

With less than 10 colonies per sq cm count the

colonies in 13 squares with representative

distribution of colonies. Select 7 consecutive

horizontal squares and 6 consecutive vertical

STANDARD PLATE COUNT

105

image:

squares for counting. Sum the colonies in

these 13 sq cm, and multiply by 5 to estimate

the colonies per plate for glass plates (area of

65 sq cm) or multiply by 4.32 for plastic plates

(area of 57 sq cm). With more than 10 colonies

per sq cm, count 4 representative squares,

average the count per sq cm, multiply by the

number of sq cm/plate (usually 65 for glass

plates and 57 for plastic plates) to estimate the

colonies per plate. Then multiply by the

reciprocal of the dilution to determine the

count/rnl. When bacterial counts on crowded

plates are greater than 100 colonies per sq

cm, report the result as Estimated Standard

Plate Count greater than (>) 6,500 times the

highest dilution plated.

5.6,4 Spreaders: Plates containing

spreading colonies must be so reported on the

data sheet. If spreaders exceed one-half of the

total plate area, the plate is not used. Report as:

No results, spreaders.

Colonies can be counted on

representative portions of plates if spreading

colonies constitute less than one-half of the

total plate area, and the colonies are well-

distributed in the remaining portion of the

plate.

(a) Count each chain of colonies as a single

colony.

(b) Count each spreader colony that

develops as a film of growth between the agar

and the petri dish bottom as one colony.

of water at the edge or over the surface of the

agar as one colony.

(d) Adjust count for entire plate and report

as: Estimated Standard Plate Count/ml.

5.6.5 Remarks ori Data Sheet: Any

unusual occurrences such as missed dilutions,

loss of plates through breakage,

contamination of equipment, materials, media,

or the laboratory environment, as shown by

sterility control plates, must be noted on the

data sheet. Report as: Lab Accident, etc.

6. Reporting Results

Report Standard Plate Count or Estimated

Standard Plate Count as colonies per ml, not

per 100ml.

Standard Plate Counts should be rounded

to the number of significant figures (S.F.)

obtainable in the procedure: 1 S.F. for 0-9

actual plate counts, 2 S.F. for 10-99 actual

plate counts and 3 S.F. for 100-300 actual

plate counts. See Part II-C, 2.8.1 of this

manual.

7. Precision and Accuracy

7.1 Prescott et an[ (7) reported that the

standard deviation of individual counts from

30-300 will vary from 0-30 percent. This

plating error was 10% for plate counts within

the 100-300 range. A dilution error of about

3% for each dilution stage is incurred in

addition to the plating error. Large variations

can be expected from high density samples

such as sewage for which several dilutions are

necessary.

7.2 Laboratory personnel should be able

to duplicate their plate count values on the

same plate within 5%, and the counts of others

within 10%. If analysts'counts do not agree,

review counting procedures for analyst error.

106

MICROBIOLOGICAL MANUAL 1978

image:

REFERENCES

1. Geldreich, E. E,, H, D. Nash, D. J, Reasoner and R. H. Taylor, 1972. The necessity of controlling

bacterial populations in potable waters: Community Water Supply, J. Amer. Water Works Assoc.

64:596. • • • .

2. Geldreich, E. E., H. D. Nash, D. J. Reasoner and R. H. Taylor, 1975. The necessity of controlling

bacterial populations in potable waters: Bottled Water and Emergency Water Supplies. J. Amer.

Waterworks Assoc. 67:117. .

3, Geldreich, E. E., 1973. Is the total count necessary? 1st AWWA Technology Conference

Proceedings, Amer. Waterworks Assoc. VII-1, Cincinnati, Ohio.

4. Clark, D. S., 1971. Studies on the surface plate method of counting bacteria. Can. J. Microbiol.

•V7:943 ' ' < "> . . ''

5. Klein, D. A, and S. Wu, 1974. Stress: a factor to be considered in heterotrophic microorganism

enumeration from aquatic environments. Appl. Microbiol. 27:429.

6. Van Soestbergen, A. A. and C; H. Lee, 1969. Pour plates or streak plates? Appl. Microbiol.

1^8:1092.

7. Prescott, S. C,. C-E. A. Winslow, and M. H. McCrady, 1946. Water Bacteriology. (6th ed.) John

Wiley and Sons, Inc., p. 46-50. '

STANDARD PLATE COUNT 107

image:

PART III. ANALYTICAL METHODOLOGY

Section B Total Coliform Methods

This section describes the enumerative

techniques for total coliform bacteria in water

and wastewater. The method chosen depends

upon the characteristics of the sample. The

Section is divided as follows:

Definition of the Coliform

Group

Single-Step, Two-Step

and Delayed-lncubation

Membrane Filter (MF)

Methods

2. Single-Step, Two-Step and Delayed-

lncubation Membrane Filter Methods

2,1 Summary: An appropriate volume of a

water sample or its dilution is passed through

a membrane filter that retains the bacteria

present in the sample.

In the single-step procedure the filter retaining

the microorganisms is placed on M-Endo agar,

LES M-Endo agar or on an absorbent pad satu-

rated with M-Endo broth Jn a petri dish. The

test is incubated at 35 C for 24 hours.

3. Verification

4. Most Probable Number

(MPN) Method

Differentiation of the

Coliform Group by Further

Biochemical Tests

1. Definition of the Coliform Group

The cotiform or total coliform group

includes ail of the aerobic and facultative an-

aerobic, gram-negative, nonspore-formlng,

rod-shaped bacteria that ferment lactose in

24-48 hours at 35 C. The definition includes

the genera: Escherichia, Citrobacter, Entero-

bactar, and Klebsietla.

In the two-step enrichment procedure the filter

retaining the microorganisms is placed on an

absorbent pad saturated with lauryl tryptose

(lauryl sulfate) broth. After incubation for 2

hours at 35 C, the filter is transferred to an

absorbent pad saturated with M-Endo broth,

M-Endo agar, or LES M-Endo agar, and incu-

bated for an additional 20-22 hours at 35 C.

The sheen colonies are counted under low

magnification and the numbers of total coli-

forms are reported per 100 ml of original

sample.

In the delayed-incubation procedure, the

filter retaining the microorganisms is placed

on an absorbent pad saturated with M-Endo

preservative medium in a tight-lidded petri

dish and transported from field site to the

laboratory. In the laboratory, the filter is trans-

ferred to M-Endo growth medium and incu-

bated at 35 C for 24 hours. Sheen colonies are

counted as total coliforms per 100 ml.

108

•SEPA MICROBIOLOGICAL MANUAL 1978

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2,2 Scope and Application; The total coli-

form test can be used for any type of water or

wastewater, but since the development of the

fecal coliform procedure there has been in-

creasing use of this more specific test as an

indicator of fecal pollution. However, the total

coliform test remains the primary indicator of

bacteriological quality for potable water, distri-

bution system waters, and public water sup-

plies because a broader measure of pollution

is desired for these waters. It is also a useful

measure in shellfish-raising waters.

Although the majority of water and waste-

water samples can be examined for total coli-

forms by the single-step MF procedure,

coliforms may be suppressed by high back-

ground organisms, and potable water samples

may require the two-step method.

If the membrane filtration method is used

to measure total coliforms in chlorinated

secondary or tertiary sewage effluents the

two-step enrichment procedure is required.

However, it may be necessary to use the MPN

method because of high solids in the wastes or

toxicity from an industrial waste (see Part II-D,

this Manual).

The delayed-incubation MF method is

useful in survey monitoring or emergency

situations when the single step coliform test

cannot be performed at the sample site, or

when time and temperature limits for sample

storage cannot be met. The method eliminates

field processing and equipment needs. Also,

examination at a central laboratory permits

confirmation and biochemical identification of

the organisms as necessary. Consistent results

have been obtained with this method using

water samples from a variety of sources (1, 2).

The applicability of this method for a specific

water source must be determined in

preliminary studies .by comparison with the

standard MF method.

2.3 Apparatus and Materials

2,3.1 Water jacket, air, or heat sink incu-

bator that maintains 35 + 0.5 C. Temperature

is checked against an NBS certified thermom-

eter or equivalent. Incubator must have

humidity control if loose-lidded pertri dishes

are used. See Part II-B, 1.2.

2.3.2 A binocular (dissection) microscope,

with magnification of 10 or 15x, and a day-

light type fluorescent lamp angled to give max-

imum sheen appearance.

2.3.3 Hand tally.

2.3.4 Pipet container of stainless steel,

aluminum or pyrex glass for glass pipets.

2.3.5 Sterile 50-100 ml graduated cylin-

ders covered with aluminum foil or kraft paper,

2.3.6 Sterile, unassembled membrane fil-

tration units (filter base and funnel), glass, plas-

tic or stainless steel, wrapped with aluminum

foil or kraft paper. Portable field filtration units

are available.

2.3.7 Vacuum source.

2.3.8 Vacuum filter flask with appropriate

tubing. Filter manifolds which hold a number

of filter bases can also be used.

2.3.9 Ultraviolet sterilizer for MF filtration

units (optional).

2.3.10 Safety trap flask between the filter

flask and the vacuum source.

2.3.11 Forceps with smooth tips.

2.3.12 Methanoiorethanol, 95%, in small

vial, for flaming forceps.

2.3.13 Bunsen/Fisher burner or electric

incinerator.

2.3.14 Sterile TD bacteriological or Mohr

pipets, glass or plastic, of appropriate size.

2.3.15 Sterile petri dishes with tight-

fitting lids, 50x12 mm or loose-fitting lids 60

X 15 mm, glass or plastic.

TOTAL COLIFORMS

109

image:

2.3.16 Dilution bottles (milk dilution), py-

rex, marked at 99 ml volume, screw cap with

neoprene rubber liner.

2.3.17 Membrane filters, white, grid-

marked, 47 mm diameter, with 0.45 pm ±

0.02 pm pore size, or other pore size, as recom-

mended by manufacturer for water analyses.

2.3.18 Absorbent pads.

2.3,19 Inoculation loops, at least 3 mm

diameter, or needles, nichrome or platinum

wire, 26 B&S gauge, in suitable holder.

2.3.20 Disposable applicator sticks or

plastic loops as alternatives to inoculation

loops.

2.3.21 Shipping tubes, labels, and packing

materials for mailing delayed incubation

plates.

2.4 Media: Media are prepared in pre-

sterilized erlenmeyer flasks with metal caps,

aluminum foil covers, or screw caps.

2.4.1 M-Endo broth or agar (See Part II-B,

5.2.2).

2.4.2 LES M-Endo agar (See Part ll-B,

5.2.4).

2.4.3 Lauryl tryptose broth (See Part II-B,

5.3.1).

2.4.4 Brilliant green lactose bile broth

(See Part II-B, 5.3.2).

2.4.5 M-Endo holding medium (See Part II-

B, 5.2.3).

2.4,6 Sodium benzoate, U.S.P., for use in

the delayed incubation procedure (See Part II-

B, 5.2.3),

2.4.7 Cycloheximide (Actidione - Upjohn,

Kalamazoo, Ml) for use as antifungal agent in

delayed incubation procedure (See Part II-B,

5.2.3).

2.5 Diiution Water (See Part Il-B, 7 for

preparation).

2.5.1 Sterile dilution water dispensed in

99 i 2 ml amounts in screw-capped dilution

bottles.

2.5.2 Sterile dilution water prepared in 1

liter or larger volumes for wetting membranes

before addition of small sample volumes and

for rinsing the funnel after sample filtration.

2.6 Procedure: Refer to the general proce-

dure in Part II-C for more complete details.

2.6.1 Single-Step Procedure

(a) Prepare the M-Endo broth, M-Endo agar

or LES M-Endo agar as-directed in Part II-B.

(b) Place one sterile absorbent pad in the

bottom half of each petri dish. Pipet 1.8-2.0 ml

M-Endo broth onto the pad to saturate it. Pour

off excess broth. Alternatively, pipet 5-6 ml of

melted agar into each dish (2-3 mm) and allow

to harden before use. Mark dishes and bench

forms with sample identities and volumes.

filter base, grid-side up and attach the funnel to

the base of the filter unit; the membrane filter

is now held between the funnel and the base,

(d) Shake the sample bottle vigorously

about 25 times and measure the desired vol-

ume of sample into the funnel. Select sample

volumes based on previous knowledge to

produce membrane filters with 20-80 coli-

form colonies. See Table ll-C-1. If sample vol-

ume is < 10 ml, add 10 ml of sterile dilution

water to the filter before adding sample.

It is desirable to filter the largest possible

sample volumes for greatest accuracy. How-

ever, if past analyses of specific samples have

resulted in confluent growth, "too numerous to

count" membranes, or lack of sheen from

excessive turbidity, additional samples should

be collected and filtration volumes adjusted to

provide isolated colonies from smaller volumes.

See 2.7.2 in this Section for details on adjusting

sample volumes for potable waters.

110

MICROBIOLOGICAL MANUAL 1978

image:

The suggested method for measuring

sample volumes is described in Part II-C, 3.4.6.

(e) Filter sample and rinse the sides of the

funnel at least twice with 20-30 ml of sterile

dilution water. Turn off the vacuum and re-

move the funnel from the filter base. Asepti-

cally remove the membrane filter from the

filter base and place grid-side up on the agar or

pad.

(f) Filter samples in order of increasing

sample volume, filter potable waters first

(g) If M-Endo broth is used, place the filter

on an absorbent pad saturated with the broth.

Reseat the membrane, if air bubbles occur, as

evidenced by non-wetted areas on the mem-

brane. Invert dish and incubate for 24 ± 2

hours at 35 + 0.5 C in an atmosphere with

near saturated humidity.

(h) If M-Endo agar or LES M-Endo agar is

used, place the inoculated filter directly on the

agar surface. Reseat the membrane if bubbles

occur. Invert the dish and incubate for 24^2

hours at 35 -j- 0.5 C in an atmosphere with

near saturated humidity.

(i) If tight-lidded dishes are used, there is

no requirement for near-saturated humidity.

(j) After incubation remove the dishes from

the incubator and examine for sheen colonies.

(k) Proceed to 2.7 for Counting and

Recording Colonies.

2.6.2 Two-Step Enrichment Procedure

(a) Place a sterile absorbent pad in the top

of each petri dish.

(b) Prepare lauryl tryptose broth as di-

rected in Part 11-8. Pipet 1.8-2,0 ml lauryl tryp-

tose broth onto the pad to saturate it. Pour off

excess broth.

(e) Place a sterile membrane filter on the

filter holder, grid-side up and attach the funnel

to the base of the filter unit; the membrane.

filter is now held between the funnel and the

base.

(d) Shake the sample bottle vigorously

about 25 times to obtain uniform distribution

of bacteria. Select sample volumes based on

previous knowledge to produce membrane fil-

ters with 20-80 coliform colonies. See Table

II-C-1. If sample volume is < 10ml, add 10ml

of sterile dilution water to filter before adding

sample.

(e) Filter samples in order of increasing

sample volume, rinsing with sterile buffered

dilution water between filtrations. The me-

thods of measurement and dispensation of the

sample into the funnel are given in Part ll-C,

3.4.6.

(f) Turn on the vacuum to filter the sample

through the membrane, rinse the sides of the

funnel at least twice with 20-30 ml of sterile

dilution water. Turn off vacuum and remove

funnel from base.

(g) Remove the membrane filter asepti-

cally from the filter base and place grid-side up

on the pad in the top of the petri dish. Reseat

MF if air bubbles are observed.

(h) Incubate the filter in the petri dish with-

out inverting for 1 1/2 - 2 hours at 35 ± 0.5 C

in an atmosphere of near saturated humidity.

This completes the first step in the Two-Step

Enrichment Procedure.

(i) Prepare M-Endo broth, M-Endo agar, or

LES M-Endo agar as directed in Part II-B.

If M-Endo broth is used, place a new sterile

absorbent pad in the bottom half of the dish

and saturate with 1.8-2.0 ml of the M-Endo

broth. Transfer the filter to the new pad. Reseat

MF if air bubbles are observed. Remove the

used pad and discard.

If M-Endo or LES M-Endo agar is used,

pour 5-6 ml of agar into the bottom of each

petri dish and allow to solidfy. The agar me-

dium can be refrigerated for up to two weeks.

TOTAL COLIPORMS

111

image:

(j) Transfer the filter from the lauryl tryp-

tose broth onto the Endo medium. Reseat if air

bubbles are observed.

(k) Incubate dishes in an inverted position

for an additional 20-22 hours at 35 + 0.5 C.

This completes the second step in the Two-

Step Enrichment Procedure.

{!•) Proceed

Recording.

to 2.7 Counting and

2.6.3 Delayed Incubation Procedure

{a} Prepare the M-Endo Holding Medium or

LES Holding Medium as outlined in Part II-B,

5.2.3 or 5.2.5. Saturate the sterile absorbent

pads with about 2.0 ml of holding broth. Pour

off excess broth. Mark dishes and bench forms

with sample identity and volumes.

(b) Using sterile forceps place a mem-

brane filter on the filter base grid side up,

filter unit; the membrane filter is now held

between the funnel and base.

(d) Shake the sample vigorously about 25

times and measure into the funnel with the

vacuum off. If the sample is < 10 ml, add 10

ml of sterile dilution water to the membrane

filter before adding the sample.

(1) Select sample volumes based on previ-

ous knowledge to produce counts of 20-80

coliform colonies. See Table ll-C-1.

(2) Follow the methods for sample mea-

surement and dispensation given in Part II-C,

3.4.6

(e) Filter the sample through the mem-

brane and rinse the sides of the funnel walls at

least twice with 20-30 ml of sterile dilution

water.

(f) Turn off the vacuum and remove the

funnel from the base of the filter unit.

(g) Aseptically remove the membrane filter

from the filter base and place grid side up on

an absorbent pad saturated with M-Endo Hold-

ing Medium or LES Holding Medium.

(h) Place the culture dish in shipping con-

tainer and send to the examining laboratory.

Coliform bacteria can be held on the holding

medium for up to 72 hours with little effect on

the final counts. The holding period should be

kept to a minimum.

(i) At the examining laboratory remove the

membrane from the holding medium, place it

in another dish containing M-Endo broth or

agar medium, and complete testing for coli-

forms as described above under 2.6.1.

2.7 Counting and Recording Colonies:

After incubation, count colonies on those

membrane filters containing 20-80 golden-

green metallic surface sheen colonies and less

than 200 total bacterial colonies. A binocular

(dissection) microscope with a magnification

of 10 or 15x is recommended. Count the

colonies according to the general directions

given in Part II-C, 3.5.

2.7.1 The following general rules are used

in calculating the total coliform count per 100

ml of sample. Specific rules for analysis and

counting of water supply samples are given in

2.7.2.

(a) Countable Membranes with 20-80

Sheen Colonies, and Less Than 200 Total Bac-

terial Colonies: Select the plate counts to be

used according to the rules given in Part II-C,

3.6, and calculate the final value using the

formula.

Total Coliforms/100 ml:

No. of Total Coliform Colonies Counted

Volume in ml of Sample Filtered

X 100

(b) Counts Greater Than the Upper Limit of

80 Colonies: All colony counts are above the

recommended limits. For example, sample vol-

umes of 1, 0.3, and 0.01 ml are filtered to

112

MICROBIOLOGICAL MANUAL 1978

image:

produce total coliform colony counts of TNC,

150, and 110 colonies.

Use the count from the smallest filtration

volume and report as a greater than

count/100 ml. In the example above:

110

0.01

X 100 = 1,100,000

or > 1,100,000 coliforms/100 ml,

W Membranes with More Than 200 Total

Colonies (Coliforms plus Non-coliforms).

(1) Estimate sheen colonies if possible,

calculate total coliform density as in (a) above.

Report as: Estimated Count/100 ml.

(2) If estimate of sheen colonies is not

possible, report count as Too Numerous to

Count (TNTC).

(d) Membranes with Confluent Growth

Report as: Confluent Growth and specify

the presence or absence of sheen.

2.7.2 Special Rules for Potable Waters

(a) Countable Membranes with 0-80 Sheen

Colonies, and Less than 200 Total Colonies

Count the sheen colonies per volume filter-

ed. Calculate and report the number of Total

Coliforms/100 ml.

(b) Uncountable Membranes for Potable

Water Samples

If 100 ml portions of potable water sam-

ples cannot be tested because of high back-

ground counts or confluency, multiple volumes

of less than 100 ml can be filtered. For example,

if 60 colonies appear on the surface of one

m,embrane through which a 50 ml portion of

the sample was passed, and 50 colonies on

a second membrane through which a second

50 ml portion of the sample was passed, the

colonies are totaled and reported as 110 total

coliforms per 100 ml.

If filtration of multiple volumes of less than

100 ml still results in confluency or high back-

ground count, the coliforms may be present

but suppressed. These samples should be ana-

lyzed by the MPN Test. This MPN check should

be made on at least one sample for each prob-

lem water once every three months.

For potable water samples, confluence

requires resampling and retesting.

(d) Verification. Because unsatisfactory

samples from public water supplies containing

5 or more coliform colonies must be verified,

at least 5 colonies need to be verified for each

positive sample. Reported counts are adjusted

based on verification.

(e) Quality control procedures are speci-

fied by EPA under the law, and described in

Appendix C in this Manual.

2-7.3 Reporting Results: Report total coli-

form densities per 100 ml of sample. See

Figure ll-C-3 for an example of a bench form for

reporting results. A discussion on significant

figures is given in Part II-C, 2,8.

2,8 Precision and Accuracy: There are

no established precision and accuracy data

available at this time.

3. Verification

Verification of total coliform colonies from

M-Endo type media validates sheen as evi-

dence of coliforms. Verification of representa-

tive numbers of colonies may be required in

evidence gathering or for quality control pro-

cedures. The verification procedure follows:

3.1 Using a sterile inoculating needle, pick

growth from the centers of at least 10 well-

isolated sheen colonies (5 sheen colonies per

plate for potable waters). Inoculate each into a

tube of lauryl tryptose broth and incubate 24-48

hours at 35 C ± 0.5 C. Do not transfer exclu-

sively into brilliant green bile lactose broth.

However, colonies may be transferred to LTB

and BGLB simultaneously.

TOTAL COLIFORMS

113

image:

3,2 At the 24 and 48 hour readings, con-

firm gas-positive lauryl tryptose broth tubes by

inoculating a loopful of growth into brilliant

green lactose bile broth and incubate for

24-48 hours at 35 ± 0.5 C. Cultures that are

positive in BGLB are interpreted as verified

coliform colonies (see Figure ill-B-1).

3.3 If questionable sheen occurs, the

worker should also verify these colonies.

4. Most Probable Number (MPN) Method

4.1 Summary: This method detects and

estimates the total coliforms in water samples

by the multiple fermentation tube technique.

The method has three stages: the Presumptive,

the Confirmed, and the Completed Tests. In

the Presumptive Test, a series of lauryl tryp-

tose broth fermentation tubes are inoculated

with decimal dilutions of the sample. The for-

mation of gas at 35 C within 48 hours consti-

tutes a positive Presumptive Test for members

of the total coliform group. However, the MPN

must be carried through the Confirmed Test

for valid results. In this test, inocula from posi-

tive Presumptive tubes are transferred to tubes

of brilliant green lactose bile {BGLB) broth. The

BGLB medium contains selective and inhib-

itlve agents to suppress the growth of all non-

coliform organisms. Gas production after incu-

bation for 24 or 48 hours a,t 35 C constitutes a

positive Confirmed Test and is the point at

which most MPN tests are terminated. The

Completed Test begins with streaking inocu-

lum from the positive BGLB tubes onto EMB

plates and incubating the plates for 24 hours

at 35 C. Typical and atypical colonies are

transferred into lauryl tryptose broth fermenta-

tion tubes and onto nutrient agar slants. Gas

formation in the fermentation tubes and pres-

ence of gram-negative rods constitute a posi-

tive Completed Test fortotalcoliforms. See

Figure III-B-2, The MPN perl 00 ml is calculated

from the MPN table based upon the Con-

firmed or Completed test results.

4.2 Scope and Application

4,2.1 Advantages: The MPN procedure is

a tube-dilution method using a nutrient-rich

medium, which is less sensitive to toxicity and

supports the growth of environmentally-

stressed organisms. The method is applicable

to the examination of total coliforms in chlori-

nated primary effluents and under other

stressed conditions. The multiple-tube proce-

dure is also better suited forthe examination of

turbid samples, muds, sediments, or sludges

because particulates do not interfere visibly

with the test.

4.2.2 Limitations: Certain non-coliform

bacteria may suppress coliforms or act syner-

gistically to ferment lauryl tryptose broth and

yield false positive results. A significant num-

ber of false positive results can also occur in

the brilliant green bile broth when chlorinated

primary effluents are tested, especially when

stormwater is mixed with the sewage (3). False

negatives may occur with waters containing

nitrates (4). False positives are more common

in sediments.

4.3 Apparatus and Materials

4.3.1 Water bath or air incubator set at 35

± 0.5 C.

4.3.2 Pipet containers of stainless steel,

aluminum, or pyrex glass for glass pipets.

4.3.3 Inoculation loops, at least 3 mm

diameter and needles of nichrome or platinum

wire, 26 B & S gauge, in suitable holders.

4.3.4 Disposable sterile applicator sticks

or plastic loops as alternatives to inoculating

loops.

4.3.5 Compound microscope, oil

immersion.

4.3.6 Bunsen/Fisher burner or electric in-

cinerator unit.

4.3.7 Sterile TD Mohr or bacteriological

pipets, glass or plastic, of appropriate size.

114

MICROBIOLOGICAL MANUAL 1978

image:

Pick 10 sheen colonies

from each sample

Lauryl Tryptose Broth

24 hours at 3BC

Gas

Gas-

Reincubate

24 hours at 35 C

Gas +

Gas-

Negative

Test

Brilliant Green Lactose Bile Broth

24 hours at SBC

Gas +

Verified

Coliform

Colony

Gas-

Reincubate

24 hours at 35 C

Gas

Gas-

Verified

Coliform

Colony

Negative

Test

FIGURE II l-B-1. Verification of Total Coliform Colonies on the Membrane Filter

TOTAL COLIFORMS

115

image:

»-

Sample

Lauryl Tryptose Broth

35 ± 0.5 C

Gas -

48 hr

Negative Test

Brilliant Green Lactose Bile Broth

35 ± 0.5 C

Gas -

Negative Test

Eosin Methylene Blue Agar

24hrat3i±0,5C

Nutrient Agar Slant

24hrat35±0.5C

Lauryl Tryptose Broth Tube

24-48 hr at 35 ± 0.5 C

Gram 4-

Sporeforrners

Negative

Test

Gram

Nonspore-forming

Conforms

Present

Gas +

Conforms

Present

Gas -

Negative

Test

FIGURE lll-B-2. Flow Chart for the Total Coliform MPN Test

116

MICROBIOLOGICAL MANUAL 1978

image:

4.3.8 Pyrex culture tubes, 150 x 25 mm

or 1 50 X 20 mm, containing inverted fermen-

tation vials, 75 X 10 mm with caps.

4.3.9 Culture tube racks to hold fifty, 25

mm diametertubes.

4.3.10 Dilution bottles (milk dilution) py-

rex glass, 99 ml volume, screw cap with neo-

prene rubber liners.

4.4 Media

4.4.1 Presumptive Test: Lauryl tryptose

broth. See Part II-B, 5.3.1 . Lactose broth is not

used because of false positive reactions.

4.4.2 Confirmed Test: Brilliant green bile

broth. (See Part II-B, 5.3.2).

4.4.3 Completed Test:

(a) Eosin methylene blue agar (see Part II-B,

5.3.3).

(b) Nutrient agar or plate count agar slants

(see Part II-B, 5.1.1 and 5.1.5).

4.5 Dilution Water: Sterile dilution water

dispensed in 99 + 2 ml amounts preferably in

screw-capped bottles. (See Part II-B, 7).

4.6 Procedure: Part II-C describes the gen-

eral MPN procedure in detail.

4.6.1 Prepare the media for Presumptive,

Confirmed or Completed Tests selected. (See

Part II-B, 5.3).

4.6.2 Presumptive Test (See Figure lll-B-2):

To begin the Presumptive Test, arrange fer-

mentation tubes of lauryl tryptose broth in

rows cf 5 tubes each in the tube rack. Select

sample volumes and clearly label each bank of

tubes to identify the sample and volume

inoculated.

(a) For potable waters, five portions of 10

ml each or five portions of 100 ml each are

used.

(b) For relatively-unpolluted waters the

sample volumes for the five rows might be

100, 10, 1,0.1 and 0.01 ml, respectively; the

latter two volumes delivered as dilutions of

original sample.

sample inoculations might be 0.1, 0.01,

0.001, 0.0001, and 0.00001 ml of original

sample delivered as dilutions into successive

rows each containing five replicate volumes.

This series of sample volumes will yield deter-

minate results from a low of 200 to a high of

16,000,000 organisms per 100 ml.

(d) Shake the sample and dilutions vigor-

ously about 25 times. Inoculate each 5-tube

row with replicate sample volumes in increas-

ing decimal dilutions and incubate at 35 C ±

0.5 C.

(e) After 24 + 2 hours incubation at 35 C,

gently agitate the tubes in the rack and exam-

ine the tubes for gas. Any amount of gas

constitutes a positive test. If there is no gas

production in the tubes, reincubate for an

additional 24 hours and reexamine for gas.

Positive Presumption tubes are submitted

directly to the Confirmed Test. Results are

recorded on laboratory bench forms.

(f) If a laboratory using the MPN test on

water supplies finds frequent numbers of Pre-

sumptive test tubes with heavy growth but no

gas, these negative tubes should be submitted

to the Confirmed Test to check for suppression

of coliforms.

(g) If The Presumptive Test tubes are gas-

negative after 48 + 3 hours, they are dis-

carded and the results recorded as negative

Presumptive Tests. Positive Presumptive tubes

are verified by the Confirmed Test.

(h) If the fecal colifrom test is to be run,

(Part III-C), the analyst can inoculate growth

from positive Presumptive Test tubes into EC

medium at the same time as he inoculates the

Confirmed Test Medium.

4.6.3 Confirmed Test (See Figure lll-B-2)

(a) Carefully shake each positive Presump-

tive tube. With a sterile 3 mm loop or a sterile

TOTAL COLIFORMS

117

image:

applicator stick, transfer growth from each

tube to BGLB. Gently agitate the tubes to mix

the inoculum and incubate at 35 +_ 0.5 C.

(b) After 24+2 hours incubation at 35 C

examine the tubes for gas. Any amount of gas in

BGLB constitutes a positive Confirmed Test.

If there is no gas production in the tubes

{negative test) reincubate tubes for an addi-

tional 24 hours. Record the gas-positive and

gas-negative tubes. Hold the positive tubes for

the Completed Test if required for quality con-

trol or for checks on questionable reactions.

{e) After 48 +_ 3 hours reexamine the

Confirmed Test Tubes. Record the positive and

negative tube results. Discard the negative

tubes and hold the positive tubes for the Com-

pleted Test if required as in (b) above.

(d) In routine practice most sample ana-

lyses are terminated at the end of the Con-

firmed Test. However, the Confirmed Test data

should be verified by carrying 5% of Confirmed

Tests with a minimum of one sample per test

run through the Completed Test.

(e) For certification of water supply labora-

tories, the MPN test is carried to completion

{except for gram stain) on 10 percent of positive

confirmed samples and at least one sample

quarterly.

4,6.4 Completed Test (See Figure lll-B-2)

Positive Confirmed Test cultures may be

subjected to final Completed Test identifica-

tion through application of further biochemical

and culture tests, as follows:

(a) Streak one or more EMB agar plates

from each positive BGLB tube. Incubate the

plates at 35 +_ 0.5 C for 24 + 2 hours.

(b) Transfer one or more well-isolated typi-

cal colonies {nucleated with or without a metal-

lic sheen) to Iduryl tryptose broth fermentation

tubes and to nutrient or plate count agar

slants. Incubate the slants for 24 +j 2 or 48 ±

3 hours at 35 +_ 0.5 C. If no typical colonies are

present, pick and inoculate at least two atypi-

cal (pink, mucoid and unnucleated) colonies

into lauryl tryptose fermentation tubes and

incubate tubes for up to 48 +j 3 hours.

the fermentation tubes and presence of gram

negative'rods constitute a positive Completed

Test for total coliforms,

4.6.5 Special Considerations for Potable

Waters

Sample Size- For potable waters the stan-

dard sample shall be five times the standard

portion which is either 10 milliliters or 100

mifliliters as described in 40 CFR 141 (5).

• Confirmation - If a laboratory using the

MPN test on water supplies finds frequent

numbers of Presumptive test tubes with heavy

growth but no gas, these negative tubes

should be submitted to the Confirmed Test to

check for suppression of coliforms.

Completion — In water supply laboratories,

10% of all samples and at least one sample

quarterly must be carried to completion but

no gram stain of cultures is required.

4.7 Calculations: The results of the Con-

firmed or Completed Test may be obtained

from the MPN table based on the number of

positive tubes in each dilution. See Part II-C,

4.9 for details on calculation of MPN results.

4.7.1 Table II-C-4 illustrates the MPN in-

dex and 95% Confidence Limits for combina-

tions of positive and negative results when five

10 rnl, five 1.0 ml, and five 0.1 ml volumes of

sample are tested.

4,7.2 Table ll-C-5 provides the MPN indi-

ces and limits for the five tube, single volumes

used for potable water supplies.

4.7.3 When the series of decimal dilutions

is other than those in the tables select the MPN

value from Table II-C-4 and calculate accord-

ing to the following formula:

MPN (From Table) X

Largest Volume Tested

= MPN/100 ml

118

SERA, MICROBIOLOGICAL MANUAL 1978

image:

4.8 Reporting Results: Report the MPN

values per 100 ml of sample. See an example

of a reportform in Figures IIl-D-2 and lll-D-3.

4.9 Precision and Accuracy: The preci-

sion of the MPN value increases .with in-

creased numbers of replicates tested. A five

tube, five dilution MPN is recommended for

natural and waste waters. Only a five tube,

single volume series is required for potable

waters.

5. Differentiation of the Coliform Group by

by Further Biochemical Tests

5.1 Summary: The differentiation of the

members of the colifprm group into genera and

species is based on additional biochemical and

cultural tests (see Table lll-B-1). These tests

require specific training for valid results.

5.2 Apparatus and Materials

5.2.1 Incubator set at 35 ± 0.5 C.

5.2.2 Pipet containers of stainless steel,

aluminum or pyrex glass for glass pipets.

5.2.3 Inoculation loop, 3 mm diameter

and needle.

5.2.4 Bunsen/Fisher type burner or elec-

tric incinerator.

5.2.5 Sterile TD Mohr and bacteriological

pipets, glass or plastic, of appropriate volumes.

5.2.6 Graduates, 25 - 500 ml.

5.2.7 Test tubes, 100 x 13 mm or 1 50 x

20 mm with caps, in racks.

5.2.8 Reagents

(a) Indole Test Reagent: Dissolve 5 grams

para-dimethylamino benzaldehyde in 75 ml

isoamyl (or normal amyl) alcohol, ACS grade,

and slowly add 25 ml cone HCI. The reagent

should be yellow and have a pH below 6.0. If

the final reagent is dark in color it should be

discarded.

Some brands are not satisfactory and oth-

ers become unsatisfactory after aging. Both

amyl alcohol and benzaldehyde compound

should be purchased in as small amounts as

will be consistent with the volume of work

anticipated. Store the reagent in the dark in a

brown bottle with a glass stopper.

(b) Methyl Red Test Reagent: Dissolve 0.1

gram methyl red in 300 ml of 95% ethyl alco-

hol and dilute to 500 ml with distilled water.

(1) Naphthol solution: Dissolve 5 grams

purified alphanaphthol (melting point 92.5 C

or higher) in 100 ml absolute ethyl alcohol.

This solution must be freshly prepared each

day.

(2) Potassium hydroxide solution: Dissolve

40 grams KOH in 100 ml distilled water.

(d) Oxidase Test Reagents

(1) Reagent A: Weigh out 1 gram alpha-

napthol and dissolve in 100 ml of 95%

ethanol.

(2) Reagent B: Weigh out 1 gram para-

aminodimethylaniline HCI (or oxylate) and

dissolve in 100 ml of distilled water. Prepare

frequently and store in refrigerator.

5.3 Media

5.3.1 Tryptophane broth for demonstrat-

ing indole production in the Indole Test. (See

Part II-B, 5.1.9 (a) for preparation).

5.3.2 MR-VP broth (buffered glucose) to

demonstrate acid production by methyl red

color change in the Methyl Red Test and to

demonstrate acetyl methyl carbinol

production in the Voges-Proskauer test. (See

Part II-B, 5.1.9 (b)for preparation).

5.3.3 Simmon's Citrate Agar to demon-

strate utilization of citrate as a sole source of

carbon. (See Part II-B, 5.1.9 (c) for preparation).

TOTAL COLIFORMS

119

image:

TABLE Ili-B-1

Differentiation of the Coliform and Related Organisms Based

Upon Biochemical Reactions

i MICROBIOLOGICAL MANUAL 1978

Bacterium

Escherichia coli

Citrobacter freundii

Enterobacter aerogenes

Klebsiella

Pseudomonas

Tests

Methyl Voges- _, Cytochrome Ornithine Lysine Arginine

Indole Re(j Prague,. Citrate rjxidase Decarboxylase Decarboxylase Dehydroiase MotllltY

4-4---- V V V +

_+_+_ v V V +

-- + •)• - 4- 4- - ' 4-

4. _ 4- +. _ _ 4- _-

. .

Aeromonas

V = variable

>1 -

- reaction of P. aeruginosa

image:

5.3,4 Nutrient agar slant for oxidase test.

(See Part II-B, 5.1.1 for preparation).

5.3.5 Decarboxylase medium base con-

taining lysine HCI, arginine HCI or ornithine

HCI to demonstrate utilization of the specific

amino acids. (See Part II-B, 5.5.14 for

preparation).

5.3.6 Motility test medium (Edwards and

Ewing). (See Section II-B, 5.1.10 for

preparation).

5.3.7 Multitest Systems (optional to Sin-

gle Test Series)

(a) API Enteric 20 (Analytab Products, Inc.).

(b) Enterotube (Roche Diagnostics).

Wilson Pharmaceutical and Chemical Corp.).

(d) Minitek (Baltimore Biological Labora-

tories, Bioquest).

(e) Pathotec Test Strips (General Diagnos-

tics Division of Warner-Lambert Company).

(f) r/b Enteric Differential System (Diagn-

ostic Research, Inc.).

5.4 Procedure

5.4.1 Biochemical tests should always be

performed along with positive and negative

controls. See Table IV-A-5.

5.4.2 Indole Test

(a) Inoculate a pure culture into 5 ml of

tryptophane broth.

(b) Incubate the tryptophane broth at 35 +

0.5 C for 24±2 hours and mix well.

hour culture, shake, and allow the mixture to

stand for 10 minutes. Observe and record the

results.

(d) A dark red color in the amyl alcohol

layer on top of the culture is a positive indole

test; the original color of the reagent, a nega-

tive test. An orange color may indicate the

presence of skatole and is reported as a +

reaction.

5.4.3 Methyl Red Test

(a) Inoculate a pure culture into 10 ml of

buffered glucose broth.

(b) Incubate for 5 days at 35 C.

drops of methyl red indicator.

(d) A distinct red color is positive and dis-

tinct yellow, negative. Orange color is dubious,

may indicate a mixed culture and should be

repeated.

5.4.4 Voges Proskauer Test: This procedure

detects the production of acetyl methyl carbinol

which in the presence of alphanapthol and

potassium hydroxide develops a reddish color.

(a) Use a pure culture to inoculate 10 ml of

buffered glucose broth or 5 ml of salt peptone

glucose broth or use the previously inoculated

buffered glucose broth from the Methyl Red

Test.

(b) Incubate the inoculated salt peptone

glucose broth or the buffered glucose broth at

35 +_ 0.5 C for 48 hours.

ml KOH solution to 1 ml of the 48 hour salt

peptone or buffered glucose broth culture in a

separate clean test tube. Shake vigorously for

10 seconds and allow the mixture to stand for

2-4 hours.

(d) Observe the results and record. A pink

to crimson color is a positive test. Do not read

after 4 hours. A negative test may develop a

copper orfaint brown color.

TOTAL COLIFORMS

121

image:

5.4.5 Citrate Test

(a) Lightly inoculate a pure culture into a

tube of Simmon's Citrate Agar, using a needle

to stab, then streak the medium. Be careful not

to carry over any nutrient material.

(b) Incubate at 35 C for 48 hours.

change. A distinct Prussian blue color in the

presence of growth indicates a positive test;

no color change is a negative test.

5.4.6 Cytochrome Oxidase Test

(Indophenol): The cytochrome oxidase test can

be done with commercially-prepared paper

strips or on a nutrient agar slant as follows:

(a) Inoculate nutrient agar slant and incu-

bate at 35 C for 18-24 hours. Older cultures

should not be used.

(b) Add 2-3 drops of reagent A and reag-

ent B to the slant, tilt to mix and read reaction

within 2 minutes.

slant or paper strip) occurs in 30 seconds.

Ignore weak reactions that occur after 2

minutes.

5.4.7 Decarboxylase Tests (lysine, argi-

nineandornithine)

(a) The complete decarboxylase test series

requires tubes of each of the amino acids and a

control tube containing no amino acids.

(b) Inoculate each tube lightly.

broths to make 3-4 mm layers on the surface

and tighten the screw caps.

(d) Incubate for 18-24 hours at 35 C and

read. Negative reactions should be re-

incubated up to 4 days.

(e) Positive reactions ark purple and

negative reactions are yellow. Read the control

tube without amino acid first; it must be yellow

for the reactions of the other tubes to be valid.

Positive purple tubes must have growth as

evidenced by turbidity because uninoculated

tubes are also purple; nonfermenters may

remain alkaline throughout incubation.

5.4.8MotilityTest

(a) Stab-inoculate the center of the tube of

Motility Test Medium to at least half depth.

(b) Incubate tubes 24-48 hours at 35 C.

reincubate at room temperature for 5 more

days.

(d) Non-motile organisms grow only along

the line of inoculation. Motile organisms grow

outward from the line of inoculation and

spread throughout the medium producing a

cloudy appearance.

(e) Addition of 2, 3, 5 triphenyl tetrazolium

chloride (TTC) will aid recognition of motility.

Growth of microorganisms reduces TTC and

produces red color along the line of growth.

5.4.9 Additional Biochemical Tests: If

other biochemical tests are necessary to fur-

ther identify enteric bacteria, for example spe-

cific carbohydrate fermentation, see the Table

lll-E-5, Biochemical Characteristics of Entero-

bacteriaceae.

5.4.10 Multitest Systems: Multitest sys-

tems are available which use tubes containing

agar media that provide numerous biochemi-

cal tests, plastic units containing a series of

dehydrated media, media-impregnated discs

and reagent-impregnated paper strips. Some

of the systems use numerical codes to aid

identification. Others provide computerized

identification of bacteria. A number of inde-

pendent investigators have compared one or

more multitest systems with conventional or

traditional biochemical tests. Some of the ear-

lier systems have been improved. Most of the

recent studies report the correct identification

of high percentages of isolates. The systems

are described in Part III-E, 5.6.

122

MICROBIOLOGICAL MANUAL 1978

image:

REFERENCES

1. Geldreich, E. E., P. W. Kabler, H. L. Jeter and H. F. Clark, 1955. A delayed incubation membrane

filter test for coliform bacteria in water. Amer. Jour. Public Health 45:1462.

2. Brezenski, F. T. and J. A. Winter, 1969. Use of the delayed incubation membrane filter test for

determining coliform bacteria in sea water. Water Res. 3:583.

3. Geldreich, E. E., 1 975. Handbook for Evaluating Water Bacteriological Laboratories (2nd ed.),

EPA-670/9-75-006. U.S. Environmental Protection Agency, Cincinnati, Ohio.

4. Tubiash, H., 1951. The Anaerogenic Effect of Nitrates and Nitrites on Gram-negative Enteric

Bacteria. Amer. Jour. Public Health 41:833.

5. National Interim Primary Drinking Water Regulations, 40 Code of Federal Regulations (CFR) Part

141.14 (b) and (c), Published in Federal Register, 40, 59566, December 24, 1975.

TOTAL COLIFORMS 123

image:

PART III. ANALYTICAL METHODOLOGY

Section C Fecal Coliform Methods

The direct membrane filter (MF), the

delayed-incubation MF and the multiple-tube,

most probable number (MPN) methods can be

used to enumerate fecal conforms in water and

wastewater. For a general description of the

fundamental laboratory techniques refer to

Part II-C. The method chosen depends upon

the characteristics of the sample. The Section

ts divided as follows:

1. Definition of the Fecal Coliform

Group

2. Direct Membrane Filter (MF)

Method

3. Delayed-incubation Membrane

Filter Method

4. Verification

5. Most Probable Number (MPN)

Method

1. Definition of the Fecal Coliform Group

1,1 The fecal coliforms are part of the total

coliform group. They are defined as gram-

negative nonspore-forming rods that ferment

lactose in 24 ± 2 hours at 44.5 ±_ 0.2 C with

the production of gas in a multiple-tube proce-

dure or produce acidity with blue colonies in a

membrane filter procedure.

1.2 The major species in the fecal coliform

group is Escherichia coli, a species indicative

of fecal pollution and the possible presence of

enteric pathogens.

2. Direct Membrane Filter (MF) Method

2.1 Summary: An appropriate volume of a

water sample or its dilution is passed through

a- membrane filter that retains the bacteria

present in the sample. The filter containing the

microorganisms is placed on an absorbent pad

saturated with M-FC broth or on M-FC agar in a

petri dish. The dish is incubated at 44.5 C for

24 hours. After incubation, the typical blue

colonies are counted under low magnification

and the number of fecal coliforms is reported

per 100 ml of original sample.

2.2 Scops and Application

2-2-1 Advantages: The results of the MF

test are obtained in 24 hours. Up to 72 hours

are required for the multiple-tube fermentation

method. The M-FC method provides direct enu-

meration of the fecal coliform group without

enrichment or subsequent testing. Over 93%

of the blue colonies that develop in this test

using M-FC medium at the elevated tempera-

ture of 44.5 C ± 0.2 C are reported to be fecal

coliforms (1). The test is applicable to the ex-

amination of lakes and reservoirs, wells and

springs, public water supplies, natural bathihg

waters, secondary non-chlorinated effluents

from sewage treatment plants, farm ponds,

stormwater runoff, raw municipal sewage, and

feedlot runoff. The MF test has been used with

varied success in marine waters.

2-2-2 Limitations: Recent data (2, 3)

indicate that the single-step MF fecal coliform

procedure may produce lower results than

those obtained with the fecal coliform

124

MICROBIOLOGICAL MANUAL 1978

image:

multiple-tube procedure, particularly for

chlorinated effluents. Since chlorination

stresses fecal coliforms and significantly

reduces recovery, this method should not be

used with chlorinated wastewater.

Disinfection and toxic materials such as

metals, phenols, acids or caustics also affect

recovery of fecal coliforms on the membrane

filter. Any decision to use this test for stressed

microorganisms requires parallel MF/MPN

evaluation based on the procedure described

inPartlV-C, 3.

Recently-proposed solutions to problems

of lower recovery (2, 4, 5, 6) include the use of

two-step incubation, two-step incubation over-

lay and/or enrichment techniques and modifi-

cation of membrane filter structures.

2.3 Apparatus and Materials

2.3.1 Water bath, aluminum heat sink, or

other incubator that maintains a stable 44.5 +

0.2 C. Temperature is checked against an NBS

certified thermometer or one of equivalent

accuracy.

2.3.2 Binocular (dissecting type) micro-

scope, with magnification of 10-15x and

daylight-type fluorescent lamp.

2.3.3 Hand tally.

2.3.4 Pipet containers of stainless steel,

aluminum or pyrex glass for glass pipets.

2.3.5 Graduated cylinders, covered with

aluminum foil or kraft paper before

sterilization.

2.3.6 Sterile, unassembled membrane fil-

tration units (filter base and funnel), glass, plas-

tic or stainless steel, wrapped with aluminum

foil and kraft paper.

2.3.7 Vacuum source.

2.3.8 Vacuum filter flask, with appropriate

tubing. Filter manifolds which hold a number

of filter bases can also be used.

2.3.9 Safety trap flask between the filter

flask and the vacuum source.

2.3.10 Forceps with smooth tips.

2.3.1 1 Ethanol, 95% or methanol, in small

vial, for sterilizing forceps.

2.3.12 Bunsen/Fisher burner or electric

incinerator.

2.3.13 Sterile TD bacteriological or Mohr

pipets, glass or plastic, of appropriate size.

2.3.14 Sterile petri dishes, 50 x 12 mm

plastic with tight-fitting lids.

2.3.15 Dilution bottles (milk dilution), py-

rex glass, marked at 99 ml volume, screw-cap

with neoprene rubber liner.

2.3.16 Membrane filters, white, grid

marked, 47 mm diameter, 0.45 + 0.02/xm pore

size or other pore size recommended by manu-

facturer for water analyses. The Millipore

HC MF, not the HA, is recommended.

2.3.17 Absorbent pads.

2.3.18 Water-proof plastic bags.

2.3.19 Inoculation loops, 3 mm diameter,

or needle of nichrome or platinum wire, 26

B&S gauge, in suitable holder.

2.3.20 Disposable applicator sticks or

plastic loops as alternatives to inoculation

loops.

2.3.21 Ultraviolet sterilizer for MF filtra-

tion units (optional).

2.4 Media

2.4.1 M-FC broth or agar prepared in pre-

sterilized erlenmeyer flasks (See Part II-B, 5.2.1).

2.4.2 Lauryl tryptose broth prepared in 10

ml volumes in fermentation tubes (see Part II-B,

5.3.1) for verification.

FECAL COLIFORMS

125

image:

2.4.3 EC medium prepared in 10 ml vol-

umes in fermentation tubes (see Part II-B,

5.3.4) for verification.

2.5 Dilution Water (See Part

preparation).

I,B, 7 for

2.5.1 Sterile buffered dilution water or

peptone water dispensed in 99+2 ml amounts

in screw-capped dilution bottles.

2.5.2 Sterile buffered water or peptone

water prepared in 500 mi or larger volumes for

wetting membranes before addition of the

sample, and for rinsing the funnel after sample

filtration.

2.6 Procedure: The general membrane fil-

ter procedure is described in detail in Part II-C.

2.6.1 Prepare the M-FC broth or agar me-

dium as outlined in Part ll-B, 5.2.1. Saturate the

sterile absorbent pads with about 2.0 ml of

broth or add 5-6 ml of M-FC agar to the bottom

of each 50 X 12 mm petri dish (to a depth of

2-3 mm). Pour off excess liquid from broth-

saturated pads. Mark dishes and bench forms

with sample identity and sample volumes,

2.6.2 Using a sterile forceps place a sterile

membrane filter on the filter base, grid side up.

Attach the funnel to the base of the filter unit;

, the membrane filter is now held between the

funnel and base.

2,6.3 Shake the sampie vigorously about

25 times and measure the sample into the

funnel with the vacuum off. If sample volume is

< 10ml, add 10 ml of sterile dilution water to

the filter before adding the sample.

2.6.4 Sample volumes for fecal coliform

enumeration in different waters and wastewa-

ters are suggested in Table lll-C-1. These vol-

umes should provide the recommended count

of 20-60 colonies on a membrane filter. Fecal

coliform levels are generally lower than total

coliform densities in the same sample; there-

fore larger volumes are sampled.

2.6.5 Do not filter less than 1,0 ml of

undiluted sample.

2.6.6 Filter the sample and rinse the sides

of the funnel walls at least twice with 20-30 ml

of sterile dilution water,

2.6.7 Turn off the vacuum and remove the

funnel from the filter base.

2.6.8 Aseptically remove the membrane

filter from the filter base. Place the filter, grid

side up, on the absorbent pad saturated with

M-FC Broth or on M-FC agar, using a rolling

motion to prevent air bubbles.

2.6.9 Incubate the petri dishes for 24 +_ 2

hours at 44.5 +_ 0.2 C in sealed waterproof

plastic bags submerged (with the petri dishes

inverted) in a waterbath, or without plastic bag

in a heat-sink incubator. MF cultures should be

placed in incubator within 30 minutes of

filtration.

2.6.10 After 24 hours remove dishes from

the incubator and examine for blue colonies.

2.7 Counting and Recording Colonies:

Select those plates with 20-60 blue (some-

times greenish-blue) colonies. Non-fecal

colonies are gray, buff or colorless and are

not counted. Pinpoint blue colonies should be

counted and confirmed. The colonies are

counted using a microscope of 10-15x and

a fluorescent lamp. Use of hand lens or other

simple optical devices of lower magnification

make difficult the identification and differentia-

tion of typical and atypical blue colonies.

2.7.1 The general counting rules are given

in Part II-C, 3.5. The following rules are used

in calculating the fecal coliform count per

100 ml of sample:

(a) Countable Membranes with 20-60

Blue Colonies. Count all blue colonies using

the formula:

No. of Fecal Coliform Colonies Counted

Volume in ml of Sample Filtered

X 100

fecal coliform

count/100 ml

126

MICROBIOLOGICAL MANUAL 1978

image:

TABLE lll-C-1

Suggested Range of Sample Volumes for Fecal Coliform Tests

Using the Membrane Filter Method

FECAL COUFOFIMS

Sample Source 100 30 10 3 1

Swimming Pools X

Wells, Springs XX

Lakes, Reservoirs X X X

Water Supply Intakes XXX

Bathing Beaches - XX X

River Water X

Chlorinated Sewage Effluent X

Raw Sewage

0.3 0.1 0.03 0.01 0.003 0.001 0.0003 0.0001

X X

XXX X

X X X X

X X X X X

image:

For example, if 40 colonies are counted

after the filtration of 50 ml of sample, the

calculation is:

X 100 = 80 fecal coliforms/100 rnl.

fa) Countable Membranes With Less Than

20 Blue Colonies. Report as: Estimated

Count/100 ml and specify the reason.

(°) Membranes With No Colonies. Report

the count as: Less than (calculated value)/100

ml, based upon the largest single volume

filtered.

For example, if 10, 3 and 1 ml are filtered

and all plates show zero counts, select the

largest volume, apply the general formula and

report the count as a < (less than) value:

X 100 = 10

or < 10 fecal coliforms/100 ml.

(d) Countable Membranes With More Than

60 Blue Colonies. Calculate count from high-

est dilution and report as a > value.

(&} Uncountable Membranes With More

Than 60 Colonies. Use 60 colonies as the

basis of calculation with the smallest filtration

volume, e.g., 0.01 ml:

0.01

X 100 = 600,000

Report as: >

coliforms/100 mi.

600,000 fecal

2.7.2 Reporting Results. Report fecal coli-

form densities per 100 ml. See discussion on

significant figures in Part ll-C, 2.8.1.

2.8 Precision and Accuracy

2.8.1 Ninety-three percent of the blue col-

onies that develop on M-FC medium at the

elevated temperature of 44.5 ± 0.2 C were

verified as fecal coliform (1).

2.8.2 Laboratory personnel should be able

to duplicate their own colony counts on the

same plate within 5%, and the counts of other

analysts on the same plate within 10%.

3. Delayed-lncubation Membrane Filter (MF)

Method

3.1 Summary: Bacteria are retained on

0.45 um filters after passage of selected sam-

ple volumes through the filters. The filters are

placed on M-VFC broth (a minimum growth

medium) and transported from field sites to the

laboratory. In the laboratory, the filters are

transferred to the M-FC medium and incubated

at 44.5 C for 24 hours. Blue colonies are coun-

ted as fecal coliforms.

3.2 Scope and Application

3.2.1 Advantages: The delayed incubation

MF method is useful in survey monitoring or

emergency situations when the standard fecal

coliform test cannot be performed at the sam-

ple site, or when time and temperature limits

for sample storage cannot be met. The method

eliminates field processing and equipment

needs. Also, examination at a central labora-

tory permits confirmation and biochemical

identification of the organisms as necessary.

Consistent results have been obtained with

this method using water samples from a vari-

ety of sources (7).

3-2.2 Limitations: The applicability of this

method for a specific water source must be

determined in preliminary studies by compari-

son with the standard MF method. For exam-

ple, limited testing has indicated that the

delayed-incubation method is not as effective

in saline waters (7).

3.3 Apparatus and Materials

3.3.1 Water bath, aluminum heat sink, or

equivalent incubator that maintains a 44.5 ^

0.2 C temperature.

3.3.2 Binocular (dissection) microscope,

with magnification 10 or 15x, binocular,

wide-field type. A microscope lamp producing

128

«>EPA MICROBIOLOGICAL MANUAL 1978

image:

diffuse daylight from cool white fluorescent

lamps.

3.3.3 Hand tally.

3.3.4 Pipet containers of stainless steel,

aluminum or pyrex glass for glass pipets.

3.3.5 Graduated cylinders, covered with

aluminum foil or kraft paper before

sterilization.

3.3.6 Sterile unassembled membrane fil-

tration units (filter base and funnel), glass, plas-

tic or stainless steel wrapped with aluminum

foil or kraft paper.

3.3.7 Vacuum source.

3.3.8 Filter flask to hold filter base, with

appropriate tubing. Filter manifold to hold a

number of filter bases can also be used. In the

field, portable field kits are also used.

3.3.9 Safety trap flask between the filter-

ing flask and the vacuum source.

3.3.10 Forceps with smooth tip.

3.3.11 Ethanol, 95% or methanol, in small

vial, for sterilizing forceps.

3.3.12 Bunsen/Fisher type burner.

3.3.13 Sterile TD bacteriological or Mohr

pipets, glass or plastic, in appropriate volumes.

3.3.14 Sterile petri dishes, 50 x 12 mm

plastic with tight-fitting lids.

3.3.15 Dilution bottles (milk dilution), py-

rex glass, 99 ml volume, screw-caps with neo-

prene rubber liners.

3.3.16 Membrane filters, white, grid mark-

ed, 47 mm in diameter, 0.45±0.02pm pore size,

or other pore size recommended by the manu-

facturer for water analyses. The Millipore HC

MF, not the HA is recommended.

3.3.17 Shipping tubes, labels, and packing

materials for mailing delayed incubation plates.

3.3.18 Ultraviolet sterilizer for MF filtra-

tion units (optional).

3.4 Media: The following media are pre-

pared in pre-sterilized erlenmeyer flasks with

metal caps, aluminum foil covers, or screw-

caps:

3.4.1 M-VFC holding media (see Part II-B,

5.2.6).

3.4.2 M-FC broth or agar (see Part II-B,

5.2.1).

3.5 Dilution Water

3.5.1 Sterile dilution water dispensed in

99 + 2 ml volumes in screw-capped bottles.

3.5.2 Sterile dilution water prepared in

large volumes for wetting membranes before

the addition of the sample, and for rinsing the

funnel after sample filtration.

3.6 Procedure: The general membrane fil-

ter procedure is described in detail in Part II-C.

3.6.1 Prepare the M-VFC holding medium

as outlined in Part II-B, 5.2.6. Saturate the

sterile absorbent pads with about 2.0 ml of M-

VFC broth. Pour off excess broth. Mark dishes

and bench forms with sample identity and

volumes.

3.6.2 Using sterile forceps place a mem-

brane filter on the filter base grid side up.

3.6.3 Attach the funnel to the base of the

filter unit; the membrane filter is now held

between the funnel and base.

3.6.4 Shake the sample vigorously about

25 times and measure into the funnel with the

vacuum off. If the sample is < 10 ml, add 10

ml of sterile dilution water to the membrane

filter before adding the sample.

(a) Sample volumes for fecal coliform enu-

meration in different waters and wastewaters

are suggested in Table II1-C-1. These volumes

should produce membrane filters with a re-

commended count of 20-60 colonies.

FECAL COUFORMS

129

image:

(b) Follow the methods for sample mea-

surement and dispensation given in Part II-C,

3.4.6.

3.6.5 Filter the sample through the mem-

brane and rinse the sides of the funnel walls at

least twice with 20-30 ml of sterile dilution

water.

3.6.6 Turn off the vacuum and remove the

funnel from the base of the filter unit.

3.6.7 Aseptically remove the membrane

filter from the filter base and place grid side up

on an absorbent pad saturated with VFC

medium.

3.6.8 Place the culture dish in shipping

container and send to the examining labora-

tory. Fecal coliform bacteria can be held on the

VFC holding medium for up to 72 hours with

little effect on the final counts. The holding

period should be kept to a minimum.

3.6.9 At the examining laboratory remove

the membrane from the holding medium,

place it in another dish containing M-FC broth

or agar medium, and complete testing for fecal

coliforms as described above under 2.6.

4. Verification

Verification of the membrane filter test for

fecal coliforms establishes the validity of col-

ony differentiation by blue color and provides

supporting evidence of colony interpretation.

The verification procedure corresponds to the

fecal coliform MPN (EC Medium) test.

4.1 Pick from the centers of at least 10

well-isolated blue colonies. Inoculate into lauryl

tryptose broth and incubate 24-48 hours at

35 + 0.5 C.

4.2 Confirm gas-positive lauryl tryptose

broth tubes at 24 and 48 hours by inoculating

a loopful of growth into EC tubes and incubat-

ing for 24 hours at 44.5 ± 0.2 C. Cultures that

produce gas in EC tubes are interpreted as

verified fecal coliform colonies (see Figure III-

C-1).

4.3 A percent verification can be deter-

mined for any colony-validation test:

No. of colonies meeting verification test

No. of colonies subjected to verification

X 100

= Percent verification

3.7 Counting and Recording Colonies:

After the required incubation select those

plates with 20-60 blue (sometimes greenish-

blue) colonies. Gray to cream colored colonies

are not counted. Pin-point blue colonies are

not counted unless confirmed. The colonies

are enumerated using a binocular microscope

with a magnification of 10 or 15*.

Refer to 2.7.1, for rules used in reporting

the fecal coliform MF counts.

3.8 Reporting Results: Record densities

as fecal coliforms per 100 ml. Refer to Part II-C,

2.8, for discussions on the use of significant

figures and rounding off values.

Example: Twenty blue colonies on M-FC

medium were subjected to verification studies

shown in Figure lll-C-1. Eighteen of these colo-

nies proved to be fecal coliforms according to

provisions of the test:

Percent verification = X 100 = 90%

4.4 A percent verification figure can be

applied to the direct test results to determine

the verified fecal coliform count per 100 ml:

Percent verification

100

count per

100 ml

Verified fecal

coliform count

3.9 Precision and Accuracy: As reported

in 2.8, this Section.

Example: For a given sample, by the M-FC

test, the fecal coliform count was found to be

130

<3>ERA MICROBIOLOGICAL MANUAL 1978

image:

Pick 10 blue colonies

Lauryl Tryptose Broth

24 hours at 35 C

Gas +

Gas-

Reincubate

24 hours at 35 C

Gas +

EC Broth

24 hours at 44.5 C

Gas + Gas-

Verified Negative

Fecal Coliform ,-

Colony rest

Gas-

Negative

Test

FIGURE lll-C-1. Verification of Fecal Coliform Colonies on the Membrane Filter

FECAL COLIFORMS

131

image:

42,000 organisms per 100 ml. Supplemental

studies on selected colonies showed 92%

verification.

Verified fecal

coliform count

100

X 42,000 = 38,640

Rounding off = 39,000 fecal coliforms

per 100 ml

The worker is cautioned not to apply per-

centage of verification determined on one

sample to other samples.

5. Most Probable Number (MPN) Method

5.1 Summary: Culture from positive tubes

of the lauryl tryptose broth (same as presump-

tive MPN Method, Part lll-B) is inoculated into

EC Broth and incubated at 44.5 C for 24 hours

(see Figure lll-C-2). Formation of gas in any

quantity in the inverted vial is a positive reac-

tion confirming fecal coliforms. Fecal coliform

densities are calculated from the MPN table on

the basis of the positive EG tubes (8).

5.2 Apparatus and Materials

5.2.1 Incubatorthat maintains 35 ± 0.5 C.

5.2.2 Water bath or equivalent incubator

that maintains a 44.5 ^ 0.2 C temperature.

5.2.3 Pipet containers of stainless steel,

aluminum or pyrex glass for glass pipets.

5.2.4 Inoculation loop, 3 mm diameter

and needle of nichrome or platinum wire, 26 B

& S gauge, in suitable holder. Sterile applica-

tor sticks are a suitable alternative.

5.2.5 Sterile pipets T.D., Mohr or bacterio-

logical, glass or plastic, of appropriate size.

5.2.6 Dilution bottles (milk dilution), pyrex,

99 ml volume, screw-cap with neoprene liners.

5.2.7 Bunsen or Fisher-type burner or elec-

tric Incinerator unit.

5.2.8 Pyrex test tubes, 150 x 20 mm,

containing inverted fermentation vials, 75 x

10 mm, with caps.

5.2.9 Culture tube racks to hold fifty, 25

mm diameter tubes.

5.3 Media

5.3.1 Lauryl tryptose broth (same as total

coliform Presumptive Test medium) prepared

in 10 ml volumes in appropriate concentration

for sample volumes used. (Part II-B, 5.3.1).

5.3.2 EC medium prepared in 10 ml vol-

umes in fermentation tubes (Part II-B, 5.3.4).

5.4 Dilution Water: Sterile buffered or

peptone dilution water dispensed in 99 ± 2 ml

volumes in screw-capped bottles.

5.5 Procedure: Part II-C describes in detail

the general MPN procedure. See Figure lll-C-2.

5.5.1 Prepare the total coliform Presump-

tive Test medium, (lauryl tryptose broth) and

EC medium. Clearly mark each bank of tubes,

identifying the sample and the volume

inoculated.

5.5.2 Inoculate the Presumptive Test me-

dium with appropriate quantities of sample

following the Presumptive Test total coliform

procedure, (Part lll-B).

5.5.3 Gently shake the Presumptive tube.

Using a sterile inoculating loop or a sterile

wooden applicator, transfer inocula from posi-

tive Presumptive Test tubes at 24 and 48

hours to EC confirmatory tubes. Gently shake

the rack of inoculated EC tubes to insure mix-

ing of inoculum with medium.

5.5.4 Incubate inoculated EC tubes at

44.5 ± 0.2 C for 24 ± 2 hours. Tubes must be

placed in the incubator within 30 minutes after

inoculation. The water depth in the water bath

incubator must come to the top.level of the

culture medium in the tubes.

5.5.5 The presence of gas in any quantity

in the EC confirmatory fermentation tubes af-

132

<&EF9\ MICROBIOLOGICAL MANUAL 1978

image:

Sample

Lauryl Tryptose Broth

35 ±0.5 C

Q-

Gas +

24 hr

Gas-

24 hr

Reincubate

24 hr

Gas

Gas-

Negative

Test

Elevated Temperature Test

EC Medium at 44.5 ± 0.2 C

Gas +

24 hr

Fecal Coliforms Present

Calculate

Fecal Coliform

MPN

Gas-

24 hr

Negative

Test

FIGURE III-C-2. Flow Chart forthe Fecal Coliform MPN Tests.

FECAL COLIFORMS

133

image:

ter 24 ± 2 hours constitutes a positive test for

fecal conforms.

5.6 Calculations

5.6.1 Calculate fecal coliform densities on

the basis of the number of positive EC fermen-

tation tubes, using the table of most probable

numbers (MPN).

5.6.2 The MPN results are computed from

three dilutions that include the highest dilution

with all positive tubes and the next two higher

dilutions. For example, if five 10 ml, five 1.0 ml,

and five 0.1 ml sample portions are inoculated

initially into Presumptive Test medium, and

positive EC confirmatory results are obtained

from five of the 10 ml portions, three of the 1.0

ml portions, and none of the 0.1 ml portions,

the coded result of the test is 5-3-0. The code

is located in the MPN Table ll-C-4, and the MPN

per 100 ml is recorded. See Part II-C, 4.9 for

rules on selection of significant dilutions.

5.7 Reporting Results: Report the fecal

coliform MPN values per 100 ml of sample.

5.8 Precision and Accuracy: The preci-

sions of the MPN counts are given as confi-

dence limits in the MPN tables. Note that the

precision of the MPN value increases with

increased numbers of replicates per sample

tested.

REFERENCES

1. Geldreich, E. E, H. F. Clark, C. B. Huff, and L. C. Best, 1965. Fecal-coliform-organism medium for

the membrane filter technique. J. Amer. Water Works Assoc. 57:208.

2. Bordner, R. H., C. F. Frith and J. A. Winter, (Editors), 1977. Proceedings of the Symposium on the

Recovery of Indicator Organisms Employing Membrane Filters. U.S. Environmental Protection

Agency, Environmental Monitoring and Support Laboratory, Cincinnati, OH. (EPA-

600/9-77-024).

3, Lin, S. D., 1973. Evaluation of coliform tests for chlorinated secondary effluents. «J. Water

Pollution Control Federation 45:498.

4. Lin, S, D., 1976, Evaluation of Millipore HA and HC membrane filters for the enumeration of

indicator bacteria. Appl. Environ. Microblol. 32:300.

5. Lin, S. D.,1976. Membrane filter method for recovery of fecal conforms in chlorinated sewage

effluents. Appl. Environ. Microbiol. 32:547.

6. Green, B, L., E. M. Clausen, and W. Litsky, 1975. Two-temperature membrane filter method

for enumeration of fecal coliform bacteria from chlorinated effluents, Appl. Environ.

Microblol. 33: 1259. ~

7. Taylor, R. H., R. H. Bordner, and P. V. Scarpino, 1973. Delayed-incubation membrane-filter test for

for fecal conforms. Appl. Microbiol. 25:363.

8. Geldreich, E. E., 1966. Sanitary Significance of Fecal Coliforms in the Environment, U.S. Dept, of

the Interior, FWPCA, WP-20-3, 122 pp.

134

MICROBIOLOGICAL MANUAL 1978

image:

PART III. ANALYTICAL METHODOLOGY

Section D Fecal Streptococci

The membrane filter (MF), most probable

number (MPN), and direct pour plate proce-

dures can be used to enumerate and identify

fecal streptococci in water and wastewater.

Fora general description of these fundamental

techniques refer to Part II-C. The method se-

lected depends upon the characteristics of the

sample. The Section is divided as follows:

1. The Fecal Streptococcus Group

2. Membrane Filter Method

3. Verification

4. Most Probable Number Method

5. Pour Plate Method

6. Determination of FC/FS, Ratios

7. Identification to Species

1. The Fecal Streptococcus Group

1.1 Fecal Streptococci and Lancefield's

Group D Streptococcus: The terms "Fecal

Streptococcus" and "Lancefield's Group D

Streptococcus" have been use synonymously.

When used as indicators of fecal contamina-

tion the following species and varieties are

implied: S. faecalis, S. faeca/is subsp. liquefa-

ciens, S. faecalis subsp. zymogenes, S. fae-

cium, S. bovis and S. equinus. For sanitary

analyses, media and methodology for quantifi-

cation are selective for these organisms.

1.2 Fecal Streptococcal Intermediates

and Biotypes: Current information indicates

that other streptococci belonging to Lance-

field's serological Group Q occur in the feces

of humans and other warm-blooded animals,

especially chickens. S. avium is characteristi-

cally found in the feces of chickens and occa-

sionally in the feces of man, dogs and pigs. The

Group Q antigen is found in the cell wall of

these organisms, and in addition, the Group D

antigen is located between the cell wall and

the cytoplasmic membrane where it occurs

naturally in the established Group D species.

These common antigens indicate a relation-

ship between Group D and Group Q orga-

nisms. The Group Q Streptococcci may ac-

count for the occurrence of the "intermediate

strains or biotypes" of Group D Streptococci

(1). Group Q organisms grow on media com--

monly used for the isolation and enumeration

of enterococci. Kenner ert aL (2) observed that

enterococcus biotypes occur more commonly

in the feces of fowl than of pigs, sheep, cows

and humans. Forty percent of the streptococci

isolated from the feces of fowl by Kenner et aL

were enterococcus biotypes with the rest be-

ing the enterococcus group. It is probable that

some of the biotypes they described were

Group Q Streptococci. The Group Q Strepto-

cocci, with a type species, should be consid-

ered in the Fecal Streptococcus Group.

1.3 Definition of the Fecal Streptococ-

cus Group: The term, fecal streptococci, will

be used to describe the streptococci which

indicate the sanitary quality of water and

FECAL STREPTOCOCCI

135

image:

wastewater. The fecal streptococci group in-

cludes the serological groups D and Q.

Fecal Streptococci

" a

O 3

§ 8

S. faecalis

5. faeca//ssubsp. liquefaciens

S. faeca/issubsp, zymogenes

S1. faecium

S. bovis

S, equinus

S. avium

1.4 Viridans Streptococci: The viridans

streptococci, primarily S. salivarius and S. mi-

tis are not considered as part of the fecal

streptococci as defined in 1.2 and 1.3. These

inhabitants of the nasopharyngeal tract have

been reported by a few workers in feces and

do grow on some fecal streptococci media.

However, their low numbers when present the

low frequency of occurrence and the limited

data available at this time concerning their

presence, have resulted in their exclusion from

the classification of fecal streptococci.

1.5 Scope and* Application: Fecal strepto-

cocci data verify fecal pollution and may pro-

vide additional information concerning the re-

cency and probable origin of pollution. In com-

bination with data on coliform bacteria, fecal

streptococci are used in sanitary evaluation as

a supplement to fecal conforms when a more

precise determination of sources of contami-

nation is necessary. The occurrence of fecal

streptococci in water indicates fecal contami-

nation by warm-blooded animals. They are not

known to multiply in the environment. Further

identification of streptococcal types present in

the sample may be obtained by biochemical

characterization. (See Figure III-D-2 "Isolation

and Identification of Fecal Streptococci").

Such information is useful for source investi-

gations. For example, S. bovis and S. equinus

are host specific and are associated with the

fecal excrement of non-human warm-blooded

animals. High numbers of these organisms are

associated with pollution from meat process-

ing plants, dairy wastes, and run-off from feed-

lots and farmlands. Because of limited survival

time outside the animal intestinal tract their

presence indicates very recent contamination

from farm animals.

2. Membrane Filter (MF) Method

2,1 Summary: A suitable volume of sam-

ple is passed through the 0.45 ym membrane

filter which retains the bacteria. The filter is

placed on KF Streptococcus agar and incu-

bated at 35 C for 48 hours. Red and pink

colonies are counted as streptococci (1,2).

2.2 Scope and Application: The mem-

brane filter technique is recommended as the

standard method for assaying fecal strepto-

cocci in fresh and marine waters and in non-

chlorinated sewage. Wastewaters from food

processing plants, slaughter houses, canner-

ies, sugar processing plants, dairy plants, feed-

lot and farmland run-off may be analyzed by

this procedure. Colonies on a membrane filter

can be transferred to biochemical media for

identification and speciation to provide infor-

mation on the source of contamination. The

general advantages and limitations of the MF

method are given in Part II-C.

2.3 Apparatus and Materials

2.3.1 Water bath, aluminum heat sink or

air incubator set at 35 ±0.5 C. Temperature

checked with an NBS thermometer or one of

equivalent accuracy.

2.3.2 Stereoscopic (dissection) micro-

scope, with magnification of 10 to 15x, pref-

erably wide field type. A microscope lamp with

diffuse light from cool, white fluorescent tubes

is recommended.

2.3.3 Hand tally.

2.3.4 Pipet containers of stainless steel,

aluminum or pyrex glass for glass pipets.

136

S»iPA MICROBIOLOGICAL MANUAL 1978

image:

2.3.5 Sterile graduated cylinders, covered

with aluminum foil or kraft paper.

2.3.6 Sterile, unassembled membrane fil-

tration units (filter base and funnel), glass, plas-

tic or stainless steel, wrapped with aluminum

foil or kraft pa per.

2,3.7 Vacuum source.

2.3.8 Filter suction flask to hold filter base,

with appropriate tubing. Filter manifolds that

hold a number of filter bases can also be used.

2.3.9 Safety vacuum flask.

2.3.10 Forceps, with smooth tips.

2,3.11 Ethanol, 95%, or methanol, in

small vial for sterilizing forceps.

,2.3.12 Bunsen/Fisher burner or electric

incinerator.

2.3.13 Sterile T.D. bacteriological or Mohr

pipets, glass or plastic, of appropriate size.

2,3.14 Sterile petri dishes, 50 x 12 mm

plastic or 60 x 15 mm glass or plastic.

2,3.15 Dilution bottles (milk dilution), py-

rex glass, marked at 99 ml volumes, screw-cap

with neoprene rubber liner.

2.3.16 Membrane filters, manufactured

from cellulose ester materials, white, grid

marked, 47 mm in diameter, 0.45 H^ 0.02 um

pore size or other pore size as recommended

by the manufacturer for fecal streptococci

analyses.

2.3.17 Ultraviolet sterilizer for MF filtra-

tion units (optional).

2.4 Media: KF Streptococcus agar pre-

pared as described in Part II-B, 5.4.1.

2.5 Dilution Water

2.5.1 Sterile buffered dilution water or

peptone water dispensed in 99 ± 2 ml vol-

umes in screw-capped dilution bottles.

2.5.2 Sterile buffered water or peptone

water prepared as described in Part II-B, 7, in

large volumes for wetting membranes before

the addition of the sample, and for rinsing the

funnel after filtration.

2.6 Immediate MF Procedure: The gen-

eral membrane filter procedure is described in

detail in Part II-C, 3.

2.6.1 Clearly mark each petri dish and

aseptically add 5-6 ml of the liquified agar

medium (to each dish to a depth of 2-3 mm).

2.6.2 Place a sterile membrane filter on

the filter base, grid-side up and attach the

funnel to the base of the filter unit; the mem-

brane filter is now held securely between the

funnel and base.

2.6.3 Shake the sample vigorously about

25 times and measure the sample into the

funnel with the vacuum off.

2.6.4 Filter appropriate volumes of water

sample through the sterile membrane to ob-

tain 20-100 colonies on the membrane

surface.

At least 3 sample increments should be

filtered in order of increasing volumes. Where

no background information is available, more

may be necessary. The methods of measure-

ment and dispensation of the sample into the

funnel are given in Part II-C, 3.4.6.

2.6.5 Filter the sample and rinse the sides

of the funnel at least twice with 20-30 ml of

sterile buffered dilution water. Turn off the

vacuum and remove the funnel from the filter

base.

2.6.6 Aseptically remove the membrane

from the filter base and place grid-side up on

the agar.

2.6.7 Incubate the petri dishes in the in-

verted position at 35 + 0.5 C for 48 hours.

2.6.8 After incubation, remove dishes and

examine for red to pink colonies and count.

FECAL STREPTOCOCCI

137

image:

2.7 Counting and Recording Colonies:

Select those plates with 20-100 pink to dark

red colonies. These may range in size from

barely visible to about 2 mm in diameter. Colo-

nies of other colors are not counted. Count the

colonies as described in Part II-C, 3.5, using

low-power (10-15X) microscope equipped

with overhead illumination.

Fecal streptococcal density is reported as or-

ganisms per 100 ml. Use the general formula

to calculate fecal streptococci densities:

Fecal Streptococci/ 1 00 ml =

No, of Fecal Streptococcus Colonies Counted

Volume of Sample Filtered, ml

X 100

For example, if 40 colonies are counted

after the filtration of 50 ml of sample the

calculation is:

Fecal Streptococci/ 100 ml

100 = 80.

See Part ll-C, 3.6 for calculation for results.

Reporting Results: Report fecal strepto-

coccal densities per 100 ml of sample. See

discussion on significant figures in Part It-C

2.8.

2,8 Precision and Accuracy

2.8.1 Extensive precision and accuracy

data are not available, however, KF Strepto-

coccus agar has been reported to be highly

efficient in the recovery of fecal streptococci

(2, 3, 4). In the analyses of feces, sewage and

foods, KF yielded a high recovery of fecal

streptococci with a low percent (18.6) of non-

fecal streptococci.

2.8.2 Laboratory personnel should be able

to duplicate their colony counts on the same

plates within 5%, and the counts of others

within 10%.

2.9 Delayed MF Procedure: Because of

the stability of the KF agar and its extreme

selectivity for fecal streptococci, it is possible

to filter water samples at a field site, place

membranes on the KF agar medium in tight-

lidded petri dishes and hold these plates for up

to 3 days. After the holding period, plates are

incubated for 48 hours at 35 C and counted in

the normal manner. This 72 hour holding time

can be used to air mail the membranes on KF

agar to a central laboratory for incubation and

counting. (National Pollution Surveillance Sys-

tem FWPCA, data collected from geographical

locations around the Nation; and Kenner et aL,

Kansas City data.)

3. Verification

Periodically, typical colonies growing on

the membrane filter should be verified. When

a survey is initiated or a new body of water is

being sampled, it is recommended that at

least 10 typical colonies from the membrane

or agar plate used in computing the final

density be picked and transferred into BHI

broth or onto BHI agar slants. After 24-48

hours incubation, subject the cultures to a

catalase test. Catalase activity indicates the

nonstreptococci. Atypical colonies should also

be verified to determine false negative reac-

tions on the membrane filter. Final confirma-

tion of fecal streptococci is achieved by

determining growth of catalase negative

isolates in BHI broth at 45 C and in 40% bile

within two days (see Figure lll-D-1).

3,1 Apparatus and Materials

3.1.1 Incubators set at 35 ± 0.5 C and 45

± 0.5 C.

3.1.2 Inoculating needle and loop.

3.1.3 Bunsen/Fisher burner or electric

incinerator.

3.1.4 Solution of 3% hydrogen peroxide.

3.1.5 Glass microscope slides, 2.5 x 7.6

cm(1 x 3 inches).

138

&EPA. MICROBIOLOGICAL MANUAL 1978

image:

3.1.6 Media

(a) Brain heart infusion (BHI) agar. (See Part

!!-B, 5,4.6).

(b) Brain heart infusion (BHI) broth. (See

Part II-B, 5.4.5).

3.2 Procedure

3.2.1 Plates for verification should con-

tain 20-100 colonies. Pick at least 10 typical

colonies from the selected membrane or agar

plate and inoculate into a BHI agar slant and

into a BHI broth tube.

3.2.2 After 24-48 hours incubation at 35

+0.5 C, transfer a loopful of growth from the

BHI slant to a clean glass slide and add a few

drops of freshly tested 3% hydrogen peroxide

(H2O2) to the smear. If the catalase enzyme is

present, it cleaves the H202 to water and visible

oxygen gas. Bubbles constitute a positive cata-

lase test and indicate non-streptococcal spe-

cies. Confirmation need not be continued. Use

a platinum loop, not nichrome, to avoid false

positive reactions.

3.2.3 If a negative catalase reaction occurs,

transfer a loopful of growth from the BHI broth

to fresh BHI broth and BHI broth + 40% bile and

incubate at 45 C and at 35 C. Growth within two

days indicates fecal streptococcal species (see

Figure lll-D-1 and lll-D-2).

3.2.4 Further identification of streptococ-

cal types present in the sample may be ob-

tained by biochemical characterization. (See

Figure III-D-2 to ill-D-4 for identification of fecal

streptococci. Such information is useful for

investigating sources of pollution. See Part II-B

for preparation of media used in the schematic

outlines).

4. Most Probable Number Method

4.1 Summary: The multiple-tube proce-

dure estimates the number of fecal strepto-

cocci by inoculating decimal dilutions of the

sample into broth tube media. Positive tubes in

the Presumptive Test are indicated by growth

(turbidity) in azide dextrose broth after incuba-

tion at 35 C for 24-48 hours. To confirm the

presence of fecal streptococci, a portion of the

growth from each positive azide dextrose

broth tube is streaked onto PSE or equivalent

esculin-azide agar and incubated at 35 C for

24 hours (5). The presence of brownish-black

colonies with brown halos confirms fecal

streptococci. The MPN is computed on the

basis of the Confirmed Test results read from

and MPN table.

4.2 Scope and Application: This method

can be used for detection of fecal streptococci

in water, sewage or feces, but is more time-

consuming, less convenient and less direct

than the other procedures. The MPN must be

used for samples which cannot be examined

by the MF or direct plating techniques because

of turbidity, high numbers of background bac-

teria, metallic compounds, the presence of co-

agulants, the chlorination of sewage effluents

or sample volume limitations of the plating

technique.

4.3 Apparatus and Materials

4.3.1 Water bath or air incubator set at 35

± 0.5 C. Temperature checked with an NBS

thermometer or one of equivalent accuracy.

4.3.2 Pipet containers of stainless steel,

aluminum or pyrex glass for pipets.

4.3.3 Culture tube racks to hold fifty 25

mm diameter tubes.

4.3.4 Sterile T.D. bacteriological or Mohr

pipets, of appropriate sizes.

4.3.5 Dilution bottles (milk dilution), pyrex

glass, 99 ml volume, screw-capped, with neo-

prene rubber liner.

413.6 Test tubes, pyrex, culture 150 X 25

or 150 x 20 mm, with caps.

4.3.7 Inoculating loop, 3 mm diameter, in

holder or disposable applicator sticks or loops.

4.3.8 Bunsen/Fisher burners or electric

incinerator.

FECAL STREPTOCOCCI

139

image:

10 Colonies Typical in Appearance of

Fecal Streptococci on Isolation Media

Brain Heart Infusion Broth and Agar Slant

(24-48Hoursat35C)

Catalase Negative

Growth at 45 C

Growth in 40% Bile

Verification of Fecal Streptococci

FIGURE IH-D-1. Verification Procedure for Fecal Streptococci.

140

MICROBIOLOGICAL MANUAL 1978,

image:

Sample

PSE Agar

(Brownish-black colony

with brown halo)

KF Agar

(pink-red colony)

Growth at 45 C and 10 C

Growth in 6.5% NaCi

and pH 9,6 BHI Broth

0.1% Methylene Blue in Milk

Reduction

Enterococcus

Group

No Reaction

Tentative

Group Q

Growth at 45 C only

Positive Starch Hydrolysis

Lactose

Fermentation

Acid only

No change

S. bovis S. eguinus

(Livestock and

Poultry Sources)

See Figures IH-D-3

and lll-D-4

FIGURE IH-D-2. Isolation and Identification of Fecal Streptococci, General Scheme

FECAL STREPTOCOCCI

141

image:

4.4 Media

4,4.1 Azide dextrose broth prepared in 10

ml volumes in test tubes without fermentation

via! (see Part Il-B, 5.4.2 for preparation). Ethyl

violet azide broth is not used because of false

positive reactions.

4.4.1 Azide dextrose broth prepared in 10

ml volumes in test tubes without fermentation

vial (see Part Il-B, 5.4.2 for preparation).

4.4.2 Pfizer Selective Enterococcus (PSE)

or equivalent esculin-azide agar in pour plates

(see Part Il-B, 5.4.4 for preparation).

4.5 Dilution Water: Sterile dilution water

dispensed in 99 ml ^ 2 ml amounts in screw-

capped bottles.

4.6 Procedure: The general MPN proce-

dure is described in detail in Part Il-C, 4.

4.6.1 Prepare the media for the .Presump-

tive Test, (azide dextrose broth) and the Con-

firmed Test, (PSE Agar plates), (see Part ll-B,

5.4,2 and 5.4.4 respectively).

4.6.2 Mark culture tubes to identify sam-

ples and sample volumes.

4.6.3 Shake the sample vigorously about

25 times.

4.6.4 Inoculate the azide dextrose broth

with appropriate sample volumes for the Pre-

sumptive Test. The number of fecal strepto-

cocci in a water polluted with municipal

wastes is generally lower than the number of

coliforms. Therefore, larger sample volumes

must be used to inoculate the MPN tubes for

fecal streptococci than for coliforms. For ex-

ample, if sample volumes of 1.0,0.1,0.01, and

0.001 ml are used for the coliform test, a

series of 10,1.0,0.1, and 0.01 ml volumes are

inoculated for the fecal streptococci test. Use

single-strength broth, 10 ml tubes for inocula

of 1.0 ml or less, and double-strength broth, 10

ml tubes for inocula of 10 ml. Sample volumes

from feedlots, meat packing plants, and storm-

water run-off with more fecal streptococci

than coliforms must be adjusted accordingly.

4.6,5 Shake the rack of inoculated culture

tubes to mix well and incubate them at 35 +

0.5 C for 48 hours + 3 hours. Examine tubes

for turbidity after 24 ± 2 hours and 48 ± 3

hours.

4.6.6 Read and record the results from

each tube. A positive Presumptive Test shows

growth consisting of turbidity in the medium or

a button of sediment at the bottom of the

culture tube, or both.

4.6.7 For the Confirmed Test, streak

growth from each positive azide dextrose

broth tube onto PSE Agar plates, making cer-

tain that the label on the plate corresponds to

the positive azide dextrose tube used,

4.6.8 Incubate the PSE Agar plates at 35

+ 0.5 C for 24 hours.

4.6.9 Read and record the results of each

plate corresponding to the positive azide dex-

trose tube. A positive Confirmed Test is evi-

denced by the presence of brownish-black col-

onies with brown halos. The number of posi-

tive confirmed azide tubes in each dilution is

used to compute the density from an MPN

table.

4.7 Calculations

4.7,1 Calculate fecal streptococci densi-

ties on the basis of the number of positive

Confirmed Tests from the PSE agar plates,

using the Table of Most Probable Numbers

(MPN) in Table ll-C-1,

4.7.2 The MPN results are computed from

3 usable Confirmed Test dilutions. For exam-

ple, if positive Confirmatory Test results are

obtained from 5 of the 10 ml portions, three of

the 1.0 ml portions, and none of the 0.1 ml

portions, the coded results of the test is 5-3-0.

The code is located in the MPN Table II-C-4,

and the MPN per 100 ml is recorded. See Part

Il-C, 4 for details.

4.8 Precision and Accuracy: The precision

of the MPN value increases with increased

numbers of replicates tested. Five tubes are

recommended for each dilution.

142

MICROBIOLOGICAL MANUAL 1978

image:

S. Pour Plate Method

5.1 Summary: Aliquots of the water sam-

ple or diluted sample are delivered to the bot-

tom of a petri dish, and liquified Pfizer Selec-

tive Enterococcus (PSE) agar, equivalent

esculin-azide agar or KF agar is added and

thoroughly mixed with the water sample. Fecal

streptococci on PSE agar are 1 mm in diameter

and brownish-black with brown halos after

18-24 hours at 35 C. On KF agar fecal strepto-

cocci are red or pink after 48 hours at 35 C.

5.2 Scope and Application: The pour

plate method is recommended as an alternate

procedure to the MF technique when chlori-

nated sewage effluent and water samples with

high turbidity are encountered. PSE agar, the

medium of choice, has several advantages: (1)

it requires only 24 hours incubation compared

to 48 hours for other media, and (2) it exhibits

consistent recovery, regardless of sources.

With the pour plate technique, only small

volumes of sample may be analyzed. This is a

disadvantage when the fecal streptococcal

density is low and a large volume of sample

would be required for an accurate density de-

termination. Consequently, the MF technique

should be used unless the water is so turbid

that filtration is impossible.

5.3 Apparatus and Materials

5.3.1 Air incubator set at 35 ± 0.5 C.

Temperature is checked against a National

Bureau of Standards thermometer or one of

equivalent accuracy.

5.3.2 Water bath for tempering agars set

at 44-46 C.

5.3.3 Colony Counter, Quebec darkfield

model or equivalent.

5.3.4 Pipet containers of stainless steel,

aluminum or pyrex glass for glass pipets.

5.3.5 Petri dish containers for glass or

plastic petri dishes.

5.3.6 Sterile T.D. bacteriological or Mohr

pipets of appropriate sizes.

5.3.7 Sterile 100 mm

dishes, glass or plastic.

X 15 mm petri

5.3.8 Dilution bottles (milk dilution), pyrex,

99 ml volume, screw-capped, with neoprene

rubber liners.

5.3.9 Bunsen/Fisher burner or electric

incinerator.

5.3.10 Hand tally.

5.4 Media: Sterile Pfizer Selective Entero-

coccus agar (PSE), equivalent esculin-azide

agar, (Part II-B, 5.4.4) or KF Streptococcus agar

(Part II-B, 5.4.1) are prepared in pre-sterilized

erlenmeyer flasks or bottles with metal foil

covers, or screw-caps.

5.5 Dilution Water: Sterile dilution water

dispensed in 99 ± 2 ml amounts preferably in

screw-capped dilution bottles (see Part II-B, 7).

5.6 Procedure

5.6.1 Shake the sample bottle vigorously

about 25 times to disperse the bacteria. Take

care that the closure is tight to prevent leakage

of sample during shaking.

5.6.2, Dilute the sample to obtain final

plate counts between 30-300 colonies. The

number of colonies within this range gives the

most accurate estimation of the microbial pop-

ulation. Because the magnitude of the micro-

bial population in the original water sample is

not known beforehand, a range of dilutions

must be prepared and plated to obtain a plate

within this range of colony counts.

5.6.3 Transfer 0,1 and 1.0 ml from the

undiluted sample to each of 2 separate petri

dishes.

5.6.4 Prepare the initial 1:100 or 10~2

dilution by pipetting 1 ml of the sample into a

99 ml dilution water blank using a sterile 1.1

ml pipet (see Part II-C, 1.4 "Preparation of

Dilutions," and Figure ll-C-1).

FECAL STREPTOCOCCI

143

image:

5.6.5 Vigorously shake the 1:100 dilution

bottle to obtain uniform distribution of

bacteria.

5.6.6 Pipet 0.1 and 1.0 ml of this 1:100

dilution into each of 2 separate petri dishes

using another sterile pipet.

5.6.7 Make additional dilutions as re-

quired for raw wastes or stormwater run-off

and prepare pour plates containing the dilu-

tion aliquots.

5.6.8 Prepare duplicate petri dishes for

each sample increment (Figure IH-A4). Mark

each petri dish with the number of the sample,

the dilution, the date, and any other necessary

information. Deliver the liquid into the dish,

and touch the tip once against a dry area in the

petri dish bottom while holding the pipet

vertically.

5.6.9 Pour 12-15 ml of liquified cooled

agar medium into each petri dish containing

the sample or its dilution. Mix the medium and

the sample thoroughly by gently rotating and

tilting the petri dish. Not more than 20 minutes

should elapse between dilution, plating, and

addition of the medium. Refer to Part II-C, 2.6,

for further information.

5.6.10 Allow agar to solidify as rapidly as

possible after pouring, and place the inverted

PSE plates at 35 ± 0.5 C for 18-24 hours and

KF plates at 48 ± 3 hours.

5.7 Counting and Recording Colonies

5.7.1 After the specified incubation period,

select those plates with 30-300 fecal strepto-

coccal colonies. Fecal streptococci on PSE agar

are brownish-black colonies, about 1 mm in

diameter with brown halos. On KF agar, fecal

streptococci are pink to red and of varying

sizes.

5.7.2 Count colonies in the plates with the

aid of a colony counter (10-15* magnification)

equipped with a grid.

5.7.3 Observe the following rules for re-

porting the fecal streptococcal plate counts.

(a) Plates with 30-300 Fecal Streptococ-

cal Colonies: Count all colonies for each plate

within the 30-300 range. Calculate the aver-

age count for these plates correcting for the

dilution as follows:

Sum of Colonies

Sum of Volumes tested, ml

x100 = FS/100ml

^b^ All Plates Greater than 300 Colonies:

When counts for all dilutions contain more

than 300 colonies, e.g., >500 for 1.0 ml,

>500 for 0,1 ml, and 340 for 0,01 ml; com-

pute the density by counting the plate having

nearest to 300 colonies. In this case use the

0:01 ml plate.

340

0.01

x 100 = 3,400,000

Report as: Estimated Fecal Streptococcal

Count, 3,400,000/100 ml

If all plates are less than 30 colonies, record

the actual number of colonies on the lowest

dilution plated and report the count as the

Estimated Fecal Streptococcal Plate Count

per 100 ml.

(d) Plate with No Colonies: If plates from all

dilutions show no colonies, assume a count of

one (1) colony; then divide 1 by the largest

volume filtered and report the value as a less

than {<) count. For example, if 0.1, 0.01 and

0.001 ml were filtered with no reported colo-

nies, the count would be:

— x 100 = < 1000

0.1

Report the count as: < 10/100 ml

(e) When all plates are crowded, it is possi-

ble to use the square divisions of the grid on

the Quebec or similar counter to estimate the

numbers of colonies on the plate. See Part III-

A, 5.6.3 for details.

^•^ Precision and Accuracy: Replicate

plate counts from the same sample deviate

144

•&EPA MICROBIOLOGICAL MANUAL 1978

image:

because of errors introduced from a variety of

sources.

Prescott et al. (6) reported for the Standard

Plate Count (but applicable here) that the stan-

dard deviation of individual counts from

30-300 will vary from 0-20%. This plating

error was 10% for higher plate counts within

the 100-300 range. The authors pointed out

that a dilution error of about 3% for each

dilution stage is incurred in addition to the

plating error. Therefore, large variations can

be expected from high density samples such

as sewage from which several dilutions are

made.

Laboratory personnel should be able to

duplicate their plate count values for the same

plate within 5%, and the counts of others

within 10%.

6. Determination of Fecal Coliform/Fecal

Streptococcus Ratios (FC/FS)

The relationship of fecal coliform to fecal

streptococcus density may provide informa-

tion on the potential source(s) of contamina-

tion. Estimated per capita contributions of in-

dicator bacteria for animals were used to de-

velop FC/FS ratios (7, 8). These ratios are as

follows:

FC/FS Ratios

Man

Duck

Sheep

Chicken

Pig

Cow

Turkey

4.4

0.6

0.4

0.2

0.1

From the data, it was reasoned that ratios

greater than 4:1 were indicative of pollution

derived from domestic wastes composed of

man's body wastes. Ratios of less than 0.7

suggested that contamination originated from

livestock and poultry wastes, milk and food

processing wastes or from stormwater run-off

{non-human source). Further speciation of the

fecal streptococci provides more specific

source information. There are several precau-

tions to be observed when ratios are being

used.

(a) Bacterial densities can be altered dras-

tically wheh the pH of the sample is below 4.0

or above 9.0.

(b) Due to limited survival capability of

some of the fecal streptococci, it is essential to

sample close to the pollution source to obtain

reliable ratios. This is especially true for the

highly sensitive S. bovis and S. equinus

species.

when mixed pollution sources are present.

(d) In marine waters, bays, estuaries, and

irrigation returns, FC/FS ratios have been of

limited value in accurately defining major pol-

lutional source's.

(e) If fecal streptococcal counts are

< 100/100 ml, ratios should not be applied.

7. Identification of Fecal Streptococci to

Species

7.1 Summary: Although the fecal st pto-

cocci are enumerated as described in the >re-

vious sections and are verified with simple

biochemical tests in Part III-D, 3 above, it is

important at times to identify the fecal strepto-

cocci to species to further verify animal and

human sources of pollution and to determine

the sanitary significance of isolates. This iden-

tification to speeies is performed using the

additional biochemical tests described to dif-

ferentiate and confirm the Group Q strepto-

cocci, the bovis-equinusGroup and the entero-

cocci. The enterococci can be separated as S.

faec/umand S. faeca/isvarteties or into groups

according to original source.

7.2 Scope and Application: The initial

biochemical test confirms that the isolates are

fecal streptococci by negative catalase reac-

tion. Group Q streptococci and enterococci are

separated from the bovis-equinus group by

positive growth at 10 and 45 C and are then

FECAL STREPTOCOCCI

145

image:

verified by growth in 6.5% NaCI and at pH 9.6.

The enteroeoeei are separated from the Group

Q streptococci by reduction of methylene blue

milk. Subsequently, the enteroeoeei are either

speciated or separated by origin using addi-

tional biochemical tests. The Streptococcus

bovis and equinus are verified by hydrolysis

of starch and separated by lactose fermenta-

tion. These tests require specific training for

valid results.

7.3 Apparatus and Materials

7.3.1 Incubators set at 10 ± 0.5 C, 35 +

0.5 C, and 45 ^ 0.5 C (water baths recom-

mended for 10 and 45 C). Temperatures

checked with a National Bureau of Standards

thermometer or one of equivalent accuracy.

7.3.2 Pipet containers of stainless steel,

aluminum or pyrex glass for glass pipets.

7.3.3 Sterile T.D. bacteriological or Mohr

pipets, of appropriate sizes.

7.3.4 Sterile petri dishes 100 x 15 mm or

60 x 15 mm, glass or plastic.

7.3,5 Dilution bottles (milk dilution), pyrex,

99 ml volume, screw cap, with neoprene rub-

ber liners.

7.3.6 Inoculation loop, 3 mm diameter, or

needle.

7.3.7 Bunsen/Fisher burner or electric

incinerator.

7.3.8 Media

(a) Brain heart infusion (BHI) broth. (Part II-

B, 5.4.i).

(b) Brain heart infusion (BHI) agar. (Part II-B,

5.4.6).

6.5% NaCI. (Part II-B, 5.4.7).

(d) Brain heart infusion (BHI) broth, pH 9.6.

(Part II-B, 5.4.8).

(e) Brain heart infusion (BHI) broth

with 40% bile. (Part II-B, 5.4.9).

(f) Starch agar plates. (Part II-B, 5.4.10).

(g) Starch liquid medium. (Part II-B,

5.4.11).

(h) Nutrient gelatin. (Part II-B, 5.4.12).

(i) Litmus milk. (Part II-B, 5.4.13).

(j) Skim milk with 0.1% methylene blue.

(Part II-B, 5.4.14).

(k) Potassium tellurite in brain heart infu-

sion. (Part It-B, 5.4.15).

(1) Potassium tellurite in blood agar

(optional). (Part II-B, 5.4.16).

(m) Tetrazolium glucose (TG) agar or 2, 3,

5-triphenyl tetrazolium chloride (TTC) agar.

(Part II-B, 5.4.17).

(n) Blood agar with 10% blood. (Part II-B,

5.4.18).

(o) 1 % D-sorbitol solution in purple broth

base. (Part II-B, 5.1.7).

(p) 1 % glycerol in purple broth base. (Part

11-6,5.1.7).

(q) 1 % L-arabinose solution in purple broth

base. (Part II-B, 5.1.7).

(r) 1 % lactose solution in purple broth

base. (Part II-B, 5.1.8).

(s) 1 % sorbose solution in purple broth

base. (Part II-B, 5.1.8).

(t) 1% sorbose solution in purple broth

base at pH 10. (Part II-B, 5.1.8).

7.4 Procedure: Follow the schematic out-

lines in Figures lll-D-2 to 4 for the identification

of fecal streptococcal species.

7.4.1 Isolation and Confirmation of Fecal

Streptococci (Figure lll-D-2)

146

SERA MICROBIOLOGICAL MANUAL 1978

image:

(a) Pick colonies typical of fecal strepto-

cocci from the membranes or the pour plates,

and inoculate them onto BHI agar slants and

into BHI broth tubes.

(b) After 24-48 hours incubation at 35 +

0.5 C, transfer a loopful of growth from the BHI

slant to a clean glass slide, and add a few

drops of freshly-tested 3% hydrogen peroxide

to the smear. A positive control such as staphy-

lococcus and a negative control such as 5.

faecalis should be used for testing the 3%

H202.

cleaves the H202 to produce water and visible

oxygen gas bubbles. The presence of bubbles

constitutes a positive catalase test that indica-

tes non-streptococcal species. Verification

need not be continued.

(d) If the catalase test is negative, a separa-

tion of the enterococcus and Q gtoup orga-

nisms from 5. bovis, and 5. equinus can be

made by testing for growth at 10 and 45 C.

7.4.2 Separation of Enterococci and

Group Q Streptococci (Figure lll-D-2)

(a) Transfer 1 drop of the growth from the

BHI broth tube from 7.4.1 to each of 2 BHI

broth tubes.

(b) Place 1 tube in a 45±0.5 C water bath

and observe for growth (as evidenced by tur-

bidity) within 2 days. Place the other tube in a

10 + 0.5 C water bath and check for growth

within 5 days. Growth at 10 and 45 C indicates

that the culture is a potential member of the

enterococcus or Q groups. On the other hand,

S. equinus and S. bovis exhibit growth at 45 C

but not at 10 C. (See 7.4.7 for these

speciations).

7.4.3 Confirmation of Enterococcus Group

(Figure lll-D-2)

This is done by testing for growth in 6.5%

NaCI and at pH 9.6 in BHI broth and observing

for reduction of 0.1% methylene blue in milk.

Positive reactions in all cases confirm the

presence of the enterococcus group. Positive

reactions in 6.5% NaCI and pH 9.6 BHI broths

and no reaction in 0.1 % methylene blue indicate

the tentative identification of group Q.

(a) Growth Test ir^ 6.5% NaCI-BHI Broth:

Transfer 1 drop of 24 hour BHI broth culture to

a tube of BHI broth containing 6.5% sodium

chloride. Incubate at 35 + 0.5 C, and check for

growth as evidenced by turbidity within a 3-7

day period. Growth is a positive test.

(b) Growth Test ir^ BHI Broth of pH 9.6:

Transfer 1 drop of the 24 hour BHI broth cul-

ture to a tube of BHI broth adjusted to pH 9.6.

Incubate at 35 + 0.5 C, and check for growth

as evidenced by turbidity at 1, 2,3, and 7 days.

Growth is a positive test.

Skim Milk: TransfeTl drop of the 24 hour BHl

broth culture to a tube of sterile skim milk

containing 0.1% methylene blue. Positive re-

duction of methylene blue is evidenced by the

color change from blue to white.

7.4.4 Separation of Enterococcus Group

by Species (Figure lll-D-3)

The enterococci can be separated into

species as described in 7.4.4 or into groups by

original source as described in 7.4.5. The

enterococcus group can be separated into

species by observing the reduction of potassium

tellurite and 2, 3, 5-triphenyl tetrazolium chlo-

ride and the fermentation of D-sorbitol and

glycerol.

(a) Streak the 24 hour BHI broth culture

onto an agar plate containing a final concen-

tration of 0.4% potassium tellurite. Invert the

plates, incubate at 35 -± 0.5 C, and observe

the plates each day for 7 days. Colonies reduc-

ing the potassium tellurite will appear black on

this medium.

(b) Streak the 24 hour BHI broth culture

onto tetrazolium glucose agar (TG). Reduction

of tetrazolium to formazin is observed after 48

hours at 35 + 0.5 C. The degrees of reduction

are indicated as follows:

FECAL STREPTOCOCCI

147

image:

Enterococcus Group

Enterococcus, by Species

Redjction of KjTeOs.Tatrazoliu

and Fermentation of D-sorbitol

and Glycsrol

Enterocoeci by Origin

Se paration of Vegetation

Insect and Animal

Sources

Potitlv*

Hydrolysis

of Gelatin

Positive

S, ft tct Us

Sublp. liquadcians

Negative

Beta-Hemolyais

Negative

Fermentation

of L-Arabinose

Positive

S, fatcium

(See Figure lll-D-4)

Pojitlvo

S, ft act Us

Subsp, zymogtnas

Negative

S. faecalis

FIGURE lll-D-3. Identification of Fecal Streptococci, Separation of Enterococcus

Group by Species and by Original Source of Culture.

148

MICROBIOLOGICAL MANUAL 1978

image:

Strong Reduction (+4 to +3) = Red cen-

tered colony.

Moderate Reduction (+2) = Pink centered

colony.

Weak Reduction (+1) = Pale pink cen-

tered colony.

No reduction (0) = White colony.

each 24 hour BHI agar slant culture to sepa-

rate purple broth base tubes containing 1 % D-

sorbitol and 1% glycerol. Be careful not to

penetrate the agar slant and carry over agar to

the carbohydrate medium. Incubate the inocu-

lated carbohydrate media at 35 ± 0.5 C, and

note acid production over a 4 day incubation

period. A negative reaction in D-sorbitol or

glycerol shows that S. faecium is present. The

indicator will be unchanged (purple in color).

(d) Reduction of potassium tellurite (black

colonies), reduction of tetrazolium to formazan

on TTC agar and the fermentation of glycerol

and D-sorbitol indicate S, faeca/isand its varie-

ties. The determination of S. faecalis subspe-

cies is performed as follows:

(1) Stab-inoculate gelatin with a small

amount of growth from a 24 hour BHI agar

slant. Incubate the culture at 35 ± 0.5 C for

2-14 days, according to the rate of growth.

After incubation, place the tube in a cold water

bath or refrigerator 15-30 minutes to deter-

mine whether or not the gelatin will still solid-

ify. Uninoculated controls must be run in paral-

lel, especially when prolonged incubation peri-

ods are encountered. Liquefaction dictates S.

faecalis subsp. Hquefaciens, whereas solidifi-

cation indicates S. faecalis or S, faecalis

subsp. zymogenes. The latter 2 strains are

separated by their hemolysis reactions.

(2) The hemolytic properties of the fecal

streptococci are determined by streak or pour

plates. Melt blood agar base, cool and add

10% sheep blood. Inoculate streak plates or

prepare pour plates and incubate for 48 hours.

After incubation, read plates. Overnight refrig-

eration may enhance the hemolytic reactions.

Hemolysis is classified as 3 types:

Alpha-Hemolysis - Some streptococci par-

tially lyse red blood cells and reduce hemoglo-

bin to methemoglobin producing a discolor-

ation of the red blood cells. This appears as

greenish zones around the colonies.

Beta Hemolysis- Enzymes of fecal strepto-

cocci completely lyse red blood cells produc-

ing yellowish hue, or clear, colorless zone in

the blood agar surrounding the colony. S, fae-

calissubsp. zymogenes demonstrates beta he-

molysis of the blood.

Gamma-Hemolysis - Some streptococci

produce no hemolysis which is designated as

gamma hemolysis, S. faecalis is alpha or

gamma hemolytic.

It is important to note that upon serial

transfer of the culture in the laboratory, hemol-

ysis may be lost. This is especially true for S.

faecalis subsp. zymogenes where beta hemol-

ysis may not occur after serial transfer.

(e) Negative reactions in potassium tellur-

ite and tetrazolium media and failure to fer-

ment D-sorbitol and glycerol indicate S,

faecium.

(f) Transfer a small amount of growth from

the 24 hour BHI agar slant to a purple broth

base tube containing 1 % L-arabinose. Be care-

ful not to penetrate the agar slant surface and

carry over agar to the carbohydrate medium.

Incubate the inoculated carbohydrate medium

at 35 ± 0.5 C, and note acid production over a

4 day incubation period. If S. faecium is

present, the L-arabinose is fermented, acid will

be produced and the medium will turn yellow.

7.4.5 Separating Enterococci by Origin

(Vegetation, Insect and Animal Sources)

In contrast to the separation of entero-

cocci by species described in 7.4.4, the mem-

bers of the enterococcus group can be sepa-

rated according to original source of culture.

The starch hydrolysis tests separate the enter-

ococci originating on vegetation from those

typically found in insects and animals; the pep-

tonization of litmus milk test separates the

FECAL STREPTOCOCCI

149

image:

insect-origin from warm-blooded animal-

source enterococci, as shown in Figure lll-D-4.

S. faecalis from vegetation hydrolyzes

starch while 5. faecalis subsp. liquefaciens

from insects and enterococci from warm-

blooded animals do not. The starch hydrolysis

test can be performed satisfactorily with

starch agar plates or in a liquid starch tube

medium (9).

(a) Starch Hydrolysis Plate Test

(1) Pour the starch agar medium into petri

dishes and allow to solidify. With a wax pencil

divide the bottoms of the petri dishes into 6

individual areas to allow the testing of 6

isolates.

(2) Streak each isolate from a 24 hour BHI

agar slant onto one of the areas and incubate

at 35 ± 0.5 C for 24 hours.

(3) After incubation, flood the agar me-

dium with Lugol's iodine solution (Part II-C,

5.3). Streaks showing clear hydrolytic zones

with absence of the usual blue-black color of

the starch-iodine complex in the zones sur-

rounding the colonies are considered positive.

(b) Starch Hydrolysis Tube Test

(1) The starch test may also be performed

using a liquid medium instead of starch agar

plates. Inoculate tubes of sterile liquid medium

with the test organisms. After 18 hours of

Incubation at 35 ± 0.5 C, test the tubes for

starch hydrolysis using a modification of the

iodine test (8). For this modification, 0.2 ml of

2% FeCI solution and 0.2 ml of Lugol's iodine

solution are added to 5 ml of the inoculated

medium and to 5 ml of the uninoculated me-

dium (control).

(2) Hold the tubes for 3 hours at room

temperature. Compare the inoculated tubes to

the control tube. The control tube (negative

test) maintains a violet color, but positive test

cultures hydrolyze the starch and decolorize

the medium to a reddish-violet hue.

litmus milk is used to separate 5. faecalis

subsp. liquefaciens (from insect sources) from

enterococci derived from warm-blooded ani-

mal sources.

(1) Transfer 1 drop of the isolate growing

in a 24 hour BHI broth culture to a tube of

sterile litmus milk.

(2) Incubate the tube at 35 ± 0.5 C, and

observe at 1,2,3 and 7 days.

(3) A positive peptonization includes liqui-

faction and clearing of the milk with the devel-

opment of a brownish color. Peptonization in-

dicates S. faecalis subsp. liquefaciens. Color

changes without clearing, or coagulation are

negative reactions and indicate enterococci

from warm-blooded animal sources are

present.

7.4.6 Identification

Streptococci

of Group Q

The group Q Streptococci are initially sep-

arated from the Enterococcus Group and tenta-

tively identified by growth in 6.5% NaCI in BHI

and in BHI broth at pH 9,6, but no growth in

0.1 % methylene blue in milk. See Figure III-D-

Other pertinent physiological characteris-

tics of Group Q Streptococci are: fermentation

of sorbose, growth in sorbose medium at pH

10, but no growth in tellurite medium and

negative hydrolysis of starch and gelatin. The

tests may be carried out as follows:

(a) Inoculate a sorbose fermentation tube

with a small amount of growth from an isolate

on a 24 hour BHI slant. Be careful not to

penetrate the agar slant surface and carry over

agar to the carbohydrate medium. Incubate

the carbohydrate medium at 35 ± 0.5 C and

note acid production over a 4 day incubation

period. Sorbose fermentation is indicated by

acid production and change in the medium

colorfrom purple to yellow,

(b) Transfer a small amount of growth from

the 24 hour BHI slant to the 1 % sorbose me-

150

MICROBIOLOGICAL MANUAL 1978

image:

Separation of

Enterococcus Group

by Original Source,

Vegetation, Insect

and Animal Sources

Starch Hydrolysis

Positive

Negative

Peptonization of

Litmus Milk

Positive

Negative

Atypical

S. faecalis

(Vegetation sources)

S. faecalis

Subsp. liquefaciens

(Insect Sources)

Enterococci

(Warm-blooded

Animal Sources)

FIGURE lll-D-4. Identification of Fecal Streptococci, Separation of Enterococci from

Vegetation, Insect and Animal Sources.

FECAL STREPTOCOCCI

151

image:

dium (described in Part II-B, 5.1.8 above) which

has been adjusted to pH 10 with sterile 38%

sodium phosphate solution (see Part II-B,

5.4.8). Incubate the inoculated medium at 35

± 0.5 C for at least 4 days. Growth indicates a

positive test.

BHl slant into BHI broth containing 0.04%

potassium tellurite and incubate at 35 ± 0.5 C

for 7 days. Group Q organisms do not grow on

this medium.

(d) Stab-inoculate gelatin with a small

amount of growth from the 24 hour BHI slant.

Incubate the culture at 35 ± 0.5 C for 2-14

days, according to the rate of growth. After

incubation, place the tube in a cold water bath

or refrigerator for 15-30 minutes to determine

whether or not the gelatin will solidify. Unino-

culated control must be done in parallel, espe-

cially when prolonged incubation periods are

used.

(e) Group Q organisms do not hydrolyze

starch. Perform starch hydrolysis test as in

7.4.5 (a) or (b) above.

(0 Streptococcus avium sp. constitute

Lancefield's Group Q. Consequently, Group Q

antiserum may be used in the precipitin test to

provide further identification. However, the Q

antigen is not demonstrable in all strains.

therefore, identification in those cases will de-

pend solely on physiological characteristics.

7.4.7 Separation and Speciation of 5. bo-

vis and S. equinus

(a) 5. bovis and S. equinuswere separated

from the Enterococci and Group Q Strepto-

cocci by growth at 45 C but no growth at 10 C

(see Figure lll-D-2). 5. bow's and S. equinus can

be separated by the lactose fermentation test

in which 5. bovisproduces acid and 5. equinus

produces no change,(c)lbelow.

(b) Starch Hydrolysis Test: Perform starch

hydrolysis test as in 7.4.5 (a)or(b).

Positive starch hydrolysis test confirms 5.

bovis and S. equinus.

tiate between 5. bovis and S. equinus by lac-

tose fermentation, transfer a small amount of

growth from the 24 hour BHI agar slant to a

purple broth base tube containing 1 % lactose

and an inverted fermentation tube. Do not pen-

etrate the agar slant surface and carry over

agar to the carbohydrate medium. Incubate

the inoculated carbohydrate medium at 35 ±

0.5 C and observe the reaction over a 4 day

incubation period. 5. bovis gives an acid reac-

tion only; 5. equinus shows no change.

152

MICROBIOLOGICAL MANUAL 1978

image:

REFERENCES

1. Nowlan, Sandra and R, H, Diebel, 1967. Group Q streptococci I. Ecology, serology, physiology

and relationship to established enterococci. J. Bacteriol. 94, (2):291.

2. Kenner, B. A., H. F. Clark and P, W. Kabler, 1960. Fecal streptococci. II. Quantification of

streptococci in feces. Am. J. Public Health. 50:1553.

3. Kenner, B. A., H. F. Clark and P. W. Kabler, 1961. Fecal streptococci. I. Cultivation and

enumeration of streptococci in surface water. Appl. Mlcrobiol. 9:15.

4, Pavlova, M. T., F. T. Brezenski and W. Litsky, 1972. Evaluation of various media for isolation,

enumeration and identification of fecal streptococci from natural sources. Health Lab. Scj. 9:289.

5. Clausen, E. M., B. L Green and W. Litsky, 1977. Fecal Streptococci: Indicators of Pollution, pp.

247-264. In: A. W. Hoadley and B. J. Dutka, Eds., Bacterial Indicators/Health Hazards Associated

with Water, ASTM STP635, American Society for Testing and Materials, Philadelphia, PA.

6. Prescott, S. C., C-E. A. Winslow and M. H. McCrady, 1946. Water Bacteriology. (6th ed.) John

Wiley and Sons, Inc., p. 46-50.

7. Geldreieh, E. E., H. F. Clark and C. B. Huff, 1964. A study of pollution indicators in a waste

stabilization pond. J. WPCF, 36 (11): 1372.

8. Geldreieh, E. E., 1976. Fecal coliform and fecal streptococcus density relationships in waste

discharges and receiving waters. Irr, CRC Critical Reviews in Environmental Control, p. 349.

9. Pavlova, M. T., W. Litsky and F. J. Francis, 1971. A comparative study of starch hydrolysis by fecal

streptococci employing plate and tube techniques. Health Lab. Sci. 8:67.

FECAL STREPTOCOCCI 153

image:

PART Hi. ANALYTICAL METHODOLOGY

Section E Salmonella

Recommended methods are presented for

recovery of Salmonella from water and waste-

water and their subsequent identification. The

methods are particularly useful for recreational

and shellfish-harvesting waters. No single

method of recovery and identification of these

organisms from waters and wastewaters is

appropriate for all sampling situations. The

method selected depends on the character-

istics of the sample and the microbiologist's

experience with the techniques. Multiple

option techniques are described for sample

concentration, enrichment, isolation and

identification. The Section is divided as

follows:

1. The Genus, Salmonella

2. Methods for Concentration of

Salmonella

3. Primary Enrichment for

Salmonella

4. Isolation of Salmonella

5, Biochemical Identification of

Salmonella

6. Serolog ica I Test f or Salmonella

7. Quantitative Techniques

8, Optional Fluorescent Antibody

Screening Technique

1. The Genus, Salmonella

1.1 Definition

The genus Salmonella is comprised of a

large number of serologically related, gram-

negative, nonspore-forming bacilli that are

0.4-0.6 urn in width * 1-3 /um in length, and

which occasionally form short filaments. They

are motile with peritrichous flagella or are non-

motile. Ordinarily salmonellae do not ferment

lactose, sucrose, malonate or salicin but do

ferment numerous carbohydrates including

glucose, inositol and dulcitol. These bacteria

are positive for lysine and ornithinedecarboxy-

lase and negative for urease and phenylalanine

deaminase. Usually they produce hydrogen

sulfide and do not liquify gelatin. All of the

known Salmonella species are pathogenic for

warm-blooded animals, including man. They

cause enteritis, (via contaminated water, food

or food products) enteric fevers and are found

in reptiles, amphibians and mammals. Edwards

and Ewing have published an authoritative

.work on the isolation and characterization of

Salmonella (1).

In Bergey's 8th edition (2), the salmonellae

have been reclassified tentatively into 4 sub-

genera containing II subdivisions. However,

the problem of Kaufman's listing of manysero-

types, and the lack of agreement as to what

constitutes the genus Salmonella or its spe-

cies, leaves the taxonomy in a fluid state.

1.2 Identification Schemes

A comprehensive scheme of the recom-

mended isolation, detection and identification

154

MICROBIOLOGICAL MANUAL 1978

image:

methods is outlined in sequence in Figure III-E-

When space and equipment are limited,

the number of options at each stage depicted

in Figure lll-E-1 can be reduced and salmonel-

lae isolated successfully. One such simplified

scheme is outlined in Figure ill-E-2.

The procedures outlined in these schema

are described in the following subsections

2-8.

2. Methods for Concentration of Salmonella

The initial steps for detection of salmonel-

lae in water and wastewater require concen-

tration of the organisms by one of several

methods: the gauze swab, diatomaceous

earth, the cartridge filter, or the membrane

filter technique. The volume of sample tested

is directly related to the level of pollution.

2.1 Swab Technique Modified After

Moore's Method (3)

2.1.1 Summary: In this method sterile

gauze swabs are immersed for about 5 days

just below the surface of a water or wastewa-

ter. After the exposure period during which

bacterial concentration occurs, the gauze

swabs are retrieved, placed in sterile bags,

iced and returned to the laboratory for exami-

nation. The swab, portions of the swab, or the

expressed liquid from the swab are added to

enrichment media for selective growth of sal-

monellae and suppression of coliforms and

other non-salmonellae.

2.1.2 Scope and Application

(a) Advantages: The gauze swab technique

is superior to grab sampling, because salmo-

nellae concentration occurs in the swab

permitting improved detection. Although this

technique is not quantitative, it has proved

effective in detection of low numbers of salmo-

neilae in waters and wastewater. The tech-

nique is simple and inexpensive.

{b) Limitations: This is not a quantitative

procedure, since some salmonellae may pass

through the swab, others may desorb from the

swab during the exposure period, and the

water volume sampled is unknown. It is not

possible to predict the salmonellae concentra-

tion in,the water or wastewater from the con-

centration in the swab nor does the procedure

reflect changes or cycling of salmonellae con-

centrations at the sample site.

2.1.3 Apparatus and Materials

(a) Cheesecloth roll, 23 cm wide.

(b) Paper cutter or large pair of shears.

(d) Sterile 250 ml flasks or jars', screw-cap,

containing enrichment media. Part III-E, 3,3.10.

(e) Sterile plastic bags (e.g., Whirl-pak or

heavy-duty food freezer bags),

(f) Insulated container with ice (optional).

2.1.4 Procedure

(a) Prepare a swab from a length of cheese-

cloth 180 cm long * 23 cm.wide by folding

the length 5 times to form a pad 36 cm * 23 cm.

Cut the folds at one end. From this end, cut

the pad into 5 parallel strips, 4.5 cm wide and

26 cm long, leaving an uncut top section of

10 cm (see Figure'lll-E-3).

(b) Bind the top of the gauze swab with 16

gauge wire to form a mop-head shape, with the

strips hanging free (see Figure III-E-4).

autoclave.

(d) Place the swab just below the surface

of the water or wastewater to be examined and

secure with the wire to a solid support.

(e) Leave the swab in place for about 5

days.

SALMONELLA

155

image:

SAMPLE

CONCENTRATION OF SAMPLE

Gauze

Swab

Diatomaceous

Earth Plug

Cartridge

Filter

Membrane

Filter

ENRICHMENT

Dulclto!

Selenite

Broth

Selenite

Broth

Tetrathionate

Broth

Tetrathionate

Brilliant

Green Broth

FA Screening Option

ISOLATION PLATING

Xylose Lysine

Desoxycholate

Agar

Xylose Lysine

Brilliant

Green Agar

Brilliant

Green Agar

Bismuth

Sulfite

Agar

BIOCHEMICAL IDENTIFICATION

(Primary Screening)

Triple Sugar

Iron Agar

Urea

Agar

Lysine

Iron Agar

Cytochrome

Oxidase Test

ADDITIONAL BIOCHEMICAL IDENTIFICATION

(Minimal Biochemical Set by Single Tube or Multitest Systems)

Lysine

Decarboxylase

Broth

Tryptophane

Broth

Malonate

Broth

Phenylalanine

Deaminase

Broth

Phenol Red

Dulcitol

Broth

SEROLOGICAL VERIFICATION

Polyvalent 0

Polyvalent H

Confirmation by a

Reference Laboratory

FIGURE lll-E-1. Schemeforthe Concentration, Isolation and Identification of Salmonella.

156

MICROBIOLOGICAL MANUAL 1978

image:

SAMPLE

CONCENTRATION OF SAMPLE

ENRICHMENT

Dulcitol

Selenite

Broth

Tetrathionate

Broth

ISOLATION PLATING

Xylose Lysine

Desoxycholate

Agar

Brilliant

Green

Agar

BIOCHEMICAL IDENTIFICATION

(Primary Screening)

Triple Sugar

Iron Agar

Urea

Agar

ADDITIONAL BIOCHEMICAL IDENTIFICATION

(Multitest System)

SEROLOGICAL VERIFICATION

Polyvalent O

FIGURE lll-E-2. Simplified Scheme for Concentration, Isolation and Identification

of Salmonella.

SALMONELLA

157

image:

23 cm

FIGURE lll-E-3. Dimensions of the Gauze Swabs.

158

MICROBIOLOGICAL MANUAL 1978

image:

16, GAUGE WIRE

FINGERS

OF SWAB

FIGURE III-E-4. The Gauze Swab in Position.

SALMONELLA

159

image:

(f) After the exposure period, retrieve the

swab and place it directly into a sterile plastic

bag (Whirl-Pale), ice and return to the labora-

tory for examination. See enrichment step

3.4.1. Alternatively, the swabs may be placed

in enrichment media and incubated on-site,

then iced and returned to the central

laboratory.

2.2 Diatomaceous Earth Technique

2.2.1 Summary: The filtering action of dia-

tomaceous earth is used to concentrate the

organisms (4). Diatomaceous earth is loosely

packed on top of an absorbent pad in the

funnel of an assembled membrane filtration

unit One to two liters of a water sample are

passed through the diatomaceous earth using

vacuum and portions of the diatomaceous

earth plug are added aseptically to enrichment

broth.

(e) Safety trap flask between the filtering

flask and the vacuum source.

(f) Sterile spatula.

(g) Forceps.

(h) Sterile graduated cylinders, 1000 ml

size, covered with aluminum foil or kraft paper.

(i) Containers for glass pipets.

0) Sterile absorbent pads of cellulosic pa-

per, 47 mm in diameter.

(k) Sterile T.D., 10 ml Mohr pipets, glass or

plastic.

(1) Rinse water, sterile phosphate buffered

water, prepared in large volumes. See Part II-B,

7 for preparation.

2.2.2 Scope and Application

2.2.4 Procedure

(a) Advantages: Although it is not enumer-

ative, this method is quantitative in the sense

that known volumes of water or wastewater

are filtered through the diatomaceous earth.

(b) Limitations: The diatomaceous earth

filter Is easily clogged with the suspended

material found in turbid waters. This slows the

filtering process but may not prevent its use.

Also salmonellae may pass through due to

improper formation of the diatomaceous filter.

^•2.3 Apparatus and Materials

(a) Diatomaceous earth (Johns-Manville's

"Celite" or equivalent).

(b) Sterile membrane filter unit (filter base

and funnel), plastic, glass or stainless steel,

wrapped with aluminum foil or kraft paper.

(d) Vacuum flask, 2-liter, to hold the filter

base, with appropriate tubing. An alternative is

a commercially-available manifold.

(a) Assemble the membrane filter unit, sub-

stituting an absorbent pad for the membrane

filter.

(b) With the funnel in place, loosely pack

approximately 2.5 cm thickness of diatoma-

ceous earth on top of the absorbent pad.

a 10 ml pipet to saturate the diatomaceous

earth. Draw water through filter under vacuum

(d) Filter 1 liter or more of the sample

under vacuum. The volume of sample filtered

will depend upon the estimated amount of

pollution in the water or wastewater.

(e) Rinse the funnel at least twice after

sample filtration with 20-30 ml of the rinse

water.

(f) Remove the funnel. Add the absorbent

pad, the diatomaceous earth plug or halves

of each to flasks of selected enrichment broths,

See the enrichment step 3.4.2.

160

&EPA MICROBIOLOGICAL MANUAL 1978

image:

2,3 Membrane Filtration of Samples

2-3-1 Summary: The water or wastewater

sample is passed under positive pressure

through a 0.45 urn pore, 142 mm diameter

filter in a pressure filtration unit. After filtra-

tion, the membrane filter is aseptically divided

into portions that are added to enrichment

broths.

2.3.2 Scope and Application

(a) Advantages: Membrane filtration is

used when the sample volume desired is larger

than the 1-2 liters employed with the diatoma-

ceous earth technique, when the 5 day sample

period for the swab technique is not feasible,

or when the sample is not too turbid to permit

passage of the desired volume. The method is

useful for water of very low organic and panic-

ulate matter content. It is quantitative in that it

retains all cells from the filtration of a known

volume of water or wastewater.

^k) Limitations: Use of this technique is

somewhat limited because the sample may

clog pores and prevent filtration. Also, the limi-

tations of membrane filtration cited in Part II-C,

3.2.2, are applicable.

2.3.3 Apparatus and Materials

(a) Membrane filter holder, stainless steel,

142 mm, autoclavable for use in pressure fil-

tration. (Gelman Disc, Filter Holder, 11872,

Millipore SS Filter Holder, YY22 14200, or

equivalent).

(b) Dispensing pressure vessel, 10/12 liter

size. (Gelman 15108, Millipore XX67 00003,

or equivalent).

high pressure necessary for pressure filtration

(7 kg/cm2 maximum pressure).

(d) Forceps, with smooth tips.

(e) Sterile shears to divide membrane.

(f) Dilution water, phosphate buffered, pre-

pared in large volumes.

(g) Membrane filter, white, grid-marked,

142 mm in diameter with 0.45 + 0.02 pn

pore size.

(h) Prefilter (Millipore AP15-142-50 or

equivalent).

2.3.4 Procedure

(a) Aseptically add the sterile membrane

filter to the sterile filter holder.

(b) Add a 2-20 liter sample to the dispens-

ing pressure vessel. The sample size is limited

by the level of solids or turbidity in the sample.

of the membrane filter.

(d) Using positive pressure, force the sam-

ple through the membrane filter.

(e) After filtration, the membrane and pre-

filters are added to enrichment media. See

enrichment step 3.4.3.

2.4 Cartridge Filter Technique (5)

2.4.1 Summary: The water or wastewater

sample is drawn under negative pressure

through a filter of borosilicate glass microfi-

bers bonded with epoxyHresin. The volume

filtered may be measured. After filtration, the

filter is placed aseptically into enrichment

broth.

2.4.2 Scope and Application

(a) Advantages: Because this technique

can be used to filter 20 liters or more of sam-

ple, it is applicable to waters with low concen-

trations of organisms. It can be combined with

an MPN procedure for a quantitative estimate

of bacterial density.

(b) Limitations: The presence of high num-

bers of background organisms may make re-

covery of Salmonella very difficult by this tech-

nique. The autoclave must be sufficiently large

to hold sample reservoir containers. As with

other filtration procedures, high turbidity in the

sample lengthens filtration time.

SALMONELLA

161

image:

2.4.3 Apparatus and Materials

(a) Vacuum purnp, capable of operation at

a pressure differential up to 69 kN/m2.

(b) Vacuum gauge.

with at least a 5-liter capacity) closed by a 2-

holed rubber stopper, with 2 short pieces of

glass tubing inserted.

(d) Glass and rubber vacuum tubing, with

shut-off valve.

(e) Optional for MPN technique: manifold,

capable of 5 simultaneous filtrations.

(f) Sterile, 20 liter, heavy-walled pyrex

glass carboy, calibrated from 1-20 liters (Corn-

Ing CWG No. 434490).

(g) Balston (Lexington, MA) type AA car-

tridge filter, 2.5 x 6.4 cm or equivalent and a

type 90 filter holder. Filters, filter holders and

tubing are wrapped and sterilized by autoclave

at 121 C for 15 minutes.

2,4.4 Procedure

(a) Connect the parts of the filtration appa-

ratus as depicted in Figure lll-E-5.

(b) Collect or transfer water sample to ster-

ile, calibrated container.

container. Protect container from contamina-

tion by sealing glass tubing in neck of con-

tainer with sterile cotton and aluminum foil.

(d) Start vacuum pump and filter desired

volume of water. To avoid rupture of filter, the

pressure differential should not exceed 69

kN/m2.

(e) After filtering sample, aseptically re-

move filter and filter holder. Separate filter and

place in appropriate enrichment medium. See

3.4.4.

(f) Place another sterile filter in the filter

holder and repeat filtration procedure.

(g) When a new sample is to be filtered,

replace glass tubing, filter, filter holder and

sample container with new sterile units.

3. Primary Enrichment for Salmonella

Selenite and tetrathionate broths are used

for primary enrichment. The selenite may be

combined with dulcito! to improve the selectiv-

ity for salmonellae. The tetrathionate may be

used as a broth base alone or combined with

brilliant green dye which enhances its selectiv-

ity for salmonellae other than S. typhi.

3.1 Summary: After concentration of sal-

monellae, the gauze swabs, diatomaceous

earth plugs, membrane filters or cartridge fil-

ters are placed in flasks of selenite, tetrathion-

ate or other selected broth. The broth encour-

ages salmonellae to grow white inhibiting

other bacteria. Multiple flasks of the enrich-

ment broth are incubated for 3-4 days at se-

lected temperatures. After incubation, cultures

are streaked onto differential plating media for

salmonellae isolation. These differential plates

should be streaked from the enrichment media

every 24 hours over a 3-4 day period.

3.2 Scope and Application

3.2.1 Advantages: The enrichment me-

thods described provide an optimal environ-

ment for salmonellae and other enteric patho-

gens, and to some extent suppress the other

organisms that are present.

3.2.2 Limitations: Although enrichment

broths provide an optimal environment for sal-

monellae development, recovery is not quanti-

tative.

3.3 Apparatus and Materials

3.3.1 Incubators set at 35 ± 0.5 C, 41.5 ±

0.5 C, and optionally at 37 ± 0.5 and 43 ± 0.5

3.3.2 Sterile shears and spatula.

3.3.3 Sterile forceps.

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MICROBIOLOGICAL MANUAL 1978

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3.3.4 Sterile beakers, 500 ml size covered

with aluminum foil or kraft paper,

3.3.5 Sterile erlenmeyer flasks, 125 ml

size, to hold 50 ml of enrichment broth.

3.3.6 Bunsen, Fisher burner or electric

incinerator.

3.3,7 Sterile petri dish.

3.3.8 Sterile aluminum foil.

3.3.9 Alcohol, 95% ethanol, in a vial

3.3.10 Media

(a) Selenite broth (Difco 0275, BBL

11608, or equivalent), (See Part II-B, 5.5,1).

(b) Tetrathionate broth (Difco 0104, BBL

11706, or equivalent). (See Part II-B, 5.5.2).

5.5.3).

(d) Tetrathionate brilliant green broth

(same as (b) above with the addition of 0.01

gram of brilliant green per liter of medium).

(See Part II-B, 5,5.4).

3.4 Procedures for Enrichment

Select at least two enrichment media,

preferably one selenite and one tetrathionate

type, for each sample, The actual choice of

medium is based on the experience of the

analyst.

Selenite and tetrathionate enrichment

broths are useful for all Salmonella including

S. typhi. Although the addition of brilliant

green to tetrathionate broth base increases its

selectivity for salmonellae, it inhibits the recov-

ery of S, typhi. It is reported that tetrathionate

broth is toxic to salmonellae at a temperature

of 43 C (6). Dulcitol selenite medium may not

completely recover S. typhi, S. cholerae-suis,

S. enteritidis bioserotypes Paratyphi A and

Pullorum, from some samples because these

species ferment dulcitol slowly (7).

3.4.1 The Gauze Swab: Squeeze the gauze

swab in the plastic bag to express the liquid

from the swab into a sterile 500 ml beaker.

After this, the entire swab or portions of it may

be placed directly into the enrichment broth

using sterile forceps and shears to divide the

swab if necessary into the number of portions

required for the enrichment media to be used.

For example, 2 enrichment media incubated at

2 temperatures, 35 C and 43 C, would require

4 portions. The pieces of swab and measured

volumes of the liquid can be apportioned into

125 ml flasks containing 50 ml of enrichment

broth made double strength to compensate for

the dilution effect of liquid.

3-4.2 The Diatomaceous Earth Plug: Re-

move the diatomaceous earth plug from the

membrane filtration funnel by carefully tap-

ping the funnel on a sheet of sterile metal foil.

Tap the sides of the funnel and shake it gently

to dislodge the plug. Transfer the entire plug to

an enrichment broth or divide the plug into

halves using a sterile spatula. Add one half of

the plug to each 125 ml flask of medium used.

Inoculate at least 2 media in flasks containing

50 ml of sterile single-strength broth. Incubate

at 2 temperatures. Halve the absorbent pad

with sterile shears, and add a portion to each

of the previously inoculated flasks.

3.4.3 The Membrane'Filter: Remove the

membrane filter aseptically from the filter base

with sterile forceps, and hold over the bottom

of a sterile petri dish. Cut the membrane in

quarters with the sterile shears, and let the

quarters fall grid-side up into the petri dish

bottom. Insert aseptically each quarter of a

membrane filter into a 125 ml flask containing

50 ml of single-strength medium.

3-4.4 The Cartridge Filter: Remove the car-

tridge filter from the holder aseptically and

place in the selected enrichment medium.

More than I filter may be used in succession on

a single sample and placed in the same culture

medium. Repeat for the second enrichment

medium.

3.4.5 After inoculation in 3.4.1, 3.4.2,

3.4.3 or 3.4.4, incubate enrichment flasks at

35 C, 41.5 C and other selected temperatures

SALMONELLA

163

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for at least 24 hours. However, some salmonel-

lae are slow-growing and recovery may be

increased by incubating for successive 24-

hour periods up to 96 hours before streaking

on isolation agars,

3.4.6 If a sample must be collected in an

area some distance from the central laboratory

responsible for Salmonella identifications, the

scheme can be interrupted at different points.

First, the samples can be concentrated, iced

and transported back to the central laboratory.

Second, the samples can be concentrated,

placed in enrichment media, incubated at the

selected temperature for 18-24 hours at a field

laboratory then iced and transported to the

central laboratory.

3.5 Incubation Temperature as a Selec-

tion Technique for Salmonellae

3.5,1 Historically 37 C has been used for

the isolation of enteric microorganisms be-

cause it Is representative of the gut tempera-

ture of humans and other warm-blooded

animals. This temperature was considered

optimal for the detection and isolation of

these enterics.

3.5.2 Some workers have used 35 C in

place of 37 C because the latter temperature is

close to the upper tolerance limit for the group

and might prevent the growth of some desired

species. This inhibitory effect would be most

significant if the 37 C temperature were not

well-controlled and might be exceeded by 2-3 C.

Consequently, double temperatures of 35 C and

37 C came into common usage (1, 8) for isola-

tion and identification of Enterobacteriaceae.

3.5.3 Because the large populations of

normal gut microorganisms interfere with the

Isolation of pathogens such as Salmonella

from humans and other warm-blooded ani-

mals, temperatures above 37 C were proposed

to reduce the background microorganisms.

3.5.4 The 43 C temperature was proposed

as an aid to the isolation of Salmonella, partic-

ularly from various heavily contaminated ma-

terials (9, 10). The 41.5 C temperature has

been recommended by others for the detec-

tion and isolation of Salmonella from water

(11, 12, 13),

3.5.5 Although investigators have used

different elevated temperatures, the results

are inconclusive for a single incubation tem-

perature. The general conclusion of most stud-

ies on comparison of recoveries of salmonel-

lae at different incubation temperatures is that

a greater number of isolates and more species

are isolated at 41-43 C than at 35-37 C (9,

14-19). This manual recommends two temper-

atures in enrichment and isolation procedures

for Salmonella, 35 C and 41.5 C.

Optional Fluorescent Antibody Screening

Technique

The FA technique is a rapid screening me-

thod that can be used after primary enrich-

ment but it is tentative and optional. The de-

tailed procedure Is described in Part III-E, 8

following the conventional methods for detec-

tion and identification of Salmonella,

4. Isolation of Salmonella

4.1 Summary: The organisms that de-

velop in the primary enrichment broth media

are isolated and differentiated on solid media.

The enriched cultures are streaked every 24

hours for 3-4 days onto the surface of XLD,

BG, XLBG and bismuth sulfite media. After

incubation, the plates are examined for typical

colonies of salmonellae which are picked and

characterized biochemically and serologically.

4.2 Scope and Application

4.2.1 Advantages: Pure cultures of Salmo-

nella can be isolated by the careful selection

and use of plating media and incubation tem-

peratures. Brilliant green agar is favored for

the development and identification of salmo-

nellae except for S. typhi and a few other

species whereas bismuth sulfite agar allows

the growth of most Salmonella including S.

typhi.

4.2.2 Limitations: Bacteria other than sal-

monellae may compete with the salmonellae

164

©EPA MICROBIOLOGICAL MANUAL 1978

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on the secondary differential media, thus inter-

fering with their isolation and identification.

The use of brilliant green agar at an elevated

temperature of 41.5 C reduces the number of

interfering organisms, but also inhibits devel-

opment of some serotypes of Salmonella.

4.3 Apparatus and Materials

4.3.1 incubators set at 35 ± 0.5 C, 41.5 ±

0.5 C, and optionally at 37 ± 0.5 C and 43 ±

0.5 C.

4.3.2 Water bath set at 44-46 C for tem-

pering agar.

4.3.3 Petri dish canisters for glass petri

dishes.

4.3.4 Thermometer certified by National

Bureau of Standards or one of equivalent

accuracy.

4.3.5 Inoculating needle and 3 mm loop.

4.3.6 Colony Counter, Quebec darkfield

model orequivalent.

4.3.7 Bunsen/Fisher burner, or electric

incinerator.

4.3.8 Sterile 100 mm x 15 mm petri

dishes, glass or plastic.

4.3.9 Sterile phosphate buffered or

peptone dilution water in bottles, 99 ± 2 ml

volumes (see Part II-B, 7 for preparation).

4.4 Media: The following agar media are

dispensed in bulk quantities in screw-capped

bottles or flasks. (See Part II-B, 4 for

preparation).

4.4.1 Xylose lysine desoxycholate (XLD)

agar(see Part II-B, 5.5.7).

4.4.2 Brilliant green (BG) agar (see Part II-

8,5.5.5).

4.4.3 Xylose lysine brilliant green (XLBG)

agar (see Part II-B; 5.5.6).

4.4.4 Bismuth sulfite agar (see Part II-B,

5.5.8).

4.S Procedure

4.5.1 Prepare two selected media {XLD,

BG, XLBG or bismuth sulfite agars) in petri

dishes. As a minimum xylose lysine desoxy-

cholate (XLD) and brilliant green (BG) or xylose

lysine brilliant green (XLBG) agars are recom-

mended. Bismuth sulfite agar permits the pre-

sumptive detection of S. typhi and/or S.

enteriditis.

4.5.2 Streak the surface of a previously

poured and solidified agar with a loopful of the

enrichment culture.

4.5.3 Duplicate plates should be streaked

from each enrichment culture every 24 hours

for 3-4 days.

4.5.4 Inoculate duplicate plates from each

enrichment culture and incubate, one each, at

35 C and 41.5 C (and optionally at 37 C and 43

C). Incubate the XLD and XLBG plates for 24

hours, and the BG agar and bismuth sulfite

agar plates for 48 hours.

4.5.5 After incubation, examine plates for

colony appearance. Table lll-E-1 describes the

appearance of colonies on XLD, XLBG, BG and

bismuth sulfite agars. The salmonellae colo-

nies on BG agar are pinkish white with a red

background. Lactose fermenters will form

greenish colonies or other colorations. Occa-

sionally, slow lactose fermenters such as Pro-

teus, Citrobacter, Pseudomonas and Aeromo-

nas mimic Salmonella.

4.5.6 Pick growth from the centers of well-

isolated colonies that have the characteristic

appearance of salmonellae, and streak onto

the screening media described in This Section,

5.4.1 and 5.5.1. Isolated, single colonies from

a plate where all colonies appear alike may be

assumed to be pure. At least 2 colonies of each

type suspected to be Salmonella should be

picked.

4.5.7 The suspected colonies of Salmo-

nella should now be characterized by the sin-

SALMONELLA

165

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TABLE III-E-1

Colonial Appearance of Salmonella and Other Enterics on Isolation Media

Colony Appearance

Salmonella

Other Enterics

1, Bismuth Sulfite Agar

Round jet black colonies

with or without sheen

5. typhi

S. enteritidis ser

Enteritidis

S, enteritidis ser

Schottmuelleri

Flat or slightly raised

green colonies

S. enteritidis ser

Typhimurium

S. enteritidis bioser

Paratyphyi

5. choleraa-suis

Proteus spp.

2. Brilliant Green Agar

Slightly pink-white,

opaque colonies surrounded

by brilliant red medium

Salmonella spp

Yellow-green colonies

surrounded by yellow-green

zone

Escherichia, Klebsiella,

Proteus spp. (lactose or

sucrose fermenters)

3. XLD or XLBG Agar

Red, black centered colonies

Red colonies

Yellow colonies

Salmonella spp.

Shigella spp.

Escherichia spp.

and biotypes

Citrobacter spp.

Klebsiella spp.

Enterobacter spp.

Proteus spp.

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MICROBIOLOGICAL MANUAL 1978

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gle biochemical tests or muititests in Partlll-E,

5.6. An 0-1 bacteriophage screening test may

also be used on the isolate for a rapid (4-5

hours) determination of the tentative identifi-

cation of Salmonella, Results must be verified

(20,21).

5. Biochemical Identification of Salmonella

5,1 Summary: Salmonellae can be identi-

fied to genus by determining their reactions in a

series of biochemical tests. These tests can be

made in single tube media or in commercial

multitest systems. This Section offers the mini-

mal required set of tests, a series of additional,

optional tests and a brief description of available

multitest systems. These tests require specific

training for valid results.

5.2 Scope and Application

5.2.1 Advantages: The biochemical reac-

tions characterize the Salmonella-like isolate,

permit a separation from closely related bacte-

ria, and provide presumptive identification as

Salmonella. Confirmed identification requires

additional serological tests.

The multitest systems have a number of

advantages over single tube media prepared

in the laboratory:

(a) The different .designs of these systems

permit the user to select tests to fit his needs,

facilities and budget.

(b) The multitest systems may be used with

confidence in the laboratory or in field situa-

tions as rapid and convenient screening me-

thods for cultures suspected to be Salmonella.

Large numbers of cultures can be examined in

a relatively short time; and those tentatively

identified as Salmonella can then be held for

later serological confirmation.

minimal space requirements, immediate avail-

ability, and economy when compared with the

preparation and use of tubed media. They are

ideally suited to field work and to small labora-

tories that do not routinely perform these iden-

tification tests.

(d) The systems offer these same advan-

tages for the identification and differentation of

other enteric bacteria such as E. cofi, Klebsiella,

Enterobacter, Citrobacter and Shigella.

5.2.2 Limitations: Whether accomplished

by individual tube or multitest methods, bio-

chemical identification of large numbers of

cultures is expensive and time-consuming. It

should not be attempted independently with-

out previous training and experience in read-

ing reactions and interpreting results.

5.3 Apparatus and Materials

5.3.1 Incubator set at 35 ±0.5 C.

5.3.2 Bunsen/Fisher burner, or electric

incinerator.

5.3.3 Inoculating needle and loop.

5.3.4 Culture tubes, 100 * 13 or 150 x

20 mm.

5.3.5 Fermentation tubes, 75 X 1Omm.

5.3.6 Closures to fit culture tubes.

5.3.7 Thermometer certified by National

Bureau of Standards, or one of equivalent

accuracy.

5.4 Media

6.4.1 Screening Media

(a) Triple Sugar Iron Agar (TSI) (Difco

0265, BBL 1 1749). See Part II-B, 5.5.9.

(b) Lysine Iron Agar (LIA) (Difco 0849, BBL

11363). See Part II-B, 5.5.10.

See Part II-B, 5.5.16,

(d) Urea Agar Base Concentrate (Difco

0284). See Part II-B, 5.5.11.

SALMONELLA

167

image:

(e) Cytochrome Oxidase Test Reagents

5.4.3 Optional Biochemical Tests

Reagent A. Weigh out 1 gram alpha-

napthol and dissolve in 100 ml of 95%

ethanol.

Reagent B. Weigh out 1 gram of para-

aminodimethylaniline HCI (or oxylate) and dis-

solve in 100 ml of laboratory pure water. Pre-

pare frequently and store in refrigerator.

5.4.2 Minimal Biochemical Set

(a) Phenylalanine Agar {Difco 0745, BBL

11537). See Part 11-8,5.6.12.

(b) Ferric chloride reagent: Prepare a 10%

solution (w/v) of ferric chloride in laboratory

pure water. Store in a brown bottle in the

refrigerator.

11-8,6.1.9.

(1) Tryptone (Difco 0123).

(2)Tryptiease(BBL 11921).

(3) Indole Test Reagent: Dissolve 5 grams

paradimethylamino benzaldehyde in 75 ml iso-

amyl or normal amyl alcohol, ACS grade.

Slowly add 25 ml cone HCI. The reagent

should be yellow and have a pH below 6.0; if

the final reagent Js dark in color it should be

discarded. Examine the reagent carefully dur-

ing preparation because some brands are not

satisfactory after aging. Both amyl alcohol and

benzaldehyde should be purchased in a small

amount consistent with the volume of work

anticipated. Refrigerate the reagent in a glass-

stoppered bottle.

There has been some difficulty in obtain-

ing amyl alcohol. If this problem occurs, Gillies

describes an alternate paper strip test for in-

dole production (22).

(d)Malonate Broth, Modified (Difco 0569,

BBL 11436). See Part II-B, 5.5.13.

(a) Carbohydrate Utilization

Purple Broth Base (Difco 0092, BBL

11506). See Part II-B, 5.1.7 containing:

Duicitol(Difco0162),

Lactose (Difco 015 6), or

lnositol(Difco0164)

(b) Decarboxylase Activity

Decarboxylase Medium Base (Difco

0872). See Part II-B, 5.5.14 containing:

Lysine HCI (Difco 0705),

Arginine HCi (Difco 0583), or

Ornithine HCI (Difco 0293)

(1) Monosodium Phosphate Solution

Dissolve 6.9 grams NaH2Po4.HOH in 45

ml of laboratory pure water. Add 3 ml of 30%

NaOH and adjust pH to 7.0. Bring to 50 ml with

laboratory pure water and store in refrigerator.

(2) ONPG Solution

Dissolve 80 grams of o-nitrophenyl-6-D-

galactopyranoside (ONPG) in 15 ml pure water

at 37 C. Add 5 ml of 1 M NaH2PO4 from (1)

above. This 0.75 M solution of ONPG should

be colorless. Store in a refrigerator. A portion

of the buffered solution sufficient for the num-

ber of tests to be done should be warmed to 37

C before use,

5.4.4 Multitest Systems (optional to Sin-

gle Test Series)

(a) API Enteric 20 (Analytab Products, Inc.).

(b) Enterotube (Roche Diagnostics).

Wilson Pharmaceutical and Chemical Corp.)

(d) Minitek (Baltimore Biological Labora-

tories, Bioquest).

168

MICROBIOLOGICAL MANUAL 1978

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(e) Pathotec Test Strips (General Diagnos-

tics Division of Warner-Lambert Company).

(f) r/b Enteric Differential System

(Diagnostic Research, inc.)

5.5 Procedures: Since cultures are found

which react atypically, an isolate should not be

eliminated because of a single anomalous re-

action, rather the biochemical reactions

should be considered as a group. For example,

LIA tubes not showing H2S production, but

having alkaline slants and alkaline butts may

be atypical S. typhi. These tubes should be

retained for further characterization.

Further, to insure that the media are yield-

ing proper reactions, the analyst is urged to

incorporate both positive and negative control

cultures into Single Test and Multitest

Procedures.

5.5.1 Screening Tests: Pick growth from

the center of a single isolated colony on a

selective plating medium and inoculate into

the primary screening medium.

Fermentation tube reaction code for TSI

and LIAAgars:

Report slant/butt where K, A and N

indicate alkaline, acid and neutral reactions

respectively; G, g indicate large and small

amounts of gas production, respectively; and

H2S 1+ to 4+ indicate levels of blackening

due to hydrogen sulfide production. For

example, K/Ag is an alkaline slant and an acid

butt with a small amount of gas.

* (a) Triple Sugar Iron Agar

(1) Inoculate by stabbing the butt and

streaking the slant.

(2) Incubate at 35 C for 18-24 hours with

cap loose.

(3) Read and record reactions. Color of

slant or butt is yellow for an acid reaction or

red for an alkaline reading. Gas production is

evidenced by bubbles in the medium, and H2S

production by blackening of the medium.

(4) Typical reactions:

Salmonella: K/Ag with H2S, 1 + to 4+.

S. typhi: K/A with H2S, trace to 1 +.

Citrobacter: K/Ag or A/Ag with H2S, 1 +

to3 + .

(5) Atypical reactions: TSI tubes showing

alkaline slants and acid butts without H2S

production should be inoculated into Motility

Sulfide Medium to verify the negative H2S

reaction. If still H2S negative, perform serolog-

ical testing to confirm an atypical Salmonella.

• (b) Lysine Iron Agar

(1) Inoculate by stabbing the butt twice

and streaking the slant.

(2) Incubate for 18-24 hours, and if nega-

tive for an additional 24 hours, at 35 C.

(3) Read and record reactions. The slant or

butt is yellow from an acid reaction and

blue/purple for an alkaline reading. Gas

production is evidenced by bubbles in the me-

dium and H2S production by blackening of the

medium along the stab line. Proteus has a

distinctive red slant caused by oxidative deam-

ination and a yellow butt.

(4) Typical reactions:

Salmonella: K/K or K/N with H2S +(-).

S. typhi: K/K with H2S -(+).

Citrobacter: K/A with H2S - or +.

Proteus; R(red)/A with H2S-{+).

* (c) Urea Agar (Christensen)

(1) Inoculate slant only, using a heavy

inoculum.

(2) Incubate for 18-24 hours at 35 C with

cap loose. A positive reaction for Proteus may

be recorded in 2-4 hours, but all negative tests

at 2-4 hours must be held for 18-24 hours.

(3) Reactions are red for urease positive

and yellow for urease negative. Salmonella

give negative urease reactions. Cultures of

SALMONELLA

169

image:

Citrobacter may yield weak delayed positive

reactions at 18-24 hours.

* (d)CytochromeOxidaseTest

The cytochrome (indophenol) oxidase test

can be done with prepared paper strips or the

following test on a nutrient agar slant:

(1) Inoculate nutrient agar slant and incu-

bate at 35 C for 18-24 hours. Older cultures

should not be used.

(2) Prepare reagents as in 5.4.1 (e).

(3) Add 2-3 drops of reagent A and reag-

ent B to the slant, tilt to mix and read reaction

within two minutes.

(4) A strong positive reaction (blue color

slant or paper strip) occurs in 30 seconds.

Ignore weak reactions that occur after two

minutes. Pseudomonads, aeromonads and

vibrios are positive. Salmonella is negative.

5.5.2 Minimal Biochemical Set: After read-

ing the TSI reaction, use growth from the slant

to inoculate the minimal biochemical set (in

5.4.3 above) with a straight wire needle. Suffi-

cient culture to inoculate all of the minimal

biochemical media is provided by one applica-

tion of the tip of the needle to the TSI growth.

» (a)PhenylalanineAgar

(1) Inoculate surface of slant heavily.

(2) Incubate for 18-24 hours at 35 C with

cap loose. A positive reaction for Proteus may

be recorded in 4 hours but negative tests must

be held for 18-24 hours.

(3) Test for phenylalanine deaminase by

allowing 4-5 drops of a 10% solution of ferric

chloride to run down over the growth on the

slant.

(4) A dark green color on the agar slant

and in the fluid is a positive reaction. A yellow

or brown color is negative. Salmonella and

Citrobacter give negative reactions.

* (b)lndoleTest

(1) Inoculate the tryptophane broth lightly

from the TSI agarslant culture.

(2) Incubate the broth at 35 + 0.5 C for 24

± 2 hours, with cap loose.

(3) Add 0.2-0.3 ml indole test reagent to

the culture, shake and allow the mixture to

stand for 10 minutes.

(4) Observe and record the results.

(5) A dark red color in the amyl alcohol

layer on top of the culture is a positive indole

test; the original yellow color of the reagent is

a negative test.

(6) With rare exceptions, Salmonella and

Citrobacter are indole-negative,

» (c) M a Ion ate B roth Test

(1) Inoculate from the 18-24 hours TSI

agar slant culture.

(2) Incubate for 48 hours at 35 C. Observe

tubes after 24 and 48 hours. Positive reactions

are indicated by a change in color of the me-

dium from green to a deep blue. Lots of malo-

nate medium should be checked with positive

and negative cultures.

(3) Salmonella arizonae and some strains

of Citrobacter utilize malonate. Other Salmo-

nella do not.

• (d) Fermentation of Dulcitol in Phenol Red

Broth Base

(1) Inoculate the Dulcitol broth lightly us-

ing a 24-hour culture.

(2) Incubate at 35 C and examine daily for

7 days.

(3) A positive reaction is production of acid

with yellow color.

(4) Most salmonellae and some Citrobac-

ter utilize dulcitol. Some that do not use it or

170

MICROBIOLOGICAL MANUAL 1978

image:

use it slowly include: S. typhi, S. cholerae-suis,

S, enteritidis bioser Paratyphi A and Pullorum,

and S. enterf/cf/sserTyphimurum.

5.5.3 Optional Biochemical Tests: If the

minimal set of biochemical tests has not satis-

factorily identified cultures as Salmonella, or

variable reactions have been observed, pro-

ceed with the following optional tests.

• (a) Fermentation of Lactose in Phenol Red

Broth and Inositol in Phenol Red Broth

(1} Inoculate the broth lightly using a 24-

hour culture.

(2) Incubate at 35 C and examine daily for

7 days.

(3) A positive reaction is production of acid

and yellow color with or without gas

production.

(4) Salmonella do not utilize lactose, but

some strains of Citrobacterdo.

Some Salmonella do utilize inositol.

• (b) Decarboxylase tests (lysine, arginine

and ornithine)

(1) The complete decarboxylase test series

requires tubes of each of the amino acids and a

control tube containing no amino acids.

(2) Inoculate each tube lightly.

(3) Add sufficient sterile mineral oil to the

broth to make 3-4 mm layer on the surface

and tighten the screw cap.

(4) Incubate for 18-24 hours at 35 C.

Negative reactions should be reincubated up

to 4 days.

(5) Positive reactions are deep purple and

negative reactions, remain yellow. Read the

control tube without amino acid first; it must

be yellow for the reactions of the other tubes ta

be valid. Positive purple tubes must have

growth as evidence by turbidity because

uninoculated tubes are also purple, nonfer-

menters may remain alkaline throughout

incubation.

(6) Salmonella are positive for lysine; posi-

tive or delayed positive for arginine; and posi-

tive for ornithine. S. typhi and bioser Gallina-

rum are negative for ornithine. Cltrobacterand

bioser Paratyphi A are negative for lysine.

Strains are variable for arginine and ornithine.

• (c) Gelatin Liquefaction

(1) Inoculate nutrient gelatin tube by

stabbing.

(2) Incubate at 35 C for 5 days.

(3) Cool tubes to 20 C and inspect. Failure

to solidify is a positive reaction.

(4) All Group I Enterobacteriaceae are

negative, except for S. arizonae which shows a

delayed positive reaction (see Table lll-E-5),

« (d) ONPG Test,

galoctopyranoside (23)

o-nitrophenyl-3-D-

(1) Emulsify a large loopful of growth from

each culture in 0.25 ml of physiological saline

in a 10 x 75 mm fermentation tube.

(2) Add one drop of toluene to each tube

and shake well. Let tubes stand for 5 minutes

in 35 C water bath.

(3) Add 0.25 ml of buffered O.75 M ONPG

solution to each tube (see 5:4.3, (c) (1)) and

incubate again in 35 C water bath.

(4) Read tubes at ¥2,. 1 and 24 hours. A

positive result is development of yellow color.

Lactose fermenters have P galactosidase so

Citrobacter are positive and most Salmonella

negative.

» (e> Motility Test: A test for motility is used

in serology but is not recommended in the

biochemical reactions because:

(1) Most members of the family Enterobac-

teriaceae are motile, so the test would not add

much to a characterization of the isolate.

SALMONELLA

171

image:

(2) A negative test cannot be considered

conclusive until the culture is passed once or

twice through a motility or broth medium and

the isolate retested.

5.6 Multitest Systems: Multitest systems

are available which use tubes containing pre-

pared agar media, plastic units containing de-

hydrated media, media-impregnated discs and

reagent-impregnated paper strips. Some of

the systems use numerical codes to aid identi-

fication of bacteria. Others provide computer-

ized identification of bacteria. A number of

independent investigators have compared one

or more multitest systems with conventional

biochemical tests. Some of the earlier systems

have been improved. Most of the recent stud-

ies report the correct identification of high

percentages of isolates (24-29).

5.6.1 The following systems for the identi-

fication of Enterobacteriaceae are commer-

cially available. These have been quality tested

by the manufacturers and others and can be

used with confidence.

<a) API Enteric 20 consists of 20 small

chambers (called cupules) in a plastic strip,

each containing dehydrated medium. An iso-

lated colony is used to prepare a cell suspen-

sion to inoculate the media. The inoculated

media are incubated for 18 hours at 35 C in a

special plastic chamber. A numerical identifi-

cation system based on thousands of reaction

combinations is available. The identification

system is updated periodically. Computer serv-

ices may be obtained which are more compre-

hensive and accurate than the manual system

(27,28).

(b) Improved Enterotube with 8 compart-

ments of agar media in a single plastic tube

provides tests for 11 biochemical reactions.

The media are inoculated by touching one end

of a wire to an isolated colony and drawing the

wire containing the inoculum through the me-

dia. The Enterotube is incubated for 18-24

hours at 35 C. A manual numerical identifica-

tion aid, ENCISE, is part of the system.

comprised of a test card unit containing 10

reagent-filled capillary chambers. A single iso-

lated colony is picked into broth and cultured

for 3Vz hours at 35 C. After incubation, the

broth tube is centrifuged, the cells resus-

pended in water and inoculated into each cap-

illary chamber on the card. Each card is incu-

bated for 3 hours at 35 C in its own plastic

container. Isolates can be identified in 7 hours

unless additional tests are required. A numeri-

cal binary code named Var-ident is part of the

system.

(d)The r/b Enteric Differential System con-

sists of 4 Beckford tubes, 2 basic and 2 ex-

pander tubes. The 4 tubes contain agar media

and are constricted to form upper and lower

compartments which provide 14 biochemical

reactions. The tubes are stabbed, streaked and

incubated at 35 C for 24 hours. A color chart of

typical tube reactions is provided for

identification.

(e) The Pathotec Test Strips are individual

biochemical paper strips impregnated with

reagents that test for enzymes or end products

characteristic of certain bacteria. A cell sus-

pension prepared from isolated colonies is

used as the inoculum to demonstrate the bio-

chemical reactions. An incubation period of 4

hours at 35 C may be required for the cell

suspension. The earlier Rapid I-D System has

been discontinued. A new identification sys-

tem for enterics is planned for marketing in

1978-1979.

(f) The Minitek Microorganism Differentia-

tion System consists of 10 impregnated discs

that test for 12 biochemical reactions. A single

colony suspended in special broth serves as

the inoculum. The 10 basic discs are part of 34

discs and 37 reactions offered to identify aero-

bic and anaerobic bacteria. The inoculated

discs are contained in a plastic tray within a

humidor and incubated at 35 C for 18-24

hours. Accessory equipment required to proc-

ess the discs includes plastic inoculum plates,

a 10-disc dispenser, a special pipetter, pipet

tips, a pipet tips organizer, an incubation humi-

dor, a color comparator card set and inoculum

broth. The system stresses the biochemical

options and flexibility of the system. A flow

diagram is provided for identifications.

172

MICROBIOLOGICAL MANUAL 1978

image:

5.6.2 Factors for Selection: The 6 multi-

test systems briefly described above were

designed for the biochemical identification of

members of the family, Enterobacteriaceae.

Most of the isolates suspected as Salmonella

could be identified by any of the multitest

systems. Some of the factors that should be

considered in selecting a multitest system are:

(a) Biochemical Reactions h^ the Multitest

System: Since analysts working with Salmo-

nella develop a series of tests that yield good

results forthem, they should consider a system

which fits their preferred test pattern.

(b) Need to Identify Atypical Salmonella:

Because it is important for the laboratory to

identify typical and atypical Sa/mone/fairom a

series of samples, systems that use numerical

identification should be selected (API, Entero-

tube, and Inolex). Further, systems containing

the most tests have a better chance for identifi-

cation of typical and atypical Salmonella. Only

one system, API, requires no additional tests.

time span required to identify typical

Salmonella vanes with the system as shown in

Table lll-E-2.

(d) Refrigerated Storage: Refrigerated

space is required for some systems. This can

be a problem in purchase of a large supply.

Table lll-E-3 shows the reported shelf-life of the

multitest systems, with and without

refrigeration.

(e) Purchase of Special Equipment: Some

of the test systems contain all materials and

equipment necessary to do the analyses.

Others require purchase of special items of

equipment for full use. For example, Inolex

requires a small centrifuge; API can be sight-

read or can utilize a profile register for easy,

rapid identification of atypical and typical

strains; Minitek uses a starter kit which

includes special plates, broth, pipetter, pipet

tips, color comparator card set, incubation

humidor, pipet tips, organizer and dispenser.

(f) Safety Considerations: The probability

of laboratory-acquired infections is directly

proportional to the amount and number of

exposures to pathogens. Some multitest sys-

tems are more dangerous to handle than oth-

ers because they require more opening, clos-

ing and manipulating of the test container

which may pose added hazards to the worker.

Some systems such as Enterotube and r/b use

direct colony picks for reactions. API, Inolex,

Pathotec and Minitek require culturing and

additional handling of cell suspensions which

are greater hazards.

(g) Cost and Source of Multitest Systems:

The per unit cost of the multitests varies with

the system, as shown in Table lll-E-4.

5.7 Biochemical Characteristics of

Enterobacteriaceae: The analyst may wish to

differentiate salmonellae from the other

Enterobacteriaceae by applying additional

biochemical tests. Table lll-E-5 is a chart of

these biochemical reactions.

5.8 Serological Verification: The analyst

should understand that completion of the bio-

chemical tests does not yield identification of

Salmonella. The cultures that have been bio-

chemically confirmed should be verified

serologically.

6. Serological Testing for Salmonella

6.1 Summary: Serological typing of Sal-

monella strains is done by using slide-

agglutination for somatic (0) antigens and tube

testing for flagellar (H) antigens.

6.2 Scope and Application: Serological

testing completes the identification of Salmo-

nella. It is the only testing which identifies to

serotype and bioserotype levels. However, se-

rological testing is an expensive, complex pro-

cedure that should be carried out only by

trained personnel.

6.3 Apparatus and Materials

6.3.1 Small inverted fluorescent lamp.

6.3.2 Incubator set at 35 + 0.5 C, and a

water bath set at 50 C.

SALMONELLA

173

image:

TABLE lll-E-2

Production Rate and Time Requirements of Multitest Systems*

Multitest

System

API

Enterotube

Inotex

r/b Diff.

Pathotec

Minitek

Analyst's Time

Per Culture in Min.

Time Span of

Test in Hours

18-22

18-24

Cultures

Per Day Per Analyst

100

10-15

20-30

'Based on experience in EMSL-Cincinnati.

TABLE lll-E-3

Reported Shelf-Life of Multitest Systems With or Without Refrigeration

System

API

Inolsx

Pathotoc

Enterotube

r/b

Minitek

Refrigeration No

Required Refrigeration*

1 2 months —

— 12 months

3 years** 12 months

7 months —

6-12 months —

2 years —

'Store in cool, dark place, ambient temperature.

"Refrigeration not required, but will extend the shelf-life.

174

<*EFV\ MICROBIOLOGICAL MANUAL 1978

image:

TABLE lll-E-4

Cost and Source of Multitest Systems1

Multitest

System

Cost

per Unit

Cost

per Box

Address of

Manufacturer

API Enteric 20

Inolex

r/b Enteric Differ-

ential System

Pathotec Test

Strips

Mlnitek

$2.052 $51.25 (25/box)

Improved Enterotube $2.16

$0.91

$1.90

3 tubes/

set

$0.20

$54.00 (GSA) {25/box}

$22.80 (25/box)

.$38.60 (20 sets/box)

$20.00 (100 tests/

box)

$1.803 $90.00 (50 tests/kit)

Analytab Products, Inc.

200 Express Street

Plainview, NY 11803

Roche Diagnostics

Div. of Hoffmann-La Roche, Inc.

Nutley, NJ 07110

Inolex Biomedical Division

Inolex Corporation

3 Science Road

Glenwood, IL 60425

Diagnostic Research, Inc.

25 Lumber Road

Roslyn, Long Island, NY 11576

Warner-Lambert Company

General Diagnostics Division

Morris Plains, NJ O795O

Baltimore Biological Laboratories

Cockeysville, MD 21030

1. As of October, 1977.

2. Plus $99.OO/year, if the numerical identification system. Analytical Profile

Inolex Service, is used.

3. Requires one time purchase of accessories for $94.10.

SALMONELLA

175

image:

TABLE lll-E-5

Biochemical Characteristics of the Enterobacteriaceae

Reaction

Catalaso

Oxidise

8-Q»!»closid«sc

Gas Irom Glucose

»I35C

KCN (growth on)

Mucate (»cid)

NiDate reduction

Carbohydrates:

(acid produclion)

AdomtcH

Arabinose

Outeitol

Esculin

Inosllol

Lactose

Maltose

Manmtol

Satlcin

Sorbilol

Sucrose

Trehalose

Xylose

Related C sources:

Ciliato

Gluconato

Malonate

cT-Tartrala

VP,

Protein reactions;

Arginine

Gelatin hydrolysis

H}S Jrom TSI

Indote

Lyilno

docarboxylaie

Orntthme

Uie* hydrolysis

Glutimic icld

Phenylatanlne

Group 1

' 1

—

••-or*

—

•f

•~

Eetwardstella

—

CItrobacter

—

+ or x

—

(*)

—

! Salmonella

—

-S

•c

—

- •

—

- D

—

Group II

Klebsie/la

—

(d)

—

Enterobacter

(4-)

—

(d)

- or x

—

Serratia

—

Group III

Proteus

—

- •

Group IV

Yersinia

—

*D * diltofonl rtactions given by different species of a genus; d = different reactions given by different strains of a species or serotype;

X » late end irregularly positive (mutative).

From Sergey's Manual of Determinative Bacteriology, Eighth Ed. (2)

176

MICROBIOLOGICAL MANUAL 1978

image:

6.3.3 Bunsen/Fisher burner or electric

incinerator.

6.3.4 Inoculating loop (3 mm) and needle.

6.3.5 McFarland Barium Sulfate Standard

#10 (Difco 0691).

6.3.6 Test tubes, 150 x 25 or 150 x 20

mm and 100 x 13mm.

6.3.7 Glass microscope slides, 5.0 x 7.6

cm (2" x 3") cleaned to remove grease and oil.

6.3.8 Salmonella Q Antiserum Poly (Difco

2264, BBL 40707, Sylvana 27-108A or

equivalent).

6.3.9 Salmonella Vi Antiserum {Sylvana

27-106A, BBL 40708, Difco 2827, or

equivalent).

6.3.10 Salmonella 0 Antisera Set A-l

{Difco 2892) includes 1 vial each of Groups, A,

B, C1f C2, D, E1( Ev E2, E4, F, G, H and I; Poly A-l

and Vi.

Salmonella Grouping Serum Kit {BBL

40709) includes one vial each of Groups A, B,

C!, C2, D and E Polyvalent and Vi.

6.3.11 Salmonella H Antiserum (BBL

407-99, Difco 2406-47 or equivalent).

6.3.12 Capillary pipets with rubber bulb.

6.4 Media

6.4.1 Brain heart infusion (BHI) broth {BBL

11058, Difco 0027 or .equivalent) {Part ll-B,

5.4.5).

6.4.2 Brain heart infusion (BHI) agar slant

(BBL 11064, Difco 0418 or equivalent) (Part II-

8,5.4.6),

6.4.3 Motility medium {Difco 0869, BBL

11436, or equivalent) (Part ll-B, 5.5.16).

6.4.4 H-broth (Difco 0451, BBL 11289, or

equivalent) (Part ll-B, 5.5.17).

6.4.5 Blood agar base (without blood)

(Difco 0045-02, BBL 11036, or equivalent)

(Part ll-B, 5.4.18).

6.4.6 Nutrient agar {Difco 0001-02, BBL

11471, or equivalent) (Part II-D, 5.1.1).

6.4.7 Phenolized saline (0.6 grams phenol

in 100 ml of 0.5-0.85% NaCI solution).

6.4,8 Formalinized saline {0.6 ml formalin

in 100 ml of 0.5-0.85% NaCI solution).

6.5 Procedure: as described in Figure III-E-

1, there are serological procedures for the 3

antigen groups, O, Vi and H. However, it is

usually necessary only to test for the O and Vi

antigens for verification of the Salmonella

identification.

6.5,1 Sljde Agglutination Test for 0^

Grouping (30)

(a) Prepare a dense suspension of organ-

isms from a fresh 24 hour BHI slant in 0,5 ml

of phenolized saline solution. The suspension

should be homogeneous and at least as

concentrated as that of McFarland Barium

Sulfate Standard #10, which corresponds to

3 x 1Q9 cells/ml.

(b) Mark rectangular areas on an alcohol-

cleaned glass slide with a wax pencil. Mark

heavy continuous lines to prevent flow of sus-

pension from one section to another as shown

in the example below. For safety, it is recom-

mended that the outside perimeter be in-

scribed with a wax line to prevent flow off the

edge of the slide. Note that slide sections 1, 2

and 3 are controls and section 4 is the one

complete test:

1 1

• i

1 1

!',

SALMONELLA

177

image:

Section 1 -Add antiserum alone.

Section 2 - Combine antiserum and

0.85% Nad.

Section 3 - Combine bacterial suspension

and 0,85% NaCI.

Section 4 - Combine bacterial suspen-

sion, 0.85% NaCI and antiserum.

For large numbers of cultures, the 2x3

Inch (50 x 75 cm) glass slide can be used to

accommodate 12 or more sections per slide in

2'rows. It is recommended that only one cul-

ture be tested at one time against the series of

sera to avoid premature drying. As in the fol-

lowing example:

tu O

K ^

isidsns

nnno

1 1

f I

1 1

antiserum near the top of a rectangle and a

drop of saline near the bottom.

(d) Using a wire loop, suspend a sufficient

amount of growth from an isolated colony or a

BHI slant into the drop of saline to produce a

milky suspension.

(e) Mix the suspension with the antiserum,

using the wire loop. Make a long, narrow track

ratherthan a circular pool.

(f) Flame loop well between uses to pre-

vent cross-reactions from contamination of

sera.

(g) Mix the antigen and antiserum further

by tilting the slide back and forth until aggluti-

nation (or clumping) is apparent. If agglutina-

tion is not evident or if it is weak at the end of 1

minute, consider the reaction negative. Com-

pare reactions with controls. As a QC function,

test antisera against cultures of known reac-

tions, monthly or as indicated.

(h) If the polyvalent test is positive, test the

culture with Salmonella 0 groups A, B, C, D

and E antisera. Kits containing additional 0

group antisera are described in 6.3.

6.5.2 Slide Agglutination Test for Vi Anti-

gen (S. typhi) (30): Occasionally S. lyphTwltt be

isolated without the capsule-like Vi antigen.

The procedure described in 6.5.1 will identify

it as Salmonella, O group D, If the biochemical

reactions are characteristic of S. typhi, record

the results presumptively as S. typhi, no Vi

antigen detected and in this case, check other

colonies from same plate for Vi antigen. If Vi

antigen is present, it will block O agglutination

and the procedure described in 6.5.1 will not

identify the serogroup. If S. typn/ls suspected,

proceed as follows:

(a) To three rectangles on a glass slide,

add respectively:

(1) Polyvalent Salmonella antiserum.

(2) Group D 'SalmonellaQ antiserum.

(3) Vi antiserum.

(b) Mix with antigen as in procedure 6.5.1.

(1) No agglutination in any rectangle - not

a common Salmonella,

(2) Agglutination in polyvalent antiserum

only - possibly a Salmonella other than Group

D. Check antigen in other 0 groups.

(3) Agglutination in Vi antiserum only or in

Vi and weakly in polyvalent - presumptive S.

typhi.

178

MICROBIOLOGICAL MANUAL 1978

image:

(d) Follow-up on reactions (3) immediately

above:

(1) Make a heavy suspension of antigen in

0.5 ml of phenolized saline.

(2) Heat the suspension for 1 5 minutes in a

boiling water bath.

(3) Cool and retest suspension in the

above three antisera. Include the three con-

trols as in 6.5.1 (b).

(4) Compare results of slide agglutination

obtained with live and heated antigens. S. ty-

phi will give the following reactions:

Antiserum

Antigen

Live Heated

Polyvalent Salmonella

0 group D

+ or -

6.5.3 Alternative Procedure for Salmo-

nella, Including S. typhi: Edwards & Ewing and

Douglas & Washington describe this alternate

procedure as a more rapid serological method

(1,30).

(a) Slide test the organism for agglutina-

tion in polyvalent 0, Vi, and all of the common

O group antisera at the same time. When indic-

ated, heat antigen as described in 6.5.2 (d).

(b) Interpret results as outlined in 6.5.1

and 6.5.2.

6.5.4 Tube Test for H Antigen (1): Before

tests are made for H (flagellar) antigen, test for

motility by inoculating the pure cultures into

Motility Test Medium (5.5.1 6), incubating and

reading. If the tests are negative, transfer the

cultures again through the motility medium

before performing the flagellar antigen test.

Motility medium in large diameter tubes or

small petri dishes may be inoculated on one

side and motile descendants picked on the

other side after 24 hours. The test procedure

follows:

(a) Inoculate H-broth from an 18-24 hour

pure culture and incubate 18 hours at 30 C or

with heavy inoculum in 35 C water bath for 4

hours.

(b) Dilute the 18 hour culture 1:1 with

formalinized saline and mix. Allow to stand at

room temperature for 1 hour.

to small test tubes (13 x 100 mm), 1

tube/antiserum.

(d) Prepare dilution of polyvalent H antis-

era according to directions of manufacturer

(usually 1:1000). Add 0.5 ml to each tube

using a fresh pipet for each tube. Mix by pipet-

ting the solution up and down several times.

(e) Incubate tubes for 1 hour in 50 C water

bath. If the H-antigen is present,

flocculation/agglutination may occur in 5-10

minutes but wjll occur within 1 hour. The posi-

tive test is indicated by a diffused, fluffy sedi-

ment in the bottom of the tube. A negative

reaction gives a tight "button-like" group of

cells in the bottom of the tube.

6.5.5 Confirmation of Salmonellaio sero-

type by an official typing center or state health

laboratory is recommended when required for

tracing sources of contamination, for enforce-

ment or for other Agency actions. This service

is usually available if cultures are of public

health significance. See II-C, 6 for details on,

proper shipment procedures.

7. Quantitative Techniques

7.1 Summary: These quantitative me-

thods are time-consuming, but necessary

when it is desirable to determine salmonellae

densities in recreational waters, shellfish-

raising waters, stormwater run-off, wastewa-

ters and sludges.

7.1.1 Samples are concentrated if neces-

sary by the standard techniques described in

this Section.

SALMONELLA

179

image:

7.1.2 Samples or sample concentrates are

Inoculated into enrichment media in the stan-

dard 5-tube, 3-dilution sequence of the MPN.

See Part II-C.

7.1.3 After incubation, each tube culture

is streaked on selective plating media.

7.1.4 Colonies reacting as Salmonella are

picked and confirmed biochemically or

serologically.

7.1.5 Based on the confirmation above,

the number of tubes confirmed as positive for

Salmonella are tabulated and the MPN index

calculated from the regular MPN table.

7.2 Concentration by MPN (15)

The need for concentration and sample

volumes required vary with the anticipated

salmonellae densities. High density samples

such as sewage may be inoculated directly as

10 ml portions into double-strength, or as 1 ml

or less portions into single strength

enrichment media in MPN tubes (15). Low

density samples may be concentrated by MF,

dlatomaceous earth, or cartridge filters before

analysis by the MPN procedure.

7.3 Concentration by Membrane Filter

or Diatomaceous Earth Filter (12,31, 32,33)

Another quantitative technique uses either

a large MF (142 mm diameter, 0.45 nm pore)

with filter aid (12) or diatomaceous earth with

a support pad. One liter or larger sample is

either mixed with 1%Celiteandfilteredthrough

an MF or is filtered through the diatomaceous

earth and pad. After filtering, the Celite/MF

or diatomaceous plug and pad are placed in

a sterile 1 pint (473 ml) blender jar containing

100 ml sterile 0.1% peptone water and blended

at about 5000 RPM for 1 minute on a Waring

blender (see Part ll-C, 1.3). Beginning with

10 ml of the homogenate, serial tenfold

dilutions (5 replicates per dilution) are inocu-

lated into enrichment media. Verification tests

are done on each positive enrichment tube

and the MPN is calculated from the number of

tubes which verify.

7.4 Concentration by Cartridge Filter (5)

The cartridge filter is particularly useful for

concentrating large sample volumes (5). The

sample is placed in a sterile, calibrated

container and measured volumes (e.g. 10, 1

and 0.1 liters) are passed through separate

filters as described in this Section, 2,4. To

speed the analyses, 5 replicate portions are

filtered simultaneously through 5 cartridge

filters using a manifold. If turbidity is a

problem, successive filters may be used. The

filters are removed aseptically and placed in

the selected enrichment media. The

concentration procedure is repeated for each

sample volume. Verification tests are done on

each enrichment culture and the MPN is

calculated from the positive verification tests.

8. Optional Fluorescent Antibody Screening

Technique

The fluorescent antibody (FA) technique

may be used as a rapid screening method for

the detection of salmonellae particularly from

large numbers of samples, after primary

enrichment cultures (18-24 hours). Because

this technique does not require pure cultures

or serological typing, positive FA results should

be confirmed by the isolation procedures

described in this Section, 4.5. If the FA test

is negative, the salmonellae detection pro-

cedure may be terminated.

8.1 Summary: An optional FA screening

technique concentrates bacteria from a water

sample in diatomaceous earth ( Celite' or

equivalent). See this Section, 2.2. The diato-

maceous earth with the entrapped bacteria is

added to enrichment broth. After incubation

of the broth, loopfuls of enrichment culture

are transferred to selective plating media

to produce spot cultures on the agar sur-

faces. After the plates are incubated for 3

hours, slide impression smears are prepared

from the micro-colonies and stained with a

Salmonella polyvalent antiserum labeled with

fluorescent dye. Fluorescent cells indicate a

positive reaction and the possible presence of

Salmonella.

180

MICROBIOLOGICAL MANUAL 1978

image:

8.2 Scope and Application

8.2.1 Advantages: Rapid screening of

large numbers of samples for Salmonella

eliminates negative samples from further

testing.

8.2.2 Limitations: Careful interpretation of

fluorescence is critical for this technique but

difficult to attain. Positive FA results must be

confirmed by the conventional cultural

techniques described above (see Part III-E, 4, 5

and 6). The cost of equipment for fluorescence

microscopy is approximately $5,000.

8.3 Apparatus and Materials

8.3.1 Equipment

(a) Light microscope and low

autofluorescence optics suitable for UV

microscopy.

(b) Condenser, Cardioid Dark-Field, oil

immersion objective 95x, with iris diaphragm

and with a numerical aperture (N.A.) at least

0.05 less than N.A. of objective lens.

Heat Filters - BG22, KG-1, KG-2 or

equivalent.

Excitation Light Filters - UG1 and BG1 2 or

equivalent.

Barrier Filters - GG-9 and OG1 or

equivalent.

(d) Intense Light System: Fluorescence

illuminator with power source, voltage

regulator and mercury arc, quartz iodide or

tungsten light source.

(e) Incubator set at 35 C + 0.5 C.

8.3.2 Materials

(a) Non-drying low fluorescence immer-

sion oil.

(b) Fluorescent antibody pre-cleaned

micro slides, 2.5 X 7.6cm.

thick.

(d) FA Kirkpatrick fixative (Difco 3188, or

equivalent).

(e) Petri dishes, 100 x 15 mm, pyrex

glass.

(f) Nail polish.

(g) Phosphate buffered saline, pH 8.0. Add

10 grams of dry FA buffer (Difco 2314-33, or

equivalent) to 1 liter of fresh laboratory pure

water. Dissolve completely and adjust pH to

8.0 with NaOH.

(h) FA mounting fluid (Difco 2329-57, or

equivalent), reagent grade glycerine adjusted

to pH of 9.0-9.6.

(i) Laboratory pure water.

(j) Methanol orethanol, 95%,forsterilizing

forceps.

(k) Staining assembly consisting of jar,

cover and slide rack with handle. At least 5 are

needed: I each for fixative, ethanol and

laboratory pure water and 2 for buffered saline

rinses.

(I) FA Salmonella polyvalent conjugate is

a fluorescein conjugated anti-salmonellae

globulin (Difco 3187, 3185; Sylvana

27-1OOA, or equivalent).

When the conjugate is rehydrated, pre-

pare 0.2-0.3 ml aliquots and freeze for future

use. Rehydrated conjugates stored in the re-

frigerator are not stable. (See 8.4.5 fortitration

of conjugates).

(m) Moist chamber used to hold slides

containing conjugated-stained smears. A sim-

ple chamber consists of a culture dish bottom

(150 x 20 mm) placed over wet toweling. A

larger dish for this purpose may be prepared

by placing moist towels onto a flat tray, then

SALMONELLA

181

image:

placing the slides face-up on this surface and

covering the tray with an inverted glass baking

pan orsimilar metal pan.

8.3.3 Media

(a) Brain heart infusion (BHI) broth (Difco

0038, BBL 11059, or equivalent). See Part II-B,

5.4.5..

(b) Brilliant green agar (BG) (BBL 11073,

Difco 0285-01). See Part II-B, 5.5.5.

(BBL 11838, Difco 0788-01). See Part II-B,

5.5.7.

(d) Xylose lysine brilliant green agar

(XLBG) XL agar base (BBL 1 1836, Difco 0555).

See Part II-B, 5.5.6.

8.4 FA Staining Techniques (13, 34, 35)

8.4.1 Preparation

Collect samples and concentrate them by

cartridge filter, membrane filter or diato-

maceous earth technique as described in this

Section, 2.2, 2.3 and 2.4.

After concentration, place whole or half

plugs and pads, cartridge filters or membrane

filters into flasks of selenite broth, tetrathionate

broth, dulcitol selenite broth or tetrathionate

brilliant green broth. Incubate flasks for

18-24 hours at 35 and 41.5 C. See this

Section, 3.

8.4.2 Spot Culture Plates: After the primary

enrichment has been incubated 18-24 hours,

prepare spot culture plates on the differential

media: brilliant green (BG) agar, xylose lysine

desoxycholate (XLD) agar, and xylose lysine

brilliant green (XLBG) agar as follows:

(a) Mark the bottom of the plate to locate

the drops of inoculum then place 1 drop of

enrichment culture at 4 separate points on the

surface of each solid medium.

(b) Space the drops 6-7 cm apart on the

plate so that when an FA microscope slide is

later placed over 2 of the drops, both inocula-

tion points will be included on the slide. This is

essential, since glass slide impression smears

of the inoculated points will be made after

incubation of the plates.

agar surface of the differential plates, incubate

the plates at 35 C for 3 hours.

(d) Remove the plates after incubation and

make impression smears.

8.4.3 Impression Slides: Place a clean FA

1x3 inch glass slide (nonfluorescent) over 2

of the inoculated points on the agar. Press

down lightly, without moving the glass slide to

either side. Too much pressure will cause

movement of the slide and accumulation of

agar on the slide. Repeat the process for the

remaining 2 inoculation points with another

clean slide. Make impression slides for each

differential plate.

8.4.4 Fixing Slides

(a) Air dry the smears.

(b) Fix for 2 minutes in Kirkpartick's Fixa-

tive (mix 60 ml absolute ethanol, 30 ml chloro-

form, and 10 ml formalin).

(d) Air dry. Do not blot.

8.4.5 Titration of Conjugates

After the slides have been fixed, stain with

Salmonella FA conjugate. However, each lot

number of conjugate must be titered before

use.

Based on the previous experience of the

analyst or other workers, the conjugate and

test components must have been proven with

water samples. Sources of polyvalent conju-

gates known to be acceptable (CDC Approved

List) are: Burroughs Wellcome & Company,

Clinical Sciences Inc., Difco Laboratories and

Sylvana Company.

182

dEPA MICROBIOLOGICAL MANUAL 1978

image:

The Difco Bacto-FA Poly Conjugate con-

tains antibodies against the major 0, Vi and H

antigens. The Sylvana Salmonella Polyvalent

OH Globulin represents the somatic antigens

of Salmonella O groups A through S. The Difco

Panvalent conjugate is not recommended be-

cause of excessive cross-reactions.

Titer the conjugate as follows:

(a) Inoculate a known Salmonella culture

into BHI broth. Incubate at 35 C for 18-24

hours.

(b) From this broth culture, make smears

on clean FA glass slides. Prepare enough

smears for each conjugate dilution and con-

trols. Include a known 4 + Salmonella control

to be used as a reading standard. Air dry the

smears and follow the instructions for fixing

slides. Part III-E, 8.4.4.

conjugate in buffered saline: 1:2, 1:4, 1:6, 1:8

and 1:10. Most batches are effective at the 1:4

dilution.

(d) Cover a smear with one of the conju-

gate dilutions or the undiluted conjugate.

Place slides in moist chamber and proceed as

in Part III-E, 8.4.6 (b)-(f) and 8.4.7 (a)-(b). Use the

known 4 + Salmonella control as the standard.

(e) The titer of conjugate to be used is the

second highest dilution which gives 4 +

fluorescence. For example, if the conjugate

dilutions outlined above in 8.4.5 (c) above gave

fluorescence intensity ratings (in order) 4 + ,

4 + , 4+, 2 + , 1 +, the conjugate dilution to be

employed would be 1:4. This value should be

marked on the conjugate bottle. Prepare an

amount of chosen conjugate dilution sufficient

for each day's run.

8.4.6 Staining Impression Slides: After the

slides have been fixed, they are stained with

Salmonella FA conjugate.

(a) Cover each smear with one drop of the

predetermined dilutions of conjugate (Difco

FA Salmonella Polyvalent, or Sylvana Polyva-

lent OH Globulin). Include a known 4+ Salmo-

nella culture as a procedure control. Place the

slides in a moist chamber to prevent evapora-

tion of the staining reagent. Tap off excess

reagent onto paper towel.

(b) After 30 minutes, wash away the ex-

cess reagent by dipping the slides into a bath

of saline buffered at pH 7.2.

buffered saline for 10 minutes.

(d) Replace the rinse solutions in (b) and (c)

above, after each use.

(e) Rem'ove slides, rinse in a laboratory

pure water bath and allow to drain dry. Do not

blot

(f) Place a small drop of FA mounting fluid

(Difco 2329-57) onto the smear and cover

with a coverslip. Seal the edges of the cover-

slip with nail polish. Such slides may be stored

for up to 1 year in a freezer at -70 C with

minimal loss of fluorescence.

(g) Examine the slides under a

fluorescence microscope (900-1 OOOx) fitted

with the proper filters. Use a known 4+ slide

for comparison. Read slides within 2% hours if

slides have not been sealed and stored in a

deep-freeze.

8.4.7 Interpretation of Fluorescence, Re-

porting Results

(a) Fluoresence results are recorded as

shown in the following table. The number of

fluorescing cells and the degree of

fluorescence/cell are the criteria on which

positive results are based. Smears with large

numbers of strongly fluorescent cells (3+ or

4+) are positve; weakly fluorescent cells are

negative. Smears with few fluorescent cells

should be examined carefully.

(b) Cultures giving positive FA reactions

(4+ or 3+) must be isolated on differential

plating media, confirmed by biochemical iden-

tification, and verified serologically. Cultures

displaying negative FA results may be

discarded.

SALMONELLA

183

image:

Rating

Positive

Negative

Intensity

24-

14-

Description

Brilliant yellow-green fluorescence, cell sharply-outlined

Bright yellow-green fluorescence, cell sharply outlined with

dark centers

Dull yellow-green fluorescence, cells not sharply outlined

Faint green discernible in dense areas, cells not outlined

No fluorescence

REFERENCES

1. Edwards, P. R. and W. H. Ewing, 1972. Identification of Enterobacteriaceae (3rd ed}. Burgess

Publishing Co., Minneapolis, MN.

2. Buchanan, R. E. and N. E. Gibbons, 1974. Sergey's Manual of Determination Bacteriology {8th

ed). The Williams and Wilkins Company, Baltimore, MD.

3. Moore, B., 1948. The detection of paratyphoid carriers in towns by means of sewage examina-

tion. Monthly Bulletin of the Ministry of Health and the Public Health Laboratory Service (Q. Brit.)

.1:241.

4. Brezenski, F. T. and R. Russomanno, 1969. The detection and use of salmonellae in studying

polluted tidal estuaries. J. WPCP 41:725.

5. Levin, M. A., J. R. Fischer and V. J. Cabelli, 1974. Quantitative large-volume sampling technique.

Appl. Microbiol. 28:51 5.

6. McCoy, J.H., 1962. The isolation of Salmonella. J. Appj. Bacteriol. 25:213.

7. Raj, H., 1966. Enrichment medium for selection of Salmonella from fish homogenate. Appl.

Microbiol. 14:12.

8. Dunn, C. and W. J. Martin, 1971. Comparison of media for the isolation of salmonellae and

shigellae from fecal specimens. Appl. Microbiol. 22:17.

9. Harvey, R. W, S. and T. H. Price, 1968. Elevated temperature incubation of enrichment media for

the Isolation of Salmonella from heavily contaminated materials. J. Hyg. Camb. 66:377.

10. Kampelmacher, E. H. and L. M. van Norle Jansen, 1971. Reduction of Salmonella in compost in a

hog-fattening farm oxidation vat. J.Wat. Poll. Cont. Fed. 43:1541.

11. Spino, D. F., 1966. Elevated temperature technique for the isolation of Salmonella from streams.

Appl. Microbiol. 14:591.

12. Cheng, C. M., W. C. Boyle and J. M. Goepfert, 1971. Rapid, quantitative method for Salmonella

detection in polluted water. Appl. Microbiol. 21:662.

13, Cherry, W. et al., 1972. Salmonellae as an index of pollution of surface waters. Appl. Mierobiol.

24:334. ~

14. Buffer, J. R., 1971. Comparison of the isolation of salmonellae from human feces by enrichment

at 37 C and at 43 C. Zbl. Bakt. 1. Abt. 217:35.

15. Phirke, P. M., 1974. Elevated temperature technique for enumeration of salmonellae in sewage.

Indian J. Med. Res. 62:6:938.

184 <&Ef¥k MICROBIOLOGICAL MANUAL 1978

image:

16. Harvey, R. W. and T. H. Price, 1976. Isolation of salmonellae from sewage-polluted river water

using selenite F and Muller-Kauffmann tetrathionate. J. Hyg. Cajnb. 77:333.

17. Edel, W. and E. H. Kampelmaeher, 1939. Comparative studies on Salmonella isolation in eight

European laboratories. Bull. World Health Org. 39:487.

18. Kampelmaeher, E. H. and L. M. Van Noorle Jansen, 1973. Comparative studies on isolation of

Salmonella from effluents. Zbl. Bakt. Hyg., I. Abt. Org. 157:71.

19. Vanderpost, J. M. and J. B. Bell, 1975. A bacteriological investigation of meat-packing plant

effluents in the Province of Alberta with particular emphasis on Salmonella. Environmental

Protection Service, Environment Canada, Report No. EPS 3-WP-7 6-9.

2O. Cherry, W. et aL, 1954. A simple procedure for the identification of the genus Salmonella by

means of a specific bacteriophage. J. Lab. Clin. Med. 44:51,

21. Wolkos, S., M. Schreiberand H. Baer, 1974. Identification of Salmonella with O-1 bacteriophage.

Appl. Microbiol. 28:618.

22. Gillies, R. R., 1956. An evaluation of two composite media for preliminary identification of

Shigella and Salmonella. J. Clin. Pathol. 9:368.

23. Media and Tests for Differentiation of Enterobacteriaceae, 1970. Center for Disease Control,

USDHEW, PHS, Atlanta, GA.

24. Nord, C. E., A. A. Lindberg and A. Dahlback, 1974. Evaluation of five test—kits—API, Auxotab,

Enterotube, Pathotec and r/b—for identification of Enterobacteriaceae. Med. Microbiol.

159:211.

25. Shayegani, M., M. E, Hubbard.T. Hiscottand D. McGlynn, 1975. Evaluation of the r/b and minitek

systems for identification.of Enterobacteriaceae. J. of Clin. Microbiol. Jj,6:504.

26. Tomfohrde, K. M., D. L. Rhoden, P. B. Smith and A. Balows, 1973. Evaluation of the redesigned

Enterotube—a system for the identification of Enterobacteriaceae. Appl. Microbiol. 25:301.

27. Kiehn, T. E., K. Brennan and P. D. Ellner, 1974. Evaluation of the minitek system for identification

of Enterobacteriaceae. Appl. Microbiol. 28:668.

28. Robertson, E. A. and J. D. MacLowry, 1974. Mathematical analysis of the API enteric 20 profile

29. Robertson, E. A. and J. D. MacLowry, 1975. Construction of an interpretive pattern directory for

the API 10 S kit and analysis of its diagnotstic accuracy. J. of Glin. Microbiol. W3:515.

30. Douglas, G. W. and J. A Washington, 1973. Identification of Enterobacteriaceae in the clinical

laboratory. Center for Disease Control, Public Health Service, USDHEW, Atlanta, GA.

31. Presnell, M, N. and W. H. Andrews, 1976. Use of the membrane filter and a filter aid for concen-

trating and enumerating indicator bacteria and Salmonella from estuarine waters. Water

Research 10:549.

32. Kaper, J. B., G. S. Sayler, M. M. Baldini and R. R. Colwell, 1977. Ambient-Temperature primary

nonselective enrichment for isolation of Salmonella spp. from an estuarine environment. Appl. &_

Environ. Microbiol. 33:4:829.

33. Olivieri, V. P. and S. C. Riggio, 1976. Experience on the assay of microorganisms in urban runoff,

in proceeding of workshop on microorganisms in urban stormwater, EPA-60012-76-244,

Municipal Environmental Research Laboratory, EPA, Cincinnati, OH.

34. Thomason, B. M., 1971. Rapid detection of Salmonella microcolonies by fluorescent antibody.

Appl. Microbiol. 22:1064.

35. Thomason, B. M. and J. G. Wells, 1971. Preparation and testing of polyvalent conjugates for

fluorescent-antibody detection of salmonellae. Appl. Microbiol. 22:876.

SALMONELLA 185

image:

PART III. ANALYTICAL METHODOLOGY

Section F Actinomycetes

1. Summary of Method

The pour-plate technique is the prinicipal

method for measuring actinomycete popula-

tions. Selective media are employed that favor

actinomycete development over fungi and

other bacteria. The mass of branching fila-

ments characteristic of this bacterial group

offers distinctive features for identification. To

facilitate identification and counting of the ac-

tinomycete colonies, plates are prepared by

the two-layer agar technique. Since only the

upper layer is inoculated, the method assures a

predominance of surface colonies.

2. Definition

2.1 The actinomycetes are a group of

microorganisms with cells ranging from

0.5-2.0 /«m diameter but normally less than

1.0 pm diameter, which usually develop as

non-septate hyphae in branching mycelial

masses. The actinomycetes are generally

saprophytic but some are parasitic or patho-

genic to plants, animals and man.

2.2 The actinomycetes are fungal in mor-

phology and in spore formation, but lack a

membrane around nuclear materials. They

have a sensitivity to bacterial antibiotics, are

susceptible to specific phage and have other

biochemical characteristics which class them

as fllamentuous, branching bacteria (1). Al-

though actinomycetes are found in water and

sediments, the greatest natural reservoir for

these organisms is the soil.

3. Scope and Application

3.1 Actinomycetes are of interest in water

treatment and waste treatment facilities be-

cause of the taste and odor problems they

cause in potable waters and fish, and the foam-

ing problems they can cause in waste treat-

ment plants.

3.2 The taste and odor problems result

from volatile products characterized by an in-

tense earthy-musty odor {2, 3). Evidence points

to 2 highly odoriferous metabolites, geosmin

and 2-methylisoborneol, as the sources of the

problem (4, 5, 6). It appears that the relative

abundance of these 2 metabolites in natural

waters is linked to an ecological factor not yet

resolved. Traces of these products can impart

a disagreeable persistent odor to a municipal

water supply, which is extremely difficult to

treat. These natural odorants, prevalent in

many parts of the world, can also affect com-

mercial fishing.

3.3 Actinomycete distribution in waters

with an earthy-musty odor shows a correlation

between actinomycete counts and odor levels,

indicating such enumeration to be a useful

parameter in measuring quality of water. The

genus of most interest is Streptomyces.

3.4 Actinomycetes have also been recog-

nized as the cause of disturbances in the oper-

ation of activated sludge wastewater treat-

ment plants where massive growths of these

186

&EPA MICROBIOLOGICAL MANUAL 1978

image:

organisms can produce thick foams (7, 8). The

genus involved is Nocardia.

3.5 Because of diverse nutrient require-

ments, no single medium has been devised

that will support the growth of all actinomy-

cetes. Moreover, the culture media that have

proven useful in their isolation are not neces-

sarily the preferred media for encouraging

abundant growth. The isolation media are re-

strictive and nutritionally poor. They act by

depressing growth of other microorganisms

and by favoring a higher proportion of actino-

mycete colony development.

3.6 The pour-plate method does not indic-

ate whether the isolated colony originated

from individual spores, spore aggregates,

small mycelial fragments or a mycelial mat.

4. Apparatus and Materials

4.1 Incubator set at 28 ±0.5 C.

4.2 Water bath set at 44-46 C for temper-

ing agar.

4.3 Electric oven set at 105-110 C.

4.4 Hand tally or electronic counting de-

vice (optional).

4.5 Thermometer which has been

checked against a National Bureau of

Standards-Certified Thermometer.

4.6 Pipet containers of stainless steel, alu-

minum or pyrex glass for glass pipets.

4.7 Petri dish canister of stainless steel or

aluminum for glass dishes.

4.8 Erlenmeyer flasks, pyrex, screw-cap,

250 and 500 ml volume.

4.9 Sterile T.D., bacteriological or Mohr

pipets, glass or plastic, of appropriate size.

4.10 Sterile 100 mm x 15 mm petri

dishes, glass or plastic.

4.11 Screw-cap culture tubes, borosilicate

glass, 25 x 150mm.

4.12 Dilution bottles (milk dilution), pyrex,

marked at 99 ml, screw-cap with neoprene

rubber liner.

5. Media

5.1 Sterile starch-casein agar or equiva-

lent agar prepared in 17 ml volumes in screw-

cap tubes and in 250-300 ml volumes in 500

ml, screw-cap bottles or flasks. See Part II-B,

5.6.

5.2 Sterile buffered dilution water in bot-

tles containing 99 + 2 ml volumes. See Part II-

B,7. ,

5.3 Cycloheximide Stock Solution. Weigh

out 100 mg cycloheximide (antifungal

antibiotic), bring to 100 ml with distilled water,

pour into a screw-cap flask and mix until dis-

solved. Sterilize for 15 minutes at 121 C (15

Ibs. pressure). Cycloheximide is available as

Actfd/onefrom the Upjohn Company.

6. Sample Preparation

6.1 Water Samples

6.1.1 Fill sample bottle only 3/4 full so

that ample airspace is left in the bottle for

thorough mixing of the water sample.

6.1.2 Mix water sample by shaking vigor-

ously about 25 times. Using a pipet, transfer

11 ml immediately after mixing to a 99 ml

water blank.

6.1.3 Repeat for, desired dilutions. A dilu-

tion of 10~3 is usually sufficient for plating

water samples.

6.2 Soil Samples

6.2.1 Mix soil sample thoroughly and

weigh out a 50 gram sample in a tared weigh-

ing pan. Dry at 105-110 C to constant weight.

ACTINQMYCETES

187

image:

The final weight is used in calculating numbers

of organisms/gram dry soil.

6.2.2 Prepare the initial dilution by weigh-

ing out 11 grams of soil and adding to a 99 ml

volume of buffered dilution water for a 1:10

dilution. Shake dilution bottle vigorously for 1

minute.

6.2.3 Transfer a 11 ml sample of the 1:10

dilution to a second dilution bottle containing

99 ml buffered dilution water and shake vigor-

ously about 25 times. Repeat this process until

the desired dilution is reached. Dilution of

10~3to 10~8are usually necessary for enumer-

ation of soil samples.

7. Plating Procedures

Prepare triplicate plates for each test dilu-

tion using the two-layer agar technique as

follows:

7.1 Cool flask of starch casein agar to

44-46 C and pour 15 ml layer in each petri

dish. Allow to harden.

7.2 Melt starch-casein agar in tubes and

cool to 44-46 C. Add 2 ml of sample or sample

dilution and 1 ml of cycloheximide solution.

Mix tube contents well.

7.3 Pipet immediately 5 ml of the inocu-

lated agar over the solid agar base layer in

each plate with gentle swirling to evenly dis-

tribute the inoculated agar. Each 5 ml contains

0.5 ml of the particular dilution used. This 0.5

factor must be taken into consideration in cal-

culating the final dilution.

7.4 After plates are solidified, invert and

Incubate at 28 C for approximately 7 days and

count.

8. Counting of Colonies

8.1 Select plates for counting with

30-300 colonies.

8.2 Rules for making plate counts are

giveninPartlll-A,5.6.2.

8.3 Examine plates macroscopically hold-

ing toward a light source. Actinomycete colo-

nies appear dull or chalky when covered with

aerial mycelium. The edges of the colonies are

less dense producing a halo effect. Colonies

adhere strongly to the agar and have a tough,

leathery texture. In contrast, bacterial colonies

appear shiny or opalescent with a soft texture,

adhere weakly to the agar, and show no gen-

eral distinction between the edge and the col-

ony as a whole (see Figure lll-F-1).

8.4 Actinomycete colonies can be verified

at a magnification of 100x. Because of their

filamentous growth, they typically have fuzzy

borders which contrast sharply to the smooth

borders characteristic of bacteria. (See Figures

lll-F-2and3).

8.5 Addition of cycloheximide to the isola-

tion medium suppresses development of fun-

gal colonies. If fungi do develop, they can be

differentiated from actinomycetes by their

woolly appearance and much larger cell diam-

eter. With a little experience in examining fun-

gal and actinomycete colonies, it is fairly easy

to distinguish them macroscopically.

9. Reporting Results

9.1 Calculate the actinomycete density in

water samples in counts/ml according to the

following equation:

Sum of Replicate Plate Counts

Total Volume of Original

Sample Tested, in ml

Sum of Replicate Plate Counts

No of

Replicates

Sample Dilution

Tested

Agar Plate

Dilution

Factor (See 7.3)

Actinomycete

Count/ml

188

V*EPA MICROBIOLOGICAL MANUAL 1978

image:

FIGURE lll-F-1. A plate containing bacterial and actinomycete colonies.

A—The dull, powdery appearance of a sporulating actinomycete colony.

B—The smooth, mucoid appearance of a bacterial colony.

ACTINOMYCETES

189

image:

FIGURE III-F-2. An actfnotnycete colony showing the branching filaments

that cause the fuzzy appearance of its border. X 225

FIGURE Ill-F-3, A bacterial colony with its relatively-distinct, smooth

border. X 225

190

MICROBIOLOGICAL MANUAL 1978

image:

For example, if the triplicate sample vol-

umes tested at a 1:10 dilution yielded 45, 39

and 42 actinomycete colonies, the calculation

would be:

126

3(rep) X 0.1 (dil) x 0.5 (A.P.D. factor)

126

0.15

840

Actinomycetes/ml

9.2 Correct the actinomycete counts from

soil and mud samples for water contents. See

6.2, this Section. Calculate the counts as

follows:

wt. of collected soil

wt. of dried soil

= conversion factor

conversion factor x count/gm collected

soil = count/gm dry soil

Determine count/gm collected soil by the

equation given in 9.1.

REFERENCES

1. Lechevalier, H. A. and M. P. Lechevalier, 1967. Biology of Actinomycetes. Am. Rev. Microbiol.

21_:71.

2. Adams, B. A., 1929. Cladothrixdichotoma and allied organisms as a cause of an "indeterminate"

taste in chlorinated water. Water & Water Eng. 31:327.

3. Thaysen, A. C., 1936. The origin of an earthy or muddy taint in fish. Ann. Appl. Biol. 23:99.

4. Rosen, A. S., C. I. Mashni and R. S. Safferman, 1970. Recent developments in the chemistry of

odour in water: the cause of earthy/musty odour. Water Treatment Exam. 19:106.

5. Piet, G. J., B. C. J. Zoeteman and A. J. A. Kraayeveld, 1972. Earthy-smelling substance in surface

waters of the Netherlands. Water Treatment Exam. 2Jj28 1.

6. Yurkowski, M. and J. L. Tabachek, 1974. Identification, analysis and removal of geosmin from

muddy-flavored trout. J. Fish. Res. Board Can. 31:1851.

7. Lechevalier, H. A., 1975. Actinomycetes of sewage-treatment plants. U.S. Environmental Protec-

tion Agency, Environmental Protection Technology Series, EPA-600/2-75-031, Cincinnati, OH.

8. Lechevalier, M. P. and H. A. Lechevalier, 1974. Nocardia amarae, sp. nov., an actinomycete

common in foaming activated sludge. Int. J. Syst. Bacteriol. 24:278.

ACTINOMYCETES

191

image:

PART IV. QUALITY ASSURANCE

Regulatory agencies making decisions on water quality standards and wastewater discharge

limits require formal analytical quality control programs for their laboratories and program

participants to assure validity of their data. For example, water quality regulations include

provisions for quality control and testing procedures (the Safe Drinking Water Act, National Interim

Primary Drinking Water Regulations published in Federal Register, Title 40 Part 141, 59566,

December 24, 1975). Further, the Act states that analyses for maximum contaminant levels must

be conducted by laboratories approved in a formal certification program. The quality control

procedures specified in the Manual for the Interim Certification of Laboratories Involved in

Analyzing Public Drinking Water Supplies (Appendix B) are a recommended minimal program and

are not equivalent to the comprehensive system described in Part IV of this Manual.

A laboratory quality control program is the orderly application of the practices necessary to

remove or reduce the errors that occur in any laboratory operation due to personnel, equipment,

supplies, sampling procedures and the analytical methodology in use.

A quality control program must be practical, integrated and require only a reasonable amount

of time or it will be by-passed. When properly administered, a balanced, conscientiously applied

quality control program will assure the production of uniformly high quality data without inter-

fering with the primary analytical functions of the laboratory. This within-laboratory program

should be supplemented by participation of the laboratory in an interlaboratory quality control

program such as that conducted by EPA and detailed in Part V. The major considerations for quality

control are discussed under three separate Sections:

Section A Laboratory Operations

1. Sample Collection and Handling

2. Laboratory Facilities

3. Laboratory Personnel

4. Laboratory Equipment and Instrumentation

5. General Laboratory Supplies

6. Membrane Filters

7. Culture Media

Section B Statistics for Microbiology

1. Measures of Control Tendency

2. Measures of Dispersion

3. Normal Distribution

4. Poisson Distribution

5. Measures of Performance

192

&ERA MICROBIOLOGICAL MANUAL 1978

image:

Section C Analytical Quality Control Procedures

1. Quality Control on Routine Analyses

2. Quality Control in Compliance Monitoring

3. Comparative Testing of Methodologies

QA/LABORATORY OPERATIONS 1 93

image:

PART IV. QUALITY ASSURANCE

Section A Laboratory Operations

Section A describes the checks and moni-

toring procedures that should be performed

on materials, supplies, instrumentation and

the physical facility. These checks should be

documented completely and recorded as per-

formed. See Part V-A, 1.2 for details on this

documentation.

Sample Collection and

Handling

Laboratory Facilities

Laboratory Personnel

Laboratory Equipment and

Instrumentation

General Laboratory Supplies

Membrane Filters

Culture Media

1. Sample Collection and Handling

The acquisition of valid data begins with

collection of a representative water sample or

other environmental material being tested.

Samples must be maintained as closely as

possible to original condition by careful han-

dling and storage.

Sample sites and a sampling frequency

are selected to provide data representative of

the characteristics and the variability of the

water quality at that station. The most important

quality control factor in sampling is the

immediate analysis of the sample. If the sample

cannot be analyzed at once, it should be

refrigerated and analyzed within six hours.

Recommended procedures for collecting,

transporting and handling water and waste-

water samples are described separately in

Part II-A of this Manual.

2. Laboratory Facilities

2.1 Ventilation: Laboratories should be

well-ventilated and free of dust, drafts and

extreme temperature changes. Central air con-

ditioning has advantages: 1) The incoming air

is filtered, reducing contamination of the labo-

ratory and culture work. 2) The uniform tem-

perature control of air conditioning permits

stable operation of incubators. 3) With closed

windows, drafts and air currents which can

cross-contaminate surroundings and work

areas during warm weather are minimized. 4)

Low humidity reduces moisture problems with

media, chemicals, analytical balances and

other instrumentation.

2.2 Space Utilization: Ideally the areas

provided for the preparation and sterilization

of media, glassware and equipment should be

separated from the laboratory working area

but located close enough for convenience. In

public health laboratories that analyze many

194

MICROBIOLOGICAL MANUAL 1978

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different types of samples a separate work

area is desirable for water analysis. Special

work areas such as an absolute barrier or

vented laminar flow hood (see Part V-C) are

often used for dispensing and preparing sterile

media and tissue cultures, for transferring

microbial cultures or for working with

pathogenic materials. In smaller laboratories it

may be necessary to carry out these separate

activities in different sections of the same

room. However, limited facilities and restricted

work space may seriously hamper the quality

of the work and influence the validity of

results. Visitors and through traffic should be

discouraged in work areas. Through traffic can

be prevented by laboratory design.

2.3 Laboratory Bench Areas: Sufficient

clean bench space should be available for the

analyses to be performed efficiently. For rou-

tine work, 6 linear feet is the recommended

minimum work area for each analyst. Research

work or other analyses using specialized

equipment may require significantly more

space per worker. These estimates of bench

space are exclusive of work areas used for

preparatory and supporting activities. Labora-

tory lighting should be even, screened to re-

duce glare, and provide about 100 footcandle

light intensity at working surfaces.

Bench tops should be set at heights of

36-38 inches with a depth of 28-30 inches.

This height is comfortable for work in a stand-

ing or sitting position. Desk tops or sit-down

benches are set at 30-31 inches height to

accommodate microscopy, plate counting, cal-

culations and writing activities. Bench tops

should be stainless steel, epoxy plastic or

other smooth impervious material which is in-

ert, corrosion-resistant and has minimum

seams.

2.4 Walls and Floors: Walls should be

covered with waterproof paint, enamel or

other surface material that provides a smooth

finish which is easily cleaned and disinfected.

Floors should be covered with good quality

tiles or other heavy duty material which can be

maintained with skid-proof wax. Bacteriostatic

agents contained in some wall or floor finishes

increase the effectiveness of disinfection.

2.6 Monitoring for Cleanliness in Work

Areas: High standards of cleanliness should

be maintained in work areas. The laboratory

can be monitored for cleanliness by one or

more of the procedures described below.

Since these monitoring procedures cannot re-

cover all of the microorganism populations

present, absolute limits are difficult to develop.

Rather, the tests should be used regularly on a

.weekly or other basis to monitor counts In the

same work areas over time or to make compar-

isons between different work areas.

2.5.1 RODACAgar Plates (1)

Work areas and other surfaces can be

checked by RODAC plates which contain gen-

eral growth media for total counts or selective

media for conforms, enteric pathogens, strep-

tococci, staphylococci, or other microorga-

nisms cultured in the laboratory. RODAC is an

acronym for Reproducible OrganismDetection

and Counting. The RODAC dish has a test area

of about 25 cm with Quebec style grids em-

bossed in the plate. It is specifically designed

to enumerate the microbial population of flat

solid surfaces by contact techniques.

(a) Purchase RODAC plates prefilled with

desired test medium or prepare plates by fill-

ing the center well with about 16 ml of appro-

priate agar. When preparing agar in the labora-

tory, add 0.07% soy lecithin or 0.5% polysor-

bate (Tween 80) to the agar to neutralize the

effect of the disinfectant on test surfaces.

Cooling leaves a raised bed of agar about 1

mm higher than the rim of the dish, allowing

contact of the sterile agar to the test surface

for direct counts.

(b) To sample an area, remove the plastic

cover and carefully press the agar to the solid

surface being sampled. It is important that the

entire agar layer contacts the test surface. Use

a rolling motion with uniform pressure on the

back of the plate to insure complete contact

Replace the cover and incubate in an inverted

position for the appropriate time and

temperature.

ony counter and report as the number of colo-

MICROBIOLOGICAL MANUAL 1978

195

image:

nles per RODAC plate or number of colonies

par 25 sq. cm.

2.5.2 Swab Method (1)

(a) The swab contact method can be used

to monitor the contamination of work areas

and especially those with cracks, corners,

crevices and rough surfaces. Dacron swabs

rinse out more easily than cotton ones. These

may be purchased from Econ Microbiological

Laboratory, 2716 Humboldt Avenue, South,

Minneapolis, MN. Rayon swabs are available

from Fuller Pharmaceutical Company, Minne-

apolis, MN, and Consolidated Laboratories

Inc., Chicago Heights, IL. Swabs made of cal-

cium alginate which is soluble in water, can

also be used. These are available from Consoli-

dated Laboratories, Inc., Chicago Heights, IL.

Rinse Solution Vials - Add 1.25 ml stock

phosphate buffer solution, 5 ml of 10% aque-

ous sodium thiosulfate, 4 grams Asolectin (As-

sociated Concentrates, 32-30 61st Street,

Woodside, Long Island, NY 11377) and 10

grams Tween 20 or Tween 80 (Hilltop Re-

search, Inc., Miamiville, OH 45147) to 500 ml

distilled water; heat to solution in boiling water

bath. Cool and make up to 1 liter. Dispense in

screw-capped vials in 10 ml or other volumes

and sterilize for 15 minutes at 15 Ib. pressure

(121 C). Because Asolectin is hygroscopic,

store it in a desiccator and weigh quickly.

(b) To sample an area, open a sterile swab

container, grasp the end of the stick and re-

move aseptically. Open a 10 ml vial of neutral-

izing buffer, moisten swab head, and press out

excess solution against the inside of the vial.

Hold the swab handle at a 30° angle against

the sampling area. Rub the swab head slowly

and throughly over approximately 8 sq. in. of

surface area. Repeat procedure 3 times over

the same area, turning the swab and reversing

directions. Place the swab head in the neutral-

izirjg buffer vial, rinse briefly in solution and

press out the excess liquid.

Test four more 8 sq. in. areas of surface,

rinsing the swab in solution after each swab-

bing. After the fifth area has been swabbed,

remove the excess liquid, place swab head in

vial and break or cut with a sterile scissors

leaving the swab head in the vial. Replace the

cap and store vial at 4 C or on ice until analysis.

When using alginate swabs, rinse solution

vials should contain 4.5 ml after sterilization.

Prepare a 10% solution of sodium

hexametaphosphate in appropriate vials and

steam sterilize. Follow the above swabbing

procedures but after swabbing the fifth 8 sq.

in. area, deposit the swab head in the rinse vial

and add 0.5 ml of sterile hexametaphosphate

solution.

To begin plating, shake the vial vigorously

to dislodge the bacteria from the swab. Pipet 1

ml and 0.1 ml aliquots of the rinse solution.

The 1 ml portions represent a 1:10 dilution

and the 0.1 ml portions represent a 1:100

dilution. Pour the plates with Standard Me-

thods Agar. Mix and cool to solidify and incu-

bate at 35 C for 48 hours.

the number/ml which equals the count per 8

sq. in. of area.

2.5.3 Air Density Plates (1)

The number of microorganisms in the lab-

oratory air is directly proportional to the

amount and kind of activity. These organisms

affect results when the suspended cells con-

tact materials and equipment or when they

settle on exposed test materials and surfaces.

(a) The numbers and types of airborne

microorganisms can be determined by expos-

ing petri plates for a specified time at points

where inoculating, filtering, plating and trans-

fer work is done. This exposure method can be

used to monitor total bacteria or the specific

organisms being tested such as coliforms, en-

teric pathogens, streptococci, staphylococci,

yeasts or molds.

(b) Pour the petri dishes with the appropri-

ate agar and allow to harden. Store poured

plates in the refrigerator if they are not used on

the same day.

dishes top side up on sterile towels. Expose the

196

QA/LABORATORY OPERATIONS

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plates in selected work sites for 15 minutes

and mark them with sample site identification.

Replace covers. Incubate Standard Methods

Agar at 35 C for 48 hours and other media for

the specified time periods,

(d) Count the colonies and report the num-

ber per square foot. The number of organisms

which settle in 15 minutes of exposure on a

petri dish is equivalent to that for 1 sqt

ft./minute because the area of a standard-size

petri dish is approximately 1/15 sq. ft. The

microbial density should not normally exceed

15 colonies per sq. ft. Air density plates should

be taken weekly during peak work periods for

routine monitoring.

2.6 Laboratory Maintenance: Laboratory

benches, shelves, floors and windows should

be cleaned on a scheduled basis. Proper main-

tenance is evidenced by lack of dust and soil

build-up on shelves, in corners, etc. Floors

should be wet-mopped and treated with a dis-

infectant solution to reduce contamination of

air in the laboratory- Sweeping or dry-mopping

should not be permitted in a microbiology

laboratory. Work benches should be wiped

down with disinfectant before and after each

use and at the end of the day.

Every attempt should be made to maintain

the laboratory areas free of clutter. Laboratory

benches, space under benches, shelves and

drawers accumulate equipment materials and

supplies at a steady rate. The disorder can

interfere with laboratory operations. It can be

controlled by making a directed effort to clean

up work areas immediately after each use and

by conducting a weekly clean-up of the labora-

tory- Discard unneeded materials and store

equipment and supplies not in current use. The

most important step in maintaining an orderly

laboratory is to have ample storage space

near-by.

3. Laboratory Personnel

Microbiologists, technicians and support

personnel in the microbiology laboratory must

have training and experience appropriate to

the laboratory's analytical program. The vari-

ety and complexity of the tasks and tests per-

formed determine the professional and on-the-

job-training required.

3.1 Professional Microbiologist: The mi-

crobiologist directs and participates in the col-

lection and storage of samples, the prepara-

tion of glassware, equipment and media, the

analysis of a variety of waters and wastewa-

ters and the evaluation of procedures as re-

quired. He takes part in the quality control

program. He counts, tabulates and summa-

rizes data and prepares or helps to prepare

reports from the results. He should have at

least a BS degree in microbiology or a BS/BA

degree in biology with a minor in

microbiology.

The professional microbiologist initially

carries out routine tasks and progresses to

more difficult work as he gains experience.

When he reaches the senior level he performs

the most complex duties.

3.2 Senior Grade and Supervisory Micro-

biologists: A senior grade microbiologist per-

forms the duties described in 3.1 but carries

out a wider range of assignments and more

difficult tests. He participates in program plan-

ning and laboratory management and solves

significant microbiological problems that re-

quire a higher than bench-level skill and knowl-

edge. The experienced microbiologist consults

and advises on procedural problems and trains

personnel. He provides expert testimony,

interprets results, prepares reports and

recommends microbiological standards or

actions for regulatory programs.

The supervisory microbiologist performs

administrative functions of: planning and

directing water quality monitoring programs,

designing field surveys, advising administrative

officers on policy matters related to micro-

biology and inspecting laboratory facilities. He

is recognized for his authoritative scientific

competence.

3.3 Technicians: The technician performs

semi-professional and professional duties of

limited scope and complexity. Typically, he

assists professionals by doing the routine tests.

QA/LABORATORY OPERATIONS

197

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Under supervision, he performs tasks involving

a series of steps. Technicians learn through

on-the-job instruction and by performing

standard tasks repetitively.

Technicians' tasks begin at the simplest'

level but can progress to the more, detailed

procedures performed by the professional mi-

crobiologist. However, these higher levels of

skill and knowledge are generally limited to

the specific areas in which they have received

on-the-job-training.

3.4 Support Personnel: Laboratory aides

and clerks provide the necessary support serv-

ices to the laboratory. Aides prepare glass-

ware, make media and sterilize materials. One

or, more aides are recommended for every

bench microbiologist. Clerical duties include

recording data, filing records, typing reports,

ordering supplies, maintaining inventories,

distributing mail and answering the telephone.

3.5 On-the-Job-Training and Experience:

In addition to the formal academic training, the

professional microbiologist should have

experience in aquatic or environmental

microbiology. Technical or on-the-job-training

for at least two weeks is required for each

parameter tested in water microbiology. The

formal training of the technician ranges from

high school to technical or academic training

short of a degree in microbiology. Technical

and scientific experience is often substituted

for advanced formal training.

The microbiologist and technician should

be encouraged to attend courses at centers of

expertise such as universities, commercial

manufacturers, US EPA facilities, the Center

for Disease Control (CDC) of USPHS, FDA and

other federal and state governmental agen-

cies. Courses might include fluorescent anti-

body techniques, isolation and identification

of the Enterobacteriaceae and laboratory

safety.

3.6 Laboratory Supervison: Ideally, the

laboratory should be directed by a profes-

sional microbiologist. However, in a small

laboratory where the staff consists of a single

non-professional technician, an approved con-

sultant microbiologist should be available for

guidance and assistance.

Work assignments in the laboratory

should be clearly defined. The analyst should

be trained in basic laboratory procedures and

should perform well at a given level of respon-

sibility before new assignments are made. The

supervisor should periodically review proce-

dures such as sample collecting and handling,

media and glassware preparation, sterilization,

routine testing procedures, counting, data han-

dling, and quality control techniques. Problem

areas should be identified and solved by the

staff. Improved laboratory results will be the

measure of effective personnel practice and

training.

4. Laboratory

Instrumentation

Equipment

and

Quality control of laboratory apparatus in-

cludes servicing and monitoring the operation

of incubators, waterbaths, hot-air sterilizing

ovens, autoclaves, water stills, refrigerators,

freezers, etc. Each item of equipment should

be tested to verify that it meets the manufac-

turer's claims and the user's needs for accu-

racy and precision. Maintenance should be

performed on a regular basis by a tebhnician

who is familiar with the equipment.

4.1 A summary of recommended monitor-

ing procedures for laboratory equipment is

given in Table IV-A-1.

4.2 Monitoring Procedures for UV

Lamps

4.2.1 Spread Plate Irradiation

This test should be run when a new UV

lamp is installed and rerun quarterly to mea-

sure continuing effectiveness of the UV

irradiation.

(a) Prepare 100 ml or more of plate count

agar. Pour 10-15 ml of the melted agar into

each 100 mm petri dish as needed. Keep cov-

ers opened slightly until agar has hardened

198

SEPA MICROBIOLOGICAL MANUAL 1978

image:

and moisture and condensation have evapo-

rated. Close dishes and store in refrigerator

until use.

(b) Prepare a series of dilutions of a coli-

form culture so that 0.5 ml of inoculum will

give a 200-250 colony count (see Geldreich

and Clark).

each agar plate.

(d) Remove a glass spreader-rod from the

alcohol container and ignite by passing

through flame. Let burn completely and cool

for 15 seconds. Test glass rod on edge of agar

to verify safe temperature before use. Glass

spreaders may also be autoclaved.

(e) Place sterile, cool glass spreader on

agar surface next to inoculum. Position

spreader so that the tip forms a radius from the

center to the plate edge. Holding stick motion-

less, rotate plate several revolutions, or hold

the plate and move the stick in a series of

sweeping arcs to spread the inoculum uni-

formly over the entire surface of the agar.

(f) Lift the glass spreader from the agar and

place in disinfectant solution.

(g) Repeat the spread plate procedure with

other petri plates.

(h) With cover removed, place agar spread

plates under UV lamp at points where sterility

is desired.

(i) Place one inoculated plate under ordi-

nary laboratory lighting as a control.

(j) Expose plates for two minutes.

(k) Close plates and incubate at 35 C for

24 hours. Remove and examine for growth.

(1) Count all plates and record results.

Control plate should contain 200-250 colo-

nies. UV-irradiated plates should show 99%

reduction in the count of the control plate. If

count reduction is less than 80%, replace

lamp.

4.2.2 Measurements of UV Light Intensity

To monitor performance of UV lamps, it is

necessary to measure UV light intensity using

the UV light meter manufactured for this pur-

pose. The shortwave UV meter Model J-225 is

available from UV Products, Inc., San Gabriel,

CA 91778 for about $ 125. This instrument or

equivalent is recommended.

(a) Measure and average light intensities

of a new UV lamp at proper distances of use.

Record results in QC log, noting readings and

dates of installation.

(b) Monitor lamp intensity quarterly there-

after. Replace bulb when light is down to 80%

of original intensity.

of replacement lamp at proper distances.

Record readings and date of installation.

5. {^eneral Laboratory Supplies

5.1 Laboratory Glassware

5.1.1 A summary of procedures for main-

tenance of glassware is given in Table IV-A-2).

5-1.2 Glassware pH Check: Traces of

some cleaning solutions are difficult to remove

completely. Before using a batch of clean

glassware, test several pieces for an alkaline

or acid residue by adding and dispersing a few

drops of bromthymol blue or other pH indica-

tor and observing the color reactions. Brom-

thymol blue is particularly advantageous for

this check because it shows color changes of

yellow to blue green to blue, in the pH range

6.5 to 7.3.

To prepare bromthymol blue indicator,

add 16 ml of 0.01 N NaOH to 0.1 gram of

bromthymol blue. Dilute to 250 ml with dis-

tilled water (0.04% solution).

5.1.3 Test Procedure for Suitability of De-

tergents Used in Washing (2)

QA/LABORATORY OPERATIONS

199

image:

(a) Wash and rinse six, 100 mm diameter

petri dishes in the usual manner. These are

Group A.

(b) After normal washing, rinse a second

group of 6 petri dishes 12 times with succes-

sive portions of non-toxic distilled water.

These are Group B.

wash water using detergent concentrations

normally employed, and dry without rinsing.

These are Group C.

(d) Sterilize dishes in the usual manner.

(e) Add the proper dilution (usually two

different dilutions are used) of a water sample

yielding 30-300 colonies to triplicate petri

dishes from each Group (A, B and C). Proceed

according to the Standard Plate Count Proce-

dure in Partlll-A.

(f) The results are interpreted as follows:

{1} Differences in bacterial counts of less

than 15% among all Groups indicate the deter-

gent has no toxicity or inhibitory effect.

(2) Differences in bacterial counts of 15%

or more between Group A and B demonstrate

that Inhibitory residues are left on glassware

after the normal washing procedure used.

(3} Disagreement in averages of less than

15% between Groups A and B, and greater

than;15% between Groups A and C indicates

that detergent used h,as inhibitory

properties which are eliminated during routine

washing.

5.1.4 Sterility Checks on Glassware

After sterilization of a load of bottles,

flasks or tubes, test items of each type for

sterility by adding to one of each aerobic or

anaerobic broth medium (e.g., lauryl tryptose

or fluid thioglycollate broth). Incubate and

check for growth.

5.2 Pure Water Quality: Pure water sys-

tems are meant to produce the best possible

water; however, the potential quality of the

water will vary with the type of system used.

The quality of water obtainable from a

pure water system differs with the system used

and its maintenance. The acceptable limits are

given in Table IV-A-3.

5.3 Water Suitability Test

5.3.1 Summary: The water suitability

procedure of Geldreich and Clark (3) is a

sensitive test for determination of toxic or

stimulatory effects of distilled or deionized

water on bacteria. It is based on the growth of

Enterobacter aerogenes in a chemically defined

medium. Reduction of 20% or more in the

bacterial population compared to a control, is

judged toxic. Increased growth greater than

300% is called stimulatory.

5.3.2 Scope and Application: The test

is recommended for periodic use and as a

special measure of water suitability. This

test called the Test for Bacteriological

Properties of Distilled water, in the 14th

edition of Standard Methods is required

annually of laboratories in the Interim Certi-

fication Program for Drinking Water Supplies.

However, the test is not easily done on

an infrequent basis because it requires: work

over a four day period to complete, an ultra-

pure control water and very pure reagents,

and absolute cleanliness of culture flasks,

petri dishes, test tubes and pipets, etc. It is

a complex method that requires skill and

experience, is very sensitive to toxicants and

cannot be related directly to routine analytical

results.

5.3.3 Apparatus and Materials

(a) Incubator set at 35 ± 0.5 C.

(b)Water bath for tempering agar set at

44-46 C.

model or equivalent.

(d) Hand tally or electronic counting de-

vice (optional),

(e) Pipet containers of stainless steel,

aluminum or pyrex glass for glass pipets.

(f) Petri dish containers of stainless steel

or aluminum for glass petri dishes.

200

<&EPA MICROBIOLOGICAL MANUAL 1978

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(g) Thermometer certified by National

Bureau of Standards or one of equivalent

accuracy, with calibration chart,

(h) Sterile TD (To Deliver) bacteriological

or Mohr pipets, glass or plastic of appropriate

volumes, see Part II-B, 1.8.1.

(i) Sterile 100 mm x 15 mm petri dishes,

glass or plastic.

(j) Sterile pyrex glass flasks, 100 ml,

500 ml, and 1000 ml volume.

(k) Dilution bottles (milk dilution), pyrex

glass, marked at 99 ml volume, screw cap

with neoprene rubber liner.

(I) Bunsen/Fisher gas burner or electric

incinerator.

(m) Inoculation loops, at least 3 mm

diameter, or needles, nichrome or platinum

wire, 26 B&S gauge, in suitable holder.

5.3.4 Media

(a) Sterile Plate Count Agar (Tryptone

Glucose Yeast Agar) dispensed in tubes (15

to 20 ml per tube) or in bulk quantities in

screw cap flasks or dilution bottles. See

Part II-B, 5.1.5.

(b) Sterile buffered dilution water, 99 ± 2

ml volumes, in screw capped dilution bottles.

See Part II-B, 7.

imately 6.3 cm (21/2 in) length in 125 x 16 mm

screw cap tubes.

5.3.S Reagents: Sensitivity of the test

depends on the purity of the reagents used.

Use reagents of the highest purity and

prepare them in water freshly redistilled from

a glass still.

(a) Sodium citrate solution: Dissolve

0.29 g sodium citrate, Na3CeHgO7-2H20, in

500 ml redistilled water.

(b) Ammonium sulfate solution: Dissolve

0.60 g ammonium sulfate, (NH^SO^ in

500 ml redistilled water.

magnesium sulfate, MgSCU • IHaO; 0.17 g

calcium chloride, CaCl2'2H2O; 0.23 g ferrous

sulfate, FeS04 • 7H20; and 2.50 g sodium

chloride, NaCI, in 500 ml redistilled water.

(d) Phosphate buffer solution: Stock

phosphate buffer solution, diluted 1:25 in

redistilled water, see Part II-B, 7.1 in this

Manual.

Boil reagent solution 1-2 min.to kill vege-

tative cells. Store in sterilized glass-stoppered

bottles in the dark at 5 C for several months but

test for sterility before each period of use.

Prepare the salt-mixture solution without the

ferrous sulfate for long-term storage. To use

the mixture, add an appropriate amount of the

freshly prepared and freshly boiled iron salt.

Solutions with a heavy turbidity should be

discarded and a new solution prepared. Bac-

terial contamination may cause turbidity in the

phosphate buffer solution. Discard if this

occurs.

5.3.6 Culture: Isolate a pure culture of

Enterobacter aerogenes (IMViC type —++)

from a polluted stream or sewage sample.

5.3.7 Procedure

(a) Collect 200 ml each, of the unknown

Test Water and the Control Water (laboratory

pure reference water) in sterile 500 ml screw

cap flasks. Boil for 2 minutes and cool to

room temperature.

(b) Label five, sterile 200 ml screw cap

flasks as A, B, C, D, and E. Add the Test

Water, Control Water, and reagents to each

flask as described in the following table:

Reagents

Sodium citrate solution

Ammonium sulfate solution

Salt-mixture solution

Phosphate buffer (7.3 ± 0.1)

Test Water

Control Water

Total volume

Control

Water

2.5

1.5

21.0

30.0

Test (ml)

Unknown

Test

Water

2.5

2,5

2.5

1.5

21.0

30.0

Optional Tests

Nutrient

Available

2.5

1.5

21.0

5.0

30.0

Nitrogen

Source

2.5

1.5

21.0

2.5

30.0

(ml)

Carbon

Source

2.5

1.5

21.0

2.5

30.0

QA/LABORATORY OPERATIONS

201

image:

prepared reagents. Control Water, and Test

Water as a check on contamination.

(d) On the day before performing the

distilled-water suitability test, inoculate a

strain of E. aerogenes onto a nutrient agar

slant. Streak the entire agar surface to

develop a continuous-growth film and

incubate 18-24 hours at 35 C. To harvest

viable cells, pipet 1-2 ml sterile dilution water

from a 99 ml water blank onto the 18-24

hour culture. Emulsify the growth on the

slant by gently rubbing the bacterial film

with the pipet, being careful not to tear the

agar; then pipet the suspension back into the

original 99 ml water blank.

(a) Make a 1:100 dilution of the original

bottle into a second water blank, a further

1:100 dilution of the second bottle into a

third water blank, then 10 ml of the third

bottle into a fourth water blank, shaking

vigorously after each transfer. Pipet 1.0 ml of

the fourth dilution (1:10"8} into each of Flasks

A, B, C, D, and E. This procedure should

result in a final dilution of the organisms to

a range of 30-80 viable cells from each ml

of test solution.

(f) Variations among strains of the same

organism, different organisms, media, and

surface area of agar slopes will possibly

necessitate adjustment of the dilution pro-

cedure in order to arrive at a specific density

range between 30 and 80 viable cells. To

establish the growth range numerically for a

specific organism and medium, make a series

of plate counts from the third dilution to

determine the bacterial density. Then choose

the proper volume from this third dilution,

which, when diluted by the 30 mi in Flasks A,

B, C, D, and E, will contain 30-80 viable

cells/ml. If the procedures are standardized

as to surface area of the slant and laboratory

technic, it is possible to reproduce results on

repeated experiments with the same strain of

microorganism.

(g) Add a suspension of Enterobacter

aerogenes (IMViC type —++} of such density

that each flask will contain 30-80 cells/ml,

prepared as directed above. Cell densities

below this range result in ratios that are not

consistent, while densities above 100

cells/ml decrease sensitivity to nutrients in

the Test Water. Make an initial bacterial

count by plating triplicate 1 ml portions from

each culture flask in plate count agar. Mix

well and incubate Test A through E at 35 C

for 24+2 hours. Prepare final plate counts

from each flask, using dilutions of 1, 0.1,

0.01, 0.001, and 0.0001 ml.

5.3.8 Calculations

(a) For growth-inhibiting substances:

colony count/ml Flask B

Ratio =

colony count/ml Flask A

A ratio of 0.8 to 1.2 (inclusive) shows no

toxic substances; a ratio of less than 0.8

shows growth-inhibiting substances in the

water sample.

(b) For nitrogen and carbon sources that

promote growth:

Ratio =

colony count/ml Flask C

colony count/ml Flask A

growth:

Ratio =

colony count/ml Flask D

colony count/ml Flask A

(d) For carbon sources that promote

bacterial growth:

Ratio =

colony count/ml Flask E

colony count/ml Flask A

{e) Do not calculate ratios b, c, or d when

ratio a indicates a toxic reaction. Ratios b, c,

or d, in excess of 1.2 indicate an available

source for bacterial growth.

5.3.9 Interpretation of Results

(a) The colony count from Flask A after

20-24 hours at 35 C, will depend on the

number of organisms initially planted in

Flask A and on the strain of E. aerogenes

used in the test procedures. This is the

reason the control. Flask A, must be run for

each individual series of tests. However, for

a given strain of E. aerogenes under identical

environmental conditions, the terminal count

202

©EPA MICROBIOLOGICAL MANUAL 1978

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should be reasonably constant when the

initial plant is the same. The difference between

30 and 80 cells in the initial plant in Flask A

will produce a three-fold difference in the final

counts, providing the growth rate remains

constant. Thus, it is essential that the initial

colony counts on Flask A and Flask B be approx-

imately equal to secure accurate data.

(b) When the ratio exceeds 1.2, it may be

assumed that growth-stimulating substances

are present. However, this procedure is ex-

tremely sensitive and ratios up to 3.0 would

have little significance in actual practice.

Therefore, when the ratio is between 1.2 and

3.0, Tests C, D, and E do not appear to be

necessary except in special circumstances.

Flasks D and E will have a ratio of less than 1.2

when the ratio of Flask B to Flask A is between

0.8 and 1.2. The limiting factors of growth in

Flask A are the nitrogen and organic carbon

present. An extremely large amount of

ammonia nitrogen with no organic carbon

could increase the ratio in Flask D above 1.2, or

the absence of nitrogen with high carbon con-

centration could give ratios above 1.2 in Flask E,

with a B:A ratio between 0.8 and 1.2.

(d) A ratio below 0.8 indicates that the

water contains toxic substances, and this ratio

includes all allowable tolerances. As indicated

in the preceding paragraph, the ratio could go

as high as 3.0 from 1.2 without any undesirable

consequences.

5.4 Use Tests for Media, Membranes and

Laboratory Pure Water

The Use Test, a pragmatic approach to

evaluation of materials and supplies, uses the

routine methods of analysis to compare current

and new batches or lots. Such tests operate

on the theory that if a stimulatory or toxic effect

cannot be demonstrated in actual test use,

there is no effect.

When a new shipment or lot of culture

medium, membrane filters or a new source of

laboratory pure water is to be used, or at annual

testing period for water, conduct comparison

tests of the current lot in use (reference lot)

against the new lot (test lot) as follows:

5.4.1 Use a single batch of pure water,

glassware, membrane filters, or other needed

materials as specified to control all other vari-

ables except the one under study.

5.4.2 Use the reference lot and the test lot,

to conduct parallel pour plate or MF plate count

tests on five natural or treated water samples

according to standard procedures in this

Manual.

When comparing sources of pure water,

conduct the tests in parallel using the reference

water and test water separately for all water

purposes in the tests (dilution, rinse, media

preparation).

5.4.3 After incubation, compare the

bacterial colonies from the two lots for size

and appearance. If the colonies on the test lot

are atypical or noticeably smaller than colonies

on the reference lot plates, record the evidence

of inhibition or other problem, regardless of

ctiunt differences.

5.4.4 Count plates and calculate the indi-

vidual counts per ml or per 100 ml, as required

for final reporting values.

5.4.5 Transform the final reporting values

to logarithms.

5.4.6 Compile the log-transformed results

for the two lots in parallel columns and calculate

the + or - difference, d, between the two trans-

formed results for each sample.

5.4.7 Calculate the mean, d, and the

standard deviation sd of these differences.

5.4.8 Calculate the Student's t statistic,

using the number of samples as n:

t =

Sd/i/fT

5.4.9 Use the critical value, 2,78, selected

from a Student's t table at the .05 significance

level for five samples (4 degrees of freedom), for

comparison against the calculated value.

5.4.10 If the calculated t value does not

exceed 2.78, the lots do not produce signifi-

QA/LABORATORY OPERATIONS

203

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cantly different results and the test lot is

acceptable.

5.4,11 if the calculated t value exceeds

2.78, the lots produce significantly different

results. If the test lot results exceed reference

lot results, the test lot is more stimulatory. If the

test lot results are less than the reference lot,

the test lot is less stimulatory.

5,4.12 If condition 5.4.3 or 5.4.11 occurs,

review test conditions, rerun the test and/or

obtain different lots for testing and use.

5.5 Reagents in General: The quality of

test reagents must be assured. They must be

correctly prepared and properly stored. The

following general rules should be followed:

5.5.1 Use ACS or AR grade chemicals that

meet ACS specifications for preparing reagents.

Impurities in uncertified or lesser grades of

chemicals may inhibit bacterial growth, provide

nutrients or fail to produce the desired reaction.

5.5.2 Date chemicals and reagents when

received and when opened for use.

5.5.3 make reagents up to volume in volu-

metric flasks. For storage, transfer to good

quality plastic (polyethylene, polypropylene or

tetrafluoroethylene) or borosilicate glass bot-

tles with polyethylene or other inert plastic

stoppers or caps.

5.5.4 Identify prepared reagents with the

generic name, the concentration, the date pre-

pared and the initials of preparer.

5.5,5 Store reagents under the conditions

recommended by the manufacturer.

5.5.6 Run positive and negative controls

with each series of cultural or biochemical

tests.

5.6 Serological Reagents

5.6.1 Evaluate serological antisera against

known antigens and compare with antisera that

have demonstrated acceptable reactivity. The

quality of commercial serological reagents is

subject to methodology changes and new

knowledge in manufacturing processes. Con-

tinuity of reagent quality depends on com-

pliance of each new reagent with minimum

specifications.

5.6.2 Repeat quality control procedures

each time that reagent batches are prepared,

regardless of the expiration date given by the

manufacturer.

5.6.3 Discard sera or antigens if contami-

nation is discovered.

5.6.4 Select another working dilution if the

level of activity has dropped.

5.7 Fluorescent Antibody Reagents

Highly specific reagents, antigens of high

purity and very specific potent antibodies are

required for fluorescent antibody techniques.

The antisera must exhibit high staining intensi-

ties. Some sera may have high titers in one type

of serological test, but demonstrate poor

staining titer and vice versa.

5.7.1 Store desiccated fluorescent anti-

body (FA) reagents at 4 C, Prepare aliquots of

the rehydrated conjugate in screw-cap glass

vials, freeze and store at -20 C until working

dilutions are prepared.

5.7.2 Test FA reagents for correct reactions

with positive and negative controls before each

use. Results of positive controls should be

within one dilution of the average titer.

5.8 Dyes/Stains: Organic chemicals are

used as selective agents (e.g., brilliant green in

brilliant green lactose bile broth), as indicators

in bacteriological media (phenol red lactose),

and as bacteriological stains (gram stain). Dyes

from any commercial supplier vary from lot to

lot in percent dye, dye complex, insolubles and

inert materials present. Because dyes for micro-

biological uses must be of proper strength and

stability to produce the correct reactions, pur-

chase only dyes which have been certified by

the Biological Stain Commission for biological

use.

5.8.1 Fluorescent Dyes: An important fac-

tor in the preparation of antisera-dye conju-

204

MICROBIOLOGICAL MANUAL 1978

image:

gates is the purity of the fluorescein dye.

Infrared studies reveal that the purity of fluor-

escein isothiocyanate (FITC) commonly used

in FA tests, ranges from 30 to 100% in com-

mercial products. Ideally 100% pure FITC

preparations are used. Dyes of less purity may

be satisfactory if the weight used to label the

protein component of the serum allows for the

impurities, and if the impurities do not increase

non-specific staining.

5.8,2 Check bacteriological stains before

use with at least one positive and one negative

control culture.

6. Membrane Filters

6.1 Government Specifications

6.1.1 The quality and performance of

membrane filters vary with the manufacturer,

type, brand, lot number and storage conditions.

Membranes considered for purchase by federal

agencies must conform to the government

specifications published in 1974 Military

Specifications (4). The detailed specifications

required of the manufacturer by the federal

government are an important advantage to

other users of membrane filters because by

meeting federal requirements, manufacturers

realize it is most efficient to make all mem-

branes to these specifications. Hence, mem-

brane filters purchased from companies selling

to federal agencies meet the federal specifi-

cations.

6.1.2 The specifications list requirements

for pore size, porosity, flow rate, diffusibility,

autoclavability, sterility, diameter, thickness,

bacterial retention and bacterial cultivation.

The gridlines must be easily visible, permanent

and not imprinted into the surface so as to

cause channelling of growth. The grid ink must

not stimulate or inhibit growth.

6.1.3 The pads must have a specified di-

ameter and thickness, limited acidity, no

waxes, sulfites, and other stimulating or inhib-

itive materials, and must absorb uniformly a

specified volume of medium.

6.2 Manufacturer's Quality Assurance

6.2.1 Some manufacturers certify that

their membranes meet stated specifications

on sterility, retention, recovery, pore size, flow

rate, pH, total acidity, phosphate and other

extractables.

6.2.2 Most manufacturers sell sterile

membranes and those packaged for steriliza-

tion by the user. Membranes may be sterilized

by autoclaving, ethylene oxide or irradiation.

6.3 User's Quality Assurance

6.3.1 In addition to the quality assurance

provided by the manufacturer, the user should

determine that the membranes perform satis-

factorily by inspecting and testing each lot he

orders. The membranes should not be mis-

shapen or the gridlines distorted after

autoclaving.

6.3.2 A membrane should allow good dif-

fusibility of medium and be completely wetted

within 15 seconds after it is placed on the

medium substrate with no dry areas indicated

by colorless or lighter-colored areas.

6.3.3 After incubation, the colonies

should be of the expected size, with defined

shape and clearly delineated edges. The color

and morphology of the colonies must be typi-

cal of those defined by the test procedure.

6.3.4 The analyst should observe that the

gridline ink does not "bleed" across the sur-

face, or restrict colony development on or ad-

jacent to the cross-hatching. Colonies should

be evenly distributed across the membrane

surface. Membranes containing sizable areas

with no colony development are questionable.

6.4 ASTM Test Procedures

Because of variable recoveries of micro-

organisms on different membrane filters, there

is a critical need for standard procedures for

evaluating membrane filters. Procedures are

being developed by Committee D-19.08.04 of

ASTM for physical, chemical and microbiologi-

cal characteristics of membrane filters. The

procedures are intended for the larger labora-

tory and the manufacturer to test new batches

QA/LABORATORY OPERATIONS

205

image:

or lots of MF's. Brief descriptions of these tests

for bacterial retention, inhibitory effects, re-

covery, extractables and flow rate follow.

6.4.1 Bacterial Retention Test

The test is based on filtration of a standard

culture through a 0.45 /urn MF into a broth.

Sterile equipment and aseptic techniques must

be used. Five randomly-selected membranes

from five randomly-selected packages should

be tested. Control membranes should be taken

from the same package as the test mem-

branes.

(a) Add 140 ml of double strength Trypti-

case soy or Tryptic soy broth to a liter vacuum

flask and attach vacuum tubing. Wrap flask in

kraft paper and sterilize.

(b) Using an 18 hour broth culture of

Serratfa marcescens, prepare a final dilution in

0.1% peptone, containing 1000 cells per

100 ml.

ratus using a sterile vacuum flask containing

the broth. Insert the test filter, turn on vacuum

and pour in 20 ml of sterile rinse water to set

the membrane. Filter 100 ml of the culture

suspension. Rinse funnel twice with 20 ml of

peptone water.

{d) After the filtration remove funnel and

transfer the membrane filter to a Trypticase

soy or Tryptic soy agar plate. Then aseptically

remove membrane filteration base from the

flask and insert a sterile stopper. Repeat

filtrations four more times with sterile MF

assemblies, culture flasks and membranes.

(e) Incubate agar plates and flasks for 48

hours at 35 C. Examine membranes for growth

outside of filtering areas which indicates a

leaky filtration assembly that could cause a

false positive test. Turbidity in the culture flask

indicates bacterial growth and failure of the

membrane to retain the 0.2-0.3 um sized bac-

teria, but does not indicate the numbers of

organisms passing the filter.

(f) Turbidity indicates filter failure. Repeat

the test and control procedures. •

6.4.2 Inhibitory Effects: The test for inhibi-

tory effects compares membrane filter counts

on one or more test lots of membrane filters,

with spread plate counts on Plate Count Agar

using a specific pure culture of Escherichia coli

(IMViC + H ).

(a) Prepare a dilution of the stabilized 24-

hour culture in 0.1 % peptone dilution water so

that 0.2 ml contains 30-150 viable cells. Add

about 20 ml of sterile dilution water to the

funnel before filtration.

(b) Filter five replicate sample volumes of

0.2 ml through five replicate membrane filters

of each MF test lot and place each on a plate of

Tryptic soy agar.

plates in random fashion to avoid a time effect,

following a randomization table.

(d) With the same pipet, deliver five addi-

tional 0.2 ml volumes to the surface of five

plates of Tryptic soy agar. The samples are

distributed evenly over the surfaces of the

plates with sterile glass rods.

(e) Incubate the membrane filters at 44.5 C

and agar plates at 35 C for 24 hours. Count the

colonies. Compare recoveries on spread plates

and membrane filters. Acceptable filters should

recover some set percentage of the spread

plate count. This percentage should be estab-

lished by each laboratory based on previous

performance by a known acceptable lot.

6.4.3 Recovery (5): The procedure for re-

covery compares the fecal coliform counts on

test membranes to the counts on spread plates

using M-FC agar substrate. Four polluted

waters and one raw sewage sample are

analyzed.

(a) To determine sample test volumes, pre-

pare serial dilutions of each sample in 0.1%

peptone water to produce a suspension con-

taining approximately 20-60 fecal coliforms

per 0.1 ml. Hold original samples at 4 C. Deter-

mine the fecal coliform density of each of the

samples or dilutions by membrane filter or

spread plate test. Read the results after 22-24

hours incubation at 44.5 -±_ 0.2 C.

206

•SEPA MICROBIOLOGICAL MANUAL 1978

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(b) If the fecal coliform density in the raw

water samples is less than 10 per 0.1 ml, seed

the sample with raw sewage, allow to stabilize

at 4 C for 24 hours and test as in (a). Then

proceed with the standard test procedure.

for the test membranes and the spread plate

controls.

(d) Perform the membrane filter tests ac-

cording to the procedure described in Part II-C,

3 of This Manual.

(e) Prior to beginning the spread plate

tests,air drythesurfaceof the M-FC agar con-

tained in 100 ml petri dishes. Aseptically de-

liver 0.1 ml of the selected sample dilution to

the agar surface and spread with a sterile bent

glass rod. Allow the sample aliquot to be com-

pletely absorbed before inverting the dish.

(f) Alternate MF tests with spread plate

controls to randomize systematic errors.

(g) Insert petri dishes into waterproof bags

or seal with waterproof tape, and submerge in

the waterbath incubator. Incubate for 22-24

hours at 44.5 + 0.2 C. Record the temperature

continuously during the incubation period. Af-

ter incubation, remove plates and examine.

(h) Count the blue colonies. If more than

one dilution was prepared, select the plates

with between 10 and 100 colonies, but prefer-

ably with 20-60 colonies. Calculate the arith-

metic mean of the five replicate fecal coliform

counts and the five replicate spread plates.

Determine percent recovery:

or suspected to have a high concentration of

pseudomonads or aeromonads, perform the

cytochrome oxidase test. As in the verification

procedure, a minimum negative oxidase test

confirmation of 80% should be achieved for

the membranes to be considered acceptable.

6.4.4 Extractables

(a) Total Extractables

(1) Dry filters for 15 minutes at 70 C then

bring to room temperature in a desiccator.

Weigh to constant weight on a four-place ana-

lytical balance.

(2) Boil filters in 100 ml of distilled water

for 20 minutes. Remove the filters and dry at

70 C for 60 minutes. Bring filters to room

temperature in a desiccator and reweigh to

constant weight. Weighings shall be to the

nearest 0.1 milligram.

Original Weight - Weight After Extraction

Original Weight

X 100 =

Percent

Extractables

(b) Specific Extractables

(1) Immerse filter in ASTM Type 1 reagent

grade water for 24 hours (6).

(2) Remove filter and assay the extract for

metals, total organics, phosphorus and

ammonia.

MF X Count

Spread Plate X Count

X 100 =

Recovery

(i) Verification: Pick 20 blue colonies from

each of 2 randomly-selected filters and 2

spread plates. If plates contain less than 20

colonies, pick all blue colonies. Verify the colo-

nies in EC media as described in Part III-C, 4 of

This Manual. To be' acceptable, 80% of the

colonies must verify. If the samples are known

6.4.5 fjow .Rate

Water flow rate shall be determined by

timing the passage of 500 ml of particle-free

distilled water through a filter at 25 C and at a

differential pressure of 70 centimeters of mer-

cury. Particle-free water for this test is produced

by passage of a high purity water through a

0.2 /im membrane filter, three times in succes-

sion, at 25C and at a differential pressure of

177 cm of mercury.

QA/LABORATORY OPERATIONS

207

image:

The flow rate =

X 500 ml

EFA cm2

•= ml/min/cm

Where: t = experimental time in minutes for

filtering 500 ml of particle-free dis-

tilled water

and

EFA - effective filtering area of a 47 mm diam-

eter filter

An average flow rate reported by manufac-

turers for 0.45 jum pore size, 47 mm diameter

filters Is 65 ml/min/cm2.

7. Culture Media

Since even the best cultural procedure is

ineffectual if the medium is not prepared cor-

rectly, it is important to train personnel to use

the best materials and techniques in media

preparation, storage and application. Some

factors that must be considered follow;

7.1 Ordering Media

7.1.1 Order media in quantities to last up

to one year. Always use oldest stock first.

7.1.2 Whenever possible and practical,

order media in 1/4 pound multiples rather

than one pound bottles to insure sealed pro-

tection of the supply as long as possible. Most

deterioration nf media occurs after bottles are

opened.

7.1.3 Maintain an inventory record of me-

dia: the dates received, sizes, number of units,

etc. Review the inventory quarterly for neces-

sary reordering. Date each bottle when re-

ceived and when opened. Bottles should be

inspected for color changes, caking or other

indication of deterioration. Discard such bot-

tles and reorder.

7.2 Holding Time Limits for Media

Because of the myriad of environmental

conditions affecting media, and the unique

composition and sensitivities of different

media, it is impossible to establish universal

time limits for holding unopened bottles of

media. Therefore, a conservative and protec-

tive recommendation is to limit the storage of

unopened bottles of cultural media to two

years. This limit should insure good perfor-

mance of media with prop'er storage

conditions.

7.3 Preparation of Media

7.3.1 In high humidity areas, store opened

bottles of media in a large hinged-door desic-

cator. Open bottles as briefly as possible during

the weighing process and return to the desic-

cator immediately after use.

7.3.2 Discard opened bottles of media 6

months after initial use.

7.3.3 Weigh media to the nearest 0.1

gram on a single pan, top loader balance as

quickly as possible.

7,3.4 Keep balance out of drafts and away

from high humidity. Use a plastic shield around

the balance to protect from drafts.

7.3.5 Clean the balance and surrounding

area immediately after weighing media.

7.4 Solution of Media

7.4.1 Check cleanliness of glassware. Use

bromthymol blue indicator to spot-check pH of

glassware (5.1.2 in this Section).

7.4.2 Prepare media in a deionized or dis-

tilled water of proven quality. Ultra-pure water

from a recirculating deionization system is re-

commended. Measure volumes with the great-

est accuracy possible using the proper pipet or

graduate (Part II-B, 1.8).

Check the pH of media after solution and

after sterilization using a laboratory model pH

meter. Enter results in the QC record book.

The reading should be within 0.2 units of the

stated value. If not, discard batch and remake.

If the pH is still incorrect, use another bot-

tle or batch. pH differences indicate a prob-

lem of distilled water quality, deterioration of

208

V>EPA MICROBIOLOGICAL MANUAL 1978

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medium or improperly-prepared medium. The

problem must be identified and corrected. The

problem should be carefully documented in

the quality control record book and reported to

the manufacturer if the data indicate the me-

dium as the source of error.

7.4.3 Note in the QC record any unusual

color development, darkening, or precipitation

of media. Check the sterilization time and tem-

perature. If there is a drastic change in appear-

. ance, discard medium and remake it. If the

problem still exists, remake the batch using a

different lot of medium.

7.4.4 Containers for preparation of

batches of broth or agar should be twice the

volume of the medium being prepared.

7.4.5 Media should be stirred continu-

ously while being heated to avoid burning.

Agar media are particularly susceptible to

scorching and boilover. The only insurance

against scorching or boilover is use of a boiling

water bath for small batches of media or con-

stant attention while heating larger batches on

a hot plate or burner. A combination hot plate

and magnetic stirrer is recommended for solu-

tion of media.

7.4.6 Bottles, tubes or plates of prepared

media are identified and dated.

7.5 Sterilization by Autoclave

7.5.1 Media should be sterilized for the

minimal time specified by the manufacturer.

The amount of time required to sterilize a me-

dium in an autoclave will vary with the type

and volume of medium and the size and shape

of containers. See the table in Part II-B, 4.3

on sterilization.

7.5.2 Since the potential for damage to

media increases with increased exposure to

heat, the amount of lag time before the auto-

clave is at full pressure and temperature can

be a critical factor in whether media are dam-

aged. The danger from an extended heating

period is reduced with use of a double-walled

autoclave which allows the operator to main-

tain full pressure and temperature in the jacket

between loads. As soon as the autoclave is

loaded and closed, steam can be admitted to

the chamber and in a relatively short time, full

pressure and temperature are developed in the

chamber. The total exposure time for media

sterilized 15 minutes at 121 C should not

exceed 45 minutes.

7.5.3 Avoid overcrowding in an autoclave

which reduces its efficiency. Large volumes of

media should be preheated to reduce the lag

period before placing them in the autoclave.

7.5.4 Remove sterile media from the auto-

clave as soon as pressure is at zero.

7.5.5 Media must be discarded if contami-

nation is suspected. Reautoclaving is^ not

permitted.

7.5.6 A preventive maintenance contract

is recommended for autoclaves. It should in-

clude checks on the accuracy of pressure and

temperature gauges and recorders and opera-

bility of safety valve.

7.5.7 Check the effectiveness of steriliza-

tion weekly, using strips or ampuls of Bacillus

stearothermophilus spores. Commercial pack-

ages of these spores are available in ampuls of

growth-indicator media. Sterilization at 121 C

for 12-15 minutes will kill the spores. Incubate

the autoclaved cultures at 55-60 C and if

growth occurs, sterilization is inadequate.

7.6 Sterilization by Filtration

7.6.1 Non-autoclavable solutions can be

sterilized by membrane filtration. With careful

preparation of the sterile filtration and receiving

apparatus, passage of a solution through a

0.2 pm membrane filter will produce a sterile

solution.

7.6.2 The filtration and subsequent sterile

dispensations should be performed in a safety

cabinet or bio-hazard hood.

7.7 Gaseous Sterilization

Equipment, supplies or other solid or dry

materials which are sensitive to heat can be

QA/LABORATORY OPERATIONS

209

image:

sterilized by long exposure to ethylene oxide

gas (ETO) using available commercial equip-

ment. Check sterility with culture of B, subtilus

var. nfgerspores,

7.8 Use of Agars, Broths, and Enrich-

ment Media

7.8.1 Agars

{a) Agar plates to be used for streaking or

spread plates are kept open slightly for 15

minutes after pouring or after taking out of

refrigerated storage to evaporate free mois-

ture which would cause confluent growth on

streak plates.

(b) Agar plates used for MF and spread

plate work must be free of lumps, uneven

surfaces, pock marks, bubbles or foam which

prevent good contact between the agar and

the membrane or uniform growth on spread

plates.

pering water bath at 44-46 C but no longer than

three hours. As a safety precaution against the

use of agars which are too hot and might kill

cells, place a bottle of agar in the same boiling

water or under the same autoclave conditions

as the agars to be used. After the agar is melted,

transfer agar to a tempering bath. Insert

thermometer in the agar bottle and use it to

determine when the temperature of the agars

is at approximately 44-46 C and safe for use

in pour plates.

7.8.2 Broths

(a) Handle sterile MPN fermentation tubes

of lauryl tryptose broth or brilliant green bile

broth carefully prior to use. Shaking can en-

trap air In the inner tubes and produce a false

positive. Examine fresh tubes before use and

discard any with a bubble,

(b) Reduced media such as thioglycollate

broth oxidize in storage. Before use, these

broths must be heated in boiling water for

20-30 minutes to reduce the medium.

7.8.3 Enrichments

(a) Bring the base medium to 44-46 C

before addition of a labile constituent.

(b) Warm enrichments such as blood or

serum to room temperature before adding to a

base medium.

dium, prepare plates or tubes as soon as possi-

ble. Do not hold the batch medium in a water

bath for more than 10 minutes.

7.9 Storage of Media

7.9.1 The recommended time limits for

holding prepared media in the laboratory are:

MF broths in screw-cap

flasks at 4 C

MF agars in plates with

tight-fitting covers

at 4 C

Agar or broths in loose-

cap tubes, at 4 C

Agar or broths in screw-

cap tubes, tightly

closed, at 4 C

Agar plates (non-MF) with

loose-fitting covers, in

sealed plastic bags, at

4 C

Large volumes of agar

screw-cap flasks or

bottles tightly-closed,

at 4 C

hours

(Work Week)

Two

Weeks

One

Week

Three

Months

Two

Weeks

Three

Months

7.9.2 Store fermentation tube media in

the dark at room temperature or 4 C. If refriger-

ated, incubate overnight at room temperature

to detect false positive gas bubbles.

7.9.3 Since loss of moisture is a major

problem of storage, screw-capped tubes and

flasks are recommended. Prepoured agar

210

MICROBIOLOGICAL MANUAL 1978

image:

plates can be sealed in plastic bags to retain

moisture and refrigerated.

7.9.4 A simple check for loss of moisture

in broth tubes can be made by marking the

original level in several tubes of each batch

and then observing the loss of moisture over

time. If the estimated moisture loss exceeds

ten percent, discard the tubes of broth.

7.9.5 Protect media containing dyes from

light. If color changes are observed, discard

the medium.

7.9.6 Prepared sterile broths and agars

are available from commercial sources. Their

use may be advantageous when analyses are

done intermittently, when staff is not available

for such preparation work, or when cost of

their use can be balanced against other factors

of laboratory operation. However, purchase of

prepared media does not reduce the responsi-

bility of the laboratory for checking the perfor-

mance of the media, regardless of the stated

quality control practices of the manufacturer.

7.10 Quality Control of Prepared Media

7.10.1 Maintain a book with a'complete

record of each batch of medium prepared.

Include the date, the name of the medium

and lot number, amount of medium weighed

out and volume of medium prepared, the

record of sterilization, pH measurements,

pH adjustments made, special handling or

preparation techniques, e.g., use of heat-

sensitive compounds or components, and

name of preparer.

7.10.2 Incubate five percent of each batch

of medium for 2 days at 35 C and inspect for

growth.

7.10.3 Check each batch of medium when

used by inoculating 2 tubes or plates with pure

cultures of species producing positive and

negative reactions for that medium.

7.10.4 Test new batches of differential

media by inoculating with organisms of known

fermentative or other biochemical ability. Simi-

larly, enrichment and selective media are

tested for productivity of the desired microor-

ganisms and inhibition of other microorga-

nisms. Tables IV-A-4 and IV-A-5 list a group of

organisms with the broths, agars, biochemical

tests and reactions to which they can be

applied.

7.10.5 Record sterility and positive/nega-

tive performance checks in the media prepara-

tion portion of the quality control log.

QA/LABORATORY OPERATIONS

211

image:

TA1LE IV-A-1

Monitoring Laboratory Equipment

Item

Monitoring Procedure

1. Balance

a. An analytical balance with a sensitivity of 1 mg or less at a

10 g load should be used for weighing 2 g or less. For larger

quantities, a balance with accuracy of 50 mg at a 150 g load

should be used,

b. Check balance monthly with a set of certified class S weights.

c. Wipe balance and weights clean after each use.

d. Protect weights from laboratory atmosphere and corrosion.

e. Contract with a qualified expert for balance maintenance on an

annual basis.

2. pH Motor

a. Compensate for temperature with each use.

b. Date standard buffer solutions when first opened and check

monthly with another pH meter. Discard the buffer solution if

the pH is more than ±0.1 pH unit from the manufacturer's

stated value or if it is contaminated with microorganisms.

c. Standardize with at least one standard buffer (pH 4.0, pH 7.0,

or pH 10.0) before each use.

d. Do not re-use buffer solution.

e. Have meters inspected at least yearly as part of a maintenance

contract.

3, Water Daionlzer

a. Monitor water for conductance daily. Monitor trace metals and

other toxic or nutritive compounds monthly. See Table IV-A-3.

b. Replace cartridges as indicated by manufacturer or as indicated

by analytical results.

c. Monitor bacterial counts at exit point of unit Replace the

cartridges when standard plate count exceeds 10,000/ml.

4. Watar Still

a. Drain and clean monthly according to instructions from the

manufacturer.

b. Drain and clean distilled water reservoir quarterly.

c. Check distilled water continuously or daily using a conductance

meter. See Table IV-A-3.

d. Conduct chemical tests on water to detect toxicity or stimulation

effect. See Table IV-A-3.

e. Conduct standard plate counts monthly on stored water and

clean out reservoir if count > 10,000/ml.

212

SEPA MICROBIOLOGICAL MANUAL 1978

image:

TABLE IV-A-1

(continued)

Monitoring Laboratory Equipment

Item

Monitoring Procedure

i. Dispensing Apparatus

a. Check accuracy of dispensation with an NBS class A, graduated

cylinder at the start of each volume change and periodically

throughout extended runs,

b. Lubricate moving parts according to manufacturer's instructions

or at least once per month,

c. Correct immediately any leaks, loose connections or malfunctions,

d. After dispensing each type of medium, pass a large volume of

hot distilled water through dispenser to remove traces of agar

or medium,

e. At the end of the work day, break down unit into parts, wash

well, rinse with distilled water and dry.

6. Ultraviolet

Sterilizer

a. Remove plug from outlet and clean ultraviolet lamps monthly by

wiping with a soft cloth moistened with ethanol.

b. Test ultraviolet lamps with light meter quarterly; if they emit

less than 80% of their rated initial output, replace them.

c. Perform spread plate irradiation test quarterly, see This

Section, 4,2.1.

7. Membrane Filter

Equipment

a. Check funnel support for leaks.

b. Check funnel and funnel support to make certain they are

smooth. Discard funnel if inside surfaces are scratched.

c. Clean thoroughly after each work day.

8. Spectrophotometer

a. Maintain quality control and calibration check as recommended

by the manufacturer.

b. Have inspected yearly by a factory maintenance man.

9. Centrifuge

a. Check brushes and bearings for wear every six months.

b. Check rheostat control against a tachometer at various loadings

every six months to ensure proper gravitational fields.

QA/LABORATORY OPERATIONS

213

image:

TABLE IV-A-1

(continued)

Monitoring Laboratory Equipment

Item

Monitoring Procedure

10. Microscope

a. Allow only trained technicians to use. • '

b. Appoint one laboratory worker to be responsible for the care

of the microscope.

c. Clean optics and stage after every use. Use only lens paper

for cleaning.

d. Keep covered when not in use.

e. Establish annual maintenance on contract.

f. Maintain in one location if possible.

11. Microscope,

Fluorescence

a. Allow only trained technicians to use microscope and light

source.

b. Keep a log of lamp operation time.

c. Monitor lamp with meter. See Section 4.2.2. Replace the

lamp when < 80% of original fluorescence is observed.

d. Check lamp alignment particularly if bulb has been changed.

Realign the fluorescent light source if necessary.

e.. Use known 4+ fluorescence slides as controls.

12. Safety Cabinet

(Hood)

a. Check filters monthly for plugging or obvious dirt accumulation.

Clean or replace filter as needed.

b. Check cabinet for leaks and for rate of air flow every three

months.

c. Expose blood agar plates to air flow for one hour once per

month to measure contamination.

d. Remove plug from the outlet and clean ultraviolet lamps every

two weeks by wiping with a soft cloth moistened with ethanol.

e. Test ultraviolet lamps quarterly with a light meter. If lamp

emits less then 80% of the rated output, replace lamp.

f. Perform maintenance as directed by the manufacturer.

g. Purchase and use a pressure monitor control device to measure

efficiency of air flow.

214

<&EFA MICROBIOLOGICAL MANUAL 1978

image:

TABLE IV-A-1

(continued)

Monitoring Laboratory Equipment

Item

Monitoring Procedure

13. Thermometers and

Recording Devices

a. Check the accuracy of thermometers and temperature recording

instruments, in the monitoring range, at least annually against a

certified thermometer or one of equivalent accuracy. Thermometer

graduations should not exceed the 0.2 or 0.5 C deviation permitted

in the analytical method. Check mercury columns for breaks.

b. Record calibration checks in quality control (QC) record. Mark

NBS calibration correction on each thermometer or on the outside

of the incubator, refrigerator or freezer containing the thermometer.

c. Record daily temperature checks on charts and keep for at least

three years. A simple, one year chart is shown in Figure IV-A-1.

14. Water Bath

a. Check and record temperature daily. Bath must maintain the uniform

temperature needed for the test in use.

b. Maintain accurate thermometer completely immersed in water bath.

c. A recording thermometer and alarm system are recommended.

d. Clean monthly.

e. Use only stainless steel, rubber, plastic-coated, or other

corrosion-proof racks.

15. Refrigerator at 4 C

a. Check and record temperature daily.

b. Clean monthly.

c. Require identification and dating of all material.

d. Defrost unit and discard outdated materials in refrigerator and

freezer compartments every three months.

16. Hot Air Oven

8. Test performance with spore strips or suspensions quarterly.

b. Equip and monitor sterilization with a thermometer accurate in

160-180 C range.

17. Freezers

a. Check temperature and record daily.

b. Use of recording thermometer and alarm system recommended.

c. Require identification and dating of all materials.

d. Clean and defrost freezer every six months. Discard outdated

materials.

QA/LABORATQRY OPERATIONS

215

image:

TABLE 1V-A-1

(continued)

Monitoring Laboratory Equipment

Item

Monitoring Procedure

18. Autoclave

a. Record temperature and pressure for each run. Recording

thermometer recommended.

b. Verify that autoclave maintains uniform operating temperature.

e. Check operating temperature with a min/max thermometer on a

weekly basis.

d. Test performance with spore strips or suspensions weekly. If

evidence of contamination occurs, check until the cause is

identified and eliminated.

e. Procure semi-annual preventive maintenance inspections.

19. Incubator (Air/Water-Jacket)

a. Check and record temperature daily.

b. If partially-submersible glass thermometer is used, bulb and stem

must be immersed in water to the mark on stem.

c. Measure temperatures daily on top and bottom shelves.

Periodically measure temperature on all shelves in use.

d. Expand test points proportionately for walk-in incubators,

e. Recording thermometer and alarm system are recommended.

f. Locate incubator where room temperature does not go outside

of the 16-27 C range.

216

MICROBIOLOGICAL MANUAL 1978

image:

Instrument

Temperature

Room

Read daily.

Record temperature in space provided.

Date Jan. Feb.-Mar. Apr. May June Jul.Aug. Sep. Oct. Nov. Dec.Date

•9-

• 1

• 19- •

FIGURE IV-A-1. Equipment Operation Temperature Record.

OA/LABORATORY OPERATIONS

217

image:

TABLE IV-A-2

Glassware Maintenance

Item

Monitoring Procedure

1, Utensils and

Containers for

Media Preparation

Use utensils and containers of non-corrosive and non-contaminating

materials such as pyrex glass, stainless, steel or

aluminum.

2. Glassware (Reusable)

a. With each use, examine glassware especially screw-capped dilution

bottles and flasks, for chipped or broken edges and etched

surfaces. Discard chipped or badly-etched glassware.

b. Inspect glassware after washing. If water beads excessively on the

cleaned surfaces, run the glassware through again.

c. Test for acid or alkaline residues by adding bromthymol blue indicator

to representative glassware items (see 5.1.2 in This Section).

d. Test for residual detergent by the test in 5.1.3, This Section.

218

MICROBIOLOGICAL MANUAL 1978

image:

TABLE IV-A-3

Laboratory Pure Water for Bacteriological Testing

Parameter

Ideal

Monitoring

Frequency

Limit

Chemical Tests

Conductivity

Total Organic

Carbon

Trace Metal, Single

Trace Metals, Total

(Od, Cr, Cu Ni, Pb, Zn)

Ammonia/Amines

Free chlorine

Continuously

or with each

use

With each use

Monthly

With each use

1-2 £f mhos/cm

at 25 C

5.5-7.5

<1.0 mg/liter

<0,05 mg/liter

<1.0 mg/liter

<0.1 mg/liter

Biological Tests

Standard Plate Count

Fresh Water

Stored Water

Water Suit-

ability Test

Monthly

Yearly and when

conditions change

< 1000 bacteria/ml

< 10,000 bacteria/ml

Ratio: 0.8-3.Q

Water Use-Test

Yearly and when

conditions change

Calculated

t value

< 2.78

QA/LABORATORY OPERATIONS

219

image:

TABLE IV-A-4

Quality Control of Media

Medium

Control Cultures

Expected Results

M-Endo MF Broth

or Agar

Escherichia coli

Enterobecter aerogenes

Achromobacter sp

Pseudomonas sp

Salmonella sp

Golden green metallic sheen

Red colonies

Red colonies if medium overheated

M-FC Broth

or Agar

Brilliant Green Bile

Lactoie Broth

Lauryl Tryptose

Broth

E coll

K. pneumonlae

£ aerogenes

£ coli

£ aerogenes

C. freundil

Staph. aureus

£ coli

£ aerogenes

S. typhlmurlum

S. aureus

Blue colonies

No growth

Growth with gas

No growth

Growth with gas

Marked to complete inhibition

Levlne's Eosln

Methylana Blua Agar

£ coli

£ aerogenes

C. freundli

Salmonella sp

Klebsiella sp

Nucleated black colonies with

golden green metallic sheen

Pink colonies with dark centers

Colorless colonies

Large brown mucoid colonies

Xylose Lysine

Deioxycholata Agar

(XLD)

Salmonella sp

Klebsiella sp

£ coli

£ aerogenes

Red colonies, to red with black centers

Yellow colonies

Bismuth Sulfito Agar

Salm. typhosa

Other Salmonella sp

Coliforms

Black colony with black or brownish-

black zone, with or without sheen

Raised green colonies

Green colonies

Brilliant Green Agar

Salmonella sp

£. coli

P. vulgarls

Pink-white opaque colonies surrounded

by brilliant red zone

Inhibition or yellow green colonies

Marked to complete inhibition or red

colonies

220

4»EF¥V MICROBIOLOGICAL MANUAL 1978

image:

TABLE IV-A-4

(continued)

Quality Control of Media

Medium Control Cultures Expected Results

KF Streptococcus Agar Strep, faecalis Pink to red colonies

Strep, pyogones No growth

S. auraus No growth

£ coll No growth

PSE Agar S. faecalis Black colonies

£ coll No growth

S. auraus No growth

QA/LABORATORY OPERATIONS 221

image:

TABLE IV-A-5

Quality Control of Biochemical Tests

Test

BH1 Broth at pH 9,6

BHI Broth with 6.5%

NaC1

Arginlne Dehydrolase

(Monitor's medium)

Lysino Decarboxylase

(Mooltar's medium}

Ornlthlne Docarbox-

ytase (Moollar's

medium}

Indolo Production

(Tryptophana Broth)

Methyl Red (Buffered

Peptone Glucose Broth)

Voges-Proskauer

(Buffered Peptone

Glucose Broth)

Citrate Utilization

(Simmons Citrate Broth)

Urease Production

(Christansen's Urea

Ag«r)

Catalase

(BHI agar slant)

Control Culture

Strep, faecalis

Strep, mltis-salivarius

S. faecalis

S. mitis-salivarius

Salm. typhlmurium

Satm. flexneri

S. typhlmurium

S. flexneri

S. typhimurium

S. flexneri

Escherichia coti

Salmonella sp

Enterobacter aorogenas

E coli

£. aerogenes

£ aaroganes

£ col/

£ aeroganes

E. coli

Proteus mirabilis

Salmonella sp

S. aureus

S. faecalis

Positive:

Negative:

Positive:

Negative:

Positive:

Negative:

Positive:

Negative:

Positive:

Negative:

Positive:

Negative:

Positive:

Negative:

Positive:

Negative:

Positive:

Negative:

Positive:

Negative:

Positive:

Negative:

Expected Results

growth

no growth

growth

no growth

alkaline reaction

reddish violet color

yellow color

alkaline reaction

reddish violet color

acid, yellow reaction

alkaline reaction

reddish violet color

yellow color

red color

orange/yellow color

red color

no change

pink color

no color change

growth, change to

blue color

no color change,

no growth

color change, pink

to red

no color change

bubbles

no bubbles

222

MICROBIOLOGICAL MANUAL 1978

image:

TABLE IV-A-5

(continued)

Quality Control of Biochemical Tests

Test

Control Culture

Expected Results

Cytochrome Oxidase

(Alpha-napthol and para-

amlno-dimethylaniline

oxalate)

Phenylalanine

Oeaminase

(Pfienylalanine Agar)

Malonate Utilization

(Malonate Broth)

Milk, Methytene

Blue, 0.1%

Nitrate Reduction

(Potassium Nitrate

Broth)

2, 3, 5-Triphenyl

Tftrajzotium Chloride

In TG Agar

Tel I u rite Agar

Beta-Hemolysis

in Blood Agar

Hydrogen Sulfide

(Triple Sugar

Iron Agar)

Pseudomonas aeruginosa

£ coli"

S. aureus

P. mirabilis

Salmonella sp

£ coli

K. pneumoniae

£ coli

S, faecalis

Group Q Streptococci

Strep, salivarius

P, aeruginosa

C. perfringens >

£ coli

P, aeruginosa

S. faecalis

Strep, faecium

S. faecalis

S. faecium

S. faecalis

var. zymogenes

S. faecalis

•£. coli

P. vulgaris

S. typhimurium

Positive: blue color

Negative: no change

Positive: green color

Negative: no color change

' Positive: blue color change

Negative: no growth or color

change

Positive: reduction of

methylene blue

Negative: no growth

Peptonization and digestion

Acid, coagulation and gas

Positive: red color change

Negative: no color change

Positive: reduction of TTC

(red color)

Negative: no color change

Growth

No growth

Positive: lysis of red blood cells

Negative: no lysis of red blood cells

Slant Butt H2S Production

A A G

A A G +

K A G +

QA/IABORATORY OPERATIONS

223

image:

TABLE IV-A-5

(continued)

Quality Control of Biochemical Tests

Test

Lyilna

(Lytino Iron Agar)

Golatin Liquefaction

ot 20 C

(Nutrient Gelatin)

Control Culture

S. typtiimurium

S. flexneri

S. marcascens

S. faecalis

var. liquefaciens

S. faecalis

C, perfringens

E coli

Expected Results

Slant

Positive:

Negative;

Positive:

Negative:

Butt H25

liquefaction

no liquefaction

liquefaction

no liquefaction

> Production

•f

REFERENCES

1. American Public Health Association, 1972. Standard Methods for the Examination of Dairy

Products, (13 ed.) American Public Health Association, Inc., Washington, DC. RODACfp. 192;

Swab: pp. 43-44; and Air: p. 44.

2. American Public Health Association, 1975. Standard Methods for the Examination of Water and

Wastswater (14 ed.) American Public Health Association, Inc., Washington, DC. p. 885,

3. Geldretch, E. E. and H. F. Clark, 1965. Distilled water suitability for microbiological applications.

J. Milk and Food Tech. 28:351.

4. Military specifications for Disk, Filtering, Microporous, Hydrosol Type, 47 mm diameter 100s.

MlL-D-37005 (DSA-DM) 5 September, 1973. Directorate of Medical Materiel, Defense Personnel

Supply Center, Dept. of Defense, Philadelphia, PA.

5. Tentative method for evaluating water testing membrane filters for fecal coliform recovery.

D3508-76T. 1978 Annual Book of ASTM Standards, Part 31, p. 1127.

6. Standard specification for reagent water. D1193-77. 1978 Annual Book of ASTM Standards,

Part 31, p. 20.

224

MICROBIOLOGICAL MANUAL 19f6

image:

PART IV. QUALITY ASSURANCE

Section B Statistics for Microbiology

The Section is divided into five major

areas of statistical measure:

1. Measures of Central Tendency

2. Measures of Dispersion

3. Normal Distribution

4. Poisson Distribution

In this Section the computational formats

for the more commonly used measures in sta-

tistics are described. In the following discus-

sion let Xj denote a typical observed result so

that (x ^ x2,....,xn) represents a sample of n ob-

servations. A good reference bookf or further

details on these parameters is Dixon and

Massey (1).

1. Measures of Central Tendency

1.1 The Arithmetic Mean: The most com-

monly used measure of central tendency is the

arithmetic mean which is often simply called

"the mean". Denote the sample mean by X and

the population mean, of which X is an esti-

mate, by u. The computational formula is:

An example of microbiological data is shown

in Table IV-B-1.

TABLE IV-B-1

Microbiological Results, Count/100 ml

110

130

170

220

Arithmetic

Geometric

220

230

280

330

Mean, "X

Mean, Xg

Median

Mode

Standard Deviation (S)

Range

330

490

790

950

1100

= 372.7

= 287.8

= 305

= 330

= 293.2

= 1021

The sample mean is calculated as follows:

where . I x, = x^

4- ... Xn

X =

6709

= 372.7

QA/STATISTICS

225

image:

1.2 The Geometric Mean: A second mea-

sure of central tendency that is preferred for

certain distributions such as the Poisson. It will

be discussed later, in 4.3 but is defined as:

is tedious. It is relatively simple to show the

following relationship:

S =

- 1

n(n - 1)

The geometric mean of the data in Table IV-B-1

is 287.8.

1.3 The Median: Another measure of cen-

tral tendency is the median. The median is the

value such that half of the other values are

greater and half are less. To find the sample

median, the data are arranged in ascending or

descending order and the middle value is

picked. When there is an even number of ob-

servations, the average of the two middle val-

ues Is taken. For the data in Table IV-B-1, the

median is 305.

1.4 The Mode: The mode, one other mea-

sure of central tendency, is the most frequently

occurring value. In Table IV-B-1 the mode is

330, since this value occurs four times. The

population mode is the value corresponding to

the peak of the frequency distribution curve.

Frequency distributions with more than one

peak are called multimodai. In a symmetrical

frequency distribution, the mean, median, and

mode are all equal.

2. Measures of Dispersion

2.1 The Standard Deviation: Of the sev-

eral measures of dispersion, the most common

is the standard deviation. Denote the sample

standard deviation by S and the population

value by o"(of which S is an estimate), when the

computational formula is:

The derived formula is preferable because of

its adaptability to the desk calculator. The sam-

ple standard deviation of the microbiological

data in Table IV-B-1 is calculated as follows:

S =

(18) (3961541) - (6709r

_____

'71307738 - 45010681

306

' 26297057

306

= y85939 = 293.2

Confidence Limit (95% and 99%): The range

of values within which a single analysis will be

included, 95% or 99% of the time.

95% C. L =

1.96S

99% C. L = X ± 2.58S

2.2 The Variance: The sample value S2 is

referred to as the sample variance and is

merely the square of the sample standard devi-

ation. Often it is more convenient in conversa-

tion as well as computation to refer to the

variance. This should not cause confusion if

the above relationship is kept in mind.

The population variance is represented by

a2. Its formula is;

n ,

£ (X- — U)

This is the same as the formula for S

However, the computation using this formula except that the true population mean u is used

226

MICROBIOLOGICAL MANUAL 1978

image:

rather than its estimate X and the numerator is

divided by n instead of n - 1.

In calculating the sample variance the true

mean is not known and the estimate of the

mean from the data is used instead. Because

the sample mean is being used to calculate the

variance of the same data, only n - 1 of the

squared difference terms are independent. It

can be shown that the estimate of the variance

must be based upon the sum of independent

squared terms, thus indicating the division by

n - 1, which is called the number of degrees of

freedom (d.f.) in the sample. As a rule, in any

calculation, for every parameter that must be

estimated, one degree of freedom is lost.

2.3 The Range: The range is also used as a

measure of dispersion. It is the difference be-

tween the highest and lowest values in a set of

data.

R = max(Xj)-min{Xj)

For the data in Table IV-B-1 the range is then:

R= 1100-79= 1021

A rough estimate of S can be made by dividing

the range of the sample by the square root of n,

the numberof observations, when n.<_ 10:

Use of the range as a measure of dispersion is

generally limited to instances where the labor

of computing the standard deviation is

impractical.

3. The Normal Distribution

3.1 The most important theoretical distri-

bution in statistics is the familiar bell-shaped

normal distribution which is symmetric about

its peak (see Figure IV-B-1). The following as-

sumptions give rise to this distributional form:

3.1.1 Values above or below the mean are

equally likely to occur.

3.1.2 Small deviations from the mean are

extremely likely.

3.1.3 Large deviations from the mean are

extremely unlikely.

3.2 The normal distribution is completely

defined by its mean,y, and its standard devia-

tion 0 in the following manner:

3.2.1 The area under the normal curve

betweeny minus 0 and y plus 0 is 68 percent of

the total area, to the nearest 1 percent.

3.2.2 The area .under the normal curve

between y minus 20 and y plus 20 is 95 per-

cent of the area, to the nearest 1 percent.

3.2.3 The area under the normal curve

between y minus 30 and y plus 30 is 99.7

percent of the total area, to the nearest 0.1

percent.

3.3 If the frequency distribution of a sam-

ple is a good approximation to the normal

curve, these characteristics of the normal

curve can be used to develop a great deal of

information about the underlying population.

4.Non-symmetric Distribution

4.1 Asymmetry: In some investigations

one encounters distributions which are not

symmetric. For example, distributions of

bacterial counts are often characterized as

having a skewed distribution because of the

many low and a few extremely high counts. This

characteristic leads to an arithmetic mean

which is considerably larger than the median

or the geometric mean. The frequency curves of

these distributions have a long right tail, as

shown in Figure IV-8-2, and are said to display

positive skewness.

4.2 Logarithmic Transformation: For

practical and theoretical reasons, statisticians

prefer to work with symmetric distributions

like the normal curve. Therefore, it is usually

necessary to convert skewed data so that a

symmetric distribution resembling the normal

distribution results. An approximately normal

QA/STATISTICS

227

image:

-3cr -2cr -la u +lcr +20 +3cr

QUANTITY MEASURED

FIGURE IV-B-1. Normal Distribution Curve.

228

QUANTITY MEASURED

FIGURE IV-B-2. Positively-Skewed Distribution Curve.

MICROBIOLOGICAL MANUAL 1978

image:

distribution can be derived from positively-

skewed distributions by expressing the original

data as logarithms. An example of conform

counts and their logarithms are shown in

Table IV-B-2, A comparison of the frequency

tables for the original data and their logs in

Tables IV-B-3 and 4, shows that the logarithms

more closely approximate a symmetric

distribution.

4.3 The Best Measure of Central Ten-

dency for Microbiological Data: Assuming

that the microbiological data has been normal-

ized through a logarithmic transformation, the

arithmetic mean is the best estimate of central

tendency. However, there is a direct relation-

ship between this mean and the geometric

mean of the original data:

= log

>y = log X

Therefore, the best measure of central tend-

ency for microbiological data is the log-

transform.

The mean of tne log MPN data in Table IV-

B-2 is:

log X =

ill'00 X"

32.737

= 2.1825

and the true mean of the MPN data is:

(log X) =

antilog (2.1825) = 152

REFERENCES

1. Dixon, W. J. and F. J. Massey, Jr., 1969. Introduction to Statistical Analysis, 3rd Edition, McGraw-Hill,

Inc., New York, NY.

TABLE IV-B-2

Coliform Counts and Their Logarithms

Coliform

Count/ 100 ml

MPN

120

130

138

161

317

601

760

1020

3100

log MPN

1.041

1.431

.1.556

1.681

1.903

1.929

2.080

2.114

2.134

2.207

2.501

2.779

2.881

3.009

3.491

QA/STATISTICS

229

image:

TABLE IV-B-3

Comparison of Frequency of MPN Data

Class Interval Frequency JMPN)

0 to 400 11

400 to 800 2

800 to 1200 1

1200 to 1600 0

1600 to 2000 0

2000 to 2400 0

2400 to 2800 0

2800 to 3200 1

TABLE IV-B-4

Comparison of Frequency of Log MPN Data

Class Interval Frequency (log MPN)

1.000 to 1.300 1

1.300 to 1.600 2

1.600 to 1,900 1

1.900 to 2.200 5

2.200 to 2.500 1

2.500 to 2.800 2

2.800 to 3.100 2

3.100 to 3.400 0

3.400 to 3.700 1

230 «yERA MICROBIOLOGICAL MANUAL 1978

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PART IV. QUALITY ASSURANCE

Section C Analytical Quality Control Procedures

The Section on Analytical Quality Control

is divided into three major areas of statistical

usage:

1. Quality Control on Routine

Analyses

2. Quality Control in Compliance

Monitoring

3. Comparative Testing of

Methodology

4. Method

Characterization

1. Quality Control on Routine Analyses

Each laboratory must establish quality

control over the microbiological analyses in

use. Fifteen percent of total analyst time

should be spent on quality control practices

discussed in this Manual,

1,1 Duplicate Analyses: Run duplicate

analyses on 10% of the known positive sam-

ples analyzed and a minimum of one per

month. The duplicates may be run as split

samples by more than one analyst.

1.2 Positive Control Samples: Test a min-

imum of one pure culture of known positive

reaction per month for each parameter tested.

1.3 Negative (Sterile) Control: Include

one negative control with each series of sam-

ples using buffered water and the medium

batch at the start of the test series and follow-

ing every tenth sample. When sterile controls

indicate contamination, data on samples af-

fected should be rejected and a request made

for immediate resampling of those waters

involved.

1.4 MF Colony Counting by More than

One Analyst: At least once per month, two or

more analysts should count the colonies on the

same membrane from a polluted water source.

Colonies on the membrane should be verified

and the analysts' counts compared to the veri-

fied count.

1.5 Check Analyses on Water Supply

Program by State Laboratories: In a local

laboratory, a minimum number of the water

supply samples should be analyzed by the

State laboratory. For example, laboratories

that are required to test less than 100 samples

per month should submit an additional 10% of

the number to the State laboratory for analysis.

Water systems with sample requirements

above 100 per month should submit an addi-

tional 2% to the State Laboratory for analysis.

1.6 Reference Sample: Laboratories

should analyze reference samples quarterly

when available for the parameters measured.

1.7 Performance Sample: Laboratories

should analyze at least one unknown perfor-

mance sample per year when available, for

parameters measured.

Q~A/'ANALYTICAL PROCEDURES

231

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1.8 MF Verification: Five percent of the

analyses performed should be verified.

1.8.1 Total Coliforms: Pick at least 10

isolated sheen colonies from each sample.

Transfer into lauryl tryptose broth. Incubate

and read. Transfer positive tubes into brilliant

green bile broth for verification of coliforms.

Since samples from public water supplies

with 5 or more sheen colonies must be verified,

at least 5 colonies are picked from each positive

potable water sample.

The laboratory should make every effort to

detect coliforms from samples with excessive

non-coliforms on the membrane filter. Any

sheen colonies appearing in mixed confluent

growth must be verified (see Part III-B).

1.8.2 Fecal Coliforms: Pick at least 10

isolated colonies containing blue to blue-green

pigment and transfer to lauryl tryptose broth.

Incubate and read. Transfer positive tubes to

EC broth where gas production verifies fecal

coliform organisms (see Part III-C).

1.8.3 Fecal Streptococci: Pick at least 10

isolated pink to red colonies from MF or pour

plates. Transfer to BHI agar or broth. After

growth, perform catalase test. If negative (pos-

sible fecal streptococci) transfer growth to BHI

and 40% bile BHI broth tubes and incubate at

45 C and 35 C respectively. Growth at both tem-

peratures verifies fecal streptococci (see

Part lll-D).

1.9 MPN Completion of Total Coliform Test

1.9.1 For routine analyses, complete the

MPN test on five percent of the positive con-

firmed samples and a minimum of one sample

per test run.

1.9.2 For potable waters, complete the

MPN test once each quarter on 10 percent of

positive confirmed samples. If insufficient pos-

itive tubes result from potable water samples,

perform the completed test on positive source

waters.

1.10 Measurement of Analyst Precision:

If the routine work of the laboratory includes

samples from different wastewaters, surface

waters, water supplies or finished waters, the

following steps should be accomplished for

each type.

Step 1

Perform duplicate analyses on the first 15

typical samples with positive responses.

Although each set of duplicates must be

run by the same analyst, all analysts per-

forming routine analyses should contrib-

ute a share of this initial data.

Step 2

Calculate the logarithms of results. If ei-

ther of a set of duplicate results is zero,

add 1 to both values before calculating the

logarithms.

Step 3

Calculate the range (R) for each pair of

transformed duplicates and the mean (R)

of these ranges.

Step 4

Thereafter, run 10% of routine samples in

duplicate. Transform the duplicates as in

Step 2 and calculate their range. If this

range is greater than 3.27 W, analyst preci-

sion is out of control and all analytical

results since the last precision check must

be discarded. The analytical problem must

be identified and resolved before doing

further analyses (1).

Step 5

In order that the criterion used in Step 4

be kept up-to-date, periodically repeat

Steps 2 and 3 using the most recent sets

of 15 duplicate results.

232

MICROBIOLOGICAL MANUAL 1978

image:

2. Quality Control in Compliance Monitoring

(National Pollution Discharge Elimination

System)

2,1 For any legal assurance of non-

compliance within a permit, analytical results

must exceed the permit limit by a statistically

significant amount. This requires allowance of

the analytical deviation known to occur at a

level equal to the permit limitation.

2.2 It is apparently common for many

monitoring agencies to judge compliance with

microbiological permit limitations based upon

one analysis each for single grab samples

taken at about the same time on 3 consecutive

days. The largest of these 3 results is then

compared to a maximum discharge limitation,

while the arithmetic mean is compared to a

daily average limitation. Judgment based

upon such a sampling program is only valid

under certain assumptions; namely that there

is no relationship between the discharge level

and the time of sampling, and that the variabil-

ity among analytical results for samples taken

simultaneously is either inconsequential or

can be properly estimated from previous data.

The first assumption is usually invalid be-

cause the discharge level is dependent upon

production or processing operations which are

not uniform, but rather follow some daily or

weekly cycle. To keep such periodic cycles

from biasing the results, the grab samples

should be taken at randomly-selected times

during the study or, if there is some knowledge

of the cycle, at times selected systematically to

define its extremes.

The second assumption is important in

order to make a compliance judgment. As

noted in 2.1, this judgment must take into

account the estimated analytical standard de-

viation at a level equal to the permit limit. If an

appropriate estimate of this standard devia-

tion is not already available then it must be

estimated during the compliance study, either

from multiple analyses of each grab sample or

from single analyses of multiple grab samples

taken simultaneously.

2.3 Whenever the analytical standard de-

viation must be estimated from the study data

the following procedure should be followed

for each major type of discharge.

Step 1

Take 3 simultaneous grab samples at each

of K (at least 3} randomly or systematically

selected sampling times which are

expected to represent the range of

discharge throughout the study period.

Step 2

. Analyze the samples and convert the re-

sults to logarithms.

Step 3

Using these logarithms, calculate the fol-

lowing statistics:

K 3

S =

X,f -

Xi = { I Xij)/3

j=1-

_ ' K _

X = ( Z Xj)/K

where: S = the pooled estimate of the

analytical standard deviation

Xjj = the analytical result for the jth

grab sample taken at

sampling time i,

5?i = the mean result for the 3 grab

samples taken at

sampling time \,

X = the overall mean for the K

sampling times

throughout the study

period.

QA/ANALYTICAL PROCEDURES

233

image:

Step 4

3. Comparative Testing of Methodologies

maximum discharge limitation (Dmax)

has been violated if for any sampling time

Xr - t,95{2K){SA/3J > Dmax

where t gg(2K) is the Student's t value at a

95 percent confidence level and 2K de-

grees of freedom.

Step 5_

An average limitation (D) over the study

period (daily, weekly, monthly, etc.) has

been violated if:

3.1 Protocol for Testing: If a laboratory

proposes a new or modified method for accept-

ance certain minimal testing should be

completed.

3.1.1 Collect and test at least 10 samples

from each sample source or type for which the

method is intended, i.e., fresh surface water,

sewage treatment plant influent or effluent,

industrial effluents, saline water, etc. Source

waters should be chosen which are represent-

ative and which span the relevant range of

concentrations.

- t.95(2K)(SA/3T<)

2,4 If an estimate of the analytical stan-

dard deviation (S) is available that has at least

30 degrees of freedom, and if the estimate

applies to the same type of waste and was

generated from samples at a level near the

discharge limitation of the permit under study,

then Steps 1-5 can be modified as follows:

Stepl

Take one grab sample at each of K (at least

3} sampling times selected as before.

Step 2

Analyze samples and convert results to

logarithms.

Step 3

Calculate:

Step 4

Xj)/K

Dmax has been violated if Xj > Dmax + 2S

for any sampling time i.

Step 5

D has been violated if X > 15 + 2SA/IC

3.1.2 Collect the samples and perform the

analyses over 1 week of plant processing, but

not less than 5 calendar days.

3.1.3 Take an even number of aliquots (at

least 8) from each sample and simultaneously

analyze half of them using the accepted me-

thod and the other half using the proposed

alternative method. Throughout this protocol,

the analysis of aliquots from a single sample

using one method will be referred to as "repli-

cate analyses" and the resulting data will often

be referred to as "replicates."

3.2 Statistical Analyses: Compile the

data and perform the following steps for each

sample type:

Step 1: Transform the Data

Calculate the logarithms of the basic data

and use these transformed data as the

basis for all subsequent statistical

analyses.

The characteristics of microbiological

data have been discussed by many

authors. Of particular importance to this

procedure is the effect that a logarithmic

transformation has on such data (2, 3).

234

MICROBIOLOGICAL MANUAL 1978

image:

Specifically, a logarithmic transformation

normalizes the distribution of results from

replicate analyses of the same sample and

equalizes the within-sample standard

deviations among samples with different

microbiological densities. These

conditions are required assumptions for

many of the statistical procedures that

follow.

Step 2: Calculate the Basic Statistics

Calculate the arithmetic mean (X) and

standard deviation (S) for each set of

replicates, and tabulate the data and their

basic statistics.

Step 3: Test for Suspected Outliers

For each set of n replicates, test for outli-

ers by calculating the Extreme Studen-

tized Deviate as T = (XE - X)/S, where XE

= the maximum or minimum replicate,

whichever is farthest from X.

This is done by applying Cochran's Test (4)

as follows:

max

K = the number of samples

Sj = the standard deviation estimate from

the set

sample,

of replicates for the i

Smax = the largest value for Sj i =1, 2,..., K

Look up the critical C value for n-1 degrees

of freedom on each of K standard devia-

tions and a .01 significance level, i.e., C gg

(n-1, K) in Dixon and Massey (5),

If C < C gg (n-1, K), then pool the standard

deviations for the method (M) as follows:

Sjy. is the pooled within-sample variance

estimate for the method.

Look up the critical T value for the number

of replicates on each sample and a .01 (1

percent) significance level in Standard

D2777-72, 1975 Book of ASTM Stan-

dards, Part 31, Water, page 15. If T is

greater than the critical value, reject all

data for that sample with 99 percent conf i-

dence that XE is an outlier.

This unfortunate loss of data is necessary

because the following statistical proce-

dures have been simplified and only apply

to the unique situation where, for each of

K sample, there are exactly n replicates by

each of the two methods.

Step 4£ Test for Equality Among the

Within-Sample Standard Deviation

Estimates

For each method, test for equality of the

standard deviation estimates (S) calcu-

lated in Step 2 from the replicates for each

sample.

If C > C gg (n-1, K), then the within-sample

standard deviation of this method is not a

constant over the concentration range

represented in the data. To correct for this,

the data must be stratified, i.e., subdi-

vided, into sets of samples which have a

common standard deviation among the

results of replicate analyses by this me-

thod. Care should be taken to minimize the

number of strata to those that are required

in order to justify the development of

pooled within-sample standard deviation

estimates. If both methods require stratifi-

cation, common strata should be devel-

oped which are jointly suitable. Step 4

should be repeated to verify the proposed

stratification. Subsequently, Step 5

should be carried out for each stratum

independently. If you have any problems

with stratifying the data, please seek the

assistance of a qualified statistician/data

analyst before proceeding.

Step &_ Test for Equality of the Pooled

Within-Sample Variance Estimates

QA/ANALYTICAL PROCEDURES

235

image:

Having developed a pooled within-sample

variance estimate for each method, within

common strata if necessary, test the

equality of these estimates by performing

the following Ftest:

, where

sj,

Look up the critical F value for K(n-1)

degrees of freedom for each variance and

a .05 significance level, i.e., F.g5 (K(n-1),

K(n-1)), in any standard statistical

reference. If F £ F95 (K{n-1), K(n-1)), then

the two methods produce results with

within-sample variances which are not

significantly different. If F > Fa5 (Kfn-1),

K{n-l)), then. there is a statistically

significant difference between the

variances.

Step 6£ Test for Equality of the Method

Means

Finally, calculate the difference between

the method means for each of the K sam-

ples as:

d| ** Xiuj — XM , i = 1, 2,...,K,

Using the mean (d) and standard deviation

(Sj) of these differences, calculate the Stu-

dent's statistic as:

sewage treatment effluents. Recognized prob-

lems in Method 1 include: deviation among

replicate results is considered excessive, aver-

ages show a positive bias of about 40 percent,

and the procedure is difficult and time-

consuming.

Suppose that Method 2 is quicker and

easier than Method 1. Then, if the following

statistical analysis shows the performance of

Method 2 to be at least equal to Method 1, EPA

may designate Method 2 as an alternative to

Method 1. If Method 2 offered no operational

advantage over Method 1, the statistical analy-

sis would have to show the performance of

Method 2 to be significantly superior to Me-

thod 1.

Steps 1 and 2

Transform the Data (Table IV-C-1) and Cal-

culate the Basic Statistics:

The logarithmic transformation was found

most appropriate for the data in Table IV-

C-1, see the results in Table IV-C-2.

Step 3

Test for Suspected Outliers In Table IV-C-

T •—

Refer to a statistical reference for the crit-

ical Student's t value at the .05 signifi-

cance level and K-1 degrees of freedom,

i.e,t%95(K-1). Ift > t>95(K-1), then the means

for the two methods are significantly

different.

3.3 Example of the Statistical Analysis

Procedure

Statement of the Problem

Method 1 is the accepted method for de-

termining fecal coliform levels in chlorinated

Sample 2, method 2, replicate 1:

XE = 3.3711

3.3711 - 3.6773)

T =

.1738

Sample 4, method 2, replicate 1:

XE = 2.7497

[2.7497 - 2.5299I _

.1301

236

MICROBIOLOGICAL MANUAL 1978

image:

TABLE IV-C-1

Raw Sample Data from the Analysis of Chlorinated Sewage

Treatment Plant Effluents

Sample

No. Date Method

1 3/11 1

2 3/11 1

3 3/17 1

4 3/18 1

5 3/24 1

6 3/25 1

7 3/26 1

Replicates (XR)

in counts/ 100 ml

1850,

1200

7800,

2350,

4000,

2400,

625

562

3870,

2730,

7200,

4400,

1200,

700

1350,

, 850,

8100,

5300,

4400,

2150,

, 512,

, 350,

2330,

3930,

4400,

4000,

1000,

, 600,

1050,

650,

5900,

5650,

2650,

2050,

512,

300,

3330,

2530,

7800,

4400,

1000,

900,

1800

500,

5450

6350

3000

1600

462,

275,

3670

2200

6600

3600

1200

900,

,1150

1050

, 7150

, 5450

, 3650

, 2200

700

275

, 2530

, 1730

, 5200

, 6200

, 1600

900

2Xp Xp

,200

,250

,400

,100

,700

,400

,811

,762

,730

,120

,200

,600

,000

1440

850

6880

5020

3540

2080

562

352

3146

2624

6240

4520

1200

800

368

285

1163

1545

715

297

121

685

822

1410

996

245

141

QA/ANALYTICAL PROCEDURES

237

image:

TABLE IV-C-2

Logarithmic Transformation of the Data in Table IV-C-1

Sample

No. Method

1 1

2 1

3 1

4 1

5 1

8 1

7 1

.2672,

.0792,

.8921,

.3711,

.6O21,

.3802,

.7959,

.7497,

.5877,

.4362,

.8573,

.6435,

.0792,

.8451,

Transformed Replicates

(X = log XR)

3.1303,

2.9294,

3.9085,

3.7243,

3.6435,

3.3324,

2.7093,

2,5441,

3.3674,

3.5944,

3.6435,

3.6021,

3.0000,

2.7782,

.0212,

.8129,

.7709,

.7520,

3.4232,

3.3118,

.7093,

.4771,

.5224,

.4031.

.8921,

.6435,

.0000,

.9542,

3.2553,

2.6990,

3.7364,

3.8028,

3.4771,

3.2041,

2.6646,

2.4393,

3.5647,

3.3424,

3.8195,

3.5563,

3.0792,

2.9542,

3.0607

3.0212

3.8543

3.7364

3.5623

3.3424

2.8451

2.4393

3.4031

3.2380

3.7160

3.7924

3.204 1

2.9542

.7347

.5417

.1622

.3866

.7082

.5709

.7242

.6496

.4453

.0141

.9284

.2377

.3625

.4860

3.1469

3.9083

3.8324

3.6773

3.5416

3.3142

2.7448

2.5299

3.4891

3.4028

3.7857

3.6475

3.0725

2.8972

.1115

.1544

.0756

.1738

.0903

.0664

.0735

.1301

.0984

.1310

.1033

.0886

.0836

.08 16

238

MICROBIOLOGICAL MANUAL 1978

image:

Sample 5, method 2, replicate 2;

XE = 3.5944

13.5944 - 3.40281

T = J - = 1.46

.1310

Sample 6, method 2, replicate 5:

XE = 3.7924

_ 13.7924 - 3.64751

~~ .0886 ~~

The critical T value for sets of 5 replicate

and a .01 significance level is 1.76. Since

none of the T values is greater than 1.76,

we must accept all of the suspected

values.

Step 4

Test for Equality Am.ong the Within-

Sample Standard Deviation Estimates (S)

for Each Method in Table IV-C-2:

Recall that C=

For Method 1: C = {. 115)2/.0590 = .2106

For Method 2: C = (.1738)2/.1071

= .2822

The critical Cochran statistic for 7 vari-

ances with 4 degrees of freedom each and

a .01 significance level equals .5080.

Since both the calculated C values are less

then the critical value, the within-sample

variances are equal and we can proceed to

calculated pooled variance and standard

deviation estimates for each method:

= .0084 .%

= 0153

= -0918

= 1237

Step 5

Test for Equality of the Pooled Within-

Sample Variance Estimates

= .0153/.0084 = 1.82

The critical F value at .05 significance

level, when both variance estimates have

7(4) or 28 degrees of freedom, equals

1.88. Since the critical value is not ex-

ceeded, these methods do not produce

significantly different within-sample vari-

ances. The same statement can be made

regarding within-sample standard

deviations.

Step 6

Test for Equality of the Method Means

First, for each sample, calculate the differ-

ence between the means in Table IV-C-2.

These differences (dj) are shown in Table

IV-C-3 along with their mean (3) and stan-

dard deviation (S^). Then calculate:

t = d/SdA/nj = .1765/(.0547/Vf)

= .1765/.0207 = 8.54

The critical Student's t value at a ,05 sig-

nificance level and 6 degrees of freedom

is 2.45. Since the calculated value ex-

ceeds the critical value, the methods do

produce significantly different means.

To Summarize the Results

(a) The withinTsampIe standard deviations

for these methods are not significantly differ-

ent at the .05 significance level.

(b) The mean for Method 1 is significantly

higher than the mean for Method 2 at well over

the .05 significance level.

QA/ANALYTICAL PROCEDURES

239

image:

Discussion of the Results

Referring to Table 1V-C-3, it can be easily

seen that Method 1 produces the larger mean.

The difference can be calculated as:

antilog(3.3733) - antilog(3.1967)

antilog(3,1967) ,

100

100 = 50.2%

100

trary degree, for example, three orders of mag-

nitude, compared with growth and recovery on

non-selective media.

4.3 Precision is a measure of the deviation

among multiple measurements of a single

quantity. The most widely used expression of

precision is the standard deviation (a) which is

equal to the square root of the variance and

indicates the deviation of the values about the

mean.

Like accuracy, the true precision of a me-

thod must be generated in a collaborative

study among at least 15'Iaboratories.

Since Method 1 results are known to be

about 40% greater than the true value, it can

be estimated that Method 2 results are about 7

percent less than the true value. Therefore,

besides being quicker and easier. Method 2

offers comparable precision with improved ac-

curacy. Such results would qualify Method 2

for approval as an alternative to Method 1.

4. Method Characterization

The choice of a method of analysis among

several considered should be based on com-

parison of relative performance using these

measureable characteristics.

4-1 Specificity is the ability of a method to

recover the desired organisms identified by a

selective or differential characteristic and veri-

fied by additional tests. A method is judged

specific if the recovered microorganisms ver-

ify as the desired organism, and the colonies

designated as "other organisms" do not verify

as the desired organism when picked and

tested. The acceptable level of specificity for a

method cannot be set absolutely, for example

as 90%, but rather must be established for

standard procedures or for new parameters on

a best judgment basis by comparison with the

accepted methods.

4.2 Selectivity is the ability of a method to

encourage growth of the desired organism

while reducing other organisms by some arbi-

4.4 Accuracy is a measure of the close-

ness of observed values to a known true value.

The lack of available standards for comparison

of microbiological methodology has resulted

in methods with known precision, but with

limited accuracy information.

In microbiology, accuracy has been deter-

mined by applying the test method to a sus-

pension of a pure culture while independently

determining the cell number in the suspension

using a non-selective medium. Natural water

samples are tested with and without the pure

culture spike and the recovery determined by

difference. The recovery from the spiked sam-

ple is compared with the count on the non-

selective medium (assumed to be true value)

and expressed as a percent of this true value.

True accuracy of a method must be gener-

ated in a collaborative study among at least 15

laboratories using known levels of organisms.

4.5 The Section on Sanitary and Health

Effects Microbiology in the American Society

for Testing and Materials (ASTM) Committee

D-19:24 has also proposed a characteristic

called Counting Range. The Counting Range is

described as the range from the lowest num-

ber to the highest number of colonies that can

be measured on a single agar plate or mem-

brane filter, without affecting the reliability of

the method.

240

«»EPA MICROBIOLOGICAL MANUAL 1978

image:

TABLE IV-C-3

Analysis of Difference Between Means

Sample Mean from Table

No. Method 1{XMl)

1 3.1469

2 3.8324

3 3.5416

4 2.7448

5 3.4891

6 3,7857

7 3.0725

XM^ = 3.3733 XM,

IV-C-2 for

Method 2 (XM2)

3,9083

3.6773

3.3142

2.5299

3.4028

3.6475

2,8972

= 3.1967

Difference Between

Means, (di = X^ — Xy^)

.2386

.1551

.2274

.2149

,0863

.1382

.1753

d =. .1765 Sd = .0547

QA/ANALYTICAL PROCEDURES

241

image:

REFERENCES

1. Grant, E. L and R. S. Leavenworth, 1972. Statistical Quality Control. Fourth Edition. McGraw-Hill, Inc.,

New York, NY. p. 87.

2, Eisenhart, C. and P. W. Wilson, 1 i43. Statistical Methods and Control in Bacteriology. Bacteriological

Reviews 7:57.

3. Velz, C.'J., 1951. Graphical Approach to Statistics, Part 4: Evaluation of Bacterial Density. Water and

Sewage Works. 98:66.

4. Dlxon, W. J. and F. J. Massey, Jr., 1969. Introduction to Statistical Analyses, Third Edition. McGraw-Hill,

Inc., New York, NY. p. 310,

5. ibid. p. 537.

242 &EF¥\ MICROBIOLOGICAL MANUAL 1978

image:

PART V LABORATORY MANAGEMENT

Part V addresses those laboratory activities which supplement the analytical methodology and

are primarily the responsibility of the laboratory manager.

Section A Development of a Quality Control Program

Section B Manpower and Analytical Costs

Section C Safety

Section D Legal Considerations

MANAGEMENT OF QUALITY CONTROL 243

image:

PART V. LABORATORY MANAGEMENT

Section A Development of a Quality Control Program

1. Intralaboratory Quality Control

1.1 To insure a viable quality assurance

effort, management must recognize the need

for a formal program and require its develop-

ment. Management must commit itself to the

program by setting aside 15% of the labora-

tory man-years for quality control activity. It

must meet with supervisors and staff to estab-

lish levels of responsibility for management,

supervisors and analysts. Laboratory person-

nel should participate in the planning and

structuring of the QA program.

1.2 Once the QA program is functioning,

supervisors review laboratory operations and

quality control with analysts on a frequent

(weekly) basis. Supervisors use the results of

the regular meetings with laboratory person-

nel to inform management on a regular

(monthly) basis of the status of the QA pro-

gram. These meetings are important to identify

the problems at the laboratory level and to get

the backing of management in the actions

necessary to correct problems.

2. Documentation of the Intralaboratory

Quality Control Program

Unless a record is made of the quality

control checks and procedures described in

Part IV of this Manual, there is no proof of

performance, no evidence for future reference

and for practical purposes, no quality control

program in operation. The following documen-

tation should be made and maintained.

2.1 An Operating Manual is prepared

which describes the sampling techniques, ana-

lytical methods, laboratory operations, mainte-

nance and quality control procedures. Specific

details are given on all procedures and quality

control checks made on materials, supplies,

equipment, instrumentation and facilities. The

frequency of the checks, the person responsi-

ble for each check (with necesssary back-up

assignments), the review mechanism in the QC

program to be followed, the frequency of the

review and the corrective actions to be taken

are specifed. A copy is provided to each

analyst.

2.2 A Sample Log is maintained which

records chronologically information on sam-

ple identification and origin, the necessary

chain of custody information, and analyses

performed.

2.3 A Written Record is also maintained of

all analytical QC checks: positive and negative

culture controls, sterility checks, replicate ana-

lyses by an analyst, comparative data between

analysts, use-test results of media, membrane

filters and laboratory pure water, replicate ana-

lyses done to establish precision of analysts, or

of methodology used to determine non-

compliance with bacterial limits and water

quality standards.

3. Interlaboratory Quality Control

An inter-laboratory quality control program

consists of: 1) formal collaborative method

244

MICROBIOLOGICAL MANUAL 1978

image:

studies to establish precision and accuracy of unknown samples, 5) follow up on problems

selected methodology, 2) specific minimal detected in onsite inspections and perfor-

standards for personnel, sampling and sample mance evaluations,

preservation procedures, analytical methodol-

ogy, equipment, instrumentation, facilities and

within-laboratory quality control programs, 3) EPA has established such a interlabora-

verification of acceptable standards through tory quality control program in response to

annual on-site inspections, 4) periodic perfor- the FWPCA Amendments of 1972 and the Safe

mance tests of analytical capabilities using Drinking Water Act of 1974.

MANAGEMENT OF QUALITY CONTROL 245

image:

PART V. LABORATORY MANAGEMENT

Section B Manpower and Analytical Costs

Laboratories planning to begin or increase

microbiological activities have difficulty in de-

termining their added manpower, equipment

and supply needs. This Section provides esti-

mates of added costs:

Supplies

Time Expenditures

Specialized Equipment and

1. Time Expenditures for Microbiological

Analyses

The following estimates of time required

for membrane filter (MF) and most probable

number (MPN) tests were prepared in re-

sponse to information requests from Regional

Offices and States planning new or additional

microbiological work. The estimates are

presented as guidelines only.

The Microbiological Methods Section,

8MB and the Quality Assurance Branch, both

of EMSL-Cincinnati prepared the estimates

based on average performance of one techni-

cian qualified by short-course or on-the-job

training and experience in the specific tech-

niques. The procedures are those described in

This Manual.

1.1. MF Analyses

If only fecal coliform bacteria are being

tested, thirty (30) samples (estimating 3

dilutions/sample) can be prepared by one ana-

lyst on day one. Two hours are required to

complete the counting, calculation of results

and verification on day two.

If fecal and total coliform tests are per-

formed using 3 dilutions, twenty (20) samples

can be analyzed in one day by one analyst. On

the second day, counting of plates takes an

estimated 2 1/2 hours. An estimated four

hours are needed for preparation of media,

dilution water, dishes and pipettes for the test

period if analyses are performed over five

days. The time estimates include 10% devoted

to quality control procedures.

1.2 MPN Analyses

After preparation of tubed media, one

worker can process 15 samples for MPN anal-

yses for total and fecal coliforms in an 8-hour

day. The procedures include 5 tube x 5 or more

dilutions to assure the positive and negative

tubes result in 3 significant dilutions needed to

obtain the optimum MPN index. If 15 samples

are analyzed each day for a 5 day week, prepar-

ation and clean-up of tubed media and supplies

for the test period would require an estimated

8 hours. Estimates include 10% of time devoted

to quality control procedures {Part IV of this

Manual).

Since the MPN requires reading and trans-

fer of growth from positive tubes of lauryl

tryptose broth to brilliant green bile broth and

EC broth at 24 and 48 hours, the time span for

one day's samples may cover four days. These

times are indicated as 8, 4, 2 and 1 hours on

the four successive days. New samples tested

on the 2, 3, 4th day, etc., add to each day's

246

&ERA MICROBIOLOGICAL MANUAL 1978

image:

workload. This add-on effect results in 8 hours

+ 4 hours + 2 hours + 1 hour for a workload

of 15 hours from the 4th day onward. Either

the sample load must be reduced or one must

provide a second technician. The accumulative

load is shown in Table V-B-1.

2. Specialized Equipment and Supplies

The following tables of materials and

equipment are provided as guidance to labora-

tories. Tables V-B-2, 3 and 4 are designed for a

minimal program of 2 samples/day, 7 days a

week. Estimates of expendable supplies are

based on those used in 1 year. Tables V-B-5,6,

7 and 8 are designed for laboratories planning

a program of 15 samples per day, 5 days per

week. Estimates for expendable supplies are

based on those used in 1 week.

The following assumptions are made:

The basic laboratory facility and equipment

are available (space, benches, lighting, utilities,

chemicals and standard glassware).

The laboratory will do membrane filtrations

as single analyses at three dilution levels or

MPN's as five tube, five dilution tests.

The amounts of supplies or numbers of

items are based on needs, then adjusted to

take advantage of quantity discounts by the

dozen, box, 100's, etc. For both laboratory

efforts, the expendable items are based on 2

weeks usage, while consumable media are

based on usage/week in the high effort labora-

tory and usage/year in the low effort laboratory.

Laboratories doing primarily drinking

water analyses may require fewer items; other

laboratories analyzing only polluted waters may

require more items.

Costs are current figures provided to give

rough estimates only. The necessary character-

istics of equipment and materials are cited to

assist the purchaser in his choices.

ANALYTICAL COSTS

247

image:

Day J_

First Series

Samples Stan

15 MPNS

8 hours

TABLE V-B-1

Estimated Time Required for Fifteen MPN Analyses/Day*

Day 2_ Day 3_ Day 4_ Day 5_ Day 6_

4 hours

2 hours

1 hour

Day 7_ Day 8_

*•*

Second Series

15 MPNs

8 hours

4 hours

2 hours

1 hour

Third Series

15 MPNs

8 hours

4 hours

2 hours

1 hour

Fourth Series

15 MPNs

8 hours 4 hours

2 hours

1 hour

Fifth Series

15 MPNs

8 hours

4 hours

2 hours

1 hour

•The time required per MPN analysis is not 1/15 of the time estimated because there is a time savings

in preparing larger numbers of samples.

image:

TABLE V-B-2

General Equipment and Supplies

Minimum Program

Incubator,

DWH, 18

Incubator,

Item

gravity convection,

X 19 x 28", (46

waterbath, 44.5 +

35 ± 0.5 C,

X 48 X 71 cm)

0.2 C for fecal coliforms,

Quantity

Cost

Quantity*

$360.00

380.00

LWH 18 x 12 x 7'/2", (46 x 31 X 19 cm)

Sterilizer, steam, bench top, electric heat with temperature

and pressure controls and gauges I.D.: 9" (23 cm)

diameter chamber, 16" (41 cm) deep

pH meter, analog, 0-14 pH range, accuracy + 0.1 pH

units, temperature compensated, with electrode

Qven, double wall, gravity connection RT to 225 C,

automatic temperature control, I.D.: LHD

19 X 18 X 16", (48 X 46 X 41 cm)

Refrigerator, 3 cubic ft. (.19 M3) with freezer

compartment

Thermometer, mercury, range 0-50 C graduated in 0.1 C.

Meets NBS specs.

Cylinder, graduated, 100 ml, 1 ml graduations

Cylinder, graduated, 50 ml, 1 ml graduations

Bottles, dilution, 99 ml mark, screw-cap, Pyrex glass

Bottle, sample, polypropylene, wide mouth, screw-capped

autoclavable.

1500.00

225.00

575.00

425.00

40.00

66.00

85.00

50.00

125 ml

250 ml

500 ml

Bottle, sample, glass, wide mouth, screw-cap, autoclavable

130 ml

210 ml

Beaker, stainless steel, 4 qt. waterbath for media

preparation

Pump, vacuum, polypropylene, water powered, 11.5

liters/min capacity

5.90

9.00

13.00

6.80

8.32

15.00

2.92

*1978 prices.

ANALYTICAL COSTS

249

image:

TABLE V-B-2

General Equipment and Supplies

Minimum Program (continued)

Item

Burner, Bunsen, utility

Needle, inoculating, in holder

Tongs, flask

Pen, ink, felt-tip, waterproof

Balance, torsion, two pan, 200 g capacity, accurate

to 10 mg, with 100 g weight

Hot Plate/Magnetic Stirrer, variable speed and heat,

6 X 6", (15 X 15 cm) top

Plpettor, automatic, volume of. 5-50 ml, speed- of

10-60 deliveries/minute with glass syringe

Cost

Quantity Quantity*

1 5.25

2 2.50

. 1 pr. 5.25

12 11.00

1 3 1 0.00

1 125.00

1 485.00

•1978 prices.

250 4>EFA MICROBIOLOGICAL MANUAL 1978

image:

TABLE V-B-3

Equipment and Supplies for MF Analyses

Minimum Program

Item

Filters, membrane, white, gridded 47 mm, 0.45 ym

or equivalent pore size

Pads absorbent, 47 mm (optional)

Dishes, petri, plastic, tight-lid, 50 mm X 12 mm

Bag, plastic, waterproof for 44.5 C waterbath

incubator

Pipet, glass Mohr or bacteriological, 10 ml

in 0.1 ml increments

Pipet, glass Mohr or bacteriological, 2.0 or

2.2 ml, in 0.1 increments

Pipet, glass Mohr or bacteriological, 1.0 or

1.2 ml, in 0. 1 increments

Can, pipet, stainless steel, 2.5 X 2.5 X 16"

6,5 X 6.5 x 41 cm

Jar, polypropylene, for disinfection of pipets.

6.5 X 20" (16.5 X 50 cm)

Flask, vacuum, pyrex glass, 1 liter

Water Trap, glass bottle, stoppered with glass tube

inlet/outlet

Tubing, rubber, vacuum, 3/6" O.D. & 3/32" I.D.

(1.3 cm O.D. & .24 cm I.D.)

Pinchclamp, flat jaw

Forceps, blunt with smooth tip

Microscope, dissecting, binocular, 15 power

Illuminator, microscope, fluorescent, fits round tube

microscope

Quantity

2100 MFs

in pkgs of 100

2100 MFs pads

in pkgs of 100

2100 dishes ,

in boxes of 500

1000 in boxes ,

of 500

1,8-

Cost

Quantity*

$400,00

32.00

210.00

60,00

45.00

28.00

25.00

42.00

20.00

6.00

1.00

9.00

2.00

3.00

200.00

62.00

•1978 prices.

ANALYTICAL COSTS

251

image:

TABLE V-B-3

Equipment and Supplies for MF Analyses

Minimum Program

(continued)

Item

M Endo MF broth (optional)

LES Endo agar (optional)

M-FC broth

Rosollo Acid (for M-FC broth)

Methanol, 95%, in small vial for forceps disinfection

Ethnnol, 95%, unadulterated, for M-Endo, MF broth

and LES Endo agar

Quantity

8 x i/4#/yr

8 x «/4#/yr

54 x 1 g/yr

1 pt/yr

500 ml/yr

Cost1

$60.00

72.00

64.00

90.00

1.00

5.00

Equipment and Supplies for Verification of MF Analyses

Minimum Program

Item Quantity Cost1

Tubas, Culture, w.o. Up, borosilicate glass, 8 x 24 $30.00

1 BO x 20 mm, reusable.2

Tube, fermentation, borosilicate, w.o. lip, 8 x 24 1S.OO

75 x 10 mm, reusable.2

Rack, wire, for 10 x 4 culture tubes

Basket, wire, rectangular galvanized,

10 x 6 x 6", (25 x 15 x 15 cm)

Caps, culture tube, aluminum 22 mm I.D.

Lauryl Tryptose broth

Brilliant Green Bile broth

EC broth

16 x 12

1#/yr

48.00

35.00

40.00

13.80

17.90

16.45

11978 prices.

2Available as disposables.

image:

TABLE V-B-4

Equipment and Supplies for MPN Analyses

Minimum Program

Item

Tubes, Culture, w.o. lip, borosilicate glass,

150 X 20 mm, reusable,2

Tubes, Culture, w.o. lip, borosilicate glass,

150 X 25 mm, reusable.2

Tube, fermentation, borosilicate, w.o. Hp>

75 x 10 mm, reusable.2

Rack, wire, for 10 X 5 culture tubes

Basket, wire, rectangular galvanized,

10 X 6 X 6", (25 X 15 X 15 cm)

Caps, culture tube, aluminum 22 mm I.D.

Caps, culture tube, aluminum 27 mm I.D.

Lauryl Tryptose broth

Brilliant Green Bile broth

EC broth

Amount

2 X 576

288

2 X 720

15 X 1

10 X 1

60 X 12

24 X 12

14 X 1#/yr

16 x 1#/yr

15 X 1#/yr

Cost1

$170.00

70.00

105.00

225.00

80.00

1 50.00

60.00

200.00

300.00

250.00

11978 prices,

2Available as disposables.

ANALYTICAL COSTS

253

image:

TABLE V-B-S

General Equipment and Supplies

Full Program in Microbiology

Item

Quantity

Cost

Quantity*

Pipets, glass, Mohr or bacteriological T.D., 10 ml

in 0.1 ml graduations

Pipets, glass, Mohr or bacteriological T.D., 2 or 2.2 ml

In 0.1 ml graduations

Pipets, glass, Mohr or bacteriological T.D., 1 ml

in 0.1 ml graduations

Can, pipet, stainless steel, 2.5 x 2.5 x 16"

(6.5 X 6.5 X 41 cm)

Jar, polypropylene, for disinfection of pipets,

6.5 X 20" (16.5 X 50 cm)

Cylinder, graduated, 100 ml

Cylinder, graduated, 50 mi

Bottle, dilution, pyrex glass, marked at 99 ml,

screw-cap

Bottle, sample, glass, wide mouth, screw-cap,

autoclavable

125 ml

250 ml

Bottle, sample, polypropylene, wide mouth,

screw-cap, autoclavable

125 ml

2BO ml

500 ml

1000 ml

Thermometer, mercury, 0-50 C, graduated in 0.1 C,

meets NBS specifications

Beaker, stainless steel, 4 quart, waterbath for

media preparation

Flask, vacuum, aspirator, pyrex glass, 1 liter

Water-trap, glass (glass bottle, stoppered, with glass

tube inlet and outlet)

70.00

60.00

55.00

210.00

40.00

132.00

170.00

100.00

42.00

50.00

72.00

108.00

52.00

38.00

40.00

15.00

18.00

1.00

*1978 prices.

254

MICROBIOLOGICAL MANUAL 1978

image:

TABLE V-B-5

General Equipment and Supplies

Full Program in Microbiology {continued)

Item

Quantity

Cost

Quantity*

Tubing, rubber, vacuum, 3/16' I.D. x 3/32' O.D.

(1.3 cm I.D. X 2.4 cm O.D.)

Needle, inoculating, with holder

Tongs, flask

Pen, ink, felt tip, waterproof

Balance, single pan, electric, top loader, 200 g capacity,

sensitivity 0.02 g for weighings of 2-200 grams

pH meter, analog, 0-14 pH range accuracy + 0.1 pH

units, temperature compensated line voltage, with

comb, electrode

pH meter, same as above, with accuracy ^ 0.05

pH units

Burner, gas, Bunsen

Hot plate-magnetic stirrer combination, variable

speed and heat, 15 X 15 cm. top

Incubator, 35 C + 0.5 C, water jacketed radiant heat

18 X 38 X 27", (46 X 96 X 68 cm)

Incubator, 35 C + 0.5 C, forced air, large, single chamber

I.D.: DWH 18 X 36 X 27", (46 x 96 X 68 cm)

Incubator, 44.5 C + 0.2 C water bath circulating,

I.D., LWD 36 X 18 X 91A", (91 X 46 X 24 cm)

Sterilizer, large rectangular, double-wall steam, electric

automatic, I.D., LWH 38 x 20 x 20"

(97 X 51 x 51 cm)

Oven, double wall, gravity convection RT to 225 C

I.D., LWH 24 X 20 X 20" (61 X 51 X 51 cm)

Refrigerator, 13 cubic foot, with freezer compartment,

automatic defrost

48'

1 pr

9.00

6.00

5,25

11.00

700.00

225.00

500,00

10.50

125.00

3380.00

2300.00

720.00

14000.00

750.00

500.00

*1978 prices.

ANALYTICAL COSTS

255

image:

TABLE V-B-6

Equipment and Supplies for MF Analyses

Full Program in Microbiology

Item

Quantity

Cost

Quantity*

Membrane filtration assembly, stainless steel, for

47 mm filters

Membrane filtration assembly, plastic, autoclavable,

for 47 mm filters

Membrane filtration assembly, pyrex, for 47 mm filters

UV sterilizer unit for sterilizing MF filtration assemblies

Manifold, PVC, 3 place, for multiple filtrations

Microscope, binocular, dissecting type, 15 power

Lamp, fluorescent, microscope illuminator, fits round

tube microscope

Filters, membrane, white, 47 mm 0.45 ym or equiv.

pore size, gridded

Pads, absorbent, 47 mm

Dishes, petri, plastic, 50 x 12 mm, tight lid

Bags, plastic, waterproof for submersion of M-FC dishes

in waterbath

Bottle, rinse water, round, wide-mouth, screw-cap,

polypropylene, 1000 ml

Forceps, blunt with smooth tip

Counter, mechanical, hand type

Pump, vacuum, polypropylene, water powered, 11.5

liters/rnin capacity

Pump, vacuum/pressure, electric, portable, produces

20" mercury vacuum

Plnchclamp, flatjaw

300

500

S3 60.00

24.00

120.00

550.00

200.00

62.00

57.00

4.50

50.00

30.00

38.00

4.00

9.00

3.00

130.00

6.00

*1978 prices.

256

MICROBIOLOGICAL MANUAL 1978

image:

TABLE V-B-7

Equipment and Supplies for MPN Analyses

Full Program in Microbiology

Basket, wire

10 x 6 x

Rack, wire.

Item

, rectangular galvanized,

6" (25.4 X 15 x 15 cm)

for 10 x 5 culture tubes.

Cost

Quantity Quantity1

190 $1520.00

150 2250.00

150 X 25 mm

Tubes, culture w.o. lip, borosilicate glass,

150 x 20 mm, reusable.2

Tubes, culture w.o. lip, borosilicate glass,

150 X 25 mm, reusable.2

Tubes, fermentation w.o. lip, borosilicate glass,

75 x 10 mm, reusable.2

Caps, culture tube, aluminum, 22 mm I.D.

Caps, culture, tube, aluminum, 27 mm I.D.

30 X 576

17 x 288

21 X 720

1250 x 12

408 X 12

2560.00

1200.00

1100.00

3125.00

1020.00

11978 prices.

2Available as disposables.

ANALYTICAL COSTS

257

image:

TABLE V-B-8

Media for Full Program in Microbiology Laboratory

Usage for each Week/100 Samples

Medium

MF Analyses:

M Endo MF Broth

M-FC Broth

LES Endo Agar

Rosollc Acid (for M-FC Broth)

MPN Analyses:

Lauryl Tryptose Broth

Brilliant Green Bile Broth

EC Broth

EMB Agar

Lauryl Tryptose Broth

Brilliant Green Bile Broth

EC Broth

Usage/Week

0.1 Ib/week

0.07 Ib/week

0.17 Ib/week

1 g/week

' 1.96 Ib/wk2

2.2 Ib/wk

2.04 Ib/wk

variable

Verification Costs on 5 Samples/Week

20 Colonies/Sample

.07 Ib/week

Cost/lb1

$17.90

24.85

20.95

1.70/g

13.80

17.90

16.4B

18.70

13.80

17.90

16.45

11878 prices.

2For single strength medium.

258

<8>ERk MICROBIOLOGICAL MANUAL 1978

image:

PART V. LABORATORY MANAGEMENT

Section C Laboratory and Field Safety

Introduction

1. Administrative Considerations

This Section has been compiled from the

best available sources. The procedures given

for general laboratory safety follow the OSHA

regulations (1, 2). Specific recommendations

for microbiology were selected from literature

of the Center for Disease Control, the National

Institute for Occupational Safety and Health of

the Public Health Service, the U.S. Environ-

mental Protection Agency and other sources

(3-12).

Safety procedures should be performed

as an integral part of the analytical methods

and should be included in program planning

on a day to day basis.

The objectives of a laboratory and field

safety program are 1)to protect the laboratory

worker, the laboratory environment and the

surrounding community from microbial agents

studied and 2) to protect the integrity of the

microbiological studies. The program is dis-

cussed as follows:

1. Administrative Considerations

2. Sources of Hazard

3. Field Guidelines

4. Laboratory Guidelines

5, Biohazard Control

6. Safety Check List

1.1 Development of a Safety Program

1.1.1 If a laboratory safety program is to

be effective, management must know the

causes of infections in order to develop and

upgrade safety procedures, equipment and

rules, and to reduce incidence of infection. The

lack of information on sources of laboratory

infection (Table V-C-1 and 2) prevents improve-

ment in safety programs and emphasizes the

need for reporting all accidents. A preponder-

ance of evidence in the literature indicates that

if the known causes of labpratory infections

were eliminated, the remaining infections

could be considered to be caused by airborne

transmission.

Some common microbiological proce-

dures shown to produce aerosols include pi-

petting into a petri plates or flasks, opening

lyophilized culture ampuls, opening culture

containers, inserting a hot loop into a culture

container and removing the cover from stan-

dard blender after mixing a sample.

1.1.2 Job attitudes can be the cause of

laboratory accidents: overly rigid work habits,

failure to recognize dangerous situations,

work at excessive speed and deliberate viola-

tions of rules. These attitudes can only be

overcome by the development of safe work

habits through continued education and

training.

SAFETY

259

image:

TABLE V-C-1

Laboratory-acquired Infections Related to Personnel and Work*

No.

Distribution

of cases

of deaths

Total # of

Infections

2262

Bacteria

1303

Type

Viruses

519

of Infective Agent

Rickettsia

293

Parasites

Fungi

Type of Personnel (where known)

Trained Scientific

Personnel"

Students

Animal Caretakers

Clerks, occasional

visitors, maintenance

Types of Work (where known)

Diagnostic

Research

Biological Reagent

Production

Classwork

Combination of

Activities

1534

221

525 ,

726

§1

856 358

80 —

137 3i

48 —

393 96

289 230

19 31

37 —

206

155

—

599

335

133

•Reference 3.

"Includes research assistants, professional and technical workers and graduate students.

260

&ERA MICROBIOLOGICAL MANUAL 1978

image:

TABLE V-C-2

Sources of Laboratory-acquired Infections*

Source of

Infection

Work Situation but No Known. Incident

Clinical specimens

Autopsy, including known

accidents

Aerosols

Handled infected animals

and ectoparasites

Work with agent

Discarded glassware

Known accidents

Needle and syringe

Pipetting

Spilling and splattering

Type of Infective Agent

Bacteria Parasites

103 17

95 —

135 1

126 5

412 16

21 ' — '

66 3

66 1

41 5

Fungi

—

Injury with broken glass,

etc.

Bite of animal or

ectoparasite

Centrifuge

. 28

"Reference 3.

SAFETY

261

image:

1.1.3 Safety training of laboratory and

field personnel, formulation of safety regula-

tions, and the establishment of mechanisms

for reporting and investigating accidents are

prime responsibilities of the laboratory and

field supervisors and higher management.

First aid courses should be provided to the

laboratory supervisor and at least one other

permanent employee.

Each employee should have a copy of the

safety program. Joint supervisor-employee

safety committees should identify potential

laboratory hazards and formulate workable

safety regulations. Laboratory safety regula-

tions should stress the protection of the labora-

tory personnel, janitorial and maintenance

staff and others who might come in contact

with the laboratory and its personnel.

1,2 Reporting Laboratory Infections and

Accidents

1.2.1 Because many laboratory accidents

are not reported, most laboratory infections

are never traced to a specific cause. To im-

prove safety, it is important to maintain good

records of laboratory accidents and infections.

It is necessary to know the pathogens involved

and the circumstances under which the infec-

tion or accident occurred. It is recommended

that each laboratory set up a formal system for

reporting accidents as they occur so that ap-

propriate prophylatic measures may be insti-

tuted. A record of accidents with after effects,

particularly those resulting in infection, can be

of considerable value.

1.2.2 In EPA, laboratory-acquired

infections oY accidents must be reported to the

Immediate supervisor. EPA Form 1440,

Supervisor's Report of Accident, and Form CA

1, "Federal Employee's Notice of Traumatic

Injury and Claim for Continuation of

Pay/Compensation," must be completed by

the employee and the supervisor and filed

within two working days. Form CA 2, "Federal

Employee's Notice of Occupational Disease

and Claim for Compensation" is to be

completed by the employee and submitted

within 30 days.

It is recommended that the safety officer

at each installation conduct a quarterly safety

inspection of the facilities to identify and cor-

rect dangerous conditions or procedures. He

should make full use of the safety check lists

developed for the laboratory. An example of a

safety check list is given at the end of this

Section.

2. Sources of Hazard

2.1 Causative Agents

Only a small percentage of microorgan-

isms are capable of producing disease in

man; they include bacteria, fungi, yeasts,

protozoa, actinomycetes, animal and human

viruses, and rickettsiae (3, 4). Because

innocuous and infectious microorganisms

cannot be differentiated in natural materials

and because microorganisms considered

harmless may produce disease in man under

favorable conditions, it is good practice to

treat all microorganisms and materials as if

they are pathogens or disease carriers.

2.2 Sources in Sampling

Laboratory and field personnel collecting

samples and isolating cultures from natural

sources must be made aware that pathogens

are present in environmental samples.

Disease-causing organisms are found in natu-

ral waters, municipal effluents and sludges,

industrial wastewaters from packing plants, in

soils and runoff from feedlots and from septic

tank systems. These pathogens have also been

found in inadequately-treated finished water

systems and in ground water supplies. Before

working with environmental samples, field

personnel should have thorough training in

aseptic technique and handling pathogens.

2.3 Sources in the Laboratory

Table V-C-1 and 2 present data on

laboratory-acquired infections in the United

States which were collected in a survey of

5000 laboratories over a 20-year period (3).

Table V-C-1 shows the distribution of

laboratory-acquired infections according to

262

MICROBIOLOGICAL MANUAL 1978

image:

personnel and their work. It is significant that

trained scientific personnel had 1534 of the

1924 infections identified by type of person-

nel. Among laboratory workers, researchers

led with 726 of 1938 infections. These data

suggest that the majority of infections occur

because of carelessness of trained and knowl-

edgeable workers and not because of igno-

rance. However, new technicians should be

made aware that culturing microorganisms

from natural sources develops extremely large

numbers of cells which could cause disease if

not properly handled.

Table V-C-2 presents data on the sources

of laboratory-acquired infections. This table

shows that the five most common accidents

causing laboratory infection were:

1. Accidental inoculation with syringes

and needles.

2. Accidental oral aspiration of infectious

material through a pipet.

3. Cuts or scratches from contaminated

glassware.

4. Spilling or splattering of pathogenic

cultures on floors, table tops, and other

surfaces.

5. Bites of animals or ectoparasites.

It is significant that ir^ about 80% of the

laboratory infections studied ir^ Table V-C-2

the mechanism was not known. It was only

known that the individual had worked with the

agent or had tended infected animals.

3. Field Safety Guidelines

The sample collector or investigator must

also consider safety in his work. The potential

for accidents in field work is much greater than

in the laboratory. The following rules on field

safety were extracted from a comprehensive

safety manual developed by EPA's National

Enforcement and Investigations Center at

Denver (5). They are intended as guidelines to

assist the laboratory in developing its own

protocol.

3.1 Automotive Safety

3.1.1 The driver should make certain that

he has valid state and agency driver licenses

on his person before operating a vehicle.

3.1.2 If the driver observes a questionable

or unsafe condition when first operating a ve-

hicle, he should return it directly to the carpool

regardless of work demands.

3.1.3 Continuous driving in excess of ten

hours, in any 24-hour period, is not

recommended.

3.1.4 Occupants of vehicles should wear

seat belts and shoulder harnesses, where pro-

vided, whenever vehicles are in motion. The

driver should carry a kit provided by the labor-

atory that includes fire extinguisher, flares,

reflectors, and a first-aid kit.

3.1.5 Safety screens should be installed in

carryall and van-type vehicles to separate the

cargo and passenger compartments. If safety

screens are not installed, cargo should not be

stacked higher than the back of the seat.

3.1.6 Employees required to tow a trailer

should be instructed in the proper handling of

the equipment involved.

3.1.7 Vehicles used to tow any kind of

trailer should be equipped with west-coast

type mirrors and the necessary connections

for trailer signal, tail lights, brakes, and safety

chains.

3.1.8 If a boat is transported in a pickup

truck, it should not obstruct the vision of the

driver or extend over the vehicle cab.

3.1.9 A driver backing a vehicle with a

trailer in tow should have someone outside the

vehicle to direct him.

3.2 Boat Safety

3.2.1 Only qualified employees should op-

erate watercraft. Boat operators must have

completed advanced emergency first-aid

training.

SAFETY

263

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3,2.2 The boat operator must not operate

the boat without a second person on board.

3.2.3 The boat operator is responsible for

the safety of persons and equipment on board.

He should provide a a boat safety briefing to

occupants before embarking.

3.2.4 Occupants must wear life jackets

onboard. No exceptions are permitted. Soft-

soled, non-skid shoes are recommended.

3.2.5 Flare gun, fire extinguishers and first

aid kit should be kept on each boat.

3.2.6 Boats operated in estuaries or open

seas should be equipped with depth-finding

instruments, navigational aids and two-way ra-

dios adequate to communicate with at least

one shore station. Boats with marine radios

should monitor distress frequency except

when transmitting.

3.2.7 Boats should not be operated in high

winds, storms, heavy rain, fog, etc. Boats

should not be operated more than one half

mile from launch point on estuaries, large

lakes, and large rivers until acquiring reliable

weather forecasts.

3.2.8 The operator should attach a red

pennant to the radio mast when operating at

slow speeds (e.g., sampling, dredging, towing).

3.2.9 The operator must install and use

lights according to established practice for

night operations.

3.3 General Rules for Sampling

3.3.1 Require two people on each gauging

or night sampling crew or on other hazardous

projects.

3.3.2 Wear safety glasses, safety shoes,

hard hats, respirators, gas masks, and ear-

protection devices, as appropriate in hazard-

ous areas.

3.3.3 Wear fluorescent vests or jackets

while sampling from roadways and bridges.

During sampling, post a yellow flasher on ap-

proaches at each end of the bridge. On heavily

traveled roads post flagmen or warning de-

vices at each end of bridge that lacks 24-inch

walkways. Such sampling points should be

avoided where possible.

3.3.4 Wear rubber gloves while handling

samples that might be toxic or corrosive. Wear

work gloves while handling sampling equip-

ment. During collection and transport, the field

worker should store containers to prevent

spilling or splashing of samples. Disinfect

hands immediately after handling sewage

samples and equipment for sampling sewage.

3.3.5 Do not sample from railroad bridges

unless there is an adequate walkway or the

railroad dispatcher has been contacted and it

has been positively determined that no trains

will run during sampling period.

3.3.6 Equip vehicles for sampling and as-

sociated work with rotary amber caution

lights. Operate such lights whenever vehicles

are driven slowly on roadways or are parked

near roadways. Do not park vehicles on

bridges.

3.3.7 Inform employees of the safety rules

in force within industrial sites. Employees

must conform to rules promulgated by the

industry while on-site.

3.3.8 Properly ground electrical apparatus

employed in field operations and use battery

straps to handle or move wet-cell batteries.

3.4 Sampling from Manholes

3.4.1 Erect barricades around manholes

where samples are being collected. Do not

leave manholes uncovered while unattended

orunbarricaded.

3.4.2 Do not enter a manhole until it is

cleared by using a blower for at least five

264

&EPA MICROBIOLOGICAL MANUAL 1978

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minutes. Following the five-minute ventilation,

use a lead acetate swab to check for I-^S. If

H2S is present, wear a respirator when enter-

ing manhole. Substitute a respirator for blower

ventilation where explosive gases are present

(i.e., in storm sewer or domestic sewer).

3.4.3 A sample collector entering man-

holes must wear safety lines handled by two

persons outside of manholes. Keep safety lines

taut at all times. Fifteen minutes is the maxi-

mum time allowed in manhole. Keep a vehicle

at hand in case of emergency.

3.4.4 Do not enter sewer lines for any

reason.

3.5 Sampling Channels and Streams

3.5.1 Sample collector must work from

behind a barricade or wear a safety line at-

tached to a secure object when sampling fast-

moving channels or streams from shore, walk-

way, etc. The same rule applies to any open

channel when footing is questionable, i.e.,

snow, steep bank, etc.

3.5.2 Wade only to knee-depth In swift

waterstreams, or to hip-depth in placid water.

When wading in fast moving water secure

safety lines to shore and have at feast two

other persons in attendance.

3.5.3 Attach lines from sampling devices

!°_ a secure object but never to sampling

personnel.

3.6 Sampling Under Ice

3.6.1 Two people are a minimum crew for

operations involving ice cover. One person

must remain on solid footing until thickness of

ice is known.

3.6.2 Do not sample on ice if the ice thick-

ness is less than four inches.

3.6.3 Wear life preservers and secure

safety lines to an object on shore when sam-

pling on ice-covered water.

4. Laboratory Safety Guidelines

The following safety rules are intended as

guidelines. They were developed from the

available safety literature (3-9) and have con-

sidered the Occupational Safety and Health

Administration (OSHA) regulations (1/2). Us-

ing such source materials, the laboratory direc-

tor, laboratory supervisor or senior profes-

sional should develop rules that are specific

for the laboratory program and the organisms

involved.

4.1 Personal Conduct and Clothing

4.1.1 Store coats, hats/jackets, and other

items of personal clothing outside of the mi-

crobiology laboratory. Do not mix laboratory

and street clothes in the same locker.

4.1.2 Wear a non-flammable laboratory

gown or coat in the laboratory. If clothing

becomes contaminated, autoclave before

laundering. Laboratory clothing should not be

worn in clean areas or outside the building.

Open-toed shoes, or extreme shoe styles

should not be worn, since they provide little

protection or are unstable.

4.1.3 Wear goggles or safety glasses to

protect eyes from UV irradition.

4.1.4 Wash hands carefully after labora-

tory and field duties, using a germicidal soap.

4.1.5 Use forceps or rubber gloves when

there is a significant danger of contamination

such as during the clean-up of pathogenic

material.

4.1.6 Do not touch one's face, lick labels

or put pencils and other materials in one's

mouth.

4.1.7 Don't smoke, eat, drink or chew gum

in the laboratory or while sampling. Do not

keep food or drinks in the lab refrigerator or

cold room. Do not brew coffee or tea in the

laboratory area.

SAFETY

265

image:

4.1.8 Keep conversation to an absolute

minimum during bench work to prevent self-

infection or loss of analytical data.

4.1.9 Keep reading matter, surplus materi-

als and equipment out of the laboratory area.

4.1.10 Laboratory and field personnel

handling polluted samples should be vacci-

nated against typhoid, tetanus and polio.

4.2.10 Lyophilization procedures can be a

source of laboratory infection. When vacuum

is applied during lyophilization, the contami-

nated air is withdrawn from the ampuls

through the pump and into the room. Use bio-

logical air filters or air decontamination proce-

dures to reduce hazard. Aerosols are also often

created by opening lyophilized ampuls. Re-

duce this hazard by wrapping the ampul in a

disinfectant-soaked pledget of cotton before

breaking.

4.2 Laboratory Equipment

4.2.1 Limit traffic through the work areas.

4.2.2 Treat all cultures and samples as if

they are potentially pathogenic. The degree of

risk Is increased greatly in culture work be-

cause the microorganisms are produced in

very large numbers.

4.2.3 Do not mouth-pipet polluted water,

wastewater or other potentially infectious or

toxic fluids; use a bulb or other mechanical

device. See Part II-B, 1.8.2.

4.2.4 For potable waters, plug pipets with

non-absorbent cotton. Do not use pipets with

wet plugs.

4.2.5 Use a hooded bunsen burner or

shielded electric incinerator to protect against

splattering during culture work.

4.2.6 Maintain benches in a clear and un-

cluttered condition for maximum efficiency

and safety.

4.2.7 Perform all culture work in a

biohazard hood to protect cultures and

workers.

4.2.8 Do not use the kitchen type blender

for mixing materials containing infectious

agents. Safety blenders are available in which

infectious materials may be mixed without dis-

semination of infectious aerosols.

4.2.9 When a vacuum line is used, inter-

pose suitable traps or filters to insure that

infectious agents do not enter the system.

4.2.11 Read II-C-6 for instructions on

proper packing of cultures for mail shipment

before sending any isolates to a central labora-

tory for confirmation.

4.2.12 Periodically clean out freezers, ice

chests and refrigerators to remove any broken

ampuls, tubes, etc., containing infectious ma-

terials. If units contain pathogenic cultures,

use rubber gloves during this cleaning. Use

respiratory protection if actinomycetes, fungi

or other easily disseminated agents are

involved.

4.3 Disinfection/Sterilization

4.3.1 Disinfect table tops and work carts

before and after laboratory work. A bottle of

disinfectant and gauze squares or towelling for

washing and wiping purposes should be avail-

able in laboratory for routine and emergency

use.

4.3.2 Use a disinfectant which specifies

germicidal activity against the organisms most

often encountered in the laboratory. Organo-

iodine complexes, quaternary ammonium

compounds, phenolics and alcohols which are

effective against vegetative bacteria and

viruses are recommended for general use.

However, these disinfectants are not sporo-

cidal. If spore-forming bacteria are encounter-

ed, formaldehyde or formaldehyde/alcohol

solution is recommended. See Table V-C-3.

Mercury salts, chlorine-containing com-

pounds or home-use products are not recom-

mended for the laboratory.

266

&EFW MICROBIOLOGICAL MANUAL 1978

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TABLE V-C-3

Normal Use Concentration of Disinfectants

Use

Compound Concentration

mg/Iiter

Organo-lodine Complexes

Quaternary Ammonium

Compounds

Phenoltes

Alcohol, 70% w/v

Formaldehyde

Formaldehyde in

70% Alcohol Solution

100-150

700-800

water

solution

4.3.3 If a culture or infective material is

spilled, notify the laboratory supervisor at

once, then disinfect and clean up the area.

4.3.4 Never pour viable cultures or con-

taminated materials in the sink. Never leave

infectious material or equipment unattended

during use.

4.3.5 Immediately after use, place con-

taminated pipets in a disinfectant container

which allows complete immersion; place cul-

tures and contaminated materials in color-

coded biohazard bags and seal. Disinfectant

containers of pipets and sealed bags of materi-

als are autoclaved as units.

4.3.6 Place used glassware in special cans

marked for autoclaving. Keep broken glass-

ware in a separate container. Place plastic

items in separate cans to prevent fusing of

plastic around glass items.

4.3.7 Mark contaminated items as Con-

taminated before removal from the laboratory

for autoclaving. Use temperature-sensitive

tapes which indicate exposure to heat. Pre-

printed tapes or tags simplify this task.

4.3.8 Check autoclaves with the use of

spore strips or spore suspensions of B. stear-

othermophilus and maximum-minimum

recording thermometers. Ideally autoclaves

are equipped with temperature recording de-

vices so that a permanent record may be

maintained.

Check hot air ovens and gas sterilizers

periodically with spore strips or the indicator,

B. subt/'/isvar. niger.

4.3.9 Wet-mop floors weekly, using water

containing a disinfectant. Dry or wet pickup

vacuum cleaners with high-efficiency exhaust

air filters are recommended. Wax floors with

bacteriostatic floor waxes if available..

4.4 Chemicals and Gases

4.4.1 Label containers plainly and perma-

nently. Dispose of material in unlabelled con-

tainers carefully. Wipe or rinse residual ma-

terial from the external surfaces of reagent

containers after use.

4.4.2 Store flammable solvents in an ap-

proved solvent storage cabinet or a well-

ventilated area.

4.4.3 When opening bottles which may be

under pressure i.e., hydrochloric acid, ammo-

nium hydroxide, cover the bottle with a towel

to divert chemical spray.

4.4.4 Use bottle carriers to transport bot-

tles containing hazardous chemicals (acids,

corrosives, flammable liquids). Large cylinders

are transported only by means of a wheeled

cart to which the cylinder is secured. Store and

transport compressed gas cylinders with ship-

ping caps on, in an upright position, always

securely clamped or chained to a firm support

and away from heat.

4.4.5 Reagents and chemical which might

react in water drains or be dangerous to the

environment must be disposed of in other

ways. Examples are 1) sodium azide which

reacts with metal drains to produce very explo-

sive lead or copper azides and 2) mercury and

its salts which should not be returned to the

SAFETY

267

image:

environment Consult reference texts to deter-

mine the proper disposal procedure for each

chemical (8,9),

4.5 Handling Glassware

4.5.1 Discard broken, chipped or badly

scratched glassware. Use gloves or sweep up

broken glass, do not use bare hands. Pick up

fine glass particles with wet paper towelling.

4.5.2 Fire polish tubing and rods.

4.5.3 Protect hands with gloves, towel, or

tubing holder when inserting tubing into stop-

pers. Lubricate the tubing with water or glycer-

ine. Handle tubing close to the stopper and out

of line with end of the tube.

4.5.4 Use asbestos-centered wire gauze

when heating glass vessels over a burner.

4.5.5 Do not attempt to catch falling

glassware.

4.6 Electrical Equipment

4.6.1 Keep materials, tools and hands dry

while handling electrical equipment.

4.6.2 Use grounded outlets only.

4.6.3 Do not use electrical equipment near

flammable solvents.

4.6.4 Use only carbon dioxide or dry pow-

der fire extinguishers in case of fire in or near

any electrical equipment.

4.7 Emergency Precautions

4.7.1 Install and maintain both foam and

carbon dioxide fire extinguishers within easy

access of the laboratory.

4.7.2 Fire exits should be clearly marked

and accessible.

4.7.3 Install and maintain a complete first

aid kit and an oxygen respiration unit in the

laboratory.

5. Biohazard Control

5.1 Safety Cabinets

5.1.1 The safety cabinet is the most impor-

tant primary barrier available to the microbiol-

ogistfor isolation and containment of microor-

ganisms and for protecting the laboratory envi-

ronment, and the surrounding area from con-

tamination. Transfers of cultures especially

pathogenic fungi, actinomycetes and yeasts

should be conducted in the safety cabinets.

5.1.2 UV lamps are commonly used in

biohazard hoods to maintain sterility of the

work area. Goggles should be worn to protect

the worker and cultures should be protected

from undesirable exposure (see Part IV-A, 4 in

this Manual).

5.1.3 There are several types of ventilated

cabinets available for use (10,13):

(a) Partial Barrier Cabinet

The open or closed front cabinet is usually

referred to as a partial barrier ventilated cabi-

net. This cabinet can be used with the glove

panel removed, depending upon an inward

flow of air of at least 100 linear ft. per min. to

prevent escape of airborne particles. It can

also be used with the glove panel in place and

arm-length gloves attached, in which case it

will be maintained under a reduced air pres-

sure of about one inch of water gauge. When

operated closed, the partial barrier needs an

attached air lock for movement of materials. A

third mode of operation consists of using a

cabinet with glove panel attached, but with

gloves removed.

(b) Absolute Barrier Cabinet

The second type of ventilated cabinet is

the gas-tight cabinet system, referred to as an

absolute barrier cabinet. Absolute barrier cabi-

nets are connected to form a modular cabinet

system with enclosed refrigerators, incuba-

tors, etc. Air is drawn into the cabinet system

through ultrahigh efficiency filters and is ex-

hausted through ultrahigh efficiency filters.

268

MICROBIOLOGICAL MANUAL 1978'

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This third type of safety cabinet is not gas-

tight. It relies on high efficiency filters to pro-

tect the worker and the environment. In the

Figure V-C-1, the blower (1) set into the bottom

of the cabinet pushes air up through a rear

duct into the top of the cabinet (2) and causes a

slight negative pressure in the work area (3). In

the top of the cabinet (2), part of the air is

forced out the exhaust through a High Effi-

ciency, Particulate, Ah* (HEPA) filter (4). How-

ever, the major part of the air is forced down

through a large HEPA filter (5), in a vertical flow

into the work area (6). This vertical flow (lam-

inar flow) combines with the negative pressure

from the blower to draw in enough make-up

from the room to replace that exhausted

above. The make-up air (7) is pulled through

vents at the edge of the hood opening and is

drawn down to the blower without contacting

the work area. It combines with the filtered air

and is recirculated through the HEPA filters.

The laminar flow draws off any contaminating

particles emanating from the work area. HEPA

filters are 99.i9% efficient in removing parti-

cles 0.3 jjm diameter or larger, by the OOP test.

5.1.4 Selection of a Cabinet

The partial barrier cabinet with open front

gives some protection to the worker and the

laboratory environment but does hot protect

the cultures. With the glove panel in place, it

protects the worker and adjacent laboratory

area. The absolute barrier and laminar flow

cabinets provide the greatest protection to the

worker and the cultures.

For routine water bacteriology and limited

work with pathogens, a partial barrier cabinet

with glove panel can be used. If a significant

portion of the workload involves pathogenic

microorganisms, a gas-tight absolute barrier

cabinet with glove ports or laminar flow cabi-

net is recommended. These provide protection

of cultures as well as the worker and the work

environment. Because laminar flow cabinets

do not require glove openings, yet do protect

personnel and culture work and are easy to

use, they are recommended for all but the most

hazardous microbiological operations.

5.2 Biohazard Identification

5.2.1 The revised Federal Occupational

Safety and Health Act of 1972 requires biolog-

ical hazard signs and tags to signify the actual

or potential biological hazard (1, 10). The haz-

ards are defined as infectious agents that

present a risk to human well-being. These

signs and tags are used to identify equipment,

containers, rooms, materials, experimental an-

imals, or combinations of the above, which

contain or are contaminated with viable haz-

ardous agents.

5.2.2 The biological or biohazard symbol

design is shown in Figure V-C-2. It has a fluo-

rescent orange or orange-red color, and may

contain appropriate wording to indicate the

nature or identity of the hazard, the name of

the individual responsible for its control, pre-

cautionary information, etc. The wording must

not be superimposed on the symbol. Back-

ground color is optional, but enough contrast

must be provided so that the symbol can be

clearly defined.

6. Safety Check List

The laboratory safety check list that fol-

lows is provided as a guide for a laboratory to

incorporate wholly or in part into its own safety

program.

SAFETY

269

image:

FIGURE V-C-1. Laminar Flow Cabinet.

270

MICROBIOLOGICAL MANUAL 1978

image:

CAUTION

BIOLOGICAL

HAZARD

FIGURE V-C-2. Example of Biohazard Sign.

SAFETY

271

image:

Safety Check List

fcr Microbiological Water Laboratories

Survey By:

Laboratory:

Location:

Date:

Code: S=Satisfactory, U —Unsatisfactory

1. Administrative Considerations

(a) Laboratory has a formal documented safety program.

(b) Each worker has a copy of the safety program.

and unsafe conditions.

(d) New employees are instructed on laboratory safety.

(e) Joint supervisor-employee safety committee has been estab-

lished to identify potential laboratory hazards.

(f) Records are maintained of accidents and consequences.

(g) Name and phone number of the supervisor and an alternate are

posted at door of the laboratories so he may be contacted

in case of an emergency.

(h) Laboratory supervisor and at least one other permanent

employee have attended appropriate first aid courses. If

so, when:

(date)

{i} Emergency telephone numbers for fire, ambulance, health

centers, and poison control center are placed in a conspic-

uous location near the telephone.

(j) Employees know the location of first aid supplies.

(k) Emergency first aid charts, and hazardous agents charts

are posted in the laboratory.

(1) Fire evacuation plan is established for the laboratory

and is posted In a conspicuous location.

2. Personal Conduct

(a) Personal clothing is stored outside of the microbiology

laboratory.

(b) Lab coats and street clothes are kept in separate

lockers.

272 SEr¥\ MICROBIOLOGICAL MANUAL 1978

image:

id) Germicidal soap or medicated surgical sponges are available

for employees' use.

(e) Preparing, eating or drinking food and beverages are not

permitted in the laboratory.

(f) Smoking or chewing gum are not permitted in the laboratory.

(g) Food or drink are not stored in laboratory refrigerators.

(h) Reading materials are not kept in the laboratory.

(i) Laboratory coats are not worn outside the lab.

(j) Employees who have cuts, abrasions, etc. on face, hands,

arms, etc. do not work with infectious agents.

3. Laboratory Equipment

(a) Bulb or mechanical device is used to pipet polluted water,

wastewater or other potentially infectious or toxic fluids.

(b) Pipets are immersed in disinfectant after use.

(d) Centrifuge cups and rubber cushions are in good condition.

(e) A suitable disinfectant is available for immediate use.

(f) Blender is used with sealed container assembly.

(g) Microscopes, colony counters, etc. are kept out of the work

area.

(h) Water baths are clean and free of growth and deposits.

(i) Employees are instructed in the operation of the autoclave

and operating instructions are posted near the- autoclave.

(j) Autoclaves, hot air sterilizing ovens, water distilling

equipment, and centrifuges are checked routinely for safe

operation.

Give frequency and last date

Autoclave , - .,

Water still

Centrifuge L.

Hot Air Oven

(k) No broken, chipped or scratched glassware are in use.

(1) Broken glass is discarded in designated containers.

SAFETY 273

image:

(m) Electrical circuits are protected against overload with

circuit breakers or ground-fault breakers.

(n) Power cords, control switches and thermostats are in good

working order.

(o) Water taps are protected against back-siphoning.

4. Disinfection/Sterilization

(a) Proper disinfectant is used routinely to disinfect table

tops and carts before and after laboratory work.

(b) Receptacles of contaminated items are marked.

(cj Performance checks of autoclaves, gas sterilizers and hot air

ovens are conducted with the use of spore strips, spore

ampuls, indicators, etc.

Item Frequency Last Date

(d) Safety glasses are provided to employees.

(e) Safety glasses are used with toxic or corrosive agents and

during exposure to UV irradiation.

5. Biohazard Control

(a) Biohazard tags or signs are posted in hazardous areas.

(b) Safety cabinets of the appropriate type and class are

provided.

and polio.

(d) Floors are wet-mopped weekly, with a disinfectant solution.

(e) Personnel are trained in the proper procedures for handling

lyophilized cultures where used.

6. General Handling and Storage of Chemicals and Gases

(a) Containers of reagents and chemicals are labelled properly.

fb) Flammable solvents are stored in an approved storage

cabinet or well-ventilated area away from oil burners,

hot plates, etc.

{c} Bottle carriers are provided for hazardous substances.

274 SEPA MICROBIOLOGICAL MANUAL 1978

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(d) Gas cylinders are securely clamped to a firm support.

(e) Toxic chemicals are clearly marked poison or toxic.

7. Emergency Precautions

(a) Foam and carbon dioxide fire extinguishers are installed

within easy access to laboratory and are properly

maintained. Frequency

(b) Eye wash stations , showers , oxygen

respirators , and fire blankets are available

within easy access.

(d) First aid kits are available and in good condition.

(e) At least one full-time employee is trained in first aid.

(f) Source of medical assistance is available and known to

employees.

8. Suggested Areas of Improvement:

9- General Comments:

(Signature of Installation Officer) (date)

SAFETY

275

image:

REFERENCES

1. 29 Code of Federal Regulations (CFR) Part 1910, "Occupational Safety and Health Standards"

and Amendments.

2. Occupational Health and Safety Administration, 1974. Provisions for Federal Worker Safety and

Health, OSHA(October, 1974), Reference File 41:6241

3, American Public Health Association, 1963. Diagnostic Procedures and Reagents, (4th ed.),

APHA, Inc. pp. 89.

4. Pike, R. M., 1976. Laboratory-associated infections: summary and analysis of 3921 cases. Health

Laboratory Science, 13:105.

6. National Field Investigation Center, 1973. NFIC-Denver Safety Manual, NFIC-Denver, EPA,

Denver, CO. EPA 330/9-74-002 (In revision).

6. Wedum.A, G., 1961. Control of laboratory airborne infection. Bacterial Reviews 25.

7. Reitman, M. and A. G. Wedum, 1971. Infectious hazards of common microbiological techniques.

In: Handbook of Laboratory Safety. (N. V. Steere, ed.). The Chemical Rubber Co., Cleveland, OH

pp.~63~3~I

8. Shapton, D. A. and R. G. Board, Ed. 1972. In:Safety in Microbiology. Academic Press, New York,

NY.

9. Steere, N.V., editor, 1971. Handbook of Laboratory Safety. The Chemical Rubber Co., Cleveland,

OH.

10. Manufacturing Chemists Association, 1975. Laboratory Waste Disposal Manual, Washington,

DC. •

11. US Public Health Service, 1974. National Institutes of Health, Biohazards Safety Guide, USPHS,

DHEW, GPO 1740-00383.

12. U.S. Environmental Protection Agency, 1977. Occupational Health and Safety Manual, TN5

(9-12-77).

13. Runkle, K. S. and G. B. Phillips, Ed. 1969. Mlcrobial Contamination Control Facilities. Van

Nostrand Reinhold Co., New York, NY.

276 EBI\ MICROBIOLOGICAL MANUAL 1978

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PART V. LABORATORY MANAGEMENT

SECTION D LEGAL CONSIDERATIONS

This Section Is intended to guide practic-

ing microbiologists in assessment of their re-

sponsibilities and role under the three federal

laws on water quality. It is not intended as

formal legal guidance or as representative of

official legal position. It is based on the three

Federal laws on water quality, (1, 2, and 3), on

A Primer on the Law, Evidence and Manage-

ment of Federal Water Pollution Control Cases,

Legal Support Division, US EPA May 1972 (4)

and on "Enforcement Activities," David I. She-

droff, in Proceedings of the First Microbiology

Seminar on Standardization of Methods, US

EPA, March 1973 (5). Lawyers should be con-

sulted for the exact interpretation of the laws

and their applications.

The Section describes the portions of the

Federal laws on water quality that are relevant

to microbiologists and relates analytical me-

thods and record-keeping to these laws. It also

shows how the analytical results become evi-

dence in administrative or court proceedings

and will help the analyst to understand his role

as a witness. A brief outline of the contents of

the Section follows:

1. Enabling Legislation (Federal

Laws Dealing with Water

Quality)

1.1 Scope and Application

1.2 Federal Laws Dealing

with Water Quality

1.3 Federal Water Pollution

Control Act Amend-

ments of 1972 Public

Law 92-500

1.4 The Marine Protection,

Research and Sanctu-

aries Act of 1972,

Public Law 92-532

1.5 The Safe Drinking Water

Act of 1974, Public

Law 93-523

2. Application of the Laws

to Microbiology

2.1 Gathering and Preserving

Evidence

2.2 Admissibility of Evidence

2.3 Preparation for

Testimony

2.4 Testimony in Court

1. Enabling Legislation

1.1 Scope and Application: To under-

stand the role of microbiology in environmen-

tal and compliance monitoring, it is necessary

to briefly describe EPA's responsibilities for

development of methodology, assistance to

the States, promulgation of criteria and guide-

lines, establishment of compliance with per-

mits and conduct of enforcement actions.

Much of the work of the microbiologist in EPA

will involve generation of data to determine

compliance with the Federal laws on water

quality. These laws with related regulations

limit the choice of analytical technique and

sometimes require more documentation than

the analyst might otherwise provide.

LEGAL ASPECTS

277

image:

1.2 Federal Laws Dealing with Water

duality (6)

Congress has passed three principal laws

on water quality which concern the microbiol-

ogist: The Federal Water Pollution Control Act,

as amended; the Marine Protection, Research

and Sanctuaries Act, commonly known as the

Ocean Dumping Law; and the Safe Drinking

Water Act. These acts have a common theme:

1.2.1 Legislation is passed by Congress

providing the general framework of Federal

Interest and control of an area of the environ-

ment. EPA promulgates general rules describ-

ing, requiring and/or limiting the qualities of

wastewater discharges or drinking water.

1,2.2 EPA or appropriate state agencies

issue permits placing specific limitations on

discharges, establish Maximum Contaminant

Levels in drinking and ambient water, and pro-

vide general rules controlling underground

injections.

1.2.3 Permittees or others subject to the

particular act may be required to self-monitor

their discharges, and report findings to the

State and/or EPA.

1.2.4 Provisions are made for enforce-

ment actions when permit, variance, or abate-

ment schedules are violated.

1.3 Federal Water Pollution Control Act

Amendments of 1972, Public Law 92-500

(See Appendix A for fisting and summary

of pertinent sections of the law, and the related

microbiological activities).

1.3.1 Background and Summary (6): This

Is the most comprehensive program ever en-

acted to prevent, reduce, and eventually elimi-

nate water pollution. The two general goals of

the Act are: To achieve wherever possible b*y

July 1, 1983, water that is clean enough for

recreational uses and for the protection of

aquatic life; and by 1985 to have no dis-

charges of pollutants into the Nation's waters.

The law extends the Federal program to all

U.S. surface waters, not just interstate waters.

The States have submitted water quality stan-

dards for intrastate waters to EPA for approval

or revision. While the States retain primary

responsibility to prevent, reduce, and elimi-

nate water pollution they must now do so

within the framework of a new national pro-

gram. The law sets forth guidelines for the

control of industrial and municipal water pollu-

tion, expands water quality standards, estab-

lishes a new system of permits for discharges

into the Nation's waters, and creates stringent

enforcement machinery and heavier penalties

for violations.

1.3.2 The Regulatory Scheme (6): Under

this act the States establish the minimum

water quality standards for streams and these

standards are approved by EPA. The Adminis-

trator determines minimum acceptable efflu-

ent limits for municipal treatment plants and

for specific industries based on current tech-

nology! These limits become more restrictive

overtime. Using the more stringent water qual-

ity or treatment limitations, the Administrator

or State determines specific limits for a dis-

charge. These limits set forth in a'permit for a

direct discharge are enforceable by civil pen-

alty, civil or criminal process, or revocation of

permit.

A microbiologist may be called as a wit-

ness to prove the violation of the permit by

direct discharge or to prove the violation of

specific limitations placed on industrial firms

discharging to municipal plants. Generally

these microbiological limitations will relate to

discharges from municipal treatment plants or

from industries such as food processors.

Other enforcement activities for the micro-

biologist under the FWPCA include a permit

program covering sewage sludge discharges

and standards for discharges from marine san-

itation devices and their performance (40

CFR/40 Amendments, 41 No. 20, January 29,

1976). If such permits or standards contain

microbiological limitations, the microbiologist

may be called on to prove or disprove a viola-

tion (Section 405). Standards have been set

for discharges from marine sanitation devices

278

MICROBIOLOGICAL MANUAL 1978

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and their performance (Federal Register 41,

January, 1976).

1.3.3 Analytical Guidelines and Criminal

Sanctions (6)

Analytical guidelines and criminal sanc-

tions for improper analysis or furnishing false

results are included in the Act. Section 304 (g)

calls for the Administrator to promulgate

"...guidelines establishing test procedures for

the analysis of pollutants...". The initial guide-

lines for monitoring the National Pollution Dis-

charge Elimination System (NPDES) (7) contain

test procedures for total coliforms, fecal eoli-

forms, and fecal streptococci using methods

referenced from the 14th Edition of Standard

Methods(8). The guidelines defining second-

ary treatment originally included fecal coli-

form limitations, and permits issued to munici-

pal plants until July, 1976 contain such limita-

tions. These regulations were amended in

July, 1976 and no longer contain microbiolog-

ical limitations. Such limitations may still be

required in permits issued after that date if

required for compliance with water quality

standards or if such parameters are required in

order to comply with State law. Guidelines for

certain food related industries include fecal

coliforms as a limiting parameter. The

amended guidelines for municipal and indus-

trial wastewaters place restrictions on the

measurement of fecal coliforms in chlorinated

or toxic wastewaters. For these wastewaters

the membrane filter or most probable number

(MPN) methods may be used, but the MPN is.

the method of choice when the results may be

involved in controversy. (Refer to the amend-

ments to 40 CFR Part 136 (9)). The methods

are described in Part III, B, C and D,

Microbiologists performing analyses re-

quired under this Act should be aware of the

specialized enforcement procedures in Sec-

tion 309 (c) of FWPCA relating to analyses and

reports of results: "Any person who knowingly

makes any false statement, representation, or

certification in any application, record, report,

plan, or other document filed or required to be

maintained under.this Act or who falsifies,

tampers with, or knowingly renders inaccurate

any monitoring device or method required to

be maintained under this Act, shall upon con-

viction, be punished by a fine of not more than

$10,000, or by imprisonment for not more

than six months, or by both."

1.3.4 Alternative test procedures are per-

mitted for use in NPDES, 40 CFR Part 136 and

in the Drinking Water regulations under 40

CFR Part 141. Information on application for

use of alternative test procedures is given in

these issues of CFR. The details of the compar-

ative testing which may be required are given

in this Manual, under Quality Control, Part IV-C.

1.4 The Marine Protection, Research

and Sanctuaries Act of 1972, (Ocean-

Dumping) Public Law 92-532 (2)

(See Appendix A for listing and summary

of pertinent sections of the law, and the related

microbiological activities).

1.4.1 The Regulatory Scheme: The Marine

Protection, Research and Sanctuaries Act of

1972 regulates the dumping into ocean

waters of all types of materials which would

adversely affect human health and welfare, the

marine environment, ecological systems or

economic development

This Act bans dumping of radiological,

chemical or biological warfare agents and

high-level radioactive wastes. With one excep-

tion, permits are required for transporting ma-

terials for ocean dumping and for the dumping

itself. The Corps of Engineers issues permits

for dredge spoils; EPA issues permits for all

other materials (see Appendix A). The excep-

tion is fish-processing wastes. Since they are a

natural ocean waste product, no permit is re-

quired unless harbors or other protected

waters are involved as the receiving waters, or

unless the EPA Administrator finds that such

deposits in certain offshore areas could endan-

ger health, the environment or ecological

systems.

In evaluating permit applications, EPA and

the Corps of Engineers must consider:

The need for the proposed dumping.

LEGAL ASPECTS

279

image:

The effect on human health and welfare,

including economic, aesthetic and recrea-

tional values.

The effect on fisheries, resources, plank-

ton, fish, shellfish, wildlife, shorelines,

beaches and marine ecosystems.

The effect of dumping particular volumes

and concentrations of materials and the

persistence of the effect.

The effect on other uses such as scientific

study, fishing andtrther resource exploitation.

Appropriate locations and methods of dis-

posal or recycling, including land-based

alternatives and the probable impact of

requiring the use of such alternate loca-

tions or methods.

The law charges the Secretary of Com-

merce with responsibility for a comprehensive

and continuing research program involving

the possible long-range effects of pollution,

overfishing or man-induced changes in ocean

ecosystems. Research efforts are to be coordi-

nated with EPA and the Coast Guard.

The basic research objective of the law is to

find ways to minimize or to end all ocean dumping

within five years. It will cover the effects of dump-

Ing materials into ocean or coastal waters and

into the Great Lakes or their connecting waters.

1-4-2 Civil and Criminal Sanctions: The

law provides for both civil and criminal penal-

ties for violations but there is no penalty for

dumping materials from a vessel as emer-

gency action to safeguard life at sea. Any indi-

vidual may initiate a civil suit to enjoin any

person, including Federal, State and local gov-

ernment or agency, who is alleged to be violat-

ing any prohibition, limitations, criterion or

permit established or issued under this law.

1.5 The Safe Drinking Water Act of

1974, Public Law 93-523 (3)

(See Appendix A for listing and summary

of pertinent sections of the law, and the related

microbiological activities).

1.5.1 National Objectives: This Act has as

its main objective the establishment and en-

forcement of primary drinking water stan-

dards. These standards, which are to be en-

forced by the States, will apply to public water

systems and specify maximum levels of: 1)

Those contaminants which may have adverse

health effects {primary standards) and, 2) those

contaminants which should be limited to pro-

tect the public welfare (secondary standards).

The protection of underground drinking water

sources by regulation of State underground

injection control programs and the appoint-

ment of a 15-member National Drinking Water

Advisory Council are also provided for under

this Law.

1.5.2 The Regulatory Scheme

Interim Primary Drinking Water Regula-

tions: The Administrator of EPA proposed na-

tional interim primary drinking water regula-

tions 90 days after enactment of the Law. The

interim regulations were promulgated in De-

cember, 1975(10).

Secondary Drinking Water Regulations:

Following the enactment date of this Law, pro-

posed secondary regulations will address the

aesthetic characteristics of water such as

taste, appearance, etc. Ninety days later these

secondary regulations will be promulgated,

but will not be enforceable.

Study of Maximum Contaminant Levels in

Drinking Water: The National Academy of Sci-

ences conducted a study to determine the

Maximum Contaminant Levels which should

be recommended to protect human health.

The study investigated contaminants that

might have adverse health effects but the lev-

els of which could not be determined in drink-

ing water. The results of the above study are

reported to Congress. EPA then publishes pro-

posals in the Federal Register for recom-

mended maximum levels, which will subse-

quently be promulgated.

Revised Primary Drinking Water Regula-

tions: Following the National Academy of Sci-

ences study, EPA proposes revised primary

drinking water regulations to be adopted in

280

&EPA MICROBIOLOGICAL MANUAL 1978

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180 days and to be effective 18 months after

promulgation. These primary drinking water

regulations specify a Maximum Contaminant

Level or require the use of treatment tech-

niques for each contaminant in lieu of

Maximum Contaminant Levels.

Regulation of State Underground Injec-

tion Control Programs: The Act provides for

regulations which contain minimum require-

ments for effective (State) programs to prevent

underground injection which endangers drink-

ing sources. To be approved, a State program

must prohibit underground injection without a

state permit within three years after the enact-

ment date of the Law. Applicants for under-

ground injection permits must satisfy the State

that the injection will not endanger drinking

water sources. No regulations are promul-

gated that allow underground injection which

endangers drinking water sources.

1.5.3 Quality Control Requirement

In the Safe Drinking Water Act, the Admin-

istrator can specify analytical methodology

which includes quality control and testing

procedures.

1.5.4 Enforcement

(a) State Primary Enforcement Responsi-

bility: A State has the primary enforcement

responsibility for protection of drinking water

if it has a program acceptable to EPA. The EPA

Administrator must determine that the State

has drinking water regulations no less strin-

gent than Federal regulations. The State may

permit variances and exemptions as pre-

scribed in the Law, and must have an adequate

plan for providing safe drinking water under

emergency circumstances. In addition, the

State must have monitoring programs that

comply with Federal requirements and must

possess sufficient enforcement authority.

Approval by the EPA Administrator of the

State's underground injection program gives

to the State the primary enforcement responsi-

bility until such time as the Administrator de-

termines that the State no longer meets its

requirements underthe Act.

C3) fgJ.eJTJ.[ Enforcement: If a State fails to

assure enforcement of drinking water regula-

tions, the EPA Administrator notifies the State

concerning the violation, and provides advice

and technical assistance to the State and to

the public water system that is in non-

compliance, to bring the system into

compliance.

1.5.5 Civil and Criminal Sanctions: The

EPA Adiministrator may bring a civil action to

require compliance with either the national

primary drinking water regulations or with any

requirement of an applicable underground in-

jection control program. A maximum penalty

of $5,000 per day may be imposed by the

court for each day in which a violation occurs.

*2. Application of the Laws to Microbiology

2.1 Gathering and Preserving Evidence

2.1.1 Stream Standards: To establish a

standard violation, it is necessary to show the

navigable waterway is below approved water

quality standards. Water quality criteria spec-

ify permissible levels of chemical and biologi-

cal constituents for receiving waters. It must

be demonstrated that the defendant's dis-

charge caused or contributed to a reduction in

receiving water quality below one or more

applicable standards. Samples should be col-

lected 1) upstream of the discharge, 2) at the

point of discharge and 3) downstream of the

discharge at a point after a reasonable mixing

zone.

Although all State water quality standards

include criteria for the same basic parameters,

there are differences among the states as to

the sampling and test procedures which must

be followed in order to establish a standards

violation. It is thus imperative that only the

testing method specified be used in order to

show that a particular state water quality stan-

dard has or has not been violated.

2.1.2 Effluent Standards: The Agency has

not provided specific guidance in sampling

LEGAL ASPECTS

281

image:

and test protocol to microbiologists working

with the effluent standards and compliance

monitoring. A protocol must vary with the way

in which the permits are written. The stan-

dards may set maximum values and average

values by the day, week, month, and year with-

out defining the number and kinds of sample

required to establish compliance with a given

standard. Specifications of numbers and types

of samples are needed to provide weekly,

monthly or yearly maxima or averages.

Each Regional Office of EPA has devel-

oped an approach to compliance monitoring.

Uniformity of sampling and testing schemes is

desirable for data validation and for compari-

son of data between different laboratories.

The need for uniformity is even more impor-

tant with the transfer of the responsibility to

the states for issuance of permits, as legally

permitted. Some states are following EPA-

Regional guidance while others are develop-

ing their own plans for compliance monitoring.

The sampling plan should select the sam-

pling techniques, volumes, frequency, replica-

tion, etc., to meet the standard being chal-

lenged. All sampling and analyses to verify

compliance with a particular standard should

be performed in the same fashion and with the

same frequency. Three rules for a good sam-

pling plan are: 1) fit the design to meet the

effluent standard, i.e., use a reasonable means

to obtain statistical validity 2) apply the plan

uniformly and 3) document the plan, indicate

its source and record its use.

2.1.3 Drinking Water Standards: The Safe

Drinking Act has established Maximum Contami-

nant Levels (MCL's) on an interim basis for com-

munity and non-community systems. The MCL's

are based primarily on the 1962 Public Health

Service Standards. For microbiology, the minimal

sampll/ig frequency per month is specified in

Title 40 Part 141. Samples shall be taken at

regular time intervals and in numbers propor-

tional to the populations served. Samples shall

also be taken at points representative of the

conditions in the distribution systems.

2.1.4 Constitutional Protections: Sample

evidence taken from the defendant's (indiv-

idual or corporate) property without his con-

sent cannot be introduced into evidence in

either a civil or a criminal case because of the

Fourth Amendment guarantee against unrea-

sonable searches and seizures. Consent need

not be obtained to take samples on the public

portions of a waterway, usually up to the ordi-

nary high water level (11). Almost all Fourth

Amendment objections can be prevented by

sending an advance, written notification of the

time, scope, and purpose of any proposed EPA

inspection, or sampling visit and by obtaining

the written consent of the party to be in-

spected (12). If a search warrant has not been

obtained, unannounced investigatory inspec-

tions may be made only if the voluntary con-

sent of a person in authority is secured (13).

2.2 Admissibility of Evidence

2.2.1 Types of Legal Action: Violations of

Public Laws 92-500, 92-532, or 93-523 can

result in civil penalties assessed by the Admin-

istrator, a hearing board, or a court; criminal

sanctions by a court; or a court order requiring

a discharge source to take or cease a particular

action. Except for civil penalties imposed by

the Administrator, formal hearing will be

required.

2.2.2 Authentication of Testimony: Au-

thentication as a condition precedent to ad-

missibility may require testimony under oath

(14). Witnesses are subject to cross-

examination. The form and admissibility of evi-

dence presented in Federal courts are clearly

defined in the Federal Rules of Civil Procedure

(15) and the Federal Rules of Evidence (14).

These formal rules of evidence may be par-

tially annulled for administrative hearings. Lab-

oratory records may not be acceptable evi-

dence without proof of authenticity.

The hearsay rule is one of the most direc-

tive statutes. It states that generally persons

may only testify to what they know personally

and that they must be subject to cross-

examination. However, some exceptions to the

hearsay rule are allowed. Evidence that would

normally be hearsay is admissible in the ad-

ministrative hearing, if it has probative value in

the opinion of the hearing officer. Also, evi-

282

**EPA MICROBIOLOGICAL MANUAL 1978

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dence may be presented in written form in a

hearing, but this is more likely when the pre-

ceeding is not a full-fledged trial-type hearing.

Additional exceptions to the hearsay rule

are cited in the recently-enacted Federal Rules

of Evidence (14). These rules state that under

certain circumstances a witness does not have

to be present for his statement to be admissi-

ble. In addition, records of regularly-conducted

business activities, public reports, reports pre-

pared by law enforcement personnel and fac-

tual findings resulting from legal investiga-

tions may be admitted without the testimony

of the person or persons involved.

2.2.3 Admissibility of Records: Under Rule

803 (6) of the Federal Rules of Evidence (14),

written records made in the regular course of

any business (i.e., laboratory operation) may

also be introduced into evidence in civil ac-

tions without the testimony of the person(s)

who made the record. Prior to enactment of

the Federal Rules of Evidence, this authority

was contained in the Federal Business

Records Act, 29 U.S. Code, Section 1732A.

Although preferable, it is not always possi-

ble to have the individuals who collected, kept,

and analyzed samples testify in court. In addi-

tion, if the opposing party does not intend to

contest the integrity of the sample or testing

evidence, admission under the Business

Records Act can save much trial time. For

these reasons, it is important that the proce-

dures followed in evidence, sample collections

and analyses be standardized and described in

an instruction manual which can be offered as

evidence of the standard operating procedure

followed by the laboratory.

2.2.4 Limitations ori the Admissibility of

Records: Although the statutes do not specifi-

cally cover the point, it is clear from the exami-

nation of cases that one of the requirements

for admissibility is that the document has in-

herent probability of trustworthiness. Thus, a

trial judge has discretion in allowing or not

allowing a document into evidence if there is

doubt as to its trustworthiness. One criterion

for the judge to consider is whether the partic-

ular analysis was done as a routine matter or

whether it was specifically done in anticipa-

tion of litigation. This caution in admitting evi-

dence is an indication of distrust of the situa-

tion, not of the individuals involved.

2.2.5 Contacts with Parties to Adjudica-

tory or Adversary Proceedings: The following

statements are quoted directly from the May 5,

1975, memorandum of the Acting Assistant

Administrator for Enforcement, US EPA(16):

As we are now becoming involved in more

and more adjudicatory hearings on

NPDES (National Pollution Discharge Elim-

ination System) permits and in enforce-

ment actions, both through Administrative

Orders and in the Courts, it is very impor-

tant that our staffs clearly understand that

contacts and discussions with parties to

these proceedings be carefully controlled.

We have recently had inquiries about

cases in which requests for adjudicatory

hearing had been granted and in which

EPA technical staff members, without the

knowledge of either the attorney assigned

or of the Enforcement Director, met with

company representatives to discuss the

pending case. In each case, the merits of

EPA's position as compared to that of the

company were discussed, as were possi-

ble areas of compromise with respect to

EPA's position.

Case preparation and decisions on strat-

egy for adjudicatory hearings are the re-

sponsibility of our regional attorneys with

assistance from Headquarters Counsel for

Adjudicatory Hearings. Accordingly, I

would appreciate your instructing your

staff members not to discuss permit ques-

tions or technical issues applicable to a

particular industrial facility which is the

subject of an adjudicatory hearing or an

enforcement action until the appropriate

Enforcement Division attorney, either in

the Regional Office or in Headquarters is

notified.

The foregoing is not to be construed as

discouraging settlement discussions in

pending cases, but is only intended to

LEGAL ASPECTS

283

image:

provide for orderly resolution of matters

which can be negotiated.

2.3 Preparation for Testimony

^•^' ^ Gathering and Preserving Evidence

in Water Pollution Enforcement Actions: !n ev-

ery water pollution suit, expert testimony will

be of primary importance. To meet its burden

of proof, the Government may have to present

expert testimony on sampling, laboratory ana-

lyses, test results and the harmful effect attrib-

utable to the defendant's discharge. If the Gov-

ernment's expert witnesses do not testify ef-

fectively, the lawsuit may be jeopardized.

2.3.2 Testimony on Sampling: In the order

of proof in a trial concerning pollution there

will be testimony by witnesses who have taken

samples. The samples may be effluents, receiv-

ing waters, potable waters, sludges or sedi-

ments. These witnesses will explain how,

where and when the samples were taken. The

choice of sampling location and what to sam-

ple depends to a large extent on the type of

legal action contemplated.

2.3.3 Documentation of Procedures: In an-

ticipation of possible court presentation of evi-

dence, laboratories must maintain an orderly,

complete and permanent record-keeping and

filing system. A laboratory operating manual

should be used in all laboratories. The manual

formalizes the operation of the laboratory by

describing in detail the sampling procedures,

the line of technical responsibility, specific

analytic methods followed, data handling pro-

cedures, the continuous quality control pro-

gram established for daily operations, partici-

pation in interlaboratory and intralaboratory

quality control programs and safety guide-

lines. Since routines change, personnel should

sign dated receipts that indicate they have

received the operating instructions and modifi-

cations when issued.

Complete records of samples received

must be kept in a separate log and official

chain of custody requirements must be ob-

served. The laboratory data records, analytical

results and computations should be written,

preferably in a bound book or on bench cards,

that can be incorporated into a permanent

record log. Provision should be made for the

signatures of sample collectors, analysts and

direct line supervisors in the sample log and In

the data log so that the laboratory data are

authenticated. As described in Part V-A of this

manual, a quality control log book should be

maintained on a day-to-day basis. It should

record quality control checks on: media and

supplies, equipment and instrumentation, the

actual analyses, data handling and storage.

Training of analysts should include familiariza-

tion with the quality control book and identifi-

cation of their responsibilities in the program.

Each analyst should have a personal copy of

the manual as a guide.

2.3.4 Pre-Trlal Discovery: Whenever an

agency is a party to any federal court litigation,

it will be subject, under the Federal Rules of

Civil Procedure, to pre-trial discovery. The

agency will be required to answer the oppos-

ing party's questions and to produce re-

quested documents. Technical personnel re-

sponding to a motion to produce documents

should deliver related documents to the

agency attorney handling the case. Docu-

ments should not be withheld because they

appear to be damaging to the government's

case. The responsible government attorneys

will determine, on the basis of the law of dis-

covery, which documents must be submitted

to the opposing party.

A sensible filing system should be set up

and followed. The objective of a filing system

is to store information so that it can be found

quickly. Information which is known but is not

reflected in the file is of no use and will not be

available when needed. However, the files

should be examined regularly and outdated or

superfluous information discarded to maintain

manageability. Critical or outspoken com-

ments on notes, route slips or in margins,

should not be retained unless the originator

and recipient are prepared to defend them in

court.

2.3.5 Testimony or\ Methodology: A wit-

ness may be required to provide testimony on

methods of analyses and test results. For ac-

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MICROBIOLOGICAL MANUAL 1976

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ceptance in court, the witness must be able to

testify that the analytical method or procedure

employed has wide use in the microbiological

community. For example, the procedures out-

lined in Standard Methods {&) and in This Man-

ual are recognized and accepted. In court

cases on record, results obtained using Stan-

dard Methods have been admitted into evi-

dence while deviations from Standard Me-

thodshave had to be explained and justified.

It may be necessary to present testimony

on parameters that are not included in these

publications or on special types of samples to

which the methods described are not applica-

ble. In such cases effective testimony may be

based upon the best methodology currently

available, utilizing as substantiating evidence.

published reports, other method manuals, etc.

to demonstrate that the methods do have rec-

ognition in the scientific community.

The specific test methods to be used in the

application of the Federal Water Pollution Con-

trol Laws may be identified in the Code of

Federal Regulations (CFR). For example, the

procedures required for Section 304 (g) of the

Federal Water Pollution Control Amendments

of 1972 appear in 4O CFR, Part 136. These

guidelines establish the methodology to be

used for compliance monitoring and the me-

thods become those that are acceptable as

standards in court. Part 136 of 40 CFR also

provides a mechanism and rule for obtaining

approval for any alternate procedure that may

be proposed when the recommended method

is not appropriate.

2.3.6 Testimony by Expert: A court may

require that an expert witness' opinion be

based on studies and tests conducted or su-

pervised by him personally. However, experts

are frequently permitted to offer testimony in

the form of an opinion in the area of compe-

tence or based on someone else's work. Such

testimony can be developed through the use of

hypothetical questions and objections tend to

add weight to the expert's testimony rather

than to cast doubt on the witness'

competency.

2.4 Testimony in Court

2.4.1 General Instructions for a Witness:

The following suggestions are made for pros-

pective witnesses to lessen the apprehensions

everyone feels when first testifying before a

board, commission, hearing officer, or in court.

Even veteran witnesses often experience

some anxiety. However, if a witness is properly

prepared on the subject matter of his testi-

mony and his conduct on the witness stand, he

is much more confident about testifying. The

witness will be required to take an oath to tell

nothing but the truth. The important point is

that there are two ways to tell the truth—one is

in a halting hesitant manner, which makes the

board member, hearing officer, judge or jury

doubt that the witness is telling all the facts in

a truthful way, and the other is in a confident

straight forward manner, which gives cre-

dence to the witness' words.

If a scientist is a witness in a case involv-

ing testimony concerning the appearance of

an object, place or condition, he should refresh

his recollection by inspecting the object, place

or condition, etc., before the hearing or trial.

Later he should try to picture the item and

recall the important points of his testimony. He

should repeat this procedure until he has thor-

oughly familiarized himself with the points that

will be made in the testimony.

Before testifying, the witness should visit

a court trial or board hearing and listen to other

witnesses testifying. This will familiarize him

with such surroundings and help him to under-

stand court protocol and the problem of testi-

mony. The scientist should arrive at the hear-

ing in time to listen to other witnesses testify

before taking the witness chair himself.

A good witness listens to the question and

then answers it calmly and directly in a sincere

manner. He knows the facts and can communi-

cate them. He testifies in this manner on cross-

examination as well as on direct examination.

The witness should wear neat, clean

clothes when he testifies and should dress

conservatively.. He should speak clearly and

not chew gum while testifying.

LEGAL ASPECTS

285

image:

2.4.2 Direct Examination

(a) In a discussion on administrative proce-

dures, E. Barrett Prettyman, Retired Chief

Judge, U.S. Court of Appeals for the District of

Columbia, gave the following advice (4):

The best form of oral testimony is a series

of short, accurate, and complete state-

ments of fact. It is to be emphasized that

the testimony will be read by the finder of

the facts, and that he will draw his findings

from what he reads...confused, discursive,

incomplete statements of fact do not yield

satisfactory findings.

(b) The witness should stand upright when

taking the oath, pay attention, say "I do"

clearly, and not slouch in the witness chair. If

the witness has prepared answers to possible

questions, he should not memorize them. It is,

however, very important that he familiarize

himself as much as possible with the facts

about which he will be called to testify.

may elaborate and respond more fully than is

advisable on cross-examination. However,

when volunteering information, he should not

ramble or stray from the main point raised in

his lawyer's question. Testimony is a dialogue,

not a monologue. If testimony concerns a spe-

cialized technical area, the court or hearing

board will find it easier to understand if it is

presented in the form of short answers to a

logical progression of questions. In addition,

by letting his lawyer control the direction of his

testimony, the witness will avoid making re-

marks which are legally objectionable or tacti-

cally unwise.

(d) The witness should be serious at all

times and avoid laughing or talking about the

case in the building where the hearing or trial

is being held.

(e) While testifying, the witness should talk

to the board member, hearing officer or jury,

looking at him or them most of the time, and

speaking frankly and openly as if to a friend or

neighbor. He should speak clearly and loudly

enough so that anyone in the hearing room or

courtroom can hear him easily. The witness

makes certain that the reporter taking the ver-

batim record of his testimony is able to hear

him and record what he says. The case will be

decided entirely on the words that are re-

ported as the testimony given at the hearing or

trial. The witness must give complete state-

ments in sentence form; half statements or

incomplete sentences may convey the thought

in the context of the hearing, but be unintelligi-

ble when read from the cold record months

later.

2.4.3 Cross-Examination

(a) Concerning cross-examination, the fol-

lowing advice is given to prospective wit-

nesses (4):

Don't argue. Don't fence. Don't guess.

Don't make wisecracks. Don't take sides. Don't

get irritated. Think first, then speak. If you do

not know the answer to a question, say so. If

you do not know the answer but have an opin-

ion or belief on the subject based on informa-

tion, say exactly that and let the hearing officer

decide whether you shall or shall not give such

information as you have. If a 'yes or no' answer

to a question is demanded but you think that a

qualification should be made to any such an-

swer, give the 'yes or no' and at once request

permission to explain your answer. Don't

worry about the effect an answer may have.

Don't worry about being bulldozed or embar-

rassed; counsel will protect you. If you know

the answer to a question, state it as precisely

and succinctly as you can. The best protection

against extensive cross-examination is to be

brief, accurate and calm.

The hearing officer, board member or jury

wants only the facts, not hearsay, conclusions,

or opinions. The witness usually will not be

allowed to testify about what someone else

has told him.

(b) The witness must be polite, even to the

attorney for the opposing part. He should not

be a cocky witness. This will lose him the

respect and objectivity of the trier of the facts

286

MICROBIOLOGICAL MANUAL 1978

image:

in the case. He should not exaggerate or em-

broider his testimony.

the judge, hearing officer or board member

interrupts, or when the other attorney objects

to what is said. He must not try to sneak the

answer in or nod his head for a "yes" or "no"

answer. The reporter has to hear an answer to

record it. If the question is about distances or

time and the answer is only an estimate, he

must say that it is only an estimate.

the questions asked. No matter how friendly

the other attorney may seem on cross-

examination, he may be trying to damage the

testimony. He must understand the question

completely and should have it repeated if nec-

essary, then give a thoughtful answer. He must

not give a snap answer. He cannot be rushed

into answering, yet taking too much time

would make the board member, hearing offi-

cer or jury think the witness is making up the

answers.

(d) The witness must answer the question

that is asked—not the question that he thinks

the examiner (particularly the cross-examiner)

intended to ask. The printed record shows only

the question asked, not what was in the exam-

iner's mind and a non-responsive answer may

be very detrimental to the case. This situation

exists when the witness thinks "I know what

he is after but he hasn't asked for it." Answer

only what is asked. The witness must explain

his answers if necessary.

(e) If by chance one's answer is wrong,

correct it immediately; if the answer was not

clear, clarify it immediately. The witness is

sworn to tell the truth. Every material truth

should be readily admitted, even if not to the

advantage of the party for whom he is testify-

ing. He must not stop to figure out whether the

answer will help or hurt his side.

(f) The witness must give positive, definite

answers when at all possible and avoid saying

"I think", "I believe", "in my opinion." If he

does no.t know, he must say so and not make

up an answer. One can be positive about the

important things which he naturally would re-

member. If asked about little details which a

person naturally would not remember it is best

to say that one does not remember, but he

must not let the cross-examiner place him in

the trap of answering question after question

with "1 don't know."

(g) The witness must not act nervous. He

should avoid mannerisms which will make him

appear frightened, not telling the truth, or not

telling all that he knows. Above all, it is most

important that the witness not lose his temper.

Testifying at length is fatiguing. Fatigue will be

recognized by crossness, nervousness, anger,

careless answers and a willingness to say any-

thing or answer any questions in order to leave

the witness stand. When the witness feels

these symptoms, he must recognize them and

strive to overcome these feelings. Some attor-

neys on cross-examination try to wear out the

witness so he will lose his temper and say

things that are not correct, or that will hurt the

testimony. The witness must not let this

happen.

(h) If the witness does not want to answer

a question, he should not ask the judge, hear-

ing officer or board member whether he must

answer it. If it is an improper question, hjs

attorney will object for him. One must not ask

the presiding officer, judge or board member

for advice or help in answering a question. The

witness is on his own. If the question is an

improper one, his attorney will object. If the

judge, hearing officer, or board member then

directs the witness to answer it, he must do so.

He cannot hedge or argue with the opposing

attorney.

(i) There are trick questions which may be

asked and which, if answered, signify "yes" or

"no", ancl will damage the credibility of the

testimony. Two examples follow:

(1) "Have you talked to anybody about this

matter?" If you say "no", the hearing officer or

board member, or a seasoned jury, will know

that is not correct because good lawyers al-

ways talk to the witnesses before they testify.

If one says "yes", the lawyer may try to infer

LEGAL ASPECTS

287

image:

that you were told what to say. The best thing

to say is that you have talked to Mr ,

your lawyer, to the appellant, etc., and that you

were just asked what the facts were. All that is

wanted is the truth.

(2) "Are you getting paid to testify in this

appeal?" The lawyer asking this hopes your

answer will be "yes", thereby inferring that

you are being paid to say what your side wants

you to say. Your answer should be something

like "No, I am not getting paid to testify, I am

only getting compensation for my time off

from work, and my expenses incurred in being

here." A witness should never be paid a contin-

gency fee as it indicates strongly that since his

compensation depends upon the results, he

may be inclined to overstate the case.

REFERENCES

1. Federal Water Pollution Control Act Amendments of 1972, Public Law 92-500, October 18,

1972,86 Stat. 816,33 United States Code (USC) Sec, 1151.

2. Marine Protection, Research and Sanctuaries Act of 1972, Public Law 92-532, October 23,

19 72,86 Stat. 1052.

3. Safe Drinking Water Act, Public Law 93-523, December 16, 1974, 88 Stat 1660, 42 United

States Code (USC) 300f.

4. U.S. Environmental Protection Agency, Legal Support Division, 1972. A Primer on the Law,

Evidence, and Management of Federal Water Pollution Control Cases, Washington, D.C. pp.

43-52,54^55.

5. Shedroff, D. I., 1973. Enforcement activities. In: Proceedings of the First Microbiology Seminar

on Standardization of Methods, EPA-R4-73-O22, Office ofResearch and Monitoring, U.S.

Environmental Protection Agency, Washington, D.C., pp. 1-11.

6. Shedroff, D. I., 1976. Personal Communication. Office of Enforcement US EPA, Washington, DC.

7, Guidelines Establishing Test Procedures for Analysis of Pollutants, 40 Code of Federal Regula-

tions {CFR) Part 136, Published in Federal Register, 38, p. 28758, October 16,1973.

8. American Public Health Association. 1976. Standard Methods for the Examination of Water and

Wastewater{14th ed.) American Public Health Association, Inc., Washington, DC. p. 874

9. Guidelines for Establishing Test Procedures, 40 Code of Federal Regulations (CFR) Part 136,

Published in Federal Register, 40,52780, Dec. 1,1976.

10. National Interim Primary Drinking Water Regulations, 40 Code of Federal Regulations (CFR) Part

141, Published in Federal Register, 40,59566, December 24,1975.

11. Borough of Ford City vs. United States, 345 F. 2d 645(3rdCir. 1965),

12, Camara vs. Municipal Court, 387 U.S. 523 (1967); See vs. Seattle, 387 U.S. 547 (1967).

13. United States vs. Hammond Milling Co., 413 F. 2d 608 (5th Cir. 1969), cert. den. 396 U.S. 1002

(1970); and United States vs. Thriftimart, Inc. 429 F. 2d 1006 (9th Cir. 1970) cert. den. 400 U.S.

926(1970).

14. The Federal Rules of Evidence, Public Law 93-595, January 2, 1975,88 Stat. 1926,28 United

States Code (USC) App.

15. Federal Rules of Civil Procedure, Rule 43, adopted by the U.S. Supreme Court pursuant to Title

28, U.S. Code (USC) Section 2072, as amended effected July, 1975.

16, Johnson, R. H., Acting Assistant Administrator for Enforcement, EPA Office of Enforcement. May

5,1975. "Contacts with Parties to Adjudicator/ or Adversary Proceeding", Memorandum to EPA

Assistant and Regional Administrators.

288

•SERA MICROBIOLOGICAL MANUAL 1978

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APPENDIX A

TABLE-1

FEDERAL WATER POLLUTION CONTROL ACT AMENDMENTS Of 1972, PUBLIC LAW 92-500

Microbiological Activities Under Relevant Sections of the Law

Sections of Law

104(a)(5)

Water Quality Surveillance

System

106 (c)

Grants for State Pollution

Control. Program

108 (a)

Pollution Control in the

Great Lakes

301 (b) and 402

Permits for Publicly Owned

(Municipal) Treatment Works

Summary of Sections

The Administrator is required to establish and maintain

a water quality surveillance system with States and

other Federal Agencies, Agencies in the system will

collect and disseminate basic data on the chemical,

physical and biological effects of varying water quality.

They are to develop new methods for identifying and

measuring the effects of pollution on the chemical,

physical and biological integrity of the water.

EPA is to provide assistance and guidance to the States

on the development and operation of procedures and

systems to monitor water quality, including biological

monitoring.

EPA is to conduct projects in cooperation with other

agencies for demonstrating new methods and developing

plans for their use in controlling pollution on the

Great Lakes.

Municipal treatment plants must attain "... second-

ary treatment", as defined by Administrator, or

treatment necessary to meet water quality

standards, whichever is more stringent, by 1977,

and "... best practicable waste treatment tech-

nology over the life of the works" by 1983.

Microbiological Activity

Conduct research, develop methodology

and technology, complete necessary

analyses, perform surveys, and provide

expertise in microbiology.

Provide necessary assistance in

microbiological expertise and

consultation to the States.

Analyze water samples to support method

and development plans for Great Lakes

pollution control.

Analyses to determine compliance or

non-compliance with microbiological

portions of permit requirements.

image:

TABLE-1

(Continued)

FEDERAL WATER POLLUTION CONTROL ACT AMENDMENTS OF 1972, PUBLIC LAW 92-500

Microbiological Activities Under Relevant Sections of the Law

Sections of Law

301 (b) and 402

Permits for Non-Publicly Owned

(Industrial) Treatment Works

301 (b) and 307 (b)

Pretreatment Standards for Dis-

charges by Non-Publicly Owned

Enterprises into Publicly-Owned

Plants

304 (a) (b) and (g)

Information and Guidelines

Summary of Sections

(1) Existing Plants: Must attain "... best practicable

control technology currently available", or water

quality standards, whichever is more stringent, by

1977, and "... best available technology economi-

cally achievable" by 1983.

(2) New Plants: Must comply with "... National Indus-

trial Standards of Performance" which for a particular

industry reflect "... the greatest degree of effluent

reduction . . . achievable through the application of

the best available control technology, processes,

operating methods, or other alternatives."

Private industry discharging into public treatment plants

must demonstrate compliance with pretreatment standards

which are determined by the type of waste source and whether

the plant is already existing or is new since the passage of

the Act. Standards for both new and old plants are designed

to prevent the discharge through publicly-owned treatment

works of pollutants which "... interfere with, pass

through, or (are) otherwise incompatible with such works."

Existing sources must comply by three years after promulga-

tion of applicable pretreatment standards.

EPA must develop water quality criteria which reflect know-

ledge of the effects on plankton, fish, shellfish, wildlife,

plant life, esthetics and recreation which may be expected

from presence of pollutants in any body of water or in

ground water. Information must be developed on what factors

are needed to restore and maintain the chemical, physical

and biological integrity of navigable waters, ground waters,

coastal waters and oceans. The Administrator is also re-

quired to issue guidelines for identifying and evaluating

the nature and extent of nonpoint sources of pollutants.

Microbiological Activity

Analyses to determine compliance or

non-compliance with microbiological

portions of permit requirements.

Analyses to determine compliance or

non-compliance with microbiological

portion of the pretreatment standard.

Develop microbial water quality

criteria based on the analyses of

all navigable, ground and coastal

waters and the ocean.

image:

TABLE-1

(Continued)

FEDERAL WATER POLLUTION CONTROL ACT AMENDMENTS OF 1972, PUBLIC LAW 92-500

Microbiological Activities Under Relevant Sections of the Law

Sections of Law

304 (b)

Publication of Effluent

Limitation Guidelines

Summary of Sections

The Administrator shall publish regulations providing

guidelines for effluent limitations.

Microbiological Activity

Provide advice, technical assistance

and analyses required to establish

effluent limitations.

304 (g)

Guidelines for Test

Procedures

307 (a)

Toxic Pollutants

308

Inspections, Monitoring and

Entry

309

Federal Enforcement

The Administrator shall promulgate guidelines estab-

lishing test procedures for the analyses of

pollutants.

Discharge limitations are established or materials are

prohibited that are designated by the Administrator as

toxic, taking into account ". . . toxicity . . . persist-

ence . . . degradability . . . presence of the affected

organisms and the nature and extent of the effect of the

toxic pollutant on such organisms."

Owners and operators of pollution point sources shall

establish and maintain records; make reports; install,

use, and maintain monitoring equipment or methods, in-

cluding biological monitoring methods; and sample effluents

at locations, intervals, and with methods prescribed by

the Administrator.

On the basis of any information available that indicates

non-compliance of the requirements of a permit issued by

a State, the Administrator may notify the person in

alleged violation and the State of such findings. If

after the thirtieth day after notification the State has

not commenced appropriate enforcement action, the Adminis-

trator may issue a compliance order or bring civil action

to enforce the permit conditions or limitations.

Provide advice, technical assistance

and analyses required to establish

microbiological procedures.

Identification and quantification of

pollutants, including viruses,

designated as toxic by the Adminis-

trator.

Inspection of microbiological portions

of records, microbiological equipment

or methods used by owner or operator

of pollution point source; sampling

and analysis of effluents required to

be sampled by the owner or operator

of the pollution point source.

Provide advice and technical assistance

to the State and persons in non-compliance

to bring them into compliance; provide

analytical data, expertise and testimony

as required to establish EPA's case.

image:

TABLE-1

(Continued)

FEDERAL WATER POLLUTION CONTROL ACT AMENDMENTS OF 1972, PUBLIC LAW 92-500

Microbiological Activities Under Relevant Sections of the Law

Sections of Law

310

International Pollution

Abatement

311

Oil and Hazardous Substance

Liability

312 (b)

Marine Sanitation Devices

403 (c)

Ocean Discharge Criteria

Summary of Sections

The Administrator may call a hearing when he has reason

to believe pollution is occurring from U. S. sources

". . . which endangers the health or welfare of

persons in a foreign country."

This section bans the discharge of oil and any other

"... elements and compounds which, when discharged

in any quantity into , » , waters . . . present an

imminent and substantial danger to the public health or

welfare, including, but not limited to, fish, shellfish,

wildlife, shorelines, and beaches," The ban applies

to any substance which fits this description and that

the Administrator designates as "hazardous."

Vessel sanitation devices must conform to performance

standards issued by the Administrator. New vessels must

comply within two years of promulgation; existing

vessels have five years in which to comply.

The Administrator is required to promulgate guidelines

for determining the degradation of territorial waters,

coastal waters and oceans.

Microbiological Activity

Microbiological surveys to determine

if domestic pollution is adversely

affecting a foreign country; survey

results provide the Administrator

with data to assist him in deciding

whether to call a hearing.

Performance of degradability tests

and experiments under actual or

simulated conditions.

Analyses to confirm compliance with

microbiological portions of per-

formance standards. Analyses to

determine if the device operates

in conformity with the standards;

the Coast Guard is responsible

for such testing of the devices.

Conduct required analyses to

establish guidelines for monitor-

ing' the degradation of territorial

and coastal waters and the

oceans.

image:

TABLE-1

(Continued)

FEDERAL WATER POLLUTION CONTROL ACT AMENDMENTS OF 1972, PUBLIC LAW 92-500

Microbiological Activities Under Relevant Sections of the Law

Sections of Law

405

Permits for Disposal of

Sewage Sludge

504

Emergency Powers

Summary of Sections

Permits are required for disposal of sewage sludge

(including removal of in-place sewage sludge from one

location and its deposit in another location) where

disposal "... would result in any pollutant . . .

entering . . . waters."

An injunction prohibiting discharge by a particular

source may be issued on proof of ". . . imminent and

substantial endangerment to the health of persons or

to the welfare of persons where such endangerment is

to the livelihood of such persons,"

Microbiological Activity

Analysis of sludges at tine of

transport and at disposal site to

determine compliance or non-compliance

with permit requirements.

Detection of pathogens in water and

from their sources; and enumeration

of indicators authorizing closure

of shellfish beds and identifica-

tion of pollutant sources.

image:

APPENDIX A

TABLE-2

MARINE PROTECTION, RESEARCH AND SANCTUARIES ACT OF 1972, PUBLIC LAW 92-532

Microbiological Activities Under Enforcement and Compliance Monitoring Sections

Sections of Law

102

EPA Permits

Dumping

for Ocean

103

Corps of Engineer Permits

Summary of Sections

Establishment of a program for the issuance of EPA permits

based on criteria which consider the effects of ocean

dumping on human welfare, shellfish and fisheries

resources, plant and animal life, shorelines, beaches,

and marine ecosystems.

The responsibility for issuing permits (based on the

criteria in Section 102 above) for the ocean dumping

of dredged materials is under the jurisdiction of the

Army Corps of Engineers,

Microbiological Activity

Analyses to determine compliance or

non-compliance with microbiological

portions of permit requirements.

Possible conduct of microbiological

analyses to determine that the

proposed dumping ". . .will not un-

reasonably degrade or endanger human

health, welfare, or amenities, or

the marine environment, ecological

systems, or economic potentialities."

(Regulations in 40 CFR 227 set forth

criteria for evaluation of permit

applications for materials containing

living organisms. See especially 227.36.)

Analyses to determine compliance

or non-compliance with microbiolog-

ical portions of permit requirements.

image:

APPENDIX A

TABLE-3

SAFE DRINKING WATER ACT OF 1974, PUBLIC LAW 93-523

Microbiological Activities Under Enforcement and Compliance Sections

Sections of Law

1412 (a) (1)

Establish Primary Interim

Standards

1412 (c)

Proposed National Secondary

Drinking Water Regulations

1412 (e)

Study by Independent

Organizations

Summary of Sections

EPA has the responsibility for establishing national interim

primary drinking water regulations which will protect human

health by using the technology which is generally available.

The Agency promulgates National Secondary Drinking Water

regulations which are mostly related to the aesthetic

characteristics of drinking water.

The Administrator shall arrange studies with the National

Academy of Sciences or other independent scientific

organization to determine maximum contaminant levels of

known or anticipated contaminants, and to identify those •

contaminants in drinking water which are at levels too

low to measure.

Microbiological Activity

Provide the expertise and advice to

assist in the establishment of the

required safe interim primary standards

for drinking water.

Provide the expertise and advice to

EPA for establishing National Second-

ary Standards relating to micro-

biology.

Provide input as needed for estab-

lishment, performance and evaluation

of studies by the National Academy

of Sciences or other independent

scientific organization.

1413 (b)

Recommended Maximum

Contaminant Levels

1414 (a) (1) (A)

Failure by State to Assure

Enforcement of Standards

The Agency must also establish for each contaminant a

maximum contaminant level which will produce no known

adverse effects and allows an adequate margin of safety.

If the Administrator finds that a State with primary en-

forcement responsibility has not maintained compliance in

its public water systems, he shall so notify the State and

provide assistance in achieving compliance.

Conduct research and monitoring analyses

to establish acceptable levels and

maximum levels for bacterial indicators,

pathogens and viruses.

Provide advice and technical assistance

to the State and water systems to

bring them into compliance.

image:

TABLE-3

{Continued}

SAFE DRINKING WATER ACT OF 1974, PUBLIC LAW 93-523

Microbiological Activities Under Enforcement and Compliance Sections

»*

Sections of Law

1414 (a) (1) (B)

Civil Action

1414 (f)

Non-Coinplianee/Public Hearings

1421

Underground Injection Control

Systems

1431 (a)

Emergency Powers

410

Amendments to the Bottled

Drinking Water Standards

(Section 4. Chapter IV of the

Federal Food, Drug, and

Cosmetic Act)

Summary of Sections

If the Administrator determines that the State abused its

discretion in carrying out its primary enforcement responsi-

bility, the Administrator may commence a civil action to

enforce the standards.

If the Administrator finds non-compliance by a public

water system in a State with primary enforcement responsi-

bility, he may hold hearings to gather information from

technical and other experts and may issue recommendations

on actions which will achieve compliance.

The Administrator shall publish and promulgate regulations

for State underground injection control systems after

public hearing. The States will issue permits for under-

ground injection which will not endanger drinking water

sources; and will inspect, monitor and keep records of

the permitted underground injection wells.

EPA may obtain an injunction against a non-complying water

system on proof of the presence of a contaminant which

presents an imminent and substantial endangerment to human

health and if the appropriate State or local authority has

not acted.

After the promulgation of drinking water regulations, the

Food and Drug Administration must either promulgate amend-

ments to the bottled drinking water standards or publish

reasons for not making amendments.

H i crob i o 1 og i c a 1 Activity

Provide analytical data, expertise

and testimony as required to establish

EPA's case.

Provide confirming analytical data,

recommendations and expert advice on

microbiological aspects demonstrating

non-compliance.

Determine the feasibility of micro-

biological criteria and perform

analyses of injected wastewaters and

of ground waters if required.

Proof of presence of bacterial indica-

tors, pathogens or viruses in suffi-

cient numbers to pose a danger to

human health.

Conduct analyses to determine if the

quality of bottled drinking water

meets drinking water regulations.

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APPENDIX B

FROM; Manual for the Interim Certification of Laboratories Involved in Analyzing Public Drinking Water Supplies, EPA

600/8-78-008, May, 1978. OMTS, Office of REsearch and Development, U.S. Environmental Protection Agency,

Washington, DC 204 60

Chapter V

MICROBIOLOGY: CRITERIA AND PROCEDURES FOR INTERIM

CERTIFICATION OF LABORATORIES INVOLVED

IN ANALYSIS OF PUBLIC WATER SUPPLIES

The criteria and procedures described herein, shown in bold, are minimum requirements consid-

ered essential for laboratories seeking certification for microbiological analysis of public water sup-

plies. The requirements include laboratory equipment and supplies, laboratory practices, methodology,

sample collection, and certain quality control measures. The other items, involving personnel, facili-

ties, additional quality control procedures, data reporting, and action response, are optional. For a

commercial laboratory to qualify for certification in microbiology, it must process a minimum of 20

potable water samples per month using either the multiple tube procedure or membrane filter test.

Until National Revised Primary Drinking Water Regulations require certification of water supply

laboratories, all specifications will be considered as guidelines to be used by certification officials.

At that time, minimal requirements will be essential to certification of laboratories involved in anal-

ysis of public water supplies.

The minimum requirements must be in compliance, or action must be taken to correct defi-

ciencies prior to certification. A laboratory that exceeds these minimum requirements is encouraged

to maintain and improve those higher standards for facilities, equipment, methodology, and quality

control, as well as to continue the upgrading of personnel through training efforts to ensure routine

production of reliable data.

The required methods of analyses are referenced in "Standard Methods for the Examination of

Water and Wastewater," 13th edition; however, some criteria in this document are more specific and

permit fewer variations than "Standard Methods."

The guidelines for quality assurance procedures are those in EPA's quality assurance propam as

cited in the EPA Manual, "Microbiological Methods for Monitoring the Environment" (EMSL EPA

Cincinnati). A valuable source of further detail and background information for the laboratory eval-

uator is available in EPA's "Handbook for Evaluating Water Bacteriological Laboratories" (EPA-

670/9-75-006, August 1976).

Minimum requirements are shown throughout in bold.

PERSONNEL1 (OPTIONAL REQUIREMENTS)

Analyst

The analyst performs microbiological tests with minimal supervision in those specialties for

which he is qualified by education and/or training and experience.

1 Exceptions will be made for those persons employed by the laboratory and currently doing the required analy-

ses prior to promulgation of the interim regulations provided that within 2 years after June 24, 1977, they receive a

minimum of 2 weeks of additional training in water microbiology.

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• Academic training: Minimum of high school diploma in academic or laboratory-oriented

vocational courses.

• Job training: Minimum of 30 days on-the-job training plus one week of supplementary train-

ing acceptable to the Federal and State regulatory agency or agency responsible for primacy.

Personnel should take advantage of courses available to Federal and State regulatory

agencies.

* Supervision: Supervision by an experienced professional scientist. In the small water plant

laboratory consisting of a single analyst, the services of a State-approved outside consultant

must be available.

Supervisor I Consultant

The supervisor directs technical personnel in the proper performance of laboratory procedures

and the reporting of results. If no technical supervisor is available, a consultant should be available.

* Academic training: Minimum of a bachelor's degree in microbiology, biology, chemistry, or

a closely related field. Exceptions will be made for employees of laboratories that serve

communities with populations of 50,000 or less if they receive at least 2 weeks of additional

training in water microbiology from a Federal agency, State agency, or university.

» Job training: Technical training in water microbiology for a minimum of 2 weeks from a

Federal agency, State agency, or university in the parameter to be tested. Consultant must

have 1 year of bench experience, approved by the State, in total coliform analysis. State

laboratory expertise would be the most desirable source of outside consultation.

• Experience: One year of bench experience in sanitary (water, milk, or food) microbiology.

LABORATORY FACILITIES (OPTIONAL REQUIREMENTS)

Laboratory space should be adequate (200 ft2 and 6 linear ft of bench space per analyst) to

accommodate periods of peak work load. Working space requirements should include sufficient

bench-top area for processing samples; storage space for media, glassware, and portable equipment

items; floor space for stationary equipment (incubators, waterbaths, refrigerators, etc.); and associ-

ated area for cleaning glassware and sterilizing materials. The space required for both laboratory

work and materials preparation in small water plant laboratories may be consolidated into one

room, with the various functions allocated to different parts of the room.

Facilities should be clean, air-conditioned, and with adequate lighting at bench top (100 ft-

candles).

Laboratory safety, which must be an integral and conscious effort in laboratory operations,

should provide safeguards to avoid electric shock, prevent fire, prevent accidental chemical spills,

and minimize microbiological dangers, facility deficiencies, and equipment failures. While safety

is not an aspect of laboratory certification, the evaluation should point out on an informal basis,

potential safety problems observed during an on-site visit.

298 <SEPA MICROBIOLOGICAL MANUAL 1978

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LABORATORY EQUIPMENT, SUPPLIES, AND MATERIALS (MINIMUM REQUIREMENTS)

The laboratory must have available or access to the items required for the total coliform mem-

brane filter or most probable number procedures as listed below.

» pH Meter: Accuracy must be ±0.1 units.

• Balances—top loader or pan: Balance must be clean, not corroded, and be provided with ap-

propriate weights of good quality. Balance must tare out and detect 50-mg weight accurate-

ly: this sensitivity is required for use in general media preparation of 2g or larger quantities.

• Temperature-monitoring devices:

—Glass or metal thermometers must be graduated in 0.5°C increments.

—Continuous temperature recording devices must be sensitive to within 0.5°C.

—Liquid column of glass thermometers must have no separation.

—A certified thermometer or one of equivalent accuracy must be available.

• Air (or water jacketed) incubator/incubator rooms/waterbatfts/aluminum block incubators:

—Unit must maintain internal temperature of 35.0° ± 0.5°C in area of use at maximum

—When aluminum block incubators are used, culture dishes and tubes must be snug-fitting in

block.

• Autoclave:

—Autoclave must be in good operating condition when observed during operational cycle or

when time-temperature charts are read. Vertical autoclaves are not recommended. For

most efficient operation, a double-walled autoclave constructed of stainless steel is sug-

gested (optional).

—Autoclave must have pressure and temperature gauges on exhaust side and an operating

safety valve.

—Autoclave must reach sterilization temperature (121°C) and be maintained during steriliza-

tion cycle: no more than 45 minutes is required for'a complete cycle.

—Depressurization must not produce air bubbles in fermentation media.

• Hot-air oven: Oven must be constructed to ensure a stable sterilization temperature. Its use

is optional for sterilization of glass pipets, bottles, flasks, culture dishes, etc. (optional).

• Refrigerator: Refrigerator must hold temperature at 1° to 4.4°C (34° to 40°F).

• Optical/counting/lighting equipment: Low power magnification device (preferably binocular

microscope with 10 to 15x) with fluorescent light source must be available for counting MF

colonies. A mechanical hand tally can be used for counting colonies (optional).

• Inoculation equipment:

—Loop diameter must be at least 3 mm and of 22 to 24 gauge Nichrome, chromel, or

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platinum-indium wire. Single-service metal loops, disposable dry heat-sterilized hardwood

applicator sticks, pre-sterilized plastic, or metal loops may be used (optional).

Membrane filtration equipment:

—Units must be made of stainless steel, glass, or autoclavable plastic. Equipment must not

leak and must be uncorroded.

—Field equipment is acceptable for coliform detection only when standard laboratory MF

procedures are followed.

Membrane fitters and pads:

—Membrane filters must be manufactured from cellulose ester materials, white, grid-marked,

47-mm diameter, 0.45 /am pore size. Another pore size may be used if the manufacturer

gives performance data equal to or better than the 0.45-Atm membrane filter.

—Membranes and pads must be autoclavable or presterilized.

Laboratory glassware, plastic ware, and metal utemiJs:

—Except for disposable plastic ware, items must be resistant to effects of corrosion, high

temperature, and vigorous cleaning operations. Metal utensils made of stainless steel are

preferred (optional).

—Flasks, beakers, pipets, dilution botfles, culture dishes, culture tubes, and other glassware

must be of borosilicate glass and free of chips, cracks, or excessive etching. Volumetric

glassware should be Class A, denoting that it meets Federal specifications and need not be

calibrated before use.

—Plastic items must be of clear, inert, nontoxic material arid must retain accurate calibration

marks after repeated autoclaving.

Culture dishes:

—Sterile tight or loose-lid plastic culture dishes or loose-lid glass culture dishes must be used.

—For loose-lid culture dishes, relative humidity in the incubator must be at least 90 percent.

—Culture dish containers must be aluminum or stainless steel; or dishes may be wrapped in

heavy aluminum foil or char-resistant paper.

—Open packs of disposable sterile culture dishes must be reseated between uses.

Culture tubes and closures:

—Culture tubes must be made of borosilicate glass or other corrosion resistant glass and

must be of a sufficient size to contain the culture medium, as well as the sample portions

employed, without being more than 3/4 full. It is desirable that the fermentation vial

extend above the medium (optional).

—Caps must be snug-fitting stainless steel or plastic; loose-fitting aluminum caps or screw

caps are also acceptable.

300 4*ER^ MICROBIOLOGICAL MANUAL 1978

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• Measuring equipment:

—Sterile, glass or plastic pipets must be used for measuring 10 ml or less.

—Pipets must deliver the required volume quickly and accurately within a 2,5 percent toler-

ance.

—Pipets must not be badly etched; mouthpiece or delivery tips must not be chipped; gradua-

tion marks must be legible.

—Open packs of disposable sterile pipets must be resealed between uses.

—Pipet containers must be aluminum or stainless steel.

—Graduated cylinders must be used for samples larger than 10 ml; calibrated membrane

filter funnel markings are permissible provided accuracy is within a 2.5 percent tolerance.

GENERAL LABORATORY PRACTICES (MINIMUM REQUIREMENTS)

Sterilization Procedures

• The following times and temperatures must be used for autoclaving materials:

Material Temperature!Minimum Time

Membrane filters and pads 121°C/10min.

Carbohydrate-containing media 121°C/12-15min.

(lauryl tryptose, brilliant green

lactose bUe broth, etc.)

Contaminated materials and discarded 121 C/30min.

tests

Membrane filter assemblies (wrapped), 121 C/30min.

sample coDection bottles (empty),

individual glassware items

Rinse water volumes of 500 ml to 1,000 121°C/45 min.

Rinse water in excess of 1,000 ml 121°C/time adjusted for volume; check

for sterility

Dilution water blank 121°C/30 min.

Membrane filter assembles must be sterilized between sample filtration series. A filtration series

ends when 30 minutes or longer elapse between sample filtrations. At least 2 minutes of UV light or

boiling water may be used on membrane filter assembly to prevent bacterial carry-over between

filtrations (optional).

Dried glassware must be sterilized at a minimum of 170°C for 2 hours.

Laboratory Pure Water (Distilled, Deionized, or Other Processed Waters)

• An analyst must test the quality of the laboratory pure water or have it tested by the State

or by a State-authorized laboratory.

MICROBIOLOGICAL MANUAL 1978 301

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» Only water determined as laboratory pure water (see quality control section) can be used

for performing bacteriological analyses.

Although processed water may be acceptable for routine chemistry, there is a good chance that

it contains enough of some constituent to be toxic or stimulatory to microorganisms (optional).

Rinse and Dilution Water

Stock buffer solution must be prepared according to "Standard Methods" using laboratory pure

water adjusted to pH 7.2. Stock buffer must be autoclaved or filter-sterilized, labeled, dated, and

stored at 1° to 4.4°C. The stored buffer solution must be free of turbidity.

Rinse and dilution water must be prepared by adding 1.25 ml of stock buffer solution per liter

of laboratory pure water. Final pH must be 7.2 ± 0.1.

Media Preparation and Storage

The following are minimum requirements for storing and preparing media:

» Laboratories must use commercial dehydrated media for routine bacteriological procedures

as quality control measures.

• Lauryl tryptose and brilliant green lactose bile broths must be prepared according to

"Standard Methods"; lactose broth is not permitted.

• Dehydrated media containers must be kept tightly closed and stored in a cool, dry location.

Discolored or caked dehydrated media cannot be used.

• Laboratory pure water must be used; dissolution of the media must be completed before

dispensing to culture tubes or bottles.

• The membrane filter broth and agar media must be heated in a boiling water bath until com-

pletely dissolved.

» Membrane filter (MF) broths must be stored and refrigerated no longer than 96 hours. MF

agar media must be stored, refrigerated and used within 2 weeks.

• Most probable number (MPN) media prepared in tubes with loose-fitting caps must be used

within 1 week. If MPN media are refrigerated after sterilization, they must be incubated over-

night at 35° C to confirm usability. Tubes showing growth or gas bubbles must be discarded.

* Media in screw cap containers may be held up to 3 months, provided the media are stored in

the dark and evaporation is not excessive (0.5 ml per 10 ml total volume). Commercially

prepared liquid and agar media supplies may be used (optional).

• Ampouled media must be stored at 1° to 4.4°C (34° to 40°F); time must be limited to man-

ufacturer's expiration date.

METHODOLOGY (MINIMUM REQUIREMENTS)

The required procedures, which are mandatory, are described in the 13th edition of "Standard

Methods": standard coliform MPN tests (p. 664-668), single step or enrichment standard total

coliform membrane filter procedure (p. 679-683). Tentative methods are not acceptable. All other

procedures are considered alternative analytical techniques as described hi section 141.27

302 -&EPA MICROBIOLOGICAL MANUAL 1978

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of the National Interim Primary Drinking Water Regulations. Application for the use of alternative

methods may require acceptable comparability data.

The membrane filter procedure is preferred because it permits analysis of large sample volumes

in reduced analysis time. The membranes should show good colony development over the entire

surface. The golden green metallic sheen colonies should be counted and recorded as the coliform

density per 100 ml of water sample. The following rules for reporting any problem with MF results

must be observed:

» Confluent growth; Growth (with or without discrete sheen colonies) covering the entire

filtration area of the membrane. Results are reported as "confluent growth per 100 ml, with

(or without) coliforms," and a new-sample requested.

» TNTC (Too numerous to count): The total number of bacterial colonies on the membrane is

too numerous (usually greater than 200 total colonies), not sufficiently distinct, or both. An

accurate count cannot be made. Results are reported as "TNTC per 100 ml, with (or with-

out) coliforms," and a new sample requested.

* Confluent growth and TNTC: A new sample must be requested, and the sample volumes

filtered must be adjusted to apply the MF procedure; otherwise the MPN procedure must be

used.

• Confirmed MPN test on problem supplies: If the laboratory has elected to use the MPN test

on water supplies that have a continued history of confluent growth or TNTC with the MF

procedure, all presumptive tubes with heavy growth without gas production should be sub-

mitted to the confirmed MPN test to check for the suppression of coliforms. A count is

adjusted based upon confirmation and a new sample requested. This procedure should be

carried out on one sample from each problem water supply once every 3 months.

SAMPLE COLLECTION, HANDLING, AND PRESERVATION (MINIMUM REQUIREMENTS)

When the laboratory has been delegated responsibility for sample collecting, handling, and pres-

ervation, there must be strict adherence to correct sampling procedures, complete identification of

the sample, and prompt transfer of the sample to the laboratory as described in "Standard Meth-

ods," 13th edition, section 450, p. 657-660.

The sample must be representative of the potable water system. The sampling program must

include examination of the finished water at selected sites that systematically cover the distribution

network.

Minimum sample frequency must be that specified in the National Interim Primary Drinking

Water Regulations, 40 CFR 141.21.

The collector must be trained in sampling procedures and approved by the State regulatory

authority or its delegated representative.

The water tap must be sampled after maintaining a steady flow for 2 or 3 minutes to clear serv-

ice line. The tap is free of aerator, strainer, hose attachment, or water purification devices.

The sample volume must be a minimum of 100 ml. The sample bottle must be filled only to the

shoulder to provide space for mixing.

The sample report form must be completed immediately after collection with location, date and

time of collection, chlorine residual, collector's name, and remarks.

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Sample bottles must be of at least 120 mi-capacity, sterile plastic or hard glass, wide mouthed

with stopper or plastic screw cap, and capable of withstanding repeated sterilization. Sodium thio-

sulfate (100 mg/1) is added to all sample bottles during preparation. As an example, 0.1 ml of a 10

percent solution is required in a 4-oz (120-mI) bottle.

Date and time of sample arrival must be added to the sample report form when sample is re-

ceived in the laboratory.

State regulations relating to chain-of-custody, if required, must be followed in the field and in

the laboratory.

Samples delivered by collectors to the laboratory must be analyzed on the day of collection.

Where it is necessary to send water samples by mail, bus, United Parcel Service, courier service,

or private shipping, holding/ transit time between sampling and analyses must not exceed 30 hours.

When possible, samples are refrigerated during transit and during storage in the laboratory (optional).

If the laboratory is required by State regulation to examine samples after 30 hours and up to 48

hours, the laboratory must indicate that the data may be invalid because of excessive delay before

sample processing. Samples arriving after 48 hours shall be refused without exception and a new

sample requested. (The problem of holding time is under investigation by EPA.)

QUALITY CONTROL PROGRAM

Minimum Requirement

A written description for current laboratory quality control program must be available for

review. Management, supervisors, ahd analysts participate in setting up the quality control pro-

gram, Each participant should have a copy of the quality control program and a detailed guide

of his own portion. A record on analytical quality control tests and quality control checks on

media, materials, and equipment must be prepared and retained for 3 years.

Analytical Quality Control Tests for General Laboratory Practices and Methodology

Minimum and optional requirements for analytical quality control tests for general practices

and methodology are:

» Minimum requirements;

—At least five sheen or borderline sheen colonies must be verified from each membrane con-

taining five or more such colonies. Counts must be adjusted based on verification. The

verification procedure must be conducted by transferring growth from colonies into lauryl

tryptose broth (LTB) tubes and then transferring growth from gas-positive LTB cultures to

brilliant green lactose bile (BGLB) tubes. Colonies must not be transferred exclusively to

BGLB because of the lower recovery of stressed coliforms in this more selective medium.

However, colonies may be transferred to LTB and BGLB simultaneously. Negative LTB

tubes must be reincubated a second day and confirmed if gas is produced. It is desirable to

verify all sheen and borderline sheen colonies (optional).

—A start and finish MF control test (rinse water, medium, and supplies) must be conducted

for each filtration series. If sterile controls indicate contamination, all data on samples

affected must be rejected and a request made for immediate resampling of those waters

involved in the laboratory error.

304 < MICROBIOLOGICAL MANUAL 1978

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—The MPN test must be carried to completion, except for gram staining, on 10 percent of

positive confirmed samples. If no positive tubes result from potable water samples, the

completed test except for gram staining must be performed quarterly on at least one

positive source water.

—Laboratory pure water must be analyzed annually by the test for bactericidal properties

for distilled water ("Standard Methods," 13th edition, p. 646). Only satisfactorily tested

water is permissible in preparing media, reagents, rinse, and dilution water. If the tests do

not meet the requirements, corrective action must be taken and the water retested.

—Laboratory pure water must be analyzed monthly for conductance, pH, chlorine residual,

and standard plate count. If tests exceed requirements, corrective action must be taken

and the water retested.

—Laboratory pure water must not be in contact with heavy metals. It must be analyzed

initially and annually thereafter for trace metals (especially Pb, Cd, Cr, Cu, Ni, and Zn). If

tests do not meet the requirements, corrective action must be taken and the water re-

tested.

—Standard plate count procedure must be performed as described in "Standard Methods,"

13th edition, p. 660-662. Plates must be incubated at 35° ± 0.5°C for 48 hours.

—Requirements for laboratory pure water:

pH 5.5 - 7.5

Conductivity Greater than 0.2 megohm as resistivity or

less than 5.0 mieromhos/cm at 25°C

Trace metals:

A single metal Not greater than 0,05 mg/1

Total metals Equal to or less than 1.0 mg/1

Test for bactericidal properties of dis-

tilled water {"Standard Methods,"

13th edition, p. 646) 0.8 - 3.0

Free chlorine residual 0.0

Standard plate count Less than 10,000/ml

—Laboratory must analyze one quality control sample per year (when available) for param-

eter^) measured.

—Laboratory must satisfactorily analyze one unknown performance sample per year (when

available) for parameter(s) measured.

Optional requirements:

—Duplicate analyses should be run on known positive samples at a minimum frequency of

one per month. The duplicates may be run as a split sample by more than one analyst,

with each split being a 50-ml sample.

—Water plant laboratories should examine a minimum of one polluted water source per

month in addition to the required number of distribution samples.

—If there is more than one analyst in laboratory, at least once per month each analyst

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should count the sheen colonies on a membrane from a polluted water source. Colonies on

the membrane should be verified and the analysts' counts compared to the verified count.

—A minimum number of the official water supply samples required for each system should

be analyzed by the State laboratory. For example, systems that are required to have less

than 10 samples examined per month should submit one additional sample to a State

authorized laboratory. Water systems with sample requirements above 10 per month

would submit two additional samples to a State authorized laboratory.

Quality Control Checks of Laboratory Media, Equipment, and Supplies

Minimum and optional requirements for quality control checks of laboratory media, equipment,

and supplies are:

• Minimum requirements:

—pH meter must be clean and calibrated each use period with pH 7.0 standard buffer. Buf-

fer aliquot must be used only once. Commercial buffer solutions must be dated on initial

use.

—Balances (top loader or pan) must be calibrated annually.

—Glass thermometers or continuous recording devices for incubators must be checked year-

ly and metal thermometers quarterly (or at more frequent intervals when necessary)

against a certified thermometer or one of equivalent accuracy.

—Temperature in air (or water jacketed) incubator/incubator room/waterbaths/aluminum

block incubators must be recorded continuously or recorded daily from in-place thermom-

eter^) immersed in liquid and placed on shelves in use.

—Date, time, and temperature must be recorded continuously or recorded for each steriliza-

tion cycle of the autoclave.

—Hqt air oven must be equipped with a thermometer calibrated in the range of 170°C or

with a temperature recording device. Records must be maintained showing date, time/, and

temperature of each sterilization cycle. It is desirable to place the thermometer bulb in

sand and to avoid overcrowding (optional).

—Membrane filters used must be those recommended by the manufacturer for water analy-

sis. The recommendation must be based on data relating to ink toxicity, recovery, reten-

tion, and absence of growth-promoting substances.

—Washing processes must provide clean glassware with no stains or spotting. With initial use

of a detergent or washing product and whenever a different washing product is used, the

rinsing process must demonstrate that it provides glassware free of toxic material by the

inhibitory residue test ("Standard Methods," 13th edition, p. 643).

—At least one bottle per batch of sterilized sample bottles must be checked by adding ap-

proximately 25 ml of sterile LTB broth to each bottle. It must be incubated at 35 ± 0.5°C

for 24 hours and checked for growth.

—Service contracts or approved internal protocols must be maintained on balances, auto-

clave, water still, etc., and the service records entered in a log book.

306 &EPA MICROBIOLOGICAL MANUAL 1978

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—Records must be available for inspection on batches of sterilized media showing lot num-

bers, date, sterilization time-temperature, final pH, and technician's name.

* Optional requirements:

—Positive and negative cultures should be used, and testing should be carried out to deter-

mine recovery and performance compared to a previous acceptable lot of medium,

—Media should be ordered on a basis of 12-month needs. Bottles should be dated on receipt

and when opened initially. Except for large volume uses, media should be purchased in 1/4-

Ib bottles. Bottles of media should be used within 6 months after opening; however, in no

case should opened media be used after one year. Opened bottles should be stored in a des-

iccator to extend storage time beyond 6 months. Shelf life of unopened bottles is 2 years.

—Testing should be carried out in media and membranes to determine recovery .and per-

formance compared to previous acceptable lot.

—Lot number of membrane filters and date of receipt should be recorded.

—Heat sensitive tapes and spore strips or ampoules should be used during sterilization. Maxi-

mum registering thermometer is recommended.

DATA REPORTING (MINIMUM REQUIREMENTS)

Where the laboratory has the responsibility for sample collections, the sample collector should

complete a sample report form immediately after each sample is taken. The information on the

form includes sample identification number, sample collector's name, time and date of collection,

arrival time and date in the laboratory, direct count, MF verified count, MPN completed count,

analyst's name, and other special information.

Results should be calculated and entered on the sample report form to be forwarded. A careful

check should be made to verify that each result was entered correctly from the bench sheet and

initialed by the analyst.

All results should be reported immediately to the proper authority.

Positive results are reported as preliminary without waiting for MF verification or MPN comple-

tion. After MF verification and/or MPN completion, the adjusted counts should be reported.

A copy of the sample report form should be retained either by the laboratory or State program

for 3 years. If results are entered into a computer storage system, a printout of the data should be

returned to the laboratory for verification with bench sheets.

ACTION RESPONSE TO LABORATORY RESULTS (MINIMUM REQUIREMENT)

When action response is a designated laboratory responsibility, the proper authorities should be

promptly notified on unsatisfactory sample results, and a request should be made for resampling

from the same sampling point.

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SAMPLE FORMS FOR ON-SITE EVALUATION OF LABORATORIES INVOLVED IN

ANALYSIS OF PUBLIC WATER SUPPLIES-MICROBIOLOGY

LABORATORY:.

STREET;.

CITY: STATE:

TELEPHONE NUMBER:,

SURVEY BY:

AFFILIATION:.

DATE;.

CODES FOR MARKING ON-SITE EVALUATION FORMS:

S- Satisfactory

U—Unsatisfactory

NA- Not Applicable

308 &ERA MICROBIOLOGICAL MANUAL 1978

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Laboratory Evaluator.

Location Date

PERSONNEL

The form dealing with personnel can be found on the following page.

LABORATORY FACILITIES

Space in laboratory and preparation room is adequate for needs during peak work periods

(200 ft2 and 6 linear ft of usable bench space per analyst).

Facilities are clean, with adequate lighting (100 ft-candles) and air conditioning.

NOTE: Material on pages 53-65, except where indicated, are minimum requirements.

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PERSONNEL

Laboratory.

Location

Date

Evaluator

Position/title

Laboratory director

Supervisor/consultant

Professionals

(note discipline)

Technician/analyst

Name

Academic training

BA/BS

SA,

MA/MS

sA[p)[l If?

Ph.D.

F©

Present

specialty

&M €

Experience

(years/area)

MIY

image:

Laboratory . Evaluator

• Location Date

LABORATORY EQUIPMENT, SUPPLIES, AND MATERIALS

L pHMeter

Manufacturer Model

Clean, calibrated to 0.1 pH units each use period; record maintained .

Aliquot of standard pH 7.0 buffer used only once

2. Balance—Top Loader or Pan

Manufacturer .,; ' - ,' :—\—: Model

Clean. Detects a 50-mg weight accurately (for a general media preparation of >2-g

quantities)

Good quality weights in clean condition

3. Temperature-Monitoring Devices

Accuracy checked annually against a certified thermometer or one of equivalent

accuracy . : .

Legible graduations in 0.5°C-increments ,

No separation in liquid column

4. Incubator or Incubator Room

Manufacturer, ; Model.

Sufficient size for daily work load

Uniform temperature maintained on shelves in all areas used (35.0° ± 0.5°C)

Calibrated thermometer with bulb immersed in liquid and located on shelves in use.

Temperature recorded daily or recording thermometer sensitive to ± 0.5°C

5. Autoclave

Manufacturer Model

Reaches sterilization temperature (121°C), maintains 121°C during sterilization

cycle, and requires no more than 45 min for a complete cycle

Pressure and temperature gauges on exhaust side and an operating safety valve ..

No air bubbles produced in fermentation vials during depressurization

Record maintained on time and temperature for each sterilization cycle ,

311

image:

6. Hot-Air Oven

Manufacturer Model

Operates at a minimum of 170 C

Thermometer inserted or oven equipped with temperature-recording thermometer

device

Time and temperature record maintained for each sterilization cycle

Thermometer bulb in sand (optional)

7. Refrigerator

Temperature maintained at 1° to 4.4° C (34° to 40°F)

8, Optical Equipment

Low power magnification device (preferably binocular microscope with 10 to 15X)

with fluorescent light source for counting MF colonies

Colonies counted with a mechanical hand tally (optional);

9. Inoculation Equipment

Sterilized loops of at least 3-mm diameter, 22 to 24 gauge Nichrome, Chromel, or

platinum-iridium wire

Disposable dry heat-sterilized hardwood applicator sticks or presterilized loops....

JO. Membrane Filtration Equipment

Manufacturer Model

Made of stainless steel, glass, or autoclavable plastic

Nonleaking and uncorroded

22. Membrane Filters and Pads

Manufacturer Model

Filters recommended by manufacturer for water analyses

Filters and pads presterilized or autoclavable

12. Glass, Plastic, and Metal Utensils for Media Preparation

Washing process provides glassware free of toxic residue as demonstrated by the

inhibitory residue test

Glass items of borosilicate, free of chips and cracks

Utensils clean and free from foreign residues or dried medium

Plastic items clear with visible graduations

312 £EPA MICROBIOLOGICAL MANUAL 1978

image:

13. Sample Bottles

Wide-mouth hard glass bottles; stoppered or plastic screw-capped; capacity at least

120 ml

Glass-stoppered bottles with tops covered with aluminum foil or kraft

paper

Screw-caps have leakproof nontoxic liners that can withstand repeated sterilization

(30 min at 121°C)

Sterile sample bottles contain 10 mg of dechlorinating agent per 100 ml of sample .

14. Pipets

Brand ; Type

Sterile; glass or plastic; with a 2.5 percent tolerance

Tips unbroken; graduations distinctly marked

15. Pipet Containers

Aluminum, stainless steel

Pipets wrapped in quality kraft paper (char-resistant)

Open packs of disposable sterile pipets resealed between uses.

16. Culture Dishes

Brand. , Type.

Sterile plastic or glass ,

Open packs of disposable sterile plastic dishes resealed between uses .

Dishes are in containers of aluminum or stainless-steel with covers or

are wrapped with heavy aluminum foil or char-resistant paper

/ 7. Culture Tubes and Closures

Sufficient size to contain sterile medium and sample without danger of spillage.

Metal or plastic caps; plastic plugs

Borosilicate glass or other corrosion-resistant glass

313

image:

Laboratory Evaluator,

Location Date

GENERAL LABORATORY PRACTICES

L Sterilization Procedures

Satisfactory sterilization procedures and/or records ,

Tube .broth media and reagents sterilized at 121°C 12 to IS min

Tubes and flasks packed loosely in baskets or racks for uniform heating and cooling

MF presterilized or autoclaved at 121°C for 10 min with fast exhaust

MF assemblies and empty sample bottles sterilized at 121°C for 30 min.

MF assemblies sterilized between sample filtration series

Rinse water volumes of 500 to 1,000 ml sterilized at 121°C

for 45 min ,

Dilution water blanks autoclaved at 121°C for 30 min

Wire loops, needles, and forceps sterilized

Total exposure of MPN media to heat not over 45 min

Timing for sterilization begins when autoclave reaches 121 °C

Individual glassware items autoclaved at 121°C for 30 min ,

Individual dry glassware items sterilized 2 hours at 170°C (dry heat)

Pipets, culture dishes, and inoculating loops in boxes sterilized at 170°C for 2 hours,

MPN media removed and cooled as soon as possible after sterilization and stored in

cool dark place (optional)

UV light or boiling water for at least 2 min may be used on membrane filter assem-

blies to reduce bacterial carry-over between each filtration (optional)

2. Laboratory Pure Water

Only laboratory pure water used in preparing media, reagents, rinse water, and dilu-

tion water

Laboratory pure water not in contact with heavy metals

Source: Laboratory-prepared Purchased.

If laboratory-prepared:

Still manufacturer

Deionizer manufacturer

Record of recharge frequency.

Production rate and quality adequate for laboratory needs

Inspected, repaired, cleaned by service contract or in-house service

a. Chemical quality control

Record of satisfactory annual analyses for trace metals

A single metal not greater than 0.05 mg/1

314 -SEPA MICROBIOLOGICAL MANUAL 1978

image:

Total metals: equal to or less than 1.0 mg/1.

Record of monthly analyses of laboratory pure water

Conductance: >0.2 megohm resistivity or <5.0 microhmos/cm .

pH: 5.5-7.5

Standard plate count: <10,000/ml.

Free chlorine residual: 0.0

b. Microbiological quality control

Test for bactericidal properties of distilled water (0.8-3.0) performed at least

annually

Testing laboratory Date Ratio _

3. Rinse and Dilution Water

Stock buffer solution prepared according to "Standard Methods," 13th edition.. . .

Stock buffer solution adjusted to pH 7.2

Stock buffer autoclaved at 121°C, stored at 1° to 4.4°C (34° to 40°F) or filter

sterilized . .

Stock buffer labeled and dated

Stock potassium phosphate buffer solution (1.25 ml) added per liter distilled water

for rinse and dilution water

Final pH 7.2 ± 0.1

4. Media

Dehydrated media bottle kept tightly closed and protected from dust and excessive

humidity in storage areas .

Dehydrated media not used if discolored or caked

Laboratory pure water used in media preparation

Dissolution of media complete before dispensing to culture tubes or bottles

MPN tube media with loose-fitting caps used in less than 1 week

Tube media in screw-capped tubes held no longer than 3 months

Ampouled media stored at 1° to 4.4°C and time limited to manufacturer's expira-

tion date :

Media stored at low temperatures are incubated overnight prior to use and

tubes with air bubbles discarded

Media protected from sunlight

MF media stored in refrigerator; broth medium used within 96 hours, agar within

two weeks if prepared in tight-fitting dishes

315

image:

5. Lauryl Tryptose Broth

Manufacturer Lot No.,

Single strength composition, 35.6 g per liter pure water

Single strength pH 6.8 ± 0.2; double strength pH 6.7 ± 0.2

Not less than 10 ml per tube

Media made to result in single strength after addition of sample portions.

6. Brilliant Green Lactose Bile Broth

Manufacturer Lot No.

Medium composition 40 g per liter pure water

Final pH 7.2 ± 0.2

7. M-Endo Media

Manufacturer Lot No.

Medium composition 48.0 g per liter pure water optionally 15 g agar added/1

Reconstituted in laboratory pure water containing 2 percent ethanol (not

denatured)

Final pH 7.2 ± 0.2

Medium held in boiling water bath until completely dissolved

8, Standard Plate Count Agar

Correct composition, sterile and pH 7.0 ± 0.2

Sterile medium not remelted a second time after sterilization

Culture dishes incubated 48 hours at 35° ± 0.5°C

No more than 1.0 ml or less than 0.1 ml sample plated (sample or dilution).

Liquefied agar, 10 ml or more; medium temperature between 44° to 46°C.

Melted medium stored no longer than 3 hours before use

Only plates with between 30 to 300 colonies counted; when 1 ml of undiluted

sample is plated, colony density may be less than 30

Only two significant figures recorded and calculated as standard plate count/

1.0ml «

316 <e>5ft MICROBIOLOGICAL MANUAL 1978

image:

Laboratory P^__ Evaluator.

Location : _^_ Date

METHODOLOGY

Methodology specified in "Standard Methods," 13th edition, or EPA manual

M-Endo broth, M-Endo agar, or Les Endo agar used in a single step procedure,

In two-step Les M-Endo procedure, MF incubated on lauryl-tryptose-broth-saturated

absorbent pad for 1.5 to 2 hours at 3.5° ± 0.5°C; then on M-Endo broth or Les

Endo agar for 20 to 22 hours at 35° ± 0.5°C

/. Total Coliform Membrane Filter Procedure

Samples containing excessive bacterial populations (greater than 200), confluency,

or turbidity retested by the MPN procedure

Filtration assembly sterile at start of each series

Absorbent pads saturated with medium, excess discarded; or 4.0 ml of agar medium

can be used per culture dish instead of a pad

Sample shaken vigorously immediately before test

Jest sample portions measured and not less than 100 ml

Funnel rinsed at least twice with 20- to 30-ml portiqns of sterile buffered water

MF removed with sterile forceps grasping a*ea outside effective filtering area

MF rolled onto medium pad or agar so air bubbles are not trapped

2, Incubation of Membrane Filter Cultures

Total incubation time 22 to 24 hours at 35° ± 0.5'C

Incubated in tight-fitting culture dishes or loose-fitting dishes incubated in high

relative humidity chambers

3, Membrane Filter Colony Counting

Samples repeated when coliforms are "TNTC" or colony growth is confluent, possi-

bly obscuring coliform development and/or detection

Total coliform count calculated in density per 100 ml

Samples containing five or more coliforms per 100 ml are resampled and tested . . .

Low power magnification device with fluorescent light positioned for maximum

sheen visibility

4. Verification of Total Coliform Colonies

All typical coliform (sheen) colonies or at least five randomly selected sheen colo-

nies verified in lauryl tryptose broth and BGLB

Counts adjusted based on verification

All atypical coliform (borderline sheen) colonies or at least five randomly-selected

colonies verified in LTB and BGLB

Counts adjusted based on verification ,. ,

317

image:

5. MF Field Equipment

Manufacturer , Model _

Only standard laboratory MF procedures adapted to field application

6. Total Coliforrn Most Probable Number Procedure

a. Presumptive Test

Five standard portions, either 10 or 100 ml

Sample shaken vigorously immediately before test

Tubes incubated at 35° ± 0.5°C for 24 ±2 hours

Examined for gas (any gas bubble indicates positive test) ,

Tubes that are gas-positive within 24 hours submitted promptly to confirm test .

Negative tubes returned to incubator and examined for gas within 48 ±3 hours;

positives submitted to confirm test

Public water supply samples with heavy growth and no gas production con-

firmed for presence of suppressed coliforms

Adjusted count reported based upon confirmation

Adequate test labeling and tube dilution coding (optional)

b. Confirmed Test

Presumptive positive tube gently shaken or mixed by rotating

One loopful or one dip of applicator transferred from presumptive tube to

BGLB

Incubated at 35°C ± 0.5°; checked within 24 hours ± 2 hours for gas production.

Positive confirmed tube results recorded; negative tubes reincubated and read

within 48 ± 3 hours

Unsatisfactory sample defined as three or more positive confirmed tubes

c. Completed Test

Applied to 10 percent of all positive samples each quarter

Applied to all positive confirmed tubes in each test completed

Positive confirmed tubes streaked on EMB plates for colony isolation

Plates adequately streaked to obtain discrete colonies

Incubated at 35° ± 0.5°C for 24 ± 2 hours

Typical nucleated colonies, with or without sheen, on EMB plates selected for

completed test identification

If typical colonies absent, atypical colonies selected for completed test

identification

If no colonies or only colorless colonies appear, confirmed test for that particu-

lar tube considered negative

An isolated typical colony or two atypical colonies transferred to lauryl tryptose

broth

Incubated at 35° ± 0.5°C; checked for gas within 48 ± 3 hours

Cultures producing gas in lauryl tryptose broth within 48 ± 3 hours are consid-

ered coliforms

318 wEPA MICROBIOLOGICAL MANUAL 1978

image:

Laboratory Evaluator.

Location , Date

SAMPLE COLLECTION, HANDLING, AND PRESERVATION

Representative samples of potable water distribution system ,.. , ,

Minimal sampling frequency as specified in the National Interim Primary Drinking Water

Regulations

Sample collector trained and approved as required by State regulatory authority or its

. delegated representative

1. Sample Bottles

Sterile sample bottles of at least 120 ml; able to withstand repeated sterilization....

Ample air space remains after sample collected to allow for adequate mixing.

Sodium thiosulfate, 100 mg/1, added to sample bottle before sterilization :...

2. Sampling

Sample collected after maintaining a steady flow for 2 to 3 min to clear service

line

Tap free of aerator, strainer, hose attachment, water purification, or other devices ..

Samples refrigerated when possible during transit and storage periods in the labora-

tory (optional)

3. Sample Identification

Sample identified immediately after collection

Identification includes, water source, location, time and date of collection, and col-

lector's name; insufficiently identified samples discarded.

Chlorine residual where applicable

4. Sample Transit Time

Transit time for potable water samples sent by mail or commercial transportation,

not in excess of 30 hours

No sample processed after 48-hour transit/storage

Samples delivered to laboratory by collectors examined the day of collection ....

Data marked as questionable on samples analyzed after 30 hours

5. Sample Receipt in Laboratory

Sample logged in when received in laboratory, including date and time of arrival and

analysis

Chain-of-custody procedures required by State regulations followed .............

319

image:

Laboratory Evaluator,

Location Date

QUALITY CONTROL

A written laboratory quality control program is available for review.

/. Analytical Quality Control

A record containing results of analytical control tests available for review

a. Verification of MF Colonies

At least five coliforms verified from each positive sample

Sheen colonies in mixed confluent growth reported and verified (optional)

b. Negative Coliform Controls

A start and finish MF control test (rinse water, medium, and supplies) run with

each filtration series

When controls indicate contamination occurred, all data on affected samples

rejected and resampling requested

c. Total Coliform Confirmed Test

Presumptive tubes with heavy growth but no gas production submitted to con-

firmed test to check for suppression of coliforms. Confirmation procedure

carried out every 3 months on one sample from each problem water supply ..

d. Duplicate analyses (optional)

Duplicate analyses run on positive polluted samples not to exceed 10 percent

but a minimum of one per month (optional)

e. Positive Control Samples (optional)

One positive control sample (polluted water) run each month (optional)

f. Colony Counting (If More Than One Analyst in Laboratory) (optional)

Two or more analysts count sheen colonies; all colonies are verified; analysts'

counts compared to verified counts; procedure is carried out at least once per

month (optional)

g. Check Analyses by State Laboratories (optional)

A minimum of samples, proportional to the local laboratory work load, proc-

essed by State laboratory (see criteria for recommendations) (optional)

320 «*EFft MICROBIOLOGICAL MANUAL 1978

image:

2. Quality Control of Equipment, Supplies and Media

a. Records

Satisfactory records containing complete quality control checks on equipment,

supplies, and media available for inspection

b. Equipment and Supplies

Service contracts or approved internal protocol maintained on balance, auto-

clave, water still, etc.; service records entered in a log book _

Glass thermometers calibrated annually against a certified thermometer; metal

thermometers checked quarterly _

pH Meters standardized with pH 7.0 buffer , _

Laboratory pure water analyzed as described in criteria _

Lot numbers and dates of receipt of membrane filters recorded (optional)

Heat-sensitive tapes and/or spare strips/ampoules used during sterilization (optional)

c. Media Quality Control-

Laboratory chemicals of Analytical Reagent Grade _

Dyes certified for bacteriological use _

pH checked and recorded on each batch of medium after preparation and

sterilization _

Causes for deviations beyond ± 0.2 pH units specified _

Media ordered on a basis of 12-month need; purchased in 1/4-lb. quantities,

except those used in large amounts (optional)

Bottles dated on receipt and when opened (optional)

Opened bottles of routinely used media discarded within 6 months (if stored in

desiccator storage may be extended) (optional)

Shelf life of unopened bottles not in excess of 2 years (optional)

New lots of media quality tested against satisfactory lot using natural water

samples (optional)

321

image:

Laboratory • Evaluator.

Location , Date

DATA REPORTING

Sample information and laboratory data fully recorded

Direct MF counts and/or confirmed MPN results reported promptly

After MF verification and/or MPN completion, adjusted counts reported

One copy of report form retained in laboratory or by State program for 3 years

Test results assembled and available for inspection (optional)

322 <8-ER<V MICROBIOLOGICAL MANUAL 1978

image:

Laboratory , Evaluator.

Location : Date

ACTION RESPONSE TO LABORATORY RESULTS

Unsatisfactory test results given action response and resampled as defined in National

Interim Primary Drinking Water Regulations , . .

State and responsible local authority notified within 48 hours after check samples cpji-

firm coliform occurrence

All data reported to State and local authorities within 40 days

323

image:

APPENDIX C

BIBLIOGRAPHY

1. Kittrell, R. W., 1969. A Practical Guide to Water Quality Standards of Streams, Publ. No.

CWR-5. U.S. Department of the Interior, FWPCA, U.S. Government Printing Office,

Washington, DC.

2. American Public Health Association, 1975. Standard Methods for the Examination of

Water and Wastewater, (14th ed.) Washington, DC.

3. Society of American Bacteriologists, 1957. Manual of Microbiological Methods,

McGraw-Hill Book Company, Inc., New York, NY.

4. Geldrelch, E. E., 1975. Handbook for Evaluating Water Bacteriological Laboratories,

(2nd ed.) U.S. Environmental Protection Agency, Cincinnati, OH. EPA-670/9-75-006.

5. Scarpino, P. V,, 1971. Bacterial and viral analysis of water and wastewater. Chapter 12.

In: Water and Water Pollution Handbook. Volume 2. (L L Ciaccio, ed.) Marcel Dekker,

Inc., New York, NY. 2^639.

6. Mitchell, Ralph (ed.), 1972. Water Pollution Microbiology. John Wiley and Sons, Inc.,

New York, NY.

7. Current Practices in Water Microbiology. 1973. U.S. Environmental Protection Agency,

Office of Water Program Operations, National Training and Operational Technology

Center, Cincinnati, OH.

8. Greeson, P.E., T. A. Ehlke, G. A. Irwin, B. W. Lium, and K. V. Slack, 1977. Techniques

of Water Resources investigations of the United States Geological Survey. Chapter A4,

Methods for Collection and Analysis of Aquatic Biological and Microbiological Samples.

Book 5, Laboratory Analysis. U.S. Dept., of the Interior, Superintendent of Documents,

U.S. Government Printing Office, Washington, D.C. 20402.

9. Bordner, R. H., C. F. Frith and J. A. Winter, Eds., 1977. Proceedings of the Symposium

on Recovery of Indicator Organisms Employing Membrane Filters. U.S. Environmental

Protection Agency, EPA-60019-77-024, EMSL-Cincinnati, Cincinnati, OH 45268.

324 &EFA MICROBIOLOGICAL MANUAL 1978

image:

INDEX

Absorbent pads, 74, 205

Accidents, reporting, 262

Accuracy, method, 240

Actinomycetes, analytical method for, 186

Agar, nutrient, 39

Agglutination test

for H antigen, 179

for O grouping, 177

for O and Vi, alternate procedure, 179

for Vi antigen, 178

Air density plates in quality control, 196

Alcohol

for sterilization procedures, 71, 74

in WIF media, 43-45

Alternate test procedures, 92

Analytical cost, 246

Analytical quality control

comparative testing of methodologies, 236

in compliance monitoring, 239

in routine analyses, 231

Antisera, for serological testing of

Salmonella, 177

Applicator sticks, 32, 74, 80

Arabinose (L) fermentation, 41, 149

Arabinose, (L), Solution in purple broth

base, 41

Arginine decarboxylase test, 122, 171

ASTM tests for membrane filters

bacterial retention, 206

extractables, 207

flow rate, 207

inhibitory effects, 206

recovery, 206

Asymmetry of data, 227

Autoclave, steam

quality control, 216

specifications, 36, 37, 38

temperature control, 216

Automotive safety, 263

Azide dextrose broth, 46

Balances

quality control, 212

specifications, 33

Bacteria

see total coliforms, 108

fecal coliforms, 124

fecal streptococci, 135

Salmonella, 154

standard plate count, 101

Bathing beaches, sampling, 29

BHI tests

10 C and 45 C, 146

6.5% NaCI and pH 9.6, 147

with 0.4% potassium tellurite, 147

Bench forms

for membrane filter analyses, 60

for MPN analyses, 60

Biochemical characterization of the

Eriterobacterlaceae, 176

Biochemical identification of Salmonella, 167

Biochemical tests

coliforms, 119

fecal streptococci,

multitest systems,

Salmonella, 167

146

172

325

image:

Biohazard, control of, 268

Bismuth sulfite agar, 53

Blending, high solids samples, 62

Blood agar base, with 10% blood, 50

Blood agar with 0,4% potassium tellurite, 50

Boat safety, 263

Bottles, sample, 36

Brain heart infusion

agar, 47

•gar with potassium tellurite, 49

broth, 47

broth pH 9.6, 48

broth with 40% bile, 48

broth with 6.5% NaCI, 48

Brilliant green agar, 52

Brilliant green lactose bile broth, 45

Brom thymol blue, pH check on glassware,

199

Buffered glucose medium, see MR-VP Broth,

Buffered water

peptone, 57

phosphate, 57

Calculating results

MF analyses, 75

MPN analyses, 81

spread plates, 69

Catalase test, 147

Celita (diatorrtaceous earth), 160

Centrifuge, quality control of, 213

Certification program. Appendix B, 297

Citrate test, 122

Chain of custody, 17

Characterization of Enterobacterlaceae, 176

Check list, safety, 269

Chelating agent, 6

Chemicals and gases, safe use of, 267

Chlorinated effluents, 6, 96

Chlorination of wastes, 6, 96

Chlorine, damage to cells, 6, 96

Citrobacter, 108

Cleaning glassware, 36

Coliforms, differentiation of, 119

Conforms, fecal

definition, 124

MF test, delayed incubation, 128

MF test, direct, 124

MF test, verification, 130

MPN test, 132

Coliform test limitations

fecal coliforms, MF, 124

total coliforms, MF, 108

total coliforms, MPN, 114

Coliforms, total

differentiation of, 119

MF test, immediate two-step, 111

MF test, single step, 110

MF test, delayed , 112

MF verification, 113

MPN test, 114

Colony

counting, pour plate, 69

counting, spread plate, 69

counting, membrane filter, 75

counting by more than one analyst, 231

spreader colonies, 106

Colony counters, 33

Conjugate

approved list, 182

fluorescent antibody, 182

titration of, 182

Comparative testing of methodologies, 234

Completed MPN, 81

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