METHODS FOR MICROBIOLOGICAL ANALYSES
OF SEWAGE SLUDGES
March 1993
Prepared for:
Office of Science and Technology
Health and Ecological Criteria,D1vision
U.S. Environmental Protection Agency
401 M Street, SH
Washington, DC 20460
Prepared by:
Dynamac Corporation
The Dynamac Building
2275 Research Boulevard
Rockvllle, MO 20850
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METHODS FOR MICROBIOLOGICAL ANALYSES
OF SEWAGE SLUDGES
March 1993
Prepared for:
Office of Science and Technology
Health and Ecological Criteria-Division
U.S. Environmental Protection Agency
401 M Street, SW
Washington, DC 20460
Prepared by:
Dynamac Corporation
The Dynamac Building
2275 Research Boulevard
Rockville, MO 20850
-------
DISCLAIMER
The- mention of a manufacturer, trade name, or commercial product does
not constitute endorsement or recommendation for use by EPA.
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CONTENTS
Page
LIST OF FIGURES v
LIST OF TABLES vi
I. INTRODUCTION M
II. SAMPLING AND TRANSPORTATION II-l
III. MICROBIOLOGICAL METHODOLOGIES FOR IDENTIFICATION AND
QUANTIFICATION Ill 1
A. Dry Weight Analysis Ill- 2
B. Dilution of Solid and Semi-Solid Sewage Sludge Samples . Ill- 3
C. Fecal Coliforms Ill- 3
D. Salmonella Ill- 7
1. Sample Collection and Concentration Ill- 7
2. Primary Enrichment/Isolation Plating Ill- 9
3. Biochemical Identification 111-12
4. Serological Identification '........ 111-17
5. Quantitative Methodology 111-17
6. Regrowth 111-19
E. Viruses 111-20
1. Sample Collection and Storage of Samples
for Viral Analysis . . 111-21
2. Elution/Concentration 111-23
3. Assay Identification '111-30
F. Ascaris Ova ; . .' 111-36
e>
IV. STATUS OF PUBLIC AND PRIVATE LABORATORIES CAPABLE OF . '
CONDUCTING ANALYSIS OF MICROORGANISMS IN SEWAGE SLUDGE ... IV-1
A. Availability of Laboratories IV-1
B. Laboratory Certification IV-2
C. Standardization of Analytical Methods IV-4
D. Quality Assurance IV-6
E. Cost of Analyses IV-7
V. NEEDS AND PROBLEMS OF POTW OPERATORS V-l
A. Capabilities of POTWs to Perform Microbiological Analyses V-l
B. Need for Outside/Private Laboratories V-2
C. Storage and Transport to Outside Laboratories V-3
D. Uniformity of Methodology V-3
iii
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CONTENTS (cont.)
VI. SUMMARY AND RECOMMENDATIONS VI-1
VII. REFERENCES VIM
APPENDICES
Appendix A: Analytical Methodologies for Performing Microbiological
Testing of Sewage Sludge
Appendix B: Sources of Laboratories For Microbiological
Testing
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• LIST OF FIGURES
Figure No. Page
II-l Sample Collection Form Sent to Participating Facilities . . . II-3
II-2 Instructions for Completing Sample Collection Form Sent to
Participating Facilities II-4
III-l Schemes Used for Estimation of Density of Fecal Coliforms
in Sewage Sludge 111-4
III-2 Scheme Used for the Isolation and Identification Procedures
for Salmonella 111-8
II1-3 Scheme for the Elution, Concentration, Decontamination/
Detoxification and Assay of Vjruses in Sewage Sludge .... 111-22
III-4 Zinc Sulfate Density Gradient Separation Method for
Identification of Ascaris Ova in Sewage Sludge ....'... 111-38
A-l Flow Chart for the Fecal Coliform MPN Tests A-13
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LIST OF TABLES
Table No.
III-l Colony Appearance of Salmonella and Other Enterics
on Isolation Media ..................... III-ll
III-2 Production Rate and Time Requirements of Multitest
Systems I II- 14
III-3 Reported Shelf-Life of Multitest Systems With or
Without Refrigeration .................... 111-14
III-4 Costs and Sources of Multitest Systems ........... 111-16
III-5 Biochemical Characteristics of Salmonella .......... 111-18
III-6 Viral Recovery From Sewage Sludge As a Function of
Processing Technique .................... 111-25
III-7 Relative Recoveries of Indigenous Viruses from
Raw Sewage Sludge by Different Extraction Methods ...... I II -29
III-8 Relative Plaguing Efficiencies of Extracted Viruses
on Different Cell Lines ................... 111-31
*
III-9 Types of Sludges Examined and Their Physicochemicat
Characteristics ...................... ' . . 111-32
111-10 Comparisons of EPA and Glass Methods for Virus Recovery
From Sewage Sludge ........ .... ......... 1 1 1-33
III-ll Serum Neutralization of Rotavi ruses ............. 1 11-35
A-l MPN Itidex and 95%° Confidence Limits for Various
Combinations -of Positive and Negative Results
When Five 20-mL Portions Are Used .............. A- 10
A-2 MPN Index and 95% Confidence Limits for Various
Combinations of Positive and Negative Results
When Ten 10-mL Portions Are Used ........... "... A- 10
A-3 MPN Index and 95% Confidence Limits for Various
Combinations of Positive Results When Five Tubes
Are Used per Dilution (10 mL, 1.0 mL, 0.1 mL) ........ A-ll
A-4 Detection Limits for Membrane Filtration and MPN Analyses . . A-17
A-5 Colony Appearance of Salmonella and Other Enterics
on Isolation Media ..................... A-26
vi
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I. INTRODUCTION
The U.S. Environmental Protection Agency's Office of Water is developing
a comprehensive regulation to control the use or disposal of sewage sludge under
section 405(d) of the Clean Water Act. This regulation will establish standards
for various sewage sludge use or disposal practices. A primary concern is the
potential public health risk posed by pathogens in sewage sludge.
technical documents, as well as on discussions with experts involved in testing
sewage sludge for microorganisms and parasites: research scientists, wastewater
treatment plant operators, and analytical laboratory personnel experienced in
development and application of the mo-st recent methodologies.
1-1
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II. SAMPLING AND TRANSPORTATION
The objective of sampling sewage sludge or any other material for
laboratory analysis is to collect a uniform portion of material small enough in
volume to be transported and handled in the laboratory while still accurately
representing the material being sampled. This implies that the relative
concentration of all components will be the same in the sample as in the material
being sampled, and that the sample will be handled in a manner that will cause
no significant changes in composition before the appropriate tests are completed.
This chapter presents a general review of optimal and currently used
representative sampling and transportation methodologies for sewage sludge. The
numerous, differences in these sampling practices indicate the lack of
standardization for these procedures. Specifics for sampling various
microorganisms are discussed in appropriate sections of Chapter III.
A. SAMPLING
Harding et al. (1981) collected liquid sewage sludge samples into 1-liter
polypropylene containers for microbiological.analyses; samples were cooled to 4*C
in an ice-water bath, packed in an insulated container with-frozen Kool Pac®, and
shipped to the testing laboratory via an airline parcel service. The t\me
between sample collection and microbiological assay was no longer than 24 hours.
Samples were collected from an irrigation system located between the storage
lagoon and spray irrigation nozzles or as the sewage sludge was loaded onto a
tanker truck for use or disposal. Vortex mixing with glass beads for 2 minutes
was determined to be the best method for obtaining a uniformly dispersed sewage
sludge sample; 75% of tests used for this determination showed higher recovery
of viable bacterial cells as a result of this procedure.
Sterile 2- to 5-gallon tubs with scalable tight-fitting covers and wide
mouths have been used for ease of handling to accommodate collection of turbid
samples (Craun et al., 1990). Dewatered sewage sludge can be sampled using a
sterilized trowel, scoop, shovel, or -auger. A shovel or auger is better suited
for sampling from a deeper bed of material. Dewatered sewage sludge must be
II-l
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diluted prior to processing, e.g., total and fecal coliform bacteria analyses,
1 to 10 dilution (Hunger, 1992).
Most sampling for sewage sludge utilizes single grab samples (Clancy.
1992). Bordner and Winter (1978) indicate that grab samples collected from
treatment works and industrial waste treatment operations should be obtained at
evenly spaced, selected intervals for a 3- to 5-day period when plant treatment
efficiency varies considerably; fewer samples should be collected when the
process displays little variation. It was indicated that composite samples that
might obscure minimal and maximal results should not be collected for
bacteriological examination (Bordner and Winter, 1987). Greenberg et al. (1992)
in Standard Methods for the Examination of Uater and Hastewater, 18th edition,
indicated, however, that time-composite samples are useful for calculating
efficiency of publical.ly owned treatment works (POTWs). Composite samples also
represent a substantial savings in laboratory effort and expense.
Sample volumes have not been specified for sewage sludge; however, a sample
of sufficient size should be collected to allow for necessary subdivision to
isolate or separate various microorganisms for Identification (Clancy, 1992).
Minimum collection volume for water and dilute wastewater is. 2 L for bacterial
analyses a^d 400 L for viruses' and parasites (Craun et al., 1990). Similarly,-
specific numbers of samples for sewage sludge were not Indicated. Sufficient
numbers of samples should be collected to satisfy National Pollutant Discharge
Elimination System (NPOES) permits or to provide statistically sound data and to
give an accurate representation of the microbiological- quality of the discharge
(Bordner and Winter, 1978).
Often 1n wastewater -determinations, the testing laboratory prescribes the
sampling program, which 1s determined 1n consultation with the treatment works.
Simplified detailed sampling instructions should be sent to the treatment works
with the sampling device, 1ce packs, cooler for return transportation, and proper
collection forms (Yanko, 1988; Clancy., 1992). Examples of the sample collection
form and instructions are presented in Figures II-l and 11-2. Space should be
left in the collection device for aeration and mixing. An air space of
II-2
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SAMPLE COLLECTION FORM
I. Sjmplq Information
1. Sample 1.0. (Code) Collection Date Time
2. Sampling Site _
3. Field Sampling Manager (on site)
4. Contractor L.A. County Sanitation Oist. Contract No. CR-812589-010
EPA Project Officer y. JattubowsM Program Nai
Distribution and Marketing .of Municipal Sludges
5. EPA Project Officer y. JattubowsM Program Name Occurrence of Pathogens jn
6. Source Sampled
7. Quantity Sampled/Units.
8. Sample Description
9. Other Information as Applicable Air Temp. Pile Temp..
Weather Other
II. Handling and Shinning
1. Describe Sample Treatment Prior to Shipping
Field Storage and Shipping Conditions
Container Temperature
Whirl Pak Bag Ambient
Glass with Teflon Lid Liner Packed with Freeze Pak
3. Date and Tlmt Shippad _J '..
-i
4. Comments ' • . _
5. .Mode and Carrier for Shipping
6. Sample Shipped to: San Jose Creek Mater Quality Laboratory
1965 So^ Workman Hill Rd..Whittier.CA 90601
Attention! M.A. Yanko
^
III. Arrival (Lab use onTy) Date /___ Tin* * By •__
Lab Job Mo. Charge No. Proj. No.
Requested by: Report To:
Date and Tim* - Grab Sample: L 7
Sample Location Type
Description
Figure II-l. Example of sample collection form .sent to participating facilities.
Source: Yanko (1988).
II-3
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INSTRUCTIONS FOR COHPLETING SAMPLE COLLECTION FORM
(Use a separate form for each sample)
I Sample Information
1. Sample I.0.: The identifying information written on the label on the sample
container. This information includes site code, sample number if more than
one sample collected on same day, date, and time sample collected. Your
site code is: .
2. Sampling Site: The name of treatment plant and/or city and state.
3. Field Sampling Manager: The name of person collecting sample.
4. and 5. Already completed.
6. Source Sampled: The point the sample was collected from, such as,
'stockpile at treatment plant* or 'bag of Grow Fast garden food" or "drying
bed", etc.
7. Quantity Sampled: e.g., "2/3 of whirl pak bag" and/or "1 qt. jar".
8. Sample Description: e.g"., "air dried digested sludge" or "windrow composted
sludge" or "in-vessel composted sludge". Also indicate if composite sample-
or single grab sample.
9. Other Information:' Air temperature: Self-explanatory,
Weather: Brief description of prevailing weather conditions when samples
collected, e.g., "cold, snow* or "Intermittent rain" or "hot humid" etc.
Pile temperature: if sludge Is in a stockpile and Is self heating, or if
sludge Is above ambient temperature for any reason, measure sample
temperature at a representative point or depth. Other Is any other
information you think may be pertinent regarding samples, such as, "flocks
of seagulls feeding on tops of stockpiles", or "extremely heavy rains three
days ago". Any Information about conditions that could affect the sample
would be helpful.
II. Handling and Shipping
1. Describe Sample Treatment Prior to Shipping: Briefly describe how you
collected the sample.For example,"Using a clean shovel, removed
approximately one foot of material from surface of stockpile. With clean
scoop, collected sample and placed in sample containers".
2. and 3. Self-explanatory.
4. Comments: Any comments or observations concerning sampling that might
-influence laboratory results.
5. UPS, Federal Express, etc.
6. Already completed.
III. Arrival
Thts section 1s filled out by laboratory.
Figure II-2. Instructions for completing sample collection form sent to
participating facilities.
Source: Yanko (1988).
II-4
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approximately 1% of the container capacity should be allowed for thermal
expansion of shipped samples.
The following sampling procedures are recommended based on a review of the
literature. Samples should be representative of the bulk material from which
they are collected, and proper quality assurance procedures should be followed
(Greenberg et al., 1992). Physical characteristics of the sewage sludge should
be considered when selecting a sampling device or method. The sampling device
should be sterile and constructed of an inert or unreactive substance such as
stainless steel or Teflon. The sampling bottle should be sealed until filled
with the sample. Sample ports should be disinfected, and aseptic techniques
should be used to avoid sample contamination. All procedures employed relative
to sample collection should be documented in a study plan or field log. All
samples should be properly labeled and packaged prior to shipment. The
collection device should be labeled with the site code, name of sample collector,
date and time of sample collection, and any other specifics needed for sample
correlation (e.g., wastewater samples should be identified by process producing
waste stream). Ambient temperature and temperature of the sample at the time of
sampling should be noted. Location of sampling sites should be indicated by
description or maps, and the use of stakes or landmarks would permit future
identification of sites if necessary. It is necessary to have the ability to
trace sample handling from time of collection .through analysis and final
disposition.
B. TRANSPORTATION .
The adherence to sample preservation and holding time limits is critical
to the.production of valued data. Bordner and Winter (1978) indicate that
bacteriological samples should be iced or refrigerated at 1 to 4*C in insulated
containers during transit to the laboratory. Samples for parasite analysis may
be stored at 4'C in 3.7% formaldehyde (Craun et al., 1990). Samples should not
be held longer than 6 hours between collection and initiation of analyses
(Bordner and Winter, 1978). However, Clancy (1992) indicates that in practice
a hotd time of 6 hours is impractical for transport of samples to the laboratory;
hold times of up to 24 hours (overnight courier) are more realistic and are used
II.-5
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routinely. Craun et al. (1990) indicated that bacterial samples should be
assayed within 24 hours and stored at 4*C until that time. The treatment
facility should coordinate availability of laboratory personnel to initiate
processing upon receipt of samples. Samples that are not processed within the
specified time and under the proper conditions can yield erroneous results,
especially with the less stable microorganisms (i.e., bacteria).
II-6
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III. MICROBIOLOGICAL METHODOLOGIES FOR
IDENTIFICATION AND QUANTIFICATION
Microbiological methodologies for identification and quantification of
fecal coliforms, Salmonella, viruses, and ascarids are discussed in this chapter.
Specific descriptions of the methods are presented in Appendix A of this
document. These methodologies have been collected from literature review and
from discussions with POTW operators and microbiological laboratory personnel.
All methodology pertinent to water and wastewater is discussed in general;
methodologies specific to wastewater and sewage sludge are considered in detail.
To ascertain whether microbiological recovery methods are adequate,
positive and negative controls (i.e., growth or no growth) are processed with the
test samples. These controls generally indicate if the analysis is working but
do not show the level of sensitivity that may be achieved. It is necessary that
the positive controls used are viable organisms.
In all cases, the type of sewage sludge being sampled and th« percentage
of solids will influence the specific method used. Anaerobically digested sewage
sludge contains 2-5% solids; when >5% solids exist in the sewage sludge, the
sewage will form a gel-like matrix. Samples with solids concentrations of 2-3%
are the most useful. All results of sample methodologies are based on analysis
per gram of solids or total solids based on dry weight. For this reason, watered
sewage sludge arid wastewater samples must be converted from a liquid to solid
basis., Applicable dry weight methodologies as determined from literature review
•' '" -
are discussed in this- chapter.
Yanko (1988) conducted a study of the occurrence of microorganisms in
distributed1 and marketed sewage sludge to determine the levels of indicator and
pathogenic organisms that might be present. Seven sewage sludge compost products
were sampled weekly for 1 year; in addition, sewage sludge from 24 municipalities
was sampled bimonthly. Results for sewage sludge products, which included
composts, air-dried sewage sludges,, and heat-treated sewage sludges, varied
widely. Indicator microorganisms were detected at high concentrations with large
variability between samples. The only pathogens defected were bacterial (e.g.,
• III-l
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Salmonella). Total and fecal coliforms and fecal streptococci were found to be
good predictors of the presence of Salmonella. Air-dried sewage sludge contained
the lowest concentration of pathogenic bacteria. In addition, significant
increases in pathogenic (Salmonella) and indicator bacteria occurred during
production of commercial soil amendments, which was consistent with a regrowth
phenomenon. No protozoan cysts were found. Helminth ova were detected but were
not viable. Enteric viruses were confirmed at low levels in only two samples.
The methodologies and techniques used by Yahko (1988) are discussed in this
chapter.
A. DRY WEIGHT ANALYSIS
To standardize microbiological sample methodology within one laboratory or
between laboratories, sample results must be expressed per gram of solids, and
sewage sludge and wastewater sample measurements must be converted from
milliliter to gram. Greenberg et al. (1992), 1n Standard Methods for the
Examination of Hater and Uastewater, 18th edition, Indicate the suitability of
one method for the determination of total solids in sewage sludges. The
technique, entitled 'Total, Fixed, and Volatile Solids in Solid and Semi sol id
Samples," is subject to negative error due to loss of ammonium carbonate and
volatile organic matter from the sample during drying. Clesceri et al. (1989)
indicated that this effect tends to be more pronounced with sewage sludges
because the mass of organic matter recovered from* sewage sludge Requires a long
drying time, The specified drying time, and temperature must be carefully
observed to control losses of volatile Inorganic salts. All weighings should be
performed quickly to prevent weight loss through evaporation prior to drying and
to prevent absorption of moisture from the air after drying. A detailed
expUnat1on-of this method 1s-Included 1n Appendix A, section A, of this
document.
To determine the number of m1croorgan1sms/g (dry weight) of dewatered
sewage sludge or compost, Yanko (1988) suspended samples 1n appropriate diluents
(50 g sample to 500 ml sterile phosphate buffered dilution water containing 0.1%
Tween 80 used as a dlspersant) and blended the mixture at medium to high speed
in 1-quart stainless .steel Waring blender jars for 1 minute. Separate
III-2
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suspensions were prepared for bacterial and parasite analyses. The Clescari et
al. (1989) methodology indicated above was used for the final dry weight
analysis. Following analysis, numbers of organisms found were expressed on a dry
weight basis. Other diluents for dewatered sewage sludge or compost include
peptone (10 g/L, 1:1 dilution, or 1% solution), peptone + 2 g/L Calgon 3, or
peptone + 0.01 mL/L Tween 80 (Olivieri et al., 1989). Samples were blended at
high speed for 1 minute using a Waring blender.
B. DILUTION OF SOLID OR SEMI-SOLID SEWAGE SLUDGE SAMPLES
Solid or semi-sol id sewage sludge samples to be processed for analysis must
first be diluted.. Greenberg (1992) and Ma (1992) suggest blending 50 g sieved
samples with 450 mL sterile buffered water containing 0.1% Tween 20, which equals
a 10"1 dilution. The mixture is homogenized in a blender for 1 minute.
Alternatively, 50 g of the sample should be blended with 250 mL of the buffer
containing 0.1% Tween 20 foV 1 minuter the resulting homogenate is added to an
additional 200 ml of buffer containing 0.1% Tween 20 and blended again for
5 minutes (Ma, 1992). Dilutions of the homogenates should be prepared using
18-mL blanks (sterile buffer). Appropriate dilutions are based on the sample
.being processed.
C. .FECAL COLIFORMS
• *
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SEWAGE SLUDGE SAMPLE
MOST PROBABLE NUMBER (MULTIPLE TUBE)
PROCEDURE
PRESUMPTIVE TEST
LAURYL TRYPTOSE
BROTH
LAURYL SULFATE
BROTH
CONFIRMED TEST
ESTIMINATION OF BACTERIAL DENSITY
(MPN/100Q WET WEIGHT FOR SEWAGE SLUDGE)
MEMBRANE FILTER
PROCEDURE
FILTRATION OF SAMPLE
ESTIMINATION OF BACTERIAL DENSITY
(f COLONY FORMING UNITS/G DRY WEIGHT)
Figure III-l. Schemes used for estimation of density of fecal conforms
in sewage sludge.
Source: Gretnberg *t iK (1992); Oliveri et al. (1989); Bordner and
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U.S. EPA Part 503 regulation for Use or Disposal of Sewage Sludge (U.S. EPA,
1993). Gas production in a fermentation tube within 24 hours or less is
considered a positive MPN reaction indicating coliforms of fecal origin. Failure
to produce gas constitutes a negative reaction indicating a source other than the
intestinal tract of warm-blooded animals (Bordner and Winter, 1978; Clesceri et
al., 1989; Craun et al., 1990; Greenberg et al., 1992). A positive test on m-FC
medium is indicated by the appearance of dark blue colonies; all other colonies
are light blue, gray, or cream-colored.
Results of the multiple tube fermentation procedure are reported as an
MPN/100 ml index, which is not a direct colony courrt or actual' enumeration.
By contrast, direct plating methods such as the MF procedure permit a direct
count of coliform colonies/100 ml. The MF technique is highly reproducible, can
be used to test large volumes of sample, and yields numerical results more
rapidly than the multiple-tube procedure. However, the MF technique has
limitations when testing waters with high turbidity. For such waters, e.g.,
sewage sludge, it is more appropriate to use the multiple-tube fermentation
technique. However, with proper dilution, the fecal coliform membrane filter
procedure can-be used to test sewage sludge. This method is described in
Appendix A, section B.
A positive, confirmed fecal coliform identification using the multiple-tube
fermentation method is the formation of gas i'n three to five tubes (10- to 100-mL
•samples); for sewage sludge, using EC broth., this positive reaction indicates the
need for additional examination and confirmation. The procedure* is a single-step
method when using A-l broth .and does not require confirmation (Greenberg et al.,
1992). The absence of gas in five 10-mL sample tubes, (equivalent to a-n MPN of
<2.2 coliforms/100 mL) is a negative indication for the presence of fecal
coliforms. Up- to 72 hours is required for this procedure; detailed .descriptions
of this method as described bV Greenberg et al. (1992), Bordner and Winter
(1978), and Yanko (1988) are presented in Appendix A, section B. The lowest
detection estimate for solid or semi-solid sewage sludge samples diluted 10"1 as
described above would be an MPN of 2.2 fecal col i form/10 g wet weight or 23 fecal
coliform/100 ml (Meckes, 1992).
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Greenberg et al. (1992) indicate the use of several rapid detection methods
for fecal coliforms: the 7-hour fecal coliform test, the bioluminescence test,
the colorimetric test, and radiometric detection. All methods may be completed
within 1 to 7 hours. However, the membrane filter technique is used in
conjunction with the procedures. In addition, the procedures require reagents
not generally available, they are tedious to perform, and they require special
handling or incubation schemes incompatible with most laboratory schedules.
For the above reasons, these methods will not be described further.
The presence-absence (P-A) test for fecal col 1 forms is a simple
modification of the multiple-tube procedure; it may be used if a quantitative
assessment is not required. The P-A test includes the advantage of examining a
large number of samples per unit of time; the opportunity for further screening
of the sample may follow. This procedure may be used on routine sample
submissions, collected from distribution systems or water treatment plants
(Greenberg et al., 1992; Clesceri et al., 1989). These short-term tests are not
discussed in detail because they are not applicable to the Identification of
fecal conforms in sewage sludge.
Harding et al. (1981) Indicated the use of direct plating on selective
media for assay of fecal conforms 1n sewage .sludge samples. Handling time was
lessened considerably with this technique, although recoveries were not optimal.
Additional Incubation times were found to be needed to Improve recovery. Sewage
sludge samples were diluted 1n sterile phosphate-buffered saline, and" 0.1-mL
allquots were Inoculated onto m-FC agar. Plates were Inoculated ai 35*C for 4 to
5 hours.and transferred to 44.5'C for an additional 20 hours of Incubation.
Results were reported as colony-forming units per gram of total suspended solid*.
When Harding et al. (198.1) compared the recovery potential of fecal conforms
using the direct"plating technique, the multiple-tube fermentation method, and
membrane filtration method, the recovery of the microorganisms was 20 to 50%
higher with the multiple-tube fermentation technique.
As indicated, the Part 50.3 regulation requires that representative samples
of sewage sludge be collected and analyzed for fecal coifforms. Either the Most
Probable Number (multiple-tube) procedure using A-l broth (Fecal Coliform Direct
III-6
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test) or EC broth, or the Membrane Filter method may be used to test sewage
sludge for fecal coliforms. These methods are described in Appendix A, section 8
(Greenberg, 1992; U.S. EPA 1993).
D. SALMONELLA
Kenner and Clark (1974) reported an incidence of 100% of Salmonella in
wastewater and in sewage sludges. In many cases, total and fecal coliforms were
shown to be good predictors for the presence of these potential pathogens.
However, Salmonella may be found in the absence of fecal conforms. This is a
situation where negative indicator tests may provide a false indication about the
presence or absence of Salmonella. There are limitations and variations in both
•the sensitivity and selectivity of accepted Salmonella isolation procedures.
Methods for detection and quantification of Salmonella in sewage sludge involve
sample collection, enrichment, isolation plating, biochemical identification, and
serological verification. Either the method developed by Kenner and Clark (1974)
for detection and enumeration of Salmonella in sewage sludge or the multiple tube
enrichment technique of Greenberg et al. (1992), Standard Methods for the
Examination of Mater and Hastewater, 18th edition, may be used to analyze sewage
sludge for Salmonella as Indicated in the Part 503 regulation. The regrowth
phenomenon has been found to be a potential problem with Salmonella. This issue
should be considered when determining appropriate sampling times. Figure III-2
•presents the scheme used for the current Isolation and Identification procedures
for Salmonella.
1. Sample Collection .and Concentration
The initial steps for detection of Salmonella in wastewater or watered
sewage sludge may require concentration of the organisms by one of several
methods: the swab technique, the diatomaceous earth technique, the cartridge
filter, or the membrane filter technique (Greenberg et al., 1992; Bordner and
Winter, 1978). However, when sewage sludge 1s sampled, sample turbidity may limit
use of these techniques. The sample may clog pores, or the high numbers of
background organisms may make recovery of Salmonella difficult. For these
reasons, concentration techniques are not appropriate, as an initial step for the
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SEWAGE SLUDGE SAMPLE
1:10 DILUTION
Stenle Buffered Water
. Tween 20
ENRICHMENT
Oulcitol
Selenite Broth
Selenite
Broth
Tetrathionate
Broth
Tetrathionate Brilliant
Green Broth
ISOLATION PLATING
Eosin Methylene Xylose Lysine Xylose Lysine Brilliant Green Bismuth Sulfite
Blue Agar Desoxycnolate Agar Brilliant Green Agar Agar Agar
BIOCHEMICAL DIFFERENTIATION
COMMERCIAL DIFFERENTIAL
MEDIA Krrs
API Enterotube Inolex
Enteric 20
SlNQLf TUM DIFFERENTIATION MEDIA
(PRIMARY SCREENING)
Triple Sugar . Lysine
Iron Agar Iron Agar
ADDITIONAL BIOCHEMICAL IDENTIFICATION
(MINIMAL BIOCHEMICAL SET BY SINGLE TUBE OR MULTTTEST SYSTEMS)
Carbohydrate Lysine Phenylalanine
Fermentation Decarboxylase Tryptopnane Malonate Deaminase Phenol Red
Test Brpth Broth Broth Broth Dulcitol Broth
SEROLOGICAL VERIFICATION
PolyvaJentO Vi | Polyvalent H
REFERENCE LABORATORY CONFIRMATION
Figure .111-2, Scheme used for the isolation and identification procedures for
Salmonella.
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identification of Salmonella in sewage sludge. Therefore, concentration
techniques are not described in Appendix A.
Sampling of dewatered sewage sludge is accomplished by collecting grab
samples that may be analyzed as single or composite samples. These samples must
be diluted prior to processing (see section III.B, Dilution of Solid or
Semi-Solid Sewage Sludge Samples). Kenner and Clark (1974) have indicated that
sufficient sample volumes (dependent on the sample source) are necessary and must
be collected to ensure accuracy and representativeness.
Greenberg et al. (1992) recommends a 6-hour storage-transit time" for
Salmonella. Samples should not be transported in enrichment media because
ambient transport temperature may cause sufficient proliferation of competitive
organisms to mask Salmonella. If a sample must be collected in an area distant
-to the laboratory responsible for Salmonella identification, the samples should
be concentrated, iced, and transported (Bordner and Winter, 1978).
2. Primary Enrichment/Isolation Plating
f
Following sample*collection, the diluted samples are placed in flasks of
broth (e.g., selehite broth of tetrathionate broth) for .primary enrichment
(Bordner and Winter, 1978) (Figure III-2). The enrichment broth provides an
optimal environment for Salmonella and encourages growth while inhibiting growth
of other bacteria (e.g., conforms); this also enhances the probability of
isolating Salmonella. Sample enrichment is also necessary because solid
selective media for colony isolation (Figure III-2, Isolation Plating) are toxic
to the Salmonella and may eliminate the organism if the organism is hot present
in larger numbers (Greenberg et al., .1992). Once enrichment procedures are
performed, the detection and identification of Salmonella becomes a qualitative
procedure.
The selenite enrichment broth may be combined with dulcitol to improve
selectivity for Salmonella by allowing rapid growth while inhibiting, many
nonpathogenic enterobacteria and causing cultures containing Salmonella to
develop a distinct orange or red color (Craun et al., 1990). Similarly, the
III-9
-------
tetrathionate broth may be used alone or combined with Brilliant Green dye to
enhance selectivity for Salmonella (other than 5. typhi) (Craun et al., 1990).
Multiple flasks of the enrichment broth containing sample organisms are
incubated from 24 hours to 3 to 4 days at selected temperatures, after which
organisms that develop in the primary enrichment broth are inoculated onto
differential plating media for Salmonella isolation (Greenberg et al., 1992;
Bordner and Winter, 1978) (Figure III-2). This process is known as secondary
differentiation. Solid media used for enteric pathogen detection may be
classified into (1) differential media with little inhibition toward
nonpathogenic bacteria (e.g., Eosin Methylene Blue agar containing sucrose);
(2) selective media containing bile salts or sodium desoxycholate as inhibitors
(e.g., xylose lysine desoxycholate agar); and (3) selective media containing
Brilliant Green dye (e.g., Brilliant Green agar; bismuth sulfite agar) (Greenberg
et al., 1992). The selective solid media most commonly used are Brilliant Green
agar (BG), bismuth sulfite agar (BS), or xylose lysine desoxycholate agar (XLD)
(Bordner and Winter, 1978). Salmonella colonies may appear pinkish-white with
a red background on BG, black (with or without a metallic sheen, which may extend
beyond the colony to give a "halo" effect) on BS, and red with black centers on
XLO. Table III-l Indicates the appearance of Salmonella on various Isolation
media. Overgrowth of other organisms may be reduced by using1 agar plates dried
to reduce surface moisture.
Differential plates should be Inoculated with enrichment media every
24 hours over a 3- to 4-day period. Inoculation should occur from broth cultures
that develop turbidity and orange-red'color as a result of selenlte reduction.
Preferably two enrichment media should be used for each sample. After
incubation, the plates are examined for typical colonies of Salmonella that are
characterized biochemically and serologically.
The recoveries of Salmonella at different Incubation temperatures have been
compared; it appears that a greater number of Isolates and more species are
isolated at 41 to 43*C than at 35 to 37*C (Bordner and Winter, 1978; Craun et
al., 1990). The use of a temperature of 41.5*C also reduces the numbers
of interfering organisms that may hinder the Isolation and identification of
111-10
-------
Table 111-1. Colony Appearance of Salmonella and Other Enterics on Isolation Media
Colony Appearance
Salmonella
Other Enterics
1. Bismuth Sutfite Agar
Round jet black colonies
with or without sheen
Round jet black colonies
with or without sheen
Round jet black colonies
with or without sheen
S. typhi
S. enteritidis ser
Ententidis
S. enteritidis ser
SchottmueJIeri
Rat or slightly raised
green colonies
Rat or slightly raised
green colonies
Rat or slightly raised
green colonies
S. enteritidis ser
Typhimurium
S. enteritidis bioser
Paratyphy!
S. choierae-suis
Proteus spp.
2. Brlliant Green Agar
Slightly pink-white,
opaque colonies surrounded
by brilliant red medium •
Salmonella spp.
Yellow-green colonies
surrounded by yellow-green
zone
Escherichia, Klebsfella,
Proteus spp. (lactose or
sucrose fermenters)
3. XLD or XLBG Agar
Red, black centered colonies
Red colonies
Yellow colonies
Yellow colonies
Yellow colonies
Yellow colonies
Yellow colonies
Salmonella spp.
Shigella spp.
Escherichia spp.
and biotypes
Citrobacter spp.
KJetsiella spp.
Enterobacter spp.
Proteus spp.
Source: Bocdner and Winter (1978).
III-ll
-------
Salmonella. Brilliant Green agar is favored for the development and
identification of Salmonella except for 5. typhi and a few other species; bismuth
sulfite agar allows for growth of most Salmonella including 5. typhi. These
procedures and media preparation methods are discussed in detail in Appendix A,
section C.
A modified xylose lysine Brilliant Green medium was proposed by Hussong et
al. (1984) to control the loss of selectivity by heating the Brilliant Green
component. The use of unheated Brilliant Green dye (7 ppm) in xylose lysine agar
was proposed for detecting H2S-positive Salmonella from samples with low pathogen
concentration; only Salmonella were detected with black-centered colonies. The
Agricultural Research Service (1986) suggested that 6-9 ppm of the Brilliant
Green'dye should be added to the autoclaved xylose lysine agar base (2% agar)
i
after cooling to increase the effectiveness of discrimination for Salmonella in
sewage sludge and sewage-sludge compost.
Harding et al. (1981) Indicated that concentration of sewage sludge samples
by filtration through diatomaceous earth was not feasible during this sampling.
As a result, sensitivity of procedures was reduced; sewage sludge was inoculated
directly into enrichment media. Specifics of this modified method are presented
in Appendix A, section C.
The Optional Fluorescent Antibody Screening technique is a rapid screening
method that can be used after primary enrichment of water samples in d.iatomaceous
earth (Bordner and Winter, 1978). Careful Interpretation of fluorescence is
critical for this technique, 'and conventional cultural techniques must be used
to confirm positive results. Since this technique is not normally used for
sewage sludge samples, it is not described in further detail in this-document
(Bordner'and Winter, 1978).
3. Biochemical Differentiation
Since other enteric organisms share major biochemical characteristics with
Salmonella, it 1s necessary to confirm the identity of suspect colonies by
biochemical tests and serotyplng (Figure III-2). Biochemical reactions
111-12
-------
characterize the Salmonella, permit a separation from closely related bacteria,
and provide presumptive identification. Confirmed identification requires
additional serological tests. However, biochemical identification of large
numbers of cultures is expensive and time-consuming.
Biochemical differentiation can be performed in single tube media or in
commercial multitest systems (Figure III-2). Commercially available differential
media kits give 95 to 98% agreement with conventional tests, prior to serological
confirmation (Greenberg et al., 1992; Bordner and Winter, 1978; Craun et al.,
1990). Commercially available systems for identification of Salmonella include
the API Enteric 20 (Analytab Products, Inc.), Enterotube (Becton, Dickinson and
Co.), and Inolex (Inolex Biomedical Division of Wilson Pharmaceutical and
Chemical .Corp.). Other systems, including Minitek (Baltimore Biological
Laboratories), Pathotec Test Strips (General Diagnosis Division of Warner
Lambert), and the r/b Enteric Differential System (Diagnostic Research, Inc.),
are available for other enteric bacteria (Bordner and Winter,
1978). The commercially available methods for identificati.on of Salmonella are
discussed in detail in Appendix A, section C, of this document. Selection of the
specific test system should include consideration of the following factors:
1. The test system should fit the biochemical test pattern of the
Salmonella.
2. The system should use numerical identification, and the largest
* .
number of tests should be selected to identify typical and'atypical
* i *
Salmonella. The API Enteric 20, Enterotube, and Fnolex systems
listed above for identification of Salmonella are the most
appropriate.
3. The time required to perform the test and production rate vary with
'the system (see Table 111-2).
4. Refrigeration is required for maintenance of some systems.
Table III-3 indicates shelf-life of systems with and without
refrigeration.
111-13
-------
Table 111-2. Production Rale and Time Requirements of Multttest Systems
Multitest
System
API
Enterotube
Inolex
r/b Diff.
Pathotec
Mlnitek
Analyst's Time
per Culture in Mln.
10
6
30
8
15
10
Time Span of
Test in Hours
18-22
18-24
7
24
4
18-24
Cultures
per Day per Analyst
80
100
10-15
80
20-30
80
Source: Bordner and Winter (1978).
Table III-3. Reported Shetf-Ufe of Multitest Systems With or Without Refrigeration
System
Refrigeration
Required
No
Refrigeration*
API
Inolex
Pathotec
Enterotube
r/b
Mlnitek
12 months
3 years0
7 months
6-12 months
2 years
12 months
12 months
'Store In a cod, dark place at ambient temperature.
"No Information provided.
'Refrigeration not required, but w* extend the shelf-life.
Source: Bordner and Winter (1978).
111-14-
-------
5. Some test systems contain all required materials and equipment
necessary for analysis; others require additional items for use
(e.g., the API system can be sight-read or can utilize a profile
register for rapid identification of strains; the Inolex system
requires a small centrifuge).
6. The probability of laboratory-acquired infections is directly
proportional to the number of exposures to pathogens. The
Enterotube system uses direct colony picks for reactions; the API
and Inolex systems require culturing and additional handling of cell
suspensions.
7. The unit cost may vary from $0.91 to $2.16 for the Salmonella test
systems (see Table III-4).
When commercially available media kits are not used, the first phase of
biochemical identification is primary screening and the determination of
phenylalanlne deaminase activity (Figure III-2). Primary screening for
Salmonella is usually performed on triple sugar iron (TSI) agar or lysine iron
agar (LIA). Color determination is made for acid or alkaline reactions, as well
as gas and H2S production. TSI media also indicate presence of o-nitrophenyl B-
D-glactos1de (ONPG) for B-D-galactosidasS (Greenberg et al., 1992). Specifics
for this methodology are'presented in Appendix A| section G.
Isolates from TSI or LIA agar are inoculated onto phenylalanine agar and
incubated for 24 h at 35* to 37*C (Greenberg et al., 1992). Phenylalan.ine
deaminase activity is indicated by a green zone that develops around the colony;
a yellow or brown color is negative. Salmonella gives a negative reaction.
(See Appendix A, section C, for specific methodology.) A positive indole test
is indicated by a dark red color in the amyl alcohol (top) layer of the
culture; the original yellow color is negative. Salmonella is indole-negative.
111-15
-------
Table 111-4. Costs and Sources of Multitest Systems
Multitest
System
Cost
per Box
Address of
Manufacturer
API Enteric 20
Improved Enterotube
Index
$111.00 (25/box)
$128.25 (GSA)
(25/box)
NA*
r/b Enteric Differential System NA
Pathotec Test Strips
Minrtek
NA
NA
Anaiytab Products, Inc.
200 Express Street
Plalnview, NY 11803
Becton Dickinson & Co.
Microbiological Systems
Support Canter
250 Schilling Clrde
CockeysvHIe, MO 21030
Index Corporation
Blomedlcal Division
3 Science Road
Glenwood, IL 60425
Diagnostic Research, Inc.
25 Lumber Road
Roalyn, Long Island, NY 11576
Warner-Lambert Company
General Diagnostics Division
Morris Plains, NJ 07950
Baltimore Biological Laboratories
CockeysvUe, MD 21030
" Prices currently unavaiabie.
111-16
-------
A positive malonate broth test is indicated by a change of media color from green
to deep blue. 5. an'zonae utilize malonate; other strains are negative.
Conformance to typical biochemical patterns of Salmonella determines
whether to process cultures further. Reactions should be reviewed as a group and
not on the basis of a small number of apparent anomalies, since aberrant cultures
may not conform to classic reactions. Fermentation reactions in dextrose,
mannitol, maltose, dulcitol, xylose, rhamnose, and inositol broth may further
determine physiological characteristics of isolates (see Figure III-2, Additional
Biochemical Identification, and Table III-5). This reduces the possible number
of positive cultures to be processed for serological confirmation. If the
testing laboratory is equipped for serological identification, extensive
biochemical tests can be eliminated since serological testing is more accurate
and greater correlation exists between serological types and pathogenicity
(Greenberg et al., 1992).
4. Serological Identification
Final verification of Salmonella and determination of pathogenicity are
based on serological identification. • It is the only testing that identifies to
*
the serotype and bioserotype level. Serotyping is a more accurate indication of
pathogenicity than biochemical identification. Typing of Salmonella is performed
by using slide-agglutination for somatic (0) antigens and tube testing for
flagella.r (H) antigerts. SerologicaT testing is an expensive, complex procedure
that should be carried out only by trained personnel. Serological methodology
is presented in Appendix A, section C, of this document.
\
The Salmonella methodologies of Yanko (1988) and Oliverl et al. (1989) that
incorporate all of the above procedural steps are described.in Appendix A,
section C.
5. Quantitative Methodology
.When it is desirable to determine Salmonella densities in sewage sludge,
quantitative methods are utilized. The quantitation identification method for
III-171
-------
Table 111-5. Biochemical Characteristics of Salmonella
Reaction Salmonella
Cataiase +a
Oxidase
S-Galactosidase Dc
Gas from glucose at 35" C +
KCN (growth on) D
Mucate (acid) D
Nitrate reduction +
Carbohydrates:
(acid production)
Adonftol
Arabinose +
Dulcitoi D
Esculin
Inositol dd
Lactose' D
Maltose +
Mannrtol +
Sallcin
Sorbitoi -t-
Sucrose
Trehaiose +
Xylose +
Related C sources:
Citrate +
Gluconate *
Malonate D
d-Tartrate D
M.R. +
V.P.
Protein reactions:
Arginine +
Gelatin hydrolysis D
HjSfromTSI +
Indoia
Lysine decarboxyiase +
Omithine +
Urea hydrolysis
Glutamic acid
Phenvlalanine .
1 -i- - Positive biochemical reaction.
°- « Negative bkxhemteal reaction.
c D - Different reactions given by different species of a genus.
a d - Different reactions given by different strains of a species or serotype.
Source: Sneath et'al. (1986).
TTT.Ifl
-------
Salmonella indicated in Greenberg et al. (1992) uses an MPN (multiple-tube)
procedure that proportions homogenate into five tubes with three dilutions using
dulcitol selenite or tetrathionate broth for enrichment. Following incubation
for 24 h and 48 h at 35*C, plates of Brilliant Green and xylose lysine
desoxycholate agars are inoculated for isolation plating. Plates are incubated
for 24 h. Primary biochemical screening of Salmonella for identification and
serological verification is performed. The MPN/100 ml of original sewage sludge
sample is calculated from the negative and positive identification of Salmonella.
Additional quantitative methodology is presented in Appendix A, section C.
6. Regrowth
Monitoring of the Los Angeles County sanitation district's composting
operation has indicated that dewatered, anaerobically digested sewage sludge
contains an average of 10s Salmonella per gram of total solids (Russ and Yanko,
1981). Composting was considered to reduce the -Salmonella population to a level
below the detection limits of the MPN test system (<0.02 MPN per g of total
solids). However, regrowth of Salmonella was demonstrated with an increase of
more than 3 orders of magnitude within 5 days; the effect was transient, and
growth- returned to background, levels within 3 weeks.
Depopulation of sewage sludge by Salmonella may. occur as a result of
regrowth of organisms existing in the sewage sludge at an undetectable
concentration, through regrowth.of organisms that may have survived treatment
(e.g., failure to obtain a lethal time and temperature regimen), or through the
growth of organisms introduced from an outside source. A possible outs.ide source
may be feces from Salmonella-infected birds or other animals (Surge et al.,
1986). Salmonella regrowth requires the appropriate moisture level (20% or
greater), temperature (20-40'C), and nutrient content [carbon-nitrogen (C/N)
ratio in excess of 15:1} and growth despite competing coliforms (Russ and Yanko,
1981; Jaeger and Ward, 1981; Burge et al., 1986). Hussong (1985) noted that
moist compost would support regrowth for approximately 6 weeks. When the C/N
ratio was less than 15:1, repopulation did not occur. The C/N ratio is
considered to be the long-term nutritional indicator of Salmonella repopulation
111-19
-------
potential (Russ and Yanko, 1981). Studies have also indicated that Salmonella
can regrow extensively only if the sewage sludge has been sterilized, indicating
that the presence of other microflora (primarily bacteria) in sewage sludge that
is not sterilized prevent Salmonella regrowth (Yanko, 1988; Yeager and Ward,
1981; Hussong et al., 1985).
Both aerobic and anaerobic incubation conditions showed equal growth
potential; however, the aerobic system was more efficient in C/N reduction and
Salmonella die-off. It was suggested that Salmonella repopulation can be
minimized or eliminated by using volatile solids content and C/N ratios as
determining factors for assessing adequacy of treatment (Russ and Yanko, 1981).
The quantisation of Salmonella in sewage sludge using the MPN assay at
specific intervals should indicate regrowth (Hussong et al., 1985). Considering
the time needed to complete the various steps of the assay (enrichment in
buffered peptone broth, selective enrichment in tetrathionate broth with added
Brilliant Green, selective differentiation on XLB6 agar, presumptive screening
on triple sugar iron agar, and confirmation with slide agglutination), it would
be most expedient if sampling were to occur as close as possible to the time of
use or disposal of the sewage sludge while allowing 6 to 9 days for completion
of test results. Salmonella has been found to persist in sludge-amended soil for
up to 5 months, but' a 90% reduction may occur within 3 weeks (Yanko, 1988).
Ward et al. (198.4) Indicated that regrowth of Salmonella is not a problem once
sewage sludge is applied to the soil.
The Part 503 regulation requires the analysis of Salmonella in sewage
sludge using the quantitative method of Kenner and Clark (1974) or the multiple
tube enrichment technique of'Greenberg et al. (1992) (U.S.-EPA, 1993). These
methods are described in Appendix A, section C.
E. VIRUSES (INCLUDING ENTERIC VIRUSES)
Viruses found in digested sewage sludges and dewatered sewage sludges
originate in the gastrointestinal tract of humans and are excreted with the feces
of infected individuals. Because viruses multiply only in living cells, their
111-20
-------
numbers cannot increase in sewage sludge and, in fact, will decrease in varying
degrees during wastewater treatment processes and subsequent treatment of sewage
sludges. Nevertheless, viruses do survive sewage sludge treatment processes and
may be found in the processed sewage sludges, although in somewhat reduced
numbers.
Viruses known to be excreted in fairly large numbers in feces include
enteroviruses, such as polioviruses, coxsackieviruses, and echoviruses, as well
as hepatitis A virus, adenoviruses, reoviruses, rotaviruses, and Norwalk-type
viruses. Of these,' only some of the enteroviruses and reoviruses are readily
cultivated in cell culture by skilled technicians and trained specialists.
Hepatitis A, rotaviruses, and enteric adenoviruses are cultivated only with
difficulty, and Norwalk-type viruses have not been grown and are detectable only
by immunoassay, immunochemical, or molecular methods.
It is. very important to assay sewage sludges for enteric viruses before use
or disposal because sewage sludge can contain potentially large numbers of
enteric viruses that can survive in varying numbers in treated sewage sludges and
persist in soil for long periods. Unlike bacteria, regrowth of viruses is not
a problem following sewage sludge treatment because enteric viruses are unable
to multiply in the absence of susceptible host cells.
ANALYTICAL TECHNIQUES
Methods used for detection and quantification of viruses in sewage sludge
and other environmental samples involve three main steps: sample collection,
elution/concentratlon, and assay/identification. A disinfection step, is also.
employed to eliminate interfering microbial contaminants. A detoxification step
also may be necessary for some concentrated eluates where metals also have'been
concentrated to toxic levels. The various steps are indicated in Figure III-3.
1. Sample Collection and Storage of Samples for Viral Analysis
Because sewage sludge has a high sol Ids content as compared with water and
wastewater, filtration and adsorption on pads are not satisfactory methods of
w , t
111-21 -
-------
SEWAGE SLUDGE SAMPLE
ELUTION OF VIRUSES
ASTM-2
(SONICAT1ON METHOD)
ASTM-1
(ADSORPTION METHOD)
CONCENTRATION OF VIRUSES
CONCENTRATION OF VIRUSES
DECONTAMINATION/DETOXIFICATION
DECONTAMINATION/DETOXIFICATION
VIRAL ASSAY
VIRAL ASSAY >
Figure III-3. Scheme for the elution, concentration, decontamination/
detoxification and assay of viruses in.sewage sludge.
Source: ASTM Designation: D 4994-89.' "Standard Practice for Recovery of
Viruses from Wastewater Sludges."* 1992 Annual Book of ASTM
Standards: Section 11.
-------
sample collection. Most sewage sludge samples are obtained as grab samples taken
directly from the digestor (watered sludge) or, more likely, taken directly from
the centrifugal drier or from the belt drier as dewatered sludge. Regardless of
sampling site, all samples should be taken from at least three sampling points
to ensure a representative sample. Samples should be collected aseptically in
closed sterile containers to prevent drying en route to the laboratory. Most
POTVIs polled collected composite samples for viral analysis either as a 24-hour
composite or as a composite sample taken from multiple sites.
Temperature is the single most important factor in virus survival during
transport and storage. Samples should be placed on cracked ice immediately after
collection and transported to the laboratory as soon as possible. If samples
cannot be processed immediately, they may be held in a closed sterile container
for several days at 4*C, although it is best to process samples within 24 hours
whenever possible. If storage must be prolonged, samples should be held at
temperatures between -20 and -100'C (Farrah, 1982). Samples that must be shipped
over long distances to reach the laboratory should be frozen at -70*C and shipped
in dry ice, then either processed as soon as possible after receipt or held
frozen without thawing until processed.
2. Elution/Concentration
Almost all the viral particles, in sewage sludge samples are adsorbed to
sewage sludge solids rather than associated with the aqueous phase. For this
reason, solid-bound viruses must be dissociated/eluted to permit their assay
and/or identification. Once they have been partitioned into an aqueous phase and
separated from the solids, the resulting sample can be concentrated.prior to
viral assay. The* concentration, step is^ necessary because the numbers of
plaque-forming units (PFUs) of.viruses are often too few for direct viral assay
of the eluates. Because of the virus-sludge sol Ids association, the methods
commonly used to elute samples from high-volume filters or to concentrate viruses
from water with low solids content, such as adsorption-elution
methods/microporous filters, hydroxyextract1on-d1alys1s with polyethylene
glycol, reverse osmosis, ultracentrifugatIon/continuous flow ultracentrffugation,
electrophoresis, electroosmosis, and freeze concentration (Berg et al., 1967;
111-23
-------
Bitton, 1980), are of no value during the elution step. Variations of some of
these methods are useful in the concentration step after elution by other means
such as blender homogenization, mixing by mechanical or magnetic stirring, and
sonication (Bitton, 1980; Harding et a1., 1981).
Harding et al. (1981) compared three elution methods. Equal volumes of
liquid sewage sludge and suspending medium were combined prior to sample
processing for elution in all three methods:
1. Blender homogenization for 3 min.
2. Magnetic stirring for 30-60 sec.
3. Sonication at 60 W output for 5 min.
Viral separation from solids determines recovery of Viruses. Prior to
dispersion, the sewage sludge sample was divided into three equal volumes and
mixed with the given eluant (Table III-6). The results Jndicate that high
recovery of viruses was seen using sonication and 3% beef extract as the eluant
medium, whereas blender homogenization produced high recovery when distilled
water was the eluant. Magnetic mixing did not provide sufficient dispersion of
solids, particularly in the case of*primary sewage sludge samples using beef
extract as the eluant. Viral recovery with glycine buffer was poor except when
blender homogenization was used. High pH glycine buffers are not usefuLbecause.
a large amount of organic matter is released from sewage sJudge under these
conditions. Also, Indigenous viruses were unstable when sonicated at high pH.
»
Glass et al. (1978, as cited in Yahko, 1988). detected small amounts of
poliovirus in anaerobic digester sewage sludge. The method detected one
infective unit 1n the final concentrated sample. They indicated that the limit
of detection depends on the amount of solids processed; for example, a detection
limit of 0.10 per gram of digester solids is obtained using 30 g (dry weight) of
solids eluted with 600 ml of 3% beef extract. 'In their study, they used
dewatered and partially composted digester solids artificially contaminated with
poliovirus type 1. Approximately 104 PFU/mL were added to the water and assayed
for the original virus dose; this step was followed by the addition of solids.
111-24
-------
Table 111-6. Viral Recovery From Sewage Sludge As a Function of Processing Technique
Eluting Medium .
Distilled water
Glycine buffer,
pH 11°
Beef extract, pH 9*
Sewage
Sludge Type
Primary
Digested
Digested
Primary
Digested
Digested
Primary
Primary
Digested
Magnetic
(30-60
pfu/g TSS*
5.4
8.2
6.6
28
1.0
10.2
2.8
0.5
2.6
Mixing
sec)
% Max.5
51
17
29
67
37
100
9
6
2
96
Blender
Homoqenization
pfu/g TSS* %
10.5
47
23
42
2.7
2.6
35
13
0.5
(3 min)
Max.b
100
100
100
100
100
26
73
57
20
Sonication
(60 W - 5 mini
pfu/g TSS* %
8.2
31
6.2
40
ND
ND
48
23
2.7
Max
78
66
27
95
ND
ND
100
100
100
a pfu/g Total Suspended Solids (TSS) assayed on HeLa cell mondayers.
"% Max. = Percent of maximum recovery.
CA1I samples concentrated with bentonite.
dAII samples concentrated by organic flocculation.
ND = Not done.
Source: Harding et al. (1981).
IM-25
-------
The resulting slurry was homogenized and stirred to allow adsorption of virus to
the solids. The homogenate was centrifuged, the supernatant was assayed for
virus that did not adsorb to the solids, and the pellet was suspended in 3% beef
extract, homogenized, and subjected to one of three elution methods. Samples of
original dose, unadsorbed viruses, and eluted viruses were assayed by the plaque
method on monolayers of monkey kidney (BGMK) cells. The results showed 95-99%
of dosed viruses adsorbed to the solids. The best elution method was that
conducted at neutral pH using disruption by sonic treatment. Enteric viruses are
present at very low concentration in digester sewage sludge; to concentrate these
viruses, the authors used organic flocculation methods. This method, however,
was toxic to mammalian cell monolayers. The toxlcity was related to high levels
of heavy metals such as chromium, mercury, ztnc, nickel, and lead. To alleviate
this problem, the concentrated eluates were treated with dith-izone in chloroform
and directly inoculated onto the BGMK monolayers and HEp-2 cells. It was found
that dithizone extraction rendered'the formerly, cytotoxic concentrates nontoxic
for mammalian cells. The authors reported that this method 1s a reliable and
sensitive test not only for poliovlrus but also for coxsackievlruses and
echoviruses; reoviruses were more resistant.
The following five methods of recovering indigenous viruses from raw sewage
sludge were compared by Brashear and Ward (1982).
Viral recoveries from sewage sludge for all methods were measured by
the plaque assay using one of the following cell lines: BGM (African
green monkey kidney), RO {human rhabdomyosarcoma), or MA-.104 (rhesus
monkey kidney) cells. Samples were thawed and sonicated for 15 sec
using a 100 U sonic probe, and appropriate sample dilutions were
made Into-nutrient broth and layered onto cells in culture flasks
(5 replicates/dilution). Following viral adsorption, each flask was
a
overlain with nutrient agar, incubated at 37*C, and, for 2 weeks,
checked dally for plaque formation.
1. Raw sewage sludge, 100 ml, was mixed with 0.05M A1C13; the pH was
adjusted to 3-3.5 with 5N HC1, and the mixture was stirred for 30
min. The mixture was centrifuged at 2,500xg for 10 m1n,. and the
111-26
-------
pellet was suspended in 10% buffered beef extract. Foil-owing
centrifugation at lO.OOOxg for 30 min, the supernatant was filtered
through a series of membrane filters of decreasing porosity (5-0.45
^m). The filtrate was divided into two fractions; one was stored
at -80'C, and the second was diluted with water to 3% beef extract
and concentrated by flocculation and stored at -80*C. All steps of
this method were conducted at room temperature.
Raw sewage sludge, 200 mL, was blended for 2 min at high speed with
beef extract (4.8 g) and then treated with antifoam agent 10. The
sample was then shaken and sonicated at 100 U for 2 min in an ice
bath and centrifuged at 10,000xg for 30 min. The supernatant was
recovered, and the pH was adjusted to 3.5 with 6N HC1. The
supernatant was mixed for 30 .min and centrifuged again as above.
The supernatant was discarded, • and the pellet was dissolved in
sodium phosphate and detoxified. Detoxification involved mixing
equal volumes of the virus sample with chloroform solution
containing 10 nq dithizone per ml, and centrifugation for 15 min at
40,000xg. The upper aqueous layer was mixed with 0.1% CaCl2 and
aerated for 10 min to remove the excess chloroform. The final
sample was treated with antibiotics and stored at -80*C. All steps
•
were performed at room temperature.
A 100-ml sewage sludge sample was mixed with an equal volume of
Freon* and blended at high speed (not specified) for 3 min; the
mixture temperature was kept below 10'C. The blended mixture was
centrifuged at 600xg for 15 mi-n, the upper layer was.removed, and
the lower layer and the interface were reextracted with an equal
volume of phosphate-buffered saline. The upper layer of the second
extraction was combined with that from the first extraction and
divided into two portions for concentration. Viruses in one
fraction were concentrated by centrifugation at 140,000xg for 2
hours followed by suspension in balanced salt solution. Viruses in
the second fraction were concentrated by flocculation with 20%. beef
• ,
extract to give a final concentration of 3%; the- pH Was adjusted to
111-27
-------
3.5 with 5N HC1. The flocculated material was centrifuged at lOOOxg
for 3 min and dissolved in sodium phosphate. Both concentrated
fractions were disinfected with ether, and samples were stored at
-80*C until analysis.
4. Raw sewage sludge, 100 ml, was blended with an equal volume of 20%
beef extract at high speed for 3 min and centrifuged. The
supernatant was divided into two fractions and concentrated with
either centrifugation or flocculation with beef extract to a final
concentration of 3%. The samples were disinfected with ether and
stored at -80*C to await analysis.
5. Raw sewage sludge, 100 ml, was blended at high speed for 3 min with
an e°qual volume of water and centrifuged at 2500xg for 15 min, and
the supernatant was divided Into two fractions. The fractions were
concentrated either by high-speed centrifugation or flocculation
with beef extract as Indicated in method 1, above. The fractions
were then disinfected with ether and stored at -80*C to await
analysis.
The last three methods differ from each other in the type of eluant us.ed:
water, 10% beef extract, or Freon. All five methods were tested with raw sewage
sludge obtained from a POTVMn Ohio, and all contained 4% solids by weight and
a large number of enteric viruses. Each method Included the following steps:
elution, concentration, and disinfection (descriptions of methods 1 and 2 are,
found in Appendix A,1 section D). The highest viral recovery was found 1n the
Freon elution method using high-speed centrifugation and BGM cells for culture
(Table III-7). Using flocculation as the concentration method, viral recovery
by the Freon method declined to an average of only 61% for the three cell lines
(Table 111-7). The Freon method has the advantage of detoxification;
cytotoxicity was not observed in any sample treated with Freon. However, because
111-28
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Table 111-7. Relative Recoveries of Indigenous Viruses From Raw Sewage Sludge by Different Extraction Methods
1.
2.
3.
4.
5.
Elution/Concentration Method
AJO3 and beef
extract/unconcentrated
A1Q3 and beef
extract/ftoccuiation
Beef extract, blending, and
sonication/flocculation
Freon/centrifugation
Freon/flocculation
Beef extract/centrifugation
Beef .extract/ftoccuiation
Water/centrifugatlon
Water/ffocculation
BGM
0.55
0.42
0.34 •
1.00
0.73
0.41
0.34
0.58
0.09
Relative recover/* on cell line:
RD
0.52
0.34
0.32
1.00
0.61
0.48
0.35
0.67
0.11
MA- 104
0.63
0.44
0.15
1.00
0.49
0.87
0.48
0.94
0.05
'Relative to recoveries obtained by the Freon/centrifugation technique.
Source: Brashear and Ward (1982).
IM-29
-------
of the environmental consequences of Freon use, this method is not considered
further and is not described in detail in Appendix A. Table III-7 presents viral
recoveries using each of the three cell lines. Highest plaguing efficiency is
found in the BGM cells (Table III-8).
Goyal et al. (1984) found that the EPA/low pH-aluminum chloride method
[method 4 of Brashear and Ward (1982)] is slightly more efficient in viral
recoveries than the Glass method [method 5 of Brashear and Ward (1982)] using
various types of sewage sludge, except the dewatered sewage sludge. Of the total
102 samples, 69 had higher viral recoveries for the EPA method than those assayed
by the Glass method. However, the precisions for both methods were not
significantly different. Both methods are recommended as tentative ASTM standard
techniques for viral detection in sludge. Table III-9 presents the types of
sewage sludges used in the two methods, and Table III-10 compares the advantages
and disadvantages of both methods. The sewage sludge samples, used in the EPA/low
pH-aluminum chloride method were tested at 4*C as compared with the method of
Brashear and Ward (1982), which was conducted at room temperature.
Additional methods for recovery of viruses from suspended solids in water
and wastewater are given in Section 9510F of Greenberg et al. (199.2). Methods
for concentration of viruses following elution from sewage sludge solids are also
given in Sections 9510B, 0, and E.
v
3. Assay Identification
»
The plaque assay 1s the common method used to detect and quantify virus
recoveries (detailed description 1s found in Appendix A, section D). Viruses are
isolated by Infecting cell monplayers overlain with a sem1-soT1d medium. .This
leads to gradual-and progressive destruction of cells forming plaques. When
viruses are Inoculated into cell monoUyers maintained under liquid medium, the
method is called the cytopatMc (cytopathogenlc) method or CP. This latter
method, although simple, 1s unlikley to detect more than, one type of virus in a
given inoculum. The plaque method, although more complex and less likely, to
detect as broad a range of viruses' as the CP method, allows precise
quantification of viruses.
111-30
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Table III-S. Relative Plaquing Efficiencies of Extracted Viruses on Different Cell LJnes
Relative plaguing efficiency on cell line:
El ution /Concentration Method
1.
2.
3.
4.
5.
AJC1, and beef
extract/unconcentrated
AiCij and beef extract/flocculation
Beef extract blending, and
sonication/flocculation
Freon/centrifugation
Freon/flocculatlon
Beef extract/centrifugation
Beef extract/flocculation
Water/centrifugatlon
Water/flocculation
BGM
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00.
RD
0.68
0.57
0.68
0.66
0.53
0.93 .
0.85
0.71
0.69
MA- 104
0.57
0.43
0.22
0.49
0.30
0.70
0.55
0.75
0.28
'\/aliu»« an» tfriA •n/araj-iA \Afal ra«viuariae nn oo<«h rait Una anH ara HatorminoH fnr oflrh matlwH ralath/A tn
recoveries obtained on BGM cells. The average relative recoveries on cell lines BGM, RD, and MA-104
were 1.00, 0.70, and 0.48, respectively.
Source: Brasher and Ward (1982).
111-31
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Table 111-9. Types of Sewage Sludges Used to Evaluate the EPA and Glass Methods
and Their Physicochemical Characteristics
Sewage Sludge Type
pH
Alkalinity
(mg/L)
as CA CO;
Suspended Solids
fg/U
Total
Volatile Source
1. Anaerobic, high rate digested 7.4 5,200 31.4 14.6
(mesophUlc, 29to3ff)
2. Anaerobic standard rate 7.0 3,630 39.4 22.7
digested (mesophflic, 29 to 3ff)
3. Aerobic, digested (17) 5.7 174 24.5 16.5
4. Primary, undigested 5.1 1,535 41.2 31.0
5. Anaerobic, digested 6.9 900 116.0 76.0
Dayton, OH
Clbolo, TX
Gainesville, PL
Las Cruces, NM
Houston, TX
Source: GoyaJ et al. (1984).
111-32
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Table 111-10. Comparisons of EPA and Glass Methods for Virus Recovery From Sewage Sludge
Procedure
Advantages
Disadvantages
EPA
No special or unusual equipment is required. Final sample volume is too large
(ca. 50 to 100 mL).
Easier to process several samples
simultaneously.
Problem with bacterial and fungal
contamination.
FWratton step is difficult and time
consuming to perform, particularly
with primary sewage sludge.
Most investigators found the primary
method cumbersome.
Glass
Final concentrate volume to be assayed for
virus Is small.
The method Is relatively simple with single
samples.
No problem with bacterial or fungal
contamination.
Needs sophisticated equipment such as
sonteator.
Resuspension of organic floe proves
difficult sometimes.
Excessive foaming occurs with
aerobically digested sewage sludge.
The anaerobic sewage sludges do not
pack well after sonication and
centrifugatton.
It is difficult to obtain the top aqueous
layer after dithizone treatment.
Source: Goya! et al. (1964).
111-33
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A modified plaque assay was used to analyze 10 primary sewage sludge
samples of two wastewater treatment plants in Cincinnati, Ohio, to detect
enterovirus (Williams and Hurst, 1988). Continuous African green monkey kidney
cell line monolayers were incubated with 5-iodo-2'-deoxyuridine (IDU) (50 jig/ml)
for 4 days prior to use. This modification enhanced the recovery of viruses by
up to 160% from those assays without IDU. This enhancement was found for sewage
sludge samples from both wastewater treatment plants despite the
characteristically different wastewater sources. The degree of enhancement
varied from sample to sample, and reflected variability in the cell cultures
used. The disadvantage of using the IDU-treated cells was its higher sensitivity
to cytotoxic agents present in the sewage sludge concentrates. Using
immunofluorescent assay, adenoviruses were also detected in these sewage sludge
samples. The results also showed that adenoviruses are more abundant than
enteroviruses (detected by the IDU-treated or untreated BGM plaque method).
An immunofluorescent method was used to detect human rotavirus, and the
plaque assay was used to detect simian SA11 rotavirus in the study by Smith and
Gerba (1982) reported in the elution/concentration section mentioned earlier.
No reaction was noted .for reoviruses or other common enteroviruses. To further
characterize the sewage sludge isolates, a neutralization test was used.
Domestic sewage sludge Isolates were neutralized by a convalescent serum from a
human adult and Infant who had been infected by rotavirus; however, no
significant neutralization was noted by the preillness/preimmune serum
(Table III-ll). Neutralization also occurred by a hyper immune serum prepared in
guinea pigs against purified human rotavirus. The eluted viruses in the sewage
sludge isolates were Identified to be of a human strain rotavirus.
The nucleic acid hybridization assay is a recently developed method
operationally similar to immunofluorescence and enzyme immunoassay (Craun et al.,
1990; Margolin, 1992). The main difference is that the hybridization assay uses
nucleic acid strands instead of antibodies as probes. The nucleotide sequence
of the probe nucleic acid strands is complementary to the sequence of the
targeted progenyviral nucleic acid material. After the addition of a nucTelc
acid probe solution to .the virally infected*cells, the probe and target viral
111-34
-------
TABLE III 11. Serum Neutralization of Rotaviruses
Rotavinis
SA11
Bovine
Pordne
Human
Sewage Isolate A
Sewage Isolate B
Gradient purified-
sewage Isolate
*ExDressed as number of I
Normal
guinea
pig serum
(1:200)b
106
116
132
3° .
24
18
56
Guinea pig
anttsbnian
rotavbus
(1:10.000)
0
102
119
38
26
V
16
48
Guinea pig
antibovina
rotavirus
(1:1.000)
*,
80
0
NT
30
• 28
NT
NT
Porcine
antlporclne
rotavirus
(1:10.000)
100
NT
0
24
20
NT
NT
Fluorescent Fod"
t
Guinea pig
anti-human
rotavirus
(1:10.000)
100
100
140
0
0
NT
NT
uorescent lod per 0.1 mL after treatment wtth the Indicated antiserum. Equal volumes of virus and
Human adult
Preiness
(1:200)
78
120
117
28
16
0
21
62
Convalescent
(1:200)
92
112
126
0
0
0
0
serum were Incubated tor 1 h at 37* C and
Human Infant.
convalescent
(1:500)
102
NT
NT
1
0
NT
0
then assayed by
11F In duplicate.
"Serum dilution.
NT • Not tested against the particular virus.
Source: Smith and Gerba (1962).
-------
nucleic acid will form "hybrid" DNA molecules. These bound DNA molecules can
then be visualized either by radiometric or colorimetric detection systems.
The advantages of the antibody-based and DNA-based assays are that they
require short periods of time for completion and are capable of detecting viruses
that are not cytopathogenic.
Additional methods and comments on assay and identification of viruses in
sample concentrates may be found in Section 9510G of Standard Methods for the
Examination of Hater and Uastewater, 18th edition (Greenberg et al., 1992).
The method required by the the Part 503 regulation (U.S. EPA, 1993) is ASTH
Method D 4994-89 "Standard Practice for Recovery of Viruses from Wastewater
Sludges." The ASTM-1 and ASTM-2 methods lis.ted in Figure III-3 and described in
Appendix A, section D, are specified as Procedure A--Adsorption, and
Procedure B--Sonication, respectively, in ASTM Method D 4994-89.
F. ASCARIS OVA
Ascaris ova (helminths) are reported to present a particularly severe
problem because of the ability of this organism to pass through sewage sludge
treatment processes and to persist in soils for several years (Burge et al.,
1980; Fox et al., 1981). In addition, they may appear to be destroyed by heat
within the composting temperature, or they may appear to be nqnviable after
passage-in sewage sludge through an anaerobic digester, but they may actually be
viable and embryonate when removed from the presence of sdwage sludge
(Fitzgerald and Ashley, 1977; Bunge et al., 1980; Fox et al. 1981). Ascaris are
considered to be the most resistant of all pathogens. There 1s also some
indication that some unknown factor present in anaerobically digested sewage
sludge provides a degree of protection for these organisms (Fox et al., 1981).
Heating and drying seem to be the greatest deterrents to long-time survival of
ova or parasites.
No standardized methods presently exist for the analysis of ascaris ova
(Clancy, 1992); however, the Zinc Sulfate Density .Gradient Separation method
111-36
-------
developed by Yanko (1988) is the required methodology for the identification of
ascaris ova by the EPA Part 503 regulation. Helminth methods have many complex
steps that depend on skilled analysts. Microorganisms can be lost at each step
in the process. Quality assurance/quality control is critical in these analyses
to ensure that reporting of negative results reflects the detection limit of the
test rather than the capability of the analyst to perform the procedure
adequately. In addition, of particular concern is the lack of a reliable
commercial source of ascaris ova to be used as a positive control in each assay.
Most laboratories are unable to locate live ascaris ova for use in assay
procedures; this problem needs to be addressed (Clancy, 1992).
The ascaris ova procedures documented by Yanko (1988) for dewatered sewage
sludge samples consist of a zinc sulfate density gradient separation followed by
an acid-alcohol/ether sedimentation. The steps of these procedures are
delineated in Appendix A, section E, of this document and are shown in
Figure III-4. Recovery experiments using these methods with seeded ascaris ova
were conducted with the range of recovery between 84 and 92% (average
88%). A hemocytometer was found to be adequate for counting cysts.
Fox et al. (1981) Indicated that flotation analysis procedures are
effective in isolation of ascaris ova from sewage sludge. o Many ova float in
high-density solutions with a specific gravity of 1.18 or greater. Some of the
•
more common compounds used for flotation are sucrose, sodium chloride, sodium
nitrate, sodium sal icy!ate, sodium or potassium dichromate, zinc sulfate, and
cesium chloride. Unc sulfate 1s the best solution known for floating the more
o . .
delicate cysts. The sucrose flotation methods of Fox et al. (1981) are specified
for watered and dewatered sewage sludge, anaerobically digested sewage sludge,
and raw sewage, and are delineated in Appendix A, section E. The procedures
provided rapid analysis of samples and dependable results, and proved to be
consistent between samples. The methods were slightly modified to compensate for
the type of sample examined. The salt flotation method was designed for counting
ascaris ova in composed sewage sludge. The zinc sulfate flotation method, also
incorporated by Yanko (1988), was specifically developed; for sewage sludge
analysis and incorporates a hyperchlorite and detergent that dissolve the sticky
outer coating of ascarrs ova.
111-37
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SEWAGE SLUDGE SAMPLE
HOMOGENIZE/FILTER
SETTLE (12 MRS.)
PELLET
ZINC SULFATE
ACID-ALCOHOL-ETHER
SOLUTION
SULFURICACID
(0.1% SOLUTION) *
INCUBATION
(3-4 WKS. AT 26°C)
MICROSCOPIC EXAMINATION
(# OVA/G DRY WEIGHT)
Figure III-4. Zinc sulfate density gradient separation method for
identification of ascaris ova .1n°sewage sludge.
Source: Yanjto (1988).
-------
Oliveri et al. (1989) reported the use of the zinc sulfate flotation method
for the concentration of ascaris ova from thick sewage sludge samples. This is
a modification of the Fox et al. (1981) methodology; this method differs only
slightly from the methods of Fox et al. (1981) and Yanko (1988). Specific
details of the method are described in Appendix A, section E, of this document.
Lugol's iodine stain has been used to stain ascaris eggs; the iodine is
able to penetrate the outer shell of the ascarids and provides good detail of the
nucleus (Fox et al., 1981). It is reported to be most efficient in staining
sewage sludge samples. A more detailed description.of the stain is reported in
Appendix A, section E, of this document.
A new method for the recovery and enumeration of ascarids in sewage sludge
is under development by ASTM committee 0*19.24 (Clancy, 1992). The method of
Yanko (1988) is used as the strawman. Method development, round-robin testing,
and ASTM methodology adoption are proceeding; no further information is available
at this time.
Yanko (1992) has indicated that because ascaris ova are hardier than
protozoa and easier to detect, the County Sanitation District of Los Angeles
County assumes that if ascarids have been eliminated from sample sewage sludge,
protozoa are also, considered to have been eliminated. Ascaris ova'are an example
of a parasitic ova with thick shells capable o.f resisting penetration of toxic
materials and resistant to temperature change or other adverse environmental
% •
changes. It is reasonable, -therefore, to assume .that factors influencing the
survival of these ova would also significantly affect the survival of less
resistant forms. The survival of the ascarid ova, therefore, may be considered
to be an indicator of pathogenic survival, direct microscopic examination would
provide for observation of movement as identification of viability.
The Part 503 regulation requires the analysis of ascan's ova in sewage
sludge using the method of Yanko, 1988. This method is described in Appendix A,
section E (Yanko, 1988; U.S. EPA, 19.93).
111-39
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IV. STATUS OF PUBLIC AND PRIVATE LABORATORIES
CAPABLE OF CONDUCTING ANALYSIS OF MICROORGANISMS IN SEWAGE SLUDGE
The information in the following sections is based on a report recently
prepared for Dynamac by Dr. Jennifer Clancy, the former director of a private
commercial laboratory with direct experience in working with POTWs in the
sampling and analysis of microbiologicals in sewage sludge. Dr. Clancy is also
a member of the American Society for Testing and Materials committee currently
involved in developing methodologies for analysis of microorganisms in
wastewater. The following issues are discussed:
Availability of laboratories qualified to analyze microorganisms in
sewage sludge
. Laboratory certification
Standardization of analytical methodologies
Quality assurance
Analytical costs
A. AVAILABILITY OF LABORATORIES
The number of qualified laboratories in the United States capable of
analyzing sewage sludge for the presence of microorganisms varies, depending on
the microbe being analyzed. Public and private laboratories in all States have
• * *
the capability to perform analyse.; of the common bacterial parameters, including
fecal conforms. Some*, but not all, of these labs are able to analyze for the
bacterial pathogen Salmonella.
Very few laboratories have the capability to analyze for enteric viruses
and helminth ova. These tests are much more complex and require specialized
equipment and training to be conducted properly. The majority of the
laboratories with these capabilities are university research laboratories. As
such, they have limited capacity-for accepting samples.on a commercial basis.
The turnaround time and reporting of results are often unacceptable for treatment
plants requiring these analyses for compliance.
IV-1
-------
A list of laboratories capable of conducting the standard bacteriological
tests can be obtained from the State agencies that certify laboratories for water
or wastewater testing. In some States, this list can be obtained from the agency
responsible for overseeing implementation of the 1987 Water Quality Act. Some
State health departments also will have a listing of these laboratories. A list
of State agencies is provided in Appendix B.
A list of laboratories capable of performing the more sophisticated
analyses is given in Appendix B. This list is not necessarily inclusive of all
laboratories. It represents those currently known by parties interviewed for
this report to have demonstrated expertise in these complex analyses, primarily
because they have been involved in the development of the current methodologies.
In 1991, Viar & Company performed, for the U.S.'Environmental Protection
Agency, a survey of 91 laboratories known to possess biological testing
capabilities. Of these, only 20 laboratories responded, with 7 indicating that
they could perform Salmonella testing. None of the seven was capable of
t
performing enterovlrus testing; one laboratory offered helminth ova testing.
These laboratories are listed in Appendix B.
Some State and Federal laboratories as well as some of the larger POTW
laboratories are capable of performing specific microbiological analyses.'
Generallyx they do not accept outside samples; however., discussions conducted
with POTW operators for this report Indicated that laboratories at two treatment
•
plants are performing analyses for other, smaller POTWs. One of these was able
to offer testing for bacteria and viruses; the other offered only fecal conform
analysis. (See Chapter V, section A)
•
*
B. LABORATORY CERTIFICATION
Laboratory certification for sewage sludge analysis for microorganisms
currently exists only for the bacterial parameters, total and fecal coliforms,
and fecal streptococci. Some States offer specific certification for wastewater;
others combine water and wastewater certification. Some States require no
specific certification for wastewater analyses. Certification for private
IV-2 -
-------
laboratories sometimes requires that analysts demonstrate a certain level of
competency (technical or university degrees in biology or credit hours in these
subjects).
Laboratories in wastewater treatment facilities in some States have few
requirements for laboratory personnel. In some cases, they perform routine
testing without any actual knowledge of the science behind the test procedures.
This lack of. knowledge can lead to problems in testing procedures and in their
interpretation. It is difficult to troubleshoot problems that can occur in
laboratory technique, reagent integrity, glassware processing, etc., when the
analyst does not understand the science behind the testing method.
For the more complex analyses (viruses, helminth ova), no certification
programs exist. Some States have virus monitoring guidelines, but none currently
requires certification. California certifies laboratories based on the ability
to demonstrate competence for each specific parameter. It is expected that this
will be required for the viruses and helminths.
A survey of eight States found the following regarding certification
requirements for wastewater analysis:
New Hampshire, Vermont, North Carolina, and Florida have
no certification requirements . for wastewater analysis ,for
microbiologicals.
New Jersey and New York require certification for microbiological
analysis of wastewater; Arizona also requires certification and has
guidelines for virus monitoring in wastewater.
California dropped its-program for wastewater certification in the
early 1980s, but began certifying laboratories again last year.
Not all laboratories that perform wastewater analysis have been
recertified yet. California certifies laboratories for each
parameter based on the analyst's demonstrated competence.
IV-3
-------
C. STANDARDIZATION OF ANALYTICAL METHODS
Approved, standardized methods have been developed for the recovery and
enumeration of bacterial parameters in sewage sludge. The methods of Clesceri
et al. (1989) and Greenberg et al. (1992) for the recovery of total and fecal
coliforms, fecal streptococci, and Salmonella are the generally accepted for
these bacterial parameters.
The section on Salmonella isolation and identification in Greenberg et al.
(1992) begins with the following disclaimer, which indicates the need for further
methods development and testing:
The methods presented below for the isolation of Salmonella from
water or wastewater are oot standardized. The procedures may need
modification to fit a particular set of circumstances and methods
comparisons are encouraged.
Several methods for concentration, enrichment, and selective growth are
described. Generally, a combination of several techniques should be employed
before a sample is declared negative for Salmonella because of its relatively low
numbers in comparison, to col 1 form bacteria.. Although methods for bacterial
recovery are generally well developed as compared with those for the viruses and
helminths, the Salmonella method(s) is one that needs considerable work before
a reliable standard can be developed.
The laboratories conducting enteric virus analyses are using a variety of
methods, including"the methods described ia Berg et al. (1984), other published
methods -(including Greenberg et al., 1992), and as yet unpublished methodologies
developed in their own or other laboratories. Some virologists have developed
variations on standard methods that they use to Improve recoveries of enteric
viruses.
A committee of the American Society for Testing and Materials (ASTM) is
currently developing several methodologies for analysis of microorganisms in
wastewater:
IV-4
-------
1. Standard Test Method for the Isolation and Enumeration of Fecal
Coliform Bacteria in Water by the Membrane Filter Procedure
Three methods are described: a single-step method and a two-step
method utilizing m-FC medium for the selection of fecal coliforms,
and a two-step method using m-TEC medium for fecal coliform
isolation. Type of treatment and percentage of solids or toxicants
will influence the specific method to be used.
2. Standard Practice for the Recovery of Enteroviruses from Waters
This method describes the recovery of low levels of enteric viruses
from waters and wastewaters using negatively charged cartridge
filters. The viruses are adsorbed to. the filter, eluted using beef
extract, and concentrated. The concentrates are assayed using the
USEPA Manual of Methods for Virology.
3. Recovery and Enumeration of Helminth Ova in Sewage Sludge
A task group has been formed to develop a new method based on the
method developed by William Yanko at the Los Angeles Sanitation
District (EPA* Publication No. 600/1 -87-Olf). .
Methods 1 and 2 are under development and will be published by ASTM when
they have been round-robin tested by a panel of laboratories as required by the
ASTM prior to designation as an ASTM method, these methods are all well along
in development and should be round-robin tested by J994.. The helminth ova task
group had not yet met at the time of this report, but the task group leader
• 3
expects method development-and round-robin testing to proceed.
a
The methods currently required under the Part 503 regulation include
analyses for fecal coliform, Salmonella, enteric viruses, and helminth ova..
These methods are detailed in Appendix A.
IV-5
-------
One problem that needs to be addressed in all of the methodologies is the
question of percent solids in the wastewater/sewage sludge sample. The percent
solids in a sample can indicate which method may be better suited to maximal
recovery of the specific microbiological parameter. As discussed in the
following section, Quality Assurance, the matrix composition can play a
significant role in microbiological recovery. (See also Chapter III.A, Dry Weight
Analysis.)
D. QUALITY ASSURANCE
Unlike chemical analyses, in which spike samples are a routine part of the
analysis and regularly scheduled instrument calibration ensures precision ami
accuracy, microbiological analyses are often less precise. This is due to the
nature of microbiological samples. The combination of microorganisms interacting
with the components of the matrix can have a variety of-effects. Microorganisms
can interact antagonistically, resulting in one species suppressing or
eliminating the others. The metabolic byproducts of one group may serve as
substrates for another group, leading to proliferation of one or more genera.
Matrix components can affect the microbiologicals. Particulates to which
microbes can adhere can confer protection from toxic compounds. The percent of
water available can affect the growth rate of certain microorganisms. The
chemical composition of-the matrix can affect recovery of microorganisms from the
matrix itself.
To ascertain whether, microbiological recovery methods are working, controls
are run with the analyses. The positive control 1s done.to show that the testing
media, incubation procedures, etc., are capable of allowing growth of the target
microorganism. The negative control indicates that none of the reagents has been
contaminated with the test organism, *h1ch could lead to false-positive results.
However, these control procedures can lack sensitivity. For example, a stock
Escherichia coll culture used as a positive control for a chlorinated effluent
sample may show good growth, wh1l<; the sample, which may contain Injured £*. coli
unable to withstand the stress of culture, shows no growth. Although £. coli
were present in the sample, a negative result based on the testing will be
reported.
IV-6
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Good laboratory practice as well as many State certification requirements
call for quality assurance/quality control (QA/QC) procedures. These criteria
usually involve instrument and equipment calibration, pH of reagents, dating of
materials for expiration, use of positive and negative controls with analyses,
and reporting methods. However, very few environmental laboratories practice
QA/QC procedures for the quantifiable recovery of microorganisms on a routine
basis. With relatively simple methods (bacteria), this is less of a problem than
with the more complex methods (viruses, helminths). The virus and helminth
methods have many complex steps that depend on skilled analysts. QA/QC is
critical in these analyses to ensure that reporting of negative results reflects
the detection limit of the test rather than the inability of the analyst to
perform the test procedure adequately.
E. ANALYTICAL COSTS
A telephone survey of the six laboratories that perform microbiological
analyses on sewage sludge resulted in the following price ranges for each
analysis:
Analysis Cost H992 $1
Total coliforms $25 to J60
Fecal conforms $2S. .to $150
Fecal streptococci
(Enterococci) $50 to $200
Salmonella $100 to $200
Helminth ova $250 to $500
Enteric viruses $300.to $700
The prices quoted by private, commercial laboratories were generally higher
than those quoted by university research laboratories. In some cases, university
laboratories have longer turnaround times and Inconsistent reporting procedures
because they are generally not set up to handle high volumes, of samples.
Semester breaks, examination periods, etc., can alter the production of data in
university laboratories whose primary goal Is generation of research data. The
• *
IV-7
-------
overhead expenses for university personnel are usually not included in the price
of the analyses because these personnel would be employed whether or not
additional outside sample analyses were performed. Also, university researchers
may be more willing, for no additional cost, to do additional work on a
particular sample as a learning exercise.
Private, commercial laboratories factor in all overhead expenses plus
profit when setting prices. Private laboratories are equipped to handle large
numbers of samples and to process them within a specified time frame. Reporting
procedures are usually standardized and tailored to each client's needs. The
laboratory director or senior scientists should be readily available to answer
questions about the report, e.g., data interpretation, regulatory significance,
future testing needs. The general availability of highly qualified personnel for
consultations is a plus when dealing with a private laboratory. Private
laboratories are in the business to serve their clients and generally have a much
better reputation for service than do university laboratories, whose primary role
is scientific research.3
IV-8
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V. NEEDS AND PROBLEMS OF POTW OPERATORS
Personnel at 10 publicly owned treatment works (POTWs) were contacted to
discuss the needs and problems they encounter in the testing of sewage sludge for
microorganisms. The flow rate of the treatment works, located in seven States
and the District of Columbia, ranged from 0.23 million gallons per day (MGD) to
350 MGD. Four of the treatment works were large, metropolitan sites with flow
rates ranging from 270 to 350 MGD; three treatment works had flow rates of 15 to
20 MGD; and the flow rates of the three smallest treatment works ranged from 0.23
to 2.5 MGD.
Comments also were solicited from the former director of a private
microbiological laboratory with direct experience working with POTW staff in the
sampling and analysis of sewage sludge for microbiologicals.
The problems and needs expressed in these discussions fell 'into the,
following four areas:
Capabilities of POTV/s to perform microbiological analyses
Need for outside/private laboratories qualified to perform analyses
Storage/transport to outside laboratories
Uniformity of methodologies used by different laboratories
A. CAPABILITIES OF POTWs TO PERFORM MICROBIOLOGICAL ANALYSES
Two individuals felt tfiat except for the major metropolitan treatment
*
works, most treatment works do not currently possess the capability to perform
microbiological testing other than for indicator organisms. Of the 10 POTWs
contacted for this report, 3 of the 4 largest are able to analyze sewage sludge
samples for Indicator organisms (e.g., fecal conforms), Salmonella, enteric
viruses, and helminth ova. .All of these tests are conducted in-house except for
helminth analysis, which is performed in-house by two treatment works and sent
to a private, outside laboratory by one POTW. The fourth large POTW performs
testing for fecal conform, fecal streptococci, enterococci, and salmonella, but
V-l
-------
not for viruses or helminths; the laboratory notes that its staff does not have
the expertise required to perform these tests, and that without sufficient
experience the results can be misinterpreted.
Of the remaining treatment works, three perform routine testing for fecal
coliforms, and one tests for this indicator as a process control check. Two
treatment works do not perform any testing for microbiologicals.
Two individuals commented on the need for proper training of POTW staff if
they are to perform testing for microorganisms other than indicator organisms.
One commenter noted that this training is usually not available to POTW staff
except at the larger metropolitan sites. Another emphasized the importance of
proper training for both the performance of testing procedures and the
i
interpretation of results, and added that lack of knowledge can also make it
difficult to troubleshoot problems of laboratory technique, reagent integrity,
and glassware processing.
B. NEED FOR OUTSIDE/PRIVATE LABORATORIES
9
Staff at 4 of the 10 POTWs contacted Indicated that they would contract
with outside laboratories if they were required to perform any testing other than
what they are currently conducting. Another three POTWs would consider using an
outside laboratory for any additional testing, but would weigh thfs against the
possibility .of establishing the-techniques ia-house. The latter option would
depend on factors such as cost, complexity of analysis, and availability of
training and methodologies. None of the POTVs contacted who might use an outside
laboratory knew of any that are currently qualified to perform pathogen analysis,
specificallyvirus and helminth analysis, on sewage sludge. However; two POTW
operators Indicated that private, commercial laboratories would be expected to
respond to testing needs that might arise.
Two of the.POTWs contacted are currently providing microbiological analyses
for other, smaller POTVs and Indicated the willingness to expand these services.
(See Chapter IV, section A, for a discussion'of the availability of laboratories
qualified to perform microbiological testing of sewage sludge.)
V-2
-------
C. STORAGE AND TRANSPORT TO OUTSIDE LABORATORIES
Discussion of the use of outside laboratories to perform microbiological
testing on sewage sludge led contacts at three POTWs to note their concern over
the short holding times (e.g., 6 hours) allowed for samples being analyzed for
certain microbiologicals. This time limit is impractical for transportation of
samples to outside laboratories where overnight express for analysis the next day
is the norm. (See also Chapter II, Sampling and Transportation, section B.)
D. UNIFORMITY OF METHODOLOGY
Uniformity of the methods being used for microbiological testing of sewage
sludge was a concern of three POTW staff contacted. They felt that disparity
*
exists among the methodologies being used as well as in the capabilities of
different laboratories to perform tests that require specialized skills and
experience, e.g., virus and helminth 'analyses. The need for standardized or
recognized methods was stated by two'POTWs and by the former director of a
private microbiological laboratory. One POTW contact related his experience of
calling several people, including laboratory and EPA staff, to obtain information
on performing the MPN calculations for fecal coJiform analysis. He received
differing answers from each person and was left with the impression that each was
"finding his own way" in performing the analysis.
Other problems specific to methodology included testing for parasites. Two
POTW staff commented on the difficulty of analyzing for helminth ova, and the
problem of determining ova viability was specifically raised by one POTW. The
need for a reliable source of live helminth*ova for positive controls was also
noted .•
Another specific problem with methodology concerned the issue of choosing
methods appropriate for the solids content of the sewage sludge being tested.
One comment indicated that the percent solids in a sample can determine which
method may be better suited to maximal recovery of the specific microbiological
parameter. As an example, most of the POTWs contacted who test for fecal
coliform use. the multiple-tube, or MPN, method. Two POTWs, however, were using
V-3 •
-------
the membrane filter technique for fecal coliform analysis of sewage sludge
samples. The contact at one of these indicated they ran the test on sewage
sludge (with solids content of 40 to 50%) at high dilutions, and that they were
not set up to perform the multiple tube method.
V-4
-------
VI. SUMMARY AND RECOMMENDATIONS
Routine examination of sewage sludge for pathogens is limited by factors
such as lack of qualified laboratories, untrained personnel, inadequate
standardization of methodology, insufficient time for completion of analyses,
and high costs. In general, the methodology for microbiological analyses is
somewhat developed for water and wastewater; the methodology for dewatered sewage
sludge and compost is deficient. Survey results indicate a need for intensive
research in this area.
Many methods are currently under development and require round-robin
testing by a panel of laboratories to meet the requirements of ASTM methodology.
These methods should be available for testing during 1993 and 1994 and will at
that time be designated ASTM methods. The following specific deficiencies have
been ident'ified:
1. Quantitative methodology should be developed for fecal coliform
bacteria in turbid water such as wastewater or sewage sludge.
2. Methodology should be developed for detection of noncultivatable and
stressed bacteria.
3. The cell culture techniques for many adenoviruses and rotoviruses
require development. Viral elution/concentration/assay techniques
must be standardized for enteroviruses in sewage sludge.
4.- ' Methodology. should be developed to determine the°viability of
helminth ova.
5. While many laboratories are available to analyze sewage sludge for
the common bacterial indicators, only a few laboratories have the
capability to analyze for enteric viruses or helminths.
6. Viable positive controls (e.g., helminth ova) may be difficult to
obtain for testing .laboratories.
VI-1
-------
Standard methods for the recovery of bacteria exist, and newer methods are
under development and should be available soon. Methods exist for the recovery
of enteric viruses, but no standard to be used by all laboratories has yet been
determined. No standard methods exist for recovery of helminth ova.
Certification programs exist in only some States for laboratories and treatment
works that analyze wastewater for microorganisms; some States require no special
certification. Certification can range from very lax (filling out forms only),
to moderate (laboratory inspections, educational requirements for personnel), to
strict'criteria (require demonstration of ability to perform specific analyses).
Methods for the microbiological analysis of sewage sludge required under
the Part 503 regulation have been documented in this report. To provide a
comprehensive overview, other commonly used methods have also been included.
Examination and comparison of these methods should continue. Methods should be
round-robin tested to determine precision and bias. QA/QC procedures and
detection limits should be Included in each methodology. Standard methods should
be published and made readily available to laboratories. Public and private
laboratories that wish to offer testing should be certified by demonstrating
competence in each methodology. The USEPA should offer training in each of these
methodologies through workshops to ensure that laboratory personnel are properly
trained. -Attempts'must-be made to curtail the testing service offered and
conducted by*unqualified laboratories.
VI-2
-------
VII. REFERENCES
ASTM. 1992. Standard Practice for Recovery of Viruses from Wastewater Sludges.
1992 Annual Book of ASTM Standards: Section 11 - Water and Environmental
Technology. ASTM Designation: D4994-89. Philadelphia, PA.
Berg, G., R.S. Safferman, D.R. Dahling, D. Berman, and C.J. Hurst. 1984. U.S.
EPA Manual of Methods for Virology. Environmental Monitoring and Support
Laboratory. U.S. EPA Office of Research and Development, Cincinnati, Ohio. EPA
Pub. No. EPA-6001 4-84-013.
Berman, D., G. Berg, and R.S. Safferman. 1981. A method for recovering viruses
from sludges. J. Virol. Meth. 3:283-291.
Bordner, R.H. 1985. Quality assurance for microbiological analyses of water.
In Taylor, J.K. and T.W. Stanley (eds.) Quality Assurance for Environmental
Measurements. A symposium sponsored by ASTM Committee D-19 on Sampling and
Analysis of .Atmospheres, Boulder, Colorado, pp. 133-143.
Bordner, R. and J. Winter (eds.) 1978. Microbiological Methods for Monitoring
the Environment: Water and Wastes. U.S. EPA, Office of Research and
Development, Washington, DC. EPA Pub. No. EPA/600/8-78/017.
Brashear, D.A. and R.L. Ward. 1982. Comparison of methods for recovering
indigenous viruses from raw wastewater sludge. Appl. Environ. Hicrobiol.
43:1413-1418.
Buras, N. 1975. Concentration of enteric viruses in wastewater and effluent:
a two year survey. Hater Res. 10:295-298.
Burge, W.D., P.O. Mi liner, N.K. Enkiri, and 0. Hifcsong. 1986. Regrowth of
Salmonellae in Composted Sewage. Sludge. Prepared for EPA by U.S. Agricultural
Research Service, Beltsville, Maryland. Water Engineering Research Laboratory,
U.S. EPA Office of Research and Development. Cincinnati, Ohio. .EPA Pub. No.
EPA/600/2-86/106.-
Clancy, J.L. 1992, The Status of Public and Private Laboratories Capable of
Conducting Analyses of Microorganisms in Sewage Sludge. Report to Dynamac
Corporation.
Clescerl, L.S., A.E. Greenberg,.and R.R. Trussell (eds.) 1989. Standard Methods
for the Examination of Water and Wastewater, 17th Ed. American Public Health
Association,. Washington, DC.
Craun, G.F- (ed.) 1990. Methods for the Investigation and Prevention of
Waterborne Disease Outbreaks. U.S. EPA, Office of Research and Development,
Washington, DC. EPA Pub. No. EPA/600/l-90/005a.
Dahling, D.R. and B.A. Wright. .1984. Processing and transport of environmental
virus samples. Appl. Environ. Hicrobiol. 47:1272-1276.
VIM
-------
Farrah, S.R. 1982. Isolation of viruses associated with sludge particles.
In C.P. Gerba and S.M. Goyol (eds.) Methods in Environmental Virology. Marcel
Dekker, Inc., New York, NY.
Fitzgerald, P.R. and B.F. Ashley. 1977. Differential survival of Ascan's ova
in wastewater sludge. J. HPCF', July:1722-1724
Fox, J.C., P.R. Fitzgerald, and C. Lee-Hing. 1981. Sewage Organisms: A Color
Atlas. Metropolitan Sanitary District of Greater Chicago, Chicago.
Glass, J.S., R.J. Van Sluis, and W.A. Yanko. 1978. Practical method for
detecting poliovirus in anaerobic digester sludge. Appl. Environ. Microbiol.
35:983-985.
Goyal, S.M., S.A. Schaub, F.M. Wellings, D. Berman, J.S. Glass, C.J. Hurst, D.A.
Brashear, C.A. Sorber, B.E. Moore, G. Bitton, P.H. Gibbs, and S.R. Farrah. 1984.
Round robin investigation of methods for recovering human enteric viruses from
sludge. Appl. Environ. Microbiol. 48:531-538.
Greenberg, A.E., A.D., Eaton, and L.S. Clesceri (eds). 1991. Standard Methods
for the Examination of Water and Wastewater. Supplement to the 17th Ed.
American Public Health Association, Washington, DC.
Greenberg, A.E., L.S. Clesceri, and A.D. Eaton (eds). 1592. Standard Methods for
the Examination of Water and Wastewater. 18th Ed. American Public Health
Association, Washington, DC.
Greenberg, A.E., J.S. Thomas, T.W. Lee, and W.R. Gaffey. 1967. Interlaboratory
comparisons in water bacteriology. AW A 59:237-244.
• . %
Harding, H.J., R.E. Thomas, D.E. Johnson, and C.A. Sorber. 1981. Aerosols
Generated by Liquid Sludge Application to Land. U.S. EPA, Office of Research and
Development, Health Effects Research Laboratory, Cincinnati, Ohio.
* f <»
Hurst, J.H., and T. Goyke. 1986. Improved method for recovery of entire viruses
from wastewater s-ludge. Hater Res. 209:1321-1324
*
Hussong, D., N.K. Enkiri, and W.O. Burge. 1984. Modified agar medium for
detecting environmental Salmon'ellae by the most-probable-number method. Appl.
Environ. Hicrobiol. 48(5):1026-1030.
Hussong, 0., W.O. Burge, and N.K. Enkiri. 1985. Occurrence, growth, and
suppression of Salmonellae 1n composted sewage sludge. Appl. Environ. Microbiol.
50(4):887-893.
Jorgensen, P.H. and E. Lund. 1986. Transport of viruses from sludge application
sites. Comm. Eur. Communities, Process Use Organic Sludge Liquid Agricultural
Wastes pp. 215-224. (no vol. no. only "August" Issue).
Kenner, B.A. and H.P. Clark. 1974. Detection and enumeration of Salmonella and
Pseudomonas aeruglnosa. J. HPCF 46(9).-2163-2171.
VII-2
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Ma, G. 1992. Personal Communication. Municipality of Metropolitan Seattle,
Seattle, Washington.
Meckes, M. 1992. Personal Communication. U.S. EPA. Cincinnati, Ohio.
Munger, S. 1992. Personal Communication. Municipality of Metropolitan
Seattle, Seattle, Washington.
Olivier'i, V.P., M. Sakai, S.L. Sykora, and P. Gavaghan. 1989. Selected
Indicator and Pathogen Microorganisms Removal During Conventional Sludge
Treatment Processes. U.S. EPA Office of Research and Development, Risk Reduction
Engineering Laboratory, Cincinnati, Ohio. EPA Project No. CR B135 98.
Reimers, R.S., M.D. Little, T.G. Akers, W.D., Henriques, R.C. Badeaux, O.B.
McDonnell, and K.K. Mbela. 1990. Project Summary: Persistence of Pathogens in
Lagoon-Stored Sludge. U.S. EPA, Risk Reduction Engineering Laboratory,
Cincinnati, OH: EPA Publication No. EPA/600/S2-89/015.
Russ, C.F. and W.A. Yanko. 1981. Factors affecting Salmonellae reproduction in
composted sludges. Appl. Environ. Hicrobiol. 41(3):597-602.
Slanetz, L.W. and C.H. Bartley. 1957. Numbers of enterococci in water, sewage,
and feces determined by the membrane filter technique with an improved medium.
J. Bacterial. 74:591-595.
Smith, E.M. and Gerba, C.P. 1982. Development of a method for detection of
human rotavirus in water and sewage. Appl. Environ. Microbiol. 43:1440-1450.
Sneath, P.H.A., N.S. Ma1r, M.i. Sharpe, and J.G. Holt (eds.) 1986. Sergey's
Manual of Systematic Bacteriology. Williams and Wilkins Publ., Baltimore,
Maryland.
Sobsey, M.D. 1975. Enteric viruses and drinking water supplies. J. Am. Hater
Horks Assoc. (no vol. specified). 414-418.
Sobsey, M.D.,. P.A. Shields, F.H. Hauchman, R.L. Hazard, and L.W. Canton. 1986.
Survival and transport of hepatitis A virus in soils, groundwater, and
wastewater. Hater Sci. Tech. 18:97-106.
Sorber, C.A. and B.E. Moore. 1987. Project Summary: Survival and Transport of
Pathogens 1n Sludge-Amended Soil: A Critical Literature Review. U.S. EPA,
Office of Research and Development, Engineering Research Laboratory, Cincinnati,
Ohio: EPA Pub. No. 'EPA/600/S2-87/028.
U.S. EPA. 1989. Environmental Regulations and Technology. Control of Pathogens
in Municipal Wastewater Sludge for Land Application Under 40 CFR Part 257.
EPA/625/10-89/006. Pathogen Equivalency Committee. Cincinnati, OH.
U.S. EPA. 1993. Criteria for Classification of Solid Waste Disposal Facilities
and Practices. Final Rule. Subpart A. General Provisions §503. Office of
Science and Technology. Health and Ecological Criteria Division. U.S. EPA.
Washington, DC. •
VII-3
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Ward, R.L., G.A. McFeters, and J.G. Yeager. 1984. Pathogens in Sludge:
Occurrence, Inactivation, and Potential for Regrowth. Sandia National
Laboratory, Albuquerque, New Mexico. Sandia Report No. Sand 83-0557.
Williams, F.P. Jr. and C.J. Hurst. 1988. Detection of environmental viruses in
sludge: Enhancement of enterovirus plaque assay titers with 5-iodo-2'-
deoxyuridine and comparison to adenovirus and coliphage titers. Vater Res.
22:847-851.
Yanko, W.A. 1988. Occurrence of Pathogens in Distribution and Marketing
Municipal Sludges. Office of Research and Development, U.S. EPA, Health Effects
Research Laboratory, Research Triangle Park, North Carolina.
Yanko, W.A. 1992. Personnel communication. Los Angles County Sanitation
District, Whittier, California.
Yeager, J.G. and R.L. Ward. 1981. Effects of moisture content on long-term
survival and regrowth of bacteria in wastewater sludge. Appl. Environ.
Microbiol. 41(5):1117-1122.
VII-4
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APPENDIX A
Analytical Methodologies for Performing
Microbiological Testing of Sewage Sludges
Page
A. Dry Weight Analysis A-l
B. Fecal Coliform Procedures A-3
* •
C. Salmonella Identification and Quantification A-18
0. Virus Methodology A-37
E. Ascaris Ova Methodology A-42
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ANALYTICAL METHODOLOGIES
A. DRY WEIGHT ANALYSIS
The following apparatus and procedures are used in dry weight analysis.
Total, Fixed, and Volatile Solids in Solid and Semi sol id Samples
(Greenberg et al., 1992).
1. Apparatus
a. Evaporating dishes: Dishes of 100-mL capacity made of one of the
following materials:
1) Porcelain, 90-mm diameter.
2) Platinum, generally satisfactory for all purposes.
3) High-silica glass (Vycor, Corning Glass Works, Corning, NY).
b. Muffle furnace for operation at 550 ± 50*C.
c. Steam bath.
d. Desiccator, provided with a desiccant containing a color indicator
of moisture concentration.
e. Drying oven, for operation at 103 to 105*C.
f. Analytical balance, capable of weighing to 0.1 mg.
2. Procedure
a. Total solids:
1) Preparation of evaporating dish--If volatile solids are to be
measured, Ignite a clean evaporating dish at 550 ± 50'C for
1 h in a muffle furna.ce. If only total solids are to be
measured, heat dish at 103 to 105*C for 1 h in an oven. Cool
in desiccator, weigh, and store in desiccator until ready for
use.
2) Sample analysis
a) • Fluid samples — If the sample contains enough moisture to
flow more or. less readily, stir to homogenize, place 25
to 50 g in a prepared evaporating dish, and weigh.
Evaporate to dry ness in a water bath, dry at 103 to
105*C for 1 h, cool to balance temperature in. an
Individual desiccator containing fresh desiccant, and
weigh.
A-l
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b) Solid samples — If the sample consists of discrete pieces
of solid material (dewatered sewage sludge, for
example), take cores from each piece with a No. 7 cork
borer or pulverize the entire sample coarsely on a clean
surface by hand, using rubber gloves. Place 25 to 50 g
in a prepared evaporating dish, and weigh. Place in an
oven at 103 to 105"C overnight. Cool to balance
temperature in an individual desiccator containing fresh
desiccant, and weigh.
Fixed and volatile solids: Transfer to a cool muffle furnace, heat
furnace to 550 ± 50*C, and ignite for 1 h. (If the residue from
sample analysis contains large amounts of organic matter, first
ignite the residue over a gas burner and under an exhaust hood in
the presence of adequate air to lessen losses due to reducing
conditions and to avoid odors in the laboratory.) Cool in
desiccator to balance temperature, and weigh.
3. Calculation
% total solids - (A - B) x 100
C - B
% volatile solids - (A - Dl x 100
A - B
% fixed solids - (D - Bl x 100
A - B
where:
A - weight of dried residue + dish (ing).
B * weight of dish.
C - weight of wet sample + dish (mg).
0 » weight of residue + dish after ignition- (mg).
A-2
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B. FECAL COLIFORM PROCEDURES
The fecal coliform test differentiates between coliforms of fecal origin
(intestines of warm-blooded animals) and coliforms from other sources.
Use EC medium or, for a more rapid test of the quality of treated
wastewaters, use A-l medium in a direct test. Additional procedures
include the fecal coliform membrane filter procedure, and MPN
methodologies for the estimation of bacterial density.
1. Fecal Coll form Test (EC Medium) (Greenberg et al., 1992; Olivieri et al.,
1989; Bordner and Winter et al., 1978) (required under EPA Part 503
Regulation for Use or Disposal of Sewage Sludge; U.S. EPA, 1993).
a. EC medium:
Tryptose or trypticase 20.0 g
Lactose . 5.0 g
Bile salts mixture or bile salts No. 3 1.5 g
Oipotassium hydrogen phosphase, K-HP04 4.0 g
Potassium dihydrogen phosphate, KH2P04 1.5 g
Sodium chloride, NaCl 5.0 g
Reagent-grade water 1.0 L
Add dehydrated ingredients to water, mix thoroughly, and heat to
dissolve. The pH should be 6.9 ± 0.2 after sterilization.
Before sterilization, dispense in fermentation tubes, each with an
inverted vial, and with sufficient medium to cover the inverted vial
at least partially after sterilization. Close tubes with metal or
heat-resistant plastic caps.
b. Procedure: Submit to the confirmed test all presumptive
fermentation tubes or bottles showing any amount of gas, heavy
growth, or acidity within 48 h of incubation.
1) Gently, shake or rotate presumptive fermentation tubes or
bottles showing gas, heavy growth, or acidity. With a sterile
3--or 3.5-mm-diam metal loop or sterile wooden applicator
stick, transfer growth from each presumptive fermentation tube
or bottle to EC .broth.
2) Incubate Inoculated EC broth tubes in a water bath at 44.5 t
0.2*C for 24 t 2 h. Place .all EC tubes 1n water bath within
30 m1n after inoculation. Maintain a sufficient water depth
in water bath incubator to Immerse tubes to upper level of the
medium.
c. Interpretation: Gas production with growth in an EC broth culture
within 24 h or less .is considered a positive fecal coliform
reaction. Failure to produce gas (with little or no growth)
constitutes a negative reaction indicating a source other than the
intestinal tract of warm-blooded animals. If multiple tubes are
used, calculate Most Probable Number (MPN) from the number of
A-3
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positive EC broth tubes as discussed in procedure 4, MPN Estimation
of Bacterial Density. When using only one tube for subculturing
from a single presumptive bottle, report as presence or absence of
fecal coliforms.
2. Fecal Coliform Direct Test (A-l Medium) (Greenberg et al., 1992; Oliveri
et al., 1989) (required under EPA Part 503 regulation? U.S. EPA, 1993).
a. A-l broth: This medium may not be available in dehydrated form and
may require preparation from the basic ingredients.
Lactose 5.0 g
Tryptone 20.0 g
Sodium chloride, NaCl 5.0 g
Salicin 0.5 g
Polyethylene glycol p-isooctylphenyl
ether 1.0 ml
Reagent-grade water .1.0 L
Heat to dissolve solid ingredients, add polyethylene glycol
p-isooctylphenyl ether, and adjust to pH 6.9 t 0.1. Before
sterilization, dispense in fermentation tubes with an inverted vial
and sufficient medium to cover the inverted vial at least partially
after sterilization. Close with metal or heat-resistant plastic
caps. Sterilize by autoclaving at 121'C for 10 m1n. Store in the
dark at room temperature for not longer than 7 d. Ignore formation
of precipitate.
Make A-l broth of such strength that adding 10-mL, sample portions to
medium will not reduce'Ingredient concentrations'below those of the
standard medium. For 10-mL samples, prepare double-strength medium.
i-
b. Procedure: Inoculate tubes of A-l' broth. Incubate for 3 h at 35 t
0.5'C. Transfer tubes to a water bath at 44.5 t'O.Z'C, and incubate
for an additional 21 ± 2 h.
c. Interpretation: Gas production 1n any A-l broth culture within 24 h
or less Is a positive reaction Indicating conforms of fecal origin.
Calculate MPN from the number of positive A-l broth tubes as
described In procedure 4, MPN Estimation of Bacterial Density. This
procedure does not require confirmation.
3. Fecal Coll fora Moafarane Filter Procedure (Greenberg «t al., 1992)
(optional requirement under EPA Part 503 regulation; U.S. EPA. 1993).
a. Materials and Culture Medium
1) M-FC medium: The need for uniformity dictates the use of
dehydrated media. Never prepare media from basic ingredj-ents
when suitable dehydrated media are available. Follow
manufacturer's- directions for rehydratlon. Commercially
A-4
-------
prepared media in liquid form (sterile ampule or other) also
may be used if known to give equivalent results.
m-FC medium:
Tryptose or biosate 10.0 g
Proteose peptone No. 3 or polypeptone 5.0 g
Yeast extract 3.0 g
Sodium chloride, NaCl 5.0 g
Lactose 12.5 g
Bile salts No. 3 or bile salts mixture 1.5 g
Aniline blue 0.1 g
Agar (optional) 15.0 g
Reagent-grade water 1.0 L
Rehydrate in water containing 10 mL 1% rosolic acid in 0.2N
NaOH. (Rosolic add reagent will decompose if sterilized by
autoclaving. Store stock solution in the dark at 2 to 10*C,
and discard after 2 weeks or sooner if its color changes from
dark red to muddy brown). Heat to near boiling, promptly
remove from heat, and cool to below 50*C. Do not sterilize by
autoclaving. If agar is used-, dispense 5- to. 7-mL quantities
to 50- x 12-mm petri plates and let solidify. Final pH should
be 7.4. Store finished medium at 4 to 8*C, preferably in
sealed plastic bags or other containers to reduce moisture
loss, and discard unused broth after 96 h or unused agar after
2 weeks.
Test each medium lot for satisfactory productivity by
preparing dilutions-of a culture of f. co7; and filtering
appropriate volumes to give 20 to 80 colonies per filter.
With each new lot of medium, verify 10 or more colonies
obtained from several natural samples to establish the absence
of false positives. For most samples, m-FC medium may be used
without the 1% rosolic add addition, provided there is no
interference with background growth.
. v
Culture dishes. Use tight-fitting plastic dishes because the
MF cultures are submerged in water bath during Incubation.
Enclose groups of fecal col 1 form cultures in plastic bags, or
seal Individual dishes with waterproof (freezer) tape to
prevent leakage during submersion.
Irtcubator. The specificity of the fecal col 1 form test is
related directly to the incubation temperature. Static air
Incubation may be a problem in some types of incubators
because of potential heat layering within the chamber and the
slow recovery of temperature each time the Incubator is opened
during dally operations. To meet the need for greater
temperature control, use a water bath, a heat-sink incubator,
or a properly designed and constructed Incubator giving
equivalent results. A temperature tolerance of 44.5 t 0.2*C
A-5
-------
can be obtained with most types of water baths that also are
equipped with a gable top for the reduction of water and heat
losses. A circulating water bath is excellent but may not be
essential to this test if the maximum permissible variation of
0.2'C in temperature can be maintained with other equipment.
b. Procedure
1) Selection of sample size. Select a volume of sample to be
examined in accordance with the information in the table
below. Use sample volumes that will yield counts between 20
and 60 fecal coll form colonies per membrane. When the
bacterial density of the sample 1s unknown, filter several
decimal volumes to establish fecal coliform density. Estimate
a volume expected to yield a countable membrane, and select
two additional quantities representing one-tenth and 10 times
this volume, respectively.
Suggested Sample Volumes for Membrane Filter Fecal Coll form Tut
Volume (XI to be Filtered (ml)
Water Source
100
SO
1
0.1
0.01
0.001
Lakes, reservoirs
Wells, springs
Water supply Intake
Natural bathing waters
Sewage treatment plant, secondary
effluent
Farm ponds, rivers
Stonnwater runoff
Raw municipal sewage
Feedlot runoff
X X
X X
XXX
XXX
XXX
XXX-
XXX
0
X X
X X
X
X
2) filtration of sample. Using sterile forceps, place a sterile
membrane filter (grid side up) over porous plates of a
receptacle. Carefully place a matched funnel unit over the
receptacle and lock it fn place. Filter the sample under a
partial vacuum. With the filter still In place, Hnse the
funnel by filtering three 20- to 30-mL portions of sterile
dilution water. Upon completion of the final rinse and the
filtration process, disengage the vacuum, unlock and remove
the funnel, Immediately remove the membrane filter with
sterile forceps, and place it on selected medium with a
rolling motion to avoid entrapment of air. Insert a sterile
rinse water sample (100 ml) after f1Hrat1on-of a series of 10
A-6
-------
samples to check for possible cross-contamination or
contaminated rinse water. Incubate the control membrane
culture under the same conditions as the sample.
Use sterile' filtration units at the beginning of each
filtration series as a minimum precaution to avoid accidental
contamination. A filtration series is considered to be
interrupted when an interval of 30 min or longer elapses
between sample filtrations. After such interruption, treat
any further sample filtration as a new filtration series and
sterilize all membrane filter holders in use. Decontaminate
this equipment between successive filtrations by using an
ultraviolet (UV) sterilizer for 2 min, flowing steam, or
boiling water for 5 min. Do not expose membrane-filter
culture preparations to random UV radiation leaks that might
emanate from the sterilization cabinet. Eye protection is
recommended; either safety glasses or prescription-ground
glasses afford adequate eye protection against stray radiation
from a UV sterilization cabinet that is not light-tight during
the exposure interval. .Clean the UV tube regularly and check
it periodically for effectiveness to ensure that it will
produce a 99.9% bacterial kill in a 2-min exposure.
3) Preparation of the culture dish. Place a sterile absorbent
pad in each culture dish and pipet approximately 2 mL m-FC
medium, prepared as directed above, to saturate the pad.
Carefully remove any excess liquid from the culture dish.
Place the prepared filter on a medium-Impregnated pad.
As a substrate substitution for the nutrient-saturated
absorbent pad, add 1.5% agar to m-FC'broth.
4) Incubation. Place prepared cultures in waterproof plastic
bags or seal petri dishes, submerge in a water bath, -and
Incubate for 24 t 2 h at 44.5 ± 0.2*C. Anchor dishes below
the water surface to maintain critical temperature
requirements. Place all prepared cultures in the water bath
wjthln 30 m1n after filtration. Alternatively, use an
appropriate, accurate solid heat sink or equivalent incubator.
5) Counting. Colonies produced by fecal coll form bacteria on
m-FC medium are various shades of blue. Pale yellow colonies
may be atypical E.° coli; verify for gas. production in mannit&l
at 44.5*C. Nonfecal coliform colonies are gray to cream-
colored. Normally, few nonfecal coliform colonies will be
observed on m-FC medium because of selective action of the
elevated temperature and' addition of rosolic acid salt
reagent. Elevating the temperature 45.0 t 0.2'C may be useful
in eliminating environmental Klebsiell* from the fecal
coliform population. Count colonies with a low-power (10 to
15 magnifications) binocular wide-field dissecting microscope
or other optical device.
A-7
-------
c. Calculation of Fecal Colifortn Density
Compute the density from the sample quantities that produced MF
counts within the desired range of 20 to 60 fecal coliform colonies.
This colony density range is more restrictive than the 20 to 80
total coliform range because of the larger colony size on m-FC
medium. Record densities as fecal colifortns per 100 ml.
Compute the count by the following equation:
Coliform colonies/100 mL - Co11f?nn c.9lo.n.ie$ counted, x 100
mL sample filtered
For verified coliform counts, use the following equation.:
Percentage of . Number of verified colonies x 100
verified coliforms Total number of coliform colonies
subjected to verification
*
4. Most Probable Number (MPN) Estimation of Bacterial Density (Greenberg «t
al., 1992).
To calculate coliform density, compute in terms of the Most Probable
Number. The MPN values for a variety of plating series and results are
given in Tables A-l, A-2, and A-3. Included 1n these tables are the 95%
confidence limits for each MPN' value determined. If the sample volumes
used are found in the tables, report the value corresponding to the number
for positive and negative results in the series as the MPN/100 ml, or
report as total or fecal col 1 form presence or absence.
The sample volumes indicated on Tables A-l and A-2 relate more
specifically to finished waters. Table A-3 Illustrates MPN values for
combinations of positive and negative results when five.lO-mL, five
1.0-mL, and five 0.1-mL volumes of samples are tested. When the series of
decimal dilutions 1s different from that 1n the table, select the HPN
value from Table A-3 for the combination of positive tubes, and calculate
according to the following formula:
MPN value (from table) x Ifl - MPN/100 mL
largest volume tested
When more than three dilutions are used 1n a decimal series of dilutions,
use the results from only three of these in computing the MPN. To select
the three dilutions to be used in determining the MPN Index, choose the
highest dilution that gives positive results in all five portions tested
(no lower dilution giving any negative results) and the two next
succeeding higher dilutions. Use the results at these three volumes in
computing the MPN Index. In the examples given below, the significant
dilution results are Indicated with an asterisk. The number in the
numerator represents positive tubes; that 1n the denominator, the total
A-8
-------
tubes plated; the combination of positives simply represents the total
number of positive tubes per dilution:
Volume of Samole (mU:
Example
a
b
c
1
5/5
5/5*
0/5*
0.1
5/5*
4/5*
1/5*
0.01
2/5*
2/5*
0/5*
0.001
0/5*
0/5
0/5
Combination
of positives
5-2-0
5-4-2
0-1-0
MPN
Index/100
ml
5000
2200
20
In c, select the first three dilutions so as to include the positive
result in the middle dilution.
When a case such as that shown below in line d arises, where a positive
occurs in a dilution higher than the three chosen according to the rule,
incorporate it in the result for the highest chosen dilution, as in e:
Example
d
e"
1
5/5*
5/5*
Volume of
0.1
.3/5*
3/5*
Samole (mU:
0.01
1/5*
2/5*
0.001
1/5- '
0/5
Combination
of Positives
5-3-2
5-3-2
MPN
Index/100
1400
1400
When it is preferable to summarize with a single MPN value the results
from a series of samples, use the geometric mean or the median.
Table A-3 shows the most likely positive tube combinations. If unlikely
combinations occur with a frequency greater than 1%, it is an indication
that the technique 1s faulty or that the statistical assumptions
underlying the MPN estimate are not being fulfilled. The MPN for
combinations not appearing in- the table, or for other combinations of
tubes or dilutions, may be estimated by Thomas' simple formula:
in> nl PUMIIN'O Uibo • l|Hi
MI'N um ml. —
I mL >jinplc in mL Dimple in |
v \ ncKidvt: mho .ill mlv> '
While the MPN tables and calculations are described for use in the
coliform test, they are generally applicable to determining the MPN of any
other organisms, provided suitable test media are available.
A-9
-------
Table A-l. MPN Index and 95% Confidence Limits for Various Combinations
of Positive and Negative Results When Five 20-mL Portions
Are Used
No. of Tubes
Giving Positive
Reaction Out of
5 of 20 ml Each
0
1
2
3
4
5
Source: Greenberg et al
MPN
Index/
100 ml
<1.1
1.1
2.6
4.6
8.0
>8.0
. (1992).
Table A- 2. MPN Index and 95% Confidence Lirai
of Positive and Negative Results
No. of Tubes
Giving Positive
Reaction Out of
10 of 10 mi. Each
0 .
1
2
3
4
5
6
. 7.
8
9
10
MPN Index/
100 ml
<1.1
1.1
2.2
3.6
5.1
6.9
9.2
12.0
16.1
23.0
>23.0
95%
Lower
0
0.05
0.3
0.8
1.7
4.0
Confidence Limits
(Aooroximate)
Upper
3.0
6.3
9.6
14.7
26.4
Infinite
ts for Various Combinations
When Ten 10-ml Portions Are Used
95%
Lower
0
0.03
0.26
0.69
1.3
2.1
3.1
4.3
5.9
8.1
13.5
Confidence Limits
(Aooroximate)
Upper
•3.0
5.9
8.1
10.6
13.4
16.8
21.1
27.1
36.8
59.5
Infinite
Source: Greerrberg et al. (1992).
A-10
-------
Table A-3. MPN Index and 95% Confidence Limits for Various Combinations of Positive
Results When Five Tubes Are Used per Dilution (10 ml, 1.0 mL, 0.1 ml)
Combination
of Positives
0-0-0
0-1-0
0-1-0
0-2-0
1-0-0
1-0-1
-1-1-0
1-1-1
1-2-0
2-0-0
2-0-1
2-1-0
2-1-1
2-2-0
2-3-0
3-0-0
3-0-1
3-1-0
3-1-1
3-2-0
3-2-1
4-0-0
4-6-1
4-1-0
4-1-1
4-1-2
MPN
Index/
100 mL
<2
2
2
4
2
4
4
6
6
4
7
7
9
9
12
8
11
11
14
14
17
13
17
,17
21
26
95%
Lower
-
1.0
1.0
1.0
1.0
1.0
1.0
2.0
2.0
1.0
2.0
2.0
3.0
3.0
5.0
3.0
4.0
4.0
6.0
6.0
7.0
5.0
7.0
7.0
9.0
12
Confidence
Limits
Upper
-
10
10
13
11
15
15
18
18
17
20
31
24
25
29
24
29
29
35
35
40
38..
45
46
55
63
Combination
of Positives
4-2-0
4-2-1
4-3-0
4-3-1
4-4-0
5-0-0
5-0-1
5-0-2
5-1-0
5-1-1
5-1-2
5-2-0
5-2-1
5-2-2
5-3-0
5-3-1 •
5-3-2
5-3-3
5-4^}
5-4-1
5-4-2
5-4-3
5-4-4
5-5-0
5-5-1
5-5-2
5-5-3
5-5-4
5-5-5
MPN
Index/
100 mL
22
26
27
33
34
23
30
40
30
50
60
50
70
90
80
110°
140
170
130
170
220
280
350
240
300
500
900
1600
>1600
95%
Confidence
Limits
Lower Upper
9.0
12
12
15
16
9.0
10
20
10
20
30
20
30
40
30
40
60
80
50
70
100
. 120
160
100
100
200
300
600
-
56
65
67
77
80
86
110
140
120
150
180
170
210
250
250-
300
360
410
390
480
580
690
820
940
1300
2000
2900 .
5300
-
A-ll
-------
5. Host Probable Number (HPN) Method for Fecal Coliform (Bordner and Winter,
1978) (Figure A-l).
Apparatus and Materials
a. Incubator that maintains 35 ± 0.5'C.
b. Water bath or equivalent incubator that maintains a 44.5 t 0.2'C
temperature.
c. Pipet containers of stainless steel, aluminum, or Pyrex; glass for
glass pipets.
d. Inoculation loop, 3-mm diameter, and needle of nichrome or platinum
wire, 26 B&S gauge, in suitable holder. Sterile applicator sticks
are a suitable alternative.
e. Sterile pipets (T.O., Mohr, or bacteriological, glass or plastic),
of appropriate size.
f. Dilution bottles (milk dilution), Pyrex, 9^-mL volume, screw-cap
with neoprene liners. •
g. Bunsen or Fisher-type burner or electric incinerator unit.
h. Pyrex test tubes, 150 x 20 mm, containing Inverted fermentation
vials, 75 x 10 mm, with caps.
1. Culture tube racks to hold fifty 25-mm diameter tubes.
j. Media
1) lauryl tryptose broth (same as total col 1 form Presumptive Test
medium) prepared in 10-raL volumes in appropriate concentration
for sample volumes used.
Laurvl Sulfate Broth (BBL 11338)
Laurvl Trvotose Broth (D1fco 0241-02)
Use: Primary medium for the Presumptive Test for the total
conform group.'
a .
Composition:
Tryptose or tryptlease
peptone 20.00 g
Lactose 5.00 g
01 potassium hydrogen.
phosphate 2.75 g
Potassium d1hydrogen
phosphate 2.75 g
A-12
-------
Sample
a.
«i
LU
oc
a.
o
u
Lauryl Tryptose Broth
35±O.SC
Gat 4-
24 hr
Gas-
24 hr
Reincubate
24 hr
Gas
Gas-
Negative
T«st
El«v«t«d T«mp«ratur« T«$t
EC Medium at 44.5^0.20
Gas-i-
24hr
F«c«l Coliforms Present
Ctleulttt
Ftcil Conform
MPN
Gas-
24 hr
T9st
Figure A-l. Flow chart for the fecal coliform MPN tests,
Source: Bordner and Winter et al. (1978).
A-13
-------
Sodium chloride 5.0 g
Sodium lauryl sulfate 0.1 g
Final pH: 6.8 ± 0.2
Preparation: Add 35.6 grams of the medium to 1 liter of
laboratory pure water and mix to dissolve. Dispense 10-mt
volumes in fermentation tubes (150 x 20 tubes containing 75 x
10 mm tubes) for testing 1 ml or less of samples. For testing
10-mL volumes of samples, add 71.2 grams of the medium per
liter of laboratory pure water and mix to dissolve. Dispense
in 10-mL amounts in fermentation tubes (150 x 25 mm tubes
containing 75 x 10 mm tubes). Sterilize for 15 minutes at
12TC (15 Ib pressure). The concentration of the medium
should vary with the size of the sample according to the table
below.
Compensation in Lauryl Tryptose Broth (LIB)
for Diluting Effects of Samples
LTB Medium/
Tube (mL)
10
10 '
20
Sample Size/
Dilution
(mL)
0.1 to 1.0
10
10
Medium
Concentration
Ix
2x
l.Sx
Dehydrated -
LTB
(g/D
35.6
71,2
53.4'
2) EC medium prepared in 10-mL volumes in fermentation tubes
(see EC media description at beginning of section B, Fecal
CoHform Procedures).
3) ' Dilution Water: Sterile buffered or peptone dilution water
dispensed in 99 t 2 mL volumes in screw-capped bottles.
Procedure
a. Prepare the total conform Presumptive Test medium {lauryl tryptose
broth) and EC medium. Clearly mark each bank of tubes, Identifying
the sample and the volume Inoculated.
b. Inoculate the Presumptive Test medium with appropriate quantities of
sample.
A-14
-------
c. Gently shake the Presumptive Test tube. Using a sterile inoculating
loop or a sterile wooden applicator, transfer inocula from positive
Presumptive Test tubes at 24 and 48 hours to EC confirmatory tubes.
Gently shake the rack of inoculated EC tubes to ensure mixing of
inoculum with medium.
d. Incubate inoculated EC tubes at 44.5 i 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.
e. The presence of gas in any quantity in the EC confirmatory
fermentation tubes after 24 ± 2 hours constitutes a positive test
for fecal col iforms.
f. Alternatively, double strength presumptive media, could be used for
enrichment of 10 g solid or semi sol id samples, this would increase
the detection limit but could affect growth of organisms if toxic
agents are present (Meckes, 1992).
Calculations
a. Calculate fecal coliform densities on the basis of the number of
positive EC fermentation tubes, using the table of Most Probable
Numbers.
b. 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 MPN per
100-mL 1s recorded. (See the. MPN Estimation of Bacterial Density,
, Greenberg et al., 1992*)
c. Report the fecal col 1 form MPN values per 100 ml of sample.
-» *
•
d. The precisions of the MPN counts are given as confidence limits in
the MPN tables. Note that the precision of the MPN value increases
with Increased numbers of replicates per sample tested.
6. Test for Standard Indicator Organisms for Fecal Conforms (Yanko, 1988).
Procedure
a. Tubes of appropriate media for MPN tests are inoculated from the
sample suspension.
b. Dilutions to be Inoculated are selected based on experience with the
anticipated range of bacteria in the samples.
A-15
-------
c. Tests are performed and MPN-computed as described in the previously
documented MPN method of Greenberg et al. (1992).
d. Using Total Solids (TS) data, final results are calculated and
expressed as MPN/g dry weight.
Aerobic and Anaerobic Plate Count
a. First, 0.1 mL of dilutions from the sample suspension are inoculated
onto duplicate plates of pre-dried plate count agar for each
dilution.
b. Dilutions to be inoculated are selected based on experience with the
anticipated range of bacteria in the samples.
c. Spread plate method tests are performed as described in the above
methodology of Greenberg et al. (1992).
d. Anaerobic plates are incubated in "Gas Pak" anaerobic jars, per
manufacturer's instructions.
•
e. Plates are incubated at 35'C for 48 hours.
f. Colonies are counted per methodology of Greenberg et al. (1992).
g. TS results are used to calculate and report final results as
colony-forming units (CFU)/g.
7. Test for Fecal Conforms (Ma, 1992).
• a
a. Inoculate five appropriate-dilutions Into lauryl sulfate broth
fermentation tubes set up as five-tube WPN,'
b. Incubate for 48 hours at 35'C.
c. At 24 and 48 hours, transfer positive lauryl sulfate tubes, using
wooden applicator sticks to brilliant green bile broth (BGB) 'and EC
broth. Record positive tubes on coll form MPN forms.
d. Incubate BGB tubes at 35'C. Read and record positive results at 24
and 48 hours.
e. Incubate EC tubes at 44.5'C in a water bath for 24 hours. Read and
record positive tubes at 24 hours, only.
f. Confirm one sample per set via the completed col 1 form tests.
g. Report conforms as HPN per 100 g wet weight for sewage sludge and
soil samples, and as MPN per 100 ml for water samples.
A»16
-------
Table A-4. Detection Limits for Membrane Filtration and MPN Analyses
Membrane Filtration -
mL or g
102
101
10°
10*2
lO'3
io-4
10
IO*6
5-Tube MPN (FDA)
Dilution Range
10i. o, -i
in-l. -2. -3
10
1Q-2. -3. -4
1U , . e
iQ-3. -4.. -5
1Q-4. -5. -6
1Q-5. -6. -7
Fecal col i forms
Low
60
>600
>6,000
> 60, 000
> 600, 000
>6, 000, 000
> 60 , 000,000
> 600, 000, 000
>6, 000, 000, 000
High
22,400
224,000
2240,000
22,400,000
' 224,000,OdO
2240,000,000
22,400,000,000
Source: Ma (1992).
'A-17
-------
SALMONELLA IDENTIFICATION AND QUANTIFICATION
The following procedures are used for Salmonella identification and
quantification.
Detection and Enumeration of Salmonella (Kenner and Clark, 1974) (required
under EPA Part 503 regulation; U.S. EPA, 1993).
a. A multiple tube (MPN) procedure is used in which dulcitol selenite
(DSE) broth is used for primary enrichment modified by the use of
sodium acid selenite. The constituents include:
Proteose peptone 0.4%
Yeast extract 0.15%
Oulcitol 0.4-0.5%
Na,HPO. 0.125%
KH'PO. 0.125%
Distilled water
Constituents are heated to 88*C in a water bath to obtain clear
sterile medium. Productivity for Salmonella is enhanced by addition
of an 18-h, 37*C culture of Salmonella paratyphi A (10% by volume)
in single-strength DSE broth killed by heating to 88'C.
b. .When concentration of bacteria Is not usually required, as in sewage
sludge, the transfer of 10 ml of .sample to each tube in the first
row of the setup into 10 mL of double-strength DSE is made, 1 ml of
sample in 9 mL of single-strength DSE in the second row, and so on.
•
c. Incubation temperature of 40'C for 1 and 2 days is critical to
obtain optimum recovery of Salmonella sp. when DSE broth ,1s used for
primary enrichment..
*
d. After primary Incubation at 40*C, surface-loopfuls (7 mm platinum or
nichrome wire loop) are removed from each multiple-tube culture and
streaked on each of two sections of a divided plate of xylose Vysine
desoxycholate (XLD) agar to isolate colony growth. The numbered
plates are Inverted and incubated at 37*C for a period not to exceed
24 h.
e. Positive Incubated XLD plate cultures contain clear, pink-edged,
black-centered Salmonella colonies. These colonies are picked to
Kllgler Iron agar (KIA) or triple sugar Iron agar slants for typical
appearance, purification, and identi'ty tests.
f. Salmonella slant cultures Incubated overnight at 37'C give an
unchanged or alkaline red-appearing slant; the butt is blackened by
H.S, is add yellow, and has gas bubbles, except in rare species.
Typical-appearing slant cultures are purified by transferring them
to XLD agar plates for the development of Isolated colonies.
A-18
-------
g. The flat or umbonated-appearing colonies with large black centers
and clear pink edges are then picked to KIA slants, incubated at
37'C, and urease tested before the identification procedure.
Urease-negative tubes are retained for presumptive serological tests
and serotype identification.
2. Quantitative Salmonella Procedures (Greenberg et al., 1992) (required
under EPA Part 503 regulation; U.S. EPA 1993).
a. Multiple-tube enrichment technique. Dilute (1:10 dilution) sewage
sludge samples are proportioned into the five-tube, three-dilution
multiple-tube procedure using either dulcitol selenite or
tetrathionate broth as the selective enrichment medium. Incubate
for 24 h at 35*C, and streak from each tube to plates of brilliant
green and xylose lysine desoxycholate agars. Incubate for 24 h at
35*C. Select from each plate at least one colony suspected of being
Salmonella, inoculate a slant each of triple sugar iron (TSI) and
lysine iron agars, and incubate for 24 h at 35'C. Test cultures
giving a positive reaction for Salmonella by the serological
techniques described above. From the combination of Salmonella
negative and positive tubes, calculate the MPN/100 ml of original
sample of sewage sludge.
b. Membrane Filter Procedure for 5. typhl. Not applicable for sewage
sludge.
3. The following steps of the Salmonella Identification process are presented
for. detail of Materials and procedures.
a. 'Prlnry Enrichment (Bordner and* Winter. 1978; Greenberg et al.,
1992).
g
Apparatus and Materials
1) Incubators set at 35 ± 0.5*C, 41.5-± 0.5'C, and optionally at
37 ± 0.5 and 43 ± 0.5'C.
2) Sterile shears and spatula.
3) Sterile forceps.
4) Sterile beakers, 500-ml size, covered with aluminum foil or
kraft paper.
5) Sterile Erlenraeyer flasks, 125-mL size, to hold 50 mL of
enrichment broth.
6) Bunsen/Fisher burner, or electric incinerator.
7) Sterile petri dish.
A-19
-------
8) Sterile aluminum foil.
9) Alcohol, 95% ethanol, in a vial.
10) Media
a) Selenite broth (Difco 0275, BBL 11608).
Cpmposition:
Tryptone or polypeptone 5.U g
lactose 4.0 g
Disodium hydrogen phosphate 10.0 g
Sodium selenite 4.0 g
Final pH 7.0 ± 0.2
Preparation: Add 23 grams of selenite broth to 1 liter
of laboratory pure water. Mix and warm gently untr
dissolved. Dispense in tubes to depth of 6 cm, and
expose to flowing Steam for 15 minutes. Avoid excessive
heating. Do not autoclave. Sterilization is
unnecessary if broth 1s used immediately.
b) Tetrathionate broth (Difco 0104, BBL 11706).
Composition:
Proteose peptone or
polypeptone 5.0 g
Bile salts . 1.0 g
Calcium carbonate 10.0 g
Sodium thlosulfate 30.0 g
Final pH 7.8 t 0.2
•
Preparation: Add 46 grams of tetrathionate 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.
(The Iodine solution 1s prepared by dissolving 6 grams
Iodine crystals and 5 grams potassium Iodide in 20 ml of
distilled water.) Mix and dispense in 10-ml volumes
Into screw-cap tubes. Do not heat after the addition of
the Iodine. Do.not autoclaves The tetrathionate broth
base with Iodine may be stored for later use. The
complete medium (with iodine) should be used on the day
it is prepared.
A-20
-------
c) Dulcitol selenite broth.
(Medium may not be commercially available.)
Composition:
Proteose peptone 4.00 9
Yeast extract 1.50 g
Dulcitol *•.00 g
Sodium selenite 5.00 g
Disodium hydrogen
phosphate 1.25 g
Potassium dihydrogen
phosphate 1-25 g
Final pH: 6.9 ± 0.2
Preparation:. Add 16.5 grams of dulcitol selenite broth
to 1 liter of laboratory pure water and heat carefully
to dissolve ingredients. Do not boil.. The prepared
medium should be buff-colored. Dispense into screw-cap
tubes to a depth* of 6 cm. Do not autoclave.
d) Tetrathionate Brilliant Green broth (same as
C.3.a.lO)b), above, Tetrathionate broth) with the
addition of 0.01 gram of Brilliant Green per liter of
medium).
Composition: Same as tetrathionate broth base with
addition of 0.01 gram of Brilliant Green per. liter.
Procedures for Enrichment
After inoculation, Incubate enrichment flasks at 35*C, 41.5*C, and other
selected temperatures for at least 24 hours. However, some Salmonella are
slow-growing, and recovery may be Increased by Incubating for successive
24-hour periods up to 96 hours before streaking on Isolation aqars.
b. Isolation Plating (Bordn«r and Winter, 1978; Gr««nberg et al.,
1992),
Apparatus and Materials
1) Incubators set at 35 t 0.5'C, 41.5 ± 0.5'C, and optionally at
37' ± 0.5'C and 43 ± 0.5'C.
2) Water bath set at 44-46'C for tempering agar.
3) Petri dish canisters for glass petri dishes.
4) Thermometer certified by National Bureau of Standards or one
of equivalent accuracy.
A-21
-------
5) Inoculating needle and 3-mm loop.
6) Colony counter, Quebec darkfield model or equivalent.
7) Bunsen/Fisher burner, or electric incinerator.
8) Sterile 100-mm x 15-mm petri dishes, glass or plastic.
9) Sterile phosphate buffered or peptone dilution water bottles,
99 ± 2 mi volumes.
10) Media: The following agar media are dispensed in bulk
quantities in screw-capped bottles or flasks.
a) Xylose lysine desoxycholate (XLD) agar
(Salmonella differentiation)
Composition:
SBL 11837
Xylose 3.50 g
L-Lysine . 5.00 g
Lactose 7.50 g
Saccharose (sucrose) 7.50 g
Sodium chloride • 5.00 g
Yeast extract 3.00 g
Phenol Red 0.08 g
Agar • 13.50 g
Sodium desoxycholate 2.50 g
Sodium thiosulfate 6.80 g
Ferric ammonium citrate 0.80 g
D1fco 0788-02
Xylose 3.75 g
L-Lys1he . 5.00 g
Lactose 7.50 g
Saccharose (sucrose) 7.50 g
Sodium chloride • 5.00 g
Yeast extract 3.00 g
Phenol Red 0.08 g .
Agar . .15.00 g
Sodium desoxycholate 2.50 g
Sodium'thiosulfate ' 6.80 g
Ferric ammonium citrate 0.80 g
Final pH: 7.4 t 0.2
Preparation:-'. Add 55 or 57 grams of XLD agar 1n 1 liter
of cold laboratory pure water, heat to boiling with
mixing. Do not overheat, and do not autoclave. Pour
Into sterile petri dishes.
A-22
-------
Note: Better recoveries have been reported by using XL
agar base (BBL 11835 or Difco 9555) (used for XLBG agar)
and, adding separately, sterile solutions of the last
three ingredients.
b) Brilliant Green (BG) agar (BBL 11072, DIFCO 0285-02)
(Salmonella isolation)
Composition:
Yeast extract 3.00 g
Polypeptone or proteose
peptone 10.00 g
Sodium chloride 5.00 g
Lactose 10.00 g
Saccharose (sucrose) 10.00 g
Phenol Red 0.08 g
Brilliant Green 0.0125 g
Agar 20.00 g
Final pH: 6.9 ± 0.2
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 nlnutes at 121'C (15 1b pressure). Pour Into sterile
petrl dishes.
Warning: A longer period of sterilization will reduce
the selectivity of the medium.
c), • Xylose lysine Brilliant Green (XLBG) agar
(Salmonella differentiation)
Composition of XL agar base:
BBL 11836
XyTose 3.5 g
L-Lys1ne 5.0 g
Lactose 7.5 g
Saccharose (sucrose) 7.5 g
Sodium chloride 5.0 g
Yeast extract 3.0 g
Phenol Red 0.08 g
Agar 13.5 g
Difco 0555-02
Xylose 3.75 g
L-Lys1ne 5.0 g
Lactose 7.5 g
A-23
-------
Saccharose (sucrose) 7.5 g
Sodium chloride 5.0 g
Yeast extract 3.0 g
Phenol Red 0.08 g
Agar 15.0 g
Final pH: 7.4 ± 0.2
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 Ib
pressure). Cool the sterilized medium to about 45-50*C,
and add 20 ml .of a solution containing 34% sodium
thiosulfaie and 4% ferric ammonium citrates Pour into
sterile petri dishes.
d) Bismuth sulfite agar Difco 0073-02, BBL 11030
(Salmonella differentiation)
*
Composition:
Polypeptone or proteose
peptone 10.00 g
Beef* extract 5.00 g
Dextrose 5.00 g
01 sodium hydrogen.
Phosphate 4.00 g
Ferrous sulfate 0.30 g
Bismuth sulfite indicator 8.00 g
Brilliant Green 0.025 g
Agar 20.00 g
Final pH 7.6 ± 0.2
Preparation: £dd 52 grams of bismuth sulfate agar to 1
liter of cold laboratory pure water and heat to boiling.
Do-not autoclave or overheat. Twirl the flask prior to
pouring plates to evenly dispense the characteristic
precipitate. Use the plated medium on the day prepared.
Procedure
1) Prepare two selected media (XLD, BG, XLBG, or bismuth sulfite
agars) in petri dishes. As a minimum, xylose lysine
desoxycholate (XLD) and Brilliant Green (BG) or xylose lysine
Brilliant Green (XLBG) agars are recommended. Bismuth sulfite
agar permits the presumptive detection of 5. typhi and/or
S. mttrftfft/s.
A-24
-------
2) Streak the surface of a previously poured and solidified agar
with a loopful of the enrichment culture.
3) Streak duplicate plates from each enrichment culture every 24
hours for 3-4 days.
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.
5) After incubation, examine plates for colony appearance.
Table A-5 describes the appearance of colonies on XLD, XLBG,
BG, and bismuth sulfite agars. The Salmonella colonies on
BG agar are pinkish white with a red background. Lactose
fermenters will form greenish colonies or other colorations.
Occasionally, slow lactose fermenters such as Proteus,.
Citrobacter, Pseudomonas> and Aeromonas mimic Salmonella.
*
6) Pitk growth from the centers of well-isolated colonies that
have the characteristic appearance of Salmonella, and streak
onto the screening media. A description of the screening
media follows. Isolated, single colonies from a plate where
all colonies appear alike may be assumed to be pure. At least
two colonies of each type suspected to be Salmonella should be
picked.
7) The suspected colonies of Salmonella should now be
characterized by the single biochemical tests or multitests
described as follows. An 0-1 bacteriophage screening test may
also be* used on the Isolate for a rapid (4-5 hours)
determination of the tentative identification of Salmonella.
Results must be verified.
c. Biochemical Identification (Bordner and Winter, 1978; fireenberg
et al., 1992).
1) Commercially available biochemical test systems for
Identification-of Salmonella Include the following:
a) API Enteric 20 consists of 20 small Chambers (called
cupules) in a plastic strip, each containing dehydrated
medium. An isolated colony 1s used to prepare a cell
suspension to Inoculate the media. The Inoculated media
are Incubated for 18 hours at 35*C in a special plastic
chamber. A numerical Identification system based on
thousands of reaction combinations 1s available. The
Identification systen 1s updated periodically. Computer
services that are more comprehensive and accurate than
the manual system may be obtained.
A-25
-------
Table A-5. Colony 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
Round jet black colonies
with or without sheen
Round jet black colonies
with or without sheen
S. typhi
S. enteritidis ser
Enteritidis
S. enteritidis ser
Schottmueileri
Rat or slightly raised
green colonies
Flat or slightly raised
green colonies
Rat or slightly raised
green colonies
S. enteritidis ser
Typnimurium
S. enteritidis bioser
Paratypnyi
5. cholerae-suis
Proteus spp.
2. Brlliant Green Agar
Slightly pink-white,
opaque colonies surrounded
by brilliant red medium
Salmonella spp.
Yellow-green colonies
surrounded by yefiow-greefj
zpne
Escherichfe, KJebsietla,
Proteus spp. (lactose or
sucrose fermenters)
3. XLD or XLBG Agar
Red, black centered colonies
Red colonies
Yellow colonies
Yellow colonies
4
Yellow colonies
Yellow colonies
Yellow colonies
Salmonella spp.
Shigella spp.
Escherichia spp.
and btotypes
C/trooacfer spp.
KJebsieila spp.
Enterobacter spp.
Proteus spp.
Source: Bordner and Winter (1978).
A-26
-------
b) The Improved Enterotube with eight compartments 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
media. The Enterotube is incubated for 18-24 hours at
35'C. A manual numerical identification aid, ENCISE, is
part of the system.
c) The Inolex system (formerly Auxotab) consists of a test
card unit containing 10 reagent-filled capillary
chambers. A single isolated colony'is picked into broth
and cultured for 3t hours at 35'C. After incubation,
the broth tube is centrifuged, and the cells are
resuspended in water and inoculated into each capillary
chamber on the card. Each card Is incubated for 3 hours
at 35'C in its own plastic container. Isolates can be
identified in 7 hours unless additional tests are
required. A numerical binary code named Var-ident is
part of the system.
2) Primary Biochemical Screening Agar
a) Triple Sugar Iron (TSI) Agar
Use: Differentiation of gram negative enterics by their
differing ability to ferment dextrose, lactose, and
sucrose, and ability to produce hydrogen sulfide.
Composition:
.Dlfco 0265-02
Beef extract 3.00 g
Yeast extract 3.00 g
Peptone • 15.00 g
Proteose peptone 5.00 g
Lactose 10.00 g
Saccharose (sucrose) 10.00 g
Dextrose f.OO g
Ferrous sulfate 0.20 g
Sodium chloride 5.00 g
Sodium thiosulfate 0.30 g
Agar 12.00 g
Phenol Red 0.024 g
fifiL 11748
Peptone 20.00 g
Lactose 10.00 g
Saccharose (sucrose) 10.00 §
Dextrose ' 1.00 g
A-27
-------
Ferrous sulfate 0.20 g
Sodium chloride 5.00 g
Sodium thiosulfate 0.20 g
Agar 13.00 g
Phenol Red 0.025 g
Final pH: 7.3 ± 0.2
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 dissolve the agar. Dispense into screw-cap
tubes and sterilize for 15 minutes at 118*C (12 Ib
pressure). Slant tubes for a generous butt. Inoculated
. TSI slants must be Incubated with loosened caps to
prevent complete blackening of the medium from H2S.
b) Lysine Iron Agar
Use: Differentiation of Proteus, Citrobacter, and
Shigella from Salmonella based on deamination of lysine
and hydrogen sulfide production. Salmonella cultures
produce large amounts of hydrogen sulfide and lysine
decarboxylase.
Composit1on:
Difco 0849-02
Peptone 5.00 g
Yeast extract 3.00 g
Dextrose 1.00 g
L-Lysine 10.OQ g
Ferric ammonium citrate 0.50 g
Sodium thiosulfate 0.04 g
Broro Cresol Purple 0.02 g
Agar ' 15.00 g
BBL 11362 •
Peptone 5.00 g
Yeast extract 3.00 g
Dextrose 1.00 g
L-Lys1ne 10.00 g
Ferric ammonium citrate 0.50 g
Sodium thiosulfate 0.04 g
Brom Cresol Purple 0.02 g
Agar 13.50 g
Final pH: 6.7 f 0.2
A-28
-------
Preparation: Add 34.5 or 33 grains, depending on
manufacturer, of lysine iron agar to 1 liter of
laboratory pure water. Heat in a boiling water bath to
dissolve the agar. Dispense in 4-mL amounts in
screw cap tubes, and sterilize for 12 minutes at 12TC
(15 Ib pressure). Cool to give a deep butt and short
slant. Inoculated LIA slants must be incubated with
loosened caps.
3) Biochemical 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 LIA Agars:
3 *
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
add butt with a small amount of gas.
a) Triple Sugar Iron (TSI) 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 1s yellow for an add reaction or red for an
alkaline reading. Gas production 1s evidenced by
bubbles. 1n the medium, and H2S .production by
blackening of the medium.
(4) Typical reactions:
Stlaonellt: K/Ag with H.S, 1+ to 4+.
S. typhi: K/A with H,S, trace .to H.
C/tro6acter: K/Ag or A/Ag with H2S, 1+ to 3*.
(5) Atypical reactions: TSI tubes showing alkaline
slants and add butts without H.S production
should be Inoculated Into Motility Sulfide Medium
to verify the negative H2S reaction. If H2S is
still negative, perform serological testing to
confirm an atypical Salmonella.
b) Lysine Iron Agar
(1) Inoculate by, stabbing the butt twice and
streaking the slant.
A-29
-------
(2) Incubate for 18-24 hours, and if negative for an
additional 24 hours, at 35*C.
(3) Read and record reactions. The slant or butt is
yellow for an acid reaction and blue/purple for
an alkaline reading. Gas production is evidenced
by bubbles in the medium and H2S production by
blackening of the medium along the stab line.
Proteus has a distinctive red slant caused by
oxidative deamination and a yellow butt.
(4) 'Typical reactions:
Salmonella: K/K or K/N with H.S +(-).
5. typhi: K/K with H.S -(+).
Citrobacter: K/A with H2S - or +.
Proteus: R(red)/A with H.S -(+).
V '
L) Additional Biochemical Identification
a) Phenylalanine Agar (BBL 11536, Difco 0745-02)
Use: Differential tube medium for the separation of
members of the Proteus and Providencia genera from other
members of the Enterobacteriaceae based on deaminase
activity.
Composition:
Yeast extract 3.0 g
OL-Phenylalanine 2.0 g
01 sodium phosphate 1.0 g
Sodium chloride 5.0 g
Agar 12.0 g
•
.Final pH: 7.3 t 0.2
Preparation: Add 23 grams of phenylalanlne agar to 1
liter of cold laboratory pure water. Heat 1n 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 Ib pressure). Slant and cool tubes.
* a • a
Phenylalanine agar test
(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.
A-30
-------
(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) Indole Test Reagent
Preparation: Dissolve 5 grams paradimethyl ami no
benzaldehyde in 75 mL isoamyl or normal amyl alcohol,
ACS grade. Slowly add 25 mi. cone HC1. The- reagent
should be yellow and have a pH below 6.0; if the final
reagent is dark in color it should be discarded.
Examine the reagent carefully during preparation because
some brands are not satisfactory after ao>1ng. 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 obtaining amyl
alcohol. If this problem occurs, an alternative paper
strip test for Indole production can be used.
Indole Test
(1) Inoculate the tryptophane broth lightly from the
TSI agar slant culture.
(2) Incubate the broth at 35 ± 0.5'C for 24 t 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 1s a positive Indole test; the
original yellow color of the reagent is a
negative test.
(6) With rare exceptions, Salmonella and Citrobacter
are indole-negatlve.
A-31
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c) 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 1.00 g
Ammonium sulfate 2.00 g
Dipotassium phosphate 0.60 g
Monopotassium phosphate 0.40 g
Sodium chloride 2.00 g
Sodium malonate 3.00 g
Dextrose 0.25 g
Brom Thymol Blue 0.025 g
Final pH: 6.7 ± 0.2
Preparation: Dissolve 9.3 grams in 1 liter of
laboratory pure water. Dispense into tubes, and
sterilize for 15 minutes at 12TC (15 Ib pressure).
Malonate Broth'Test
(1} Inoculate from the 18- to 24-hour TSI agar slant
culture.
o
(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 medium from
green to a deep blue. . Lots of malonate medium
should be checked with positive and negative
cultures.
(3) S, arizonae and some strains of Citrobacter
utilize malonate. .Other Salmonella do not.
Fermentation of Dulcitol in Phenol Red Broth Base
(1) .Inoculate the Dulcitol broth lightly using a
24-hour culture.
(2) Incubate at 35*C, and examine daily for 7 days.
(3) -A. positive reaction 1s production of acid with
yellow color".
(4) Most Salmonella and some Citrobacter utilize
dulcitol. Examples of some that do use it or use
A-32
-------
it slowly include 5. typhi, S. cholerae-suis,
5. enteritidis bioser Paratyphi A and Pullorum,
and 5. entertidis ser Typhimurium.
d. Genus Identification of Salmonella by Serological Techniques
(Greenberg et al., 1992).
Upon completion of the reconmended biochemical tests, inoculate the
suspected Salmonella pure culture onto a brain-heart in-fusion agar
slant, and incubate for 18 to 24 h at 35*C. With wax pencil, divide
an alcohol-cleaned glass slide into four sections. Prepare a dense
suspension of test organism by suspending growth from an 18- to 24-h
agar slant in 0.5 ml 0.85% NaCl solution. Place a drop of
Salmonella "0" polyvalent anti-serum in the first section and
antiserum plus 0.85% NaCl in the second section. Using a clean
inoculating loop, transfer a loopful of bacterial suspension to the
third section containing 0.85% NaCl solution plus antlserum. Gently
rock slide back and forth. If agglutination is not apparent irv the
fourth section at the end of 1 min, the test is negative. All other
sections should remain clear.
If the biochemical reactions are characteristic of 5. typhi and the
culture reacts with "0" polyvalent antlserum, check other colonies
from the same plate for vi antigen reaction. If there is no
agglutination with Salmonella v1 antlserum, the culture 1s not
5. typhi. Identification of Salmonella serotypes requires
determination of H antigens and phase of the organism. Isolates
yielding biochemical reactions consistent for Salmonella and
positive, with polyvalent "0" antlserum may be Identified
as 'Salmonella sp., serotype or bloserotype undetermined."
4. Salmonella Methodology for Sewage Sludge (Ma, 1992).
Presumptive Salmonella colonies are picked Into trypticase soy broth
supplemented with 0.5% yeast extract (TSB/YE), allowed to grow overnight
at 35*C, then streaked for purity to MacConkey agar (without Crystal
Violet). Pure cultures are then transferred to urea agar. (Mixed
cultures are purified by selecting for the lactose-negative calonies, and
passaging again through TSB/YE and MacConkey agar). Urease-negatlve
isolates are further tested 1n Klingler's Iron agar (KIA) (we prefer KIA
to TSI, but the reactions are the same), lysine Iron agar, malonate broth,
and ONPG broth. Isolates that conform to the reactions listed below for
KIA (TSI), LIA, and urease, and are either malonate and ONPG positive or
malonate and ONPG negative (++ or --) are presumptively identified as
Salmonella spp. (See p. A-34). Confirmation is performed by using the
commercial test kit 20E marketed by API. Any Isolates with API's notation
other than "Excellent ID" or "Good ID" are tested 1n the full tubed
biochemical screen described above. In the past 3 years, this protocol
for identification has been successful with 99% of our Isolates, and we
have not found it necessary to test many isolates in the full conventional
media.
A-33
-------
IS1
K/A-
K/A+
K/A-
K/A+
K/A-
A/A+
L1A
K/A- (Shigella spp.)
K/K+ or K/N+
K/K- or K/N or K/A-
(5. paratyphi A)
K/K- or K/N
K/K + K/N+
K/K+ or K/N-i-
(Occasional-S. arizonae, 'rarely
other Salmonella spp.)
UREASE
K
A
N
HS
Alkaline
Acid
No change
Biochemical test
Purple broth base-glucose
(acid/gas)
Catalase/oxidase (TSA)
ONPG
Phenylalanine deaminase
Salmonella RX
Vd
V-
Biochemical Test
)oxylase
:arboxylase
^drolase
»
Malonate
Indol
Salmonella RX
Biochemical Test
MR/VP
Sucrose
Salmonella RX
A-34
-------
Rhamnose +
Dulcitol d
Raffinose
Cellobiose d
Motility +
5. Salmonella Methodology (Yanko, 1988).
•
a. Tubes of SBG sulfa enrichment (D1fco) are inoculated for MPN tests
from the sample suspension. SBG sulfa enrichment broth was prepared
fresh by heating in a water bath to 60-70*C for 30 min instead ;f
boiling for 10 min as indicated in manufacturer's instructions.
b. SBG inoculated tubes are incubated 20-24 h at 37*C.
c. Growth in SBG is inoculated^to plates of xylose-lysine desoxycholate
(XLO) agar (Difco) and modified lysine iron agar (MLIA) (Difco base,
modified).
d. SBG enrichment broths are reincubated an additional 24 h.
e. Isolated colonies exhibiting typical Salmonella morphology are
picked to slants of triple sugar Iron agar (TSI) and lysine iron
agar (LIA) (both"Difco) and urease test broth (BBL).
f. Cultures showing the correct biochemical reactions are confirmed by
agglutination with Salmonella polyvalent 0 antiserum (Difco).
g. When examining the primary Isolation plates (XLO, HLIA), it is
determined-whether the pattern of presumptive Salmonella isolations
followed a logical- dilut-ion distribution.
• , • t
*
1) If misses occurred, 1 mL from the corresponding 48 h SBG
enrichment tube is inoculated. Into a fresh tube of SBG
enrichment and incubated 24 h at 37*C.
2) These secondary enrichment tubes are streaked to isolate
Salmonella as described above.
f
3) MPNs are computed from enrichment tubes confirmed to contain
Salmonella and reported as HPN/g dry weight.
6. Salmon*}la Methodology (011v1er1 et al., 1989).
A multiple-tube dilution technique was used to determine the level of
Salmonella. Enrichment media, dulcitol -selenite (DSE) and tetrathionate
broth, and Isolation media, xylose lysine desoxycholate (XLD), Brilliant
Green agar (BGA), and bismuth sulfile agar were evaluated at 37, 40., 41,
A-35
-------
and 45*C. Suspected Salmonella colonies (black and red) were picked and
run through a biochemical screen consisting of phenylalanine deaminase,
oxidase, mannitol, malonate, and lysine decarboxylase. Isolates with
biochemical results matching that of a typical Salmonella species were
considered presumptive Salmonella. These isolates were then transferred
to triple sugar iron agar (TSI) and lysine iron agar (LIA) slants, and
confirmed with polyvalent antisera.
Salmonella Methodology (Harding et al., 1981).
Processed sewage sludge samples, were inoculated directly into two
enrichment media: selenite broth and GN broth. Volumes of 25 ml, 10 mL,
1 ml, and 0.1 mL or 10 ml, 1 ml, 0.1 ml, and 0.01 ml were inoculated as
appropriate. Enrichment media were held at 37'C for approximately 18
hours. •
After enrichment, an aliquot from each bottle was dilution plated onto
appropriate media for selection of isolated colonies. Following
incubation at selective temperatures, colonies were subcultured and tested
for oxidase reactivity. Oxidase negative isolates were then subjected to
appropriate biochemical test screens consisting of triple sugar iron
(TSI), lysine-lron agar (LIA), or motmty-indole-ornithine medium (MIO).
Presumptive Salmonella isolates were confirmed with commercially available
polyvalent antisera.
A-36
-------
D. ENTERIC VIRUS METHODOLOGY
The following methods are used for viral recovery from sewage sludge.
1. ASTM Methods
The following two methods have been round robin tested by the American
Society for Testing and Materials (ASTM) for efficiency of viral recovery
from sewage sludge. Method 1 is designated as ASTM-1 and is the EPA
procedure (Berman et al., 1981) basically adapted and given in Section
9510G of Standard Methods for the Examination of Water and Wastewater
(Greenberg et al., 1992). Method 2, designated ASTM-2, is the method of
Glass et al. (1978). The final version of these methods has been
published by the ASTM and designated D 4994-89, "Standard Practices for
Recovery of Viruses from Wastewater Sludges." This is the required method
designated by the EPA under the EPA Part 503 regulation (U.S. EPA, 1993).
Please refer to the published ASTM method for full details on apparatus
required, reagents and materials preparation, and for a complete detailed
description of both procedures (adsorption and sonicatlon).
*
a. ASTM-1 (Adsorption Method) "
This method has been round robin tested for all wastewater sewage
sludge except primary sewage sludge.
1) While stirring a 300-mL sample of sewage sludge using a
magnetic stirrer, adjust the pH to 3.5 with 5N HC1.
2) Add sufficient 0.05 M A1C13«6H20 to bring the final A1C13
concentration to 0.0005M, and continue stirring for 30 min.
!»
3) Check pH ano* readjust to 3.5 if necessary, then centrifuge at
2,500xg for 15 m1n at 4'C.
7
4) Discard supernatant and resuspend conditioned solids in 300 ml
of 10% buffered beef extract solution (30 g beef extract
powder, 4.02 g Na.HPO..*7H?0, and 0.36 g citric acid dissolved
1 n 300 ml d 1 stfl 1 led- water).
5-) Stir using a magnetic stirrer for 30 min at sufficient speed
to develop a vortex without excessive foaming.
6) Centri fuge'at 10,000xg for 30 m1n at 4*C, decant supernatant,
and discard sediment.
7) Filter supernatant fluid through a stack of 3.0-, Q.45-, and
0.25-Mm porosity Filterite filters (Duo-Fine series; Filterite
Corp., Tlmcnlum, Maryland),, and dilute filtrate-with sterile
distilled water it a ratio of 7 parts water per 3 parts beef
extract. • • '
A-37
-------
8) Adjust 7:3 filtrate to pH 3.5 with 1M HC1, stir for 30 min
(magnetic stirrer), and centrifuge at 2,500xg for 15 min at
4'C.
9) After measuring its volume, discard supernatant and resuspend
precipitate in 0.15 M Na,HPO. = 7H20 using 5 ml of buffer per
100 ml of diluted filtrate.
10) Adjust final mixture to pH 7.0-7.5, and store at -70*C until
assayed.
As a result of round robin testing (Goyal et al.), this method was
judged slightly superior (as compared with ASTM-2) for viral
analysis of watered sewage sludge.
A modification of this method exists for use with primary sewage
sludge; however, primary sludge is not a consideration for this
report.
b. ASTM-2 (Sonication Method)
This method has been round robin tested and has been found to be
slightly more consistent for viral analysis of dewatered sewage
sludge than ASTM-1,
1) For watered sewage sludge, pour 800 ml Into a blender jar and
add 0.4 ML of Ant1 foam B and 19.2 g of beef extract powder;
For dewatered sewage sludge, mix 40 g dried sludge with 800 ml
of 3% beef extract containing 0.4 ml Ant1 foam B.
2) Blend mixture at a low speed for 1 m1n, then at high speed for
2 m1n.
9
3) Transfer to a beaker, adjust pH to 9.0 with 2N NaOH, and stir
for 25 min.
4) Pour-Into centrifuge bottles In an ice bath and sonicate each
for 2 n1n at 100 W using a sonic'ator probe (Labeline; model
9100 with 9106 probe or equivalent)-.
5) Centrifuge at 10,000xg for 30 n1n, discard sediment, and
adjust pH of supernatant to 3.5 with 2N HC1.
»
6) Stir for 30 m1n, recentrlfuge at 10,000xg for 30 min, and
discard supernatant.
7) Resuspend beef extract precipitant (floe) in 10 ml of 0.15 M
Na,P04, adjust pH to 7.5, and pour Into glass bottle for
detoxification. •
A-38
-------
icat ion/disinfect ion procedure
a) Dissolve 100 mg diphenylthiocarbazone (dithizone;
Eastman Chemical Products, Inc.; Kingsport, TN; No. 3092
or equivalent) in 1,000 ml chloroform, and store in
amber bottles at 4*C (shelf .life 1 month).
b) Before use, dilute stock dithizone solution 1:10 in
chloroform and add 10 ml of this working solution to the
suspended floe.
c) Blend at high speed for 1. min, and centrifuge at
10,000 x g for 30 min.
d). • Carefully remove upper aqueous layer, place in sterile
test tubes containing 0.5 ml of 0.1% CaCl., and gently
aerate (approximately 1 bubble/sec) with a sterile
Pasteur pipette attached to a filtered air line for 10
min to remove residual chloroform.
e) Treat final sample with antibiotics, split into three
subsamples, and store frozen at -70*C until assayed.
2. Other Methods
The following method of elution/concentration, found to be most effective
for viral recovery from dewatered sewage sludges, was reported by
Ma (1992).
Glvcine-Aluminum Hydroxide Method for Watered and Dewatered Sewage
Sludge
•
a. Blend 100 ml (g) watered sewage sludge with* 100 ml 0.1 M glycine
buffer (pH 11.5) for 1 minute. Use 200 ml buffer with dewatered
sewage sludges.
b. Pour into 250-mL centrifuge bottles,, and centrifuge at 7500xg for
10 minutes. Pour supernatant "into small sterile beaker, and discard
sediment.
c. Neutralize (pH 6.5-6.8) with HC1. Use 6N HC1, adding dropwise while
stirring vigorously. As the endpoint is appraachexk use. IN HC1.
Add one mL 0.3 M A1C13 per 100 mL-ehiate: Stir until pH stabilizes.
d. Raise pH to 7 with 1.0 M Na2C03. Make sure pH stabilizes. Stir
gently for 30 minutes.
e. Centrifuge at 2,000xg for 20 minutes at 4*C. Discard supernatant
and save floe.
f. Add 10 mL of 3% beef extract/EDTA solution (pH 11.5) to sample.
Dissolve the floe by pipetting vigorously back "and forth. If
A-39
-------
necessary, continue adding B.E. until the pH reaches a maximum of
9.5.
Neutralize the sample. Freeze at -20*C if processing will be
continued the next morning. If processing will be delayed longer,
freeze at -85'C. Follow with detoxification.
Detoxification Procedure
a. Add 5 ml of working stock (see below) to 5 ml of sample (or any
similar proportionate volumes). Mix at high speed with vortex mixer
for 1 minute.
b. Centrifuge at 6,500xg for 30 minutes. Remove upper phase with pipet
and save; discard lower phase. Do not allow precipitate at
interface to contaminate upper phase. Place sample in plastic petri
plate, and incubate in refrigerator for at least 1 hour.
c. Oialyze samples against IX PBS for 48 .hours at 4*C. Change buffer
three times.
«
d. Treat dialyzed samples again with chloroform without dithizone.
Follow steps a and b above.
e. 'Add 1 ml ant1b1ot1c-ant1mycot1c solution (GIBCO *600r5240) per 10-ml
sample. Record final volume at this point.
f. Store 1n glass 4-dram vials at -85*C until Inoculation.
Ch1orofonn-D1th1zone Solution Preparation
a. Dissolve 100 mg dlthizone in 1,000 mLof chloroform for concentrated
stock. Store at 4*C 1n amber glass bottle for no longer than 1
month.,
b. For working stock, dilute concentrated stock (step 3 above) to 1:9
in chloroform dally.
Wral Assay -
A complete description of cell line maintenance and. selection and the
materials and methods for conducting viral assays 1s beyond the scope of
this report. For this purpose, .standard textbooks and handbooks of
virology and cell culture are available. Likewise, viral assays should be
performed by trained vlrologists and/or highly skilled technicians,
trained in virology working in suitably equipped laboratories. Virus
assay is, therefore, beyond the capability of most POTW wastewater
laboratories. Currently, only a few of the larger POTWs routinely sample
sewage sludges for viral analysis. Some have expressed a willingness to
A-40
-------
perform viral analyses under contract to smaller POTWs. A few commercial
labs are also available to perform viral assays.
In practice, BGM (African green monkey kidney) cells, HEK (human embryonic
kidney) cells, MA-104 (rhesus monkey kidney) cells, and HeLa cells have
been used for assay of viruses in sewage sludge. HEK and BGM cells have
been used most frequently for recovery of most enteroviruses,
adenoviruses, and reoviruses.
Cell lines are inoculated with three replicates per dilution of
appropriate sample volumes (no more than 0.06 ml sample/cm2 of cell layer
surface). After a 2-hr adsorption period with agitation at 37*C, the
moriolayers are overlain with agar and incubated for 14 days with periodic
observation for plaque formation.
A-41
-------
ASCARIS OVA METHODOLOGY
The following methods have been found to be efficient for ascaris ova
recovery from sewage sludge.
Zinc Sulfate Density Gradient Separation Method Followed by
Acid-Alcohol/Ether Sedimentation (Yanko, 1988, adapted from Reimers et
al., 1981) (required under the EPA Part 503 regulation; U.S. EPA, 1993).
a. The homogenized sample (see homogenization of dewatered sewage
sludge, steps a-c, p. A-45, method of Fox et al., 1981) is measured
and poured through a 48-mesh sieve placed in a large funnel over a
2-L beaker.
b. The sample is washed through the sieve with several rinses of warm
tapwater catching the washings in the beaker.
c. The washed sample in the beaker is allowed to settle overnight.
d. The supernatant is siphoned off to just above the settled layer of
solids in the beaker.
e. The settled material in the beaker is mixed by swirling, and poured
into two 100-mL centrifuge tubes.
f. The beaker is rinsed two or. three times, and rinsings are poured
into two 100-mL centrifuge tubes.
*
g. The tubes are balanced and centrifuged at 1,250 rpm (400 x g) for
3 rain.
•
h. The supernatant is poured off and pellet resuspended thoroughly in
zinc sulfate solution (Sp. Gr. 1.20).
i. Zinc sulfate 1s centrifuged at 1,250 rpm for 3 min.
j. The zinc- sulfate supernatant is poured Into a 500-mL Erlenmeyer
flask, diluted with deionized water, covered, and allowed to settle
3 h or overnighti
k. The supernatant 1s aspirated off to just above the settled material.
1. The sediment is resuspended by swirling .and pipetted into two to
four 15-mL conical centrifuge tubes.
m. Tlie flask is rinsed with deionized water two to three times, and
rinse water 1s pipetted into tubes.
n. Tubes are centrifuged at 1,400 (480 x g) rpm for 3 min.
o. Pellets are combined into one tube and centrifuged at 1,400 rpm (480
x g) for 3 m1n.
A-42
-------
p. Pellets are resuspended in a tube I full of acid-alcohol solution
(0.1 N sulfuric acid in 35% ethanol solution); approximately 3 mi of
ether is added.
q. The tube is capped with a rubber stopper and inverted several times,
venting each time.
r. The tube is centrifuged at 1,800 rpm (660 x g) for 3 min.
s. The acid-alcohol, ether, and plug are poured off, and the tube is
inverted over a paper towel to prevent the reagent from running back
into the tube.
t. The pellet is resuspended 1n 0.1% sulfuric add and poured into
Nalgene tubes with loose caps.
u. The tubes are incubated in a slant rack at 26*C for 3 to 4 weeks.
1) Control ova dissected from an adult Ascaris lumbricoides,v*r.
suum were also incubated.
2) When the majority of control ova had embryonated, samples were
examined.
v. Concentrates were examined microscopically using a Sedgewick Rafter
cell to enumerate detected ova.
1) - Variability was noted based on presence of embryonated ova and
on whether or not larval forms could be Induced to move.
2) Ova were Identified'and reported as ova/g dry weight.
Flotation Methods
a. Sucrose Flotation (Fox et al., 1981).
1) Anaeroblcally Digested Sewage Sludge
3
a) Dilute a volume of homogeneous sewage sludge 1:1 with
H20 (direct from digesters and strained through a 20-
mesh sieve).
b) Place duplicate 10- or 20-mL allquots on separate fresh
20-mL continuous sucrose gradients in 50-ml tubes.
(See Preparation of Gradients, pg. A-45.)
c) Centrifuge at 2,900 G for 10 m1n.
d) Aspirate off the upper 15 rat of sucrose gradient using
a syringe, and transfer to 15-mL centrifuge tubes.
A-43
-------
e) Fill the tubes to a meniscus with concentrated
Sheather's solution, and top with a covers!ip.
f) Centrifuge at 500 G for 4-6 min.
g) Carefully lift coverslips and place them side by side on
a microslide.
h) Scan with lOx objective for the presence of parasite ova
or cysts.
i) A 100-mL sample of sewage sludge is oven-dried, and the
residue is weighed to determine the dry weight of solid
materials. Calculation of ova/g of dried sewage sludge
can then be made.
2) Raw Sewage
a) One to 10 gallons of formal in-fixed1 raw sewage are
sedimented in a large Imhoff cone (or large bucket) for
8 hours (or overnight).
b) One gallon of the sedimented material is removed and
concentrated to a 500-mL volume and fixed in 10%
formalin.
c) Duplicate 25-mL aliquots are passed through gradients as
described under Section E.2.a.l)b), above.
d) The upper 15-mL gradient 1s transferred to centrifuge
tubes, centrifuged, and cqverslip preparations are
prepared as described under 'Section E.2.a.l)c) through
e), above.
« .•
3) Watejfed Sewage Sludge (3-5% solids)
«
a) • Strain a quantity of sewage sludge through a 20-mesh
sieve.
b) Mix ID ml of strained sewage sludge with 10 ml of 10%
"7x" anlonic detergent.
c) thoroughly stir the re-suiting 20-mL solution, place into
a 50-ml plastic centrifuge tube, and centrifuge at 500 G
for 3 m1n.
d) Aspirate and discard the supernatant. Resuspend the
• residue with 25 ml of distilled water. Centrifuge this
mixture at 500 G for 3 minutes.
e) Repeat the above step two more times.
A-44
-------
f) Aspirate and discard the supernatant from the final
washing. Add distilled water to the residue until a
20-mL volume is obtained.
g) Thoroughly mix the resulting 20 mi of washed sewage
sludge solution, and follow the sucrose gradient
procedure for parasite ova under Section E.2.a.l)b),
above.
4) Dewatered Sewage Sludge (>40% solids)
a) Place 20 mi H20 into a 50-mL conical centrifuge tube.
b) Add Nu Earth (air-dried sewage sludge) until the volume
is 30 mL.
c) Pour material into a Waring blender, add approximately
• 100 ml H20 or sufficient H
homogenize for 15-30 seconds.
100 ml H.O or sufficient H.O to cover blades, and
d) Pour'homogenized material through a 100-mesh sieve, and
rinse with water to a volume of 1 liter.
e) Place strained material Into a 1,000-ml graduated
cylinder and allow to settle for 1 hour.
f) Aspirate to a 100-ml volume and transfer to a 250-mL
Erlenmeyer flask.
g) Wash 20 mL of strained material twice with a 10% 7x
anionic detergent solution. Resuspend to 10 mL.
h) Pass duplicate washed 10-mL samples (2 g) through a
continuous gradient, and examine as. in sewage sludge
procedure (Sections E.2.a.l)b) through f), above).
5)- Preparation of Gradients
Density gradients are effective tools for separation of
parasite ova and cysts from other organic materials. As the
solids pass through the gradient, ova and cysts will remain in
a band at a level where the density of the solution
approximates their onn. Gradients give much better separation
of tna-terials than simply mixing the sample with levitation
media because there 1s more than one density level. After the
desired band is located, that portion of the gradient can be
removed and analyzed for content. Continuous sucrose
gradients can.be prepared on an ISCO model 570 Gradient Former
(ISCO, Lincoln, Nebraska). This consists of 12 mL of 80%
Sheather's sucrose and 8 mL of distilled water prepared in
50-mL round-bottom centrifuge tubes.- The specific gravity
ranges from 1.3 at the bottom to 1.0 at the top.
A-45
-------
Discontinuous gradients can be made by preparing several
concentrations of levitation solution (dependent on the number
of layers desired). An example is to prepare 80%, 50%, 35%,
and 20% sucrose solutions. Carefully and slowly layer 5-mL
portions of these solutions one above the other in a 50-mL
centrifuge tube using a pipette. Start with the most dense
solution and finish with the least dense. Be careful that
well-defined interfaces are formed. When completed, push a
glass stirring rod very slowly through the interfaces, and
then remove it to break the interfaces (do not stir the
gradient). Concentrations of solution can be varied according
to needs. This process is very difficult to perform, but the
gradients produced are very good.
b. Salt Flotation (Peterson, 1971, as cited in Fox et al., 1981)
Composted Sewage Sludge
1) Place a 10-g sample of composted sewage sludge .mixture in a
500«-mL flask containing 100 ml physiological salt solution
(PSS) and glass beads.
2) Shake the flask 5 minutes on a rotary shaker at medium speed.
4) Filter the material through a 20-mesh sieve, and rinse with 1G
ml PSS.
5) Adjust' volume to 150 mi with PSS.
6) Place a 30- or 50-mL aliquot in a 50-mL tube, and centrifuge
at 2,000 rpm for 1 minute.
7) Remove supernatant fluid.
8) Resuspend sediment in saturated salt solution, and allow to
stand 30 to 60 minutes..
*
9)' Remove surface film with a sterile wire loop, and place on a
microscope .slide.
10) Add covers!ip and examine under microscope at lOx
magnification for ova.
Note: • An alternative method for counting the ova and cysts would be
to transfer the material to 15-mL test tubes, filling the tubes with
PSS, then placing a covers! 1p on the top of the tube. After
standing for 15 minutes, the coversltp is then carefully lifted,
placed on a micro-slide, and examined as previously described.
A-46
-------
Zinc Sulfate Flotation (Meyers, Kirk and Kaneshiro, 1978, as cited
in Fox et al., 1981).
Sewage Sludge
1) Add 75 g of sewage sludge to 100 mL of 2.62% hypochlorite
solution and mix well.
2) Allow the foam to disperse for 5-10 minutes, and adjust the
volume to 225 ml with hypochlorite.
3) Allow the hypochlorite to work on the material for 50 minutes
at room temperature (20-25'C).
4) Centrifuge for 2 minutes at 800 G, and remove the supernatant
by aspiration.
5) Resuspend the pellet in 200 ml of an anionic detergent
solution (7x, Limbro Sci., Inc., Homden, Connecticut),
readjust the volume to 225 ml with distilled water, and
centrifuge again.
6) Remove the supernatant and wash two more times in distilled
water.
7) Resuspend the pellet in 75 ml of saturated zinc sulfate
solution and centrifuge again for 2 min.
8) Pour the supernatant (now containing the ova) onto a membrane
filter (0.45 urn), arid remove the fluid by negative pressure
. filtration.
9) Remove the filter disc, and rinse the ova and debris from,the
membrane into a petri dish using a wash bottle.
10) Ova are counted by scanning the petri dish with a dissecting
microscope. A grid should be placed under the dish to
facilitate counting.
Zinc Sulfate Flotation Method (Oliveri et al., 1989).
0
1) A sample (.50-100 mL) 1s placed frt a 1,000-mL low form beaker
with 250-300 ml of 0.1X solution of Tween 20 and mixed on a
magnetic stirrer for a minimum of 5 minutes.
2) The homogenized sample is strained through a series of sieves
(10, 20, 50, 100, and 150); it is allowed to settle for at
least 1 hour in a tall form 1,000-mL beaker.
3) The supernatant is decanted and discarded. The sediment is
placed Into a 50-mL centrifuge tube and centrifuged for 5 min
at 2,000 rpm.
A-47
-------
4) The supernatant is discarded, and the sample is resuspended in
tapwater; centrifugation and decantation are repeated.
5) If the packed sediment is more than 8 mL, the sediment is
resuspended in tapwater and evenly divided between 'two tubes
and centrifuged. If not, zinc sulfate solution is added to
the remaining sediment and'thoroughly mixed with an applicator
stick.
6) The volume is increased to 50 ml; the sample is centrifuged
for 3-5 minutes at 2,000 rpm.
7) • The top 30- to 40-mL supernatant is poured into a 400-mL
beaker and diluted to 250 ml with tapwater. The diluted
supernatant is centrifuged at 2,000 rpm for 2 min.
8) The supernatant is discarded; the sediment is transferred.to
a 15-mL centrifuge tube, which is filled to the top with
tapwater, and centrifuged for 2 min at full speed in a
clinical centrifuge.
9) The supernatant is discarded, and a 10 ml alcohol -acid'
solution added. Three ml of ethyl acetate is added
immediately, and the sample is mixed by 15 sec of vigorous
shaking, followed by 2 min of full speed centrifugation.
10) An applicator stick 1s used to loosen the plug at the
interface between the ethyl acetate and alcohol-acid solutions,
and the sediment and plug are aspirated off.
11) The sediment remaining on the bottom 1s stained, -diluted to 10
ml, vortexed, and centrifuged a final time at 2,000 rpm for
5 min.
e. Formal in-Ether Procedure for-Parasites (Ma, 1992)."
*
1) Weigh 100 gram wet weight sample into sterile beaker. Label
with sample name and number. '.Cover with foil to prevent
'contamination.
2) Carefully add 100 ml APHA water to the sample. Homogenize
with a sterile spatula or tongue blade.
o •
o
3) Allow to stand for at least 1 hour, preferably overnight in
the refrigerator.
4) Pour through two-layers of wetted gauze (cheesecloth) into a
funnel/beaker assembly. Carefully and slowly express all
liquid out of sample.
5) Collect liquid extract and place into centrifuge tube(s).
*
A-48
-------
6) Concentrate by centrifugation at 1,800 rpm (300-400xg) for 7-8
minutes.
7) Pour off supernatant to disposal bucket.
8) Resuspend pellet in 10 ml 10% buffered formalin. Allow to
stand for 5 minutes. (This is a convenient holding point.)
9) Add up to 40 mL ethyl acetate and shake tube. Carefully vent
tube, and shake again.
10) Centrifuge at 2,000 rpm (450-500xg) for 3.5-4 minutes.
11) Carefully remove cap;, remove floating debris with cotton
applicator swab along the sides of the tube. Carefully pour
off layers of debris, formalin, and ethyl acetate, taking
pains to preserve the pellet.
12) Examine the pellet under a microscope using a maximum of two
slides. Iodine (Gram's or 1/5 J»ugol's) may be used as stain
to provide contrast.
•
f. Lugol's Iodine Stain for Ascarls Ova.
4
1) Stain: Lugol's Iodine
Potassium Iodine 10 g
Iodine crystals 5 g
. Distilled -H20 100 g
This stain has a short shelf life and must be stored in a
brown bottle.
2) Add stain drop-wide to sample until supernatant fluid is dark
brown. 0The Lugol's stain works well with wet mount
preparations; however, much of the debris stains similar to
the organisms. Helminth eggs stain very well because the
Iodine 1s able to penetrate through the shell.
A-49
-------
APPENDIX B
Sources of Laboratories For Microbiological Testing
-------
A. STATE AGENCIES INVOLVED IN CERTIFICATION OF LABORATORIES THAT PERFORM
WATER/WASTEWATER TESTING
ARKANSAS
Dick Cassat
Arkansas Dept. of Pollution- Control 4 Ecology
Technical Services Division
P.O. Box 8913 •
Little Rock, AK 72219-8913
501-570-2131 or 501-562-7*44, ext.131
CALIFORNIA
Department of Health Services
Southern California Laboratory Section
1449 West Temple Street
Room 101
Los Angeles, CA 90026
213-620-3564
.George C. Kulasingam, Manager
Environmental Laboratory Approval Program
Department of Health Services
2151 Berkeley Way
Berkeley, CA 94704
415-540-2800 -
COLORADO
Drinking Water Unit
Department of Health
4210 East llth Avenue
Denver, CO 80220
303-331-4732
CONNECTICUT
Nicholas Macelletti, Jr.
Connecticut State Department of Health Services Laboratory
P.O. Box 1689
Hartford, CT 06144
203-566-2438 or 203-566-4045
B-l
-------
DELAWARE
Division of Public Health
Health Systems Protection
P.O. Box 637
Dover, DE 19903
302-739-5410
FLORIDA
Dr. Carl ICircher
Department of Health and Rehabilitative Services
Laboratory 'Services
1217 Pearl Street
Jacksonville, FL 32202
904-359-6454
GEORGIA
Georgia Board of Examiners for Certification of
Water and Wastewater Treatment Plant Operators
and Laboratory Analysts
166 Pryor Street, SH
Atlanta, GA 30303-3465
404-656-3933
(certifies analysts who perform bacteriological or chemical testing of
water/wastewater)
Loretta M. Lambert
Laboratory Certification Coordinator
Drinking Water Program .
Environmental Protection Division
Department of Natural Resources
Twin Towers East, Suite 1066
205 Butler Street, SE
Atlanta, GA 30334
HAWAII
Environmental Protection and Health Services Division
Department of Health
P.O. Box 3378
1250 Punchbowl Street
Hololulu, HI 96801
808-548-6345
8-2
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IDAHO
Chuck Brokopp, Laboratory Director
Bureau of Laboratories
2220 Old Penitentiary Road
Boise, ID 83712
208-334-2235
ILLINOIS
Illinois Environmental Protection Agency
Division of Laboratories, No. 4
2200 Churchill Road
Springfield, IL 62794-9276
217-782-6562
Bob Slaber
Laboratory Certification Officer
Illinois Environmental Protection Agency
Division of Laboratories
P.O. Box 19276
Springfield, IL 62794
INDIANA
Indiana Department of Health
Laboratory Improvement Branch
1330 West'Michigan Street
P.O. Box 1964
Indianapolis, IN 46206-1964
317-233-3442
IOWA
Charlotte Henderson
Water Supply Section
Department of Natural Resources
Henry A. Wallace Building
900 East Grand Avenue
Qes Moines, IA 50319
515-281-8914
B-3
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KANSAS
Aurora Shields/Jack McKenzie
Department of Health and Environment
Laboratory Division
Forbes Field, Bldg. 740
Topeka, KS 66620
913-296-1639
KENTUCKY
Division of Environmental Services
Department of Environmental Protection
18 Reilly Road
Frankfort Office Park
Frankfort, KY 406"01
502-564-3410 -
LOUISIANA
Department of Health and Hospitals
Office of Public Health Services
Division of Laboratory Services
325 Loyola Avenue
Room 709
New Orleans, LA 70112
504-568-5375
MAINE
Gardner Hunt
Department of Human Services
Health and Environmental Testing Laboratory
'State House Station 112
Augusta, ME 04333
202-289-2070-
MARYLAND
Laboratories Administration
.Maryland Department of Health and Mental Hygiene
201 West Preston Street
Baltimore, MO 21201
410-225-6150
B-4
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MASSACHUSETTS
Laboratory Certification Officer
Lawrence Experiment Station
Department of Environmental Protection
37 Shattuck Street
Lawrence, MA 01843
617-292-5529
MICHIGAN
John Bloemker
District Engineer
Division of Public Water Supply
Department of Public Health
3423 North Logan Street
Lansing, MI 48909
517-335-8319
MINNESOTA
Division of Public Health Laboratories
Department of Health
717 Delaware Street, S.E.
Minneapolis, MN 55440
612-623-5301
John Ikeda
Laboratory Certification and Development
Division of Public Health Laboratories
717 Delaware Street, S.E.
Minneapolis, MN 55440
612-623-5681
MISSISSIPPI
Public Health1 Laboratory
State Department of Health
P.O. Box 1700
Jackson, MS 39215-1700
601-960-7582
B-5
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MISSOURI
Water Pollution Control
Department of Natural Resources
P.O. Box 176
Jefferson City, MO 65102
314-751-1300 -
Barrel 1 Osterhoudt
Public Drinking Water Program
Department of Natural Resources
P.O. Box 176
Jefferson City, MO 65102
314-751-5331
MONTANA
John Hawthorne
Chief, Chemistry Laboratory
Department of Health i Environmental Sciences
Cogswell Bldg.
Helena, MI 59620
406-444-5262
(conduct performance audits of laboratories that do wastewater testing)
NEBRASKA
John Blosser, Director
Division of Laboratories
Department of Health
P.O. Box 2755 -
Lincoln, NE 68502
402-471-2122
NEVADA
State Health Laboratory
State Health Department
1660 North Virginia Street
Reno, NV 89503
702-789-0335
B-6
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HAMPSHIRE
Charles Dyer
Department of Environmental Services
P.O. Box 95
•6 Hazen Drive
Concord, NH 03301
603-271-2991
NEW JERSEY
Jerry Bundy
New Jersey Department of Environmental Protection and Energy
9 Ewing Street
Trenton, NJ 08625
609-292-3950
NEW MEXICO
Department of .Environmental Health
P.O. Box 965
Las Cruces, NM 88004
503-827-2784
NEW YORK
Environmental Laboratory Approval Program (ELAP)
Wadsworth Center for Laboratories and Research
Department of Health
Empire State Plaza, Room D224
Albany, NY 12201-0509
518-474-8519
NORTH CAROLINA
Don Beesley
Department of Environmental Health and Natural Resources
Environmental Sciences Section
P.O. Box 28047
Raleigh, NC 27611
919-733-8695
B-7
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NORTH DAKOTA
Department of Health
Consolidated Laboratories Branch
P.O. Box 937
Bismark, NO 58502
701-221-6177 or 221-6140
OHIO
Division of Public Drinking Water
18 WaterMark Drive
P.O. Box 1049
Columbus, OH 43266-1049
614-644-2752
OKLAHOMA
State Environmental Laboratory
Department of Health
P.O. Box 24106
Oklahoma City, OK 73124
405-271-5240
Water Resources Board
Water Quality Division
600 North Harvey
Oklahoma City, OK 73101
405-231-2500
OREGON
William C. Miller
Certification Coordinator
Public Health Laboratory
1717 SW 10th Avenue
Portland, OR 97201
503-229-5882
PENNSYLVANIA
Department of Environmental Resources
Bureau of Laboratories
Evan Press Building
P.O. Box 1467 •
Harrisburg, PA 17105-1467
717-783-7150
B-8
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RHODE ISLAND
Department of Health
Health Laboratory Building
50 Orms Street
Providence, RI 02904
401-274-1011
SOUTH CAROLINA
Department of Health & Environmental Control
Laboratory Certification Section
P.O. Box 72
State Park, SC 29147
803-935-7025
SOUTH DAKOTA
Department of Environmental and Natural Resources
523 East Capital' Avenue
Room 412
Pierre, SO 57501
605-773-3754
TENNESSEE
Charles Mickle
Department of Health
630 Ben Allen Road
Nashville, TN 37247-0801
615-262-6354
TEXAS
Environmental Analytical Section
Bureau of Laboratories
Department of Health
1100 West 49th Street
Austin, TX 78756-3199
512-468-7580
Texas Water Commission
15531 Kirkendall-Rankin
Houston, TX 77092
B-9
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UTAH
State Health Laboratory
Microbiology and Chemistry
44 Medical Dr.
Salt Lake City, UT 84113
801-584-8300
VERMONT
Public Health Laboratory
Department of Health
P.O. Box 70
195 Colchester Avenue
Burlington, VT 05401
802-863-7336
VIRGINIA
Dr. Albert W. Tiedemann, Jr., Director
Division of Consolidated Laboratory Services
One North 14th Street
Richmond, VA 23219
804-786-7905
WASHINGTON
Department of Ecology
Manchester Laboratory
P.O. Box 488
Manchester, WA 98353
206-895-4698 or 895-4649
Robert Gunther
department of Health
Seattle, WA 98155
206-361-2891
Richard Cunningham, Program Manager
Department of Ecology
Technical Services
7272 Cleanwater Lane
Olympia, WA 98504
B-10
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Microbiology, Environmental
Chemistry Laboratory
Department of Health
1610 NE 150th Street
Seattle, WA 98155
206-361-2822 '
WEST VIRGINIA
Don Caldwell, Steve Wright
Office of Laboratory Services
Department of Health
167 llth Avenue
South Charleston, WV 25303
304-348-3530
WISCONSIN
Technical Services Section
Department of Natural Resources
101 South Webster Street
Box 7921
Madison, WI 53707
608-267-7633
Judy Courtney, CMef '
Microbiological Laboratory
Certification Section
Department of Health and Social Services
P.O. Box 309
Madison, WI 53701
608-266-5753
Paul Harris, Chairman
Certification Standards Review Council
Davy's Laboratories
Box 2076
LaCrosse, WI 54601
608-782-3130 .
WYOMING
Department of Environmental Quality
Water Quality
Herschler Building, 4th Floor West
Cheyenne, WY 82002
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B. LIST OF LABORATORIES QUALIFIED TO ANALYZE VIRUSES, HELMINTHS, AND
BACTERIA IN SEWAGE SLUDGE
1. Private Laboratories
IEA, Inc.
P.O. Box 626
Essex Junction, VT 05453
800-723-4432
Contact: Scott Tighe, Senior Microbiologist
Michele Eisenstein, Senior Microbiologist
James-M. Montgomery Laboratories
Pasadena, CA
818-568-6490
Contact: DeeAnne Bryant, Microbiology Supervisor
2. University Research Laboratories
Or. Aaron Margolin
University of New Hampshire
Purham, NH
603-862-2252
Ors. Joan Rose, Sam Farrah, and Boo Kwa
University of South Florida
Tampa, FL
813-974-6627
Dr. Charles Gerba
University of Arizona
Phoenix, AZ
602-621-6163
Dr. Mark Sobsey . •
University of North Carolina
Chapel Hill, NC
919-966-7303
Source: Clancy (1992) '
B-12
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C. ADDITIONAL PRIVATE LABORATORIES PERFORMING MICROBIOLOGICAL TESTING
Aquatech Inc.
75 Green Mountain Drive
South Burlington, VT 05401
(Salmonella) •
Arthur 0. Little
25 Acorn Park
Cambridge, MA 02140
(Salmonella'}
•>
Industrial Laboratories
3001 Cull en Street
Fort Worth, TX 76107
(Salmonella)
Lancaster Labs
2425 New Holland Pike
Lancaster, PA 17601
(Salmonella)
Pace Laboratory
1710 Douglas Drive North
Minneapolis, MN 55422
(Salmonella)
South Eastern Analytical Services
[Address not available]
(Salmonella)
Twin City Testing
662 Cromwell Avenue
St. Paul, MN 55114
(Salmonella and helminth ova)
Source: VIar & Company (Respondents to survey)
B-13
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