EPA-R4-73-022 Environmental Monitoring Series
MARCH 1973
Proceedings of the
First Microbiology Seminar
on Standardization of Methods
Office of Research and Monitoring
U.S. Environmental Protection Agency
Washington. D.C. 20460
-------
RESEARCH REPORTING SERIES
Research reports of the Office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
categories were established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technology
transfer and a maximum interface in related
fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental studies
This report has been assigned to the ENVIRONMENTAL
MONITORING series. This series describes research
conducted to develop new or improved methods and
instrumentation for the identification and
quantification of environmental pollutants at the
lowest conceivably significant concentrations. It
also includes studies to determine the ambient
concentrations of pollutants in the environment
and/or the variance of pollutants as a function of
time or meteorological factors.
-------
EPA-R4-73-022
March 1973
PROCEEDINGS OF THE FIRST MICROBIOLOGY SEMINAR
ON STANDARDIZATION OF METHODS
San Francisco, California
January 1973
Sponsored by
Quality Assurance Division
Office of Research and Monitoring
U.S. Environmental Protection Agency
Washington, D.C. 20460
For «le by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 30402
Price $2.60 domestic postpaid or SJ.2S OPO Bookstore
-------
EPA Review Notice
This report has been reviewed by the Environmental
^potection Agency and approved for publication.
Approval does not signify that the contents necessarily
reflect the views and policies of the Environmental
Protection Agency/ nor does mention of trade names or
commercial products constitute endorsement or recommenda-
tion for use.
ii
-------
EDITORIAL BOARD
Editor-in-Chief
S. Sidney Verner
Editorial Staff
Gerald Berg, Ph.D.
Robert H, Bordner
Edwin E. Geldreich, Jr.
Leonard J. Guarraia, Ph.D.
David Shedroff
Kathleen G. Shimmin
William Stang
ORM/ Headquarters
NERC-Cincinnati
NERC-Cincinnati
NE RC-C inc innat i
OAWP, Headquarters
NFIC, Cincinnati
Region IX
NFIC, Denver
iii
-------
FOREWORD
The Seminar on Standardization of Microbiological
Methods was organized to develop within the Environmental
Protection Agency (EPA) common methodological techniques
and to obtain broad Agency acceptance of a standardization
program in microbiology. A meeting limited to EPA
microbiologists was held to serve as a forum for airing
common problems and by opening channels of communication
among Agency microbiologists, help to clarify, unify and
strengthen the standardization program.
These proceedings are not intended to present the
Agency's complete standardization plan in microbiology.
The Seminar was necessarily of limited scope and additional
meetings will be held to complete the program. The Office
of Monitoring welcomes comments on this publication as
well as suggestions for future activities.
Willis B. Foster
Deputy Assistant Administrator
for Monitoring
IV
-------
Table of Contents
Foreword • iv
Preface ...... vi
Standardization Processes
Enforcement Activities 1
Office of Research Activities 12
Regional Concern and Activities 17
Microbiological parameters
Total Col if orms 20
Fecal Col if orms 25
Discussion . . 34
Klebsiella pneumoniae 43
Discussion 46
Fecal Streptococci 47
Discussion 69
Viruses • 72
Zoomicrobial Indicators 90
Pseudomonas aeruginosa 99
Yeast, Molds and Fungi 106
Salmonella 112
Discussion 125
Special Problems 132
procedures
Sampling 143
Discussion 166
Quality Control 170
Discussion 186
Summarization
Research Needs 195
Index of Authors 202
Appendix A
Seminar Agenda A-l
Appendix B
Roster of Attendees B-l
-------
PREFACE
This document contains the proceedings of the "Seminar
on Standardization of Microbiological Methods" held in
January, 1973. The Seminar and these proceedings are
representative of a continuing effort by the Office of
Monitoring to highlight and alleviate, through Agency-wide
cooperative efforts, areas and issues of monitoring
requiring technical or administrative support.
This Seminar was organized to focus on priority
problem areas and was divided into four segments, viz.,
standardization processes as related to enforcement, research
activities, and regional problems; microbiological parameters
which consumed the major portion of the meeting; analytical
procedures as related to sampling and quality control; and
a final paper summarizing research requirements prior to
standardization. In addition, the meeting was structured
to permit free discussion of the topic parameter after
each formal presentation and, where available, verbatum
or summary discussions are presented following each
respective paper in these proceedings.
The publication of these proceedings will be followed
by a compilation (compendium) of candidate methods based on
the suggested recommendations of the authors. The compila-
tion will be written by a committee of experts for each of
the microbiological parameters and will serve as a data
base of analytical methods in microbiology.
The Seminar brought together EPA microbiologists from
all program elements, offices and Regions to discuss problems
of mutual concern in methodology. The appendix lists the
agenda for the Seminar and the names of those attending the
meeting. It is anticipated that future Seminars will be
held to complement this one by focusing on issues not
considered here.
VI
-------
STANDARDIZATION PROCESSES
-------
ENFORCEMENT ACTIVITIES
*
David I. Shedroff
In order to present the role of standardization of
microbiological sampling and analysis in Federal enforcement,
it will be necessary to briefly describe the enforcement pro-
visions of the new Federal water pollution control bill
(see Figure 1), particularly as they affect the work of the
microbiologist, and then describe how expert opinions of
microbiologists concerning quantitative or qualitative pre-
sence of particular indicators or pathogens becomes admissible
evidence in an administrative or court proceeding. Finally/
some of the areas of impact of the new law and the methods
of introducing evidence on further standardization of micro-
biological sampling and analysis will be related,
A. New Law
The Federal Water Pollution Control Amendments of 1972 (1)
became law in October of last year. This statute strengthens
Federal and joint Federal-State enforcement authority over
dischargers and gives added responsibilities to microbiologists
in the enforcement area.
1. Emergency Authority
For the first time Congress has given authority to
the government to obtain a court order to immediately stop
discharges which present "an immiment and substantial en-
dangerment to the health of persons or to the welfare of
persons where such endangerment is to the livelihood of such
persons, such as the inability to market shellfish" (2).
One of the means of proving health hazards will obviously be
to prove the presence of pathogens. If past history of
closure of shellfish beds gives a preview of possible use
of the "endangerment of welfare" portion of this new section,
part of the proof will often turn on the validity of results
of analyses for total coliform, which are the basis for
orders closing the beds and on proof of the sources of the
coliform. Here is a prime example where EPA microbiologists
may well have to serve as government witnesses.
2. Toxic Pollutants and Hazardous Substances
Closely related to the emergency provisions of the
Act are those covering toxic pollutants (3) and hazardous^
substances (4). The Administrator will shortly publish his
*Enforcement Division, National Field Investigation Center,
Cincinnati
-------
TABLE I. KICMJBIOLOCICAL ACTIVITIES UNDER ENTORCEMEKr AKD RELATED PROGRAMS 0? FEDERAL UATER POLLUTION CONTROL ACT, AS AMENDED
Section of Law Activity Covered
504 toergency Authority
307 (a)
Toxic Pollutant*
311
Hazardous Substance*
402 and 301 (b) Permits for publicly owned
(municipal) treatment work*
402 and 301 ft) Permit* for non-publlcly
(Industrial) treatment vork*
a) Existing rlsnts
b) tat rim*
307
3010") Pretreaement standard* for
discharges try non-publlcly owned
enterprises into publicly-owned
405
312
no
Permit* for disposal of swage
sludge
Harlae Sanltatloo Device*
International pollutlem
Anticipated Activities for MlcrobioloRJats
Proof of presenc* of pathogen! In waters and fro*
particular sources); and in the case of shellfish
beds presence of and sources of indicator* author-
izing closure.
Activity Description
Injunction nay be issued on proof of "bnainent
and substantial fcndangennent to the health of
persons or to the welfare of persons where
such endangerment it to the livelihood of such
persona'*
Discharge limitations, including zero If
materials designated as toxic taking into
acqount "toxlcity . . . persistence, . .
degradability, . . presence of affected
organisms, .. Importance of affected organ-
isms and the effect of the toxic pollutant
oo such organism"
"Elements and compounds which, when discharged
In any quantity. . .Into waters,. . .present an
imlnent and substantial danger to the public
health, including but not United to, fish.
shellfish, wildlife, shorelines, and beaches."
Attainment of "secondary treatment", as defined Analyses to determine compliance or non-compliance
Identification and quantification of pollutants
designated aa toxic by Administrator (ma; include
viruses)
Performance of degradabllity tests and experiment*
under actual or simulated conditions
by administrator, or treatment necessary to
neet water quality standards, whichever it ,
more stringent by 1977. and "best practicable
waste treatment technology over the life of
the vork*" by 19(3.
Attainment of "best practicable control
technology currently available", or water
quality standards, whichever is oore stringent
by 1977, and "best available technology
ecnomlcally achievable" by 1983.
Compliance with "National Industrial Stand-
ard* of performance" which for a particular
industry "reflects the greatest degree of
effluent reduction, . . achievable through
the application of the best available con-
trol technology, processes, operating methods,
or other alternatives"
Compliance with standards of pretreataent by
category or categories of sources from
existing plants or new plants. Standard*
for both categories of plant must prevent
by discharge through a publicly-owned
treatment vorks of pollutants which
"interfere vith, pass through, or (are)
otherwise Incompatible with such works*
Standard* for existing sources will Inclode
tine for compliance which may not excmmj
three years from date of promulgation.
Permit* required for disposal of sevage sludge
(including removal of in-place seuace sludge
from one location and its deposit in another
location) where disposal "would result In any
pollutant. . .entering. . .waters."
Vessel sanitation devices aust conform to
performance standards issued by Administrator.
Mew vessels must comply within tvo rears of
promulgation; existing vessels have five
year* In which to comply.
Administrator may call hearing when he has
reason to believe pollution Is occurring
from U. S. source* "which endanger the health
or welfare of persons in • foreign country"
with microbiological portions oŁ permit requirement*.
Analyses to determine compliance or non-compliance
with microbiological portions of permit requirements
Analyses to determine compliance or non-compliance
with microbiological portion of the pretreatment
standard.
Analysis of sludges at time of transport and at
disposal site to determine compliance er non-
compliance with permit requirement*.
Analyses to confirm compliance with microbiological
portions of perfomtance standards. Analyses (for
Coast Guard) to determine if device in place 1*
operable in conformity with standard*.
Microbiological surveys to determine If pollution
being caused In a foreign country as aid to Admin-
istrator in deciding whether or not to call hearing
and as evidence at hearing, if called.
-------
initial list of toxic pollutants for which effluent limits,
including zero discharge, will thereafter be set. After the
limits are set by regulation, following public hearings, a
violation of those limitations will subject the discharger
to civil fine or criminal penalties, or a civil action to
stop the discharge, but without the right to immediate
cessation as in the case of discharges subject to the
emergency provisions (5). Comparable enforcement provisions
will be applicable to discharges of hazardous substances (6)
after these substances are designated by the Administrator
and the President specifies quantity limitations.
The statute contains technical definitions which
define and limit what may be classified as toxic pollutants
(7) or hazardous substances (8). Because the hazardous
definition limits its applicability to elements and compounds,
it is unlikely that living organisms will be included in that
list; however, since penalties for violations depend on the
severity of pollution (degradability of the substance) (9),
testimony may be required describing results of experiments
on degradability under actual or simulated spill conditions.
The toxic pollutant definition specifically states that it
includes "disease-causing agents" (10), so that organisms
identified or identifiable by microbiologists may well be
classified as toxic pollutants. To assure that viruses are
included in the list of toxic pollutants, the Senate report
on the bill specifically states "the presence of pathogenic
organisms, including viruses" is one criterion for classifying
a substance as toxic.
3. Permits for Discharges from Municipal and Industrial
Plants
Perhaps the most sweeping reform of the new Act is
in the point source discharge licensing system. Under the
Act, municipal treatment plants and new and existing industrial
plants discharging to any surface body of water must obtain
a permit (12). All existing municipal and industrial plants
must minimally attain "secondary treatment" and "best
practicable control technology currently available," re-
spectively, by July 1, 1977 (13). By 1983, municipal plants
must attain "best practicable water treatment technology,"
and industrial plants must attain "best available technology
economically achievable" (14). New industrial plants, when
built must meet "national industrial standards of performance"
(15), and plants discharging to municipal plants must conform
to "pretreatment effluent standards" (16).
-------
The Administrator must issue regulations defining
each of the above terms within the next few months. The
regulations on "best practicable treatment/' "best technology,"
"pretreatment/" and "required treatment" for new industrial
sources will vary from industry to industry reflecting the
nature of the wastes produced and the current availability
of treatment methodology. Except in the case of pretreatment
effluent standards/ permits will then be issued requiring
the discharges to conform with the requirements of the ap-
propriate treatment requirements regulations/ or to attain
v/ater quality standards (17), if that requires a higher
degree of treatment than the 1977 requirement. These permits
will place limits on discharges by parameters and will include
a requirement of self-monitoring and reporting (18). Viola-
tion of permit conditions or failure to obtain a permit can
subject the violator to stringent civil or criminal penalties
whether the permit is issued by EPA (19), or by a State or
Inter-State agency (20).
It is almost certain that the requirements of
"secondary treatment" will require the inclusion of micro-
biological parameters in permits for municipal treatment
plants, and that permits issued to industries where the
presence of microbiological organisms is a valid indicator
of the quality of treated effluent will include such para-
meters. Some of these industries are pulp and paper/ tanneries/
and the processing of food and dairy products/ including
sugar plants. The nature of wastes from manufacturers of
Pharmaceuticals will almost certainly require permits from
discharges by this industry to contain one or more micro-
biological parameters. In addition/ the proximity of a dis-
charge to a watercourse for which there are microbiological
standards may require the inclusion of microbiological para-
meters on other discharges/ or the inclusion of more stringent
numbers than those required under the treatment requirement
regulations, e.g., discharges to or near water intakes,
bathing beaches/ and shellfish beds.
The primary source of information on whether or not
there is compliance with permit conditions will be reports
made by the discharger. The discharger's data will pre-
sumptively be admissible in court proceedings as '"'business
records" (21).
4. Miscellaneous Provisions
The new bill has several other enforcement provisions
which will bring microbiological testing into play. There is,
for example/ a special provision requiring persons depositing
-------
sewage sludge which might reach waterways to obtain permits
(22). The regulations on issuance of such permits will no
doubt include microbiological criteria.
Likewise, the performance standards for vessel
sanitation devices (23) will undoubtedly include a maximum
total coliform or fecal coliform criterion. In addition,
the Act contains an absolute prohibition against discharge
of biological warfare agents (24) .
5. Abatement of International Pollution
Finally/ the Act gives the Administrator the
authority to call a hearing on alleged pollution in the
United States believed to be endangering health or welfare
of persons in a foreign country (25). The matter is heard
first by a hearing board whose recommendations are trans-
mitted to the Administrator for implementation. The re-
commendations may include suggesting the commencement of
legal action. In any such legal action, the presentations
before the hearing board are not automatically admissible
as evidence, and such evidence and any later determinations
must be admitted by the court. Microbiological evidence,
e.g., relating to discharges from municipalities, beet sugar
factories, and pulp mills can be expected to be part of
hearings and trials under the section of the Act relating to
International Pollution.
B. Admissibility of Microbiological Evidence
1. Courts, Agencies and Boards as Finders of Fact
Violations of the type described previously can
usually result in the imposition of civil penalties by the
Administrator, a hearing board, or a court; the imposition
of criminal sanctions by a court, or the entry of a court
order requiring a discharger to take or cease a particular
action. Except for civil penalties imposed by the Administra-
tor, it is clear that formal hearings will be required and
that a final report as such will not be acceptable evidence
without a satisfactory showing of authenticity (26). The
Federal Rules of Civil Procedure define the form and ad-
missibility of evidence in Federal courts, and the same
general rules will probably apply to testimony before
hearing boards. Rule 43 of the Federal Rules states, "In
all trials the testimony of witnesses shall be taken orally
-------
in open court unless otherwise provided by these rules. All
evidence shall be admitted which is admissible under the
Statutes of the United States." Two of these statutes relate
to exceptions to the hearsay rule. The hearsay rule states
that generally persons may only testify to what they know
personally, and that they must be subject to cross-examination.
2. Business Records and Government Records
a. Business Records
Under some circumstances business records are
admissible without requiring the maker of the record appear.
The Federal Act (27) says that such records may be admitted
in evidence as the record of an act/ occurrence, or event
"if made in the regular course of ... business and if it
was the regular course of such business to make such memo-
randum or record at the time of such act . . . occurrence
or event or written a reasonable time thereafter." Thus,
the results of microbiological examinations may be admitted
if there is a normal routine and it is followed. In order
to establish that there is a set routine, it is wise to put
it down on paper; as an example the simple sentence "after
this date, all microbiological examinations will be made
using appropriate directions as contained in part 400, 13th
Edition Standard Methods" (28) may be used. Instruction
sheets or operating manuals are used in some EPA microbiology
laboratories at the present time. Since routines do change
from time to time, it is recommended that all personnel sign
dated receipts for the instruction materials as originally
distributed and for modifications as issued from time to
time. The statute also requires that the documents be pre-
pared at or within a reasonable time after a particular
occurrence. This suggests that it is a wise procedure to
put all information directly into the log book or on bench
cards rather than on scraps of paper and later copying over
from these scraps of paper.
b. Government Records
Bench cards and other indicators of raw data
may also be admissible as government records (29) . Here
again there is a requirement that there be an official record;
the preparation of appropriate instructions defining exami-
nations to be performed will be of value in getting the data
admitted.
-------
c. Limitations on the Admissibility of Business
and Government Records
Although the statutes do not specifically cover
the point, it is clear from the examination of cases under
the two acts that one of the requirements for admissibility
is that the document has inherent probability of trust-
worthiness. Thus/ a trial judge has discretion in allowing
or not allowing a document into evidence if there is doubt
as to its trustworthiness (30) . One criterion for the judge
to consider is whether the particular analysis was done as
a routine matter, or whether it was specifically done for
or in anticipation of litigation (31). This is an indication
of distrust of the situation, not of the individuals involved.
3. Direct Testimony as to Microbiological Analyses
Even where records may be admitted under the special
acts described above, many government attorneys prefer to use
direct testimony of the participants. They point out that
there is a greater psychological impact involved when the
trier of facts relates a particular event or datum to a
person rather than to a piece of paper. This is particularly
true where one side relies on documents and the other side
uses convincing witnesses.
When testifying as an expert witness, you are
in a position to give opinions as to the absence or pre-
sence of microbiological growths; most other witnesses are
limited to stating what they themselves saw-or heard. Rule
702 of Rules of Evidence for the U. S. Courts and Magistrates
which will become applicable to all Federal trial courts
regardless where located puts it this way:
"Rule 702. Testimony by Experts: "If scientific,
technical, or other specialized knowledge will
assist the trier of fact to understand the evidence
or to determine a fact in issue, a witness quali-
fied as an expert by knowledge, skill experience,
training, or education, may testify thereto in
the form of an opinion or otherwise."
These rules have been promulgated by the U. S. Supreme Court
and will become effective July 1, 1973 unless voided by
Congress. Thus the expert microbiology witness must be in
a position either on direct examination or on cross-examina-
tion to justify his conclusions in all ways. This includes
both, the general acceptance of the test methodology and
that the methodology was followed to the letter.
-------
On a parallel plane, testimony on results using a
particular method may be inadmissible as irrelevant to the
issues of the lawsuit if the offense is described as including
a particular test methodology and the stated method is not
followed (32). The probable inadmissibility of MF results
to prove a violation of an MPN limit in shellfish waters is
an obvious example.
Assuming the correct methodology is used, the witness
will still have to prove the validity and accuracy of his
results. If the question concerns details of the particular
analysis, the presence of documents showing who did what and
when will obviously be of great benefit. Having such documents
will often forestall cross-examination questions on how well
you recall the details of a particular analysis when hundreds
are performed over a period of time. Such documentation is
also a great value in convincing a trier of fact that your
result is more probably correct than the one testified to
by the other party's microbiologist.
Of course/ just being more accurate and precise
does not mean that the trier will accept your results. How
to project yourself as a witness is a topic you should discuss
with your counsel or someone experienced in presenting legal
testimony. Giving an opinion in a field outside your area
of expertise and shown to be wrong impinges on the
credibility of all of your testimony and should be avoided
at all costs.
C. Conclusion
In closing, there are four areas of standardization to
consider in light of enforcement requirements of the new law
and the methods of translating data into evidence:
1. The area of standardization of equipment, reagents,
media, and filters.
2. The area of defining the preferable method for a
given type of sample—and having the yardstick included in
a discharger's permit where required.
3. Modifying Standard Methods where presently published
techniques are inadequate, e.g., in connection with ef-
fluents, particularly untreated or particularly treated
effluents, viz., should procedures be changed to compensate
for the effects of highly chlorinated effluents?
8
-------
4, The area of defining standard tests for identifica-
tion and quantification of pathogens which may be included
in the list of toxic pollutants or which may become the
subject of action under the emergency provisions of the Act,
9
-------
REFERENCES
1. PL 92-500, 86 Stat. 816, 33 United States Code (U.S.C.)
Sec. 1151 et sea.
2. Section 504 of Federal Water Pollution Control Act,
as amended (FWPCA)
3. Section 307(a), FWPCA
4. Section 311, FWPCA
5. Section 309, FWPCA
6. Section 311, FWPCA
7. Section 307(a), FWPCA and Section 502(13) FWPCA
8. Section 311(b)(2), FWPCA
9. Section 311(b)(2)(B)(ii), FWPCA and Section 311(b)(2)
(B)(iii), FWPCA
10. Section 502(13), FWPCA
11. Senate Report 92-414, P. 77
12. Section 402, FWPCA
13. Section 301(b)(l), FWPCA (Secondary treatment
may be deferred until four years after approval of
construction grants approved during FY 1975)
14. Section 301(b)(2), FWPCA; with economic exceptions, 301(c)
15. Section 306, FWPCA
16. Section 307(b), (c) and (d), FWPCA
17. Section 301(B)(i)(c), FWPCA
18. Section 402, FWPCA
19. Section 309, FWPCA
20. Forty Code of Federal Regulations (C.F.R.) Section 124.73,
published in 36FR28399, Dec. 22, 1972
10
-------
21. See Section B 2.a. of this paper
22. Section 405, FWPCA
23. Section 312, FWPCA
24. Section 301(f), FWPCA
25. Section 310, FWPCA
26. The regulations regarding public hearings under FWPCA
prior to 1972 amendments are contained in 40CFR Part
106. This part requires testimony under oath and
makes it clear that witnesses are subject to cross-
examinat ion.
27. Twenty-eight U.S.C. Sec. 1732
28. APHA, et. al. Standard Methods for the Examination of
Water and Wastewater, 13th edition, 1971. Where_
several alternative techniques are permissible with
respect to a particular determination, the specific
alternative(s) should be listed.
29. Twenty-eight U.S.C. Sec. 1733
30. LeRoy v. Sabena Belgian World Airlines, CA 2d, 344F2d 266,
1965; cert, den., 382 U.S. 878
31. cf. Hoffman v. Palmer, CA 2d, 129F2d 976, 1942; affd.
318 U.S. 109; rehearing denied, 318 U.S. 800
32. See U.S. EPA Water Quality Criteria Digest: (a) a
compilation of Federal/State Criteria on Bacteria,
August 1972 and (b) a compilation of Federal/State
Criteria on Water Quality Sampling and Analytical
Methods, August 1972.
11
-------
OFFICE OF RESEARCH ACTIVITIES: MICROBIOLOGICAL METHODS
Louis G. Swaby, Ph.D.
The current microbiological methodology research in
EPA's Office of Research covers two program elements
associated with (a) natural waters and (b) with water
supplies including recreational waters. To some extent
che methodology requirements of both programs are similar
and the recent incorporation of the water supply research
program into the Office of Research and Monitoring should
result in a more efficient use of resources. A description
of our ongoing work as taken from the official work plans
follows.
Bacterial Methodology
This program is divided into two distinct areas:
(a) methodology associated with the use of indicator
organisms and (b) methods for the detection and identifica-
tion of pathogenic bacteria.
Indicator Organisms
Water Supply Health Effects
(1) Evaluate and adapt existing techniques for the
quantitative enumeration of total and fecal coliforms as
indicators of fecal pollution to marine and estuarine
waters.
(2) Evaluation of fecal streptococci as an indicator
of fecal pollution in the marine and estuarine environments
(3) Adapt existing techniques for Staphylococcus
aureus to marine and estuarine waters for use as indicators
of human health hazard associated with primary contact
recreational activities.
Methods Development for Identification of Pollutants
(4) Improved methods for the detection of fecal
streptococci.
(5) Investigation of the use of the fecal coliform
test for industrial effluents.
*Processes and Effects Division, Office of Research and
Monitoring
12
-------
The first two tasks are aimed at obtaining the
information needed for establishing the traditional
indicator organisms as indicators of fecal pollution in
the marine environment. This is consistent with the
emphasis on recreational waters and on estuarine waters
frequently used for disposal of treated or untreated
wastes. Little is known about the characteristics of
coliforms and streptococci in the marine environment and
it may be necessary eventually to look for more appro-
priate indicators of fecal pollution, for instance a
chemical indicator.
The third task, of course, bears on the public
health hazard in recreational areas. The choice of
Staphylococcus aureus is associated with its high occur-
rence in the human environment, particularly the skin,
and consequently it should be prevalent in recreational
waters used for swimming.
The significance of the fourth task is obvious. An
important aspect of the routine use of indicator organisms
is to have rapid and accurate methods for the detection
and quantification of the organism.
The fifth task is an extremely important one. Many,
if not all, water quality standards include a fecal coliform
standard. The permit program requirements also includes a
fecal coliform measurement and it is quite likely that the
effluent guidelines and standards will also include such a
measurement. Yet, there is some question concerning its
applicability to certain effluents or to waters receiving
these effluents. In some cases regrowth of the coliforms
has been noted and in other cases one might expect the
opposite, a very rapid die off due to toxic substances.
In either case, the significance of coliform measurements
would be doubtful and challengeable.
Pathogenic Organisms
(1) Establish the potential pathogenicity of bacteria
that proliferate in the marine and estuarine environments.
(2) Adapt existing quantitative techniques, for the
organisms identified in the first task, to marine and
estuarine waters.
13
-------
(3) Investigate and evaluate cultural/ biochemical
and serological methods for Salmonella.
(4) Evaluate and adapt existing qualitative and
quantitative procedures for Salmonella.
(5) Investigate fluorescent antibody - membrane
filter techniques for enteropathogenic Escherichia coli,
Salmonella and other pathogens.
(6) Develop the reverse phage technique.
The first two tasks at present emphasize Klebsiella
which normally occurs in humans and can cause severe in-
fections. These Klebsiella-caused diseases are very dif-
ficult to treat and in one Klebsiella-related disease
mortality is still as high as 50% inspite of antibiotics.
The next two tasks are aimed at evaluating and im-
proving the methods for Salmonella. The first will inves-
tigate enrichment media, biochemical confirmation procedures
and serological methods. The results should be the defi-
nition of the most useful approach to the identification of
Salmonella in water. The second task emphasizes the
quantitative aspects of Salmonella methodology and will
investigate both MPN and membrane filter techniques.
The fifth task is an investigation of the fluorescent
antibody - membrane filter technique for enteropathogenic
E. coli. The initial effort will evaluate commercially
available antibody preparations made primarily for clinical
application. Hopefully these preparations will prove use-
ful and allow the development of a valuable technique for
the water related problem.
The last task looks at a less well known method. The
aim is to develop a screening technique which will identify
the more prevalent pathogenic bacteria in a water sample.
The technique involves plating a water sample mixed with
phage specific for the bacteria of interest. The presence
of the bacteria in the water sample will be indicated by
clear plaques where the phage have lysed the bacteria.
The technique should be rapid and easy to use and there is
some possibility that it can be made quantitative.
14
-------
Viral Methodology
The research on viral methodology is conducted under
the same program elements as the bacterial methodology
research. The emphasis is on methods to concentrate and
recover the viruses in large volumes of water. The present
tasks are:
(1) Survey water supplies in different geographical
regions for the occurrence of viruses.
(2) Investigate, improve or develop viral methodology.
(3) Quantitative detection of small quantities of
viruses in large volumes of water.
(4) Quantitative recovery of viruses from solids in
water.
(5) Preservation of field-concentrated virus samples
during transit.
The first task is for the purpose of defining the scope
of the viral problem in water supplies. The information
obtained will be of value in epidemiological studies/ in
establishing adequate treatment procedures and for identifying
the detection techniques suitable for use in monitoring
water supplie s.
The second and third tasks/ which represent the major
portion of the viral research program/ are aimed at developing,
evaluating and improving methods for sampling all types of
water for viruses. Viruses occur in water most frequently
at concentrations less than one particle per gallon. To
detect/ identify and quantify requires that/ at lease in
some cases, the viruses in hundreds of gallons of water be
removed and then recovered in appropriately small volumes
of suspending media.
Several methods of recovering viruses from large
volumes of water are being studied. Ultrafiltration is
being investigated and so far does appear to have some
applicability. The major difficulty of ultrafiltration is
its inability to handle large volumes of water in a reason-
able time. This project should be completed in the near
future. This approach will not be the answer for all
situations; however, it may prove applicable as a final
concentration procedure. The use of absorbants is also
being investigated. Of particular interest are the
15
-------
polyelectrolytes which have been shown to be very efficient
for recovering poliovirus from water. For other viruses/
recovery has been generally very low and the present effort
is aimed at finding the conditions for increased recovery
of these viruses.
*
The Melnick-Wallace Sampler developed under this
program is presently undergoing initial field evaluation
which should be completed in the near future. This device
is capable of processing 300 gallons of water per hour and
is adaptable to heavily polluted as well as to relatively
clean waters. If the initial evaluation is successful we
will undertake a complete evaluation which will include
the recovery of viruses from marine and estuarine waters.
In the fourth task attemps are being made to solve the
problem of detecting viruses which are adsorbed to solids.
A true assay of viral concentrations must include adsorbed
viruses unless it can be shown that these viruses are never
available for infection.
Lastly/ regardless of the method of sampling, the
samples must be returned to a virology laboratory to be
assayed. The fifth task seeks a method of preventing loss
of viral activity while the sample is being transported to
the laboratory.
The research activities just described are recognized
as minimal. Increased funding would allow the exploration
of new approaches in addition to the refinement of traditional
methods.
*Water Research/ Vol. 6, PP 1249-1256 (1972)
16
-------
REGIONAL CONCERN AND ACTIVITIES
RE; STANDARDIZATION OF MICROBIOLOGICAL METHODOLOGY
*
Kathleen G. Shimmin
A major Regional concern has been to demonstrate a
coordinated approach toward analytical methodology whenever
one of the various segments of the EPA Organization is
dealing with other Federal agencies and States. There is
the continuing problem of interpreting and effecting Head-
quarters' directives, as well as Regional, and yet appearing
to remain consistent.
Consistencey of Methodology
It is essential to use the same approach, not a number
of widely divergent paths when dealing with other Federal
agencies and the States within the Region. If inconsist-
encies were to become apparent, they would serve as a
cource of irritation to other agencies and render them less
than cooperative.
One example of how this has recently been a problem,
as in the field of chemistry, has been EPA's concern with
the Corps of Engineers and EPA's national dredge criteria.
The Corps spends millions of dollars on dredge projects,
with increased expenses being incurred if open water
disposal of the dredge spoils is prohibited. However, the
criteria which were issued nationally did not contain
methodology which was applicable for all the waters covered
by the criteria. Thus, the very real possibility exists
that the level of a given parameter analyzed and interpreted
in one Region might be significantly different from that
in another, even if the true values were similar. This
means that a Corps District in oae EPA Region might be
treated quite differently from that in another Region -
all because of a difference in methodology and interpretation,
With consistent methodology results and precedents from
one Region can be applied to another. Data can be inter-
changeable (which is currently not possible - even with
STORET data). The possibility of applying court decisions
from one area to another becomes increased. Pollution
originating from a State in one Region and impinging upon
waters of a neighboring State in another Region would be
identified by means of the same (or equivalent) methodology.
Laboratory Support Branch, Region IX
17
-------
With the creation of EPA, many agencies were con-
solidated into one organization. Two of these agencies were
Water Supply (Public Health Service) and Federal Water Quality
Administration (FWQA). With the consolidation came methodology
from each of these two organizations - a situation which is
still in the process of resolution. However, this could
creat a potential difficulty if both the Water Supply and
former FWQA deal with a given State and give conflicting
advice. Furthermore, if methodology were consistent, both
groups could use the same quality control program. At the
present time, these two former agencies each have separate
ongoing analytical quality control programs.*
Improvement of Methodology
With a standardized approach, mechanisms for round-robin
testing of methodology could be employed in a fashion similar
to that used in inorganic chemistry. Problems such as inter-
ference from local water would come to light. These dif-
ficulties might not surface otherwise.
With a concerted effort toward methodology standardiza-
tion a common basis for discussion would be established.
In this way, microbiologists from all the programs within
the Agency could have a chance to express their views and
to modify methods which were inadequate. Without standardiza-
tion, lack of uniform guidelines could lead to adoption of
less desirable methods.
Methodology Standardization
It has been stated that established methods might be
very slowly changed and would not keep up with current
thinking and research. Therefore, a mechanism must be built
into the standardization process to insure updating at an
acceptable frequency.
Within a Region there might be a situation in which
the standardized method might not apply. Mechanisms for
alternatives should exist. For example, in Region^IX there
are areas in Micronesia which, if they have electricity at
all, have an insufficient power supply. This means that
at times the elevated-temperature fecal coliform tests
cannot be run because the power supply is not consistent
enough to provide * reliable 44.5 i 0.2° C.
All EPA analytical quality control programs have been unified
under the Quality Control Program guidelines approved by the
Deputy Administrator on February 13, 1973.
18
-------
However, there is a concomitant fear that if alternative
methods exist, the wrong one may be chosen. This is a matter
of competency of the laboratory. With the standardization
effort, round-robin testing and exchange of information
should reduce this problem.
It is essential that there be local Regional involvement
in method evaluation. Often the Headquarters or other
centralized administration are not aware of necessary modi-
fications required by local situations. When this concept
is ignored, disastrous conditions can arise. The case of
EPA's original dredge criteria being based upon levels ob-
served in fresh water in the Great Lakes Area and then being
applied to the marine waters of other geographical areas
is a familiar example.
Regional Standardization
In addition to intra-laboratory procedures, standardiza-
tion activities of Region IX have been directed primarily
towards training State and other Federal agencies to perform
microbiological testing, quality control, and field studies
by methods compatible with those used in the Regional
Laboratory. Training has been conducted for the State of
Nevada, USGS, and Micronesia. In the spring of this year,
special courses will be given to the State of Hawaii and to
the Navy. This approach has proven to be very useful for
Region IX. It seems timely now for all the Regions and
laboratory centers to develop appropriate strategy to deal
with the problems of consistency of procedures and improve-
ment of methodology.
19
-------
MICROBIOLOGICAL PARAMETERS
-------
TOTAL COLIFQRMS
Harold L. Jeter*
Introduction
It must be assumed that all participants in this seminar
have, within the past few weeks—or days—studied the bac-
teriology portion of the current (13th) edition of Standard
Methods. Therefore, this will present an overview of the
methodology for total coliforms, not so much in terms of a
detailed account of step-by-step procedures as in terms of
identifying the nature and number of options that are avail-
able in the testing procedures. From its first edition in
1905, Standard Methods has included tests for the bacteriol-
ogical quality of water. Attention was not directed to the
group now called "coliforms" until the third edition, which
appeared in 1917.
Standard Methods
A terminology assigned to the coliform group has been
somewhat variable, though the nature of the tests makes it
clear that the tests are applied to the same group of
microorganisms. The term "coliform group" has been used
since the appearance of the 9th edition, in 1946. Prior to
that time the group was termed the "coli-aerogenes group"
in Standard Methods editions which were current between
1925 and 1946 (6th through 8th editions). Prior to that,
tests for these organisms were covered under the term
"Bacterium coli group" in the third through fifth editions,
which were current between 1917 and 1925.
A definition of the coliform group has remained relatively
stable through the various editions of Standard Methods.
Prior to 1955 the text read "It is recommended that the
coliform group be considered to include all aerobic and
facultative anaerobic Gram-negative nonspore-forming bacilli
which ferment lactose with gas formation." With the 1955
(10th) edition of Standard Methods the coliform group
definition was amended to read "The coliform group shall
include all of the aerobic and facultative anaerobic Gram-
negative nonspore-forming bacilli which ferment lactose with
gas formation within 48 hours at 35 C. " With minor work
changes this definition has been continuous to the present
edition.
*National Training Center, MDS, WPO
20
-------
With acceptance of membrance filter methods for coliform
tests, the Standard Methods Committee introduced a supplemental
definition of coliforms/ as applied to membrane filters:
"In the membrane filter procedure, all organisms that produce
a dark (purplish green) colony with a metallic sheen in 20-2
hr. of incubation are considered members of the coliform
group ." Some refinements of this definition have been
necessary; the current (13th) edition of Standard Methods
says "All organisms which produce a colony with a golden
green metallic sheen within 24 hr. of incubation are considered
members of the coliform group ."
Analytical methodology accepted as "standard" in Standard
Methods comprises two different groups of procedures; these
are the multiple tube and membrane filter methods.
Far from offering a choice of only two methods, each of
these approaches to total coliform determination comprises a
system of methodology with several identifiable options
related to the extent to which the test is to be carried,
media, and inoculation patterns. The result is that any one
of a wide range of methods or "protocols" may be selected.
For example, consider the multiple tube method. The
worker has a choice as to whether he will perform a Pre-
sumptive Test, a Confirmed Test, or a Completed Test. For
the Presumptive Test there are two choices of media standard
lactose broth or lactose lauryl sulfate tryptose broth.
Thus there are two different methods for performing the
Presumptive Test.
If the Confirmed Test has been selected, it is conducted
as a continuation from the Presumptive Test. The Confirmed
Test offers a choice of three different media - brilliant
green lactose bile broth, eosin methylene blue agar, or
Endo's agar. Based on the two identified Presumptive Test
Methods and the three Confirmed Test media, there are six
ways whereby a sample examination could be carried to the
Confirmed Test.
Continuing this line of reasoning it is demonstrable
that the six variants on the Confirmed Test would lead to a
total of 8 different methods for the Completed Test of
Standard Methods. Adding the variants of each, there are
16 ways in which coliform tests could be conducted by the
multiple tube method.
21
-------
To this, add the further complication of the number of
different available tube inoculation patterns (six are
shown in Standard Methods) and the number of different avail-
able protocols for total coliform determination soars to 96.
In fairness to the authors of Standard Methods, the
number of available options recommended for a particular
purpose is considerably lower than the above total. For
example, for potability tests of drinking water, only the
5-tube test is designated by the authors as standard.
Furthermore, in drinking water potability testing there is
no provision for terminating the analysis at the Presumptive
Test stage if positive tubes are found. The examination
must be continued to the Confirmed Test or to the Completed
Test stage. Thus there may be a total of approximately 14
different analytical pathways for drinking water testing.
Similarly, for water of other than drinking water quality,
in which quantitative data are desired, the authors of
Standard Methods have reserved their recommendations to an
inoculation scheme of 5 tubes in each of 3 or more sample
volumes decreasing by decimal increments. The alternate
use of 3 tubes for each sample volume is acknowledged but
is not strongly recommended. Thus, for water of other than
drinking water quality, a worker might have to select from
32 different test protocols.
The number of variations in standard membrane filter
procedures is not nearly so great. There is a choice between
direct incubation on the differential Endo-type medium or a
preliminary enrichment on lactose lauryl sulfate tryptose
broth; and there is a choice of whether a liquid or an agar-
solidified version of the Endo-type medium should be used.
Thus there are four variations on the standard membrane
filter test for total coliforms.
In a separate section, under the definition of coliforms
as recovered on membrane filters, a procedure is given for
verifying colong interpretation on the filter. In effect,
this comprises a Confirmed Test on individual colonies
selected for verification of interpretation.
Prior to the 13th edition of Standard ^Methods accept-
ance of membrane filter methods was conditional. Acceptance
required demonstration of suitability of the membrane filter
method by each user laboratory, through a series of parallel
tests of the membrane filter method and multiple tube method
for the waters being tested. With the 13th edition this re-
quirement has been softened and presently stands only as a
recommendation.
22
-------
Recommendations
1. It is recommended that EPA develop a manual of its
own official methods for microbiological examination of
environmental specimens. Let it be clearly noted that this
does not constitute recommendation of any proposal to abandon
Standard Methods in favor of such an agency manual.
2. As applied to tests for total coliforms, a re-
commended agency manual of microbiological methods would —
a. To the extent possible, identify the purposes
of all total coliform tests which the manual is intended to
encompass;
b. Sharply reduce the number of options in tests
for total coliforms in environmental specimens;
c. On the basis of the identified purpose of
total coliform tests, stipulate the normal first-choice
agency method;
d. Describe all test procedures in simple
language, in step-by-step fashion;
e. Supplement each test description with graphic
representation of the test protocol. Do not show optional
test procedures within any one graphic representation of a
test protocol;
f. Provide for necessary departures from the
designated procedures through stipulation of the nature of
supportive evidence demonstrating need for such departures,
and which demonstrate that the introduced substitution is
better suited to the purpose at hand. It is here emphasized
that professional judgment alone is not sufficient grounds
for departure from standardized procedures. Hard evidence
should be provided in any such case; and,
g. Provide a basis for professional consultation
with qualified personnel within or without the Agency in
matters not covered by the gency's manual of methods.
3. In my view, an EPA manual should not —
a. Designate as agency official method any procedure
which is contradictory to, or not included in Standard Methods.
23
-------
This does not imply that the Agency should avoid research into
new methods, it is perhaps better-qualified and better-
equipped to conduct such investigation than any other agency.
However, it should not unilaterally try to impose its own
agency methods on the field as a whole. The rational method
here is to work within the standard Methods system, and seek
orderly introduction of new methods and acceptance only after
adequate testing and evaluation by such laboratories as the
Standard Methods Committee leadership may prescribe. This
undoubtedly opens the door to the entire question of the
interrelationships between this Agency and the Standard
Methods leadership.
b. Seek to achieve brevity for its own sake, and
particularly, it should not seek to achieve brevity at the
expense of clarity; and finally, the manual should not —
c. Be the product of only a few microbiologists
in the Agency. All major installations' and organizational
elements of the Agency which conduct total coliform tests
of environmental specimens should be represented on any
group developing such a manual of methods. Ratification of
such a body of methods should, on a technical basis, be
limited to professional microbiologists of the Agency.
Ratification on policy, legal, and/or administrative grounds
should be performed by appropriate personnel, not necessarily
professional microbiologists.
24
-------
FECAL COLIFORM5
*
Edwin E. Geldreich
In 1904 Eijkman (1) discovered that coliform bacteria
derived from the gut of warmblooded animals would produce
gas from glucose broth at 46°C while the coliform strains
from non-fecal sources failed to grow. Subsequent research
into this concept by many investigators produced a variety
of modifications in methodology which have resulted in
increased sensitivity and selectivity. Major imporvements
include the change to a modified lactose broth fermentation
(2, 3) ; reduction in the elevated temperature requirement
from 46° to 44.5°C; the use of primary enrichment for the
multiple tube procedure (4, 5); and development of a routine
(24-hour) membrane filter procedure (6), a rapid (7-hour)
method for use either in emergency testing of water supplies
or in quick appraisal of bathing water quality (7), and a
delayed incubation technique where sample transit time
exceeds 8 hours (8).
Standard Methods
Standard Methods (9) presents several procedures for
detecting fecal coliforms in water. The fecal coliform
test is easily accomplished by a multiple tube procedure
(10) with minimal laboratory effort per analysis. The
multiple tube procedure is the preferred method for the
quantification of fecal coliforms when examining turbid
waters or chlorinated primary effluents because of the
difficulties of applying the membrane filter (MF) procedure
to these waters.
The most common approach is to inoculate EC broth
tubes from each Presumptive Test gas-positive tube using
either a transfer loop or applicator stick. EC broth tubes
are incubated at 44.5°C (+ 0.2°C) for 24 hours and then
examined for gas production. Positive-negative tube com-
binations can then be determined and the MPN calculated.
As an alternate procedure, each Presumptive Test gas-
positive tube is confirmed in Boric Acid Lactose Broth and
incubated at 43 C (+ 0.2 C) for 48 hours. Data^developed
from testing both procedures in parallel on a wide variety
Water Supply Research Laboratory, NERC-Cincinnati
25
-------
of water sources, fecal specimens, and environmental samples
indicate that Boric Acid Lactose Broth has essentially the
same selectivity and sensitivity as the EC medium. However,
the use of this medium does require 48 hours incubation to
achieve results equivalent to the 24-hour confirmed EC test.
Thus, EC broth is the medium of choice.
The direct inoculation of sample aliquots into EC tubes
without preliminary enrichment in a presumptive test medium
is unsatisfactory. Unpublished data from our laboratory show
the direct application of the selective EC medium and
immediate incubation at 44.5 C resulted in the detection of
an average of only 24 percent of the coliform population in
88 fecal specimens obtained from human and farm animals.
Parallel examination with the use of the recommended EC
confirmation procedure resulted in 90 percent recovery of
fecal coliforms from the same specimens.
The need for presumptive enrichment was also demonstrated
in studies of the minimum Escherichia coli cell density
necessary to produce gas in EC broth. The majority of 24
IS. coli strains tested required from one to 20 viable cells
to yield a gas positive reaction in EC broth, incubated for
24 hours at 44.5°C. However, three E_. coli strains required
500 or more viable cells per inoculum, thus demonstrating
that significant variability may occur. An optimal cell
density, generally in excess of 1,000 viable organisms, is
ensured when culture transfers are made from the Presumptive
Test gas-positive tubes incubated at 35 C to the more selective
EC broth incubated at the elevated temperature.
Standard Methods also includes a MF procedure for detecting
fecal coliforms. In this procedure, appropriate sample
volumes are filtered through the MF, then placed on an
absorbent pad saturated with M-FC broth (6), and contained
in tight fitting plastic Petri dishes. These cultures are
then inserted in nlastic bags and submerged in a water bath
at 44.5 C (+ 0.2^::) for 24 hours. Following incubation,
the MF cultures are then examined under low-power magni-
fication for fecal coliform colony occurrences; all blue
colored colonies are then counted; and the fecal coliform
density per 100 ml are calculated. Test accuracy has been
found to be approximately 93 percent for a variety of samples.
Other Candidate Methods
The International Standards for Drinking Water (11)
recognizes confirmatory fecal coliform tests utilizing a
26
-------
.o
choice of brilliant green bile (BGLB) broth (12), formate-
ricinoleate broth (13) , or MacConkey broth (14, 15) in the
multiple tube procedure. Fecal coliforms are considered to
be present if gas production occurs with 6 to 24 hours incubation
at 44 C (16). These confirmatory media offer no greater
convenience over EC broth and somewhat lower sensitivity for
the fecal coliform population. Preliminary studies comparing
the use of EC and BGLB media in the elevated temperature
confirmatory test (44.5 C) indicated that BGLB detected only
72.2 percent of the fecal coliforms in 24 hours whereas 92.1
percent were detected after 48 hours incubation when 24-hour
EC results were used as the base line. Significantly lower
recoveries can also be expected for lactose ricinoleate broth.
In contrast, MacConkey broth used in the elevated temperacure
test has been reported to yield some false positive reactions
because of growth of spore-forming anaerobic bacteria (14).
With respect to MF procedures, alternative methods have
also been proposed. One technique employs an initial 2-hour
enrichment on nutrient broth at^37 C followed by transferrin
the MF culture to a modified MacConkey broth for 16 hours
incubation at 44CC (16). In another method, 0.4 percent
Teepol broth is the sole test medium but cultures are Q
initially incubated at 3C°C for 4 hours and then at 44 C for
a final 14-hour period. MF cultures from chlorinated samples
are incubated at 25°C for 6 hours followed by incubation at
44°C for 18 hours (17). This more complicated incubation
temperature scheduling can be accomplished either by trans-
ferring cultures from one incubator to another or by using
a water bath that provides a programmed temperature change
at the appropriate time.
The use of programmed media and temperature changes
locks the laboratory into a very limited and rigid schedule
that can severely restrict time for processing samples^
during working hours. Alternatives include extending labora-
tory hours, using two work shifts to cover the critical
transfer time periods, or developing a dependable series of
automatic water baths capable of programmed temperature
changes.
Investigation of the modified MacConkey medium revealed
an occasional difficulty with development of a consistent
discrete yellow color confined to individual colonies.
Heavy colony concentrations and long-term incubation may
make accurate counts more difficult. Colony counts should
be made promptly after removal from the incubator to reduce
false positive occurrences.
27
-------
Tryptose bile agar (TBA) medium combined with an indol
test has also been used to detect fecal coliforms (18). In
this method, the MF isQplaced on the TBA medium and incubated
20 to 24 hours at 44.5 C; then, the MF is transferred to an
absorbent pad saturated with indol reagent. Indol positive
colonies, 12. coli Type I, become dark red upon reaction with
this reagent. Such a procedure is not strictly a fecal
coliform test since it detects only those fecal coliforms
that are indol positive. In addition, verifying the results
of this procedure has presented some difficulty because the
indol reagent is toxic and prevents colony transfer. Dis-
cernment of reaction on adjacent colonies is also difficult
because of wide zone of color development around indol
positive colonies.
Rapid Methods
Rapid assessment of the sanitary quality of water is
often needed for emergency or temporary potable water supplies,
bathing beaches whose quality may have deteriorated following
storms, and shellfish growing areas subject to sewage pollution.
Some approaches, to the quick determination of water quality
have utilized C -labeled sodium formate in a rapid (4 hr)
test for total coliforms (19, 20) . The procedure shows
considerable promise when used for fecal coliform detection
but must be refined for greater reproducibility and increased
sensitivity to coliforms at concentrations below 100 organisms
per 100 ml. A membrane filter-fluorescent antibody (MF-FA)
technique has also been proposed for rapid identification of
fecal coliforms (21, 22). Before the MF-FA test for fecal
coliforms can be considered practical, however, commercial
polyvalent antisera must be developed that include all 145
"O" antigens and 86 "K(B)" antigens identified with the
E. coli group (23) plus a few Enterobjacter and Klebsiella
strains also defined as fecal coliforms. To date, the three
commercial polyvalent antisera contain only 20 "O" and "B"
serotypes.
These rapid methods may not be adaptable to true emergency
situations where skilled personnel and specialized equipment
are not available. At present the most promising approach
involves use of a new MF procedure utilizing a lightly
buffered, lactose-based medium containing an acid-sensitive
indicator system and incubation in a water bath at 41.5 C
for 7 hours. Colonies should be examined at 20 to 30X
magnification and all yellow colonies, both pale and bright,
should be counted as fecal coliforms. In a study of colony
28
-------
verification, 94.3 percent of 4,082 yellow colonies from
the 7-hour medium were verified as fecal coliforms and, from
the same samples, 93.7 percent of 4,034 blue colonies on the
M-FC medium after 24 hour incubation, were verified as fecal
coliforms. These data indicated that both media are measuring
essentially the same population of organisms. Advantages
of this procedure include the 7-hour time factor versus the
24-hour normal test time and the knowledge that all necessary
test equipment is normally available in most bacteriological
laboratories.
Delayed Incubation Procedure
Sample transit time (8-hour limit) is especially critical
for bacteriological examination of waters from stream pol-
lution investigations or from the monitoring of remote
sampling locations. Where special courier service or the
use of mobile laboratory units is not feasible, the use of
a delayed incubation test for fecal coliforms is the solution.
In this procedure, MF cultures are held on vitamin-free
casitone (VFC) holding medium during transport to the pro-
cessing laboratory (8). Laboratory and field results have
shown that inoculated MF can be held on VFC holding medium
for up to 72 hours with little effect on fecal coliform
counts. Upon arrival in the laboratory, the MF cultures are
promptly transferred from the VFC holding medium to m-FC broth
and incubated, submerged, in a 44.5 C water bath for 24+2
hours. Following incubation, fecal coliform colonies are
counted as in the standard fecal coliform test. Correlation
coefficients between the delayed MF procedure and the im-
mediate fecal coliform test for 1, 2 and 3 days were 0.93,
0.95 and 1.09, respectively.
Suggested Recommendations
Fecal coliform methodology must include both a multiple
tube method and a MF procedure for flexibility in applications
to field investigations and monitoring requirements. Where
sample transit time is beyond an 8-hour limit, a suitable
delayed MF procedure is needed as well as some simplified
emergency procedure for rapid detection of fecal coliforms.
Methods chosen should be reproducible, have a high order of
specificity for the fecal coliform group, and utilize
equipment and materials that are readily available.
To meet these criteria it is proposed that methodology
for the multiple tube test be restricted to EC confirmation
at 44.5°C for 24 hours after transfer of culture from gas
29
-------
positive 24-hour or 48-hour tubes of lauryl tryptose lactose
broth incubated at 35 C. The recommended MF procedure should
involve the use of M-FC broth, with incubation of cultures
in a water bath at 44.5 C for 24 hours. These two tests are
included in Standard Methods for the Examination of Water
and Wastewater (9).For those special cases involving
emergency testing of water supplies suspected of being con-
taminated and decisions on beach closings or openings, the
7-hour, rapid, FC test must be considered. Finally, a
delayed procedure that employs VFC holding medium has proven
practical and fills a definite need in the quantitation of
fecal coliforms.
30
-------
REFERENCES
1. Eijkman, C. Die garungsprobe bei 46° als hilsmittel bei
der trink-wasseruntersuchung. Centr. Bakteriol.
Parasitink., Abt. I;, orig. 32:742-752 (1904).
2. Hajna/ A. A., and Perry, C. A. Comparative Study of
Presumptive and Confirmatory Media for Bacteria of
the Coliform Group and for Fecal Streptococci.
Amer. Jour. Pub. Health, 35_i 550-556 (1943).
3. Vaughn, R. N., Levine, M., and Smith, H. A. A Buffered
Boric Acid Lactose Medium for Enrichment and Pre-
sumptive Identification of Escherichia coli. Food
Research, .16:10-19 (1951).
4. Clark, H. F., Geldreich, E. E., Kabler, P. W., Bordner,
R. H., and Huff, C. B. The Coliform Group I. Boric
Acid Lactose Broth Reaction of Coliform IMViC Types.
Appl. Microbiol., 5/.396-400 (1957).
5. Geldreich, E. E., Clark, H. F., Kabler, P. W., Huff,
C. B., and Bordner, R. H. The Coliform Group II.
Reactions in EC Medium at 45 C. Appl Microbiol.,
Ł: 347-348 (1958).
6. Geldreich, E. E., Clark, H. F., Huff, C. B., and Best,
L. C. Fecal-Coliform-Organism Medium for the Membrane
Filter Technique. Jour. Amer. Water Works Assoc.,
Ł7:208-214 (1965).
7. Van Donsel, D. J., Twedt, R. M., and Geldreich, E. E.
Optimum Temperature for Quantitation of Fecal Colifonus
in Seven Hours on the Membrane Filter. Bact. Proc.,
American Soc. Microbiol., page 25 (1969).
8. Taylor, R. H., Bordner, R. H., and Scarpino, P. V.
A Delayed Incubation Membrane Filter Test for Fecal
Coliforms. Appl. Microbiol. (In Press)
9. Standard Methods for the Examination of Water and Waste-
water. 13th edition Amer. Pub. Health Association.
New York (1971).
10. Geldreich, E. E. Sanitary Significance of Fecal Coliforms
in the Environment. U. S. Dept. of Interior, F.W.P.C.A.
Water Pollution Control Research Series Publication No.
WP-20-3. Nov. 1966, 122 pp (Cincinnati).
31
-------
11. World Health Organization, International Standards for
Drinking Water. 3rd edition, WHO, Geneva, 70 pp. (1971)
12. Mac Kenzie, E. F. W., Taylor, E. W., and Gilbert, W. E.
Recent Experiences in the Rapid Identification of
Bacterium coli type I. Jour. Gen. Microbiol., J2:
197-204 (1948).
13. Public Health Laboratory Service, Standing Committee on
the Bacteriological Examination of Water Supplies.
Confirmatory Tests for Coliform Organisms. Jour. Hyg.
(Camb.) 66:641-647 (1968).
14. Public Health Laboratory Service, Water Sub-Committee.
The Effect of Anaerobic Spore-Bearing Organisms on the
Validity of the Presumptive Coliform Test as Used in
the Bacteriological Examination of Water. Jour. Hyg.
(Camb.), J31: 268-277 (1953).
15. Bonde, G. J. Bacterial Indicators of Water Pollution.
422 pp., Copenhagen (1962).
16. Windle Taylor, E., Burman, N. P., and Oliver, C. W.
Membrane Filtration Technique Applied to the Routine
Bacteriological Examination of Water. Jour. Inst.
Water Engrs., 2:248-263 (1955).
17. Burman, N. P., Oliver, C. W., and Stevens, J. K.
Membrane Filtration Techniques for the Isolation from
Water, of coli-aerogenes, E s che r i chi a coli, Faecal
Streptococci, Clostridium perfringens,, Actinomycetes
and Microfungi. pp. 127-134 in Isolation Methods for
Microbiologists, Technical Series No. 3, edited by
Shapton, D. A. and Gould, G. W. Academic Press,
178 pp. (1969).
18. Delaney, J. E., McCarthy, J. A., and Grasso, R. J.
Measurement of E_. coli Type I by the Membrane Filter.
Water & Sewage Works, 109;289-294 (1962).
19. Levin, G. V., Harrison, V. R., Hess, W. C., and Gurney,
H. C. A Radioisotope Technic for the Rapid Detection
of Coliform Organisms. Amer. Jour. Pub. Health,
46:1405-1414 (1956).
20. Scott, R. M., Siez, D., and Shaughnessy, H. J. I.
Rapid Carbon-14 Test for Coliform Bacteria in Water.
Amer. Jour. Public Health, J34:827-833 (1964).
32
-------
21. Danielsson, D. A Membrane Filter Method for the
Demonstration of Bacteria by Fluorescent Antibody
Technique. 1. A Methodological Study. Acta Path.
et Microbiol. Scandinav., j53:597-603 (1965).
22. Ginsburg, W., Crawford, B., and Knipper, J. J. Filter-
fluorescent Antibody Technique for Rapid Screening of
Indicator Organisms. Jour. Amer. Water Works Assoc.,
j54:499-505 (1972).
23. Edwards, P. R., and Ewing, W. H. The Escherichia coli
Group in Identification of Enterobacteriaceae.
Burgess, Minneapolis (1962).
33
-------
FECAL COLIFORM DISCUSSION
Bordner:
Geldreich:
Bordner:
Geldreich;
Berg:
Knittel:
Stang:
Geldreich:
Will you make a comment on the possibility of
applying the PC test on industrial effluents
in light of Klebsiella pneumoniae occurrences?
At the Denver Conference we concluded that
(1) Klebsiella pneumoniae is an inhabitant of
the intestinal tract, (2) inclusion with the
FC group as defined at 44.5°C is not detrimental
to test interpretation, and (3) the presence of
Klebsiella in the fecal material of over 40%
of humans, indicates we can't ignore it.
Well there was some talk about elevating the
incubation temperature to 45.0 C.
Elevation of the test to 45.0° was proposed not
so much to eliminate Klebsiella detection, but
to screen out non-fecal coliform organisms
that, at times, do cause interferences in fecal
coliform colony discernment.
Do any of the Klebsiella that are growing out
on M-FC medium actually appear as fecal coliforms?
Yes, we have recovered several strains that
give typical dark blue colonies on M-FC medium
and they are EC+.
Will raising the temperature from 44.5 to
45.0°C cause some loss of fecal coliforms?
I see no problem in this change to 45.0 C.
However, when incubation temperatures exceed
45.7 C, we are in trouble because of the sharp
die off of fecal coliforms. We had originally
set the temperature tolerance at ± 0.5 C.
However, the shellfish program felt that this
was too broad a tolerance. Thus, to be com- o
patible, we agreed to make the tolerance io.2 C
since there are many labs along the coast that
examine both shellfish waters and fresh waters.
Having the same temperature tolerance limits for
both types of sample circumvents the need for
two different water baths, differing by less
than 0.5 C.
34
-------
Geldreich:
Cabelli: We have data from several different estuarine
areas along the east coast where we have used
M-FC medium. We get sometimes as low as 40%
recovery using the MF procedure compared to
another unselective method where the colonies
are confirmed. So I would caution, in terms of
estuarine waters at least, that the MF fecal
coliform test may produce underestimates.
I would like to zero in a little closer on that
problem by warning that before condemning a
procedure, an investigation of the commercial
media supply must be made. Don't comdemn a
procedure until you are certain that the problem
is not with a bad lot of commercial media. I
have become so alarmed about the problem of
commercial media quality that I am seriously
considering preparing a questionnaire for the
response of all EPA and State Laboratory
Directors, as well as all interested bacteri-
ologists in various federal, State, county,
city, private, water treatment and sewage
treatment laboratories, to determine what
problems they have had with the quality of
commercial media supplies. I have recently
been in several State Health laboratories where
I was informed of low recovery and poor colony
differentiation with one brand of M-Endo medium.
The manufacturer did exchange the bad lot for
another lot of M-Endo medium but no effort was
made to recall the defective lot form lab
suppliers or unknowing consumers. Similar
problems have been noted with lots of M-FC
medium, BGLB broth, and standard plate count
agar. What Bordner and his group must include
in their activities is a media evaluation program
or some mechanism to assist with the standardiza-
tion of media, materials (MF) and instrumentation.
Cabelli: This problem is entirely possible. However, at
least in our own studies, we used two different
lots and two different manufacturers and ran
into the same problem. It may be that this
problem is widespread in estuarine waters, and
I would, therefore, like to offer a word of
caution when using this procedure with estuarine
waters.
35
-------
Geldreich:
Cabelli:
Geldreich:
Cabelli:
Geldreich;
Cabelli:
Geldreich:
Knittel:
If this is true then we must use the multiple
tube procedure in these instances.
Concerning Klebsiella and industrial wastes;
we took a look at textile finishing plants
where there is the same kind of problem as
with paper mill effluents. In particular,
there was one plant where there was no con-
ceivable way they had fecal pollution. In
fact/ what they do is use sewage as a starter
or fermenter to degrade the starch materials.
There was never any feces in the "starter" and
this fermenter has been in use for two years.
There is no history of feces in this material.
Once you introduce this sewage "starter" they
[fecal coliforms] will persist for long periods.
We had this problem with sugar beet wastes.
The point is how do you justify that there is
in fact fecal pollution? There is no history
of feces being in this material.
If those organisms can survive in the "starter"
for two years we are concerned that pathogenic
organisms could also survive during this period.
Obviously there is no indication there are
viruses present in this "starter," so now we
must be talking about Salmonella.
— or other pathogens, not necessarily just
Salmonella, that are capable of persisting in
that fermenter environment for long periods.
[We have been doing studies] using a continuous
flow system composed of primary settled pulp
mill effluent inoculated with sewage. After
three months, Klebsiella are beginning to take
over. A profile of coliforms has been done.
At the start Klebsiella occurred in less than
one percent of the samples; now it occurs in
more than 80 percent. As far as any significance
to fecal pollution, [I feel] as long as they
can grow other pathogens could also grow.
36
-------
Cabelli:
Geldreich:
Cabelli:
Shedroff:
Brezenski;
Berg:
Can you define fecal pollution from a legalist
point of view in this case?
Yes, it started with fecal pollution and we
are working with something that has persisted
in culture; now it is an entirely different
menstruum.
Now you're talking about pathogens that are
surviving on nutrient pollution, but can you
say this is fecal pollution, per se?
I don't know the answer to that. What we are
talking about in development of waste discharge
permits is a limit that will indicate that there
is no fecal pollution; that the material has
been treated properly under the definition of
best practicable treatment of the waste. It is
not necessary to make that tie in with pathogens.
It is only necessary to say that if you have
properly treated you will have a certain level
of quality and this is the quality we are
expecting. You just don't have to demonstrate
pathogens. However, the legislation being
developed to define quality of industrial
effluents may require a valid indicator to show
that a particular effluent has been treated
properly, as required.
Fecal pollution implies a hazard and I don't
care if those organisms are from recent feces
or have been in the system for some time. The
point is that they are organisms that indicate
a public health hazard.
The point is, these organisms were recovered
from a supply of water of some sort and, in terms
of a positive reaction at 44.5 C, are indicators
of fecal pollution.
Are there other organisms that have been shown
to grow at 44.5°C on M-FC medium which are not
fecal coliforms?
37
-------
Geldreich;
Berg:
Geldreich:
Berg:
Geldreich;
There is a recent report in Applied Microbiology
of finding one "odd-ball" organism in hot
spring water from Yellowstone National Park.
There is a possibility that some false positive
organisms could grow on M-FC medium at 44.5 c,
such as some thermophiles. The key is the
requirement for lactose fermentation at the
elevated temperature. This eliminates a good
portion of the thermophilic population that
could otherwise cause a problem.
I recall reading reports that Aeromonas will
grow on M-FC medium and this organism is found
in streams.
Aeromonas has been reported in this year's
German literature to be capable of growth at
an elevated temperature. These authors had
suggested some modifications in their medium
(not M-FC medium) to suppress Aeromonas, which
was giving a problem of interference. I don't
know what it would look like on the medium we
developed. I have not had a chance to check out
the growth response of Aeromonas on M-FC medium,
and, therefore, am not aware of the colony
color or possible problems of interference.
Of all of the organisms we have studied on
M-FC medium (over 10,000 strains), we have never
identified an Aeromonas to date, so that is
all I can say, but that is not all inclusive.
All I am trying to point out is that in a
court of law this question could be brought up.
Aeromonas is becoming a real problem in fish
hatcheries as a fish pathogen. There is some
evidence of this problem in the recent literature.
In discussions with Bobby Carrol, this morning,
he expressed a concern about this organism as
a fish pathogen in the Region IV area. Thus,
although Aeromonas may not be related to a human
wealth hazard, it may be a problem in a very
unique way.
38
-------
Carrol:
Cabelli:
Geldreich:
Cabelli:
The contact we have had with Aeromonas was
primarily in a wildlife area. Aerompnas
liquifaciens was a secondary invader causing
haemorrhagic septicemia in fish. This was
attributed to be the casue of a fish kill.
We have been looking at methods for Aeromonas
detection in marine and fresh waters and have
looked at Aeromonas occurrence in natural
waters. in our studies we have not found
Aeromonas growing on M-FC medium. We have
found Aeromonas growing on M-Endo broth which
presents a real problem. Aeromonas can be
isolated in large numbers; 1 or 2 orders of
magnitude less than total colifonus in raw
and treated sewage.
Aeromonas has been reported in the current
literature to occur in very high numbers when
the total coliform population is very low. There
may be, therefore, some difficulty in interpre-
ting the sanitary significance of Aeromonas.
Their occurrence is a problem in fish culture/
but whether we can use this group as an
indicator of human health hazard in bathing
waters is debatable. There is beginning to
appear a tremendous amount of literature
indicating Aeromonas may be present in natural
waters in high densities, but what is the
significance?
I think I can address myself to that question.
First of all, we have looked at Aeromonas in
Narragansett Bay and as you go away from a
pollution source the Aeromonas levels decrease.
Near the pollution source Aeromonas densities
are high. Secondly, it is capable of multi-
plying, at least in fresh water environment.
Thirdly, Aeromonas is a pathogen, as can be
cited in the literature, being isolated from
urine, feces and even some systemic illnesses.
We are speaking not of an indicator but a
pathogen which has the ability to multiply in
the aquatic environment. This being so, you
can get higher counts in the stream than were
39
-------
shown to be discharged. It is in the feces
of a certain number of individuals, and,
from the work we did, it seems its erratic
appearance in sewage suggests it is multiply-
ing in the course of the treatment process.
Furthermore, it can multiply in the aquatic
environment, which means the numbers you find
may be disproportionate to the numbers one
would expect out of feces. Bonde, I thinly, has
suggested we consider Aeromonas as a possible
indicator. I would suggest we may have to
worry about Aeromonas as a potential pathogen
for humans as well as fish. The primary
Aeromonas fish pathogenic strain will not
grow at the elevated temperature. In fact,
it does not grow well at 37 C unless you
employ a very enriched medium.
Geldreich: In discussions with Dr. Bonde last March, in
a WHO Conference on methodology for measuring
bathing water quality in coastal areas, I was
convinced he is not that strong about the use
of Aeromonas. Dr. Bonde would prefer that the
fecal coli test be used, which is identical to
our definition of a fecal coliform test. That
is, most European bacteriologists don't run
out IMViC's on the gas positive results at the
elevated temperature. He did say that he can
find Aeromonas present in large populations
in the estuarine environment, but does not
know how to interpret their significance.
This WHO Conference chose not to consider
Aeromonas as a parameter for bathing water
quality at this time because of the confusion
as to their significance.
You know what this differnece really is
between the 12. coli test and a fecal coliform
test—only a 5 to 7% difference. Thus, if we
were to narrow the definition of fecal coli-
form to E, coli and exclude Klebsiella
pneumgniae, we are going to be accepting a
larger health risk. The British are actually
using an enrichment procedure, then confirma-
tion in a lactose type broth at 44 C; generally
ending the test there. It is not always true
40
-------
Cabelli:
Geldreich:
Cabelli:
Resi:
Berg:
Resi:
that an indole test is being done in those
laboratories as part of the confirmatory
procedure. Much of their monitoring of
water quality by the elevated temperature
test is very similar to our fecal coliform
test, but involves glutamic acid broth at
37 C, with confirmation in brilliant green
broth or MacConkey broth at 44 C.
The IMViC test is not adequate and we should
be looking at other biochemical tests such
as the decarboxylase reaction. It is now
time to go back and look at all of these
kinds of environments and look at the composi-
tion of the coliform population; i.e., whether
they are influenced by certain kinds of pollu-
tion; what is the Klebsiella portion of the
population, what proportion of the population
is Aerobacter aerogenes, or Enterobacter
cloacae or E_. coli?
This would be like discovering the wheel all
over again—going back to repeat so much
old research ground.
Well, you know environments change, societies
change and the nature of pollution changes.
Thus, those things that were true at one time
may not be true now.
One of the things I want to bring up is the
temperature requirement for the fecal coliform
test. The use of - 0.2 for temperature
tolerance is difficult to obtain in the field.
Ed seems to think this temperature tolerance
is obtainable very readily, but I question
that it is.
I can tell you there is no problem in main-
taining 0.2 C in a system if you use a system
capable of doing it. We do it all the time.
I agree with this but how many people do this
all the time?
41
-------
Geldreich: Critical factors in maintaining close temperature
tolerance include gable tops over baths/ some
method to defeat temperature stratification,
bi-metallic strips in the thermostatic control
that are sensitive to the subtle changes in
temperature and an accurate thermometer.
Gordon: Small commercial units are very good in the
laboratory but you go out into a field labor-
atory where we don't have temperature control-
ability. With a small water bath that can be
put in a plane you get stratification, and
fluctuations in temperature become a problem.
Geldreich: Now we are talking about the real world of
field laboratory work where these problems can
be difficult to surmount.
Shedroff: Now we are talking about not what a research
lab can obtain in temperature tolerance and
other refinements but primarily what an
industry laboratory or state laboratory can
obtain on a regular basis.
42
-------
ISOLATION AND IDENTIFICATION OF KLEBSIELLA PNEUMONIAS
M. D. Knittel, Ph.D.*
It has been considered for many years that coliform
bacteria with an IMViC profile of h+ were of soil or plant
origin and when found in water were of no sanitary significance,
All of the tests of microbiological potability of water were
arrived at to separate the fecal coliforms from the non-fecal
colifonus. The IMViC determination represents the most used
tool to accomplish this task.
Recent findings have shown that some waste water
effluents contained high numbers of coliform bacteria with
IMViC formulae of —++ (Bauer, unpublished results, EPA,
Region X, Seattle, Washington). Further work demonstrated
that these coliforms were non-motile and did not decarboxylate
ornithine. These cultures were submitted to the U. S. Public
Health Center for Disease Control and their finding was that
these coliforms were Klebsiella pneumoniae.
It has been found (Knittel, unpublished data) that in
spent sulfite liquor wastewaters, K. pneumoniae can constitute
up to 90 percent of the coliforms present. At the same time,
the elevated temperature and medium of the fecal coliform
test will allow some K. pneumoniae to grow. Although most of
these colonies are atypical, a few will appear as typical
fecal coliform colonies, i.e., dark blue, flat colonies.
The significance of the occurence of K. pneumoniae in
certain industrial effluents has been questioned by the
National Council for Stream and Air Improvement(1). Whether
2S- pneumoniae is a primary health hazard or only an indicator
of fecal pollution is immaterial; clearly, the presence of
K. pneumoniae is itself an indication of conditions detri-
mental to human health.
The methods of isolation and enumeration of K. pneumoniae
are not included in the recent edition of Standard Methods of
Water and Wastewater Analysis. The methods used in that text
are devoted to the selection and separation of fecal coliform
bacteria. Therefore, any coliform bacterium that has an
IMViC formula of M- is discarded.
*Pacific Northwest Environmental Research Laboratory,
NERC-Corvallis.
43
-------
Quantitative methods of isolation and enumeration of
*Ł• pneumoniae do not appear in the literature either. Most
media that are inhibitory for coliforms are also inhibitory
for K. pneumoniae and thus there is not a medium available
for the direct enumeration of K. pneumoniae. The author
has employed a basal salts lactose medium that was deficient
in nitrogen. The lack of nitrogen was designed to take
advantage of the ability of K. pneumoniae to fix atmospheric
nitrogen(2). Preliminary evaluation failed to support the
concept and further work was discontinued.
The following procedure is used at this laboratory to
isolate and identify K. pneumoniae from samples of water and
wastewater: Appropriate dilutions of the sample are filtered
through a 0.45 Millipore membrane filter. After washing,
the membrane is placed in a petri dish containing M. Endo
LES agar (Difco, Detroit, Michigan) and incubated at 35 C
for 24 hours. At the end of the incubation period a count
of the sheen producing colonies is made and recorded.
A representative number of these colonies are picked
at random and transferred to triple sugar iron agar slants
(TSIA) and nutrient agar slants (NA) .
These slants are incubated at 35°C for 24 hours. The
reaction of the TSIA are recorded and indophenol oxidase
test is performed on the nutrient agar slant (3). The TSIA
slants that are indophenol oxidase negative and show a TSIA
reaction of: acid slant and acid butt with or without gas
and no H?S production are considered to be coliforms. Transfers
are made into tryptone, buffered glucose, citrate, malonate,
motility, urea agar, KCN, Lysine, ornithine decarbonylase and
arginine dihydrolase media. The above cultures are incubated
up to five days at 35 C with daily observations.
Cultures isolated from a sample which are IMViC -(+) -
++, non-motile, lysine decarboxylase positive but arginine
dihydrolase negative and ornithine decarboxylase negative,
which are urea; KCN and malonate positive are K. pneumoniae.
Further confirmation can be done by passing the culture in
a medium to enhance capsule production such that of Hoogerheide
(4) or Wbrfel and Purgeson (5) and then typing the K or
capsular antigen.
The estimation of the percentage of K. pneumoniae can
be calculated by dividing the number of K. pneumoniae identified
by the number of isolates originally picked. This percentage
times the total coliform count will give the number of K.
pneumoniae in the original sample. Of course this is a
relative and somewhat arbitrary figure. However, in leu
of a selective medium for K. pneumoniae this procedure, at
least, will give a first approximation of numbers of K.
pneumoniae present in samples tested.
44
-------
LITERATURE CITED
1. Blosser, R. O. 1971. Experience with indicator
organism tests in determining the bacteriological
quality of pulp and paper mill effluents and their
receiving waters. National Council of the Paper
industry for Air and Stream Improvement. Tech.
Bull. No. 244. 56p.
2. Mahl, M. C., P. W. Wilson, M. A. Fife, and W. H. Ewing
1965. Nitrogen Fixation by Members of the Tribe
Klebsiellae. J. Bact. 89: 1482-1487.
3. Edwards, P. R. and W. H. Ewing. 1972. Identification
of Enterobacteriaceae. Burgess Publishing Co.
Minneapolis, Minn. 362p.
4.
Hoogerheide, J. C. 1939. Studies on capsule Formation,
1. The conditions under which Klebsiella pneumoniae
(Friedlanders Bacterium) Forms Capsules. J. Bact. 38
367-371.
5. Worfel, M. T. and W. W. Ferguson. 1951 American J.
Clin. Path., 21, 1096.
45
-------
DISCUSSION; A SUMMARY
The selection of colonies from the primary culture
for identification of K. pneumoniae is necessary because
of the lack of a selective or a differential medium for
the bacterium. This approach does allow one to determine
the number of K. pneumoniae in the original sample by
calculating the percentage of the isolates that it repre-
sents. In addition, it also provides information as to
what other types of coliforms make up the total population.
This information could be useful in determining the source
or fate of coliforms in water.
The classification of coliforms has undergone some
changes from what is listed in Berqy's Manual of Determinitive
Bacteriology. The separation of K. pneumoniae from Enter-
obacter aeroqenes (Aerobacter aerogenes) was not certain
and was based primarily on source, encapsulation and
motility. Thus, a gram negative rod-shaped bacterium,
that fermented lactose and was non-motile isolated from
soil, plants or water was classified as E. aerogenes
(A. aerogenes).
The development of a medium and procedures for determining
the ability of bacteria to decarboxylate amino acids (Moeller
1965)* has provided a means to separate K. pneumoniae from
E. aeroqenes (A. aeroqenes). This development has shown that
not all of the IMViC —++ coliform bacteria are E_. aeroqenes
(A. aeroqenes). The presence of K. pneumoniae in certain
industrial wastewaters raises the question whether these
organisms are primary pathogens or indicators of fecal
pollution.
* Moeller, V. 1955. Simplified Tests for Some Amino
Acid Decarboxylases and for the Arginine Dihydrolase
System. Acta Path, et Microbiol. Scandin. 36, 158*-172
46
-------
FECAL STREPTOCOCCI
*
Francis T. Brezenski
In the past it has been the tendency to think in terms
of a universal indicator system - one system which would
apply to the highest percentage of cases or conditions that
exist. It is apparent that as technology has increased, so
has the diversity of waste materials. Problems with in-
dustrial wastes have arisen which indicate that bacterial
parameters will have to be more specialized and show the
closest compatibility or relevancy to the substrate being
tested. Simplicity can no longer be a major or decisive
consideration in choosing testing procedures. The most
accurate, descriptive and rapid system is desirable. It
is from this frame of reference that pertinent methodology
is being chosen.
For a number of years the fecal streptococci have been
one of the most controversial groups of bacteria. Micro-
biologists have had great difficulty in defining, utilizing,
and interpreting data collected from fecal streptococcus
assays. It is true that the fecal streptococci possess
desirable characteristics which warrant consideration and
further development. These are: host specificity, lack of
multiplication in the aqueous medium, serological character-
istics which demonstrate potential application to automated
detection and identification systems and finally, an increased
interest in the association of the Group D Streptococci in
specific disease processes. On the other hand, many problems
exist such as lack of standardization in biochemical testing.
This is crucial since present identification is primarily
based on biochemical characterization.
Standard Methods for the Examination of Water and Waste-
water (SM), 13th Edition, 1971 lists three methods for the
assay of fecal streptococci: Multiple Tube Technic (MPN),
Membrane Filter Technic (MF) and a tentative Fecal Strep-
tococcal Plate Count procedure. The MPN procedure involves
a Presumptive Test using Azide-dextrose broth and a Confirmed
Test utilizing Ethyl violet azide broth. The MPN test was
developed to determine the density of enterococci in sewage.
Density determinations are estimates based on statistical
probability measurements. The more rapid MF technique,
unlike the MPN procedure, is a direct measurement of fecal
streptococci on a membrane filter. The test can be used to
*
Technical Support Branch, Region II
47
-------
assay fecal streptococci in water and non-chlorinated sewage.
A choice of either M-Enterococcus or KF agar medium is offered
to the analyst. The Fecal Streptococcal Plate Count (FSPC)
is a pour plate technique using M-Enterococcus or KF Strep-
tococcus agar. It is suggested as an alternate method for
the MF procedure when highly turbid waters containing low
numbers of fecal streptococci are encountered. At the present
time it is listed as a tentative procedure.
In order to choose the most effective and expedient
methodology, one must first define what the detection system
is and what it is detecting. Secondly, one must know the
sensitivity and reproducibility of the method. The first -
definition of the detection system - needs to be made clear
and agreed upon. To understand this, one must look at the
major developments and present status of the fecal strep-
tococcus group.
The fecal streptococci were proposed as indicators of
fecal contamination as early as 1910. However, the United
States application of this group of bacteria to pollution
detection did not take place until the post World War II
period. Since that time numerous reports have appeared in
the literature concerning methods and media for the isolation
and enumeration of the fecal streptococci. Sixty-eight (68)
different types of media have been proposed in one form or
another for the selective isolation of these organisms (1).
Such a list demonstrates the difficulty and complexity in
selecting out the microbes from the existing heterogeneous
microflora. The diversity of this group of organisms
created serious problems with taxonomy and classification.
To further complicate the problem, a number of terms -
(some used synonymously) appeared in the literature. Fecal
streptococci, enterococci, Lancefield Group D streptococci,
Enterococcus Group etc. were used to denote the bacteria
in question. Numerous divisions in groups and species,
involving biochemical reactions or serological analysis
and/or other characteristics have been proposed.
The initial task therefore was to establish which
streptococci are significant in determining the sanitary
quality of the substrate being tested. Sherman (2) divided
the streptococci into four basic groups: Pyogenic, Viridans,
Lactic/ and Enterococcus. This division represented a
practical application to pollution investigation. The
Viridans and Enterococcus Groups contained species which
-vere associated with the alimentary tract of warm-blooded
animals. Organisms from both groups were combined to form
48
-------
the Fecal Streptococcus Group. Hartman et al. (3) in 1966
defined the various species of the Fecal Streptococcus Group.
The Enterococcus Group consisted of S_. faecalis and its
varieties liquefaciens and zymogenes, j5. faecium and its
variety durans. These organisms shared a common antigen,
hence were serologically classified as Group D organisms.
The Viridans Group organisms consisted of _S. bovis, j3. eguinus/
S.. mitis/ and _S. salivar-ius.
The most important bond of the fecal streptococci was
the fact that all of the species were associated with fecal
material. It was not long, however, when Mundt (4) found
that Enterococcus Group organisms may be chance contaminants
of plants. Such organisms were recovered from a number of
different plants in areas remote from man. This finding
seriously challenged the group as strict indicators of fecal
.contamination.
A later report by Geldreich and co-workers (5) indicated
that the presence of these organisms on vegetation may, at
least in part, have been derived from insect contact. This
seemed plausible since previous reports in the literature
demonstrated the presence of Enterococcus Group organisms
in various species of insects.
Besides establishing the levels and streptococcal types
on vegetation and insects, Geldreich and co-workers performed
concurrent coliform and fecal coliform determinations.
Results suggested that typical fecal colifonus, as measured
by the fecal coliform test, contributed only a small per-
centage of the organisms associated with insects and
vegetation. Therefore, it was concluded that the contribu-
tion from warm-blooded animal pollution was a minor factor
in both vegetation and dissemination by insects.
The higher densities of streptococci found on vegetation
were explained as being streptococcal types not of warm-
blooded origin. These strains hydrolyzed starch, even
though all other biochemical criteria were typical for the
Enterococcus Group as defined by Sherman (2). Strains of
S,- faecalis, however, isolated from water, warm-blooded
animals, and cold-blooded animals have never displayed this
characteristic. The starch hydrolyzing strains were called
atypical S.. faecalis and were interpreted as being of
vegetative source.
A very recent report by Martin and Mundt^(6) showed
high numbers of Enterococcus Group organisms in insects
49
-------
collected from non-urban, wild and cultivated fields and
woods. More than 93% of the cultures of S_. faecal is isolated
digest casein milk from the top downward, following the
production of soft flowing curd. This property is not a
characteristic of S_. faecal is isolated from humans. The
litmus milk reaction therefore discriminates between cultures
associated with insects and vegetation and cultures derived
from human origin.
The above has served to illustrate the need to specify
which organisms form the basis of the detection system and
how they can be discriminated from other forms. Discrimina-
tion was based on biochemical characterization. The techniques
used for biochemical characterization are long, involved,
quite vulnerable to error during laboratory manipulations and
lack standardization. For these reasons, and the fact that
the fecal streptococci share a common antigen, fluorescent
antibody techniques have been proposed for the detection
and identification of species belonging to the group.
In order to develop fluorescent antibody (FA) techniques
for the identification of Group D streptococci, it was first
necessary to isolate strains from a number of sources.
Strains would then be used in the preparation of the conjugate
which will form the basis of the detection system. The
University of Massachusetts (7), in concert with the EPA
Region II laboratory at Edison, New Jersey isolated a number
of fecal streptococcal strains from various sources and
developed a conjugate for the identification of Group D
Streptococci. In order to ensure isolation of all fecal
streptococcal strains present, five (5) different media
containing different classes of inhibitory agents were
used: M-Enterococcus, KF-Streptococcus, Thallous-Acetate,
Azide-Sorbitol, and Pfizer Selective Enterococcus (PSE)
Agars. KF and M-Enterococcus agar contain sodium azide and
2,3,5 triphenyltetrazolium chloride (TTC), while PSE in-
corporates bile and sodium azide as main constituents to
attain selectivity. Domestic sewage and the feces of sheep,
cattle, horses, rabbits, chickens, geese, and rats were
analyzed for streptococcal species. Additional sources
that were investigated included frozen and non-frozen foods.
The lowest level of fecal streptococcus recovery
occurred on M-Enterococcus and Azide-Sorbitol Agars.
Thallous-Acetate agar was the most productive, however, it
was the least selective since 58% of the isolates were non-
fecal streptococci. KF and PSE Agars yielded the highest
recovery of fecal streptococci while exhibiting the lowest
50
-------
percent of non-fecal streptococci. PSE Agar recovered a
higher percent of non-fecal streptococcal types (23%) than
did KF (19%) , The advantage of PSE is that it only requires
half the incubation time required by KF. As a result/ fecal
streptococcal counts are available after 24-hours incubation.
In summary, PSE exhibited a consistently higher recovery
from a wide range of sources which included foods, animal
feces, and domestic sewage. The medium allowed the growth
of all types of fecal streptococci while exhibiting a
slightly higher percent of non-fecal streptococci than did
KF. Results from PSE plates are available after 18 to 24-
hours incubation.
The fresh isolates obtained from sewage/ feces of sheep,
cattle, horses, rabbits, chickens, geese, and rats were
used to develop FA techniques for the identification of
Group D Streptococci. The species isolated were initially
identified by conventional, physiological, biochemical, and
serological tests. Both whole and disrupted cells of re-
presentative strains of each species were used for the
preparation of the Group D Streptococcus vaccine. Globulin
fractions of individual and pooled antisera were labeled
with fluorescein isothiocyanate dye (FITC), and the resulting
conjugates were tested with homologous and heterologous
antigens. Conjugate specificity was determined by adsorption
and inhibition tests using controls with homologous and
heterologous antigens. Using the direct staining method and
individual and pooled conjugates, it was possible to obtain
84 and 85% positive FA reactions/ respectively, with Group D
Streptococcal strains. Non-group D Streptococci and Staphy-
lococci (FA cross-reactants) were eliminated by trypsiniza-
tion of the smears prior to staining.
The indirect FA staining technique v/as also evaluated
for use with the Group D conjugates used above in the direct
staining procedure. Results indicate that the indirect
method is more sensitive than the direct in its ability to
identify the Group D Streptococci. However, the number of
false positives due to cross reactants increases tremendously,
Trypsinization did not remove the cross reactions. The more
sensitive indirect technique had to be sacrificed in favor
of the more specific direct method.
At the present time, the FA system for Group D Strep-
tococcal identification is ready for field testing. Reagent
standardization, optimum conditions for preparation of
globulin to be labeled/ the determination of the most ^
optimum labeling conditions, the determination of optimum
51
-------
storage conditions of the conjugates and the application
of photometric techniques for direct fluorescent measurements
of specimens have been initiated. The data at the present
time indicate that the PA method/ using a direct staining
technique, will be of value in the rapid identification of
Group D Streptococci.
Summary
On the basis of the above information and experience
gained to date/ the following recommendations are being made:
1. The MF technique, using KF agai; be used to assay
fecal streptococci in water samples and non-chlorinated sewage,
The pour plate technique is recommended as an alternate
procedure for the MF technic when chlorinated sewage effluent
and water samples with high turbidity are encountered. PSE
and KF agars are recommended as the pour plate media.
2. High recoveries of fecal streptococci from human
and animal feces/ a shorter incubation period (24 hrs.) and
a low percentage recovery of non-fecal streptococci cause
considerable interest in the PSE medium. Unlike the PSE
pour plates/ poor colony distinction occurs on PSE membrane
filters. Efforts are presently underway to solve this
di s crepancy.
3. The identification of fecal streptococcal strains
provide valuable data in determining the source of contamina-
tion. Biochemical tests used to achieve strain identifica-
tion lack standardization and are the chief sources of error
in strain identification.
52
-------
REFERENCES
1. Pavlova, M. T., Brezenski, F. T. and Litsky, W. 1972
"Evaluation of Various Media for Isolation, Enumera-
tion and Identification of Fecal Streptococci from
Natural Sources". Health Lab. Science, 9_, No. 4,
p. 289-298.
2. Sherman, J. M. 1937 "The Streptococci". Bacti Reviews,
1:3-97.
3. Hartman, P. A., Johnson, R. H. , Brown, L. R., Jacobson,
N. L., Allen, R. S., Shellenberger, P. R., and Van Horn,
H. H., Jr., 1962. Cited from Hartman et al 1966.
4. Mundt, J. O. 1963 "Occurrence of Enterococci on Plants
In A Wild Environment". Applied Microbiol., 11, 2:141.
5. Geldreich, E. E., Kenner, B. A. and Kabler, P. W. 1964
"Occurrence of Coliforms, Fecal Coliforms and Strep-
tococci on Vegetation and Insects". Applied Microbiol.,
12, 1:63.
6. Martin, J. D. and Mundt, O. "Enterococci in Insects".
1972 Applied Microbiol., 24, 4:575.
7. Pavlova, M. T., Beauvais, E., Brezenski, F. T., and
Litsky, W. 1972 "Fluorescent-Antibody Techniques for
the Identification of Group D Streptococci: Direct
Staining Method". Applied Microbiol. 23, 3:571.
53
-------
THE USE AND ABUSE OF FECAL STREPTOCOCCI
IN WATER QUALITY MEASUREMENTS*
Edwin E. Geldreich**
The fecal streptococcus group does merit consideration
in water quality criteria since it is now being recommended
and used in many stream pollution measurements. Generally/
the occurrence of fecal streptococci in water suggests
fecal pollution and their absence indicates little or no
warm-blooded animal contamination (1). Although fecal
streptococci may persist for extended periods in irrigation
waters of high electrolyte content/ they rarely multiply
in polluted water.
This group encompasses a wide spectrum of strains
that have diverse survival rates and specific fecal origins
and also includes several biotypes that are of limited
sanitary significance (1-3) . The ubiquitous S_. faecalis
var. liquifaciens (Figure 1) may frequently represent a
substantial portion of any fecal streptococcus population
in natural waters of good quality. These waters (Table 1)
may be devoid of recent fecal pollution, receive only small
additions of contamination/ or contain minute vestiges of
some pollutional discharge remote in time or place. Until
better methodology is available to selectively exclude such
strains of limited sanitary significance, the use of fecal
streptococcus limits in recreational waters must be pegged
to some maximum density above 100 organisms per 100 ml (4) .
An alternate approach for bathing waters would be to confirm
the validity of very low densities of fecal streptococci by
parallel examination for fecal coliform bacteria before
making a decision to temporarily restrict use of that water.
In contrast, analysis of data on the distribution of
subgroups of fecal streptococci in warm-blooded animal
discharges reveal that S_. bovis and S_. equinus are specific
indicators of non-human warm-blodded animal pollution
(Figure 2). This is a particularly useful differential
characteristic in pollution investigations involving cattle
feedlot runoff, farm land drainage, and discharges from meat
and duck processing operations/ and dairy plant wastes. In
addition, S. bovis and S_. eguinus are the indicator organisms
showing the most rapid die-off outside the animal intestinal
tract (Figure 3) . Therefore the detection of these two
strains in water indicates very recent farm animal contamination.
* This paper submitted in writing for the record.
** Water Supply Research Laboratory, NERC-Cincinnati.
54
-------
-^ r ' r r< -• '•
; •" ' "* C
CATS and DOGS i
LIVESTOCK
POULTRY
RODENTS g""~
jiSl?^^^;
INSECTS
VEGETATION
FARM SOILS
-------
HUKiAH
CHICKED
CATS!
TURKEY pj
r
0
20
40
60
80
100
S. BOVIS - S. EQUINUS PERCENTAGE OF
FECAJ-, STREPTOCOCCUS POPULATION IN
\V A RM - B LOODE D A NI MA L FE C E S
FIGURE 2.
56
-------
100,
[
10
*V D*k X<>
"A X'
"?!
*
i- to
'r~-* • *
kv\ %
uVi *
\Jl
.'-~. v- >.\
4* * . ,
•» •• •. \
u «• ••
u X -
.
e- *
1/1 \
CT '
o
'
• /^,/'.
'V
K
^
a
1 2 3 4 5 6 7 8 9 10 11 12 13
length of Time--doys
Fig. 3. Persistence of Selected Enteric Bacteria in Storm Water Stored at 20°C
14
57
-------
TABLE 1. DISTRIBUTION OF S. FAECALIS LIQUIFACIENS IN PRAIRIE
WATERSHEDS, RECREATIONAL WATERS AND PRIVATE WELL SUPPLIES
Ui
oo
WATER GIOriRPP1
PRAIRIE WATERSHEDS
Cherry Creek, Wyo.
Saline River, Kan.
Cub River/ Idaho
Clear Creek, Colo.
RECREATIONAL WATERS
Lake Mead
Lake Moo va lay a
Colorado River
Whitman River
Merriraack River
PRIVATE WELL SUPPLIES
Gilman
Miami
Baas
Kilb
Hopper
DENSITIES PER 100 ML*
FECAL
COLIFORMS
90
95
110
170
2
9
4
32
100
0.8
1.0
1.1
2.8
5.0
FECAL
STREPTOCOCCI
83
180
160
110
444
170
256
88
96
8,800
18
32
106
134
Percent
Occurrence..
S, FAECALIS
LIOJJIFACIENS „
35.3
24.3
NONE
4.3
4.0
22.0
NONE
NONE
4.8
100.0
NONE
26.7
26.5
NONE
* • •• •• i_ j j -I*.
All bacterial densities for prairie watersheds and recreational waters
were based on median values. Data for private well supplies were
developed from single samples only.
-------
TABLE 2.
FECAL COLIFORM TO FECAL STREPTOCOCCUS RELATIONSHIPS
IN DOMESTIC SEWAGES AND HUMAN FECES
SOURCE
DOMESTIC SEWAGE
PRESTON, IDAHO
FARGO, N. D.
MOOREHEAD, MINN.
CINCINNATI, OHIO
LAWRENCE, MASS.
MONROE, MICH.
DENVER, COL.
HUMAN FECES
43 SAMPLES
DENSITIES PER
EFFLUENT OR 1
FECAL
COLIFORMS
340,000
1,300,000
1,600,000
10,900,000
17,900,000
19,200,000
49,000,000
13,000,000
100 ML *
GM FECES
FECAL
STREPTOCOCCI
64,000
290,000
330,000
2,470,000
4,500,000
700,000
2,900,000
3,000,000
RATIO
FC/FS
5.3
4.5
4.9
4.4
4.0
27.9
16.9
4.4
MEDIAN VALUES
59
-------
Another valuable application of the fecal streptococcus
indicator system in stream pollution investigations has been
through correlation with the fecal coliform group. Fecal
coliform bacteria are more numerous than fecal streptococci
in domestic sewages, with a fecal coliform to fecal strep-
tococcus ratio always greater than 4.0(Table 3). As would
be expected, similar ratios are common to the feces of man.
Conversely, fecal streptococci are more numerous than fecal
coliforms in the feces of farm animals, cats dogs, and
rodents (Table 4). In feces from these animals, the fecal
coliform to fecal streptococcus ratios are less than 0.7.
Similar low ratios are common to urban stormwater and
farmland drainage.
These fecal coliform to fecal streptococcus ratios must
be applied carefully. Correlations are most meaningful when
developed from bacterial densities in water samples taken
at waste outfalls into a stream. Once these organisms are
diffused into the receiving stream, various ecological
forces may alter the interrelationship between these indicator
systems during flowtime downstream. Under these conditions,
the distribution of subgroups of fecal streptococcus strains
within this pollution indicator group can be drastically
altered by dilution with organisms in the receiving water
or through selective adaption to the water environment by
only a few vigorous strains. For these reasons, ratios
for stream samples will be valid only during the initial
24-hour travel downstream from point of pollution discharge
into the receiving river.
Areas of Research Development
In contrast to the milk and food environment, water is
a severe menstruum for the survival and subsequent detection
of bacterial indicator systems and any associated pathogens.
Until recent years, fecal streptococci were considered by
early investigators to be present in stream water in rela-
tively low magnitude approaching a density of one tenth that
of the total coliform population and always dying rapidly
outside the intestinal tract (5-7). More recent media
developments (8-11) have shown that the fecal streptococcus
densities in polluted water approach the magnitude of the
coliform population or at times exceed it by a factor of 10,
depending upon the source of fecal pollution. Fecal
streptococci are now observed to persist for long periods
in stormwater and irrigation water containing large concen-
60
-------
TABLE 3 .
FECAL COLIFORM TO FECAL STREPTOCOCCUS RELATIONSHIPS
IN STORMWATER RUNOFF, ANIMAL PETS, RODENTS, AND FARM ANIMALS
I
1
SOURCE
:
STORMWATER RUNOFF
BUSINESS DISTRICT
RESIDENTIAL
RURAL
ANIMAL PETS
CAT
DOG
RODENTS
LIVESTOCK
COW
PIG
SHEEP
j POULTRY
DUCK
CHICKEN
TURKEY
DENSITIES PER^
OR 1 GM FECES
FECAL
COLIFORMS
13,000
6,500
2,700
7,900,000
23,000,000
160,000
230,000
3,300,000
16,000,000
33,000,000
1,300,000
290,000
i
100 ML RUNOFF
;
FECAL
STREPTOCOCCI
51,000
150,000
58,000
27,000,000
980,000,000
4,600,000
1,300,000
84,000,000
38,000,000
54,UuO,000
3,400,000
2,800,000
RATIO
FC/FS
0.26
0.04
0.05
0.3
0.02
0.04
0.2
0.04
0.4
0.6
0.4
0.1
MEDIAN VALUES
61
-------
TABLE 4.
Ov
K>
FECAL STREPTOCOCCUS MEDIA EVALUATION ON OHIO RIVER WATERS'
RIVER STATION
FEBRUARY 1970
OHIO RIVER
Mile 640
Mile 626
Mile 502
Mile 491
Mile 450
Mile 421
LICKING RIVER
12th St. Bridge
LITTLE MIAMI RIVER
Beechmont Bridge
GREAT MIAMI RIVER
Seller Road
Liberty-Fairf ield
American Mat*
Lost Bridge
WHITEWATER RIVER
River Mouth
DENSITIES PER 100 ml STREAM SAMPLE
FECAL
COLIFORM
800
1,100
1,100
3,200
800
1,700
1,400
6,800
6,400
11,000
9,200
9,000
120
FECAL STREPTOCOCCI
KF AGAR
2,700
2,300
13,000
3,900
1,700
1,500
4,800
22,000
17,000
17,000
9,000
14,000
1,600
M-ENTEROCOCCUS
1,000
660
1,300
1,100
660
780
1,300
6,100
5,800
4,700
6,800
4,800
720
b
Data courtesy of the Analytical Quality Control Laboratory, EPA
-------
trations of electrolyte and a favorable water temperature (1) .
Mundt et al. (12) have found some evidence of S_. faecal is
multiplication in waste waters of vegetable processing
plants.
These increases in media sensitivity have not come
about without some compromise in suppression of other
organisms present in the water environment. Strains of
Corynebacterium, Pediocossus, Streptococcus 1actis and
other organisms common to irrigation water, sugar beet
plant effluents and other food processing wastes can develop
substantial growth in either the liquid media or selective
agars now available (12-15),
Maximum suppression of interfering organisms can, in^
part, be achieved through careful media preparation. Sodium
azide, a frequent selective inhibitor in these media, does
deteriorate with age in dehydrated media held for two years
or more prior to reconstitution. Sterilization in auto-
claves with long time cycles or contact with a chemically
contaminated boiler steam source can cause a decomposition
of sodium azide to form toxic acid products. Addition of
a sodium carbonate buffer is sometimes necessary to prevent
undesirable shifts in pH during sterilization. Poor
differential colony color on both KF and M-Enterococcus
agars can result from either an unsatisfactory grade of
tetrazolium indicator or from its exposure to excessive
sterilization temperatures. Laboratory experience indicated
that a five minute exposure in a boiling water bath after
the agar has melted, and addition of filter-sterilized
tetrazolium after cooling will eliminate much of the
irregularities observed.
Lack of uniformity in media sensitivities has been
observed during comparative studies using stream samples.
Data presented in Table 5, which were supplied by courtesy
of the Analytical Quality Control Laboratory, FWPCA, revealed
significant differences in the fecal streptococcus densities
detected by M-Enterococcus and KF agars. The question of
which medium is more accurately detecting the fecal
streptococcus density along a 220 mile reach of the Ohio
River and its tributary streams can only be resolved
through verification of the colonies by selected biochemical
tests. Further colony confirmation would indicate whether
KF medium needed to be incubate at 44.5 C to suppress non-
fecal streptococcus organisms or whether M-Enterococcus
agar needed prior enrichment to increase recovery of all
63
-------
strains of fecal streptococci. Other comparative studies
frequently have shown both media to yield essentially
equivalent results on sewage effluents. However, KF agar
recoveries were higher on samples examined from cattle
feedlots, meat packing house wastes, and farm drainage
because KF agar apparently gave better recovery of the
-S. bovis and Ł3. equinus strains.
Normally there is no need for species identification
of fecal streptococci in stream pollution studies. Density
relationships with fecal coliforms are adequate to assign
the probable source of waste discharge as being domestic
or from farm animals and wild life. However, special
applications involving: tracer organism identification,
confirmation of sanitary significance of very low fecal
streptococcus densities, and media evaluations will require
further biochemical identification (1, 19-21). Practical
application of identification procedures demands a simpli-
fication of the tests and more specific biochemical
reactions. Further development of a serological schema,
which currently includes 39 serotypes, could be an important
breakthrough in this problem (21, 22-27).
SUMMATION
The true sanitary significance of fecal streptococci
has been confused somewhat by controversies concerning
procedures for quantitation, definition of the group, and
differing concepts as to their occurrence in the water
environment. It is true that there are problems in
methodology yet to be resolved which include: improvements
in media formulations, possible application of elevated
temperature incubation, and simplification of biochemical
tests or development of a serological procedure for use in
species or subgroup identification. However, recognition
of these areas of future research should not detract from
use of the fecal streptococcus group in stream pollution
measurements.
64
-------
Ul
SAMPLE
t
KF agar
Pink-red colonies
Growth in jrain-heart infusion broth within 2 days at 45° and 5 days at 10°C with
confirmation as catalase-negativs and positive for growth in 40% bile broth
t I
Growth at !5° and 10°C
I
Ccr.fi rm wi th growth in
6.5% Nad in brain-heart
infusi ;n broth
i
Lancefield group'D Streptococci
Y
Starch hydrolysis
X" \
Positive Negative
Y
Atypical s. faecal is
(Vegetation source)
Peptonization of
I i trnus mi Ik
Growth at 45°C only
S. bovis - S, equinus
I
Starch hydrolysis
Posi tive
Lactose fermentation
/ \
Acid only No change
5. bovis S. equinus
(Livestock and poultry sources)
Posi tive
Negative
I
S. faecal is var. liquefaciens Enterococci
(Insect source) (Y/arm-blooded
anirnal -sources)
Note: A small percentage of unclassified strains (biotypes) will be found since fecal
streptococci vary greatly in their biochemical reactions.
Figure \i Schematic outline for identification of fecal streptococci.
-------
REFERENCES
1. Geldreich, E. E., and Kenner, B. A. Concepts of Fecal
Streptococci in Stream Pollution. Water. Poll. Contr.
Fed. 41: R336 (1969).
2. Mundt, J. O., Coggin Jr., J. H., and Johnson, L. F.
Growth of Streptococcus faecalis var. liquifaciens on
Plants. Appl. Microbiol., 10: 552 (1962).
3. Mundt, J. O., and Graham, W. F. Streptococcus faecium
var. casseliflavus, nov. var. Bacteriol., 95: 2005 (1968)
4. Geldreich, E. E. Applying Bacteriological Parameters to
Recreational Water Quality. Amer. Water Works Assoc.,
62.: 113 (1970) .
5. Houston, A. C. On the Value of Examination of Water for
Streptococci and Staphylococci with a View to Detection
of its Recent Contamination with Animal Organic Matter.
Supplement 29th Annual Report Local Government Board
Containing Report of Medical Officer 1899-1900, London
County Council, England, 458 (1900).
6. Winslow, C. E., and Hunnewell, M. P. Streptococci
Characteristics of Sewage and Sewage-Polluted Waters.
Sci., 15: 827 (1902).
7. Winter, C. E., and Sandholzer, L. A. Studies of Fecal
Streptococci. U.S. Dept. Int., Fish & Wildlife Ser.,
Fishery Leaflet 201(1946).
8. Mallmann, W. L., and Seligmann, E. B. A Comparative
Study of Media for the Detection of Streptococci in
Water and Sewage. Amer. Jour. Pub. Health, 40: 286(1950).
10. Slantez, L. W., and Bartley, C. H. Numbers of Enterococci
in Water, Sewage, and Feces Determined by the Membrane
Filter Technique with an Improved Medium. Jour.
Bacteriol., 74: 591(1957).
11. Kenner, B. A., Clark, H. P., and Kabler, P. W. Fecal
Streptococci. Cultivation and Enumeration of
Streptococci in Surface Waters. Appl. Microbiol.,
2: 15 (1961).
66
-------
12. Mundt, J. o., Larsen, S. A., and McCarty, I. E. Growth
of Lactic Acid Bacteria in Waste Waters of Vegetable-
Processing Plants. Appl. Microbiol., 14: 115 (1966).
13. Lelliott, R. A. The Plant Pathogenic Coryueform Bacteria.
Jour. Appl. Bact., 29: 114 (1966).
14. Mundt, J. O., Beattie, W. G., and Wieland, F. R.
Pediococci Residing on Plants. Jour. Bact., 98; 938(1969)
15. Stark, J., and Sherman, J. M. Concerning the Habitat
of Streptococcus lactis. Jour. Bacteriol. 30: 639(1935).
16. Rose, R. E., and Litsky, w. Enrichment Procedure for
Use with the Membrane Filter for the Isolation and
Enumeration of Fecal Streptococci in Water. Appl.
Microbiol., 13: 106 (1965).
t
17. Burman, N. P. Some Observations on Coli-Aerogenes
Bacteria and Streptococci in Water. Jour. Appl. Bact.,
24: 368 (1961) .
18. Mead, G. C. Faecal Streptococci in Water Supplies and
the Problem of Selective Isolation. Proc. Soc. Water
Treat. Exam. 15: 207(1966).
19. Deibel, R. H., Lake, D. E., and Niven Jr., C. F.
Physiology of the Enterococci as Related to their
Taxonomy. Jour. Bacteriol., 86: 1275 (1963).
20. Hartman, P.A., Reinbold, G. W., and Saraswat, D. S.
Indicator Organisms - A Review. I. Taxonomy of the
Fecal Streptococci. Internat. Jour. System. Bacteriol.,
I6_: 197(1966) .
21. Tilton, R. C., and Litsky, W. The Characterization of
Fecal Streptococci. An Attempt to Differentiate
Between Animal and Human Sources of Contamination.
Jour. Milk Food Technol., 30; 1(1967).
22. Lancefield, R. C. A Serological Differentiation of
Human and other Groups of Hemolytic Streptococci.
Jour. Exptl. Med., 57: 571(1933).
23. Sharpe, M. E., and Shattock, P. M. F. The Serological
Typing of Group D Streptococci Associated with Outbreaks
of Neonatal Diarrhoea. Jour. Gen. Microbiol. 6_: 150(1952).
67
-------
24. Sharpe, M. E. Occurrence of a Common Type Antigen in
Streptococci of Groups D and N. Gen. Microbiol.,
7: 192(1952).
25. Sharpe, M. E., and Fewins, B. G. Serological Typing
of Strains of Streptococcus Faecium and Unclassified
Group C Streptococci Isolated from Canned Hams and
Pig Intestines. Jour. Gen. Microbiol. 23: 621(1960).
26. Medrek, T. F. , and Barnes, E. M. The Physiological and
Serological Properties of Streptococcus bovis and
Related Organisms Isolated from Cattle and sheep.
Appl. Bact., 25: 169(1962).
27. Deibel, R. H, The Group D. Streptococci. Bact. Rev.,
28: 330(1964).
68
-------
FECAL STREPTOCOCCI - DISCUSSION
The membrane filter method or agar pour plate technique
are recommended over the multiple tube procedure (MPN) for
the enumeration of fecal streptococci because:
(a) Recoveries on current MF media are higher and are
less affected by interference organisms.
(b) A higher number of false positive reactions occur
in broth MPN systems.
(c) When group/or species identification are required,
it is necessary that preliminary isolation be made on solid
media. MF and agar pour plates readily allow for primary-
isolation of fecal streptococcus colonies.
However, certain environmental conditions prevent the use of_
the membrane filter technique, e.g., waters with high turbidity
and waters with high concentrations of heavy metals or other
toxic compounds. Under those conditions, the AD-EVA multiple
tube technic is recommended. (Azide - Dextrose - Presumptive
and Ethyl Violet Azide - Confirmatory Tests.) It must be
realized that a high number of false positives may occur,
especially in seawater, and streaking of a percentage of the
positive tubes on agar medium for subsequent colony verifica-
tion is mandatory.
Data on fecal streptococcus recovery from animal feces,
domestic sewage, frozen and non-frozen foods indicate KF and
PSE agars to be the most productive. These findings were
confirmed by work done at the EPA Narragansett laboratory.
For environmental samples (fresh water) good recoveries were
noted. PSE and KF gave over 80% confirmation. However, for
marine water samples it was noted that one can expect a one
log decrease in recovery. It was concluded that additional
data would be required to further substantiate this dis-
crepancy and isolate the problem.
At this time, a two-stage testing system was proposed.
This proposal was based on a pre-enrichment system with
subsequent transfer to M-Enterococcus agar developed by Rose
and Litsky. Their data showed significant increases in
fecal streptococcus recovery when using this two-stage
system. Although increased recoveries were obtained, it
was the consensus of those present that two-stage tests may
pose additional problems. For example, test results would
be delayed because of the pre-enrichment incubation period.
69
-------
Results therefore/ would not be available for three days.
Secondly/ additional manipulation and equipment would be
required. It was agreed that the single-stage test is more
appropriate especially where results are available 24 to 48
hrs. after sample processing. Therefore/ PSE appears to be
the medium of choice/ since the incubation period is only
24 hrs. However, the application to a membrane filter^
technique has not been completed. Work on this phase is
in progress at the University of Massachusetts and at the
EPA narragansett laboratory. KF agar using the MF technique
remains at least for the present time/ the medium of choice
for processing environmental samples.
The use of the fecal streptococcus test was discussed
with the following points being made:
(a) The presence of fecal streptococci in water
indicates fecal contamination. However, there are some
strains which have limited sanitary significance. These are
the atypical Streptococcus fecalis strains which are associated
with vegetation and S,. fecalis var. liouefaciens which has
been found to be ubiquitous. With the latter organism/
interpretation of data becomes more difficult when the counts
fall below 100 per 100 ml. On the other hand, strains such
as S.. bo vis and S. eouinus can be used to denote animal waste
or agricultural runoff because of host specificity.
(b) Fecal coliform correlations with fecal strepto-
cocci may be useful in determining the source of contamination.
Ratios must, however, be applied carefully because of the
survival rates of the fecal streptococcus strains. When
travel downstream from the point of discharge exceeds 24 hrs.,
ratios cannot be used. Also, it becomes difficult to establish
ratios when fecal streptococcus densities are below 100 per
100 ml.
(c) It was pointed out that the fecal coliform to
fecal streptococcus ratios (ratios of 4.0 and above indicating
domestic sources while ratios of 0.7 and less indicate animal
wastes) were based on fecal samples and not on environmental
samples. Ratios developed on actual field samples may not
indicate the same results. It was pointed out that ratios
may be meaningful and subject to interpretation providing
that the following factors be taken into consideration:
water temperature, pH, presence of toxic materials, organic
nutrients and travel time downstream from the point of dis-
charge.
70
-------
(d) The use of fecal streptococcus standards to
indicate the quality of bathing and recreational waters is
not recommended. The following factors discourage its use:
- lack of sensitivity of the medium, especially
at a density of 25 per 100 ml. which was
proposed by one agency.
- fecal streptococci may survive for a long-
time in some waters because of electrolytic
content; this persistence may unduly restrict
the use of these waters.
(e) Fecal streptococcus data can best be used as
supplemental information when run in parallel with the fecal
coliform test. The detection of S_. bovis and Ł. equinus
indicates non-human animal pollution. Therefore, identifica-
tion as to subgroups or species may provide valuable source
information. The routine use of fecal streptococcus assays
alone is not advocated for recreational waters.
Special Problem
Bacterial aerosols were considered as a special problem.
No degree of significance was assigned to the problem due to
the lack of information available on the subject. Reports,
however, do imply that sewage treatment facilities are
potential sources of setting-up aerosols which may harbor
pathogenic microorganisms. Work on New York Sewage Treat-
ment Plants indicate microbial emissions into the atmosphere.
In conjunction with aeration units, aerosols were found to
contain Shigella, Salmonella, beta-hemolytic streptococci,
bacillus and acid fast organisms which resemble tubercle
bacilli. Concentrations of organisms at various distances
away from the plants were not determined. Apparently there
is a paucity of data on the dispersion patterns from the
treatment facilities. Since there will be a need for such
information, methodology will be required. It is recommended
therefore that bacterial aerosols be given special attention
and methodology for bacterial air sampling be included in
the EPA Microbiological Manual.
71
-------
METHODS FOR DETECTING VIRUSES IN ENVIRONMENTAL
WATERS - A STATUS REPORT
*
Gerald Berg, Ph.D.
Introduction
Viruses are in essence alive only when they infect,
for infection normally results in multiplication and with
it the opportunity for the virus to manifest that character-
istic of living things -multiplication - and mutation, the
only other characteristic of life that the virus is capable
of manifesting. Outside of living cells, the virus is
inert. Its essential viability in the hostile environment
outside the cell is time-marked. Outside of living cells,
few survive for long.
Yet, even as their numbers diminish, among those
viruses excreted into sewage by infected people, sufficient
numbers survive to reach the water intakes of downstream
communities. The smallest amounts of viruses detectable in
cell cultures, the most sensitive hosts for many viruses,
are sufficient to infect susceptible individuals who consume
them (Table 1) (1).
Thus, the smallest amount of virus that reaches a
water intake or that can be contacted by a recreationalist
is a potential hazard. Methods to detect such small amounts
must be developed even when detection requires concentrating
viruses from large volumes of water.
Over the past several years, a growing awareness within
the scientific community of the waterborne virus problem
has resulted in the development of a number of techniques
for recovering viruses from waters of various qualities.
These waters range from sewage to totally renovated. The
techniques include membrane adsorption, ultrafiltration,
polyelectrolyte adsorption, aluminum hydroxide adsorption,
protamine precipitation, two-phase separation, and alginate
membrane filtration. Some of these methods appear fairly
efficient in limited circumstances. None of them appears
to have universal potential at present. The greatest
problem, one which may long be with us, is the endless
change of the chemical quality of waste and receiving waters,
and the unpredictable effects of such change on the efficiency
of methods for quantitatively concentrating viruses from
water. For this reason alone, the usefulness of standard
*
Virology Section/ Advanced Water Treatment Research
Laboratory, NERC-Cincinnati
72
-------
TABLE 1
Infective Doses of Viruses for Man
Virus
Poliovirus 1
Poliovirus 3
Dose
2 PFU
1 TCD50
Route of
Inoculation
Oral
(gelatin capsule)
Gavage
No. Inoculated
3
10
% Infected
67
30
-------
procedures is doubtful. Methods may always require selection
and flexibility to meet the needs of changing situations.
Quantities of Viruses in Environmental Waters
The detectable quantities of viruses in sewage vary
from several hundred (Table 2) to ten thousand or .more per
gallon (2). The numbers generally increase in the warmer
months and decrease in the colder months, reflecting in-
fection and excretion patterns in the community. Since
viruses do not multiply outside of susceptible living cells,
dilution and the toll of time in hostile waters reduce the
concentrations of viruses downstream of outfalls to levels
barely detectable by the best techniques available even
when 50- and 100-gallon quantities of water are tested.
Yet these quantities, in terms of the daily water require-
ments of even small communities, are not small. Table 3
shows the quantities of viruses recovered at or near water
intakes of several cities. The data are given for one
million gallons of water, an apparently large amount, but
in fact only the amount consumed in one day by a community
of only 10,000 people.
When one considers that some of the methods used for
concentrating these viruses had efficiencies of less than
one percent, that the cell culture systems used for detecting
the viruses were sensitive to less than 50% of the
viruses that are excreted by man, and that there are un-
doubtedly viruses in sewage that have not yet been detected
and identified, it is easy to surmise that the numbers of
viruses we now detect are probably several orders of magnitude
below the quantities actually present there. Thus, the
quantities of viruses that reach intakes downstream of out-
falls must be very large indeed.
Recommendations in Standard Methods for the Examination of
Water and Wastewater (13th edition) for Methods for Detection
of Viruses in Various Waters
Because of its high volume capability and thereby its
potential sensitivity, the gauze pad method is recommended
for qualitative studies by Standard Methods and the pH 7
membrane adsorption procedure is tentatively recommended for
quantitative studies. No recommendations are made for
routine examinations of environmental waters.
Methods for Detecting Viruses in Sewage
In the mid-sixties, technology for the detection of
viruses in sewage moved from the non-quantitative and
74
-------
TABLE 2
Concentrations of Viruses in Sewage (1970)
Site No.
Viruses recovered
(PFU/100 gallons)
11,900
38,600
40,500
31,800
13,400
-------
TABLE 3
Recovery of Viruses at or Near Water Intakes
8/28/70 10/1/70 11/12/70 11/19/70 11/19/70 4/1/71
(PFU/million gallons )
Viruses recovered
from water
160,000
40,000
640,000
0
Viruses recovered
from solids
20,000
20,000
50,000
20,000
30,000
«
Actual samples tested were 50 and 100 gallons and solids from those volumes.
-------
relatively insensitive grab sample and gauze pad techniques
to the relatively sensitive and quantitative aluminum
hydroxide adsorption, protamine precipitation (3, 4), and
phase separation procedures (5, 6).
The aluminum hydroxide and protamine precipitation
procedures, as modified in our laboratory, are a combined
procedure arranged in tandem whereby aluminum hydroxide gel
is mixed with sewage, filtered out, and assayed for adsorbed
viruses while the viruses remaining in the filtrate are pre-
cipitated with protamine sulfate, eluted, and also assayed.
Aluminum hydroxide originally was thought to adsorb only the
smaller viruses, the enteroviruses, and the protamine was
believed to precipitate only the larger viruses, the reoviruses
and adenoviruses. In fact, most of the enteroviruses are
adsorbed and removed with aluminum hydroxide and protamine
does remove most of the larger viruses, but aluminum hydroxide
is now known to remove some of the larger viruses and the
protamine is now known to precipitate some of the entero-
viruses. The^original use of this combination method, one of
parallel testing of two sewage samples with each method,
clearly must have resulted in high estimates of the amount
of viruses present in sewage. The modified system of tandem
operation obviates this error.
The aluminum hydroxide-protamine precipitation procedure
appears to be an efficient recovery system, but newer
methodology has resulted in better virus recoveries.
The two-phase system (5, 6) has also yielded good virus
recoveries from sewage, perhaps equal to those reported for the
aluminum hydroxide-protamine sulfate procedure. In two-phase,
polyethylene glycol and dextran are mixed with sewage and
allowed to separate overnight at 4°C into two immiscible phases,
The virus concentrates in the lower dextran phase and in the
interphase and can be precipitated out and assayed. This
procedure may be sensitive and quantitative, but the over-
night separation requirement makes it rather cumbersome.
The most sensitive method available today for recovering
viruses from sewage is also the simplest (7). Sewage, when
the pH is lowered to 3 and filtered through a 0.45 ;um cell-
ulose nitrate membrane filter, yields most of those viruses
to the filter. The adsorbed viruses are eluted by calf serum
in borate buffer (pH 8) when the elutant is passed through
the membrane (8, 9).
Other techniques have been less effective.
77
-------
Recovery of Viruses from Seeded Distilled Water
A membrane filter (MF) method for detecting viruses was
first described some years ago (10) and modified to the point
where it is 100% efficient for recovering some viruses from
distilled water (and perhaps from other very clean water)
when volumes up to 25 gallons are sampled (11). The method
is one of filtering water buffered at pH 7 (thus adding salt)
through a 0.45^im cellulose nitrate membrane onto which the
virus adsorbs, and eluting the virus by sonicating the mem-
brane in beef extract.
Comparative experiments have shown that viruses in
distilled water adsorb much better at pH 7 than at pH 3, since
much better recoveries are experienced- when adsorption is
accomplished at the higher pH (Table 4). The presence of
organics, which interfere markedly with virus adsorption to
the membrane at pH 7, seems not to interfere with adsorption
at pH 3 and may actually favor it. Critical experiments to
clarify the role of organics in adsorption at low pH are
now under way.
Adsorption of viruses onto the insoluble polyelectrolyte
PE 60, also interfered with by organics (12), is not an
efficient method for recovering viruses from distilled water
either (Table 5). Some viruses, such as polioviruses, are
recovered with modest efficiency, but other viruses are not.
Adenoviruses (not shown in Table) are difficult to recover
at all.
Recovery of Viruses from Seeded Tap Water
Efforts to recover viruses from seeded tap waters by
the MF and the insoluble polyelectrolyte (PE 60) techniques
have yielded erratic results, probably reflecting an erratic
chemical composition of the tap waters (Table 6). The pH 7
method resulted in good recovery of poliovirus 1 from one-
liter quantities of water in two of three tests, yielding
more than 80% of the virus in both. At pH 3, recovery of the
virus from one liter of water never exceeded 52% and twice
was 5% or less. With 50-gallon volumes, the efficiencies
of these procedures were always reduced markedly. Even with
the pH 7 method, they never exceeded 44%. In one test, the
PE 60 gave poor results comparable to those with the membrane
at pH 7 in the poorest test of the series with that method.
There is clearly a long way to go in the development
of methods for recovering viruses quantitatively from tap
water.
78
-------
Table 4
Recovery of Viruses from Seeded Distilled Water
by MF Technic at pH7 and 3
Virus
Sample
Volume
Recovery
Method
% Virus
Recovered
Poliovirus 1
i
Reovirus 1
Coxsackie-
virus A9
{
! Echovirus 7
I
i
}
1
f
1 Liter
50 Gal
1 Liter
50 Gal
1 Liter
50 Gal
1 Liter
50 Gal
pH7-MF
pH3-MF
pH7-MF
pH3-MF
pH7-MF
pH3-MF
pH7-MF
pH7-MF
pH3-MF
pH7-MF
pH3-MF
pH7-MF
104
53
45
17
16
115
37
90
70
115
129
51
79
-------
Table 5
00
o
Recovery of Viruses Seeded Into Distilled Water
by Adsorption Onto Polyelectrolyte PE 60
Virus
Poliovirus 1
Echovirus 7
Reovirus 1
Control
(PFU)
75
79
105
84
PE
(PFU)
38
24
33
14
60
(% recovery)
51
30
31
17
-------
Table 6
Recovery of Poliovirus 1 from Seeded Tap Water
by MF and PE 60 Technics
Test No. Sample Volume Recovery Method Virus Recovered J
1
50 Gal
1 Liter
50 Gal
1 Liter
50 Gal
1 Liter
50 Gal
I
pH7-MF
29
| i
pH3-MF
pH7-MF !
pH3-MF
pH7-MF
pH3-MF
pH7-MF
pH3-MF
pH7-MF
pH3-MF
pH7-MF
pH3-MF
PE 60
pH7-MF
pH3-MF
PE 60
0
i
102 |
5
31
0
84
52
44
36
33
2
24
18
0
18
81
-------
Recovery of Viruses from River Water
Recovery of viruses from river water by the membrane
adsorption method is relatively poor at pH 7 probably because
of the organic matter present/ but even poorer at pH 3
perhaps because enough organic matter is not available
(Table 7). The critical experiments clearly have yet to
be done.
The PE 60 method has been used successfully with river
waters (Table 8) and has been a major factor in demonstrating
the existence of the virus problem, for it was with this
method that viruses were demonstrated at water intakes.
The few comparative' data available with PE 60 and the membrane
method indicate that the membrane procedure produces superior
results with river water. Here/ tob, effective methodology
is at an early stage of development.
Recovery of Viruses from Ocean Water
In a limited series, the membrane procedure at pH 3
was clearly superior to the PE 60 techniques for re-
covering viruses from ocean water (Table 9). More viruses
were recovered from 15-and 25-gallon quantities of ocean
water (all that could be passed through the membranes in
these tests) by MF, than from 25-and 100-gallons of the same
water by the PE 60 method. The factors in ocean water that
affect these recovery systems may hardly be surmised.
Recovery of Viruses from Solids
It has been apparent for some time that much, and
perhaps most/ of the viruses in the water environment are
adsorbed on solids in the water. Generally, the more solids
there are in a water the more viruses one can expect to find
adsorbed on the solids, although rates of adsorbtion differ
for different solids.
In our early efforts, even as we found viruses with
equal or greater frequency on the solids in water than in
the water itself, and often in greater quantities as well,
we found also that our method for recovering adsorbed
viruses had an efficiency of less than 1% (Table 10) .
Clearly, the amounts of viruses adsorbed must have been
several orders of magnitude greater than what we were able
to recover. At the time, viruses were recovered from solids
simply by stirring the solids in 3% beef extract for one
hour and assaying the extract after centrifugation to remove
the solids.
82
-------
Table 7
Recovery of Viruses From River Water
by MF Technic at pH7 and 3
Sample
Size
(Gal)
50
50
Recovery
Method
pH7
pH3
PFU
Recovered
29
1
83
-------
Table 8
Recovery of Viruses at Selected Sites Along the Mississippi River
00
Viruses Recovered
(PFU/50 Gal)
. -
I
24
2
76
I,«LJII ,11.. .—.'• ~-r, -.. "
Site
3
32
1
No.
4
10
— -
5
*+
30
6
*
20
.. .. .,„ ,-, . „ ....... .
*Recovered from 100-gallon water samples,
^Recovered near water intake.
-------
Table 9
Recovery of Viruses From Ocean Water
by PE 60 and pH3-MF Technics
Test
No.
1
2
Sample
Size
(Gal)
25
15
100
25
Recovery
Method
PE 60
pH3-MF
PE 60
pH3-MF
PFU
Recovered
0
3
0
11
85
-------
Table 10
Efficiency of Recovery of Seeded Poliovirus 1 From River Solids
00
Grams of
River
Solids
10
Virus mixed
with solids
(PFU)
492
492
Virus recovered
from supernate
(PFU)
78
28
Virus recovered
from solids
(PFU)
Virus recovered
from solids
0.6
0.3
-------
Efficiency of recovery from solids has improved to
about 25% in recent months, but here too we are only at the
beginning of the problem.
Current Status of Methodology for Detection of Viruses in the
Water Environment
The present status of methodology research in water-
borne virus detection is one of concurrent new methods
development, improvement of recent and sometimes older methods,
and comparative evaluation of the new with the old. Methods
available today allow detection of viruses in situations
where they could not have been detected just a few years ago.
Yet, we know that we miss a large number of viruses
present in the waters that we sample. And we have only the
beginnings of an inkling into the essential and comparative
effectiveness of methods presently under development. There
are many other methods under study than those discussed
herein which, although not highly promising at this moment,
may see significant development in the future.
87
-------
REFERENCES
1. Plotkin, S. A. and Katz, M. (1967). Minimal Infective
Doses of Viruses for Man by the Oral Route/ In
"Transmission of Viruses by the Water Route," edited
by G. Berg. John Wiley and Sons, Inc., New York, NY,
p. 151.
2. Shuval, H. I. (1970). Detection and Control of Entero-
viruses in the Water Environment, In "Developments in
Water Quality Research," edited by H. I. Shuval.
Ann Arbor-Humphrey Science Publishers, Ann Arbor,
Michigan (London, England), p. 47.
3. Wallis, C. and Melnick, J. L. (1967). "Concentration of
Viruses on Aluminum and Calcium Salts," Amer. J.
Epidemiol., 85:459-468.
4. England, B. (1972). "Concentration of Reoviruses and
Adenoviruses from Sewage and Effluents by Protamine
Sulfate (Salmine) Treatment," Appl_.__M_icrobiol.,
24(3):510-512.
5. Shuval, H. I., Fattal, B., Cymbalista, S./ and Goldblum, N.
(1969). "The Phase-Separation Method for Concentration
and Detection of Viruses in Water," Water Research,
3:225-240.
6. Grindrod, J. and diver, D. O. (1970). "A Polymer Two-
Phase System Adapted to Virus Detection," Archiv.
qesamte Virusforsch., 31:365-372.
7. Rao, V. C. and Chandorkar, U., Rao, N. U., Kumaran, P.,
and Lakhe, S. B. (1971). "A Simple Method for Con-
centrating and Detecting Viruses in Wastewater."
Presented at the Second International Congress for
Virology, Budapest (Hungary), June 27-July 3.
8. Rao, V. C., Chandorkar, U., Rao, N. U., Kumaran, P.,
and Lakhe, S. B. (1972). "A Simple Method for Con-
centrating and Detecting Viruses in Wastewater,"
Water Research, 6(12):1565-1576.
9. Wallis, C., Henderson, M., and Melnick, J. L. (1972).
"Enterovirus Concentration on Cellulose Membranes,"
Appl. Microbiol., 23(3):476-480.
88
-------
10. Cliver, D. O. (1967). Enterovirus Detection by Membrane
Chromatography, In "Transmission of Viruses by the
Water Route," edited by G. Berg. John Wiley and Sons,
Inc., New York, N.Y., p. 139.
11. Berg, G., Dahling, D. R., and Berman, D. (1971).
"Recovery of Small Quantities of Viruses from Clean
Waters on Cellulose Nitrate Membrane Filters,"
Appl. Microbiol., 22(4):608-614.
12. Wallis, C., Melnick, J. L., and Fields, J. E. (1971).
"Concentration and Purification of Viruses by Adsorp-
tion to and Elution from Insoluble Polyelectrolytes,"
Appl. Microbiol., 21(4):703-709.
89
-------
ZOOMICROBIAL EXAMINATION OF WATER;
A STATE-OF-THE-ART
Shih L. Chang, M. D.*
INTRODUCTION
The use of zoomicrobes as pollution indicators may
seem antiquated in today's approach to develop methodology
for a more complete microbiological examination of water
and water supplies. The methods that have been used in
some European countries, as described in the 2nd edition
of the International Drinking Water Standards (W.H.O., 1962),
require identification and enumeration of protozoa of
different species or genera to which numerical values have
been assigned according to the "saprobity system" (1) for
their relative significance as pollution indicators.
Such examination, commonly referred to as biological,
is very time-consuming and calls for a competent aquatic
protozoologist. Furthermore, the "saprobity system" was
developed on the basis that different species/genera of
protozoa occur in predominance in different stages or zones
of recovery of a body of water from pollution by raw sewage.
Since only a few cities and small number of small towns in
this country have no sewage treatment facilities, and since
the 1972 Amendments to the Federal water Pollution Control
Act make it mandatory for all cities and towns to treat
their sewage by 1977, any method using criteria based on
pollution by raw sewage would soon be obsolete.
Pollution of surface waters by effluent from secondary
or more advanced sewage treatment processes produce certain
microbiological changes in the receiving water that are
indicative of the event because of the qualitative as well
as quantitative differences between the effluent-bound
microbes and those that are "native" in the aquatic envir-
onment. It has been these differences that provide criteria
for the bacteriological methods for examination of water.
Zoomicrobes As Pollution Indicators
For the purpose of methodological development the
effluent-bound microbes may arbitrarily be placed in two
major categories: namely, the fecal microbes and microbes
attributed to biological treatment processes.
* Water Supply Research Laboratory, NERC-Cincinnati.
90
-------
The fecal microbes include enteric bacteria, viruses
and protozoa. The helminth ova are too few and too restricted
in geographic distribution to justify their use as pollution
indicators. Microbes of any of these three subgroups are
indicators of fecal pollution; their presence has direct
bearing on the health hazard potential of the water in
transmitting enteric infections. Among the indicators of
fecal pollution enteric bacteria are the largest in popu-
lation/ of which the coliforms, streptococci and, to a
lesser degree, Clostridium perfrinqens have been used as
pollution indicators in water quality control.
The enteric viruses are even more specific as pollution
indicators than fecal bacteria. The relative concentration
of enteric viruses to fecal coloforms has been estimated at
approximately 1:100,000 (2); therefore, very large volumes
of water have to be used in virological examination if a
reasonable degree of sensitivity is desired. The methods
for such examination are sophisticated and complex.
Very little information is available on enteric
protozoa in sewage and sewage effluent. In reporting a
method for the examination of cysts of Endamoeba histolytica
in water in times of disease outbreak, Chang and Kabler (3)
approximated the relative concentration of cysts to fecal
coloforms in raw sewage under normal conditions is about 1:105,
Since sewage treatment removes amoebic cysts more easily than
enteric bacteria, the cyst concentration in secondary
effluents is likely to be lower. This observation (4) sub-
stantiated the later study made in the Chanute, Kansas
water supply during an emergency use of reclaimed water.
Only one positive culture for E. histolytica and two for
intestinal flagellates were obtained from secondary efflu-
ent out of a total of ten examinations of five gallon
samples of finished water and one gallon samples of efflu-
ents from various stages of treatment. It is apparent that
the concentration of intestinal protozoa is too low to
justify their use as pollution indicators.
The microbial flora in a biological treatment system
can be grouped into saprophytic bacteria and zoomicrobes
and fungi. They play a role as links in a food chain even
in a biological treatment process and are found in large
numbers in the secondary effluent. The ubiquity of the
saprophytic bacteria in natural waters and soils reduces
the usefulness of these bacteria to nonspecific indicators
of water quality as ascertained by the plate count method
described in the Standard Methods (5).
91
-------
The microbes that are particularly concerned in this
report are the zoomicrobes. They are free-living micro-
scopic animals and comprised of protozoa (ciliates, amoebae
and flagellates)/ nematodes, rotifers, bristle worms and
minute arthropods. These organisms are unrelated to fecal
pollution; they are found in low numbers in raw sewage but
their numbers increase tremendously during the biological
treatment process. Even a primary settling with aeration
has been found to result in a significant increase in the
nematode population (6), As links in the food chain, the
larger zoomicrobes, such as ciliates, nematodes, rotifers
and bristle worms, increase in numbers at the expense of
the small ones, such as flagellates and small amoebae.
In a secondary effluent, the larger zoomicrobes are usually
found in greater numbers than the smaller organisms.
Zoomicrobes are useful indicators of pollution by
secondary effluent. They are normally members of_the
aerobic fauna in soil and benthos whenever bacteria-food
is available and flourish in the biological treatment
because of the rich bacterial growth and relatively aerobic
environment provided by such a treatment.
On the other hand, the planktonic microbes in non-
polluted to slightly polluted rivers, lakes and impoundments
are predominantly algae (including phytoflagellates). Most
zooplankton are algae-feeders. This composition of the
microbial distribution has been confirmed by findings from
surveys of algae (7,8,9), rotifers (10), nematodes (11,12),
and nematodes and protozoa (13) in surface waters in the
United states.
When a secondary effluent is discharged into a surface
water the microbes it carries are intermingled with those
native in the receiving water. The use of secondary efflu-
ent flora as pollution indicators would be impractical if
the method requires species/genus identification. The
concept of their usefulness as pollution indicators was
based on a significant quantitative difference between the
effluent-bound and most of the native zoomicrobes (13).
The zoomicrobiological method for judging water quality
has to be an adjunct to those indicating fecal pollution.
In this capacity the zoomicrobial results can supply in-
formation for a more complete interpretation of the state _
of pollution in the aquatic environment. The fecal bacterial
92
-------
or virological results provide a cross-section of the
sanitary quality of water. Zoomicrobial results reflect
not only the degree of effluent pollution but also the
interaction between the effluent and the receiving water.
For instance, the very actively-feeding zoomicrobes, such
as the ciliates and bristle worms, generally perish and
disintegrate in a matter of hours due to the sudden decrease
in food bacteria. Presence of these zoomicrobes indicates gross
and very recent pollution by secondary effluents. Very
low nematode counts associated with moderately high fecal
bacteria counts indicate very slow flow-rate which promotes
nematode settling. Very low fecal bacterial counts associated
with low protozoa counts but high nematode counts indicate
that the effluent has been exposed to disinfection. High
fecal bacterial counts associated with very low protozoa
and nematode counts suggest the occurrence of a number of
unusual events such as transient exposure to anaerobiosis.
Anerobic conditions are detrimental to the survival of
zoomicrobes but not to enteric bacteria or viruses. Other
unusual events which may be adverse to nematodes or protozoa
are relatively short residence of secondary effluent in
stabilization ponds, or presence of animal respiration
poison accidently introduced or slowing accumulating in the
receiving water. Zoomicrobial findings can provide some
useful information on the state of the water quality in_its
capacity as a carrier of effluent, as well as to pollution
indication.
Methods of Zoomicrobial Examination
1. Development of a zoomicrobial pollution index (ZPI).
A formula was proposed for determining the ZPI (13):
ZPI = A + B + 1,OOOC
A
where A is the total phytoflagellate count of a sample, B
is the total protozoan count, and C is the total nematode
count. To make the formula applicable to a wider range of
pollutional conditions, C is expanded to cover both nematode
and bristle worm counts.
The formula excludes the rotifers and small arthropods
and stresses the importance of the presence of worms. Rotifers
and small arthropods are present in relatively small numbers
in comparison with the planktonic rotifers and arthropods
in open waters.
93
-------
The weighting factor assigned to the nematode count
was based on the presence of thousands to tens of thousands
of them in secondary effluents (6, 14) and their relatively,
long survival in the receiving waters and absence in % to
one gallon samples of unpolluted or relatively unpolluted waters
(11,12,13).
When the water is no more than moderately polluted, the
phytoflagellates are present in greater numbers than the
protozoa and nematodes, the value for A is larger so the
ZPI approaches 1.0. As the degree of pollution increases,
there is a corresponding increase in value of B and C and
a subsequent increase in the, ZPI. Thus, the higher the
ZPI the greater the degree of pollution.
The data obtained in a study'of the Missouri and Ohio
rivers (13, 15) demonstrated a close correlation among the values
for ZPI, nematode count, and the total and fecal coliform
counts. These results provided the basis for the quanti-
tative relationship between the values of ZPI and levels of
pollution and are reproduced in Table 1.
Table 1 reveals a correlation between the total and
fecal coliform counts and the values for ZPI at all levels
of pollution. The sensitivity of the ZPI is demonstrated
by the significant increase in its value at a point several
miles downstream from an effluent discharge. Another
point of interest is the continuous decrease in both nematode
count and ZPI value as the sampling point was moved further
and further downstream from the point of discharge, while
the total and fecal coliform counts reached a stationary
state" when the hydraulic conditions became stabilized.
The procedure for determining the ZPI is relatively
simple. The 13th edition of the Standard Methods suggests
in the Section on Biological Examination the grouping of
microbes into categories of flagellated protozoa, nonfla-
gellated protozoa, phytoflagellates and other algal organ-
isms in a plankton analysis. Rotifers are included but
nematodes are not mentioned. Values for A and B, therefore,
are supplied by the data from a routine plankton analysis.
To obtain the value for C, a sample of water of 1/4 - 1
gallon is filtered through one or more 8-10 microm
millipore membrane filters and the particulate concen-
trates, nematodes included, are washed off the membrane
filters with a few mis of dilution water. The washings
are pooled and transferred to Sedgwick-Rafter counting
chambers. Enumeration of nematodes is done under low power
magnification.
94
-------
Table 1. Correlation between Total and Fecal Coliform Counts and Nematode Counts
vp
Ol
Distance from point
of discharge (miles)
Missouri River
-------
Data from zoomicrobial examination offers information
on the extent of pollution, interaction between effluent
and receiving water when the interpretation is made in the
light of bacteriological data. For instance, a secondary-
effluent that has had a residence in stabilization ponds
would yield a low value for ZPI and very few nematodes due
to the rich algae growth and settling of nematodes during
the retension period. A stream used as carrier of such
effluent would show relatively high coliform counts but
very low value for ZPI.
2. Nematode Pollution Index
In the absence of plankton analysis data, the nemato-
logical examination can easily be made and the nematode
pollution index (NPI) used as a substitute for the ZPI.
The nematode count obtained with the procedure described
above can be used alone as a quick determination of the
pollution level of a surface water. The counts are expressed
as the number of nematodes per gallon.
Summa ry and Conclus ion
The use of zoomicrobes as pollution indicators was
described and discussed on the basis of historical back-
ground and present pollutional conditions. From these
discussions a ZPI was worked out from zoomicrobial examin-
ation data obtained in field studies made on 2 polluted
and 7 clean or relatively non-polluted rivers. For a
quick assessment of pollutional condition in a surface
water, the NPI may be used as a substitute for ZPI. A
procedure for nematological examination of water has been
described.
96
-------
REFERENCES
1. Liebmann, H. Handbuch der Frischwasser- und
Abwasserbiologie, Bd. I, 2 Auf1. Michen, Verlag
Olderbourg, 1962.
2. Clark, N. A., Berg, G., Kabler, P. W., & Chang, S. L.
Human enteric viruses in water: Source, survival,
and removability. Int. Conf. Wtr. Pollu. Res.,
Lond. Pergamon Press, 1964.
3. Chang, S. L. & Kabler, P. W., Detection of cysts of
Endamoeba histolytica in water by the use of membrane
filter. Am. Jl. Hyg. 64:170, 1956.
4. Metzler, D. F. , Gulp, R. L. Stoltenberg, H. A.,
Woodward, R. L. , Walton, G., Chang, S. L., Clarke,
N. A. Palmer, C. M., & Middleton, F. M. Emergency
use of reclaimed water for potable supply at Chanute
Kan. Jl. Am. Wtr Wks Assoc., 50:0121, 1958.
5. American Pub. Health. Ass'n, American Wtr Wks Ass'n,
and Wtr Pollut. Cont. Fed. Standard Methods for the
Examination of Water and wastewater, 13th Ed., Amer.
Pub. Hlth Ass'n, New York, 1971.
6. Chang, S. L. & Kabler, P. W. Free-living nematodes in
aerobic treatment plant effluent. Jl. Wtr. Pollut.
Contr. Fed. 34:1256, 1962.
7. Palmer, C. M. Biological aspects of water supply
arid treatment in Virginia with reference to algae.
Va. Jl. Sci., 18:6, 1967.
8. Ibid., Algae and associated organisms in West Virginia
waters: Problems and Control, Castanea 32:123, 1967.
9. Ibid., Algae in relation to water quality in Pennsylvania,
Pa. Acad. Sci.7 41, 1967.
10. Williams, L. G. Dominant planktonic rotifers of major
waterways of the United States. Fed. Wtr. Pollu.
Contro. Ass'n. Limnol. & oceanog. 11:83, 1966.
97
-------
11. Chang, S. L., Woodward, R. L., and Kebler, P. W.
Survey of free-living nematodes and amoebas in
municipal supplies. Jl. Am. Wtr Wks Ass'n., 52:613,
1960.
12. Chaudhur, N., Siddigi, R., & Engelbrecht, R. S.
Source and Persistance of nematodes in surface waters
Ibid., 56:73, 1964.
13. Chang, S. L. Zoomicrobial indicators of water pollution,
Presented at the Annual Meeting of the American
Society for Microbiology in Philadelphia, Pa.,
April 23-28, 1972.
14. Chaudhuri, N., Engelbrecht, R. S. & Austin, J. H.
Nematodes in an aerobic waste treatment plant. Am.
Wtr. Wks Ass'n. 57:1561, 1965.
98
-------
METHODOLOGY FOR THE ENUMERATION OF PSEUDQMQNAS AERUGINOSA
*
V. J. Cabelli, Ph.D. and M. A. Levin, Ph.D.
Standard Methods
Two methods are described for the enumeration for
Pseudomonas aeruqinosa in the section on "Tests for Swimming
Pool and Bathing Water Places" in Standard Methods for the
Examination of Water and Wastewater (1) . Both appear to be
modifications of procedures described by Drake (2). One is
a membrane filter (MF) procedure utilizing the "tech" medium
containing 9.05% hexadecyltrimethyl ammonium bromide; the
results obtained therewith are described as qualitative.
The second is a most probable number (MPN) method; it is
recommended in Standard Methods (1) that all presumptive-
positive tubes be confirmed.
Other media for the isolation and cultivation of P_.
aeruqinosa have been described by King and Raney (3) and
Goto and Bromoto (4). According to Drake (2), most of
these media require large inocula and do not yield quantita-
tive recovery of the organism.
Other Methods
Two other methods which are amenable to the examination
of environmental and potable water samples have been described.
Both are MPN methods. The first is Drake's (2) original
method and the second is the Salmonella - P.. aeruqinosa
method described by Kenner et al. (5). Drake's method requires
confirmation of questionable tubes on an acetamide agar
medium; it was examined by Levin and Cabelli (6) and found
to be deficient in several regards: recoveries were less
than half that on mPA; was not useable with marine waters
because a floculate precipitate developed; and the confirmation
frequency was poor. The Kenner procedure, probably because
of its dual purpose, required that the tubes be streaked
on XLD agar (7), followed by confirmation of typical colonies.
The quantity and types of positive data required for
the acceptance of any of the four of the above procedures
as reference or candidate methods are unavailable from
published reports.
*Northeast Water Supply Research Laboratory
Narragansett, Rhode Island, NERC-Cincinnati
99
-------
Candidate Method
A membrane filter procedure (mPA) developed by Levin
and Cabelli (6) appears to be the best candidate for a
reference or standard method for the enumeration of P_.
aeruqinosa from recreational and potable waters. The medium
(mPA agar) is prepared by adding the ingredients (Table 1)
to distilled water, autoclaving the mixture at 121°C for
15 min., cooling the medium to 55-60°Q adjusting its pH to
7.2+ 0.1 and adding the dry antibiotics thereto. Minimal
quantities of lactose/ sucrose and xylose and a pH indicator
(phenol red) are included in the medium so that those
coliforms which grow in the presence of the inhibitors can
be differentiated from the non-fermentative P.. aeruqinosa.
The H2S indicator system permits the differentiation of
P_. aeruqinosa from most Salmonella and Proteus species.
The ferric ammonium citrate, sodium thiosulfate and phenol
red also are essential for the development of the tan to
brown color characteristic of P_. aeruqinosa colonies on
mPA medium. The inhibitors used are those to which JP.
aeruqinosa is insensitive relative to most Gram-negative
organisms. Actidione is incorporated into the medium to
prevent fungal overgrowth; the concentration used was shown
not to affect the recovery of P_. aeruqinosa from field
samples. Membrane filters, through which the water samples
are passed, are placed on the surface of mPA agar plates
and incubated at 41.5 + 0.5°C to suppress background
organisms including most pseudomonas other than P_. aeruqinosa
as well as many of the organisms indigenous to the aquatic
environment. Forty-eight hour incubation is required for
the appearance of distinctive P_, aeruqinosa colonies.
Typically, the colonies are 0.8-2.2 mm in diameter and flat
in appearance with light outer rims and brownish to greenish-
black centers. The medium is dispensed in 3 ml quantities
to sterile 50 x 12 mm petri plates. An agar medium is used
since variable recoveries were obtained when liquid media
were used to impregnate filter pads. Poured plates of the
medium can be stored at 6 C for one month without affecting
recovery or selectivity.
Confirmation of typical colonies can be accomplished
by the method of Brown and Scott Foster (8) , in which the
isolate is transferred to a milk agar plate - when trans-
ferring from isolated colonies, a single streak on a portion
of the plate is sufficient. Following incubation for 24
hours at 35 C, _P. aeruqinosa hydrolyzes the casein and
produces a yellowish-green to green diffusible pigment.
Verification of colonies typical of I?, aeruqinosa on mPA
100
-------
Table 1. Composition of mPA medium
Component Quantity in
gm/100 ml
L-lysine HCl 0.5
Yeast extract (Difco) 0.2
Xylose 0.25
Sucrose 0.125
Lactose 0.125
NaCl 0.5
Phenol red 0.008
Sodium thiosulfate 0.68
Ferric ammonium citrate 0.08
Agar 1.5
Distilled water 100 ml
Add ingredients to distilled water; mix, autoclave at 121 C
for 15 min.; cool to 55-60 C; adjust pH to 7.1 + 0.1;
and add the dry antibiotics.*
Sulfapyridine (Nutritional biochemicals) , 17.5 mg;
Kanamycin (Bristol-Myers), 0.85 mg; Nalidixic acid
(Cal Biochemicals), 3.7 mg; and Actidione, (Upjohn),
15.0 mg per 100 ml or medium.
101
-------
medium will not be required routinely. However/ verification
of a number of typical P_. aeruqinosa and other bacterial
colonies is necessary not only when an operator is being
trained in the mPA procedure but also when the method is
used at a new location. The above considerations notwith-
standing, in the absence of verification/ estimates of P_.
aeruqinosa densities should be designated as "probable."
Following verification by the method of Brown and Scott
Foster (3), the estimates would be considered as "confirmed."
The mPA procedure was evaluated by Levin and Cabelli
(6) against the following acceptability criteria: (a)
accuracy - the recovery of at least 75% of the "viable"
Ł. aeruqinosa cells from estuarine and fresh water samples
artificially seeded with the organism and stressed by
storage in these suspending media; (b) selectivity - the
reduction of "backgrouns organisms" in naturally polluted
water samples by at least three orders of magnitude (1000
fold); (c) specificity - when assaying field samples/ at
least 90% of those colonies designated as Ł. aeruqinosa
should verify as such; and no more than 10% of those
colonies not designated as JP. aeruqinosa should/ in actuality/
be this organism; (d)precision - that/ with field samples,
the distribution of D value estimates of assay variability,
calculated according to Eisenhart and Wilson (9)/ approximates
that expected by chance; and (3) comparability - that the
accuracy and sensitivity of the method be equal to or
greater than existing methods. The mPA method satisfied
all the aforementioned criteria. Subsequent to its develop-
ment and evaluation/ the mPA procedure was used at^several
other laboratories for the enumeration of P. aeruqinosa in
potable and recreational waters and in sewage samples. It
was found amenable to routine use/ and confirmation of
typical colonies approached 100 percent.
102
-------
REFERENCES
1. American Public Health Association. 1971. Standard
Methods for the Examination of Water and Wastewater,
Thirteenth ed., p. 710, Am. Publ. Hlth. Assn., New York
2. Drake, C. H. 1966. Evaluation of culture media for the
isolation and enumeration of Pseudomonas aeruqinosa.
Health Lab. Sci. 3_-. 10-19
3. King, E. O., M. K. Ward, and A. E. Raney. 1954.
Two simple media for the demonstration of pyocyanin
and fluoracein. J. Lab. and Clin. Med. 44;301-307
4. Goto, S., a*nd S. Bromoto. 1970. Nalidixic acid
Cetrimide agar. Jap. J. Microbiol. 14:65-72.
5. Kenner, B. A., Dotron, G. K., and J. E. Smith. _ 1971
Simultaneous Quantitation of Salmonella Species and
Pseudomonas aeruqinosa; Environmental Protection
Agency, National Environmental Research Center,
Cincinnati, Ohio.
6. Levin, M. A., and V. J. Cabelli. 1972. Membrane Filter
Technique for Enumeration of Pseudomonas aeruginosa.
App. Microbiol. 24:864-870.
7. Taylor, W. I. 1965. Isolation of Shigellae I.
Xylose lysine agars; new media for isolation of
enteric pathogens. Tech. Bull. Reg. Med.
Technologists, 35:161-165.
8. Brown, M. R. W., and J. H. Scott Foster. 1970.
A simple diagnostic milk medium for Pseudomonas
aeruqinosa. J. Clin. Pathol. 23:176-177.
9. Eisenhart, C., and P. W. Wilson. 1943. Statistical
methods and control in bacteriology. Bacteriol.
Rev. 7:57-137.
103
-------
DISCUSSION
Chang
Cabelli
Chang
Cabelli
Did you test the reproducibility of the method
by determining the 95% confidence limits?
o
No, the D method as described by Eisenhart
and Wilson was used for this purpose. It
is a measure of plate to plate variability
across samples.
2
Are not 30 samples required for a D analysis?
We were told that 24 would be sufficient.
QUESTION
Cabelli
Did you use the OF test to confirm the identity
of suspected P> aeruqinosa colonies?
Yes, it was used initially as one of seven
tests performed for the confirmation of
"typical" colonies. When comparability was
established between the results obtained
with the seven tests and those obtained by
the method of Brown and Scott Foster, the
latter procedure was used exclusively.
QUESTION
Cabelli
What do you substitute for the skim milk
product used by Brown and Scott Foster?
We used a Carnation skim milk product called
"Starlac".
QUESTION -
Cabelli
Geldreich -
Did you try hexachlorophene as an inhibitor?
No, we did not. Incidently, we did try
several chemically defined media with no
success.
We are proposing to delete the MF procedure
for P. aeruqinosa in the present edition of
Standard Methods and to substitute the mPA
procedure.However, there is still a need
for an MPN method for situations in which an
MF procedure is not appropriate. What
would you recommend?
104
-------
Cabelli
Geldreich -
Cabelli
Kenner
Geldreich -
Brezenski -
Cabelli!
Brezenski -
Cabelli -
There are two methods, Kenner's and Drake's.
We feel certain both are superior to the one
in Standard Methods, although we did not
examine the Kenner procedure.
There is a need for a round robin to evaluate
MPN methods for P_. aeruqinosa for. use in
situations/ such as turbid waters, where MF
methods can not be used. We could also
examine pour plate methods if such are
available.
You are correct.
I have evaluated my method on a variety of
samples from soils to potable water. In
fact, we have used glass fiber filters
wherein we could test up to 10 gallons
of potable water. Others have tried it
with good results.
Jay Vascoucelos found that, in the ground
water samples he examined, ]?. aeruqinosa
generally was isolated when the Standard
Plate Count exceeded 500/ml.
Vic, when you examined the samples from the
bathing beaches in New York, did you use
the mPA method?
Yes
In general, what were the P.. aeruqinosa
densities relative to those of the
streptococci and fecal coliforms?
We haven't completely analyzed the data;
however, they were about 1- 1% orders
of magnitude less.
105
-------
FUNGI: A State-of-the-Art
*
Leonard J. Guarraia/ Ph.D.
The outline for the talk, as stated in the agenda/ calls
for, first, a review of standard Methods. In the case of
the fungi, this is a relatively easy task, since there are
no standard methods available. Secondly, a review of candi-
date methods is called for. Again, this is a relatively
easy task, since there are a variety of methods and no clear
choice for a standard method, with the exception of the
hemp seed bait technique and rose bengal medium which will
be discussed later. Finally, suggested recommendations are
called for. The most important point here is to develop
the techniques and the correlation between specific fungal
populations as water pollution indicators.
Fungi are ubiquitous, achlorophyllous, plant-like
organisms capable of growing under almost every conceivable
condition. In contrast to bacteria, fungi possess a true
nucleus and nucleoli, but differ from higher plants in that
they do not possess cross walls. Rather the mycelia contain
septa which permit the nucleus to float freely within the
cytoplasm. Both parasitic and free living forms have been
found living in air, soil and water, and will grow on such
diverse substrates as preserved specimens in comparative
anatomy laboratories, shoes, bread and preserves.
Taxonomically there are four classes of fungi: the
Phycomycetes, Ascomycetes, Basidiomycetes and Deuteromycetes
or Fungi Imperfecti. Classification is based upon where the
spores are formed. Phycomycetes possess no septa and bear
endogenous asexual spores. A common Phycomycete is water
mold. Ascomycetes develop spores in an ascus or sac-like
structure, Allomyces and Neurospora being examples. Basid-
iomycetes form external spores on a basidium or stalk type
structure. The mushroom is a common example. Deuteromycetes
are characterized as those fungi for which a completed sexual
cycle has not been demonstrated. It is believed that the
majority of the Deuteromycetes belong to the Ascomycetes.
A more complete review of classification of fungi has been
completed by Cooke (13) and Alexopoulos (14). Yeasts are
fungi which are said to have lost the mycelial habit of
growth and have become unicellular and are found in all
classes of fungi.
Division of Water Quality and Non-Point Source Control,
Office of Water Programs Operations
106
-------
As relating to water pollution the significance of
specific fungi to specific pollutants remains an area open
to question. To date there are no data which relate
specific general or species to water pollution. Various
species are capable of degrading complex compounds such as
polysaccharides; cellulose, chitin and glycogen; proteins;
casein albumin and keratin; hydrocarbons such as kerosene;
and certain pesticides. Most species are aerobic; however,
some species are capable of an anaerobic growth.
Fungi occur in all waters and various reviews have
detailed their occurrence in both the marine (1-4) and fresh
water environments (5). Cooke (6, 7) has described fungi in
fresh water streams and rivers. Also, many workers have
demonstrated (8-11) that yeasts occur in greater numbers in
organically enriched waters than in less enriched systems.
The presence of certain yeasts in water has been suggested
as a potential indicator of the presence of proteins, hydro-
carbons, straight and branched chained alkyl-benzene sulfo-
nates, fats, metaphosphates, and wood sugars (12) by virtue
of the fact that the species are capable of degrading these
compounds.
Certain microbes have been termed "sewage fungi" based
upon their common occurrence in polluted situations and
their morphology. One such organism, Sphaerotilus. natans
is, in fact, a sheathed iron bacterium in the Order
Chlamydobacteriales. J3. natans characteristically forms
chains of cells within a protein-polysaccharide-lipid
sheath attached to a substrate. The ability to attach to
a substrate provides the organism with a decided growth
advantage in flowing streams (16).
Another "sewage fungus" is a phycomycete, Leptomitus
lacteus, which produces slimes and floes in fresh waters.
This organism can be found in organically enriched cold
waters (5 -22°C) and cannot assimilate simple sugars. Best
growth is found in the presence of organic nitrogenous
wastes (12). The nomenclature and distribution of the
organism has been reviewed by Yerkes (16) and Emerson and
Weston (17).
A group of fungi which may more nearly reflect pollution
is the coprophilic fungi. These organisms are associated
with the feces of animals but rarely of man. Examples are
found in the genera Mucor and Pilobolus. These fungi
produce spores which attach to plant leaves and are in turn
107
-------
ingested by grazing animals, pass through the alimentary
canal, germinate, and use the feces as food. However, few
if any fungi are associated with human feces. Sordaria
humana has been associated with human wastes under certain
conditions (5).
Fungi can adapt to a variety of substrates and require
an organic carbon substrate and therefore can be expected to
be found in sewage and other organically enriched wastes.
As the concentration or organic material increases the
number of fungi isolated also increases. This is true not
only in water, but also on land; as the organic load of the
soil increases the numbers of fungi found als6 increase (18).
The significance of a specific species of fungi as an
indicator of water pollution has not been established.
Pathogenic fungi, once introduced into the aquatic environ-
ment, are difficult, if not impossible, to reisolate.
Associations of enteric pathogens with fecal associated
fungi do not have the same meaning as isolation of fecal
bacteria (5).
The enumeration of fungi is not equivalent^to that of
bacteria. The reason for this is that while, with bacteria,
one colony usually develops from a single cell a fungal or
yeast colony may result from a single cell, from a piece of
mycelium, or from a number of cells. Thus no one-to-one
relationship exists for easily quantifying the numbers in
water.
Sampling for the presence of fungi in the aquatic
environment is done in much the same way as for bacteria.
Grab samples can be taken using either plastic or glass
containers. A minimum volume of 45 milliliters should be
collected. Mud, soil or sand samples can also be used.
Once the sample is taken, the container should not be
tightly sealed (13) so as to permit an exchange of air.
Another technique available to the sampler is the use
of baits. A cylinder made of wire mesh stopped at both ends
is often used. The bait can be any of a variety of sub-
stances, examples being a chunk of beef, apple, rose hip,
date and hawthorn fruit. The most common bait used, however,
is hemp seed. The bait is allowed to remain in the water
for a week and is then returned to the laboratory in a
container with the water obtained from the point at which
the baits were exposed (13) . Once back in the laboratory
the baits are placed on the appropriate sterile medium.
108
-------
Media used for the isolation of fungi are usually
similar to those used for bacteria in that there is a carbon
source, inorganic salts, agar and peptone. Additionally a
dye, usually Rose Bengal, and a broad spectrum antibiotic
are included. These are added to prevent or decrease
bacterial growth. A thorough discussion of the types of
isolation media has been prepared by Cooke (13). The most
common fungi found in polluted waters are terrestrial fungi.
For this reason the media employed for an aquatic sample
are similar to those used for earth samples.
Once growth occurs in the isolation medium,transfers
can be made to agar slants. This, however, does not provide
any quantitative data. It is possible to isolate many but
not all of the yeasts present in the original medium.
Distinct colonies can be picked for further identification.
Preliminary screening for identification can best be made
using a suitable guide such as that devised by Cooke (6) ,
and depends upon knowledge of the spore-bearing structures
and general morphology upon staining (13) .
The salient points are:
1. Currently there are no fungal indicator species
for pollution,
2. Generally an increase in organic material on
water is mirrored by an increase in the fungal
populations and
3. Present techniques for isolation of fungi give
only qualitative answers.
The role of airborne fungal spores in relationship to
human health with respect to Coccidioidomycosis, Crytococcocis
and Histoplasmosis not addressed in the text is an area
requiring consideration.
109
-------
REFERENCES
1. Johnson, J. W. and Sparrow, F. K., Fungi on Oceans and
Estuaries. Weinheim, Germany. p668 (1961).
2. Johnson, T. W., Saprobic Marine Fungi, In: Ainsworth,
G. C. and Sussman, A. S. The Fungi, III. Academic
Press, pp 95-100 (1968).
3. Meyers, S. P., Observations on the Physiological
Ecology of Marine Fungi, Bull, Misaki. Biol. Inst.
12.; 207-225 (1968) .
4. VanUden, N. and Fell, J. W. 1968. Marine Yeasts.
pp 167-210. In: "Advances in Microbiology of the
Seas." (Editors M. R. Droop and E. J. F. Wood*)
Academin Press, New York.
5. Cooke, W. B. Our Mouldy Earth - A study of the fungi
in our environment with emphasis on water-Indicator
Organisms. U. S. Department of Interior, FWPCA.
pp 329-339.
6. Cooke, W. B. 1961 Population effects on the fungus
population of a stream. Ecology 42: 1-18.
7. Cooke, W. B. 1967. Fungal populations in relation to
pollution of the Bear River, Idaho-Utah. Utah Acad.
Proced. 44: 298-315.
8. Cooke, W. B.; Phaff, H. J.; Miller, M. W.; Shifrine, M.;
Knapp, E. 1960. Yeast in polluted water and sewage.
Mycologia 52: 210-230.
9. Cooke, W. B., and Matsuura, G. S. 1963. A Study of
Yeast Populations in a Waste Stabilization Pond
System. Protoplasma 57: 163-187.
10. Ahearn, D. G.; Roth, F. J.; and Myers, S. P. 1968.
Ecology and Characterization of Yeasts from Aquatic
Regions of South Florida. Marine Biology .1: 291-308.
11. Cooke, W. B. 1965. The Enumeration of Yeast Populations
in a Sewage Treatment Plant. Mycologia 57.: 696-703.
12. Ahearn, D. 1968. Fungi in Keys to Water Indicative
Organisms, Department of Interior, FWPCA. pp C-l -
C-8.
110
-------
13. Cooke, W. B. 1963. A Laboratory Guide to Fungi in
Polluted Waters, Sewage, and Sewage Treatment Systems.
U. S. Department of Health, Education and Welfare.
Cincinnati. pp 6-171.
14. Alexopoulos, J. C. 1962. Introductory Ecology. 2nd
Edition. John Wiley and Sons, New York. p613.
15. Stockes, J. L. 1954. Studies on the Filamentous
Sheathed Iron Bacterium Sphaerotius natans.
J. Bacteriol. 67; 278-291.
16. Gerbes, W. D. 1966; Observations on the Occurrence of
Septomitus lactus in Wisconsin. Mycologia 58_:
976-978.
17. Emerson, R. and Weston, W. H. 1967. Aaualinderella
fermentous. A Phycomycete Adapted to Stagnant Waters.
I. Morphology and Occurrence in Nature. Amer. J.
Bot. 14: 702-719.
18. Michaels, G. E. and McClung, N. M. 1966. The Microflora
of Deeply Buried Midden Layers. Bull. Ga. Acad. Sci.
24: 44-54.
Ill
-------
SALMONELLA
Donald F. Spino*
INTRODUCTION
This discussion is primarily limited to the isolation
and identification of the Salmonella from water. The
presentation is in three parts: General review of methods
in Standard Methods, 13th edition, available method as a
candidate method; and discussion and suggested recommenda-
tions on methodology.
Brief Review of Methods in Standard Methods,
(13th edition, pp. 697-707)
Standard Methods, when recognizing the presence of
Salmonella in the environment, states that the isolation
techniques involve relatively complicated procedures that
exceed the capabilities of all but a few water laborator-
ies (1). Recent studies have shown that salmonella can be
detected with relative ease in many surface waters(2,3,4,5);
one study detected the organism in water that was otherwise
of high quality(6).
Primary Enrichment Broth
Tetrathionate broth is among the enrichment broths
discussed in Standard Methods. Its excellence for the
primary enrichment of Salmonella is noted.
Standard Methods states that when using the
tetrathionate broth, incubation should extend beyond 48 hours,
with repeat streaking from the same tube daily up to 5 days
to ensure recovery of all the Salmonella serotypes that
may be present. Stationary growth should be completed
between 18 and 24 hours. H. Raj, for example, performed
viable counts on 13 different serotypes of Salmonella at
24, 40, 48, and 72 hours (7). For S_. tvphimurium, the
viable count is shown in Table 1(7).
Time 24 hrft 40 hr 48 hr4 72 hr2
Cell Concentration 15xlOb 93x10 24x10 43x10
Per Unit Volume
Table 1
Survival of S. typhimurium vs time
* Solid Waste Research Division, NERC-Cincinnati.
112
-------
For the remaining 12 serotypes studied there was no
increase in the viable count after 24 hours. It would
appear that the increased time did not increase the count.
Solid Media (differential)
Brilliant green agar. Standard Methods advises that
the recovery efficiency of given lots of this medium should
be checked with the use of several recently isolated strains
of Salmonella. Personal observations have shown that if a
mixed culture of Escherichia, Proteus, and Salmonella is
used, then one can observe the efficiency with which non-
Salmonella are suppressed and salmonella are selected and
proliferated. Standard Methods states that Salmonella typhi
and a few other species of Salmonella grow poorly on
brilliant green agar because of its brilliant green dye
content. Difco states that this medium is not recommended
for isolation of S. typhi(8). For those species that do
grow well on brilliant green agar, incubation to 24 hours
should be sufficient; in some cases the plates need to
stand for an additional 5 to 6 hours at room temperature
for full development of some colonies.
Xylose lysine deoxycholate agar. When discussing the
color of colonies grown on xylose lysine deoxycholate agar,
Standard Methods states that after incubation for 24 hours
colonies of Shigella are red, whereas Salmonella and Arizona
organisms produce black-centered red colonies. Coliform
bacteria,- Citrobacter, Proteus, and most paracolons produce
yellow colonies. One researcher found that yellow colonies
may also be Salmonella? if incubated at room temperature
for an additional 6 hours (30 hours total) they should turn
pink with dark centers(9).
Temperature Options
When discussing the proper temperature for incubation,
Standard Methods explains how pathogens can be further sep-
arated from the surviving nonpathogenic bacterial population
with the proper choice of incubation temperature for primary
enrichment and secondary diffenentiation on selective solid
media. The two factors of temperature and choice of media
are interrelated. Great emphasis must be placed on this
latter interrelationship.
113
-------
Biochemical Reactions
For identifying the genus Salmonella, Standard Methods
suggests a three-phased schedule. At the end of phase three,
16 tests are required to identify this genus. The candidate
method presented here for consideration requires three
different media/ two reagents/ and one antiserum.
CANDIDATE METHOD FOR SALMONELLA IDENTIFICATION
The Haina Method/ with a Modification
In 1951, Hajna found that the reactions for most typical
Salmonella are as shown in Table 2(10).
"H"
Motilitv Sulfide Media Triple Sugar Iron Broth Serologjcal
Motility H2S Urease Slant/Butt H2S Indol Antigen
+ + - alkaline/acid + - +
gas
Table 2
Selected biochemical and serological reactions for typical
Salmonella.
For this method/ three different media, two reagents/ and
one polyvalent "H" antiserum are needed.
A suspected Salmonella colony may be picked and inocu-
lated into Motility Sulfide Media (MSM) , Triple Sugar Iron
(TSI), and "H" Broth; incubated 12-18 hours at 35 C. If
TSI shows that it is a non-lactose fermenter, then the MSM
shguld be topped with 1 ml of urea broth and incubated at
35 C for six hours. Antigen should be prepared from "H"
broth by placing 1 ml of "H" broth culture into an equal
volume of 0.6% formalinized saline. The remaining "H"
broth culture can be used for the indol test. The culture
is discarded if indol-positive. Serological reaction is
determined by placing: 0.5 ml of polyvalent "H" antiserum
with 0.5 ml of the formaldehyde-treated "H" broth culture/
mixed, and placed in a 50 C water bath for one hour. Urease
activity is observed on the MSM. Typical reactions of the
Salmonella have been given. One may save time by discarding
the non-Salmonel1a earlier than six hours by checking indol
production and floculation before reading urease activity.
114
-------
In 16 water quality surveys of rivers throughout the
United States from 1964 to 1972, a total of 317 cultures
were identified by this method.* All were sent to the
National Center for Disease Control for serological typing.
One of them was an Arizona and the remaining 316 were
verified as Salmonella of which there were 41 different
seretypes.
SUGGESTED RECOMMENDATION
Identification and the Proposed Methods
The simplicity and accuracy of Hajna's method makes
it a feasible approach to identifying Salmonella. The
simplicity of the technique makes it possible for any
technician to do the job in a small laboratory; the 16
tests described in Standard Methods are not needed. Media
preparation is reduced by 80 percent when compared with
Standard Methods. Urea broth, Kovac's reagent, and the
antiserum prepared in 100 ml volumes is sufficient for 200
tests of each. Time for media preparation is saved as
well as time consumed for testing.
Media
E_nr_ichme_nt_ Broths
Tetrathionate, GN broth, and Dulcitol selenite broth
are recommended because none are very toxic to the Salmonella
and due selectively encourage the growth of Salmonella.
Selective Plating Media
Brilliant green agar. About 90 percent of the colonies
that are white with a pink background are Salmonella. The
lots of agar should be checked for toxicity with various
strains of Salmonella(11).
Galton and Boring(12) state citing references that,
The most widely used selective media are bismuth sulfite
agar, deoxycholate citrate agar, SS agar, MacConkey Brilliant
Green agar, and the brilliant green-phenol red agar.
*These investigations were performed by the National
Field Investigation Center-Cincinnati.
115
-------
These same authors believe that, "Although Salmonellae
may be isolated on all these media, the BG agar, when
properly prepared, is more inhibitive for enteric organisms
other than Salmonellae, and Salmonella colonies are detected
with greater ease than on other selective media."
Brilliant green agar was used as the solid selective
medium in the 16 surveys previously noted, with the isolation
of 316 salmonella of 41 different serotypes. The Salmonella
found downstrean from a source of fecal pollution, were
always recovered on brilliant green agar.
Xylose lysine agar, with the option of preparing
xylose lysine deoxycholate (XLD) described by Taylor, is
also recommended(13) .
Temperature
For isolating Salmonella from the environment, various
investigators have used a variety of temperatures:
43.0°C - Harvey and Price(14).
4JL^5°C - For both the enrichment broths and
differnetial plates for sensitivity of
isolation of many of the 900 to 1200
serotypes of Salmonellae(3,5,11,15,16,17).
40.0°C - Specifically for the isolation of S_. typhi,
Livingstone (Johannesburg) personal
communication.
39.5°C - Peterson(19).
37.0°C - Could be used in conjunction with elevated
temperatures for comparison; perhaps just
the first and second days of survey.
Kenner, Dotson, and Smith report an 85 percent recovery
of selected Salmonella serotypes at 40.0 C, 25 percent
recovery of Salmonella at 41.5 C, and 10 percent recovery
at 37.0 C using dulcitol selenite broth (DSE) (7) and
xylose lysine deoxycholate plates(18).
116
-------
One cannot explain successful isolation of Salmonella
in terms of temperature alone. There appears to be a
marked interdependency of temperature and type of medium
used6 Livingstone(20) (via correspondence) claims that
41.5 C is too high for isolation of j3. typhi. Wun et al.,
grew S. typhi at 41.5 C in GN broth(20).
Kraft et al(16) states: "The selective inhibitors,
sulfathiazole and brilliant green, were added to the
tetrathionate broth intended for use at 37.0°C, but
brilliant green was omitted from the 41.5°C broth because
at this temperature the dye severely inhibited the growth
of the live Salmonella species tested."
It is recommended that each laboratory be free to choose
a combination of media which would enrich for and selectively
differentiate the Salmonella bacteria at incubation tempera-
tures ranging from 37.0 C to 43.0 C. Results should be
submitted to an appropriate evaluation committee to arrive
at a consensus on methodology.
117
-------
REFERENCE LIST
1. Anon. Standard Methods for the Examination of Water
and Wastewater. 13th edition sec. 411, pp 697-704.
Am. Public Health Assoc. New York, N.Y. (1971).
2. Brezenski, F. T. , and R. Russomanno. 1969. The
Detection and Use of Salmonellae in Studying
Polluted tidal estuaries. J. Water Pollut. Contr.
Fed. 41: 725-737.
3. Gallagher, T. P., and D. -F Spino," The Significance of
Numbers of Coliform Bacteria as an indicator of
Enteric Pathogens. Water Res. 2:169-175 (1968).
4. McCoy, J. H» , Salmonella in Crude Sewage, Sewage
Effluent and Sewage-Polluted Natural Waters, p 205-219,
B. A. Southgate (ed) , Advances in Water Pollution
Research, vol. 1, Pergamon Press, Ltd., Oxford,
England (1962).
5. Spino, D. F., Elevated temperature technique for the
isolation of Salmonella from streams. Appl. Microbiol.
14:591-596 (1966).
6. Fair, J. F. , and S. M. Morrison, Recovery of Bacterial
Pathogens from High Quality Surface Water. Water
Resour. Res. 3:799-803 (1967).
7. Raj, H. , Enrichment media for selection of Salmonella
from fish homogenate. Applied Microbiol. 14:12 (1966).
8. Difco Manual, 9th edition, Difco Laboratories, Inc.
Detroit, Mich, pp 144-145.
9. Wun, Chun K. , Joel R. Cohen, Warren Litsky, Evaluation
of Plating Media and Temperature Parameters in the
Isolation of Selected Enteric pathogens. Health Lab.
Science Vol. 9, No. 3 (1972).
10. Hajna, A. A., A Proposed Rapid Method of Differenitating
and Identifying Bacteria of the Intestinal Group in
State Public Health Laboratories, The Public Health
Laboratory 9:23-30 (1951).
118
-------
11. Read, R. B., and A. L. Reyes, Variation in Plating
Efficiency of Salmonellae on Eight Lots of Brilliant
Green Agar. Appl. Microbiol. 16:746-748 (1968).
12. Galton, B. M. , and J. R. Boring III, Public Health
Service Publication 1142. Examination of Foods for
Enteropathogenic and Indicator Bacteria: Review of
Methodology and Manual of Selected Procedures, pp
26-32, (1964).
13. Taylor, W. I., Isolation of Shigellae I. Xylose
Lysine Agars; New Media for Isolation of Enteric
Pathogens, American Journal of Clinical Pathology,
Copyright 1965 by Williams and Wilkins Company, Vol.
44 No. 4 Reprinted from: Technical Bulletin of the
Registry of Medical Technologist, Vol. 35, No. 9,
1965.
14. Harvey, R. W. S., and T. H. Price, Elevated Temperature
Incubation of Enrichment Media for the isolation of
Salmonellae from Heavily Contaminated Materials.
J. Hyg- Comb. 66:377 (1968).
15. Grunnet on B. Brest Nielsen, Salmonella Types Isolated
from the Gulf of Aarhus Compared with Types from
Infected Human Beings, Animals, and Feed Products
In Denmark. Appl. Microbiol. 18:985-990 (1969).
16. Kraft, D. J. , Carolyn Olechowski-Gerhardt, J. Berkowitz
and M. S. Finstein, Salmonella in Wastes Produced
at Commercial Poultry Farms. Appl. Microbiol.
18:703-707 (1969).
17. Van Donsel, D. J., and E. E. Geldreich, Relationship
of Salmonellae to Fecal Coliforms in Bottom Sediments,
Water Research 5:1079-1087 (1971).
18. Kenner, Bernard A. G., Kenneth Dotson, and James E.
Smith, Simultaneous Quantitation of salmonella Species
and pseudomonas Aeruginosa. Environmental Protection
Agency, National Environmental Research Center,
Cincinnati, Ohio (September, 1971).
19. Peterson, M. and A. Klee, Studies on the Detection of
Salmonellae in Municipal Solid Waste and incinerator
Residue, International Journal Environmental Studies,
2:125-132 (1972).
119
-------
20. Livingstone/ D. J., Improved Method for Isolating
Salmonellae from Polluted Water, Public Health,
Vol. 65, pp 87-88 (1965).
120
-------
AQCI/ MICROBIOLOGY SECTION REPORT*
,, **
Herbert Manning, Ph.D.
In the Analytical Quality Control Laboratory at NERC-
Cincinnati we have been evaluating enrichment and plating
media for detecting Salmonella from natural water samples.
Using the gauze swab sampling technique we examined Ohio
River sites above and below Cincinnati, as well as a
recreational lake.
Our procedure consisted of retrieving the swab after
five days, adding it to tetrathionate-brilliant green or
dulcitol selenite enrichment media, and incubating it in
a water bath at 41.5 C. Rappaport's medium was also in-
cluded in a split sample comparison with the other two
enrichment media. After incubating the primary enrichment
broth culture for 7 1/2 hours, a 150 ml volume was added
to 150 ml of fresh broth to establish a secondary enrich-
ment culture. At the end of 24 hours both enrichment
cultures were streaked to xylose-lysine-desoxycholate,
Hektoen Enteric, brilliant green, and MacConkey's agars.
These plates were incubated in sealed plastic bags in a
water bath at 41.5 C for 24 hours. Twenty typical Salmonella
colonies from each medium were picked to TSI agar slants
and subcultured to urea, phenylalanine, lysine iron agars,
and lactose, dulcitol, and H broths. Isolates with bio-
chemical characteristics typical of Salmonella were then
serologically confirmed with Difco Poly A-I O antiserum
and Difco Poly a - z H antiserum. Each isolate was also
tested with specific O-group antisera. A limited number of
isolates from brilliant green agar were identified by CDC-
Atlanta and the Ohio Department of Health Laboratory,
Columbus, Ohio.
The results, based upon 1,414 isolates, indicated that
of the enrichment media tested, dulcitol-selenite was most
selective, that is, yielded fewer false positives, and
tetrathionate-BG much less selective, that is, yielded more
false positives. Preliminary evidence indicates that
Rappaport's medium did not enrich for Salmonella when all
three media were compared with a split sample. Among_the
plating media tested the order of decreasing selectivity
was found to be brilliant green, Hektoen enteric, xylose-
lysine-desoxycholate agar, and MacConkey's agars. The
16 1/2 hour secondary enrichment in tetrathionate-BG broth
Submitted in writing for the record,
**
Analytical Quality Control Laboratory, NERC-Cincinnati,
121
-------
markedly increased the percent confirmation of Salmonella
over that of the primary enrichment. Secondary enrichment
in dulcitol selenite broth did not increase the percent
confirmation.
122
-------
CONCENTRATION OF SAMPLE
**
Kathleen G. Shimmin
Recovery of Salmonella from water is facilitated by
employing an initial sample-concentration step. Concentra-
tion may occur over several days to a week by suspending a
gauze pad into a flowing body of water; after the desired
suspension time the pad is placed into enrichment broth
and incubated one to five days. Discrete samples (one to
two liters) may be concentrated by filtration through either
diatomaceous earth or membrane filters (a prefilter may be
necessary for waters which are very turbid).
*
**
Submitted in writing for the record
Laboratory Support Branch, Region IX
123
-------
FLUORESCENT ANTIBODY SCREENING
**
Kathleen G. Shimmin
A fluorescent antibody Salmonella screening procedure
has been developed for detection of Salmonella in salt and
fresh water samples. The procedure basically involves the
concentration of bacteria from a water sample in a diato-
maceous earth filter aide (Celite). The Celite with the
entrapped bacteria is then immersed in Salmonella enrichment
broth. After incubation of the broth spot plates for primary
isolation are prepared. The primary plates are incubated
for three hours; then slide impression smears are prepared
and stained with a Salmonella Panvalent Antiserum (Difco).
Positive fluorescence indicates the possible presence of
Salmonella.
Submitted in writing for the record
**
Laboratory Support Branch, Region IX
124
-------
THE SALMONELLA: DISCUSSION
Geldreich;
Spino:
Geldreich:
Spino:
Geldreich:
Dan, I believe that after you reviewed the
current edition of Standard Methods, you
basically came around full circle to saying
the same thing. Actually the Standard Methods,
13th ed. , is a state-of-the-art approach. it
has not locked in on any method because there
is no one method. Apparently you agree with
this. I am a little confused about your objec-
tions to Standard Methods and what direction
you think the methodology should take. You
did mention incubation temperature and time.
Data as yet unpublished which Fran Brezenski
accumulated at Edison, New Jersey indicates
there are certain advantages to carrying out
the incubation of these organisms beyond the
24 hour limit. Some investigations have pro-
posed as long as five days of incubation.
We can't assume that 24 hours is satisfactory.
I did refer to Raj who performed viable counts
on 13 different Salmonella species. There was
no increase in viable counts after 24 hours.
The ability to recover additional species may
be related to the volume of sample or enriched
growth actually streaked. Limited numbers of
selective agar plates are streaked from the
enrichment growth and the loopfuls of growth
represent very limited sample volumes. Further
plate streaking at any time interval may allow
the recovery of additional species.
We need flexibility of methodology which could
eventually come to the same conclusion reached
in Standard Methods. We have proposed some
limitations in the types of media used. Standard
Methods has recognized that some media don't
recover Shigella and has proposed an alternative
choice. You don't propose any specific procedure
for Shigella.
I did limit my presentation primarily to Salmonella,
We have to go beyond Salmonella of course;
we all agree on that.
125
-------
Spino:
Geldreich:
Shimmin:
Geldreich:
Bordner:
Spino:
Geldreich:
Brezenski:
My point was that I felt that these two groups
of organisms even though they are in the same
tribe, should be treated separately for isolation.
Perhaps the next revision of s t and a rd Methods
should include a separate section on Shigella.
I think the statement Dan made that was different
was that each regional or other laboratory should
try this range of methods that he suggested and
send in the results to a central committee.
This is a little different than found in Standard
Methods. I think it is a good point.
I think this is something we have to do with
other media also. I go for that.
Methods evaluation is an approach that we don't
have in Standard Methods. I don't really think
Standard Methods should be the place for it.
An EPA manual that we might develop could pro-
mote this kind of evaluation. Also Dan gave
us a candidate method, which we asked for, but
we don't have to accept it. It is an interest-
ing viewpoint and we could use it as a basis
for comparison and method development.
Standard Methods states: "Salmonellae are
extremely common in the environment and are
probably responsible for most water-borne out-
breaks. Unfortunately isolation techniques
even for these ubiquitous organisms involve
relatively complicated procedures that will
exceed the capabilities of all but a few water
laboratories." I say that anybody can do this.
I don't agree with you, I think our laboratories
are exceptionally high caliber laboratories that
can handle pathogen isolation; but this may
not be true of small or branch laboratories
which use Standard Methods. standard Methods
covers more than just EPA needs.
To me, "once a pathogen, always a pathogen"; that
means that people with certain recognized quali-
fications must handle them, not just anybody.
126
-------
Spino:
Geldreich:
Brezenski:
Spino:
Brezenski
I have shown this identification method to a
technician once, and after that he could do
it as well as I. I don't think you need a
high-powered microbiologist to do it.
This procedure takes more judgment to perform
properly than any of the others. One has to
have an understanding of all the choices and
judgments of the methodology and media, for
example the interpretation of colonies on
various selective media.
I think what we are driving at is that the
methodology per se is a set of mechanical
manipulations which are relatively easy and a
technician can be trained to do this, but the
decision, the choices of media, etc., will have
to be made by someone qualified. I use as an
example, the scheme which includes motility
sulfide medium which is supposed to give you
the hydrogen sulfide reaction of the salmonella.'
I have data and there is some data in the lit-
erature showing that the hydrogen sulfide system
in that particular medium is not amenable to
all the salmonella. For example, we have had
to use Sims medium in conjunction because we
got negatives with that particular system.
If the technician saw a negative H2S, would he
have the knowledge to use the Sims medium or
would he disregard it because it is not a H^S
positive strain. There are some H2S negative
Salmonella strains.
I think that if the procedure is spelled out
in cookbook form and the technician follows
directions, he should be able to do the tests.
He could follow all the way through to the "H"
antisera floculation tests and check all the
reactions.
Look at the manual which NCDC puts out on the
Enterobacteriaceae and at the section on
Salmonella alone. There are many pages devoted
to salmonella. NCDC has not restricted their_
people to specific methods from a clinical point
of view, or an ecological point of view. Also
in the food analysis section they devote much
127
-------
Bordner:
Brezenski:
Shimmin:
Spino:
Brezenski:
space to the presentation of different types
of media which are available to the technician.
Someone has to know that (1) these are avail-
able and (2) when to use them. I think we
are minimizing this methodology or simplifying
it to a degree which we shouldn't be.
On the other hand Fran, can you conceive of
simplifying the methodology to some extent for
our use if we are talking about Salmonella from
water as opposed to the clinical diagnostic
viewpoint where they must get right down to
the exact species? Can you see some inter-
mediary position where we could suggest certain
choices of media but assure we don't want our
microbiologists to go all the way and "count
the species" in every case.
We are not doing this now. At the same time I
feel we shouldn't be too abbreviated. For
example, I have a bone to pick with the philo-
sophy of going to extreme simplicity all the time.
I think it is about time we started to add a
little more complexity because we have to not
because we want to. It requires additional
methodology, expertise, and I think our micro-
biologists are capable of providing this
expertise. Let's bring the level up, not down.
Did anyone say anything about serological testing;
in the beginning did you mention anything about
fluorescent antibody screening?
No, I did not.
I would like to make a recommendation, if I may.
As a candidate method, fluorescent antibody
technique for screening should be included. We
have enough data available and we are presenting
the paper for publication right now. The FDA,
I understand, has adopted the procedure and
they are going to be using their routine FA
technique to screen practically all their food
samples and some others. Import samples tested
by the Department of Agriculture are to be
screened by FA. A technique and conjugates are
available from at least three different manu-
facturers and they are fairly good. Two of
these have been evaluated. NCDC has just come
out with a publication in Applied Microbio1ogy
128
-------
Shimmin:
Spino:
Shimmin:
Geldreich:
Shimmin:
Brezenski
showing that this conjugate works. I definitely
feel that EPA should consider the FA as a can-
didate system and I plan to submit a write-up
for consideration.
Have you ever tried the Enterotube? How do you
feel about that as another type of identifica-
tion of Salmonella?
I haven't tried it—I haven't done this type
of work recently.
We find it is rather useful for quick and
numerous parameters to be tested. We have
compared it with individual biochemical media
and it worked out pretty well. It is commonly
referred to in the literature as well.
Kathy, there are a lot of these methods being
released in the literature. I think this is
another area where we ought to evaluate a lot
of these devices. There are a lot of them that
just don't work. People are taking them on
faith that they are performing as advertised
and again I think our EPA group should really
screen a lot of these things out and find what
we will accept and what we will absolutely
refuse to consider.
I think that is a good idea.
As far as the Enterotube is concerned, I have
seen two papers which evaluated the Enterotube
system and also the r/b Enteric Differential
system. Our laboratory ran them simultaneously
with the FA technique and the cultural-biochemical
tests; they both showed the same high quality
of performance. Let me first say that both
these systems are inclined to tell you that
you have certain kinds of enteric bacteria
present and are supposed to be able to give
you enough biochemical criteria to identify
the isolate. The systems will tell you if you
have a Salmonella or not, and this is what we
want to know.
Kathy, were you speaking of the improved Enter-
otube which is relatively recent?
129
-------
Shimmin:
Brezenski
Shimmin:
Cabelli:
Jeter:
Spino:
Question
from the
floor:
Jeter:
No.
We plan to evaluate the modified one which I
think is appreciably better than the original.
At the time we began using it all that was
available commercially was the old one. We
did test it with test results that were
assembled independently and they checked out
practically 100%.
I think we are going to have to get numbers
(quantitation) of Salmonella. Quantitation is
needed to fight enforcement cases. Present
methods only tell if the sample did or did not
contain Salmonella.
I would like to ask a question of the group as
a whole. As several of you know I head up a
training unit at Cincinnati and we are always
interested in the identification of training
needs. This question of Salmonella opens up
this point loud and clear to you as a group.
Over the years we have relied on NCDC to meet
the training requirement for identification of
Salmonella. Is it time for us now to offer
training on Salmonella collection and identi-
fication? If so how far should this be carried?
To whom should we address this training? We
reach quite a large clientile.
The candidate method I propose has a simplicity
and clarity that anybody who attends the course
would be able to use for the identification of
the Salmone1la.
Can you handle hazardous material in your
training program?
They are giving botulism training in that same
laboratory. I don't think I would hesitate to
use Salmonella with proper safeguards. Are
we talking about incorporating Salmonella
training in the one course which we call
"Current Practices" or should an alternative
two or three day workshop specifically be
addressed to this one topic?
130
-------
Brezenski: I think you would have to do the latter because
look at the NCDC courses which they give on
Salmonella. These are one or two week courses.
I don't think incorporation into the "Current
Practices" is the way.
Jeter: I would agree.
131
-------
SPECIAL PROBLEMS; DISCUSSION
*
Kathleen G. Shimmin
During the course of this seminar a number of special
problems were brought up - special subjects which are not
currently covered by existing methodology. Topics mentioned
include the following:
1. Cultivation procedures for airborne pathogens
*
2. Special considerations for industrial effluents
3. Cultivation of non-pathogenic organisms (iron/
sulfur/ photosynthetic, filamentous bacteria,
yeasts/ fungi)
4. Microbial testing for toxicity
5. Cultivation procedures for waterborne pathogens
other than Salmonella (e.g. Shiqella, Leptospira/
Vibrio/ Clostridia)
6. Rapid serological identification procedures
7. Evaluation procedures for special media and
identification systems
8. Evaluation of safety and efficacy of seed cultures
and microbiological pesticides
9. Evaluation of automated equipment, electronic
counters, video scan systems
10. Investigation of low-temperature bacterial
indicators
11. Indicator microorganisms for sludge
12. Evaluation of emergency and field test kits
Excerpts from the discussion period follow.
Laboratory Support Branch, Region IX
132
-------
Borquin:
Brezenski:
Geldreich;
Bourquin:
Roessler:
Shimmin:
Roessler:
Engler:
Special Problems
I would like to discuss the impact of seed
cultures on the aquatic environment. A recent
Atlanta workshop conference addressed this.
There are some seed cultures for oil degradation
which contain pathogens. Standard testing pro-
cedures should be recommended to demonstrate
safety and reliability of claims.
With respect to oil degraders/ it is time we
had a set of procedures to be used on a routine
basis to test degradation rates. We had a
research topic currently on seed cultures to
degrade oil in oil spills. The seed cultures
contained Salmonella; hence we couldn't recommend
their use. Things like this are cropping up.
Tests for biodegradability are being requested
by the Permit Program. Should we include such
items in our manual or just mention them?
The Advanced Waste Treatment group has looked
at biodegradable organisms (especially with
respect to detergents). They may be able to
supply us with some background material.
I would like to expand the seed culture concern
to include the impact of microbial seed cultures
on the environment/ especially in reference to
microbial pesticides (such as those for control
of cabbage boll and control of the isopod which
preys upon mangroves in south Florida).
This probably falls within the pesticide program.
In this regard products must be registered for
use. There is no standard degradation test set
up for new chemicals. The manufacturer must
supply data on that new chemical.
How do you evaluate?
set of studies?
Do you prescribe a specific
No. We just require that the manufacturer per-
form some tests.
I think two different things are being discussed:
133
-------
Shedroff;
Unknown:
Lewis:
Brezenski:
Shimmin:
1. Chemicals which are degraded by the
environment;
2. Microorganisms which effect this and which
may combat this. We should look at spreading
and persistence of microbial pesticides
(bacteria, fungi, viruses), expecially after
they have killed the pest.
Fort Detrick Army Chemical Base was doing
testing on chemical warfare. They probably
won't give their results, but they might give
protocols which might be very appropriate.
When with the ultimate disposal unit, I identi-
fied the bacteria at three locations: Houston,
Illinois, and St. Louis areas. I found that a
Pseudomonas sp. broke down oil in oil ponding
waste. In work on cutting oils from about ten
oil companies, I found that the organism most
prominent in breaking down oil was Pseudomonas
sp. If organisms were produced in large amounts
and dried, perhaps the preparation could be
used in breaking down oil slicks; I don't know.
We have a paper on that that was given at the
International Meeting in 1970.
In Richmond, California, at a major Chevron
Research Center they have more than 100 different
isolates able to degrade various hydrocarbons.
We should check with other oil company laboratories,
There is a great amount of microbiological study
being conducted by these companies themselves.
A lot of money is being spent on this. This
is a long-term research. For rapid clean up
physical and chemical means to clean up oil
spills are now being investigated. They are
looking at the biological degraders as something
way in the future - a long research program.
We're going to have to wait for information to
be developed. Let's let this topic lie for
the time being until we have more material
developed in the next few years.
Do you feel your remarks apply to the entire
field of degradation?
134
-------
Brezenski;
Shimmin:
Vasconcelos;
Brezenski:
Bordner:
Shimmin:
Brezenski;
Shimmin:
Chang:
We shouldn't give ourselves an undue concern;
other topics have higher priority and should
be addressed. We can get input on degradation
from other agencies. The information is coming
slowly.
The purpose of this section is not to put some-
thing into a manual but to outline special
problems we should be aware of.
Standard Methods doesn't address itself to
topics such as the single delivery pipet system
or rapid plate count techniques, e.g. the
video-scan system.
We should give special attention to this.
Automatic scanners, electronic counters, and
bacterial counters are available now. These
have more direct application to us than the
degradability topic. Some provision should be
made to evaluate these things. Right now it
would fall into Bob Bordner's shop; but I'm
not sure Bob is equipped to do such evaluation.
We are talking about a lot of instrumentation,
which is coming up - some of which has almost
immediate application to us.
I propose that we take up some of the topics
which are coming up now and make them the
subject of a future meeting similar to the one
we have had today. We should establish some
types of priorities and then given time for
evaluation. Can we consider items for an
agenda?
Is there a general interest in this?
Yes, I second that motion.
All right, we'll note that.
I'd like to mention use of monolayer film, an
interest in the early part of the 1960's. I
was involved in investigating the cause of
degradation. The film was used in water
reservoirs and resulted in a great increase
of bacterial population. Invariably a
135
-------
Gordon:
Geldreich:
Gordon:
Pseudomonas was involved first. Others would
live on byproducts, e.g. Aerobacter, Entero-
bacter/ Klebsiella/ or others. These would
multiply frequently to very large numbers. I
believe now no one in the United States is
using this film to suppress evaporation. It
is being used in places such as Australia and
India.
There are two areas which might be considered
for future meetings:
*
1. Indicator microorganisms in sludges:MPN
seems to be the answer but recommendations
seem to be away from MPN.
2. The plate count in Standard Methods is
inadequate. It is unrealistic to look at
35 C plate counts when your water only gets
to 15 C. What significance is 35 C?
What is in Standard Methods with regard to
plate counts was meant to apply to potable
water and as it relates to interference with
indicator organisms. In revision of Drinking
Water Standards/ we are proposing an upper
limit of 500/ml using Standard Plate Count
at 2 days/ 35°C. I agree with you/ Ron. There
should be provision for study of some tempera-
tures for environments such as the one you
work in. These have a place in an EPA manual/
but not necessarily in Standard Methods. In
Standard Methods there should be Plate Count
information only on quality of treated water.
In the natural environment we have found it
is very difficult to interpret a Standard
Plate Count and to tie in with some need or
some kind of sanitary quality. For these
reasons for the past 20-30 years people have
been rather cool on the subject. Everything
cycles back, so perhaps this should be re-
investigated.
I find that organisms which grow at 0 C change
in population composition and numbers when you
pass through an area of pollution. In the area
of Fairbanks, there is a large increase in
136
-------
Geldreich;
Gordon:
Shimmin:
Geldreich;
Gordon:
Lewis:
numbers and change in population types. These
organisms grow at 0 C. They don't grow in two
days. I grow them for 43 days.
What's the significance of this?
The 35°C counts receive wider usage than what
they were intended for in Standard Methods.
-1 S~\ «M^V^MW»^M-~MM_M—^BB^W.**^^—»»• • "II If
I get at 35 C counts which are very low
(e.g. 5/ml). Counts at 20 C and 0 C may be
several thousand/ml. I'm not the only one
that's got water at low temperature (the upper
States of the United States may get down to
0 C). These are important for oxygen consump-
tion under the ice cover. One has to be
accurate in streams. You don't have a static
population which you can go back and measure.
Organisms have to be growing somewhere at these
temperatures. You get organisms year round.
We don't get sterile streams just because water
gets cold. We need to develop methods to look
at this methodology which can be used to compare
one study with another.
Ed, has the WHO group concerned themselves at
all with Arctic conditions for drinking water
methodology.
No. There have been a few international meetings
on this topic. The one a few years ago in
Alaska on waste is the last one I know of.
The military has had some.
Another meeting which is coming up at the
University of Saskatchewan, will be on waste
treatment in the Arctic.
One part that will enter into this too, when
you talk about waste treatment and evaluation
of the effectiveness, is the development of
some of the new techniques such as the ATP
meter (it measures the amount of adenosine
triphosphate released upon breakage of a cell and
provides rapid identification). I've seen^the
second generation instrument already, and it's
quite probable that this could be developed
into an on-line instrument which could give you
137
-------
Geldreich:
Shedroff:
Geldreich:
Lewis:
Geldreich:
Lewis:
indication of relative effectiveness of dis-
infection within minutes of sampling. The
technique needs evaluation in comparison to
standard quantification methods.
The whole field of rapid methods is a good
subject to be included, and many of us are,^
working on it one way or another - ATP & C ,
etc. It might be a good subject for our next
meeting.
On this I'd like to mention something on permits,
Some of the people who are going to have to
report on their permits are not going to have
all the equipment to do some of these tests.
We should consider the use of quick and dirty
tests and then a requirement to do something
further when the quick and dirty shows up bad.
This gets down to the emergency and field test
kits that are on market but have never been
approved for Standard Methods. The concept
might have some limited value but would have
to be evaluated.
Since I came to EPA, I have worked from time to
time on some nuisance bacteria (filamentous
forms, iron, sulfur bacteria, Gallionella,
Sphaerotilus, Crenothrix and nuisance Nocardia
forms that can cause trouble in sewage treat-
ment plants) . In many, cases there are no
cultural identification factors or quantitative
methods•
We need this not only for waste treatment but
also for finished waters and for reservoir
waters (e.g. taste and odor organisms). This
is very important for early consideration and
should perhaps be on the next program. You
would be a likely subject to give us some input
on the topic.
Since Sphaerotilus grows so well in sugar beet
and pulp mill wastes, instead of trying to get
rid of it maybe we could make modifications in
systems for separating solids from the liquids
and use it in waste treatment.
-JQ
-------
Chang:
Bordner:
Knittel:
Geldreich;
Chang:
Ron, maybe you should mention Actinomyces in
taste and odor problems/ too.
The Biological Methods Committee for AIBS is
discussing methods for Actinomyces and including
it in their manual. Does this show a precedent
for cross-interests in certain areas?
We should consider adopting one key for classi-
fication of enteric bacteria [I propose we
adopt Ewing's work, because in Sergey's it will
be the major one.] We should adopt a standard
set of media. During the Salmonella discussion
many media were mentioned. We should test H^S
production on TSI and should use urea agar for
urease production (if broth is used, some weak
urease producers are missed such as Citrobacter
and Klebsiella).
I'm going to try in the next Standard Methods
to have the section on enteric pathogens ex-
panded at least to state-of-the-art presentations,
Leptospira are found in urine. So perhaps you
should say "pathogens found in the enteric
tract and urine".
139
-------
IMPACT OF MICROBIAL SEED CULTURES ON
THE AQUATIC ENVIRONMENT*
Al W. Bourquin**
The tremendous use of oil for energy in the United
States has caused rapid increase in oil imports on large
cargo carriers. These large tankers, with capacities
equal to or greater than 100,000 dead-weight tons capacity,
and increased shipping, has enhanced greatly the danger
of major oil spills. With the impending danger ot cata-
strophic spills, technology of clean-up is extremely
limited. Present clean-up methods include adsorption
and recovery, chemical dispersion, and physical removal.
Each technique has limitations due to quantity and type
of oil spilled, extent of the slick, and nature of the
environment where the spill occurred or where the slick
floated. Some authors believe no efficient and safe method
exists for clean-up of a spill in shallow estuaries (1,2).
Extensive research is being conducted for the purpose
of increasing microbial oil degradation by seeding oil
slicks with hydrocarbonoclastic microorganisms. It may
be possible that large quantities of selected microorganisms,
under proper environmental conditions, could hasten degra-
dation and ultimate removal of pollutant hydrocarbons (1).
The need for standardization of testing procedures for
commercially available microbial formulations was pointed
out at a recent international workshop held in Atlanta,
Georgia. Papers were presented to show that at least two
commercial products are completely ineffective or have
very little hydrocarbonoclastic activity—below that of
natural seawater (3). Other evidence, presented by EPA^
representatives, demonstrated that at least one commercial
formulation contained at least four species of pathogenic
microorganisms (4).
A panel, "Environmental Considerations in Microbial
Degradation of Oil", at the Atlanta workshop recommended
that a committee be formed to study the problems of
effective and safe use of microbial seed cultures in the
environment. The committee should be composed of members
of a governmental agency, members of API—representing the
petroleum industry, and members of the academic community
who are active in oil pollution research (5).
"*submitted in writing for the record.
** Gulf Breeze Environmental Research Laboratory, Associate
Laboratory of NERC-Corvallis
140
-------
Microbial seed cultures are currently being studied
for application to the environment as microbiological
pesticides. Viruses have been isolated which attack
selectively the cabbage boll; a bacterium has been
isolated as a specific pathogen of mosquitoes; and
chitinoclastic bacteria have been proposed as agents
against plant predators in estuarine areas. The range
of impact on the aquatic environment by seed cultures
must be investigated adequately before they are used
on a large scale.
141
-------
LITERATURE CITED
1. Haxby, L. P., and E. Cotton, Joint Conference on
Prevention and Control of Oil Spills/ 544 pp.,
American Petroleum Institute, Washington, D.C. (1971).
2. King, Kerryn, and L. P. Haxby, Proceedings - Joint
Conference on Prevention and Control of Oil Spills -
API and FWPCA. 345 pp., American Petroleum Institute,
Washington, D.C. (1969).
3. Atlas, R. M., and R. Bartha. Effects of some commercial
oil herders, dispersants and bacterial inocula on
biodegradation of oil in sea water. In: Ahearn, D. G.,
and S. P. Meyers (eds.), The Microbial Degradation of
Oil Pollutants, p. 32-34, Georgia State University,
Atlanta, Georgia (1972) .
4. Personal Communication. Environmental Protection Agency,
Edison Water Quality Laboratory, Edison, New Jersey (1972).
5. Ahearn, D. G., and S. P. Meyers. The Microbial
Degradation of Oil Pollutants. 69 pp., Georgia State
University, Atlanta, Georgia (1972).
142
-------
PROCEDURES
-------
COLLECTION AND HANDLING OF
BACTERIOLOGICAL SAMPLES: STATE-OF-THE-ART
William J. Stang*
INTRODUCTION
One of the first considerations in an examination of
any water supply is the collection and handling of samples.
This presentation discusses the current status of standard
Methods regarding the collection and handling of water
samples for bacteriological examination. Investigators
will agree that the integrity of any sample cannot be
maintained if not collected and stored properly. Every
research, enforcement, and surveillance effort entails
something unique or different about the type of sample
desired.
Water Quality Investigations
Potable Water
The 1962 Public Health Service Drinking Water Standards (1)
is usually mentioned in any state-of-the-art discussion
concerning Standard Methods (2). Responsibility for further
development of these standards now lies with an inter-
departmental advisory committee that is composed of
representatives of various city and state water quality
enforcement agencies and representatives of EPA.
The Federal Drinking Water Standards emphasizes
collection of samples from representative points through-
out a distribution system. Frequency of sampling and the
location of sampling points are determined after the quality
fo both the treated and untreated water supply has been
determined, i.e., the quality of the finished water being
controlled, in part, by the quality of the raw water source
and, therefore, by the need for treatment.
The monthly minimum number of samples to be examined
has been established by the Federal Drinking Water Standards
and is based upon the population served by the distribution
system. For example, for a population of 1,000-2,000, two
samples should be evaluated monthly; for a population of
10,000, twelve samples; for a population of 25,000, thirty
samples; and so on, for a population of 100,000, a minimum
* National Field Investigations Center-Denver,
Denver, Colorado
143
-------
of 100 samples is required. The number selected from the
graph should be "rounded off" in accordance with the
following: for a population of 25,000 and under, to the
nearest one; 25,001 to 100,000, to the nearest five; more
than 100,000, to the nearest ten.
Minimum Number of Samples Per Month
100
1000
10,000
FIGURE 1
Recommended minimum monthly samples per population served by
water supply - 1962 Public Health Service Drinking Water
Standards
144
-------
Standard Methods states that it is important to
examine samples from widely distributed sampling sites,
as well as repetitive samples from any single point (2).
Daily samples taken after an unsatisfactory sample has
been detected should not be counted in the overall total
of monthly samples. Additionally, in the event of an
unsatisfactory sample, daily samples should be collected
from the sampling point until two consecutive samples yield
satisfactory quality water.
The 1962 federal drinking water sampling requirements
are now being re-evaluated. it would seem that two samples
per month for a city of 2,000 or less population are too
few to maintain adequate surveillance of the system. In
any surveillance activity of a drinking water system the
importance of a well planned and efficient sampling program
cannot be over-emphasized. in many cities, the samples are
taken from the same locations year after year. These
locations are selected for convenience rather than thorough-
ness, such as a favorite tavern, restaurant, or even the
sanitarian's home (3). Samples should be collected from other
points that might prove to be a more meaningful measure of
the water quality in the distribution system. Additional
aspects that should be considered are the qualifications,
competence, and integrity of the sample collector. Over-
filling sample bottles, inadequate dechlorination of
samples, improper sample bottles, prolonged or improper
sample transit and storage time are other deviations
from Standard Methods that often occur in a water-sampling
program (3,4).Therefore, sample collectors should authen-
ticate the samples.
Other variables that might be considered when determin-
ing sampling requirements might include: frequency of
unsatisfactory samples from supplies serving various popu-
lation levels, repeat sampling occurrence, and the time
interval for repeat sampling. The quality of the raw water
supply must be considered; those having poorer water quality
should be supervised more closely. When establishing
sampling requirements, seasonal population changes, plant
capacity, raw water source, and the application of
chlorination must be considered (4) .
An important subject, concerning sampling of potable
water supplies is the continuing development of an extensive
evaluation program (4,5). A certifying agency must be
145
-------
responsible for inspecting the laboratory techniques and
sampling procedures used by individual laboratories. This
combination of internal and external safeguards will provide
assurance that sampling techniques, laboratory procedures
and equipment will continue to be as sensitive as necessary.
The Technical Advisory Committee on Potable Water
Standards is responsible for determining, per unit popu-
lation, the number and frequency of samples to be collected.
Locations of the various sampling points in each municipality
are determined by the reporting agency and the regional
water supply representative of EPA. The Federal Drinking
Water Standards (1) should be closely referenced in any
EPA manual that is to be developed.
Surface Water
Surface-water studies are usually efforts to determine
source and extent of pollution and are intended either for
baseline data collection or potential enforcement action.
Representative samples are taken by considering the site,
method, and time of sampling. The frequency of sampling
is determined by the survey objectives, i.e., does one
wish to measure cyclic pollution, duration of peak pollu-
tion, or the probable average pollution? state of water
quality is measured by collecting samples immediately
downstream from the source of pollution. One approach
might be an hourly collection during a certain period of
time or by advancing sampling intervals one hour each
24-hour period to obtain data for a 7-to-10-day study. To
measure state of water quality, samples are collected down-
stream from mixing zones. Samples from downstream sites are
collected less often than those needed for source detection.
Considerations in the choice of bacteriological sampling
sites should include base-line locations upstream of the
pollution sources, industrial- and municipal-waste outfalls
in the stream study area, impact of tributaries, municipal
and industrial treatment-plant intake points, and downstream
recreational areas affected by certain streamflow time
intervals (4). In potential enforcement cases essential
consideration should.be given to legal requirements.
Cross-sectional sampling or multidepth sampling of
the stream may sometimes be necessary in order to determine
mixing patterns. Non-representative water areas should be
.46
-------
avoided. Another factor to consider in any surface water
quality investigation is water use. For example, the
water quality at a specific point of water intake or at
a restricted water use area is of more value than the
average quality of the total pollution loads passing
through the cross-section (4). This may be true of source-
water intakes, shellfish-harvesting areas, recreational
areas or for other restrictive requirements specified
for a particular use. Lack of mixing is of no concern
in measuring water quality in these situations, and the
sampling station is located at the points or areas of
actual use. For monitoring stations the frequency of
sampling may be seasonal where it relates to recreational
waters; daily where it measures raw water used in water
treatment; hourly where waste treatment control is erratic;
and on a continuous basis for wastewater re-use.
Recreational Water
In the case of swimming beaches or other recreational
areas selected sites should include upstream peripheral
areas and locations adjacent to natural drains that would
discharge stormwater collections, or runoff areas draining
septic wastes from boat marinas, or garbage collection
areas. Samples of bathing-beach water should be collected
at locations and times where the use is heaviest. The
optimum frequency during the season would be daily, pre-
ferably in the afternoons. Weekends and holidays definitely
would be included as periods of highest use. Swimming-pool
water should also be monitored during maximum use periods.
High chlorine levels in swimming pools rapidly reduce
bacterial counts when pools are not in use. Residual
chlorine tests are necessary in order to check neutrali-
zation of chlorine in the sample.
Data on the water quality of all estuarine bathing
beaches should be obtained at high-tide, ebb-tide, and low-
tide in order to determine the cyclic water-quality deter-
ioration that must be controlled during the swimming season.
Waters overlying shellfish harvesting areas should be
collected during periods of most unfavorable hydrographic
conditions, most probably at low-tide after heavy precipi-
tation. Procedures for sampling of shellfish and shellfish-
growing areas are governed by the National Shellfish
Sanitation Program's Manual of Operations (6,7).
147
-------
Bacteriological sampling stations in reservoirs,
lakes, estuaries, and oceans are generally located in
grid networks or along transects extending across the
long axis of the water bodies. Limited information can
be obtained by sampling from the draw-off area of small
lakes or impoundments. Samples are usually collected at
the same depth over a large area near the outlet.
Municipal and Industrial Wastes
It is becoming increasingly important to sample
secondary treatment wastes from municipal waste treatment
plants and various industrial waste treatment operations.
In situations where the plant treatment efficiency varies
considerably, grab samples are usually collected around
the clock at intervals for a 3 to 5 day period. If it is
known that the process displays little variation, then
fewer samples are needed. In no case should composite
samples be collected for bacteriological examination.
Sample Transit and Storage Time
Sample transit and storage time are perhaps the most
important cautions cited by Standard Methods (2). The bacteria
population in a stored sample may change over a period of
time. Cold storage minimizes these effects. In circum-
stances where samples must be mailed, the use of thermos
type containers by sample collectors has been suggested (2,8).
Maximum use of special delivery or special handling by the
postal service must be required. Because most state
laboratories and U. S. postal services are minimal on
weekends, sample collection schedules should be arranged
so as to assure sample delivery to the laboratories at
times other than weekends or holidays. This must be a
strong recommendation or a requirement in any methods manual
that is to be developed.
In the case of secondary treated sewage effluents and
industrial waste effluents, where nutrients and/or toxic
substances are likely to be present in higher concentrations,
the opportunity for bacterial populations to change in a
sample is increased. In these types of samples standard
Methods allows a storage time of 6 to 8 hours. The National
Field investigations Centers, working in mobile laboratories
can usually process any sample in 4 hours or less.
148
-------
One consideration that should be presented is the use
of commercial airlines or other forms of transit to transport
samples contained in ice chests to the laboratory. Often
air transport will meet the sample transit time requirements
and offset the high costs involved in setting up a field
laboratory.
Sampling Devices and Sampling Techniques
Sampling Equipment
Sampling bottles should have a capacity for at least
100 ml of sample, plus an air space for mixing. The bottle
and cap must be of bacteriologically inert materials such
as glass or heat-resistant plastics. The bottles must be
clean, sterile, and contain a proper concentration of
sodium thiosulfate so that there are 100 mg/1 in the sample.
Also, at the time of sterilization the top and cap of the
glass-stoppered bottle must be protected from contamination
with paper or metal-foil hoods (2).
Most often the type of sample collected is a "grab
sample" involving the use of a 250 ml wide mouth bottle.
Aseptic technique must be employed to ensure that nothing
but the sample water comes in contact with the inside of
the bottle or cap.
In sampling from a distribution system, the water
faucet is first opened full for several minutes to purge
the service line. The flow is then reduced, and the bottle
is filled without splashing.
To sample surface water, the bottle is held at the
base, pushed rapidly about 6 inches into the water, mouth
down, and tilted towards the current to fill. If there is
no current, the bottle is moved through the water away
from the hand. A small amount is spilled intentionally
in order to provide a proper air space (2).
Emmett samplers have been used to collect bacterial
samples (9). In this collection device a sterile glass tube
is inserted through a one-hole stopper in the cover of the
sampler, with the lower end extending inside and near the
bottom of the sample bottle. The bottle is filled through
this tube, and a fresh sterile tube is used for each sample.
The air relief tube extends through the cover into the
bucket far enough to reach below the top of the sample so
that the sampler stops filling before the neck of the
bottle is submerged.
149
-------
Other sampling devices simply involve placing the
bottle in a weighted frame that holds the bottle securely.
The bottle is then opened and lowered into the water with
a cord or string. Care must be taken not to contaminate
the bottle by dislodging dirt or other material from the
structure where the collector is standing.
There are various types of sampling devices available
where access to the sample point is either difficult, or
where depth samples are desired. The general problem is
to place the bottle in position at the desired depth, open
it, close it, and return it to the surface. No bacteria
but those in the sample must enter the bottle.
The most popular depth-sampling device is the J-Z
sampler, described by ZoBell in 1941 (10,11). The J-Z sampler
includes a metal frame to hold the sample bottle, with a
glass tube-breaking device which is activated by a heavy
messenger. Either glass or collapsable neoprene rubber
bulbs serve as the sample containers. A variation of the
J-Z sampler that contains a collapsable rubber bulb is
known as the Cobet modification.
Another depth sampler sometimes used is the "Woods
Hole" sampler. This device consists of a vane-and-lever
mechanism which lifts the cap as water inertia is applied
by tugging on the line. As the cap is lifted, water pours
into the sample bottle.
The Niskin sampler (sometimes called a sterile-bag
sampler or "Book" sampler) is suitable for obtaining 1.5
liters of water aseptically. It can be operated singly,
or several devices may be attached in series and activated
by additional messengers (12). Other samplers exist with
lever or cord attached for pulling the stopper.
The Van Dorn and Kemmerer samplers have been used in
deep water without pre-sterilization between sampling.
Cross contamination between stations that would signifi-
cantly alter bacteriological results probably occurs. For
enforcement situations involving court litigations this type
of sampler is unacceptable.
Peristaltic pumping devices employing sterilizable
neoprene or plastic tubing have been used successfully.
150
-------
Sediment Sampling
Until recently sediment samplers designed strictly
for microbiological work were not available. Coring devices
with sterilizable plastic liners were the best compromise
of sediment samplers available on the market (13). However,
contamination with surface water cannot be avoided because
these devices must be sent down open. For lack of some-
thing more suitable, the Petersen, Ekraan, and some shell-
fish dredges have been used occasionally. However, use of
these is not suitable for bacteriological studies in the
strictest sense.
A bacteriological sediment sampler designed to collect
muds or sediment with use of a sterile, 6 oz. plastic bag
is now available—a solution to one very large and serious
problem in aquatic microbiology (14).
Sampling for pathogens
Isolation methods for analyses of water samples for
bacterial pathogens usually involve various concentration
techniques. Three of the choices available are: the
sterile gauze pad placed in flowing water for a l-to-7 day
period (15,16,17) (The exposure time is left to the
judgement of the investigator); collection of a large
volume sample ranging from one-half to several liters and
filtration of the bacteria; centrifugation.
Photographs
Colored photographs are often useful in reports to
help not only the general public but the courts as well to
better understand scientific material. If photographs are
to be used as evidence/ these should be well documented on
the reverse side. Written documentation should include the
signatures of the photographer and witnesses, locations,
dates, time, and direction of flow. The photographs should
contain suitable annotation and should describe the pollu-
tion situation with appropriate landmarks whenever possible.
Survival Studies
Sometimes it is necessary to establish the presence
and persistance of coliforms and pathogens. A technique
that is becoming increasingly important in enforcement
151
-------
surveys is bacterial survival studies. Microbiologists
have employed use of sterile collodian bags in order to
entrap a bacterial suspension in situ and remove aliquots
of sample from this container for analyses after varied in-
tervals of time. The collodian bag allows small molecules
of water and other substances to pass through it, but
retains the organisms of interest in an environment (pH,
temperature, etc.) very similar to that of the stream.
Another approach is to collect a sample of water,
place the sample in a controlled environmental chamber,
such as a BOD incubator and examine aliquots for bacteria
at predetermined time intervals (18,19,20). The sample
may be sterilized and then inoculated with a pure culture
of bacteria. This simulated sample is then examined
periodically during its exposure to an artificial environ-
ment for bacterial survival.
Planning the StreamStudy
Workloads imposed on the laboratory facilities and
personnel should be planned well in advance of the survey.
The principle microbiologist should be consulted in the
selection of bacteriological sampling stations as well as
the determination of total numbers and types of analyses
required.
Chain of Custody
Chain-of-custody procedures should be followed on a
sufficient number of samples to prove a water-quality
standard violation or a permit violation. For any poten-
tial enforcement case, if there is any anticipation of a
criminal action, for every sample which may be offered as
evidence the chain-of-custody procedures must be followed
as a precautionary measure.
The Office of Enforcement now has suggested chain-of-
custody procedures (21). Other EPA elements have incorporated
these procedures in their own more detailed instructions.
152
-------
REFERENCES
1. Public Health Service Drinking Water Standards,
Publication No. 956, U. S. Public Health Service.
Washington, D.C. 1962.
2. Standard Methods for the Examination of Water and
Wastewater,13th ed., American Public Health
Association. New York, 1971.
3. E. E. Geldreich, Application of Bacteriological Data
in Potable Water Surveillance, Jour. Am. Water Works
Assoc., Vol, 63, No. 4. April, 1971.
4. E. E. Geldreich and E. F. McFarren, Evaluation of
Water Laboratories, 2nd Ed., Water Supply Programs
Division, Environmental Protection Agency, Cincinnati,
Ohio. 1973 [in preparation],
5. Manual for Evaluating Public Drinking Water Supplies,
U. S. Department of Health, Education, and Welfare,
Public Health Service, Environmental Control Admin-
istration, Bureau of Water Hygiene. Cincinnati, Ohio.
1969.
6. National Shellfish Sanitation Program, Manual of
Operations, Part I, Sanitation of Shellfish Growing
Areas, 1965 Revision, ed. by Leroy S. Houser, U. S.
Department of Health, Education, and Welfare, Public
Health Service, Shellfish Sanitation Branch.
Washington, D.C., 1965.
7. Recommended Procedures for the Examination of Seawater
and Shellfish, 4th ed., American Public Health
Association, 1970.
8. E. E. Geldreich, Standard Methods for the Bacteriological
Examination of Water and Wastewater: Purpose, Changes,
and Future Developments, presented at the Annual
Milk and Water Laboratory one-day seminars, sponsored
by the Illinois Department of Public Health. November
9-11, 1971. Springfield, Illinois.
153
-------
9. W. H. Frost, J. K. Hoskins, H. W. Streeter, and
R. E. Tarbett, A Study of Pollution and Natural
Purification of the Ohio River, II. Reports on
Surveys and Laboratory Studies. Public Health
Bulletin No. 143, U. S. Public Health Service,
Shellfish Sanitation Branch. Washington, D.C. 1924.
10. C. E. ZoBell, Apparatus for Collecting Water samples
from Different Depths for Bacteriological Analysis.
Journal of Marine Research, 4:3, 1941.
11. C. E. ZoBell, Marine Microbiology, Chronica Botanica.
Waltham, Mass., 1946.
12. S. Niskin, Deep Sea Resources, Vol. 9, pp 501-503. 1962.
13. R. R. Colwell and M. S. Zambruski, Rodina-Methods in
Aquatic Microbiology. Univeristy Park Press.
Baltimore, Maryland. 1972.
14. D. J. Van Donsel and E. E. Geldreich, Relationships
of Salmonella to Fecal Coliforms in Bottom Sediments,
Water Research, P^rgamon Press. Vol. 5, pp 1079-1087.
Oxford, New York, London, Paris, 1972.
15. B. Moore, The Detection of Paratyphoid Carriers in
Towns by Means of Sewage Examination. Min. Health Lab.
Serv., Vol. 7:241-248. 1948.
16. D. E. Spino, Elevated Temperature Technique for the
Isolation of salmonella from streams, Appl. Micro-
biology, Vol. 14:591. 1966.
17. F. T. Brezenski, R. Russomano, and P. DeFalco, Jr.,
The Occurrence of Salmonella and shigella in Post
Chlorinated and Nonchlorinated Sewage Effluents
and Receiving Waters, Health Laboratory Science,
Vol. 2, No. 1. January, 1965.
18. Proceedings of the Conference in the Matter of Pollution
of the Interstate Waters of the Red River of the
North, North Dakota-Minnesota, September 14, 1965.
Fargo, North Dakota, U. S. Department of Health,
Education and Welfare, Public Health Service
Washington, D.C.
154
-------
19. Federal Water Pollution Control Administration, Report
on the Beet Sugar Industry—the Water Pollution
Problem and Status of Waste Abatement and Treatment,
U. S. Department of the Interior. South Platte
River Basin Project. Denver, Colorado. June, 1967.
20. E. E. Geldreich, L. C. Best, B. A. Kenner, and
D. J. Van Donsel, The Bacteriological Aspects of
Stormwater Pollution, Jour. Water Pollution Control
Federation. Vol. 40. No. 11. November, 1968.
21. A Primer on the Law, Evidence, and Management of
Federal Water Pollution Control Cases, Prepared by:
The Legal Support Division, Environmental Protection
Agency. Washington, D.C. May, 1972.
155
-------
BACTERIAL SURVIVAL SAMPLER*
Louis A. Resi**
The survival of microorganisms has been important in
assessing water quality. Municipal and industrial wastes
contain high concentrations of bacterial nutrients, com-
pounds which may be toxic, enteric pathogens, and other
microorganisms. Survival studies of waste-associated
microorganisms yield additional information in determining
the sanitary significance. Rates of die-off and growth
have been determined by laboratory incubation procedures.
More recently attempts have been made to study bacterial
survival under the natural stresses of the aquatic environ-
ment .
The Bacterial survival Sampler is described as having:
cylinder body with a screw-cap glass sample port; mounted
on each end of the cylinder are 250-ml plastic Millipore
MF filter funnels which hold the 47-mm diameter membrane
filters, 0.45 um pore size; a metal retainer assembly
having fins on one end to give aqua-dynamic stability and
maintain the seal of the funnel ends to the body.
Bacterial survival studies are initiated by isolating
significant indicator and pathogenic microorganisms from the
aquatic environment being studied. Known densities of the
sample organisms are inoculated into 300 ml of micro-filtered
water. This sample is then placed into the sterile bac-
terial survival sampler and suspended into the body of
water being studied. The sample organisms are retained in
the sampler by the 0.45 um membrane filters at each end of
the sampler body. The filters allow exchange of the water
in the sampler. Aliquots of sample are periodically removed
from the sampler through the sample port, and the rates of
growth and die-off are determined to show the bacterial
survival of the test organisms under the natural stresses
of the aquatic environment. The growth-stimulating sub-
stances from organic wastes could result in increasing
bacterial numbers. Rapidly decreasing bacterial populations
could result from toxic wastes.
*This paper submitted~Tn writing for the record.
** National Field Investigation Center-Cincinnati.
156
-------
BACTERIAL SURVIVAL SAMPLER
FINS
METAL RETAINER
FUNNEL FILTER ENDS
-------
SEDIMENT SAMPLER
**
Edwin E. Geldreich
The sampler I wish to discuss was developed by Dale
Van Donsel and myself several years ago. The mechanics
of the sampler will be described in an article to be submitted
to Water Research. An advance copy of the publication will
appear in the proceedings of the symposium entitled: The
Aquatic Environment: Microbial Transformations and Water
Quality Management Implications. We have had blueprints
made up which are available to those of you who want them.
The sampler involves the use of 6 oz. Whirl-pak plastic
bags. The bag is placed over a nose piece and it remains
shut until the sample is collected. The sampler is weighted
so that it can be lowered to the bottom of a stream or lake.
As the sampler contacts the bottom surface, a mud plate
releases the mechanism and the bag is opened. As the weight
of the sampler bears down, the bag sinks into the mud and
is filled. After the sampler has stopped sinking, a double
noose that is tied to the bottom of the bag is pulled. This
seals the sample in the sterile bag and it can be removed
to the surface.
Some advantages of the sampler are that all kinds of
bottom materials up to about one inch in size may be
sampled. Some pretty large stones will go into the bag
if a gravel bottom is encountered. Some of the other
samplers now available on the market will jam with anything
larger than fine sand.
Different types of mud plates have been tried with this
sampler. We have found that if one is working in deep
water, a perforated mud plate helps to assure that the
sampler will be lowered down straight and will not contact
the bottom at a perpendicular angle. In shallow waters a
solid plate may be used. Two types of devices are avail-
able. With one of them we can attach a rod to the sampler
and just ram it down through the shallow water into the
bottom sediment. Working from a boat in deep water we
attach a double set of cords to the sampler so that it can
be released by use of a messenger device. A safety chain
is attached so that the sampler will not be lost.
**
This paper submitted in writing for the record.
Water Supply Research Laboratory, NERC-Cincinnati
158
-------
The sampler has worked down to depths of 60 feet in
Lake Erie. Its maximum depth is unknown. We do have
people studying oceanography who are very interested in
the sampler at this time. Dr. Colwell of the University
of Maryland is already trying to modify it for her use in
ocean studies.
159
-------
FOERST KEMMERER SAMPLER
For Routine Bay Sampling
**
Francis T. Brezenski
The kemmerer sampler is cylindrical in shape and
is made of brass. It contains two rubber stoppers at
either end connected to a rod which passes through the
center of the cylinder. It has an automatic device for
locking the stoppers open previous to lowering into the
water. By dropping the messenger, a mechanism is activated
and the valves are closed, thus locking the sample of water.
When the unit is closed, the entire weight of the sampler
and contents is carried upon the lower valve and there-
fore is water tight. Under these conditions water from
the upper layers are not allowed to enter during the
retrieving process. The unit contains an outlet at the
bottom which allows for drawing off the water contained
in the sampler. This type of sampler has a capacity of
200 ml and is able to collect samples at a wide variety
of depths.
Although this sampler has the advantage of collecting
a large volume of water at a wide variety of depths and
needs no elaborate preparation, it has certain character-
istics, which at least theoretically, violate basic bacter-
iological notions. The Foerst sampler is made of brass
and because of the slight solubility of metals in sea
water, there may be a bactericidal or bacteriostatic effect
on the microorganisms present in the sample. The copper
used in the manufacturing process is of prime interest.
This becomes even more significant when the water is to be
analyzed by a membrane filtration technique. Shipe and
Fields (1) reported that samples containing 1 mg per liter
or more of zinc or copper compounds interfere with coliform
results in membrane filter tests. The metallic ions are
adsorbed on the membrane sufficiently to prevent bacterial
growth. It is possible, however, that if the sample is
collected and transferred quickly to a sterile glass con-
tainer and the water filtered through the membrane and the
membrane immediately placed on the medium, the effect may
be minimized. This presupposes excess rinsing of the mem-
brane after sample filtration with sterile buffered distilled
water.
Submitted in writing for the record.
**Technical Support Branch, Region II.
160
-------
Another factor that requires consideration is the
use of a nonsterile sampler. The Foerst sampler is not
sterilized prior to sampling and is not sterilized after
each individual sample is taken. The sampler depends
upon a rinsing or flushing type of action for the removal
of material that might adhere on to the sampler from the
previous sample. When the sampler is lowered into the
water it passes through several layers until it reaches
the desired sampling depth. During this process, the
sampler is in the open position and is being rinsed as it
passes through the layers of water. Theoretically, the
rinsing action should remove materials clinging to the
sides of the sampler from the previous sample. Raritan
Bay samples are normally taken five feet from the surface
and five feet from the bottom. Therefore, in the shallow
sample—the sampler passes through at least five feet of
water before it is closed and the new sample trapped in the
chamber. With the deep samples, a much more vigorous
rinsing is achieved because of the increased depth. The
average depth in Raritan Bay is about 35 feet.
The Foerst sampler is used routinely in marine
microbiology for qualitative analyses. It has its limit-
ations when assays for total bacteria are required. The
most obvious reason for this limitation is the absence of
sterile conditions. In the Raritan Bay study, total
bacterial densities are not considered. The main considera-
tion, from a bacteriological point of view, is the detection
and enumeration of bacterial indices which demonstrate
fecal pollution. Due to the nature of the bacterial
parameters being used in the study, this type sampler seems
feasible, especially when large volumes of water are needed
for chemical and bacteriological analyses, (especially when
both types of analyses are to be performed on water from
the same ^sample container); where approximately 90 samples
are being collected on the same day and where samples are
needed from greater depths. The sterile bottle sampler
does not lend itself easily to the three demands just
mentioned. It was from a point of real need, as imposed
by the lack of a practical sampler that this type of unit
was approached and studied to see if the problem of mass
aquatic sampling could be solved.
The following data are presented on Raritan Bay samples
collected simultaneously by the kemmerer and sterile bottle
sampler. Coliform densities reported are the average of
triplicate plates. Although a limited amount of data was
available—it was, however, possible to make several
observations.
161
-------
MF TOTAL COLIFORM/100 ML
Sterile
Keiranerer (K) Bottle ( s
11,400 12,000
11,000 14,000
2,600 1,900
4,500 3,300
8,400 7,800
5,300 11,000
540 460
3,200 4,000
33,000 110,000
110,000 180,000
29,000 28,000
49,000 32,000
180 250
170 260
5,400 5,800
5,000 4,100
3,200 3,600
1,200 1,400
4,500 3,000
1,500 1,500
500 300
120 100
8,000 6,700
5,000 4,900
26,000 17,000
32,000 26,000
1,700 2,400
2,800 3,600
Keiranerer
No. of samples =
Coliform Density
- Total
Mean =
Median =
Ratio
) K/s
0.95
0.78
1.36
1.36
1.10
0.48
1.17
0.80
0.30
0.61
1.03
1.53
0.72
0.65
0.93
1.21
0.88
0.85
1.50
1.00
1.61
1.20
1.19
1.02
1.52
1.23
0.70
0.77
55
793,490
14,427
3,200
Kemmerer
2,200
560
680
100
2,900
23,000
220,000
1,800
6,700
1,600
900
2,400
45,000
20,000
2,800
5,200
200
3,800
2,400
900
60
10,000
5,000
1,000
80
45,000
24,000
Sterile
(K) Bottle (s)
2,200
800
900
400
4,400
25,000
350,000
1,400
4,800
1,600
700
2,000
28,000
24,000
4,500
7,000
300
4,000
1,400
1,000
90
9,000
4,800
790
80
27,000
23,000
Sterile Bottle
55
1,014,530
18,446
4,100
Ratio
K/S
1.00
0.70
0.75
0.25
0.66
0.92
0.62
1.28
1.40
1.00
1.28
1.20
1.60
0.83
0.62
0.74
0.66
0.95
1.71
0,90
0.66
1.11
1.04
1.26
1.00
1.66
1.04
Sampler
Inspection of the data shows that during the study
coliform densities of less than 80 and greater than 300,000
were encountered. The median ratio was 1.21 and when the
data were ranked according to three density ranges, the
following was observed:
162
-------
Values Coliform Range Median Ratio
14 80 - 1000 0.66
26 1300 - 9000 1.11
15 11,000 - 350,000 1.23
55
In order to check for the possible contribution of copper
by the kemmerer through a leaching process, copper assays
were performed on bay samples held in the kemmerer for 20 to
30 seconds and 10 minutes.
Analysis for Cu by Atomic Absorption Spectrophotometry
Cu
Sample Holding Time (mq/1)
1. (Bay Water) 20-30 sec. 0.195
2. (Bay Water) 10 min. 0.195
3. (Bay Water) 20-30 sec. 0.195
4. (Bay Water) 10 min. 0.195
5. (Distilled H2O) 20-30 sec. 0.075
(Bay water collected in glass container assayed at
0.110 mg/1)
The data indicate that minute amounts of copper are
leaching from the sampler. Concentration to 1 mg/1 on the
membrane does not occur since small volumes of sample water
are being filtered.
Conclusions
1. Fifty-two percent of the time, the kemmerer produced
values equal to or greater than those received from samples
collected with the sterile bottle. The fact that^negligible
amounts of copper were recovered from water held in the
kemmerer enhance the idea that severe toxicity due to metal
contamination is not being exerted since concentration values
do not approach 1 mg/1.
2. The data in the table are presented in the order
that the samples were collected. There is no_evidence that
carry-over is a severe problem - even when shifting from
high to low coliform density areas.
3. At this time there appears to be more variability
at the low coliform ranges rather than at the higher ranges
encountered in the study. This, however, needs further study
163
-------
employing a larger number of samples—both at the high and
low ranges. Consequently, for this and other reasons, the
sterile bottle sampler should be used when lower coliform
densities are anticipated ; (e.g. approved bathing beach
and shellfish harvesting areas.)
4. For the study of Raritan Bay, where gross pollu-
tion exists, the above data illustrates that the kemmerer
can be used. It is hopeful that a sterile sampler will be
available shortly, that will operate at a variety of depths,
provide larger volumes of sample water and does not involve
elaborate preparation.
5. A continuing comparison of these two samplers be made
so as to fully evaluate performance and ascertain limitations
at all density ranges.
References
1. Shipe, E. F. and Fields, A. 1954. Comparison of the
Molecular Filter Technique with Agar Plate Counts for
Enumeration of Escherichia coli in Various Aqueous
Concentrations of Zinc and Copper Sulfate. Applied
Microbiol. 2:382-384.
164
-------
BACTERIOLOGICAL VERTICAL WATER SAMPLER*
**
Robert E. Becker
We have used this particular sampler (1) in the Mobile
Bay area several times. It seems to work quite well. The
sampler can be modified to suit your needs. It has a base
plate similar to that of the sediment sampler which was
discussed earlier.
The handle of the sampler consists of six foot lengths
of galvanized pipe. The end of one pipe section is attached
to the base plate. If it is desired, three lengths of pipe
may be coupled for longer extension and greater sampling
depth. The sample collection device consists of the usual
250 ml^sample bottle attached near the lower end of the
pipe with stainless steel clamps. The mouth of the sample
bottle is sealed with a sterile solid rubber ball. The
ball is held in place by elastic rubber tubing threaded
through the ball and stretched around the bottom of the
sample bottle where it is anchored to the sampler. The
bottle is filled by raising the rubber ball from the mouth
of the bottle. This is accomplished by pulling on a wire
leader attached to the ball and extending to the top of
the handle. After filling, the pressure is released, the
sampler is raised to the surface and the full sample bottle
is recovered.
This paper submitted in writing for the record.
**
Water Supply Research Laboratory, Dauphin Island, Alabama.
(l)This sampler was designed and constructed by Clinton A.
Collier of the Gulf Coast Water Supply Research Laboratory.
165
-------
SAMPLING: DISCUSSION
Brezenski:
Geldreich:
Brezenski:
Geldreich:
Scotten:
Geldreich:
A chamber similar to what Mr. Resi described
was being developed for in situ bacterial
survival studies. On main difference was that
the chamber contained an additional port so
that access could be made to study changes in
temperater, pH/ dissolved oxygen and chlorides
within the chamber. Of primary concern was the
exchange of materials from the outside into the
chamber. From the initial—Dr. Litsky (U. of
Massachusetts)—found that by using certain
dyes and other tracers there was not an adequate
exchange of material between the inside and
outside of the chamber. The chamber would seem
to be selective. Additional criticism of the
chamber came from Mitchell of Harvard University
who has done many of the marine environment
studies, particularly with the indigenous flora
of marine waters. Mitchell's whole theory is
based on one thing: the die-off and survival
rate of the enteric, bacteria depends upon the
micro-flora present in the water because of
the antibiotic type substances that they produce.
Wait a minute, Mr. Brezenski, what we are
planning to do is to use a natural sample, and
add Salmonella to this natural sample. I have
to entrap the Salmonella in the chamber to study
its survival, but the chamber will have other
organisms present.
I see, what you are going to do is use "natural"
water.
Yes, we didn't make this clear to you.
How do you take the sample?
Well, we will anchor it in the water. The
chamber has a sampling port large enough to
accomodate a 10 ml pipet. The chamber will be
taken from the water, sampled from this port,
and returned to the lake or stream. The chamber
will consist of pyrex glass and other autoclav-
able materials.
We are in the preliminary stages of testing
the device. It is likely that problems will
arise that we are not aware of yet.
166
-------
Cabelli:
Geldreich:
Cabelli:
Geldreich:
Kenner:
Geldreich:
Shimmin:
Gordon:
John Seaburg at the marine laboratory at the
University of Rhode Island has developed a
device like this to study salmonella. One
might contact him. I believe he has solved
some of the problems. He has a mixing
arrangement inside the chamber.
In a recent publication in Applied Microbiology
the investigators are using a very small chamber.
It has a membrane on each side of it also. Their
limited data indicates that the device may work.
Seaburg1s device is larger, I believe.
It must be larger for our purposes, because
we are sampling over a long period, seven days
at least. We are going to have to do additional
work on this membrane device.
Mr. Geldreich, do you really think that you will
get enough exchange of materials between the
interior and the outside of the chamber?
Well, we have to investigate the practicality
of the device. It may not work, but we will
find out.
I have some comments on some things mentioned
before we discussed the sampling devices.
Based upon data developed in Region II labor-
atory, basically we do not process any sample
from sea water that is older than four hours.
We try to analyze the sample within two hours.
In the case of samples collected from a fresh
water source we do not process samples older
than four to five hours. Our studies are
planned upon these time considerations.
In our area we have trouble with samples freezing.
This creates a serious problem which is opposite
to that of the usual regrowth or aftergrowth
problem encountered if samples are not refrig-
erated. We know that ice crystal formation
kills bacteria, but its not the same from sample
to sample, or from one collection time to the
next.
What about sterile polypropylene containers?
167
-------
Geldreich:
Gordon:
Geldreich;
Gordon:
Geldreich;
Gordon:
Scotten:
Geldreich:
Scotten:
Geldreich:
Yes, those are alright.
They are good sample bottles because they are
difficult to break.
These are okay as long as they have a screw
cap consisting of the same material as the
bottle.
The cap is polyethylene, and the bottle is
polypropylene.
These have been shown to leak after they have
been autoclaved.
No, these are considered to be disposable.
They are discarded after each use.
I have seen some evidence in the literature
where pure cultures of E. coli have been in-
cubated at 10°C and then elevated to somewhere
in the neighborhood of 45 C which demonstrated
considerable killing of these cells.
Well, when we work on mixed populations we
encounter a different set of circumstances
than we do with pure cultures. I use pure
cultures for a starting point as everybody
does. I do not even like to evaluate media
with use of pure cultures. I prefer to use
natural samples if I wish to study the
selectivity and sensitivity of a particular
medium. I think that many of the studies
in the literature refer to pure cultures.
The conclusions may be valid for one parti-
cular organism, but they might not work for
a mixed population of microorganisms.
Well, that is true, of course, I am only
pointing out that E. coli is susceptible to
that kind of treatment.
If anybody would observe this, it would be
our people from Alaska. Mr. Gordon, when
samples are switched from a very cold
environment to the elevated temperature test,
do you observe evidence of thermal shock
causing a low recovery of fecal coliforms?
168
-------
Gordon: I have not noticed this to be occurring.
Geldreich: I have not observed data indicating a detri-
mental affect of thermal shock, except some
studies relating to food freezing tempera-
tures with E. coli involved; but this is a
different environment.
169
-------
QUALITY CONTROL: A STATE-OF-THE-ART
Robert H. Bordner*
Introduction
An established quality control program is essential to
maintain a high level of performance for technical procedures
and to ensure reliable and valid data from the microbiological
analyses of water and wastewater. Media of substandard pro-
ductivity, toxic distilled water, incubation temperatures
that are too high or too low, nonsterile supplies, and care-
less recording and reporting of laboratory results are examples
of poor quality control in routine laboratory procedures that
may drastically affect microbiological data.
Microbiological determinations measuring living organisms
that are continuously adapting to a changing environment are
not as adaptable to precise, statistical quality control as
the analyses performed in other disciplines. Personal judg-
ment is frequently required to determine the next step in a
microbiological procedure. Some of the basic tools of quality
control in other disciplines such as analytical standards,
spiked samples, and quality control charts are not yet suit-
ably adapted to the needs of the microbiologist. Therefore,
it is even more important that a high level of control be
maintained over laboratory and field procedures and personnel
performance.
There is a real need to ensure the quality of microbio-
logical data in view of the increasing emphasis upon water
quality standards, enforcement and monitoring activities, and
the requirements for evidentiary data in litigation.
Quality Control Programs
All laboratories carry out forms of quality control.
Most quality control measures have evolved from common sense
practices and the principle of controlled experiments intro-
duced in early bacteriological investigations. Sound practices
can be built into a continuous quality control program, from
sample collection to data reporting, and conscientiously
followed. An effective program should include all the con-
trollable factors that ultimately influence the results. It
is especially important that laboratories that only sporadi-
cally perform microbiological tests exercise rigid quality
control.
"Analytical Quality Control Laboratory, NERC-Cincinnati
170
-------
Recently laboratories performing analyses in several
different fields of microbiology have reported on the need
for and benefits of a quality control program (1-5).
Divergent practices and sources of errors were uncovered
and improved reliability and reproducibility were accom-
plished. The Laboratory Division of the National Center for
Disease Control (NCDC) and the Food and Drug Administration
(FDA) are presently developing formal quality control programs.
After thorough review, the proposed procedures will be incor-
porated into quality control manuals. The NCDC program in-
cludes a well-operated proficiency testing system that evaluates
performance of over 500 clinical laboratories and a pre-
market evaluation program for testing serological reagents
according to NCDC specifications. The Division of Micro-
biology, FDA, publishes a Bacteriological Analytical Manual,
which is periodically updated. The Division of Microbiology
also operates a proficiency testing program for shellfish
and food and dairy products utilizing split samples. Micro-
biology laboratories in the U.S. Department of Agriculture
and U.S. Department of the Interior participate in the FDA
proficiency testing program.
The U.S. Environmental Protection Agency (EPA) is devel-
oping a formal quality control program for microbiology. The
development of such a program will be accomplished by a com-
mittee of qualified and experienced personnel within the
Agency. A good source of quality control procedures that
have been recommended for water quality determinations is
Public Health Service Publication No. 999-EE-l entitled
Evaluation of Water Laboratories (6), which was originally
published in 1966 and is currently being revised.
This presentation, which reflects in part work performed
by EPA's National Environmental Research Center (NERC) in
Cincinnati, is intended as a review of practices which may
be considered for such a program. Some commonly accepted but
often neglected measures are highlighted, and other less
established practices are proposed.
Sample Collection and Storage
The reliability of results begins with the quality of
the sample. A detailed review and full discussion of sample
collection and handling was presented in the preceding paper.
Sample treatment is an example of many areas where the ideal
practice, performing the analysis of the sample immediately
after collection, has to be compromised with the practical
situation where time is required to transport the samples
to the laboratory bench.
171
-------
Equipment and Supplies
Quality control of laboratory apparatus involves ser-
vicing and monitoring laboratory equipment, including the
operating temperatures of incubators, waterbaths, hot-air
sterilizing ovens, autoclaves, refrigerators and freezers.
Thermometers must be checked against a certified National
Bureau of Standards thermometer or one of equivalent accuracy.
Recording thermometers provide a continuous record of the
operating temperatures of test equipment. All temperatures
should be recorded on weekly or monthly charts and records
should be retained for 2 years. Some laboratories such as
the NCDC, Atlanta, Georgia, and Microbiological Associates,
Inc., Bethesda, Maryland, have sophisticated remote moni-
toring systems for controlled environments which are operated
from a central control room with an alarm system that sounds
when temperatures go beyond the allowable limits (7). Tables
1 and 2 indicate proposed examples of the monitoring of labora-
tory equipment; the monitoring practices are similar to those
used by NCDC (8).
The evaluation of membrane filters is a quality control
activity that the Analytical Quality Control Laboratory (AQCL)
periodically undertakes. The specifications for the purchase
of membrane filters and absorbent pads have for some time
included over 20 chemical and physical as well as performance
characteristics. Currently there are plans to standardize
the chemical and physical tests in the appropriate committee
of the American Society for Testing and Materials.
There have been recent reports from EPA laboratories of
performance differences in membranes from different sources
and of variations in membranes with different lot numbers from
the same manufacturer. This type of quality control problem,
common to all user laboratories, demonstrates the need for a
designated EPA laboratory to continuously monitor such materials
and supplies.
Filtering equipment should be kept clean and checked to
make certain that it is leakproof and uniformly smooth, and
that no toxic metal or corroded surfaces are exposed. Upon
one occasion, the chrome-nickel plating covering the bases of
about 40 filter funnels the Analytical Quality Control Laboratory
had purchased wore off and exposed the brass underneath. All
of these funnels were replaced by the manufacturer.
172
-------
Table 1. Examples of Monitoring Laboratory Equipment
pH Meter
Spectrophotometer
Centrifuge
Microscope
Sterile Air Cabinets
Monitoring Procedure
a. Check with certified weights
weekly.
b. Wipe balance and weights clean
with ethanol monthly.
c. Discard weights if they do not
weigh accurately.
a. Compensate for temperature
on each run.
b. Date buffer solutions when
first opened and check monthly
with another pH meter. Discard
the buffer solution if the pH
is more than ±0.4 from the
manufacturer's stated pH or
if it is contaminated with
microorganisms.
c. Standardize with at least 2
standard buffers (e.g., pH
4.0 and pH 7.0) before each
test or series of tests.
d. Have inspected every 6 months.
a. Have inspected every 6 months.
b. Check transmittance weekly at
a specified wavelength.
a. Check brushes and bearings
every 6 months.
b. Check rheostat control against
a tachometer at various loadings
and frequently enough to ensure
proper gravitational fields.
a. Clean after each use.
a. Clean filters every 3 months.
b. Check for leaks and rate of
air flow every 3 months.
c. Expose air flow to blood agar
plates for 1 hour every month.
173
-------
Table I. Examples of Monitoring Laboratory Equipment (Contd)
Sterile Air Cabinets
Water Stills
Dispensing Apparatus
d. Clean ultraviolet lamps every
2 weeks by wiping with a soft
cloth moistened with ethanol.
e. Test ultraviolet lamps every
6 months; if they emit less
than 70% of their rated initial
output, they should be replaced.
a. Drain and clean monthly according
to instructions from the manu-
facturer.
b. Clean distilled water reservoir
concurrently with the stills.
c. Check distilled water contin-
uously against suitable standards.
a. Check accuracy of dispensation
with a graduated cylinder at
the start of each volume change.
b. Lubricate moving parts according
to manufacturer's instructions
or at least once per month.
c. Correct immediately any leaks,
loose connections, or malfunctions
174
-------
Table 2
Monitoring of Controlled Temperature Laboratory Equipment
Autoclave
Each run
Incubators
Recording thermometers a,
and alarm system
recommended
Water Baths
Refrigerators
2-8°C
Daily before use
Daily
Hot Air Oven
Freezers
Each run
Daily
Also record pressure
once during each run.
Use peak temperature
thermometer weekly.
Use spore strips or
spore suspensions
monthly.
If evidence of con-
tamination occurs,
check frequently until
the cause is determined
and eliminated.
If recording thermo-
meters are not used,
record temperature
daily.
a. Clean monthly.
a. Clean monthly.
b. Defrost or check
refrigerator and
freezer compartment
every 3 months.
a. Use spore strips or
spore suspensions
monthly.
a. Connect to alarm
system.
b. Clean every 6 months,
175
-------
The test for the suitability of distilled water out-
lined in Standard Methods (9) is provided to evaluate water
from new or repaired stills and as a periodic check on dis-
tilled or demineralized water quality. Some laboratories
find this test cumbersome and do not run it frequently.
From time to time manufacturers have made special
products incorporating the membrane filter or other tests
designed for use in field applications. The Millipore Field
Monitor and Coli Count Water Tester are examples. In a
series of comparisons with the standard membrane filter
procedure, the Field Monitor produced a rough estimate of
water quality. The Coli Count Water Tester, which has been
marketed more recently, did not always yield coliform counts
comparable to the count from the Standard Methods procedure
when tested at AQCL. However, when representative numbers
of blue colonies were picked from the Coli Count for verifi-
cation, percentages comparable to those from the standard
membrane filter (MF) procedure did confirm. The Coli Count
will absorb only 1 ml of sample and has been proposed as a
rapid field method for preliminary estimates or screening
of the sanitary quality of water at new sampling sites.
Neither the Field Monitor nor the Coli Count Water Tester
are accepted as standard methods. The limitations of these
and other specially promoted products should be recognized;
data resulting from these products cannot be used for official
purposes such as enforcement cases.
Culture Media and Reagents
Culture media should provide good sensitivity, selec-
tivity, and differentiating capability. The use of dehydrated
media, when available, is required for more uniform results.
Such media should have lot-to-lot equivalence and long-term
stability. Purchasers expect to buy a certain amount of
quality control when they purchase commercially prepared
products. Although some commercial sources state that they
routinely test their products with pure cultures of known
reactions, these tests may not be directly applicable to
water quality analyses and variations from lot to lot are
possible. There is a lack of communication between the users
and manufacturers; the user's problems and expectations of
performance are often not reported to the manufacturer.
Media manufacturers indicate an interest in working with
the user agencies on improved media standardization.
176
-------
Prepared media employed in water analyses may be tested
with natural samples of the type being investigated and with
pure cultures, preferably cultures of representative micro-
organisms recently isolated from water. The importance of
including natural samples in the "use" type of evaluation is
emphasized. It is wise, particularly if results from new
lots are questionable, to perform a comparative evaluation
using as a control a medium lot previously tested and of
known capabilities. For example, an evaluation of M-Endo MF
medium compares sheen production, colony size, and recovery
of coliforms from natural samples, using a previously satis-
factory lot as a control.
It is good procedure to test the media prior to and
concurrent with use, utilizing cultures with known, stable
characteristics. Each laboratory can maintain a collection
of positive, negative, and known reaction cultures for the
various media used. The amount of inoculum should be stan-
dardized by an established technique each time the medium
is checked. Stock cultures which have been maintained by
frequent transfer, lyophilized, or fast frozen in replicate,
are often used. Convenient and standardized reference
cultures are commercially available/ such as Bact-Check
(Roche Diagnostics) or Dri-Bac (Warner-Chilcott Laboratories) .
The objectives of media testing are to make certain
that nutrient media will support growth from small inocula.
Biochemical media must produce the expected positive and
negative reactions from control organisms; for example,
MR VP tests are checked by Escherichia coli and Enterobacter
aerogenes cultures. Differential media are tested with
organisms of known reactions to demonstrate the physiological
differences among morphologically identical organisms. An
example is lactose broth utilized to separate lactose fer-
menters as pollution indicators from those that are not. When
a new batch of these media is prepared, it should be tested
with organisms of known fermentative ability. Similarly,
enrichment and selective media are tested both for productivity
of the desired organisms and the inhibition of others. Bril-
liant green and xylose lysine deoxycholate agar are examples
of plating media that should be tested before use for the
growth of typical Salmonella strains and the inhibition of
coliforms and gram positive organisms.
Media standardization is an area that needs additional
investigation. Published data derived from carefully designed
studies are required to evaluate media performance and to
177
-------
define media capabilities related to their use in specific
procedures. Also, there are no established specifications
for the performance of many selective media.
Among organizations involved in writing specifications
or planning media standardization are the APHA Subcommittee
on Standardization of Culture Media, the National Committee
for Clinical Laboratory Standards, the Interagency Task Force
on Media Standardization, the Defense Medical Support Center,
the U.S. Pharmacopeia, The National Formulary, The Association
of Official Analytical Chemists, and the U.S. Department of
Agriculture's Consumer Protection Programs. !
The supply and quality of commercial reagents are sub-
ject to methodology changes and new knowledge in manufacturing
processes. Continuity of reagent quality must depend on
compliance of each new reagent with minimum specifications
and evaluation of the performance of that reagent compared
with previous usage. All reagents can be tested for correct
reactions with positive and negative controls before they are
used and retested at appropriate intervals. An example is
the rosalic acid reagent added to the M-FC medium.
Serological reagents should be evaluated against known
antigens and antisera whose reactivity has been confirmed
by previous experience. Quality control procedures should
be repeated each time reagent batches are prepared, regard-
less of the expiration date designated by the manufacturer.
Unacceptable levels of reagent activity often result from
poor storage conditions or from contamination.
The following controls may be incorporated into the
test procedures: all reagents should be dated when received
and when put into routine use; new lots of reagents should
be tested in parallel with old lots of reagents to eliminate
any unsatisfactory reagents; positive and negative controls
or reference preparations, where available, should be used
each time a serologic test is performed; positive controls
should be within one dilution of the average titer; if con-
tamination is discovered in sera or antigens, or if the potency
has deteriorated, they must be discarded immediately.
Preparation Services
The preparation of culture media must follow prescribed
procedures for weighing, measuring, pH adjustment, and steri-
lization. Frequently, media preparation is in the hands of
junior or unskilled personnel; experience, training, and
continuing supervision should be provided.
178
-------
The laboratory should check each prepared, batch of
medium for clarity, color, pH, and sterility. Growth-
promoting capacity may be adversely affected by oversteri-
lization, burning or charring, contamination with metallic
salts from unclean glassware, and repeated melting of solid
media. Darkening may result from oversterilization. Pre-
cipitates may indicate chemical incompatability or over-
heating. The pH should be taken electrometrically at room
temperature and be within ±0.2 units of that specified. If
a medium has been overheated at any time, the pH is likely to
be lowered. To check sterility, representative tubes or
plates are incubated for 2 or more days at 35°C.
Store sterile culture media in a clean, cool, and dark
area free from dust, contamination, and excessive evaporation,
Many laboratories store media in the refrigerator. Strong
light may break down some media such as brilliant green
lactose bile broth. Excessive evaporation alters the con-
centration of ingredients. If changes from exposure to
light or evaporation are observed, the medium should be
discarded. If media are stored at low temperatures, they
must be incubated overnight and any tubes with air bubbles,
discarded. Sterile media should be prepared in amounts
that will be used within a month.
It is good practice to date dehydrated media when
received, rotate in use, store in tightly-closed, screw-
capped containers at less than 25°C, and discard if signs
of deterioration, such as caking or discoloration, are
observed. All dehydrated media in sealed containers should
be used within 2 years and thoroughly checked before use.
The media should be purchased in amounts that meet the needs
of the laboratory for 6 to 12 months. An example is the
purchase of membrane filter media in 1/4-pound packages to
ensure that the media do not remain unsealed for prolonged
time periods before use.
Ideally, an opened container should not be in use longer
than 2 or 3 weeks. Such media deteriorate at different
rates varying with formulation, humidity, temperature, and
other factors. Significant variability in differential and
selective plate media resulting from improper preparation
and storage have been reported in a laboratory evaluation by
Barry, et al (4).
Laboratory personnel should make certain that the glass-
ware cleaning procedures used are adequate and that there is
no residual chemical or detergent film. Standard Methods
179
-------
recommends a procedure for the bacteriological testing of
glassware for bacteriostatic or inhibitory residues.
Test Procedures
Methods selection is an important application of quality
control. A set of guidelines or a procedural manual that
describes EPA methodology for microbiological analyses has
been suggested. It is reasonable that methods must be clearly
defined before a quality control program can be effective.
Such a manual would select the best methods for specific
applications and identify the options, if options are neces-
sary, that will provide the most reliable and defensible
results. Participation and recommendations should come from
all EPA microbiologists. The selection of fecal rather than
total coliforms, MF as opposed to Most Probable Number (MPN)
procedures, and methods for the determination of Klebsiella
are examples of microbiological issues for which EPA should
establish policy.
Adherence to Agency-recognized procedures is an important
factor in quality control. Any deviation from recognized
procedures should be carefully noted in the laboratory. A
laboratory practicing such digressions should be prepared
to demonstrate by quantitative methods that the deviations
have not adversely affected test results.
An active program for evaluating new methods and reevalu-
ating the present methods is a function of any laboratory
truly interested in improving its performance. Such a pro-
gram can logically be assigned to quality control.
Quality control also implies the comparison of methods.
For example, when changing from the MPN to MF procedure, it
is necessary to run a series of samples in duplicate by both
methods and to compare results.
Replication of MF analyses is proposed as an aid to
quality control. Some laboratories perform all MF analyses
in duplicate or triplicate. For research projects using
membrane filters some laboratories, including AQCL, filter
five replicates for each analysis.
Comparison counts of colonies provide information on
precision. Such counts are particularly valuable for the
total coliform MF test, in which more than one technician
periodically counts the same membranes to reinforce the
interpretation of sheen colonies on Endo MF media. At AQCL
comparison counts are made on 10% of the membranes.
180
-------
One of the most frequently used quality control pro-
cedures for MF tests is the confirmation of colonies. At
least 20 typical, well-isolated colonies for each sample are
picked and inoculated into tube media. After incubation the
tube media are observed for positive results. The percentage
confirmation in tube media is calculated for coliforms from
the number of positive tubes. Fecal streptococcal colonies
can also be confirmed by inoculation into the appropriate
biochemical media.
The confirmation procedure is also used for enteric
pathogens isolated from water. Typical colonies from selec-
tive media are purified and tested biochemically and serolo-
gically. Additional confirmation by NCDC or a qualified state
health laboratory is desirable, but usually possible only for
limited numbers of isolates. There is a need for EPA to
provide this expertise in an Agency laboratory or by contract
with an authoritative laboratory such as NCDC.
Comparison of results from field tests with those from
the laboratory is necessary. If the delayed coliform MF
tests are used in the field, the results should be compared
with those from the immediate test.
Data Handling and Validation
Permanently recording analytical data in meaningful,
exact terms and reporting it in proper form to some information
storage facility for future use implies quality control.
Precise rules for the use of significant figures, rounding
off of numbers, and arithmetic operations should be agreed
upon. An example is the rules to follow for reporting MF
results when more than one increment is within acceptable
colony count range, but when there does not appear to be a
proportionate relationship among the results. A system for
maintaining neat, accurate, and legible records and for
reporting results is mandatory. Bound laboratory records
books and preprinted report forms are recommended. Some
programs use multi-copy forms to record all information from
sample collection to calculation of results. These forms
are then forwarded to the appropriate office for direct trans-
fer of the data to computer programs. Laboratory records
should be readily available for inspection and held on file
for a period of at least 2 years.
A research group at the Robert S. Kerr Water Research
Center at Ada, Oklahoma, has investigated the application of
the Cu Sum Quality Control Technique to microbiological
results for the purpose of producing a reliable method of
validating the data (10).
181
-------
The conclusions of this validation study were that pre-
cision control charts are a useful tool for precision but
they cannot measure accuracy; the data can be precise and
still inaccurate. The control charts have to be constructed
on data from the same waters under study. Tests must be
performed at least in duplicate. To obtain maximum benefits,
the data must be plotted daily and problems must be rectified
immediately.
Performance of Personnel
The specificity and selectivity of microbiological tests
can be closely controlled by some of the techniques suggested,
but the care with which the test is performed cannot be as
easily controlled. Formal training in microbiology and in
performing environmental analyses is strongly recommended.
The degree of professional training required is related to
the number and variety of tests performed. Ideally, the
microbiology laboratory should be under the supervision of
a professional microbiologist. When this is not possible,
personnel performing the microbiological tests should have
the readily available support of a professional microbiologist
who is experienced in environmental analyses. If the data
will be used to support legal action, analyses must be super-
vised by a professional microbiologist who may be called upon
as an expert witness.
On-Site Laboratory Inspection
For years microbiologists in the water supply program
have performed evaluations by on-site inspections of labora-
tories that test the quality and safety of interstate carrier
water supplies. Often special problems have been solved,
inapparent discrepancies have been brought to light, and more
efficient techniques have been developed as a result of this
activity. An established laboratory visitation program for-
mally recognizes the capabilities of the laboratory and its
personnel. Such a program should be considered for EPA
microbiology laboratories.
Intra-laboratory Quality Control
The Analytical Quality Control Laboratory is responsible
for providing reference samples to quality control programs
in Regional laboratories, NERCs, other federal, state, and
local agencies and the private sector. Although this program
began with the development of samples for chemical parameters,
it includes biological and microbiological parameters as well.
182
-------
A development of a sample preparation system for micro-
biological analyses is planned. The problems involved in
the development of stable replicate samples are recognized.
There is presently no method of effectively preserving water
samples for this purpose. The use of replicate membrane
filters which have been inoculated, incubated, and dried has
been investigated. The rapid distribution of quick-frozen
samples and the use of lyophilized cultures are also being
considered.
Inter-laboratory Studies
Formal inter-laboratory studies for evaluating all EPA
analytical methods for water are also carried out by the AQCL.
Stable replicate samples for chemical and physical parameters
are provided to participants with exact instructions for
sample preparation and analysis within a limited period of
time. Results are evaluated and reported in a formal EPA
report that provides the precision and accuracy statements
of method performance.
For inter-laboratory method studies in microbiology,
standardized media and supplies should be prepared in the
issuing laboratory and sent to other laboratories for the
evaluation of a procedure. A method of sample preservation
such as rapid freezing or lyophilization would be required.
An alternative approach would bring the participating
microbiologists and technicians together in one laboratory
where they could analyze sample aliquots at the same time
with the use of the same media, materials, and procedures.
EPA and its predecessor organizations have conducted
field tests for the evaluation of delayed total and fecal
coliform MF procedures. The microbiology laboratory sends
the prepared media and materials to participating laboratories
in different geographic locations that perform replicate
filtrations for the immediate and delayed tests. The repli-
cates on holding media are mailed to AQCL for completion of
the delayed test. Results are compared and evaluated.
Summary
An effective quality control program is the continuous
systematic practice of accepted procedures coupled with the
correct performance of competent technicians. Such a program,
when properly administered, will produce data of uniformly
high quality without unduly interfering with the primary
analytical functions of the laboratory. A well defined EPA
183
-------
quality control program for microbiological analyses will
ensure reliable and valid microbiological data and a high
level of performance.
Such a program should include the following:
1. A set of guidelines or a procedural manual that
selects and clearly describes EPA methodology with appro-
priate references to recognized microbiological journals/
manuals or books such as Standard Methods and with provisions
for periodic updating.
2. An efficient internal quality control protocol in
each laboratory to monitor sampling techniques, equipment,
supplies, sterilization methods, glassware washing procedures,
media, and reagent preparation and data handling.
3. Intra-laboratory methods and performance evaluation
programs in each laboratory based on reference samples.
4. A single EPA microbiology laboratory responsible
for evaluating and monitoring the quality of equipment and
supplies in general use throughout Agency laboratories
(e.g., water baths, incubators, field kits, membrane filters
and large lots of media for routine tests).
5. An on-site visitation program for EPA laboratories
conducted by experienced microbiologists.
6. Participation in all EPA inter-laboratory performance
evaluations and method validation studies.
7. Improved EPA specifications for media and supplies.
8. Formal training programs for new laboratory personnel
and refresher courses for more experienced technicians.
9. Maintenance of accurate, legible, laboratory data and
quality control records.
10. Periodically scheduled meetings of Agency microbio-
logists to continuously reevaluate methodology and research
needs.
184
-------
References
1. Vera, H. D. Quality Control in Diagnostic Microbiology.
Health Laboratory Science, 8:3, 1971.
2. Vera, H. D. Quality Control in Dairy Microbiology. J.
Milk Food Tech. 34:7, 1971.
3. Russell, R. L., R. S. Yoshimori, T. F. Rhodes, J. W.
Reynolds, and E. R. Jennings. A Quality Control Program
for Clinical Microbiology, Tech. Bull. 39:195, 1969.
4. Barry, A. L., and K. L. Feeney. Quality Control in
Bacteriology through Media Monitoring. Am. J. Med. Tech.
33:387, 1967.
5. Halstead, E. G., R. A. Quevado, and W. H. Gingerich. A
Quality Control Program for the Bacteriology Laboratory.
Am. J. Tech. 37:15, 1971.
6. Evaluation of Water Laboratories. PHS Publication No.
999-EE-l, DHEW, PHS, Washington, D.C., 1966.
7. Remote Monitoring for Controlled Environments. Laboratory
Management 10:27, 1972. (No author)
8. Personal communication, Dr. Robert J. Ellis, Laboratory
Division, National Center for Disease Control, Atlanta,
Georgia, December, 1972.
9. Standard Methods for the Examination of Water and Waste-
water, 13th Ed., Am. Public Health Assn.1971.
10. Cumiford, H. F., J. L. Kingery, R. D. Harkins and J. N.
Jones. Analytical Quality Control Methods for Use in
Validating Microbiological Data. Robert S. Kerr Water
Research Center, Office of Technical Programs, Ada,
Oklahoma, April 16, 1970.
185
-------
QUALITY CONTROL: DISCUSSION
Geldreich: May I supplement your material by describing
the Laboratory Evaluation Program? The program
was initially established about 1943 for
laboratories that are involved with the exam-
ination of public water supplies and to some
extent private water supplies. We have through
the years developed a systematic approach by
going to each of the State Health Department
Laboratories and spending a day and a half
evaluating their procedures in the areas of
sampling, monitoring, laboratory apparatus,
glassware, plastic ware, metal utensils,
materials and media preparation, culture media,
controls on culture media, multiple tube pre-
cedures, membrane filter tests and supplemental
bacteriological analyses. We take an in-depth
look at the laboratory facilities, laboratory
management and staff problems. We work right
with them at the bench level while they're
doing samples and if necessary roll up our
sleeves, show them what they're doing wrong
and explain how it should be done.
At the end of the laboratory visit we review
the evaluation with the laboratory director,
the administrator in charge, or his designated
representative. We clearly outline any concerns
we have, such as deviations in procedures, and
suggest improvements; for example, specifications
for equipment. The follow-up written report
that goes through the EPA Regional Offices to
the laboratory director and the state health
department officials holds no suprises. This
approach has been very rewarding. We've received
tremendous cooperation from state health depart-
ment laboratory people. We have to do this at
the state level about once every three years;
and at the smaller laboratories it should
actually be done every two years. After more
than three years the turnover of personnel is
such that procedures could change and the quality
of the data could deteriorate. Because we cannot
begin to cover all the laboratories in this
country, we have in turn checked out one individual
in each state health department to be the certi-
fying laboratory officer for all water analysis
Jaboratories within his state.
186
-------
More recently in EPA we have been placing more
emphasis on all water supplies, including re-
creational waters, and looking at the methodo-
logies used in monitoring programs, so that
laboratory evaluation has spread out consider-
ably from its original inception.
Some years ago this program culminated in the
development of the manual called Evaluation of
Water Laboratories. We are rewriting this manual
in two volumes, one devoted to chemistry and the
other to bacteriology. Associated with the
manual is a survey form, a working check list,
which we use when we go in to work with labora-
tories. It covers all the items that we've
just briefly discussed here in general and
relates to Standard Methods or good laboratory
practices. There are some items that we con-
sider good laboratory practices and have been
demonstrated to be equal to or better than
Standard Methods. In essence, we're trying to
put together, chapter by chapter, an opportunity
for the user to study his own laboratory and
see where he could perhaps use some improvements.
We have provided references to original publi-
cations in each chapter and have placed a part
Of this new survey form at the end of each
chapter. The reader can expose himself to some
of these problems, see what he is doing wrong
and hopefully correct it.
We do feel that until Bob and his group can
work out a split sampling program, evaluate the
media and provide specifications for materials
and equipment which we can all take advantage
of, the only thing we can do is to visit the
laboratories and work with them. We believe
very strongly that we need to supplement these
laboratory evaluation visits with a split-
sampling program. Marty, I think you mentioned
yesterday something in this area. Would you
like to comment?
Knittel: These are some comments on experience in the
aerospace industry. In their quality assurance
program, in building a space craft and speci-
fically in the planetary quarantine program in
187
-------
which I was involved, each item had to be
checked by another individual whom we called
a quality assurance officer. All quality
assurance information had to be logged in
each day. It was not enough to state that
media had been sterilized at 121 C for a certain
length of time; they wanted proof that it was
indeed sterile. Related to some of the quality
control principles that Bob pointed out, pro-
cedures were practiced such as obtaining selected
portions of a batch of prepared media and actually
incubating it to prove that it was sterile.
Autoclaves were checked periodically. We used
a lot of laminar flow hoods which had to be
certified at different points in time. We were
dealing with very low levels of contamination
on space craft surfaces; we would get perhaps
10 or 20 microorganisms per square inch of
surface. When a person was actually involved
in doing plate counts or similar determinations,
he was indeed following the procedure set forth
and someone would be logging in all of this
information.
Geldreich: I have one question, Bob. You and I have had
this discussion privately, but for the record
I would like to say that I am always nervous
when we have to concentrate our evaluation of
media primarily on pure cultures. I don't
believe that you can adequately evaluate a
medium only on pure cultures. A greater emphasis
should be placed on using natural samples. My
reasoning here is that it may be that you have
an organism that can do tricks for you and will
produce gas or whatever the characteristic is;
it works great when you're checking it out on
that medium. However, in the real world we're
working with mixed floras and you have to not
only know something about survival rates and
whether the microorganism gives gas or not,
but also how it interracts with other organisms.
So, I would like the group here to understand
that I think we shouldn't put all our marbles
• in evaluations with pure cultures. We need pure
cultures to a certain extent, but we must relate
more to natural samples and evaluation of media.
188
-------
Berg:
Geldreich;
Berg:
Geldreich:
Bordner:
Geldreich:
Bordner:
Can we apply laboratory quality control methods
other than laboratory evaluation?
Can we? We wish we could. Some years ago/
Gerry, we tried to get into a media certifi-
cation program. Harold F. Clark, Luther Black
and several other people actually had a program
started but the lawyers in the DHEW said at
that time that federal agencies could not get
involved in this type of a certification pro-
gram; yet NCDC has come up with some arrange-
ment.
NCDC is the only one?
And they're federal. We need to know the proce-
dure, find that mechanism and use it.
It was explained to me at NCDC that the certi-
fication program for serologicals is voluntary.
NCDC asks the various manufacturers to volun-
tarily submit samples of every lot number that
they put out for NCDC evaluation. The manu-
facturers don't have to do this and some of them
don't; but if they do, NCDC evaluates the products
and publishes the results. NCDC also publishes
the test procedures.
NCDC finds then that people in the state health
and other laboratories who are using these
products will check the ratings of the products
evaluated. The manufacturers find that it is
greatly to their advantage to have their products
tested because the state health representatives
and other user laboratories check NCDC's evalu-
ation list and a great many of them call NCDC
for such information.
We need something in our own organization. If
you're doing all of this work, I would very much
like to see some of this data so that I can
disseminate it and use it myself, and I don't
see this. The data you showed today is the
first time I've ever seen anything like this.
What was that, Ed?
189
-------
Geldreich:
Shimmin:
Geldreich:
Shimmin:
Geldreich:
Resi:
Shimmin:
Well, data that you have on evaluations,
particularly those on your replicates. I think
all of us here need to have this kind of infor-
mation; you should supply it through some mechanism
such as your Newsletter.
There's one point that bothered me both in your
presentation today and yesterday in Bill Stang's
presentation; perhaps I'm not understanding it
correctly. You said today that in field studies
there are some laboratories that only run one
filter per sample because they have a problem
running any more, and yesterday Bill Stang
implied at one point that the microbiologist
just has to take more or less what happens along.
I think that we should really make an effort in
this manual to stress the fact that the micro-
biologist should be involved in planning studies.
The microbiologist should be completely in control
of what kind of samples are coming into his
laboratory, so that if he has to adjust the
numbers of samples in order to get adequate
quality control, he can.
Is Andy Sidio here? This will be a chance for
him to make a comment. You know this is a
problem we have with engineering groups, getting
together.
I know that. I've not been with EPA very long
and I certainly had the problem when I first
came, but microbiology in Region IX doesn't
participate in a study unless they have been
involved in planning it and regulating the
frequency of the samples and the timing of the
samples.
Lou, would you like to say something?
Well, my comment is that this is the ideal
situation; what you say is being idealistic
rather than a realist.
Well, it might be idealistic for the rest of
the country but it's not for Region IX.
190
-------
Stang:
Shimmin:
Bordner:
Shimmin:
Bordner:
I would like to say that at NFIC, Denver, we
do get in on the planning and lots of times
there are other things involved like the
amount of time and money that can be spent.
Of course, an engineer is going to ask for the
most samples that he possibly can in the short-
est amount of time; this is a continuing fight
that we have.
Yes, I think that's true, but we should be
definite in this manual and say just precisely
what kind of errors we're talking about. If
we're taking our time giving an inaccurate
report of data, then why waste our time doing
it?
It seems to me that we could come up with some
better precision data than I'm familiar with
now for duplicates or triplicates or whatever
number is required, maybe as a result of the
fifty replicate date which AQCL has produced or
some other source data, we could say to the
engineer, "this is the amount of precision we
can give you for triplicates as opposed to single
determinations. It will cost extra time or
perhaps less overall samples will be examined."
We need statistical data to prove the advantage
of replicates. How do you feel about that?
Yes, I think that's true but, certainly the
chemists are asked how many samples they can
run in a day and they add their quality control
needs. They feel that maybe they can run this
many samples and also they can say this is the
amount of time the samples can be stored; if
you would exceed that amount of time, we can't
do it. So if that's good enough for the
chemists, it ought to be good enough for us.
Except that they have better quality control
data for most of their determinations than we
have. So you show the engineers these duplicate
or triplicate analyses and you're going to come
up with a pair that is split pretty far apart
once in awhile and the others are close; is
the engineer going to say, "I don't think this
is worth it."
191
-------
Shimmin:
Bordner:
Geldreich;
Sidio:
Well, I don't think the engineer should be
making that decision. That's a microbiologist's
decision and the laboratory's decision.
I think it's a combination.
Andy, when you plan a survey, and you have a
certain amount of bacteriological work to be
done, how do you go about making a decision
on .what you're going to do? The microbiologists
have asked a question here. They don't always
have an opportunity to say, "we can do this
much," but, the engineering section says "we
need more than that" and there's just a limit-
ation to what the microbiologist can do. How
do you resolve your problem?
First of all I think that anybody that sets up
a survey and doesn't consult each of the chemists,
biologists, and microbiologists, prior to the
starting of the study, is making a big mistake
right off the bat. First of all he's doing
a poor job of planning that study. That is
part of working with those people, using them
as your consultants. I don't care whether its
an engineer planning the study or whoever it
might be. There is no one who knows every con-
sideration that is to be involved in that study.
Whoever is planning it must deal with micro-
biologists and other professional people to
assist him in planning that study.
It is true in many cases that the person
planning that study may say, "I need this much
bacteriological data," or whatever it might be.
Let's take an example: you tell us you can
only take 20 samples a day and we've got to
have 40. We have to do one of two things; we
either double the crew that we've got, double
the facilities, or we double the length of time
that we need to get the data. We might put in
a ten or twelve hour shift for a period of two
weeks or whatever the period of time is. This
is part of proper planning of a survey. There
has to be a mutual understanding that you're
in this thing as a team.
192
-------
You have to do the survey as a team; you have
to work together. You as a bacteriologist
cannot go out and do a field study by yourself.
You contribute to a team effort. The better
the people work together, the better the study
and the better the results. The smoother the
working relationship together, the better the
ability of that group to get a job done. For
the past few years we have even taken the
attorneys into account. There has to be con-
sideration now that every one of those samples
may wind up in court. We now have to bring in
that legal member of the team. The whole
point is teamwork.
Shimmin: Andy, I'd like to ask you a question—how well
does this cooperative effort work at NFIC,
Cincinnati?
Sidio: If we have had any success at NFIC, and I'm
sure this is true of any other group, I think
it is because we have a team effort there.
Shimmin: Do you always involve everyone who's going to
be in on a study in the planning of it?
Sidio: There are certain instances where you get a
call and you have to move immediately, such as
the floods last fall in Pennsylvania. They
flew a C124 in from Utah to fly one of our
bacteriology labs into the Wilkes-Barre-
Scranton Airport. In a situation like that
you don't do a lot of planning before you
start acting. Usually, however, each of the
crew members is included as part of the plan-
ning. You can't plan a field study from behind
a desk. It's very important to know what you're
planning a survey for and to have all of those
people there. All of them? No, but all of
those people who are going to have responsibil-
ities out there and that includes the field
crew that is going to collect the samples.
Every person who is part of that field crew is
just as important as any other person, non-
professional as well as professional. Everyone
has to know he is an important part of the team.
I think this teamwork approach in Cincinnati
is excellent.
193
-------
Jeter:
Lewis:
Bordner:
I can vouch for this, Andy. I have been
pressed into service with the predecessor
organizations of NFIC where we actually sat
down around a table and I noticed that every
sample collector, and everyone else who was
going out on that survey, was around that
table and spoke very freely about their
responsibilities. I saw it actually working
under those field conditions. I can support
this very strongly.
Bob, I just wanted.to bring up the fact that
for hospitals and clinical laboratories,
unknown specimens are sent out by NCDC and
other laboratories. FDA in their milk program
does use split samples.
Yes, they do. I'm not too familiar with the
previous ones in milk. As I understand the
program, they've put out split samples using
pure cultures and mixtures of pure cultures in
milk samples which they sent out frozen. More
recently they wanted to get closer to food and
they used mashed potatoes. I believe they send
out mixtures of cultures for which they can
anticipate a certain die-off rate for the
cultures they're using. They ask the parti-
cipating laboratories to warm up and test the
samples all at the same time and then return
the results. Whether this is practical for a
water quality program I don't know.
We've talked a little about round robins in
some of our previous discussions. This may be
a possibility if we're talking about sending
out a medium which is controlled from some
central source to be used in participating
laboratories with their natural waters and then
the results sent back to Cincinnati for evalu-
ation. Does anybody else have any ideas on a
round robin or reference sample study?
(No comment s)
194
-------
SUMMARIZATION
-------
MICROBIOLOGICAL RESEARCH NEEDS
*
Kenneth M. Mackenthun
I accepted this assignment with certain trepidations
that, as a result, I too might inhabit the proverbial pit-
falls of professional pronouncements. As these are related
to research needs, they include:
1. Casting every unknown solution to conceived problems
into research needs regardless of their national priority
relevancy, or reasonable probability of successful attainment;
2. Displaying noticeable naivete of literature or state-
of-the-art as they may be associated with a particular need
concept; and
3. Fostering trivia over priority needs by assuming
that by mass of need numbers, or by repeating a particular
need in a number of different sentence structures, some of
the suggestions will stimulate action. Perhaps I may stand
accused before this paper's end.
Thirty years ago the romanticist of the era had an ear
tuned weekly to the Wednesday night radio programs where
swing and sway music was directed by Sammy Kaye and Rubinoff
and his magic violin were noteworthy predecessors to Liberace.
A principal source of radio entertainment of the time was
listening to the Hit Parade. The Hit Parade featured the
ten top tunes of the week and the program had a certain
repetitiveness from week to week. As a result, many of the
same tunes stayed on the program for a period of several
weeks and a length-of-time score was kept on the tunes that
persisted in the No. 1 spot. But that was part of the lure
and drawing power of this program — it featured the familiar
tunes.
A direct analogy can be drawn between the Hit Parade
and a list of microbiological research needs. Research
needs, too, have become familiar through time and the
priority items have been restated on many occasions. And
the needs will not be consumated in the foreseeable futurei
Research needs generally are couched in language that
encourages a seeking of the ultimate. successful research,
to the contrary, results usually in the cohesion of facts
for a segment of the whole puzzle — not in an immediate
^Director, Technical Support Staff, Office of Air and
Water Programs
195
-------
total solution of the puzzle itself. As facets of a problem
become known in our increasingly complex environment, new
questions requiring new solutions accelerate clearly into
focus. Research proceeds ad infinitum. We need, and
we will continue to need, definite answers to particular
environmental problems.
Needs
Because of the implied interests of the attendees at
this seminar, I have limited my research needs remarks to
public health-related organisms. I have categorized the
research needs proffered into the ten groups following:
1. Detection
I suppose there will be a need always for more refined
detection techniques. We need more sensitive rapid quantita-
tive methods to detect pathogenic organisms. In the develop-
ment of appropriate technology, costs should be held to^a
minimum, technical training requirements for investigative
personnel should be of a reasonable order of magnitude, the
time lag in obtaining results should be four hours or less,
and procedures should be developed for concentrating viruses
and other pathogens from large water volumes. At the present
time we are able to recover only a percentage of the viral
numbers believed to be within a sample. When a positive
answer is obtained from our present detecting procedures, a
positive statement can be given; when a negative answer^is
obtained, there is always an element of doubt. Always_it is
a search for the elusive few who may be the problem children
of their generation.
A corollary concern in detection is media quality control,
Specifications for acceptable media are needed. Commercial
media now are not getting sufficient quality control and
technicians may be forced to rely on media that may be
questionable and to produce results that could affect public
health. A media certification program might be desirable.
2. Sampling
The time-worn truism is that the results obtained can
be no more representative of prevailing conditions than the
validity and representability of the sample analyzed. We
need tp develop and refine techniques to collect large
samples from which few organisms may be detected. Bacteriol-
ogical procedures need to be standardized to become more
196
-------
responsive for enforcement actions. Methods for j.n situ
examination should be developed to complement the need for
bench cultures. Automatic monitoring of bacteriological
constituents is a need that should receive considered
attention. Two ever-present questions are: How many samples
do we need? How do we best use these samples in a monitoring
program to determine quality of materials sampled?
3. _P_e rsis te nee
Persistence often is a key toward successful mission
accomplishment even in the world of the microbes. We need
to determine the possible role of water in the transmission
of viral diseases from man to man or from animals to manf
the degree to which other pathogens are waterborne, and the
role of water in the spread of cancer viruses. Do human
pathogens decrease in biological wastewater treatment
systems at the same relative rate as fecal coliform bacteria?
Where sewage-contaminated water is used for crop irrigation/
what is the incident and survival of known human and animal
pathogens? Such pathogens would include the Mycobacteria on
crops irrigated for livestock consumption, various mammalian
viruses, as well as the potential for parasitic infestations,
including livestock liverflukes or human intestinal parasites.
4. Indication
There is always an element of doubt associated with the
use of coliform bacteria as indicators of water quality.
Investigations are needed to determine the potential of
other organisms either as a virtual replacement of the
coliform group or as an adjunct test to more precisely
define water quality. A corollary need is better correlation
of expected prevalence of indicator and pathogenic organisms
in recreational waters. Do human enteric pathogenic organisms
increase proportionately to fecal coliform bacteria under
environmental conditions where the latter can increase?
5. Dj.sj.nfe ct ion
An ever-present management problem is the adequate
disinfection of wastewater effluents. The environmental
effects of the present use of chlorine to disinfect_waste-
waters is of contemporary concern. Viral disinfection and
removal capabilities of existing and proposed water and
wastewater treatment processes need further evaluation..
The effectiveness, efficiency, and economy of disinfection
197
-------
procedures are interrelated factors for consideration in
disinfection research. The natural purification processes
of surface and ground waters and the effects of nutrients,
particularly excess nitrogen and phosphorus on these
processes are matters for continuing investigation.
6. Good Looks;
There is much circumstantial evidence which indicates
that aesthetically pleasing waters generally meet require-
ments for bacteriological safety. Is it possible to define
aesthetic quality to the extent that it could be correlated
with bacteriological safety?
7. Sensation
The more specific role of particular microorganisms
in imparting organoleptic sensations to water should be
determined. There is need for an objective chemical test
or tests to identify compounds that cause tastes and odors
and to correlate the quantity and quality of such compounds
to ambient microorganism populations. Toxicological studies
need to determine the relationship between organoleptic
sensation-producing organisms and compounds and human health.
As discussed under the detection item/ it is often necessary
to examine large quantities of water in order to identify
its taste and odor-producing components. The techniques
employed in the collection and examination of mega samples
require refinement.
8. Interaction
There are a myriad of interrelationships among the
physical, chemical, and biological environments that influence
the survival, persistence, and population development of
pathogenic and nonpathogenic microbes. The role of various
pollutants in these natural phenomena needs more precise
definition. Auto-inhibitors or extracellular metabolites
that serve as antibiotics to portions of the microbiological
community are phenomena related to survival and persistence,
which may be quite significant to the potential for disease
transmission. The role of sediments and the potential effects
of the associated microbiological community on the overlying
water is an area that has not received past abundant research.
What is the microbial mechanism in both aerobic and anaerobic
sediments and the implications to a water supply source, to
naturally-occurring shellfish beds or to aquacultural
activities? Of a corollary nature, there is a need to
198
-------
establish guidelines to determine the intensities of
different types of recreational pursuits that can be
sustained concurrently on the same body of water without
impairing the relative public use.
9. Risks
Often the professional comment arises that epidemiol-
ogical data are insufficient for a particular cause-effect
relationship. Especially this is true for the transmission
of diseases, actual or potential, through recreational-use
waters. The eye, ear, nose, and throat infections associated
with swimming need a more positive epidemiological correlation
with particular water quality. The risks of enteric infection
to those associated with water contact sports of varying
microbiological water quality should be determined. What
is the relationship between the volume of water swallowed,
deliberately or accidentally, and the infective dose of
pathogenic organisms?
10. Finale
Because of its association with human swimming risks
in many nations, amoebic meningoencephalitis, which is
thought to be caused by a strain of Naegleria qruberi, has
stimulated much concern recently. We need to know much
more about this organism and its virulent strains. Needs
include methods for identifying pathogenic and nonpathogenic
forms, additional studies on the organisms' pathogenicity,
and the chemical nature of the cytotoxic substance produced
by the pathogenic strains.
Although not of the highest priority, there is a need
to determine the prevalence in various mammalian and avian
populations of pathogenic organisms that could affect man.
Responses
It would be inaccurate to state the EPA research efforts
are not attuned to these microbiological problem areas and
research needs. Likewise it would be inaccurate to state
that the needs in a general sense, will soon be met. They
will remain with us, demanding additional research, for
many years to come. Research efforts have been chipping
away at the visible portion of the iceberg, but they have
engaged also in occasional forays into the less evident
needs realm. Some of these positive research efforts include
the development of:
199
-------
. Rapid methods for the detection and enumeration of
pathogenic bacteria including cultural, biochemical,
and serological methods for the evaluation of
quantitative and qualitative determinations on
Salmonella;
. Improved and more rapid methods for the detection,
identification, and enumeration of pollution
indicator organisms;
. Methods for the concentration, recovery/ and
identification of viruses from water;
. Microbiological criteria for recreational waters,
for field evaluation of total and fecal coliform
and other indicator organisms in marine and estuarine
waters;
. Techniques for studying the pathogenic strains of
the causative organism of amoebic meningoencephalitis;
. Improved methods of identifying and isolating
viruses, identifying diseases transmitted by
drinking water, and investigating the endemic
occurrence of diseases known to be waterborne.
. Methods for the identification of pathogenic
bacteria in rennovated wastewaters and to demonstrate
any detrimental effects on human health from water
reuse activities;
. Generally applicable chemical and physical treatment
methods for the removal or inactivation of micro-
organisms from municipal wastewaters to any desired
degree, which would include a quality suitable for
water reuse.
A continuing effort will be devoted to the validation
of methods for chemical, biological and microbiological
analyses. Efforts to overcome a suppression of coliform
organism detection caused by excessive bacterial populations
will be researched. The analyses of data indicate that
non-coliform bacterial populations in excess of 1,000
Standard Plate Count organisms per 1 ml may affect adversely
the detection of the coliform indicator group. This_is a
problem associated with older water supply distribution
networks. The bacteriological quality of bottled water will
200
-------
be examined concurrently to define acceptable criteria.
Studies will be made on viral and bacterial pathogens as
they occur naturally to determine the influence of environ-
mental conditions, particularly turbidity on disinfection.
A corollary problem is to identify acceptable disinfecting
procedures without causing consumer rejection of the finished
drinking water.
From the above discussion it is apparent that many of
the ten itemized research categories are being addressed,
in part, through current research. As answers to contemporary
questions are found, new questions will arise. Research
priorities must be assessed and reevaluated at periodic
intervals with an annual appraisal as a minimal goal.
Research must continue ad infinitum.
201
-------
INDEX OF AUTHORS
-------
Index of Authors
Becker, R.
Bacteriological Depth Sampler
Berg, G.
Methods for Detecting Viruses In Environmental
Waters-A Status Report .......................... -72
Bordner, R. H.
Quality Control : A State-of-the-Art ............ 170
Bourquin, A. W.
Impact of Microbial Seed Cultures on Aquatic
Environment
Brezenski, F. T.
Fecal streptococci .......................... • • • • 47
Foerst Kemmerer Sampler for Routine Bay sampling 160
Cabelli, V. J. and M. A. Levin
Methodology for the Enumeration of Pseudomonas
Aeruqinosa ................... • .................. "
Chang , S . L .
Zoomicrobial Examination of Water: A State-
of-the-Art ...................................... 90
Geldreich, E. E.
Fecal Coliforms ................................. "
The Use and Abuse of Fecal Streptococci In
Water Quality Measurements ...................... 54
Sediment Sampler ................................ 158
Guarraia, L. J.
Fungi: A State-of-the-Art ...................... iub
Jeter, H. L.
Total Coliforms
Knittel, M. D.
Isolation and Identification of Klebsiella
Pneumoniae ......................................
Mackenthun, K. M.
Microbiological Research Needs
Manning, H.
AQCL Microbiology Section Report
202
-------
Resi, L. A.
Bacterial Survival sampler
Shedroff, D. I.
Enforcement Activities
Shimmin, K. G.
Regional Concern and Activities
Re: Standardization of Microbiology
Methodology
Fluorescent Antibody Screening .................. 124
Concentration of Sample
Special Problems
Spino, D. F.
Salmonella
112
Stang, W. J.
Collection and Handling of Bacteriological
Samples: State-of-the-Art 143
Swaby, L. G. . .
Office of Research Activities: Microbiological
Methods 12
203
-------
APPENDIX A
-------
AGENDA
SEMINAR ON STANDARDIZATION OF
MICROBIOLOGICAL METHODS
TIME: January 9-11, 1973
PLACE: Region IX, San Francisco, California
100 California Street
MODERATOR: Mr. S. Sid Verner
Technical Support Branch
Quality Assurance Division
Office of Research and Monitoring
Headquarters
Tuesday, January 9, 1973
Registration
Welcome - Statement of Problems
Mr. Paul DeFalco, Jr.
Regional Administrator, Region IX
STANDARDIZATION PROCESSES
Enforcement Activities
Mr. David L. Shedroff
Enforcement Division
Office of General Counsel
Research Activities
Dr. Louis G. Swaby
Measurements and Instrumentation Branch
Processes and Effects Division
Office of Research and Monitoring
Headquarters
Regional Activities
Ms. Kathleen Shimmin
Laboratory Support Branch, Region IX
MICROBIOLOGICAL PARAMETERS
Formal presentations will address these
topics:
1. General review of method(s) in
"Standard Methods"
A-l
-------
AGENDA (Cont inued)
Tuesday, January 9, 1973
2. Available methods as Candidate
Methods
3. Suggested recommendations
Discussion:
Leader:
Discussion:
Leader:
Discussion:
Leader:
Discussion:
Leader:
Discussion:
Leader:
Discussion:
Leader:
Discussion:
Leader:
Discussion:
Total Coliforms
Mr. Harold Jeter
National Training Center,
Cincinnati
Fecal Coliforms
Mr. Edwin E. Geldreich
Water Supply Research Laboratory
NERC-Cincinnati
Klebsiella
Dr. Martin D. Knittel
Pacific Northwest Water
Laboratory
Wednesday, January 10, 1973
Fecal Streptococci
Mr. Francis T. Brezenski
Technical Support Branch
Region II
Viruses
Dr. Gerald Berg
Virology, AWTRL
NERC-Cincinnati
Zoomicrobial Indicators
Dr. Shih Lu Chang
Water Supply Research Laboratory
NERC-Cincinnati
Pseudomonas aeruginosa
Dr. Victor J. Cabelli
Northwest Water Supply Research
Laboratory
Yeasts, Molds and Fungi
Dr. Leonard J. Guarraia
Office of Water Programs
Headquarters
A-2
-------
Thursday, January 11, 1973
Discussion:
Leader:
Discussion:
Leader:
PROCEDURES
Salmonella
Mr. Donald J. Spino
S.W.R. Bio-Engineering and
Science Branch, NERC-Cincinnati
Special Problems
Ms. Kathleen Shimmin
Laboratory Support Branch,
Region IX
Formal presentations will address these
topics:
1. Current problems and state-of-the-art
2. Need for standardization in sampling
and quality control
3. Benefits of standardization in sampling
and quality control
Discussion:
Leader:
Discussion:
Leader:
Sampling
(Collection, Storage,
Chain of custody)
Mr. William J. Stang
National Field Investigation
Center, Denver
Quality Control
(media, equipment, supplies,
performance, data management)
Mr. Robert H. Bordner
Analytical Quality Control Lab
NERC-Cincinnati
SUMMARIZATION
Research Needs
Mr. Kenneth M. Mackenthun
Direcotr, Technical Support Staff
Office of Air and Water Programs
Adjourn
A-3
-------
APPENDIX B
-------
List of Attendees
Name
James H. Adams
Elizabeth Anderson
Frederick Au
Robert Becker
G. Berg
Bob Bordner
Al Bourquin
Francis T. Brezenski
Victor Cabelli
Bobby J. Carrol
Shih Lu Chang
Howard Davis
William C. Dierkshield
Reto Engler
Edwin E. Geldreich
Ronald C. Gordon
Leonard Guarraia
Harold Jeter
Bernard A. Kenner
Martin Knittel
Robert Laidlaw
Office
Region V, INDO
OEGC, Washington, DC
NERC-Las Vegas, NV
Water Supply Research
Lab. Dauphin Island, AL
NERC-Cincinnati
AQCL, Cincinnati
GBERL, NERC-Corvallis
Gulf Breeze
Tech, Support, Region II
Edison, NS
NE Water Supply Resf
Lab. Naragansett, RI
Region IV S/A Athens r GA
RA Taft NERC-Cincinnati
4676 Columbia PKWY
NERL, Reg. I, Needham Ht
Mass .
Permits Branch, Reg IX
Off. Pesticide Prog.
Washington, DC
Water Supply Research
NERC-Cincinnati
AERL, College, AK
OWP, WQNPS, Washington, DC
Water Training, NERC-Cincinnati
NERC-Cincinnati
NERC-Corvallis
NFIC-Denver
B-l
-------
Name
Ronald F. Lewis
Ken Mackenthun
John P. Manhart
Robert Manning
Robert B. Medz
Don Nash
Stephen Poloncsik
Louis A. Resi
William G. Roessler
Robert F. Ruhl
Harold Scotten
David Shedroff
Kathleen G. Shimmin
Andy Sidio
Don Spino
William J. Stang
G. J. Vascoucelos
Sidney Verner
William H. Winders
S. C. Yin
Ho Lee Young
Office
NERC-Cincinnati, AWTRL
Region VIII, Denver S/A
NERC-Cincinnati
ORM, Washington, DC
Water Supply Research
Lab. Cincinnati
ORM, Reg. V, Chicago, IL
NFIC-Cinc innat i
"OPP, Criteria & Evaluation
Div., Washington, DC
ATB, Washington, DC
Region IX, San Francisco
NFIC-Cincinnat i
Lab. Support Branch,
Region IX, Alameda, CA
NFIC-Cincinnat i
NFIC-Denver
NWWSRL, NERC-Cincinnati
Gig Harbor, Wash. 98335
Office of Monitoring
Washington, DC
Region VI, Enforcement
Dallas, TX
Robert S. Kerr, Environ.,
Lab, Ada OK
Lab. Branch, Alameda,
Region IX
B-2
GOVERNMENT MINTING OFF!CE:1973 5U-155/304 1-3
-------
SELECTED WATER
RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
tNo.
05A
4. Title proceedings of the First Microbiology
Seminar on Standardization of Methods
•V-; 5. R -.rtD
.T •;••.•;::•;• -y 6.
• 8, Pe~fortait'< Orgar'ution
7. Author(s)
S. S. Verner, Editor-in-Chief
9. Organization . • _ - '-;',;.
Office of Research and Monitoring
Environmental Protection Agency
'''.'•-''•-1$. Type'. Repo* . nd
._." '_! Perioo Covered
11.
IS. Siipphnieatary Notes ,•.':..... • -' : ";-:
Activity performed under .Program Element 1H1327
EPA Report No. EPA--R4-73-022
. ,.( ., . ,
This doctiment contains the proceedings of the7|1 Seminar on standardiza-
tion of Microbiological Methods" held in January,.1973. The Seminar
brought together EPA microbiologists from all program elements,
offices and Regions to discuss problems of mutual •concern in
methodology.
.This Seminar was organized into four segments, vizy, standardization
processes as related to enforcement, research activities, and
regional problems; microbiological parameters Which consumed the
major portion of the meeting; analytical procedures as related to
sampling and quality control; and a final paper summarizing research
requirements prior to standardization. In additipn> the meeting was
structured to permit free discussion of the topic parameter after
each formal presentation and, where available, yerbatum or summary
discussions are presented following each respective paper in these
proceedings.
I7a. Descriptors
Microbiology in Aquatic Ecosystems, Aquatic Pathogens
17b. Identifiers
17c. COWRR Field & Gronp
]g. Availability
GPO
| 19. S .'ifityC :ss. j
, • (*i*-pOŁt)
1 201 Sfcutft-y Class. -
11, LCofi
i -i Pages >, *
Ł2.^ Price ^ -,
' V^ . i- ) ,
Send To:
WATER RESOURCES SCIENTIFIC INFORMATION CENTER
U.S. DEPARTMENT OF THE INTERIOR
WASHINGTON. D. C. ZO24O
Abstrsctor
10; 'REV JUNE
------- |