Current Practices In Water Microbiology: This Course Is Designed For Engineers, Chemists, Biologists, Bacteriologists, And Other Administrative Personnel Responsible For The Planning And Conduct Of Water Pollution Surveys


CURRENT PRACTICES IN WATER
MICROBIOLOGY
TRAINING MANUAL
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF WATER PROGRAMS

-------
EPA-4 30/ 1-75-002
April 1975
CURRENT PRACTICES IN WATER MICROBIOLOGY
This course is designed for engineers, chemists,
biologists, bacteriologists, and other administrative
personnel responsible for the-planning and conduct
of water pollution surveys.
fc.
.r
ENVIRONMENTAL PROTECTION AGENCY
Office of Water Program Operations
TRAINING PROGRAM

-------
CONTENTS
Title or Description	Outline No.
Bacteriological Indicators of Water Pollution	1
Bacteria and Their Survival in the Aquatic' Environment	2
Bacteriological Pathogens in the Aquatic Environment	3
Transmission of Viruses by Water	4
Filamentous Bacteria	5
Water Quality Criteria for Recreation and Aesthetics	6
Water Quality Surveys - Organizing the Stream Survey (Part	1) "
Role of Bacteriologist (Part 2)	7
Presentation and Interpretation of Bacteriological Data (Part	3) 7
Preparation of Survey Reports (Part 4)	7
Bacteriological Tests in EPA Pollution SurveiLLance Systems	8
Examination of Water for Coliform and Fecal Streptococcus Groups	9
Media and Solutions for Multiple Dilution Tube Methods	10
Use of Tables of Most Probable Numbers (Part 1)	11
Most Probable Number Theory (Part 2)	11
The Membrane Filter in Water Bacteriology	12
Membrane Filter Equipment and Its.Preparation for Lab Use	13
Membrane Filter Equipment for Field Use	14
Principles of Culture Media for use with Membrane Filters	15
Selection of Sample Filtration Volumes for Membrane Filter Methods	16
Detailed Membrane Filter Methods	17
Colony Counting on Membrane Filters	18
Verified Membrane Filter Tests	19
Determining Acceptability of Membrane Filter Methods in Water
Quality Tests	20
120.4. 75

-------
Contents
Title or Description	Outline No.
Collection and Handling of Samples for Bacteriological Examination	21
Recovery and Identification of Salmonella and Shigella From
Environmental Waters	2 2
Testing the Suitability of Distilled Water For The Bacteriology Laboratory	2 3
Identification of the Fecal Streptococci	24
Introduction to Statistics (Part 1) - (Part 2)	2 5
Biology and Ecology of Bivalves (Part 1)	26
Contaminants of Shellfish (Part 2)	2 6
The Development of Bacteriological Standards for Shellfish Growing Areas	27.
Shellfish Growing Area Surveys	2 8
Biological Aspects of Natural Self Purification	2 9
Biota of Wastewater Treatment Plants	3 0
Wastewater Treatment - The Result of Natural Phenomena (Part 1)	31
Aerobic Bacterial Systems for Industrial Wastes (Part 2)	31
Unit Operations in Waste Treatment	32
2

-------
BACTERIOLOGICAL INDICATORS OF WATER POLLUTION
, Part 1. General Concepts
I INTRODUCTION
A Bacterial Indication of Pollution
1	In the broadest sense, a bacterial
indicator, of pollution is any organism
which, by its presence, would demon-
strate that pollution has occurred, and
often suggest the source of the pollution.
2	In a more restrictive sense, bacterial
indicators of pollution are associated
primarily with demonstration of con-
tamination of water, originating from
excreta of warm-blooded animals
(including man, domestic and wild
animals, and birds).
B Implications of Pollution of Intestinal
Origin
1	Intestinal wastes from warm-blooded
animals regularly include a wide
variety of genera and species of
bacteria. Among these the coliform
group may be listed, and species of
the genera Streptococcus, Lactobacillus,
Staphylococcus, Proteus, Pseudomonas,
certain spore-forming bacteria, and
others.
2	In addition, many kinds of pathogenic
bacteria and other microorganisms
may be released in wastes on an inter-
mittent basis, varying with the geo-
graphic area, state of community
health, nature and degree of waste
treatment, and other factors. ¦ These
may include the following:
a Bacteria: Species of Salmonella,
Shigella, Leptospira, Brucella,
Mycobacterium, and Vibrio comma.
b Viruses: A wide variety, including
that of infectious hepatitis, Polio -
viruses, Coxsackie virus, ECHO
viruses (enteric cytopathogenic
human orphan -- "viruses in search
of a disease"), and unspecified
viruses postulated to account for
outbreaks of diarrheal and upper
respiratory diseases of unknown
etiology, apparently infective by
the water-borne route.
c Protozoa: Endamoeba histolytica
3	As routinely practiced, bacterial
evidence of water pollution is a test
for the presence and numbers of
bacteria in wastes which, by their
presence, indicate that intestinal
pollution has occurred. In this con-
text, indicator groups discussed in
subsequent parts of this outline are .
as follows:
a Coliform group and certain sub-
groupings
b Fecal streptococci and certain
sub groupings
c Miscellaneous indicators of pollution
4	Evidence of water contamination by
intestinal wastes of warm-blooded
animals is regarded as evidence of
health hazard in the water being tested.
II PROPERTIES OF AN IDEAL INDICATOR
OF POLLUTION
A An "ideal" bacterial indicator of pollution
should:
1 Be applicable in all types of water
W. BA. 48g. 2. 75

-------
Bacteriological Indicators of Water Pollution
2 Always be present in water when
pathogenic bacterial constituents of
fecal contamination are present.
Ramifications of this include --
a Its density should have some direct
relationship to the degree of fecal
pollution.
b It should have greater survival time
in water than enteric pathogens,
throughout its course of natural
disappearance from the water body,
c It should disappear rapidly from
water following the disappearance
of pathogens, either through natural
or man-made processes,
d It always should be absent in a
bacteriologically safe water.
3 Tests for compliance with established
standards in cases involving the pro-
tection or prosecution of municipalities,
industries, etc.
B Treatment Plant Process Control
1	Water treatment plants
2	Wastewater treatment plants
C Water Quality and Pollutant Sou rce Monitoring
1	Determination of intestinal pollution
in surface water to determine'type and
extent of treatment required for com-
pliance with standards
2	Tracing sources of pollution
3	Lend itself to routine quantitative
testing procedures without interference
or confusion of results due to extra-
neous bacteria
4	Be harmless to rrtan and other animals
B In all probability, an "ideal" bacterial
indicator does not exist. The discussion
of bacterial indicators of pollution in the
following parts of this outline include
consideration of the merits and limitations
of each group, with their applications in
evaluating bacterial quality of water.
IE APPLICATIONS OF TESTS FOR
POLLUTION INDICATORS
A Tests for Compliance with Bacterial
Water Quality Standards
1	Potability tests on drinking water to
meet Interstate Quarantine or other
standards of regulatory agencies.
2	Determination of bacterial quality of
environmental water for which quality
standards may exist, such as shellfish
waters, recreational waters, water
resources for municipal or other
supplies.
3 Determination of effects on bacterial
flora, due to addition of organic or
other wastes
' D Special Studies, such as
1	Tracing sources of intestinal pathogens
in epidemiological investigations
2	Investigations of problems due to the
Sphaerotilus group
3	Investigations of bacterial interference
to certain industrial processes, with
respect to such organisms as Pseudo-
monas, A chromobacter, or others
IV SANITARY SURVEY
The laboratory bacteriologist is not alone in
evaluation of indication of water pollution of
intestinal origin. On-site study (Sanitary
Survey) of the aquatic environment and
adjacent areas, by a qualified person, is a
necessary collateral study with the laboratory
work and frequently will reveal information
regarding potential bacteriological hazard
which may or may not be demonstrated
through laboratory findings from a single
sample or short series of samples.
1-2

-------
Bacteriological Indicators of Water Pollution
Part 2. The Coliform Group and Its Constituents
I ORIGINS AND DEFINITION
A Background
1	In 1885, Escherich, a pioneer bacteri-
ologist, recovered certain bacteria from
human feces, which he found in such
numbers and consistency as to lead him
to term these organisms "the charac-
teristic organism of human feces. "
He named these organisms Bacterium
coli-commune andJ3. lactis aerogenes.
In 1895, another bacteriologist,
Migula, renamed B. coli commune as
Escherichia coli, which today is the
official name for the type species.
2	Later work has substantiated much of
the original concept of Escherich, but
has shown that the above species are
in fact a heterogeneous complex of
bacterial species and species variants.
a This heterogeneous group occurs not
only in human feces but representatives
also are to be found in many environ-
mental media, including sewage,
surface freshwaters of all categories,
in and on soils, vegetation, etc.
b The group may be subdivided into
various categories on the basis of
numerous biochemical and other
differential tests that may be applied.
B Composition of the Coliform Group
2 The term "coliforms" or "coliform
group" is an inclusive one, including
the following bacteria which may
meet the definition above:
a Escherichia coli, E. aurescens,
E. freundii, E. intermedia
b Aerobacter aerogenes, A. cloacae
c Biochemical intermediates between
the genera Escherichia and Aero-
bacter
5 The above terminology is in accordance
with the current editions of Standard
Methods and Bergey's Manual of Deter-
minative Bacteriology and will be
consistent throughout this manual until
these sources are modified.
3 There is no provision in the definition
of coliform bacteria for "atypical" or
"aberrant" coliform strains,
a An individual strain of any of the above
species may fail to meet one of the
criteria of the coliform group,
b Such an organism, by definition, is
not a member of the coliform group,
even though a taxonomic bacteriologist
may be perfectly correct in classifying
the strain in one of the above species.
II SUBDIVISION OF COLIFORMS INTO
"FECAL" AND "NONFECAL"
CATEGORIES
1 Current definition
As defined in "Standard Methods for the
Examination of Water and Wastewater"
(13th ed): "The coliform group includes
all of the aerobic and facultative
anaerobic. Gram-negative, nonspore-
forming rod-shaped bacteria which
ferment lactose with gas formation
within 48 hours at 35°C. "
A Need
Single-test differentiations between
coliforms of "fecal" origin and those of
"nonfecal" origin are based on the
assumption that typical E. coli and
closely related strains are of fecal
origin while A. aerogenes and its close
relatives are not of direct,fecal origin.
(The latter assumption is not fully borne
out by investigations at this Center.
See Table 1, IMViC Type --++). A
number of single differential tests have
been proposed to differentiate between
"fecal" and "nonfecal" coliforms.
1-3

-------
Bacteriological Indicators of Water Pollution
Without discussion of their relative merits,
several may be cited here:
B Types of Single-Test Differentiation
1	Determination of gas ratio
Fermentation of glucose by E. coli
results in gas production, with
hydrogen and carbon dioxide being
produced in equal amounts.
Fermentation of glucose by A.
aerogenes results in generation of
twice as much carbon dioxide as
hydrogen.
Further studies suggested absolute
correlation between W^lCO ratios
and the terminal pH resulting from
glucose fermentation. This led to the
substitution of the methyl red test.
2	Methyl red test
Glucose fermentation by E. coli
typically results in a culture medium
having terminal pH in the range 4.2 -
4. 6 (red color a positive test with the
addition of methyl red indicator).
A. aerogenes typically results in a
culture medium having pH 5.6 or
greater (yellow color, a negative test).
3	Indole
When tryptophane, an amino acid, is
incorporated in a nutrient broth,
typical E_. coli strains are capable of
producing indole (positive test) among
the end products, whereas A. aerogenes
does not (negative test).
In reviewing technical literature, the
worker should be alert to the method
used to detect indole formation, as the
results may be greatly influenced by
the analytical procedure.
4	Voges-Proskauer test (acetylmethyl
carbinoltest)
The test is for detection of acetylmethyl
carbinol, a derivative of 2,3, butylene-
glycol, as a result of glucose
fermentation in the presence of%
peptone. A. aerogenes produces
this end product (positive test)
whereas E. coli gives a negative test;
a Experience with coliform'cultures
giving a positive test has shown a
loss of this ability with storage on
laboratory media for. 6 months to
2cyears, in 20 - 25% of cultures
(-103 out of 458 cultures).
b Some workers consider that all
coliform bacteria produce acetyl-
methyl carbinol in glucose metab-
olism. These workers-regard
acetylmethyl carbinol,-negative
cultures as those which have
enzyme systems capable of further
degradation of acetylmethyl
carbinol to other end products
which do not give a positive test
with the analytical procedure.
Cultures giving a positive test for
acetylmethyl carbinol lack this
enzyme system.
c This reasoning leads to a hypothesis
(not experimentally proven) that the
change of reaction noted in certain
cultures in 4.a above is due to the
activation of a latent enzyme system.
5	Citrate utilization
Cultures of _E. coli are unable to use
the carbon of citrates (negative test)
in their metabolism, whereas cultures
of_A. aerogenes are capable of using
the carbon of citrates in their metab-
olism (positive test).
Some workers (using Simmons Citrate
Agar) incorporate a pH indicator
(brom thymol blue) in the culture
medium in order to demonstrate the
typical alkaline reaction (pH 8:4 - 9.0)
resulting with citrate utilization.
6	Elevated temperature (Eijkrhan) test
a The test is based on evidence that
E. coli and other coliforms of fecal
1-4

-------
Bacteriological'Indicators of Water Pollution
origin are capable of growing and
fermenting carbohydrates (glucose
or lactose) at temperatures signif-
icantly higher than the body tem-
perature of warm-blooded animals.
Organisms not associated with direct
fecal origin would give a negative
test result, through their inability
to grow at the elevated temperature.
b While many media and techniques
have been proposed, EC Broth, a
medium developed by Perry and
Hajna, used as a confirmatory
medium for 24 hours at 44.5 ±
0,2 oc are the current standard
medium and method.
While the "EC" terminology of the
medium suggests "E. coli" the
worker should not regard-this as a
specific procedure for isolation of
_E. coli.
c A similar medium. Boric Acid
Lactose Broth, has developed
by Levine and his associates. This
medium gives results virtually
identical with those obtained from
EC Broth, but requires 48 hours of
incubation.
d Elevated temperature tests require
incubation in a water bath. Standard
Methods 13th Ed. requires this
temperature to be 44. 5 + 0.2°C.
Various workers have urged use of
temperatures ranging between
43. 0OC and 46. 0o c. Most of these
recommendations have provided a
tolerance of + 0. 5° C from the rec-
ommended levels. However, some
workers, notably in the Shellfish
Program of the Public Health Service,
stipulate a temperature of 44. 5 +
0.2°C. This requires use of a water
bath with forced circulation to main-
tain this close tolerance. This
tolerance range was instituted
in the 13th Edition of Standard Methods
and the laboratory worker; should
conform to these new limits.
e The reliability of elevated temper-
ature tests is influenced by the
time required for the newly-
inoculated cultures to reach1 the
designated incubation temperature.
Critical workers insist on place-
ment of the cultures in the water-
bath within 30 minutes, at most,
after inoculation.
7 Other tests
Numerous other tests for differentiation
between coliforms of fecal vs. nonfecal
origin have been proposed. Current
studies suggest little promise for the
following tests in this application:
uric a.cid test, cellobiose fermentation,
gelatin liquefaction, production of
hydrogen sulfide, sucrose fermentation,
and others.
C IMViC Classification
1 In 193 8, Parr reported on a review of
a literature survey on biochemical tests
used to differentiate between coliforms
of fecal vs. nonfecal origin. A summary
follows:
No. of times
Test	used for dif-
	ferentiation
Voges-Proskauer	2 2
reaction
Methyl red test	2 0
Citrate utilization	20
Indole test	15
Uric acid test	6
Cellobiose fermentation	¦ 4
Gelatin liquefaction	3
Eijkman test	2
Hydrogen sulfide	1
production
Sucrose fermentation	1
a-Methyl-d-glucoside	1
fermentation
1-5

-------
Bacteriological Indicators of Water Pollution
2	Based on this summary and on his own
studies, Parr recommended utilization
of a combination of. tests, the indole,
methyl red, Voges-Proskauer, and the
citrate utilization tests for this differ-
entiation. This series of reactions is
designated by the mnemonic "iMViC".
Using this scheme, any coliform culture
can be described by an "IMViC Code"
according to the reactions for each
culture. Thus, a typical c\ilture of
_E. coli would have a code	and a
typical A. aerogenes culture would
have a code —H-.
3	Groupings of coliforms into fecal,
non-fecal, and intermediate groups,
as shown in "Standard Methods for the
Examination of Water and Wastewater"
are shown at the bottom of this page.
D Need for Study of Multiple Cultures
All the systems used for differentiation
between coliforms of fecal vs. those of
nonfecal origin require isolation and study
of numerous pure cultures. Many workers
prefer to study at least 100 cultures from
any environmental source before attempting
to categorize the probable source of the
coliforms.
Ill NATURAL DISTRIBUTION OF COLIFORM
BACTERIA .
A Sources of Background Information
Details of the voluminous background of
technical information on coliform bacteria
recovered from one or more environ-
mental media are beyond the scope of
this discussion. References of this
outline are suggested routes of entry
for workers seeking to explore this
topic.
B Studies'on Coliform Distribution
1 Since 1960 numerous workers
have engaged in a' continuing study of
the natural distribution of coliform
bacteria and an evaluation of pro-
cedures for differentiation between
coliforms of fecal vs. probable non-
fecal origin. Results of this work
have special significance because:
a Rigid uniformity of laboratory
methods have been applied through-
out the series of studies
b Studies are based on massive
numbers of cultures, far beyond
any similar studies heretofore
reported
Groupings of Coliforms into Fecal, Nonfecal and Intermediate Groups
Organism
Indole
Methyl
red
\ oges Citrate
Proskauer
E. coli, Variety I
Variety II
+
+
+

E. freundii
(Intermediates)



Variety I

+
+
Variety, II
+
+
+
A. aerogenes



Variety I
-
-
+ +
Variety II
+
-
+ +
1-6

-------
Bacteriological Indicators of Water Pollution
c A wider variety of environmental and
biological sources is being studied
than in any previous series of reports.
d All studies are based on freshly
recovered pure culture isolates
from the designated sources.
e All studies are based on cultures
recovered from the widest feasible
geographic range, collected at all
seasons of the year. It is believed
that no more representative series
of studies has been made or is in
progress.
2 Distribution of coliform types
Table 1 shows the consolidated results
of coliform distributions from various
biological and environmental sources.
a The results of these studies show a
high order of correlation between
known or probable fecal origin and
the typical _E. coli IMViC code
(++t-). On the other hand,
human feces also includes
numbers of_A. aerogenes and other
IMViC types, which some regard as
"nonfecal" segments of the coliform
group. (Figure 1)
b The majority of coliforms attributable
to excretal origin tend to be limited
to a relatively small number of the
possible IMViC codes; on the other
hand, coliform bacteria recovered
from undisturbed soil, vegetation,
and insect life represent a wider
range of IMViC codes than fecal
sources, without clear dominance of
any one type. (Figure 2)
c The most prominant IMViC code
from nonfecal sources is the inter-
mediate type,	which accounts
for almost half the coliform cultures
recovered from soils, and a high
percentage of those recovered from
vegetation and from insects. It
would appear that if any coliform
segment could be termed a "soil
type" it would be IMViC code -+-+.
d It should not be surprising that
cultures of typical E. coli are
recovered in relatively smaller
numbers from sources judged,
on the basis of sanitary survey,
to be unpolluted. There is no
known way to exclude the influence
of limited fecal pollution from small
animals and birds in such environ-
ments.
e The distribution of coliform types
from human sources should be
regarded as a representative value
for large numbers of sources.
Investigations have shown that there
can be large differences in the
distribution of IMViC types from
person to person, or even from an
individual.
3 Differentiation between coliforms of
fecal vs. nonfecal origin
Table 2 is a summary of findings
based on a number of different criteria
for differentiating between coliforms
of fecal origin and those from other
sources.
a IMViC type ++-- is a measurement
of E_. coli, Variety I, and appears
to give reasonably good correlation
between known or highly probable
fecal origin and doubtful fecal origin,
b The combination of IMViC types,
+H—, +	, and - + gives
improved identification of probable
fecal origin, and appears also to
exclude most of the coliforms not
found in excreta of warm-blooded
animals in large numbers.
c While the indole, methyl red,
Voges Proskauer, and citrate
utilization tests, each used alone,
appear to give useful answers when
applied only to samples of known
pollution from fecal sources, the
interpretation is not as clear when
applied to coliforms from sources
believed to be remote from direct
fecal pollution.
1-7

-------
Table 1. COLIFORM DISTRIBUTION BY IMViC TYPES AND ELEVATED TEMPERATURE
TEST FROM ENVIRONMENTAL AND BIOLOGICAL SOURCES
IMViC
type
Vegetation
Insects
Soil
Fecal sources
Poultry
Undisturbed
Polluted
Hun-
lan
Livestock
i\To.
strains
% of
total
No.
strains
i
% of
total
No.
strains
% of
total
No.
strains
% of
total
No.
strains
% of
total
No.
strains
% of
total
No.
strains
1 % of
total
++--
128
10. 6
134
12. 4
131
5. 6
' 536
80. 6
3932
87. 2
2237
95. 6
185 7
i
97. 9
- -++
237
19. 7
113
10. 4
443
18. 8
13
2. 0
245
5. 4

<0. 1
1
0. 1

23
1. 9
0
<0. 1
78
3. 3
1
0. 2
99
¦ 2. 2
14
0. 6
20
1.1
+++-
2
0. 2
0
<0. 1
7
0. 3
0
<0. 1
106
2. 4
59
• 2.5
0
<0. 1
-+- +
168
14. 0
332
30. 6
1131
48. 1 *
87
13. 0
50
1. 1
1
<0. 1
5
0. 3
++- +
116
9. 6
118
10. 9
87
3. 7
22
3. 3
35
0. 8
27
...
11
0. 6
-+++
32
2. 7
28
2. 6
181
7. 7
5
0. 7
21
0. 5
0
<0. 1
0
<0. 1
++++
291
24. 2
254
23. 4
159
6. 8
0
<0. 1
6
0. 1
0
<0. 1
o
<0. 1
+-++
88
7. 3
46
4. 2
67
2. 9
0
<0. 1
14
0. 2
0
<0. 1
0
<0.1
+
87
7. 2
42
3. 9
4
0. 2
1
0. 2
2
<0. 1
0
<0. 1
0
<0. 1
! -++"
5
0. 4
0
<0. 1
1
<0. 1
0
<0. 1
0
<0. 1
0
<0. 1
0
<0. 1

19
1. 6
0
<0. 1
53
2. 3
0
<0. 1
0
<0. 1
0
<0. 1
o'
<0. 1
+-+-
2
0. 2
0
<0. ]
6
0. 3
0
<0. 1
0
<0. 1
0
<0. 1
0"
<0. 1
+-- +
5
0. 4
8
0. 7
0
<0. 1
0
<0. 1
0
<0. 1
»
-------
Bacteriological Indicators of Water Pollution
HUMAN
EC ® 96.4
BALB © 94.7
SUMMARY
Type
Percent positive
* + —
91.8
-+—
J.5
f
O.J
-- + 4
2.8
EC ©
96.3
BALB
95.3
FIGURE 1
COLIFORMS
67 Soil Samples
(Gelilreu-h. el. al.)

EH3
tZHD
Undisturbed
Soil
Polluted
Soil
1-9
-------
Bacteriological Indicators of Water PoLlunon
Table 2. COMPARISON OF COLIFORM STRAINS ISOLATED FROM WARM-BLOODED ANIMA L
FECES, FROM UNPOLLUTED SOILS AND POLLUTED SOILS WITH USE OF THE
IMYiC REACTIONS AND THE ELEVATED TEMPERATURE TEST IN EC MEDIUM
AT 44.5° C (+0.5°) (12th ed. 1965; Standard Methods for the Examination of Water
and Wastewater)
Test
War m-blooded
animal feces
Soil.
Unpolluted
Soil.
Polluted
Vcge -
tation
Insects
- - - -
91. 8'-*
5. 6 ro
80. G'-'o
10. Gir0
12. 4iro
)
— - - and
93. 3ro
8. 9'rc
80. ic
12. 5^
13. 2%
Indole positive
94. 0'ro
19. 4%
82. 7%
5 2. 5%
52 .'4^1
Methyl red posit-ive
96. 9tr0
75. 6tro
97. 9^
63. 6^0
79. 9^0
Yoees-Proskauer positive
5. Ko
40. 7r0
97. 3°"o
56. 3%
4 0. 6%
Citrate utilizers
3. 6^0
88. 2%
19. 2ro
85. l^o
86. 7ro
Elevated temperature (EC)
positive
96. 4
-------
Bacteriological Indicators of Water Pollution
e Coliforms are more persistent in
the aquatic environment than are
pathogenic bacteria of intestinal
origin.
f Coliforms are generally harmless
to humans and can be determined
quantitatively by routine laboratory
procedures.
2 Limitations
a Some of the constituents of the
coliform group have a wide environ-
mental distribution in addition to
their occurrence in the intestines
of warm-blooded animals.
b Some strains of the coliform group
may multiply in certain polluted
waters ("aftergrowth"), of high
nutritive values thereby adding to
the difficulty of evaluating a pollution
situation in the aquatic environment.
Members of the A. aerogenes section
of the coliform are commonly
involved in this kind of problem.
c Because of occasional aftergrowth
problems, the age of the pollution
may be difficult to evaluate under
some circumstances.
d Tests for coliforms are subject to
interferences due to other kinds of
bacteria. False negative results
sometimes occur when species of
Pseudomonas are present. False
positive results sometimes occur
when two or more kinds of non-
conforms produce gas from lactose,
when neither can do so alone
(synergism).
B The Fecal Coliform Component of the
Coliform Group (as determined by elevated
temperature test)
1 Merits
a The majority (over 95% of the coli-
form bacteria from intestines of
warm-blooded animals grow at the
elevated temperature.
b These'organisms are of relatively
infrequent occurrence except in
association with fecal pollution.
c Survival of the fecal coliform group
is shorter in environmental waters
than for the coliform group as
whole. It follows, then, that high
densities of fecal coliforms is
indicative of relatively recent
pollution.
d Fecal coliforms generally do not
multiply outside the intestines of
warm-blooded animals. In certain
high-carbohydrate wastes, such as
from the sugar beet refineries,
exceptions have befen noted.
e In some wastes, notably those from
pulp and paper mills, "Klebsiella has
been found in large numbers
utilizing the elevated temperature
test. There has been much contro-
versy about whether the occurrence
of Klebsiella is due to aftergrowth
due to soluble carbohydrates in such
wastes. The significance of
Klebsiella as an indicator of direct
discharge of intestinal wastes thus
is under challenge. The issue is
still further complicated by questions
over whether Klebsiella is in and of
itself a pathogenic organism or is
potentially pathogenic. This is a
serious problem which is the subject
of current intensive research by this
Agency.
2 Limitations
a Feces from warm-blooded animals
include some (though proportionately
low) numbers of coliforms which do
not yield a-positive fecal coliform
test when the elevated temperature
test is used as the criterion of
differentiation. These organisms
are E. coli varieties by present
taxonomic classification.
b There is at present no established
and consistent correlation between
1-11
-------
Bacteriological Indicators of Water. Pollution
ratios of total coliforms/fecal
coliforms in interpreting sanitary
quality of environmental waters.
In domestic sewage, the fecal
coliform density commonly is
greater than 90% of the total
coliform density. In environmental
waters relatively free from recent
pollution, the fecal coliform density
may range from 10-30% of the total
coliforms. There are, however,
too many variables relating to
water-borne wastes and surface
water runoff to permit sweeping
generalization on the numerical
relationships between fecal- and
total coliforms.
c Studies have been made
regarding the survival of fecal
coliforms in polluted waters
compared with that of enteric
pathogenic bacteria. In recent
pollution studies, species of
Salmonella have been found in the
presence of 220 fecal coliforms per
100 ml (Spino), and 110 fecal
coliforms per 100 ml (Brezenski,
Raritan Bay Project).
3 The issue of the Klebsiella problem
described in an earlier paragraph
may ultimately be resolved as a
merit or as a limitation of the value
of the fecal coliform test.
V APPLICATIONS OF COLIFORM TESTS
A Current Status in Official Tests
1 The coliform group is designated, in
"Standard Methods for the Examination
of Water and Wastewater" (13th ed.,
1971), through the Completed Test
MPN procedure as the official test
for bacteriological potability of water.
The Confirmed Test MPN procedure
is accepted where it has been demon-
strated, through comparative tests,
to yield results equivalent to the
Completed Test. The membrane filter
method also is accepted for examination
of waters subject to interstate regulation.
2 The 12th edition of Standard Methods
introduced a standard test for fecal
coliform bacteria. It is emphasized
that this is to be used in pollution
studies, and does not apply to the
evaluation of water for potability.
This procedure has been continued in
the 13th Edition.
B Applications
1 Tests for the coliform group as a
whole are used in official tests to
comply with interstate drinking water
standards, state standards for shell-
fish waters, and1 in most, if not all,
cases where bacterial standards of
water quality have been established
for such use as in recreational or
bathing waters, water supplies, or
industrial supplies. Laboratory
personnel should be aware of possible
implementation of the fecal coliform
group as the official test for recreational
and bathing waters.
2 The fecal coliform test has application
in water quality surveys, as an adjunct
to determination of total coliform
density. The fecal coliform test is
being used increasingly in all water
quality surveys.
3 It is emphasized that no responsible
worker advocates substitution of a
fecal coliform test for total coliforms
in evaluating drinking water quality.
1-12
-------
Bacteriological Indicators of Water Pollution
Part 3. The Fecal
I INTRODUCTION
Investigations regarding streptococci
progressed from the streptococci of medical
concern to those which were distributed in
differing environmental conditions which,
again, related to the welfare of man. The
streptococci were originally reported by Laws
and Andrews (1894), and Houston <1899, 1900)
considered those streptococci, which we now
call "fecal streptococci," as . . . "indicative
of dangerous pollution, since they are readily
demonstrable in waters recently.polluted and
seemingly altogether absent from waters above
suspicion of contamination.
From their discovery to the present time the
fecal streptococci appear characteristic of
fecal pollution', being consistently present in
both the feces.of all warm-blooded animals
and in the environment associated with animal
discharges. As early as 1910 fecal strepto-
cocci were proposed as indicators to the
Metropolitan Water Board of London.
However, little progress resulted in the
United States until improved methods of
detection and enumeration appeared after
World War II.
Renewed interest in the group as indicators
began with the introduction of azide dextrose
broth in 1950, (Mallmann & Seligmann, 1950).
The method which is in the current edition
of Standard Methods appeared soon after.
(Litsky, et al. 1955).
With the advent of improved methods for
detection and enumeration of fecal strep-
tococci, significant body of technical
literature has appeared.
This outline will consider the findings of
various investigators regarding the fecal
streptococci and the significance of discharges
of these organisms into the aquatic environment.
II FECAL MATERIALS
A Definition
reptococci
The terms "enterococci, "."fecal
streptococci, " "Group D streptococci, "
"Streptococcus fecalis, " and even
"streptococci1 have been used in a loose
and interchangeable manner to indicate
the streptococci present in the enteric
tract of warm-blooded animals or of the
fresh fecal material excreted therefrom.
Enterococci are characterized by specific
taxonomic biochemistry. Serological
procedures differentiate the Group D
streptococci from the various groups.
Although they overlap, the three groups,
fecal streptococcus, enterococcus, and
Group D streptococcus, are not synonymous.
Because our emphasis is on indicators of
unsanitary origin, fecal streptococcus is
the more appropriate term and will include
the enterococcus as well as other groups.
A rigid definition of the fecal streptococcus
group is not possible with our present
knowledge. The British Ministry of Health
(1956) defines the organisms as "Gram-
positive" cocci, generally occurring in
pairs or short chains, growing in the
presence of bile salt, usually capable of
development at 45° C, producing acid but
not gas in mannitol and lactose, failing to
attack raffinose, failing to reduce nitrate
to nitrite, producing acid in litmus milk
and precipitating the casein in the form of
a loose, but solid curd, and exhibiting a
greater resistance to heat, to alkaline
conditions and to high concentrations of
salt than most vegetative bacteria. "
However, it is pointed out that "streptococci
departing in one or more particulars from
the type species cannot be disregarded
in water. "
For the proposes of this outline, and in line
with the consensus of most water micro-
biologists in this country, the definition
of the fecal streptococci is:
. . . "The group composed of Group D
species consistently present in
significant numbers in-fresh fecal
excreta of warm-blooded animals,
which includes all of the enterococcus
group in addition to other groups of
streptococci."
1-13
-------
Bacteriological Indicators of Water Pollution
B Species Isolated
1 Findings
a Human feces
Examination of human fecal specimens
yields a high percentage of the
enterococcus group and usually
demonstration of the S. salivarius
which is generally considered a
member of the human throat flora
and to be surviving in human fecal
materials rather than actively
multiplying in the enteric tract.
Also present would be a s^all
percentage of variants or biotypes
of the enterococcus group.
b Nonhuman Feces
1) Fecal material which are from
nonhuman or not from fowl will
yield high percentages of the
S. bovis and/or S. equinus
organisms with a concomitantly
reduced percentage of the
enterococcus group.
2) Fowl excreta
Excrement from fowl characteris-
tically yields a large percentage
of enterococcal biotypes as well
as a significant percentage of
enterococcus group.
2 Significance
Species associations with particular
animal hosts is an established fact and
leads to the important laboratory
technique of partition counting of colonies
from the membrane filter or agar
pour plates in order to establish or
confirm the source of excretal
pollution in certain aquatic investi-
gations.
It is important to realize that a suitable
medium is necessary in order to
allow all of the streptococci which
we consider to be fecal streptococci
to grow in order to give credence to
the derived opinions. Use of liquid
growth media into which direct
inoculations from the sample are
made have not proven to be successful
for partition counting due to the differing
growth rates of the various species of
streptococci altering the original
percentage relationships. Due to the
liriiited survival capabilities of some
of the fecal streptococci it is necessary
to sample fresh fecal'material or water
samples in close proximity to the
pollution source especially when
multiple sources are contributing to a
reach of water. Also the pH range
must be within the range of 4.0-9.0.
Ill FECAL STREPTOCOCCI IN THE
AQUATIC ENVIRONMENT
A General
From the foregoing it is appears that
the preponderant human fecal streptococci
are composed of the enterococcus group
and, as this is the case, several media are
presently available which will detect only
the enterococcal group will be suitable
for use with aquatic samples which are
known to be contaminated or potentially
contaminated with purely domestic
(human) wastes. On the other hand,
when it is known or suspected that other-
than-human wastes have potential egress
to the aquatic environment under investi-
gation, it is necessary to utilize those
media which are capable of quantitating
the whole of the fecal streptococci group.
B Stormwaters and Combined Sewers
1 General
Storm sewers are a series of pipes
and conduits which receive surface
runoffs from the action of rainstorms
and do not include sewage which are
borne by a system of sanitary sewers.
Combined sewers receive both the storm
runoff and the water-borne
wastes of the sanitary system.
1-14
-------
Bacteriological Indicators of Water Pollution
Both storm water and combined
sewer flows have been found to
usually contain large quantities of
fecal streptococci in numbers which
generally are larger than those of
the fecal coliform indicator organisms.
2 Bacteriological Findings
Table 1 represents, in a modified form,
some of the findings of Geldreich and
Kenner (1969) with respect to the
densities of fecal streptococci when
considering Domestic sewage in contrast
to Stormwaters:
Table 1
DISTRIBUTION OF FECAL STREPTOCOCCI
IN DOMESTIC SEWAGES AND STORMVVATER
RUNOFFS
Fecal Streptococci
per 100 ml Ratio
Water Source median values FC/FS
Domestic Sewage
Preston, ID 64,000 5.3
Fargo, ND 290, 000 4. 5
Moorehead, MN 330, 000 4.9
Cincinnati, OH 2,470,000 4.4
Lawrence, MA 4, 500, 000 4. 0
Monroe, MI 700,000 27.9
Denver, CO 2, 900,000 16.9
Stormwater
Business District 51,000 0.26
Residential 150, 000 0. 04
Rural 58, 000 0. 05
The Ratio FC/FS is that of the
Fecal coliform and Fecal streptococci
and it will be noted that in each case,
when considering the Domestic
Sewage, it is 4. 0 or greater while
it is less than 0. 7 for stormwaters.
The use of this ratio is useful to
identify the source of pollution as
Table 2. ESTIMATED PER CAPITA CONTRIBUTION OF INDICATOR MICROORGANISMS.
FROM SOME ANIMALS*
Average indicator Average contribution
density per gram per capita per 24 hr
of feces
Animals
Avg wt of
Feces/24 hr,
wet wt, g
F ecal
coliform,
million
Fecal
streptococci,
million
Fecal
coliform,
million
Fecal
streptococci,
million
Ratio
FC/FS
Man
150
13. 0
3.0
2, 000
450
4.4
Duck
336
CO
CO
o
54. 0
11, 000
18, 000
0.6
Sheep
1, 130
16. 0
38.0
18, 000
43,000
0. 4
Chicken
182
1. 3
3.4
240
620
0.4
Cow
23,600
0. 23
1.3
5, 400
CO
O
O
o
0.2
Turkey
448
0. 29
2.8
130
1, 300
0. 1
Pig
2, 700
3. 3
84.0
8, 900
230, 000
o
o
^Publication WP-20-3, P. 102
1- 15
-------
Bacteriological Indicators of Water Pollution
being human or nonhuman warm-
blooded animal polluted. When the ratio
is greater than 4. 0 it is considered to be
human waste contaminated while a ratio
of less than 0. 7 is considered to be
nonhuman. It is evident that the storm-
waters have been primarily polluted by
excreta of rats and other rodents and
possibly domestic and/or farm animals.
Species differences are the main cause
of different fecal coliform-fecal
streptococci ratios. Table 2 compares
fecal streptococcus and fecal coliform
counts for different species. Even
though individuals vary widely, masses
of individuals in a species have charac-
teristic proportion of indicators.
C Surface Waters
In general, the occurrence of fecal
streptococci indicates fecal pollution and
its absence indicates that little or no
warm-blooded fecal contribution. In
studies of remote surface waters the fecal
streptococci are infrequently isolated and
occurrences of small numbers can be
attributed to wild life and/or snow melts
and resultant drainage flows.
Various examples of fecal streptococcal
occurrences are shown in Table 3 in
relation to surface waters of widely varying
quality. (Geldreich and Kenner 1969).
IV FECAL STREPTOCOCCI: ADVANTAGES
AND LIMITATIONS
A General
Serious studies concerning the streptococci
were instituted when it became apparent
that they were the agents responsible or
suspected for a wide variety of human
diseases. Natural priority then focused
itself to the taxonomy of these organisms
and this study is still causing consternation
as more and more microbiological techniques
have been brought to bear on these questions.
The sanitary microbiologist is concerned
with those streptococci which inhabit the
enteric tract of warm-blooded animals,
their detection, and utilization in develop-
ing a criterium for water quality standards.
Table 3
INDICATOR ORGANISMS IN SURFACE
WATERS
Densities / 100 ml
Fecal Fecal
Water Source coliform streptococci
Prairie Watersheds
Cherry Creek, WY 9 0 83
'Saline River, KS 95 "180
Cub River, ID 110 160
Clear Creek, CO 170 110
Recreational Waters
Lake Mead 2 444
Lake Moovalaya 9 170
Colorado River 4 256
Whitman River 32 88
Merrimack River 100 96
Public Water Intakes
Missouri River (1959)
Mile 470.5 11, 500 39, 500
Mile 434.5 22, 000 79, 000
Mile 408.8 14,000 59, 000
Kabler (1962) discussed the slow acceptance
of the fecal streptococci as indicators of
pollution resulting from:
1 Multiplicity and difficulty of laboratory
procedures
2 Poor agreement between methods of
quantitative enumeration
3 Lack of systematic studies of ... .
a sources
b survival, and
c interpretations, and
4 Undue"attention to the S. faecalis group.
1-16
-------
Bacteriological Indicators of Water Pollution
Increased attention to the fecal streptococci,
especially during the last decade, have
clarified many of the earlier cloudy issues
and have elevated the stature of these
organisms as indicators of pollution.
Court precedents establishing legal status
and recommendations of various technical
advisory boards have placed the fecal
coliform group in a position of primacy
in many water quality applications. The
fecal streptococci have evolved from a
position of a theoretically useful indicator
to one which was ancillary to the coliforms
to one which was useful when discrepancies
or questions evolved as to the validity of
the coliform data to one where an equality
status was achieved in certain applications.
In the future it is anticipated that, for
certain applications, the fecal streptococci
will achieve a position of primacy for
useful data, and, as indicated by Litsky
(1955) "be taken out of the realm of step-
children and given their legitimate place
in the field of santiary bacteriology as
indicators of sewage pollution. "
B Advantages and Limitations
1 Survival
In general, the fecal streptococci have
been observed to have a more limited
survival time in the aquatic environment
when compared to the coliform group.
They are rivaled in this respect only
by the fecal coliforms. Except for cases
of persistence in waters of high electro-
lytic content, as may be common to
irrigation waters, the fecal streptococci
have not been observed to multiply in
polluted waters as may sometimes be
observed for some of the coliforms.
Fecal streptococci usually require a
greater abundance of nutrients for sur-
vival as compared to the coliforms and
the coliforms are more dependent upon
the oxygen tension in the waterbody.
In a number of situations it was concluded
that the fecal streptococci reached an
extinction point more rapidly in warmer
waters while the reverse was true in the
colder situations as the coliforms now
were totally eliminated sooner.
2 Resistance to Disinfection
In artificial pools the source of
contamination by the bathers'is
usually limited' to throat and skin
flora and thus increasing attention
has been paid to indicators other
than those traditionally from the
enteric tract. Thus, one of the
organisms considered to be a fecal
streptococci, namely, S. salivarius,
can be a more reliable indicator
when detected along with the other
fecal streptococci especially since
studies have confirmed the greater
resistance of the fecal streptococci
to chlorination. This greater
resistance to chlorination, when
compared to the fecal coliforms, is
important since the dieoff curve
differences are insignificant when
the curves of the fecal coliforms
are compared to various Gram
negative pathogenic bacteria which
reduces their effectiveness as
indicators.
3 Ubiquitous Strains
Among the fecal streptococcus are
two organisms, one a biotype and
the other a variety of the S. faecalis,
which, being ubiquitous (omnipresent)
have limited sanitary significance.
The biotype, or atypical, S. faecalis
is characterized by its ability to
hydrolyze starch while the varietal
form, liquefaciens, is nonbeta
haemolytic and capable of liquefying
gelatin. Quantitation of these organisms
in anomalous conditions is due to their
capability of survival in soil or high
electrolytic waters and in waters with
a temperature of less than 12 Degrees C.
Samples have been encountered which
have been devoid of fecal coliforms
and yet contain a substantial number of
"fecal streptococci" of which these
ubiquitous strains constitute the majority
or all of the isolations when analyzed
biochemically.
1-17
-------
BacterioLogical Indicators of Water PolLution
V STANDARDS AND CRITERIA
Acceptance and utilization of Total Coliform
criteria, which must now be considered a
pioneering effort, has largely been supplanted
in concept and in fact by the fecal coliforms
in establishing standards for recreational
waters.
The first significant approach to the utiliza-
tion of the fecal streptococci as a criterium
for recreational water standards occurred in
1966 when a technical committee recommended
the utilization of the fecal streptococci with the
total coliforms as criteria for standards
pertaining to the Calumet River and lower
Lake Michigan waters. Several sets of
criteria were established to fit the intended
uses for this area. The use of the fecal
streptococci as a criterium is indicated to
be tentative pending the accumulation of
existing densities and could be modified in
future standards.
With the existing state-of-the-art knowledge
of the presence of the fecal streptococci in
waters containing low numbers of fecal
coliforms it is difficult to establish a specific
fecal streptococcus density limit of below
100 organisms/100 ml when used alone or
in conjunction with the total coliforms.
1-18
-------
Bacteriological Lndicators of Water Pollution
Part 4. Other Bacterial Indicators of Pollution
I TOTAL BACTERIAL COUNTS
A Historical
1 The early studies of Robert Koch led
him to develop tentative standards of
water quality based on a limitation of
not more than 100 bacterial colonies
per ml on a gelatin plating medium
incubated 3 days at 20° C.
2 Later developments led to inoculation
of samples on duplicate plating media,
with one set incubated at 37° C and the
other at 20° c.
a Results were used to develop a ratio
between the 37° C counts and the
20°C counts.
b Waters having a predominant
count at 37° C were regarded as
being of probable sanitary signifi- •
cance, while those giving
predominant counts at 20° C were
considered to be of probable soil
origin, or natural inhabitants of
the water being examined.
B Groups Tested
There is no such thing as "total" bacterial
count in terms of a laboratory determination.
1 Direct microscopic counts do not -
differentiate between living and dead
cells.
2 Plate counting methods enumerate only
the bacteria which are capable of using
the culture medium provided, under the
temperature and other growth conditions
used as a standard procedure. No one
culture medium and set of,growth
conditions can provide, simultaneously,
an acceptable environment for all the
heterogeneous, often conflicting,
requirements of the total range of
bacteria which may be recovered from
waters.
C Utilization of Total Counts
1 Total bacterial counts, using plating
methods, are useful for:
a Detection of changes in the bacterial
composition of a water source
b Process control procedures in
treatment plant operations
c Determination of sanitary conditions
in plant equipment or distributional
systems
2 Serious limitations in total bacterial
counts exist because:
a No information is given regarding
possible or probable fecal origin
of bacterial changes. Large numbers
of bacteria can sometimes be
cultivated from waters known to be
free of fecal pollution.
b No information of any kind is given
about the species of bacteria
cultivated.
c There is no differentiation between
harmless or potentially dangerous
forms.
3 Status of total counts
a There is no total bacterial count
standard for any of the following:
Interstate Quarantine Drinking
Water Standards
PHS regulations for water
potability (as shown in
"Standard Methods" Public
Health Service Drinking
Water Standards of 1962.)
b The most widely used current
application of total bacterial counts
in water bacteriology today is in
1-19
-------
Bacteriological Indicators of Water Pollution
water treatment plants, where some
workers use standard plate counts
for process control and for deter-
mination of the bacterial quality of
distribution systems and equipment.
c Total bacterial counts are not used
in PHS water quality studies, though
extensively used until the 1940's.
B Spore-Forming Bacteria (Clostridium
perfringens, or C. welchii)
1 Distribution
This is one of the most widely distributed
species of bacteria. It is regularly
present in the intestinal tract of warm-
blooded animals.
2 Nature of organism
C. perfringens is a Gram-positive,
spore-forming rod. The spores cause
a distinct swelling of the cell when
formed. The organism is extremely
active in fermentation of carbohydrates,
and produces the well-known "stormy
fermentation" of milk.
3 Status
The organism, when present, indicates
that pollution has occurred at some
time. However, because of the ex-
tremely extended viability of the spores,
it is impossible to obtain even an
approximation of the recency of pollution
based only on the presence of
C. perfringens.
The presence of the organism does not
necessarily indicate an unsafe water.
C Tests for Pathogenic Bacteria of Intestinal
Origin
1 Groups considered include Salmonella
sp, Shigella sp, Vibrio comma,
Mycobacterium sp, Pasteurella sp,
Leptospira sp, and others.
2 Merits of direct tests-
Demonstration of any pathogenic
species would demonstrate an
unsatisfactory water quality, hazardous
to persons consuming or coming into
contact with that water.
3 Limitations
a There is no available routine pro-
cedure for detection of the full
range of pathogenic bacteria cited
above.
b Quantitative methods are not avail-
able for routine application to any
of the above.
c The intermittent release of these
pathogens makes it impossible to
regard water as safe, even in the
absence of pathogens.
d After detection, the public already
would have been exposed to the
organism; thus, there is no built-in
margin of safety, as exists with
tests for the coliform group.
4 Applications
a In tracing the source of pathogenic
bacteria in epidemiological investi-
gations
b In special research projects
c In water quality studies concerned
with enforcement actions against
pollution, increasing attention is
being given to the demonstration of
enteric pathogenic bacteria in the
presence of the bacterial indicators
of pollution.
D Miscellaneous Indicators
It is beyOnd this discussion to explore the
total'range of microbiological indicators
of pollution that have been proposed and
1-20
-------
Bacteriological Indicators of Water Pollution
investigated to some extent. Mention can
be made, however, of consideration of
tests for the following.
1 Bacteriophages specific for any of a
number of kinds of bacteria
2 Serological procedures for detection
of coliforms and other indicators, a
certain amount of recent attention has
been given to applications of fluorescent
antibodies in such tests
3 Tests for Pseudomonas aeruginosa
4 Tests for viruses, which may persist
in waters even longer than members
of the coliform group.
REFERENCES
1 Standard Methods for the Examination of
Water and Wastewater, 13th ed.,
APHA. .AWWA, WPCF. Published by
American Public Health Association,
1790 Broadway, New York, N. Y. 1971.
2 Prescott, S. C., Winslow, C.E.A., and
McCrady, M. Water Bacteriology.
John Wiley & Sons, Inc. 1946.
3 Parr, L.W. Coliform Intermediates in
Human Feces. Jour. Bact. 36:1.
1938.
4 Clark, H.F. and Kabler, P.W. The
Physiology of the Coliform Group.
Proceedings of the Rudolfs Research
Conference on Principles and Appli-
cations in Aquatic Microbiology. 1963.
5 Geldreich, E. E., Bordner, R.H., Huff,
C.B., Clark, H. F. , and Kabler, P.W.
Type Distribution of Coliform Bacteria
in the Feces of Warm-Blooded Animals.
JWPCF. 34:295-301. 1962.
6 Geldreich et al. The Fecal Coli-Aerogenes
Flora of Soils from Various Geographic
Areas. Journal of Applied Bacteriology
25:87-93. 1962.
7 Geldreich, E.E., Kenner, B.A., apd
'Kabler, P.W. Occurrence of
Coliforms, Fecal Coliforms, and
Streptococci on Vegetation and Insects.
Applied Microbiology. 12:63-69. 1964.
8 Kabler, P.W., Clark, H.F., and
Geldreich, E.E. Sanitary Significance
of Coliform and Fecal Coliform
Organisms in Surface Water. Public
Health Reports. 79:58-60. 1964.
9 Clark, H.F. and Kabler, P.W.
Re-evaluation of the Significance of the
Coliform Bacteria. Journal AWWA.
56:931-936. 1964.
10 Kenner, B. S., Clark, H.F., and
Kabler, P.W. Fecal Streptococci.
II. Quantification in Feces. Am. J.
Public Health. 50:1553-59. 1960.
11 Litsky, W., Mailman, W.L., and Fifield,
C.W. Comparison of MPN of
Escherichia coli and Enterococci in
River Water. Am. Jour. Public Health.
45:1949. 1955.
12 Medrek, T. F. and Litsky, W.
Comparative Incidence of Coliform
Bacteria and Enterococci in
Undisturbed Soil. Applied Micro-
biology. 8:60-63.. 1960.
13 Mailman, W.L., and Litsky, W.
Survival of Selected Enteric Organisms
in Various Types of Soil. Am. J.
Public Health. 41:38-44. 1950.
14 Mailman, W.L,, and Seligman, E.B., Jr.
A Comparative Study of Media for
Detection of Streptococci in Water and
Sewage. Am. J. Public Health.
40:286-89. 1950.
15 Ministry of Health (London). The
Bacterial Examination of Water Supplies.
Reports on Public Health and Medical
Subjects. 71:34.
1-21
-------
Bacteriological Indicators of Water Pollution
16 Morris, W, and Weaver, R.H.
Streptococci as Indices of Pollution
in Well Water. Applied Microbiology,
2:282-285. 1954.
17 Mundt, J.O., Coggin, J.H., Jr., and
Johnson, L. F. Growth of
Streptococcus fecalis var. liquefactens
on Plants. AppliedMicrobiology.
10:552-555. 1962.
22 Geldreich, E. E. Applying Bacteriological
Parameters to Recreational Water
Quality. J. AWWA. 62:113. 1970.
23 ueldreich, E.E , Best, L.C., Kenner, B A
and Van Donsel, D. J. The Bacteriolog-
ical Aspects of Stormwater Pollution
J. WPCF. 40:1860. 1968
24 FWPCA Report of Water Quality Criteria
Calumet Area - Lower Lake Michigan,
Chicago, IL Jan. 1966.
18 Geldreich, E.E. Sanitary Significance
of Fecal Coliforms in the Environment.
TJ. S. Department of the Interior.
FWPCA Publ. WP-2 0-3. 1966.
19 Geldreich, E.E. and Kenner, B.A.
Concepts of Fecal Streptococci in
Stream Pollution. J. WPCF. 41:R336.
1969.
2 0 Kabler, P.W. Purification and Sanitary
Control of Water (Potable and Waste)
Ann. Rev. of Microbiol, 16:127. 1962,
21 Litsky, W., Mailman, W.L., and Fifield,
C.W. Comparison of the Most Probable
Numbers of Escherichia coli and
Enterococci in River Waters. A. J. P. H.
45:1049. 1955,
This outline was prepared by H. L Jeter,
Director, National Training Center and
revised by R. Russomanno, Microbiologist.
National Training Center, WPO, EPA,
Cincinnati, OH 45268.
Descriptors: Coliforms,' Escherichia coli ,
Fecal Coliforms, Fecal'Streptococci, Indicator
Bacteria, Microbiology, Sewage Bacteria,
Water Pollution
1-22
-------
BACTERIA AND THEIR SURVIVAL IN THE AQUATIC ENVIRONMENT
I INTRODUCTION
The bacteriology of the aquatic environment
may be considered from two viewpoints, the
first dealing with natural history and the
second concerned with man's welfare. The
first consideration concerns itself with the
naturally occurring or true water bacteria
while the second ccinsiders the bacteria
commonly found but not indigenous to the
aquatic environment. This outline will
primarily deal with the second consideration
and place emphasis upon the bacterial
indicators of pollution,
II BACTERIA IN THE AQUATIC
ENVIRONMENT
A Bacterial Content of Various Waters
count. Miquel (1886) observed counts
of 4.3 bacteria per milliliter in the
country and 19 bacteria per milliliter
In the city of Paris. Tissandier later
observed that the dust in the air
amounted to 4 mg/cu m. in the open
country and 23 mg/cu m. in Paris.
Pollution indicator counts for rainfall
are generally less than 1/100 milliliters.
The bacterial content of natural ice is
usually very low unless it is formed
from heavily polluted water trapped on
the surface of formed ice. Instances
of disease caused by polluted ice are a
matter of record. Samples of ice from
Quebec rivers have given the results
shown in Table 1 and these are typical
of those found in the average marketed
product.
1 Precipitation
The part of the hydrologic cycle which
ultimately supplies us with freshwater
is rain, snow, hail, sleet or dew.
These sources are by no means free
from bacterial contamination and there
is a direct relationship between the
dust content and the total bacterial
Ice
Sample
Bacteria/ ml
Table 1. BACTERIA IN NATURAL ICE
Ice
Coliforms/100 ml Sample Bacteria/ml
Coliforms/100 ml
1
1
0
9
16
0
2
2
0
10
16
0
3
4
0
11
16
0
4
4
1
. 12
18
0
5
10
1
13
18
0
6
14
0
14
19
0
7
14
0
15
19
0
8
16
0
16
26
0
W. BA. 58. 11. 72
2-1
-------
Bacteria and their Survival in the Aquatic Environment
2 Land runoff
Bacteriological numbers increase
tremendously as the waters of pre-
cipitation contact the earth's surface
and flow over the varied topography.
Total counts from rivulets in roadways
or ploughed land can exceed several
hundred thousand per milliliter.
Availability of nutrients and attendant
physical and chemical conditions pro-
vide a complex "medium" in which
bacterial counts can vary in an
unpredictable manner. An example
of.these complexities are provided by
studies of stormwater. pollution. In
an abridged form Table 2 shows
differences between pollution indicators
from sewer overflows from the cities
of Detroit and Ann Arbor during
monthly monitoring. Significant
differences between the two cities can
be attributed mainly to the differences
in populations, the type of systems
(Detroit: Combined; Ann Arbor:
Separate), amounts of rainfall, and the
species of warm-blooded animal wastes
predominating in the sewage con-
tribution (human vs non-human).
Table 2. DETROIT AND ANN ARBOR OVERFLOWS
Overflows
Sucessfully
Monitored
Ann
Arbor
Detroit
Analysis
Range of Geometric Means
Count/100 ml
Ann Arbor
Detroit
0
11
11
10
11
Tot.
Fee.
Fee.
Tot.
Fee.
Fee.
Tot.
Fee.
Fee.
Tot.
Fee.
Fee.
Tot.
Fee.
Fee.
Tot.
Fee.
Fee.
Tot.
Fee.
Fee.
Tot.
Fee.
Fee.
Col.
Col.
Strep.
Col.
Col.
Strep.
Col.
Col.
Strep.
Col.
Col.
Strep.
Col.
Col.
Strep.
Col.
Col.
Strep.
Col.
Col.
Strep.
Col.
Col.
Strep.
120, 000-

7,400-

12,000-

139,000-

29,000-

73,000-

190, Q00-
10,
24, 000-

42,0007

390, 000-
15,
60, 000-

180,000-

220, 000-
34,
160, 000-

120,000-

350, 000
17,000
31, 000
880, 000
73,000
320, 000
000,000
130, 000
330, 000
000, 000
560, 000
670, 000
000, 000
750, 000
480, 000
1, 100, 000- 8, 500,000
570, 000- 8, 200, 000
190,000- 2, 600,000
1, 900, 000-
410, 000-
280, 000-
1, 300, 000-
470, 000-
660, 000-
12, 000, 000-
1, 900, 000-
1'80, 000-
12,000,000-
1, 400, 000-
410, 000-
200, 000-
200, 000-
200, 000-
900,000
900, 000
1.
24, 000, 000
3,600, 000
1, 500, 000
41, 000, 000
8, 700, 000
980, 000
45, 000, 000
10, 500, 000
490, 000
45, 000, 000
16, 000, 000
790,000
110, 000, 000
20, 000, 000
900, 000
- 85, 000, 000
- 5, 300, 000
100, 000
2-2
-------
Bacteria and their Survival in the Aquatic Environment
In addition to the differences previously
observed from the overflows of two
large communities it is common to
observe seasonal variations. Table 3
indicates the seasonal variations in
stormwater. It is well at this point to
compare these pollution indicator counts
with those obtained from domestic
sewage. The following typical median
values have been noted by the same
author: *
Source Total coliform Fecal coliform Fecal streptococci
Domestic 33, 000,000 10, 900,000 2,470, 000
Sewage
Table 3. Seasonal Variations (Median Values) for Bacterial Discharges
in Stormwater and Rainwater from Suburban Areas, Cincinnati, Ohio,
and in Agricultural Land Drainage, Coshocton, Ohio (Count/100 ml)
Source
Date
Total
Samples
Season
Total
Coliform
Fecal
Colifrom
Fecal
Streptococcus
Wooded
hillside
Feb. 62 to
Dec. 64
278
Spring
Summer
Autumn
Winter
2, 400
79,000
180, 000
260
190
1, 900
43 0
20
940
27,000
13,000
950
Street
gutters
Jan. 62 to
Jan. 64
177
Spring
Summer
Autumn
Winter
1, 400
90, 000
290, 000
1, 600
230
6, 400
47,000
50
3, 100
150, 000
140,000
2, 200
Business
district
Apr. 62 to
Jul. 66
294
Spring
Summer
Autumn
Winter
22, 000
172,000
190, 000
46, 000
2, 500
13,000
40, 000
4, 300
13,000
51, 000
56,000
28,000
Rural
Jan. 63 to
Aug. 64
94
Spring
Summer
Autumn
Winter
4, 400
29,000
18,000
58,000
55
2, 700
210
9, 000
3,600
58, 000
2, 100
790, 000
Rainwater
Jun. 65 to
Feb. 67
49
Spring
Summer
Autumn
Winter
<1.0
<1.0
<0.4
<0.8
<0.3
<0.7
<0. 4
<0.5
<1. 0
<1. 0
<0. 4
<0. 5
1 References for each outline table and figure are
provided at end of outline.
2-3
-------
Bacteria and their Survival in the Aquatic Environment
3 Surface waters
Rivers in inhabited regions contain
several hundreds to thousands of
total bacteria per milliliter. These
bacterial contents are likely to show
sudden fluctuations due to a variety
of factors such as stream flow and
rainfall. An example of this striking
fluctuation was observed by Gage
(1906) where the bacterial content
of the Merrimac was highest when
the stream was lowest and, therefore,
when its sewage content was less
subject to dilution. (Table 4)
When a stream of fair quality is
compared to a highly polluted one
the effects of surface contamination
due to rainfall are informative.
A stream study by Kisskalt (Table 5)
compares the Lahn (fair quality)
with the Wieseck (highly polluted).
In the Lahn, as is a general rule with
streams of fair and high quality,
fluctuations are most pronounced as
runoff contamination enters the stream.
On the other hand, the Wieseck shows
less pronounced fluctuations since the
constant influx of sewage damps out
the surface runoff contributions.
Table A. MERRIMAC RIVER
flow of stream
(cu ft/sec/Sq mi
of watershed)
Bacteria/ml
B. coli
*/ml
Canal
Intake
Canal
Intake
less than 1
7, 500
10, 800
66
88
1 - 2
6, 800
6, 200
50
51
2 - 4
3, 600
5, 600
29
39
over 4
3, 400
3, 100
16
29,
*The Bacillus coli of the earlier water bacteriologists corresponds
approximately to our present species Escherichia coli.
Table 5
Monthly Variations of Bacteria in a Normal and in a Polluted Stream
bacteria per milliliter (1904- 1905)

Lahn
Wieseck
July
318
104, 000
July
132
156, 000
Aug
840
98, 400
Oct*
1, 235
28, 400
Oct*
420
5 8, 000
Nov
2, 340
39, 200
N ov*
1, 740
52, 000
Dec*
780
28, 600
Dec*
1, 220
21, 200
Jan*
3, 668
29, 920
Feb*
5, 380
11, 900
Mar*
1, 210
8, 250
Apr*
4, 925
5, 910
May
570
14, 800
June
686
50, 180
*Rain or high water due to previous thaw,
2-4
-------
Bacteria and their Survival in the Aquatic Environment
The total bacterial content of large
reservoirs, lakes, or ponds which do
not have sewage or nutrient contam-
ination ordinarily have only a few
hundred per milliliter and in many
cases are less than one hundred.
The 20° counts and the coliform
counts' of Western Canadian Lakes
were examined by the QuebecMinistry
of Health in the 1930's and these are
tabulated in Table 6. Note that the
total bacterial count is per milliliter
while the coliform count is per
100 milliliters.
Table 6. Bacteria in Eastern Canadian Lakes
Table 7. Bacteria in the Atlantic Ocean
Bacteria' per milliliter
(Otto and Neumann, 1904)
Nearest Land
5
Depth in Meters
50 " 100 200
Canary
Islands
120
76
20
1
Cape Verde
Islands
5 8
16
64
6
St. Paul
Island
20
480
54
4
Pernambuco
48
168
83
14
. Bacteria Coliforms
"ia 8 per ml per 100 ml
1 9 0
2 13 0
3 31 2
4 39 0
5 46 0
6 55 15
7 80 8
8 110 3
9 110 6
10 120 0
11 130 2
12 200 9
13 240 27
14 300 7
15 350 5
16 500 7
17 550 1
18 650 0
19 650 11
20 850 2
In the estuarine and marine environ-
ment, as well as in fresh waters, the
ability and/ or disposition of bacteria
to migrate to the sediment layers has
been well recognized. Table 8
indicated the extremely high counts
which may be found in these environ-
ments as well as their biological
capabilities for life in the sea.
This tendency to. sediment can be seen
in Figure 1 which is a. profile of densities
of coliform bacteria in the Hyperion
outfall (city of Los Angeles, California).
It is important to note the coliform
"sag" in the. left of the figure and the
current flow is from right to left. The
fresh water effluent has a tendency to
rise in the saline waters of the bay.
which further emphasizes this
"sedimentation" sag.
4 Estuarine and marine waters
It has been estimated that the number
of viable bacteria occurring in the sea
range from 10° to 10® cells/ml. In
general the amount of bacterial life
decreases as we proceed outward from
the shore and downward from the
surface. Table 7 indicates this trend
as one samples to a greater depth.
2-5
-------
Bacteria and their Survival in the Aquatic Environment
READINGS TAKEN EVERY 1.000 FEET ON A LINE PARALLEL
To|THE COAST AT 0,15.304 45 FEET DEPTHS APPROXIMATE
COLIFORM CONTOURS SHOWN AS COLIFORMS PER MILLILITER
CH LOR IN AT ION .OF HYPER IOf"J EFFLUENT HAD BEEN INTENTION-
ALLY INTERUPTED PRIOR TO AND DURING THIS SURVEY
t ¦ i
LEGEND
N = UPCOAST STATION
S = DOWNCOAST STATION
O = OVER OUTLETS
J-
J.
DISTANCE FROM HYPERION OUTFALL, FEET
FIGURE 1 PROFILE OF DENSITIES OF COLIFORM ORGANISMS ABOUT ONE MILE OFF
SHORE, VICINITY OF HYPERION OUTFALL ON JAN 12, 19S6
Table 8
Sediment Sample
8160
8330
9309

Station Location
32051. 2'N
330 25.9 'N
33044.
2'N

117°28. 3'W
118O06.5'W
118°46
. l'W
Depth of overlying water (meters)
780
505
1, 322
Bacteria per gram of sediment (wet basis)

Total aerobes (plate count)
930, 000
31, 000, 000
O
O
00
%
00
000
Total anaerobes (oval-tube count)
190, 000
2, 600, 000
1,070,
000
Ammonification (peptone —~ NH^)
100, 000
1, 000, 000
»—1
O
O
O
000
Ammonification (nutrose-* NH )
10, 000
1,000,000
100,
000
Urea fermentation (urea -*NHj)
100
+
1,
000
Proteolysis (gelatin liquefaction)
100, 000
10, 000, 000
I—1
0
0
0
000
Proteolysis (peptone-* H S)
10, 000
1, 000, 000
100,
000
Denitrification (NO„ N„)
Nitrate reduction (NO^ "^NO^)
100
10, 000
10,
000
100, 000
10, 000, 000
10,
000
Nitrogen fixation
0
0

0
Nitrification (NH NO )
0
0

0
Sulfate reductionist}^—* H^S)
1, 000
0
0
0
rH
10,
000
Dextrose fermentation
10, 000
100, 000
1.
000
Xylose fermentation
10, 000
+
10,
000
Starch hydrolysis
10, 000
100, 000
10,
000
Cellulose decomposition
1, 000
+
1,
000
Fat hydrolysis (lipoclastic)
1, 000
+
+

Chitin digestion
100
+
+

From C.E, Zobell, J. Sedimentary Petrology, 8:10, 1938
2-6
-------
Bacteria and their Survival in the Aquatic Environment
B Factors Influencing Reduction of Bacteria
Density
The forces which tend to decrease the
numbers in stored samples are important.
Some of these forces have been extensively
studied and much is known of their actions.
Others are more obscure and as little as
mere postulations have been advanced for
their actions of presence. The following
discussion will deal with some of these
forces and, it will be evident from the
individual topic, that some of these will
only be found in a particular environment
as, for instance, salinity in ocean waters.
1 Natural self-purification
Natural self-purification can be
described as the result of the com-
bined effects of all of the forces which
tend to diminish the numbers of
bacteria in a given time interval.
These combined influence include
physical, chemical, and biological
factors. The following two tables
(Table 9 and 10) show the bacterial
reductions which occur with the passage
of time. In the first case the
reductions occur as one obtains counts
at successive points away from the
highly polluted mouth of the River
Brathay. In the second case successive
passages to three reservoirs from a
a polluted river show a marked decrease
in bacteria.
Table 9. Numbers of Bacteria at Various Distances from the Mouth
of the River Brathay, Windermere, at Depth of 1 Meter
(Taylor, 1940)
Distance from
River Mouth
(meters).
Bacteria*
per ml
22 July, 1938
Distance from
River Mouth
(meters)
Bacteria
per ml
2 November, 1939
0
I—1
4^
CO
O
O
67
15, 260
133
12, 120
536
4, 720
757
4, 400
30 November,
1938
2, 800
4, 300
3, 200
3, 900
4, 000
4, 150
4, 800
00
o
5, 300
3, 420
6, 000
3, 260
River
20, 200
0
18, 500
191
620
351
1, 060
542
960
670
960
861
650
990
700
1, 212
640
1, 467
570
1, 690
550
4, 180
580
*On special agar, 20° C, 15 days
2-7
-------
Bacteria and their Survival in the Aquatic Environment
Table 10. Reduction of Bacteria in Washington Reservoirs
Bacteria per milliliter. Monthly Average, 1907

Potomac
Dalecarlia
Georgetown
Washington

River
Reservoir
Reservoir
City Reservoir
January
4, 400
2,400
2, 200
950
February
o
o
o
iH
950
1, 000
750
March
11, 500
8, 300
7, 200
3', 600
April
3, 700
2, 100
1, 400
475
May
750
350
325
130
June
2, 300
950
600
100
July
2, 700
600
350
160
August
3, 000
275
425
80
September
6, 200
. . .
1, 900
230
These same forces are naturally in
effect when the bacterial indicators of
pollution are considered. Figure 2
indicates the curves for coliform
reduction in streams in winter. This
curve is flatter than that of the summer
reductions indicating a greater survival
at lower temperatures, which, in gener;
can be said to be the case for the
majority of bacteria.
Figure 2. Decrease in coliform bacteria in
streams in winter
A study of pollution indicators and their
survival when compared to a pathogen,
in this case Salmonella typhimurium,
was done by Geldreich, et al, utilizing
stormwater stored at different temper-
atures.
Figure 3 indicates the survival, or
persistence, of these enteric bacteria
as compared to the "dotted line" of the
pathogen. It is important to note that
the apparent longer persistence of the
pathogen over two of the three standard
indicators does not imply that a number
of them will be found after complete
dieoff of the indicators - in other-words,
the percent survival is based upon
vastly differing numbers of original
inoculum of bacteria.
2 Predation
The predators of bacterial populations
belong to the group of microphagic
organisms which draw part of their
nutrient from ingested microbes which
themselves have transformed organic
matter. Numerous in vitro studies
have confirmed the voracity of these
predators in rapidly reducing micro-
bial populations. At the present time
investigators vary in their consensus
of the importance of predation in
reducing the bacterial populations and
claims vary from negligible effect to
a first order importance.
2-8
-------
Bacteria and their Survival in the Aquatic Environment
FIGURE 3 PERSISTENCE OF SELECTED ENTERIC BACTERIA IN
STOIHWDTER STORED AT 20° C
3 Sunlight
Although considerable information
regarding the bactericidal properties
of sunlight has been generated rel-
atively little has been done regarding
this property in relation to water.
The major consensus is that it is a
contributory factor in bacterial
destruction and of secondary importance.
It can be readily seen that in waterbodies
of shallow depth it assumes greater
importance than if deep ocean depths
are considered. Figure 4 illustrates
this thought as it can be seen that the
coliform count diminishes at a greater
rate for the container closer to the
surface (0. 18 meter) than for the
seawater sample at the greater depth
(4 meters).
4 Adsorption and sedimentation
Although a few investigators have
discounted the effects of adsorption
and sedimentation upon removal of
bacteria from the aqueous environ-
ment the majority of studies have
shown varying degrees of removal
and deposition of bacteria to the
bottom stratum. Bacterial adsorption
is found to have a direct relationship
to particle size and physiochemical
nature of the particle. Marine muds
have been found to be highly adsorptive
while sand particles are only slightly
adsorptive. Table 8, previously
presented, indicates the high counts
which can be found in the sediments.
2-9
-------
Bacteria and their Survival.inthe Aquatic Environment
CUMULATIVE SOLAR RADIATION, S(cal/cm2) DURING EXPOSURE
FIGURE 4
5 Antibiotics and toxins
These factors are ordinarily con-
sidered as agents of the marine
environment but one cannot restrict
them from the fresh water environment
since some'common bacteria have this
property. Numerous studies have been
conducted in estuarine and oceanic
environments in which the presence of
an antibiotic or toxic principle has been
apparent. One method of measuring
the bactericidal action of seawater is to
compare the differences in survival when
using untreatedandtreatedportions of
seawater.
An example of this type of study is pro-
vided in Table 11 where the survival
of E. coli is compared when untreated,
filtered, and autoclaved seawater from the
same sample point are used as 48 hour
diluents. Notice that the untreated
seawater has the greatest bactericidal
activity which has been appreciably
' diminished or eliminated by the-
treatment.
Table 11. Survival of Escherichia coli in
untreated, filtered, and autoclaved portions
of six seawater samples collected during
July and August,- 1957
Treatment
Survival after 48 hr in
seawater sample no.

1.
2
3 '
4
5
6
None
Filtered
Autoclaved
%
2.2
8. 3
8.4
%
0.7
3.8
30.9
%
4.6
4.8
64. 6
%
22. 8
30. 1
53. 6
%
4.4
0. 6
69.6
io
2.7
39.6
38.5
2-10
-------
Bacteria and their Survival in the Aquatic Environment
It is interesting to compare the same
treatment given to waters having a
subsequent inoculum of coliphage
(Table 12). Again the untreated sea-
water is viricidal (phage are virus
particles which are capable of infecting
and/or killing bacteria of a specific
species) and the phage are rapidly
eliminated while the treatments allow
for a greater persistence of the coli-
phage (phage active against coliforms).
Table 12
Persistence of coliphage in various waters
Water
Survival
10 days
2 0 days
30 days

%
%
%
Untreated seawater
2.0
<0. 01
<0. 01
Filter sterilized
56. 0
37.0
6.4
seawater

Autoclaved seawater
102. 0
68.0
58. 0
0. 85% NaCl in
1.2
0.4
0.05
deionized water

6 Salinity
The salinity of surface seawater
averages about 3.5 percent and this
concentration may be considerably
reduced in areas where interactions
with fresh water occur. These
inorganic salts may adversely affect
the survival of bacteria by either
specific ion toxicity or osmotic effect.
Although some investigators have
reported that sea waters do not
appreciably affect enteric bacteria
in this environment there is general
agreement that toxic effects are exerted
to enteric bacteria in ocean environments
but not to a degree of primary importance.
Tables 13 and 14, in an abridged form,
indicate this effect. Note that at a 25
percent concentration of sea water the
affect is stimulatory when compared to
a 0 percent or concentrations above the
25 percent sea water. Table 12 enforces
this concept as sea water salinities are
compared with NaCl solutions of equal
salinities. (Note: the 0. 85 percent
roughly equates with the 25 percent
sea water concentrations. )
Table 13
Influence of concentration of sea water
on the survival of Escherichia coli
Concentration of Survival after 48 Hr
Sea Water
%

%
0

59. 9
25

74. 5
50

34. 6
75

22. 5
100

8. 2

Table 14

Survival of Escherichia coli in sea water
and NaCl solutions of equal salinity
Solution
Salinity
Survival after 48 Hi

%
%
Sea water
0.85
40. 4

2. 50
7.6

5. 00
<0. 01
NaCl
o
CO
en
41. 3

2.50
2. 1

5.00
<0. 01
2-11
-------
Bacteria and their Survival in the Aquatic Environment
7 Temperature
It is a general rule that bacteria
survive somewhat longer at lower
temperatures. An example of this
generality can be seen in Figure 5
which shows the effect of temperature
on coliform bacteria in Pacific Ocean
water.
Figure 5. Effect of temperature on coliform
survival in Pacific Ocean water
As an example to confirm that forces
acting on bacterial survival are
complex and in unison affect the
population viable at each specific time
interval, Figure 6 is presented.
Here the variable is still the tem-
perature (as was Figure 5) but to each
test sample was added an equal amount
of organic matter (120 ppm lactose
broth) and this drastically altered the
survival times for the different tem-
peratures. In many instances, an
adequate food supply can overcome
detrimental conditions to the bacterial
populations.
TIME - OATS
Figure 6. Effect of temperature on
coliform survival in sea water containing
organic matter.
2-12
-------
Bacteria and their Survival in the Aquatic Environment
8 Other factors
There are a multitude of other biolog-
ical, physical, and chemical factors
which affect the bacterial populations.
Such other factors are pH, turbidity,
chlorination, industrial wastes, etc.
Some factors are still obscure and
others are postulations. Factors in
bacterial survival may be simply over-
looked as may be the case shown in
Figure 7 where channel characteristics
can profoundly effect survival rates.
Ill RATES OF BACTERIAL DISAPPEARANCE
FROM RECEIVING WATERS
A Or lob (1956) graphically summarized
data from several investigations of
bacterial survivals in sea waters.
(See Figure 8)
Based on these observations, Orlob
expressed bacterial survivals on"
several different equations, shown
and plotted on Figure 9.
_L
TIME-DAYS
Figure 7. Effect of channel characteristics
on the rate of coliform decrease
/CROf I
tsi IOATORQ
2-13
-------
Bacteria and their Survival in the Aquatic Environment
SURVIVAL TIME - DAYS
Figure 9. Typical survival curves
for bacteria in sea water
The value of k is found as follows:
N^= bacterial'population at time
at end of lag period
N2= bacterial population at time t
t = time in days at end of lag period
(at onset of logarithmic rate of
decrease)
t = subsequent time t in days
1 Curve A is a "typical" survival curve
when the receiving environmental water
permits extended survival, or even'
limited growth, prior to onset of rapid
disappearance of the sewage organisms.
This lag period is not included in the
rate calculation. Results of this type
are not uncommon, particularly with
pure cultures in restricted environ-
mental laboratory studies or in con-
tainers placed in the'marine environment.
Explanation of symbols:
N = number of survivors at time t (days)
Nq = initial population at t = O
k = rate constant (usually in the range
0.4 - 1.6)
k1 = special rate constant for equation C
t = lag period in days before onset of
'logarithmic decrease
2 Curve B is a pattern of bacterial
disappearance when the receiving
environment is totally unfavorable
to the introduced bacteria. Many
of the curves in Figure 9 begin in this
type of pattern. Curves of this type
can be expected in many laboratory
studies of bacterial survival in marine
water. Pure cultures often are used
in such studies. With all cells
representing the same species, and
being at approximately the same level
of physiological activity, the rate of
disappearance should be uniform under
unfavorable environmental conditions.
2-14
-------
Bacteria and their Survival in the Aquatic Environment
3 Curve C is to be expected when
multiple species are introduced
into the marine environment.
Coliform bacteria, representing
several species and levels of
physiological activity, can be expected
to represent different susceptibilities
to the adverse factors in a marine
environment. Similar overall patterns
are apparent in Figure 9, some of
which represent pure culture studies.
4 Curve D represents an increasing rate
of bacterial disappearance with time,
and has been interpreted by some as
indicative of increasing susceptibility
of the bacteria to an unfavorable
environmental factor such as a toxic
agent. Such a pattern of bacterial
disappearance might also occur with
increasing populations of predatory
biota.
B Evidence of bacterial disappearance from
saline waters is not' restricted to that
environment. Freshwater also is not a
suitable habitat for extended survival of
sewage bacteria. See Figure 10 (Kittrell
and Furfari). Comparison with Figure 9,
with respect to bacterial disappearance
from sea waters, reveals a generally
similar pattern, with the differences
being largely a matter of degree.
IV SUMMARY AND CONCLUDING REMARKS
A Sea waters do not represent a favorable
environment for extended survival- of
sewage bacteria. Factors bringing about
bacterial disappearance are numerous, -
interrelated, and many also are applicable
to bacterial removals from the freshwater
environment. These common factors'
include dilution, sedimentation, predation,
and some chemical factors.
B Some factors influencing bacterial
disappearance appear to be more specific
to sea waters. This includes salinity and.
the presence of soluble toxic substances.
Figure 10. Rates of coliform decrease
below five selected cities
C Because each factor which influence
bacterial survival is a variable, in any
given situation in which the fate of
sewage organisms is in question, it will
be necessary to conduct.special
investigations to determine the overall
effects of the factors leading to bacterial
disappearance in that particular
environment.
D Of primary interest to the water
microbiologist is that the concept
of the indicator-pathogen dieoff curve
(i.e., gradual dieoff of indicator
bacteria but yet at a slower rate and
for a greater period than any pathogen)
is maintained in the aquatic environment.
As yet, there have been no observable
deviations from this concept which would
seriously challange established criteria.
2-15
-------
Bacteria and their Survival in the Aquatic Environment
REFERENCES
OUTLINE REFERENCES
PUBLICATION TABLE FIGURE
1 The Bacteriological Aspects of Stormwater 3 3
Pollution. Jour. WPCF, Vol. 40:1861-1872,
1968. E.E. Geldreich, L. C. Best,
B.A. Kenner, and D.J. Van Dousel.
Water Bacteriology, 6th Edition. John Wiley 1, 4, 5, 6, 7, 9, 10
& Sons, Inc. S. C. Prescott, C.E.A. Winslow
and M.H, McCrady.
Bacteriological Comparison Between Combined 2
and Separate Sewer Discharges in Southeastern
Michigan. Jour. WPCF, Vol 38:400-409.
R. J. Burm and R. D. Vaughan.
J. Sedimentary Petrology. 8:10, 1938. 8
C. E. Zobell.
Sewage Effluent Dilution in Sea Water. Water 1
and Sewage Works. March 1958. C.H. Lawrence.
Observations of Coliform Bacteria in Streams. 2, 7, 10
Jour. WPCF, Vol. 35:1361- 1385, 1963.
F. W. Kittrell and Santo A. Furfari.
Field Studies on Effect of Daylight on Mortality 4
of Coliform Bacteria Water Research.
1:279-295, 1967. A.L.H. Gameson and
J.R. Saxon.
8 Evaluation of Factors Affecting Survival of 11
Escherichia coli in Sea Water, V. Studies
with Heat- and Filter-Sterilized Sea Water-.
Appl, Microbiol. 9:400-404, 1961.
A.F. Carlucci, P. V. ¦ Scarpino and D. Pramer.
9 An Evaluation of Factors Affecting Survival of 12
Escherichia coli in Sea Water. IV Bacteriophages,
Appl. Microbiol. 8:254-256, 1960. A.F.
Carlucci and D. Pramer.
10 An Evaluation of Factors Affecting the Survival of 13, 14
Escherichia coli in Sea Water. II Salinity, pH,
and Nutrients. Appl. Microbiol. 8:247-250, 1960.
A.F. Carlucci and D. Pramer.
11 Viability of Sewage Bacteria in Sea Water. Sewage 5, 6, 8, 9
and Industrial Wastes. Vol.28: 1147-1167, 1956.
Gerald T. Or lob.
This outline was prepared by R. Russomanno,
Microbiologist, National Training Center,
WPO, EPA, Cincinnati, OH 45268.
2-16
-------
BACTERIOLOGICAL PATHOGENS IN THE AQUATIC ENVIRONMENT
I INTRODUCTION
Of the large group of microorganisms which
have been implicated with outbreaks by orga-
nisms in the aquatic environment this outline
will deal with only those of a bacteriological
nature.
It must be realized that any of the pathogenic
bacteria are capable of initiating a disease
process in an individual or group when
waterborne and viable. This outline, however,
will emphasize only those which through the
history of mankind have been implicated
epidemiologically and clinically with water-
borne outbreaks.
II BASIC DEFINITIONS
In order to follow the descriptions of the
disease process it is necessary to be
knowledgeable of some basic definitions
which follow:
CARRIER. .. .An individual who harbors a
pathogenic organism which may manifest
itself either as an observable clinical
disease or be undetectable. In both cases
the individual can initiate disease outbreaks
by continual or sporadic passing of the
organisms to the environment,
ENDEMIC.... The continual occurrence of
a disease entity to a specific geographical
area which serves as a focus for future
invasions to widespread areas.
EPIDEMIC. .. .The occurrence of an illness
clearly in excess of the normal expectancy
and arising from a common source.
INCUBATION PERIOD The time interval
between the acquisition of the disease
producing organism and when the first
observable signs of the disease are evident.
Ill
RESERVOIR. . . .Reservoirs for the
infectious diseases may be man, animal,
plant, soil, etc., which are hosts upon
which the disease agent lives and
multiplies. Man is the most frequent
reservoir for these organisms which are
transmitted to man.
DESCRIPTIONS OF COMMUNICABLE
DISEASES
The following descriptions of the various
diseases are written with an overview of the
aquatic environment. In many cases the
individual diseases can be spread just as
well by person-to-person contact, arthropod
transfer, inanimate object contamination, etc.
This must be kept in mind when observing
morbidity and mortality data in this outline
as the source of infection may be any of the
above.
A Cholera
1 General
Cholera is a severe acute intestinal
infection which is usually characterized
by a sudden onset, vomiting, profuse
watery diarrhea, dehydration, and
possible collapse. An epidemic is
usually explosive and fatalities can
vary from 5 to 75% with deaths occurring
within a few hours of disease onset.
It is endemic in parts of India and
although absent in epidemic-proportions
in Europe and the Western Hemisphere
for many years it has repeatedly'
invaded. Since 1962 two Americans,
both tourists to foreign countries,
developed Cholera while overseas.
W. BA. 57a. 11.72
3-1
-------
Bacteriological Pathogens in the Aquatic Environment
One can see the explosive nature of the disease by referring to Table 1 .(Abridged from
G.I. Forbes)"of the incidence of Cholera since it first appeared in Hong Kong in 1961 and
is inclusive to the year 1966.
TABLE 1
Year
1961
1962
1963
1964
1965
1'966
Cases
77
11
115a
34b
-
1
Contacts . . .
748
120
22 10c
391
-
4
Carriers . . .
53
20
119d
29e
-
-
Deaths
15
1
6
4
-
-
a. ... includes 17 cases from a common source
b. . . . includes 16 cases from a common source
c. ... includes 231 contacts of 10 "nightsoil" carriers
d. ... includes 14 "nightsoil" carriers and 34 carriers from a restaurant
e. .. .includes 3 "nightsoil" carriers and 18 carriers from a restaurant
2 Infectious agent
Vibrio cholerae. ... A gram-negative
"comma" shaped bacteria.
3 Reservoir
The reservoir is an infected person and
the source is infected feces and
vomitus of patients.
4 Transmission
In the initial wave of an epidemic the
spread is regularly by the contaminated
water route. The carrier has not been
a significant factor in this disease.
This transmission period lasts as long
as viable organisms are being excreted
and this is usually about 7 to 14 days
for feces.
5 Incubation period
This can vary from a few hours to 5 days
with the usual period of 3 days.
B Dysentery, Bacillary (Shigellosis)
1 General
Shigellosis (this is now-the preferred
nomenclature for reporting to the.
National Communicable Disease Center)
is an acute infection of the intestinal
tract and is characterized in severe
cases by bloody stools with mucous and
pus and accompanied by malaise, fever,
toxemia, and cramps. Some cases
may be inapparent or accompanied by
mild symptomology. This disease is
rarely fatal and in the cases which
are, it is usually of the very young,
very old, or debilitated. In 1969 there
were a total of 9, 054 isolations of
Shigella reported to NCDC^ and this
released total does not include 1, 943
clinical cases reported from California
during 1969. Figure 1 includes the
reported isolations of Shigella in the
United States during the periods 1964
to 1969.
3-2
-------
REPORTED ISOLATIONS OF SHIGELLA IN THE UNITED STATES
'ALASKA, ARIZONA. HAWAII, ILLINOIS, KANSAS, MARYLAND, NEW JERSEY, NEW MEXICO, NORTH CAROLINA, NORTH DAKOTA, OHIO.
OKLAHOMA, OREGON, SOUTH DAKOTA. TENNESSEE, TEXAS AND VERMONT
"ADJUSTED TO FOUR-WEEK MONTHS
MMWR Vol. 19, No. 14 NCDC
Figure 1
co
I
CO
-------
Bacteriological Pathogens in the Aquatic Environment
2 Infectious agent
Various species of the genus Shigella:
Sh. dysenteriae, Sh, sonnei, Sh.
flexneri, Sh. boydii, and others.
Table 2 lists the six most frequently
reported serotypes of Shigella from
humans in 1969.
3 Reservoir
The reservoir is man and the source
of infection is feces from an infected
person.
4 Incubation period
One to 7 days but usually less than
4 days.
5 Period of communicability
The infected person is able to transmit
the disease during the acute stages of
the affliction and whenever the bacteria
is present in the feces.
Unless the carrier state is present the
infectious agent- is present for only a
few weeks. Carriers have been known
to harbor the bacteria for a year or
two, rarely longer.
C Leptospira
1 General
Leptospirosis is an acute systemic
infection characterized by fever,
headache, malaise, chills, vomiting,
muscular aches and less frequently
juandice, renal insufficiency and
hemorrhage in the skin and mucous
membranes. The acute illness lasts
from 1 to 3 weeks and relapses may
occur.,. Case fatalities are generally
low but increase with advancing age
levels and may reach 20% or more in
the severe cases.
Table 2
The Six Most Frequently Reported Serotypes of Shigella from Humans, 1969
Rank
Serotype
Reported
Number
Calculated-*
Number
Calculated
Percent
Rank
1961
1
s.
sonnei
5, 584
5, 513
60. 9
1
2
s.
flexneri 2a
668
1, 308
14.4
2
3
s.
flexneri 3a
303
768
8. 5
3
4
s.
flexneri 6
3 09
3 86
4.3
4
5
s.
flexneri 2b
170
333
3.7
5
6
s.
flexneri 4a
154
295
3. 3
6
**Calculated number is derived by distributing the unspecified isolations in each group to
their subgroups in the same proportions as the distribution of the specified isolations of
that group.
MMWR Vol. 19, No. 14 NCDC
3-4
-------
Bacteriological Pathogens in the Aquatic Environment
Table 3 lists the numbers of cases
and deaths attributed to this disease
for the years indicated:
Table 3
Reported Cases and Deaths from Leptospirosis: United States
1968 1967 1966 1965 1964 1963 1962 1961 1960
No. of Cases
Reported 69 67 72 84 142 89 79 71 53
No. of Deaths 4 9 11 14 14 10 .9 14
Source: National Center for Health Statistics, Vital Statistics of the United .States
2 Infectious agent
Many species of the Genus Leptospira,
of which there are at least 21
(serogroups containing 59 serotypes),
have been isolated from the aquatic
environment. Some nonpathogenic
leptospires have been found in water.
3 Reservoir
Reservoirs include cattle, dogs, and
swine. The wide spectrum of animals
and birds implicated with infection also
include rats and other rodents, foxes,
skunks, racoons, opossums, and a
variety of water birds. The source of
infection is the urine of the infected
animals.
4 Incubation period
Four to 19 days with the usual period
of 10 days.
D Paratyphoid ±< ever
1 General
Paratyphoid fever is a generalized
infection which is characterized by
an abrupt onset, with continued fever
and usually accompanied by diarrhea.
Fatality rates are much lower than
for typhoid fever and its incidence in
the United States has dropped along
with that of typhoid fever.
2 Infectious agent
Salmonella paratyphi, S. schottmuelleri,
S. hirshfeldii (paratyphoid bacilli A,
B, and C).
3 Reservoir
The'reservoir for paratyphoid fever is
man either as a patient or a carrier.
The source of the infectious agent is
urine or feces.
3-5
-------
Bacteriological Pathogens in the Aquatic Environment
4 Incubation period
One to 10 days and usually somewhat
longer for paratyphpid A than for B
and C.
Preventative measures, epidemic
measures and control of patients
contacts, and the immediate environ-
ment closely parallel that for typhoid
fever.
E Salmonellosis
1 General
Of the many clinical syndromes for
salmonellosis the three main types
are enteric fever, acute gastroenteritis,
and septicimia. Every Salmonella
strain is capable of initiating a disease
process. Table 4 lists the number of
cases and deaths attributed to
Salmonelloses during the years indicated.
2 Infectious agent
Numerous species of the genus Salmonella
of that group pathogenic for man and
animal. For reporting purposes the
primary human pathogens (typhoid and
paratyphoid fevers) are excluded from
this group by the national reporting
agencies although they are also
taxonomically part of the salmonellae.
The more common species in the
United States are S. typhimurium,
S. choleraesuis, S. newport,
S. oranienburg, S. montevideo,
S. panama, and S. anatum. On a
world evaluation ,S. typhimurium is
the most prevalent and this trend is
evident in Table 5 as reported by the
Salmonellosis Unit, NCDC.
3 Incubation period
In epidemics this is from 6 to 48 hours
and usually about 12 hours. In sporadic
cases this time is believed to be about
1 to 7 days.
Table 4
Reported Cases and Deaths Attributed to Salmonellosis
(excluding typhoid fever) in the United States

1968
1967
1966
1965
1964
1963
1962
1961
1960
No. of Cases
Reported
16514
18120
16841
17161
17144
15390
9680
8542
6929
No. of deaths. ..

63
73
87
67
72
62
64
82
3-6
-------
Bacteriological Pathogens in the Aquatic Environment
Table 5
10 Most Frequently Reported Salmonella
Serotypes Isolated from Humans and
Nonhumans
October, November, and December 1969
Serotype
Number
Percent
Human

typhi-murium *
1, 590
25. 1
enteritidis
534
8.4
newport
498
7.9
heidelberg
422
6.7
thompson
306
4. 8
saint-paul
3 03
4. 8
infantis
246
3.9
javiana
176
2.8
typhi
165
¦2.6

153
2.4
Subtotal
4, 393
69.5
Total all serotypes
6, 324

Nonhuman

typhi-murium**
354
12. 3
heidelberg
300
10. 5
saint-paul
192
6.7
anatum
188
6.6
cholera-suis K
172
6.0
thompson
101
3.5
derby
92
3.2
senftenberg
91
3.2
montevideo
87
3.0
infantis
85
3. 0
Subtotal
1, 662
57.9
Total all serotypes
2, 868

* Includes var. Copenhagen 73 1. 2
** Includes var. copenhagen46 1. 6
F Typhoid Fever
1 General
A systemic infection characterized by
continued fever, involvement of
lymphoid tissue, and constipation
more often than diarrhea. Many mild
inapparent infections remain
unrecognized and this group con-
tributes to the carrier population.
A fatality rate of 10% can be reduced
to 2 to 3% by proper antibiotic therapy.
An unusual feature of this disease is
that the typhoid bacilli are found in the
blood during the first two weeks and in
the. feces and urine after the second
week. Table 6 lists the number of
cases and deaths attributed to typhoid
fever in the United States for the
intervals shown.
2 Infectious agent
Salmonella typhi is the infectious bacilli
for typhoid fever. About 50'types are
distinguishable by phage typing'and this
procedure has proved invaluable in the
epidemiology of this disease.
Table 6
Reported Cases and Deaths attributed to Typhoid Fever in the United States
1968 1967 1966 1965 1964 1963 1962 1961 1960 1959
No. of Cases 395 396 373 454 501 566 608 814 816 859
Reported
No. of Deaths 12 15 6 14 21 15 17 21 22
3-7
-------
Bacteriological Pathogens in the Aquatic Environment
3 Reservoir
The reservoir is man in the patient or
carrier state. Infected urine and feces
are the sources of infection. The
carrier state is more prevalent in
persons over 40 years of age and
females are afflicted to a greater
extent. About 10% of patients will
excrete the bacilli as long as 3 months
after onset and about 2 to 5% will
become permanent carriers.
4 Incubation period
The usual range is from 1 to 3 weeks
and averages about 2 weeks.
IV RELATIONSHIPS OF PATHOGENS TO
WATER-BORNE OUTBREAKS
As was previously mentioned disease
processes cannot be strictly categorized
as solely water-borne.- Typhoid fever can
just as well, for instance, be transmitted
via food contamination, person-to person
contact, mechanical transfer by insects, etc.
The following ipformation is given to provide
a means of comparison for reported outbreaks.
It will be noted that in some cases the
infectious agent is unknown and it is common
to record this fact in several ways:
"unknown etiology"; gastroenteritis";
"unknown", and "other".
The following three tables (tables 7,-8, and 9)
are taken from Weibel, et al (6) and show
the incidence of water-borne disease, types
of illness and its relationship to various
water systems, and the principal causes of
water-borne disease outbreaks in the
United States. It will be noted that some of
the infectious agents are not bacterial but
are either viral or protozoan.
Table 7
Average Annual Number of Reported Disease Outbreaks in the United States, 1938-60
Years
Water-Borne Disease
All Other Sources of Disease
Outbreaks
Cases per
Outbreak
Outbreaks
Cases per
Outbreak
1938-40
45
583
201
34
1941-45
39
201
330
45
1946-50
23
121
360
33
1951-55
8
139
211
44
1956-60
7
121
254
41
3-8
-------
Bacteriological Pathogens in the Aquatic Environment
Table 8
Water-Borne Disease Outbreaks in the United States, 1946-60, by Type of Illness and System

Private or Semi-
Public Utilities
All Systems
Illness
Public Systems

Outbreaks
Cases
Outbreaks
Cases
Outbreaks
Cases
Gastroenteritis
92
'4,233
34*
9, 397
126
13,630
Typhoid
33
403
6
103
39
506
Infectious hepatitis
14
430
9
5 00
23
930
Diarrhea
7
320
9
4, 840
16
5, 160
Shigellosis
4
596
7
5, 057
11
5, 653
Salmonellosis
3
22
1
2
4
24
Amebiasis
2
36
0
0
2
36
Other
3
16
4
29
7
45
TOTAL
158
6, 056
70
19, 928
228
25, 984
* One gastroenteritis outbreak also included a typhoid case.
Table 9
Principal Causes of Water-Borne Disease Outbreaks in the United States, 192 0-36
Causes
No. of
Outbreaks
Percentage
of Outbreaks"
No. of
Cases
Percentage
of Cases**
Surface pollution of shallow wells
52
13
3, 403
3
Cross-connection with polluted supply
40
10
10, 636
9
Contamination of spring or infiltration
gallery by pollution on watershed
31
8
1, 185
1
Contamination of stream by pollution
on watershed
27
7
3, 283
3
Untreated water from river or irrigation
ditch
25
6
668
1
Inadequate chlorination when the only
treatment
23
6
4, 5 00
4
Inadequate control of filtration and
allied treatment processes
22
5
49, 410
12
Seepage of surface water into gravity
13
3
11, 354
10
conduits

TOTAL (principal causes)
TOTAL (all causes)
233
39?
58
100
84,439
116,000
73
100
* All outbreaks (399) from all causes (31).
**A11 cases (116, 000) from all causes.
3-9
-------
Bacteriological Pathogens in the Aquatic Environment
REFERENCES
1 MMWR, Vol. 12, No. 46, National
Communicable Disease Center.
2 Bull. Wld. Health Org. 1968, 39; 381-
388. G.I. Forbes and others.
3 MMWR, Vol. 19, No. 14, National
Communicable Disease Center.
4 MMWR Annual Supplemental Summary
1968. National Communicable Disease
Center.
5 Control of Communicable Diseases in
Man. 1965. American Public Health
Association.
6 Water-Borne Disease Outbreaks, 1946-60.
S. R. Weibel, F.R. Dixon, R. B.
Weidner and L. J, McCabe, JAWA,
Vol.-56, No. 8. August 1964.
This' outline was prepared by Rocco
Russomanno, Microbiologist, National
Training Center, WPO, EPA,
Cincinnati, OH 45268.
3-10
-------
TRANSMISSION OF VIRUSES BY WATER
I INTRODUCTION
A Certain viruses which are capable of pro-
ducing human disease are excreted into
domestic sewage in large quantities with
the feces of numerous individuals, many,
of whom are not ill. .With the demand for
water becoming increasingly acute in many
sections of the country, the reuse of sewage
is becoming a common practice and pre-
sents an imposing problem in public health.
B Only limited data are available regarding
the survival of viruses in various waters
and little more is known about their re-
sistance to disinfectants. Under certain
conditions, it may be possible for water
to be safe in terms of coliform density
and residual chlorine level and yet be
capable of initiating an enormous epidemic
of a viral disease.
C In rural areas small explosive outbreaks
of infectious hepatitis occur from time to
time which are traced to the consumption
of accidentally contaminated small water
supplies (e. g., wells, ponds, streams).
D The risk of transmitting viral diseases
through water under field conditions is
real. For this reason, disinfectants
prepared for use in drinking water and
wash water must be capable of quickly
destroying viruses.
II DEFINITION AND CHARACTERIZATION
OF VIRUSES
A Size - About 10 - 300 rruu.
B Structure - Nucleic acid core surrounded
by a protein coat (capsid) and sometimes
by an envelope, sperm-like (phage).
C All viruses are obligate, intracellular
parasites, many of which multiply only
in certain cell types of certain hosts. They
do not reproduce by themselves. Instead,
they channel the cells complex bio-
chemical systems into the production of
new viruses.
D Chemical composition - Some smaller
viruses may be composed of nothing but
nucleic acid and protein while some of
the larger viruses may contain more
complex substances in addition.
E Sensitivity to antibiotics - Except for the
larger more complex viruses of the parrot
fever group, viruses are completely
insensitive to all known antibiotics. In the
laboratory large quantities of antibiotics are
added to viral suspensions in order to
minimize hactprial contamination.
Ill TECHNICS AND PROBLEMS IN
GROWING VIRUSES IN THE LABORATORY
A Cell Culture
1 Simple to obtain
2 Various types are highly susceptible to
many viruses
3 Relatively inexpensive
B Animals
1 Some species are more susceptible to
some viral infections than known types
of cell cultures.
2 Much is known about animal susceptibility
to certain viral infections which have not
yet been studied to any great.extent in
cell cultures.
C Chick Embryos
This is an extensively used and very
sensitive host especially for members of
the influenza-mumps group of viruses.
D Human Volunteers
The obvious risk involved precludes the
use of this host except in very special
circumstances.
W. BA. 51a. 10. 71
4-1
-------
Transmission of Viruses by Water
E Detection of Virus in an Infected Host
1 Cell culture - Characteristic cell
destruction is produced by viruses and
is observable microscopically and
sometimes macroscopically.
2 Animals - Typical disease is produced
by viruses. Also an antibody rise
usually occurs.
3 Chick embryos - Death of the embryo
may result. Certain viruses can be
detected by mixing embryonic fluid's
with red blood cells. The presence of
certain viruses causes a typical aggluti-
nation of the red blood cells. Some
viruses produce pocks on the embryonic
membranes which are easily detectable
and countable.
4 Man - Same as for animals
F Problem of Latency
In cell cultures and in animals, latent
virus infections may occur. In this
circumstance, the presence of the virus is
not detected because cell destruction and
disease do not occur. Under some conr
ditions, however, the virus may suddenly
multiply actively and produce disease. If
this occurs subsequent to the inoculation
into cell culture or animals of a specimen
suspected of containing virus, the pre-
viously latent virus present in the host
may appear to have been present in the
specimen.
IV THE ENTERIC VIRUSES
A Definition of Enteric Viruses
For our purposes, enteric viruses may be
defined as those which may be demon-
strable in large quantities in the feces of
infected individuals and in sewage. They
include infectious hepatitis virus(es), the
enteroviruses (polioviruses, coxsackie-
viruses, echoviruses), the reoviruses,
and the adenoviruses. Most of these
viruses probably multiply in the gastro-
intestional tract.
Other viruses, such as influenza, mumps,
and Herpes simplex (cold or fever sore
virus) are found occasionally in the feces
as a result of being swallowed, and arc
probably not significant.
B Epidemiology of Enteric Virus Infections
1 Infections with these agents are wide-
spread in the normal population especially
during the summer and early fall.
Infection rates in any given area often
are greatly dependent on hygienic
conditions. Thus, infants and young
children of the lowest socioeconomic
groups usually suffer the highest attack
rate. This may range from under 5 to
60% or higher. In only a very few cases
does overt disease accompany infection.
Illness, when it occurs, may be as mild
as a "slight cold" or gastrointestinal
upset, or as severe as paralytic polio-
myelitis. Factors such as virulence
of the virus and individual resistance
play important roles in determining the
course of infections. Large quantities
of virus usually are shed in the feces of
infected individuals and are detectable
in domestic sewage.
2 Viruses have never been isolated from
adequately treated drinking water. It
is possible that small amounts of certain
viruses survive treatment and produce
sporadic cases or endemic foci of infec-
tion. Usually an explosive water-borne
disease outbreak occurs only when a
breakdown of good sanitary practices
occurs.
C Character of the Enteric Viruses
1 Infectious hepatitis virus
a Significance
This virus has not yet been propagated
demonstrably in laboratory animals or
in cell cultures. It can be demonstrated
only by producing disease in human
volunteers after oral inoculation.
4-2
-------
Transmission of Viruses by Water
Infectious hepatitis was a major
medical problem in World War II.
In recent years, tens of thousands
of cases have been reported annually
in the United States.
b The role of water in the transmission
of infectious hepatitis.
Infectious hepatitis appears to be
transmitted by the fecal-oral route,
usually by personal contact though
occasionally through the ingestion of
water contaminated by infectious feces.
Numerous documented waterborne
outbreaks of infectious hepatitis have
occurred in the U.S. and Alaska,
none of which involved more than
350 people. In one case, the pre-
sence of the virus in the drinking
water was demonstrated. These
drinking water supplies were either
unchlorinated or inadequately
chlorinated. The infectious hepatitis
outbreak in New Delhi', India, which
occurred in December 1955 and
January 1956, involved tens of thou-
sands of cases. The raw water
source, during the period of infection,
was heavily contaminated with sewage.
Contamination of the raw water was
known to have occurred and was com-
pensated for at the water treatment
plant. It is significant that during
the epidemic of infectious hepatitis
there was no concomitant increase
in any enteric bacterial disease.
This suggests that treatment of drink-
ing water adequate for eliminating the
enteric bacterial pathogens is not
necessarily adequate for destroying
the virus of infectious hepatitis.
2 Poliovirus
There are three serologically distinct
types of poliovirus, all of which are
easily propagated in cell cultures. The
intensive study of these viruses during
the last two decades hae resulted in the
Salk formalinized vaccine and in the
Sabin live-virus vaccine.
The polioviruses are responsible
primarily for paralytic poliomyelitis,
some aseptic meningitis (nonparalytic
poliomyelitis), and possibly for some
cases of mild gastrointestinal upsets
which occur in the summer and fall.
Poliovirus infection apparently is
transmitted by the fecal-oral route
primarily by personal contact. Only
two suspected water-borne outbreaks
have been reported to date. In both
cases, fecal contamination of the water
and inadequate chlorination were
believed responsible.
3 Coxsackieviruses
The coxsackieviruses are classified
into Group A (32 human serotypes)
and Group B (6 human serotypes) de-
pending upon the lesions they produce
in suckling mice. Some of the Group A
strains cause herpangina and aseptic
meningitis. The Group B strains
are responsible for pleurodynia, aseptic
meningitis and infantile myocarditis.
Both groups possibly cause diarrheal
disease in infants and young children
during the summer and fall. Trans-
mission of these viruses is probably
by the fecal-oral and respiratory
routes. No water-borne outbreaks due
to these agents have been reported.
4 Echoviruses (enteric cytopathogenic
human orphan viruses)
This is a group now composed of 32
serologically distinct agents which by
original definition are found in the feces
of infected individuals, produce cyto-
pathic changes in cultures of human and
simian renal tissues, and are not
associated with known human disease
(orphans in search of a disease). Since
the original classification, however,
several members of this group have
been clearly associated with many cases
of aseptic meningitis and "summer
rash". Members of this group, too,
may be responsible for cases of
diarrheal disease in the summer and
early fall. These viruses are transmitted
4-3
-------
Transmission of Viruses by Water
by the fecal-oral route but no known
water-borne outbreaks of disease due
to the echoviruses have been reported.
5 Adenoviruses
The classification of these 30 odd (human
strains) viruses is based on their com-
mon soluble complement fixing antigen.
Agents of this group produce a number
of acute respiratory diseases of the
cold-influenza type and at least two
types of eye infection. These viruses
appear to be responsible also for some
of the upper respiratory disease in
infants and young children. Trans-
mission of these viruses is probably by
the respiratory route. Several out-
breaks of a disease now called
pharyngoconjunctival fever, caused by
adenovirus 3, have occurred with which
chlorinated swimming pools have been
associated.
2 Gauze pad method
Gauze pad is immersed in flowing
sewage for varying lengths of time.
Virus is trapped in cotton and can be
eluted. Controlled tests have de-
monstrated that this method yields
more positive samples than the grab
sample technic but because it is impossible
to estimate accurately the amount of
sewage that flows through the pad, the
gauze pad method cannot be used for
quantitative determinations.
B Concentration Technics
1 Cliver technic (membrane filtration)
2 Shuval technic (two-phase separation)
3 Bier technic (forced-flow electrophoresis)
See References for details
6 Diarrheas and upper respiratory
disease of unknown etiology.
Apparently water-borne outbreaks of
diarrheal diseases have occurred in
tourists visiting summer resorts.
While not proved responsible, viruses
have been suggested as a possible cause.
It is probable that viruses produce
many sporadic and epidemic outbreaks
of gastrointestinal and upper respiratory
illnesses, and it is difficult to tell
presently how many of these outbreaks
are water-borne.
V ISOLATION OF VIRUSES FROM
SEWAGE
A Isolation
1 Grab samples
Sampling bottle is immersed in sewage
and sample is removed. Because a
definite sample volume is collected, this
method is useful in quantitative studies.
. C Results of Isolation Studies
Polioviruses, coxsackieviruses, echo-
viruses, reoviruses and adenoviruses have
been isolated from sewage. Peak
isolations occur during the summer and
early fall. The seasonal peak corresponds
well with the peak isolations of viruses
from healthy children and with the peak
occurrences of illnesses apparently
caused by viruses of these groups.
D Problems in Technics of Isolation and
Concentration
1 Technics are not sufficiently sensitive
and quantitative
2 Concentration technics may be specific
for certain viruses.
VI VIRUS SURVIVAL STUDIES
A A number of laboratory studies have de-
monstrated that several viruses of the
enteric group survive storage in certain
cool waters for as long as 6 to 7 months.
4-4
-------
Transmission of Viruses by Water
B Laboratory studies have shown that
flocculation, sedimentation, and filtration
do not completely eliminate several enteric
viruses from processed water.
C Chlorine
Chlorine, in the form of hypochlorous acid
(HOCI), is one of the; fastest disinfectants
available. However, chlorine exists as
HOCI only at relatively low pf-J and in the
absence of ammonia and organic materials.
As the alkalinity of a solution increases
beyond 1, HOC1 ionizes to form hypochlorite
ion(OCl") which possesses relatively little
germicidal activity. At pH 9 almost all
free chlorine is present as OC1".
Furthermore, when ammonia is present,
chlorine exists as some form of chlor-
amine. Figure 1 demonstrates the relative
bactericidal efficiencies of HOC'l, OC1",
and monochloramine. These data were
obtained by us from an analysis of avail-
able literature. At 2 - 6°C, at a given
concentration, OC1" requires about 66 times
as much time as HOC1 to destroy the same
amount of Escherichia coli. Monochlor-
amine requires about 300 times as much
time as HOC1 to achieve the same amount
of destruction.
MINUTES
RELATIONSHIP BETWEEN CONCENTRATION AND
TIME FOR 99% DESTRUCTION OF E COLI
BY 3 FORMS OF CHLORINE AT 2r6°C
Figure 1
While the rate ol destruction increases
considerably with rising temperature,
data at precise temperatures are not
available.
Figure 2 demonstrates the relative rates of
destruction of several viruses and E. coli.
These data were obtained by us from an an-
alysis of the studies of scve ral investigators
who worked under dissimilar conditions. The
different data can be compared directly with
each other only on a c rude approximation basis.
MINUTES
RELATIONSHIP BETWEEN CONCENTRATION AND
TIME FOP 99% DESTRUCTION OF E COL)
ANO 3 VIRUSES BY HYPOCHLOROUS
ACID ( HOCI) AT 0-6° C
i*3 ¦> c »* ' c:w*n 'i5 ¦
Figure 2
'The data with E. coli suggest that this
bacterium is destroyed more slowly than
adenovirus 3 by HOCI at 0 - 6°C. How-
ever, the poliovirus took more than 5 times
longer to destroy than the E. coli and the
coxsackievirus took more than 24 times
longer to destroy than the E. coli.
D Iodine
Elemental iodine (I2), too, is a very rapid
disinfectant though not as rapid as HOCI.
However, iodine, under most conditions,
does not react with ammonia to form
iodamines and is generally less reactive
than HOCI with organic material. In ad-
dition, the presence of color, in the absence
of excess iodide, is a. good indicator of the
presence of disinfecting potential.
Care must be taken in the preparation of
iodine solutions not to use any more iodide
4-5
-------
Transmission of Viruses by Water
ion (I~, usually in the form of KI or Nal)
than necessary to solubilizc elemental
iodine which is soluble only to the extent
of about 300 ppm at room temperature.
Both I" and triodide ion (1^ , which results
from the interaction of I2 and I ) have
little, if any, virucidal capacity.
As the pH of iodine solution increase from
6 to 8, I2 hydrolyzes (reacts with water)
progressively to form hypoiodous acid
(HOI) which is more virucidal than I2.
However, while more HOI forms as the
pH increases beyond 8, it also decom-
poses to form non-virucidal iodate ion
(IO3 ). Figure 3 demonstrates the
relative rates of destruction of several
viruses by elemental iodine.
REFERENCES
1 Berg, G. , ed. , Transmission of Viruses
by the Water Route. Intersciencc
Publishers, New York ( 1967).
2 Berg, G. Virus Transmission by the
Water Vehicle: I. Viruses. Health
Laboratory Science. 3:86 -89.' (1966).
3 Berg, G. Virus Transmission by the
Water Vehicle: II. Virus Removal by
Sewage Treatment Procedures. Health
Laboratory Science. 3:90- 100. (1966).
4 Berg, G. Virus Transmission by the
Water Vehicle: III. Removal of Viruses
by Water Treatment Procedures.
Health Laboratory Science. 3:170-181.
(1966).
This outline was prepared by G. Berg, Ph. D.
Chief, Virology Section, Advanced Waste
Treatment Research Laboratory, NERC,-
EPA, Cincinnati, OH 45268.
CONCENTRATION-TIME RELATIONSHIP FOR 99*
DESTRUCTION OF ENTEROVIRUSES BY 1, AT I5*C
Figure 3
4-6
-------
FILAMENTOUS BACTERIA
,1 INTRODUCTION
There are a number of types of filamentous
bacteria that occur in the aquatic environment.
They include the sheathed sulfur and iron
bacteria such as Beggiatoa, Crenothrix and
Sphaerotilus, the actinomycetes which are
unicellular microorganisms that form chains
of cells with special branchings, and
Gallionella, a unicellular organism that
secretes a long twisted ribbon-like stalk.
These filamentous forms have at times
created serious problems in rivers,
reservoirs, wells, and water distribution
systems.
II BEGGIATOA
Beggiatoa is a sheathed bacterium that grows
as a long filamentous form. The flexible
filaments may be as large as 25 microns wide
and 100 microns long. (Figure 1)
Transverse separations within the sheath
indicate that a row of cells is included in
one sheath. The sheath may be clearly
visible or so slight that only special staining
will indicate that it is present.
The organism grows as a white slimy or
felted cover on the surface of various objects
undergoing decomposition or on the surface
of stagnant areas of a stream receiving
sewage. It has also been observed on the
base1 of a trickling filter and in contact
aerators.
It is most commonly found in sulfur springs
or polluted waters where H^S is present.
Beggiatoa is distinguished by its ability to
deposit sulfur within its cells; the sulfur
deposits appear as large refractile globules.
(Figure 2)
containing granules of
sulphur
When H S is no longer present in the environ-
ment, tne sulfur deposits disappear.
Dr. Pringsheim of Germany has recently
proved that the organism can grow as a true
autotroph obtaining all its energy from the
oxidation of HgS and using this energy to fix
CO into all material'. It can also use
certain organic materials if they are present
along with the HgS.
Faust and Wolfe, and Scotten and Stokes
have grown the organism in pure culture in
this country. Beggiatoa exhibits a motility
that is quite different from the typical
flagellated motility of most bacteria, the
filaments have a flexible gliding motion.
BA. 8a. 12. 72
5-1
-------
Filamentous Bacteria
The only major nuisance effect of Beggiatoa
known has been overgrowth on trickling filters
receiving waste waters rich in The
normal microflora of the filter was suppressed
and the filter failed to give good treatment.
Removal of the H^S from the water by blowing
air through the water before it reached the
filters caused the slow decline of the
Beggiatoa and a recovery of the normal
microflora. Beggiatoa usually indicates
polluted conditions with the presence of HgS
rather than being a direct nuisance.
Figure 3 Filaments of Actinomycetes
Their filamentous habit and method of
sporulation is reminiscent of fungi. However,
their size, chemical composition, and other
characteristics are more similar to bacteria.
(Figure 4)
S 0 \
- \ Figure 4
U v, \
q O \ albumin isolation plate
0 O I 'A' an actinomycete colony,
® / and 'B' a .bacterial colony
^ o
-------
system, the odors often are present before
the water enters the plant. Use of perman-
ganate oxidation and activated carbon filters
have been most successful of the methods
tried to remove the odors from the water.
Control procedures to prevent the odorous
material'from being washed into the water
supply by rains or to prevent possible develop-
ment of the actinomycetes in water rich in
decaying organic matter is still needed.
IV FILAMENTOUS IRON BACTERIA
The filamentous iron bacteria of the
Sphaerotilus- Lept othrix group, Crenothrix,
and Gallionella have the ability to either
oxidize manganous or ferrous ions to manganic
or ferric salts or are able to accumulate
precipitates of these compounds within the
sheaths of the organisms. Extensive growths
or accumulations of the empty, metallic
encrusted sheaths devoid of cells, have
created much trouble in wells or water dis-
tribution systems. Pumps and back surge
valves have been clogged with masses of
material, taste and odor problems have
occurred, and rust colored masses of
material have spoiled products in contact
with water.
Crenothrix polyspora has only been examined
under the microscope as we have never been
able to grow it in the laboratory. The orga-
nism is easily recognized by its special
morphology. Dr. Wolfe of the University of
Illinois has published photomicrographs of
the organism. (Figure 5)
Organisms of the Sphaerotilus- Leptothrix
group have been extensively studied by many
• investigators (Dondero et. al., Dondero,
Stokes, Waitz and Lackey, Mulder and van
Veen, and Amberg and Cormack, ) Under
different environmental conditions the mor-
phological appearance of the organism varies.
The usual form found in polluted streams or
bulked activated sludge is Sphaerotilus natans.
(Figure 6)
-------
Filamentous Bacteria
This is a sheathed bacterium consisting of
long, unbranched filaments, whereby individual
rod-shaped bacterial cells are enclosed in a
linear order within the sheath. The individual
cells are 3-8 microns long and 1. 2 -1 .-8
microns wide. Sphaerotilus grows in great
masses; at times in streams or rivers that
receive wastes from pulp mills, sugar
refineries, distilleries, slaughterhouses,
or milk processing plants. In these conditions,
it appears as large masses or tufts attached
to rocks, twigs, or other projections and the
masses may vary in color from light grey to
reddish brown. In some rivers large masses
of Sphaerotilus break loose and clog water
intake pipes or foul fishing nets. When the
cells die, taste and odor problems may also
occur in the water.
Amberg, Cormack, and Rivers and McKeown
have reported on methods to try to limit the
development of Sphaerotilus in rivers by
intermittant discharge of wastes. Adequate
control will probably only be achieved once
the wastes are treated before discharge to
such an extent that the growth of Sphaerotilus
is no longer favored in the river. Sphaerotilus
grows well at cool temperatures and slightly
low DO levels in streams receiving these
wastes-and domestic sewage. Growth is slow
where the only nitrogen present is inorganic
nitrogen; peptones and proteins are utilized
preferentially.
Gallionella is an iron bacterium which appears
as a kidney-shaped cell with'a twisted ribbon-
like stalk emanating from the concavity of the
cell. Gallionella obtains its energy by
oxidizing ferrous iron to ferric iron and uses
only CC>2 and inorganic salts to form all of
the cell material; it is an autotroph. Large
masses of Gallionella may cause problems
in wells or accumulate in low-flow low-
pressure water mains. Super chlorination
(up to 100 ppm of sodium hypochlorite for
48 hours) follow,ed by flushing will often
remove the masses of growth and periodic
treatment will prevent the nuisance effects
of the extensive masses of Gallionella.
(Figure 7)
REFERENCES
Beggiatoa
1 Faust, L. and Wolfe, R.S. Enrichment
and Cultivation of Beggiatoa Alba.
Jour. Bact. , 81:99- 106. 1961.
2 Scotten, H. L. and Stokes, J. L.
Isolation and Properties of Beggiatoa.
Arch Fur, Microbiol. 42:353-368.
1962.
3 Kowallik, U. and Pringsheim, E. G.
The Oxidation of Hydrogen Sulfide by
Beggiatoa. Amer. Jour, of Botany.
53:801-805. 1966.
A ctinomycetes and Earthy Odors
4 Silvey, J. K. G. etal. A ctinomycetes and
Common Tastes and Odors. JAWWA,
42:1018-1026. 1950.
5 Safferman, R. S. and Morris, M. E.
A Method for the Isolation .and
Enumeration of Actinomycet.es Related
to Water Supplies. Robert A. Taft
Sanitary Engineering Center Tech.
Report W-62- 10. 1962.
5-4
-------
Filamentous Bacteria
6 Gerber, N.N. and Lechevalier, H.A.
Geosmin, an Earthy-Smelling Substance
Isolated from Actinomycetes. Appl.
Microbiol. 13:935-938. 1965.
Filamentous Iron Bacteria
7 Wolfe, R.S. Cultivation, Morphology, and
Classification of the Iron Bacteria.
JAWWA. 50:1241- 1249. 1958.
8 Kucera, S. and Wolfe, R. S. A Selective
Enrichment Method for Gallionella
ferruginea. Jour, Bacteriol, 74:344-
349. 1957.
9 Wolfe, R.S. Observations and Studies
of Crenothrix polyspora. JAWWA,
52:915-918. I960.
10 Wolfe, R.S. Microbiol. Concentration
of Iron and Manganese in Water with
Low Concentrations of these Elements.
JAWWA. 52:1335-1337. 1960.
11 Stokes, J. L. Studies on the Filamentous
Sheathed Iron Bacterium Sphaerotilus
natans. Jour. Bacteriol. 67:278-291.
1954.
14 Dondero, N. C. Sphaerotilus, Its Nature
and Economic Significance. Advances
Appl. Microbiol. 3:77- 107. 1961.
15 Mulder, E.G. and van Veen, \V. L.
Investigations on the Sphaerotilus-
Leptothrix Group. Antonie van
Leewenhoek. 29:121-153. 1963.
16 Amberg, H. R. and Cor mack, _ J. F.
Factors Affecting Slime Growth in
the Lower Columbia River and
Evaluation of Some Possible Control
Measures. Pulp and Paper Mag. of
Canada. 61:T70-T80. 1960.
17 Ambei'g, H. R., Cormack, J. F. and
Rivers, M.R. Slime Growth Control
by Intermittant Discharge of Spent
Sulfite Liquor. Tappi. 45:770-779.
1962.
18 McKeown, J.J, The Control of
Sphaerotilus natans. Ind. Water
and Wastes. 8:(3) 19-22 and
8:(4)30-33. 1963.
12 Waitz, S. and Lackey, J. B. Morphological
and Biochemical Studies on the
Organism Sphaerotilus natans. Quart.
Jour. Fla. Acad. Sci. 21(4):335-340.
1958.
13 Dondero, N.C., Philips, R.A. and
Henkelkian, H. Isolation and
Preservation of Cultures of Sphaerotilus.
Appl. Microbiol. 9:219-227. 1961.
This outline was prepared by R. F. Lewis,
Bacteriologist, Advanced Waste Treatment
Research Laboratory, NERC, EPA,
Cincinnati, OH 45268.
-------
WATER QUALITY CRITERIA FOR RECREATION AND AESTHETICS
I INTRODUCTION
A Legal Aspects
The following discussion is based upon the
Water Quality Act of 1965 wherein a partial
statement of Paragraph 3, Section 10
states:
"Standards of quality established
pursuant to this subsection shall be
such as to protect the public health
or welfare, enhance the quality of
water and serve the purposes of this
act. . . "
Recommended criteria in'relation to the
above is stated in this outline from the
Report of the Committee on Water Quality
Criteria of the Federal Water Pollution
Control Administration dated April 1, 196 8.
B Definitions
For a knowledgeable evaluation of these
recommendations, it is necessary to be
aware of the use of the following terms:
1 STANDARD - A plan that is established
by governmental authority as a program
for water pollution prevention and
abatement.
2 CRITERIA - A scientific requirement
on which a decision or judgment may be
based concerning the suitability of water
quality to support a designated use.
3 PRIMARY CONTACT RECREATION -
Activities in which there is a prolonged
and intimate contact with the water
involving considerable risk of ingesting
water in quantities sufficient to pose a
significant health hazard.
4 SECONDARY CONTACT RECREATION -
Activities not involving significant risks
of ingestion.
C Preliminary Objectives
The subcommittee recommendations were
based upon the preliminaiy objectives of:
1 Recommend water quality criteria for
recreation and aesthetic uses, and
2 Identify research needs and priorities
relating to water quality for recreation
and aesthetic uses.
D Microbiological Considerations
The basis for the microbiological con-
siderations for the Water Quality criteria
have been due to:
1 Careful consideration of the lengthy
historical background of the total
coliforms and its relationships to the
fecal coliform group of organisms.
2 Epidemiological studies of-
a Great Lakes (Michigan), and
b Inland River (Ohio)
These studies have indicated an
epidemiologically detectable health
effect at levels of 2, 300 - 2, 400
coliforms per 100 ml and a later
study of the Inland River established
the fecal coliform load at 18% of the
total coliform. Based upon these
findings the indications are that the
detectable health effects may occur
at a fecal coliform level of about 400
per 100 ml and a factor of safety would
use this level as a starting point
c Santee project ¦
This work correlated the prevalence
of virus with fecal coliform con-
centrations following sewage treat-
' ment. Following secondary treatment
W. Q. STO. 3a. 11. 72
6-1
-------
Water Quality Criteria For Recreation and Aesthetics
the virus levels can be expected to
be 1 PFU (plaque forming unit) per
ml with a ratio of one virus particle
per 10, 000 fecal coliforms.
II CRITERIA DEVELOPMENT
A Aesthetics
Since natural conditions vary widely,
recommendations are based upon a series
of descriptive rather than numerical
criteria. The following requirements
have been recommended:
1 General requirements
a All surface waters should be capable
of supporting life forms of aesthetic
value
b Surface waters should be free of
substances attributable to discharges
or wastes as follows:
1) Materials that will settle to form
objectionable deposits
2) Floating debris, oil, scum, and
other matter.
3) Substances producing objectionable
color, odor, taste, or turbidity
4) Materials, including radionuclides,
in concentrations or combinations
which are toxic or which produce
undesirable physiological responses
in human, fish, and other animal
life and plants.
5) Substances and conditions or
combinations thereof in concen-
trations which produce undesirable
aquatic life.
2 Desirable additional requirements
a The positive aesthetic values of
water should be attained through
continuous enhancement of water
quality. This is based upon the
concept that water quality standards
are designed to enhance the quality
of water and consequently the
aesthetic values are enhanced by
continuing improvement in quality
conditions in microbiological,
chemical, and physical terms.
b The aesthetic values of unique or
outstanding waters should be
recognized and protected by develop-
ment of appropriate criteria for
each individual case.
Aesthetic considerations should be
continually desired objective and
the recommended standards should
be borne in mind when waters are
examined for recreation as they are
not a separate entity from the overall
desired water quality
3 Research needs
a Greater evaluation of key factors
for limited waters within restricted
areas' Extensive research on a
national level would be unwarranted
due to the variance in numerical
values from community to community
b Improved control over discharges of
materials producing objectionable
changes.
c Studies on the economical and
sociological aspects as they relate
to land values and human acceptance
of water for various usage.
B Recreation
1 Considerations
Historical developments and sub-
committee recommendations have
been guided by the precepts of health
and safety for the recreation user.
Criteria have been recommended to:
a Provide for and enhance general
recreation use of surface waters,
b Enhance recreation value of waters
designated for recreation use, and
6-2
-------
Water Quality Criteria for Recreation and Aesthetics
c Provide special protection for the
recreation user where significant
body contact with water is involved,
2 Surface waters
Surface waters should be suitable for
use in secondary contact recreation
without reference to official designation
of recreation as a water use. It should
be noted that this consideration will
provide an impetus for the enhancement
of water quality in many waterbodies
which fall below this desired surface
water quality Table 1 lists the
recommended criteria for general
recreational use of surface waters.
It will be noted, from previous
discussion of microbiological con-
siderations, that the microbiological
criteria is based upon fecal coliform
densities which further consider that
these surface waters are devoid of
primary contact recreation and there-
fore have a risk considered to be one-
tenth that which has primary contact
recreation.
TABLE 1
GENERA L RECREATIONAL USE OF SURFACE WATERS
(Without reference to specific designation of recreation as a water use)
Enjoyment of Recreation
Microbiological Activities Harvest Species

Fecal Coliform:

Average not to exceed
Recommendations of the
Sanitation of Shellfish

2000 per 100 ml
National Technical
Growing Areas ( 1965)
Recommended
Criteria
Maximum Value (except
in specific mixing zones
adjacent to outfalls)
4000 per 100 ml
Advisory Subcommittee
on Fish, Other Aquatic
Life, and Wildlife
U. S. Public Health
Service
3 Waters designated for recreation uses
Waters within this category are those
which, for water quality management
purposes, have been placed in an area
designated for recreation uses but where
primary recreation does not occur.
This category is deemed necessary to
keep water quality at a high level and
yet not have the stringent criteria which
is necessary to those waters providing
primary recreation contact. Table 2
lists the recommended criteria for
Waters Designated for Recreation Uses.
Recommended
Criteria
TABLE 2
WATERS DESIGNATED FOR RECREATION USES
{Other than primary contact recreation)
Microbiological
Enjoyment of Recreation
Activities
Fecal Coliform:
Should not exceed a log
mean of 1000 per 100 ml
Never equal or exceed
2000 per 100 ml in more
than 10% of the samples
Recommendations of the National
Technical Advisory Subcommittee
on Fish, Other Aquatic Life, and
Wildlife
6-3
-------
Water Quality Criteria for Recreation and Aesthetics
Waters designated for primary contact
recreation
Based upon the Microbiological
Considerations previously mentioned
the following recommendations in
Table 3 apply to Waters Designated
for Primary Contact Recreation.
Among the additional criteria for these
waters, which are deemed to be desirable
but not mandatory, are the ones relating
to clarity and temperature.
TABLE 3
Clarity - The basis for this rec-
ommendation is that of visual appeal,
recreational enjoyment, and safety.
The clarity should be such that a
Secchi disc is visible at a minimum
depth of 4 feet except in the learn-
to-swim areas where it should be
visible when resting on the bottom.
Temperature - Experience with
military personnel indicates that
exposure to warm water continuously
for several hours can be safely
tolerated with,a maximum tempera-
ture of 30oc (85°F) and these limits
are recommended.
WATERS DESIGNATED FOR PRIMARY CONTACT RECREATION
Microbiological

Fecal Coliform:

Shall not exceed a log mean
Within the range of 6 5-8 3

of 200 per 100ml (based on a
except when due to natural

minimum of not less than 5
causes and in no case shall

samples taken over not more
be less than 5. 0 nor more
Recommended
than a 30-day period)
than 9. 0
Criteria
Not more than 10% of total
When the extended ranges of

samples during any 3 0-day
the natural causes are con-

period shall exceed 400 per
sidered, any discharges

100 ml.
which increase the buffering

capacities are to be limited
5 Research needs
a Additional research on indicator
organisms from specific sources
to more effectively monitor water
quality control programs.
b Development of techniques that will
indicate potential presence of
pathogenic viruses.
c Studies of the marine or estuarine
environment associated with
indicator organisms.
d Work on pathogenic organisms in the
applied research area.
e Degree to which the various pathogens
are waterborn and, as such, the
infective doses which will initiate
the disease processes.
f Temperature and injuries as they
relate to recreation users.
g Managing water quality for secondary
contact recreation users as related
to its impact on health
h Treatment and control of wastes
affecting recreation uses of
receiving waters.
6-4
-------
Water Quality Criteria for Recreation and Aesthetics
i Development of improved methodology
for disinfection as these effluents
effect recreational waters.
IH CONCLUSIONS
A Criteria
It must be realized that the problem facing
the establishment of any criteria relating
to water quality must be that of balancing
reasonable safeguards for public health
while guarding against placing undue
restrictions upon the available water-
source in present or future planning.
The most pressing problem which directly
effects the setting of such standards is the
lack of precise knowledge regarding
microbiological pollution and its relation-
ship to public health. Since this is the
present situation and much remains to be
ascertained in future epidemiological
studies the criteria is based on the three
previously mentioned studies and, in the
light of future developments, the standards
caln be revised to reflect more complete
knowledge.
B Subcommittee Membership
The conclusions relating to the establish-
ment of Criteria for Water Quality in
Aesthetics and Recreation were drawn by
the following group of distinguished
individuals:
Mr. Leland J. McCabe, Assistant Program
Chief for Disease Studies and Basic Data,
Water Supply and Sea Resources Program,
Public Health Service, U.S. Department
of Health, Education, and Welfare,
Cincinnati, Ohio.
Mr. John C. Merrell, Jr., Chief, Southern
California Field Station, Federal Water
Pollution Control Administration, U. S.
Department of the Interior, Garden Grove,
California.
Mr. Eric W. Mood, Assistant Professor of
Public Health, Chief, Environmental
Health Section, Department of Epidemiology
and Public Health, Yale University School
of Medicine, New Haven, Conn.
Mr. Harold Romer, Director, Department
of Air Pollution Control, City of New York,
and Professor of Environmental Pollution
Control, Long Island University, Brooklyn,
New York.
Mr. Roy K. Wood, Chief, Division of Water
Resources Studies, Bureau of Outdoor
Recreation, U. S. Department of the
Interior, Washington, D.C
Dr. Richard T. Gregg, Technical Executive
¦ Secretary, Federal Water Pollution
Control Administration, U.S. Department
of the Interior, Washington, D.C.
Mr. R. Frank Gregg, Chairman, New England
River Basins Commission, Boston, Mass.
Dr. Leonard Duhl, Special Assistant to the
Secretary of Housing and Urban Develop-
ment, Washington, D.C.
Mr. Clarence W. Klassen, Chief Sanitary
Engineer, Illinois Department of Public
Health, Springfield, 111.
Mr. William J. Lucas, Assistant Director,
Division of Recreation, National Forest
System, Forest Service, U.S. Department
of Agriculture, Washington, D. C.
6-5
-------
Water Quality Criteria for Recreation and Aesthetics
C Bibliography
The following lists a selected bibliography
which have been instrumental in the con-
clusions rendered by the subcommittee:
Bureau of Outdoor Recreation. 1967.
Superintendent of Documents, U.S.
Government Printing Office, Washington, D. C.
Coetze, O.J. 1961. Comments on sewage
contamination of coastal bathing waters.
South African Med. J. 35:261.
Committee on Science and Astronautics,
Subcommittee on Science, Research, and
Development. 1966. U.S. House of
Representatives, 89th Cong., 2nd sess.
Geldreich, E. E. 1966. Sanitary significance
of fecal coliforms in the environment.
U. S. Department of the Interior, Federal
Water Pollution Control Administration.
WPC Res. Ser. Publ. No. WP-20-3.
Moore, B. 1959. Sewage contamination of
coastal bathing waters in England and
Wales. A bacteriological and epidemiological
study. J. Hyg. 57(4):435.
Outdoor Recreation Resources Review
Committee. 1962. Outdoor recreation
for America, Superintendent of Documents,
U.S. Government Printing Office,
Washington, D.C.
Public Health Activities Committee, Sanitary
Engineering Division. 1963. Coliform
standards for recreational waters. Amer.
Soc. Civil Engr. , Proc. 57-94.
Smith, R.S., Woolsey, T.D. and
Stevenson, A.H. 1961. Bathing water
quality and health, I. Great Lakes.
U. S. Public Health Service,
Environmental Health Center, Cincinnati,
Ohio.
Smith, R.S., and Woolsey, T.D 1952.
Bathing water quality and health, II. Inland
river. Environmental Health Center,
Cincinnati, Ohio.
Smith, R S. , et al. 1961. Bathing water
quality and health, III. Coastal waters.
Environmental Health Center, Cincinnati,
Ohio.
Stevenson, A.H. 1953 Studies of bathing
water quality. Amer. J. Pub. Health
43(5): 5 29.
A CKNOW LEDGMENT:
The majority of this outline must be con-
sidered an abridgement of the Report of the
Subcommittee on Water Quality Criteria,
FWPCA, USDI, April 1, 1968, Section 1,
Recreation and Aesthetics, which should be
consulted for more complete rationale.
This outline was prepared by R. Russomanno,
Microbiologist, National Training Center,
WPO, EPA, Cincinnati, OH 45268.
6-6
-------
WATER QUALITY SURVEYS
ORGANIZING THE STREAM SURVEY
I INTRODUCTION
A Theoretical Approach
1 This discussion will present as a base,
the procedures for the conduct of an
ideal water quality survey.
B Practical Limitations
1 In practice, limitations of personnel,
facilities, time and money, always
will require compromise between the
ideal and the possible.
C Objectives of Quality Studies
1 The four basic objectives behind the
almost limitless number of possible
reasons for water quality studies are:
a Determination of the natural water
quality of the stream
b Measurement, in a selected and
limited period of time, of the
existing effects of wastes on water
quality and uses
c Procurement of data on waste loads,
water quality and stream charac-
teristics that will permit projection
of the data to describe probable
water quality and effects on uses
under a variety of conditions other
than those that prevailed during the
study.
d Determination of corrective meas-
ures needed to protect the stream
water quality for proper uses.
WP. SUR. 9f . 3. 74
II PRELIMINARY PLANNING
A Available Information
Assemble and review all readily avail-
able maps, information, and.reports
bearing on the stream under consid-
eration .
B Problem and Objectives of Study
1 Define the problem requiring study
as completely as possible on the
basis of available information.
C Tentative Plan of Study
1 Prepare a brief preliminary plan of
study for guidance in the subsequent
field reconnaissance.
2 Include in.the preliminary plan:
a Locations and strengths of known
sources of waste
b Locations of areas of water use
and a list of legitimate water
uses
c Section of major stream and
locations of important tributaries
and unique features such as dams
and points of major diversions
d Possible sampling stations
e Sampling frequency and number of
samples at each station
-------
Water Quality Surveys
f Types of laboratory determinations
g Existing stream gauging stations and
additional points where stream flow
data are needed.
h Other hydrological data needed and
possible means of procurement
i Potential laboratory locations
j Special supplies and equipment
k Personnel requirements
1 Approximate cost of field operations
III PRELIMINARY FIELD OPERATIONS
A Importance
This step is often neglected, but a small
advance party of two or three experienced
field men can obtain information and make
preparations that will save time and money
and avoid much confusion and possible
error when the subsequent field study
starts.
B Local Contacts
Contact local agencies and individuals who
use or have knowledge of the stream, and
assemble available maps, reports,
operating records, and verbal information.
C Sources of Wastes
1 Sewage
a Obtain best available estimate of
sewered population.
b Examine sewage treatment plant, if
any, and obtain copies of operating
records on volumes and characteristics
of sewage.
c Locate all points of discharge and
make any installations necessary for
sampling and gauging.
2 Industrial wastes
a . Investigate and prepare flow diagram
of process with especial reference
to points of water use and waste
discharge, and quantities of raw
materials and finished products.
b Obtain information on plant-operating
schedule, with especial attention to
daily or seasonal variations, and to
any anticipated changes in process
or increase in production.
c Obtain data on water consumption,
numbers of plant employees by work
shifts, and disposal of domestic
sewage.
d Investigate waste treatment facili-
ties, if any, and obtain operating
records on volumes and character-
istics of wastes.
e Locate all points of waste discharge,
trace origins in plant process, and
make any installations necessary for
sampling and gauging.
f Collect samples for preliminary
examination in laboratory to permit
selection of proper analytical
procedures.
D Stream
1 Observe entire reach of stream involved
by wading, walking bank, or boat.
2 Note especially points of waste discharge
and dispersion patterns, visual evidences
of pollution confluences and mixing of
tributaries, stream flow characteristics,
approximate widths and depths, and
locations of prospective sampling
stations, and of water uses.
3 Collect bottom samples of biological
organisms and bottom deposits, and
observe evidences of algae.
7-2
-------
Water Quality Surveys
a Bottom organisms serve as an index
of degree of pollution, and of length
of river that should be sampled.
b Bottom deposits indicate extent of
sludge deposits.
c Algae indicates probable significance
of photosynthesis and possible need
for night sampling or light-dark
bottle technique for full evaluation
of dissolved oxygen variations.
4 Determine times of water travel between
pertinent points on stream.
a Knowledge of times of water travel
not only will permit the most intelli-
gent conduct of study, but also is
essential to the complete analysis,
interpretation, and projection of
data.
b Time and effort involved are a minor
portion of the totals devoted to the
stream study, and once established,
need not be repeated in subsequent
studies.
c Observations should be made at three
or more stream flows to develop a
time of travel versus stream dis-
charge curve, which will permit
interpolation for any desired stream
flow.
d Methods include: tracing an inherent
or added variable stream constituent,
timing surface or submerged floats;
and determining stream cross sections
at selected intervals.
5 Select and identify sampling stations
a For DO evaluation on the majority of
streams, there should be: one station
just above each major waste source;
stations at about half day intervals for
two days time of travel below waste
sources; and stations at about one
day intervals for an additional three
to four days time of travel.
b For coliform evaluation, the stations
should be similar to those for DO
except that the downstream stations
at one day intervals should extend
to a total of 10 to 20 days time of
water travel below the source of
wastes.
c In very small or very turbulent
streams, natural purification may be
well advanced in as little as one to
two days time of water travel and
stations should be at much closer
intervals than suggested above.
d If tributary streams are important,
there should be one station near the
mouth of each tributary and one on
the main stream just above .each
confluence.
e Stations should be established as
close as practicable to points or
9.reas of important water uses.'
f At stations wnere wastes ana tributary
waters are well mixed, one sampling
point near mid-channel usually is
adequate.
g At stations where mixing is inadequate,
common practice is to sample at the
quarter points of the stream.
h Identify stations by adequate descrip-
tion and marking, and make any
necessary preparations for use by
sampler.
l Select best access routes to sampling
stations and make a round-trip to
stations to determine time required
for sampling.
E 'Preliminary Stream Samples
1 Collect one or more sets of stream
samples for preliminary examination.
a Dissolved oxygen results will be
useful in establishing sampling
stations and length of stream that
should be studied
7-3
-------
Water Quality Surveys
b Determinations of coliform organisms
and BOD will assist in selection of
proper dilutions for subsequent use
when study is started.
c Determinations of other constituents
will enable analyst to select most
acceptable laboratory procedures and
to determine sizes of sample portions
necessary for those analyses in which
concentrations of constituents govern
choice of portion sizes.
F Laboratory Location
Examine potential laboratory locations in
relation to convenience to the field opera-
tions, adequacy of space and utilities,
storage space, and revisions or additional
facilities that will be needed.
G Miscellaneous
1 Locate local sources of supplies that
will be purchased during study.
2 Determine transportation routes, and
schedules for shipments of samples,
supplies, and materials.
3 Arrange for local personnel and special
facilities that will be used.
4 Investigate rooming facilities conven-
ient to the field operations.
IV FINAL PLANNING
A Revision of Preliminary Plan
With the preliminary plan as a guide, and
with the knowledge gained from the pre-
liminary field operations it will be possible
to prepare an intelligent, workable plan
for the field study.
B Objectives of Study
Redefine, add to, or delete from, the
initial list of objectives, and prepare a
set of specific objectives which will
include provisions for all essential
answers to the problem at' hand, but will
also eliminate needless expenditure, on
nonessential matters, of effort which
might better be devoted to the principal
problem.
C Period of Field Operations
1 The overall period selected for the
study should be at the time of year when
past experience indicates that 'stream-
flows usually are relatively stable.
2 For evaluation of DO'depletion; a period
of warrrvweather and low streamilow
is desirable.
3 For evaluation of coliform contamination,
a period of intermediate flow may be
desirable.
4 A period of intensive round-the-clock
sampling for a few days generally is
preferable to a period'of a month of
sampling-daily or on alternate days, but
a combinationof both "methods may be
better than either one.
D Sampling
1 General
a If sources of wastes do not vary
significantly during period of study,
the wastes may be sampled in
advance of the stream study.
b Sampling procedures and preserva-
tion of samples should follow
"Standard Methods for the Examin-
ation of Water and Wastewater" and/or
procedures recommended by the
investigators agency.
2 Sewage
a Collect samples around-the-clock
at 15 minute to 1 hour intervals for
three to seven' days.
b Composite sample portions in portion
to flow for three to four equal time
periods of each day.
7-4
-------
Water Quality Surveys
3 Industrial wastes
a In general, the better known the
process is, and the less variable
the waste discharge, the less de-
tailed need the sampling be.
b For well known processes with little
variation, equal sample portions
collected at one-half to one hour
intervals and composited for eight
hours or for a complete cycle of
operation on three to five days should
be adequate.
c For little known and variable pro-
cesses, detailed and prolonged
sampling may be necessary, with
samples collected at 5 to 15 minute
intervals, and composited in pro-
portion to flow for six to 24 hour
periods for seven or more days or
complete cycles of operation.
4 Stream
a If streamflow, waste discharge, or
oxygen production by algae vary
widely throughout the day, sample
around-the-clock at several stations
for at least one or two days to
establish the daily cycle of variation
in waste constituents and effects.
b In most streams, grab samples each
day or on alternate days for two weeks
to a month is adequate if conditions
remain stable.
c The total number of samples collected
from a single station during a field
study generally ranges between 15
and 20. In the absence of wide
variations in streamflow, 12 to 15
sets of samples usually yield
adequate, usable results.
d If sampling is not around the clock,
sample collection should be varied
throughout the day as much as is
feasible, and as a minimum, the
direction of the sampling trip should
be reversed on alternate days.
e When more than one point is sampled
at a station, individual field deter-
minations are made and a separate
bacteriological sample is taken at
each point, but samples for other
determinations may be combined in
a single composite. Bacteriological
samples from two or more points at the
same station may be composited in the
laboratory.
E Gauging
1 Methods of waste and stream gauging are
covered in another reference outline.
2 Generally stream discharge records
are obtained from existing U. S.
Geological Survey stations, or from
special stations established and rated
by them upon request.
3 Streamflow data at each main stream
sampling station generally can be
computed with sufficient accuracy from
records for one or two main stream
gauging stations and one for each
major tributary.
F Laboratory Operations
1 Select principal determinations that
will measure pertinent waste con-
stituents and their effects, and
auxilliary determinations that will
contribute to interpretation of principal
results.
2 Reject determinations that would be
"interesting" but would not contribute
to solution of the problem.
3 Generally useful principal determinations
common to many stream studies are
those for coliform bacteria, bottom
organisms, temperature, DO, and BOD,
and auxiliary determinations frequently
are for pH, alkalinity, and turbidity.
Other determinations must be selected
for special purposes and for special
types of wastes.
5 Laboratory methods should follow
procedures recommended in "Standard
Methods for the Examination of Water
and Wastewater. "
6 Avoid excessive overload of the labora-
tory by working closely with the
7-5
-------
Water Quality Surveys
laboratory supervisor during the
planning stage and accepting his
estimate of the volume of work that
can be handled.
7 In establishing the laboratory work
load, allow time for calculation of
analytical results at the end of each dav.
8- Ship samples to the headquarters
laboratory for all determinations of
stable or preserved constituents that do
not have to be made in the field.
G Personnel
1 The field crew should possess com-
petencies for the specific tasks involved,
or should be trained in advance if
necessary.
2 Personnel needed commonly will include:
a Sanitary engineers
b Chemists
c Bacteriologists
d Biologists
e Sampling and laboratory aides
H Supplies and Equipment
1 Prepare and use a check list of needed
field supplies and equipment.
2 All operations that can be performed
in advance at headquarters, such as
training, purchases, equipment repair,
and reagent preparation will save money
and valuable time in the field.
I Cost Estimate
1 Revise the initial estimate of cost of
field operations in light of the final
plan adopted.
2 Adjust the final plan'to fit the money
available for the study, or arrange
'for th'e additional money needed.
V FIELD OPERATIONS
A Briefing
1" Assemble the field crew and explain
the problem, objectives, and details
of field operations.
2 Take at least key personnel on a tour
of pertinent physical features of the
study.
3 Specify responsibilities of individuals
for the various phases'of the operation.
B Communications
Arrange a system of communication by
which any member of the crew can be
contacted within a reasonable length of
time.
C Records
1 A few simple field and laboratory
forms will serve to systematize the
maintenance of basic records.
2 Keep all permanent field and laboratory
records in bound volumes.
3 Encourage the recording of other than
routine observations by both sampling
and laboratory personnel.
4 Write down at once, 'and do not depend
on memory for all significant
observations.
D Revision of Operations
1 Review all daily data accumulated at
the end of each day, and note especially
any omissions or unusual results.
7-6
-------
Water Quality Surveys
2 On the basis of the daily review,
consider the need to:
a Add new determinations if needed.
b Omit determinations that are not
showing significant results.
c Revise analytical methods.
d Change sampling stations or
scheduled times of sampling.
e Investigate causes of apparently
abnormal or erroneous data.
3 Do not revise operations unless the
data or other new information indicates
changes are essential to achieve the
desired objectives.
VI USE OF DATA
A Analysis and Interpretation
1 The raw data are of only limited value
until submitted to analysis and inter-
pretation by an experienced person who
is familiar with the problem involved.
2 Methods of analysis and interpretation
constitute a major subject in them-
selves and are covered by other
lectures.
3 No amount of statistical manipulation
of the data can produce sound con-
clusions unless the stream study is
soundly conceived, and carefully arrd
conscientiously executed.
C Ultimate Disposal
After use, the data should be filed, in-
dexed, and describ d so that they can
be available and understandable years
later to others not directly associated
with the study
D Maximum Usefulness
The results of a stream pollution study
yield their maximum benefit and return
their greatest satisfaction to those who
worked to obtain them only when the
results serve as a basis for actual cor-
rection of an abuse of stream water
quality.
REFERENCE
Kittrell, F.W. A Practical Guide to
to Water Quality Studies of Streams.
USD!. FWPCA. CWR-5. 1969.
This outline was prepared by Francis W,
Kittrell, former Special Consultant,
National Field Investigations Center,
--Cincinnati Office of Enforcement and
General Counsel, EPA, Cincinnati,
OH 45268
Descriptors: Baseline Studies, Data
Collections, Hydrologic Data, On-Site
Data Collections, On-Site Investigations,
Streams, Surveys, Planning, Sampling,
Stream Pollution, Water Quality
B Report
A report should be prepared if the data
are to achieve their maximum usefulness,
since raw data in dead files only rarely
benefit anyone.
7-7
-------
WATER QUALITY SURVEYS
ROLE OF BACTERIOLOGIST
I INTRODUCTION
The determination of bacteriological quality
of water should include active participation
of the bacterial analyst in all phases of the
survey, from the planning stages, through and
including the performance of the survey and
the final preparation and presentation of the
finished report.
The principal bacteriologist may be a pro-
fessional bacteriologist serving as the prin-
cipal survey bacteriologist, a bacteriological
consultant, or, in practice, these duties
sometimes may be assumed by a qualified
engineer or scientist nominally representing
some other professional discipline.
Chief responsibilities of the principal bacteri-
ologist in a survey are summarized in the
following paragraphs.
II IDENTIFICATION OF BACTERIOLOGICAL
IMPLICATIONS OF SURVEY OBJECTIVES
A Through consultation with survey manage-
ment and review of survey objectives, he
determines what specific pollution problems
are related to bacteria.
B A determination is made of the kind of
bacteriological information which must be
developed in order to meet survey
objectives.
C The related chemical and physical tests
are identified in terms of their expected
need with respect to interpretation of
bacteriological data.
Ill DETERMINATION OF PROCEDURES
In oi'der to develop the kind ,of bacteriological
information required to meet s'lrvey objec-
tives, the following decisions must be made,
and pertinent information obtained
A The specmc indicator organisms used to
detect and evaluate pollution must be
selected.
B The methods used for their
quantitative measurement must be deter-
mined.
C Consultations must be included with re-
spect to bacteriological sampLing.
1 The sample points must be established.
2 The frequency of bacteriological testing
at each point must be determined.
3 The location of proposed sampling
stations with reference to assumed
influences on bacterial levels must be
reviewed. These influences may b,e
alleged pollution sources, entrance of
diluting waters, drainage areas, or,
in some cases, industrial drainage or
outfalls.
D Any existing data on bacterial densities,
as well as water flow velocity and volume
should be obtained and studied.
E Based on the sampling plan and determin-
ations to be made, a protocol of bacteri-
ological sample examination is developed.
Figure 1 is an example of such a protocol,
used in a recent water pollution survey.
W. BA. 47a. 3. 74
7-9
-------
Water Quality Surveys
STREAM SAMPLE
Lauryl tryptosa broth (1)
(Presumjtive test)*
Incubate 2u hours at 35*C
MF on it£ Strepto-
coccal Agar (3)
Incubate U8 hours at 35°C
1
1.
Incubate 2& hours at }$*C
no gas
discard - no
conforms present
Easy.
1
Tear
rl
Elevated Tenper-
ature test (EC).
>Incubate 2li hours
at 44. 5 + 0.2 C .
Count red or pink colonies,
using 10-20 diameter magni-
fication.
I
V
Brilliant green bile
lactose broth* (2).
Incubate I18 hours at 35 "C
Record as fecal streptococcal
count per 100 ml
—)no gas
fecal coliform group
absent (negative)
^'gas - fecal coliform group
gas present (positive),
positive test record as fecal coliform
coliforms present MPN per 100 ml
no gas
negative test -
coliforas absent
calculate confirmed test MPN ¦
per 100 ml.
* Indicates procedure described as standard test In StandBrd^Methods^JFor^thi5_Exan]inatlon^of
Water and Wastewater, 13th e(j. (1971)
(1). Lactose broth may be substituted for lauryl tryptooe broth.
(2). Eosln Methylene blue or Endo agar may be substituted for the brilliant green bile lactose broth.
(3). The ty Agar pour plate count may be used interchangeably with the HF colony counting technique.
Figure 1
IV REVIEW OF BACTERIOLOGICAL PLANS
The bacteriological plan is reviewed for
adequacy to accomplish the objectives
of the survey, based on a "paper review" and
on preliminary samples which should be
examined at the selected sampling stations if
at all feasible.
A Sufficient qualified personnel for the labora-
tory load should be provided. Estimates of
work loads are shown in Part 2 of this
chapter.
B The detailed methods for all procedures
are reviewed with all personnel responsi-
ble for collection and handling, trans-
mission to the laboratory, and with the
laboratory personnel responsible for
sample testing procedures.
C The duties assigned to all personnel must
be reviewed with all concerned, work
scheduled.reviewed and understood by all.
Care must be taken to assure that assigned
work is consistent with training, ability,
and experience of individuals concerned.
D Location and quality of the laboratory
facilities must be reviewed to ascertain
that sample testing procedures will be in
accordance with established plan, standard
procedures, and to assure that adequate
work space is available.
E Laboratory equipment and supplies must
be adequate for the expected work loads.
Glassware and other supplies which must
be used on a rotational basis should, in
general, be 3^ - 4 times the expected
daily need.
V REVIEW OF LABORATORY OPERATIONS
After the established daily work of the survey
has gotten well underway, the principal
bacteriologist should review operations based
on the plan established as the final plan for
survey operations. Typical check points
include:
7-10
-------
Water Quality Sui-vev.s
Review of sample collection.and handling
procedures and assurance that there is
minimum delay between collection and
starting testing procedures on individual
samples.
Evaluation of Laboratory Work
1 Determination of whether the methods
actually used are in conformance with
plans.
2 Elimination or justification of deviations
from planned procedures.
3 Problems which have arisen during the
operations are discussed, particularly
with reference to matters not anticipated
when planning the survey.
Data are reviewed to determine validity
for use in the survey, in terms of
reliability of results.
a When membrane filter, or plate count
methods are used, check the correla-
tion between occasional duplicate or
triplicate plate count for
reproducibility.
b When multiple dilution tube methods
are used, check the percentage of the
usual codes, according to the method
described by Woodward and Walton
(JAWWA, 1957,'49: 1060- 10G8, "How
Probable is the Most Probable Number?")
Table 1. STATISTICAL EXPECTANCY OF POSITIVE TUBE COMBINATIONS FOR A SERIES OF
5 PORTIONS PLANTED IN EACH OF 3 DECIMAL DILUTIONS
Expectancy
Positive tube
combinations

Frequency of occurrence
group
constituting group

By group
Cumulative

%
%
Group I (Frequent)
100 200 300
400
500
550
67. 5
67. 5

510
551

520
552

530
553

540
554

Group II (Common)
1 10 210 310
410
511

23. 6
91.1 ,

420
521

531

541

542

Group IIT
001 101 201
301
401
501
7. 9
11
99. 0
(Uncommon)
010 120 211
311
411
502

020 220
320
421

330
430
522

431
532

440
533

543

544

Group IV (Rare)
:A11 other positive tube combinations
1. 0
100. 0
Ref.: Richard L. Woodward and Graham Walton. 195 7.
-------
Water Quality Surveys
Table 2. ACTUAL POSITIVE TUBE COMBINATIONS
CLASSIFIED BY EXPECTANCY GROUP
PLANT A. RAW WATER COLIFORM TEST
Positive tube j j j
combination j Number of l II 1 III i IV
portions p an e j (.imes found Frequent Common' Uncommon 1 Rare ¦
in each of 3 decimal ; !
dilutions) ; [
100 ! 1
110 ! 1
200 1 2
220 j 1
: 300 !. 6
1 !
1
2 : . ;
6 1 ;
1
301 1
310 3
400 8
401 ; 1
i 410 12
I
l i
3
8 ^
1
12 i i
1 ; ; !' j
j 411 ; 2 ! 2 i
j 412 1 ; 1"
! 420 4 4 !
| 430 2 ; 2
j 440 j 1 i 1 :
500 j 13
510 j 11
511 1 2
512 1
i 520 ; 9
is ; j
ii ; :
2 j i
1
9 !
; 521 ; 2
530 : 9
531 : 3
540 ¦ 3'
| 541 ; 5
2
9
1
3
3
5

; 542 1 : 1
550 2 2, ,
551 1 j 1 ¦ |
Total No. ! 108 65 33 j 9 j 1
Percent of total 1 100. 0 ; 60. 3 30. 5 J 8. 3 ; 0. 9
Cumulative percent | 100 60. 3 | 90. 8 i 99. 1 100. 0
i I 1
Ref. : Richard L. Woodward and Graham Walton. 1957.
7-12
-------
Water Quality Surveys
c Data sometimes show apparent
anomalies which necessitate addi-
tional test procedures or special
tests on selected samples in order
to account for seemingly unusual
results or to establish the validity
of the results being obtained.
VI INTERIM REVIEW OF DATA
It is essential that periodic review of data
be conducted during the survey. It is sug-
gested that such reviews be conducted daily
during the initial stages of the survey and
thereafter at weekly or biweekly intervals.
A Review can reveal the need for changes in
procedures to meet the objectives of the
survey.
B Changes needed in sampling pj.an can be
revealed. According to the type of results
being obtained; it may be necessary to add
further sampling stations, to delete certain
stations, or to modify the schedule of sam-
ple collection.
C Need for additional test procedures may be
demonstrated.
D Interim data may show that certain test
procedures are producing data of no value
in meeting survey objectives, such proce-
dures can be dropped.
VII FINAL REVIEW OF DATA AND PREPARA-
TION OF REPORT
Development of the survey plan, performance
of the planned operations to meet survey
objectives, and the final data analyses and
preparation of report are interdependent
activities.
There are many techniques available for
summarizing and presenting data for interpre-
tation of findings. This topic is discussed at
greater length in the outline titled "Presenta-
tion and Interpretation of Bacteriological Data "
7-13
-------
Laboratory Operations
I INTRODUCTION
Part 1 of this outline has been concerned with
planning the bacteriological aspects of a sur-
vey. With this part of the outline, certain
specific recommendations are presented with
respect to perspnnel qualifications and work
schedules, desired location and features of
laboratory facilities, arid the care and handling
of samples prior to starting test procedures.
II PERSONNEL
A Skill Levels
1 The principal bacteriologist should have
at least 3-5 years of professional
experience in the sanitary bacteriology
of water. The duties of the principal
bacteriologist, who may or may not
personally perform the laboratory pro-
cedures, have been identified in Part 1
of this outline.
2 Subordinate bacteriologists preferably
should have at least 1-2 years of
working experience in water
bacteriology.
The laboratory bacteriologist is re-
sponsible for the correct testing of
samples according to predetermined
plans and for preparation of accurate
records of results, in an orderly
manner. Nonprofessional laboratory
assistants, if employed, must be
closely supervised in all duties
undertaken.
3 Nonprofessional laboratory assistants
may be needed. In a short-term,
highly intensive survey, the writer is
reluctant to employ any but experienced
laboratory helpers. Such experience
can best be gained in a fixed laboratory
where close supervision and direction
are available for routine operations than
would be possible with the short-term
intensive survey operations.
Duties of nonprofessional laboratory
assistants include washing and
sterilization of glassware, preparation
of sample bottles and related supplies,
preparation and maintenance of culture
media supplies, and'related duties.
B Work Loads
1 Based on the assumption of
utilization of highly skilled,
fast, -laboratory workers, the
bacteriologist-consultants of
many water quality surveys have
recommended a maximum of 20
coliform determinations per
man-day of bacteriology labora-
tory service'. This includes
media preparation and other
laboratory support service.
2 Additional tests may require adjust-
ment of work loads.
a Fecal coliform tests require a
slight addition of time. For
planning purposes however,
coliform tests plus fecal coliform
tests have been considered to
permit the 20-samples per
man-day. Probably 16 samples
per man-day is more realistic.
b The addition of fecal streptococcus
tests to the coliform and fecal
coliform tests requires major
readjustment in the number of
daily samples. With this series
of tests, a maximum of 10-14
samples can be examined per
man-day of laboratory service.
3 When considering above work loads,
the customary 8-hour day may not
be possible.
7-14
-------
Water Quality Surveys
For a short-term, highly intensive,
survey there usually is little difficulty
in operating on this basis, provided
that personnel are motivated to the task
at hand. With long-term investigations,
personnel levels should be held at such
levels as to permit normal work-days
and work-weeks with off-duty days
provided at periodic intervals.
Ill LABORATORY FACILITIES
A Location
The requirement for prompt examination
of samples after collection demands
attention to the location of laboratory
facilities.
1 With suitable transportation arrange-
ments it may be possible to perform
examinations in established water
bacteriology laboratories.
This is to be preferred if at all possible.
Recently, increasing attention has been
given to air transport of samples as a
means of resolving this problem.
2 With limitations on available trans-
portation it more commonly is neces-
sary to establish a temporary labora-
tory. This may be temporary
space in established laboratories, as
in local or hospital laboratories, or it
may be necessary to use a mobile trailer
laboratory.
3 It is not practiced to conduct the entire
bacteriological examination of water in
stream surveys under field condi-
tions, even with membrane filter
methods. In some cases, however, it
may be necessary to inoculate samples
into primary tube media or on mem-
brane filters in the field. In such sam-
ples, temporary incubation must be
provided (no icing of inoculated sam-
ples) for transport of the sample to the
laboratory where the remainder of the
incubation and subsequent laboratory
procedures are performed.
B Space
The bacteriology laboratory requires
provision for several functions, each of
which requires a significant amount of
space. To give assurance that adequate
space is provided, the following functions
must be considered:
1 Bench space
Bench space of the type used for chemi-
cal determinations is suitable for
bacteriological work. Unless the num-
ber of bacteriological samples i's quite
small, the bench space should be re-
served for this function. About six
lineal feet of bench space per worker
is a minimum allowance.
2 Preparation area
If the number of daily samples is low,
it may be feasible to make and dispense
culture media, prepare sample bottles,
etc. , at the laboratory bench. If the
work load is large, one general area
should be reserved for media prepara-
tion, washing and sterilization of glass-
ware, and other supporting functions.
3 Incubation and sterilization
Space must be provided for certain
fixed laboratory equipment, such as
washing equipment, dry sterilizer and
autoclave, water bath, and incubator,
according to the work load anticipated
for the survey.
IV SUPPLIES
A Source
In survey planning, consideration should
be given to preparing all culture media
and reusable supplies in a central labora-
tory, and transporting such supplies to the
field laboratory site. The practice fre-
quently is more economical than efforts to
provide such support at a field site.
7-15
-------
Water Quality Surveys
B Sample Bottles
1 All sample bottles mast be clean,
sterile, and free of substances, un-
favorable to bacterial survival.
Although a wide variety of containers
is acceptable, the preferred form is
a glass-stoppered, wide-mouth boro-
silicate glass bottle, having about 250
ml capacity.
2 Some samples, such as effluent from
some waste treatment plants, may con-
tain residual chlorine. In such cases,
enough sodium thiosulfate is introduced
into the clean sample before steriliza-
tion to give 100 mg/l thiosulfate in the
sample. For example, add 0. 2 ml of a
10% solution of sodium thiosulfate to a
250 ml bottle such as described above.
This will dechlorinate samples contain-
ing up to about 23 mg chlorine per liter.
The bottle is dry or moist heat sterilized
following introduction ot the sodium
thiosulfate.
Laboratory tests have shown' that this
amount of sodium thiosulfate has no
adverse effect on the survival and
growth of coliform bacteria or fecal
streptococci.
V SAMPLE COLLECTION AND HANDLING
A Collection
1 In many cases, sampling is limited to
surface grab samples. The opened
bottle is plunged into the water to a
depth of about one foot. The sample
bottle never should be filled more than
one-half to three-fourths full. This is
to facilitate mixing by shaking when
laboratory tests are started.
2 If but one sample is taken from each
station at the selected intervals, it
usually is collected from .near the
center of the main channel of flow.
Preliminary tests may demonstrate
need for multiple samples from,each
station. In this case, it may be neces-
sary to collect samples from predeter-
mined points across the body of water
and/Or a[t'designated depths. Such
samples are not composited and
retain-the^r identity as single samples
throughout the laboratory testing
procedure.
3 The sample collection is made in such
a manner as to insure obtaining" a sam-
ple representative of the source. Thus,
if sampling is from a boat, the bottle
should be dipped into the upstream side
of the boat, with tne mouth of the bottle
also directed upstream. If there is no
discernible current, then the bottle is
moved through the water in a direction
away from the hand holding the bottle.
4 If depth sampling is indicated, it is nec-
essary to use special depth samplers,
and open them at the desired depth.
B Identification
1 All samples must be immediately
and fully identified at the time
of collection, including at least
the sample location, and the date
and time of sampling. Many workers
use a supplemental sheet to record
temperature, pH and other data.
2 The sample tag should be affixed to the
sample bottle. If the bottle has perma-
nent identifying marks, a supplemental
sheet may be used to identify the sample
3 Wax pencils are not satisfactory. In-
delible pencils are preferred. Any
marking'material that will run if wetted
will be unsatisfactory.
C Care in Transit
1 Time between sample collection and
starting tests
a The test procedures should
ideally be run immediately, or
if this'is not feasible,' should
preferably be started within 1
hour after sample collection;
it never should exceed 8 hours
(six hours transit time and two
hours laboratory).
7-16
-------
Water Quality Surveys
b With some samples, such as those
from heavily polluted streams, even
greater limitation on this interval
is necessary.
2 Temporary storage
With temporary storage of samples,
"Standard Methods" now recommends
the practice of icing samples prior
to examination.
This outline was prepared by H. L. Jeter,
Director, National Training Center, WPO,
EPA, Cincinnati, OH 45268
Descriptors: Personnel, Planning, Labor-
atory Equipment, Laboratory Tests, Micro-
biology, Sampling, Surveys, Water Quality
7-17
-------
WATER QUALITY SURVEYS
PRESENTATION AND INTERPRETATION OF BACTERIOLOGICAL DATA
I OBJECTIVES
Bacteriological data analysis consists of an
or'derly assembly and summary of the data
obtained in the investigation-, in the simplest
available manner, leading to demonstration
and explanation of the observed results in
terms of the initial survey-objectives. The
topics of summary of data and interpretation
of data are discussed separately in this
presentation; however these, are mutually
interdependent activities.
A There are many ways in which data can be
summarized and analyzed. The most
commonly used procedures are described
in this outline.
B A number of criteria apply to the selection
of procedures for data analysis:
The analysis must be -
1 Understandable by readers of the report;
2 Consistent with survey objectives,
3 Accurate summary of data, and
4 Representative of technically sound
reasoning.
II PRESENTATION OF QUANTITATIVE DATA
A Data on coliform bacteria, fecal coliforms,
and fecal streptococci must be presented
on a quantitative basis if useful interpreta-
tions are to be drawn. Partition
counting of coliforms (IMViC typing) or
streptococci (biochemical characterization
or species identification) should be done on
a quantitative basis, through study of a
representative number of ( 100 for
example) pure cultures, with determination
of percentage of occurrence of each
identified variant.
B Development of Expression of Central
T endency
1 Single central value
a Median
The median is determined by assem-
bling all data in an array of ascend-
ing or descending order. The median
value is the central value, a posi-
tional value in the array.
b Arithmetic mean, or average. This
is much influenced by individual
extremes.
c Geometric mean. (Logarithmic
mean)
Many workers prefer use of this
value, as it includes all determinate
values but minimizes the effects of
extremely high or extremely low
values.
d In selecting the form of expression
of central value, it is necessary to
know the methods of calculation re-
quired in meeting established stand-
ards. For example, geometric
mean values often are lower than
arithmetic mean values on identical
data. If the standards of quality are
based on use of arithmetic mean
values, it would be necessary to use
the arithmetic mean value in the
data analysis.
2 Distributions of values
a Data may be calculated and arranged
on a distribution table. The method
has particular usefulness when the
investigation is related to designated
standards of water quality.
WP. SUR. 30g. 3. 74
7-19
-------
Water Quality Surveys
b The central value may be obtained
by one of the above methods, plus
showing the maximum value at the
designated sampling point.
c The central value may be obtained,
plus a percentage range for the data
from a single location. Some
investigators re'commend use of the
geometric mean value, plus the
range of the middle 80 percent of all
values from the data being con-
solidated. The method permits
expression of average water quality
(geometric mean) and the maximum
and minimum quality demonstrated.
Exclusion of the extreme 10 percent
of high and low values reduces the
danger of misinterpretation by
ascribing particular significance to
the sporadic extremes of values
which may not be truly representative
of quality of the water.
C Values Used to Describe Numbers of
Organisms
1 Number per 100 ml of water. This is
direct use of data obtained in laboratory
work, and may be a Most Probable
Number value; or it may be a value
obtained by membrane filter methods
or plate count methods in which a
simple calculation is made to relate the
number of colonies to the volume of
sample tested, computing the number
of test organisms per 100 ml.
This form of data expression appears
to be most applicable to investigations
for compliance with established water
quality standards, or to trace pollution
indicators when there is a relatively
fixed volume of water.
2 In stream surveys where there may be
multiple sources of pollution with
changes in volume of the receiving
water through waste outfalls, or
juncture with other streams, it may
be necessary to determine the total
number of bacteria and trace their
fate in the investigation.
Because the numbers of organisms may
reach nearly astronomical proportions,
and to simplify presentation, consoli-
dated values often are used.
a Bacterial Quantity Unit (QU) is the
number of bacteria passing a given
point in one day if the concentration
is 1000 organisms per milliliter,
and the stream flow is 1 cubic foot
per second. One QU is equivalent
to 2. 45 X1012 bacteria.
b The Bacterial Population Equivalent
(BPE) is another useful tool for
consolidating data and representing
the total number of organisms in
the water being studied. This value
does not represent the number of
coliform bacteria discharged by an
individual but, instead, represents
a value obtained by determining the
relationship between the number of
coliform bacteria passing some down-
stream point and the number of
individuals in the community dis-
charging wastes into the flowing
waterbody. The BPE (calculated
sewered populations) are derived
by converting flows of cfs to flows
of 100 ml/day, multiplying by
density of coliforms (MPN/100 ml)
and dividing the total numbers by
400 billion/day (summer data) or
125 billion/day (winter data).
Summer (15 C. or more):
BPE = Q (cfs) XMPN/100 ml X(G. 1 X 10~ )
Winter (less than 15 C. )
BPE = Q (cfs) XMPN/100 ml X(l. 95 X 10 )
7-20
-------
Water Quality Surveys
D Methods of Presentation of Data
1 Distribution tables (Table 1)
Table 1. Data Presentation by Showing Distribution of Values
MPN over
2
10
50
100
250
500
750
1000
2500
no. tests
50
49
49
36
31
6
1
1
0
percentage
100
98
98
72
62
12
2
2
0
2 Circles, histograms, or other geometric
figures superimposed on maps to show,
by relative dimensions, relative values
obtained.
3 Graphs
4 Development of mathematical expressions
to describe death rate of the organisms.
a The method involved determination of
a peak value for coliforms at some
point ipimediately downstream
(10 - 15 hours flow time) of the point
of discharge.
b The decreasing numbers of bacteria
often can be described by the
equation
Y = A X 10_bt + C X10_dt
Where:
Y = the fraction of the peak number
of bacteria remaining after the
time, t_;
A = the fraction of the bacteria out
of the peak number which decrease
at a rapid rate;
b = the rate at which the "A" fraction
of bacteria decrease;
C = the fraction of the bacteria out
of the peak number which decrease
at a different (lesser) rate; and
d = the rate at which the "C" fraction
of bacteria decrease.
The values of the rate coefficient b
and d are determined on the basis of
the coliform data, using techniques
similar to those, used for development
of the rate coefficient k in the BOD
equation. (See Figure 1)
Figure 1 • —Rates of coliform decrease below five selected cities.
7-21
-------
Water Quality Surveys
III INTERPRETATION OF FECAL COLIFORM -
FECAL STREPTOCOCCUS RELATIONSHIPS
A Development of Ratios
Using the single central-tendency values
from each sampling station in'a survey,
it often is useful to determine the ratio of
fecal coliforms to fecal streptococci. If,
for example, at a given station the fecal
coliform value is given as 72, 000 per 100
ml and.the fecal streptococcus value is
16, 000, then the fecal coliform/fecal
streptococcus ratio is 4. 5.
B Interpretation of Ratios
1 When the fecal coliform/fecal strepto-
coccus ratio is greater than 4. 0, this
is regarded as overwhelming evidence
of pollution derived from human origin;
or that if the pollution is of mixed
origin, the majority of such pollution
is of human origin.
2 When the fecal coliform/fecal strepto-
coccus ratio is less than 0. 7, this
suggests pollution derived predominantly
or entirely from livestock or poultry
wastes. Feedlots, stockyards, and even
stormwater runoff usually produce such
ratios.
3 Ratios falling between 4.0 - 0. 7 are
not quite so certain. To be sure, a
ratio of 3.5, for example, would be
more suggestive of pollution represent-
ing predominantly human origin; and a
ratio of 0, 9 would be more suggestive
of animal origin. A truly "gray-area"
of interpretation of these ratios is in
the range 2.0 to 1.0.
a When the ratio is in this range, it
frequently represents significant
mixtures of both human and animal
contribution, or
b The source of pollution may be some-
what remote, and due to differences
in the rates of disappearance of the
two bacterial groups, the original
numerical relationships have been
obscured.
4 Limitations on interpretation of fecal
coliform - fecal streptococcus ratios.
a The ratios have greatest reliability
for samples taken not more than 24
hours flow time (or distance) from
the origin of the pollution, and
b The ratios must be based on waters
in pH range between 4.0 - 9.0.
c Total coliform counts cannot be
usea in determination or
interpretation of ratios with fecal
streptococci.
IV PRESENTATION OF QUALITATIVE DATA
A For some determinations, notably demon-
strations of pathogenic microorganisms,
quantitative methodology either is lacking
or is so expensive and time-consuming as
to make sure tests completely impractical.
B In surveys involving pollution of intestinal
origin, much attention is being given cur-
rently to the demonstration of pathogenic
bacteria, notably bacteria of the genus
Salmonella. Such organisms, when found,
are interpreted to represent positive proof
of deleterious bacteriological quality of the
water, since all members of the genus •
are disease-causing bacteria.
C The FWQA
survey report of the Red
River of the North presented data on
occurrence of Salmonella as shown in
Table 2 and Figure 2.
7-22
-------
Table 2. SALMONELLA ISOLATIONS - RED RIVER OF THE NORTH
Distance from

River
waste source
Flow time
Total
Fecal

Date Station
mile
(miles)
(days)
coli/ 100ml
coli/ 100ml
Salmonella isolated
Moorhead

sewage

treatment

Sept. 1964 plant
448
0

Not done
Not done
S.
kentucky
RR 11
436
12
0. 5
250,000 .
64,500
S.
kentucky
RR 12
426
22
1. 0
47, 600
2, 850
S.
saint paul
Nov. 1964 RR 9
462
-14

314
49
Absent
RR 10
441
7
. 3
432,000
155,000
S.
typhimurium

S.
braenderup

S.
reading
RR 11
436
12
. 4
249,000
85,600
s.
braenderup

s.
heidelburg

s.
reading
RR 12
426
22
. 8
68,000
16,800
s.
blockley

s.
braenderup
RR 16
386
62
3. 0
6, 630
1, 610
s.
heidelberg
RR 28
292
7
. 3
39,800
2, 970
s.
reading
RR 29
286
13
. 7
27, 800
1, 030
s.
reading

s.
infantis '
Jan. 1965 RR 10
441
7
. 2
162,000
61,000
s.
Chester

-S.
thompson

s.
oranienburg

-S.
st. paul
RR 11
436
12
. 4
83, 500
34,000
s.
Chester

s.
heidelburg

s.
st. paul

s.
enteritidis

s.
typhimurium
SH 13
428- 1

650
218
Absent
RR 14
416
32
1. 2
18,600
9, 170
s.
enteritidis

s.
Chester

s.
st. paul

s.
thompson
RR 16
386
62
3. 3
7, 800
5, 160
s.
st. paul

s.
enteritidis

s.
thompson
RR 18
375
73
4. 0
6, 140
2, 950
s.
st. paul'

s.
thompson
-------
Water Quality Surveys
Figure 2. Location of Sampling Stations and Station Numbers (River Miles) of Samples from the
Red River of the North, North Dakota and Minnesota
7-24
-------
Water Quality Surveys
V CASE EXAMPLES
The following studies illustrate the ways in
which certain pollution surveys became
needed, the examinations made, and the ways
in which data were summarized and presented,
leading to the final conclusion.
A Case Study #1
1 Need
To determine whether the outfalls
discharging raw sewage from a
sewered population of 3820 persons
in Villageville to River A between
miles 20 and 18, causes a deterioration
of water quality, thus constituting a
hazard to the city water plant of
Statesville with intake located at river
mile, 15.2 and the County Water Plant
with the intake located at river mile
13.0.
2 Survey and procedures
A survey was established for the study
of the reach of the river which included
the pollution in the river above the
alleged polluting city, pollution con-
tributed from a tributary creek, the
pollution contributed by Villageville
and the effect on water quality at the
water plant intakes. Samples were
collected and examined every six hours
for seven consecutive days. Bacteri-
ological procedures were the Standard
Methods multiple tube procedure by the
confirmed test for the coliform group
using three acceptable dilutions of 5/5/5;
the test for coliforms of fecal origin;
and the tentative plate count procedures
for fecal streptococcal group and their
confirmation by supporting biochemical
tests.
3 Results
These data are presented in Table 3
and Figure 3.
4 Interpretation of the data
a The bacteriological data obtained at
sample points located at river miles
24 to 21 inclusive, established the
water in the river as being of
relatively good quality, with total
coliform density of approximately
600, 20 to 30 fecal coliforms and 10 to
16 fecal streptococci per 100 ml. This
is a relatively high-quality raw-water
river supply in this area.
b The Clear Creek tributary was an
overflow from a lake with a coliform
density of approximately 2 0 per
100 ml and both fecal coliforms and
fecal streptococci were absent. In
the absence of data on the cfs of
Clear Creek and of River A, it is not
possible to partition the bacterial
densities at river miLe 2 0 but it is
evident that there is an apparent
improvement in water quality due
to the dilution factor.
c Between river mile 20 and 18, a
marked increase in pollution
occurred with 25, 000, 10, 000 and
4, 000, respectively, being the
densities of total coliform, fecal
coliform and the fecal streptococcal
groups. Periodic sampling of the
sewer outfalls indicated the domestic
waste from Viilageville was the source
of the bacterial densities.
d The bacterial data at river miles 16
to 10 inclusive demonstrated the poor
quality of the water due to the pollution
originating from the untreated wastes
entering the river from "Villageville.
7-25
-------
Water Quality Surveys
e The presence of the fecal coliform
group proves that this portion of the
total coliform group originated from
the gut of warm-blooded animals and
is present in large quantities.
f The streptococcal group was proven
by a series of biochemical reactions
to be identical with the fecal
streptococcal group found in the gut
of warm-blooded animals and there-
fore confirmed the interpretation of
the fecal coliform group.
g The presence of fecal pollution may
be at any time and frequently is
associated with enteric pathogenic
bacteria, viral agents and parasitic
organisms and by these associations,
is a hazard to health.
h The presence of fecal pollution, as
indicated by the data in Table 3,
constitutes an unnecessary and
remedial hazard and risk in the
raw water supplies to the water
plants,
Table 3. INDICATOR MICROORGANISMS PER 100 ml IN RIVER A
i i I Fecal
! River mile ¦ Total Coliform . Fecal Coliform I i Remarks
i , | ibtreptococci
24
610
i 30
26
i
23
620
! 23
16
i
I
i
22
600
: 28
10

21
590
i 23
j 10
i
20
310
!
; 13
Clear Creek enters at ;
river mile 20. 4.
19
2, 000
, 600
1 180
i
Four sewer outfalls
18
25,000
10,000
1
; 4,000
i
between river miles
19. 7 and 18.5 from
17

Villageville.
16
20, 000
o
o
O
O
i 4,200
•
15

i
Statesville water intake
at mile 15. 2
14
17,000
9, 000
! 3,900

,
County water plant
[

intake at mile 13. 0
12
j 14,000
9,200
1
: 3, 940

11
•
1
I
I
1
i
i
j
10
10,000
j 8, 900
I 3, 700
¦

EXPLANATION OF DATA
Bacterial densitities calculated as geometric mean value per 100 ml. Sample collected and
examined every six hours for seven consecutive days.
Population (sewered) of Villageville, 3, 820.
Consumers of water from city water plant, 70, 000.
Consumers from county water plant, 205, 000.
Velocity of river flow, 0.5 miles per hour.
Volume in cfs, information not available.
Report of sanitary survey by engineers: no known sources of pollution observed
between river mile 24 and 10 except as noted under remarks.
7-26
-------
Water Quality Surveys
A GRAPHIC PRESENTATION OF BACTERIOLOGICAL DATA
Figure 3
-------
Water Quality Surveys
B Case Study #2
1 Need
A study was initiated at the request of
a south western state to prepare a
supplemental report on the bacteriological
quality of a national wildlife refuge lake
based upon state gathered data'as well
as an intensive study by the FWQA
conducted during the same year. A
massive localized fish kill and observed
bacterial pollution indicator encroach-
ments on the southern half of the lake
precipitated an urgent need for this
report. Figure 4 depicts this lake,
Indian Hunt Lake, (here given a fictious
7-28
-------
Water Quality Surveys
2 Survey and procedures
Indian Hunt Lake is a man made 2000
acre wildlife refuge and recreational
area. Since there is a large population
of waterfowl (exceeding 1, 025, 000 during
resting and wintering periods) and con-
tributing polluting sources from cattle
feed lots, it was apparent that data
would be required during dry periods
and during rainfall and subsequent land
runoff periods to determine the extent
of this pollution effect. The necessity
of this runoff data became even more
manditory when it was ascertained that
Freehand Brook was not a continual
free-running tributary and this effect
was only evident during periods of
rainfall and the dry periods produced
a dry bed and storage in Oxbow Lake.
Stream flow determinations wer made
and from 14 year averages peak flows
were determined and in this manner
maximum effects upon the Indian Hunt
Lake could be more easily determined
from current data.
Previous data for this study area was
compiled by the state agency and con-
sisted only of the total coliform
indicator taken by the MPN method in
monthly intervals. A four phase plan
was established to accomplish the
survey objectives:
a Acquire bacteriological data for all
sample stations.
b Acquire additional bacteriological
data and note the effect of rec-
reational use on the bacteriological
quality of Indian Hunt Lake.
c Evaluate the effect of rainfall on the
receiving stream and Indian Hunt
Lake.
d Data analysis and report preparation,
A mobile laboratory study was initiated
and samples were analyzed by the
membrane filter technique and the
indicator organisms included the total
coliforms, the fecal coliforms, and
the fecal streptococci.
• wipa Tr*atr»»nt Pl«
F«*d1ot
P««dlol
7-29
-------
Water Quality Surveys
3 Results
After the acquisition of background data
from the study area Indian Hunt Lake
was sampled to ascertain the effect of
recreational activities on the bacteri-.
ological quality during dry periods.
Some of this data is reflected in
Figure 6 which notes the fecal coliform
median values per 100 ml for this
period and the numbers in parenthesis gives
gives the median values per 100 ml
during periods of rainfalL heavy storm-
water runoff, and subsequent encroach-
ment of runoff bacteria into the lake.
Figure 6
7-30
-------
Water Quality Surveys
4 Interpretation of data
Table 4 indicates the actual recom-
mendations given by, the final report
and the column designated as Remarks,
gives the interpretations derived from
the data.
TABLE 4
Corrective Measures to More Adequately Control Discharges from
Domestic Sewage and Cattle Feedlot Drainage into Indian Hunt Lake
Recommendations
1. Development of diversion dykes around
all cattle feedlots to channel drainage
into waste stabilization ponds.
2. Existing sewage treatment facilities
should be expanded to produce a better
quality effluent with a BOD reduction
goal of 85-90%. Post chlorination is
desirable especially during the recreation
season to further reduce pathogenic
hazards during this period.
3. Recreational restrictions will be
necessary whenever intense rainfall
occurs in a magnitude sufficient to
produce an, inflow of more than 450
acre feet of water from White Stone
Creek.
4. Restrictions to be placed'upon the, horse-
power and number of pleasure-boats in the
area of inflow from White Stone Creek.
5. A buffer zone must be continually main-
tained and enforced between the wildlife
refuge area and those areas designated
for swimming, wading, and skiing.
Remarks
Influences of the cattle feedlot wastewater
effluents are evident in Figure 7 and Figure 8
and this effect is pronounced during heavy
stormwater overflows. Fecal coliform/
fecal streptococci ratios of.less than 0.7
throughout most of the tributary and of
White Stone Creek indicate the predominance
of animal wastes other than human.
Enormous bacterial indicator contributions
from the sewage treatment plant is evident
in Figure 8 and the resultant FC/FS ratio
confirms that the efflu'ent is from domestic
wastes (greater than 4).
These inflow measurements were made during
measurements of flows occurring during
periods of intense rainfall and noting the
fecal coliform count intrusions to Indian
Hunt Lake. With continual facility improve-
ments this value could be revised upwards
as effluent values exhibit lower bacterial
parameter counts. Current standards of
200 fecal coliforms per 100 ml'would deter-
mine when restrictions could be lifted for
primary contact recreation.
Since shallow areas exist in this lake it is
necessary to include this provision to prevent
and control the resuspension of indicator and
pathogenic bacteria from the muds and deposits.
Figure 9 depicts these areas and calendar
dates. These designated buffer zones will
lessen the potential health hazards from
pathogens entrapped in the organic muck and
lake water sediments. Pathogens gain
entrance through inflow of White Stone Creek
and to a more limited extent from the waterfowl.
7-31
-------
Water Quality Surveys
A similar study was made of White Stone Creek and Freehand Brook and this is shown in Figure 7
and Figure 8. In both figures (Figure 7 - dry periods and Figure 8 - after heavy stormwater
overflow) the circular areas indicate the fecal coliform values and also the FC/FS ratios (fecal
coliform to fecal streptococci) are indicated for this sampling point.
Feed lot
80,000
FC/FS-0.4
Feed lot
Figure 7
Dry Period
Bacteriological Data
Sewage Treatment Plant
2,200
FC/FS-O 6
1,900
FC/FS-0 2
Circular areas represent fecal coliforms per
100 ml. FC/FS (fecal coliform to fecal
streptococci ratio) ratios are indicated for
each station.
630
FC/FS-0 6
7-32
-------
Water Quality Surveys
Figure 8
7-33
-------
Water Quality Surveys
Figure 9
Sw imm ing
Fishing Area. Boat
Speed Limit 5 M
Opened to boating and
fishing from
M arch 1 - O ct 31
Waterfowl Season
Nov 1 - Feb 28
caution
shallow water
Mud F lats
White Stone Creek
ACKNOWLEDGEMENT:
In the preparation of this outline the author
has made extensive use of material made
available by Mr. Harold F. Clark (deceased)
formerly bacteriological consultant to the
Enforcement Branch, DWS & PC by Mr. F. W.
Kittrell (retired) formerly Chief, Technical
Advisory and Investigations Activities, FWQA
and Mr. Edwin Geldreich, Chief Bacteriologist,
Bureau of Water Hygiene, Environmental
Protection Agency, Cincinnati, OH.
REFERENCES
1 Outline, this manual, titled "Bacteriological
Indicators of Water Pollution" and
related references.
Dam
N
A
Indian Hunt Lake
National Wildlife Refuge
S k i Jum p A rea
Kittrell, F.W. and Furfari, S. A.
Observations of Coliform Bacteria
in Streams. Jour Water Pollution
Control Federation 35:1361- 85 1963.
This outline was prepared by H. L. Jeter,
Director, and R. Russomanno, Micro-
biologist, National Training Center, WPO,
EPA, Cincinnati, OH 45268.
Descriptors: Data Handling, Evaluation,
Microbiology, Surveys, Water Pollution,
Water Quality
7-34
-------
WATER QUALITY SURVEYS
PREPARATION OF SURVEY REPORTS
I TYPE OF REPORT
The type of stream survey report to be pre-
pared depends on two basic factors.- These
are the purpose and the audience for whom
intended.
A The Purpose of the Report
1 A report of findings or basic data
2 A report of existing causes and effects
together with an explanation of how and
why.
3 An exposition of existing causes and
effects and a projection of conditions
that reasonably may occur due to natural
variations in stream flow and temperature.
4 A purpose similar to 3 above plus a pre-
diction of the effects of population growth
and industrial change.
5 The same purpose as 4 above plus an
estimate of the need to protect water
uses, and cause reduction in waste
loads.
B The Specific Audience for Whom the Report
Is Prepared
1 For the record - no expository purpose
2 Other technical agencies with compe-
tencies in.the same field
3 Other technical agencies in other fields
4 Public officials supporting or opposing
the recommendations of the report
5 The general public
C Both in style and content the report should
be adequate to serve as a basis for action
to accomplish the recommended objectives.
II ORGANIZATION OF REPORT
A Title, Authors, Contents
B Acknowledgement of Aid and Assistance
1 Can be included in a letter of trans-
mittal or submission
2 Can be incorporated in a preface or
foreword
3 Should include names of persons and
of corporations, public and private,
who assisted or aided the survey
C Authority
1 Source of Authority
2 Date of authorization
D Report Summary
1 A brief summary of the report and its
recommendations generally precedes
the report proper and should include
three topics:
a Summary of specific findings
b Conclusions drawn from findings
c. Recommendations in general terms.
2 Brevity is essential but not at the
expense of clarity.
3 A very brief but lucid description of the
stream section involved should be
included.
4 This will be the only part of the report
read by many of its audience. Conse-
quently it should be drafted with the
utmost care.
WP, SUR. 18b. 3. 74
7-35
-------
Water Quality Surveys
5 Figure 1 illustrates the principles of
written communication format. It is
important that technical reports be
presented in factual report arrange-
ment and NOT in one similar to that
of fiction.
6 Figure 2 shows how the various
portions of a survey report relate
to the generalized factual report.
7 Recommendations, although briefly
stated in general terms, should be
couched in positive, unexaggerated
language.
8 Cost estimates of compliance with
recommendations is helpful but not
essential.
9 Both tangible and intangible benefits
may be listed briefly under conclusions.
INTRODUCTION
SUMMARY
CONCLUSIONS
RECOMMENDATIONS
INTRODUCTION
METHODOLOGY
SUMMARIZED DATA
DATA ANALYSIS
CONCLUSIONS
BASIS FOR RECOMMENDATION i
BIBLIOGRAPHY
TABULAR DATA
SUBSTANCE OF
SURVEV REPORT
(INVERTED PYRAMIDTYPE'I
of writing structure
Figure 2
1 Statement of the problem
The body of the report should begin with a
statement of the problem and a discussion of
the reasons for and the location of the study.
A description of the area, empha-
sizing pertinent features, should
be included.
7-36
-------
Watci' Quality Surveys
1) The inclusion of pertinent histor-
ical data is of value for audience
orientation.
2) The relationship of this study to
other current water resource
studies.
3) Water use and economic data may
be important.
b An area map is an absolute necessity.
1) Features included should be care-
fully selected and section of the
stream involved should be
emphasized.
2) Do not include unnecessary detail.
3) A general location map usually
orients the reader to the area
map.
2 Objectives of the Survey
a A statement of the purpose by listing
the specific answers sought by the
survey to address various aspects of
the problem.
b The geographical and time scop^ of
the objectives of the survey.
F Survey Methodology
A complete description of the methods of
study employed is an important part of
the record,
1 The time period of survey should be
notec^.
2 All sampling and gauging station loca-
tions should be identified by river
mile.
3 Sampling and analytical methods
a Provide adequate description of
all non-standard methods.
b An appendix for these descripl lohs
may be required if they arc lengthy.
4 Frequency of sampling
5 Description of laboratory types and
locations
S Hydrological methods employed for:
a Times of water travel
b Stream flow data
c Any waste flow measurements
G Survey Results
1 Sources of wastes
a Computed waste loads based on
known contributing populations and
industrial waste strength
b Results of sampling and gauging
program
c Data summaries or displays suffice
for text of report.
2 Stream data
a Summarized survey m the text
b Complete tabulations including time
of collection and averages in appendix
3 Hydrological data
a Usually tabulated with analytical
results both in the text and in
appendices
b Time of water travel curve or curves
c Stream flow frequency charts
d Pertinent groundwater data
4 Aesthetic considerations are of real
importance.
7-37
-------
Water Quality Surveys
H Analysis and Interpretation of Data
1 Three fundamental procedures are
required:
a Comparison of survey results and
appropriate water quality criteria
b Projection of survey data to provide
for comparison of stream conditions
with water quality criteria under
more adverse conditions
c Estimates of permissible wastes
loads under present and future
conditions
2 Since this topic is the subject of other
outlines it will not be further developed
here.
3 A description of the methods of analysis
and interpretation belong in the report.
a Necessary assumptions should be
stated
b All statistical methods used should
be identified.
4 Results of analysis and interpretation
are best presented in chart form insofar
as possible, to support discussion and
interpretation rationale.
I Conclusions
1 Focus of attention, much of it critical,
is on this section of the report.
2 Clearness, conciseness; and positive-
ness are essential.
3 This section indicates reasoning that
leads from findings to conclusions.
J Recommendations
1 This is the crux of the report and
should answer the question, "What
needs to be done to resolve problems
delineated in the report? "
2 Cost estimates are highly desirable,
if possible, and appropriate
K Bibliography
1 Useful to the student and perhaps to
future workers
2 Entails additional time and effort in
assembling references in proper form
III TECHNIQUES AND TOOLS
A Prepare a Detailed Outline
1 This is an important step if the coverage
of the report is to be completed and its
arrangement logical. It is also a time-
saver. if properly done
2 Topics to be included in the outline
will become apparent from the fore-
going discussion of report content.
B Words are Your Tools
1 Keep dictionary, thesaurus, and
glossary available
2 Define any va'gue terms or abbreviations
3 Control superlatives and slang,
4 Avoid emphatics such as "it is to be
noted" or "it is a well-known fact".
5 Limit intensive expressions, such as
"extremely" or "undoubtedly. "
6 Use active verbs when possible.
C Regularly review the report organization
and development with a colleague.
This outline was prepared by Staff Members,
National Training Center, EPA, WPO,
Cincinnati, OH 45268
Descriptors: Reports, Surveys, Technical
Writing, Water Quality
7-38
-------
BACTERIOLOGICAL TESTS IN EPA WATER. QUALITY SURVEILLANCE SYSTEMS
I INTRODUCTION
A The Pollution Surveillance Systems
The Water Quality Surveillance Systems
Section collects chemical, physical,
biological and microbiological data on a
continuing basis from selected sampling
sites throughout the United States, for the
purpose of determining the status of water
quality and pollution control. The
Surveillance Systems also acquire and
publish specific information on problems
such as pollution-caused fish kills, the
financing and construction of sewage
treatment facilities, and inventories of the
existing sewage systems, populations
served, types of treatment and need for
additional facilities.
B Organization
The Technical Data and Information Branch
directs the surveillance program and
provides data information service, data
evaluation and computer operations. The
ten regional offices carry out the acqui-
sition of data through their own laboratory
and field support facilities and with the
cooperation of other agencies such as the
USGS, Corps of Engineers, TVA, Bureau
of Reclamation, the states and basin
authorities (e.g. ORSANCO or the Miami
River Conservancy District).
The surveillance program is designed upon
the major river basin concept. Regional,
administration of the systems places EPA
personnel in close contact with local prob-
lems and the specific sample sites.
C Objectives
The long term objectives of the pollution
surveillance program are to identify:
1 Compliance or non-compliance with water-
quality standards.
2 Baseline water quality and long term
trends.
3 Improvement in water quality produced
by abatement measures.
4 Emerging water quality problems.
The program recognizes that the sur-
veillance systems merely monitor water
quality. Additional short term con-
centrated survyes are necessary, for
instance, to acquire enforcement data
or pinpoint sources of pollution problems.
D Origin
The present surveillance systems
originated with 17 stations under the PHS
National Water Quality Network'in 1957.
The data were published in annual Water
Quality Reports through water year 1963.
The data are now entered into STORET,
the Environmental Protection Agency central
system for data storage and retrieval, and can
be rapidly retrieved to satisfy the needs
of various water quality agencies.
E Scope
Currently in the overall surveillance
program there are over 900 sample
stations monitored for various parameters
by federal agencies and 600 to 800
monitored by the states. The most
frequently tested parameters are tem-
perature and pH. Microbiological
parameters are performed on the majority
of these stations, wherever there is a
recreational use or the need to assess the
suitability of a potable water supply.
The Pollution Surveillance'Section plans
to completely integrate the state-federal
network by 1975. At that time the program
will include 900 stream and 1500 open
water federally-funded stations and an
additional 7, 000 to 10, 000 state-funded
stations are anticipated.
WP. SUR. 48a. 12. 72
-------
Bacteriological Tests in EPA Water Quality Surveillance Systems
II BACTERIOLOGICAL SURVEILLANCE
A Need
Bacteriological data are used in the
detection system:
1 To enforce water quality standards -
the computer will rapidly compare
current data to bacteriological standards
set by the states and previously entered
into STORET.
2 To monitor water quality for the
effectiveness of pollution control.
3 To provide an early warning system
for bacteriological pollution.
4 To establish water quality baselines.
5 To reflect long term trends.
6 To indicate more sudden changes or
seasonal variations.
7 To plan and manage comprehensive
water quality programs.
B Regional administration of Surveillance
Systems
The Chief of Pollution Surveillance in each
region with the support of laboratory
personnel determines the specific details
of the program according to regional and
state needs such as:
1 Selection of sampling sites.
2 Collection of samples.
3 Frequency of sampling.
4 Bacteriological parameters required.
5 Bacteriological methods.
6 Data reporting.
7 Entry of data into the STORET system :
C Bacteriological parameters and methods
The program utilizes the recognized
indicators of bacterial pollution:
1 Total coliforms
2 Fecal coliforms
3 Fccal streptococci
The regions are encouraged to use the
standard methods whenever practical.
The immediate membrane filter tests are
the method of choice'where applicable.
Field methods are necessary in some
regional programs. The original PI-TS
Basic Data Network relied upon the delayed
MF procedure for total coliform analyses.
This procedure is now used when it is
impractical to get results by conventional
methods.
D Quality Control
The following steps are taken to ensure
that the bacteriological data acquired by
the surveillance system and entered into
STORET is acceptable.
1 Standard Methods are followed wherever
possible. IJSCS Water Resources
Division has written a manual of
"Selected Interim Procedures for
Biological and Microbiological
Investigations" describing the methods
to be followed by USGS personnel.
2 Agencies and states encourage personnel
performing bacteriological surveillance
tests to acquire adequate training
including the NTC course "Current
Practices in Water Microbiology. "
3 Regional surveillance personnel and
Quality Control Officers maintain close
contact with the laboratories contributing
data to the program.
8-2
-------
Bacteriological Tests in EPA Water Quality Surveillance Systems
4 Intralaboratory quality control measures
such as colony confirmation, replicate
analyses and repeat counting .by more
than one technician are recommended.
5 Identification of data entered into the
STORET system includes the parameter
code, method and source of the data
and related information.
6 If the Delayed Incubation MF Procedure
is used, results from the same sampling
points should be compared to these
performed by the standard immediate
test procedure.
IE THE DELAYED INCUBATION PROCEDURE
(References 1-4)
A Applications
The Delayeid Incubation Method is necessary
because: bacteriological samples must be
analyzed as soon as possible after collection
before bacterial populations change
drastically and no longer represent the
count at the time of sampling. This test
is useful where:
1 It is not possible to maintain the
desired sample temperature during
transport.
2 When the elapsed time between sample
collection and analysis would exceed
the approved time limit.
3 Where the sampling location is remote
from laboratory services.
4 When it is necessary to monitor streams
or waterbodies by a standardized
procedure.
5 Other reasons which prevent the analysis
of the sample at or near the sample site.
B General Test Procedure
The delayed MF Coliform Procedure is a
modification of the Standard MF Method
for total coliforms and consists of the
following steps:
1 Sample collection.
2 Sample filtration at the collection site
or transport of the iced sample to
laboratory facilities for filtration as
soon after collection as possible.
3 Placement of the inoculated membrane
on the preservative medium in well-
marked tight-fitting plastic petri dishes.
4 Shipment in mailing containers at
ambient temperatures to Regional
laboratories (not to exceed 72 hours).
5 Receipt of membranes and transfer to
growth medium.
6 Incubation at 35° + 0. 5° C for 22 - 24
hours.
7 Counting of the characteristic coliform
colonies.
8 Computing the value per 100 ml and
recording results.
Steps 1 through 4 are performed in the
field or at a permanent laboratory by a
cooperator, participant or technician;
the remaining steps are completed in the
receiving laboratory.
C Recent Research
In a recent evaluation of the Delayed
Incubation Test, with reference-to sea
O
water sampling, BrezerisKi ana Winter
arrived at the following conclusions
following statistical comparisons of 162
samples of Immediate Incubation (IMF)
vs Delayed Incubation (DMF):
There was no apparent difference between
recovery of coliform organisms by the two
procedures at these salt water stations
and reported recoveries at fresh water
stations.
Although the data suggest lower 777^ ratios
IMF
at low pollution stations and higher
ratios at higher pollution stations,
there is no significant difference between
8-3
-------
Bacteriological Tests in EPA Water Quality Surveillance Systems
the performance of the immediate and
delay.ed tests at the 95% confidence level.
The delayed incubation procedure can be
used to quantitate coliform bacteria in salt
water samples when storage of the liquid
sample would be necessary; where
processing must be done in a central
laboratory located a great distance from
the sampling site or where immediate
field analysis cannot be accomplished.
The authors state that. .. "the delayed
incubation procedure can be expected to
provide results far superior to those
obtained from samples stored in bottles
under various conditions of time and
temperature. "
IV THE DELAYED MF TEST IN THE
SURVEILLANCE PROGRAM (Section III)
A Equipment and Supplies
Filtration equipment used for membrane
filter analysis are provided to those
participating laboratories which do not
have it available. Other laboratory
equipment and supplies necessary for
membrane filtrations are also provided
only if they are not-available. Expendable
supplies such as m-Endo broth, membrane
filters, petri dishes, etc. are provided to
participating laboratories and cooperators
at six month intervals. A sample data
sheet is provided at the end of this outline
to indicate microbiological bench data
utilizing Form FWPCA-79-12 (4-67)
which may be replaced by standard forms
utilized1 by cooperating organizations.
B Formulation
M-Endo Broth MF Code 0749.
M-Endo broth contains the following
ingredients per liter:
g
Tryptone 10.0
Thiopeptone 5.0
Casitone , 5.0
Yeast extract 1.5
Lactose 12. 5
Sodium chloride .. . 5.0
Dipotassium hydrogen phosphate. . 4. 375
Potassium dihydrogen phosphate. . 1. 375
Sodium lauryl sulfate 0. 050
Sodium desoxycholate 0.10
Sodium sulfite 2.10
Basic fuchsin 1,05
Add 3. 84 g per liter sodium benzoate
(USP Grade) or 3. 2 ml of a 12 percent _
sodium benzoate solution per 100 ml of
M Endo Broth MF.
Add 500 mg per liter Cycloheximide*
:;:Actidione manufactured by the Upjohn
Company, Kalamazoo, Michigan or equivalent.
8-4
-------
Bacteriological Tests in EPA Water Quality Surveillance Systems
REFERENCES
1 Geldreich, E. E., Kabler, P.W., Jeter,
H. L. and Clark, H. F. A Delayed
Incubation Membrane Filter Test for
Coliform Bacteria in Water. AJPH 45,
1462-1474. 1955.
2 The American Public Health Association
and the American Water Works
Association. Standard Methods for
the Examination of Water.and Sewage
(9th ed. ). New York: American
Public Health Association. 1946
3 Brezenski, Francis T. and Winter, John A .
Use of the Delayed Incubation Membrane
Filter Test for Determining Coliform
Bacteria in Sea Water. In preparation,
North Atlantic Water Quality Management
Center, Edison, N. J. - FWPCA, DI.
1968.
4 Standard Methods for the Examination
of Water and Wastewater, 13th ed
American Public Health Association,
New York, N. Y. 1971 Part VII,
Routine Bacteriologic Examinations
of Water to Determine its Sanitary
Quality.
This outline was prepared by Robert H.
Bordner, Chief, Microbiological Methods
Section, Analytical Quality Control
Laboratory, National Environmental
Research Center, EPA, Cincinnati,
OH 45268.
8-5
-------
U S. DEPARTMENT OF THE INTERIOR
FEDERAL WATER POLLUTION CONTROL A DM IN IS TR A T ION
DIVISION OF POLLUTION SURVEILLANCE
1014 BROADWAY. CINCINNATI, OHIO 45202
MICROBIOLOGICAL BENCH DATA
FORM A^-rnyvtU
BUDGET 0URE*U NO 4Z-PI489
STATIOH NO. AND LOCATION:
LABORATORY LOCATION:
SAMPLE COLLECTION BY.
DATE:
TIME:
SAMPLE RECEI
'ED DPS-
ELAPSED TIME, HOURS'
rR | *0
DA
YR
MO
0*.
TIME:
SAMPLE FILTRATION BY
DATE'
TIME-
VR MO
o»
MLS
UNOILUTlO
MLS
1: OIL
CONTAINER
SERIES NO
OPS LAB
NUMBER
" COL 1 FORM
COLONY COUNT
COL 1 FORMS
PER 100 ML
REMARKS

FOR DIV OF POLL. SURV. USE ONLY'
REMARKS
COMPUTER CODED OAT* (FOR DIVISION OF P0I1IITI0N SURVEILLANCE USE ONLT)
STATION SERIAL NUMBER YR MO OA
I
6
ITEM: Col i form-DIy-Endo. KF.'IOO ml
I I I I I I I I I I
10 9 8 7 6 5 4 3 2 1
FWPCA-79-1J (4-67) (Fommily NL-C-3)
9 10 11 12
PARAMETER CODE
~ ~
13 14 15 IB 17 If
VALUE EXPONENT RMKS

3
1
5
0
3
1 TEH
19
UNIT
23

ITEM

31
UNIT

35
30DD
27 28 29 30
•]~~~
39 40 41 42
43
47 48
~ ~~
5 1 -5 2 53 54
8-6
-------
EXAMINATION OF WATER FOR COLIFORM AND
FECAL STREPTOCOCCUS GROUPS
(Multiple Dilution Tube (MPN) Methods)
I INTRODUCTION
The subject matter of this outline is contained
in three parts, as follows:
A Part 1
1 Fundamental aspects of multiple dilution
tube ("most probable numbers") tests,
both from a qualitative and a quantitative
viewpoint.
2 Laboratory bench records.
3 Useful techniques in multiple dilution
tube methods.
4 Standard supplies, equipment, and
media in multiple dilution tube tests.
B Part 2
Detailed, day-by-day, procedures in tests
for the coliform group and subgroups
within the coliform group.
C Part-3
Detailed, day-by-day, procedures in tests
for member's of the fecal streptococci.
D Application of Tests to Routine Examinations
The following considerations (Table 1) apply
'to the selection of the Presumptive Test,
the Confirmed Test, and the Completed
Test. Termination of testing at the
Presumptive Test level is not practiced
by laboratories of this agency. It must
be realized that the Presumptive Test alone
has limited use when water quality is to
be determined.
TABLE 1
Examination Terminated at -
Type of Receiving
Water
Presumptive
Test
Confirmed Test
Completed Test
Sewage Receiving
Treatment Plant - Raw
Applicable
Applicable
Applicable
Applicable
Important where results
are to be used for control
of raw or finished water.
Application to a statis-
tically valid number of
samples from the
Confirmed Test to estab-
lish its validity in
determining the sanitary
quality.
Chlorinated
Not Done
Applicable
Bathing
Not Done
Applicable
Drinking
Not Done
Applicable
Other Information

Applicable in all
cases where Pre-
sumptive Test alone
is unreliable.
NOTE: Mention of commercial products and manufacturers does not imply endorsement by the
Environmental Protection Agency.
W. BA. 3L. 2. 75
9-1
-------
MPN Methods
II BASIS OF MULTIPLE TUBE TESTS
A Qualitative Aspects
1 For purely qualitative aspects of testing
for indicator organisms, it is convenient
to consider the tests applied to one
sample portion, inoculated into a tube
of culture medium, and the follow-up
examinations and tests on results of the
original inoculation. Results of testing
procedures are definite: positive
(presence of the organism-group is
demonstrated) or negative (presence of
the organism-group is not demonstrated.)
2 Test procedures are based on certain
fundamental assumptions:
a First, even if only one living cell of
the test organism is present in the
sample, it will be able to grow when
introduced into the primary inoculation
medium;
b Second, growth of the test organism
in the culture medium will produce
a result which indicates presence of
the test organism; and,
c Third, extraneous organisms will
not grow, or if they do grow, they
will not limit growth of the test
organism; nor will they produce
growth effects that will be confused
with those of the bacterial group for
which the test is designed.
3 Meeting these assumptions usually
makes it necessary to conduct the tests
in a series of stages (for example, the
Presumptive, Confirmed, and Completed
Test stages, respectively, of standard
tests for the coliform group).
4 Features of a full, multi-stage test
a First stage: The culture medium
usually serves primarily as an
enrichment medium for the group
tested. A good first-stage growth
medium should support growth of all
the living cells of the group tested,
and it should include provision for
indicating the presence of the test
organism being studied. A first-
stage medium may include some
component which inhibits growth
of extraneous bacteria, but this
feature never should be included
if it also inhibits growth of any
cells of the group for which the
test is designed. The Presumptive
Test for the coliform group is a
good example. The medium
supports growth, presumably, of
all living cells of the coliform
group; the culture container has a
fermentation vial for demonstration
of gas production resulting from
lactose fermentation by coliform
bacteria, if present; and sodium
lauryl sulfate may be included in
one of the approved media for
suppression of growth of certain
noncoliform bacteria. This
additive apparently has no aaverse
effect on growth of members of the
coliform group in the concentration
used. If the result of the first-stage
test is negative, the study of the
culture is terminated, and the result
is recorded as a negative test. No
further study is made of negative
tests. If the result of the first-
stage test is positive, the culture
may be subjected to further study
to verify the findings of the first
stage.
b Second stage: A transfer is made
from positive cultures of the-first-
stage test to a second culture medium.
This test.stage emphasizes provision
to reduce confusion of results due to
growth effects of extraneous bacteria,
commonly achieved by addition of
selective inhibitory, agents. (The
Confirmed Test for coliforms meets
these requirements. Lactose and .
fermentation vials are provided for
demonstration of coliforms in the
medium. Brilliant green dye and
bile salts are included as inhibitory
agents which tend to suppress growth
of practically all kinds of noncoliform
bacteria, but do not suppress growth
of coliform bacteria when used as
directed).
9-2
-------
MPN Methods
If result of the second-stange test is
negative, the study of the culture is
terminated, and the result is recorded
as a negative test. A negative test here
means that the positive results of the
first-stage test were "false positive,
due to one or more kinds of extraneous
bacteria. A positive second-stage test
is partial confirmation of the positive
results obtained in the first-stage test;
the culture may be subjected to final
identification through application of still
further testing procedures. In routine
practice, most sample examinations
are terminated at the end of the second
stage, on the assumption that the result
would be positive if carried to the third,
and final stage. This practice should be
followed only if adequate testing is done
to demonstrate that the assumption is
valid. Some workers recommend contin-
uing at least 5% of all sample examina-
tions to the third stage to demonstrate
the reliability of the second-stage results.
B Quantitative Aspects of Tests
1 These methods for determining bacterial
numbers are based on the assumption
that the bacteria can be separated from
one another (by shaking or other means)
resulting in a suspension of individual
bacterial cells, uniformly distributed
through the original sample when the
primary inoculation is made.
2 Multiple dilution tube tests for quantita-
tive determinations apply a Most Probable
Number (MPN) technique. In this pro-
, cedure one or more measured portions
of each of a stipulated series of de-
creasing sample volumes is inoculated
into the first-stage culture medium.
Through decreasing the sample incre-
ments, eventually a volume is reached
where only one cell is introduced into
some1 tubes, .and no cells are introduced
into other tubes. Each of the several
tubes ,of sampLe-inoculated first-stage
medium is tested independently,
according to the principles previously
described, in the qualitative aspects
of testing procedures.
3 The combination of positive and
negative results is used in an application
of probability mathematics to secure
a single MPN value for the sample.
4 To obtain MPN values, the following
conditions must be met:
a The testing procedure must result
in one or more tubes in which the
test organism _i£ demonstrated, to
be present; and
b The testing procedure must result
in one or more tubes in which the
test organism is not demonstrated
to be present.
5 The MPN value for a given sample is
obtained through the use of MPN Tables.
It is emphasized that the precision of
¦ an individual MPN value is not great
when compared with most physical or
chemical determinations.
6 Standard practice in water pollution
surveys conducted by this organization,
is to plant five tubes in each of a series
of sample increments, in sample
volumes decreasing at decimal intervals.
For example, in testing known polluted
waters, the initial sample inoculations
might consist of 5 tubes each in volumes
of 0.1, 0.01,0.001, and 0. 0001 ml,
respectively. This series of sample
volumes will yield determinate results
from a low of 200 to a high of 1, 60.0, 000
organisms per 100 ml.
9-3
-------
MPN Methods'
IE LABORATORY BENCH RECORDS
A Features of a Good Bench Record Sheet
1 Provides complete identification of the
sample.
2 Provides for full, day-by-day informa-
tion about all tests performed on'the
sample.
3 Provides easy step-by-step record
applicable to any portion of the sample.
4 Provides for recording of the quantitative
result which will be transcribed to sub-
sequent reports.
5 Minimizes the amount of writing by the
analyst.
6 Identifies the analyst(s).
B There is no such thing as "standard"
bench sheet for multiple tube tests; there
are many versions of bench sheets. Some
are prescribed by administrative authority
(such as the Office of a State Sanitary
Engineer); others are devised by laboratory
or project personnel to meet specific needs.
C It is not the purpose of this discussion to
recommend an "ideal" bench form; however,
the form used in this training course
manual is essentially similar to that used
in certain research laboratories of this
organization. The student enrolled in the
course for which this manual is written
should make himself thoroughly familiar
with the bench sheet and its proper use.
See Figure 1.
IV NOTES ABOUT WORKING PROCEDURES
IN THE LABORATORY
A Each bacteriological examination of water
by multiple dilution tube methods requires
a considerable amount of manipulation;
much is quite repetitious. Laboratory
workers' must develop and maintain good
routine working habits, with constant
alertness to' guard against lapses into
careless, slip-shod laboratory procedures
and "short cuts" which only-can lead to
lowered quality of laboratory work.
The student reader is urged to review the
form for laboratory surveys (PHS-875,
Rev. 1966) used by Public Health Service
personnel charged with responsibility for
accreditation of laboratories for examination
of water under Interstate Quarantine
regulations.
B Specific attention is brought to the following
by no means exhaustive, critical aspects of
laboratory procedures in multiple dilution
tube tests;
1 Original sample
a Follow prescribed care and handling
procedures before testing.
b Maintain absolute identification of
¦sample at all stages in testing.
c Vigorously shake samples (and
sample dilutions) before planting
in culture media.
2 Sample measurement into primary
culture medium
a Sample portions must be measured
accurately into the culture medium
for reliable quantitative tests to be
made. Standard Methods prescribes
that:calibration errors should not
exceed + 2. 5%.
9-4
-------
BACTERIOLOGY BENCH SHEET
Multiple Dilution Tube Tests
Project
Sample Station
Collection Data
Date £./(*/&,7 Time f. 'S0 By
Temperature /_°C PH^I
Other Observations
Analytical Record
Bench Number of Sample_
Analyst_
Test started at ///:#¦£" , By,/J/.
t
ml j
imple |
i
!
Coliform Test
Fecal
coli-
form
Fecal;
Remarks
LTB 1 BGLB
EMB
¦' LSTB
1
Gram |
¦
i°B
1 L(
.EVA
24 | 48 i 24
48
24
24
48 1
stain || 24
24
.48
24
48
to

^t><

><:

/\i X

!><£><.

p.. ... ->f
><

i <2 on/

1
I

:
i
1

!
1

1
1
1
1

Coliform MPN/100 ml
Confirmed:
Completed:
Fecal Coliform MPN:
Fecal Streptococcus MPN/100 ml
A - D - EVA:
Figure 1. SAMPLE BENCH SHEET
-------
MPN Methods
Suggested sample measuring practices
are as follows: Mohr measuring
pipets are recommended. 10 ml
•samples are delivered at the top of
the culture tube, using 10 ml pipets.
1.0 ml samples are delivered down
into the culture tube, near the sur-
face of the medium, and "touched
off" at the side of the tube when the
desired amount of sample has been
delivered. 1. 0 ml or 2. 0 ml pipets'
are used for measurement of this
volume. 0. 1 ml samples are
delivered in the same manner as 1.0
ml samples, using great care that
the sample actually gets into the
culture medium. Only 1.0 ml pipets
are used for this sample volume.
After delivery of all sample incre-
ments into the culture tubes, the
entire rack of culture tubes may be
shaken gently to carry down any of
the sample adhering to the wall of'
the tube above the medium.
Workers should demonstrate by actual
tests that the pipets and the technique
in use actually delivers the rated volumes
within the prescribed limits of error.
b Volumes as small as 0. 1 ml routinely
can be delivered directly from the
sample with suitable pipets. Lesser
sample volumes first should be diluted,
with subsequent delivery of suitable
volumes of diluted sample into the
culture medium. A diagrammatic
scheme for making dilutions is shown
in Figure 2.
b Gas in any quantity is a positive test.
It is'necessary to work in conditions
of suitable lighting for easy recog-
nition of the extremely small amounts
of gas inside the tops of some
fermentation vials.
4 Reading of liquid culture tubes for
growth as indication of a positive test
requires good lighting. Growth is-
shown by any amount of increased
turbidity or opalescence in the culture
medium, with or without deposit of
sediment at the bottom of the tube.
5 Transfer of cultures with inoculation
loop's and needles
•a Always sterilize inoculation loops
and needles in flame immediately
before transfer of culture; do not
lay it down or touch it to any non-
sterile object before making the
transfer.
b After sterilization, allow sufficient
time for cooling, in the air, to avoid
heat-killing bacterial cells on the
hot wire.
c Loops should be 3 mm in inside
diameter, with a capability of holding
a drop of water or culture.
For routine standard transfers
requiring transfer of 3 loopsful of
culture, many workers form three
3-mm loops on the same length of
wire.
3 Reading of culture tubes for gas
production
a On removal from the incubator,
shake culture rack gently, to
encourage release of gas which
may be supersaturated in the culture
medium.
C
As an alternative to use of standard
inoculation loops, the use of
"applicator sticks" have now been
sanctioned by the 13th Edition of
Standard Methods.
9-6
-------
MPN Methods :
Figure 2. PREPARATION OF DILUTIONS
Dilution Ratios:
1:100
1.10000

Tubes
Petri Dishes or Culture Tubes
Actual volume
of sample in tube
lml 0.1ml 0.01 ml 0.001ml
0.0001 ml 0.00001 ml
The applicator sticks are dry heat
sterilized (autoclave sterilization is
not acceptable because of possible
release of phenols if the wood is
steamed) and are used on a single-
service basis. Thus, for every culture
tube transferred, a new applicator
stick is used.
This use of applicator sticks is
particularly attractive in field
situations where it is inconvenient or
impossible to provide a gas burner
suitable for sterilization of the
inoculation loop. In addition, use of
applicator sticks is favored in
laboratories where room temperatures
are significantly elevated by use of
gas burners.
7 Streaking cultures on agar surfaces
a All streak-inoculations should be
made without breaking the surface
of t;he agar. Learn to use a light
touch with the needle; however,
many inoculation needles are so
sharp that they are virtually useless
in this respect. When the needle is
platinum or platinum-iridium wire,
it sometimes is beneficial to fuse
the working tip into a small sphere.
This can be done by momentary
insertion of a well-insulated (against
electricity) wire into a carbon arc,
or some other extremely hot environ-
ment. The sphere should not be more
than twice the diameter of the wire
from which it is formed, otherwise
it will be entirely too heat-retentive
to be useful.
9-7
-------
MPN Methods
When the needle is nichrome
resistance wire, it cannot be heat-
fused; the writer prefers to bend
the terminal 1/16 - 1/8" of the wire
at a slight angle to the overall axis
of the needle.'' The side of the
terminal bent portion of the needle
then is used for inoculation of agar
surfaces.
b When streaking for colony isolation,
avoid using too much inoculum. The
streaking pattern is somewhat
variable according to individual
preference. The procedure favored
by the writer is shown in the
accompanying figures. Note
particularly that when going from
any one stage of the streaking to the
next, the inoculation needle is heat-
sterilized.
8 Preparation of cultures for Gram
stain
a The Gram stain always should be
made from a culture grown on a
nutrient agar surface (nutrient agar
slants are used here) or from nutrient
broth.
b The culture should be young, and
should be actively growing. Many
workers doubt the validity of the
Gram, stain made on a culture more
than 24 hours old.
c Prepare a thin smear for the staining
procedure. Most beginning workers
tend to use too much bacterial sus-
pension in preparing the dried smear
for staining. The amount of bacteria
should be so small that the dried film
is.barely visible to the naked eye.
V EQUIPMENT AND SUPPLIES
Consolidated lists of equipment, supplies,
and culture media required for all multiple
dilutiort tube tests described in this, outline
are shown in Table 2.. Quantitative •infor-
mation is not presented; this is variable-
according to the extent of the testing pro-
cedure, the number of dilutions used, and
the number of replicate tubes per dilution.
It is noted that requirements for alternate
procedures are, fully listed and choices are
made in accordance to laboratory preference.
9-8
-------
MPN Methods
a Flame-sterilize an inoculation needle and air-cool.
b Dip the tip of the inoculation needle into the bac-
terial culture being studied,
c Streak the inoculation needle tip lightly back and
forth over half the agar surface, as in ( 1), avoid-
ing scratching or breaking the agar surface:
d Flame-sterilize the inoculation needle and air-cool
a Turn the Petri dish one-quarter-turn and streak the
inoculation needle tip lightly back and forth over one-
half the agar surface, working from area (1) into one-
half the unstreaked area of the agar.
b Flame-sterilize the inoculation needle and air-cool.
a Turn the Petri dish one-quarter-turn and streak the
inoculation needle tip lightly back and forth over one-
half the agar surface, working from area (2) into
area (3), the remaining unstreaked area.
b Flame-sterilize the inoculation needle and set it aside.
c Close the culture container and incubate as prescribed.
Figure 3. A SUGGESTED PROCEDURE FOR COLONY ISOLATION BY A
STREAK-PLATE TECHNIQUE
AREA 1 (Heavy inoc
AREA 3 (Isolated colonies)
AREA 2
(Moderate growth)
APPEARANCE OF STREAK ¦ PLATE
AFTER INCUBATION INTERVAL
-------
MPN Methods
TABLE 2. APPARATUS AND SUPPLIES FOE STANDARD
FERMENTATION TUBE TESTS
Description of Ilem
Tot*l Coliform Group
Fecal Coliform*.

Presumptive
Test
Confirmed
Test
Completed
Test
(BALB)
(EC broth)
Lauryl tryptose broth or Lactose
broth 20 ml amounts of 1.5 <
concentration medium, in 25 »" 150 mm
culture tubes with inverted fermen-
tation vialq, suitable caps.
X

Lauryl tryptose broth or Lactose
broth, 10 ml amounts of single
strength medium in 20 /150 mm
culture tubes with inverted fermen-
tation vials, suitable caps.
X

Brilliant green lactoa* bile broth, 2%
in 10 ml amounts, single strength,
in 20 X 150 mm culture tubes with
inverted fermentation vials,
suitable caps.

X
X

Eosin methylene blue agar, poured
in 100 X 15 mm Petri dishes

X
X

EndoAgar, poured In 100 /15mm
dishes
Nutrient agar slant, acrpw cap tube
Boric acid lactose broth, 10 ml
amounts of single strength medium
in fermentation tubes.

X
X
X

EC Broth, 10 ml amounts of single
strength medium in fermentation
tubes.

X
Formate ricinoleate broth
(provisional)

Culture tube racks, 10X5 openings,
each opening to accept 25 mm dia-
meter tubes.
X
X
X
X
X
Pipeites, 10 ml, Mohr type, sterile,
in suitable cans
X

Pipeites, 2 ml (optional), Morh type,
sterile, in suitable cans
X

Pipette"!, 1 i)i 1, Mohr type, sterile
in metal suitable cans
X

Standard buffered dilution water,
sterile, 99-ml amounts in screw-
capped bottles.
X

!
I
Gas burner, Bunsen type

X
X
X
^ 1
Inoculation loop, loop 3mm dia<
meter, of nichrome or platinum-
indium wire, 26 B & S gauge, In
suitable holder, (or sterile applicator
slick)

X
X
X
X
Inoculation needle, nichrome, or
platinum-iridium wire, 26 B & S
gauge, in suitable holder.

X
X

Incubator, adjusted to 35 +¦ 0. 50 Q
X
X
X

Waterbath incubator, adjusted to
43 t 0. 2°C

Waterbath incubator, adjusted to
44,5 + 0. 2°C.

X
Glass microscopic slides. l"xT'

Slide racks (optional)

Gram-stain solutions, complete set

Compound microscope, oil immer-
sion lens, Abbe1 condenser

Basket for discarded cultures
X
X
X
X
X
Container for discarded pipettes
X

-------
Part 2
DETAILED TESTING PROCEDURES FOR MEMBERS OF THE
COLIFORM GROUP BY MULTIPLE DILUTION TUBE METHODS
I SCOPE
A Tests Described
1 Presumptive Test
2 Confirmed Test
3 Completed Test
4 Fecal Coliform Test
B Form of Presentation
The Presumptive, Confirmed, and
Completed Tests are presented as total,
independent procedures. It is recognized
that this form of presentation is somewhat
repetitious, inasmuch as the Presumptive
Test is preliminary to the Confirmed
Test, and both the Presumptive Test and
the Confirmed Test are preliminary to the
Completed Test for total coliforms.
In using these procedures, the worker
must know at the outset what is to be the
stage at which the test is to be ended, and
the details of the procedures throughout,
in order to prevent the possibility of
discarding gas-positive tubes before ¦
proper transfer procedures have been
followed.
II TESTING TO PRESUMPTIVE TEST
STAGE
A First-Day Procedures
1 Prepare a laboratory data sheet for
the sample. Record the following
information: assigned laboratory
number, source of sample, date and
time of collection, temperature of the
source, name of sample collector,
date and time of receipt of sample in
the laboratory. Also show the da(te
and time of starting tests in the
laboratory, name(s) of worker(s) per-
forming the laboratory tests, and the
sample volumes planted.
2 Label the tubes of ]auryl tryptose broth
required for the initial planting of the
sample (Table 3). The label should
bear three identifying marks. The
upper number is the identification of
the worker(s) performing the test
(applicable to personnel in training
courses), the number immediately
below is the assigned laboratory num-
ber, corresponding with the laboratory
record sheet. The lower number is the
code to designate the sample volume
and which tube of a replicate series is
indicated.
Thus, if the worker knows that the test will
be ended at the Confirmed Test, he will
turn at once to Section III, TESTING'TO
THE CONFIRMED TEST STAGE, and will
ignore Sections II and IV.
The Fecal Coliform Test is described
separately, in Section V, as an
adjunct to the Confirmed Test and to the
Completed Test.
NOTE:' Be sure to use tubes containing
the correct concentrations of culture medium
for the inoculum/tube volumes. (See the
chapter on media and solutions for multiple
dilution tube methods or refer to the current
edition of Standard Methods for Water and
Wastewater).
9-11
-------
MPN Methods
Table 3. SUGGESTED LABELING SCHEME FOR ORIGINAL CULTURES AND
SUBCULTURES IN MULTIPLE DILUTION TUBE TESTS

Tube
1
Tube
2
Tube
3
Tube
4
Tube
5
Sample volume
represented
Bench number
Volume & tube
312
A
312
B
312
C
312
D
312
E
Tubes with 10 ml
of sample
Bench number
Volume & tube
312
a
•312
b
312
c
312
d
312
e
Tubes with 1 ml
of sample
Bench number
Volume & tube
312
a
312
_b
312
c
312
d
312
e
Tubes with 0.1 ml
of sample
Bench number
Volume & tube
312
la
312
lb
312
lc
312
Id
312
le
Tubes with 0.01 ml
of sample
Bench number
Volume & tube
312
2a
312
2b
312
2c
312
2d
312
2e
Tubes with 0.001 ml
of sample
Typical Example
RB
312
A .
Lab Worker
^Identification
Bench Number
Sample Volume
WJ/
Tube of Culture Medium
The labeling of cultures can be reduced by labeling only the first tube of
each series of identical sample volumes in the initial planting of the sample.
All subcultures from initial plantings should be labeled completely.
3 Place the labeled culture tubes in an
orderly arrangement in a culture tube
rack, with the tubes intended for the
largest sample volumes in the front
row, and those intended for smaller
volumes in the succeeding rows.
4 Shake the sample vigorously, approxi-
mately 25 times, in an arc of one foot
within seven seconds and withdraw the
sample portion at once.
5 Measure the predetermined sample
volumes into the labeled tubes of lauryl
tryptose broth, using care to avoid
introduction of any bacteria into the
culture medium except those in the
sample.
a Use a 10 ml pipet for 10 ml sample
portions, and 1 ml pipets for portions
of 1 ml or less. Handle sterile pipets
only near the mouthpiece, and protect
the delivery end from external con-
tamination. Do not remove the cotton
plug in the mouthpiece as this is
intended to protect the usier from
ingesting any sample.
b When using the pipet to withdraw
sample portions, do not dip the
pipet more than 1/2 inch into the
sample; otherwise sample running
down the outside of the pipet will
make measurements inaccurate.
6 After measuring all portions of the
sample into their respective tubes of
medium, gently shake the rack of. .
inoculated tubes to insure good mixing
of sample with the culture medium.
Avoid vigorous shaking, as air bubbles
may be shaken into the fermentation
vials and thereby invalidate the test.
7 Place the rack of inoculated tubes in the
incubator at 35° + 0.5°C for 24 +
2 hours.
B 24-hour Procedures
1 Remove the rack of lauryl tryptose
broth cultures from the incubator, and
shake .gently. If gas is about to appear
in the fermentation vials, the shaking
will speed the process.
-------
MPN Methods
2 Examine each tube carefully. Record,
in the column "24" under LST on the
laboratory data sheet, each tube showing
gas in the fermentation vial as a positive
(+) test and each tube not showing gas
as a negative (-) test. GAS IN ANY
QUANTITY IS A POSITIVE TEST.
3 Discard all gas-positive tubes of lauryl
tryptose broth, and return all the gas-
negative tubes to the 35°C incubator for
an additional 24 i 2 hours.
C 48-hour Procedures
1 Remove the rack of culture tubes from
the incubator, read and record gas
production for each tube.
2 Be sure to record all results under the
48-hour LTB column on the data sheet.
Discard all tubes. The Presumptive
Test is concluded at this point, and
Presumptive coliforms per 100 ml can
be computed according to the methods
described elsewhere in this manual.
Ill TESTING TO CONFIRMED TEST STAGE
Note that the description starts with the
sample inoculation and includes the
Presumptive Test stage. The Confirmed
Test preferred in Laboratories of this agency
is accomplished by means of the brilliant
green lactose bile broth (BGLB) and the
acceptable alternate tests are mentioned in
III F. In addition, the Fecal Coliform Test is
included as an optional adjunct to the procedure.
A First-Day Procedures
1 Prepare a laboratory data sheet for the
sample. Record the following infor-
mation: assigned laboratory number,
source of sample, date and time of
collection, temperature of the source,
name of sample collector, date and
time of receipt of sample in the
laboratory. Also show the' date and
time of starting tests in the laboratory.
name(s) of worker(s) performing the
laboratory tests, and the sample
volumes planted.
2 Label the tubes of lauryl tryptose broth
required for the initial planting of the
sample. The label should bear three
identifying marks. The upper number
is the identification of the worker(s)
performing the test (applicable to
personnel in training courses), the
number immediately below is the
assigned laboratory number, corres-
ponding with the laboratory record
sheet. The lower number is-the code
to designate the sample volume and
which tube of a replicate series is indicated.
NOTE: If 10-ml samples are being
planted, it is necessary to use tubes
containing the correct concentration
of culture medium^ This has previously
been noted in II A-2.
3 Place the labeled culture tubes in an
orderly arrangement in a culture tube
rack, with the tubes intended for the
largest sample volumes in the front
row,, and those intended for smaller
volumes in the succeeding rows.
4 Shake the sample vigorously, approxi-
mately 25 times, in an up-and-down
motion.
5 Measure the predetermined sample
volumes into the labeled tubes of lauryl
tryptose broth, using care to avoid
introduction of any bacteria into the
culture medium except those in the sample.
a Use a 10-ml pipet for 10 ml sample
portions, and 1-ml pipets for portions
of 1 ml or less. Handle sterile pipets
only near the mouthpiece, and protect
the delivery end from external con- .
tamination. Do not remove the cotton
plug in the mouthpiece as this is intended
to protect the user from ingesting any
sample.
9-13
-------
MPN Methods
b When using the pipet to withdraw
sample portions, do not dip the
pipet more than 1/2 inch into the
sample; otherwise sample running
down the outside of the pipet will
make measurements inaccurate.
c When delivering the sample into the
culture medium, deliver sample
portions of 1 ml or less down into
the culture tube near the surface of
the medium. Do not deliver small
sample volumes at the top of the tube
and allow them to run down inside
the tube; too much of the sample
will fail to reach the culture medium.
d Prepare,preliminary dilutions of
samples for portions of 0. 01 ml or
less before delivery into the culture
medium. See Table 1 for preparation
of dilutions. NOTE: Always deliver
diluted sample portions into the
culture medium as soon as possible
after preparation. The interval
between preparation of dilution and
introduction of sample into the
medium never should be as much
as 30 minutes.
6 After measuring all portions of the
sample into their respective tubes of
medium, gently shake the rack of
inoculated tubes to insure good mixing
of sample with the culture medium.
Avoid vigorous shaking, as air bubbles
may be shaken into the fermentation
vials and thereby invalidate the test.
7 Place the rack of inoculated tubes in
the incubator at 35° + 0. 5° C for 24 +
2 hours.,
B 24-hour Procedures
1 Remove the rack of lauryl tryptose
broth cultures from the incubator, and
shake gently. If gas is about to appear
in the fermentation vials, the shaking
will speed the process.
2 Examine each tube carefully. Record,
in the column "24" under LST on the
laboratory data sheet, each tube showing
gas in the fermentation vial as a
positive (+) test and each tube not
showing gas as a negative (-) test.
GAS IN ANY QUANTITY IS A POSITIVE
TEST.
3 Retain all gas-positive tubes of lauryl
tryptose broth culture in their place
in the rack, and proceed.
4 Select tne gas-positive tubes of lauryl
tryptose broth culture for Confirmed
Test procedures. Confirmed Test
procedures may not be required for all
gas-positive cultures. If, after 24-hours
of incubation, all five replicate cultures
are gas-positive for two or more con-
secutive sample volumes, then select
the set of five cultures representing
the smallest volume of sample in which
all tubes were gas-positive. Apply
Confirmed Test procedures to all these
cultures and to any other gas-positive
cultures representing smaller volumes
of sample, in which some tubes were
gas-positive and some were gas-negative.
5 Label one tube of brilliant green lactose
bile broth (BGLB) to correspond with
each tube of lauryl tryptose broth
selected for Confirmed Test procedures.
6 Gently shake the rack of Presumptive
Test cultures. With a flame-sterilized
inoculation loop transfer one loopful- of
culture from each gas-positive tube to
the corresponding tube of BGLB. Place
each newly inoculated culture into BGLB
in the position of the original gas-positive
tube.
7 After making the transfers, the rack
should contain some 24-hour gas-
negative tubes of lauryl tryptose broth
and the newly inoculated BGLB.
8 If the Fecal Coliform Test is included
in the testing procedures, consult
Section V of this part of the outline of
testing procedures.
9-14
-------
MPN Methods
9 Incubate the 24-hour gas-negative
BGLB tubes and any newly-inoculated
tubes of BGLB an additional 24 + 2
hours at 35° + 0. 5°C.
C 48-hour Procedures
1 Remove the rack of culture tubes from
the incubator, read and record gas
production for each tube.
2 Some tubes will be lauryl tryptose broth
and some will be brilliant green lactose
bile broth (BGLB). Be sure to record
results from LTB under the 48-hour
LTB column and the BGLB results under
the 24-hour column of the data sheet.
3 Label tubes of BGLB to-correspond with
all (if any) 48-hour gas-positive cultures
in lauryl tryptose broth. Transfer one
loopful of culture from each gas-positive
LTB culture to the correspondingly-
labeled tube of BGLB. NOTE: All
tubes of LTB culture which were
negative at 24 hours and became
positive at 48 hours are to be transferred.
The option described above for 24-hour
cultures does not apply at 48 hours.
4 If the Fecal Coliform Test is included
in the testing procedure, consult
Section V of the part of the outline
of testing procedures.
5 Incubate the 24-hour gas-negative
BGLB tubes and any newly-inoculated
tubes of BGLB 24 + 2 hours at 35° +
0. 5° C.
6 Discard all tubes of LTB and all 24-hour
gas-positive BGLB cultures.
D 72-hour Procedures
1 If any cultures remain to be examined,
all will be BGLB. Some may be 24
hours old and some may be 48 hours
old. Remove such cultures from the
incubator, examine each tube for gas
production, and record results on the
data sheet.
2 . Be sure to record the results of 24-hour
BGLB cultures in the ."24".column under
BGLB and the 48-hour results'under-the
"48" column of the data sheet.
3 Return any 24-hour gas-negative cultures
for incubation 24 + 2 hours at 35 +
0.5OC.
4 Discard all gas-positive BGLB .cultures
and all 48-hour gas-negative,cultures
from BGLB.
5 It is possible that all cultural work and
results for the Confirmed Test have
been finished at this point.' If so, codify
results and determine Confirmed Test
coliforms per 100 ml as described in
the outline on use of MPN Tables.
E 96-hour Procedures
At most only a few 48-hour cultures in
BGLB may be present. Read and record
gas production of such cultures in the "48"
column under BGLB on the data sheet.
Codify results and determine Confirmed
Test coliforms per 100 ml.
F Streak-plate methods for the Confirmed
Test, using eosin methylene blue agar or
Endo agar plates, are accepted procedures
in Standard Methods. The worker who
prefers to use one of these media in
preference to BGLB (also approved in
Standard Methods) is advised to refer to
the current edition of "Standard Methods
for the Examination of Water and Waste-
water" for procedures.
9-15
-------
MPN Methods
IV TESTING TO COMPLETED TEST STAGE
(Note that this description starts with the
sample inoculation and proceeds through the
Presumptive and the Confirmed Test stages.
In addition, the Fecal Coliform Test is
referred to as an optional adjunct to the
procedure.)
A First-Day Procedures
1 Prepare a laboratory data sheet for the
sample. Record the following information:
assigned laboratory number, source of
sample, date and time of collection,
temperature of the source, name of
sample collector, date and time of
receipt of sample in the laboratory.
Also show the date and time of starting
tests in the laboratory, name(s) of
worker(s) performing the laboratory
tests, and the sample volumes planted.
2 Label the tubes of lauryl tryptose broth
required for the initial planting of the
sample. The label should bear three
identifying marks. The upper number
is the identification, of the worker(s)
performing the test (applicable to
personnel in training courses),
the number immediately below is the
assigned laboratory number, corres-
ponding with the laboratory record
sheet. The lower number is the code
to designate the sample volume and
which tube of a replicate series is
indicated. Guidance on labeling for
laboratory data number and identification
of individual tubes is described else-
where in this outline..
NOTE: If 10-ml samples are being
plated, it is necessary to use tubes -
containing the correct concentration
of culture medium. . This has previously
been noted elsewhere in this outline
and referral is made to tables.
3 ' Place the labeled culture tubes in an
orderly arrangement in a culture tube
rack, with the tubes intended for the
largest sample volumes in the front
row, and those intended for smaller
volumes in the succeeding rows.
4 Shake the sample vigorously, approxi-
mately 25 times, in an up-and-down
motion.
5 Measure the predetermined sample
volumes into the labeled tubes of lauryl
tryptose broth, using care to avoid
introduction of any bacteria into the
culture medium except those in the
sample.
a Use a 10-ml pipet for 10 ml sample
portions, and 1-ml pipets for portions
of 1 ml or less. Handle sterile
pipets only near the mouthpiece,
and protect the delivery end from
external contamination. Do not move
the cotton plug in the mouthpiece
as this is intended to protect the
user from ingesting any sample.
b When using the pipet to withdraw
sample portions, do not dip the
pipet more than 1/2 inch into the
sample; otherwise sample running
down the outside of the pipet will
make measurements inaccurate.
c When delivering the sample into the
culture medium, deliver sample
portions of 1 ml or less down into
9-16
-------
MPN Methods
the culture tube near the surface of
the medium. Do not deliver small
sample volumes at the top of the
tube and allow them to run down
inside the tube; too much of the
sample will fail to reach the culture
medium.
d Prepare preliminary dilutions of
samples for portions of 0. 01 ml or
less before delivery into the culture"
medium. See-Table 2 for preparation
of dilutions. NOTE: Always deliver
diluted sample portions into the
culture medium as soon as possible
after preparation. The interval
between preparation of dilution and
introduction of sample into the
medium never should be as much as
30 minutes.
6 After measuring all portions of the
sample into their respective tubes of
medium, gently shake the rack of
inoculated tubes to insure good mixing
of sample with the culture medium.
Avoid vigorous shaking, as air bubbles
may be shaken into the fermentation
vials and thereby invalidate the test.
7 Place the rack of inoculated tubes in
the incubator at 35° + 0. 5° C for 24 +
2 hours.
B 24-hour Procedures
1 Remove the rack of lauryl tryptose broth
cultures from the incubator, and shake
gently. If gas is about to appear in the
fermentation vials, the shaking will
speed the process.
2 Examine each tube carefully. Record,
in the column "24" under LST on the
laboratory data sheet, each tube showing
gas in the fermentation vial as a positive
(+) test and each tube not showing gas
as a negative (-) test. GAS IN ANY
QUANTITY IS A POSITIVE TEST.
3 Retain all gas-positive tubes of lauryl
tryptose broth culture in their place in
the rack, and proceed.
4 Select the gas-positive tubes of lauryl
tryptose broth culture for the Confirmed
Test procedures. Confirmed Test
procedures may not be required for
all gas-positive cultures. If, after
24-hours of incubation, all five
replicate cultures are gas-positive for
two or more consecutive sample
volumes, then select the set of five
cultures representing the smallest
volume of sample in which all tubes
were gas - positive.' Apply Confirmed
Test procedures to all these cultures
and to any other gas-positive cultures
representing smaller volumes of
sample, in which some tubes were
gas-positive and some were eas-
negative.
5 Label one tube of brilliant green lactose
bile broth (BGLB) to correspond with
each tube of'lauryl tryptose broth
selected"for Confirmed Test procedures.
6 Gently shake the rack of Presumptive
Test cultures. With a flame-sterilized
inoculation loop transfer one loopful of
culture from each gas-positive tube to
the corresponding tube of BGLB. Place
each newly inoculated culture into
BGLB in the position of the original
gas-positive tube.
7 If the Fecal Coliform Test is included
in the testing procedure, consult
Section V of'this outline for details of
the testing procedure.
8 After making the transfer, the rack
should contain some 24-hour gas-
negative tubes of lauryl tryptose borth
and the newly inoculated BGLB.
Incubate the rack of cultures at 35° C
+ 0. 5° C for 24 + 2 hours.
C 48-hour Procedures
1 Remove the rack of culture tubes from
the incubator, read and record gas
production for each tube.
2 Some tubes will be lauryl tryptose broth
and some will be brilliant green lactose
9-17
-------
MPN Methods
bile broth (BGLB). Be sure to record
results from LTB under the 48-hour
LTB column and the BGLB results
under the 24-hour column of the data
sheet.
3 Label tubes of BGLB to correspond with
all (if any) 48-hour gas-positive cultures
in lauryl tryptose broth. Transfer one
loopful of culture from each gas-positive
LTB culture to the correspondingly-
labeled tube of BGLB. NOTE: All tubes
of LTB culture which were negative at
24 hours and became positive at 48 hours
are to be transferred. The Option
described above for 24-hour LTB
cultures does not apply at 48 hours.
4 Incubate the 24-hour gas-negative BGLB
tubes and any newly-inoculated tubes of
BGLB 24 + 2 hours at 35° + 0. 5°C.
Retain all 24-hour gas-positive cultures
in BGLB for further test procedures.
5 Label a Petri dish preparation of eosin
methylene blue agar (EMB agar) to
correspond with each gas-;positive
culture in BGLB.
6 Prepare a streak plate for colony
isolation from each gas-positive culture
in BGLB on the correspondingly-labeled
EMB agar plate.
Incubate the EMB agar plates 24 + 2
hours at 35° + 0.5°C.
D 72-hour Procedures
1 Remove the cultures from the incubator.
Some may be on BGLB; several EMB
agar plates also can be expected.
2 Examine and record gas production
results for any cultures in BGLB.
3 Retain any gas-positive BGLB cultures
and prepare streak plate inoculations
for colony isolation in EMB agar.
Incubate the EMB agar, plates 24 +
2 hours at 35 + 0.5° C. Discard the
gas-positive-BGLB cultures after
transfer.
4 Reincubate any gas-negative BGLB
cultures 24 + 2 hours al 35° + 0.5°C.
5 Discard all 48-hour gas-negative BGLB
cultures.
6 Examine the EMB agar plates for the
type of colonies developed thereon.
Well-isolated colonies having a dark
center (when viewed from the lower
side, held toward a light) are termed
"nucleated or fisheye" colonies, and
are regarded as "typical" coliform
colonies. A surface sheen may or may
not be present on "typical" colonies.
Colonies which are pink or opaque but
are not nucleated are regarded as
"atypical colonies. " Other colony
types are considered "noncolifcrm. "
Read and record results as + for
"typical" (nucleated) colonies + for
"atypical" (non-nucleated pink or
opaque colonies), and - for other types
of colonies which might develop.
7 With plates bearing "typical" colonies,
select at least one well-isolated colony
and transfer it to a correspondingly-
labeled tube of lactose broth and to an
agar slant. As a second choice, select
at least two "atypical" colonies (if
typical colonies are not present) and
transfer them to labeled tubes of
lactose broth and to agar slants. As a
third choice, in the absence of typical
or atypical coliform-like colonies,
select at least two well-isolated
colonies representative of those
appearing on the EMB plate, and trans-
fer them to lactose broth and to agar
slants.
8 Incubate all cultures transfered from
EMB agar plates 24+2 hours at 35 +
0. 5°C.
E 96-hour Procedures
1 Subcultures from the samples being
studied may include: 48-hour tubes
of BGLB, EMB agar plates, lactose
broth tubes, and agar slant cultures.
9-18
-------
MPN Methods
2 If any 48-hour tubes of BGLB are
present, read and record gas production
in the "48" column under BGLB. From
any gas-positive BGLB cultures pre-
pare streak plate inoculations for colony
isolation on EMB agar. Discard all
tubes of BGLB, and incubate EMB agar
plates 24 + 2 hours at 35 + 0. 5° C.
3 If any EMB plates are present, examine
and record results in the "EMB" column
of the data sheet. Make transfers to
agar slants and to lactose broth from
all EMB agar plate cultures. In
decreasing order of preference, transfer
at least one typical colony, or at least
two atypical colonies, or at least two
colonies representative of those on the
plate.
4 Examine and record results from the
lactose broth cultures.
5 Prepare a Gram-stained smear from
each of the agar slant cultures, as
follows:
NOTE: Always prepare Gram stain
from an actively growing culture,
preferably about 18 hours old, and
never more than 24 hours old. Failure
to observe this precaution often results
in irregular staining reactions.
a Thoroughly clean a glass slide to
free it of any trace of oily film.
b Place one-drop of distilled water on
the slide.
c Use the inoculation needle to suspend
a tiny amount of growth from the
nutrient agar slant culture in the
drop Of water.
d Mix the thin suspension of cells with
the tip of the inoculation needle, and
allow the water to evaporate.
e "Fix" the smear by gently warming
the slide over a flame.
f Stain the smear by flooding it for 1
minute with crystal violet solution.
g Flush the excess crystal violet
solution off in gently running water,
and gently blot dry with filter
paper or with other clean absorbent
paper.
h, Flood the smear with Lugol's
iodine for 1 minute.
i Wash the slide in gently running
water and blot dry with filter paper.
j Decolorize the smear writh 95%
alcohol solution with gentle
agitation for 10-30 seconds,
depending upon extent of removal
of crystal violet dye, then blot dry.
k Counterstain for 10 seconds with
safranin solution, then wash in
running water and blot dry.
¦1 Examine the slide under the
microscope, using the oil
immersion lens. Goliform
bacteria are Gram-negative,
nonspore-forming, rod-shaped
cells, occurring singly, in pairs,
or rarely in short chains.
m If typical coliform staining reaction
and morphology are observed,
record + in the appropriate space
under the "Gram Stain" column of
the data sheet. If typical morphology
and staining reaction are not
observed, then mark it + or -, and
make suitable comment in the
"remarks" column at the right-hand
side of the data sheet.
n If spore-forming bacteria are
observed, it will be necessary to
repurify the culture from which
the observations were made.
Consult the instructor, or refer
to Standard Methods, for procedures.
At this point, it is possible that all
cultural work for the Completed Test
has been finished. If so, codify results
and determine Completed Test coliforms
per 100 ml.
9-19
-------
MPN Methods
F 120-hour Procedures and following:
1 Any procedures to be undertaken from
this point are "straggler" cultures on
media already described, and requiring
step-by-step methodology already given
in detail. Such cultures may be on:
EMB plates, agar slants, or lactose
broth. The same time-and-temperature
of incubation required for earlier studies
applies to the "stragglers" as do the
observations, staining reactions, and
interpretation of results. On con-
clusion of all cultural procedures,
codify results and determine Completed
Test coliforms per 100 ml.
V FECAL COLIFORM TEST
A General Information
1 The procedure described is an elevated
temperature test for fecal coliform
bacteria.
2 Equipment required for the tests are
those required for the Presumptive
Test of Standard Methods, a water-bath
incubator, and the appropriate culture
media.
B Fecal Coliform Test with EC Broth
1 Sample: The test is applied to gas-
positive tubes from the Standard
Methods Presumptive Test (lauryl
tryptose broth), in parallel with
Confirmed Test procedures.
2 24-hour Operations. Initial procedures
are the planting procedures described
for the Standard Methods Presumptive
Coliform test.
a After reading and recording gas-
production on lauryl tryptose broth,
temporarily retain all gas-positive
tubes.
b Label a tube of EC broth to corre-
spond with each gas-positive tube
of lauryl tryptose broth. The option
regarding transfer of only a limited
number of tubes to the Confirmed
Test sometimes can be applied here.
However, the worker is urged to
avoid exercise of this option until
he has assured the applicability of
the option by preliminary tests on
the sample source. J
c Transfer one loopful of culture from
each gas-positive culture in lauryl
tryptose broth to the correspondingly
labeled tube of EC broth.
d Incubate EC broth tubes 24 + 2 hours
at 44. 5 t 0. Z°C in a waterbath
with water depth sufficient to come
up at least as high as the top of the
culture medium in the tubes. Place
in waterbath as soon as possible
after inoculation and always within
3 0 minutes after inoculation.
3 48-hour operations
a Remove the rack of EC cultures
from the waterbath,' shake gently,
and record gas production for each
tube. Gas in any quantity is a
positive test.
b As soon as results are recorded,
discard all tubes. (This is a 24-
hour test1 for EC broth inoculations
and not a 48-hour test.)
c Transfer any additional 48-hour
gas-positive tubes of lauryl tryptose
broth to correspondingly labeled
tubes of EC broth Incubate 24 +
2 hours at 44. 5 i 0. 2°C.
4 72-hour operations
a Read and record gas production for
each tube. Discard all cultures.
b Codify results and determine fecal
coliform count per 100 ml of sample.
9-20
-------
MPN Methods
TESTS FOR CONFORM GROUP
a.
3
*
3
Qe
£
2
O
o

5
*
o
o
LACTOSE OR LAURYL TRYPTOSE BROTH
FERMENTATION TUBES (SERIAL DILUTION)
T

GAS POSITIVE
(24 HR.+ 2 HR.J
GAS NEGATIVE
I
LACTOSE « LAum TRYPTOSE
MOTH ARE INTERCHANGE A Bit MEDIA
AMD ARE RKUBATED AT 35 DEG C±
OJ DIG C.
GAS POSITIVES TUBES (ANY AMOUNT
OF GAS.) CONSTITUTE A POSITIVE
PRESUMPTIVE TEST
TOTAL INCUBATION TIME FOR LACTOSE
OR LTB IS 48 HRS.t 3 HRS.
GAS POSITIVE
GAS NEGATIVE
CONFORM GROUP ABSENT
CONFKMATORY BROTH
BRILLIANT GREEN LACTOSE BILE
'4 EMB PLATES
OR
l-NDO AGAR
PIATES
GAS POSITIVE
GAS NfGATIVt
COLIFORM GROUP
NOT CONFKMEO
GRAM + AND
RODS AND/OR
SPOREFORMERS
FORMATE
RICMOLfATE
BROTH
GRAM NEGATIVE
RODS
NO SPORES
GAS POSITIVE
GAS POSITIVE
1/
CONFORM GROUP PRESENT
COMPUTED TEST
GAS NEGATIVE
I
COLIFORM GROUP ABSENT
TRANSFER TO EMB PLATE
AND REPEAT PROCESS
INCUBATE BGLB TUBES FOR 48 HRS.
± 3 HRS. AT 35 DEG. C± 0.5 DEG. C.
INCUBATE EMB OR ENDOAGAR
PIATES FOR 24 HRS. ± 2 HRS AT
35 DEG. Ct 0J DEG.C.
GAS NEGATIVE
COUFORM GROUP ABSENT
-------
Part 3
LABORATORY METHODS FOR FECAL STREPTOCOCCUS
(Day-By-Day Procedures)
I GENERAL INFORMATION
A The same sampling and holding procedures
apply as for the coliform test.
B The number of fecal streptococci in water
generally is lower than the number of
coliform bacteria. It.is good practice
in multiple dilution tube tests to start the
sample planting series with one sample
increment larger than for the coliform
test. For example: If a sample planting
series of 1.0, 0.1, 0.01, and 0.001 ml
is planned for the coliform test, it is
suggested that a series of 10, 1.0, 0. 1,
and 0. 01 ml be planted for the fecal
streptococcus test.
C Equipment required for the test is the same
as required for the Standard Methods
Presumptive and Confirmed Tests, except
for the differences in culture media.
n STANDARD METHODS (Tentative)
PROCEDURES
A First-Day Operations
1 Prepare the sample data sheet and
labeled tubes of azide dextrose broth
in the same manner as for the
Presumptive Test. NOTE: If 10-ml
samples are included in the series, be
sure to use a special concentration
(ordinarily double-strength) of azide
dextrose broth for these sample
portions.
2 Shake the sample vigorously, approxi-
mately 25 times, in an up-and-down
motion.
3 Measure the predetermined sample
volumes into the labeled tubes of azide
dextrose broth, using the sample
measurement and delivery techniques
used for the Presumptive Test.
4 Shake the rack of tubes of inoculated
culture media, to insure good mixing
of sample with medium.
5 Place the rack of inoculated tubes in
the incubator at 35° + 0.5°C for 24 +
2 hours.
B 24-hour Operations
1 Remove the rack of tubes from the
incubator. Read and record the results
from each tube. Growth is-a positive
test with this test. Evidence of growth
consists either of turbidity of the
medium, a "button" of sediment at the
bottom of the culture tube, or both.
2 Label a tube of ethyl violet azide broth
to correspond with each positive culture
of azide dextrose broth. It may be
permissible to use the same confirmatory
option as described for the coliform
Confirmed Test, in this outline.
3 Shake the rack of cultures gently, to
resuspend any living cells which have
settled out to the bottom of the culture
tubes.
4 Transfer three loopfuls of culture from
each growth-positive tube of azide
dextrose broth to the correspondingly
labeled tube of ethyl violet azide broth.
5 As transfers are made, place the newly
inoculated tubes of ethyl violet azide
broth in the positions in the rack
formerly occupied by the growth-
positive tubes of azide dextrose broth.
Discard the tubes of azide dextrose
broth culture.
6 Return the rack, containing 24-hour
growth-negative azide dextrose broth
tubes and newly-inoculated tubes of
ethyl violet azide broth, to the incubator.
Incubate 24+2 hours at 35° + 0. 5° C.
9-23
-------
MPN Methods
C 48-hour Operations
1 Remove the rack of tubes from the
incubator. Read and report results.
Growth, either in azide dextrose broth
or in ethyl violet azide broth, is a
positive test. Be sure to report the
results of the azide dextrose broth
medium under the "48" column for that
medium and the results of the ethyl
violet azide broth cultures under the
"24" column for that medium.
2 Any 48-hour growth-positive cultures
of azide dextrose broth are to be
transferred (three loopfulls) to ethyl
violet azide broth. Discard all 48-hour
growth-negative tubes of azide dextrose
broth and all 24-hour growth-positive .
tubes of ethyl violet azide broth.
3 Incubate the 24-hour growth-negative
and the newly-inoculated tubes of ethyl
violet azide broth 24 + 2 hours at 35°
+ 0.5° C.
D 72-hour Operations
1 Read and report growth results of all
tubes of ethyl violet azide broth.
2 Discard all growth-positive cultures
and all 48-hour growth-negative
cultures.
3 Reincubate any 24-hour growth-negative
cultures in ethyl violet azide broth 24
+ 2 hours at 35° + 0.5°C.
E 96-hour Operations
1 Read and report growth results of any
remaining tubes of ethyl violet azide
broth.
2 Codify results and determine fecal
streptococci per 100 ml.
REFERENCES
1 Standard Methods for the Examination of
Water and Wastewater (13th Ed).
Prepared and published jointly by
American Public Health Associatipn,
American Water Works Association,
and Water Pollution Control
Federation. 1971.
2 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. 8:347-348. 1958.
3 Geldreich, E.E., Bordner, R.H., Huff,
C.B., Clark, H.F. and Kabler, P.W.
Type Distribution of Coliform Bacteria
in the Feces of Warm-Blooded Animals,
J. Water Pollution Control Federation.
34:295-301. 1962,
4 Recommend Proc. for the Bacteriological
Examination of Sea Water and Shellfish.
3rd Edition, American Public Health
Association. 1962.
This outline was prepared by H. L. Jeter,
Director, National Training Center, Water
Programs Operations, Environmental
Protection Agency, Cincinnati, OH 45268.
Descriptors: Coliforms, Fecal Coliforms,
Fecal Streptococci, Indicator Bacteria,
Laboratory Equipment, Laboratory Tests,
Microbiology, Most Probable Number, MPN,
Sewage Bacteria, Water Analysis
9-24
-------
MEDIA AND SOLUTIONS FOR MULTIPLE DILUTION TUBE METHODS
I INTRODUCTION
A This chapter is intended to present detailed
information on preparation and management
of media and solutions needed with the tests
and observations described elsewhere in
this course manual.
B The preparation and management of
supplies of culture media and solutions
is one of the most critical aspects of a
bacteriological water quality testing
program.
1 In the same manner that the chemist
relies on correctly prepared and
standardized reagents for his analytical
work, the bacteriologist must depend
on satisfactory culture media for the
type of analysis with which he is con-
cerned.
2 In many laboratories preparation of
media is entrusted to subprofessional
personnel. Most such personnel,
properly trained and guided, are able
to perform the required tasks efficiently
and reliably.
3 The professional supervisor should
maintain close attention to all details,
however, to guard against gradual
introduction of bad habits in preparing
and preserving media and other liquid
supplies.
II GENERAL INFORMATION
A Use of Commercially Available
Dehydrated Media
1 The preparation of all media described
in this chapter is given in terms of the
individual components, and preparation
of the finished medium This is done,
even through commercially available
dehydrated media are widely used, to
acquaint the worker with the compo-
sition of the media and to indicate the
required specifications of each medium.
2 The use of commercially available
dehydrated media, requiring only
careful weighing and dissolving of the
powder in the proper quantity and
quality of distilled water, is strongly
recommended Such media are much
more likely to have uniformity at an
acceptably high level of quality than
are media compounded in the laboratory
from the individual constituents
3 It is recommended that the worker,
when using commercially prepared
dehydrated media, keep a careful
record of the lot numbers of media
being used With first use of each
new lot number of a given medium, it
is suggested that the medium be checked
for stability, pH after sterilization,
and to see that performance is satis-
factory. While rare, an occasional
lot of medium will have some unforeseen
fault which reduces or destroys its
effectiveness. Maintenance of lot
number records on medium gives
opportunity for communication with
the manufacturer to determine whether
similar problems are being encountered
in other laboratories
B Quality of General Materials
1 Distilled water
Distilled water, or demineralized water,
is required. It must be free from
NOTE: Mention of commercial products and manufacturers is for illustration and does not imply
endorsement by the Environmental Protection Agency.
W. BA. met. 19e. 12. 72
10-1
-------
Media and Solutions for Multiple Dilution Tube Methods
dissolved metals or chlorine. Freedom
from bactericidal constituents or growth
promoting substances should be dem-
onstrated through laboratory tests.
A procedure for this test is described
elsewhere in this course manual.
2 Beef extract
Any brand of beef extract is acceptable,
provided that it is known to give results
acceptable to the user. Meat infusion
is not acceptable.
3 Peptones
Peptones are Sold under a wide variety
of trade names. Any peptone shown
satisfactory by comparative tests with
an acceptable peptone, may be accepted.
4 Sugars
All sugars must be chemically pure,
and suitable for bacteriological media.
5 Agar
Any form of bacteriologic grade of
agar can be used.
6 General chemicals must be reagent
grade or ACS if used in culture media.
Chemicals used in the distilled water
quality test must be of the highest purity
available.
7 Dyes
All dyes used in culture media must be
certified by the Biological Stain
Commission; they will be so labeled
on the container.
C Quality of Equipment and Supplies Used
for Preparation of Media
1 Glassware
It is recommended that all glassware
be of borosilicate glass. Such glass
is not subject to release of soluble
products into the culture medium, as
with some of the so-called "soft glass. 11
2 Balance
A balance with sensitivity of + 2 grams
with a load of 150 grams is the minimum
acceptable standard for weighing of
culture media in dehydrated form.
3 pH meter
An electrometric meter is recommended
While a comparator block with pH
indicator solutions is useful for such
media as laur-yl tryptose broth, it
cannot be used satisfactorily with dye-
containing media such as brilliant
green lactose bile broth. Therefore
it is suggested that all pH control
work on bacteriological media be done
with an electrometric type of pH meter.
Accuracy of the meter should be estab-
lished through calibration against a
standard buffer.
4 Autoclave
The autoclave should be of sufficient
size to permit loose packing of tubed
media when normal load is being
sterilized. This is to permit free
access of steam to all surfaces.
Operation should be such that sterilizing
temperature is reached in not more than
3 0 minutes.
A pressure gauge should be present.
More important, the autoclave should
be equipped with at least 1 thermometer,
which should be located properly in the
exhaust line.
Pressure regulation should permit
operation up to and including 121C>C.
When media containing carbohydrates
are present, sterilization should be
continued 12 - 15 minutes, in media
not containing carbohydrates, normal
sterilization time should be a standard
15 minutes.
After sterilization, media should be
removed from the autoclave as soon
as possible. In no case should an
autoclave simply be turned off after
10-2
-------
Media and Solutions for Multiple Dilution Tube'Methods
the usual exposure to steam under
pressure, and allowed to stand until the
following morning before removing media.
5 Utensils for mixing and preparing media
Borosilicate glass is suggested, but
other materials, such as stainless
steel, porcelain (unchipped) containers,
or other containers free of soluble
bactericidal or bacteriostatic materials,
are acceptable. In any case, the con-
tainers must be thoroughly clean.
III CONCENTRATION OF MEDIA
A Basic formulas of all media described in
Section IV are presented as single-strength
media. Most media are used in the single-
strength concentration.
B The concentration of primary inoculation
media (media into which the measured
portions of the original sample are
delivered) requires special consideration.
1 When the amount of medium is 10 ml or
greater, and the volume of sample or
sample dilution is 1 ml or less, then
single-strength medium is satisfactory.
2 When the sample volume introduced
into the primary inoculation medium
is greater than 1 ml, then it is necessary
to compensate for the diluting effect
of the sample on the culture medium.
In such cases, it is necessary to
increase the initial concentration of
the medium so that after sample
inoculation the concentration of nutrients
in medium-plus-sample is equivalent
to the concentration of nutrients in the
single strength medium.
IV PREPARATION OF MEDIA AND SOLUTIONS
A Lauryl Tryptose Broth (Lauryl Sulfate Broth)
1 Use: Primary inoculation medium in
Presumptive Test
2 Composition:
Tryptose (or Trypticase 20. 0 g
or equivalent)
Lactose 5 0 g
Dibasic Potassium 2.75 g
Phosphate (KgHPO^)
Monobasic Potassium 2.75 g
Phosphate (KH2PO4)
Sodium Chloride 5.0 g
Sodium Lauryl Sulfate 0. 1. g
(Total Dry Constituents 35.60 g)
Distilled Water 1000 ml
Sterilization: 12 - 15 minutes at 1210C
Reaction after sterilization- pH 6. 8
approximately)
3 Compensation for diluting effect of
samples
No. ml Ml of Nominal No. grams
medium sample or concentra- dehydrated
in tube dilution tion before medium per
inoculation liter
10 0. 1 - 1.0 lx 35. 6
10 10 2x 71 2
20 10 l.5x 53.4
35 100 4x 137.3
B Brilliant Green Lactose Bile Broth
1 Use: Confirmed Test
2 Composition
Peptone (Bacto or equivalent) 10.0 g
Lactose 10. 0 g
Oxgall (dehydrated) 20.0 g
Brilliant Green 0.0133 g
(Total weight dry constituents 40. 0133 g)
Distilled Water 1000 ml
Sterilization: 12 - 15 minutes at 1210C
Reaction after sterilization: pH 7.1 to 7.4
10-3
-------
Media and Solutions for Multiple Diiution Tube Methods
C Eosin Methylene Blue Agar
1 Confirmed Test
Use: Isolation of coliform-like
colonies as a preliminary to
Completed Test procedures.
2 Composition
Peptone (Bacto or equivalent) 10 g
Lactose 10 g
Dipotassium Phosphate (K^HPO^) 2 g
Agar 20 g
Eosin Y 0. 4 g
Methylene Blue 0. 65 g
(Total weight dry constituents 43. 05 g)
Distilled Water 1000 ml
Sterilization: 12 - 15 minutes at 1210C
3 Special suggestions on preparation:
a This medium can be prepared and
dispensed into bottles or flasks in
portions of 100 ml or 200 ml each.
The sterile medium may be stored
for extended periods in cool places
out of the light.
b When ready for use of such medium,
the medium should be melted by
immersion of the bottle of prepared
medium in a boiling water bath,
after which it is dispensed into
sterile Petri dishes in portions of
approximately 15 ml. After cooling
and solidifying in the Petri dish, the
medium is ready for use. It should
be used preferably on the day it is
poured into Petri dishes, but can be
stored for a day or two in the
refrigerator.
c An alternate method of preparing
this medium requires preparation
the agar base medium which
includes all the constituents
of the medium except the dyes.
When ready to use such a preparation,
the agar base medium is melted in
a water bath, and to each 100 ml of
the melted agar base medium, 2 ml
of 2% of aqueous solution of eosin Y
and 1.3 ml of 0. 5% methylene blue
solution is delivered with a pipet.
The medium is mixed thoroughly,
poured into Petri dishes, and used
as previously described.
D Agar Slants
1 Use: This medium is used in the
Completed Test, to cultivate pure
cultures of strains of bacteria being
cultivated in preparation of a Gram-
stained smear.
2 Composition: The medium is nutrient
agar
Peptone
Beef extract
Agar
(Total weight dry
constituents
Distilled Water
5. 0 g
3-0 g
15.0 g
23.0 g)
1000 ml
Sterilization: 15 minutes at 121QC
Reaction after sterilization: pH 6.8
approximately
3 Special instructions: Dissolve the con-
stituents, using heat as needed; dispense
in amounts of approximately 8 ml per
tube. Screw-capped tubes extend shelf
life of the medium. After sterilization,
remove the melted medium from the
autoclave and place in a slanting
position until the medium has become
solidified. A routine procedure should
be established so that a uniform volume
of medium and a uniform surface of
slanted medium be present in each tube.
While this has no particular bearing on
Standard Methods procedures, certain
other laboratory procedures do require
uniform exposed surface area of the
slanted medium.
E Plate Count Agar
1 Use: This medium is used in the
distilled water test. It is not used in
other Standard Methods procedures
described in this course manual.
10-4
-------
Media and Solutions for Multiple Dilution Tube Methods
2 Composition: (Tryptone Glucose
Yeast Agar)
Peptone-tryptone (or equivalent) 5.0 g
Yeast extract 2.5 g
Glucose (dextrose) 1. 0 g
Agar 15. 0 g
(Total weight dry constituents 23.5 g
Distilled Water 1000 ml
Sterilization: 15 minutes at 121°C
Reaction after sterilization: pH 7.0 +
0:1
3 Special instructions in preparation:
Use heat as needed to dissolve and
melt the constituents. Dispense the
medium in flasks or boty.es in portions
of 100 or 200 ml each and sterilize. In
this state it can be preserved for many
months, provided that it is protected
from evaporation of the water.
When ready to use, melt the medium
by heating, and cool to 45° C. At this
temperature the medium still should be
melted, and will be satisfacotry for
preparation of pour plates for plate
counts.
F EC Broth
1 Use: Test for fecal coliform bacteria
2 Composition:
Tryptose (Bacto or equivalent) 20.0 g
Lactose 5.0 g
Bile Salts (Bacto #3 or equivalent) 1.5 g
Dipotassium phosphate (KgHPO ) 4. 0 g
Monopotassium phosphate (KH^PO^) 1.5 g
Sodium chloride 5.0 g
(Total weight dry constituents 37.0 g)
Distilled Water 1000 ml
Sterilization: 12 - 15 minutes at 121° C
Reaction after sterilization: pH 6.9
3 This medium is dispensed into culture
tubes with inverted fermentation vials
and suitable caps.
G Azide Dextrose Broth
1 Use: Primary inoculation medium for
fecal streptococcal presumptive test.
2 Composition:
Beef extract 4.5 g
Tryptone or Polypeptone 15. g
Glucose 7.5 g
Sodium chloride 7.5 g
Sodium azide 0. 2 g
(Total dry constituents 34. 7 g)
Distilled Water 1000 ml
Sterilization: 12 - 15 minutes at 1210C
Reaction after sterilization: about pH 7.2
3 Fermentation vials are not used with
azide dextrose broth.
H Ethyl Violet A zide Broth
1 Use: Confirmed test for fecal
streptococci
2 Composition:
Tryptone or Biosate 20 g
Glucose 5 g
Sodium chloride 5 g
Potassium phosphate, 2.7 g
diabasic (K2HP04)
Potassium phosphate, 2.7 g
monobasic (KHgPC^)
Sodium azide 0.4 g
Ethyl violet (certified dye . 00083 g
if available)
(Total dry constituents 35. 8 g)
Distilled Water 1000 ml
Sterilization: 12 - 15 minutes at 1210C
Reaction after sterilization: about pH 7
10-5
-------
Media and Solutions for Multiple Dilution Tube Methods
3 Fermentation vials are not used with
ethyl violet azidc broth.
I Buffered Dilution Water
1 Use: Preparation of sample dilutions
preliminary to primary inoculation, in
membrane filter work, and in plate
counts.
2 Composition
a Stock phosphate buffer solution
Monobasic Potassium 34. 0 g
Phosphate (KHgPO^)
Distilled Water 500 ml
IN NaOH solution (about 175 ml)
to give pH 7. 2
Distilled water sufficient to bring final
volume to 1000 ml
b Working solution of phosphate buffered
distilled water
Stock phosphate buffer solution 1.25 ml
Distilled water 1000 ml
3 Preparation and handling:
a Stock solution: After preparation the
stock solution should be stored in the
refrigerator until use. If at any time
evidence of mold or other contam-
ination appears, the stock solution
should be discarded and a fresh
solution prepared.
b Working solution: Dispense the
required amount into distilled water,
and deliver into screw-capped bottles
for dilution water. The amount added
should be such that, after sterilization,
the bottles will contain 99+2 ml of
the dilution water. Ordinarily this
requires initial addition of approxi-
mately 102 ml of the solution prior
to sterilization.
c Sterilization is 20 minutes at
12 lo c.
d Tightly stoppered bottles of the
dilution water, protected against
evaporation, in suitable containers,
appear to last indefinitely.
J Solutions for Gram Stain
1 Ammonium oxalate crystal violet
solution:
a Dissolve 2 g crystal violet
(approximately 85% dye content) in
20 ml of 95% ethyl alcohol.
b Dissolve 0.8 grams ammonium
oxalate in 80 ml distilled water.
c Mix solutions a and b.
d Filter through cheesecloth or coarse
filter paper.
e Problems with the gram stain
technique frequently are traceable
to the ammonium oxalate crystal
violet solution. In the event that
decolorization does not seem satis-
factory, the amount of crystal violet
in the solution can be reduced to as
little as 10% of the recommended
amount.
2 Lugol's iodine: Dissolve 1 g iodine
crystals and 2 g potassium iodide in
the least amount (usually about 5 ml)
of distilled water in which they are
soluble. After all crystals are in
solution, add sufficient distilled water
to bring the final solution to a volume
of 300 ml.
3 Counterstain: Dissolve 2.5 grams of
safranin in 100 ml of 95% ethyl alcohol.
For the working solution of counterstain,
add 10 ml of this solution of safranin to
100 ml of distilled water.
10-6
-------
Media and Solutions for Multiple Dilution Tube Methods
REFERENCES
Standard Methods for the Examination of
Water and Wastewater. 13th ed. 1971
2 Public Health Service Bacteriological
Survey Form for Water Laboratories.
PHS Form 875 (Revised 1966).
This outline was prepared by H. L. Jeter,
Director, National Training Center,
WPO, EPA, Cincinnati, OH 452G8.
10-7
-------
USE OF TABLES OF MOST PROBABLE NUMBERS
Part 1
I INTRODUCTION
A Using probability mathematics, it is
possible to estimate the number of bacteria
producing the observed result for any com-
bination of positive and negative results
in ' dilution tube tests. Because the
computations are so repetitious and time-
consuming, it is common laboratory
practice to use Tables of Most Probable
Numbers. These tables are orderly
arrangements of the possible cultural
results obtainable from inoculating various
sample increments in differential culture
media. Each possible combination of
positive and negative tube results is
accompanied by the result (MPN) of the
calculated estimate and the 95% confidence
limits of the MPN.
B The Tables of Most Probable Numbers used
in the current (13th) edition of Standard
Methods for the Examination of Water and
Wastewater were developed by Swaroop.^^
Previous editions of Standard Methods have
used the tables prepared by Hoskins.
1 Most of the tables are based on using
3 sample volumes in decreasing decimal
increments. Thus, the systems are
based on using volumes of 10 ml, 1.0 ml,
and 0.1 ml, etc. Other quantity
relationships can be used, such as
50 ml, 10 ml, and 1. 0 ml in a table.
Tables of Most Probable Numbers can
be prepared for any desired series of
sample increments.
2 In addition, tables can be devised for
different numbers of replicate
inoculations of individual sample
volumes. For example, the MPN
Table most commonly used in the
laboratories of this agency is based
on five replicate 10 ml portions, five
1. 0 ml portions, and five 0. 1 ml portions.
A separate table is required for another
combination of sample volumes, con-
sisting of five replicate 10 ml portions,
one 1. 0 ml portion, and one 0. 1 ml
portion. This is popular in bacteri-
ological potability tests on water.
MPN Tables can be prepared for any
desired combinations of replicates of
the sample increments used in a
dilution tube series.
3 An approximation of the MPN values
shown in the Tables can be obtained
by a simple calculation, developed by
Thomas. (3) The formula and
application of this calculation is shown
on a later page of this chapter.
C The method of using a Table of Most
Probable Numbers is described here,
based on the table for five 10 ml portions,
five 1. 0 portions, and five 0. 1 portions.
The principles apply equally to the other
tables presented in the current edition of
Standard Methods for the Examination of
Water and Wastewater.
II DETERMINING THE MOST PROBABLE
NUMBER
A Codifying Results of the Dilution Tube
Series
If five 10 ml portions, five 1. 0 ml portions,
and five 0. 1 ml portions are inoculated
initially, and positive results are secured
from five of the 10 ml portions, three of
the 1. 0 ml portions and none of the 0. 1 ml
portions, then the coded result of the test
is 5-3-0. The code can be looked up in
the MPN Table, and the MPN per 100 ml
is recorded directly. If more than the
above three sample volumes are to be
considered, then the determination of the
coded result may be more complex. The
examples described in Table 1 are useful
guides for selection of the significant series
of three sample volumes.
W. BA. 42g. 10. 71
11-1
-------
Use of Tables of Most Probable Numbers
Table 1. EXAMPLES OF CODED RESULTS
No. ml sample per tube 100
No. tubes per sample vol. -*¦ 5
10
5
1. 0
5
0. 1
5
0. 01
5
0. 001
5
Code
See Below
No. tubes in sample giving
5
4
1

5-4-1

positive results in test
5
4
0
0
¦ 0
5-4-0
(1)

4
1
0
0
0
o
1
1
(2)
5
5
4
1
1
0
5-4-2
(3)

5
5
5
4

5-5-4
(4)

5
5
5
5

5-5-5
(5)

0
0
0
0

o
{
• o
1
o
(6)

0
1
0
0

0
1
i
o
(7)

1
0
0
0

.1-0-0
(8)
Discussion of examples:
1 When all the inoculated tubes of more
than one of the decimal series give
positive results, then it is customary
to select the smallest sample volume
(here, 10 ml) in which all tubes gave
positive results. The results of this
volume and the next lesser volumes
are used to determine the coded result.
2 When none of the sample volumes give
positive results in all increments of
the series, then the results obtained
are used to designate the code. Note
that it is not permissible to assume
that if the next larger increment had
been inoculated, all tubes probably
would have given positive results and
therefore assign a 5-4-1 code to the
results.
3 Here the results are spread through
four of the sample volumes. In such
cases, the number of positive tubes in
the smallest sample volume is added
to the number of tubes in the third
sample volume (counting down from the
smallest sample volume in which all
tubes gave positive results).
4 Here it is necessary to use the 5-5-4
code, because inoculations were not
made of 0. 001 ml sample volumes;
and it is not permissible to assume
that if such sample volumes had been
inoculated, they would have given
negative results, or any other arbitrarily-
designated result.
5 This is an indeterminate result. Many
MPN tables do not give a value for such
a result. If the table used does not
have the code, then look up the result
for code 5-5-4, and report the result
"greater than" the value shown for the
5-5-4 code. The first number of the
5-5-4 code is based on the 1, 0 ml
sample volume.
6 Like (5), this is an indeterminate
result. If the code does not appear in
the table being used, then look up the
result for code 1-0-0, and report the
MPN as "less than" the value shown
for the 1-0-0 code.
7 The current edition of Standard
Methods stipulates this type of code
designation when unusual results
such as this occur.
11-2
-------
Use of Tables of Most Probable Numbers
8 Note the difference from (7) above.
Inoculations of 100 ml portions were
not made, and it cannot be assumed
that the result would have called for
code 0-1-0.
B Computing and Recording the MPN
When the dilution tube results have been
codified, they are read and recorded from
the appropriate MPN Table.
1 If, as in the first four of the examples
shown under (A) the first number
in the coded result represents a 10 ml
sample volume, then the MPN per 100 ml
is read and recorded directly from the
appropriate column in the table.
2 On the other hand, if the first number
in the coded result represents a sample
volume other than 10 ml, then a
calculation is required to give the
corrected MPN. For example (4) under(A)
above, the first "5" of the 5-5-4 code
represents a sample volume of 1. 0 ml.
Look up the 5-5-4 code as if the 1. 0 ml
volume actually were 10 ml, as if the
0. 1 ml volume actually were 1. 0 ml
and as if the 0.01 ml volume actually
were 0. 1 ml. The MPN obtained (1600)
then is multiplied by a factor of 10 to
give the corrected value. A simple
formula for this type of correction is
shown on a later page of this chapter.
I PRECISION OF'THE MPN VALUE
A The current edition of Standard Methods
shows for each MPN value, the 95%'
confidence limits for that value. This
draws attention to the fact .that a given
MPN value is not a precise measurement,
but an estimate. The 95% confidence
limits means that the observer will be
correct 95% of the time when he considers
that the actual number of cells producing
the observed combination of positive and
negative tubes was somewhere between
the stated upper limit and the stated
lower limit.
B The greater the number of replicates of
each sample volume in a dilution series,
the greater the precision (in other words,
the narrower the limits of the 95%
confidence range) of the test. The
precision of results, based on numbers
of tubes inoculated per sample volume,
is shown in Table 2.
(4)
C Woodward and other workers have
studied the precision of the MPN in
detail. Such reports should be studied
by those desiring further information
regarding the precision of the MPN test.
Table 2. Approximate Confidence Limits for Bacterial Densities as
Per Cent of MPN as Determined from Various Numbers of Tubes
in Three Decimal Dilutions*
Number of tubes 50% 75% 80% 90% 95%
in each dilution Lower Upper Lower Upper Lower Upper Lower Upper Lower Upper
1
33
186
18
340
15
402
10
637
6. 5
955
2
47
160
31
246
27
276
20
383
15
51 1
3
53
150
38
215
34
237
26
311
21
39 5
5
64
139
49
182
46
196
37
241
31
289
10
76
127
63
152
60
160
52
184
46
208
*The interpretation of these figures is as follows: When MPN estimates are
made on the basis of dilution tests using one tube in each of three decimal
dilutions, you will be right 50% of the time if you say that the true bacterial
density is between 33% and 186% of the MPN. If you had used 5 tubes in each
dilution you could reduce this interval to from 64% to 139% of the MPN and still
be right 50% of the time. If a greater certainty were desired, say 95%, you
would have to widen this interval to from 31% to 289%.
11-3
-------
Use of Tables of Most Probable Numbers
IV OCCURRENCE OF IMPROBABLE TUBE
RESULTS
A Many of the theoretically possible tube
^results are omitted from the MPN Table.
For example, codes 0-0-3, 0-0-4, and
0-0-5 are not included as well as many
others. These are omitted, because, in
the opinion of the authors of the tables,
the probability of occurrence of such
results is so low as to exclude them from
practical consideration.
B The frequency of occurrence of various
code results is shown in the Table 2 both
on a theoretical basis and on the basis of
actual laboratory experience.
C From the MPN tables, it can be inferred
that the codes omitted from the MPN
Ta.ble can be expected to occur up to 1%
of the time. If, in reviewing laboratory
data, the theoretically unlikely codes
occur appreciably more than 1% of.the
time, there is an indication for inquiry
into the causes. Such results can occur (1)
as a consequence of faulty laboratory
procedures, or (2) as a result of.
extraneous influences in the samples.
D The current edition of Standard Methods
does not include MPN values for many
rare combinations listed in previous
editions. By pruning out those codes listed
as Group IV in Table 3, the table has been
considerably condensed. Table 4 suggests
maximum permissible numbers of samples
for various numbers of samples tested.
Table 3
FIVE-TUBE AND THREE-TUBE CODES THAT
INCLUDE 99 PER CENT OF ALL RESULTS
Group
Theoretically Ex-
pected Percentage
of Results
Theoretically Ex-
pected Cumulative
Percentage
Observed Percentage
of 360 Samples

Five-Tube-Test
Class 1 codes
550, 551, 552. 553,
67. 5
67. 5
68.0
554, 500, 510. 520,

530, 540, 100. 200,

300, 400.

Class 2 codes
511, 521, 531. 541,
23. 6
91. 1
23. 1
542, 110, 210, 310,

410, 420.

Class 3 codes
501, 010, 532, 320,
7. 9
99. 0
7. 5
522, 220, 543. 430,

120, 533, 330, 502,

020, 544, 440, 301,

401, 431, 201, 411,

101, 311, 421. 211,

001.

Improbable codes
1.0
100. 0
1. 4

Three-Tube Test

Class 1 codes
330, 331, 332, 300,
81. 5
81. 5
81. 7
310, 320, 100, 200.

Class 2 codes
321, 311, 301, 210,
14. 9
96. 4
14. 1
110, 010.

Class 3 codes

322, 220, 201, 101
2. 7
99. 1
3. 7
312, 120.

Improbable codes
0. 9
100. 0
0.6
11-4
-------
Use of Tables of Most Probable Numbers
Table 4
MAXIMUM PERMISSIBLE NUMBERS
OF IMPROBABLE CODES FOR VARIOUS
NUMBERS OF SAMPLES TESTED
Example: From a sample of water, 5 out
of five 0. 01 - ml portions, 2 out of five
0. 001' - ml portions, and O out of five
0.0001 - ml portions, gave positive
reactions.
Number of Maximum Number
Samples of Improbable Codes
1 -
15
1
IS -
45
2
46 -
83
3
84 -
130
4
131 -
180
5
181 -
233
6
234 -
290
7
291 -
350
8
351 -
413
9
414 -
477
10
478 -
543
11
^ Table 5 is from International Standards
for Drinking-Water, published by the World
Health Organization, Geneva (1958). The
last three values, not shown in the WHO
publication, are from Woodward, "How
Probable is the Most Probable Number. "(4)
F Several theoretically possible combinations
of positive tube results are omitted in
Table 5. These combinations are omitted
because the statistical probability of
occurrence of any of the missing results
is less than 1%. If such theoretically
unlikely tube combinations occur in more
than 1% of samples, there is need for
review of the laboratory procedures and
of the nature of the samples being tested.
When the series of decimal dilutions is
other than 10, 1. 0 and 0. 1 ml, use the
MPN in Table 5, according to the following
formula:
MPN 10
(from table) Largest quantity tested
= MPN/100 ml
From the code 5-2-0 in the MPN table,
the MPN index is 49
(from (able) X"OT = 000
MPN Index = 49, 000
A simple approximation of the most
probable number may be obtained from
the following formula (after Thomas):
MPN/100 ml =
No. of Positive Tubes X 100
^ No. of ml in negative tubes) X(No. of ml
in all tubes)
Example: From a sample of water, 5 out
of five 10 - ml portions, 2 out of five
1.0 ml portions, and 0 out of five 0. 1 ml
portions gave positive results.
7 y 1 AH
MPN/100 ml ¦ ¦ ¦= 50.22
¦J (3. 5) X(55. 5)
MPN/100 ml = 50
Note that the MPN obtained from the table
on the preceding pages with these tube
results is 49. "Most probable numbers
computed by the above formula deviate
from values given by the usual methods
by amounts which ordinarily are
insignificant. The formula is not.
restricted as to the number of tubes
and dilutions used —" (Thomas)
11-5
-------
Use of Tables of Most Probable Numbers
Table 5. MPN INDEX AND 95% CONFIDENCE LIMITS FOR VARIOUS
COMBINATIONS OF POSITIVE AND NEGATIVE RESULTS WHEN FIVE
10-ML PORTIONS, FIVE 1-ML PORTIONS AND FIVE 0. 1 ML PORTIONS
ARE USED
No. of Tubes Giving

95% Con

No. of Tubes Giving

95% Con-
Positive Reaction out of
MPN
fidence Limits

Positive Reaction out of
MPN
fidence
Limits

Index
per

5 of 10
5 of 1
5 of 0.1
Lower
Upper

5 of 10
5 of 1
5 of 0. 1
Lower
Upper
ml Each
ml Each
ml Each
100 ml

ml Each
ml Each
ml Each
100 ml

0
0
0
<2

78
0
0
1
2
<0.5
7

4
2
1
26
9
0
0
1
0
2
<0.5
7

4
3
0
27
9
80
2
0
4
<0. 5
11

4
3
1
33
11
93

4
4
0
34
12
93
1
0
0
2
<0.5
7

1
1
1
1
0
1
4
<0.5
11

5
0
0
23
7
70
1
0
4
<0.5
11

5
0
1
31
11
89
1
1
6
<0.5
15

5
0
2
43
15
110
2
0
6
<0.5
15

5
1
0
33
11
93

5
1
1
46
16
120
2
0
0
5
<0.5
13

5
1
2
63
21
150
2
0
1
7
1
17

2
1
0
7
1
17

5
2
0
49
17
130
2
1
1
9
2
21

5
2
1
70
23
170
2
2
0
9
2
21

5
2
2
94
28
220
2
3
0
12
3
28

5
3
0
79
25
190

5
3
1
110
31
250
3
0
0
8
1
19

5
3
2
140
37
340
3
0
1
11
2
25

5
3
3
180
44
500
3
1
0
11
2
25

5
4
0
130
35
300
3
3
1
1
14
4
34

5
4
1
170
43
490
2
0
14
4
34

5
4
2
220
57
700
3
2
1
17
5
46

5
4
3
280
90
850
3
4
3
0
17
5
46

5
4
4
350
120
1, 000
0
0
13
3
31

5
5
0
240
68
750
4
4
4
4
4
0
1
17
5
46

5
5
1
350
120
o
O
o
1
0
17
5
46

5
5
2
540
180
1, 400
1
1
21
7
63

5
5
3
92 0
300
3, 200
1
2
26
9
78

5
5
4
1600
640
5, 800
2
0
22
7
67

5
5
5
>2400

11-6
-------
Use of Tables of Most Probable .Numbers
Table 6. MPN AND 95% CONFIDENCE LIMITS .FOR, VARIOUS
COMBINATIONS OF POSITIVE RESULTS IN A PLANTING
SERIES OF FIVE 10-ml PORTIONS OF SAMPLE
No, of Positive Tubes Out ol:
Five 10-ml Tubes
MPN per
100 ml
¦ Limits of MPN
Lower
"Upper
0
2. 2
0
6. 0
1
2. 2
0. 1
12. 6
2
5. 1
0. 5
19. 2
3
9. 2
l ". 6
29. 4'
4
16. 0
3. 3
52. 9
5
> 16
8. 0

Table 7. MPN AND 95% CONFIDENCE LIMITS FOR VARIOUS
COMBINATIONS OF POSITIVE RESULTS IN A PLANTING SERIES OF
FIVE 10-ml, ONE 1-ml, AND ONE 0.1-ml PORTIONS OF SAMPLE
No. of Positive Tubes Out of-
MPN
per
100 ml
Limits
of MPN
Five 10-ml
Tubes
One 1-ml
T ube
One 0. 1 -ml
Tube
Lower
Upper
0
0
0
<2

5.9
0
1
0
2
0 050
13
1
0
0
2. 2
0. 050
13
1
1
0
4. 4
0.52
14
2
0
0
5
0. 54
19
2
1
0
7. 6
1.5
19
3
0
0
8. 8
1. G
29
3
1
0
12
3. 1
30
4
0
0
15
3. 3
46
4
0
1
' 20
5.9
48
4
1
0
21
6.0
53
5
0
0
38
6. 4
330
5
0
1
96
12
370
5
1
0
240
12
3700
5
1
1
>240
88

11-7
-------
Use of TabLes of Most ProbabLe Numbers
IV TABLES OF MOST PROBABLE
NUMBERS
These tables consist of the MPN indices and
95% confidence limits, within which the
actual number of organisms can lie,' for
various combinations of positive and
negative tubes. Three MPN tables are
presented. Table 5 is based on five 10 ml
five 1. 0 ml and five 0. 1 ml sample portions;
Table 6 is based on five 10 ml sample por-
tions; and Table 7 is based on five 10 ml,
one 1 ml and one 0. 1 ml sample portion.
3 Thomas, H.- Al, Jr. Bacterial
¦Densities from Fermentation Tubes.
J.A.W.W.-A. 34:572. 1942.
4 Woodward, R. L. How Probable is the
Most Probable Number? J.A.W.W.A.
49:1060. 1958.
5 Standard Methods for the Examination of
Water and Wastewater. 13th Edition.
Prepared and Published Jointly by
American Public Association.
American Water Works Association,
and Water Pollution Control Federation.
REFERENCES
1 Swaroop, S. Numerical Estimation of
B. coli by Dilution Method. Indian J.
Med. Research. 26:353. 1938.
2 Hoskins, J.K. Most Probable Numbers
for Evaluation of Coli-Aerogenes Tests
by Fermentation Tube Method'. Public
Health Reports. 49:393. 1934.
This outline was prepared by H. L. Jeter,
Director, National Training Center,
Water Programs Operations, Environmental
Protection Agency, Cincinnati, OH 45268.
11-8
-------
MPN THEORY
Part 2
I DERIVATION OF THE MPN
A Assumptions
The validity of the MPN procedure is based
upon two principal assumptions.
1 In statistical language, the first is that
the organisms are distributed randomly
throughout the liquid. This means that
an organism is equally likely to be
found in any part of the liquid, and that
there is no tendency for pairs or groups
of organisms either to cluster together
or to repel one another.
2 The second assumption is that each
sample from the liquid, when incubated
in the culture medium, is certain to
exhibit growth whenever the sample
contains one or more organisms.
B The Probability Equation
Based upon these assumptions, an equation
for the probability of the observed com-
bination of positive and negative tubes can
be derived as a function of the true density
6. By solving this equation for different
values of 6 a curve can be plotted as shown
in Figure 1.
Curves of this type always have a single
maximum or peak. The value of 6, say d,
which corresponds to the peak of the curve
is called the most probable number,
commonly designated as MPN.
The MPN is "most probable" in the sense
that it is the number which maximizes the
probability of the observed results. It is
interesting to note that although the
original derivation of the MPN predates
modern statistical estimation, the MPN
procedure corresponds to the currently
accepted estimation procedure known as
the "method of maximum likelihood. "
MAXIMUM
i 1 u 1 r
d(MPN)
BACTERIA PER 100 ML
FIGURE 1
C Indeterminant Solutions
The MPN provides a meaningful estimate
of 6 only if there are both positive and
negative tubes in at least one dilution.
If all tubes are negative, the maximum
of the probability curve occurs when 6 is
set equal to zero (see Figure 2) and thus
the MPN is zero. If all tubes are positive,
the maximum of the probability curve
occurs when 6 is set equal to infinity
(see Figure 3) and thus the MPN is infinity.
ST.47.10.71
11-9
-------
MPN Theory
1.00
ALL TUBES NEGATIVE
MAXIMUM AT 8=0
BACTERIA PER 100 ML
CO
<
to
O
u
a.
SKEWED
MPN
FIGURE 2
FIGURE 4
1.00-7
ALL TUBES POSITIVE
BACTERIA PER 100 ML
FIGURE 3
II DISTRIBUTION OF MPN VALUES
A Skewed Distribution
If a very large number of independent
MPN determinations were made on the
same water sample, the distribution of
the MPN values would be such that very
high values relative to the median value
would occur more frequently than very
low values. Thus the distribution of MPN
values is skewed to the right as shown in
Figure 4.
B Logarithmically Normal Distribution
Since it is mathematically inconvenient
to work with data distributed asymmetri-
cally, it is desirable to transform the
skewed data in such a way that the trans-
formed values have a symmetric distri-
bution resembling the normal. In the
case of MPN values the logarithms of
the MPN's are approximately normally
distributed as shown in Figure 5.
CO
<
cfl
o
et
SYMMETRIC
(NORMAL)
log(MPN)
FIGURE 5
11-10
-------
MPN Theory
C Precision of MPN Estimates
The lack of precision of MPN estimates
of bacterial densities is generally recog-
nized. A measure of the precision is
given by the confidence limits on the
estimate which can be computed on the
basis of the normal distribution of the
logarithms of the MPN values. It has
been verified that three-tube and five-
tube MPN estimates are approximately
logarithmically normal and the standard
deviation of the logarithms of the MPN's
is given by the formula:
log
0. 58
j n
where o is the standard deviation of
the logarnnms of the MPN estimates and
n is the number of tubes in each dilution.
The upper and lower 95% confidence limits
of an MPN estimate are given by the
formulas:
UCL = antilog (log MPN + 1. 96a )
Notice that the confidence limits are not
symmetric about the MPN estimate.
The precision of the MPN estimate can
be increased by increasing the number
of tubes per dilution. Figure 6 shows the
width of the 95% confidence interval
expressed as a percentage of the MPN
estimate for various values of n. Notice
that the width of the confidence interval
decreases as n increases.
Ill PLANNING A DILUTION SERIES
A The Rationale
It was mentioned that the MPN procedure
provides a reasonable estimate of the true
density only if there are both positive and
negative tubes in at least one dilution.
It follows that in a series of dilutions the
expected number of organisms in the
highest sample volume (lowest dilution)
v should be at least one, otherwise all
tubes may be negative and the result will
be an indeterminant value.
= MPN • k,
LCL = antilog (log MPN - 1.96 a
= MPN -f k.
where k = antilog (1.96a , )•
log
log
log
450-|
400 -
<

>
—
CmL

*—

z
-
UJ
V
300 -
z

u.
Z

u
T
o
o
e*

u.
O

z
100-
a

*
"
I l i i ( I I I I I I I I I I I I I I
3 5 10 12 20
NUMBER OF TUBES PER DILUTION
FIGURE 6
11-11
-------
MPN Theory
Similarly, the expected number or
organisms in the lowest sample volume
(highest dilution) v should not exceed
one, to avoid the risk that all tubes will
be positive.
B The Rule
The above line of reasoning leads to the
rule that a dilution series is capable of
estimating any density between l/v and
1/v . In practice, we use the rule oy
first guessing two limits 6^ and 6 between
which we are fairly certain that trie actual
density lies. The sample volumes are
then chosen to satisfy the rules
•v-i; i
Table 1 displays the range of densities
covered by various decimal dilution;
series..
TABLE 1
SAMPLE VOLUME
(ML)
RANGE COVERED
(COLIFORMS/lOO ML)
10
io"
10
10
10
10
10
10
-1
-2
-3
-4
-5
-6
10-7J
10 1
- 10 3
102
- io4
10 3
- 10 5
104
1
o
©»
105
- 10 7
106
1
o
00
107
0>
o
1
This outline was prepared by J. H. Parker,
former Statistician, Analytical Quality
Control, Bureau of Water Hygiene, EPA,
Cincinnati, OH.
11-12
-------
THE MEMBRANE FILTER IN WATER BACTERIOLOGY
I HISTORICAL BACKGROUND
There is sometimes a tendency to look upon
membrane filters and their bacteriological
applications as newdevelopments. Both the
filters and many of their present bacterio-
logical applications are derived from earlier
work in Europe.
Some European developments prior to
1947 are as follows:
determination of bacterial counts,
coliform determinations, and isolation
of pathogenic bacteria from water and
other fluids. Most,early interest in
developing these techniques seems to
have been in Germany and in Russia.
During World War II Dr. G. Mueller
applied membrane filter techniques
to the bacteriological examination of
water, following bomb destruction of
many of the laboratories.
1 Fick is credited with application of
collodion membranes in biological
investigations in 1855.
2 Sanarelli, in 1891, reported develop-^
ment of membrane filters impermeable
to bacteria but permeable to their
toxins,
3 Bechhold, in the early 1900's made
a systematic study of the physico-
chemical properties of a number of
varieties of these membranes. After
1911 numerous investigations were
made in several countries with respect
to the properties of collodion membranes.
4 Zsigmondy and Bachmann, 1916-1918,
developed improved production methods
which were applicable on a commercial
scale. Membrane filters have been
produced for many years at the
Membranfiltergesellschaft, Sartorious
Werke, in Goettingen, Germany. In
1919 Zsigmondy applied for a U. S.
patent on his production methods; it
was granted iia 1922.
5 In the 1930's, W. J. Elford in England,
and P. Graber in France, made new
contributions in developing and teaching
methods for making collodion membranes
with controlled pore size.
B Developments m the United States .
1 In 1947, Dr. A. Goetz reported on a
mission to Germany as a scientific
consultant to the Technical Industrial
Intelligence Branch, U. S. Department
of Commerce. He obtained detailed
information about the nature, method
of preparation, and specific bacterio-
logical applications of the Zsigmondy
Membranfilter being manufactured
by the Membranfiltergesellschaft in
Goettingen.
2 After his return to this country,
Dr. Goetz developed methods for
preparing and improved type of
membrane filter from domestic
materials. On a small scale he
manufactured filters under a
government contract; afterward
membrane filter manufacture was
continued by a commercial organization.
3 In 1950, bacteriologists of the Public
Health Service began intensive study
of the applications of membrane filters
in bacteriological examinations of
water. Their first report was
published in 1951, and was followed
by numerous reports of other similar
investigations. Such studies have
been widely expanded, as indicated in
references shown elsewhe're in this
manual.
6 Before World War II filtration
procedures using the Zsigmondy
membrane had been suggested for the
"NOTE: Mention of corrfmercial products and manufacturers does not imply endorsement by the
OWP, Environmental Protection Agency.
W. BA. mem ,58h. 11.72
12-1
-------
The Membrane Filter in Water Bacteriology
4 In 1955 the 10th Edition of "Standard
Methods for the Examination of Water,
Sewage, and Industrial Wastes" included
a tentative method for coliforms by mem-
brane filter method. In the 11th and 12th
editions, the membrane filter method
for coliforms has become official. In
addition, methods for enterococcus
(fecal streptococci) are included as
tentative methods. The 13th edition
has given the fecal streptococcus test
a standard designation and the tentative
method status reserved for the "pour
plate" technique of quantitation.
5 The membrane filter is an official
method for examination of potable waters
in interstate commerce. The Public
Health Service Drinking Water Standards
(1962) state "Organisms of the coliform
group. . . All the details of technique
. . . shall be in accordance with Standard
1
Methods for Examination of Water and
Wastewater, current edition. " Thus,
acceptance by Standard Methods as
official automatically validates a method
for use with interstate waters.
II PROPERTIES OF MEMBRANE FILTERS
Membrane filters used in water bacteriology
are flat, highly porous, flexible plastic discs
about 0. 15 millimeters in thickness and usually
47-50 millimeters in diameter.
A Principle of Manufacture
The procedures described below are from
FIAT Report 1312. While the methods in-
dicated by Goetz do not necessarily describe
the current manufacturing processes, it is
assumed that similar principles of manu-
facture still apply.
1 One or more cellulose esters, such as
cellulose nitrate, is dissolved in a
suitable solvent.
2 Water, or some other liquid insoluble
in the cellulose solution, is added and
mixed, to form ah emulsion having great
uniformity in size and distribution of
droplets of the insoluble liquid.
3 The emulsion is cast on plates and dried
in an environment rigidLy controlled as to
humidity and temperature. The droplets
of insoluble fluid retain their size and
identity in the dried film, eventually
becoming the pores of the finished
membrane.
4 The dried .porous film is cut into filter
discs of the desired size. Representa-
tive discs are subjected to control tests
for accurate determination of the pore
size obtained.
5 Particle retention by membrane filters
¦ is at or very near the filter surface, by
a mechanical, sieve-like action. (This
applies to hydi'osols, not to aerosols.)
Through manufacturing control it is
possible to make membrane filters with
controlled pore size, within narrow limits.
B Some Important Characteristics of
Membrane Filters
1 The membrane filters used in micro-
biology should be flat, circular, gridded,
of uniform thickness and porosity, non-
toxic to microorganisms, wettable,
able to withstand commonly employed
sterilizing conditions, and unaffected by
the fluid to be filtered.
2 Without reference to specific manufacturers,
some particulars of their products have
included:
a ... Average pore diameter ranging
from 5 millimicrons to 10 'microns.
Thicknesses ranging from 70 to
150 microns. Can be sterilized
by autoclaving at 121°C for 10
minutes.
b ... mean flow pore size ranging from
7.5 millimicrons to 5 microns.
The pore size used in water
bacteriology having a standard
diameter, has agwater flow rate
of 70cc/'min/cm and must pass
100 ml of particle-free water
within 9 seconds.
12-2
-------
The Membrane Filter in Water Bacteriology
c ... currently produced in more
than twenty distinct pore sizes
from 14 microns to 10 milli-
microns in discs ranging from
13 mm to 293 mm in diameter.
The total range of pore size
distribution of the type used in
water microbiology of 0. 45 microns
is plus or minus 0,02 micron.
d ... membranes are offered in
graduated pore sizes ranging
from 12 microns to 5 milli-
microns. The types used in
water bacteriology have a dis-
tilled water^flow rate of 65
ml/min/cm at 700 mm Hg
differential pressure or an air ^
flow rate of 0.4 liters/min/cm
at a differential pressure of
500mm water.
4 Membrane filters are wettable. Thus,
after sample filtration, when a filter
is placed on moist culture medium the
medium diffuses through the pores and
is available to organisms collected on
the opposite surface.
5 Membrane filters are free of soluble
chemical substances inhibitory to bac-
terial growth. Water soluble plasticizers
are included in one commercially pro-
duced filter (glycerol, 2.5%). The
cellulose esters themselves have some
absorbing tendency illustrated by some
dyes and heavy metals. Total ash is
very low, less than 0. 0001%.
6 Membrane filters have a uniform index
of refraction. With membrane filters,
this index is N^: 1.5. When wetted
with a liquid having refractive index
within this range, the filters become
transparent. This property permits
direct microscopic examination of
particulate matter collected on the
filter surface.
7 Temperature resistance depends on
plastics used in the filter. The nitro-
cellulose membrane filter is stable dry
up to 125°C in air. Membranes
of cellulose triacetate are advertised to
withstand dry heat to 26G°C. In general,
however, membranes in current use
must be sterilized cautiously. Con-
sult the laboratory equipment discussion
for details. Overheating of aLl types
interferes with filtration by blocking
pores.
C Nomenclature
Membrane filters used in bacteriological
tests on water are known under several
names. Though the names are different,
the filters are similar in form, properties,
and method of use. Names commonly en-
countered are:
1 Membrane filter. This is the general
name for filters made according to
the general principles and having the
properties discussed above. The term
"membrane filter" is most used in
technical reports on filters of this type.
2 Molecular filter. This name used by
Goctz for the improved type of filter
that he and his associates developed
after study of the manufacturing methods
at the Membranfiltergesellschaft in
Goettingen, Germany.
3 Millipore filter is a trade name for
membrane filters made by the Millipore
Filter Corporation.
4- Bac-T-Flex filter is a trade name
applied to certain membrane filters
made by Carl Schleicher and Schuell
Company.
5 Oxoid filter is a trade name applied to
filters made by Oxo, Ltd., London,
England.
12-3
-------
The Membrane Filter in Water Bacteriology
6 Micropore, Polypore and Metricel
have been trade names used by the
Gelman Instrument Company.
Ill APPLICATIONS IN WATER BACTERIOLOGY
A The basic cultural procedures for bacterio-
logical tests on membrane filters are:
1 A sample is filtered through a membrane
filter.
2 The filter is placed in a culture con-
tainer, on an agar medium or a paper
pad impregnated with moist culture
medium.
3 The inoculated filter is incubated under
prescribed conditions of time, tempera-
ture, and humidity.
4 After incubation, the resulting culture
is examined and necessary interpreta-
tions and/or additional tests are made.
B With variations in such factors as culture
media, incubation time, and combinations
with other cultural and biochemical tests,
several different kinds of tests are
available.
1 Total bacterial counts are made by
cultivation of bacteria on membrane
filters using an enriched all-purpose
culture medium.
2 Tests for bacterial indicators of
pollution.
a Coliform tests
1) The direct membrane filter tests
for coliforms is one in which,
after sample filtration, the mem-
brane filter is incubated in con-
tact with one or more special
media. At least one of the media
is a selective, differential medium
including components which per-
mit coliform bacteria to develop
colonies easily recognizable by
form, color, sheen, or other
characteristics.
2) A verified membrane: filter coli-
form test can be used when needed
as a supplement to the direct mem-
brane filter test. Pure cultures
are obtained from individual
colonies differentiated on the
membrane filter and subjected
to further cultural, biochemical,
and staining tests to establish
the identity of the colonics being
studied.
3) The delayed membrane filter
coliform test was developed to
overcome bacterial changes fre-
quently occurring when there is
a delay of one to several days
between sample collection and
the initiation of laboratory tests.
The test consists of sample fil-
tration at or shortly after the time
of sample collection. The inocu-
lated filter is placed on a preser-
vative medium and taken or sent
to a laboratory, where it is trans-
ferred to a growth medium for the
differentiation of coliform colonies.
After incubation the culture is ex-
amined and the results are evalu-
ated as for the direct membrane
filter coliform test.
4) A medium and technique for
detecting and counting fecal
coliform bacteria has been
developed and is called M-FC
Broth This medium currently
is being used increasingly in
water pollution studies
b Selective, differential, culture media
have been developed for direct cul-
tural tests for members of the enter-
ococcus group of bacteria.
3 Tests for pathogenic bacteria
a Workers are currently testing new
media for the differentiation of
members of the Salmonella-Shigella
group of enteric pathogens. Avail-
able information indicates potential
usefulness of a screening medium
12-4
-------
.The Membrane Filter in,Water Bactoriology
for differentiation of nonlactose-
fermenting, non urease-producing
bacteria.
b One medium has been used
for screening tests in detection of
Salmonella typhosa.
c Further confirmatory cultural, bio-
chemical, and serological tests are
necessary to establish the identity of
bacteria differentiated with these
screening media.
C Membrane filter techniques can be applied
both in the laboratory and under field con-
ditions. Several varieties of portable mem-
brane filter field units have been developed
on a commercial basis.
IV ADVANTAGES AND LIMITATIONS
This evaluation is limited to tests for the coli-
form group. Similar, but separate evaluations
would have to be made for any other bacterio-
logical test.
A Advantages
1 Results are obtained in approximately
24 hours, as compared with 48-96 hours
required for the standard fermentation
tube method.
2 Much larger, and hence more represen-
tative samples of water can be sampled
routinely with membrane filters.
3 Numerical results from membrane
filters have much greater precision
(reproducibility) than is expected with
the fermentation tube method.
4 The equipment and supplies required are
not bulky. A great many samples can be
examined with minimum requirements
for laboratory space, equipment, and
supplies.
B Limitations
1 Samples having high numbers of non-
coliform bacteria capable of growing on
Endo type culture media sometimes
give difficulty. In such cases a high
ratio.of these noncoliform bacteria to
coliforms results in poor sheen pro-
duction, or even suppression, of the
coliform organisms.
i
2 In samples having low coliform counts
and relatively.great amounts of sus-
pended solids, bacterial growth some-
times develops in a continuous film on
the membrane surface. In such cases
the typical coliform sheen sometimes
fails to develop.
3 Some samples containing as much as
1 milligram per liter of copper or zinc,
or both, show irregular coliform bac-
terial results.
4 Occasional strains of bacteria growing
on membrane filters producing sheen
colonies prove, on subsequent testing,
to be acid but not gas-producers from
lactose. Where this occurs it may
give a falsely-high indication of coliform
density.
Such limitations as these are not frequent,
but they do occur often enough to require
consideration. In samples where these dif-
ficulties often occur, the best course of
action often is to avoid use of membrane
filter methods and use the multiple fer-
mentation tube procedures.
V SUMMARY
The development of membrane filters and
their bacterial applications has been discussed
briefly, from their European origin to their
current status in this country. Membrane
filters currently available here have been
described, and their properties have been
considered. Applications of membrane filters
in water bacteriology are indicated in general
terms. Some of the advantages and limitations
of membrane filter methods are presented for
coliform tests.
12-5
-------
The Membrane Filter in Water Bacteriology
REFERENCES
1 Goetz, Alexander. Materials, Techniques,
and Testing Methods for the Sanitation
(Bacterial Decontamination) of Small-
Scale Water Supplies in the Field Used
in Germany During and After the War.
Technical Industrial Intelligence Branch.
U.S. Department of Commerce. FIAT
Final Report 1312. December 8, 1947.
2 Clark, Harold F., Geldreich, E.E.,
Jeter, H. L., and Kabler, P. W. The
Membrane Filter in Sanitary Bacteri-
ology. Public Health Reports. 66:
951-77. July 1951.'
4 Clark, H. F., Kabler; P. W., and
Geldreich, E.E. Advantages and
Limitations of the Membrane Filter
Procedure. Water and Sewage Works.
104: 385-387. ' 1957.
5 Public Health Service Publication 956.
Public Health Service Drinking Water
Standards. 1962,
6 Windle Taylor, E., and Burman, N. P.
The Application of Membrane Filtration
Techniques to the Bacteriological Ex-
amination of Water. Journal Applied
Bacteriology, '27:294-303. 1964.
3 Goetz, A. and Tsuneishi, N. Application
of Molecular Filter Membranes to the
Bacteriological Analysis of Water.
Journal American Water Works Associ-
ation. 43:943-69. December 1951.
This outline was prepared by H. L. Jeter,
Director, National Training Center, MDS,'
WPQ EPA, Cincinnati, OH 45268.
12-6
-------
MEMBRANE FILTER EQUIPMENT AND ITS
PREPARATION FOR LABORATORY USE
I Some equipment and supplies used in the
bacteriological examination of water with
membrane filters are specific for the method.
Other items are standard in most well-
equipped bacteriological laboratories and
are readily adapted to membrane filter work.
This chapter describes needed equipment and
methods for its preparation for laboratory
use. Where more than one kind of item is
available or acceptable for a given function,
sufficient descriptive information is pro-
vided to aid the worker in selecting the one
best suited to his own needs,
H EQUIPMENT FOR SAMPLE FILTRATION
AND INCUBATION
A Filter Holding Unit
1 The filter holding unit is a device for
supporting the membrane filter and for
holding the sample until it passes
through the filter. During filtration
the sample passes through a circular
area, usually about 35 mm in diameter,
in the center of the filter. The outer
part of the filter disk is clamped
between the two essential components
of the filter holding unit. (See Plate 1)
a The lower element, called the filter
base, or receptacle, supports the
.membrane filter on a plate about
50 mm in diameter. The central
part of this plate is a porous disk
to allow free passage of liquids.
The outer part of the plate is a
smooth nonporous surface. The
lower element includes fittings for
mounting the unit in a suction flask
or other container suitable for
filtration with vacuum.
b The upper element, usually called
the funnel, holds the sample until
it is drawn through the filter. Its
lower portion is a flat ring that rests
on the outer part of the membrane
filter disk, directly over the non-
porous part of the filter support
plate.
c The assembled filter holding unit is
joined by a locking ring or by one
or more clamps
2 Characteristics of filter holding units
should include:
a The design of filter holding units
should provide for filtration with
vacuum.
(A) ASSEMBLED FILTER
HOLDING UNIT
(B) UPPER ELEMENT
PLATE 1
NOTE: Mention of commercial products and manufacturers does not imply endorsement by the
OWr and the Environmental Protection Agency.
\V. BA. mem.60k. 11.71
13-1
-------
Membrane Filter Equipment and its Preparation for Laboratory Use
b Filter holding units may be made
of glass, porcelain, plastic, non-
corrosive metal, or other impervious
material.
c Filter holding units should be made
of bacteriologically inert materials.
d All surfaces of the filter holding
assembly in contact with the water
sample prior to its passage through
the membrane filter should be
uniformly smooth and free from
corrugations, seams, or other sur-
face irregularities that could become
lodging places for bacteria.
e Filter holding units should be easily
sterilized by routine methods.
f The filter holding unit should be
easily and quickly assembled and
disassembled in routine operational
use.
g Filter holding units should be durable
and inexpensive. Maintenance should
be simple.
3 Several forms of filter holding units
have been developed for use with
aqueous suspensions.
a SS 47 Membrane Filter Holder
(Plate 2, Figure 1)
Conical-shape funnel with a 500 ml
capacity. The base section includes
a wirescreen membrane support.
Funnel and base section are evenly-
joined by a locking ring mechanism.
This assembly is designed to hold a
47 mm diameter membrane firmly in
place allowing an effective filter area
of approximately 9. 6 square centi-
meters. The entire filter unit is
made of stainless steel with the
funnel interior having a mirror-like
finish.
b The Millipore Pyrex Filter Holder
(Plate 2, Figure 2)
The unit is made of pyrex glass with
coarse grade fitted support in base
for filter The upper element of
early models of glass filter holders
had a capacity of 1 liter'. Currently
available units are supplied with
upper elements having 300 ml
.capacity The assembled filter
holder is joined with a spring
clamp which engages on flat sur-
faces encircling the upper and
lower -elements
c Millipore Standard Hydrosol Filter
Holder (Plate 2, Figure 3)
Most components of this unit are
made of stainless metal. The
porous membrane support plate is
fine-mesh stainless steel screening.
The upper element is a straight-
sided cylinder 4 to 5 inches in
diameter, constricted to a narrow
cylinder at the bottom, to fit the
lower element Capacity of the
funnel element is about 1 liter
The assembled filter holding unit is
joined by a bayonet joint and locking
ring. Accessories may be obtained
for collection of small amounts of
filtrate and for anhydrous sterilization
of the filter holding assembly.
d Gelman "Parabella Vacuum Funnel"
(Plate 2, Figure 5)
The unit is made of spun stainless
steel. The locking ring is a bayonet-
type fitting, and is spring-loaded.
The funnel element has a 1-liter
capacity
e The Sabro Membrane Filter Holder
(Plate 2, Figure 4)
The unit is mostly of stainless steel
construction. The lower element is
a combination vacuum chamber,
filtrate receiver, and filter support-
ing element. It consists of a stainless
steel cup with a metal cover The
cover is fitted with a rubber gasket
permitting airtight fit of the cover
into the top of the cup. A porous
sintered stainless steel membrane
support disk is mounted in the
center of the cover At the side
13-2
-------
Membrane Filter Equipment and its Preparation for Laboratory Use
of the beaker is a valve to which a
pumping device can be fitted. The
upper element is a stainless steel
funnel with about 500 ml capacity.
The assembled filter holding unit is
joined by a locking ring at the base
of the upper element. This engages
on three spring clamps on the covering
plate of the lower element.
f Millipore "Sterifil" filter unit
(Plate 2, Figure 6)
A funnel and flask unit of poly-
carbonate with filter base and
support of polypropylene. Manu-
facturers tables should be referred
to regarding chemicals which may
be present in the sample and their
effect on the holder and flask
elements. This unit can be safely
sterilized under steam pressure.
FILTER HOLDING UNITS
FOR AQUEOUS SUSPENSIONS
FIG. 2
B
FIG. 6
FIG. 5
PLATE 2
4 Care and maintenance of filter holding
units
a Filter holding units should be kept
clean and free of accumulated
foreign deposits.
b Metal filter holding units should be
protected from scratches or other
physical damage which could result
in formation of surface irregularities.
The surfaces in contact with mem-
brane filters should receive
particular care to avoid formation
of shreds of metal or other
irregularities which could cause
physical damage to the extremely
delicate filters.
c Some filter holding units have
rubber components. The rubber
parts may in time' become worn,
hardened, or cracked, necessitating
replacement of the rubber part
involved.
d The locking rings used in some
kinds of filter holders have two or
more small wheels or rollers
which engage on parts of the filter
holding assembly. Occasional
adjustment or cleaning is necessary
to insure that the wheels turn freely
and function properly. On some
units, the wheels are plastic, and
are not intended to turn. When
worn flat, they should be loosened,
turned a partial turn, and tightened
again.
Membrane Filters and Absorbent
Pads
1 The desired properties of membrane
filters have been discussed elsewhere.
Typical examples, commercially
available include:
a Millipore Filters, Type HA, white,
grid-marked, 47 mm in diameter
b S & S Type B-9, white, black-grid
mark, 47 mm diameter
13-3
-------
Membrane Filter Equipment and its Preparation for Laboratory Use
c Oxoid cellulose acetate membrane
filters, 4.7 cm, grid-marked
2 An absorbent pad for nutrient is a
paper filter disk, usually the same
diameter as the membrane filter.
Absorbent pads must be free of
soluble chemical substances which
could interfere with bacterial
growth. They should be of such
thickness that they will retain 1.8-
2. 2 ml of liquid culture medium.
During incubation of cultures on
membrane filters an absorbent pad
saturated with liquid culture medium
is the substrate for each filter.
Absorbent pads are supplied with
the purchase of membrane filters.
Additional absorbent pads may be
purchased separately. Sterilization
in an autoclave is recommended for
absorbent pads.
C Vacuum
Water can be filtered through a membrane
filter by gravity alone, but the filtration
rate would be too slow to be .practical.
For routine laboratory practice, two
convenient methods are available for
obtaining vacuum to hasten sample filtration.
1 An electric vacuum pump may be used
connected to a filtration apparatus
mounted in a suction flask. The pump
need not be a high-efficiency type. For
protection of the pump, a.water trap
should be included in the system,
between the filtration apparatus and
the vacuum pump.
2 A water pump, the so-called "aspirator"
gives a satisfactory vacuum, provided
there is reasonably high water pressure.
3 In emergency, a rubber suction bulb, a
hand pump, or a syringe, may be used
for vacuum. It will be necessary to
include some form of valve system to
prevent return flow of air.
D Culture Containers (Plate 3)
Most membrane filter cultures are
incubated in individual containers.
Almost any form of culture container
is acceptable if it is made of impervious
bacteriologically inert material. The
culture container should, or course, be
large enough to permit the membrane
filters to lie perfectly flat. The following
are widely used:
•1 Glass petri dishes
Conventional borosilicate glass culture
dishes are widely used in laboratory
applications of membrane filters. For
routine work, 60 mm X15 mm petri
dishes are recommended. The common
100 mm X 15 mm petri dishes are
acceptable, but are subject to difficulties.
TYPES OF CULTURE CONTAINERS
PLATE 3
13-4
-------
Membrane Filter Equipment and its Preparation for Laboratory Use
2 Plastic petri dishes
Plastic containers have been developed
for use with membrane filter cultures.
Their cost is fairly low, and single-
service use feasible. They cannot be
heat-sterilized, but are supplied
sterile. They must be free from
soluble toxic substances. They can
either be loose-fitting or of a tight lid
to base friction fit.
E Other Equipment and Supplies Associated
with Sample Filtration
1 Suction flask (Plate 4)
a Most types of filter holding apparatus
are fitted in a conventional suction
flask for sample filtration. While
other sizes may be used, the 1-liter
size is most satisfactory.
b The suction flask can be connected
to the vacuum facility with thick-
walled rubber tubing. Latex rubber
tubing, 3/16" inside diameter, with
wall thickness 3/32", is suggested.
This tubing does not collapse under
vacuum, yet it is readily closed with
a pinch clamp.
c A pinch clamp on the rubber tubing
is a convenient means of cutting off
the vacuum from the suction flask
during intervals when samples are
not actually being filtered. It is
most convenient to have the vacuum
facility in continuous operation during
sample filtration work.
d In laboratories conducting a high
volume of filtration work, the
suction flask may be.dispensed.
Filter-holding manifolds are available
to receive up to three filtration units.
The filtrate water is collected in a
trap (in series with the vacuum
source) which is periodically emptied.
e Another arrangement can be made
for dispensing with the suction flask.
In this case, the receptacle element
of the filtration unit is mounted in
the bench top. Instead of using a
suction flask, the lower element of
the filter holding unit has a dual
connection with the vacuum source
and with the laboratory drain. A
solenoid-operated valve is used to
determine whether the vacuum system
or the drain line is in series with
the filtration unit.
2 Ring stand with split ring (Optional)
(See Plate 4)
PLATE 4
When the filter holding unit is
disassembled after sample filtration,
the worker's hands must be free to
manipulate the membrane filter.
Upon disassembly of the filter holding
unit, many workers place the funnel
element, inverted, on the laboratory
bench. Some workers, to prevent
bacterial contamination, prefer a rack
or a support to keep the funnel element
from any possible source of contam-
ination. A split ring on a ring stand is
a convenient rack for this purpose.
3 Graduated cylinders
In laboratory practice, 100 ml graduated
borosilicate glass cylinders are
satisfactory for measurement of
samples greater than 20 ml.
13-5
-------
Membrane Filter Equipment and its Preparation for Laboratory Use
4 Pipettes and cans
a Graduated Mohr pipettes are needed
for many procedures, such as
measurement of small samples, and
for preparing and dispensing culture
media. Pipettes should be available
in 1 ml and 10 ml sizes.
b Holding cans may be round or square
but must not be made of copper.
Aluminum or stainless steel are
acceptable.
5 Alcohol jar with forceps (Plate 3)
a All manipulation of membrane filters
is with sterile forceps. For steri-
lization, forceps are kept with their
tips immersed in ethanol or methanol.
When forceps are to be used, they
are removed from the container and
the alcohol is burned off.
b Forceps may be straight or curved.
They should be designed to permit
easy handling of filters without
damage. Some forceps have corru-
gations on their gripping tips. It is
recommended that such corrugations
be filed off for membrane filter work.
6 A gas burner or alcohol burner is needed
to ignite the alcohol prior to use of
forceps.
7 Dilution water
The buffered distilled water described
in "Standard Methods for the Examination
of Water and Wastewater" for bacterio-
logical examination of water is used
in membrane filter methods. Dilution
water is conveniently used in 99 + 2 ml
amounts stored in standard dilution
bottles. Some workers prefer to use
9.0 + 0.2 ml dilution blanks.
8 Culture medium
Bacteriological culture media used with
membrane filter techniques are dis-
cussed at length in another part of this
manual.
F Incubation Facilities
1 Requirements
a Temperature
For cultivation of a given kind of
bacteria, the same temperature
requirements apply with membrane
filter methods as with any other
method for cultivating the bacteria
in question. For example, incu-
bation temperature for colifprm
tests on membrane filters should
be 35° C + 0, 50C.
b Humidity
Membrane filter cultures must be
incubated in an atmosphere main-
tained at or very near to 100%
relative humidity. Failure to
maintain high humidity during
incubation results in growth failure,
or at best, in small or poorly
differentiated colonies.
2 The temperature and humidity require-
ments can be satisfied in any of
several types of equipment.
a A conventional incubator may be
used. With large walk-in
incubators, it is extremely difficult
to maintain satisfactory humidity.
With most conventional incubators,
membrane filter cultures can be
incubated in tightly closed con-
tainers, such as plastic petri dishes.
In such containers, required
humidity conditions are established
with evaporation of some of the
culture medium. Because the
volume of air in a tightly closed
container is small, this results in
negligible change in the culture
medium. If glass petri dishes or
other loosely fitting containers are
used, the containers should be
placed in a tightly closed container,
with wet paper or cloth inside to
obtain the required humidity con-
ditions. A vegetable crisper, such
as used in most home refrigerators,
13-6
-------
Membrane Filter Equipment and its Preparation for Laboratory Use
is useful for the purpose. (See
Plate 3)
b A covered water bath maintaining
44. 5°C + 0.2 C is necessary for
the fecaT coliform test and this
will necessitate the use of
a water-bath having forced
circulation of water.
Ill STERILIZATION OF MEMBRANE FILTER
EQUIPMENT AND SUPPLIES
A Filter Holding Unit
1 When is sterilization necessary?
a The filter holding unit should be
sterile at the beginning of each
filtration series. A filtration series
is considered to be interrupted if
there is an interval of 30 minutes
or longer between sample filtrations.
After such interruption any further
sample filtration is treated as a
new filtration series and requires
a sterile filter holding unit.
b It is not necessary to sterilize the
filter holding unit between successive
filtrations, or between successive
samples, of a filtration scries.
After each filtration the funnel walls
are flushed with sterile water to
free them of bacterial contamination.
If properly done, the flushing pro-
cedure will remove bacteria
remaining on the funnel walls and
prevent contamination of later
samples.
2 Methods for sterilization of filter
holding unit
a Sterilization in the autoclave is
preferred. Wrap the funnel and
receptacle separately in Kraft paper
and sterilize in the autoclave 15
minutes at 121°C. At the end of
the 15 minutes holding period in the
autoclave, release the steam
pressure rapidly, to encourage
drying of the filter holding unit.
b The unit may be sterilized by
holding it 30 minutes in a flowing
steam sterilizer.
c The unit may be immersed 2 to 10
minutes in boiling water. This
method is recommended for
emergency or field use.
d Some units (Millipore Stainless Unit)
are available with accessories per-
mitting anhydrous sterilization with
formaldehyde. The method consists
of introduction of methanol into a
wick or porous plate in the sterili-
zation accessory, assembly of the
filter holding unit for formaldehyde
sterilization, ignition of the
methanol, and closure of the unit.
The methanol is incompletely
oxidized in the closed container,
resulting in the generation of
formaldehyde, which is bactericidal.
The filter holding unit is kept closed
for at least 15 minutes before use.
e Ultraviolet lamp sterilizers are
convenient to use. A device now
commercially available for ultra-
violet sterilization of membrane
filter funnel units.
B Sterilization of Membrane Filters and
Absorbent Pads
1 Membrane filters
a Membranes are supplied in units
of 10 in kraft envelopes, or in
packages of 100 membranes. They
may be sterilized conveniently in
the packets of 10, but should be
repackaged if supplied in units of
100. Large packages of filters can
be distributed in standard 100 mm
X15 mm petri dishes, or they can
be wrapped in kraft paper packets
for sterilization.
13-7
-------
Membrane Filter Equipment and its Preparation for Laboratory Use
b Sterilization in the autoclave is
preferred. Ten minutes at 121° C
or, preferably, at 116°C is
recommended. After sterilization
the steam pressure is released as
rapidly as possible, and the filters
are removed from the autoclave and
dried at room temperature. Avoid
excessive exposure to steam.
c In emergency, membrane filters may
be sterilized by immersion in boiling
distilled water for 10 minutes. The
filters should first be separated from
absorbent pads and paper separators
which usually are included in the
package. The boiling water method
•is not recommended for general
practice, as the membranes tend to
adhere to each other and must be
separated from one another with
forceps.
2 Absorbent pads for nutrient
a Unsterile absorbent pads can be
wrapped in kraft paper or stacked
loosely in petri dishes, and auto-
claved with membrane filters (ten
minutes or longer at 121°C or 116° C).
b After sterilization absorbent pads
for nutrient should be dried before
use.
C Glassware
1 Sterilization at 170° C for not less than
1 hour is preferred for most glassware
(pipettes, graduated cylinders, glass
petri dishes). Pipettes can be sterilized
in aluminum or stainless steel cans, or
they may be wrapped individually in
paper. The opening of graduated
cylinders should be covered with paper
or metal foil prior to sterilization.
Glassware with rubber fittings must not
be sterilized at 170° c, as the rubber
will be damaged.
2 Sterilization in the autoclave, 15
minutes at 121° C, is satisfactory,
and preferred by many workers. When
sterilizing pipettes it is important to
exhaust the steam pressure rapidly
and vent the containers momentarily.
This allows the vapor to leave the can
and prevents wet pipettes.
D Culture Containers
1 Glass petri dishes
a Petri dishes may be sterilized in
aluminum or stainless steel cans,
or wrapped in kraft paper or metal
foil. They can be wrapped
individually or, more conveniently,
in rolls of up to 10 dishes.
b Preferably, sterilize glass petri
dishes at 17 0° C for at least 1 hour.
c Alternately, they may be sterilized
in the autoclave, 15 minutes at
121° C. After sterilization steam
pressure should be released rapidly
to facilitate drying of the dishes.
Other suggested methods for
sterilization of plastic dishes
include exposure to ethylene oxide
vapor (0. 5 ml ethylene oxide per
liter of container volume), or
exposure to ultraviolet light.
Ethylene oxide is a dangerous
chemical being both toxic and
explosive, and it should be used
only when more convenient and
safer methods are not available.
2 Plastic culture containers
a Because of the thermo-labile
characteristics of the plastic,
these containers cannot be heat
sterilized. Manufacturers supply
these in a sterile condition.
b For practical purposes, plastic
dishes may be sterilized by
immersion in a 70% solution of
ethanol in water, for at least 30
minutes. Dishes must be allowed
to drain and dry before use, as
ethanol will influence the perform-
ance of culture media.
This outline was prepared by H. L. Jeter,
Director, National Training Center, MDS,
VVPO EPA, Cincinnati, OH 45268.
13-8
-------
MEMBRANE FILTER EQUIPMENT FOR FIELD USE
I INTRODUCTION
One of the most troublesome problems in
bacterial water analysis is the occurrence
of changes in the bacterial flora of water
samples between the time of sample collection
and the time the actual bacterial analysis is
started. Numerous studies have been made
on this problem. From these have come such
recommendations (Standard Methods, 10th ed)
as holding the sample at 0-10 C and starting
laboratory tests as soon as possible after
collection of the sample. Recommendations of
Standard Methods, 12th Edition, was to hold
the sample as close as possible to the temper-
ature of the source and to start the laboratory
tests preferably within 1 hour and always with-
in a maximum of 30 hours after collection.
Changes in the 13th edition of Standard Meth-
ods (1971) will again call for the icing of
samples, and further, that samples of envir-
onmental waters be held for not more than 8
hours total elapsed time before samples are
plated or used for microbiological testing.
The 30 hour maximum elapsed sample holding
time will still be retained for potable water
.samples.
A They would be useful in certain routine
water quality control operations. Examples
include such places as on board ships;
some airlines, particularly in overseas
operations; and some national parks. In
each example, it is seen that there is an
obvious difficulty in getting water samples
to the examining laboratory in time for
early examination.
B In addition, such units would be invaluable
in emergencies when existing laboratories
are overburdened or inoperative. Portable
kits already have proven extremely helpful
in testing many small water supplies in a
short period of time. Further there is a
predictable need for such equipment in the
event of a wartime civil defense disaster.
Experience of the Germans in the vicinity
of Hamburg during World War II lends
support to this concept.
The purpose of this discussion is to
introduce some of the portable equipment
which has been developed and to point out
noteworthy features of each. Actual
practice and experience with these units
reveal strong point and weaknesses in
each type.
The membrane filter method has been
accepted by the Federal Government for
the bacteriological examination of water
under its jurisdiction. This acceptance
was based on methods developed and
procedures applied in fixed laboratories.
While the use of field kits is not excluded,
no special concession has been made
regarding the standards of performance
of membrane filter field kits. Thus, in
planning to use a membrane filter field
kit for the bacteriological examination of
water, it is the responsibility of the
individual laboratory to establish beyond
reasonable doubt, by comparison with
Standard Methods fermentation tube tests
or established laboratory membrane
filter methods, the value of use of the
membrane filter field kit in determining
the sanitary quality of water supplies
examined.
II TYPES OF COMMERCIALLY AVAILABLE
MEMBRANE FILTER EQUIPMENT FOR
FIELD USE
A Sabro Water Laboratory
This unit represents a fixed membrane
filter laboratory in miniature, with
adaptations for special situations to be
encountered in the field. Notable features:
1 The funnel unit supplied on older units
is glass. A newer model has been
released with an all-metal funnel unit.
2 The vacuum source is a hand pump
(modified bicycle pump) or, optionally,
an all-metal syringe.
3 The manufacturer sells prepared
ampouled medium, in a liquid state.
The medium should be kept at a cool
NOTE: Mention of commercial products and manufacturers does not imply endorsement by the
OWP and the Environmental Protection Agency.
W. BA.mem, 63g. 11. 72 14-1
-------
Membrane Filter Equipment for Field Use
temperature, out of the light. Its
useful shelf life is uncertain, but
limited tests by this agency indicate
that the medium performs accept-
ably with storage up to one year.
4 Incubation of the cultures is in an
incubator drawer having a capacity of
18 1-ounce culture containers, or 36
plastic containers, and operates
electrically at 110V, and with suitable
converters, at 6V, 12V. A battery
also can be used with this unit.
5 Sterilization of the funnel unit is
carried out by a "light flaming
technique" or, optionally, by
immersion of the funnel unit in hot or
boiling water.
6 Useful accessories provided include
thermometer, alcohol lamp, measuring
cup, and forceps.
B Millipore Field Monitor Units
These units differ radically from any
other field equipment that has appeared.
Significant reductions in bulk of equipment
have been brought about through major
changes in function and design of the usual
equipment. Notable features:
1 The funnel unit has been eliminated in
its usual form. This has been done by
development of a carefully fitted,
single-use combination filtration unit
and culture container. This feature
eliminates most of the handling and use
of accessory equipment.
2 The vacuum source is an all metal
syringe with a fitting providing for
direct connection to the culture
container.
3 The culture medium provided by the
manufacturer includes M-Endo Broth,
MF, ready-to-use, in glass ampoules.
These ampoules are so designed as to
permit easy introduction of the culture
medium into the culture container.
Alternately, the manufacturer makes
available a delayed-incubation medium
in the ampoules. Other culture media
can be used at the discretion of the
user/ but some difficulty can be
anticipated in introducing the medium
without special equipment.
4 Incubation of the cultures is provided
in the field through use of an associated
portable incubator and equipment
carrying kit. This incubator has room
for about 25 cultures. It is electrically
operated, and through selection of
available switching positions, operates
at 6V, 12V, 110V, or at 220V.
5 Sterilization of components in the
field is unnecessary. The culture
containers and plastic tubes are single-
use units supplied in a sterile condition.
Samples do not come in contact with
the syringe until after they have passed
through the filter.
C Millipore Field Unit for Military Use
1 A modified Millipore field unit based
on the case and incubator described
in B, 4 above, has been adopted by the
U.S. Department of Defense. This
unit includes a miniaturized stainless
metal funnel unit instead of the Monitors.
2 The vacuum source is an all-metal
syringe.
3 Sterilization of funnel unit is by
formaldehyde generated through
incomplete combustion of methyl
alcohol.
Ill COMMON DIFFICULTIES ENCOUNTERED
IN COMMERCIALLY AVAILABLE FIELD
EQUIPMENT
A The most conspicuous problem arising
with field use of most units is their
ultimate reliance on a fixed laboratory
for essential supplies.
B These portable laboratories will permit
simultaneous incubation of up to 30 membrane
filters.
C For protracted field work, a fairly large
amount of reserve supplies and equipment
will be necessary. Such a reserve would
include culture media, membrane filters,
culture containers, fuel, and other
expendable supplies required in the field,
organized in a supplementary carrying case.
D No currently available field unit provides
illumination or optical assistance for
interpretation of results.
14-2
-------
Membrane Filter Equipment for Field Use
E Some of the sterilization methods
recommended by manufacturers are
unacceptable. If field sterilization in
boiling water is needed, then there must
be a heat source and a metal can or beaker.
Such equipment could be carried in the
case suggested in C above.'
IV IMPROVISED FIELD EQUIPMENT
The initial cost of most of the commercially
manufactured units has met some objectioa
This factor, coupled with need for additional
accessory supplies and equipment, has
aroused interest in improvised units. Such
a unit could consist largely of equipment
normally used in a fixed laboratory, packaged
in one or two fiberboard cases.
A The funnel unit could be one of the
familiar stainless steel units used in
many laboratories; or it could be specially
designed, smaller than ordinarily used,
permitting use of up to a dozen or more
upper filter holding elements in the field.
B The vacuum source could be the modified
bicycle pump (leathers reversed), and
provided with a by-pass valve. The suction
flask could be the standard side-arm glass
flask, or a metal unit could be devised.
C M-Endo Broth or LES Endo agar are
suitable media. Both are available as
dehydrated medium which must be
reconstituted and boiled in the field.
M-Endo Broth MF is now available in
liquid form, sterile, in sealed ampules.
A shelf life of approximately one year is
stated when stored under moderate
temperatures in the dark.
LES MF Holding Medium - Coliform
requires merely dissolving in distilled
water. No heating is necessary. Such
medium would be an advantage where
applicable.
D Sterilization of funnel units, graduated
cylinders, media, etc. , would be through
immersion in boiling water for 2 minutes
or longer, as indicated for the material
being sterilized. Provision for boiling
water is easy through use of a small camp
stove or other simple burner.
Improvised equipment, such as discussed
above, would have great usefulness in
emergencies, where commercially available
membrane filter field units pre not on hand.
V In a se parate outline are detailed
descriptions of procedures for use of
commercially available membrane filter
field equipment. In some cases the
suggested methods are different from
those recommended by the manufacturers.
In each case such departures are based
on a series of experimental studies made
by this agency, which suggested need
for modification of existing recommend-
ations.
REFERENCE
1 Laubusch, E. J. What You Should Know
About the Membrane Filter, Public
Works, 89: 106-13, 162-68, 1958.
This outline was prepared by H. L. Jeter,
Director, National Training Center, MDS, ¦
WPQ EPA, Cincinnati, OH 45268.
14-3
-------
PRINCIPLES OF CULTURE MEDIA FOR USE WITH MEMBRANE FILTERS
I INTRODUCTION
A Many kinds of membrane filter media
have been described for use in bacterio-
logical tests on water. This is noteworthy
in view of the relatively few years the
filters have been widely available in this
country. This discussion is to consider
several of these media in terms of their
purposes, composition, and the ways in
which they are used.
B Basic Considerations
1 Filtration of water sample through a
membrane filter results in deposition
of bacteria and particles of suspended
matter on the filter surface. The
bacteria can be cultivated in place if
suitable culture medium is made avail-
able for their growth.
2 The bacteria are cultivated by placing
the membrane on a pad of absorbent
paper saturated with liquid culture
medium, or on an agar medium. The
culture medium diffuses through the
pores of the filter, and is available to
the bacteria on the opposite surface.
Proper time, temperature, and humidity
of incubation results in development of
bacte rial colonies. In principle, each
bacterial cell multiplies to become a
single bacterial colony.
3 Some culture media, satisfactory for
tube cultures or agar plate cultures,
do not perform well when used with
membrane filters due to a selective
adsorptive property of the filter itself.
In the process of diffusion through the'
pores some components of the culture
medium may be removed completely,
or reduced in concentration. Thus, the
composition of a given culture medium
at the filter surface where it is avail-
able for bacterial growth may be dif-
ferent from its composition beneaththe
membrane filter.
There is evidence that improved cul-
tural results sometimes are obtained
with increased concentration of certain
nutritive constituents of membrane filter
culture media.
4 Pure cultures may be recovered
from membrane filters and subject-
ed to supplementary biochemical,
cultural, and serological procedures
for identification studies or for veri-
fication of interpretations based on
direct observation of membrane
filter cultures.
The same use can be made of agar plat-
ing media; however the membrane filter
offers advantages due tothe ability to
concentrate organisms from a large
volume of sample in which the organ-
isms are present in low density.
C Applications of Membrane Filter Culture
Media
The composition of bacteriological culture
media designed for tube or plate cultures
should be subjected to critical study be-
fore they are applied to membrane filter
procedures. Media based on well-known
bacteriological media have been modified
for use with membrane filters for the
following purposes in testing water.
1 Bacterial plate counts
2 Media for bacterial indicators of
pollution
a Coliform organisms
b Fecal streptococcus group
c Clostridium perfringens
3 Salmonella and other enteric bacterial
pathogens
NOTE: Mention of commercial products and manufacturers does not imply endorsement by
OWP and the Environmental Protection Agency.
W.BA. mem. 68k. 12.72
15-1
-------
Principles of Culture Media
D Constituents of Membrane Filter Culture
Me d ia
Membrane filter media for the differentia-
tion and counting of special groups of
bacteriaare based on the same principles
used in differential agar plate media. Thus,
the components of a differential medium
for membrane filter cultures include:
1 Substances favoring growth of the
organisms for which the medium is
designed. Inclusion of special peptones,
fermentable carbohydrates, yeast or
meat extracts, water and chemicals to
adjust pH to a desired level are common
methods of favoring growth of desired
organisms.
2 Differential indicator system. The
purpose of the indicator system is to
produce characteristic colonies of the
desired bacterial groups for easy
recognition when present in a mixture
with extraneous types of colonies. This
is done through inclusion of (a), a com-
ponent which is chemically changed by
the organisms to be differentiated, and
(b), indicator substances, which give
visible evidence of an intermediate or
end product resulting from a chemical
change of substance (a).
3 Selective inhibitors. Some bacterial
groups to be tested may be over-
whelmingly outnumbered by extraneous
types of bacteria. In such cases, it is
necessary that substances be included in
the medium which (a), prevent growth
of a maximum number of kinds of ex-
traneous bacteria, and (b), have mini-
mum adverse affect on growth of the
kind of bacteria for which the medium
is designed.
E Variety of Methods of Using Media Avail-
able with Membrane Filter Methods
1 Single-stage tests
After sample filtration, the membrane
filter is placed on a designated culture
medium, and left there throughout the
incubation period. The culture results
are examined and interpreted directly.
2 Multi-stage tests
A membrane filter can be transferred
from one culture medium to another
without disturbance of bacteria or
colonies on the filter. This is unique
with membrane filter methods, and
lends itself to a variety of cultural and
testing procedures.
a The membrane filter, after sample
filtration, can be incubated for a
specified time on one medium, then
transfered to a second medium. The
method permits initiation of growth
on enrichment medium, after which
the membrane filter can be trans-
ferred to a less productive medium.
With growth already begun, some
differential culture media give better
quantitative production than would
be the case without preliminary
incubation.
b After incubation on one or more
media, colonies on the membrane
filter can be subjected to bio-
chemical tests with reagents too
toxic to include in the culture
medium. Such reagents may be
flooded over the growth on the
filter, or the filter may be placed
on an absorbent pad saturated with
the reagent, in order to make such
tests.
c A third type of multi-stage test is
one in which the membrane filter,
after sample filtration, is placed
temporarily on a medium containing
a bacteriostatic agent. In the
presence of such a substance,
bacterial growth is inhibited or
slowed greatly, but the organisms
are not killed. During a limited
period, the membrane filters may
be transported or stored at ambient
temperatures. The filter can be
transferred later to a suitable
medium and incubated for develop-
ment of colonies.
15-2
-------
Principles of Culture Media
II CULTURE MEDIA FOR TOTAL BACTE-
RIAL COUNTS ON MEMBRANE FILTERS
A Concepts
1 Strictly, a "total" bacterial count
medium is nonexistent. No single
medium and incubation procedure can
provide simultaneously the full range
of oxygen requirements, needs for
special growth substances, pH require-
ments, etc. , of all the kinds of bacteria
found in water.
2 Actually, "total" bacterial counts are
counts of the bacteria developing visible
colonies on a defined culture medium
at a known pH after incubation for a
set time and temperature under aerobic
conditions.
3 Within the foregoing limitations, the
following criteria offer a useful basis
for selection of membrane filter media
to be used for estimates of the bacterial
density in water.
a The medium and its method of use
should produce a maximum number
of colonies from all types of water.
The colony yield should compare
favorably with the bacterial counts
determined as described in "Stand-
ard Methods for the Examination of
Water, Sewage and Industrial
Wastes. " 13th Ed. (1971).
b The colonies should develop rapidly
to a sufficient size to be counted
after a minimum incubation period.
At present, best results with mem-
brane filter methods are obtained
after about 18 hours incubation.
c The medium should be one which is
reproducible and routinely available
in laboratories.
B Composition of Total Count Media for
Membrane Filters
Almost any rich, general growth promoting
culture medium is acceptable for total bac-
terial counts on membrane filters. Several
such media have been suggested especially
for membrane filter methods. These ditfer
only in minor aspects, and can be discuss-
ed as a group. For details of composition
and specific applications of each, see the
media formulations elsewhere in this
manual.
1 Growth promoting substances: All the
substances included in these.media are
included to encourage growth of a maxi-
mum number of kinds of bacteria. Most
workers agree that the peptone should be
used in twice the concentration usually
found in conventional tube or agar plat-
ing media.
2 Indicator substances are unnecessary
with total count media.
3 Substances for the selective inhibition
of certain bacterial groups are not in-
cluded in total count media.
C Problems Encountered with Total Count
Media
Bacterial colonial growth habits on mem-
brane filters are similar to their surface
growth habits on similar agar plate media.
1 As with agar plate media, some species
of bacteria grow continuously, spread-
ing over the surface of a membrane
filter, tending to obscure nonspreading
colonies which otherwise could be
counted.
2 Some samples contain an appreciable
amount of particulate matter. In sam-
ple filtration, this is deposited on the
surface of the membrane filter with the
bacteria. When the culture medium
diffuses through the filter, a capillary
film of liquid culture medium accumu-
lates around the particles of extraneous
matter. Bacteria not ordinarily con-
sidered "spreaders" sometimes develop
confluent colonies due to the film of
liquid medium accumulating around such
particles.
15-3
-------
Principles of Culture Media
D What is the best totaJ count medium for
use with membrane filters?
Because of the relative ease of preparation,
most workers prefer the commercially
prepared dehydrated media. Difco M-
Enrichment Broth (B 408) or Baltimore
Biological Laboratories' M-Enrichment
Broth (No. 331) are used interchangeably.
Total colony productivity of these media
is equivalent to that of media prepared
from the individual components.
Ill CULTURE MEDIA FOR TOTAL COLIFORM
TESTS ON MEMBRANE FILTERS
A Concepts
The nature of membrane filter culture
methods imposes a different definition of
coliform bacteria than the Standard Methods
definition.
1 Standard Methods fermentation tube
method. "The coliform group includes
all of the aerobic and facultative anaero-
bic Gram-negative nonsporeforming
rod-shaped bacteria which ferment
lactose with gas formation within 48
hours at 35°C."
2 Membrane Filter Methods: "In the
membrane filter procedure, all organ-
isms that produce a colony with
a metallic sheen in 22-24 hours are
considered members of the coliform
group. The sheen may appear as a
small central focus or cover the
entire colony. " The guiding prin-
ciple is that any amount of sheen
is considered positive.
3 The Standard Methods definition of
coliforms requires demonstration of
the ability of organisms to produce gas
through the fermentation of lactose.
The membrane filter method does not
lend itself to the demonstration of gas
production. It relies instead on the
development of a particular type of
colony^on an Endo type of culture
medium. The culture medium is one
in which lactose, basic fuchsin, and
sodium sulfite comprise an indicator
system to cause differentiation of
coliform colonies. While the bacterial
groups measured by membrane filter
methods are not identical with the group
measured by Standard Methods pro-
cedures, they are believed to be es-
sentially the same, and to have equal
sanitary significance.
B Composition of Coliform Media for.
Membrane Filters
Several different media have been suggested
for coliform tests on membrane filters.
The components of these media can be
classed into three convenient groups for
general considerations.
1 Growth-promoting substances.. Growth
of bacteria on all the media is favored by
the inclusion of such components as
peptones (as Neopeptone, Thiotone Casi-
tone, Trypticase, and other-proprietary
peptones), yeast extract, dipotassium
phosphate (for adjustment of reaction of
the medium), and distilled water. Lac-
tose is included in all these media. It
serves doubly, to favor growth of coli-
form bacteria, and as an essential compo-
nent of the systems for differentiating
coliform colonies.
2 Two kinds of differential indicator
systems are available for demonstration
of lactose fermentation on membrane
filters.
a Lactose-basic fuchsin-sodium sulfite
system (Endo type media).
1) Media using this system include
lactose and a suitable concentra-
tion of basic fuchsin which has been
partially decolorized with sodium
sulfite.
2) The basic fuchsin-sodium sulfite
complex requires very careful
standardization. An excess of
either component results in an
unsatisfactory culture medium.
3) The indicator system demonstrates
lactose fermentation as follows:
15-4
-------
Principles of Culture Media
a) The coliform bacteria produce
aldehyde as an intermediate
product of the fermentation of
lactose.
b) The aldehyde is "complexed"
by the sodium sulfite-basic
fuchsin indicator. In this pro-
cess a reaction occurs in which
red color is restored to the
basic fuchsin. Colonies of
bacteria fermenting lactose
assume the color of the re-
stored fuchsin. As the restored
dye accumulates, it apparently
precipitates on the colony,
giving the colony a character-
istic green-gold surface sheen.
The reaction occurs best in an
I
alkaline medium. The culture
medium is adjusted to pH 7. 5.
4) Endo type media require very
careful standardization for suc-
cessful use in the laboratory.
Most workers prefer to use a
commercially prepared and stan-
dardized medium. M-Endo Broth
MF is the recommended coliform
medium for use with membrane
filters.
b pH indicator system
1) Media using this system rely on
detection of pH change due to the
accumulation of organic acids,
end products of lactose
fermentation.
2) Bromcresol purple, for example,
is a pH indicator, approaching
yellow at more acid pH. Colon-
ies fermenting lactose and ac-
cumulating organic acids there-
fore turn yellow.
3) Studies in England with membrane
filters for coliform tests have
been based on a modification of
MacConkey's Medium, using this
principle of colony differentiation
in coliform tests.
3 Inhibitory substances in membrane
filter coliform media
a Confusing and erroneous results in
coliform detection can be caused by
1) the overgrowth of the membrane
filter by extraneous nonlactose
fermenting bacteria, preventing
coliform colonies from developing
the characteristic color and
sheen; and
2) the development of sheen
colonies of lactose fermenting
bacteria which produce acid but
not gas in the fermentation of
lactose.
b These difficulties can be reduced
through incorporating of substances
harmless to coliform bacteria but
which have inhibitory effect on growth
of extraneous forms. Attention must
be given to the concentration of such
substances, as excessive amounts
also will reduce the productivity of
the medium for coliform colonies.
The following components of various
culture media have proven useful in
suppressing growth of noncoliform
bacteria on membrane filters.
1) Basic fuchsin-sodium sulfite. Al-
though these compounds are included
in Endo-type media for their role
in differentiating coliforms from
other types of colonies, they are
effective in preventing the growth
of many of the noncoliform bacteria
occurring in water samples.
2) Ethanol (95%. . . NOT denatured) is
included in M-Endo Broth MF. In
the concentration used, ethanol
suppresses growth of some kinds
of noncoliform bacteria, and tends
to limit the colony size of others.
In addition, the ethanol seems to
increase the solubility of some of
the other components of the media.
15-5
-------
Principles of Culture Media
3) Sodium desoxycholate or bile salts
are used in such media as M-Endo
Broth MF, and in the modified
MacConkey's Medium for membrane
filters used in British studies. They
are includedprimarily for their in-
hibitory effect against Gram-positive
cocci and spore formers.
C Methods Available for Using Coliform
Media with Membrane Filters
1 Single-stage coliform tests
a After sample filtration, the mem-
brane filter is incubated for the
desired time on a selective coliform
differentiating medium.
b The coliform colonies are counted
without further tests.
c M-Endo Broth MF and LES Endo
Agar Media are alternate standard
single-stage coliform media.
2 Two-stage coliform tests
a Immediate coliform test
1) After sample filtration, the mem-
brane filter is incubated I2 - 2
hours on the enrichment medium
of lauryltryptose broth.
2) The membrane is then transferred
to a new absorbent pad saturated
with the standard differential
medium for coliform bacteria,
and incubated for 20-22 hours at
35 + 0. 5C.
3) The coliform colonies are counted
without further tests.
4) This test procedure, based on
EHC Endo Medium, was described
in the 10th edition of Standard
Methods. With the 12th edition,
an official two-stage coliform test
has been adopted, based on LES
Endo Agar Medium.
b Delayed incibation colitorm test
1) After sample filtration, the mem-
brane filter is placed on an ab-
sorbent pad saturated with benzo-
ated Endo Medium or with LES
Holding Medium. The filter may
be preserved up to 72 hours at
ambient temperatures. During
this time it can be transported or
stored. Growth is stopped or
greatly reduced.
2) The membrane filter can be
transferred to a- fresh absorbent
pad, saturated with such a medium
as M-Endo Broth MF, or to LES
Endo Agar and incubated up to 24
hours.
3) The differentiated coliform
colonies are counted as with
other membrane filter coliform
media.
4) This test procedure makes it
possible to filter samples in the
field, place the filters on pre-
servative medium, then mail or
transport them to the laboratory
for completion of the bacteriologi-
cal examination. The procedure
is designed to eliminate the need
for maintaining sample tempera-
ture in the interval between
sample collection and initiation
of the bacteriological examination.
In addition, the method should
produce results more nearly
reflecting the quality of the source
water than is available with other
methods of collecting and testing
samples.
3 Verified membrane filter coliform
test
a This is used to verify the interpreta-
tion of differentiated colonies on any
type of membrane filter coliform
medium. The test is suggested for:
self-training of laboratory workers,
for evaluation of new or experimental
15-6
-------
Principles'of Culture Media
media, and in any water examination
in which the interpretation of results
is in doubt or likely to be involved in
legal controversy.
b The test consists of obtaining pure
cultures from differentiated coliform-
like colonies on membrane filters,
and subjected them to further cultural
and biochemical tests to establish
their identity as Gram-negative non-
sporeforming bacilli which ferment
lactose with gas production. The
technical procedures are described
elsewhere in this manual.
IV MEDIUM FOR THE FECAL COLIFORM
TEST
A Concepts
The selective effect of elevated temperature
has been the most important development
in fecal coliform tests since 1904. In that
year, Eijkman discovered that coliform
bacteria from the gut of warm-blooded
animals produced gas from glucose at
46°C, while the majority of coliform
bacteria from other sources did not.
Media variations were of only
secondary importance.
Much medium variation has resulted from
attempts to select for Escherichia coli,
only, as the fecal coliform. While E_. coli
is usually the predominant coliform in
human (and animal) feces, other types
are present, including the alleged soil
and plant coliform, Aerobacter
aerogenes in very large numbers.
All coliforms demonstrated by isola-
tion to have arisen in feces are called here
fecal coliforms and are measured empir-
ically by the fecal coliform tube test.
Membrane filter tests reflect divergence
of attitude on indicators of fecal origin.
Delaney et al. (1962) have published:
Measurement of _E. coli Type I by the
Membrane Filter. Geldreich et al. (1965)
have presented: Fecal-Coliform-Organism
Medium for the Membrane Filter Technique.
Temperatures are the same but media are
different: Because the fecal coliform test
appears more convenient, it will be
emphasized.
B Composition of Fecal Coliform Medium
MFC
1 MFC medium is a rich growth medium
containing lactose, proteose peptone
no. 3, tryptone and yeast extract. A
level of . 3% sodium chloride produces
favorable osmotic balance. Vigorous
growth results. A practical result is
shortening of test time to-24 hours.
The growth constituents are similar to
those of the tube test for fecal coliform.
Both have 0.15% bile salts to select for
coliforms but elevated temperature is
the more important selective factor.
2 The indicator system of aniline blue
results in blue fecal coliform colonies.
Nonfecal coliform colonies, generally
few, are gray to cream-colored.
C Special Problems with MFC Broth Medium
1 Temperature control must be accurate.
Current recommendations call for
44. 5 + 0.2 C and the temperature to
be maintained in a water incubator
of forced circulation.
2 Temperature equilibration must be
rapid. Nonfecal coliforms may initiate
growth at lower temperatures and sub-
sequently give false positive blue
colonies when incubated at 44 5°C
No more than 20 minutes lapse of time
is recommended from filtration to
incubation. Submergence in waterproof
plastic bags reduces actual temperature
equilibration to 10 - 12 minutes.
3 Rosolic acid presents some problems
in preparation. It is practically
insoluble in water and of limited
stability in alkaline solution. A 1%
solution in 0. 2 N NaOH should be
prepared and this added to the medium
as recommended, by the manufacturer
15-7
-------
Principles of Culture Media
V MEDIA FOR FECAL STREPTOCOCCUS
TESTS
A Introduction
1 The development of membrane filter
culture media for the fecal streptococci
reflects the continuing interest in this
group of bacterial indicators of pollution.
The productivity of enterococcus media
recently has been greatly increased
2 Standards of performance of a good
fecal streptococcus medium correspond
with those of a good coliform medium
on membrane filters. Thus, the
requirements of productivity, specificity,
ease of use, and reproducibility of the
medium, are equally applicable to
medium for the detection and enumeratior
of the fecal streptococci.
B M-Enterococcus Agar
1 In 1955, Slanetz, Bent, and Bartley
described a modification of the
Chapman mitis-salivarius medium,
and reported satisfactory results on
membrane filters for detection of
enterococci from water.
The following year, Slanetz, Bartley,
and Ray described a modification in
which 1% agar was included in the
medium. With this medium, the mem-
brane filter is placed on the agar
surface for incubation. This contrasts
with the usual practice of using fluid
media to saturate absorbent pads as the
substrate for incubating membrane
filter cultures. The authors reported
generally larger enterococcus colonies
and improved colony production with
this modification of the medium.
2 Composition of the medium
a The medium is a tryptone, glucose,
general growth media buffered with
dipotassium hydrogen phosphate.
Yeast extract is included to provide
vitamins and other growth factors.
Many bacteria would thrive if
inhibitor were not included.
b The inhibitor sodium azide is a
powerful suppressor of all except
fecal streptococci. It may act in
this manner by interfering with
aerobic respiration.,
False positives, that is red colonies
which are not fecal streptococci,
are known to occur. A verification
procedure is included in the 13th
edition of Standard Methods for fecal
streptococcus detection.
c Safety note:
Sodium azide is poisonous. In
handling dehydrated culture medium
containing sodium azide, care should
be taken to avoid any action which
might create an airborne dust of the
medium. If any powdered medium
does get blown into the air, it is
advisable to get away from the
immediate vicinity of the dust until
it settles.
d The indicator is 0. 1% 2, 3, 5,
triphenyl tetrazolium chloride in
the medium. The indicator is
colorless in the medium (oxidized
state). The metabolizing bacterial
colonies reduce the 2, 3, 5 triphenyl
tetrazolium ehloride to the insoluble
and red colored formazan (reduced
state). Colonies appear flat, light
pink to smooth raised dark red with
pink margins after 48 hours
incubation. Colonies tend small,
0.5-2 mm.
C KF Agar
1 This streptococcus medium was
developed at SEC by Kenner, et al.
and designated KF agar.
While KF medium is productive for
detection and enumeration of the
Fscal Streptococcus Group, its use
15-8
-------
Principles of Culture Media
is hampered by nonspecificity. Studies
have demonstrated that the medium
supports growth of S. bovis, and other
forms common in animals, but not
numerous in the fecal excreta of humans.
2 Composition of KF Agar
a Nutritive requirements of the fecal
streptococci are supplied by peptone,
yeast extract, sodium glycerophos-
phate, maltose, lactose, and distilled
water.
b The indicator system is phenol red
and 2, 3, 5 triphenyl tetrazolium
chloride. On KF Agar used with
membrane filters, the fecal strepto-
coccus colonies develop as small
colonies, up to 2 mm in diameter,
colored various shades from pale
pink to a dark wine color.
c The selective component of KF Agar
is sodium azide, used in 0.04%
concentration.
3 Miscellaneous applications of M-
Enterococcus Agar and KFAgar
a Both media can be used in a single
stage test for fecal streptococci.
Colonies are counted and reported
after 48 hours incubation at 35+0.5 C.
b Alternately, they can be used as a
holding medium in a delayed incu-
bation test for the fecal streptococcus
group.
c Both mediums can be used as pour
plates.
REFERENCES
1 APHA, AWWA, FSIWA. Standard Methods
for the Examination of Water and Waste-
water. 12th Edition.
2 Clark, H.F., Geldreich, E. E., Jeter, H. L.
and Kabler, P.W. The Membrane
Filter in Sanitary Bacteriology. Public
Health Reports. 66:951-77. 1951.
3 Fifield, C.W. and Schaufus, C.P
Improved Membrane Filter Medium
for the Detection of Coliform
Organisms. J. Amer. Water Works
Assn. 50:2:193-6. 1958.
4 Geldreich, E.E., Clark, H.F., Huff, C B.
and Best, L. C. Fecal-Coliform-
Organism Medium for the Membrane
Filter Technique. J. Amer Water
Works Assn 57:2:209-14. 1956.
5 Kabler, P.W. and Clark, H.F. Use of
Differential Media with the Membrane
Filter. Am. Jour. Pub. Health.
42:390-92. 1952.
6 Kenner, Bernard A., Clark, H.F. and
Kabler, P.W. Fecal Streptococci.
I. Cultivation and Enumeration of
Streptococci in Surface Waters
Applied Microbiology. 9:15-20. 1961.
7 McCarthy, J. A , Delaney, J.E. and
Grasso, R. J. Measuring Coliforms
in Water. Water and Sewage Works
108:238. 1961.
8 Rose, R. E. and Litsky, W. Enrichment
Procedure for Use with the Membrane
Filter for the Isolation and Enumeration
of Fecal Streptococci in Waters
Microbiol. 13:1:106-9. 1965.
9 Slanetz, L.W., Bent and Bartley. Use
oif the Membrane Filter Technique to
Enumerate Enterococci in Water.
Public Health Reports. 70:67. 1955.
10 Slanetz, L. W. , Bartley, C.H. and Ray,
V.A. Further Studies on Membrane
Filter Procedures for the Deter-
mination of Numbers of Enterococci
in Water and Sewage. Proc Soc. Am.
Bacteriologists. 56th General Meeting.
1956.
This outline was prepared by H. L. Jeter,
Director, and R. Russomanno, Micro-
biologist, National Training Center, MDS,
WPO, EPA, Cincinnati, OH 45268.
15-9
-------
SELECTION OF SAMPLE FILTRATION VOLUMES
FOR MEMBRANE FILTER METHODS
I INTRODUCTION
A Wide Range of Filtration Volumes
1 The membrane filter permits testing
a wide range of sample volumes, from
several hundred milliliters to as little
as 0. 0001 ml, or even less. Suitable
dilution of sample volumes smaller
than 1. 0 ml may be required for
accuracy of sample measurement.
2 While the method lends itself to a wide
range of sample-volumes, 'the filter has
limitations in the number of isolated
(or countable) differentiated colonies
which can develop on the available sur-
face area. Figure 1 illustrates a com-
mon pattern of colony counts over a
wide range of sample filtration volumes.
Figure 1
a The graph is based on coliform de-
' terminations using M Endo Broth
MF. The line designated "Total
Colonies" includes both coliform and
noncoliform colonies. The "Coliform
Colonies" line refers only to differ-
entiated colonies having the typical
color and sheen of coliform colonies
on the medium.
b For both the total colonies and the
coliform.colonies there is a pro-
portional relationship between colony
count and sample volume over much
of the range of sample volumes.
With increasing colony'counts, there
are some levels above -which the pro-
portional relationship fails, both for
total and for coliform colonies.
c In the straight lines in Figure 1,
where there is proportionality be-
tween colony counts and filtration
volumes, it is possible to compute
density of bacteria in the sample,
based on the equation:
No. organisms _ ^ x No. colonies-counted
per 100 ml No. ml of sample
filtered
The equation is not quantitatively
reliable in the curved portions of
the lines.
B Scope of this Presentation
1 To explain limitations on quantitatively
reliable colony counts on membrane
filte rs.
2 To present numbers of colonies accept-
able for quantitative tests with available,
media.
3 To demonstrate the different quantitative
bacterial density ranges determined by
single-volume filtrations, with available
media.
4 To demonstrate quantitative ranges
covered by a series of filtration volumes -
with currently-used differential media.
W. BA. mem. 75f. 11. 72
16-1
-------
Selection of Sample Filtration Volumes
5 To provide guidance for selection of
sample filtration volumes under the
following practical conditions:
a When there is need to determine
compliance with established bac-
teriological water quality standards.
b When there is need to determine
density of a specified bacterial group.
1) In the absence of prior bacterio-
logical data, and
2) When prior bacteriological data
are available.
II LIMITATIONS ON COLONY COUNTS
USED FOR QUANTITATIVE WORK
A Bacterial Density
1 The minimum sample volume should
result in production of at least 20
colonies of the bacteria being counted.
Sample volumes yielding lesser numbers
of colonies are subject to unacceptably
large random variations in the computed
bacterial density, determined as above
(I, A, 2).
2 The maximum acceptable colony density,
for quantitative determinations, is vari-
able with the bacterial group tested and
the medium used. Factors influencing
maximum acceptable colony density
include:
a Size of colonies. In principle, each
colony should represent one bacterial
cell deposited on the filter, or, con-
versely, each bacterial cell deposit-
ed on the filter should result in pro-
duction of a recognizable colony.
Madia producing relatively large
colonies (as in the fecal coliform
test) will support smaller numbers
of colonies on the filter th^n media
producing smaller colonies (such as
fecal streptococci). If the colony
size is large and the number of bac-
teria deposited on the filter is great,
some colonies will represent two or
more cells initially deposited on the
filter, and the quantitative reliability
of the test is impaired.
b Selectivity of medium. Highly
selective media permit growth of
relatively few colonies of extraneous,
unwanted, bacteria. The available
area of the filter is occupied pri-
marily by colonies of the group
tested. Thus, with a highly selective
medium such as that used for fecal
streptococci, it is reasonable to
expect good quantitative results with
relatively high eoleny counts. Con-
versely, media having limited
selectivity (such as Endo-type media
for coliforms) supports growth of
considerable numbers of extraneous
bacterial colonies, and it is' necessary
to place arbitrary limitations on the
number of colonies per membrane in
quantitative studies.
c Biochemical interference between
neighboring colonies. Associated
with the physical crowding effects
noted in (b) above, sheen production
of coliform colonies may be inhibited
by overcrowding of colonies. This
reinforces the need for restriction
of colony density on the filter.
B Suspended Matter
1 Particulate matter in the sample can
be a limitation in application of mem-
brane filters, especially when the
amount of suspended matter is relatively
great and the bacterial density is low.
2 Difficulties from suspended matter in
the sample may be apparent in several
ways.
a The pores of the filter may be
occluded, limiting the volume of
sample that can be filtered. This
problem has been noted in' waters
rich in clays and in waters containing
large populations of certain diatoms
or other' algae.
b Fibrous matter' can be troublesome,
due to the tendency for a capillary
film of liquid culture medium to form
around the fibers. Colonies in contact
with suqh fibers tend lo grow along
16-2
-------
Selection of Sample Filtration Volumes
the path of the fibers, assuming
highly irregular forms. Sometimes,
these colonies cover abnormally
large areas of the filter surface.
c A more or less continuous mat of
particles may be collected from
some samples, with each particle
soon surrounded by a film of liquid
culture medium. On such filters,
distinct colonies usually fail to
develop as discrete entities, but
grow in a more or less continuous
film over the entire surface of the
filter.
3 Problems due to particulate matter
often can be reduced by filtration of
the selected volume of water in two or
more smaller increments, through
separate filters. In effect, this is a
means of enlarging the available sur-
face area of the filter.
Prefiltration of the sample through a
coarse filter for preliminary removal
of extraneous particulate matter is not
recommended in quantitative work.
Prefiltration invariably results in re-
moval of unpredictably large numbers of
bacterial cells.
In some cases the problem of particu-
lates cannot be solved, and it must
then be conceded that the membrane
filter method is not acceptable for such
samples. It then becomes necessary to
resort to other procedures, such as the
dilution tube method or agar plating
methods.
Ill LIMITS ON NUMBER OF COLONIES ON
FILTERS WITH VARIOUS MEDIA
Referring to Figure 1, a specific number of
colonies is not shown for acceptable propor-
tionality between colony number and filtration
volume. Fixed limits cannot be stated for all
test situations, for these limits are somewhat
variable from one culture medium to another
and from one sample source to another.
The recommended limits shown in Table 1
are empirical values based on re-
search-experience. It is believed that quan-
titative determinations of acceptable statistical
reliability can be obtained if the determinations
are based on colony counts within the limita-
tions shown.
IV RANGE OF BACTERIAL DENSITIES
COVERED BY SINGLE-VOLUME
FILT RATIONS
A The equation used in Section I of this out-
line can be used with any sample filtration
volume to determine the bacterial density
range over which acceptable counts can be
made.
For example, assume that a sample of
10 ml is used for a quantitative determi-
nation of total coliforms. Based on Table
1, quantitative determinations should be
based on a filtration volume yielding 20 -
80 coliform colonies. " Compute
the coliforms per 100 ml based on 20
colonies and on 80 colonies per filter. This
will be the bacterial density range covered
by a 10 ml filtration volume, thus:
for 20 colonies:
20
No. coliforms per 100 ml = 100 X
= 200
and for 80 colonies,
No. coliforms per 100 ml n 100 X -52.
10
- 800
Thus, a 10 ml sample portion is appro-
priate for determination of total coliforms
in the range 200 - 800 per 100 ml.
B Table 2 illustrates the ranges covered for
several"filtration volumes, with colony
counts in the ranges 20 - 60, 20 - 80, and
20 - 100 per filtration volume'.
16-3
-------
Selection of Sample Filtration Volumes
Table 1. RECOMMENDED COLONY- COUNT RANGES FOR
QUANTITATIVE DETERMINATIONS WITH
MEMBRANE FILTER TESTS
Test
No. colonies
Medium
Remarks
Minimum'
Maximum
Total Coliform
20
80
M Endo'Broth MF,
LES Endo Medium
Not more than 200
colonies of all types
Fecal Coliform
20
60
M FC Broth

Fecal Streptococci
20
100
M Enterococcus
Agar, KF Agar

Total Counts
20
200
M Enrichment
Broth
Spreaders may
require adjustment
Table 2. RANGES COVERED BY REPRESENTATIVE
FILTRATION VOLUMES
Ml sample
filtered
Bacterial count per 100 ml based
on-
20 colonies
60 colonies
80 colonies
100 colonies
100
20
60
80
100
10
200
600
800
1000
1
2000
6000
8000
10, 000
0. 1
20,000
60,000
80,000
100,000
0.01
200,000
600,000
800, 000
1, 000, 000
C Application of a Series of Filtration
Volumes
1 Examination of Table 2 shows that for
quantitative work on membrane filters,
to extend the range of any test, it is
necessary to filter two or more different
sample volumes. The worker uses the
one sample volume yielding a quantita-
tively acceptable number of colonies to
compute the bacterial count per 100 ml.
2 Further, it can be seen that varying the
filtration volumes by decimal increments
will be inappropriate; there are values
within the total range covered in which
the colony number would fall outside the
critical counting range for the test being
made.
3 In order to give maximum assurance
that a series of varying filtration
volumes will yield at least one membrane
with an acceptable number of colonies,
the range of filtration volumes should
be'along these lines:
a Total coliform counts should be
based on filtration volumes varying
by a factor of 4, or less.
b Fecal coliform counts should be
based on filtration volumes varying
by a factor of 3, or less.
c Fecal streptococcus counts should
be based on filtration volumes vary-
ing by a factor of 5, or less.
V SELECTING FILTRATION VOLUMES
FOR MEMBRANE FILTER TESTS
A Total Coliform Counts
1 Determination of compliance with exist-
ing bacterial quality standards.
16t4
-------
Selection of Sample Filtration Volumes
For all tests to determine whether
water meets PHS Drinking Water
quality standards, minimum sample
sizes are prescribed as 50 ml, with
100 ml sample volumes suggested.
With tests in which it is assumed
that coliforms. are present in some
numbers, and the test is-to determine
whether some limiting standard (as
1000 per 100 ml in natural bathing
waters, prescribed by some
agencies), another approach is
suggested. Here, select the sample
filtration volume which would be
quantitatively most acceptable to
count coliforms at the limiting value.
For example, with a limiting value
of 1000 per 100 ml:
2) Polluted raw surface water, 0.02,
0. 08, 0. 15, and 0. 5 ml samples
will cover a count range of 4000
to 400, 000 per 100 ml.
3) Sewage and dilute sewage, with
filtration volumes of 0.0003,
0. 001,''-0. 003, "and 0. 01, will
cover a count range of 200,000
to 27, 000, 000 per 100 ml.
b If prior coliform data are available
Use the equation:
Basic filtration
volume in ml
= 100 X
50
Average coliform count
No. organisms _ 100 y
per 100 ml
No. colonies counted
No. ml of sample filtered
Example: Assume that prior data
indicate average coliform count of
35, 000 per 100 ml. Using the
equation:
This previously given equation can be rearranged
to:
Basic filtration volume in ml - 100 X
50
35, 000
Sample filtration
volume in ml
and from this:
= 100 X
No. colonies counted
No. organisms/100 ml
Sample filtration volume _ 50
in ml ' 1000
(The value 50 is the midrange number
of colonies for an acceptable colony
count of 20 - 80 for computing coli-
forms per 100 ml)
In quantitative work, to determine
number of coliforms per 100 ml the
worker may or may not have prior
information or standards to use as
guidance in selecting filtration volumes.
a In absence of prior bacteriological
data
1) Unpolluted raw surface water, 1,
4, 15, and 60 ml samples will
cover a count range of 33 - 8000
per 100 ml.
= 0. 143 ml
Round off the filtration volume to
0. 15 ml.
To assure a reasonable count-range,
filter increments of 0. 04 and 0. 60 ml
in addition. This will provide for
acceptable coliform counts in the
range of 3300 to 200, 000 per 100 ml.
B Fecal Coliform Counts
1 Currently, no drinking water standards
are based on fecal coliform organisms.
Many states have environmental water
quality standards which are based on
fecal coliform organisns.
2 Determination of fecal coliforms in the
absence of prior data.
a Unpolluted raw surface water:
Filter 1, 3, 10, and 30 ml sample
portions. These volumes will
cover a fecal coliform range of
67 - 6000 per 100 ml.
16-5
-------
Selection of Sample Filtration Volumes -
b Polluted raw surface water: Filter
portions of 0. 1, 0.3, 1.0, and 3.0
ml. This will cover a fecal coliform
count range of 670 to GO, 000 per 100
ml.
c Sewage and dilute sewage: Filter
sample portions of 0. 0003, 0. 001
and 0. 003 ml. This will provide for
counts of 670,000 to 20,000,000 per
100 ml.
Determination of fecal coliforms in
presence of prior data
a When previous fecal coliform counts
are available:
Filtration volume =
in ml
100 X -
40
Av. fecal coliform
count per 100 ml
Example: Prior data show 8000 fecal
coliforms per 100 ml.
Basic filtration volume in ml = 100 X
= 0. 5
40
8000
Filter volumes of 0. 15, 0. 5 and 1. 5
ml. This will be suitable for fecal
coliform counts over the range 1300
to 40, 000.
When previous total coliform data
are available but no fecal coliform
data are available, use. the total
coliform value as above, but filter
3x and 9x the computed basic volume.
Example (from above): Computed
basic value = 0. 5 ml
Filter volumes of 0. 5, 1.5 and 5. 0
ml.
C Fecal Streptococcus Determinations
1 In absence of prior data
a Unpolluted raw surface water: Filter
sample portions of 1, 5, 25 and 100
ml. This will provide for fecal
streptococcus counts in the range 20
to 10,000 per 100 ml.
b Polluted surface water: Filter
sample portions of 0. 1, 0. 5, and
2.0 ml. This will provide for fecal
streptococcus counts in the range
1000 to 100, 000 per 100 ml. Pro-
vision for rather high counts of
fecal streptococci is made because
of possible situations in which pol-
lution of the water originates from
domestic or wild animals. In the
event that such pollution is highly
improbable, a filtration series of
0.2, 1.0, and 5. 0 ml (covering a
count range of 400 to 50, 000 per
100 ml) would be more appropriate.
2 When prior data are available
a If coliform, but not fecal strepto-
coccus data are available, compute
a basic filtration volume as in A, 2
above, but use the average coliform
count as a point of reference. If
significant pollution from domestic
or wild animals is believed present,
filter 0. 2X, IX and 5X the basic
filtration volumes. If the pollution
levels are believed due primarily to
human sources, use IX, 5X and 25X
the basic filtration volumes.
b If prior streptococcus data are avail-
able, use the equation
Basic filtration = 100 X
volume in ml
60
Av. Streptococcus
count per 100 ml
and filter 0.2X, IX, and 5X the basic
filtration volume for streptococci.
REFERENCES
1 The Federal Register. October 23, 1956,
pp 8110-11; and March 1, 1957. p 1271.
2 Clark, H. F., Kabler, P. W., and
Geldreich, E. E. Advantages and
Limitations of the Membrane Filter
Procedure. Water and Sewage Works.
September 1957.
This outline was prepared by H, L. Jeter,
Director, National Training Center,
MDS, WPQ EPA, Cincinnati, OH 45268.
16-6
-------
DETAILED MEMBRANE FILTER METHODS
I BASIC PROCEDURES
A Introduction
Successful application of membrane filter
methods requires development of good
routine operational practices. The
detailed basic procedures described in
this Section are applicable to all mem-
brane filter methods in water bacteriology
for filtration, incubation, colony counting,
and reporting of results. In addition,
equipment and supplies used in membrane
filter procedures described here are not
repeated elsewhere in this text in such detail.
Workers using membrane filter methods
for the first time are urged t'o become
thoroughly familiar with these basic
procedures and precautions.
B General Supplies and Equipment List
Table 1 is a check list of materials.
C "Sterilizing" Media
Set tubes of freshly prepared medium in a
boiling waterbath for 10 minutes. This
method suffices for medium in tubes up to
25X 150 mm. Frequent agitation is needed
with media containing agar.
Alternately, coliform media can be
directly heated on a hotplate to the first
bubble of boiling. Stir the medium
frequently if direct heat is used, to avoid
charring the medium.
Do not sterilize m the autoclave.
D General Laboratory Procedures with
Membrane Filters
1 Prepare data sheet
Minimum data required are: sample
identification, test performed including
media and methods, sample filtration
volumes, and the bench numbers
assigned to individual membrane filters.
2 Disinfect the laboratory bench surface.
Use a suitable disinfectant solution and
allow the surface to dry before
proceeding.
3 Set out sterile culture containers in an
orderly arrangement.
4 Label the culture containers.
Numbers correspond with the filter
numbers shown on the data sheet.
5 Place one sterile absorbent pad* in
each culture container, unless an agar
medium is being used.
Use sterile forceps for all manipulations
of absorbent pads and membrane filters.
Forceps sterility is maintained by
storing the working tips in about 1 inch
of methanol or ethanol. Because the
alcohol deteriorates the filter, dissipate
it by burning before using the forceps.
Avoid heating the forceps in the burner
as hot metal chars the filter.
*When an agar medium is used, absorbent pads are not used. The amount of medium should be
sufficient to make a layer approximately 1/8" deep in the culture container. In the 50 mm
plastic culture containers this corresponds to approximately 6-8 ml of culture medium.
NOTE: Mention of commercial products and manufacturers does not imply endorsement by the
Office Of Water Programs, Environmental Protection Agency.
W. BA. mem. 86. 2. 75
17-1
-------
Detailed Membrane Filter Methods
Table 1. EQUIPMENT, SUPPLIES AND MEDIA

Total Coliforms

'
Item
M-Endo
Broth
L. E.S.
Coliform
Delayed
Coliform
Fecal
Coliform
Fecal
Streptococcus
Verified
Tests
Funnel unit assemblies
X
X
X
X
X

Ring stand, with about a 3" split ring, to
support the filtration funnel
X
X
,x
X
X

Forceps, smooth tips, type for
MF work
X
X
X
X
X

Methanol, In small wide-mouthed bottles,
about 20 ml for sterilizing forceps
X
X
X
X
X

Suction flasks, glass, 1 liter, mouth to
fit No. 8 stopper
X
X
X
X
X

Rubber tubing, 2-3 feet, to connect
suction flask to vacuum services, latex
rubber 3/16" I. D. by 3/32" wall
X
X
X
X
X

Pinch clamps strong enough for tight
compression of rubber tubing above
X
X
X
X
X

Pipettes, 10 ml, graduated, Mohr type,
sterile, dispense 10 per can per working
space per day. (Resterilize daily to
meet need).
X
X
X
X
X

Pipettes, 1 ml, graduated, Mohr type, .
sterile, dispense 24 per can per working
space per day. (Resterilize daily to
meet need).
X
X
X
X
X

Pipette boxes, sterile, for 1 ml and
10 ml pipettes (sterilize above pipettes
in these boxes).
X
X
X
X
X

Cylinders, 100 ml graduated, sterile,
(resterilize daily to meet need).
X
X
X
X
X

Jars, to receive used pipettes
X
X
X
X
X

Gas burner, Bunsen or similar
laboratory type
X
X
X
X
X
X
Wax pencils, red, suitable for writing
on glass
X
X
X
X
X

Sponge in dilute iodine, to disinfect the
desk tops
X
X
X
X
X.

Membrane filters (white, grid marked,
sterile,and suitable pore size for
microbiological analysis of water)
X
X
X
X
X

Absorbent pads for nutrient, (47 mm in
diameter), sterile. In units of 10 pads
per package. Not required if medium
contains agar.
Petri dishes, disposable, plastic,
50 X 12 mm, sterile
X
X
X
X
X
X
X
X
X
X

Waterbath incubator 44.5 + 0.2°C

Vegetable crispers, or cake boxes,
plastic, with tight fitting covers, for
membrane filter incubations
X
X
X

Fluorescent lamp, with extension cord.
X
X
X
X
X
X
King stand, with clamps, utility type
X
X
X
X
X

17-2
-------
Detailed Membrane Filter Methods
Table 1. EQUIPMENT, SUPPLIES AND MEDIA (Cont'd)
Total Collforms
Item
M-Endo
Broth
L. E. S.
Coliform
Delayed
Coliform
Fecal
Coliform
Fecal
Streptococcus
Verified
Testa
Half-round glass paper weights for
colony counting,with lower half of a
2-02 metal ointment box
Hand tally, single unit acceptable,
hand or desk type
Stereoscopic (dissection) microscope,
magnification of 10X or 15X, prefer-
able binocular wide field type
Bacteriological inoculating needle
Wire racks for culture tubes,
10 openings by five openings pre-
ferred, dimensions overall approxi-
mately 6" X 12"
Phenol Red Lactose Broth in 16 X-
150 mm fermentation tubes with
metal caps, 10 ml per tube
Eosln Methylene Blue Agar
(Levine) in petri plates, prepared
ready for use
Nutrient agar slants, in screw
capped tubes, 16 X 126 mm
Gram stain solutions, 4 solutions
per complete set
Microscope, compound, binocular,
with oil immersion lens, micro-
scope lamp and Immersion oil
Microscope slides, new, clean,
1" X 3" size
Water proof plastic bags
for fecal coliform culture
dish incubation
M-Endo medium, MF dehydrated
medium in 25 X 95 mm flat bottomed
screw-capped glass vials, 1.44 g
per tube, sufficient for 3 0 ml of
medium
Ethanol, 95% in small bottles or
screw-capped tubes, about 20 ml
per tube
Sodium benzoate solution, 12%
aqueous, in 25 X 150 mm screw-
capped tubes, about 10 ml per tube
L. E. S. Endo Agar MF, dehydrated
M-Endo medium, 0.36 g per 25 X
95 mm flat bottomed screw-capped
glass vial, plus 0.45 g agar, for 30 ml
Lactose Lauryl Sulfate Tryptose Broth
In 25 X 150 mm test tube without
included gas tube, about 25 ml, for
enrichment in L. E. S. method
X
X
X
X
X
X
X
X-
X
X
X
X
17-3
-------
Detailed Membrane Filter Methods
Table 1. EQUIPMENT, SUPPLIES AND MEDIA (Cont'd)

Total Cohforms

Item
M-Endo
Broth
L.E.S.
Coliform
Delayed
Coliform
Fecal
Coliform
Tecal
Streptococcus
Verified
Test
M-FC Broth for fecal coliform,,
dehydrated medium in 25 X95 mm
flat bottomed screw-capped glass
vials, 1. 11 g per tube, sufficient
for 30 ml of culture medium

Bosollc acid, 1% solution, in
0. 2N NaOH, in 25 X 150 mm flat
bottomed ecrew-capped tubes,
about 5 ml per tube, freshly
prepared

M-Enterococcus Agar, dehydrated
medium in 25 X150 mm screw-
capped tubes, sufficient for 30 ml,
1.26 g per tube

Dilution bottles, 6-oz, preferable
boro-silicate glass, with screw-
cap (or rubber stopper protected
by paper) , each containing 99 ml
of sterile phosphate buffered
distilled water
X
X
X
X
X

Electric hot plate surface
X
X
X
X
X

Beakers, 400 - 600 ml (for water-
bath in preparation of membrane
filter culture media)
X
X
X
X
X

Crucible tongs, to be used at
electric hot plates, for removal
of hot tubes of culture media for
boiling waterbath
X
X
X
X
X

17-4
-------
Detailed Membrane Filter Methods
6 Deliver enough culture medium to
saturate each absorbent pad, using
a sterile pipette.
Exact quantities cannot be stated
because pads and culture containers vary.
Sufficient medium should be applied so
that when the culture container is tipped,
a good-sized drop of culture medium
freely drains cu t of the absorbent pad.
7 Organize supplies and equipment for
convenient sample filtration. In
training courses, laboratory instructors
will suggest useful arrangements;
eventually the individual will select a
system of bench-top organization most
suited to his own needs. The important
point in any arrangement is to have all
needed equipment and supplies con-
veniently at hand, in such a pattern as
to minimize lost time in useless motions.
8 Lay a sterile membrane filter on the
filter holder, grid-side up, centered
over the porous part of the filter
support plate.
Membrane filters are extremely
delicate and easily damaged. For
manipulation, the sterile forceps
should always grasp the outer part
of the filter disk, outside the part
of the filter through which the sample
passes.
9 Attach the funnel element to the base
of the filtration unit.
To avoid damage to the membrane
filter, locking forces should only be
applied at the locking arrangement.
The funnel element never should be
turned or twisted while being seated
and locked to the lower element of the
filter holding unit. Filter holding units
featuring a bayonet joint and locking
ring to join the upper element to the
lower element require special care on
the part of the operator. The locking
ring should be turned sufficiently to
give a snug fit, but should not be
tightened excessively.
10 Shake the sample thoroughly.
11 Measure sample into the funnel with
vacuum turned off.
The primary objectives here are:
]) accurate measurement of sample;
and 2) optimum distribution of colonies
on the filter after incubation. To
meet these objectives, methods of
measurement and dispensation to the
filtration assembly are varied with
different sample filtration volumes.
a With samples greater than 20 ml,
measure the sample with a sterile
graduated cylinder and pour it into
the funnel. It is important to rinse
this graduate with sterile buffered
distilled water to preclude the loss
of excessive sample volume. This
should be poured into the funnel.
b With samples of 10 ml to 20 ml,
measure the sample with a sterile
10 ml or 20 ml pipette, and pipette
on a dry membrane in the filtration
assembly.
c With samples of 2 ml to 10 ml, pour
about 20 ml of sterile dilution water
into the filtration assembly, then
measure the sample into the sterile
buffered dilution water with a 10 ml
sterile pipette.
d With samples of 0.5 to 2 ml, pour
about 20 ml of sterile dilution water
into the funnel assembly, then
measure the sample into the sterile
dilution water in the funnel with a
1 ml or a 2 ml pipette.
e If a sample of less than 0. 5 ml is to
be filtered, prepare appropriate
dilutions in sterile dilution water,
and proceed as applicable in item c
or d above.
When dilutions of samples are needed,
always make the filtrations as soon
as possible after dilution of the
sample; this never should exceed
NOTE: Mention of commercial products and manufacturers does not imply endorsement
by the Office of Water Programs, Environmental Protection Agency.
17-5
-------
Detailed Membrane Filter Methods
3 0 minutes. Always shake sample
dilutions thoroughly before delivering
measured volumes.
12 Turn on the vacuum.
Open the appropriate spring clamp pr
valve, and filter the sample.
After sample filtration a few droplets
of sample usually remain adhered to
the funnel walls'. Unless these droplets
are removed, the bacteria cpntained in
them will be a source of contamination
of later samples. (In laboratory
practice the funnel unit is not routinely
sterilized between successive filtrations
of a series). The purpose of the funnel
rinse is to flush all droplets of a sample
from the funnel walls to the membrane
filter. Extensive tests have shoi,vn that
with proper rinsing technique, bacterial
retention on the funnel walls is negligible.
13 Rinse the sample through the filter.
After all the sample has passed through
the membrane filter, rinse down the
sides of the funnel walls with at least
20 ml of sterile dilution water. Repeat
the rinse twice after all the first rinse
has passed through the filter. Cut off
suction on the filtration assembly.
14 Remove the funnel element of the filter
holding unit.
If a ring stand with split ring is used,
hang the funnel element on the ring;
otherwise, place the inverted funnel
element on the inner surface of the
wrapping material. This requires
care in opening the sterilized package,
but it is effective as a protection of the
funnel ring from contamination.
15 Take the membrane filter from the
filter holder and carefully place it.
grid-side up on the medium.
Check that no air bubbles have been
trapped between the membrane filter
and the underlying absorbent pad or
agar. Relay the membrane if necessary.
16 Place in incubator after finishing
filtration series.
Invert the containers. The inimediate
atmosphere of the incubating membrane
filter must be at or very near 100%
relative humidity.
17 Count colonies which have appeared
after incubating for the prescribed
time.
A stereoscopic microscope magnifying
10-15 times and careful illumination
give best counts.
For reporting results, the computation
is:
bacteria/100 ml =
No. colonies counted X 100
Sample volume filtered in ml
Example:
A total of 36 colonies grew after
filtering a 10 ml sample. The
number reported is:
36 .C°l0"ieS X 100 = 360 per 100 ml
10 ml ^
Report results to two significant figures.
Example:
A total of 40 colonies grew after
filtering a 3 ml sample.
This calculation gives:
40 C3°mlieS X 100 = 1333. 33 per 100
But the number reported should be
1300 per 100 ml.
17-6
-------
Detailed Membrane Filter Methods
II MF LABORATORY TESTS FOR
COLIFORM GROUP
A Standard Coliform Test (Based on M-Endo
Broth MF)
1 Culture medium
a M-Endo Broth MF Difco 0749-02
or the equivalent BBL M-Coliform
Broth 01-494
Preparation of Culture Medium
(M-Endo Broth) for standard MF
Coliform Test
Yeast extract
1.5
g
Casitone or equivalent
5.0
g
Thiopeptone or equivalent
5.0
g
Tryptose
10.0
g
Lactose
12.5
g
Sodium desoxycholate
0. 1
g
Dipotassium phosphate
4.375
g
Monopotassium phosphate
1.375
g
Sodium chloride
5.0
g
Sodium lauryl sulfate
0.05
g
Basic fuchsin (bacteriological)
1. 05
g
Sodium sulfite
2. 1 -
g
Distilled water (containing
1000 ml
20.0 ml ethanol)

This medium is available in
dehydrated form and it is rec-
ommended that the commercially
available medium be used in
preference to compounding the
medium of its individual constituents.
To prepare the medium for use,
suspend the dehydrated medium at
the rate of 48 grams per liter of
water containing ethyl alcohol at
the rate of 20 ml per liter.
As a time-saving convenience, it is
recommended that the laboratory
worker preweigh the dehydrated
medium in closed tubes for several
days, or even weeks, at one operation.
'With this system, a large number
of increments of dehydrated medium
(e.g., 1.44 grams), sufficient for
some convenient (e.g., 30 ml)
volume of finished culture medium
are weighed and dispensed into
screw-capped culture tubes, and
stored until needed. Storage should
preferably be in a darkened disiccator.
A supply of distilled water containing
20 ml stock ethanol per liter is
maintained.
When the medium is to be used, it
is reconstituted by adding 30 ml of
the distilled water-ethanol mixture
per tube of pre-weighed dehydrated
culture medium.
b Medium is "sterilized" as directed
in I, C.
c Finished medium can be retained
up to 96 hours if kept in a cool,
dark place. Many workers prefer
to reconstitute fresh medium daily.
2 Filtration and incubation procedures
are as given in I, D.
Special instructions:
a For counting, use the wide field
binocular dissecting microscope, or
simple lens. For illumination, use
a light source perpendicular to the
plane of the membrane filter. A
small fluorescent lamp is ideal for
the purpose.
b Coliform colonies have a "metallic"
surface sheen under reflected light
which may cover the entire colony, or
it may appear only in the center. Non-
conform colonies range from
colorless to pink, but do not have
the characteristic sheen.
c Record the colony counts on the
data sheet, and compute the coliform
count per 100 ml of sample.
17-7
-------
Detailed Membrane Filter Methods
B Standard Coliform. Tests (Based on L. E. S.
Endo Agar)
The distinction of the L.E.S. count is a
two hour enrichment incubation on LST
broth. M-Endo L.E.S. medium is used
as agar rather than the broth.
1 Preparation of culture medium
(L.E.S. Endo Agar) for L. E. S.
coliform test
a Formula from McCarthy, Delaney,
and Grasso (2)
Bacto-Yeast Extract
1.2
g
Bacto-Casitone
3.7
g
Bacto-Thiopeptone
3.7
g
Bacto-Tryptose
7.5
g
Bacto- Lactose
9.4
g
Dipotassium phosphate
3.3
g
Monopotassium phosphate
1.0
g
Sodium chloride
3.7
g
Sodium desoxycholate
0. 1
g
Sodium lauryl sulfate
0. 05
g
Sodium sulfite
1.6
g
Bacto-Basic fuchsin
0.8
g
Agar
15
g
Distilled water (containing
1000 ml
2 0 ml ethyl alcohol)

b To rehydrate the medium, suspend
51 grams in the water-ethyl alcohol
solution.
c Medium is "sterilized" as directed
in I, C.
d Pour 4-6 ml of freshly prepared Agar
into the smaller half of the container .
Allow the medium to cool and solidify.
2 Procedures for filtration and incubation
a Lay out the culture dishes in a row
or series of rows as usual. Place
these with the upper (lid) or top
side down.
b Place one sterile absorbent pad in
the larger half of each container
(lid). Use sterile forceps for all
17-8
manipulations of the pads.
(Agar occupies smaller half or
bottom).
c Using a sterile pipette, deliver
enough single strength lauryl
sulfate tryptose broth to saturate
the pad only. Avoid excess medium.
d Follow general procedures for
filtering in I, D. Place filters on
pad with lauryl sulfate tryptose
broth.
e Upon completion of the filtrations,
invert the culture containers and
incubate at 35° C for 1 1/2 to 2
hours.
3 2-hour procedures
a Transfer the membrane filter from
the enrichment pad in the upper half
to the agar medium in the lower
half of the container. Carefully
roll the membrane onto the agar
surface to avoid trapping air"
bubbles beneath the membrane.
b Removal of the used absorbent pad
is optional.
c The container is inverted and
incubated 22 hours + 2 hours + 0.5 C.
4 Counting procedures are as in I, D.
5 L. E. S. Endo Agar may be used as a
single-stage medium (no enrichment
step) in the same manner as M-Endo
Broth, MF.
C Delayed Incubation Coliform Test
This technique is applicable in situations
where there is an excessive delay between
sample collection and plating. The procedure
is unnecessary when the interval be-
tween sample collection and plating is
within acceptable limits.
1 Preparation of culture media for
delayed incubation coliform test
a Preservative media M-Endo Broth
base
-------
Detailed Membrane Filter Methods
e- With a sterile pipette or sterile
absorbent pad, remove preservative
medium from the culture container.
f Place a sterile absorbent pad in
each culture container, and deliver
enough freshly prepared M-Endo
Broth to saturate each pad.
To 30 ml of M-Endo Br;oth MF
prepared in accordance with
directions in II, A, 1 of this
outline, add 1.0 ml of a sterile
12% aqueous solution of sodium
benzoate.
L. E. S. MF Holding Medium-
Coliform: Dissolve 12.7 grams in
1 liter of distilled water. No
heating is necessary. Final pH
7.1 + 0.1. This medium contains
sodium benzoate.
b Growth media
M-Endo Broth MF is used, prepared
as described in ii, A, 1 earlier in
this outline. Alternately, L, E. S.
Endo Medium may be used.
2 General filtration followed is in I, D.
Special procedures are:
a Transfer the membrane filter from
the filtration apparatus to a pad
saturated with benzoated M-Endo
Broth.
b Close the culture dishes and hold
in a container at ambient temperature.
This may be mailed or transported
to a central laboratory. The mailing
or transporting tube should contain
accurate transmittal data sheets which
correspond to properly labeled dishes.
Transportation time, in the case of
mailed containers, should not exceed
three days to the time of reception
by the testing laboratory.
g Using sterile forceps, transfer the
membrane to the new absorbent pad
containing M-Endo Broth. Place
the membrane carefully to avoid
entrapment of air between the
membrane and the underlying
absorbent pad. Discard the
absorbent pad containing pre-
servative medium.
h After incubation of 20 + 2 hours
at 35° C, count colonies as in the
above section A, 2.
i IfL.E.S. Endo Agar is used, the,
steps beginning with (e) above are
omitted; and the membrane filter is
removed from the preservative
medium and transferred to a fresh
culture container with L. E.S. Endo
Agar, incubated, and colonies
counted in the usual way.
D Verified Membrane Filter Coliform Test
This procedure applies to identification
of colonies growing on Endo-type media
used for determination of total coliform
counts. Isolates from these colonies are
studied for gas production from lactose
and typical coliform morphology. In
effect, the procedure corresponds with
the Completed Test stage of the multiple
fermentation tube test for coliforms.
c On receipt in the central laboratory,
unpack mailing carton, and lay out
the culture containers on the labora-
tory bench.
d Remove the tops from the culture
containers. Using sterile forceps,
remove each membrane and its
absorbent pad to the other half of
the culture container.
Procedure:
1 Select a membrane filter bearing
several well-isolated coliform-type
colonies.
2 Using sterile technique, pick all
colonies in a selected area with the
inoculation needle, making transfers
into tubes of phenol red lactose broth
(or lauryl sulfate tryptose lactose
17-9
-------
Detailed Membrane Filter Methods
broth). Using an appropriate data
sheet record the interpretation of
each colony, using, for instance,
"C" for colonies having the typical
color and sheen of coliforms; "NC"
for colonies not conforming to
coliform colony appearance on
Endotype media.
3 Incubate the broth tubes at 35° C+ 0. 5°C.
4 At 24 hours:
a Read and record the results from
the lactose broth fermentation tubes.
The following code is suggested:
Code
O No indication of acid or gas
production, either with or
without evidence of growth.
A Evidence of acid but not gas
(applies only when a pH indicator
is included in the broth medium)
G Growth with production of gas.
If pH indicator is used, use
symbol AG to show evidence of
acid. Gas in any quantity is a
positive test.
b Tubes not showing gas production are
returned to the 35° C incubator.
c Gas-positive tubes are transferred
as follows:
1) Prepare a streak inoculation on
EMB agar for colony isolation, and
using the same culture.
2) Inoculate a nutrient agar slant.
3) Incubate the EMB agar plates and
slants at 35° C + 0.5°C.
5 At 48 hours:
a Read and record results of lactose
broth tubes which were negative at
24 hours and were returned for
further incubation.
b Gas-positive cultures are subjected
to further transfers as in 4c.
Gas-negative cultures are discarded
without further study; they are
coliform- negative.
c Examine the cultures transferred
to EMB agar plates and to nutrient
agar slants, as follows:
1) Examine the EMB agar plate for
evidence of purity of culture; if
the culture represents more than
one colony type, discard the
nutrient agar culture and reisolate
each of the representative colonial
types on the EMB plate and resume
as with 4c for each isolation.
If purity of culture appears evident,
continue with c (2) below.
2) Prepare a smear and Gram stain
from each nutrient agar slant
culture. The Gram stain should
be made on a culture not more
than 24 hours old. Examine under
oil immersion for typical coliform
morphology, and record results.
6 At 72 hours:
Perform procedures described in 5c
above, and record results.
7 Coliform colonies are considered
verified if the procedures demonstrate
a pure culture of bacteria which are
gram negative nonspore-forming rods
and produce gas from lactose at 35° C
within 48 hours.
E Fecal Coliform Count (Based on M-FC
Broth Base)
The count depends upon growth on a
special medium at 44. 5 + 0. 2°C.
1 Preparation of Culture Medium
(M-FC Broth Base) for Fecal
Coliform Count
17-10
-------
Detailed Membrane Filter Methods
a Composition
Tryptose
10.0 g
Proteose Peptone No. 3
5. 0 g
Yeast extract
3.0 g
Sodium chloride
5.0 g
Lactose
12.5 g
Bile salts No. 3
1.5 g
Rosolic acid* (Allied
10. 0 ml
Chemical)

Aniline blue (Allied Chemical)
0. 1 g
Distilled water
1000 ml
b To prepare the medium dissolve
37.1 grams in a liter of distilled
water which contains 10 ml of 1%
rosolic acid (prepared in 0. 2 N
NaOH).
Fresh solutions of rosolic acid give
best results. Discard solutions
which have changed from dark red
to orange.
c To sterilize, heat to boiling as
directed in I, C.
d Prepared medium may be retained
up to 4 days in the dark at 2-8° C.
2 Special supplies
Small water proof plastic sacks capable
of being sealed against water with
capacity of 3 to 6 culture containers.
3 Filtration procedures are as given in
I, D.
4 Elevated temperature incubation
a Place fecal coliform count mem-
branes at 44.5 + 0.2°C as rapidly
as possible.
Filter membranes for fecal coliform
counts consecutively and immediately
place them in their culture containers.
Insert as many as six culture containers
all oriented in the same way (i.e., all
grid sides facing the same direction)
into the sacks and seal. Tear off the
perforated top, grasp the side wires,
and twirl the sack to roll the open end
inside the folds of sack. Then submerge
the sacks with culture containers in-
verted beneath the surface of a 44. 5
+ 0. 2°C waterbath.
b Incubate for 22+2 hours.
5 Counting procedures
Examine and count colonies as follows:
a Use a wide field binocular dissecting
microscope with 5 - 10X magnification.
b Low angle lighting from the side is
advantageous.
c Fecal coliform colonies are blue,
generally 1-3 mm in diameter.
d Record the colony counts on the
data sheet, and report the fecal
coliform count per 100 ml of sample.
(I, D, 17 illustrates method)
[I- TESTS FOR FECAL STREPTOCOCCAL
GROUP-MEMBRANE FILTER METHOD
A 48 hour incubation period on a choice of
two different media, giving high selectivity
for fecal streptococci, are the distinctive
features of the tests.
•'Prepare 1% solution of rosolic acid in 0. 2 N NaOH. This dye is practically insoluble in water.
17-11
-------
Detailed Membrane Filter Methods
A Test for Members of Fecal Streptococcal
Group (Tentative, Standard Methods) M-
Enterococcus Agar Medium
1 Preparation of the culture medium
a Formula (The Difco formula is shown,
but equivalent constituents from
other sources are equally acceptable).
Bacto tryptose
o
o
CM
g
Bacto yeast extract
5. 0
g
Bacto dextrose
2. 0
g
Dipotassium phosphate
4. 0
g
Sodium Azide
0.4
g
Bacto agar
10. 0
g
2, 3, 5, Triphenyl
0. 1
g
tetrazolium chloride
b The medium is prepared by
rehydration at the rate of 42 grams
per 1000 ml of distilled water. It
is recommended that the medium in
dehydrated form be preweighed and
dispensed into culture containers
(about 25 X 150 mm) in quantities
sufficient for preparation of 30 ml
of culture medium (1.26 g per tube).
c Follow I, C, for "sterilizing" medium
and dispense while hot into culture
containers. Allow plates to harden
before use.
2 List of apparatus, materials, as given
in Table I.
3 Procedure, in general, as given in I.
Special instructions
a Incubate for 48 hours, inverted,
with 100% relative humidity, after
filtrations are completed. If the
entire incubator does not have
saturated humidity, acceptable
conditions can be secured by placing
the cultures in a tightly closed
container with wet paper, towels,
or other moist material.
b After incubation, remove the
cultures from the incubator, and
count all colonies under wide field
binocular dissecting microscope
with magnification set at 10X or
20X. Fecal streptococcus colonies
are 0. 5 - 2 mm in diameter, and
flat to raised smooth, and vary
from pale pink to dark red in color.
c Report count per
100 ml of sample. This is con-
veniently computed:
No. fecal streptococci per 100 ml =
No. fecal streptococcus colonies counted ^ ioo
Sample filtration volume in ml
B Test for Members of Fecal Streptococcal
Group based on KF-Agar
1 Preparation of the culture medium
a Formula: (The dehydrated formula
of Bacto 0496 is shown, but
equivalent constituents from other
sources are acceptable). Formula
is in grams per liter of reconstituted
medium.
Bacto proteose peptone #3
10.
0
g
Bacto yeast extract
10.
0
g
Sodium chloride (reagent grade)
5.
0
g
Sodium glycerphosphate
10.
0
g
Maltose (CP)
20.
0
g
Lactose (CP)
1.
0
g
Sodium azide (Eastman)
0.
4
g
Sodium carbonate
0.
636
g
(Na^COg reagent grade)

Brom cresol purple
0.
015
g
(water soluble)

Bacto agar
20.
0
g
b Reagent
2, 3, 5-Triphenyl tetrazolium
chloride reagent (TPTC)
This reagent is prepared by making
a 1% aqueous solution of the above
chemical passing it through a Seitz
filter or membrane filter. It can
17-12
-------
Detailed Membrane Filter Methods
be kept in the refrigerator in a
screw-capped tube until used.
c The dehydrated medium described
above is prepared for laboratory
use as follows:
Suspend 7. 64 grams of the dehydrated
medium in 100 ml of distilled water
in a flask with an aluminum foil
cover.
Place the flask in a boiling water-
bath, melt the dehydrated medium,
and leave in the boiling waterbath
an addional 5 minutes.
Cool the medium to 50°-6 00 0, add
1. 0 ml of the TPTC reagent, and
mix.
For membrane filter studies, pour
5-8 ml in each 50 mm glass or
plastic culture dish or enough to
make a layer approximately 1/8"
thick. Be sure to pour plates before
agar cools and solidifies.'
For plate counts, pour as for standard
agar plate counts.
NOTE: Plastic dishes containing
media may be stored in a dark, cool
place up to 30 days without change
in productivity of the medium, pro-
vided that no dehydration occurs.
Plastic dishes may be incubated in
an ordinary air incubator. Glass
dishes must be incubated in an
atmosphere with saturated humidity.
2 Apparatus, and materials as given in
Table 1.
3 General procedure is as given in I.
Special instructions
a Incubate 48 hours, inverted with
100% relative humidity after
filtration.
b After incubation, remove the
cultures from the incubator, and
count colonies under wide field
binocular dissecting microscope,
with magnification set at 10X or
20X. ' Fecal streptococcus colonies
are pale pink to dark wine-color.
In size they range from barely
visible to approximately 2mm in
diameter. Colorless colonies are
not counted.
c Report fecal streptococcus count
per 100 ml of sample. This is
computed as follows:
No. fecal streptococci per 100 ml =
No. fecal streptococcus colonies ^ loo
Sample filtration volume in ml
C Verification of Streptococcus Colonies
1 Verification of colony identification
may be required in waters containing
large numbers of Micrococcus orga-
nisms. This has been noted
particularly with bathing waters, but
the problem is by no means limited to
such waters.
2 A verification procedure is described
in "Standard Methods for the Examination
of Water and Wastewater" 13th ed.
(1971). The worker should use
this reference for the step-by-
step procedure,
IV PROCEDURES FOR USE OF MEMBRANE
FILTER FIELD UNITS
A Culture Media
1 The standard coliform media used with
laboratory tests are used.
2 To simplify field operations, it is
suggested that the medium be sent to
the field, preweighed, in vials or
capped culture tubes. The medium
then requires only the addition of a
suitable volume of distilled water-
ethanol prior to sterilization.
17-13
-------
Detailed Membrane Filter Methods
3 Sterilization procedures in the field
are the same as for laboratory methods.
4 Laboratory preparation of the media,
ready for use, would be permissible
provided that the required limitations
on time and conditions of storage are
met.
B Operation of Millipore Water Testing Kit,
Bacteriological
1 Supporting supplies and equipment are
the same as for the laboratory
procedures.
2 Set the incubator voltage selector
switch to the voltage of the available
supply, turn on the unit and adjust as
necessary to establish operating
incubator temperature at 35 + 0.5°C.
3 Sterilize the funnel unit assembly by
exposure to formaldehyde or by
immersion in boiling water. . If a
laboratory autoclave is available, this
is preferred.
Formaldehyde is produced by soaking
an asbestos ring (in the funnel base)
with methanol, igniting, and after a
few seconds of burning, closing the
unit by placing the stainless steel
flask over the funnel and base. This
results in incomplete combustion of
the methanol, whereby formaldehyde
is produced. Leave the unit closed
for 15 minutes to allow adequate
exposure to formaldehyde.
4 Filtration and incubation procedures
correspond with laboratory methods.
5 The unit is supplied with a booklet
containing detailed step-by-step
operational procedures. The worker
using the equipment should become
completely versed in its contents and
application.
C Other commercially available field kits
should be used according to manu-
facturer's instructions. It'is emphasized
that the standards of performance are
required for field devices as for laboratory
equipment.
D Counting of Colonies on Membrane Filters
1 Equipment and materials
Membrane filter cultures to be
examined
Illumination source
Simple lens, 2X to 6X magnification
Hand tally (optional)
2 Procedure
a Remove the cultures from the
incubator and arrange them in
numerical sequence.
b Set up illumination source as that
light will originate from an area
perpendicular to the plane of
membrane filters being examined.
A small fluorescent lamp is ideal
for the purpose. It is highly
desirable that a simple lens be
attached to the light source.
c Examine results. Count all coliform
and noncoliform colonies. Coliform
and noncoliform colonies. Coliform
colonies have a "metallic" surface
sheen under reflected light, which
may cover the entire colony or may
appear only on the center.
17-14
-------
Detailed Membrane Filter Methcds
Noncoliform colonies range from
colorless to pink or red, but do not
have the characteristic "metallic"
sheen.
2 McCarthy, J. A., Delaney, J.E. and
Grasso, R.J. Measuring Coliforms
in Water. Water and Sewage Works.
1961: R-426-31. 1961.
d Enter the colony counts in the data
sheets.
e Enter the coliform count per 100 ml
of sample for each membrane having
a countable number of coliform
colonies. Computation is as follows:
This outline was prepared by H. L. Jeter,
Director, National Training Center,
EPA, WPO, Cincinnati, OH 45268.
No. coliform per 100 ml =
No. coliform colonies on MF
No. milliliters sample filtered
Descriptors: Biological Membranes,
Coliforms, Fecal Coliforms, Fecal
Streptococci, Filters, Indicator Bacteria,
Laboratory Equipment, Laboratory Tests,
Membranes, Microbiology, Water Analysis
REFERENCES
1 Standard Methods for the Examination of
Water and Wastewater. APHA,
AWWA, WPCF. 12th Edition. 1965.
17-15
-------
COLONY COUNTING ON MEMBRANE FILTERS
I INTRODUCTION
On removal of membrane filter cultures from
the incubator, the worker has several tasks
to perform, leading to the reporting of results
of the bacteriological examination. These
steps, together with the selection and use of
associated equipment, are considered in this
discussion. The following topics are included:
A Precautions on removal of membrane
filter cultures from the incubator.
B Selection of the best membrane filter for
colony counting (when more than one
membrane filter per sample was prepared,
representing a graded series of sample
increments.)
C Use of grid systems on filter surfaces as
counting aids.
D Recognition and counting of desired
colonies, including selection and use of
optical equipment.
E Calculations for reporting number of test
organisms per 100 ml of sample.
II REMOVAL OF CULTURE FROM
INCUBATOR
A Incubation time and temperature recom-
mendations should be closely adhered to.
This applies particularly to total coliform
counts. Some of our earlier training
manuals have suggested counting of colonies
after as few as 16 hours of incubation at
35°C. Currently, 22+2 hours is preferred.
B All membrane filter cultures should be
incubated in the inverted position, with
measures to avoid loss of culture medium
through leakage or evaporation. Some-
times an excessive amount of culture
medium is applied initially, or additional
moisture finds its way into the culture
container during incubation. In such
cases, when the culture is removed
from the incubator, H should be turned
"right side up" in such a way as to avoid
flooding the filter with excess liquid. If
excessive liquid is present, open the
culture container cautiously, and pour
off the excess.
C Drying Filters Before Colony Counts
1 Some workers advise opening all
cultures (especially total coliform tests
when Endo-type media are used) for
a short time (15 minutes to 1 hour)
for partial drying of coliform colonies
before counting. Advocates of this
step report that the typical surface
sheen characteristic of coliform
colonies is improved by this step.
2 Use of preliminary drying procedures
is a matter of personal preference.
In the opinion of the writer, the benefit
of preliminary drying is at best debat-
able, and at worst, may interfere with
subsequent study of the bacterial
colonies. Correct use of acceptable
lighting and optical equipment is a
far more important factor in ease and
accuracy of recognition of differentiated
colonies.
Ill SELECTION OF ACCEPTABLE MEMBRANE
FILTER CULTURE FOR EXAMINATION
A Non-Quantitative Tests
In bacteriologic examination of treated
waters, where waters meeting require-
ments result in development of very few
or no coliform colonies, the typical filtra-
tion volume is 100 ml, and but one filtra-
tion is made per sample. In this case,
there is no problem: the one membrane
NOTE: Mention of commercial products..and manufacturers does not imoly endorsement by the
Office of Water Programs, Environmental Protection Agency.
W. BA. mem. 85a. 11.72
18-1
-------
Colony Counting on Membrane Filters
filter preparation is the basis of bacteri-
ologic evaluation of the sample.
B Quantitative Tests
1 When the bacteriological water quality
standard is for some fixed limiting
value, such as 70 per 100 ml for shell-
fish waters, again only a single sample
filtration volume may be used. In such
a case, the filtration of a single portion
of 50 ml will show directly whether the
water meets bacteriologic standards,
or if the limiting standard is being
exceeded.
2 On the other hand, If the objective of the
test was to show how many coliforms
were present per 100 ml of sample,
then it is necessary to filter a series of
sample increments from each sample,
each increment being placed on a separate
membrane filter. At the end of the
incubation period, the series of mem-
brane filters representing each sample
must be inspected, with selection of the
membrane filter bearing the number of
colonies most suitable for reporting
quantitative results. This is summarized
in Table 1, below:
The lower limit of 20 is set arbitrarily,
as a number below which statistically
valid results become increasingly
questionable with smaller numbers of
colonies. The upper limits represent
numbers above which interference from
colony crowding, deposition of extrane-
ous material, and other factors appear
to result in increasingly questionable
results. It is emphasized that these
limiting values are empirical, based
on laboratory observations alone, and
do not represent results of theoretical
calculations. It follows that it is quite
possible, with some sample sources,
to obtain acceptable quantitative results
with colony.counts higher than the re-
commendations, but the minimum limit
of 20 colonies appears to' apply to the
majority of sample sources. "
3 If no membrane filter bears a number
of colonies within the recommended
limits for the test, the worker has a
choice between - a) collecting a new
sample and repeating the test; and
b) using whatever results actually were
obtained, reporting an "educated guess"
as to the number of organisms per
100 ml. In the latter case, it is most
TaEIe 1. NUMBERS OF COLOOTES ACCEPTABLE
. FOR QUANTITATIVE DETERMINATIONS
Test
Colony Counting Range
Minimum Maximum
Remarks
Total coliform
20
80
200 limit overall
Fecal coliform
20
60

Fecal streptococcus
20
100

18-2
-------
Colony Counting on Membrane Filters
strongly urged that each result of this
type be specifically identified with a
qualifying statement, such as "Estimated
count, based on non-ideal colony density
on filter. "
4 Sometimes two or more filters, of a
series of filtration volumes from a
sample, produce colony counts within
the recommended counting range.
Colony counts should be made on all
such filters. See Section VI of this
outline for calculations based on such
results. These problems may arise
from the selection of a too-close range
of sample filtration volumes, from
colony differentiation failures related
to overcrowding on the filters, or from
physico-chemical interference with
colony development related to material
in the sample deposited in or on the
filters.
USE OF GRID SYSTEMS IN COLONY
COUNTS
Most manufacturers provide grid-imprinted
membrane filters for bacteriologic use.
The ink used in such filters must be bio-
chemically inert to the test organisms,
and, of course, must be applied in such a
manner as not to degrade the quality of the
filter. Examples of such gridding have
appeared from various manufacturers as
follow s:
1 ... effective filtering area subdivided
into squares equal to 1/100 the effective
filtering area (when a filtering unit
with funnel-diameter of 35 mm is used).
2 ... grid markings which subdivide
the effective filtering area into squares
equal to 1/100 the effective filtering
area (9.6 cm^ for 47 mm diameter
filters).
3 ... filters subdivided so that each
square of the grid represents 1/60 of
the effective filtering area.
B Some special studies may require use of
membrane filters without grid markings.
For example, the ink in some filters pre-
vents growth of Brucella melitensis. In
such cases it may be necessary to impro-
vise a viewing grid-which can.be placed
over the culture after incubation and
colony development.
C Applications of Grids
1 The grid dimensions are of no particular
significance in colony counting, provided
that their size permits easy and con-
tinuous orientation in.counting of colome's.
To be sure, a rough estimate of the total
number of differentiated colonies on a
filter is possible by counting a repre-
sentative number of squares and multi-
plying colony count by the appropriate
factor. For example, with many
filters, colonies in ten squares can be
counted, multiplied by 10, and the pro-
duct is a rough estimate of the total
number on the entire filter. It is em-
phasized that such procedure is for
rough estimates only, and should not be
condoned in quantitative work with
membrane filters.
2 The primary usefulness of the grid
system is for orientation during the
counting procedure. Some colonies
will touch lines on a grid system, and
a uniform practice must be established
to avoid missing some colonies or
counting others twice. The procedure
used by the writer is as follows:
a Counts are made in an orderly back-
and-forth sweep, from top to bottom
of the filter. See Figure 1.
b Inevitably, some colonies will be in
contact with grid lines. A suggested
routine procedure for counting colonies
in contact with lines is indicated in
Figure 2. Colonies are counted in
the squares indicated by the arrows,
and no effort is made to decide
whether "most of the colony" is in
one or the other square.
18-3
-------
Colony Counting or Membrane Filters
Figure 1. The dashed circle indicates the
effective filtering area. The dashed back-
and-forth line indicates the colony counting
pathway.
V COUNTING OF COLONIES
A Equipment
1 A hand-tally is a useful device while
counts are being made,
2 Optical assistance in colony counts is
strongly recommended. Dependence
on naked-eye counts often results in
too-low results.
a Preferably, use a wide-field binocu-
lar dissecting microscope with
magnification of 10X or 15X.
b Optionally, but less desirably, a
simple lens with magnification at
least 5X can be used, provided that
acceptable illumination also is
present.
3 Lighting equipment
a For coliform counting, a large light
source is mandatory. Fluorescent
lamps in housings permitting place-
ment close to and as directly as
possible over the membrane filter is
the best lighting arrangement known
to the writer. Incandescent lamps,
whether simple light bulbs in a table

V*
4?
m

<
>-*«

1
•v.

Figure 2. Enlarged portion of grid-marked
square of filter, with various ways colonies
can be in contact with grid-lines. Colonies
are counted in squares indicated by the arrows.
lamp or in elaborate microscope
lamp housings, are not satisfactory
for coliform colony counting on
membrane filters with Endo-type
media.
b For fecal coliforms or fecal strep-
tococci, the lighting requirements
are not so severe; in this case almost
any sufficiently bright light source,
which can be placed above the filter
(either at a high or at a low angle)
will suffice.
4 Lighting arrangement and counting
a As above, for coliform counting,
the fluorescent lamp should be at a
high angle (as nearly as possible
directly over the membrane filter)
so placed that an image of the light
source is reflected off the colony
surfaces into the microscope lens
system. Properly placed, the light
will demonstrate the "golden
metallic" surface luster of coliform
colonies, which may cover the entire
colony, or-may appear only in an
area in the center of the colony.
The worker must learn to recognize
the difference between the typical
golden sheen of coliform- colonies
and the merely shiny surface of non-
conform colonies.
18-4
-------
Colony Counting or Membrane Fillers
b Other types of colonies (fecal coli-
forms, fecal streptococci, etc.) do
not require such rigid control of the
light source. Low-angle lighting
can be helpful, to give a relief of
the colony profile from the colony
surface. This is valuable with small
colonies, such as frequently en-
countered in streptococcal studies.
In such cases, almost any light
source is acceptable, provided that
it is bright enough and that it is
applied from somewhere above the
membrane filter.
c The typical appearance of various
types of colonies is related to the
culture media applied; therefore,
this is not discussed in detail at this
point. See the outlines on culture
media and on laboratory procedures
for specified indicator organisms
for such information.
d In colony counting, count all colonies
individually, even if"they are in
contact with each other (this is con-
trary to usual practice in colony
counting in agar cultures in Petri
dishes). Such colonies are recog-
nized quite easily when a microscope
is used for colony counting as re-
commended. Colonies which have
grown into contact almost invariably
show a very fine line of contact. The
worker must learn to recognize the
difference between two or more
colonies which have grown into con-
tact with each other, and single,
irregularly shaped, colonies which
sometimes develop on membrane
filters. Such colonies almost in-
variably are associated with a fiber
or particulate material deposited on
the filter, and tend to develop along
a path conforming to the shape and
size of the fiber or particulates.
VI CALCULATIONS
A Counting Units
1 In reporting densities of indicator
organisms (coliforms, fecal coliforms,
fecal streptococci), bacterial counts
always are reported in numbers per 100
ml. In standard practice, results are
expressed to' two significant figures. For
example, if the'Calculation-indicates
75, 400,or even 75,444 organisms per 100
ml, the results would be reported as
75, 000 per 100 ml in each case. (The
digits 7 and 5 are the significant figures;
the three zeros only locate the decimal
point. )
2 When "total" bacterial counts are reported,
common practice is to report in number
per ml, not the number per 100 ml.
3 Quantitative work on enteric pathogens is,
at this time, limited to reporting of
occurrence of designated enteric pathogens,
correlated with measured density of pollu-
tion indicating'bacterial groups. At such
time as the numerical determination of
enteric pathogens becomes feasible, it is
anticipated that reports will be in terms
of count per 100 ml, or even larger volume
' units.
B Typical Calculations
1 Select the membrane filter bearing the
acceptable number of colonies for re-
porting, and calculate indicators per
100 ml according to the general formula:
No. indicator organisms per 100 ml =
No. colonies of indicator organism
No. ml of sample filtered
2 Example:
a Assume that for a total coliform
count, volumes of 50, 15, 5, 1.5,
and 0.5 ml produced coliform colony
counts of 200, 110, 40, 10, and 5,
respectively.
b First, the worker actually would not
have counted coliform colonies on
all these filters. He would have
selected, by inspection, the mem-
brane filter(s) most likely to have
20-80 coliform colonies, limiting
actual counting to such colonies
(this does take some practice and
skill in making quick estimates, but
comes with experience).
18-5
-------
Colony Counting or Membrane Filters
c Having selected the membrane filter
probably most useful for reporting
purposes, coliform coloni.es are
counted according to accepted pro-
cedures, and the general formula
is applied:
40
Coliforms per 100 ml = — X 100
Coliforms per 100 ml = 800
C Special Situations in Calculating Densities
of Indicator Organisms
1 Assume a coliform count in which the
volumes of 1, 0.3, 0,1, 0.03, and
0.01 ml, respectively, produced coli-
form colony counts of TNTC, TNTC,
75, 30, and 8, respectively.
a Here, two sample volumes resulted
in production of coliform colonies
in the acceptable counting range.
b Suggestion: Compile the filtration-
volumes and colonies from both
acceptable filters, as follows:
Volume, ml Count
0. 1 75
0.03 30
0.13 105
Calculate coliforms per 100 ml from
the composite result:
105
Coliforms per 100 ml = -—— X 100
Coliforms per 100 ml = 81,000
2 Assume a coliform count in which
sample volumes of 1, 0.3, and . 01 ml
produced colony counts of 14, 3, and
0, respectively.
a Here, no colony count falls within
recommended limits.
b Suggestion: Calculate on the basis
of the most nearly acceptable value,
and report with qualifying,remark,
thus:
IJse 14 colonies from'l ml of sample:
X 100 = 1400
Report: "Estimated Count, 1400
per 100 ml, based on non-ideal
colony count"
3 Assume a contorm count lii which the
volumes 1, 0.3, and 0. 01 ml produced
coliform colony counts of 0', 0, and 0,
respectively.
a H ere, no actual calculation is possible,
even for "estimate" reports.
b Suggestion: Calculate the number
of estimated coliforms per 100 ml
that would have been reported if
there had been 1 coliform colony
on the filter representing the largest
filtration volume, thus:
Use 1'colony, and 1 ml: y- * = 100
Report: "Less than 100 coliforms
per 100 ml".
4 Assume a coliform count in which the
volumes of 1, 0. 3, and 0.01 ml pro-
duced coliform colony counts of TNC,
150, and 110 colonies.
a Here, all colony counts are above
the recommended limits.
b Suggestion: Use Example 2, above,
and report an estimated count based
on non-ideal colony counts:
jpjjy X 100 = 1, 100,000
Report: "Coliform count estimated
at 1, 100,000 per 100 ml, based on
non-ideal colony count".
18-6
-------
Colony Counting or Membrane Filters
5 Assume that, in Example 4, the volumes
of 1.0, 0.3, and 0.01 ml, all produced
too many coliform colonies to show
separated colonies, and that the labora-
tciry bench record showed TNTC (Too
Numerous to Count).
Suggestion: Use 80 colonies as the
basis of calculation with the smallest
filtration volume, thus:
X 100 = 800,000
Report: "> 800,000 coliforms per 100
ml sample. Filters too crowded. "
VII CONCLUSION
The foregoing discussion has presented a
number of factors which determine the quan-
titative reliability of membrane filter results.
It cannot be too strongly emphasized that the
correct use of acceptable colony counting
equipment is one of the most important single
factors in successful application of membrane
filter methods. Here, there is perhaps a
greater exercise of personal skill and judgment
than in any other'aspect of membrane filter
methodology. There is no substitute for prac-
tice and experience, supported by liberal use
of supporting colony verification studies, to
produce a skilled worker in colony counts
on membrane filters.
This outline was prepared by Harold L. Jeter,
Director, National Training Center, EPA,
Cincinnati, OH 45268.
18-7
-------
VERIFIED MEMBRANE FILTER TESTS
I INTRODUCTION
A The purpose of a verified membrane filter
test procedure is to establish the validity
of colony differentiation ^nd interpretation
in the test being applied. Specifically, a
verified membrane filter test may prove
useful 1) as a self-training device for new
workers, 2) as a research tool in evalu-
ation of new membrane filter media and
procedures, or 3) to provide supporting
evidence' of colony interpretation in cases
where the analytical results may be subject
to professional or official challenge.
B Reduced to essentials, a verified mem-
brane filter test consists of 1) interpre-
tation of the colonies appearing on a
selective, differential medium, 2) re-
covery of purified bacterial cultures from
differentiated colonies, and 3) application'
of supplemental test procedures to
determine the validity of the original
¦interpretation of the membrane filter
colonies.
C In this discussion, primary attention is given
to a verified membrane filter cohform test.
In addition, verification procedures are
presented for members of the fecal-con-
form group and for fecal streptococci.
II VERIFIED TEST FOR 'MEMBERS OF THE
COLIFORM GROUP
A An abbreviated procedu re corresponds to the
Confirmed Test of Standard Methods through
use of lactose broth (or lactose lauryl
tryptose broth) followed by confirmation
in brilliant green lactose bile broth. The
procedure is shown diagrammatically as
follows:
10 - 20 sheen colonies from membrane
filter (each tested separately)
J*
Lactose (or lauryl tryptose) broth
1
Incubate 24 hours at 35°C
I
X
No gas
1
Reincubate 24 hours at 35 C
i
I
No gas
Negative coliform test
Colony not coliform
Gas
~2
Gas-
Brilliant Green lactose bile broth
Incubate up to 48 hours at 35°C
*-
No gas
Negative coliform test
Colony not coliform
3,
Gas
Positive coliform test
Colony was coliform
Diagram 1. ABBREVIATED COLIFORM VERIFICATION PROCEDURE
W. BA. mem. 83a. 1. 73
-------
Verified Membrane Filter Tests
B A more elaborate verification of membrane
filter test for coliforms resembles the
Completed Test of Standard Methods. The
test is started m exactly the same way as
the abbreviated test, and may be repre-
sented diagrammatically as a continuation
from the lactose broth stage of Diagram 1.
See Diagram- 2.
C While the diagrams (1 and 2) are pre-
sented in terms of sheen colonics (inter-
preted as coliforms), the careful worker
also should subject a similarly represen-
tative number of non-sheen colonies
(judged to be noncoliforms) to the same
test procedure. This will reveal whether
the medium being studied fails to differ-
entiate appreciable numbers of colonies
which in reality are coliforms, even
though they did not demonstrate the
desired differential characteristic.
Ill VERIFICATION OF FECAL COLIFORM
TESTS ON MEMBRANE FILTERS
A The procedure described here is based
on the principle that, with use of m-FC
Broth and incubation in a water bath at
44. 5°C for 24 hours, fecal coliform
colonies on membrane filters develop a
blue color, (sometimes a greenish-blue).
Extraneous bacteria are believed to fail
to develop colonies, or else consist of
such colonies develop some color other
than the blue color of fecal coliforms
(colorless, buff- or brownish-color, or
even red colonies may develop on the
medium).
Gas-positive lactose broth tubes from
Diagram 1, above
Streak on eosin methylene blue agar
plates for colony isolation
Incubate 24 hours at 35°C
I
Transfer an isolated nucleated colony
(or at least two well-isolated representative
colonies in the absence of nucleated colonies)
to
lactose broth (or lactose
lauryl tryptose broth)
Incubate up to 48 hours
at 35°C
and to
nutrient agar slant
1
Incubate not more than 24 hours
at 35°C
4T~
No gas
Negative test
(Colony was not
coliform)
4
Prepare Gram-stained smear and
examine under oil immersion
Gas
and
Gram-negative, non-spore
forming rods, pure culture
based on morphology
Positive coliform test (Colony
originally selected was coliform)
:—i
Lack of any morpho-
logical feature
described on left
i
Negative coliform test
(Colony originally chosen
was not colifo rm)
Diagram 2. EXTENDED COLIFORM VERIFICATION PROCEDURE
19-2
-------
Verified Membrane Filter Tests
B The verified test for fecal coliforins
is indicated in Diagram 3, below:
10 - 20 blue colonies from membrane-
filter (each tested separately)
Phenol red lactose broth (or lactose
broth or lauryl tryptose broth)
Incubate 24 hours at 35°C
vt
No gas
4
Reincubate 24 hours
at 35°C
^
No gas
I
Gas
Negative test for
fecal coliforms.
Colony was not a
fecal coliform colony
Gas
¦ 1
EC Broth
|
Incubate 24 hours at 44. 5°C. ± 0. 5° C in a
water'bath,
NT ^
No gas
"~1
Gas
Negative test for fecal
coliforms. Colony was
not a fecal coliform
Positive test for
fecal coliforms.
Colony was fecal
colifo rm
Diagram 3. A VERIFICATION PROCEDURE FOR FECAL
COLIFORMjS ON MEMBRANE FILTERS
IV VERIFICATION OF FECAL STREPTO-
COCCUS COLONIES ON MEMBRANE
FILTERS
A The procedure is used in the evaluation of
results from a medium similar to the
m-Enterococcus Agar (Slanetz) described
in the current edition of Standard Methods.
The membrane filter procedure utilizes
48 hour incubation at 35°C, and colonies
which are pink to red, either in their
entirety or only in their penters, are
regarded as fecal streptococci. Most
such colonies are 1-2 mm in diameter,
and some may be larger. Occasionally,
some samples may be encountered in
which numerous extremely small colonies,
approximately 0. 1 mm in diameter, are
present in great numbers. Almost in-
variably, these are not fecal streptococci.
See diagram 4 for a representation of
a verification test.
V CALCULATIONS BASED ON VERIFICATION
STUDIES
A A percent verification can be determined
for any colony-validation test:
. Percent verification =
No. of colonies meeting verification test ^
No. of colonies subjected to verification
19-3
-------
Verified Membrane FiLter Tests
10 - 20 Pink to red colonies from
membrane fiUer(each tested separately)
Brain-heart infusion broth
or tryptose glucose broth
Incubate 24-48 hours at 35°C
i
Brain-heart infusion agar slant
Incubate 24-48 hours at 35 C
<£
No growth
Negative test; colony
not fecal streptococcus
Brain-heart
infusion broth or
tryptose glucose
broth
Incubate
Growth-
Loopful of growth from
brain-heart infusion agar
Cover with a few drops of
fresh hydrogen peroxide
J,
No growth
Negative test; colony
not fecal streptococcus
40% Bile broth
24-48
hours at 45°C
,
No growth
Negative test
Growth
Positive test
Gas released
(probably not
fecal strepto-
cocci)
Gas not released
Incubate 24-48 hours
at 45°C
I
5
No growth
Negative
test
Growth
Positive
test
I
Positive test: Demonstrates growth on brain-heart infusion broth at 3500, and
at 45°C, and growth on 40% bile broth at 45°C, and absence of
catalase enzyme
Diagram 4. FLOW SHEET AND SUGGESTED SEQUENCE OF TESTS TO PERFORM
VERIFICATION STUDIES ON COLONIES BELIEVED TO BE FECAL STREPTOCOCCI
Example: Twenty-five sheen colonies
on Endo-type membrane filter medium
were subjected to verification studies
shown in Diagram 1. Twenty-two of
these colonies proved to be coliforms
according to provisions of the test:
22
25
Percent verification =
X 100
= 88
B A percent verification figure can be
applied to a direct membrane filter
count per 100 ml to determine the veri-
fied membrane filter count per 100 ml
of the test organism.
19-4
Verified count per 100 ml
of the test organism
Percent verification
100
X
count per 100 ml
of test organism
Example: For a given sample, by a
direct membrane filter test, the fecal
coliform count was found to be 42,000
per 100 ml. Supplemental studies on
selected colonies showed 92% verification.
Verified fecal
coliform count
Rounding off:
92
X 42,000
100
= 0. 92 X 42, 000
= 38,640
= 39,000 per 100 ml
-------
Verified Membrane Filter Tests
C A percentage of false-negative tests also
can be determined (See II, C)
Percent false negative -
No. "negative" colonies found positive ^ ^qq
Total No. negative' colonies tested
Example: On a total coliform test, 25
nonsheen (coliform negative) colony
types were subjected to the coliform
verification procedure shown in Diagram
1. Two of these colonies proved to be
coliform colonies.
2
Percent false negatives » — X 100
a 8
VI SOME APPLICATIONS OF PERCENT'
VERIFICATION CALCULATIONS
A In comparisons between two or more differ-
ent membrane filter media, the medium
which has the highest percentage of veri-
fication, and the lowest percentage of
false negatives (based on a broad range
of sample types and sources) is the better
medium.
B In productivity comparisons between two
or more different membrane filter media,
the medium which produces the highest
verified membrane filter counts per 100
ml (based on a broad range of sample
types and sources) is the better medium.
C The worker is cautioned NOT to apply
percentage of verification determined
from one sample, to other samples. For
example, do not determine a percentage
verification on m-'Endo broth for a sample
taken from the Ohio River on September 6,
and then seek to apply that percentage
verification to another coliform determina-
tion from the Little Miami River, on the
same date. Even the application of the
verification percentage to another Ohio
River sample, either on the same date
from a different station, or on another
date from the same station, should be
undertaken with great caution. Such
application of verification percentages
from one sample to another should be
taken only after sufficient studies have
been made demonstrate the suitability
of such a procedure.
VII USE OF VERIFICATION STUDIES IN
MF-MPN COMPARISONS
A Comparisons of data obtained from MF
versus MPN methods have been the source
of great concern to microbiologists. For
the current basis of comparisons, see
Standard Methods (either 11th or 12th
edition) "-- with a proviso that it should
be used for determining the potability
of drinking water only after parallel
testing had shown that it afforded infor-
mation equivalent to that given by the
standard multiple-tube test. "
B Some workers have sought to apply this
requirement on the basis of statistical
calculations, based on comparisons of
numerical values from membrane filter
tests with numerical values obtained from
multiple-tube tests. Further study of this
problem, and methods different workers
have applied to the problem, can be made
on the basis of the appended reference list.
C Numerical comparisons between raw or
verified membrane filter results on split
samples, compared with multiple-tube
results, also should take into account the
question of the reliability of the multiple-
tube test. The numerical results of the
Completed Test for coliforms, for example,
can be compared with the results of the
Confirmed Test, to determine a percentage
of verificationfor the multiple-tube test:
Percent verification =
Completed Test Coliforms per 100 ml
Confirmed Test Coliforms per 100 ml 1
Example: On a given sample, the test
was carried to the Completed Test
Stage. Afterward, both a Confirmed
Test and a Completed Test coliform
result were obtained, consisting of
19-5
-------
Verified Membrane Filter Tests
Table 1. VALIDITY OF MF AND MPN "CONFIRMED TEST"*

Number
MF Coliform Test

MPN Confirmed Test
Source
of
supplies
Minimum
Maximum
Percent
verified
Minimum
Maximum
Percent
verified
Wells - Springs
16
1.0
7, 600
96. 6
7.0
11 i 000
64. 6
Lakes - Lagoons
23
1.0
420,000-
79. 6
79
490,000
-0
o
CO
Creeks
19
32
260,000
75. 8
120
4G0,000
66. 4
Rivers
22
320
890,000
69. 7
700
350,000
75. 7
Sewage
11
1,400,000
28,000,000
68. 6
460,000
49,000,000
73. 8
T otals
91

78. 1

70. 3
*A11 coliform values are per 100 ml of sample
49, 000 per 100 ml for the Confirmed
Test and 33,000 per 100 ml for the
Completed Test.
Percent verification =
33,000
49,000
67
X 100
See Table 1 for some studies of MF
verification studies, and parallel
multiple-tube verification studies
(Confirmed Test carried to Completed
Test). These studies have been con-
ducted in research laboratories of this
Center, and demonstrate the difficulty
and problems associated comparative
evaluation of membrane filter versus
multiple-tube methods. The student is
invited to study this table at leisure.
REFERENCES
1 Delaney, J. E. , McCarthy, J. A. and
Grasso, R.J. Measurement of E^ coli
Type I by the Membrane Filter. Water
and Sewage Works, 109, 289-294. 1962.
2 Geldreich, E. E. , Clark, H.F., Huff,
C. B. and Best, L.C. Fecal-Coliform-
Organism Medium for the Membrane
Filter Technique. JAWWA 57, 208-
214. 1965.
Geldreich, E. E. , Jeter, H. L. 'and Winter,
J. A. Technical Considerations in
Applying the Membrane Filter Pro-
cedures. In Press 1966.
Hoffman, D. A., Kuhns, J.H., Stewart,
R.C. and Crossley, E.l. A Comparison
of Membrane Filter Counts and Most
Probable Numbers of Coliform in San
Diego's Sewage and Receiving Waters.
JWPCF 36, 109-1 17. 1964.
McCarthy, J. A., Delaney, J. E. and
Grasso, R.J. Measuring Coliforms
in Water. Water and Sewage Works,
108, 238-243 1961.
Thomas, H.A., Jr. and Woodward, R. L.
Estimation of Coliform Density by the
Membrane Filter and the Fermentation
Tube Methods. American Journal
Public Health .45, 1431-1437. 1955.
.This outline was prepared by H. L. Jeter,
Director, National Training Center, and
A. G. Jose, Former Microbiologist,
FWQA Training Activities, SEC.
19-6
-------
DETERMINING ACCEPTABILITY OF MEMBRANE FILTER METHODS
IN WATER QUALITY TESTS
I INTRODUCTION
A Historical
The membrane filter technique was
presented in the 10th Edition of "Standard
Methods for the Examination of Water and
Waste Water" in 1955, as a tentative
method. Five years later, the method
became an accepted standard procedure,
with the 11th Edition. However, the
approval was limited by a proviso that
the membrane filter method should be
used for determining the potability of
drinking waters only after parallel testing
had shown that it afforded information
equivalent to that given by the standard
multiple-tube test. Presumably it was
intended that similar tests should be
extended in application of membrane
filter methods to bacterial tests on non-
potable waters.
B Factors in Acceptance of Membrane
Filter Methods
1 The decision to accept membrane filter
methods for determining bacterial
quality of water may be based on one
or a combination of the following:
a Applicability to the water samples
tested
b Availability and quality of special
laboratory equipment and supplies
c Technical skill of laboratory workers
d Convenience, i.e., speed of obtaining
results
e Economic factors
f Administrative attitudes
2 The demonstration of equivalent inter-
pretation of bacterial quality of water,
stipulated as a qualifying proviso of
acceptance in Standard Methods,
includes 1, a-c. On the other
hand, rejection of membrane filter
methods on the basis of noncompliance
with the limiting proviso of Standard
Methods may be due to any one of these
three basic causes with two of the three
representing rather easily corrected
deficiencies in the laboratory. Carried
to a logical conclusion, much of the
determination of "acceptability" of
membrane filter methods is, in fact,
an evaluation of the "fitness" of the
laboratory to perform the testing
procedure. This may be quite a
different matter from the inherent
applicability pf the method. Workers
seeking to evaluate the suitability of
membrane filter methods for their
own water testing programs should
undertake evaluation studies with great
care, to assure that the testing program
actually defines the factors tested.
C Scope of This Discussion
1 On the basis of the above comments,
it is worthwhile to consider some
significant factors which may influence
decisions relating to use of membrane
filter methods.
2 In addition, the discussion includes
program currently used by the State
of Ohio in certifying water treatment
quality control laboratories to use
membrane filter methods.
W. BA. mem. 84a .1.71
20- 1
-------
Determining Acceptability of Membrane Filter Methods
II MEMBRANE FILTER ACCEPTANCE
FACTORS
A Applicability to the Water Samples Tested
1 This can be determined only after
items 2, and 3, (following) have been
established at acceptable standards.
2 The validity of the multiple tube
method used in comparisons should
be established.
a There is a tendency to apply the
Confirmed Test as the standard of
comparison with results obtained
from membrane filter tests, without
demonstrating that the Confirmed
Test results are, in fact, valid.
b See Table 1. (Reference no. 4).
The table demonstrates discrepancies
between results obtained from
Confirmed Test vs the Completed
Test in a 'range of sample types
studied in the Cincinnati laboratories.
When study of the multiple tube test
shows appreciable disagreement
between Confirmed Test and Com-
pleted Test, results on the samples
tested, results from the Completed
Test should be used as the basis
of comparison with results from
membrane filter tests.
Table 1. VALIDITY OF MEMBRANE FILTER RESULTS AND OF, MULTIPLE
TUBE CONFIRMED TEST IN A SPLIT SAMPLE SERIES

Number

Average
MPN Confirmed
Average
Source
of
MF Coliform Test*
%
Coliform Test*
%

Samples
Minimum
Maximum
Verified**
Minimum
Maximum
Verified
Wells-Springs
16
1
7, 600
96. 6
7
o
o
o
64. 6
Lakes-Lagoons
23
1
420, 000
79. 6
79
490, 000
-J
o
o
Creeks
19
32
260, 000
-J
00
120
46 0, 000
66.4
Rivers
22
320
890, 000
69. 7
700
350, 000
75. 7
Sewage
11
1, 400, 000
28, 000, 000
68.6
460,000
49,000.000
73. 8
* All coliform results in count per 100 ml
** Through performance of completed test
20-2
c When individual multiple tube
results are compared with the
split-sample membrane filter
test results, the results may be
considered in agreement if the
membrane filter result falls within
the 95% confidence limits of the
bacterial count obtained'by the
parallel-test multiple tube.
There is a tendency for numerical
results from membrane filter
methods to be somewhat lower than
corresponding multiple-tube results.
This should not be surprising or
disturbing; mathematicians have
drawn attention to an inherent bias
in the computations of the Tables
of Most Probable Numbers,
estimated to range from 15 - 25%
high.
d A sufficient number of split-
sample comparisons should be
made to.establish a valid com-
parison of results. Many workers
assume that 100 split samples is
an acceptable basis of comparison,
provided that they represent
samples from all seasons of the
year.
-------
Determining Acceptability of Membrane Filter Methods
3 Numerical agreement between membrane
filter vs multiple dilution tube results
demonstrates acceptability of the mem-
brane filter method for the water samples
tested. -Lack of such agreement, when
traced to the waters tested, may be
from any of several causes, such as
high turbidity and low coliform counts,
unacceptably large numbers of non-
conform colonies and small,numbers
of coliform colonies growing on the
membrane filters, or presence of
deleterious chemicals which are
deposited in or on the filters, with
development of unreasonably low
numbers of colonies.
4 A series of verified coliform tests,
still another useful test in establishing
the validity of membrane filter methods
for the samples tested, is described
elsewhere in this-course manual.
Table 1 includes data obtained as a
result of including verified membrane
filter coliform tests in a series of
comparisons between membrane filters
vs multiple tube tests. The results of
this study suggest that results obtained
by membrane filter methods are some-
what more reliable than the results
from the Confirmed Test in the sample
series described.
B Availability and Quality of Special
Laboratory Equipment and Supplies
1 Descriptions of acceptable membrane
filter equipment, supplies, and media
are found elsewhere in this course
manual. Rigid adherence to specifications
is important in all bacteriological work,
and is vital in comparative studies of
this type.
2 Almost any single item of supply or
equipment, if misused or badly selected,
may bring about unsatisfactory results.
The following are among the most
flagrant areas of deviation from
recommended equipment and supplies:
a Use of membrane filters not meeting
recommended specifications.
b Improperly selected or prepared
culture medium.
c Lack of suitable equipment to
maintain proper levels of moisture
and recommended temperature
ranges of culture during incubation.
d Lack of recommended optical
assistance for counting colonies,
or misuse of such equipment.
e Lack of recommended illuminating
equipment for colony counts, or
misuse of recommended equipment.
C Technical Skill of Laboratory Workers
1 Membrane filter methods require
application of laboratory skills and
judgment of at least the same order
as that required for multiple-tube
tests.
2 Training is a recommended starting
point in acquisition of the required
skills. At this Training Center,
4-1/2 days of basic training includes
lectures, .demonstrations, and inten-
sive, repetitive, laboratory work under
supervision of qualified instructors.
It is recommended that this amount
of formal training should not be
appreciably reduced.
3 Extended individual practice in
membrane filter methods is rec-
ommended following a training
course. Only after such practice,
with liberal use of verified membrane
filter coliform tests to establish
reliability of colony interpretation,
should the individual undertake com-'
parisons between membrane filter vs
multiple tube tests to comply with the
limited-acceptance proviso of Standard
Methods.
D Convenience
1 The time for obtaining coliform results
is one day for membrane filter methods,
versus 2-4 days for the Confirmed
Test and up to a week for the Completed
Test.
20-3
-------
Determining Acceptability of Membrane Filter Methods
2 Space requirements for media
preparation and storage, glassware
preparation and storage, incubator
space, all are smaller with membrane
filter methods than with multiple-tube
methods.
E Economic Factors
1 Much of the convenience of membrane
filter methods, described in D above,
also covers economic implications.
2 Cost comparisons between membrane
filter methods and multiple tube methcpds
include many debatable considerations.
Personnel at this Center have tended
to regard the two methods as being
roughly equal in cost per test.
3 In a special study, workers of the
Illinois Department of Health
(McCaffrey, unpublished) reported
a saving of $0. 08 per sample with
use of membrane filter methods.
The comparison was based on more
than 30, 000 samples for each method.
F Administrative Attitudes
Decisions regarding use, or even con-
sideration of introduction of membrane
filter methods in a given laboratory may
be determined by laboratory administrative
management, without reference to any
findings within the laboratory, or even
without consultation with laboratory
personnel. Several different aspects of
this problem may develop:
1 Prejudgment without experimental data
is sometimes encountered. In candor,
it must be recognized that this is a very
real problem, and has been a significant
factor in slowness of acceptance, or
even consideration, of membrane filter
methods in some laboratories.
2 Acceptability of evidence based on
membrane filter methods in legal
procedures has concerned some
administrators. Membrane filter
methods have been used to an increasing
extent in numerous water pollution con-
trol activities of this organization.
3 Some workers have been reluctant to
convert to membrane filter methods
in water quality control investigations,
due to an obvious' contrast between the
membrane filter method and methods
used in previous investigations on the
same, or' similar, body of water based
on multiple tube methods. Often, these
workers fail to recognize that several
features of multiple tube methods today
are not identical with those in use 10
or 20 years ago. The overall acceptance
of the membrane filter method may be
just as'seriously handicapped by too-
ready an administrative acceptance of
the method without adequate equipping
or training of the laboratory and its
personnel to apply the method. In this
ca!se, the method can acquire a poor
' reputation, not due to the method itself,
but to its routine use before the neces-
sary skills and material resources
are available.
Ill State of Ohio's Procedures for Establishing
Acceptability'of Use of Membrane Filter
for Bacteriological Examination of
Drinking Water
A Background
1' The Ohio Department of Health does
accept the use of membrane filter for
bacteriological examination of potable
water.
2 In other States, the individual should
check with the regulatory agency of
the State as to acceptability; other
States may have a different position
or- conditions surrounding acceptance.
B Conditions of Acceptance
Ohio Department of Health's acceptance of
membrane filter procedure is subject to
three conditions:
1 Formal training of laboratory personnel
responsible for bacteriological
examinations
2 Laboratory survey and approval of both
equipment and procedures
20-4
-------
Determining Acceptability of Membrane Filter Methods
3 Parallel testing of raw and finished
water supplies
C Formal Training
1 This requirement is fulfilled by
successful completion of training
courses at the National Training
Center, Cincinnati, Ohio.
2 The training personnel of the Robert A.
Taft Laboratories have worked closely
with the Ohio Health Department, and
similar agencies in other states, in
providing training to meet special
requirements.
D Laboratory Survey
1 The survey is made by Ohio's main
laboratory bacteriologists.
2 The survey is made only after a period
of parallel tests of split samples using
both membrane filter and multiple tube
methods.
3 The request for survey by state
personnel must originate at the local
level.
4 The survey covers both personnel and
equipment in the laboratory.
E Parallel Testing
1 The Ohio Department of Health requires
parallel testing using the multiple tube
technique and the membrane filter
technique.
2 The test should include at least a period
of 3 months with a minimum of 100
samples of raw water and 100 samples
of finished water included in the tests.
a Practice in identifying coliform
colonies is required. Preparation
of synthetic raw water samples is
required if the raw water is free of
coliform bacteria.
b Practice in all aspects of laboratory
techniques in membrane filter tests
is required.
c The limitations of the membrane
filter must be recognized, for easy
recognition.
3 Results of parallel testing procedure
are reported on special forms designed
by the Ohio Department of Health.
4 Several precautions are presented for
those running parallel tests:
a TJse 100 ml samples for finished
waters and samples collected from
taps in the distribution system.
b Use synthetic raw water if the
natural raw water lacks coliforms.
c Clear any questions or problems
with the Central Laboratory or
Central Office personnel before the
three-month parallel testing period
is completed.
ACKNOWLEDGMENT:
This outline includes certain materials made
available to the National Training Center
by Robert S. McEwen, Engineer, Water
Supply Unit, Ohio Department of Health,
Columbus, Ohio.
REFERENCES
1 This course manual, outlines 25-32,
2 State of Ohio Department of Health.
Announcement: Acceptance of the
Membrane Filter Procedure for the
Bacteriologic Examination of Water.
1962.
3 Standard Methods for the Examination of
Water and Wastewater. 12th edition
1965. Published by APHA, AWWA,
WPCF,
4 Geldreich, E.E., Jeter, H. L. and
Winter, J.W. Technical Considerations
in Applying the Membrane Filter
Procedure, Health Lab Science
4:113-125. 1967.
20-5
-------
Determining Acceptability of Membrane Filter Methods
5 Woodward, R.L. How Probable is the
Most Probable Number? JAWWA.
49:1060. 1958.
This outline was prepared by-H, L. Jeter,
Director, National Training Center, DTTB,
MDS, WPO, EPA, Cincinnati, OH 45268.
20-6
-------
COLLECTION AND HANDLING OF SAMPLES FOR
BACTERIOLOGICAL EXAMINATION
I INTRODUCTION
The first step in the examination of a water
supply for bacteriological examination is
careful collection and handling of samples.
Information from bacteriological tests is
useful in evaluating water purification,
bacteriological potability, waste disposal,
and industrial supply. Topics covered
include: representative site selection,
frequency,'.number, size of samples,
satisfactory sample bottles, techniques of
sampling, labeling, and transport.
II SELECTION OF SAMPLING LOCATIONS
The basis for locating sampling points is
collection of representative samples.
A Take samples for potability testing from
the distribution system through taps.
Choose representative points covering
the entire system. The tap itself should
be clean and connected directly into the
system. Avoid leaky faucets because of
the danger of washing in extraneous
bacteria. Wells with pumps may be
considered similar to distribution systems
B Grab samples from streams are frequently
collected for control data or application of
regulatory requirements^ A grab sample
can be taken in the stream near the surface.
C For intensive stream studies on source
and extent of pollution, representative
samples are taken by considering site,
method and time of sampling. The
sampling sites may be a compromise
between physical limitations of the
laboratory, detection of pollution peaks,
and frequency of sample collection in
certain types of surveys. First, decide
how many samples are needed to be
•processed in a day. Second, decide
whether to" measure cycles of immediate
pollution or more average pollution.
Sites for measuring cyclic pollution are
immediately below the pollution source.
Sampling is frequent, for example, every
three hours.
A site designed to measure more ayerage
conditions is far enough downstream for
a complete mixing of pollution and water.
Keep in mind that averaging does not
remove all variation but only minimizes
sharp fluctuations. Downstream sites
sampling may not need to be so frequent
Samples maybe collected 1/4, 1/2 and
3/4 of the stream width at each site or
other distances, depending on survey
objectives. Often only one sample in the
channel of the stream is collected.
Samples are usually taken near the surface
D Samples from lakes or reservoirs are
frequently collected at the drawoff and
usually about the same depth and may be
collected over this entire surface
E Collect samples of bathing beach water
at locations and times where the most
bathers swim.
Ill NUMBER, FREQUENCY AND SIZE
OF SAMPLES
A For determining sampling frequency for
drinking water, consult the USPHS
Standards.
1 The total number, frequency, and site
are established by agreement with
either state of PHS authorities.
2 The minimum number depends upon the
number of users. Figure 1 indicates
that the smaller populations call for
relatively more samples than larger
ones. The numbers on the left of the
graph refer to actual users and not the
population shown by census
3 In the event that coliform limits of the
standard are exceeded, daily samples
must be taken at the same site.
Examinations should continue until two
consecutive samples show coliform
level is satisfactory. Such samples
are to be considered as special samples
and shall not be.included in the total
number of samples examined.
4 Sampling programs described above
represent a minimum number which
may be increased by reviewing
authority,
W. BA. sa. Id. 1. 73
21-1
-------
Collection and Handling of Samples for Bacteriological Examination
B For stream investigations the type of
study governs frequency of sampling.
C Collect swimming pool samples when use
is heavy. The high chlorine level rapidly
reduces the count when the pool is not in
use. Residual chlorine tests are
necessary to check neutralization of
chlorine in the sample.
D Lake beaches may be sampled as required
depending on the water uses
tn
~
Z
<
to
ZD
o
x
b-
z
Q
UJ
>
OC
UJ
in
Z
o
Q_
o
Q_
MINIMUM NUMBER OF SAMPLES PER MONTH
1 10 100 1000
100
1000
10,000
FIGURE 1
21-2
-------
Collection and Handling of Samples for Bacteriological Examination
E Salt water or estuarine beaches are
sampled as needed with frequency
depending on use.
F Size of samples depends upon examination
anticipated. Generally 100 ml is the
minimum size.
IV BOTTLES FOR WATER SAMPLES
A The sample bottles should have capacity
for at least 100 ml of sample, plus an
air space. The bottle and cap must be of
bacteriological inert materials. Resistant
glass or heat resistant plastic are
acceptable. At the National Training
Center, wide mouth ground-glass
stoppered bottles (Figure 2) are used.
All bottles must be properly washed and
sterilized. Protect the top of the bottles
and cap from contamination by paper or
metal foil hoods. Both glass and heat
FIGURE 2
¦resistant plastic bottles may be'
sterilized in an autoclave. Hold plastic
at 121°C for at least 10 minutes. Hot
air sterilization, 1 hour at 170°C, may
be used for glass bottles.
B Add sodium thiosulfate to bottles intended
for halogenated water samples. A quantity
of 0. 1 ml of a 10% solution provides 100
m'g per liter concentration in a 100 ml
sample. This level shows no effect upon
viability or growth.
C Supply catalogs list wide mouth ground
glass stoppered bottles of borosilicate
resistance glass, specially for water
samples.
V TECHNIQUE OF SAMPLE COLLECTION
Follow aseptic technique as nearly as
possible. Nothing but sample water must
touch the inside of the bottle or cap. To
avoid loss of sodium thiosulfate, fill the
bottle directly and do not rinse. Always
remember to leave an air space.
A In sampling from a distribution system,
first run the faucet wide open until the
service line is cleared. A time of 3-5
minutes generally is sufficient. Reduce
the flow and fill the sample bottle without
splashing. Some authorities stress
flaming the tap before collection. A
chlorine determination is often made on
the site.
B The bottle may be dipped into some waters
by hand. Avoid introduction of bacteria
from the human hand and from surface
debris. Some suggestions follow:
Hold the bottle near the base with one
hand and with the other remove the hood
and cap. Push the bottle rapidly into the
water mouth down and tilt up towards the
current to fill. A depth of about 6 inches
is satisfactory. When there is no current
move the bottle through the water
horizontally and away from the hand. Lift
the bottle from the water, spill a small
amount of sample to provide an air space,
and return the uncontaminated cap.
21-3
-------
Collection and Handling of Samples for Bacteriological Examination
C Samples may be dipped from swimming
pools. Determine residual chlorine on
the pool water at the site. Test the
sample at the laboratory to check chlorine
neutralization by the thiosulfate.
D Sample bathing beach water by wading out
to the two foot depth and dipping the
sample up from about 6 inches below the
surface. Use the procedure described in
V. B.
E Wells with pumps are similar to
distribution systems. With a hand pumped
well, waste water for about five minutes
before taking the sample. Sample a well
without a pump by lowering a sterile
bottle attached to a weight. A device which
opens the bottle underneath the water
will avoid contamination by surface debris.
F Various types of sampling devices are
available where the sample point is
inaccessible or depth samples are desired.
The general problem is to put a sample
bottle in place, open it, close it, and
return it to the surface. No bacteria but
those in the sample must enter the bottle.
1 The J - Z sampler described by Zobell
in 1941, was designed for deep sea
sampling but is useful elsewhere (Figure)
3). It has a metal frame, breaking
device for a glass tube, and sample
bottle. The heavy metal messenger
strikes the lever arm which breaks
the glass tubing at a file mark. A
bent rubber tube straightens and the
water is drawn in several inches from
the apparatus. Either glass or collapsible
rubber bottles are sample containers.
Commercial adaptations are available.
2 Note the vane and lever mechanism on
the New York State Conservation
Department's sampler in Figure 4.
When the apparatus is at proper depth
the suspending line is given a sharp
pull. Water inertia against the vane
raises the stopper and water pours
into the bottle. Sufficient sample is
collected prior to the detachment of
the stopper from the vane arm allowing
a closure of the sample bottle.
The New York State Conservation
Department's sampler is useful for
shallow depths and requires nothing
besides glass stoppered sample bottles.
FIGURE 3
Reproduced with permission of the Journal
of Marine Research 4:3, 173-188 (1941) by
the Department of Health, Education and
Welfare.
21-4
-------
Collection and Handling of Samples for Bacteriological Examination
FIGURE 4
B While a sanitary survey is an indispensable
part of the evaluation: of a water supply, its
discussion is not within the scope of this
lecture. The sample collector, could supply
much information if desired
VII SHIPPING CONDITIONS
A The examination should commence as .soon
as possible, preferably within one, hour
A maximum elapsed time between collection
and examination is 3 0 hours for potable
water samples and 8 hours for other
water samples (collection 6 hours and
laboratory procedures 2 hours).
Standard Methods (13th Edition)
recommends icing of samples between
collection and testing.
VIII PHOTOGRAPHS
A photograph is a sample in that it is evidence
representing water quality. Sample collectors
and field engineers may carry cameras to
record what they see. Pictures help the
general public and legal courts to better
understand laboratory data.
A commercial sampler is available
which is an evacuated sealed tube with
a capillary tip. When a lever on the
support rack breaks the tip, the tube
fills. Other samplers exist with a
lever for pulling the stopper, while
another uses an electromagnet.
VI DATA RECORDING
A Information generally includes: date, time
of collection, temperature of water, location
of sampling point, and name of the sample
collector. .Codes are often used. The
location description must be exact enough
to guide another person to the site.
Reference to bridges, roads, distance to
the nearest town may help. Use of the
surveyors' description and maps are
recommended. Mark identification on the
bottles or on securely fastened tags.
Gummed tags may soak off and are
inadvisable.
REFERENCES
1 APHA, AWWA, WPCF, Standard Methods
for the Examination of Water and
Wastewater. (12 Ed. ) 1965.
2 'Prescott, S. C., Winslow, C. E.A., and
McCrady, M. H. Water Bacteriology.
6th Ed. , 368 pp. John Wiley and Sons,
Inc., New York. 1946.
3 Haney, P. D. , and Schmidt, J.
Representative Sampling and Analytical
Methods in Stream Studies. Oxygen
Relationships in Streams, Technical
Report W58-2 pp. 133-42. U. S.
Department of Health, Education and
Welfare, Public Health Service, Robert
A. Taft Sanitary Engineering Center,
Cincinnati, Ohio. 1958.
Velz, C. J. Sampling for Effective
Evaluation of Stream Pollution. Sewage
and Industrial Wastes, 22:666-84. 1950.
21-5
-------
Collection and Handling of Samples for Bacteriological Examination
5 Bathing Water Quality and Health III
Coastal Water. 134 pp. U. S
Department of Health, Education, and
Welfare, Public Health Service, Robert
A. Taft Sanitary Engineering,
Cincinnati, Ohio. 1961.
6 Zobell, C. E. Apparatus for Collecting
Water Samples from Different Depths
for Bacteriological Analysis. Journal
of Marine Research, 4:3:173-88. 1941
This outline was originally prepared by
A. G. Jose, former Microbiologist FWPCA
Training Activities, SEC and updated by
the Training Staff, National Training Center,
DTTB, MDS, WPO, EPA, Cincinnati, OH
45268.
Descriptors: Bacteria, Sample, Sampling,
Water Sampling, Handling, Preservation,
Samplers, Surface Waters, Distribution
Systems
21-6
-------
RECOVERY AND IDENTIFICATION OF SALMONELLA AND SHIGELLA
FROM ENVIRONMENTAL WATERS
I INTRODUCTION
A Significant Factors Relating to Enteric
Diseases
1 Salmonellosis (species of Salmonella
other than S. typhi). An illness
characterized by the usual inception of
gastroenteritis which can further manifest
itself in an enteric fever and/or septicemi;
The usual incubation period if from 6 to
48 hours and more commonly symptoms
appear in about 12 hours. A period of
communicability can occur within the
intervals of 3 days to 3 weeks but a per-
manent carrier condition can occur in
a small percentage of cases.
2 Typhoid Fever (Salmonella typhi)
This representative of the Salmonella
genus can have a 10% mortality rate
unless immediate treatment is instituted
wherein a 2-3% rate is usual. The
organism is found in the blood during the
first two weeks and in the feces and
urine after the initial two weeks of
malady. About 50 types have been
identified by means of the Vi phage.
The usual incubation-period varies from
1 to 3 week's. The permanent carrier
rate is from 2-5% and as much as 10%
of the patients excrete the bacilli for a
period of three months.
3 Shigellosis (various species of genus
Shigella)
An acute infection of the intestine
characterized by. stools of blood,
mucous, and pus in the severe cases.
The usual incubation period is from
1 to 7 days but the usual case ocurring
in 4 days. A few individuals become
carriers for a year or two but rarely •
longer,
B Relative Incidence
1 Almost exclusive transmittance of these
enteric diseases is by the Water-Food-
Milk route as shown in figure 1.
2 Increased numbers of outbreaks have
been attributed to Salmonellosis within
recent years but an apparent disparity
exists between reported disease and
population of reporting areas. This
data is shown in figures 2 and 3.
3 Reporting of etiological agents
responsible for outbreaks has been
undetermined in a significant number of
cases. Figure 4 illustrates this for the
reporting period 1952 to 1959.
C Increased Interest in Pathogen Isolations
1 Increased desire to obtain more com-
plete knowledge of state of water
sample.
2 Development and augmentation of media
for pathogenic analysis.
3 Analysis in special areas where coli-
form isolations yield low counts.
4 Areas where pathogen analysis and
recoveries can relate pollution sources
from specific a'reas.
5 Passage and implementation of Public
Law 660 one section of which calls for
the abatement of pollution of interstate
or navigable waters which endangers
the health or welfare of any persons.
D Development of Isolation Technique
1 Early interest in the detection of
Salmonella typhi and Shigella Species
due to explosive outbreaks mainly by
the water route.
2 Development of serological techniques
which have aided in the detection and
identification of pathogens as well as
providing an impetus in taxonomic
developments.
W. DI. 2a. 1. 73
22-1
-------
Recovery and Identification of Salmonella and Shigella from Environmental Waters
320 r
230 -
240
£ 200 ¦
<
UJ
ttr 160 -
at '
i r
'•:v ttuta »» 4k »cu
"i r
l»C*r» • n»e
FOOO
(OTHER THAN MiLK PRODUCTS)
FIGURE 1
FOOV.MlLK.fiN0 WATERBORNE P/SEASE
OUTBREAKS REPORTED IN U.S.A. 1932-59
JVAT^R
MILK a OAIRY
PRODUCTS
l*Sl 1956
YEARS
FIGURE 2
**Mr mon *||*od Imr
Source MUWK Annual S^lr
i. (991. «6l.ftS<3
REPORTED INCIDENCE OF HUMAN SALMONELLOSIS
UNITED STATES. 1942-1963
*6 '48 "50 "52
YEAfl
CASES IN POPULATION
OUTBREAKS (MILLIONS)
139
45
27
1 1 1 1 1 1 1 ( 1 1 ( (
n
" *{* !C*Vt» **0 6*H0» »U|LIC HfltTti At^5*T| lltot
n
i,ro9
153
196
26
ra
1,223
166
a
1.502
95
r73
494
10 1
Z3
IIS
1 7
n
309
1 0
=3
342
4 9

769
7 2
zn
l 25
40
T \l 'J
696
25 6
ZJ'STjC;
2.1 19
204

795
349

0
24 2
i i i i i i i i i i ii
10,594 TOTAL
170 2
FIGURE 3
1>1S?A?JT y BETWEEN
REPORTEV FOGVBOME DISEASE
ANT>
^OVULATION Of REVOLTING AREAS —/959
0 2 4 6 0 10 (2
NUMBER OF STATES a DC REPORTING
now gl «i, "Ulth
FIGURE k
types of roovsoRHt must outbreaks
KfOWCt m U.S.A. 1952-5}
/
/
/ J
/
2 ,J°r
IE
/
STAPHYLOCOCCUS /
/
'./
UNDETERMINED
_ ETIOLOGY ^
/•
S SALMONELLA 8 SHIGELLA
I9SS I9M
YEARS
I99Q 1959
22-2
-------
Recovery and Identification of Salmonella and Shigella from Environmental Waters
3 Increased knowledge of the physiology
and nature,of enteric pathogens in the
following areas:
a Preservation and transport of fecal
and contaminated samples
b Enrichment procedures
c Selective procedures providing enhanced
recoveries .
d Methodology of sample concentration
of microbial flora
II RECOVERY AND IDENTIFICATION
A Procedural Sequence - Description
1 Concentration
In most cases it is necessary to provide
a relatively large volume of sample in
order to isolate pathogenic organisms
from the water sample. This is due to
the relatively small numbers of pathogens
in relation to the indicator organisms of
pollution since contribution of these
pathogens is made only by frank cases,
subclinical infections, and carriers and
even by these contributors the emissions
may be sporadic and always in smaller
¦numbers than the coliform and fecal
streptococcal organisms.
2 Enrichment
In order to provide for an optimal
opportunity of isolation it is necessary,
in most cases, to incorporate an
enrichment stage within the procedural
sequence. This is generally done by the
use of broths which are intended to
provide optimal growth conditions for
the enteric pathogens and, to some
extent, suppress the coliform and non-
pathogenic organisms.
3 Selection
Selectivity procedures separates the
pathogens from the surviving non-
pathogens, usually upon selective agar
plates, and from their, visual growth
characteristics can provide a culture
which is a presumptive pathogen to be
further scrutinized by biochemical
procedures.
4 Biochemical
Cultures isolated during the selective
procedures are further subjected to
biochemical tests to provide cultures
which can be identified as specific
pathogens. Many non-pathogenic enteric
organisms can resemble enteric patho-
gens upon selective plates and thus it
is usually necessary to provide both
selection and biochemical testing in
order to significantly reduce the numbers
of organisms which are carried to the
identification procedures which are the
serological procedures.
5 Identification
a This is a serological procedure which
identifies the more than 1, 000
serotypes of Salmonella and the more
than 30 serotypes of Shigella.
Serological identification depends
upon the antigenic pattern exhibited
by the somatic and flagellar (if
motile) components of the cell.
Identification procedures can also
include bacteriophage typing
technique.
B Procedural Sequence - Methodology
1 Concentration
a Moore Swab Technique
This technique involves the immersion
of gauze pads for varying periods
whereby passage of water in the
sample area acts to concentrate
particulate matter and microbial
flora. In most cases this technique
is used to obtain indications of
pathogens without regard to a
quantitative estimate of numbers of
species present in'a given volume.
Recently, however, a methodology
has been developed wherein a counter
22-3
-------
Recovery and Identification of Salmonella and Shigella from Environmental Waters
registers flow-through of environ-
mental waters and thereby gives
quantitative estimation. This
technique has found increased use
in estuarine and tidal waters where
organism concentration can occur
over several tidal cycles.
b Diatomaceous Earth Technique
This is a procedure whereby the
high filtration capability of
diatomaceous earth is used to con-
centrate a relatively large volume
of microbial flora. This method
has the advantage of allowing a
filtration of a known volume of water
and the quantitation of species
isolated to volume filtered can be
documented.
c Membrane Filtration
Membrane filtration is a technique
of limited use since large volumes
of sample may mechanically clog
pore surfaces and prevent filtration.
This method is useful for waters of
very low organic and particulate
matter content. Again a value can
be found for species isolated to
volume filtered if the membrane is
totally immersed in the enrichment
broth.
2 Enrichment
a Selenite Broth
This is an enrichment broth which
has the advantage of inhibiting the
nonpathogenic colon bacilli for the
first 8-12 hours of incubation while
allowing the salmonellae, particularly
_S. typhi, to multiply fairly rapidly.
b Tetrathionate Broth
This is an enrichment broth which,
again, restricts the coliform orga-
nisms and allows the salmonellae
to flourish.
While the two broths listed above
are the most generally used enrich-
ment broths, it must be mentioned
that a gram negative broth (GN Broth)
can be utilized as both a carrying
medium as well as an enrichment
broth combination. Some investi-
gators have noted goodre'sults from
the use of a non-inhibitory broth,
such as lauryl tryptose broth,
followed by polyvalent "0"antiserum
addition after incubation, centrifu-
gation of the broth, and streaking
the sediment on the primary plating
media. A multitude of modifications
have been made to the above enrich-
ment broths such as the addition of
various antibiotics, bile, brilliant
green, various amino acids, etc.,
and use of these additives should be
made only after investigation of the
literature to ascertain the reasons
and advantages.
c Traditionally, 'the temperature of
incubation for the enteric pathogens
has been 370C - that temperature
which has been considered optimal
for growth, and therefore, recovery.
Within recent years, however, the
utilization of higher temperatures
for enrichment and selection have
given indications of increased
recoveries due to the suppression
of the nonpathogens and a decrease
in the lag phase of the pathogens
upon inoculation.
3 Selection
a Brilliant Green Agar
Typical salmonellae on Brilliant
Green agar form a pinkish-white
colony with a red background when
isolation has occurred. Nonpathogenic
and lactose fermenters usually form
greenish colonial forms or may, at
times, produce other colorations.
This medium should be incubated a
full 48 hours, after a-24 hour
observation and picking of typical
colonial forms, to allow late or
partially inhibited forms to attain
macroscopic size. If no typical
forms are observed or the plate is
22-4
-------
Recovery and Identification of Salmonella and Shigella from Environmental Waters
crowded it would be well to pick a few
colonies from the plate and process
them through the biochemical schema
since it is possible that typical
colorations canbe masked by other
more numerous bordering colonies.
b SS Agar
On this medium typical colonies of
Salmonella and Shigella are colorless
to opaque and, in the case of H^S
producers, may show a blackening
within the "eye" of the colony.
Typical lactose fermenters show
various colorations which can vary
from off-pinks to deep reds. Other
non-pathogenic colonial forms may
show yellow hues. This selective
plate is, in general, more productive
in numbers of non-pathogens compared
to Brilliant Green agar and Bismuth
Sulfite agar but is a good plate to run
in conjunction with the Brilliant
Green agar plate since it allows S.
typhi and shigellae to grow which
may rot be the case with Brilliant
Green agar which is definitely toxic
to S. typhi.
c Bismuth Sulfite Agar
This medium is reputed to be the best
available medium for the isolation of
S. typhi. Luxurient growth of the
salmonellae can be expected upon
this medium and typical forms usually
have a blackening and/or metallic
sheen of which the blackening may
extend beyond the colony to give a
"halo" effect. A few of the salmonellae
may produce green colorations and
thus it would be wise to pick some of
these representatives from each
plate in addition to the typical forms
as mentioned above.
d EMB Agar
This medium is almost completely
uninhibitory to the pathogenic forms
as well as to the non-pathogens and
thus it can be seen that, in the plate
streaking process, less inoculum
should be applied to the plates or
more loop flaming intervals practiced
to obtain desired isolation. Usually
a 24 hour incubation limit is the
best that can be .expected due to over-
growth with continued incubation.
Upon this medium, the Proteus
organisms are likely to produce their
characteristic "swarming" phenomenon
and thus further reduce the isolation
capability. Typical pathogens are
colorless and can produce a "dark
eye" usually blue or purple. Typical
non-pathogens and lactose fermenters
can produce varicolored forms and
some of the coliforms can produce a
metallic sheen. , Many investigators
recommend the utilization of this
plate, despite its apparent dis-
advantages, due to the fact that it
can allow .some of the more fastidious
species of. the Salmonella and Shigella
to multiply.
In addition to the above more commonly
used media, ithas been the practice
of some investigators to utilize the
following media for primary isolation:
MacConkey Agar
ENDO Agar
Desoxycholate Citrate Agar
BCP-D Agar (Brom Cresol
Purple Desoxycholate Agar)
Again, as in the case of the enrich-
ment broths, various additives such
as antibiotics, dyes, etc. , have been
added to the standard formulation of
the various media and a .literature
review must again be necessary to
ascertain the advantages and situa-
tions where applied before utilizing
these altered formulations.
4 Biochemical Tests
At this point the pure cultures obtained
from the selective plates may number
from several to hundreds depending
upon the number of samples being
processed and the numbers of character-
istic colonial forms exhibited by the
primary isolation media. If the numbers
22-5
-------
Recovery and Identification of Salmonella and Shigella from Environmental Waters
of cultures are relatively small and the
need for prompt confirmations are
necessary the whole battery of bio-
chemical tests can be applied at once
and those showing characteristic patterns
for the Salmonella-Shigella groups can
be immediately subjected to the serol-
ogical tests and, if necessary, prompt
mailing of cultures to the Diagnostic
Centers can immediately follow.
Usually the numbers of cultures obtained
from the primary isolation media will
be numerous and the need of rapid
results is not the case and for this
situation it is recommended that a
sequential pattern of biochemical testing
be followed which will result in a great
saving of media and valuable bench
time for the laboratory personnel. If
the latter case is the prevailing situation
the following pattern of biochemical
tests are recommended:
a 1st Biochemical
Urea Agar or Urea Broth
Urea positive cultures should be
immediately discarded as indicative
of the Proteus group or other non-
pathogenic forms and the urea negative
cultures subjected to the 2nd biochemical
series. Although the Urea broth or
agar should be incubated for the
recommended time periods it will
be generally found that the 24 hour
period will be sufficient to indicate
the majority of the positive cultures
and, at this point, the cultures
which are negative should be further
processed,but should be further
incubated as the second series is
being processed.
b- 2nd Biochemical
Media
Lactose Broth
Purpose of Test
Fermentation Capability
Saccharose Broth Jt ermentation Capability
Salicin Broth
KCN Broth
SIM
Raffinose Broth
Fermentation Capability
Growth Capability in
presence of CN group
Production of Indol,
Motility, H^S Production
Fermentation Capability
Media
Purpose of Test
Decarboxylase Media Presence or absence of
enzyme system
Citrate
TSI
22-6
Utilization of Citrate
as carbon source
Fermentation pattern;
HgS production
Adherence to the biochemical patterns
established for the Salmonella -
Shigella groups will decide if the
cultures are to be further processed
to the 3rd biochemical series. The
point should be mentioned that at
times aberrant culture's will be
encountered and 'as such they will not
satisfy all of the classical reactions
attributed to occur to' each of the
pathogenic groups."' In all cases,
therefore, it will be necessary to
review all of the reactions as a
whole and not to discard cultures on
the basis of a small number of
apparent discontinuities.
c 3rd Biochemical
Dextrose Broth, Mannitol Broth,
Maltose Broth, Dulcitol Broth,
Xylose Broth, Rhamnose Broth,
Inositol Broth
This series of biochemical tests is
to establish further patterns of
fermentation capabilities of the
isolates. This series of tests is
indicated only to reduce the possible
number of positive cultures that are
sent for confirmation. For example,
if one isolates 15 positive cultures of
Salmonella group C and in completing
the 3rd Biochemical series it is found
that, on the basis of comparing
fermentative reactions, the cultures
can be roughly separated into 3
differing patterns,it is more likely
than not that in sending these three
"differing" cultures for confirmation
-------
Recovery and Identification of Salmonella and Shigella from Environmental Waters
they will confirm as three different
species within the C1 grouping. If
the testing laboratory is equipped for
flagellar analysis the elimination of
the 3rd biochemical series can be
eliminated as complete confirmation
procedures can be completed for all
of the isolates.
5 Identification
a Somatic Antigen Identification
In the serological examination of
presumptive Salmonella cultures the
somatic or "O antigens are identified
first and this is accomplished by using
a dense suspension of a fresh culture
in physiological saline and performing
a slide agglutination test first with the
polyvalent "O" antiserum and then with
each representative of the groups.
Interferences may be present due to
the presence of the Vi antigen which
is found only in a limited number of
salmonellae (including S. typhi) and
in a number of the non-pathogenic
organisms. If a positive is indicated
with the Vi antiserum, it can be
eliminated from the salmonellae
representatives by a boiling process
and the somatic grouping procedure=
resumed' for group identification.
Serological identification of the
shigellae follows the same pattern as
that of the salmonellae" somatic
analysis and it is not necessary to do
a flagellar grouping since all species
of the Shigella are non-motile.
b Flagellar Antigen Identification
Although the slide test is used for this
analysis by some laboratories, the
tube test is' recommended to eliminate
cross-reactivity. Fresh motile
cultures (there is a small number of
non-motile salmonellae) are subjected
to the tube test with specific antigens
which occur within the positive "O"
groupings previously identified and
thus a representative within these
groups can be identified as to a
specific species. Since the
salmonellae usually contain two
distinct phases of flagellar antigens
it may be necessary to isolate the
other phase than that identified in
order to obtain complete identification.
This is accomplished by the use of
semisolid medium wherein one phase
is held back by the appropriate
antiserum while the yet unidentified
phase.is allowed to "ray out1! from
the line of inoculation to be isolated"
and identified.
Ill FUTURE DEVELOPMENTS
Increased emphasis upon the recoveries of
pathogenic microorganisms from environ-
mental waters will provide an impetus in the
areas of improved utilization of current
techniques and the development of more
rapid methodolo.gies. Some of the areas, in
which these developments-are expected to
occur.are:
A Increased understanding of the physiology
of these pathogens and from this-such
developments as the recent indications
that elevated temperature, techniques
(Spino, 1966) can improve recoveries.
B Continued development of serological
procedures and the possible use of such
techniques as the fluorescent antibody
methodology to provide a rapid appraisal
of environmental waters.
C Increased capability of typing by the use
of bacteriophages for species other than
S. typhi.
22-7
-------
Recovery and Identification of Salmonella and Shigella from Environmental Waters
GROUP DIFFERENTIATION OF ENTEROBACTERIACEAE BY BIOCHEMICAL TESTS
| 1st BIOCHEMICAL I
POSITIVES OBTAINED ONLY WITH
PROTEUS-PROVIDENCE GROUP
UREA AGAR
2nd BIOCHEMICAL |
Shlgel la - Esc he r Ichia
Salmonella-
Arl2ona-Cltrob*cter

Shigella
Croup
Escherichia
Croup
Salmonella
Group
Arizona
Croup
Cttrobacrer
Groups

-
d
+
+
-
LYSINE DECARBOXYLASE
¦ •' (+)
d
(+)
(+)
(+)
ARCININE DDtYDRQLASE
.•
d
+
+
d.
ORNITHINE DECARBOXYL ASE
i
+
-
.
-
INDOL

d
+
+
+
K40TILITY
.
-
+
+
' +
llfS (TSI AGAR)
•
- 1 -
- 1 +
.KCN
-
- 1 +
+
+
SIMMONS' CITRATE
_•
~ - x| -
+ or *
+ X
LACfOSE
_•
d 1 -
*
d
SUCROSE
-
d 1 -
| d
SALlCtN
d
d
"
-
d
RAFFWOSE
3rd RIOCHEMICAL
OTHER INFORMATION
ShlgclU'EachertchU
Salmonella - Arlxona • CUroUctc r

Shigella
Croup
Escherichia
Croup
Salmonella
Croup
Arizona
Croup
Citrotactcr
Group*1*

_ •
+
+
+
+
CA*> from C1JJC06E
-
-
d
-
- "t x
INOSITOL
d
d
+
+
+
SORBITOL
d
+
+ <"
+
+
AftABlNOSF
d
d
+
+
+
fUiAMNOSE
d
+
+
+
+
MANNITOL
d
d
+ («
-
d
nuLcrroL

Shigella-Escherichia
Salmooeila -
Arlzona-CUrotecter 1

Shigella
Croup
Escherichia
Croup
Salmonella
Croup
ArUona
Croup
CUrobaccer,
Croup*1* i
GELATW (2l*c.>
-

-
(+)
- '1
Organic Acids

D-tartrate
I.-tartrate
I -tartrate
Citrate
Mucate
(1) FORMERLY ESCHERICHIA FREUNDII
(2) SCME SPECIES DO NOT FERMENT DULCITOL PROMPTLY
S.CHOLERAE SUIS DOES NOT FERMENT ARABINOSE
d DIFFERING BIOCHEMICAL TYPES
X LATE OR IRREGULARLY POSITIVE
(+) DELAYED POSITIVE
+ POSITIVE - NEGATIVE
CERTAIN BIOTYPES PRODUCE GAS
S. SONNEI FERMENTS LACTOSE & SUCROSE St DECARBOXYLATES ORNITHINE
22-8
-------
CO
to
I
CO
WATER SAMPLE
POSITIVE TEST
(DISCARD)

«y w Vi
LYSINE ARCININE ORNI fHINE
blHYLmOLASE
^ \l| \fl \l
Vi/
SALIC (N
RAFFINOSE
DECARBOXYLASE
DISCARD FRANK
NON-PATHOGENS
CAS from CLUCOSE
SORBITOL ARABINOit" RHAMNOSE MANNITOL IXjLCIIOL
OTHER TESTS
Mi
GELATIN
a e
W u/ lv
Organic Acids
REFRIGERATE
DIAGNOSTIC CENTER
CONFIRMATION *
po
0>
o
o
<
o
JD
3
a
>—i
CL
0
D
O
3
C/3
&3
3
o
3
Q
D
a
in
2T
CTQ
fD
1
O
3
t=3
D
<
i—'•
O
3
fD
3
ft)
•"S
W
-------
Recovery and Identification of Salmonella and Shigella from Environmental Waters
REFERENCES
1 Brezenski, F.T., Russomanno, R., and
DeFalco, P., Jr. The Occurrence of
Salmonella and Shigella in Post-
Chlorinated and Non-Chlorinated Sewage
Effluents and Receiving Waters.
Health Lab. Sci. 2, 1, 40. 1965.
2 Brezenski, F.T., and Russomanno, R.
The Detection and Use of Salmonellae
in Studying Polluted Tidal Estuaries.
Jour. WPCF 41, 5, Part 2. 1969.
3 Edwards, P. R. ,and Ewing, W. H.
Identification of Enterbacteriaceae.
Burgess Publishing Co., Minneapolis,
MN. 2nd Edition. 1962.
4 Harvey, R.W.S., and Thompson, S.
Optimum Temperature of Incubation
for Isolation of Salmonellae'. Min.
Health Lab- Serv. 12:149-150. >1953.
5 Jameson, J.'E. A Discussion of the
Dynamics of Salmonella Enrichment.
J. Hyg. 60:193-207. 1962
6 Spino, D.F. Elevated Temperature
Technique for the Isolation of'
Salmonella from Streams. Appl."
Microbiol! 14, 591. 1966.
This outline was prepared by R. Russomanno,
Microbiologist, National Training Center, DTTB,
MDS, WPQ EPA, Cincinnati, OH 45268.
22-10
-------
TESTING THE SUITABILITY OF DISTILLED WATER
FOR THE BACTERIOLOGY LABORATORY
I INTRODUCTION
A Standard Methods for the Examination of
Water and Wastewater (12th Edition) states;
"Only distilled water or demineralizcd
water which has been tested and found free
from traces of dissolved metals and bac-
tericidal and inhibitory compounds may
be used for the preparation of culture media
and reagents. Bactericidal compounds may
be measured by a biologic test procedure
. . .. " This outline describes a suitable
procedure.
B A need for such a test has been shown in
the lack of reproducibility of plate counts
and a possible cause of inconsistent re-
sults in split sample examinations.
II THEORY OF THE TEST PROCEDURE
A Growth of Aerobacter aerogenes in a
chemically defined minimal growth medium.
The addition of a toxic agent or a growth
promoting substance will alter the 24 hr
population by an increase or decrease of
20% or more, when compared to a control.
Ill APPARATUS AND MATERIALS
A Glassware - rinse all glassware in freshly
redistilled water from a glass still. The
sensitivity of the test depends upon the
cleanliness of the sample containers,
flasks, tubes, and pipettes. Use only
borosilicate glassware.
B Culture - any strain of coliform IMViC
type --++ (A_. ae rogenes). This can be
easily obtained from any polluted river or
sewage sample.
IV REAGENTS
A Use reagents of the highest purity. Some
brands of potassium dihydrogen phosphate
(KH2PO4) have large amounts of impurities.
The sensitivity of the test is controlled in
part by the purity of the reagents employed.
1 Carbon source - Sodium'citrate, reagent,
crystals (NagCgl^O^ • 2H2O) 0. 29 g
dissolved in 500 ml of redistilled water.
2 Nitrogen source - Dissolve 0. SO g of
ammonium sulfate, reagent, crystals,
(NH^^SO^j.) in 500 ml of redistilled
water.
3 Salt mixture solution - Dissolve the
following compounds in 500 ml of re-
distilled water.
Magnesium sulfate, reagent, crystals
(MgSO • 7H20) 0. 26 g.
Calcium chloride, reagent, crystals
(CaCl2 • 2H20) 0. 17 g.
Ferrous sulfate, reagent, crystals
(FeS04 • H20) 0. 2 3 g.
Sodium chloride, reagent, crystals
(NaCl) 2. 50 g.
4 Phosphate buffer solution - Use a 1 to
25 dilution of a stock phosphate solution
prepared by dissolving 34. 0 gm of
potassium dihydrogen phosphate
{KH2PO4) in 500 ml of distilled water,
adjusting to pH 7. 2 with 1 N NaOH and
diluting to 1 liter with distilled water.
5 Toxic control - dissolve 0.40 grams
CuSO^ • 5H2O in 100 ml of redistilled
water. Dilute 1:1000 for 1 mg per liter
Cu before use.
W. BA. lab. 12 e, 11. 7 2
23-1
-------
Testing the Suitability of Distilled Water
B Sterilization of Reagents
Unknown distilled water sample - either
boil for one minute or sterilize by mem-
brane filtration.
Prepare reagents with redistilled water
heated to boiling for 1 to 2 minutes.
Phosphate buffer solution may be sterilized
by MF filtration or boiling.
C Solutions are useful up to two weeks when
stored at 5°C in sterilized glass stoppered
bottles. The salts solution must be stored
,in the dark because sunlight results in
copious ferric ion precipitation. A slight
turbidity arising in the first 3-5 days
does not detract from the usefulness of
the reagents,
V PROCEDURE
A Collect 150 - 200 ml of water sample in a
sterile borosilicate glass flask and sterilize.
Label 3 flasks or tubes: A, B, and F
Add water Samples and redistilled
water to each flask as indicated at the
bottom of the page.
B Add a suspension of Aerobacter aerogenes
(IMViC type --++) of such density that each
flask will contain 25 - 75 cells per ml.
Make an initial bacterial count by plating
a 1 ml sample in plate count agar. Incu-
bate tests A-F at 32° or 35°C for 20 - 24
hr. Make plate counts using dilutions of
1, 0. 1, 0.01, 0.001 and.0.0001 ml.
VI PREPARATION OF A BACTERIAL
SUSPENSION
A Bacterial Growth
On the day prior to performing the distilled
water suitability test, inoculate a strain of
Aerobacter aerogenes onto a nutrient agar
slant with a slope of approximately 2 - 1/2
inches in length contained in a 125 mm X
16 mm screw cap tube. Streak the entire
agar surface to develop a continuous
growth film, and incubate 18 - 24 hrs at
35°C.
B Harvesting Viable Cells
Pipette 1 - 2 ml of sterile dilution water
from a 99 ml water blank onto the 18-24
hr culture. Emulsify the growth on the
Media
Reagents
Citrate
Ammonium sulfate
Salt mixture
Phosphate buffer (7. 3 + . 1)
Water, 1 mg per liter Cu
Unknown water ______
Redistilled water
TOTAL VOLUME
STANDARD TEST
Control Unknown Toxic
Dist. Water Control
A B F
OPTIONAL TEST
. X
. X
.21.0
X
21.0,
X ¦
30.0
30.0
.21.0
, X
• X
30.0
Food Nitrogen Carbon
Available Source Source
C D E
30.0
30.0
30.0
OPTIONAL TEST
23-2
-------
Testing the Suitability of Distilled Water
"

99
1 -2 ml
ml
slant

wash]
¦

A
ml]
B

It
III
99
ml
1

90
ml
, i
C
I . lml]
slant by gently rubbing the bacterial film
with the pipette, being careful not to tear
the agar, and pour the contents back into
the original 99 ml water blank.
C Dilution of Bacterial Suspension
Make a 1 - 100 dilution of the original
bottle into a second water blank, and a
further 1 - 100 dilution of the second
bottle into a third water blank, shaking
vigorously after each transfer. Then
pipette 0. 1 ml of the third dilution
(1:1, 000, 000) into each of the flasks A,
B, and F (see Standard Methods for
Examination of Dairy Products, 12th ed. ).
This procedure should result in a final
dilution of the organisms to a range of
25-75 viable cells for each ml of test
solution.
D Verification of Bacterial Density
Variations among strains of the same
organism, different organisms, media,
and surface area of agar slopes will
possibly necessitate adjustment of the
dilution procedure to arrive at a specific
density range between 25 - 75 viable cells.
To establish the growth range numerically
for a specific organism and medium, make
a series of plate counts from the third
dilution to determine the bacterial density.
Then choose the proper volume from this
third dilution which when diluted by the 30
ml in the flasks A, B, and F will
contain 25 - 75 viable cells per ml. If
the procedures are standardized as to
surface area of the slant and laboratory
technique, it is possible to reproduce re-
sults on repeated experiments with the
same strain of microorganisms.
E Procedural Difficulties:
1 Chlorine or chloramine distilling over
into receiver. Distilled water should be
checked by a suitable quantitative pro-
cedure like the starch-iodide titration.
If chlorine is found, sufficient sodium thio-
sulfate or sodium sulfite must be added.
2 Unknown water sample stored in soft
glass containers or glass containers
without liners for metal caps.
3 Contamination of reagents of distilled
water with a bacterial background.
4 Incorrect dilution of A. aerogenes to
get 25 - 75 cells per ml.
5 Gross contamination of the sample de-
termined by the initial colony count be-
fore incubation'.
F Calculation:
1 For growth inhibiting substances:
colony count per ml Flask B
colony count per ml Flask A
a Ratio 0.8 to 1.2 (inclusive) shows
no toxic substances.
23-3
-------
Testing for Suitability of Distilled Water
b Ratio less than 0. 8 shows growth
inhibiting substances in water sample.
2 For toxic control
colony count per ml Flask F
colony count per ml Flask A
= Ratio
OPTIONAL TEST
*For nitrogen and carbon sources that
promote growth**
colony count per ml Flask C „ ,.
—;—- - r-———- = Ratio
colony count per ml Flask A
*For nitrogen sources that promote
growth*-'
colony count per ml Flask D
colony count per ml Flask A
Ratio
*For carbon sources that promote
bacterial growth**
colony count per ml Flask E
colony count per ml Flask A
Ratio
G Interpretation of Results:
1 The colony count from Flask A after
20 - 24 hours, at 35° C will depend on
the number of organisms initially
planted in Flask A and on the strain of A.
aerogenes used in the test procedures.
This is the reason the control Flask A
must be run for each individual series
of tests. However, for a given strain
of A. aerogenes under identical
environmental conditions, the terminal
count should be reasonably constant
when the initial plant is the same.
Thus, it is essential that the initial
colony count on Flask A and Flask B
should be approximately equal to secure
accurate data.
2 When the ratio exceeds 1. 2, it may be
assumed that growth stimulating sub-
stances are present. However, this
procedure is an extremely sensitive
test and ratios up to 3.0 would have
little significance' in actual practice.
Therefore, Test C, .D, and E do not
appear necessary except in special
circumstances, when the ratio is
between 1.2 and 3.0.
3 Usually Flask C will be very low and
flasks D and E will have a ratio of less
than 1.2 when the ratio of Flask B /
Flask A is between 0. 8 and 1.2. The
limiting factors of growth in Flask A
are the nitrogen and organic carbon
present. An extremely large amount
of ammonia nitrogen with no organic
carbon could increase the ratio in
Flask D above 1. 2 or the absence of
nitrogen with high carbon concentration
could give ratios above 1.2 in Flask
E with an A/B ratio between 0. 8 and
1. 2.
4 A ratio below 0.8 indicates the water
contains toxic substances and this ratio
includes all allowable tolerances. As
indicated in item 2 (above), the 1.2
ratio could go as high as 3.0 without
any undesirable results.
5 We are unable to recommend corrective
measures in specific cases of defective
distillation apparatus. However, a
careful inspection of the distillation
equipment and a review of production
and handling of the distilled water
should enable the local laboratory
personnel to correct the cause of the
difficulty.
*Do not attempt to calculate ratios, 3, 4, or
5 when ratio 1 indicates a toxic reaction.
** Ratio in excess of 1.2 indicates available
source for bacterial growth.
23-4
-------
Testing for Suitability of Distilled Water
CASE EXAMPLES
Test results for various distilled water samples

TEST
CONTROL

SOURCE
COUNT
COUNT
RATIO
INTERPRETATION
1
< 100
120, 000

Toxic Substance
2
74,000
170,000
0. 4
Toxic Substance
3
18,000
14,000
1. 3
Excellent water
4
21,000
14,000
1. 5
Excellent water
5
310,000
60,000
5. 2
Growth Substance
6
850,000
37,000
22. 9
Growth Substance
REFERENCES 2 Geldreich, E. E. and H. F. Clark. Dis-
tilled Water Suitability for Microbiologi-
1 Standard Methods for Examination of Water cal Applications. Journal Milk and Food
and Wastewater. 12th Edition. 1965. Technical. In Press. 1965.
p 578.
This outline was prepared by E E Geldreich,
Chief Bacteriologist, Water Supply Programs
Division, WPO, EPA, Cincinnati, OH 45268.
-------
IDENTIFICATION OF THE FECAL STREPTOCOCCI
I DEFINITION
The term fecal streptococci means any
strain of streptococci commonly found in
significant numbers in the feces of humans
and other warm-blooded animals.
II THE GENERAL PROCEDURE FOR
ISOLATION OF STRAINS IS: '
A Select a natural source where streptococci
exist,
B Inoculate a portion of natural sample into
a selective streptococci growth medium.
After incubation, a nearly pure mixture
of streptococcal strains usually results
because the selective medium is designed
to inhibit the growth of other bacteria.
C Pick a single cell to obtain a pure strain.
The most common way is cultivating a
single cell on solid medium, followed by
picking the resulting colony.
Ill HISTORY
Selective media have been most successful
with the fecal streptococci. Sodium azide,
potassium tellurite, or thallium acetate
function to inhibit nonstreptococcal growth.
Sodium azide is the most common selective
agent in current use.
A As early as 1940 a broth containing . 02
percent sodium azide was suggested for
estimating sewage streptococci. Three
years later, a medium was proposed
which the authors considered almost
complete evidence for Streptococcus
fecalis when growth and acid occurred
at 45. 5°C. The medium contained 0. 05
percent sodium azide and brom cresol
purple which was to indicate pH change.
In 1951, a modification appeared, described
as a buffered glycerol glucose medium.
This medium has been found lacking in
productivity in comparative studies carried
out at this Center.
IV SELECTIVE AGENTS AND CURRENT
METHODS
A The presumptive portion of the multiple
tube test contains 0. 02 percent sodium
azide. The confirmatory portion contains
0. 04 percent sodium azide and ethyl
violet dye. Since the confirmatory ethyl
violet-azide broth is very inhibitory, the
polluted sample is inoculated first into
the presumptive medium and then, after
growth, transferred (triple 3mm loop)
into the confirmatory medium. Growth
there may be checked for the presence
of Gram-positive cocci in pairs and
chains, by the Gram-stain method.
Either broth may be streaked on brain-
heart infusion agar or other streptococcal
media.
B The membrane niter method utilizes a
general growth medium, with yeast
extract and 0. 04 percent sodium azide.
The 12th edition of Standard'Methods
indicates that confirmation of colonies
appearing may be required. In some
problem waters, selectivity is not complete.
Verified identification as listed in Standard
Methods requires four steps.
1 Isolation of a pure strain by picking
inoculum from a single colony into
broth and incubation at 35°C.
2 Testing an actively growing transfer
culture for catalase enzyme activity.
3 Testing for growth in 40 percent bile
broth.
4 Testing for growth at 45°C in broth.
Confirmed fecal streptococci should
have a negative catalase reaction and
should grow both in 40 percent bile
and at 45°C.
m-Enterococcus agar is currently the
Standard Methods 1965'medium but still
has limitations where animal pollution is
evident. First, bathing pool waters may
NOTE: Mention of commercial products and manufacturers does not imply endorsement by the
Federal Water Pollution Control Administration and the U. S. Department of the Interior
W. BA. met. 23a. 7. 70
24-1
-------
Identification of the Fecal Streptococci
contribute micrococci which are
distinguished by the presence of catalase
enzyme. Second, work by Rose and
Litsky (1965) shows 0. 04 percent sodium
azide inhibits some fecal streptococci.
They report preliminary enrichment
increases subsequent counts two-fold.
C KF liquid medium and agar have had some
use. KF is a very rich growth medium
containing 0. 04 percent sodium azide.
Also, the agar may be used for membrane
filter determinations similarly to m-
Enterococcus agar.
D Either the membrane filter technique or
the agar pour plate method offers the most
convenient method for strain isolation.
If samples are turbid or contain debris,
the pour plate is superior. Besides,
clogging the membrane filter, debris
causes colonies to grow together. A
pour plate of either KF agar or m-
Enterococcus agar aids discrete colony
formation. Total colonies appearing are
the same, whether on membrane filter
or in pour plates. After colonies are
picked into broth and purification is
certain, further biochemical characteristics
can be studied.
V ISOLATED STRAINS ARE IDENTIFIED BY
MORPHOLOGICAL AND BIOCHEMICAL
TESTS
A The fecal streptoccocci are cocci occurring
commonly in pairs or short chains. They
are nonmotile, staining purple (positive)
with the Gram stain. They average about
0. 5 to 1 micron in diameter and may
exhibit pleomorphism.
B Streptococcal colonies typically grow
slowly and colonies are small, (0. 5-2mm),
For these reasons, 40 - 48 hour incubation
times are used. Colored pigments are not
commonly produced.
C In a natural environment the biochemical
activities vary greatly. For classification,
certain activities are selected and measured.
Careful techniques and selectivity of
biochemical procedures must be exercised
in the identification of various fecal
streptococci.
D Useful differential tests are:
1 The 45°c Growth Test - Incubate
streptococci in brain heart infusion
broth at 45°C for two days and examine
for growth.
2 The 10°C Growth Test - Identical to
the 45°C test except for temperature,
and five to seven day incubation.
3 Tolerance Test in 40 Percent Bile-
Add 40 ml of sterile 10 percent Oxgall
solution to 60 ml of sterile brain heart
infusion broth. The 10 percent Oxgall
solution is equivalent in concentration
to fresh bile originally used in this test
Positive cultures show growth within
one to three days.
4 Sodium Chloride (6. 5 percent) Tests-
Inoculate brain heart infusion broth
containing 6. 5 percent sodium chloride
and observe for growth of streptococci
within three to seven day period.
5 Growth at pH 9.6- Add sterile 38
percent sodium phosphate solution
(NagPO^ 12HnO) to sterile brain
heart infusion broth to give a pH 9. 6
reaction. This usually requires
approximately 5 ml of phosphate
solution per 100 ml of medium. Observe
for growth of streptococci'at one, two,
three and 7 days.
6 Hydrolysis of Starch - Add sufficient
10% soluble starch to brain heart
infusion agar to give a 1% starch content.
Pour into petri dishes, allow to harden,
and inoculate unknown strains by
streaking. After two days, flood surface
with gram's iodine. Note clear zones
indicating starch hydrolysis.
7 Potassium Tellurite Reduction - Add
sufficient sterile warmed (50°C) 4%
K2Te03 (1ml/100 med) to brain heart
infusion agar to have a final concentration
of 1:2500 potassium tellurite. Cool,
pour and harden agar in a petri dish.
Streak unknown cultures and observe
1-7 days. Black colonies indicate if
reaction is positive.
8 Methylene Blue Test - Add sufficient
1 percent sterile solution of methylene
blue to sterile skim milk to have a
final concentration of 0. 1 percent
methylene blue. Observe reactions at
one, two, three and seven days. (Note
blue milk becoming white for positive
reduction).
9 Litmus Milk Reaction - Sterilize the
24-2
-------
Identification of the Fecal Streptococci
litmus milk at 115. 2°c (10 lb for 10
minutes). Read reactions of strepto-
cocci at one, two, three and seven
days. (Observe pink of acid formation,
white of reduction, coagulation,
proteolysis).
10 The Azide Dextrose and Ethyl Violet
Azide Growth Tests - Standard Methods
12th edition, page 620 describes
preparation and performance of these
tests.
11 The Catalase Test - Transfer growth
from a brain heart infusion agar slant
or agar plate to a glass slide. After
dropping some 3% hydrogen peroxide
on the cells, watch for bubbles.
Catalase enzyme, if present, cleaves
hydrogen peroxide to water and visible
oxygen gas.
12 Serology
Serology depends upon the presence of
specific chemical-complexes in capsules
surrounding the bacteria, in cell wall
or inside the bacteria. An individual
bacterium has an unique chemical
composition. It is more or less a
carbon copy of all other members of
the same strain which share identical
chemical-complexes. But the less
closely related a strain is, the fewer
chemical-complexes are shared.
Bacterial strains are grouped together
by their common chemical-complexes.
Specific chemical-complexes are called
antigens. The science of their
manipulation and interpretation is
serology. Much theoretical work has
been done on serology of the fecal
streptococci. Apparently, Streptococcus
fecalis, S. durans and some S.bovis, 5!
equinus share a common group antigen,
called group D. Serology has not been
useful in pollution studies.
VI By use of standard tests, the streptococci
can be separated into groups. I;or sanitary
interpretation, the groups should clearly
differentiate between strains of fecal and
nonfecal origin. Species groups are commonly
used since species are correlated to their
origin. Figure 1 lists the biochemical
characteristics of the different groups. The
enterococcus group includes Streptococcus
fecalis, its varieties, and S. durans (faecium).
A Since a fecal streptococci is defined here
as one found in significant numbers in
feces, the fecal environment determines
which group predominates. Human feces
contains chiefly the enter.ococcus group
and occasionally Streptococcus salivarius
may occur in.a small number of individual
human fecal samples. The S. equinus
and S.bovis groups are predominant
streptococci in cows, pigs and sheep,
although some enterococcal group is
present, Kenner, et_al. (1967) (Figure 2)
report S.bovis composes 48.47c of the
total streptococcal flora of cows, 41. 5%
of sheep, and 8. 8% of pigs. S. salivarius
has been observed only two times in
domestic animal feces. S. equinus occurs
in appreciable numbers in feces of cows
and pigs, although early investigators
named it as the predominent streptococci
in horse manure.
B While referring strains to species groups
is useful in some studies, the procedure
is involved and lengthy. For sanitary
meaning, a simple'test differentiating
fecal from nonfecal strains would be
useful. Geldreich et al. (1964) proposed
a simple temperature-growth test.
Presence or absence of growth at 10 or
45 C. divides the streptococci into four
different groups indicative of different
health hazards.
1 Growth at both 10 and 45°C
a Present evidence indicates the common
fecal streptococci are generally
distributed in the feces of warm-
blooded animals. These organisms
probably mean fecal pollution when
present in water.
b The term common fecal streptococci
includes the original enterococcus
group, which has at present, a
confused meaning. The common
fecal streptococci grow in 6. 5
percent NaCl and at pH 9. 6.
Specifically they would be classified
as S. fecalis, S. fecalis v.
liquefaciens, ST fecalis v7 zymogenes
and S. durans, depending on additional
biocKemical tests.
2 Growth at 45°C only. The high
temperature fecal group includes the
S. equinus strains, S. bovis strains,
¦and most strains of S. salivarius. Their
presence probably signified fecal pollution
in water.
24-3
-------
to Figure 1. BIOCHEMICAL REACTIONS OF 4, 633 CULTURES ISOLATED
^ FROM HUMANS AND OTHER WARM BLOODED ANIMALS*1

Growth at
G rowth in
Reduction of
Final pH

Litmus Milk.

Fermentation

No. of
stains

6 5%
NaCl
Broth
pH 9. 6
Methylene
blue (0. 1%)
K2Te°3
1 2500
in
1% glucose
Hydrolysis
of starch
A rginine
hydrolysis

1%
Sorbitol

10°C
450c
Acid
Coag
Reduct
Pepton
Species
2, 706
+
+
+
+
+
+
3.9-4.6
0
+
+
+
(+)
+
+
S. fccalis var.
43
+
+
+
+
+
0
4.0-4. 5
0
+
+
+
+
0
0
S. durans (faecium)
(
(+)
+
+
+
(0)
+
3.9
0
(+)
(+)
(+)
(+)
(0)
(+)
S. fecalis biotype I
j
(+)
+
+
0
+

\
0
(+)
(+)
(+)
(+>
(0)
<+)
S. fecalis biotype 11
823 1
(+)
+
0
+
+
+
\
0
(+)
<+)
(+)
(+)
(0)
(+)
S. fecalis biotype III
(
+
+
0
0
0
+
6.5
0
(+)
(+)
(+)
(+)
(+)
(+)
S. fecalis biotype IV
698
0
+
0
0
0
0
4. 0-4. 5
+
0
+
+
(+)
0
not used
S. bovis
93
0
+
0
0
0
(0)
4.0-4.5
+
0
0
0
0
0
not used
S. equinus
78
0
(+)
0
0
0
+
0
1
*>.
CJl
0
+
+
+
+
0
not used
S. salivarius
192
(+)
+
(+>
(+)
<+)

4.0-4.5
+
+
+
+
(+)
+
not used •
Atypical **
S. fecalis var.
*A11 strains were catalase-negative; all grew in 40% bile broth, except S. salivarius.
**Atypical strains usually isolated from fermenting vegetation, isolated only from cat feces (10 strains),
and dog feces (182 strains).
(+) = usually positive, (0) = usually negative; + = variable.
***Biotypes arbitrarily based on growth in 6. 5% NaCl broth and pH 9. 6 broth, allowing four types.
1B.A. Kenner and P.C. Haley
-------
Figure 2. DISTRIBUTION OF FECAL STREPTOCOCCAL GROUPS IN FECES OF HUMANS AND OTHER
WARM-BLOODED ANIMALS SOURCE ON THE FECAL SAMPLES1

Human
Cow
Sheep
Pigs
Chickens
Turkeys
Ducks
Cats
Dogs
A
Rodents
Totals
No. of fecal samples

22
12
10
10
10
9
8
10
24
22
137
Total no. of strains

1,065
438
371
360
368
349
318
268
557
606
4, 700
S. fecalis var.
No.
940
97
142
194
223
249
130
•215
160
356
2, 706

%
88. 26%
22. 15%
38. 27%
53. 89%
60. 6%
71.35%
40. 88%
80. 22%
28. 7 3%
58.75%
57. 57%
S. durans (faecium)
No.
13
13
6
2
1
0
0
0
8
0.
43

%
1. 22%
2. 97%
1.62%
0.56%
0. 27%
0%
0%
0%
1. 44%
0%
0. 91%
S. fecalis biotypes
No.
34
74
54
106
143
94
52
43
88
135
823
%
3. 19%
16.90%
14.56%
29 . 44%
38. 86%
26.93%
16.35%
16.04%
15. 8%
22.28%
17. 51%
S. bovis
No.
0
212
154
32
0
5
126
0
94
75
698

%
0%
48.4%
41.51%
8. 89%
0%
1. 43%
39. 62%
0%
16. 88%
12. 38%
14.85%
S. equinus
No.
0
41
14
26
1
1
10
0
0
0
93

%
0%
9. 36%
3. 77%
7. 22%
0. 27%
0. 29%
3. 14%
0%
0%
0%
1. 98%
S. salivanus
No.
76
1
1
0
0
0
0
0
0
0
78

%
7. 14%
0. 23%
0. 27%
0%
0%
0%
0%
0%
0%
0%
1. 66%
Atypical
No.
0
0
0
0
0
0
0
10
182
0
192
S. fecalis types*
%
0%
0%
0%
0%
0%
0%
0%
3. 73%
3 2. 68%
0%
4.09%
Streptococci X1
No.
0
0
0
0
0
0
0
0
25
40
65

%
0%
0%
0%
0%
0%
0%
0%
0%
4. 49%
6. 6%
1. 38%
S. anginosum
NO.
2
0
0
0
0
0
0
0
0
0
2

%
0. 19%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0. 04%
*See Figure 1
X - Unidentified strains
A - Rabbits (10 samples), Rat (7), Chipmunk (2), Guinea Pig (2), and Racoon (1).
1B.A. Kenner and P. C. Haley
-------
Identification of the F ecal Streptococci
Streptococcus bovis characteristically
hydrolyses starch, as does S. equinus,
when the 1% starch is incorporated into
brain heart infusion agar which contains
a small amount of dextrose. S. equinus
does not ferment lactose. S. salivarius
does not hydrolyse starch, "But does
ferment lactose.
3 Growth at 10°C only. The low
temperature group includes the lactic
acid streptococci found in soil and
plants. They have never been found in
animals or their feces. They neither
indicate fecal pollution or cause disease
themselves and do not grow in KF
streptococcus medium.
4 Growth at neither 10 or 45°c. These
are a group of no sanitary significance
and they are rare in water, but do
include the pyogenic or pathogens of
the genera.
REFERENCES
1 Geldreich, E. E. , Clark, H. and Huff,
C. B. A Study of Pollution Indicators
in a Waste Stabilization Pond. J.
WPCF 36, 1372-1379. 1964.
2 Kenner, B. A., Clark, H. F. and Kabler,
P. W. Fecal Streptococci. II.
Quantification of Streptococci in Feces.
Am. J, Public Health, 50, 1553-59.
1960.
3 Rose, R. E. and Litsky, W. Enrichment
Procedure for Use with the Membrane
Filter for Isolation and Enumeration
of Fecal Streptococci in Water.
Applied Microbiol. 13, 106-109.
1965.
4 A. P. H. A. Standard Methods for the
Examination of Water and Wastewater,
12th Ed. 1965.
This outline was prepared by B. A, Kenner,
Supervisory Research Microbiologist,
Waste Identification & Analysis Program,
Advanced Waste Treatment Research
Laboratory, RATWRC, NERC, EPA,
Cincinnati, OH 45268.
24-6
-------
INTRODUCTION TO STATISTICS
Part I
Summarization of Data
I INTRODUCTION
The body of statistical methods comprises
two groups generally called descriptive
statistics and statistical inference. Descrip-
tive statistics encompasses primarily graphical
techniques for the presentation of data while
statistical inference is basically a mathemat-
ical approach to the problem of inferring
from a part to the whole.
II BASIC GRAPHICAL TECHNIQUES
In this section we consider the non-mathematical
techniques of summarizing a set of data in
order that the meaningful information can be
extracted from it.
Consider the data in Table 1 which represent
aqueous fluoride ion concentrations determined
by a colorimetric procedure.
TABLE 1
F , mg/liter
CO
CD
O
0. 77
0. 85
0.71
0. 79
0. 85
0. 72
0.80
0. 87
0. 74
0.81
0.90
0. 75
0.82
0. 91
0. 77
0.83
0.95
A Frequency Table
As the first step in summarizing a set of
data we form a frequency table. (See
Table 2). To construct the table the data
are divided into a number of intervals of
equal length called class intervals and the
number of results falling into each interval
is recorded in the "frequency" column.
The number of class intervals chosen is
arbitrary. However, it is a rule of thumb
to choose the length of the class interval
so that 7 to 15 intervals will include all
the data under consideration.
TABLE 2
Class Interval Frequency
. 65
au*
.70
1
. 70
au
. 75
3
. 75
au
o
CO
4
o
00
au
. 85
4
m
CO
au
. 90
3
CO
o
au
. 95
2
. 95
au
1. 00
1
* and under
B Frequency Polygon
Asa further step we can graph the
frequencies recorded in the frequency
table. One way of doing this is to plot
the frequency along the ordinate (vertical
axis) and the midpoint of the class interval
along the abscissa (horizontal axis). The
line connecting the plotted points in Figure 1
form a frequency polygon.
»
6 73 7 2 5 7 7 5 115- 175 915 97S
FLUORIDE CONCENTRATION (¦ 100)
FIGURE 1
ST. 48a. 3. 74
25-1
-------
Introduction to Statistics
C Histogram
Another method of graphing the frequency
information is to construct a histogram
as shown in Figure 2. The histogram is
a two dimensional graph in which the
length of the class interval is taken into
consideration. Each class interval
becomes the base of a rectangular bar
whose height is equal to the corresponding
frequency.
" r
V .JO -
z
D
o .15 -
Bt
tM .1 0 -
>
2 o. .
tM
I 2 } 4 J 6 7 •
¦ »i 70 .71 10 .»S .90 .95 100
UNIT LENGTH AND r CONCENTRATION
FIGURE 3
>.
3
2 j -
41
45 .70 7 S .10 as .90 .9) 1.00
FLUORIDE CONCENTRATION
FIGURE 2
D Histogram of Relative Frequency
The histogram can be a very useful tool in
statistics especially if we convert the given
frequency scale to a relative scale such
that the sum of all the ordinates equals one.
Thus, each ordinate value is derived by
dividing the original frequency by the
number of observations in the sample.
For the data in Table 1 we would divide
each of the frequency values in Table 2
by 18.
The advantage of constructing a relative
frequency histogram like the one in
Figure 3 is that we can interpret areas
under the histogram as probabilities pro-
vided we assume a scale on the abscissa
such that each class interval is of unit
length. Then the probability that a given
value will fall in any one interval is the
area under the histogram in that interval.
For example, the probability that a value
will fall between . 70 and . 75 is equal to
the area under the histogram in that
interval which is 3/18X1 = 1/6.
E Cumulative Frequency Distribution
From a frequency table (See Table 2) we
qan construct a cumulative frequency
table as shown in Table 3. The cumu-
lative frequency table gives the number
of observations less than a given value.
The cumulative frequency values in
Table 3 are found by summing the
frequencies in Table 2.
TABLE 3
F Cone.
Cum. Frequency
under .70
1
. 75
4
" . 80
8
" . 85
12
. 90
15
. 95
17
o
o
18
If we convert the cumulative frequency
values to relative frequencies as we did
to construct the relative frequency histo-
gram, we can plot the relative values to
form a cumulative frequency curve as
shown in Figure 4. The probability that
a result will fall below a given value can
be read from the cumulative frequency
curve. For example, to find the
probability that a result will be less than
25-2
-------
Introduction to Statistics
0. 85 mg/liter we read up to the curve at
the point x = . 85 and across to the value
. 67 on the probability axis.
F" CONC.
FIGURE 4
25-3
-------
INTRODUCTION TO STATISTICS
Part II
Measures of Central Tendency and Dispersion
I INTRODUCTION
From a practical point of view, statistical
inference is the application of mathematical
tools to analyze and interpret the results of
investigations in the physical and social
sciences with respect to the hypotheses under
investigation. The need for a statistical
approach rests on the well-founded assumption
that any such result contains an inherent
component of error or random variation which
introduces uncertainty into any conclusions
which may be drawn from the observed
results.
A Mathematical Distributions
It is further assumed that the error com-
ponents can be described by a mathematical
function called a probability distribution.
Knowledge of the relevant type of theoret-
ical distribution provides us with the means
of assessing the uncertainty (risk of being
incorrect) in conclusions drawn from
observed results.
B Population Parameters
The concept of distribution pertains to the
totality of possible results which would be
observed in an infinite number of repetitions
of an experiment or investigation. This
theoretically infinite number of results is
called a population. Any measurable'
characteristic of a population distribution
is called a parameter. For each type of
distribution there exists a characteristic
set of parameters. Specific values for the
parameters of a given distributional form
define a particular distribution of that type.
In almost every case the entire population
can never be observed and therefore the
values for the parameters are unknown.
Population parameters are denoted by
lower case Greek letters.
C Sample Statistics
The observed results from a particular
investigation are called a sample of the
relevant population. In almost every case
the sample is a subset'of the population.
A statistic is a function of the sample
points (i. e., observed results) which
usually estimates a population parameter.
Sample statistics are denoted by English
letters.
D Classification of Statistical Measures
It is implied above that the type of
population distribution determines the
particular function of the observed results
(i. e., sample statistic) which will provide
the "best" estimate of the corresponding
population parameter. For this reason
it is advantageous to assume that any given
sample came from a population with a
specified form of distribution. The use of
graphical techniques to indicate the form
of distribution should now be.apparent.
In most situations the distributions or
frequency functions of interest have a
single peak and characteristic dispersion
of area about this peak. Parameters which
locate the distribution (by the peak, center
of gravity, midpoint, etc.) are called
location parameters and the corresponding
sample statistics are classified as measures
of central tendency. Parameters which
describe the dispersion of area are called
scale parameters and the corresponding
sample statistics are classified as measures
of dispersion. Ideally, one would like to
encounter only distributions for which one
measure of central tendency and one
measure of dispersion summarize all of
the relevant information in the sample
about the population.
25-5
-------
Introduction to Statistics
II MEASURES OF CENTRAL TENDENCY
In this section-and the next we define and lay
out the computational format for the more
commonly used measures in statistics. We
let denote a typical observed result so that
[xp Xg, ...,x ] represents a sample of n
observations.
A The Mean
The most commonly used measure of
central tendency is the mean or arithmetic
average. We denote the sample mean_by
x and the population mean, of which x is
an estimate, by/J. The computational
formula is:
x =
Ex.
x
For the fluoride data in Table 1, we
calculate the sample'mean as follows:
Ex.
1
14. 52
18
TABLE 1
0. 807
mg/liter
0. 68
0. 77
0. 85
0. 71
0. 79
0. 85
0. 72
0. 80
0. 87
0. 74
0 81
0. 90
0. 75
0 82
0.91
0. 77
0 83
0. 95
B The Median
Another common measure of central
tendency is the median. The median is
the midpoint of an array of numbers
(ordered according to value). To find the
sample median we need only arrange the
data in ascending or descending order and
pick the middle value. When there is an
even number of observations take the
average of the two middle values. For
the data in Table 1 the median is 0. 805.
C The Mode
The mode is another measure of central
tendency, although it is of little practical
importance. The mode is the most
frequently occurring value. Therefore,
the population mode is the value corres-
ponding to the peak of the frequency
distribution curve. Frequency distributions
with more than one peak are called
multimodal. In a symmetrical frequency
curve the mean, median, and mode are
all equal.'
III MEASURES OF DISPERSION
A The Standard Deviation
As with measures of central tendency,
there are several measures of dispersion,
the most common of which is the standard
deviation. We denote the sample by s and
the population value of which s is an
estimate by a The computational
formula is:
s = -J
£ (xi - x)2
n - 1
However, computation using this formula
is tedious and it is relatively simple to '
show the following relationship:
s =
' £(x - x )2
n - 1
- (Ex.;
n (n - 1)
The derived formula is preferable because
of its adaptability to the desk calculator.
We calculate the sample standard deviation
of the fluoride data in Table 1 as follows:
J
(18) ( 11.8048) - ( 14.52)'
18 (17)
- J
212.4864 - 210.8304
306

0054 = 0.073
25-6
-------
Introduction to Statistics
B The Variance
C The Range
The sample value s is referred to as the
sample variance and is merely the square
of the standard deviation. Its formula is:
2
s =
E(Xj
x)2
The population variance is represented by
. Its formula is:
E(Xi " M)
This is the same as the formula for s
except we use the true population mean m ,
rather than its estimate x and divide by n
instead n - 1.
Obviously in calculating the sample
variance the true mean is not known and
we, therefore, use the estimate of the-
mean from the data. In calculating the
sample mean and then using it to calculate
the variance of the same data we lose what
is called a degree of freedom. It can be
shown that the estimate of the variance
must be based upon the sum of independent
squared terms. We average our values
over n - 1 because this is the number of
independent squared terms that we will
have when using a mean that has been
estimated from the sample. In line with
above, we should draw the distinction
between the variance of the sample and
the sample variance. The variance of the
sample, we would calculate for its own
sake and divide by n. However, we cal-
culate the sample variance as an estimate
of the population variance and divide by
n - 1. There is no practical use of the
variance of the sample, therefore, we
always calculate the sample variance
dividing by n - L which we shall call the
number of degrees of freedom in our
sample. As a rule, in any calculation,
for every parameter that must be estimated,
one degree of freedom (d.f) is lost.
The range is also used as a measure ot
dispersion. It is the difference between
the highest and lowest values in a set of
data.
R
max(x.) - min(xj
For the data in Table 1 .the range is then:
R = 0.95 - 0. 68. = 0.27
A rough estimate of s can be made by
dividing the range of the sample by the
square root of n, the number of
observations, when n is small.
0 27 0.27
s - rw~ -Y-- °'057
The use of the range is limited to
instances where the labor of computing
the standard deviation is impractical.
IV THE NORMAL DISTRIBUTION
A The most important theoretical distribution
in statistics is the familiar bell-shaped
normal distribution which is symmetric
about its peak (See Figure 1) The
following assumptions give rise to this
distributional form:
1 Values above or below the mean are
equally likely to occur.
2 Small deviations from the mean are
extremely likely.
3 Large deviations from the mean are
extremely unlikely.
QUANTITY MEAJURtD
NORMAL DISTRIBUTION CURVE
FIGURE 1
25-7
-------
Introduction to Statistics
The normal distribution is completely
defined by its mean, n, and its standard
deviation a in the following manner:
1 The area under the normal curve ¦
between minus o and n plus a is 68
percent of the total area, to the nearest
1 percent.
2 The area under the normal curve
between m minus 2 o and fj plus 20 is
95 percent.
3 The area under the normal curve
between u minus 3 a and pi plus 3cr is
99.7 percent of the total area, to the
nearest 0. 1 percent.
If a frequency curve is a good approximation
to the normal curve, these characteristics
of the normal curve can be used to find
information about the frequency distribution.
V- TRANSFORMATION OF DATA
A Skewed Distribution
In some areas of investigation one often
encounters distributions which are not
symmetric. For example, distributions
of bacterial counts are often characterized
by many more extremely high counts
relative to the median than extremely low
counts. The frequency curves of these
distributions have a long right tail as shown
in Figure 2. Distributions of this type
display positive skewness.
COUNTS
POSITIVELY SKEWED DlST.
FIGURE 2
B Logarithmic Transformation
For many reasons, both practical and
theoretical, we prefer to work with
symmetric distributions like the normal
curve. Therefore, it is desirable to
transform skewed data in such a way that
a symmetric distribution results,
resembling the normal. One way of
deriving an approximately normal dis-
tribution from a positively skewed
distribution is by expressing the original
data in terms of logarithms. An
artificial sample of coliform counts and
their logarithms are shown in Table 2.
Comparison of the frequency tables for the
original data and the logs (Table 3 and
Table 4, respectively) clearly shows that
the logarithms more closely approximate
a symmetric distribution.
TABLE 2
Coliform
MPN/ 100 ml
MPN
log MPN
11
1.041
27
1.431
36
1. 556
48
1. 681
80
1.903
85
1. 929
120
2. 080
130
2. 1-14
136
2. 134
161
2. 207
317
2 501
601
2. 779
760
2. 881
1020
3. 009
3100
3. 491
25-8
-------
Introduction to Statistics
TABLE 3
Notice that
Class Interval
Frequency (MPN)
log x
E(log x.)
0
au
400
11
S "
400

800

so that the geometric mean of the
au
2
original data is equal to the antilog
800
au
1200
1
of the arithmetic mean of the logarithms.
1200
au
1600
0
It is of interest that the population
geometric mean is equal to the population
1600
au
2000
0
median. Thus, in the case of data from
2000

2400

a skewed distribution, the sample median
au
0
is a better estimate of central tendency
2400
au
2800
0
than the arithmetic mean of the original
2800

data. For the coliform data in Table 2
au
3200
1
we calculate:
TABLE 4
Class Interval Frequency (log MPN)
1. 000
au
1.300
1
1.300
au
1.600
2
1. 600
au
1. 900
1
1. 900
au
2.200
5
2. 200
au
2. 500
1
2. 500
au
2. 800
2
o
o
CO
CSJ
au
3. 100
2
3. 100
au
3. 400
0
CO
o
o
au
3.700
1
log x
E(log xj
32.737
15
= 2.1825
Xg = antilog (2. 1825) a, 152,
median = 130,
Ex.
6632
15
442.
C Measures of Central Tendency of Skewed
Distributions
If the logarithms of data from a positively
skewed distribution are approximately
normally distributed, we say that the
original data have a log-normal distribution.
The best estimate of central tendency of
log-normal data is the geometric mean
defined as:
This outline was prepared by John H. Parker,
Former Statistician, Analytical Reference
Service, Training Program.
Descriptors: Frequency Analysis, Histo-
grams, Statistical Methods, Statistical
Models, Statistics, Variability
g -J (x 1) (x2). . . (xr)
25-9
-------
ECOLOGY AND SANITATION OF BIVALVES
Part 1
Some important facts on anatomy, physiology
and ecology of the oyster, quahog, soft clam
and sea mussel in relation to sanitation thereof.
I THE PROBLEM: ITS .\LAJOR PHASES
A The Water Pumping and Feeding Mechanism
1 The first three bivalves (pelecypods,
lamellibranchs) are estuarine, not
marine, living in shallow, brackish
water, coastal areas subject to high
turbidity and to pollution from adjoining
lands. The mussel while chiefly marine
does invade coastal waters.
2 The oyster and "little neck" quahogs are
the only animals eaten while still alive
by civilized races.
3 All materials used in building tissue,
storage of glycogen, and shell formation
are absorbed, or strained, from sur-
rounding waters.
4 Pumpage of water through bivalves is
effected through coordinated beating of
myriads of vibrating hair-like cilia
which cover all external body surfaces,
while lining the digestive and reproductive
tracts, and interior as well as exterior
of the gills. Some cilia are under
nervous control.
5 At frequent intervals among the cilia are
unicellular mucus glands which respond
directly to a wide variety of tactile and
chemical stimuli.
6 Mucus discharged from these glands in
response to particles striking them
entangles the suspended matter, strained
from the water by one group of gill cilia,
into "food" strings which are transported
by a second and a third group of gill
cilia toward the mouth.
7 Secretion of mucus from the mucous
glands is to a high degree controlled
quantitatively by nature of the particles
in suspension: sand grains, dirt particles,
spiny diatoms, evoke copious discharge
of mucus resulting in large masses
which are mostly rejected before
reaching the mouth. Bacteria, unicellula
algae including nonspinv diatoms,
protozoa and other microscopic animals
evoke relatively less mucus hence form
thinner food strings which are largely
accepted and enter the stomach.
Separation of sand, mud particles, and
other nonfood materials from food is
thus effected to a large degree, but it
is not perfect; some sand and useless
materials enter the stomach while
considerable amounts of food are rejected.
8 The entangling of bacteria in mucus,
and elimination through ciliary action
(processes which occur on the walls of
our own respiratory tract)'make possible
cleansing of grossly polluted sea mussels,
Mytilus edulis, in 4 8 hours entirely
through conjoint action of mucous glands
and cilia. Even small traces of free
chlorine interfere seriously with this
biological process since mussels refuse
to function. Turbidity speeds bacterial
elimination through stimulating mucus
secretion. (References: Dodgson, R.W.)
Mussel not considered further in this
lecture.
SUMMARY: Topics 1-8. Mucus secretion
and ciliary activity make possible the life of
bivalves in turbid coastal waters. (Incidentally
also permit humans to exist in dusty or smoke-
filled atmospheres). They are the very kev
to purification of polluted shellfish.
B Some Basic Anatomy of Bivalves
1 Gross Anatomy
We shall consider the following:
FO. SH. bi. 2c. 9 70
26-1
-------
Ecology and Sanitation of Bivalves
a Mantle. The mantle lines the shells
or valves throughout, while the mantle
border secretes the shell. In the
oyster Crassostrea virginica the mantle
halves are separated except around
the anterior (front) end, and at one
point near the posterior (hind) end
where they unite with the gills. Water
passes in along the entire ventral
(under) side of the bivalve, leaving
it along the dorsal (upper) side. Of
chief interest to sanitarians: the
mantle collects rejected materials
concentrating them for expulsion. In
the quahog and the clam the mantle
halves are attached at two additional
points to form two tubes, the siphons,
which in the quahog, mercenaria
mercenaria, (Venus) are approximately
an inch long when extended. They are
completely withdrawn when the bi-
valve closes its shells. In the soft
clam, long necked clam, Mya arenaria,
the siphons of a large animal may be
extended a foot or more. They can
•not be withdrawn into the shell cavity
but remain mostly outside, enclosed
in a tough, leathery sheath of perio-
stracum which likewise covers the
shell in most bivalves. Water enters
through the more ventral of the two
siphons, the incurrent, and leaves
through the more dorsal, excurrent
or anal siphon. Mya cannot close its
shells completely, the mantel borders
being exposed.
The mantle border in the oyster
reveals three reduplications: the
outermost secretes the shell; the
middle one bears many tentacles
hence known as the tentacular border;
the innermost, the pallial curtain,
can be raised to block entrance or
egress of water or to direct expulsion
of water over a restricted area to
blow away encroaching mud.
In Crassostrea are two excurrent
chambers: the cloacal chamber behind
the adductor muscle, and the promyal
chamber in front of the muscle on the
right side. Water leaves over two
broad areas on the dorsal side.
b The Gills and Branchial or Gill
Chambers. There are two complete
gills or four half gills (demibranchs)
in each of the three bivalves here
considered. Each demibrarich con-
sists of two rows of gill tilaments
enclosing chimney-like excurrent
compartments, the water tubes,
between them. The gill filaments
are comparable to pickets in a 'fence
with openings of variable aperture,
the ostia, through which water is
driven into the water tubes of the
interior. In the oyster>a hormone,
designated dianthlin, relaxes the
tissue surrounding each ostium thus
enlarging it to permit passage of eggs.
The upper ends of the filaments are
attached to body of bivalve, while at
the lower ends filaments are attached
to body of marginal food collecting
furrow in which strong cilia transport
mucus strings of food and dirt toward
the mouth. Between the outermost
demibranch and the mantle on each
side is a basal food collecting furrow
with three more such furrows between
the bases of the inner demibranches,
or five basal furrows and four
marginal furrow-s in all.
The space between the mantle halves
in which the gills lie is designated
the infrabranchial, or incurrent, gill
chamber. Water passes from here
through the ostia of the gills into the
water tubes. The water tubes in
turn discharge into the epibranchial,
or excurrent, gill chamber. In the
two siphonate clams, mercenaria
and Mya, incurrent siphon passes
water to infrabranchial chamber
whence it passes through ostia into
water tubes, thence to epibranchial
chamber and out through excurrent
siphon.
c The palps, or lips, four in number,
surround mouth and extend backward
to embrace front ends' of four demi-
branchs. Mucus strings carried to
palps in nine food-collecting furrows
of gills are accepted and passed on
to mouth if small in volume, and if
26-2
-------
Ecology and Sanitation of Bivalves
of acceptable nature. Much rejection
occurs here, material in oysters
being passed over mantle edge at any
ciliary current carries particles to
this ejection point. In the two clams
powerful ciliary tracts on the mantle
carry all rejected material to a sinus
or depression at base of the incurrent
siphon whence it is ejected by quick
contraction of adductor muscles
which drives a strong stream of
water out through the siphon. These
rejected masses, commonly called
pseudo faeces, will be designated as
"rejecta" as contrasted with the
"dejecta" or true faeces which have
been through the digestive system.
Accumulation of rejecta is important
to sanitarians since a clam harvested
just before "blowing off" may contain
up to half a teaspoonfull of sediment
hence in moderately polluted waters
would be expected to contain a much
higher bacterial load than one just
recently blown. Mya should show
greater differences than mercenaria
owing to larger masses accumulated
prior to "blowing off".
2 Microscopic anatomy
The gill filaments in the oyster are
folded into folds or plicae each containing
twelve or less filaments thus greatly
increasing the number of filaments
possible in a given length of gill axis.
This folding of the walls of the gill
greatly increases the pumping power of
the oyster while aiding in separating
food from dirt on the gill surface. In
transverse section a plica shows each
filament to be hollow containing a blood
vessel. Four groups of cilia occur on
each filament, to wit:
Latera cilia: the "water pumpers",
adjacent to the ostia.
Latero-frontal cilia: the "strainers",
interlock with those of adjoining filaments.
Frontal cilia; the "food pushers" move
mucus strings across face of the filament
and into food-collecting furrows.
Abfrontal cilia: "accessory pumpers"
inside the gill aid in pushing excurrent
water toward exist.
3 SUMMARY: a quick review of water
passage through bivalves will be given.
C Ecology
1 Accumulation of rejecta; oyster may
bury itself in highly turbid water.
Mudding, lethal effects of H^S.
2 Concentration of bacteria in mud on
shells. Oyster shell difficult to clean;
ridges, perforations of boring sponge,
Cliona; retention by barnacles.
3 Effects of salinity changes: outside
limits 5 to 3 0 parts per mille. Desirable
range 12 to 28: adjustment. Tidal
influences on feeding in the oyster.
Day and night pumping in water with
low buffering potential, in swamp
water.
4 Temperature effects; oyster: reduction
of pumpage below 10oc, cessation in
most between 5-60, but .occasional
oyster may be slightly active down to
temperatures approaching Oo . Above
30° activation in northern oysters,
350 in Gulf oysters. Hibernation a
relative, not absolute term, round.
5 Water pumpage by shellfish. Deficiencies
in turbidity clearing methods. Accurate
measurement in oyster using Nelson
rubber apron method, 1935, yielded 26
liters per hour in oyster 11.5 X9 cm.
Loosanoff at Milford, Conn, obtains
average of 33 1. p. h. with maximum
of 48 1. p. h.
6 Feeding: leucocytes engulf bacteria.
Dr. Stauber has traced bacterial spores
over much of body of oyster, not digested
by the leucocytes. Much food present
in stomach of feeding oyster, very
little in mercenaria. Passage of food
into digestive gland and its digestion
there; a possible explanation of fall in
bacterial score of mercenaria during
storage.
26-3
-------
Ecology and Sanitation of Bivalves
7 Keeping quality of shellfish. Dugal's
findings on anaerobiosis; its application
to oysters; "closed respiratory system"
in mercenaria vs. "open system" in Mya.
Possible effects of oxygen and lower pH
in Mya on bacterial score. Anaesthetic
effect of CO^ on ciliary activity.
8 Elimination of mud by quahogs after
removal from water. An as yet unsolved
problem in shellfish sanitation is
explanation of consistently lower bacterial
scores in quahogs as compared with those
of clams and oysters removed simul-
taneously from the same waters. A
chance observation during late autumn
1957 offers a possible explanation.
Several dozen quahogs were raked from
exposed tidal flats of Cape May County
in front of the New Jersey Oyster
Research Laboratory. A thin layer
of finely divided mud of consistency of
thick cream covered the flats at the time.
After washing thoroughly in clean bay
water the bivalves were transported by
car six miles to my home. Several
hours later on picking up several to eat
raw the ventral margins of the shells
were found to be covered with a copious
discharge of thick mucus containing much
mud. Since the shells of the quahogs
had been thoroughly washed and drained
after harvesting this mucus and mud
must have been extruded subsequently
between the apposed ventral mantle
borders by ciliary action. Slight
relaxation of the adductor muscles to
permit escape of the material between
the valves must have occurred.
It is emphasized that this extrusion took
place out into the air and not into water
as is usual. Also, presence of the
overlying mud on the flats is significant.
Dodgson at Conway, North Wales,
found more rapid elimination of bacteria
from turbid water than from relatively
clear water correlated with heavier
mucus secretion stimulated by particles
of dirt striking the gills and mantle.
Confirmation of the above experience is
seen in the fact that only rarely have I
found any retained mud in quahogs when
opened, whereas clams which have
not been floated in clean water in-
variably have revealed small to very
large accumulations of sand and mud
in the cloacal sinus. Oysters may or
may not contain appreciable amounts of
silt when opened, hence may show wide
variations in bacterial scores.
9 Burial of oysters in their own rejecta.
In an important series of three papers
published in the Journal of the Institute
of Marine Science, No. 2, Vol. 4, for
July'1957, Dr. E.J. Lund presents
interesting data on rate of deposition
of rejecta from oysters as compared
with settling of silt due to gravity. In
one series of laboratory experiments
in relatively low turbidities, with light
transmission from 70 to 95 per cent,
a single layer of oysters covering half
the bottom area deposited sufficient
silt to cover themselves completely in
36 days. In a second series the volume
of sediment thrown down by oysters was
eight times the amount deposited by
gravity alone during the same period.
In a third series of observations this
deposit of oyster rejecta ranged from
six to twelve times the amount accumu-
lating through the force of gravity.
In open waters with good circulation on
the bottom these deposits are largely
swept away by currents. In relatively
quiet waters, however, the silt accumu-
lates, cutting off oxygen-with resultant
large production of hydrogen sulfide.
Death of oysters may follow where re-
ducing conditions of bottom muds
develop to sufficiently high levels to
result in strong hydrogen sulfide
production.
Significance of these observations to
shellfish sanitation presents two aspects.
First, at even low levels of coliforms
in the water their numbers in rejecta on
and'around the oyster's shells will be
enormously greater than in overlying
waters. Second, on removal of polluted
shellfish to clean waters it may be
expected that turbidity will hasten the
26-4
-------
Ecology and Sanitation of Bivalves
cleansing process as already demon-
strated thirty years ago by Dodgson for
the mussel, Mytilus edulis. Where
purification is carried out in taken it is
suggested that small amounts of laundry
starch be added to the water to stimulate
mucus secretion. Some by the starch
will be eaten by the shellfish with
possibility of increasing glycogen
reserves of value in anaerobic
respiration.
This outline was prepared by the late
Thurlow C. Nelson, formerly "The-Julius
Nelson Professor of Zoology", Rutgers
University, New Brunswick, New Jersey.
26-5
-------
Ecology and Sanitation of Bivalves
DESCRIPTION OF ACCOMPANYING PLATE
Figure 8
O. virginica from the right side, right
pallium dissected away exposing promyal
chamber and the right epibranchial chamber
posteriorly to its minimum size beneath
the adductor muscle at point X. The
region A to B with arrows directed inward
is the incurrent area; from B to C with
arrows pointing outward from the cloacal
and promyal chambers is the excurrent
area. Natural size.
Figure 9
O. virginica dissected from right side, X2,,
showing relation of the water tubes of the
right demibranchs to the right epibranchial,
cloacal and promyal chambers, and the
fixed position of the oral process. B.T.,
tentacular border; CH. C., cloacal
chamber;
CH.P., promyal chamber; CH. R. E. ,
right epibranchial chamber; CU.P., pallial
curtain; D., demibranch; F.PB., pallio-
branchial fusion; H.A., accessory heart
of Hopkins; J. I., interlamellar junction;
M.A., adductor muscle; P. R, , right
pallium; PE.C., pericardial cavity;
PR.O. , oral process; TU.W., water tube.
Figure 10
O. virginica from the left side to show
route of water discharged from the left
demibranchs and from posterior portion
of the right demibranchs. A roll of
black paper is inserted into the promyal
chamber. CH. L. E. , left epibranchial
chamber; H., heart; other abbreviations
as in Figure 9. Natural size.
26-6
-------
Ecology and Sanitation of Bivalves
Ref: 'I. C. Nelson, J. Morphology 63, 33 (1938)
26-7
-------
to
Oi
I
00
','c ',c<~ie, ,c
a-oicfl mooQ sesse
ro Cuooa-f e" ,
p.- -K J... '¦ Qrne.it—-¦
C*<. "Cf" O' ~ .CC —T
Abfron tai ctha
!
Bbod vessel of
pnnc.pat fi/sment
A/erae of
principal ft lament
S^eierai rod
"Than st'tonal filament
Ordinary flomg/tt
Utoter cfomber of
phca mto a/h/ch
ost/c op en
Ostium
Ostium eiponded
Lorera/ cma
Laterofrontal aha
' fine 'rontci ctho
Course f'cnr&t c '
-------
Ecology and Sanitation of Bivalves
REFERENCES
BOOKS
Cambridge Natural History Molluscs: good
for anatomy.
Dodgson, R.W. 1928. "Report on Mussle
Purification. " hTm. Stationary Office,
London, pp 498, Text figs. 12, PI. XV.
Abstracted by W.G. Savage in Bull, of
Hyg. 4(5) May 29, p. 450. "The report
is a most valuable one and should be pur-
chased and studied with close attention by
everyone who is concerned with the sub-
ject of shellfish pollution. " R.E. Tarbelt,
P.H. Eng. Abs. To which lecturer adds
that it is the outstanding work in any
language on this subject. It is understood
that volume long since out of print.
Kellogg, J. L. 1910. "Shellfish Industries. "
Henry Holt & Co. N.Y. pp. 361, Text
figs. 33, PI 67. Good general account
of anatomy of bivalves and of the shellfish
industry.
Zobell, C. 1946. "Marine Microbiology. "
pp 240, Text Figs. 12, Chronical Botanica
Co. Waltham, Mass. Excellent biblio-
graphy of 22 pages covering distribution
and role of bacteria in sea water. Parti-
cularly valuable in coverage of bacteria
in bottom deposits.
PAPERS
Bruce, J. R. 1928. "Physical Factors on
the Sandy Beach. II. Chemical Changes -
Carbon Dioxide Concentration and Sulphides."
J. MARINE BIOL. ASSN. U.K. 15:553-565.
Collier, A., Ray, S.M., Magnitzky, A.W. ,
and Bell, J.O. 1953. "Effect of Dissolved
Organic Substances on Oysters." Fishery
Bulletin #84, U.S. Fish and Wildlife
Service.
Dodgson, R.W. 1936. "Shellfish and the
Public Health. " Brit. Med. Jour. 3942:
169-173. U.S.P.H. Eng. Abs. 17:Mi 13
(4/ 10/37). 1937. Same title. U.S.P.H.
Eng. Abs. 18:Mi 37 (11/27/37). Empha-
sizes necessity of warming water to 540F
for self purification of oysters and
obstruction of process by traces of CI and
chloramines. 1938. "The Purification of
Polluted Shellfish. " Jour. Roy. San. Inst.
58:490-499. U. S. P. H. Abs.' 18 Mi 41
(10/15/38).
Reviews history of self-purification of
mussels and oysters noting differences
between them. Emphasizes role of
barnacles attached to oysters in rein-
fection of cleansed shell stock.
Dugal, L. P. 1939. "The Use of Calcaerous
Shell-to Buffer the Product of Anaerobic
Glycolysis in Venus Mercenaria. " J. Cell,
and Comp. Physiol. 12(2):235-251, Text
figs. 10: Explains superior keeping
qualities of quahogs with high glycogen
content through anaerobic glycolysis.
Acid end products thereof are buffered
by solution of calcium from inside of
shell thus keeping pH constant.
Galtsoff, P.S. 1928a. "The Effect of
Temperature on the Mechanical Activity
of the Gill of the Oyster, (Ostrea Virginica)"
J. Gen. Physiol. 11(4):451 -43 1, Text
figs. 6.
1928b. "Experimental Study of the
Function of the Oyster Gills and its
Bearing on the Problems of Oyster
Culture and Sanitary Control of the
Oyster Industry. " Bull. U.S. Bur.
Fish. 44:1-39, Text fig. 11. In both
papers discharge of water measured
in tube inserted into cloacal chamber
with promyal chamber blocked by
cotton. No flow observed below 50C,
optimum between 25 - 30OC. with
maximum discharge of 3.9 liters per
hr. at 250 C. 1936. "Biology of the
Oyster in Relation to Sanitation. " Am.
J. Pub. H. 26(3) 245-247.
Hughes, E. 1939, "A Method of,Oyster
Purification with a Note of the Breeding
of Oysters in Tanks. " Bull, of Hyg.
15(4):241. U.S.P.H. Eng. Abs. 20:Mi 45.
Purification of Oysters with sea water
stored for 10-12 days, sedimentation
reducing original coliform content to zero
in 100 ml.
26-9
-------
Ecology and Sanitation of Bivalves
Krumwied, C. et al. 1928. "The Purification
of Contaminated Oysters in Natural Waters. "
Am. J. Pub. H. 18(l):48-52. U.S.P.H.
Eng. Abs. 8 Mi 12. Conclude that polluted
oysters transferred to clean natural waters
should remain for four weeks to give wide
margin of safety.
Loosanoff, V.L., and Trommers, F. D. 1948.
"Effect of Suspended Silt and Other Sub-
stances on Rate of Feeding of Oysters. "
SCIENCE 107:69-70.
Lund, E.J. 1957. "A Quantitative Study of
Clearance of a Turbid Medium and Feeding
by the Oyster. " pp 296-312. 9 Figs.
"Self-silting, Survival of the Oyster as a
Closed System, and Reducing Tendencies
of the Environment of the Oyster, "pp 313-
319. 1 Fig.
"Self-silting by the Oyster and its
Significance for Sedimentation Geology. "
pp 320-327, 3 Figs. INSTITUTE OF
MARINE SCIENCE, 4:(2) July, 1957.
Nelson, T.C. 1918. "On the Origin, Nature,
and Function of the Crystalline Style of
Lamellibranchs. " J. Morphology 3 1:53-
111. Ph.D. Thesis. Describes the first
time formation and rotation of the crystal-
line style and its role in separation of
food from nonfood in the bivalve stomach.
Explains presence of undigested plankton
in faeces due to incomplete separation,
not to undigestibility of the organisms.
1921a. Report of the Dept. of Biology, N.J.
Agr. Exp. Sta. for 1920-21. Covers many
subjects including: world's first study of
oyster feeding habits in nature with kymo-
graph tracings of shell movements; the
importance of freshwater discharge in
maintaining nutrient salts used by oyster
food; seasonal variation in food of the
oyster.
1921b. "Some Aspects of Pollution as
Affecting Oyster Propagation. Am. J. Pub,
H. 11:498-501. Emphasizes destruction
of the biological mechanism of self-
purification of waters through industrial
effluents, thus extending zone further
seaward of bacterial contamination of
oyster beds.
1923a. "On the Feeding Habits of Oysters. "
Proc. Soc. Exp. Biol. & Med. 21:90-91 (b).
"The Mechanism of Feeding in the Oyster. "
(Same number) pp 166-168. (Both papers
covered in lecture material).
192 8. "Ciliary Activity in the Oyster. "
Science 64: ( 1646): 72.
1935. "Water Filtration by the Oyster and
a New Hormone Effect Thereon. " Abs.
Anatomical Record 64:68.
1936. "Water Filtration by the Oyster and
a New Hormone Effect on the Rate of Flow. "
Proc. Soc. Exp. Biol. &. Med. 34:189-190.
These papers describe rubber apron
method of measuring oyster's water output
with maximum of 26 liters per hour and
increased flow resulting from addition
of fresh sperm.
1937a. "Methods in the Protection and
Harvesting of Shellfish. " Pub. Health
News. N.J. Dept. Health 2 1: 76-79.
1937b. "Some Aspects of Pollution as
Affecting Shellfisheries in New Jersey. "
N.J. Municipalities 14:29 - 30.
1938. "The Feeding Mechanism of the
Oyster. I. On the Pallium (mantle) and
branchial chambers of Ostrea Virginica,
O. Edulis, and O. Angulata, with
comparisons with other species of the
genus. J. Morphology 63( 1): 1-61, 21
Figs. The fundamental anatomy of the
oyster as covered in the lecture.
Nelson, T.C. and Allison, J. B. 1940. "On-
the Nature and Action of Diantlin; a New
Hormone-like Substance Carried by the
Spermatozoa of the Oyster. " J. Exp.
Zool. 85(2) 299-338, Text figs. 12. Deals
with control of water flow through oysters.
Orton, J. H. 1.928. "The Biology of Shell-
fish in Relation to Public Health. " J. Roy.
San. Inst. 49(5) 263-274. U.S.P.H. Eng.
26-10
-------
Ecology and Sanitation of Bivalves
Abs. 9: Mi 11 (3/30/29). Discusses
structure and physiology of bivalves in
relation to purification and calls for a
clearly defined and recognizable standard
of their purity.
Round, L. A. 1914. Contributions to the
Bacteriology of the Oyster. Rpt. R. Id.
Comm. Shellfisheries, Providence, pp 88.
Found active elimination of bacteria from
oysters at 9oc but no reduction at 50 C
until after 5 days. Some slight pumping
in some oysters below 50 C hence hiber-
nation in oysters a relative term.
Yonge, C.M. 1923. "Studies on the Comparative
Physiology of Digestion. I. The Mechanism
of Feeding, Digestion, and Assimilation
in the Lamellibranch Mya. " Brit. J. Exp.
Biol. I. 15-63, Text figs. 27.
1926. "Structure and Physiology of the
Organs of Feeding and Digestion in
Ostrea Edulis. " J. Mar. Biol. Assoc.
Plymouth. 14(2): 295-386. Text figs. 42
These two papers by the foremost
malacologist (specialist on molluscs) in
the world are the outstanding contri-
butions to our knowledge of Mya and of
the European oyster.
Zobel, C.E., and Landon, W.A. 1937.
"Bacterial Nutrition of the California
Mussel. " PROC. SOC. EXP. BIO. &
MED. 36: 607-609.
26-11
-------
CONTAMINANTS OF SHELLFISH
Part 2
I INTRODUCTION
A This outline deals with the effects of
contaminants in the marine environment
on shellfish, and their uses as human
food.
B Contaminants enter shellfish from the
marine environment primarily via two
routes;
1 The body surface - gaseous exchange,
and elements in the ionic state as well
as small molecules may enter directly
through the soft body surface.
2 The digestive tract - carried in by
•feeding and absorbed either by the
mucus, as part of the food, or as
separate entities.
C Mollusks are particularly vulnerable to
salinity and other environmental chemical
changes.
1 Marine species are in osmotic equili-
brium with their environment.
1- Shellfish are a renewable, manageable
natural resource of significant economic
value to many coastal communities,
and should be treated as arc other
natural resources such as forest,
water and agricultural lands.
2 Shellfish culture and harvesting re-
presents a beneficial use of water in
the estuaries. This should be recog-
nized by State and Federal Agencies in
planning and carrying out pollution
prevention and abatement programs
and in comprehensive planning for the
use of these areas.
¦3 The goals of the National Shellfish
Sanitation Program are:
a The continued safe use of this
natural resource and
b Active encouragement of water
quality programs which will pre-
serve and restore all possible
coastal areas for this beneficial use..
2 Some resist the temporary tidal changes
in salinity by closure of the shell valves.
II ESTUARINE - SHELLFISH MANAGEMENT
According to Tarzwell's (1963) concept:
contamination is the occurrence of any
material in or any change in character of
shellfish that interferes with, lessens, or
destroys their use as a human food item.
A The need for recognition of estuaries as
a national resource, particularly the
shellfish which abound in these estuaries,
has been highlighted recently.
B The following principles will aid in estu-
arine and shellfish management.
Ill CONTAMINANTS OF SHELLFISH
A Marine Biotoxins - Among the several
types of biologically produced marine
toxins, paralytic shellfish poison (PSP),
found in shellfish of northern waters, is
the most frequent and most studied.
1 PSP is also called saxitoxin or mussel
poison.
2 Another marine biotoxin found in
shellfish and associated with "red tides"
is produced by the dinoflagellate
Gymnodinium breve. It is believed that
this poison is identical to the fish poison
known as Cignatera.
B Bacterial Accumulation b^ Shellfish
SS.62a 9. 70
26-13
-------
Contaminants of Shellfish
1 Many bacterial diseases, such as
typhoid fever, are transmitted to
humans from eating contaminated
shellfish.
a Bacterial accumulation is correlated
to pumping activity and temperature.
b Oysters may filter only 30% of known
numbers of E. coli out of the water.
2 The influence of shucking, packing
and storage upon bacterial growth in
the shucked product is of prime
consideration.
a Time and temperature are the two
prime factors; fortunately they can
be controlled to certain practical
limits.
b It is important that the shellfish
and the shucked'products be pro-
cessed as soon as possible and
stored at low temperatures.
C Effects of Pollution from Waterfowl
1 In Great South Bay, Long Island, over-
fertilization of the waters from duck
farm wastes led to great blooms of
undesirable plankton which interferred
with the feeding of oysters.
2 High counts of fecal streptococci in
certain oyster-producing tidal streams
of Delaware were attributed to the
large numbers of waterfowl nesting
in the adjacent marshes.
D Transmission of Human Viruses
1 Evidence has been established for the
transmission of infectious hepatitis by
raw shellfish.
2 The common mussel (Mytilus edulis)
can become contaminated by the polio-
myelitis virus. Someoysters can take
up the polio virus in two hours, but
self-purification did not occur in ex-
periments of 6-day's duration.
E Suspended-Silt
Water-borne sediments, as pollutants
of the shellfish environment, are important
because they interfere with the process of
water-pumping and feeding.
1 Concentrations of 0. 25 grams/l of
silt reduced pumping of oysters by
57%; 0.5 g by 68%, 1 g by 81% and
3-4 g/l by 94%.
2 In open areas, tidal and other water
currents sweep away silt; in sheltered
areas deposits accumulate, to cover
shellfish.
F Pesticides
1 DDT in sufficient quantities kills
oysters, but of more importance,
lesser amounts of DDT seriously dis-
rupt the normal activity of the oyster.
a DDT levels may be so low as to be
barely detectable in the seawater
with no apparent affect on the
oysters, but the DDT may make the
oyster unpalatable.
b Oysters may concentrate DDT in
their tissues 20,000 times greater
than that in the seawater within 7
days exposure.
2 Even at concentrations well below 1.0
ppm most chlorinated hydrocarbon
insecticides - including aldrin, DDT,
dieldrin, andrin, and lindane -
markedly inhibit shell growth.
3 There is a great difference in the
toxicity of insecticides to bivalve
larvae.
a At a concentration of 0.05 ppm,
DDT caused over 90% mortality
of oyster larvae and almost entirely
prevented growth.
b In 5.0 ppm lindane the growth of
clam larvae was somewhat faster
than in control cultures.
26-14
-------
Contaminants of Shellfish
G Reaction of Oysters to Chlorinated
Sea water
1 Oysters respond to an initial contact
with chlorine (concentrations between
0. 01 and 0. 2 ppm) by cessation of
feeding current and shell closure.
2 Apparently tolerance is developed to
repeated exposure and the valves may
remain open and pumping of water may
continue at concentrations greater than
those which caused the initial response.
H Metals, Particularly Radionuclides
Shellfish have a propensity for the uptake,
accumulation, and storage for several
months of heavy metals, including their
radionuclides.
1 Certain elements, particularly trace
metals such as copper and zinc, are
essential for maintenance of living
systems, but when they exceed normal
concentrations in the environment, as
from industrial wastes, they can be
exceedingly toxic.
2 Although mollusks may contain rela-
tively high levels of metallic radio-
nuclides, they do not constitute a
health hazard.
3 An outbreak of a severe neurologic
disorder of humans in Minamata,
Japan, during the 1950's was related
to the ingestion of seafood contaminated
by mecuric compounds.
I Effects of Hydrocarbons
1 In Louisiana, accidental oil spillage
did not kill any oysters on commercial
beds, but it did render them unpalatable
for periods from several days to several
weeks.
2 Rates of feeding are affected by
hydrocarbons.
J Pulp Mill Wastes
1 Pulp-mill waste was determined to
be the principal cause of a steady
decline since 1916 in the productivity
of oyster beds in the Upper York River,
Virginia. The pulp-mill effluent
reduced the total time the oyster shells
were open and also depressed the rate
of water-pumping.
2 Kraft (sulfate) pulp-mill wastes contain
more toxic constituents than sulfite
pulp-mill waste liquors per unit volume.
3 Three ways in which pulp-mill wastes
are harmful to marine life:
a Direct toxicity
b Indirect effect through reduction of
of oxygen in the water
c Long-term effects on the bottom
and water adjacent to it.
K Other Contaminants
1 pH - at low pH levels (4. 25), oysters
show abnormal shell movement and
reduced pumping.
2 Rhodamine B dye - hard clams showed
normal siphoning only at concentrations
of 4. 7 mg/l or less.
REFERENCES
1 Loosanoff, V.L. and Tommers, F. D.
Effect of Suspended Silt and Other
Substances on Rate of Feeding of
Oysters. Science, 107( 2768): 69-70.
1948.
2 Mason, J.O. and McLean, W. R. In-
fectious Hepatitis Traced to the Con-
sumption of Raw Oysters: An
Epidemiologic Study. American
Journal of Hygiene, 75(1):90-111.
1962.
26-15
-------
Contaminants of Shellfish
3 Nelson, T.C. Some Aspects of Pollution,
Parasitism and Inlet Destruction in
Three New Jersey Estuaries. Trans-
actions, 1959. Seminar on Biological
Problems in Water Pollution, PHS,
DHEW: 203 -211. 1960.
4 Tarzwell, C. M. Research for the De-
velopment of Water Quality Criteria
at the Narragansett Bay Marine
Laboratory Minutes, 22nd Annual
Meeting, Atlantic States Marine Fish-
eries Cpmmission, Appendix 5, 1963.
pp. 91-100.
This outline was prepared by Carl N.
Shuster, Jr., Director, Northeast Research
Center, Shellfish Sanitation Branch,
Division of Environmental Engineering and
Food Protection, BSS, PHS, DHEW,
Narragansett, Rhode Island.
26-16
-------
THE DEVELOPMENT OF BACTERIOLOGICAL STANDARDS
FOR SHELLFISH GROWING AREAS
I INTRODUCTION
A Responsibilities
The National Shellfish Sanitation Program
is a cooperative program between the
shellfish industry and both state and
federal agencies responsible for the
sanitary control of shellfish shipped in
interstate commerce. This program
has been in existence since 1925.
B Early Considerations
The possible association between the
consumption of raw shellfish and the
incidence of Typhoid Fever was sus-
pected for many years. As early as '
1902, following an outbreak of Typhoid.
Fever, Dr. Gurion stated in Public
Health Bulletin No. 86: "there is a zone
of pollution established by the mere fact
of the existence of a populated city upon
the banks of a stream or tidal estuary
which makes the laying down of oysters
and clams in these waters a pernicious
matter." The difficulty, in these early
days, of isolating pathogens and the lack
of knowledge pertaining to the indicator/
pathogen relationship resulted in the
collection of masses of data aimed at
establishing a safe level of indicator
organisms or determining the population
levels that would reduce to an insignificant
degree the health hazard potential
associated with the consumption of raw
shellfish.
II HISTORICAL
A Development of Test Procedures
The early studies of pollution indicating .
bacteria resulted in the development of
the multiple tube fermentation test for
Bacillus coli (formerly referred to as
the "coli-aerogeries" group and now known
as the coliform group of bacteria).
Historically, the development of this
test procedure can be summarized as
follows:
1 With the publication of "Elements of
Water Bacteriology" (Prescott and
Winslow^, 1904) two basic concepts
were stated which laid the ground-
work for the development of
bacteriological standards for shell-
fish growing areas:
a The detection of B. coli in a large
proportion of 1 cc volumes or
dilutions less than 1 cc is an
indication of recent pollution..
b The finding of B. coli in large
samples of water or in an
occasional small sample does
not have any special significance.
No' attempt was made to define
the term "large proportion"
however, the concept of using the
presence of the bacterial indicator
in 1 ml volumes to imply the
existence of a possible health
hazard is well established.
2
2 Eijkman proposed a test for
pollution indicator organisms in
1904 which recommended glucose
as the energy source and an incubation
temperature of 46° C. It was his
contention that "coli bacteria"
associated with warm blooded animals
would grow under these conditions
but that related organisms isolated
from soil, plants, and so called
"safe" waters would not survive at
that temperature. Thus the elevated
temperature test became the Eijkman
test, the forerunner of what is known
today in sanitary microbiology as the
"fecal coliform" test.
FO.SH.ga. 14b. 9. 70
27-1
-------
The Development of Bacteriological Standards for Shellfish Growing Areas
Prior to the development of the MPN
tables, a "score" system was devised
to denote sanitary quality. This
number system was based on the
number of positive tubes and the
dilution factors but it appears, at this
time, that the primary interest in
standards within the control agencies
was oriented toward shellfish,meats
and not the growing areas.
The table below comparing "Coliform
Organism Scores" with MPN Values,
was taken from the 1946 Manual of
Recommended Practice for Sanitary
Control of the Shellfish Industry
(Public Health Bulletin 295).
Mr. Furfari states: "I suspect this
(the score system) might have con-
tributed to a delay in the ultimate
derivation of a standard for water. "
COMPARISON OF COLIFORM ORGANISM
SCORES WITH MOST PROBABLE NUMBER
OF COLIFORM ORGANISMS PER
100 MILLILITER.
1 20
2 45
3 ¦. 78
4 130
5 230
14 330
23 490
32 790
41 1,300
50 2,400
140 3, 500
230 5,400
320 9,200
410 16,000
500 24,000
1, 400 35, 000
MPN tables for evaluating coli-
aerogenes tests were published by
Hoskins4 in 1934.
B Development of Standards
1 Following the 1924 outbreak of
Typhoid Fever a Committee on the
Sanitary Control of Shellfish in the
United States was formed .and the
c
Frost Report given in 1925, stated-
"The committee is not prepared to
recommend any precise bacterial
standards for waters from which the
taking of shellfish is permitted until
additional data which are now being
collected have been assembled and
considered. In the light of present
knowledge it would probably be unfair
and unnecessary to apply to such
waters the rigid standards which are
applied to the drinking water supplied
in interstate commerce. It is
considered, however, that the waters
should ordinarily not show the
presence of B. coli in lcc amounts,
test for B. coli being made in 10 cc,
1 cc, and 0. 1 cc amounts, according
to the Standard Methods of the
American Public Health Association. "
Had the word "ordinarily" been left
out of this statement and the rec-
ommendations, as proposed, been
accepted,the maximum allowable
count for "open" growing areas
would have been equivalent to an
MPN of 23.
2 In October 1926 a Subcommittee on
Bacteriological Examination was
formed and requested to review
recommendations relating to the
techniques, significance, and uses
of bacteriological examinations.
A tentative report of this Subcommittee
is quoted as follows:
"Areas which may be approved for
the taking of shellfish without serious
question. This class includes the
areas which are so protected against
human fecal contamination by distance
from sources of such pollution, by
27-2
-------
The Development of Bacteriological Standards for Shellfish Growing Areas
dilution and by the time afforded for
natural purification, that there is no
discoverable likelihood of dangerous
contamination. On bacteriological
examination such waters may be
expected to show the presence of
organisms of the coliform aerogenes
group more or less frequently in 10 cc
portions but usually not in 1 cc or
smaller amounts* (original italics).
The footnote reads: "*The specification
is given above as it was formulated
in conference. It has since been
proposed that it be amended to read:
but not in the majority of 1 cc portions.
Members of the sub-committee are
requested to express their preference
as between the original and the
amended forms. "
3
Furfari , at this point, states that:
"the thinking on the proportion of 1 cc
sample has progressed from not
ordinarily show to usually not (more
or less frequently) to but not in the
majority.
7
3 In 1933 Carl Green reported that
some states were specifying maxi-
mum "scores" of 2.3 and 3.6 for
water standards. Equivalent MPN
values for these "scores" would be
45 and 130/100 ml sample. He also
reported that other states were
recommending a maximum "score"
of 2. 5, an MPN equivalent of approx-
imately 62/ 100 ml, or not more than
50% of the 1 ml portions. Green also
urged that the USPHS establish a
nation wide standard. At this time
the concept of 5 0% of 1 ml portions
as a possible working standard for
shellfish growing areas was rapidly
becoming a real possibility.
g
4 Miller stated in a publication in 1935,
"The Public Health Service. . . .has
arrived at the conclusion that generally
not more than 50% of the 1 cc tubes in
an area should show the presence of
B. coli if that area is to be used for the
taking of shellfish for market. The
word "generally" was not defined.
The precedent, therefore, had been
set for "weasel words" in future
standards.
9
5 Dr. C. A. Perry , referee of the
A.P.H.A. committee on bacterio-
logical methods made the following
proposals for changes in the standard
procedure for the examination of
shellfish in 1936 ¦
a The new procedure should include
such edible mollusks as oysters,
clams and mussels.
b Escherichia coli rather than the
colon group should be the index
of pollution for both shellfish and
shellfish waters.
c A new procedure should include
methods for the examination of
shellfish waters as well as shell-
fish .
d The whole oyster rather than just
the shell liquid should be examined.
e Escherichia coli results should
be expressed as most probable
numbers rather than as a score.
f Certain recommendations should
be made in regard to amount of
pollution which should ordinarily
be tolerated.
6 In separate publications in 1937
Miller^ and Shea"'' * referred to the
"unwritten standard" of not more
than 50% of 1 cc positive tubes.
Sheas' report stated that-
"The United States Public Health
Service has arrived at the conclusion
that waters used in the production of
shellfish for the market should in
general not show the presence of coli-
aerogenes organisms in 1 cc portions
of samples more than fifty percent
of the time. "
27-3
-------
The Development of Bacteriological Standard's for Shellfish Growing Areas
It is interesting to note that the
Minimum Requirements for Endorse-
ment of State Shellfish Control
Measures and Certifications for
Shippers in Interstate Commerce ,
published in 1937, did not give a
bacteriological standard for shellfish
growing areas.
7 In 1941 a study was completed and the
results published in "A Report on the
Public Health Aspects of Clamming in
Raritan Bay. ^ By comparing
epidemiological data fo known cases
of typhoid presumed to be of shellfish
origin with coliform populations of
shellfish and shellfish waters, it was
concluded that "70 coliforms/100 ml,
on the basis of average results from
waters overlying the shellfish beds, is
recommended as the limiting allowable
standard for the taking of hard clams
to be eaten raw. "
8 The 1946 publication of the Manual
of Recommended Practice for Sanitary
Control of the Shellfish Industry^
defined the bacteriological criteria
of growing areas as follows: "The
median bacteriological content of
samples of water. . . . shall not show
the presence of organisms of the
coliform group in excess of 70/100 ml
of water. "
9 The present bacteriological standard
for approved and conditionally approved
shellfish growing areas, as stipulated
in the 1965 Revision of the National
Shellfish Sanitation Program Manual
of Operations, Part I , is as follows:
a The coliform median MPN of the
water does not exceed 7 0/100 ml.
b Not more than 10% of the samples
ordinarily exceed an MPN of
230/100 ml for a 5 tube decimal
dilution test (or 3 30/ 100 ml where
the 3 tube decimal dilution test is
used) in those portions of the area
most probably exposed to fecal
contamination during the most
unfavorable hydrographic and
pollution conditions.
c These foregoing limits need not be
applied if it can be shown by
detailed study that'the coliforms
are not of fccal origin-and do not
indicate a public health hazard.
10 A supplementary standard for an
approved growing area was recom-
mended by the 5th National Shellfish
Sanitation Workshop16 in 1964.
It states, "In an approved shellfish
growing area, a median fecal
coliform MPN of 7. 8 shall not be
exceeded and not more than 10%
of all samples tested shall exceed
an MPN/100 ml in excess of 33
(46/ 100 ml where the 3 tube decimal
dilution test is used). This recom-
mendation is still under study.
REFERENCES
1 Prescott, S. C. and Winslow, C.A.
Elements of Water Bacteriology.
John Wiley. 1904.
2 Eijkman, C. Die Gartingsprobe bei
46° als Hilfmittel bei der
Trinkwasseruntersuchung, Central-
blatt f, Bakt. Ab I (o):37:742, 1904.
3 Furfari, S.A. History of the 70/100 ml
MPN Standard. Memorandum to:
Chief, Water Resources Program,
NCUIH. 1968.
4 Hoskins, J.K. Most Probable Numbers
of Evaluation of coli-aerogenes tests
by Fermentation Tube Method.
Public Health Reports. 49:393.
Reprint 1621. 1934,
5 Frost, W.H. Report on Committee on
Sanitary Control of the Shellfish
Industry in the United States.
Supplement No. 53 to the Public
Health Reports. November 6.
(G.P.O.) 1925.
6 Subcommittee on Bacteriological
Examinations (1927a). Tentative
Draft for Report of Subcommittee
on Bacteriological Examinations.
27-4
-------
The Development of Bacteriological Standards for Shellfish Growing Areas
7 Green, Carl E. The Sanitary Control
over the Production and Handling of
Shellfish on the Pacific Coast. Am.
J. of Public Health. 23, No. 9, 895-
900. September 1933.
8 Miller, Arthur P. A Study of the
Pollution of a Shellfish Producing
Area. Paper presented at the
New Jersey Sewage Works Association
Meeting, Trenton, New Jersey.
March 19, 1935. Also in Sewage
Works Journal 8, No. 4, 634-646.
July 1936 and Am. J. of Public Health,
26, 970-978. October 1936.
9 Examination of Shellfish for Fecal
Pollution . Committee Report.
APHA Yearbook pp. 111-117. 1935-36.
10 Miller, Arthur P. Wastes Disposal as
Related to Shellfish. Paper presented
attheN.E. Sewage Works Assoc.
Meeting, Providence, R.I. April 26,
1937. Also in Sewage Works Journal
9, No. 3, 482-492. May 1937.
11 Shea, Walter J. Progress in Controlling
Pollution of Rhode Island Waters.
Paper presented at N. E. Sewage Works
Assoc. Meeting, Providence, R.I.
April 26, 1937. Also in Sew. Wks.
Journ. No. 3, 493-499. May.
12 U.S. Public Health Service. U.S. Public
Health Service Minimum Requirements
for Endorsement of State Shellfish
Control Measures and Certification
for Shippers in Interstate Commerce.
Mimeographed document forwarded
by Thomas Parran, Surgeon General,
to State Health Officers. November 5,
1937.
13 Kehr, Robert W., Levine, BenjaminS.
Butterfield, C,T. and Miller,
Arthur P. A Report on the Public
Health Aspects of Clamming in
Raritan Bay. Public Health Service
Report. 1941. Reissued in June
1954 by Division of Sanitary
Engineering Services, PHS, DHEW.
' 14 Manual of Recommended Practice for
Sanitary Control of the Shellfish
Industry. Public Health Bulletin
No. 295. 1946.
15 National Shellfish Sanitation Program
Manual of Operation, Part I. 1965.
16 Beck, W.J. Bacteriological Criteria
for Shellfish Growing Areas.
Proceedings of the 5th National
Shellfish Sanitation Workshop. 1964.
This outline was prepared by D. A. Hunt,
Chief, Standards Development Section,
Shellfish Sanitation Branch, Bureau of
Compliance, FDA.
27-5
-------
SHELLFISH GROWING AREA .SURVEYS
I INTRODUCTION
A Important Public Health Concern
The safety of a raw food resource depends
in large measure on the water quality
criteria used and its application to deter-
mine approved shellfish growing areas.
(Entire shellfish, excluding shell, of
course, is often consumed in the uncooked
state.)
B Reliability a Part of Criteria
Since the safet} of the raw food product
has to be consistently met, the water
criteria has to be one that is also con-
sistently met and not ]ust one that is satis-
fied on the average, as is the case in
many other design or survey situations.
The question of variability is covered in
the phrase, "most unfavorable hydro-
graphic and pollution condition, " which
have to be considered in the criteria and
which, unfortunately, are most often
overlooked.
C Four Types of Contamination
As indicated in previous lecture the types
of contamination which have to be con-
sidered in area studies may be considered
in four categories:
1 Bacteriological - Including virus
(infectious hepatitis), parasites (E.
histolytica), etc.
2 Biological - Paralytic shellfish poison
or other marine biotoxins
3 Chemical - Metals, pesticides, etc.
4 Radiological
D Four Groups of Area Classification
Reference should be made to Part I of the
manual on "Sanitation of Shellfish Growing
Areas, " 1965 Revision of PHS Publication
No. 33, for details of the following classes
and to Figure 1 in the publication and
Figure 8 attached which shows sche-
matically some possible interrelationships.
1 Approved areas - Survey indicates
shellfish from area maj be used as
raw food product.
2 Conditionally approved areas - Survey
indicates that shellfish from area may
be used as a raw food product provided
certain specified and controlled conditions
are met, i.e., proper control of
treatment plant effluents.
3 Restricted areas - Surve> indicates
that shellfish from the area may be
used as a raw food product only when
some further practice is employed to
improve the product before marketing.
Product may be relayed into an approved
area for a specified time, or it may be
processed in a depuration plant.
4 Prohibited areas - Survey indicates
that shellfish from the area should not
be used, except that salvage operations
may be permitted under extensive and
detailed supervision and control.
E Frequency of Survey - A Criteria
The proper area classification also
depends upon how often an area is sur-
veyed and what type of survey is made.
1 Unsurveyed - Automatically prohibited.
"All actual or potential growing areas
which have not been subjected to
sanitary surveys shall be automatically
classified as prohibited. " (Part I,
Section C, Item 2d, of manual. )
(An area is considered "guilty" until
proved otherwise. The purpose of the
survey is to determine what portion of
the area may not be guilty of con-
tributing to a public health hazard.)
FO. SH. ga. 13a. 9. 70
28-1
-------
Shellfish Growing Area Surveys
2 Ten-year intervals - Complete resurvey
required every ten years for area in an
approved category (approved, condi-
tionally approved, or restricted).
3 Two-year intervals - A reappraisal is
made of each such approved category
area every two years to determine if
there has been any change in the factors
influencing the sanitary quality1 of the
area. If any change is determined, then
a complete survey is needed to support
area classification.
4 Wet storage area - Yearly - "The
temporary storage of shellfish from
approved sources, intended for market-
ing, in tanks containing sea water or in
natural bodies of water, including
storage in floats. "
n SURVEY BASED ON BACTERIOLOGICAL
CRITERIA
A Two General Types of Pollution Sources
1 Remote^ pollution source from a
community - "The coliform median
MPN of the water does not exceed
70 per 100 ml, and not more than
10 percent of the samples ordinarily
exceed an MPN of 230 per 100 ml for
a 5-tube decimal dilution test (or 330 per
100 ml where the 3-tube decimal
dilution test is used) in those portions
of the area most probably exposed to
fecal contamination during the most
unfavorable hydrographic and pollution
conditions. " (See Figure 6)
a Margin of safety - The present'
standard has passed the test of
"it works" although the epidemiological
investigations have not established a
direct numerical correlation between
the bacteriological quality of the
water and the degree of hazard to
health.
b Background of standard and meaning -
Although the level of bacteria in
shellfish is related to the level in
the water, it varies for species and
seasons (see Figures 1, 2, and 3).
The standard is based upon the more
easily tested overlying waters and is
considered as indicating the relative
amounts of pollution and possibility
of the presence of pathogenic
organisms.
2 Direct source - For small sources
discharging directly to a growing area,
such as from boats or shoreline homes,
the bacteriological indicator is no
longer useful, numerically, in area
classification or in giving an indication
of the safety of the area. In the direct
source situation' it becomes a matter
of evaluating the chance of the problem
existing in a given situation and its
relationships to the probable amount
of product that might be affected. For
example, more distance should be
required as protection around a direct
pollution source where wet storage or
even relaying was to be practiced as
compared with a similar use of the
area as a natural bed.
B Source of Contamination - Starting Point
of Problem and Survey
1 Area study - Determine location and
type of existing and potential pollution
sources. This includes sources of
untreated wastes, treated wastes,
pumping stations, industrial waste
sources, sludge dumps, small direct
waste sources from shore, marinas,
boat anchorages, etc.
2 Determine most unfavorable pollution
condition - This has to be evaluated
for each pollution source, both existing
and potential. In some cases, as is
the case for resort areas, the seasonal
pattern has to be determined. For
sewageitreatment an analysis has to be
made of the various units and a deter-
mination made of the level of operation
that might be consistently relied upon.
28-2
-------
Shellfish Growing Area Surveys
(Any operation below this level is
cause for closure of a portion of the
approved area).
C Hydrography
1 Tidal currents - Study tidal pattern for
extreme conditions and study velocity
and direction of current relationship to
tidal phase.
2 Wind-driven currents - Determine the
effect of wind direction and magnitude
on the tidal current pattern.
3 Estuarine circulation patterns -
Determine by salinity cross-section
studies the type of estuarine circulation
pattern in the growing area.
4 Determine fresh water run-off - Study
fresh water, run-off levels for extreme
conditions.
5 Determine most unfavorable hydrographic
condition - With knowledge of the area
determine what hydrographic situation or
situations would create the most
unfavorable pollution level condition in
the shellfish growing area.
D Water Quality Study
1 Special standard methods - "Recommended
Procedures for the Bacteriological
Examination of Sea Water and Shellfish, "
3rd Edition, 1962, of the American
Public Health Association. (See
Figures 6 and 7.)
2 Location - Need to establish stations
for sampling which may be re-occupied
for serial sampling.
3 Determine efficiency of receiving water
as treatment process - Determine the
fate of a conservative (not time variable)
pollutant (Figure 4) and non-conservative
(time variable) pollutant (Figure 5) in
the body of receiving water.
a Bacteriological survey - If possible
conduct study during period when
most unfavorable conditions occur;
otherwise, extrapolate data from
survey conditions to most unfavorable
situation.
b Time in transit studies - Determine
transit time from pollution source
to various parts of shellfish area.
E Area Classification (See Figure 8)
1 Analyze data and determine pollution
levels under most unfavorable situations
(Figures 9 and 10).
2 Conditionally approved areas - The
conditions have to be built into the
criteria. ;Some example of conditions
which must be met in order for areas
to remain in an acceptable classification
for direct harvesting are:
a Operation of sewage plant - At level
of operation always in excess of
specified level for area classification.
b Alarms on pumping stations to provide
notice of changed conditions.
c Seasonal considerations such as
increase in population and boat usage.
3 Restricted areas - "The coliform
median MPN of the water does not
exceed 700 per 100 ml and not more. . . "
III BIOLOGICAL CRITERIA
A Paralytic Shellfish Poison
Epidemiological investigations have
indicated that some 200 to 600 micro-
grams of poison will produce symptoms
in susceptible persons, and a death has
been attributed to the ingestion of a
probable 480 micrograms of the poison.
This is a naturally occurring poison.
28-3
-------
Shellfish Growing Area Surveys
B Establish Conditions 3
Areas should be surveyed to establish
conditions of occurrence. When determined,
control stations should be monitored in 4
accordance with prevailing pattern.
(Figures 11 and 12)
C Closure Level
Phelps, E.B. Public Health Engineering,
John Wiley &. Sons, Inc., New York.
1948.
American Public Health Association,
Recommended Procedures for the
Bacteriological Examination of Sea
Water and Shellfish, 3rd Edition. 1962.
"A quarantine is imposed against the taking
of shellfish when the toxicity reaches 80
micrograms per 100 grams of the edible
portion of raw shellfish. "
REFERENCES
1 Public Health Service, USDHEW. Part I:
Sanitation of Shellfish Growing Areas.
National Shellfish Sanitation Program
Manual of Operations, 1965 Revision.
2 Kehr, R.W. and Butterfield, C.T.
Notes on the Relation Between
Coliforms and Enteric Pathogens.
Reprint No. 2469 from the Public
Health Reports. Vol. 58, No. 15,
pp. 589-607. April 9, 1943.
This outline was prepared by Ronald G.
Macomber, formerly, Associate Director
for Field Operations Activity, Northeast
Shellfish Sanitation Research Center,
Shellfish Sanitation Branch, Division of
Environmental Engineering and Food
Protection, BSS, PHS, USDHEW,
Narragansett, Rhode Island.
28-4
-------
Shellfish Growing Area Surveys
FIGURE I
28-5
-------
Shellfish Growing Area Surveys
10,000
"I I I I I I I I
r,Tr
1 1—I—I I r I-
1,000
63
CO
M
d
s
w
w
o
BS
TEMPERATURE
8 - 170C.
100
o
10
_i I i i i I i i I
Ji i i ¦ ¦ « • *
20
100
AVERAGE WATER MPN
1000
FIGURE II
28-6
-------
Shellfish Growing Area Surveys
AVERAGE WATER MPN
FIGURE III
28-7
-------
Shellfish Growing Area Surveys
Co (1)
Cp * FIGURE IV
28-8
-------
Shellfish Growing Area Surveys
DAYS
FIGURE V
28-9
-------
Shellfish Growing Area Surveys
PERCENTAGE
FIGURE VI
28-10
-------
Shellfish Growing Area Surveys
Variation of Mean at One
Standard Error of Mean
^"Itean = d&l£_
Vh-1
5 Tube Std.
Mean = 70 MPN/100 ml.
10% -230 MPN/100 ml.
3 Tube Std.
Mean = 70 MPN/100 ml.
10% =330 MPN/100 ml.

N
CO
a
s
OS o
O O
rH
J
O ft
o %
«
« N
« 30
- N

«
*
*

o
o
O
O
o
o
O
O
o
o
o
O
o
<£
to
Tf
CO
-------
-------
Shellfish Growing Area Survcyb
PERCENTAGE
FIGURE IX
28-13
-------
Shellfish Growing Area Surveys
PERCENTAGE
FIGURE X
28-14
-------
Shellfish Growing Area Surveys
28-15
-------
Shellfish Growing Area Surveys
1958
1,000-
TOXICITY COMPARISON
Head Harbour, N.B.
0—0
Quoddy Bar, Maine
0 0
10C
8C ¦
Minimum Detection
20
20
-L.
0
K-
10 20
30
*
10 20 30
*
10
20
30
~
10
June
July
August
September
30
-W
FIGURE XII
28-16
-------
BIOLOGICAL ASPECTS OF NATURAL SELF PURIFICATION
I INTRODUCTION
A The results of natural self purification
processes are readily observed. Did they
not exist, sewage (and other organic' ¦.
wastes) would forever remain, and the
world as we know it would long ago have
become uninhabitable. Physical, chemical,
and biological factors are involved. The
microscopic and macroscopic animals
and plants in a body of water receiving
organic wastes are not only exposed to all
of the various (ecological) conditions in
that water, but they themselves create and
profoundly modify certain of those conditions.
B Since toxic chemicals kill some of or all
of the aquatic organisms, their presence
disrupts the natural self purification
processes, and hence, will not be considered
here. The following discussion is based
solely on the effects of organic pollution
such as sewage or other readily oxidizable
organic wastes.
C This description is based on the concept of
a "stream" since under the circumstances
of stream or river flow, the events and
conditions occur in a linear succession.
The same fundamental processes occur in
lakes, estuaries, and oceans, except that
the sequence of events may become
telescoped or confused due to the reduction
or variability of water movements.
D The particular biota (plants and animals,
or flora and fauna) employed as illustrations
below are typical of central United States.
Similar or equivalent forms occur in
similar circumstances in other parts of
the world.
E This presentation is based on an unpublished
chart produced by Dr. C.M. Tarzwell and
his co-workers in 1951. Examples from
this chart are employed in the presentation.
II THE STARTING POINT
A A normal unpolluted stream is assumed
as a starting point. (Figure 1)
B The cycle of life is in reasonably stable
balance.
C A great variety of life is present, but no
one species or type predominates.
D The organisms present are adjusted to the
normal ranges of physical and chemical
factors characteristic of the region, such
as1 the following:
1 The latitude, turbidity, typical cloud
cover, etc. affect the amount of light
penetration and hence photosynthesis.
2 The slope, cross sectional area, and
nature of the bottom affect the rate of
flow, and hence the type of organisms
present deposition of sludge, etc.
3 The temperature affects both certain
physical characteristics of the water,
and the rate of biological activity
(metabolism).
4 Dissolved substances naturally present
in the water greatly affect living
organisms (hard water vs. soft water
fauna and flora).
E Clean water zones can usually be
characterized as follows:
1 General features:
a Dissolved oxygen high
b BOD low
c Turbidity low
d Organic content low
BI. ECO. nap!5c. 12. 70
29-1
-------
Biological Aspects of Natural Self Purification
THE BIOTA
VI

10 .
3 4
DAYS
12 24 36 48 60 72 84 96 108
MILES
a4
CO
o
o
o
£
O
hH
Eh
3
£
eu
o
Ph
Figure 1: Relations between variety and abundance (production) of aquatic life,
as organic pollution (discharged at mile 0) is carried down a stream. Time
and distance scales are only relative and will be found to differ in nearly every
case. -After Bartsch and Ingram. ^
29-2
-------
Biological A spects of Natural Self Purification'
e Bacterial count low
f Numbers of species high
g Numbers of organisms of each species
moderate or low
h Bottom free of sludge deposits
2 Characteristic biota includes a wide
variety of forms such as:
a A variety of algae and native higher
(vascular, or rooted) plants
b Caddis fly larvae (Trichoptera)
c Mayfly larvae (Ephemeroptera)
d Stonefly larvae (Plecoptera)
e Damselfly larvae (Zygoptera)
f Beetles (Coleoptera)
g Clams (Pelecypoda)
h Fish such as :
- Minnows (Notropid types)
- Darters (Etheostomatidae)
- Millers thumb (Cottidae)
- Sunfishes and basses (Centrarchidae)
- Sauger, yellow perch, etc. (Percidae
- Others
3 Organisms characteristic of clean lakes,
estuaries, or oceanic shores might be
substituted for the above, and likewise
in the following sections. However, it
should be recognized that no single
habitat is as thoroughly understood in
this regard as the freshwater stream.
Ill POLLUTION
A With the introduction of organic pollution
(Figure 1, day 0), a succession of fairly
well organized events are initiated.
Important items to observe in interpreting
the pollutional significance of stream
organisms are the following:
B Numbers of species present, they tend to
decrease with pollution.
C Numbers of individuals of each species
tends to increase with pollution.
D Ratios between types of organisms are
disturbed by pollution.
1 Clean water species intolerant of
organic pollution tend to become scarce
and unhealthy.
2 Animals with air breathing devices or
habits tend to increase in numbers.
3 Scavengers become dominant
4 Predators disappear
5 Higher plants, green algae, and most
diatoms tend to disappear.
6 Blue green algae often become
conspicious
E The importance of observations on any
single species is very slight.
IV THE ZONE OF RECENT POLLUTION
A The zone of recent pollution begins with
the act of pollution, the introduction of
excessive organic matter: food for
microorganisms (Figure 1, day 0)
B There follows a period of physical mixing.
C Many animals and plants are smothered
or shaded out by the suspended material.
D "With this enormous new supply of food
material, bacteria and other saprophytic
microorganisms begin to increase
rapidly.
29-3
-------
Biological Aspects of Natural Self Purification
E The elimination of intolerant predatory
animals allows the larger scavengers to
take full advantage of the situation.
F This explosive growth of organisms,
particularly fungi and bacteria, draws
heavily on the free dissolved oxygen for
respiration, and may eventually eliminate it.
G The number of types of organisms diminishes
but numbers of individuals of tolerant types
may increase.
H Zone of degeneration, or recent pollution,
can usually be characterized as follows:
1 General features:
a DO variable, 2 ppm to saturation
b BOD high
c Turbidity high
d Organic content high
e Bacterial count variable to high
f Number of species declines from
clean water zone
g Number of organisms per species
tends to increase
h Other: Slime may appear on bottom
2 Characteristic biota:
a Fewer higher plants, but rank heavy
growth of those which persist
b Increase in tolerant green, and blue
green algae
c Midge larvae (Chironomidae) may
become extremely abundant
d Back swimmers (Corbddae) and water
boatmen (Notonectidae) often present
e Sludge worms (Tubificidae) common
to abundant.
f Dragonflies (Anisoptera) often present
have unique tail breathing strainer
g Fish types, eg:
- Fathead minnows (Pimephales
promelas)
- White sucker (Catostomus
commersonni )
- Bowfm (Amia calva)
- Carp (Cyprinus carpio)-
V THE SEPTIC ZONE
A The exact location of the beginning of the
septic zone, if one occurs, varies with
season and other circumstances.
(Figure 1, day 1)
B Lack of free DO kills many microorganisms
and nearly all larger plants and animals,
again replenishing the mass of dead
organic material.
C Varieties of both macro and micro-
organisms and adjustable types (facultative)
that can live in the absence of free oxygen
(anaerobic) take over.
D These organisms continue to feed on their
bonanza of food (pollution) until it is
depleted.
E The numbers of types of organisms is now
at a minimum, numbers of individuals
may or may not be at a maximum.
F The septic zone, or zone of putrefaction
can usually be characterized as follows:
1 General features:
a Little or no DO during warm weather
b BOD high but decreasing
c Turbidity high, dark; odoriferous
d Organic content high but decreasing
29-4
-------
Biological Aspects of Natural Self Purification
e Bacterial count high
f Number of species very low
g Number of organisms may be extremely
high
h Other: Slime blanket and sludge
deposits usually present, oily
appearance on surface, rising gas
bubbles
2 Characteristic biota:
a Blue green algae
b Mosquito larvae
c Rat-tailed maggots
d Sludge worms (Tubificidae and similar
forms). Small, red, segmented
(annelid) worms seem to be character-
istic of this zone in both fresh and
salt waters, the world around.
e Air breathing snails (Physa for
example)
f Fish types: None
3 Note: Fortunately, all polluted waters
do not always degenerate to "septic"
conditions.
VI THE RECOVERY ZONE
A The septic zone gradually merges into the
recovery zone. (Figure 1, day 4)
B As the excessive food reserves diminish
so do the numbers of anaerobic organisms
and other pollution tolerant forms.
C As the excessive demand for oxygen
diminishes, free DO begins to appear and
likewise oxygen requiring (aerobic)
organisms.
D As the suspended material is reduced and
available mineral materials increase due
to microbial action, algae begin to increase
often in great abundance.
E Photosynthesis by the algae releases more
oxygen, thus hastening recovery.
F Since algae require oxygen at all times
for respiration (like animals), heavy
concentrations of algae will deplete free
DO during the night when it is not being
replenished by photosynthesis.
G Consequently this zone is characterized
by extreme diurnal fluctuations in DO.
H With oxygen for respiration and algae, etc.
for food, general animal growth is resumed.
I The stream may now enter a period of
excessive productivity which lasts until
the accumulated energy (food) reserves
have been dissipated.
J Zone of recovery may usually be
characterized as follows:
1 General features:
a DO 2 ppm to saturation
b BOD dropping
c Turbidity dropping, less color and
odor
d Organic content dropping
e Bacterial count dropping
f Numbers of species increasing
g Numbers of organisms per species
decreasing, (with the increase in
competition)
h Other: Less slime and sludge
2 Characteristic biota
a Blue green algae
b Tolerant green flagellates and other
algae
c Rooted higher plants in lower reaches
d Midge larve (Chironomids)
29-=)
-------
Biological Aspects of Natural Self Purification
e Black fly larvae (Simulium)
f Giant water bugs (Belostoma spp. )
g Clams (Megalonais)
h Fish types:
- Green sunfish (Lepomis cyanellus)
- Common sucker (Catostomus
commersonni)
- Flathead catfish (Pylodictis olivaris)
- Stoneroiler minnow (Campostoma
anomalum)
- Buffalo (Ictiobus c.yprinellus)
3 Excessive production and extreme
variability often characterize middle and
lower recovery zones.
4 Unfortunately, many waters once polluted
never completely "recover". Re-
pollution is the rule in many areas so
that after the initial pollution, clear
out delineation of zones is not possible.
Characterization of these waters may
involve such parameters as productivity,
BOD, some "index" figure, or other
value not included here.
VII CLEAN WATER ZONE
A Clean water conditions again obtain when
productivity has returned to a normal,
relatively poor level, and a well balanced
varied flora and fauna are present.
(Figure 1, day "10") Conditions may
usually be characterized as follows:
B General features: similar to upstream
clean water except that it is now a larger
stream.
REFERENCES
1 Bartsch, A.F.: and Ingram, W.M.
Stream Life and the Pollution Environ-
ment. Public Works Publications,
July 1959, Vol. 90, No. 7, pp. 104-110.*
2 Gaufin, A.R. and Tarzwell, C.M,
Aquatic invertebrates as indicators of
stream pollution. Reprint No. 3141
from PHR. 67(l):57-64. 1952.
3 Gaufin, A.R. and Tarzwell, C.M.
Environmental changes in a polluted
stream during winter. Am. Midland
Naturalist. 54:68-88. 1955.
4 Gaufin, A.R. and Tarzwell, C.M.
Aquatic macro-invertebrate communities
as indicators of organic pollution in
Lytle Creek. Sewage and Ind. Wastes.
28:906-24. 1956.
5 Hynes, H. B. N. The Biology of Polluted
Waters. Liverpool Univ. Press,
pp. 202. 1963.
6 Katz, M. and Gaufin, A.R The effects
of sewage pollution on the fish population
of a midwestern stream. Trans. Am.
Fisheries Soc. 82:156-65. 1952. *'
7 Reish, D. J. The Relationship of the
Polychaetous Annelid Capitella capitata
(Fabricius) to Waste Discharges of
Biological Origin. In: Biol. Prob.
Water Pol. - Trans. 1959 Seminar.
Robert A. Taft Sanitary Engineering
Center, USPHS, Cincinnati, OH.
pp. 195-200.
8 Biology of Water Pollution FWQA Pub.
CWA-3 (references with an asterisk
are reprinted in this publication. 1967.
C Characteristic biota: similar to upstream
clean water fauna and flora except that
species include those indigenous to a
larger -stream.
This outline was prepared by H. W. Jackson,
Chief Biologist, National Training Center,
DTTB, MDS, WPO, EPA, Cincinnati,
OH 45268.
29-6
-------
BIOTA OF WASTEWATER TREATMENT PLANTS
(MICROSCOPIC INVERTEBRATES)
I GENERAL CONSIDERATIONS
A Community rather than individual as a
unit for study of the process; quantitative
relationship among different populations -
"population dynamics".
B Sequential transformation of organic
matter through the microbial life - a
transference of materials and energy
between microbial populations led to the
development of functional synecology or
productive ecology.
C Microbes considered here include bacteria,
protozoa, and microscopic metazoa; algae
and fungi are important groups included
elsewhere in more detail.
D All microbial groups originate from
a) the waste itself, b) washing waters,
c) soil, d) dust from air, and e) incidental
sources; only those members that can
survive and establish themselves in the
community are important; some are
transient.
E Some variations in composition of the
microbial community in domestic sewage
treatment due to climatic and other
ecological factors; industrial wastes with
specific waste matter may call for develop-
ment of more restricted microbial com-
munity for degradation.
F Most active microbial groups are: True
bacteria, filamentous bacteria, fungi,
protozoa; nematodes, rotifers, oligochaetes,
and water-mites.
II BACTERIA
A No ideal method for studying distribution
and ecology of bacteria in waste-treatment.
Total bacterial counts made on nutrient
agar or gelatine reflect only a portion of
the bacterial flora present.
B Pseudomonads are probably the most
versatile in their ability to attack a great
variety of organic compounds, including
petroleum products, phenolics, cyanides.
Others, such as Achromobacter, Alca-
ligenes, Chrornobactenum, Flavobactenum,
Aerobacter, and Micrococcus, are also
important genera. Actinomyces are
prominent in wastes rich in cellulose and
Bacillus organisms are starch attackers.
Sulfur and iron bacteria are predominant
in wastes rich in respective compounds.
C Actinomyces, Bacillus spp. , Aerobacter
spp. , and nitrogen-fixation bacteria are
primarily soil dwellers and are almost
always present in any type of wastes in
small numbers.
D Parasitic and pathogenic bacteria, if
present, are transient.
E In extended aeration process with high
dissolved oxygen, predominant species
are limited to pseudomonads, Zoogloea
ramigera, and Sphaerotilus.
Ill PROTOZOA
A Classification
1 Single-cell animals in the phylum
Protozoa in the animal kingdom.
or
2 A separate kingdom, Protista, to
include protozoa, algae, fungi, and
bacteria.
a Mastigophora (flagellates) - only
the subclass Zoomastigina (non-
pigmented) included; four orders:
SE. BI. 4e. 11. 72
30-1
-------
Biota of Wastewater Treatment Plants
1) Rhizomastigina - amoeba-
flagellates with 1 or more
flagella; examples: Mastigamoeba
Actinomonas, Rhizomastix
2) Protomonadina - with 1 or 2
flagella; comprising most of the
free-living forms; examples:
Pe ran em a, Bodo, Monas,
Plcuromonas
3) Polymastigina - with 3-8 flagella;
mostly parasitic in gut of man
and animals
4) Hypermastigina - with numerous
flagella; all parasitic in insect
intestine
b Ciliophora or Infusoria (ciliates) -
largest class of protozoa; no pig-
mented members; most important
group of protozoa in waste treatment;
2 subclasses:
1) Ciliata' - cilia present during the
the entire trophic life; comprising
most of the common ciliates;
examples: Paramecium, Colpi-
dium, Colpoda, Euplotes,
Stylonychia, Vorticella, Oper-
cularia, Epistylis, Carchesium
2) Suctoria - cilia present while
young and tentacles during
trophic life
c Sarcodina (amoebae) - pseudopodia
(false feet) for locomotion and food-
capturing; cell without cell-wall;
some with test or shell; 2 subclasses:
1) Rhizopoda - pseudopodia without
axial filaments; 5 orders:
a) Proteomyxa - with radiating
pseudopodia; no test or shell
b) Mycetazoa (slime-molds)
forming Plasmodium; re-
sembling fungi in sporangium
formation.
c) Amoebina - true amoeba;
pseudopodia in the form of
lobopodia, no test or shell,
cyst formation frequent; a few
capable of flagellate trans-
formation; examples: Naegleria,
Amoeba, Hartmannella,
Endamoeba
d) Testacea - amoeba with single
test or shell; examples:
Arcella, Difflugia
e) Foramimfera - large amoeba
with calcareous shell; all
marine forms
2) Actinopoda - with spinous
pseudopodia; 2 orders:
a) Heliozoa - without central
capsule; usually spherical in
form with many radiating
axopodia; examples: Actino-
sphaerium, Actinophrys
b) Radiolaria - pelagic in various
oceans
d Sporozoa - no organ of locomotion;
all parasitic (Plasmodium, Coccidfa)
B General Morphology
1 Zoomastigina:
With the exception of Rhizomastigina
which is amoeboid, the body has defi-
nite shape (oval, leaf-like, pear-like,
etc.); most free-living forms with 1-2
flagella, some with 3 or more flagella,
few forming colonies, cytostome present
in many for feeding on bacteria; rela-
tively small size (15-40 n)
2 Ciliophora:
Most highly developed protozoa, with
few exceptions, a macro- and a micro-
nucleus; adoral zone, mouth, oral
groove, usually present in swimming
and crawling forms; stalked form with
30-2
-------
Biota of Wastewater Treatment Plants
conspicuous ciliation of a disc-like
anterior region and little or no body
cilia; cyst formed in most species
3 Sarcodina:
Cytoplasmic membrane but not cell-
wall; cytoplasm with distinct ectoplasm
and endoplasm in many common spp.;
nucleus with large nucleolus in most
of the free-living forms; some with
the body enclosed in a test or shell
and moving by protruding pseudopodia
outside of the enclosure through an
opening; few capable of temporary
transformation into flagellate; fresh-
water actinopods usually spherical
with many radiating axopodia; some
Testacea spp. containing symbiotic
algae - mistaken for pigmented amoebae;
cysts with single or double wall and
1-2 nuclei, parasitic amoebae forming
cysts with 4 or more nuclei
C General Physiology
1 Zoomastigina
Free-living forms normally holozoic;
food supply mostly bacteria; relatively
aerobic, therefore, among the first to
disappear in anaerobic conditions; re-
production by simple fission and
occasionally by budding.
2 Ciliophora:
Holozoic; true ciliates concentrating
food particles, i. e. , bacteria, by
ciliary movement around the mouth-
part; suctoria sucking through tentacles,
bacteria, small algae and protozoa
constituting main food under normal
conditions; not as aerobic as flagellates -
a few surviving under highly anaerobic
conditions, such as Metopus; repro-
ducing by simple fission, conjugation,
or encystation.
3 Sarcodina:
Mostly holozoic; feeding through engulf-
ing by pseudopodia, food supply of small
amoebae mostly bacteria; large
amoebae engulfing larger organisms;
shelled amoebae, i.e. , Arcella. feeding
on a variety of. organisms or saprozoic,
reproduction by simple fission and
encystation.
IV NEMATODA
A ^Classification
1 All in the phylum Nemata (nonsegmented
round worms); 2 subphyla:
Secernentea (phasmids) 6 orders:
Tylenchida (spear in mouth), Rhabditida
(rhabditoid eosophagus), Strongylida
(parasitic), Ascaridida (parasitic),
Spirurida (parasitic), and Camallanida
(parasitic), with the exception of
tylenchids, all with papillae on male
tail
Ad'enophora (aphasmids) 5 orders:
Dorylaimida (spear in mouth), Chromo-
dorida, Monhystenda, Enoplida, and
Diocytophymatida; no papillae on male
tail; no excretary canal
2 Nematodes encountered in polluted
water and in sewage treatment mostly
belonging to order Rhabditida and few
in orders Dorylaimida and Tylenchida;
those in Rhabditida being bacteria-
feeders and those in the latter two,
feeding on algae and other zoomicrobes;
examples of rhabditids: Rhabditis,
Diplogaster, Diplogasteroides, Mono-
choides, Cephalobus, Cylindrocorpus,
Turbatrix, examples of the other two:
Dorylaimus, Aphelenchoides
B General Morphology
Round, slender, nonsegmented (some with
markings on outside); most of the free-
living forms microscopic in size although
dorylaimids up to several mm in length,
sex separated but some parthenogenetir
complete alimentray tract with elaborate
mouth parts with or without spear (or
stylet); no circulatory or respiratory
system
30-3
-------
Biota of Wastewater Treatment Plants
C General Physiology
Most sewage treatment plant dwellers
feeding on bacteria; others preying on
protozoa, small nematodes, rotifers,
etc. ; clean water species vegetarians,
DO diffused through cuticle; rhabditids
tolerating lower DO than clean water spp;
reproduction - eggs - larvae 4 molts) -
adults
V ROTIFERS
A Classification:
1 Classified either as a class of the
phylum Aschelminthes (various forms
of worms) or as a separate phylum
(Rotifera); commonly called wheel
animalcules, on account of circular appearing
movement of cilia around head (corona);
corona contracted when crawling or
swimming and expanded when attached
to catch food.
2 Of the 3 classes, 2 (Seisonidea and
Bdelloidea) grouped by some authors
under Digononta (2 ovaries) and the
other being Monogononta (1 ovary);
Seisonidea containing mostly marine
forms.
3 Class Bdelloidea containing 1 order
(Bdelloida) with 4 families, Philodinedae
being the most important.
4 Class Monogononta comprising 3 orders:
Ploima with 14 families, Flosculariaceae
with 4 families, and Collothecaceae
with 1 family; most important genera
included in the order Ploima (l. e. ,
Brachionus, Keratella, Monostyla,
Trichocerca, Asplanchna, Polyarthra,
Synchaeta, Microcodon), common genera
under the order Flosculariaceae: Floscu-
laria, Limnias, Conochilus, and Atrochus
5 Unfortunately orders and families of
rotifers based on character of corona
and trophi (chewing organ), which are
difficult to study, esp the latter; the
foot and cuticle much easier to study.
General Morphology and Physiology
1 Body weakly differentiated into head,
neck, trunk, and foot, separated by
folds; in some, these regions are
merely gradual changes in diameter
'of body and without a separate neck,
segmentation external only.
2 Head with'corona, dosal antenna, and
ventral mouth; mastax, a chewing
organ, located in head and neck, con-
nected to mouth anteriorly by a ciliated
gullet and posteriorly to a large stomach
occupying much of the trunk.
3 Common rotifers reproducing partheno-
genetically by diploid eggs; eggs laid
in water, cemented to plants, or carried
on femals until hatching.
4 Foot, a prolongation of body, usually
with 2 toes; some with one toe; some
with one toe and an extra toe-like
structure (dorsal spur).
5 Some, like Philodina, concentrating
bacteria and other microbes and minute
particulate organic matter by corona;
larger microbes chewed by mastax;
some such as Monostyla feeding on
clumped matter, such as bacterial
growth, fungal masses, etc. at bottom;
virus generally not ingested - apparently
undetected by cilia.
6 DO requirement somewhat similar to
protozoa, some disappearing under
reduced DO, others, like Philodina,
surviving at as little as 2 ppm DO.
VI SANITARY SIGNIFICANCE
A Pollution tolerant and pollution nontolerant
species - hard to differentiate - requiring
specialist training in protozoa, nematodes,
and rotifers.
B Significant quantitative difference in clean
and polluted waters - clean waters contain-
ing large variety of genera and species
but quite low in densities.
30-4
-------
Biota of Wastewater Treatment Plants
C Aerobic sewage treatment processes
(trickling filters and activated sludge
processes, even primary settling) ideal
breeding grounds for those that feed on
bacteria, fungi, and minute protozoa and
present in very large numbers; effluents
from such processes carrying large
numbers of these zoomicrobes; natural
waters receiving such effluents showing
significant increase in all 3 categories.
D Possible Pathogen Carriers
1 Amoebae and nematodes grown on
pathogenic enteric bacteria in lab;
none alive in amoebic cysts; very few
alive in nematodes after 2 days after
ingestion; virus demonstrated in
nematodes only when very high virus
concentrations present; some free-
living amoebae parasitizing humans.
2 Swimming ciliates and some rotifers
(concentrating food by corona) ingesting
large numbers of pathogenic enertic
bacteria, but digestion rapid; no evidence
of concentrating virus; crawling ciliates
and flagellates feeding on clumped
organisms.
3 Nematodes concentrated from sewage
effluent in Cincinnati area showing
live E. coli and streptococci, but no
human enteric pathogens.
VII EXAMINATION OF SEWAGE TREATMENT
EFFLUENT, AND SLUDGE FOR MICROBES
A Bacteria - Not Included
B Zoomicrobes -
The 12th edition of the Standard Methods
(1965) has a part on Biologic Examination
of Water, Wastewater, Sludge, and Bottom
Materials, in which the sludge of sewage
treatment is discussed, but very briefly.
Much of the materials are concerned with
sediment at bottom of natural bodies of
water. Chang described a method for
examination of water for nematodes, but
the method is not applicable to sewage
treatment, sludge, or effluent.
1 Waste treatment - the method bound
to be qualitative; material scraped
from stones in trickling filters or the
floe masses in activated sludge examined
in slide-coverslip preparations for
poor, moderate, or rich zoobiota;
material relatively rich in zoobiota
indicating satisfactory treatment
process; protozoa, rotifers, and
nematodes predominant, especially
protozoa; bristle worms and watermites
in smaller numbers; springtails and
insect larvae present as grazing fauna
on top of trickling filters.
2 Sludge - representative samples sus-
pended in known quantities of dilution
water and thoroughly shaken; filtered
through bolting cloth or metal screen
of comparable pore size to remove
extraneous dead clumped matter;filtrate
examined in Sedgewick liafter (SR) counting
cell for various zoomicrobes; fresh
sludge desired or samples refrigerated.
3 Sewage effluent - samples "fixed" with
formalin, merthiolate, or similar
chemical not desirable for examination
for zoomicrobes; 50-200 ml filtered
through a 7- or 14-micron membrane
and strained material washed with a
few mis of dilution and examined in
an SR cell for zoo-microbes quantita-
tively or qualitatively.
VIII USE OF ZOOMICROBES AS POLLUTION
INDEX
A Idea not new, protozoa suggested long ago;
many considered impractical because of
the need of identifying pollution-intolerant
and pollution-tolerant species - proto-
zoologist required.
B Can use them on a quantitative basis -
nematodes, rotifers, and nonpigmented
protozoa present in small numbers in
clean water. Numbers greatly increased
30-5
-------
Biota of Wastewater Treatment Plants
when polluted with effluent from aerobic
treatment plant or recovering from sewage
pollution; no significant error introduced
when clean-water members included in the
enumeration if a suitable method of com-
puting the pollution index developed.
C Most practical method involves the
equation: (A + B)/A = Z.P.I. , where
A = number of pigmented protozoa,
B = other zoomicrobes, in a unit volume
of sample, and Z. P.I. = zoological pollu-
tion index. For relatively clean water,
the value of Z. P. I. close to 1; the larger
the value above 1, the greater, the pollution
by aerobic effluent, or sewage during
recovery. This is based on the fact that
pigmented protozoa are members of clean
water micro-fauna (stabilization pond
excluded).
IX CONTROL
A Chlorination of Effluent and'Settling
B Prolongation of Detention Time of Effluent
C Modification of Waste Treatment
¦D Elimination of Slow Sand Filters in
Nematode Control
X LIST OF COMMON ZOOLOGICAL ORGAN-
ISMS FOUND IN SEWAGE TREATMENT
PROCESS - TRICKLING FILTERS AND
ACTIVATED SLUDGE PROCESS
PROTOZOA
Sarcodina - Amoebae
Amoeba proteus; _A radiosa
Hartmanella spp.
Arcella vulgaris
Naegleria gruberi
Actinophrys sol
FLAGELLATA
Bodo caudatus
Pleuromonas jaculans
Oikomonas termo
Cercomonas longicauda
Peranema trichophorum
Swimming type
Ciliophora:
Colpidium colpoda
Colpoda cucullus
Glaucoma
Paramecium caudatum; P. bursaria
Stalked type
Opercularia spp. (short stalk
dichotomous)
Vorticella spp. (stalk single and
contractile)
Epistylis plicatilis (like Opercularia
more colonial)
Carchesium spp. (like Vorticella but
colonial, both have
spiral coiled stalk
Crawling type when contracted)
Euplotes spp.
Stylonychia mylitus
Urostyla spp.
Oxytricha spp.
NEMATODA
Diplogaster spp.
Monochoides spp.
Diplogasteroides spp.
Rhabditis spp.
Pelodera spp.
Aphelenchoides sp.
Dorylaimus sp.
30-6
-------
Biota of Wastewater Treatment Plants
Cylindrocorpus sp.
Cephalobus sp.
Rhabdolaimus sp.
Monhystera sp.
Trilobus sp.
ROTATORIA
Diglena
Monstyla
Polyarthra
Philodina
Keratella
Brachionus
OLIGOCHAETA (bristle worms)
Aelosoma hemprichi (Aelosomatidae)
Aulophorus vagus (Naididae)
Tubifex tubifex (Tubificidae)
Pachydrilus linea'tus (Enchytraeidae)
INSECT LARVAE
Metriocnemus ssp. (midge)
Orthocladius ssp. (midge)
Psychoda spp. (filter fly)
OTHER ARTHROPODA
Hydrochna sp. (Acarina, mite)
Platysieus tenuipes (A carina, mite)
Hypogastrura (= Achorutes sub-viatica)
viaticus (Collembola, Springtail)
Folsomia sp. (Collembola, Springtail)
Tomocerus sp. (Collembola, Springtail)
MOLLUSCA
Lymnaea ssp. (pulmonate snail)
Physa sp. (pulmonate snail)
XI POPULATION DYNAMICS AND THE FOOD
CHAIN IN AEROBIC SEWAGE TREATMENT
PROCESSES (Figure 1 and 2)
A Aerobic bio-oxidation of waste materials
comparable to a food chain through which
the dead organic matter is converted to
inorganic matter during the stabilization
process, e.g., waste organic matter »
bacterial phase » zoological phase >
inorganic matter.
B Systematics, physiology and biochemistry
involved in explaining the "chain reaction";
knowledge inadequate and fragmental,
ecological study limited to principles
governing the relationship of different
groups of flora and fauna with each other
and with the environment
C With adequate DO supply, bacterial popu-
lation increases rapidly, in the presence
of rich organic food; flagellates and
amoebae, which feed on bacteria and
other small'particulate matter in clumped
material (such as growth film and floe
masses), first show increase in population
size, as suspended bacterial population
increases to a high level, swimming
ciliates, which feed actively on the
suspended bacteria, also increase; in-
creased consumption of bacteria and
reduced supply of dead organic matter
results in decline-in bacterial population,
which, in turn, results in a decline in
the swimming ciliate population; the
presence of large populations of small
protozoa (ciliates, flagellates, and
amoebae) results in ah increase in
populations of rotifers, nematodes,
stalked ciliates, and crawling ciliates,
which feed on the small protozoa and
bacteria that are lodged in clumped
masses; eventually, scavengers, such
as mites, shelled amoebae, certain
nematodes, and bristle worms become
predominant, and bacteria and small
protozoa populations drop to the pre-
cycle level; rotifers that can concentrate
bacteria in suspension, (such as Rotifer
and Philodina, and nematodes), which
have long surviving time, may remain
for a long time; these zoomicrobes
appear in the effluent in proportion to
their respective population during treat-
ment - nematodes, rotifers, ciliates
predominant with small numbers of
flagellates and amoebae; bristle worms
unpredictable; mites few.
30-7
-------
Biota of Wastewater Treatment Plants
Effluent
Insects
Oligochaetes &
insect larvae
Nematodes
& rotifers

Nonpigmented .
protozoa '
4-4
Heterotrophic
bacteria
Fungi
Algae

Autotrophic bacteria ¦«
Pathogenic organisms
| -4— Raw Sewage
Suspended organic matter
(by hydrolysis)
Dissolved organic matter
(respiration,
deamination,
decarboxylation, etc.)
Inorganic C, P, N,
S comp.
(NH NO" CO" P)
(Nitrification, sulfur
& iron bacteria)
Food Chain in Aerobic Sewage Treatment Processes
Figure 1.
30-8
-------
Biota of Wastewater Treatment Plants
Organic Matter (dead)
Total Bacteria Population
V
\
\
01
(0
-o
•x,
Stalked Ciliates
Crawling Ciliates
Rotifers and
Worms (nematodes and
oligochaet.es)
Mites (Platysieus)
\
Swimming
Ciliates^
/
X
\
\
\
\
\
\
Special Characteristics:
1 More "binding" organisms
(stalked ciliates) In
activated sludge process
2.
Mites
Ollgochaetes
Shelled Amoebae
Crawling Ciliates
(Scavengers)
\
Ollgochaetes, mites,
and spring tails -
grazing fauna on
trickling filters
V
// ~.

%
\

TIKE
Population dynamics In Aerobic Sewage Treatment Process
Figure 2.
D Since sewage effluent from aerobic treat-
ment processes are rich in nonpigmented
zoomicrobes, discharge of effluent into
natural causes great increase in their
members; unpolluted waters usually have
a much higher algae-to-nonpigmented-
zoomicrobes ratio. The great increase
in the latter in water resulted from effluent
pollution is likely to change this ratio,
thus giving the basis for the Z.P.I. . This
analysis is not applicable to stabilization
ponds due to the large algal population
present in their effluents.
3 0-9
-------
Microbial Agents
Utilization
Treatment Process Pollution Indicator
Stabilization of Organic Matter
Aerobic
Anaerobic
Breakdown of Specific Industrial
Wastes
Stabilization Ponds
Aerobic and Anaerobic
Combined
Nitrification - Aerobic
Dentrification when
Anaerobic
Elimination of Nitrogen Through
N2 Formation - Special
Dentrification
Sludge Digestion
.Aerobic
Anaerobic
Phosphorus Removal
Essential in Preventing
Algae Growth
(Composting) - Solid Wastes
Total Bacteria Count
Coliform Density
MF Counts
MPN Tube Tests
E. Coli Density
Tube Test
MF Counts
Streptococci
Tube Test
MF Counts
Infrared Spectrophotometer
Specificity
Time Requirement
Enteric Pathogens
Salmonella Spp.
Quantitation in
Mixed Population
Enteroviruses
Large Volumes
Required
Coli Phage
Type Specificity To Be
Considered
Fluorescent Antibody
Against E. Coli
Against Enteric Pathogens
Gas Chromatography
on Metabolic Products
Zoological Pollution Index
Figure 3
Elimination
Removal
Detention
Settling
Limited.'to Large Organisms
Flocculation
Alum
Iron
Lime
"Coagulant Aid"
Filtration
Adsorption
Straining
Medium
Reverse Osmosis
Metal Strainer
Mesh Size Limit
Antagonistic Agent
Pseudo monads
Bacterial Feeders
Protozoa
Nematodes
Rotifers
Flotation
Destruction
or
Disinfection
Chemical
Clorine
Bromine
Iodine
Ozone
Silver
Copper
Physical
Heat
UV
Gamma
Radiation
Wave
Motion
Electro-
Hydraulic
Treatment
Aeration Cationic
Anionic Detergent Detergent
Neutral Detergent
-------
Biota of Wastewater Treatment Plants
REFERENCES
1 American Public Health Association,
American Water Works Association
and Water Pollution Control
Federation, Standard Methods for
the Examination of Water and Waste-
water, 12th ed. New York. 1965.
2 Chang, S.L., et al. Survey of Free-
Living Nematodes and Amoebas in
Municipal Supplies. J.A.W.W.A.
52:613-618.
3 Change, S.L. and Kabler, P.W.
Free-Living Nematodes in Aerobic
Treatment Plant Effluents.
J.W.P.C.F. 34:1256-1261. 1963.
4 Edmondson, W.T., et al. Ward Whipple's
Fresh Water Biology, 2nd ed.
John Wiley & Sons, New York,
pp. 368-401. 1959.
5 Hawkes, H.A. Ecology of Activated
Sludge and Bacteria Beds, (in Waste
Treatment) Pergamon Press,
pp.52-98. 1960.
6 Hawkes, H.A. The Ecology of Waste-
water Treatment, Pergamon Press.
1963.
7 Hawkes, H.A. The Ecology of Sewage
Bacteria Beds, (in Ecology and the
Industrial Society) John Wiley & Sons,
New York, pp. 119-148. 1965.
10 Calaway, W.T. and Lackey, J.B.
Waste Treatment Protozoa,
Flagellata. University of Florida,
College of Engineering, Florida
Engineering Series No. 3, pp. 1-140,
1962.
11 Calaway, W.T. The Metazoa of Waste
Treatment Processes-Rotifers.
Journal Water Poll. Cont. Fed.
4(11) part 2: pp.412-422.
12 Bick, Hartmut. An Illustrated Guide to
Ciliated Protozoa used as Biological
Indicators in Freshwater Ecology.
World Health Organization. Geneva.
1969. (includes an illustrated key)
13 Curds, C.R. An Illustrated Key to the
Freshwater Ciliate Protozoa
commonly found in Activated Sludge.
Water Research Tech. Paper 12.
Water Poll. Res. Lab. Stevenage
1969.
14 Curds, C.R. and Cockburn, A.
Protozoa in Biological Sewage
Treatment Processes - I. A
Survey of the Protozoan Fauna of
British Percolating Filters and
Activated Sludge Plants. II. Protozoa
as Indicators in the Activated Sludge
Process. Water Research
4:225-244. 1970,
8 McKinney, Ross E. and Gram, Andrew.
Protozoa in Activated Sludge.
Sew. Ind. Wastes. 28:1219-1231.
1956. (reprinted in Biology of Water
Pollution by L.E. Keup, W.M.
Ingram and Kenneth M. Mackenthun.
FWPCA Pub. No. CWA-3, pp. 252-
262. 1967.)
9 Cooke, William Bridge, Trickling Filter
Ecology. 40:273-291, pp. 269-287.
1959. (reprinted in Biology of Water
Pollution)
This outline was prepared by S. L. Chang,
M.D., Chief, Etiology, Division of Water'
Supply Programs Division, WPO, EPA
Revised by R. M. Sinclair, Aquatic
Biologist, National Training Center, DTTB
MDS, WPO, EPA, Cincinnati, OH 45268.
30-11
-------
Biota of Wastewater Treatment Plants
a. Peranema
trichoporum, 25u
£f
T>.
/"-'AK-V-^5r> >J
-------
Biota of Wastewater Treatment Plants
a. Paramecium caudatum
200 - 260u
b. P. caudatum
cyst
Side view
Top
view
e. Euplotes carcinatus
70u

Fig. 2
f. Vorticella 35-157u
S.L.Chang, 1963
30-
-------
Biota of Wastewater Treatment Plants

a. Diaptomus sp. 2 mm.
(2 egg sacs
. v ¦ "in-
£-v' wigfrM*.
•*>/

c. Philodina sp. 45u

b. Cyclops sp. 2 mm.
larva
male
female
e. Diplogaster nudicapitatus
about 1 mm.
d. Anurea cochlearis 125u
Fig. 3
S.L.Chang, 1963
30-14
-------
Biota, of Wastewater Treatment Plants
Dorsal view

b. Folsomia fimetaria (50X)

a. Hydrochna sp. (water mite)
(50X)
c. F. fimetaria (side view)
f r«-

•5^--, :./iX
i-.
;.tv
V
? *5J|
jfyf (fe
£*3
V^v
*rV>'
• 2T-V V
^. .-7

d. Typical zoological organisms
found in growth mass in
Trickling filters (50X)
*,

- *" \
'r \\
, A
- ¦ '' "V
, ; '
f} ¦ •'
;vv^
... -
-------
CO
0
1
f—*
RHABlPmS (Molt)
PHARYNX 1 0€5bPKAG£AL
nervc auue intc&tine
RING
rectal gland
(Affer CNtvoPd)4
.rwCOASTLB (Moll)
OESOPHAGEAL
BULB
OESOPHAGUS
OESOPHAGEAL
MONHVSTEWA (Ftmol«)
RHABIOITIS (F«mol»)
oesophageal
CORPUS
EXCRETORY
P0RE EXCRETORY
GLAND
(Attar CfeNeMl
Free Living Nematodes
(N>mathelminthes')
-------
WASTEWATER TREATMENT - THE RESULT OF NATURA L PHENOMENA
Part 1
I INTRODUCTION
All sewage treatment is accomplished by
application of biological, physical, chemical
processes. These processes are natural
phenomena which have been in operation since
primeval time. Man has not always under-
stood these processes and in fact we may not
have a complete understanding of them at this
time; nevertheless, it is by means of these
phenomena that sewage treatment is possible.
H PHYSICAL PROCESSES
A Specific Density
The density of waste solids, coupled with
the law of gravity, provides a physical
phenomena resulting in removal of wastes.
Sedimentation has been observed by man
for thousands of years and a study of
geologic formations reveal that sedimentation
has been continuing for millions of years.
In nature, the pools in streams, lakes, and
estuaries provide the necessary conditions
of quiescence to allow gravity separation
of settleable solids.
In using the physical laws relating to
gravity and specific density, man has
used two processes:
1 Sedimentation in tanks built to provide
quiescence, and
2 Centrifuge separation
C Reaeration
Few people have failed to take the time to
see a waterfall or to enjoy the scenic
beauty of a fast-flowing and turbulent
mountain stream. These are nature's
examples of reaeration facilities. In
addition to these dramatic aerators,
there is a constant exchange of molecules
of oxygen and other atmospheric gases
across the liquid-gas interface of rivers,
lakes, ponds, and oceans. The wind
provides mixing energy to carry the
dissolved gases to portions of the water
mass below the surface. Utilizing these
principles as treatment processes, man
injects air into the waste flow by use of
air under pressure; the making of water-
falls by pumping the liquid into the air
fountain-like; or, by creating an infinitely
large surface area with depth being
merely a thin film as the liquid trickles
downward over beds of rock.
Ill BIOLOGICAL PROCESSES
In the real world the aquatic community is
very complex, consisting of organisms of
every size from the virus to the fishes.
Each has a definite role in the community
and in a natural environment--one unaffected
by wastes from man's activities--there is a
very great variety of different kinds and
species." All are present in such numbers as
will maintain a balance with the food supply
available.
B Particle Size Distribution
Screening sewage flows to remove large
particles is merely an application of size
selection. Screens abound in nature as
settled rock deposits which prevent move-
ment of twigs, sticks, leaves and other
solids. The earth itself acts as a fine
screen and filter, removing all water-
borne material except those that are
dissolved. In treatment plants,- bar racks
and sand filters are applications of these
natural conditions.
Action by bacteria in this community breaks
down complex organic matter to simpler
molecular forms. These become the basic
building blocks for new growth by other
microorganisms. These in turn are a food
source for yet other, more complex,
organisms. This activity of decomposition
and growth is a continuous one--such that
the process is cyclic.
SE.TT.4. 1. 71
31-1
-------
Wastewater Treatment - The Result of'Natural Phenomena
All the elemental components of organic
material--carbon, nitrogen, sulfur, etc. --
are cyclic. The carbon cycle (Figure 1) is
an example. It can be seen that once elements
making up organic matter, from any source,
enter the aquatic environment they will con-
tinue in the cycle indefinitely unless they are
removed as a "Harvest" as fish or other
product.
IV CHEMICAL PROCESSES
Chemical processes in the aquatic environ-
ment are intimately connected with biological
activity and proceed simultaneously with
photosynthesis, assimilation and decomposition.
In addition, the chemistry of water is a
function of the solubility and presence of
inorganic salts in the environment.
Carbon Cycle
Figure 1
31-2
-------
Wastewater Treatment - The Result of Natural Phenomena
The salt content of the oceans, the Dead Sea,
the Salton Sea, and Great Lake are examples
which indicate that once inorganic salts enter
the aquatic environment they remain indefinitely
as an integral part. It is only by means of
evaporation that water high in inorganic salts
is returned, in the form of rain and snow, to
the fresh water state.
V LIMITATIONS OF NATURAL TREATMENT
A Although wastes discharged into the aquatic
environment enter the cycle previously
described, time is required to reach a new
balance during which time water quality
may be seriously impaired. In addition,
the new balance may not be a desirable one
as excessive nutrients may bring about
blooms of organisms causing nuisance
conditions and/or foul odors and tastes.
B It is axiomatic that elements of wastes
removed prior to discharge into the aquatic
environment do not enter these cycles and
therefore cannot cause adverse effects.
C A stream has capacity to accept organic
wastes and through natural processes to
self-purify; however, its capacity to
assimilate wastes without seriously
affecting water quality for other uses is
limited by such factors as stream flow,
reaeration rate, temperature, etc.
REFERENCES
1 Fair, G.M. and Geyer, J.C., Water
Supply and Waste-Water Disposal,
John Wiley & Sons, Inc. , New York,
(1966).
2 McKinney, R.E., Microbiology for
Sanitary Engineers, McGraw-Hill
Book Company, New York, (1962).
3 Rich, L. G. , Unit Processes of Sanitary
Engineering, John Wiley & Sons,
New York, (1963).
This outline was prepared by L. J. Nielson,
Sanitary Engineer, Regional Program
Director, Manpower & Training, PNWL,
Corvallis, OR 97205.
-------
AEROBIC BACTERIAL'SYSTEMS FOR INDUSTRIAL WASTES
Part 2
I INTRODUCTION C
Natural processes carried out by common
soil organisms under controlled conditions.
To completely understand the process one
must have some basic understanding of the
microorganisms involved.
II BACTERIA
A General Structure - See Figure 1
B Size and Shape
Cocci, about 1 micron in diameter
Rods, from 0.5 to 2 microns by 0.. 75 to
10 microns
Spirals, about 1 micron by 10 microns
C Composition - C5H7O2N (average formula)
D Growth Curve - See Figure 2
III BACTERIAL REACTIONS
A Basic Concept
Bacteria are trying to make more bacteria
from the substrate, but this requires
energy To obtain energy the bacteria
takes some of'the substrate (a minimum
amount as the bacteria wants to use as much
of the substrate as possible for new cells)
and breaks it down to end products, thereby
releasing energy, and this energy is used
to create a new cell.
B General Reaction
Organic matter + bacteria + trace
inorganics + N and P + H2
Acceptor >- end products +
energy + more bacteria (See Figure 3).
Enzymes
Organic catalysts which promote biological
reactions. True catalysts since rate of
reaction is proportional to the amount of
enzyme present, also the enzyme is always
regenerated. Most common make-up of
enzyme:
Protein + metallic activator- + coenzyme
Some common classifications of enzymes:
Extracellular - hydrolytic only.
Intracellular - all reactions', hydrolysis,
energy, and synthesis.
Constitutive - always present in cell,
whether being used or not.
Adaptive - must be continuously stimulated
by,substrate.
Oxidative Phosphorylation (See'Figure 4)
This is the. mechanism by which the bacteria
generate energy. The first step is the
removal of hydrogen from the organic
molecule-by the coenzyme DPN+.. These
hydrogens are then passed from enzyme to
enzyme until they are finally linked to
oxygen (the hydrogen acceptor in this case)
to form water. This process releases
30-50 KCal/cycle.
Energy Transfer
The question is, what does the bacteria do
withJall this energy, how does it store it and
then reuse it? The coenzyme adenosine
triphosphate (ATP) contains three high
energy phosphate bonds which store the
energy. Three ATP are formed per cycle
of oxidative phosphorylation. When a cell
needs energy for a synthesis (cell building)
reaction, some ATP is converted to ADP
with the resulting release of energy.
IN. MET. bi. 32. 1. 71
¦U-5
-------
bmm mm mi if bmtim
-------
BINERM MtmoUC HI ACTION
SYNTHESIS
OXIDATION
-*-60% OF CARBON
OXIDE
ENERGY
40% OF CARBON
fl>
d
cr
W
03
O
e-t-
(D
C/D
CO
c+-
a>
3
cn
O
t—i
D
Q-
C
w
<-+-
l-i
p'
Co
i—i
I
P
CT;
-------
CO
I
00
mem eunmi growth cmi
<
(gLU
LLI ^
»"<
uu
00 (J
u.£Z
Ouj
*2

STATIONARY
DECLINING
GROWTH
INC. DEATH
LOG DEATH
LOG GROWTH
DECLINING
DEATH
LAG
INC. GROWTH
FINAL DEATH
TIME
-------
oxwmm PHomommwN
1
REDUCED

DPIMH H

fph2

ORGANIC
. ~

4.
MATTER
W

OXIDIZED

DPN

FP
i
h2o

F«

4
Cyto
a
1
Cyto
c
1/2 0'2

Fe
REDUCED
ICOENZYME
Q
OXIDIZED
Fe
Cyto
ci
Fe
CO
I
CD
>
0>
o
cr
W
p
r>
c+
0)
in
CO
r+
CD
o
(—I
3
a
c
CO
<—h
p
cn
<—t-
(D
CO
-------
Aerobic Bacterial Systems for Industrial Wastes
F Oxidation
Complete oxidation is a rather complex
process involving many hydrogen removal
steps as well as the addition of water and
splitting of organic molecules. In general,
a given organic substrate must be biolog-
ically converted to pyruvic acid (C^H^O^)
before entering the Kreb or energy cycle.
This is a nine-step cycle during which
there is the loss of 2 CO^ and the gener-
ation of 15 ATP. Note that pyruvic acid
loses 1 COg getting into the Kreb cycle
so all three carbons in the pyruvic acid
are oxidized to CO
G Synthesis
Synthesis pathways are not the reverse of
oxidation pathways, but a complete
synthesis scheme has been worked out so
an entire cell, with all'of its complex
components, may be created from a
simple substrate.
IV FLOCCULATION
The mechanism by wnich the newly created
cells are coagulated into large enough masses
to settle out in the settling tank. Mechanism
is not well understood.
V OTHER ORGANISMS OF IMPORTANCE
IN AEROBIC TREATMENT SYSTEMS
A Autotrophic Bacteria
Nitrifying bacteria which convert ammonia
nitrogen to nitrites and nitrates. Note,
denitrifying bacteria are not autotrophic.
B Fungi
Oxidize organics well, but usually do not
settle well.
C Protozoa and Rotifers
Feed on dispersed bacterial cells and
thereby allow treatment systems to
produce a very clear effluent.
•VI PRACTICAL APPUCATION
REFERENCES
1 McKinney, R.E. Microbiology :for
Sanitary Engineers. McGraw-Hill
Book-Company, Inc. New, York. 1962.
2 Lamanna, C. and Mallette, F.M.
Basic Bacteriology. The Williams
and Wiikins Co. Baltimore, Md. 1953.
3 Prelczar, M.J., Jr. and Reid,. R. D.
Microbiology. McGraw-Hill Book
Co., Inc. New York. 1958.
4 Oginsky, E. L. and Umbreit, W..W.
An Introduction to Bacterial Physiology.
W.H. Freeman and Company, San
Francisco, California. 1954.
5 McKinney, R.'E. Biological Oxidation
of Organic Matter. Third Biological
Waste Treatment Conference.
Manhattan College. New York.
April 20-22, I960.
6 McKinney, R.'JE. and Gram, A,
Protozoa and Activated Sludge.
Sewage and Industrial Wastes.
28:1219-1231. 1956.
This outline was prepared by J.M. Symons,
Chief, Surface'. Water Protection, Water
Supply Research Laboratory, Water Supply
and Sea Resources Program, NCUIH, USPHS.
31-10
-------
UNIT OPERATIONS IN WASTE TREATMENT
I INTRODUCTION
A Definitions
1 Unit operation^ a particular kind of a
physical change that is repeatedly and
frequently used as a step in the process
for industrial chemicals and related
materials. Examples include filtration,
evaporation, .distillation, heat transfer,
fluid transfer, sedimentation and mixing.
2 Unit process^ a particular kind of
chemical reaction and equipment to
which the same basic designs and
operation may be applied. Oxidation,
coagulation, disinfection, hydrolysis
and chemical absorption are common
examples.
3 Process - a series of actions or opera-
tions conducing to an end A continuing
operation or treatment consisting of a
combination of unit operations. For
example, the activated sludge process
includes mixing, fluid and gas transfer
and clarification among unit operations;
oxidation, hydrolysis and coagulation
either biological or chemical among
unit processes.
(2)
4 Wastewater treatment any process
to which wastewater is subjected to
remove or alter its objectionable
•components.
d Unit operations for purposes of
this outline include both "unit
operations" (A 1) and "unit
processes" (A 2) to distinguish
unit process from the more
generally applied term/'process"
which may include many unit opera-
tions or unit processes.
B Increasing stress on environmental
quality means that wastewater treatment
must be upgraded. Upgrading treatment
means: removal of a larger fraction of
conventionally removed components and
removal of additional items presently not
significantly removed by conventional
treatment. This also means treatment
of a larger, fraction of collectable waste-
waters for a greater variety of used
water types and components for 24 hours
per day, 365 days per year
1 The unit operation concept tends to
focus attention upon the specific com-
ponents to be removed and upon
fundamental units most suitable for
that function The unit operation
approach offers a wider selection for
design purposes than that available
in empirical plant design. The treat-
ment therefore may be.more specific,
better tailored to the situation and show
a better cost/benefit ratio
2 Implementation oi treatment operations
requires motivated and trained man-
power. -Personnel training along the
unit operations route shortens the time
and promotes better comprehension by
focusing upon the unit operations or
tasks most commonly used. Rotation
among assignments is a smootherand
progression more likely because the
individual trained in unit operations
tends to recognize familiar unit opera-
tions in the new assignment', his learning
requirements consist of the differences,
such as a different-sequence of familiar
tasks, a smaller number of new unit
operations, and different handling
techniques because of material or
situation. Learning is split into funda-
mental units. Personal progress, job
satisfaction, and competence increase
with the recognition of proficiency of
the smaller "bits. "
a Wastewater treatment may also be
defined-as a series of unit operations
designed to produce a product "clean
water" from a raw material "waste
water. "
.b Treatment is a means to renovate
used water to meet a specific
beneficial reuse requirement.
c. Conventional treatment is commonly
classified by stage or degree of
treatment such as preliminary or
pretreatment, primary, secondary,
or advanced treatment. Processes
such as activated sludge, trickling
filtration or oxidation pond treat-
ment are commonly used. Each of
these can be more precisely
described and better understood
in terms of the unit operations
involved
PC. WAS. 4b. 11. 72
32-1
-------
C This outline considers selected unit- ..
operations of sanitary engineering '
and processes based upon them. Tables
presented later summarize interrelations
and the means whereby these are combined
into processes or stages of treatment.
1 Unit operations are the fundamental
"building blocks" of1 treatment.
2 Unit operations are the alternate routes
to a given objective. Solids-liquid
separations may be achieved by many
different operations; some are favored
in one situation, others limited by that
situation.
D The following sections consider individual
unit operations and their characteristics
as guidelines for selection or design.
These notes are general in nature and
subject to the influence of waste charac-
teristics, local conditions, practice,
economics and water quality requirements
of the situation. Each unit operation is
characterized in terms of:
1 Favorable application factors
2 Limiting application factors that may
encourage selection of an alternate
operation for a particular situation.
II PHYSICAL UNIT OPERATIONS -
SOLID-LIQUID SEPARATIONS
The separation of solids from liquid, or the
reverse, is of primary importance to
wastewater treatment. Various unit oper-
ations or adaptations of them to achieve this
objective may be used to remove objectionable
components, to protect process equipment, to
simplify subsequent operations, to increase
stability of process water, to make the water
more amenable for treatment or to complete
the process. Separations may be a part of
pretreatment, an integral process step, or
a means of upgrading process effluents. No
single operation appears more frequently, in
more numerous adaptations, at more stages
in processing, and is more critical in product
water upgrading than solids-liquid separation.
A Gravity Sedimentation
1 Favorable aspects: This unit operation
is by far the least expensive and feasible
route for a large variety of separations.
May be adapted for separation of a variety
of materials having a specific gravity
sufficiently different from that of water
and immiscible in it such as: High
dsnsity sand, gravel or scale, moderate
density organic suspended materials,
low density floatable materials.
Requires simple and generally available
equipment. Operating variables are
known and generally controllable to
favor reliable treatment.
2 Limitations: Adversely affected by
variations in wastewater characteristics
and flow. Requires a moderately large
capital, equipment and area investment.
Sludge detention conducive to solids
liquefaction and feedback Affected by
short circuiting, turbulence, distribu-
tion, temperature or density changes.
Relatively slow operation in most
situations.
B Surface Filtration
1 Coarse or fine screens
a Favorable: Inexpensive simple
operation and equipment. Reliable
removal of discrete solids larger
than the screen openings. Equipment
available and operating practice known
Simplifies subsequent operations. Low
area requirement
b Limitations: Susceptible to plugging,
large quantities of wet, difficult-to-
handle solids. Variable loading may
result in operating and performance
problems associated with higher loads.
2 Microscreens
a Favorable: Produces an effluent of
low suspended solids (<10 mg/1) and
low turbidity (2JTU) at low capital,
operating time and area cost at
rated loading Simple operating
requirements. Equipment avail-
ability good.
b Limitations: Poor tolerance for high
suspended solids feeds (>50 mg'l)
Tends to plug filter surface. Affected
by changes in waste characteristics.
Solids breakthrough at excessive
loading.
3 Diatomaceous earth filtration
a Favorable: Produces a high quality
effluent low in suspended solids and
turbidity-(0. 1 to 1.0 JTU). Low area
requirement. Pressure buildup
rather than solids breakthrough warn-
ing of overloads.
32r 2
-------
Unit Operations in Waste Treatment
b Limitations: High pressure drop
through the filter;, rapidly increases
with solids loading. Tends to plug.
Low output/ sq. ft. I unit time with
high suspended solids feed.
4 Vacuum filtration
a Favorable: Suitable for treatment
of a variety of solids-liquid
concentrates. Large choice of
filter media including string, coil,
cloth (natural or synthetic) screens.
Versatile in adaptability for varying
conditions and loading. Low area
requirement. High capacity per
unit area.
b Limitations: Complex operation,
high maintenance cost. Usually
requires chemical coagulation or
coagulant aids. High capital and
operating cost. Requires frequent
attention to maintain capacity during
¦ varying load and sludge characteristics.
High cake moisture content produces
a poor quality filtrate.
C Bed Filtration
Many options are available such as type
of media (sand, coal, gravel, synthetics,
etc. ) size of media (from fine sand to
rock or manufactured media) and flow
direction (up flow or down flow, com-
pressed or expanded bed). Fine media
and downflow operation may resemble
operational characteristics of surface
.filtration. Coarse media, multi media,
expanded beds represent filtration in
depth. In some situations such as trickling
filtration, the process is largely a
biological phenomena rather than intrinsi-
cally filtration.
1 Sand or single media filtration.
Characterized by a high rate of head
loss development with high solids
loading.
a Favorable: High quality effluents
produced. Increased solids, oxygen
demand, and organism removals
specially with low application rates.
Beneficial for upgrading reasonably
good quality treated effluents.
Dependable polishing step. Simple
operational control.
b Limitations: Large area requirement.
Usually requires pretreatment for
removal of most of the solids. High
head loss development specially for
high rate application. Usually
an intermittent operation. Low
capacity per unit time Media
replacement based upon incidence
of "balling," backwash losses,
deposition on the grains, and
nature of feed stock contamination
Possible odor'development
Backwash water may be volumi-
nous and generally requires
retreatment.
2 Soil percolation
a Favorable: Generally'a dependable
method of effluent disposal where
land is available. Returns both
water and wastewater nutrients to
the food chain. Useful for land
reclaimation purposes. Requires
simple operation and low operational
cost. Versatile for use with a wide
variety of wastewater types
b Limitations: Commonly limited wilh
respect to application, rates Requires
a large land area for intermittent
operation. Capital cost primarily
related to land area requirements
Disinfection commonly required.
Good agricultural practice needed
to support good engineering. A
cover crop, tilling and drainage
control generally required, Subject
to seasonal, soil, and topographical
factors. Odors and health hazards
tend to produce a poor public image
Ground or surface water hazard
potential.
3 Multi media filtration
The use of two or more filter media
in which both size and density are
variable makes it possible to distribute
trapped particulates in a wide zone
with respect to filter depth Usually
a larger sized lower density media
are placed over a fine high density
media. Larger particulates are
trapped in the upper zone while the
fine media upgrade effluent clarity
Head loss development occurs more
gradually to permit longer runs of high
product quality as compared with
single media filtration.
a Favorable: Head loss distributed
throughout the bed, builds up more
slowly to permit higher rate and
volume application Dependable
high quality effluent production
Generally high capacity characteristics.
32-3
-------
Unit Operations in Waste Treatment
Capable of being tailored to fit a
particular feed and effluent quality
requirement.
b Limitations: Design generally
requires more careful evaluation
of feed and product quality
requirements. Solids load and
nature are critical. Requires
more careful control during back-
washing to make it a more complex
operation than sand filtration.
Intermittent operation. Media and
equipment more expensive. Usually
requires pretreatment. Backwash
water requires retreatment--may
require more backwash water or
more time than for single media
filtration. Media replacement may
be higher.
D Pressure Floatation
1 The operation consists of aeration
of part or all of the liquid flow in a
covered tank to trap exhaust air and
provide a controlled pressure rise.
Under pressure more gas will dissolve
than can be retained at normal pressures
Discharge of the pressurized liquid to
the clarifying compartment permits
release of excess dissolved'gas. The
released gas tends to associate with
oil, scum and particulates to favor
separation from water as a floatable
concentrate. Variables include time
pressure, turbulence, air-liquid-solid
interface area and nature, and associa-
tion tendencies in both pressure'and
clarifying compartments.
a Favorable
The floatation process is highly
versatile for separation of oils
emulsions or particulates. It may
be used for thickening or clarifica-
tion with or without conditioning
chemicals such as surface active
materials, coagulants or other
separation aids. It is possible to
employ higher loading and higher
overflow rates than for sedimentation.
A higher solids concentration factor
may be achieved. More complete
clarification of hard-to-separate
materials is possible. Usually
requires less area per unit of
capacity.
1) Activated sludge concentration
by floatation is becoming
increasingly popular because the
hydrated solids are amenable to
the floatation process to a greater
extent than for sedimentation
2) Oil and surface active agents
tend to be more completely
separated by floatation to pro-
duce a better clarified product
water.
b Limitations: Usually requires very
careful design and operation for a
specific situation. The complex
operation is sensitive to feed stock
variations. Generally more com-
plex equipment requiring closer-
control. More amenable to moder-
ately concentrated feeds. Thickening
operations may require duplicate
solids handling for removal of float-
able and settleable fractions The
subnatant zone commonly has a high
solids concentration requiring
retreatment. Clarification commonly
is improved by increasing feed stock
concentration. More expensive in
capital and operating cost than
sedimentation
E Centrifugation
The centrifuge has a long history for
dependable separation of liquid-solid
suspensions according to specific gravity
differences. Solid bowl, basket, or disc
type machines are available Horizontal
solid bowl units appear to have the greatest
potential in sanitary engineering. Organic
sludge from water and grit from organic
sludge separations are attractive
Variables include feed rate, solids-liquid
characteristics, feed concentration,
temperature, chemical additives; machine
variables include bowl design, rotational
speed, contained volume, input, distribution
and takeout mechanisms.
1 Favorable: Highly versatile;" may be
designed for high sludge concentration
or high separation on a variety of feeds
High capacity-low area requirements
Low capital cost per un't of capacity
Capable of solids concentrations up to
30 to 35 percent with favorable loads
and operation; continuous performance
2 Limitations: High power requirements
(~0. 5 HP/ gpm). Usually necessary to
make a choice between high solids con-
centration and high solids recovery--
unlikely to have both. Reduced feed
32-4
-------
Unit Operations in Waste Treatment
concentration tends to reduce both con-
centration and recovery of centrifuged
solids. Centrate generally requires
retreatment. Requires suitable design
for a particular function, material and
flow and performs best at rated loading.
in CHEMICAL UNIT OPERATIONS
For purposes of this outline a chemical unit
operation refers to a particular kind of chem-
ical transformation of feed stock (I. c.). The
transformations may be inseparably associated
with both physical and biological changes, for
example, hydrolysis, oxidation and disinfection
are closely related to biological changes,
while coagulation, flocculation are closely
related to physical operations. These trans-
formations may be natural in origin or induced
by chemical additions. In a multicomponent
wastewater the control of treatment largely
is a compromise situation among operations
of biological-chemical-physical natures, some
favoring, some interfering, with the intended
function.
A Neutralization
The combination of excess acids and
alkaline materials to form a salt and
water is a recurrent natural process
called Neutralization. Among other
components, organisms release both
carbon dioxide (acidic) and ammonia
(alkaline) which neutralize each other to
form ammonium bicarbonate and water.
Under certain conditions nature tends to
inhibit itself where an excess of acid or
alkaline materials are favored such as in
low pH deep waters or high pH surface
waters. Growth may become limited
because of pressure retention of excess
CO^ or rapid assimilation of CO^
respectively. This unbalance is unlikely
to be as serious as that due to local dis-
charge of acid or alkali released from
manmade sources. Neutralization is a
common requirement.
1 Favorable: Neutralization enhances the
probability for biological and certain
chemical transformations. Commonly
reduces corrosivity of acid waters.
Increases acceptability of acid or
alkaline waters for beneficial reuse
in water supply, recreation, wildlife,
agricultural industry and esthetics.
2 Limitations: Generally high operating
• and control cost. May produce gross
quantities of solids for disposal or
increase the dissolved solids in the
water. Requires close control to
prevent excessive additions.
B Oxidation
The oxidation of organic waste components
in water is a primary consideration in
wastewater stabilization. This process is
intimately linked with solids-liquid sep-
aration. For example, organic soluble
compounds may be oxidized biochemically
to form settleable agglomerates of cell
mass, to removable gaseous CO^ and to
less reactable water. Nitrogen compounds
may be converted to the oxidized state and
reduced to less reactable and removable
nitrogen gas.
1 The use of oxygen (aeration or surface
oxygenation) from air is by far the most
used unit operation for supplying
essential oxygen for intermediate and
terminal stabilization.
a Favorable: Generally available, low
cost. Necessary pumping, cleaning
and transfer equipment available at
reasonable cost. Moderate power
cost. Transfer capability reasonably
good. Dependable supply.
b Limitations: Limited solubility of
oxygen in contact with air. Large
capital investment in tankage and
space. Air solubility and transfer
limitations generally mean a low to
moderate rate process.
2 The use of commercial oxygen instead
of air permits a five-fold increase in
oxygen partial pressures.
a Favorable: Higher oxygen partial
pressures permit higher solubility
of oxygen in water and greater
32-5
-------
Unit Operations in Waste Treatment
oxygen transfer rate in high demand
situations. More likely to maintain
a higher residual DO in high rate
situations, or through the clarifier
stage. More complete stabilization
possible in less tankage and/or time.
High oxygen tension favors sludge
oxidation--lowers solids accumulation.
b Limitations: Better design require-
ments necessary to maintain high
oxygen use efficiency. More complex
system, more costly. Covered
tanks and oxygen production facilities
nearby usually required to favor
cost/benefit ratios. Requires better
operational control.
3 Ozone is another form of oxygen used
primarily for special purposes.
a Favorable: Ozone is used primarily
for odor control because of its high
oxidizing energy and high activity in
water. Capable of reacting with
components that may not react with
oxygen under similar conditions.
b Limitations: Ozone (Ois a highly
unstable compound. Generally
cannot be stored or made in high
concentrations. Usually requires
formation on site and use in pre-
treated water. High unit cost.
Complex control.
4 Chlorine is commonly considered for
disinfection; disinfecting properties
are inherently associated with oxidizing
energy.
a Favorable: Commercially available
chemical, control equipment available,
operating controls generally known.
High energy material. Relatively
simple operation. Versatile material
capable of use in a variety of situations.
Cost higher than that for oxygen but
has a greater reactivity for many
beneficial operations. Rapid reaction
in most situations.
b Limitations: Hazardous-nonspecific
toxicity in air or water. Chlorine
reaction produces HC1 during
reaction. Usually requires neutral-
ization. Highly corrosive in water
solution or wet gas. Certain com-
ponents such as ammonia prefer-
entially react with chlorine to cause
high chlorine demands. Requires
close control. Generally requires
pretreatment to avoid excessive
chlorine dosage.
5 Peroxy-acid oxidizing agents
Permanganate and dichromate are the
most common peroxy-acids used in
sanitary engineering. Permanganate
is relatively pH independent, dichromate
is an effective oxidant only under acid
conditions. Both have high oxidizing
energy for special purposes.
a Favorable: High oxidizing energy.
Capable of being separated from
product water. Adaptable for special
requirements such as destruction of
most organic materials or color.
Permanganate may occur naturally
in water and in excess its color is
its own indicator.
b Relatively high cost per unit of
oxidizing energy. Excess reagent
contributes to poor quality water.
Close control required. Commonly
does not oxidize ammonia nitrogen.
May require catalysis to reduce
delayed reactions.
C Hydrolysis
The addition of water to split large
molecules into two or more simpler
substances is an inevitable part of
biodegradation. Cell mass may be
.hydrolyzed to form smaller component
parts that are partially oxidized to yield
energy for building another crop of cells.
The process will continue as long as
oxidation energy is sufficient for growth.
Treatment tends to produce a low energy
32-6
-------
discharged effluent in which hydrolysis
and oxidation become lower rate operations
(stabilized).
1 "Favorable: Items favoring hydrolytic
cleavage (liquefaction) include high or
low pH, hydrolase enzymes or other
catalysts, high temperatures, low or
negative oxidation reduction potential
(low oxygen tension) or anything favoring
introduction of water into a complex
molecule.
2 Limitations: Any situation favoring
resynthesis of hydrolyzed components
into larger molecules reduces the net
effect of hydrolysis. Algal photo-
synthesis, bacterial or plant growth,
absence of toxic components, and
favorable conditions for growth usually
are associated with high rate hydrolysis
in a high energy situation but growth
may be the predominant reaction.
Any situation favoring dehydration
limits hydrolysis.
IV PROCESSES USED FOR THE REMOVAL
AND DISPOSAL OF ORGANIC MATERIAL
Isolation and stabilization are the key factors
in wastewater treatment. Unstable inter-
mediates must be stabilized to be acceptable
as gaseous or solid residues. Isolation refers
to separation of gaseous and solids residues
from the recombining media--water. It is
not possible to isolate or to stabilize to "end"
products--somewhere in time recycle will
occur. Each treatment operation is intended
to hasten recycle for beneficial use and delay
other types of recycle.
A Table 1 summarizes the functions of
various stages of treatment. Certain
unit operations are repeated at each
stage in a different manner.
TABLE 1
WASTEWATER TREATMENT STAGES
Preliminary or Pretreatment:
1. Removal of roots, rocks, rags
2. Removal of sand, grit, gravel
3. To "freshen" the wastewater by short
term aeration, chlorination, grinding
or otherwise protect and promote sub-
sequent treatment
Primary Treatment
Removal of readily settleable or floatable
components
Secondary Treatment
Conversion of soluble or colloidal components
to removable form with partial stabilization
in process (commonly biodegradation).
Advanced Treatment of Wastewaters
Biological chemical or physical treatment
of used water to meet specific reuse quality
requirements. May consist of general or
specific item clean-up.
Solids Disposal
Nonpollutional takeout favoring conversion
to stable residues and separation of gas,
liquid, and solid phases.
B Tables 2 and 3 list selected unit operations
and the stages or processes in which they
may be used. Note that many of these
may occur repeatedly and that there is the
possibility of including one or more
options in any given treatment process
32-7
-------
Unit Operations in Waste Treatment
depending upon performance requirements
and nature of the problem. These lists of
physical (2) and chemical (3) operations
are not complete. It is not likely that all
of those listed may be included in any
modification of a treatment facility. The
problem is to select a series of operations
suitable to meet the performance require-
ments of the situation at a favorable cost/
benefit ratio.
Several processes are possible for
secondary treatment. Each of these have
several modifications. The most common
are based upon biological-physical unit
operations. Chemical-physical operations
may be used to upgrade the overall
removal, or to remove specific components
commonly not sufficiently removed by
treatment. The same processes may be
used for advanced treatment providing the
degree of removal is upgraded on all
components of interest to meet specific
reuse requirements,
TABLE 2
UNIT OPERATIONS BY TREATMENT STAGE OR
PROCESS - PHYSICAL TRANSFORMATIONS
Unit Operation
Stage or Process*

Pre
Pri
Sec
Adv
AS
TF
OP
SP D
Fluid Transfer

liquid pumping
X
X
X
X
X
X
X
X X
mixing
X
X
X
X
X
X
X
X X
sludge pumping

X
X
X
X
X

X X ,
process residues-scum
X
X
X
X
X
X

Gas Transfer

into process-oxygenation
X

X
X
X

X
stripping
X

X
X
X

X
mixing
X

X
X
X

X
Solids Transfer

applied chemicals

X
X
X
X

X
process residues
X
X
X
X
X
X

X X
Heat Transfer

X X
Solids-Liquid Separation
p

coarse screening
X

microscreening

X
X
X
X

gravity sedimentation
X
X
X
X
X
X
X
X X
filtration

X
X
X
X
X
X
evaporation-drying

X X
distillation

floatation
X
X
X
X
X
X

X
thickening

X
X
X
X
X

X
centrifugation

X
X
X
X
X

X
adsorption

X
X
X
X
X
X
elutriation
X
X
X
X
X
X
X
X X
flocculation

X
X
X
X
X
X
X X
32-8
-------
Unit Operations in Waste Treatment
TABLE 3
UNIT OPERATIONS BY TREATMENT STAGE OR PROCESS
CHEMICAL TRANSFORMATIONS
Unit Operation
Stage or Process*
Pre
Oxidation Reduction
wet combustion
(biol-chem)
dry combustion
corrosion
bleaching (color removal)
Disinfection
Hydrolysis (liquefaction)
'Solids-Liquid Separation
coagulation
precipitation
ion exchange
electrodialysis (phy-chem)
Pri
Sec
A dv
AS
TF
OP
SP
D
X
X
X

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

X
X
X
X
X

X
X
X
X
X
X
X

X
X
X
X
X
X
X
X
x
X
X
X
X
X
x
X
X
X
X
X
X
X
X
X
complexation
X
X
X
X
X
X
X X
assimilation (biol-chem)

X
X
X
X
X
X
absorption (phy-chem)

X
X
X
X
X
X
Neutralization
X
X
X
X
X
X
X X
* Coding Tables 1 and 2
Pre - Preliminary or pretreatment stage
Pri - Primary clarification stage
Sec - Secondary treatment stage
Adv - Advanced treatment stage
AS - Activated sludge treatment process
TF - Trickling filtration treatment process
OP - Oxidation pond treatment process
SP - Sludge processing stage
D - Disposal stage (solids)
32-9
-------
Unit Operations in Waste Treatment
1 Activated sludge
This process is based upon a mixed
fluid suspension of solids concentrates
from previous operations and raw
wastewater in the presence of excess
oxygen to rapidly stabilize the incoming
pollutants by biological growth, trans-
fer to the solids phase, agglomeration
and solids liquid separation.
a Favorable: Versatile process
capable of being adapted to high
performance on most types of
organic contaminants. Generally
capable of high efficiency in stabi-
lization and clarification. Lower
tankage and area requirements than
for most biological processes.
May be modified to achieve high
removal of nitrogen phosphorus and
solids. Adaptable to a wide range in
removal efficiency.
b Limitations: Requires close control
of load ratios and operating conditions.
High oxygen requirements. Subject
to upset by qualitative and quantitative
shock loads. Unmodified process
commonly shows poor removal of
nitrogen and phosphorus. Subject to
the variations of characteristic of
biological treatment. High operating
cost.
2 Trickling filtration
Employs an attached media of sewage
slimes on the support surface for transfer
and stabilization of organic pollutants in
the influent.
a Favorable: Versatile process capable
of being adapted for intermediate
performance on most types of organic
waste. Low operating cost. Adaptable
for a fairly wide range of removal on
substances showing good solids trans-
fer efficiency.
b Limitations: High capital cost for
land and tankage (rock or slag).
High pumping cost on manufactured
media. Generally not amenable for
coagulation and clarification. Fewer
operating controls possible.
Subject to the variations character-
istic of biological treatment even
though it may not be as noticeable
due to generally turbid effluents.
3 Oxidation ponds
Employs natural purification phenomena
of sedimentation, aerobic and anaerobic
degradation, algal photosynthesis
usually in a sacrificial pond or series
of ponds.
a Favorable: Capable of high treat-
ment efficiencies with low operational
cost. Adaptable to low or high
removal efficiencies depending on
land and capacity or time availability.
Useful on a wide variety of waste-
waters. 'Land is available for
upgrading treatment and other uses
as needed.
b Limitations; Generally limited to
application where land costs are
low. Subject to poor performance
during ice cover, overloads, spring
warmup and unexpected boil-up.
May be an odor nuisance at times.
Generally a low rate process with
poor solids recovery characteristics.
Appears to have a tendency to poorer
performance after several years of
operation.
4 Physical chemical treatment operations
Physical chemical treatment by lime
precipitation and activated carbon
adsorption is becoming recognized to
an increasing degree.
a Favorable: Is a versatile process
capable of being adapted to very
high degrees of treatment on a
variety of wastewaters. Recovery
of added lime and regeneration of
spent carbon by controlled incin-
eration permits chemical reuse and
reduces solids disposal. Relatively
low capital costs and space require-
ments. Capable of application over
32-10
-------
Unit Operations in Waste Treatment
wide flow variations with dosage
and regeneration time control.
Freedom from toxic effects.
b Limitations: Generally higher
operating cost. More complex
process requires precise operational
•control. May require pretreatment.
Chemical reuse almost mandatory to
limit solids disposal requirements.
Operating history for wastewater
applications scant.
D Sludge Processing or Disposal Routes
1 Wet combustion by aerobic biological
processes
Activated sludge, trickling filtration
and oxidation ponds involve a certain
amount of processing and disposal of
solids to a degree limited by the amount
of carbon dioxide and water formed in
process. Aerobic sludge digestion
accentuates solids disposal.
a Favorable: Generally a conventional
bio-chemical process using estab-
lished procedures. Time, oxygen
supply and favorable conditions are
basic requirements. Versatile for
a variety of wastes. Generally
capable of a high degree of stabilization.
b Limitations: Process limited to a
residue solids level containing about
40% volatile content (10 to 20% of the
feed volatile solids). High liquid
recycle of N, P, and solids content.
Generally a long term operation.
Process interference serious for low
temperatures, toxic agents, or
unfavorable pH. Generally produces
a low concentration sludge 2%).
2 Wet combustion--elevated temperature
and pressure
Wet combustion of organic-wastes at
various pressures in enclosed vessels
with liquid temperatures of 350OF or
above have been effective for separation
of a highly stable mineralized ash.
a Favorable: Capable of producing
an easily separated high-mineral
ash. ¦ Rapid process low area
requirements, low solids disposal
volume.
b Limitations: Requires complex
equipment and high heat require-
ments. Good control essential.
Residual liquid has a high color and
contaminant level.
3 Anaerobic digestion
This process is both a sludge con-
ditioning and a solids separation
process. Sludge residuals after
digestion are more concentrated in
solids content and decreased in volume
and mass due to escape of methane and
carbon dioxide gas or elutriate.
a Favorable: Versatile and dependable
process of organic stabilization for
suitable loading, mixing and tem- ¦
peratures. Low cost operation.
Produces a usable product gas.
Residual solids relatively stable,
improved in concentration, and
drainability.
b Limitations: Capital costs relatively
high for space and tankage.
Susceptible to shock loading, tem-
perature changes, poor mixing or
toxicity. Once upset--it requires
appreciable time and effort to
restore good performance. Recycle
liquids are high in dissolved and
suspended solids; are difficult to
treat.
4 Drying and incineration
Many modes of drying such as drying
beds, land spreading, flash drying are
possible with wet sludge. Incineration
in a fluidized bed, rotary kiln or
multiple hearth are used. The multiple
hearth is one example of drying and
incineration.
a Favorable: Dependable, versatile
operation for thick sludges where
32-11
-------
Unit Operations in Waste Treatment
heat release is close to heat require-
ments for water evaporation and
temperature rise. Can be controlled
to produce clean stack gases and
stable mineral solids residue. Rapid
process. Low area requirements.
Control techniques well established.
b Limitations: Solids feed of low heat
and high water content may require
excessive auxiliary fuel cost.
Generally requires stack gas
reburning and solids recovery to
meet air quality requirements.
Generally costs about $50 plus per
ton of dry solids for operation.
Requires close control of feed,
burner temperature and other
operating variables.
5 Wet disposal of sludge
¦b Limitations: Good engineering and
farming practice are required.
The local residents do not appreciate
receiving waste materials from
elsewhere unless practice and
public relations are top rate.
Possible hazards from surface
ground water and air pollution dim
the good neighbor policy.
ACKNOWLEDGMENT:
This outline contains appreciable material
from a previous outline by F. P. Nixon.
REFERENCES
1 Rose, Arthur and Elizabeth. Condensed
Chemical Dictionary, 7th Edition.
Reinhold Publishing Corporation.
New York. 1961.
Application of wet sludge to spoil areas,
stripped terrain, farm or marginal
land has been common practice for a
long time. Piping instead of truck
hauling is receiving increased con-
sideration to extend the disposal area.
2 APHA,' ASCE, AWWA, WPCF.
Glossary, Water and Wastewater
Control Engineering. 1969.
3 Rich, Linvil G. Units Operations in
Sanitary Engineering. Wiley. 1961.
a Favorable: Costs of disposal may
be reduced by avoiding costly drying
operations. Pumping of wet sludge
is more economical than hauling.
Possibilities for use of the organic
and water for reclamation of waste
land is attractive as a means of
recycling the wastes into the food
chain. Isolation possibilities are
improved by remote application
from population centers.
4 Rich, Linvil G. Unit Processes of
Sanitary Engineering. Wiley. 1963.
This outline was prepared by F. J. Ludzack,
Chemist, National Training Center,
DTTB, MDS, WPO, EPA, Cincinnati,
OH 45268.
32-12
-------
JUL 12 1992
DATE DUE
-------