&EPA
United States
Environmental Protection
Agency
National Training
and Operational
Technology Center
Cincinnati OH 45268
EPA-430/1 -79-007
August 1979
Water
Methods for the
Determination of
Bacteriological
Contaminants in
Drinking Water
Training Manual
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August 1979
EPA-430/1-79-007
METHODS FOR THE DETERMINATION OF BACTERIOLOGICAL
CONTAMINANTS IN.DRINKING WATER
This student manual was developed by the U.S. Environmental
Protection Agency, National Training & Operational Technology
Center with the Technical Support Division in response to a
request from the Office of Drinking Water.
National Training and Operational Technology Center
Municipal Operations and Training Division
Office of Water Program Operations
U.S. Environmental Protection Agency
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DISCLAIMER
Reference to commercial products, trade names, or manufacturers
is for purposes of example and illustration. Such references do
not constitute endorsement by the Office of Water Program Opera-
tions, U.S. Environmental Protection Agency.
This manual has been prepared from the National Interim Primary
Drinking Water Regulations and the references contained therein
which constitute the legal authority for these procedures. When
used within a State having been granted primary enforcement
authority, that State's regulations will then constitute the
legal authority and should be followed.
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CONTENTS
TITLE OUTLINE NO.
Federal Register - National Interim Primary Drinking
Water Regulations 1
DRINKING WATER TESTS
Collection and Handling of Drinking Water Samples 2
Coliform Test by the MPN Method for Drinking Water 3
Completed Test for the MPN Method for Drinking Water 4
Total Coliform Test for Drinking Water by the Membrane
Filter Method 5
Verified Membrane Filter Test for Drinking Water 6
SUPPLEMENTARY TEST PROCEDURES
Standard Plate Count 7
Residual Chlorine and Turbidity 8
Coliform Test by the Multiple Dilution Tube Method (MPN) 9
Completed Test for the MPN Method 10
Total Coliform Test by the Membrane Filter Method 11
Laboratory Safety Practices 12
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WEDNESDAY, DECEMBER 24, 1975
(ft
PART IV:
ENVIRONMENTAL
PROTECTION
AGENCY
WATER PROGRAMS
National Interim Primary Drinking
Water Regulations
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59566
RULES AND REGULATIONS
Title 40—Protection of Environment
CHAPTER I—ENVIRONMENTAL
PROTECTION AGENCY
SUBCHAPTER O—WATER PROGRAMS
|FRL 464-7)
PART 141—NATIONAL INTERIM PRIMARY
DRINKING WATER REGULATIONS
On March 14,1975, the Environmental
Protection Agency (EPA) proposed Na-
tional Interim Primary Drinking Water
Regulations pursuant to sections 1412,
1414.1415. and 1450 of the Public Health
Service Act ("the Act"), as amended by
the Safe Drinking Water Act ("SOW A,"
Pub. L. 93-523), 40 FR 11990 EPA held
public hearings on the proposed regula-
tions in Boston. Chicago. San Francisco,
and Washington during the month of
April Several thousand pages of com-
ments on the proposed regulations were
received and evaluated In addition, the
Agency has received comments and In-
formation on the proposed regulations
from the National Drinking Water Ad-
visory Council, the Secretary of Health,
Education, and Welfare, and from num-
erous others during meetings with repre-
sentatives of State agencies, public in-
terest groups and others.
The regulations deal only with the
basic legal requirements. Descriptive
material will be provided in a guidance
manual for use by public water systems
and the States.
The purpose of this preamble to the
final regulations Is to summarize the most
significant changes made in the proposed
regulations as a result of comments re-
ceived and the further consideration of
available information. A more detailed
discussion of the comments and of
changes in the proposed regulations Is
attached as Appendix A.
WATER SYSTEMS COVERED
The Safe Drinking Water Act applies
to each "public water system," which is
denned In Section 1401(4) of the Act as
"a system for the provision to the public
of piped water for human consumption.
If such system has at least fifteen service
connections or regularly serves at least
twenty-five individuals " Privately owned
as well as publicly owned systems are
covered. Service "to the public" Is Inter-
preted by EPA to Include factories and
private housing developments. (See gen-
erally. House Report, pp 16-17.)
The definition of "public water sys-
tem" proposed In the Interim Primary
Drinking Water Regulations sought to
explain the meaning of the statutory
reference to "regular" service. It was
proposed to interpret this term as Includ-
ing service for as much as three months
during the year. Because the proposed
definition would have excluded many
large campgrounds, lodges, and other
public accommodations which serve
large numbers of tourists but which are
open for slightly less than three months
each year, the definition In the final ver-
sion covers systems serving an average of
at least twenty-five individuals at least
60 days out of the year. The use of a
minimum number of days rather than
months also makes clear that a system
may qualify as a public water system
even If It is not open every day during a
given month.
Once "public water system" has been
defined, it is necessary to define the two
major types of public water systems—
those serving residents and those serv-
ing transients or intermittent users. The
possible health effects of a contaminant
in drinking water in many cases are quite
different for a person drinking the water
for a long period of time than for a per-
son drinking the water only briefly or in-
termittently Different regulatory con-
siderations may in some cases apply to
systems which serve residents as opposed
to systems which serve transients or in-
termittent users. Accordingly, § 141.2(e)
makes clear that all "public water sys-
tems" fall within either the category of
"community water systems" or the cate-
gory of "non-community water systems."
To make clear which regulatory require-
ments apply to which type of system, the
category covered is specifically Indicated
throughout the regulations.
The proposed regulations defined a
"community water system" as "a public
water system which serves a population
of which 70 percent or greater are resi-
dents." Reliance In the proposed defini-
tion on the percentage of water system
users who are residents would result In
treating some fairly large resort com-
munities with many year-round residents
as non-community systems Therefore.
the definition of "community water sys-
tem" has been changed to cover any sys-
tem which serves at least 15 service con-
nections used by year-round residents or
serves at least 25 year-round residents.
SMALL COMMUNITY WATER SYSTEMS
Many community water systems in the
country are quite small Since it is the
intention of the Act to provide basically
the same level of health protection to
residents of small communities as to
residents of large cities, and since a num-
ber of advanced water treatment tech-
niques are made feasible only by eco-
nomies of scale, the cost of compliance
with the requirements of the Act may
pose a serious problem for many small
communities. The regulations seek to
recognize the financial problems of small
communities by requiring more realistic
monitoring for systems serving fewer
than 1.000 persons Variances and ex-
emptions authorized by the Act can also
assist in dealing with economic problems
of small community systems in appropri-
ate cases, at least temporarily EPA will
provide technical assistance on effective
treatment techniques which can be used
by small systems
These methods of dealing with the fi-
nancial problems of some small com-
munity systems may not be sufficient in
specific Instances to make compliance
with all applicable regulatory require-
ments feasible EPA is commencing a
study of potential problems faced by
small community systems in meeting ap-
plicable requirements under the Act and
these regulations, and, If necessary, will
make additional adjustments In the In-
terim Primary Drinking Water Regula-
tions prior to their effective date.
NON-COMMUNITY SYSTEMS
"Non-community systems" are basic-
ally those systems which serve transients.
They include hotels, motels, restaurants,
campgrounds, service stations, and other
public accommodations which have their
own water system and which have at
least 15 service connections or serve
water to a daily average of at least 25
persons. Some schools, factories and
churches are also included in this cate-
gory. It Is conservatively estimated that
there are over 200,000 non-community
water systems in the country However, it
should be recognized that while their
number is large, they normally are not
the principal source of water for the
people they serve
The regulations as proposed would
have applied all maximum contaminant
levels to non-community systems as well
as to community systems This approach
failed to take into account the fact tint
the proposed maximum contaminant
levels for organic chemicals and most in-
organic chemicals were based on the
potential health effects of long-term ex-
posure. Those levels are not necessary
to protect transients or intermittent
users. Therefore, the final regulations
provide that maximum contaminant
levels for organic chemicals, and for in-
organic chemicals other than nitrates,
are not applicable to non-community
systems An exception was made for ni-
trates because they can have an adverse
health effect on susceptible infants In a
short period of time.
Even without monitoring for organic
chemicals or most inorganic chemicals.
in the initial stages of implementation
of the drinking water regulations, mon-
itoring results from tens of thousands of
non-communltv systems could over-
whelm laboratory capabilities and other
resources This could delay effective im-
plementation of the regulations with re-
spect to the community systems which
provide the water which American?
drink every day. To avoid this result.
non-community systems will be given
two years after trie effective date of the
regulations to commence monitoring In
the meantime, non-community systems
which already monitor their water are
encouraged to continue to do so, and the
States are encouraged to take appropri-
ate measures to test or require monitor-
Ing for non-community systems that
serve large numbers of people
Of course, non-community systems
which pose a threat to health should be
del If with as quickly as possible. The
maximum contaminant levels applicable
to non-community water systems there-
fore will take effect 18 months after pro-
mulgation, at the same time as levels ap-
plicable to community svstems. Inspec-
tion and enforcement authority will ap-
ply to non-community systems at the
same time as to community systems.
SANITARY SURVEYS
EPA encourages the States to conduct
sanitary surveys on a systematic basis.
FEDERAL REGISTtR, VOL. 40, NO. 2-18—WEDNESDAY, DCCCMDER 24, 19/5
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RULES AND REGULATIONS
39567
These on-slte inspections of water sys-
tems are more effective in assuring safe
water to the public than individual tests
taken in the absence of sanitary surveys.
The regulations provide that monitor-
ing frequencies for conform bacteria can
be changed by the entity with primary
enforcement responsibility for an indi-
vidual non-community system, and in
certain circumstances for an individual
community system, based on the results
of a sanitary survey.
MAXIMUM CONTAMINANT LEVELS
Numerous comments were received by
EPA on the substances selected for the
establishment of maximum contaminant
levels and on the levels chosen. Congress
anticipated that the initial Interim Pri-
mary Drinking Water Regulations would
be based on the Public Health Service
Standards of 1962. and this Congres-
sional Intent has been followed Com-
ments received on the various.levels did
not contain new data sufficient to re-
quire the establishment of levels differ-
ent from those contained In the Public
Health Service Standards
WATER CONSUMPTION
The maximum contaminant levels are
based, directly or indirectly, on an as-
sumed consumption of two liters of water
. per day. The same assumption was used
in the 1962 Standards. This assumption
has been challenged because of instances
where much higher water consumption
rates occur. EPA's justification for using
the two-liter figure is that it already
represents an above average water or
water-based fluid intake. Moreover, while
the factor of safety may be somewhat re-
duced when greater quantities of water
are ingested, the maximum contaminant
levels based on the two-liter figure pro-
vide substantial protection to virtually
all consumers. If, as has been suggested,
a water consumption rate of eight liters
per day is used as the basis for maxi-
mum contaminant level, all of the pro-
posed MCL's would have to be divided by
four, greatly Increasing the monitoring
difficulties, and in some cases challeng-
ing the sensitivity of accepted analytical
procedures. It could be expected, in such
a case, that the maximum contaminant
levels would be exceeded to a significant
degree, and that specialized treatment
techniques would be required to order
that the contaminant levels would be re-
duced. The economic Impact of a move
in this direction would be enormous. It
is not technically or economically feasi-
ble to base maximum contaminant levels
on unusually high consumption rates.
SAFETY FACTORS
A question was raised about the fact
that different safety factors are con-
tained in various maximum contaminant
levels. The levels are not Intended to
have a uniform safety factor, at least
partly because the knowledge of and the
nature of the health risks of the various
contaminants vary widely. The levels set
are the result of experience, evaluation
of the available data, and professional
judgment. They have withstood the test
of time and of professional review. They
are being subjected to further review by
the National Academy of Sciences in con-
nection with development of data for the
Revised Primary Drinking Water Regu-
lations.
MCL's BASED ON TEMPERATURE
A question was also raised as to
whether ranges of maximum contami-
nant levels should be established on the
basis of the climate in the area served
by the public water system, as was done
with fluoride EPA believes that the use
of a temperature scale for fluoride is
more appropriate than for other chemi-
cals because of the studies available on
the fluoride-temperature relationship
and because there Is a small margin with
fluoride between beneficial levels and
levels that cause adverse health effects
MCL's DELETED
Three proposed maximum contami-
nant levels have been eliminated in the
final regulations because they are not
justified by the available data One of
these is carbon chloroform extract
(CCE), which Is discussed separately
below. The others are the proposed levels
for the standard bacterial plate count
and cyanide. In the case of the plate
count, it is believed that the coliform
limits contained In the regulations, com-
bined with the turbidity maximum con-
taminant level, adequately deal with
bacterial contamination. However, EPA
continues to believe that the standard
plate count is a valid indicator of
bacteriological quality of drinking water,
and recommends that it be used in ap-
propriate cases in conjunction with the
coliform tests as an operational tool.
The proposed maximum contaminant
level for cyanide was eliminated because
the possibility of cyanide contamination
can be effectively addressed only by the
use of emergency action, such as under
Section 1431 of the Act. EPA's 1969 Com-
munity Water Supply Study did not
reveal a single Instance in which cyanide
was present in a water system at a level
greater than one-thousandth of the level
at which cyanide is toxic to humans.
Available data Indicate that cyanide
will be present in water systems at toxic
levels only in the event of an accident,
such as a spill from a barge collision.
Maximum contaminant levels are not
the appropriate vehicle for dealing with
such rare, accidental contamination.
Heptachor, heptachlor epoxlde
and chlordane have also been removed
from the list of maximum contaminant
levels at least temporarily in view of the
pending cancellation and suspension
proceedings under the Federal Insecti-
cide, Fungicide and Rodentlclde Act in-
volving those pesticides. When the re-
sults of these proceedings are available,
EPA will again consider whether maxi-
mum contaminant levels should be es-
tablished for those three pesticides.
SODIUM AND SULFATES
A number of comments were received
on the potential health effects of sodium
and sulfates. The National Drinking
Water Advisory Council has recom-
mended that consideration be given to
the monitoring of these constituents, but
has not recommended the adoption of
maximum contaminant levels because
available data do not support the adop-
tion of any specific levels. EPA has re-
quested the National Academy of Sci-
ences to include sodium and sulfates
among the contaminants to be studied
by NAS. and to include information on
the health effects of sodium and sulfates
in the report to be made by NAS in
December 1976
Since a number of persons suffer from
diseases winch are influenced by dietary
sodium intake and since there are others
who wish to restrict their sodium in-
take, it is desirable that the sodium con-
tent of drinking water be known. Those
affected can. bv knowing the sodium con-
centration in their drinking water, make
adjustments to their diets or, in extreme
cases, seek alternative sources of water
to be used for drinking and food prepara-
tion It is recommended that the States
institute programs for regular monlloi -
ing of the sodium content of drinking
water served to the public, and for in-
forming phvslcians and consumers of the
sodium concentration in drinking water.
A relatively high concentration of sul-
fate In drinking water has little or no
known laxative effect on regular users of
the water, but transcients using such
water sometimes experience a laxative
effect It is recommended that the States
institute monitoring programs for sul-
fates, and that transients be notified if
the sulfate content of the water is high
Such notification should include an as-
sessment of the possible physiological
effects of consumption of the water
PCB's AND ASBESTOS
An interagency comment expiessed
concern for asbestos and PCB's in the
environment and noted the need for tit
least a monitoring requirement, if not
for MCL's, for these contaminants. EPA
is also concerned, but for the moment
lacks sufficient evidence regarding ana-
lytical methods, health effects, or occur-
rence In the environment to establish
MCL's. The Agency Is conducting re-
search and cooperating In research proj-
ects to develop criteria for establishing
needed limits as quickly as possible. A
monitoring study on a number of organic
chemical contaminants, including PCB's,
for which MCL's are not being estab-
lished at this time, will be contained in
an organic chemical monitoring regula-
tion that is being promulgated with these
regulations. Regarding asbestos, HEW
and EPA are sponsoring a number of
studies this year at an approximate cos>t
of $16 million to establish health effects,
anayltical methods and occurrence
POINT or MEASUREMENT
Other comments on maximum con-
taminant levels focused on the proposed
requirement that such levels be tested
at the consumer's tap. Concern was ex-
pressed over the inability of the public
water system to control potential sources
FEDERAL REGISTER, VOL. 40, NO. 248—WEDNESDAY, DECEMBER 24, 1975
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59568
RULES AND REGULATIONS
of contaminants which are under the
control of the consumer.
The promulgated definition of "maxi-
mum contaminant level," § 141.2(d), re-
tains the requirement that the maxi-
mum contaminant level be measured at
the tap except in the case of turbidity,
which should be measured at the point
of entry to the distribution system. How-
ever, the definition has been expanded
to make clear that contaminants added
to the water by circumstances under the
control of the consumer are not the re-
sponsibility of the supplier of water,
unless the contaminants result from cor-
rosion of piping and plumbing resulting
from the quality of the water supplied.
It should be noted, however, that this
requirement should not be interpreted
as to discourage local, aggressive cross
connection control measures.
COLIFORM BACTERIA MCL's
The promulgated MCL's for cohform
bacteria are basically the 1962 Public
Health Service Standards, with minor
refinements and clarifications. However,
further changes may be desirable. For
example, the MCL's for the membrane
filter analytical method do not resolve
the question of how many coliform bac-
teria are assumed to be present in a
single highly contaminated sample.
Some laboratories assume an upper limit
of 50. while others seek to continue to
count Individual bacteria to a level of
100 or even higher in a single sample.
The upper limit assumed will affect the
monthly average which Is calculated to
determine compliance with the MCL's.
Another question relating to the coli-
form bacteria MCL's is the matter of
possible spurious positive samples. As the
regulations are written, all routine sam-
ples taken to determine compliance with
the MCL's must be counted, regardless
of the results of analysis of any check
samples that may be taken. The reason
for this is that bacterial contamination
is often Intermittent or transient, and as
a result negative check samples taken a
day or more after a positive sample can-
not demonstrate that the positive result
was In error. It may be possible, however,
to prescribe a means of dealing with spu-
rious positive results without compro-
mising the Integrity of the MCL's.
A third question concerning the MCL's
for coliform bacteria Is the relationship
of monthly averages of coliform bacteria
levels to monthly percentages of positive
samples. For example, the monthly av-
erage MCL for the membrane filter
method Is violated If the monthly aver-
age exceeds one coliform bacterium per
sample. However, for purposes of deter-
mining whether the monthly-percent-
age-of-positlve-samples MCL is violated,
a sample is counted as positive only if it
contains more than four coliform bac-
teria. Thus, it Is possible, particularly
when a relatively small number of sam-
ples Is taken, for a system to fail the
monthly average MCL even when no sin-
gle sample taken during the month 1s
out of compliance with the limit.
These and other questions concerning
the coliform bacteria MCL's will be re-
viewed further by EPA. If review Indi-
cates that changes In the MCL's are
desirable, those changes will be made as
soon as possible but within 6 months, in
time to take effect at the same time as
the initial Interim Primary Drinking
Water Regulations
ORGANIC CHEMICALS
The proposed maximum contaminant
levels for organic pesticides, other than
the three which are the subject of can-
cellation and suspension proceedings,
have been retained. It is anticipated that
additional organic pesticides will be
added to the regulations if surveys of
pesticides in drinking water being con-
ducted by EPA Indicate that this Is
needed.
The proposed regulations also con-
tained a maximum contaminant level for
organic chemicals obtained by the carbon
chloroform extract (CCE) method. It
was anticipated by Congress that organic
chemicals would be dealt with primarily
in the Revised Primary Drinking Water
Regulations because of the paucity of ac-
curate data on the health effects of vari-
ous organic chemicals, the large number
of such chemicals, uncertainitles over ap-
propriate treatment techniques, and the
need for additional information on the
incidence of specific organic chemicals
in drinking water supplies. EPA thought
that the CCE standard might provide an
appropriate means of dealing with or-
ganic chemicals as a class pending action
on the Revised Primary Regulations
The CCE standard was originally de-
veloped as a test for undesirable tastes
and odors in drinking water As concern
developed over the health effects of or-
ganic chemicals, the possibility of using
CCE as a health standard rather than
.an esthetic standard was considered.
As pointed out by numerous comments,
CCE has many failings as an indicator
of health effects of organic chemicals.
To begin with, the test obtains Informa-
tion on only a fraction of the total
amount of organic chemicals in the water
sampled. Furthermore, there Is serious
question as to the reliability of CCE in
identifying those organic chemicals
which are most suspected of adverse
health effects. In addition, there are no
existing data on which a specific level
for CCE cun be established on a rational
basis. To establish a maximum contami-
nant level under these circumstances
would almost certainly do more harm
than good. It could give a false sense of
security to persons served by systems
which are within the established level
and a false sense of alarm to persons
served by systems which exceed the level.
It also would divert resources from
efforts to find more effective ways of
dealing with the organic chemicals
problem.
EPA believes that the Intelligent
approach to the organic chemicals ques-
tion is to move ahead as rapidly as pos-
sible along two fronts. First, EPA Is
adopting simultaneously with these reg-
ulations a Subpart E of Part 141, con-
taining requirements for organic chemi-
cal monitoring pursuant to Sections 1445
and 1450 of the Act.
The regulations require that desig-
nated public water systems collect sam-
ples of raw and treated water for submis-
sion to EPA for organics analysis. EPA
will analyze the samples for a number of
broad organic parameters, including car-
bon chloroform extract (CCE), volatile
and non-volatile total organic carbon
(VTOC and NVTOC), total organic chlo-
rine (TOC1), ultraviolet absorbancy, and
fluorescence. In addition, monitoring will
be required for probably 21 specific or-
ganic compounds. Selection of the spe-
cific compounds has been based on the
occurrence or likelihood of occurrence In
treated water, toxlcity data and availa-
bility of practical analytical methods.
Laboratory analyses will be used to
evaluate the extent and nature of organic
chemical contamination of drinking
water, to evaluate the validity of the
general organic parameters as surrogates
for measures of harmful organic chemi-
cals, and to determine whether there is
an adequate basis for establishing maxi-
mum contaminant levels for specific or-
ganics or groups of organics.
Second, EPA is embarking on an inten-
sive research program to find answers
to the following four questions:
1. What are the effects of commonly
occurring organic compounds on human
health?
2. What analytical procedures should
be used to monitor finished drinking
water to assure that any Primary Drink-
ing Water Regulations dealing with or-
ganics are met?
3. Because some of these organic com-
pounds are formed during water treat-
ment, what changes in treatment prac-
tices are required to minimize the for-
mation of these compounds In treated
water?
4. What treatment technology must
be applied to reduce contaminant levels
to concentrations that may be specified
in the Primary Drinking Water Regu-
lations?
This research will Involve health-
effects and epldemlological studies. In-
vestigations of analytical methodology,
and pilot plant and field studies of or-
ganic removal unit processes. Some
phases of the research are to be com-
pleted by the end of this year, while
much of the remainder are to be com-
pleted within the next calendar year.
As soon as sufficient Information is
derived from the monitoring program
and related research, the Interim Pri-
mary Drinking Water Regulations will
be amended so that the organic chemi-
cals problem can be dealt with without
delay. The monitoring process will be
completed within 1 year.
During the interim period, while sat-
isfactory MCL's for organic contamina-
tion in drinking water are being devel-
oped, EPA will act In specific cases where
appropriate to deal with organic con-
tamination. If the EPA monitoring pro-
gram reveals serious specific cases of
contamination, EPA will work with State
and local authorities to Identify the
source and nature of the problem and to
FEDERAL REGISTER, VOL. 40, NO. 248—WEDNESDAY, DECEMBER 74, 1975
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RULES AND REGULATIONS
59569
take remedial action. EPA will also aid
the States in identifying additional com-
munity water supplies that require
analysis.
PUBLIC NOTICE
The public notice requirements pro-
posed In § 141.32 did not distinguish be-
tween community and non-community
public wafer systems. They would have
required that public notice of non-com-
pliance with applicable regulations be
made by newspaper, in water bills, and
by other media for all public water sys-
tems. These requirements are Inappro-
priate and Ineffective In the case of most
non-community water systems. Those
systems principally serve transients who
do not receive water bills from the sys-
tem and who probably are not exposed
significantly to the local media. A more
effective approach would be to require
notice that can inform the transient
before he drinks the system's water, and
thereby both warn the transient and
provide an incentive to the supplier of
water to remedy the violation. Accord-
ingly, Section 141.32 as adopted provides
that in the case of non-community sys-
tems, the entity with primary enforce-
ment responsibility shall require that
notice be given in a form and manner
that will Insure that the public using
the public water system is adequately
informed.
The proposed public notice require-
ments also failed to distinguish between
different types of violations of the In-
terim Primary Drinking Water Regula-
tions. Since the urgency and importance
of a notice varies according to the nature
of the violation involved, § 141.32 as
promulgated seeks to match the type of
notice required with the type of violation
Involved. Written notice accompanying
a water bill or other direct notice by
mail Is required for all violations of the
regulations, including violations of mon-
itoring requirements, and for the grant
of a variance or exemption. In addition,
notice by newspaper and notification to
radio and television stations Is required
whenever a maximum contaminant level
Is exceeded, or when the entity with
primary enforcement responsibility re-
quires such broader notice.
QUALITY CONTROL AND TESTING
PROCEDURES
Section 1401(1) of the Act defines
"primary drinking water regulation" to
Include "quality control and testing pro-
cedures." The promulgated regulations
include testing requirements for each
maximum contaminant level, including
check samples and special samples in
appropriate cases. The regulations also
specify the procedures to be followed in
analyzing samples for each of the maxi-
mum contaminant levels. These proce-
dures will be updated from time to time
as advances are made in analytical meth-
ods For example, references to "Stand-
ard Methods for the Examination of
Water and Wastewater" are to the cur-
rent, 13th, edition, but these references
will be changed to cite the 14th edition
when ft is available in the near future.
A key element of quality control for
public water .systems IE accurate labora-
tory analysis. Section 141.28 of the regu-
lations provides that analyses conducted
for the purpose of determining com-
pliance with maximum contaminant
levels must be conducted by a laboratory
approved by the entity with primary en-
forcement responsibility. EPA will de-
velop as soon as possible. In cooperation
with the States and other interested
parties, criteria and procedures for lab-
oratory certification. A State with pri-
mary enforcement responsibility will
have a laboratory certified by EPA pur-
suant to the prescribed criteria and pro-
cedures, and in turn will certify labora-
tories within the State.
Record-keeping requirements and re-
ports to the State also will assist in
quality control efforts.
RECORD-KEEPING
Adequate record-keeping Is necessary
for the proper operation and administra-
tion of a public water system. It is also
Important for providing information to
the public, providing appropriate data
for inspection and enforcement activities
and providing information on which fu-
ture regulations can be based. Accord-
ingly, a new § 141.33 has been added to
the regulations to require that each pub-
lic water system maintain records of
sample analyses and of actions to correct
violations of the Primary Drinking Water
Regulations.
ECONOMIC AND COST ANALYSIS
A comprehensive economics study has
been made of the Interim Primary Drink-
ing Water Regulations. This study esti-
mates the costs of the regulations, evalu-
ates the potential economic impact, and
considers possible material and labor
shortages. The results of this analysis are
summarized here.
Total Investment costs to community
water systems to achieve compliance
with these regulations are estimated to
be between $1,050 and $1.765 million. It
is estimated that non-community sys-
tems will invest an additional $24 million.
The range of the estimate is due to un-
certainty as to the design flow that will
be used in installing treatment facilities.
Systems not in compliance will have -to
consider sizing their new components to
reflect average dally flow conditions, or
maximum daily flow conditions in cases
where system storage is not adequate. -
This investment will be spread over
several years. Investor-owned systems
will bear about one-fourth of these costs,
and publicly-owned systems the remain-
der. It is not anticipated that systems will
have difficulty financing these capital re-
quirements.
In annual terms, national costs are ex-
pected to be wlttiln the following ranges:
In millions
Capital costs (146-247
Operations and maintenance 263-463
Monitoring (routine only) 17- 35
significantly affected. For those users in
systems serving 10,000 persons or more,
the average annual treatment cost per
capita may increase from less than $1.00
for systems requiring disinfection and
lead control, to between $15 to $35 for
control of turbidity and heavy metal re-
moval. For systems serving less than 100
persons, the average annual per capita
costs of disinfection, lead control and
fluoride/arsenic removal are estimated to
be between $2.10 and $11 80. However, If
turbidity control or heavy metal removal
were required In' a system of this size
then costs are expected to range from
$52 to $237 per year per capita. EPA Is
aware of the serious potential economic
Impact on users in these small systems.
However, the legislative history specifies
that the regulations should be based on
costs that can be reasonably afforded by
large metropolitan or regional systems.
Further economic evaluation of these
systems is being conducted, and realistic
options for these small systems are being
reviewed. Options that will be under con-
sideration include less costly treatment
technologies; formation of regional sys-
tems; and use of alternative water
sources. Industrial and commercial users,
whether providing their own water or
using public systems, are not expected
to be significantly affected by these
regulations
Possible constraints to the implemen-
tation of the Interim primary regula-
tions were examined. Although there
will be an Increase in demand for chem-
icals, manpower, laboratories, and con-
struction of treatment facilities, it Is not
anticipated that any of these factors will
be a serious obstacle to implementation
of these regulations over a reasonable
time frame.
For the reasons given above, Chapter
40 of the Code of Federal Regulations Is
hereby amended by the addition of the
following new Part 141. These regula-
tions will take effect 18 months after
promulgation.
(It Is hereby certified that the economic and
Inflationary Impacts of these regulations
bave been carefully evaluated In accordance
with Executive Order 11821)
Dated' December 10, 1975
RUSSELL E. TRAIN,
Administrator
Subpart A— General
Sec.
141.1
141.4
141.6
141.6
Applicability.
Definitions
Coverage.
Variances and exemptions
Siting requirements.
Effective date.
Total
$426-645
Although these aggregate figures are
large, most water consumers will not be
Subpart B—Maximum Contaminant Levels
141.11 Maximum contaminant levels for
inorganic chemicals.
141.12 Maximum contaminant levels for
organic chemicals.
141.18 Maximum contaminant levels for
turbidity.
141.14 Maximum microbiological contami-
nant levels.
Subpart C—Monitoring and Analytical
Requirement*
Microbiological contaminant sam-
pling and analytical requirements.
FEDERAL REGISTER, VOL. 40, NO. 248—WEDNESDAY, DECEMBER 24, 1975
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59570
RULES AND REGULATIONS
Sec
14122 Turbidity sampling and analytical
requirements
14123 Inorganic chemical sampling and
analytical requirements.
14124 Organic chemical sampling and
analytical requirements
141 27 Alternative analytical techniques.
141.28 Approved laboratories.
141.29 Monitoring of consecutive public
water systems
Subpart D—Repotting. Public Notification, and
Record-keeping
141 31 Reporting requirements.
141.32 Public notification of variances, ex-
emptions, and non-compliance
with regulations.
141.33 Record maintenance.
AUTHORITT: Sees 1412. 1414. 1445. and 1450
of the Public Health Service Act. 88 Stat. I860
(42 U.S C 300g-l. 300g-3.300J-4. and 300J-9).
Subpart A—General
§ 141.1 Applicability.
This part establishes primary drinking
water regulations pursuant to section
1412 of the Public Health Service Act. as
amended by the Safe Drinking Water
Act (Pub. L. 93-523); and related regula-
tions applicable to public water systems.
g 141.2 Definitions.
As used In this part, the term:
(a) "Act" means the Public Health
Service Act, as amended by the Safe
Drinking Water Act. Pub. L. 93-523.
(b) "Contaminant" means any physi-
cal, chemical, biological, or radiological
substance or matter in water.
(c) "Maximum contaminant level"
means the maximum permissible level of
a contaminant In water which Is de-
livered to the free flowing outlet of the
ultimate user of a public water system,
except in the case of turbidity where the
maximum permissible level Is measured
at the point of entry to the distribution
system Cbntamlnants added to the water
under circumstances controlled by the
user, except those resulting from corro-
sion of piping and plumbing caused by
water quality, are excluded from this
definition.
(d) "Person" means an Individual,
corporation, company, association, part-
nership. State, municipality, or Federal
agency.
(e) "Public water system" means a
system for the provision to the public
of piped water for human consumption.
if such system has at least fifteen service
connections or regularly serves an aver-
age of at least twenty-five individuals
dally at least 60 days out of the year.
Such term -includes (1) any collection.
treatment, storage, and distribution fa-
cilities under control of the operator of
such system and used primarily in con-
nection with such system, and (2) any
collection or pretreatment storage facili-
ties not under such control which are
used primarily In connection with such
system. A public water system is either
a "community water system" or a "non-
community water system."
(i) "Community water system" means
a public water system which serves at
least 15 service connections used by year-
round residents or regularly serves at
least 25 year-round residents.
(11) "Non-community water system"
means a public water system that is not
a community water system.
(f) "Sanitary survey" means an on-
slte review of the water source, facili-
ties, equipment, operation and mainte-
nance of a public water system for the
purpose of evaluating the adequacy of
such source, facilities, equipment, op-
eration and maintenance for producing
and distributing safe drinking water.
(g) "Standard sample" means the
aliquot of finished drinking water that is
examined for the presence of collform
bacteria.
(h) "State" means the agency of the
State government which has jurisdic-
tion over public water systems. During
any period when a State does not have
primary enforcement responsibility
pursuant to Section 1413 of the Act, the
term "State" means the Regional Ad-
ministrator, U.S. Environmental Protec-
tion Agency.
(i) "Supplier of water" means any
person who owns or operates a public
water system.
§ 141.3 Coverage.
This part shall apply to each public
water system, unless the public water
system* meets all of the following condi-
tions:
(a) Consists only of distribution and
storage facilities (and does not have any
collection and treatment facilities);
(b) Obtains all of its water from, but
is not owned or operated by, a public wa-
ter system to which such regulations
apply:
(c) Does not sell water to any person;
and
(d) Is not a carrier which conveys
passengers In interstate commerce.
§ 141.4 Variances and exemptions.
Variances or exemptions from certain
provisions of these regulations may be
granted pursuant to Sections 1415 and
1416 of the Act by the entity with pri-
mary enforcement responsibility. Provi-
sions under Part 142, National Interim
Primary Drinking Water Regulations
Implementation—subpart E (Variances)
and subpart F (Exemptions)—apply
where EPA has primary enforcement
responsibility.
g 141.5 Siting requirements.
Before a person may enter into a fi-
nancial commitment for or initiate con-
struction of a new public water system
or increase the capacity of an existing
public water system, he shall notify the
State and, to the extent practicable,
avoid locating part or all of the new or
expanded facility at a site which:
(a) Is subject to a significant risk
from earthquakes, floods, fires or other
disasters which could cause a breakdown
of the piiblic water system or a portion
thereof; or
(b) Except for intake structures, is
within the floodplaln of a 100-year flood
or Is lower than any recorded high tide
where appropriate records exist.
The U.S. Environmental Protection
Agency will not seek to override land use
decisions affecting public water systems
siting which are made at the State or lo-
cal government levels.
6 141.6 Effective dale.
The regulations set forth In this part
shall take effect 18 months after the date
of promulgation.
Subpart B—Maximum Contaminant Levels
§ 141.11 Maximum contaminant leveb
for inorganic chemicals.
(a) The maximum contaminant level
for nitrate Is applicable to both commu-
nity water systems and non-community
water systems. The levels for the other
inorganic chemicals apply only to com-
munity water systems. Compliance r-ith
maximum contaminant levels for inor-
ganic chemicals is calculated pursuant to
{141.23.
(b) The following are the maximum
contaminant levels for inorganic chemi-
cals other than fluoride •
Level.
milligrams
Contaminant per liter
Arsenic 0.05
Barium l.
Cadmium , 0 010
Chromium 0.05
Lead 0 05
Mercury 0 002
Nitrate (as N) 10.
Selenium 0 01
Silver 0.05
(c) When the annual average of the
maximum daily air temperatures for the
location in which the community water
system is situated is the following, the
maximum contaminant levels for fluoride
are.
Trmpciolme
Di^rcn
Folncnhfil
elsius
Level.
mllUrrams
per liter
S3 7 and below 120 and below 24
83810583 I2110H6 22
1)8410638 147(0170 20
639to TOfl 17710214 1 8
70710792 21510262 1 S
79.8 to 90S : 26310325 1 4
g 141.12 Maximum contaminant levels
for organic chemicals.
The following are the maximum con-
taminant levels for organic chemicals.
They apply only to community water
systems. Compliance with maximum
contaminant levels for organic chemicals
is calculated pursuant to g 141.24.
Level.
milligrams
per liter
(a) Chlorinated hydrocarbons:
Endrln (1.2.3.4.10. 10-hexachIoro- 0.0002
8.7-epoxy-l .4, 4a,5.6.7,8.8a-octa-
hydro-l,4-endo. endo-5.8 - dl-
methano naphthalene).
Llndane (1,2,3.4.6.6-hexachIoro- 0 004
cyclohexane, gamma Isomer).
Methoxychlor (1.1.1-Trlchloro- 0.1
2. 2 - bis [p-methoxyphenyl]
ethane).
Toxaphene (C10H,()Cl,-Technlcttl 0.005
chlorinated camphene, 67-80
percent chlorine)
FEDERAL REGISTER, VOL. 40, NO. 248—WEDNESDAY, DECEMBER 24, 197S
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RULES AND REGULATIONS
59571
(b) Chlorophenoxys:
2.4 -D. (2.4-Dlchlorophenoxyace- 0 1
tic acid).
2,4.6-TP Silver (8.4.5-Trlchloro- 0.01
phenoxyproplonlc acid).
g 141.13 Maximum contaminant levels
lor turbidity.
The maximum contaminant levels for
turbidity are applicable to both commu-
nity water systems and non-community
water systems using surface water
sources In whole or In part. The maxi-
mum contaminant levels for turbidity
In drinking water, measured at a repre-
sentative <*ntry polnt(s) to the distribu-
tion system, are:
(a) One turbidity unit
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S9372
RULES AND REGULATIONS
llshed by the State, until the results ob-
tained from at least two consecutive
check samples show less than one coil-
form bacterium per 100 mlUlllters.
i2> When conform bacteria occur in
three or more 10 ml portions of a single
sample . or (3) of this section
shall not be eliminated from future sam-
pling without approval of the State. The
results from all conform bacterial analy-
ses performed pursuant to this subpart,
except those obtained from check sam-
ples and special purpose samples, shall be
used to determine compliance with the
maximum contaminant level for conform
bacteria as established in 5 141.14. Check
samples shall not be Included In calculat-
ing the total number of samples taken
each month to determine compliance
with i 141 21 (b) orfc).
(e) When the presence of collform
bacteria in water taken from a particular
sampling point has been confirmed by
any check samples examined as directed
in paragraphs (d) (l), (2), or (3) of this
section, the supplier of water shall re-
port to the State within 48 hours.
(f) When a maximum contaminant
level set forth In paragraphs (a), (b) or
(c) of 1141.14 is exceeded, the supplier
of water shall report to the State and
notify the public as prescribed In 9141.31
and {141.32.
(g) Special purpose samples, such as
those taken to determine whether dis-
infection practices following pipe place-
ment, replacement, or repair have been
sufficient, shall not be used to determine
compliance with } 141.14 or § 141.21 (b)
or (c).
(h) A supplier of water of a com-
munity water system or a non-com-
munity water system may, with the
approval of the State and based upon a
sanitary survey, .substitute the use of
chlorine residual monitoring for not more
than 75 percent of the samples required
to be taken by paragraph (b) of this
section, Provided. That the supplier of
water takes chlorine residual samples at
points which are representative of the
conditions within the distribution sys-
tem at the frequency of at least four for
each substituted microbiological sample.
There shall be at least dally determina-
tions of chlorine residual. When the sup-
plier of water exercises the option pro-
vided in this paragraph Analyses for all community water
systems utilizing only ground water
sources shall be completed within two
years following the effective date of this
part. These analyses shall be repeated
at three-year intervals.
(3) For non-community water systems,
whether supplied by surface or ground
water sources, analyses for nitrate shall
be completed within two years following
the effective date of this part. These
analyses shall be repeated at intervals
determined by the State.
(b) If the result of an analysis made
pursuant to paragraph (a) indicates that
the level of any contaminant listed in
S14111 exceeds the maximum contam-
inant level, the supplier of water shall
report to the State within 7 days and
initiate three additional analyses at the
same sampling point within one month.
(c) When the average of four analyses
made pursuant to paragraph (b) of this
section, rounded to the same number of
significant figures as the maximum con-
taminant level for the substance In ques-
tion, exceeds the maximum contaminant
level, the supplier of water shall notify
the State pursuant to § 141.31 and give
notice to the public pursuant to § 14132.
Monitoring after public notification shall
be at a frequency designated by the State
and shall continue until the maximum
contaminant level has not been exceeded
in two successive samples or until a mon-
itoring schedule as a condition to a
variance, exemption or enforcement ac-
tion shall become effective.
(d) The provisions of paragraphs (b)
and (c) of this section notwithstanding,
compliance with the maximum contam-
inant level for nitrate shall be determined
on the basis of the mean of two analyses.
When a level exceeding the maximum
contaminant level for nitrate Is found,
a second analysis shall be Initiated within
24 hours, and if the mean of the two
analyses exceeds the maximum contam-
inant level, the supplier of water shall
report his findings to the State pursuant
to {141.31 and shall notify the public
pursuant to 9141.32.
(e) For the initial analyses required
by paragraph (a)(l). (2) or (3) of this
section, data for surface waters acquired
within one year prior to the effective date
and data for ground waters acquired
within 3 years prior to the effective date
of this part may be substituted at the
discretion of the State.
(f) Analyses conducted to determine
compliance with g 141.11 shall be made
In accordance with the following
methods:
(1) Arsenic—Atomic Absorption Meth-
od, "Methods for Chemical Analysis of
Water and Wastes." pp. 95-96, Envlron-
FEDERAl REGISTER, VOl. 40, NO. 246—WEDNESDAY, DECEMBER 24, 1975
-------
RULES AND REGULATIONS
59573
mental Protection Agency, Office of
Technology Transfer, Washington, D.C.
20460,1974.
(2) Barium—Atomic Absorption Meth-
od, "Standard Methods for the Exami-
nation of Water and Wastewater," 13th
Edition, pp. 210-215, or "Methods for
Chemical Analysis of Water and Wastes,"
pp. 97-98, Environmental Protection
Agency, Office of Technology Transfer,
Washington. D.C. 20460, 1974.
(3) Cadmium—Atomic Absorption
Method. "Standard Methods for the Ex-
amination of Water and Wastewater,"
13th Edition, pp. 210-215, or "Methods
for Chemical Analysis of Water and
Wastes," pp. 101-103, Environmental
Protection Agency, Office of Technology
Transfer, Washington. D.C. 20460, 1974.
(4) Chromium—Atomic Absorption
Method, "Standard Methods for the Ex-
amination of Water and Wastewater,"
13th Edition, pp. 210-215, or "Methods
for Chemical Analysis of Water and
Wastes," pp. 105-106, Environmental
Protection Agency, Office of Technology
Transfer, Washington, D.C. 20460. 1974.
(5) Lead—Atomic Absorption Method,
"Standard Methods for the Examina-
tion of Water and Wastewater," 13th
Edition, pp. 210-215. or "Methods for
Chemical Analysis of Water and Wastes,"
pp. 112-113, Environmental Protection
Agency, Office of Technology Transfer,
Washington, D.C. 20460. 1974.
(6) Mercury—nameless Atomic Ab-
sorption Method, ''Methods for Chemical
Analysis of Water and Wastes." pp. 118-
126, Environmental Protection Agency,
Office of Technology Transfer. Wash-
ington, D.C. 20460,1974.
(7) Nitrate—Brucine Colorimetrlc
Method, "Standard Methods for the Ex-
amination of Water and Wastewater."
13th Edition, pp. 461-464. of Cadmium
Reduction Method. "Methods for Chemi-
cal Analysis of Water and Wastes."
pp. 201-206. Environmental Protection
Agency, Office of Technology Transfer,
Washington. D.C. 20460,1974.
(8) Selenium—Atomic Absorption
Method. "Methods for Chemical Analysis
of Water and Wastes." p. 145, Environ-
mental Protection Agency, Office of
Technology Transfer, Washington, DC.
20460.1974.
(9) Silver—Atomic Absorption Meth-
od. "Standard Methods for the Ex-
amination of Water and Wastewater",
13th Edition, pp. 210-215, or "Methods
for Chemical Analysis of Water and
Wastes", p. 146, Environmental Protec-
tion Agency. Office of Technology Trans-
fer. Washington, DC. 20460. 1974.
(10) Fluoride—Electrode Method,
"Standard Methods for the Examination
of Water and Wastewater", 13th Edition,
pp. 172-174, or "Methods for Chemical
Analysis of Water and Wastes," pp. 65-
67, Environmental Protection Agency,
Office of Technology Transfer, Wash-
ington, D.C. 20460, 1974, or Colorimetric
Method with Preliminary Distillation,
"Standard Methods for the Examination
of Water and Wastewater." 13th Edition,
pp. 171-172 and 174-176, or "Methods for
Chemical Analysis of Water and
Wastes," pp. 59-60, Environmental Pro-
tection Agency, Office of Technology
Transfer, Washington, D.C. 20460. 1974.
§ 141.24 Organic chemical sampling
and analytical requirements.
(a) An analysis of substances for the
purpose of determining compliance with
§ 141.12 shall be made as follows:
(1) For all community water systems
utilizing surface water sources, analyses
shall be completed within one year fol-
lowing the effective date of this part.
Samples analyzed shall be collected dur-
ing the period of the year designated by
the State as the period when contami-
nation by pesticides is most likely to
occur. These analyses shall be repeated
at intervals specified by the State but
in no event less frequently than at three
year intervals.
(2) For community watei systems
utilizing only ground water sources,
analyses shall be completed by those sys-
tems specified by the State.
(b) If the result of an analysis made
pursuant to paragraph fa) of this sec-
tion indicates that the level of any con-
taminant listed in 5 141.12 exceeds the
maximum contaminant level, the sup-
plier of water shall report to the State
within 7 days and Initiate three addi-
tional analyses within one month.
(c) When the average of four analyses
made pursuant to paragraph (b) of this
section, rounded to the same number of
significant figures as the maximum con-
taminant level for the substance in ques-
tion, exceeds the maximum contaminant
level, the supplier of water shall report
to the State pursuant to § 141.31 and give
notice to the public pursuant to g 141.32.
Monitoring after puolic notification shall
be at a frequency designated by the State
and shall continue until the maximum
contaminant level has not been exceeded
in two successive samples or until a
monitoring schedule as a condition to a
variance, exemption or enforcement ac-
tion shall become effective.
(d) For the initial analysis required
by paragraph (a) (1) and (2) of this
section, data for surface water acquired
within one year prior to the effective
date of this part and data for ground
water acquired within three years prior
to the effective date of this part may be
substituted at the discretion of the State.
(e) Analyses made to determine com-
pliance with § 141.12(a) shall be made
in accordance with "Method for Organo-
chlorine Pesticides in Industrial Efflu-
ents," MDQARL, Environmental Pro-
tection Agency. Cincinnati, Ohio, Novem-
ber 28.1973.
(f) Analyses made to determine com-
pliance with 1141.12(b) shall be con-
ducted in accordance with "Methods for
Chlorinated Phenoxy Acid Herbicides In
Industrial Effluents," MDQARL, En-
vbwnmental Protection Agency, Cincin-
nati, Ohio, November 28, 1973.
§ 141.27 Alternative nnnl>lii-al tech-
niques.
With the written permission of the
State, concurred In by the Administra-
tor of the U.S. Environmental Protec-
tion Agency, an alternative analytical
technique may be employed. An alterna-
tive technique shall be acceptable only
if it is substantially equivalent to the
prescribed test in both, precision and ac-
curacy as it relates to the determination
of compliance with any maximum con-
taminant level. The use of the alterna-
tive analytical technique shall not de-
crease the frequency of monitoring re-
quired by this part.
§141.28 Approved laboratories.
For the purpose of determining com-
pliance with § 141.21 through § 141.27,
samples may be considered only if they
have been analyzed by a laboratory ap-
proved by the State except that meas-
urements for turbidity and free chlorine
residual may be performed by any per-
son acceptable to the State.
§ 1 II .29 Monitoring of consecutive puli-
li<: water systems.
When a public water system supplies
water to one or more other public water
•systems, the State may modify the moni-
toring lequirements imposed by this
pait lo the extent that the interconnec-
lon of the sysems jusifles treating them
as a single system for monitoring pur-
poses Any modified monitoring shall be
conducted pursuant to a schedule speci-
fied by the State and concurred in by the
Administrator of the U.S Environmental
Protection Agency.
Cubpart D—Reporting, Public Notification
and Record Keeping
§ 141.31 Reporting requirements.
(a) Except where a shorter reporting
peuod is specified in this part, the
suppliei of water shall report to the State
within 40 days follow ing a test, measure-
ment or analysis required, to be made by
this part, the results of that test, meas-
ui emcnt or analysis.
ib) The supplier of v.atcr shall report
to the State within 48 hours the failure
to comply with any primary drinking
water regulation (including failure to
comply with monitoring requirements)
set forth in this part
(c) The supplier of water is not re-
quired to report analytical results to the
State In cases where a State laboratory
performs the analysis and reports the
results to the State office which would
noimally receive such notification trom
the supplier.
§141.32 Public notificiilinn.
fa) If a community water system fails
to comply with an applicable maximum
contaminant level established In Subpart
B, fails to comply with an applicable
testing procedure established in Subpart
C of this part, is granted a variance or
an exemption from an applicable maxi-
mum contaminant level, falls to comply
with the requirements of any schedule
prescribed pursuant to a variance or ex-
emption, or falls to perform any moni-
toring required pursuant to Section 1445
(a) of the Act, the supplier of water shall
notify persons served by the system of
the failure or grant by inclusion of a no-
tice in the first set of water bills of the
system issued after the failure or grant
FEDERAL REGISTER, VOL 40, NO. 248—WEDNESDAV, DECEMBER 14, 1975
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59574
RULES AND REGULATIONS
and in any event by written notice within
three months Such notice shall be re-
peated at least once every three months
so long as the system's failure continues
or the variance or exemption remains In
effect If the system Issues water bills less
frequently than quarterly, or does not
Issue water bills, the notice shall be made
by or supplemented by another form of
direct mall.
(b) If a community water system has
failed to comply with an applicable max-
imum contaminant level, the supplier of
water shall notify the public of such fail-
ure. In addition to the notification re-
quired by paragraph (a) of this section,
as follows:
(1) By publication on not less than
three consecutive days In a newspaper or
newspapers of general circulation In the
area served by the system Such notice
shall be completed within fourteen days
after the supplier of water learns of
the failure.
(2) By furnishing a copy of the notice
to the radio and television stations serv-
ing the area served by the system. Such
notice shall be furnished within seven
days after the supplier of water learns
of the failure.
(c) If the area served by a community
water system is not served by a daily
newspaper of general circulation, notifi-
cation by newspaper required by para-
graph (b) of this section shall Instead be
given by publication on three consecutive
weeks in a weekly newspaper of general
circulation serving the area. If no weekly
or dally newspaper of general circula-
tion serves the area, notice shall be given
by posting the notice In post offices with-
in the area served by the system.
(d> If a non-community water sys-
tem fails to comply with an applicable
maximum contaminant level established
In Subpart B of this pert falls to comply
with an applicable testing procedure
established In Subpart C of this part. Is
granted a variance or an exemption from.
an applicable maximum contaminant
level, fails to comply with the require-
ment of any schedule prescribed pursu-
ant to a variance or exemption or falls to
perform any monitoring required pursu-
ant to Section 1445 (a) of the Act. the
supplier of water shall given notice of
such failure or grant to the persons
served by the system. The form and man-
ner of such notice shall be prescribed by
the State, and shall insure that the
public using the svstem is adequately In-
formed of the failure or grant.
(e) Notices given pursuant to this sec-
tion shall be written in a manner reason-
ably designed to inform fully the users
of the system. The notice shall be con-
spicuous and shall not use unduly tech-
nical language, unduly small -print or
other methods which would frustrate the
purpose of the notice The notice shall
disclose all material facts regarding the
subject including the nature of the prob-
lem and, when appropriate, a clear state-
ment that a primary drinking water
regulation has been violated and any pre-
ventive measures that should be taken by
the public. Where appropriate, or where
designated by the State, bilingual notice
shall be given. Notices may Include a bal-
anced explanation of the significance or
seriousness to the public health of the
subject of the notice, a fair explanation
of steps taken by the system to correct
any problem and the results of any addi-
tional sampling.
(f) Notice to the public required by
this section may be given by the State on
behalf of the supplier of water.
(g) In any Instance In which notifica-
tion by mall Is required by paragraph (a)
of this section but notification by news-
paper or to radio or television stations
Is not required by paragraph (b) of this
section, the State may order the supplier
of water to provide notification by news-
paper and to radio and television stations
when circumstances make more immedi-
ate or broader notice appropriate to
protect the public health.
§ 141.33 Record maintenance.
Any owner or operator of a puollc
water system subject to the provisions of
this part shall retain on Its premises or
at a convenient location near Its prem-
ises the following records*
(a) Records of bacteriological analyses
made pursuant to this part shall be kept
for not less than 5 years. Records of
chemical analyses made pursuant to this
part shall be kept for not less than 10
years. Actual laboratory reports may be
kept, or data may be transferred to tab-
ular summaries, provided that the fol-
lowing information Is Included:
(1) The date, place, and time of sam-
pling, and the name of the person who
collected the sample;
(2) Identification of the sample as to
whether it was a routine distribution
system sample, check sample, raw or
process water sample or other special
purpose sample;
(3) Date of analysis;
(4) Laboratory and person responsible
for performing analysis;
(5) The analytical technique/method
used; and
(6) The results of the analysis.
(b) Records of action taken by the
system to correct violations of primary
drinking water regulations shall be kept
for a period not less than 3 years after
the last action taken with respect to the
particular violation involved.
(c) Copies of any written reports.
summaries or communications relating
to sanitary surveys of the system con-
ducted by the system Itself, by a private
consultant, or by any local. State or Fed-
eral agency, shall be kept for a period
not less than 10 years after completion
of the sanitary survey Involved.
(d) Records concerning a variance or
exemption granted to the system shall
be kept for a period ending not less than
5 years following the expiration of such
variance or exemption.
'.M rccisTcR, VOL. 40. NO. :43—WEDNESDAY, C:::*'.CCR i«, icrs
-------
A PROTOTYPE FOR DEVELOPMENT OF
ROUTINE OPERATIONAL PROCEDURES
for the
COLLECTION AND HANDLING OF DRINKING WATER SAMPLES
as applied in
DRINKING WATER TREATMENT FACILITIES
and in the
DISTRIBUTION SYSTEMS OF DRINKING WATER TREATMENT FACILITIES
National Training and Operational Technology Center
Municipal Operations and Training Division
Office of Water Program Operations
U. S. Environmental Protection Agency
BA.MET.lab.WMP.la.8.79
2-1
-------
EPA 600/8-78-008, May 1978
Manual for the Interim Certification of Laboratories Involved in Analyzing
Public Drinking Water Supplies - Criteria & Procedures
Collection and Handling of Drinking Mater Samples
When the laboratory has been delegated responsibility for sample collecting,
handling, and preservation, there must be strict adherence to correct sampling
procedures, complete identification of the sample, and prompt transfer of the
sample to the laboratory as described in "Standard Methods," 13th edition,
section 450, p. 657-660.
The sample must be representative of the potable water system. The sampling
program must include examination of the finished water at selected sites that
systematically cover the distribution network.
Minimum sample frequency must be that specified in the National Interim Primary
Drinking Water Regulations, 40 CFR 141.21.
The collector must be trained in sampling procedures and approved by the State
regulatory authority or its delegated representative.
The water tap must be sampled after maintaining a steady flow for 2 or 3 minutes
to clear service line. The tap is free of aerator, strainer, hose attachment,
or water purification devices.
The sample volume must be a minimum of 100 ml. The sample bottle must be filled
only to the shoulder to provide space for mixing.
The sample report form must be completed immediately after collection with
location, date and time of collection, chlorine residual, collector's name, and
remarks.
Sample bottles must be of at least 120 ml capacity, sterile plastic or hard glass,
wide mouthed with stopper or plastic screw cap, and capable of withstanding re-
peated sterilization. Sodium thiosulfate (100 mg/1) is added to all sample
bottles during preparation. As an example, 0.1 ml of a 10 percent solution is
required in a 4-oz (120 ml) bottle.
Date and time of sample arrival must be added to the sample report form when
sample is received in the laboratory.
State regulations relating to chain-of-custody, if required, must be followed in
the field and in the laboratory.
Samples delivered by collectors to the laboratory must be analyzed on the day of
collection.
Where it is necessary to send water samples by mail, bus, United Parcel Service,
courier service, or private shipping, holding/transit time between sampling and
analyses must not exceed 30 hours.
Unen possible, samples are refrigerated during transit and during storage in the
laboratory (optional).
]MINIMUM REQUIREMENTS except where indicated as OPTIONAL.
2-2
-------
If the laboratory Is required by State regulation to examine samples after 30
hours and up to 48 hours, the laboratory must indicate that the data may be
invalid because of excessive delay before sample processing. Samples arriving
after 48 hours shall be refused without exception and a new sample requested.
(The problem of holding time is under investigation by EPA.)
At least one bottle per batch of sterilized sample bottles must be checked by
adding approximately 25 ml of sterile LTB broth to each bottle. It must be
incubated at 35 + 0.5°C for 24 hours and checked for growth.
Dried glassware must be sterilized at a minimum of 170°C for 2 hours
Aut°clave «»™»t1o. at Ul-C/30
Sample identified immediately after collection
Identification includes, water source, location, time and date of collection,
and collector's name; insufficiently identified samples discarded
Chlorine residual where applicable
2-3
-------
MATER MONITORING PROCEDURE: Collection and Handling of Drinking Water Samples
1. Analysis Objectives:
Proper technique for the collection and handling of a sample for bacteriological
examination taken from drinking water sources,
2. Brief Description of Analysis:
After assembly of necessary equipment and travel to the sample site, the
sample is collected in a manner which does not bias the sample and produces
a representative sample. Samples are collected in a suitable, labeled,
sterilized sample bottle which contains a chemical agent to inactivate the
chlorine disinfectant in the sample collected.
Controlled handling conditions maintain the integrity of the sample until its
delivery to the laboratory within specified time limitations.
3. Applicability of this Procedure:
Treatment of Interferences in Samples:
This procedure includes directions for dechlorination of samples sufficient to
act upon samples containing up to 15 mg/1 of residual chlorine.
Source of Procedure: Standard Methods for the Examination of Water and Wastewater.
14th Ed., 1975.
2-4
-------
WATER MONITORING PROCEDURE: Collection and Handling of Drinking Water Samples
General Description of Equipment and Supplies Used in the Procedure
A. Capital Equipment:
1. Autoclave, providing uniform temperatures up to, and including 121° C,
equipped with an accurate thermometer, pressure gages, safety features,
saturated steam power lines and capable of reaching required temperature
within 30 minutes. Must perform sterilization cycle within 45 minutes.
Alternately
A pressure cooker can be substituted if:
Efficient pressure gage
Thermometer bulb 2.5 cm above water level.
2. Balance, 50 mg accuracy at a load of 2 or more grams. Should be clean and
without corrosion; have good weights; and be calibrated annually.
3. Oven, drying and sterilizing. Capable of uniform temperatures and with
suitable thermometer to register accurately in the range 160 - 180° C.
4. Refrigerator (at laboratory), set for range of 1.0 to 4.4° C.
5. Distillation Apparatus, Water. In order of preference, the systems are of
stainless steel, quartz, vycor and pyrex glass. Tin-lined hardware is
acceptable but because of maintenance problems is best avoided in preference
to the above. Plumbing should be of stainless steel, pyrex or plastic PVC
material. Storage reservoirs of stainless steel and dust protected.
Produced water must be of suitable bacteriologic quality (test described
in Standard Methods. 14th Edition, p. 887).
Alternately
Distilled water meeting this quality criteria can be purchased, eliminating
the need for the distillation apparatus.
6. Washer, Glassware. Operate at 180° C during hot detergent cycle; hot
rinse cycle of 6 to 12 successive washings; and final rinse of bacterio-
logically suitable distilled or deionized water. Produces clean
sparkling glassware without spotting and meeting criterial of inhibitory
residue test as described in Standard Methods, 14th Ed., p. 885.
B. Reusable Supplies:
1. Sample Bottle. Bacteriologically inert; resistant to sterilizing conditions;
capacity at least 100 ml plus air space; containing dechlorinating agent
if a sample containing chlorine is anticipated.
2-5
-------
WATER MONITORING PROCEDURE: Collection and Handling of Drinking Water Samples
B. Reusable Supplies (Continued):
2. Sampler Device (unnecessary If bottle can be spigot or top filled which
Is the overwhelmingly usual occurrence). Line, wire, etc., if distance
to sample water Is sufficient to make line unwieldy or If sample water
Is reached with difficulty as through well covers, ports, etc. To avoid
contamination of the water supply it Is necessary to sterilize the whole
assembly (including the sample bottle) and place in, for example, an
aluminum foil bag which is opened when ready to sample.
3. Container, ice chest with cover.
C. Consumable Supplies:
1. Labels, clean and unused; non-smearing if wetted; sufficient size for
needed information; can be attached securely to sample bottle.
2. Marking Device, non-smearing if wetted; permanent marking.
3. Sodium thiosulfate (NaOBHO).
2-6
-------
HATER MONITORING PROCEDURE: Collection and Handling of Drinking Water Samples
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Presampling
Procedures
1. Sample bottle
inspection
2. Sodium thiosulfate
solution
3. Sample bottle
preparation
1. Check sample bottles for
acceptability.
1. Weigh 10.0 grams of
sodium thiosulfate.
2. Dissolve in 50-60 ml
distilled water.
3. Add distilled water to
bring final volume to
100 ml.
4. Transfer to labeled
bottle.
1. Deliver 0.1 ml or .2 ml
of 10% sodium thiosulfate
solution to each sample
bottle. (.1 ml to 4 ounce
or 120 ml size and .2 ml
to 6-8 ounce or 250 ml
size).
la. Provides at least 100 ml of sample volume and have
a suitable head space above this volume.
Ib. Suitable glass and/or plastic material.
Ic. Bottles should not be chipped, cracked or other-
wise damaged. No deposits or extensive glass
scratches or etched surfaces can be tolerated.
Bottle caps must not be cracked or otherwise
damaged.
Id. Bottles should be sparkling clean and free from
inhibitory or nutritive residues.
la. Used for dechlorination of samples.
Ib. Use of trip balance accepted.
2a. 100 ml graduated cylinder satisfactory.
la. Labeled as 10% sodium thiosulfate and stored in
refrigerator. (Indicate date of preparation and
who prepared).
Ib. It is preferable to prepare less then 50 ml and
sterilize the reagent to lessen the chance for
contamination.
la. Use sterile 1 ml pipet if the reagent is sterile.
Ib. Provides adequate sodium thiosulfate for
neutralizing chlorine in sample. (100 mg/1).
Ic. Return stock sodium thiosulfate solution to
refrigerator. Solution must not be contaminated
with microbial growth.
2-7
V.A.1.1.la
(p. 2-16)
V.A.I.Lib
(p. 2-16)
V.A.l.l.ld
(p. 2-16)
-------
WATER MONITORING PROCEDURE: Collection and Handling of Drinking Water Samples
2-8
OPERATING PROCEDURES
A. Presampling
Procedures
(Continued)
4. Preparation of
container for
sample transporta-
tion and storage
STEP SEQUENCE
2. Place cap on sample
bottle.
3. Place Kraft paper or
metal foil cover over
bottle cap or stopper.
4. Sterilize sample bottles
in sterilizing oven.
5. Store sample bottles in
clean, dry place until
used.
1. Container inspected to be
of sufficient size, leak
proof, and have tight
fitting cover.
2. Layer bottom with ice
just before departing for
sampling points.
3. Cover and assemble with
other equipment.
INFCRMATICN/OF^ATIf.'J GuALS/SFLl IF ICATIONS
Id. Do not exceed 0.4 ml of the 10% sodium thiosulfatc
to a bottle if a portion of the sample will be
used for a Standard Plate Count.
2a. A string or paper wedge is placed between cap and
neck of bottle when using ground glass stoppers
to prevent "locking" at the interface.
3a. Protects opening of sample bottle from accidental
contamination.
3b. Cover (Hood) is not required for screw cap
closures.
4a. Two hours at a minimum of 170° C.
4b. Steam sterilization (121° C for 30 minutes) can
be used but oven sterilization is preferable.
5a. At least one bottle of each "batch" prepared
should be checked for sterility and results
entered in a quality control ledger.
la. Size sufficient to hold all of required sample
bottles.
ib. Use of this means of transportation and storage
is OPTIONAL, but highly desirable if practiced.
Ic. May be mandatory in tropical or subtropical
areas where high surface temperatures prevail.
2a. To level about 1/2 of bottle height.
2b. Do not use dry ice.
TRAINING
GUIDE NOTES
V.A.3.4
(p. 2-17)
VII.A.3.5
(p. 2-18)
-------
WATER MONITORING PROCEDURE: Collection and Handling of Drinking Water Samples
OPERATING PROCEDURES
STCF SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Presampling
Procedures
(Continued)
5. Inspection of
sampling device
1. Check sampling device
for condition.
6. Assemble bottle
labels
1. Check labels for accepta-
bility.
la. May be unnecessary if particular sampling run
collects from devices which are easily acces-
sible (spigots, taps, etc.).
Ib. A number of suitable sampling devices are avail-
able and the function to (a) provide weight to
allow the sampling device to reach a depth with-
out drifting; (b) provide an anchoring point for
the sterile bottle or chamber; (c) have a
tripping mechanism to allow entry of sample to
the collector; and (d) provide a means of lowering
the device to depth and returned to surface.
Check operation of each of these areas. Some
types of samplers do not utilize a bottle but may
function with sterile bulbs, bladders, etc. It
will be necessary for the sampler to acquaint
himself to the specific device being utilized at
his facility.
la. Must be clean and unused.
Ib. Sufficient quantity for number of samples plus
a few extra labels.
Ic. Each label must have a means of attachment to
sample bottles. Mire or cord is desirable and
such attachments as scotch tape, electrical tape,
etc. must be avoided as these are affected by
moisture or water immersion.
Id. Labels can vary from that which is completely
blank to a type which is required by the facility,
agency, authority, etc.
le. Minimum required data includes:
* Sample location
* Date and time of collection
* Chlorine Residual
2-9
-------
MATER MONITORING PROCEDUKL: Collection and Handling of Drinking Water Samples
2-10
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Presampling
Procedures
(Continued)
7. Inspection of
label marker
1. Check label marker for
acceptability.
le. (Continued)
Collectors name (written out completely)
laboratory reception (date/time)
*Custody receipt (if necessary)
la. Marker must be of a non-smearing permanent type.
Ib. Marker is operable.
B. Travel from Assembly
Point to Sample Point
1. Proceed to initial sample
point.
2. Prepare sample station
for collection of sample.
(If special collection
situation. See C.2).
la. Transport equipment with care.
Ib. Upon arrival,recheck as to correctness of
designated sampling point.
2a. Remove manholes, ports, access panels, etc.,
if necessary.
2b. Note safety hazards at site. It is necessary
to have a partner if potentially hazardous con-
ditions can result in injury or death if another
person is not available for help.
VII.B.l.lb
(p. 2-18)
C. Sample Collection
Procedures
1. Spigot or tap
1. Prepare spigot for
sampling.
2. Flush spigot.
la. Must not have strainers, screens, aerators, etc.,
which can harbor bacteria or particulate matter.
Remove these attachments.
Ib. Spigot must not have leaks past gaskets, washers,
etc., and these spigots must be eliminated as
sampling points.
2a. Must have direct main connection and be
representative of system
2b. Full flow flush for 2-3 minutes or enough to
clear service line.
2c. Hand pump activated taps should be flushed for
5 minutes.
VII.C.I.1
(p. 2-18)
-------
HATER MONITORING PROCEDURE: Collection and Handling of Drinking Water Samples
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Sample Collection
Procedures
(Continued)
3. Adjust spigot
for steady flow.
4. Remove hood and cap from
sample bottle (glass type)
or screw cap from other
types.
5. Let sample run into
bottle.
6. Replace cap and hood or
screw-cap on bottle.
7. Label bottle with tag.
8. Place bottle on ice in
ice chest (optional).
3a. Avoids "scouring" of system by vigorous flow
which could cause sampling of nonrepresentative
bacterial or material particles.
4a. Remove hood and cap as unit.
4b. Discard slip of paper or string which is between
cap and bottle interface.
4c. Protect units from contamination. Usual method
is to hold cap in left hand (if right-handed)
and have bottle in right hand.
5a. No rinsing of bottle. Especially important if
bottle contains sodium thiosulfate.
5b. Fill about 3/4 full so that a mixing space is
available for thorough sample mixing prior to
laboratory operations. (At least 2.5 cm of air
space required).
5c. Sample must not be decanted (completely filled
and then a portion discarded to give the required
air space.)
6a. Secure closure but not excessively tightened or
wedged on bottle.
7a.
8a.
8b.
8c.
8d.
Fill all items
quirements and
authorities.
demanded by drinking water
others as needed by local
re-
Do not immerse bottle in excessive water volume.
Remove excessive water if present in chest.
Cover chest.
Do not use dry ice as freezing is detrimental to
sample.
Do not composite sample (mix different portions
of collected sample from different or the
same sample point).
See A.6
IMPORTANT
VII.C.I.8
(p. 2-18)
2-11
-------
WATER MONITORING PROCEDURE: Collection and Handling of Drinking Water Samples
2-12
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Sample Collection
Procedures
(Continued)
2. Special collection
situation
1. Remove sample device from
packaging.
la. Packaging (Kraft paper, aluminum foil, etc.)
preserves sterility of device and sample bottle
Ib. Remove carefully to avoid contaminating device.
See Sample
Device under
Reusable
Equipment
Also B.2
At least one foot of line
Sample bottle
Device with weight
Portion within sterile bag
-------
WATER MONITORING PROCEDURE Collection and Handling of Drinking Water Samples
OPERATING PROCEDURES
STEP SEQ'JtNCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Sample Collection
Procedures
(Continued)
2. Lower device until under
water about 6 inches.
3. Activate device to obtain
sample.
4. Retrieve sample from
device.
5. Label bottle with tag.
6. Place bottle on ice in
ice chest (optional).
Ic. Handle only beyond one foot of line to avoid
contaminating water supply.
2a. Avoid hitting sides of well, reservoir, etc.
2b. Under water to avoid collecting surface scum,
debris, etc.
3a. Many methods available to activate device
(electrical; messenger [brass plug with center
hole dropped down line]; another line pulled;
etc.).
4a. Pull up device carefully without touching sides
of well; tank; etc.
4b. Remove sample bottle from device with cap, if of
this design, and secure bottle.
4c. Some devices may not have a cap with the bottle
and may necessitate transfer of sample to another
sterile container. Shake well before transfer;
treat asceptically (without contaminating); and
be sure that new bottle has at least 2.5 cm of
head space when complete sample is contained.
5a. Fill all items demanded by drinking water re-
quirements and others as needed by local
authorities.
6a. Precautions as in C.1.8.8a-d.
VII.C.I.8
(p. 2-18 &
2-19)
0. Sample Handling
1. Transport sample to
laboratory.
la. Any of the transport modes previously discussed.
Ib. Must be in the hands of the laboratory personnel
within 30 hours (elapsed time from sample
collection to start of analysis).
VII.D.I.Ib
(p. 2-19)
2-13
-------
HATER KONITCR1NG PROCEDURE: Collection and Handling of Drinking Water Samples
2-14
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OP^TING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Sample Handling
(Continued)
Ic. If sample is delivered directly by a sampler to
the laboratory, it must be processed for analysis
on the same day.
Id. Observe log-in procedures as established by
laboratory and custody receipt procedures
observed for samples which may have legal
implications.
-------
WATER MONITORING PROCEDURE: Collection and Handling of Drinking Water Samples
TRAINING GUIDE
SECTION TOPIC
I* Introduction
II Educational Concepts - Mathematics
III Educational Concepts - Science
IV Educational Concepts - Communications
V* Field & Laboratory Equipment
VI Field & Laboratory Reagents
VII* Field & Laboratory Analyses
VIII Safety
IX Records and Reports
*Training guide materials are presented here under the headings marked*.
These standardized headings are used through this series of procedures.
2-15
-------
WATER MONITORING PROCEDURES: Collection and Handling of Drinking Water Samples
Introduction
Section I
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
The sampler must be trained and approved by the
appropriate State agency or its designated
representative.
2-16
-------
WATER MONITORING PROCEDURES: Collection and Handling of Drinking Water Samples
Field and Laboratory Equipment
Section v
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.1.1.la
A.l.l.lb
A.I.1.Id
A sample bottle size which will provide the re-
quired 100 ml volume (minimum sample volume) as
well as at least 2.5 cm of air space above this
volume for sample mixing is mandatory.
Sample bottles must be composed of a material which
is non-toxic to bacteria, resistant to solvent
action of sample, and capable of being repeatedly
sterilized without leakage occurring.
If glass-stoppered bottles are used, a strip of
paper or string should be placed in the neck of the
bottle before placing the stopper in place in
preparation for sterilization. This prevents the
glass stopper from "freezing" in place during
sterilization. The paper strip is discarded at
the time of sample collection.
Various plastics (polypropylene, Nalgene, etc.)
have been found to meet the specifications above,
and, closures of the screw cap type are acceptable
provided they are, and remain, non-toxic to the
sample and provide a tight closure. A test is
described to check new caps for toxicity (see
reference).
It is wise to purchase plastic bottles and caps of
the same material to preclude immediate or delayed
leakage problems.
Bottles can be checked for bacteriostatic or In-
hibitory residues by a bacteriological test pro-
cedure which, like the distilled water suitability
test, should be undertaken only by professional
bacteriologists or in laboratories where this test
is done on a regular basis.
All glassware (bottles, pi pets, graduates, etc.)
should be thoroughly cleaned with a suitable
detergent and hot water. Following several rinses
of hot tap water, several successive rinses with a
bacteriologically suitable deionized or distilled
water will produce a suitable bottle whether this
operation is accomplished by machine or hand
washing.
EPA-670/9-75-006
Handbook for Evaluating
Water Bacteriological
Laboratories
Std. Meth: 14:885
2-17
-------
WATER MONITORING PROCEDURES: Collection and Handling of Drinking Water Samples
Field and Laboratory Equipment
Section
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.3.4
Use of sterilizing oven ensures the drying of the
thlosulfate reagent on the bottom of the bottle
which will be visible as a tMn white film. Items
should be Inserted into a cool oven and not re-
moved until cooled after the sterilizing cycle.
Whichever method of sterilizing is used (dry heat
or steam), complete records are to be maintained
in a ledger which shows date; temperatures, time of
cycle; and laboratory workers name.
2-18
-------
MATER MONITORING PROCEDURES: Collection and Handling of Drinking Water Samples
Field and Laboratory Analyses
Section
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.3.5
B.l.lb
C.I.I
C.I.8
To check the sterility of a sterilized "batch" of
sample bottles, remove one or more bottles from
the group of bottles which were unit sterilized
and test as follows:
1. Aseptically pipet (sterile technique)
approximate!]^ 25 mts of sterile LLSTB (lactose
lauryl sulfate tryptose broth) into the sample
bottle.
2. Incubate for 24 hours at 35° C.
3. Check for results:
A. Growth (turbidity) is unsatisfactory and
indicates lactose fermenters as contaminants.
B. No growth indicates acceptability and the
bottle is sterile as regards to lactose
fermenters.
4. Record results in a bound quality control
ledger.
5. Discard any unsatisfactory batches (positives)
and investigate source of problem.
Sampling frequency and locations are stipulated
under existing governmental regulations. Estab-
lishment and changes of the above are joint
functions of the Certifying Authority (Regional
EPA Water Supply Representative); Reporting Agency
(State engineering program); and the Utility
(local administration).
Spigot or tap does not have to be "flamed" or
heated as these treatments have been found to be
of no consequence regarding bacteriological
testing and can, in addition, cause damage to
valve components.
Icing a sample prior to delivering it to the
analyzing laboratory is the most desirable holding
method. It is, however, acceptable to handle it
in a number of ways:
EPA-670/9-75-006
Handbook for Evaluating
Water Bacteriological
Laboratories
NIP DWR 40 CFR 141.21
2-19
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WATER MONITORING PROCEDURES: Collection and Handling of Drinking Water Samples
Field and Laboratory Analyses
Section
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
C.I.8
(Continued)
D.l.lb
1. If less than an hour will elapse between
collection and laboratory acceptance, icing is
not an important factor in handling.
2. Although not a desirable practice, it is accept-
able to transport a sample, uniced, in a
number of modes (mail; bus; UPS; courier
service; private shipping; etc.) as long as the
transit time is within limitations.
Another optional choice to the icing mode of
transportation is the use of thermos-type,
insulated, and sterilizable container.
Current regulations specify this 30 hour limitation
period beyond which the sample is unacceptable for
data validity. It is, however, a possibility that
the responsible State Agency may require the
laboratory to run the sample if it is received
after this period. If more than 30 hours, but
less than a 48 hour, period has elapsed (from
collection time to initiation of laboratory
procedures), the laboratory may run the sample with
the requirement that the data is indicated to be
possibly invalid. In the event that greater than
48 hours of holding time has elapsed, the
laboratory must refuse the sample as unsuitable
for analysis.
This outline was prepared by: Rocco Russomanno,
Microbiologist, National Training and Operational
Technology Center, MOTD, OWPO, USEPA, Cincinnati,
Ohio 45268.
2-20
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A PROTOTYPE FOR DEVELOPMENT OF
ROUTINE OPERATIONAL PROCEDURES
for the
COLIFORM TEST BY THE MPN METHOD FOR DRINKING WATER
as applied In
DRINKING WATER TREATMENT FACILITIES
and In the
DISTRIBUTION SYSTEMS OF DRINKING WATER TREATMENT FACILITIES
National Training and Operational Technology Center
Municipal Operations and Training Division
Office of Water Program Operations
U.S. Environmental Protection Agency
BA.MET.lab.WMP.7a.8.79
3-1
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EPA 600/8-78-008, May 1978
Manual for the Interim Certification of Laboratories Involved in Analyzing
Public Drinking Water Supplies - Criteria & Procedures
Coliform Test by the MPN Method for Drinking Mater
MPN media removed and cooled as soon as possible after sterilization and stored
in cool dark place (optional).
Tube broth media and reagents sterilized at 121°C 12 to 15 minutes with tubes and
flasks packed loosely in baskets or racks for uniform heating and cooling.
Laboratory pure water must be used; dissolution of the media must be completed
before dispensing to culture tubes or bottles.
Lauryl Tryptose Broth
(Lactose broth is not permitted.)
Single strength composition, 35.6 g per liter
pure water
Single strength pH 6.8 + 0.2; double strength
pH 6.7 + 0.2
Not less than 10 ml per tube
Media made to result in single strength after
addition of sample portions
Brilliant Green Lactose Bile Broth - Medium composition 40 g per liter pure
water. Final pH 7.2 +_ 0.2
Media stored at low temperatures are incubated overnight prior to use and
tubes with air bubbles discarded
Media protected from sunlight
MPN tube media with loose-fitting caps used in less than 1 week
Tube media in screw-capped tubes held no longer than 3 months
Presumptive Test
Five standard portions, either 10 or 100 ml
Sample shaken vigorously immediately before test
Tubes incubated at 35° +_ 0.5°C for 24 +_ 2 hours
Examined for gas (any gas bubble indicates positive test)
Tubes that are gas-positive within 24 hours submitted promptly to confirm test
Negative tubes returned to incubator and examined for gas within 48 + 3 hours
positives submitted to confirm test
Public water supply samples with heavy growth and no gas production confirmed
for presence of suppressed coliforms
Adjusted count reported based upon confirmation
Adequate test labeling and tube dilution coding (optional)
Confirmed Test
Presumptive positive tube gently shaken or mixed by rotating
One loopful or one dip of applicator transferred from presumptive tube to BGLB.
Transfer device sterile.
Incubated at 35°C +_ 0.5°; checked within 24 hours for gas production
Positive confirmed tube results recorded; negative tubes reincubated and
read within 48 + 3 hours
Unsatisfactory sample defined as three or more positive confirmed tubes
]MINIMUM REQUIREMENT except where indicated as OPTIONAL.
3-2
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Confirmed MPN test on problem supplies: If the laboratory has elected to use
the MPN test on water supplies that have a continued history of confluent
growth or TNTC with the MF procedure, all presumptive tubes with heavy growth
without gas production should be submitted to the confirmed MPN test to check
for the suppression of coliforms. A count is adjusted based upon confirma-
tion and a new sample requested. This procedure should be carried out on
one sample from each problem water supply once every 3 months.
3-3
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DRINKING WATER COLIFORM TEST
MPN METHOD FLOW SHEET
[SAMPLE
5 tubes containing an
equal sample volume
LLSTB]
Incubate 24 ± 2 hrs. at 35°C ± 0.5°C
Gas(+)
_L
Gas (-)
BGLBB
Incubate for 24 hrs ± 2 hrs.
at 35eC i 0.5°C
I
1
Reincubate for an additional
24 hrs (48 ± 3 hrs)
at 35°C ± 0.5°C
1
Gas (+)
Conforms
Present
1
1
Gas (+)
Coliforms
Present
1
nine the number
res in the 5 tube
1
Gas (-) G
1-V_
Reincubate for an
additional 24 hrs.
(48 ± 3hrs) at
35°C + 0.5°C
T
I
Gas (-)
Coliforms
Absent
of
series
1 1
as (+) Gas (-)
_| Coliforms
Absent
gas (+) - tube containing at
least one bubble
of gas
gas (— ) - complete absence
of gas in tube
LLSTB - lactose lauryl
sulfate tryptose
broth
BGLBB - Brilliant green
Lactose bile broth
Determine MPN value from
Table
I
Record results as Total
Coliforms/100 ml
I
Report results as prescribed
under regulatory requirements
3-4
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WATER MONITORING PROCEDURE: Coliform Test by the MPN Method for Drinking Water
1. Analysis Objectives:
a. In water treatment plant quality control, the objective of the test is
to determine if the effluent quality is in compliance with bacteriological
requirements as prescribed in the Federal Drinking Water Standards.
b. In distribution network and individual consumer tapping locations, the
test determines compliance with bacteriological requirements with the
above mentioned standards.
2. Brief Description of Analysis:
Five standard portions of either 100 ml or 10 ml are inoculated from a
drinking water sample into Lactose Lauryl Sulfate Tryptose broth fermentation
tubes (LLSTB) and incubated at 35°C +_ 0.5°C. After 24 hours and again at
48 hours, the LLSTB tube cultures are examined and results recorded for gas
production. Cultures showing gas production are transferred at each examina-
tion interval to B6LBB fermentation tubes and incubated at 35°C + 0.5°C. BGLBB
tubes are examined at 24 and 48 hour intervals for presence of gas and those
showing gas are considered gas (+} and containing coliforms while those com-
pletely without gas as gas (-) or not containing coliforms.
At the end of the overall incubation period, individual tubes are summarized
as positive or negative and the number positive for the five tubes is obtained.
A table of Most Probable Numbers (MPN) is used to determine the MPN value which
is given as coliforms per 100 ml. This result is reported or handled as pre-
scribed under regulatory requirements.
3. Applicability of this Procedure:
a. The range of coliform concentrations:
If this sample These ranges of coli-
volume is used forms are covered
100 ml < .22 to > 1.6
10 ml < 2.2 to > 16
b. Pretreatment of Samples: In accordance with Standard Methods,
14th ed. (p. 904)
This procedure conforms to the Standard Total Coliform MPN Tests as described
in Standard Methods for the Examination of Water and Wastewater, 14th ed. (1975).
p. 914
3-5
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WATER MONITORING PROCEDURE: Coliform Test by the MPN Method for Drinking Water
Equipment and Supply Requirements
A. Capital Equipment:
Autoclave, providing uniform temperatures up to and including 121°C, equipped
with an accurate thermometer, pressure gauges, saturated steam power lines
and capable of reaching required temperature within 30 minutes.
Balance, 0.1 g sensitivity at load of 150 g
Air incubator to operate at 35°C +_ 0.5°C
Oven, *hot-air sterilizing, to give uniform temperatures and with suitable
thermometer to register accurately in range of 160-180°C
pH meter, accurate to at least 0.1 pH unit, with standard pH reference
solution(s)
Water distillation apparatus (glass or block tin), or source of distilled
water suitable for bacteriological operations.
B. Reusable Supplies:
Apron or coat suitable for laboratory
Baskets, wire for discarded cultures
Bottles, sample*, preferred characteristics being 250 ml (6-8 oz.),
wide mouth, glass stopper
Bottle, squeeze type, with disinfecting solution
Burner, gas, Bunsen burner type
Cans, pipet, aluminum or steel; not copper (If plastic, or other type of
prepackaged disposable pi pets are used, this item is unnecessary.)
Metal caps* to fit 25 x 150 mm and 18 x 150 mm culture tubes
Pan, to receive discarded contaminated pi pets and glassware (must contain
disinfectant before use)
Inoculation loop, 3 mm diameter loop of nichrome of platinum-iridium wire,
26 B&S gauge, in holder
Pipets*, 10 ml, Mohr type preferred, sterile, cotton plugged, glass or
disposable plastic
Racks, culture type*, having at least 5 openings capable of accepting tubes
at least 20 mm in diameter
3-6
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WATER MONITORING PROCEDURE: Coliform test by the MPN Method for Drinking Water
Equipment and Supply Requirements (Continued)
Sponge, for cleaning desk top
Tubes, culture*, 150 x 25 mm and 150 x 18 mm
Tubes, fermentation*, 75 x 10 mm vials to be inverted in culture tubes
C. Consumable (must be replaced when stocks get low):
Distilled water, suitable for bacteriological cultures (note distillation
apparatus required in capital equipment)
BGLBB (Brilliant Green Lactose Bile Broth), dehydrated (recommend purchase
of 1/4-lb. units)
Lactose Lauryl Sulfate Tryptose Broth, dehydrated (recommend purchase of
1-lb. units)
Potassium Dihydrogen Phosphate (KHpPO.) (recommend purchase of 1/4 Ib. units)
Disinfectant, for bench tops. (Use household bleach solution prepared
according to instructions on bottle)
Wax pencils (recommend soft wax equivalent to Blaisdell 169T)
Sodium Thiosulfate (Na2S203-5 H20)
*Iterns marked are needed in quantities or require size or space allowances
which cannot be specified here, as they vary according to the daily analysis
schedule. As a rule-of-thumb, space/size or quantity requirements should be
at least 3 times the normal daily requirements. For further information on
specifications for equipment and supplies, see the Microbiology Section of
the current edition of "Standard Methods for the Examination of Water and
Wastewater."
3-7
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h.'VTER MONITORING PROCEDURE: Coll form Test by the MPN Method for Drinking Water
3-8
OPERATING PROCEDURES
STEP SiQ'JCNCE
INFOPMATION/OPEPATING GOAUYSPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
1. 35°C Incubator
Set-up, Adjustment
1. Place 35°C incubator in
permanent location.
2. Install thermometer.
3. Install shallow pan of
water in bottom of incu-
bator.
4. Connect incubator to
electric power source.
5. Adjust temperature until
stabilized at required
temperature.
6. Operate bacteriological
incubator continuously.
Aa. All pretest procedures completed before starting
other first-day procedures.
la. Out of drafts or places where it will be in
direct sunlight part of the day.
Ib. Location convenient to laboratory bench.
Ic. Convenient source of electric power.
2a. Thermometer functions at least in 30°-40°C range
and have intervals of 0.5° or less indicated.
Meets NBS standards.
2b. Location should be central in incubator.
2c. Mercury bulb thermometer should be fitted with
cork or rubber stopper and mounted in small bottle
filled with liquid (glycerine, water, or mineral
oil).
3a. In most laboratory incubators a pan having about
1 square foot area, with water about 1 inch deep,
is satisfactory.
3b. Maintains condition of saturated relative humid-
ity, required in bacteriological incubator.
3c. Requires daily check, with addition of water as
necessary, to keep water in pan at all times.
4a. Many incubators have pilot light to indicate
power turned on.
5a. Manufacturer's instructions for method of
temperature adjustment.
5b. Operation must be at 35 + 0.5°C.
5c. Allow about 1 hour between adjustments.
6a. Requires daily check with written temperature
record, with adjustment and water addition as
necessary.
V.A.I
V.A.1.1
(p. 31)
V.A.I.2
(p. 31)
V.A.I.3
(p. 31)
V.A.I.5
(p. 31)
V.A.I.6
(P. 31)
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HATER MONITORING PROCEDURE: Collform Test by the MPN Method for Drinking Water
OPERATING PROCEDURES
STLP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
2. Oven, Sterilizer,
Set-up
3. Autoclave Set-up
1. Place oven sterilizer in
permanent location.
2. Install thermometer.
3. Connect oven sterilizer to
power source and turn on.
4. Adjust temperature to
stabilize at required
temperature.
5. Operate oven sterilizer
only when needed. Turn
off when not in use.
1. Install and operate auto-
clave according to manu-
facturer's instructions.
la. Convenient to source of electric power; usually
on table or bench.
2a. Should indicate the 150°-180°C range, be accurate
within this interval, and be marked In 1.0 degree
intervals. Thermometer bulb is within a cylinder
filled with a fine sand and positioned on the
center shelf of the chamber.
3a. Usually has pilot light to indicate power on.
4a. Operated as near to 170°C as possible; not lower
than 160 nor higher than 180°C. Check to verify
that the 170°C temperature is reached and is
maintained within + 10°C for a 2-hour period.
5a. Turned ON in advance of need to permit reaching
required temperature before introducing material
to be sterilized.
5b. Oven sterilizer used to sterilize dry glassware,
metal objects.
5c. Oven sterilizer not used with culture media,
solution, plastics, rubber objects, or with
anything containing or including these.
5d. Paper-wrapped glass pi pets may be sterilized in
oven sterilizer.
la. Autoclaves extremely variable in design and
operation; also, potentially dangerous.
Ib. Used to sterilize objects made of, or including
liquids, rubber, culture media.
Ic. Glassware may be autoclave sterilized but must be
dried afterward.
V.A.2.1-5
(P. 31)
V.A.3.1
(p. 32)
3-9
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WATER MONITORING PROCED'jKr: Coliform Test by the MPN Method for Drinking Water
3-10
ITERATING PROCEDURES
STEP SEQUENCE:
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
4. Water Distillation
Equipment
5. pH Meter
6. Glassware
1. Install and operate in
accordance with manu-
facturer's instructions.
2. Operate continuously or
intermittently as required
to maintain adequate
supplies of distilled
water.
1. Have unit available and
operate in accordance with
procedures described in
other lab procedures.
1. Wash all glassware in hot
detergent solution;
2. Rinse at least once in hot
tap water;
3. Rinse in distilled water,
at least 6 successive times,
and,
4. Dry in air.
Id. Most plastics not sterilized in autoclave;
plastics usually require chemical sterilizers.
le. Autoclave usually operated at 121°C for 15 min.
If. Sterilized media must be removed from autoclave
as soon as possible after autoclave is reopened.
la. Must produce distilled water meeting quality
requirements for bacteriological tests.
2a. Reserve supplies kept in borosilicate glass
carboys or in plastic carboys made of material
which will not dissolve substances which will
affect growth of bacteria.
2b. Same distillation apparatus used for bacterio-
logical purposes may be used for chemical
reagents.
la. Unit for pH check on finished culture media.
Ib. Used in preparation of stock solution of
potassium dihydrogen phosphate.
la. Nontoxic detergent.
Ib. Be sure all contents and markings are washed away.
4a. No visible spots or scum; glass should be clean,
and sparkling.
4b. Glassware suitable for use in bacteriological
operations.
V.A.4.1-2
(p. 32)
V.A.5.1
(p. 33)
V.A.6.i-4a
(p. 33)
V.A.6.4b
(p. 33)
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WATER MONITORING PROCEDURE: Coliform Test by the MPN Method for Drinking Water
OPERATING PROCEDURES
STEP
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
7. Sodium Thiosulfate
Solution
8. Sample Bottle
Preparation
1. Weigh 10.0 grams of sodiun
thiosulfate.
2. Dissolve in 50-60 ml dis-
tilled water.
3. Add distilled water to
bring final volume to
100 ml.
4. Transfer to labeled
bottle.
1. Deliver 0.1 ml or .2 ml
of 10% sodium thiosulfate
solution to each sample
bottle. (.1 ml to 4 ounce
or 120 ml size and .2 ml
to 6-8 ounce or 250 ml
size)
2. Place cover on sample
bottle.
3. Place paper or metal foil
cover over bottle cap or
stopper.
la. Used for dechlorination of samples.
Ib. Use of trip balance accepted.
2a. 100 ml graduated cylinder satisfactory.
4a. Labeled as 10% sodium thiosulfate and stored
in refrigerator.
la. Use 1 ml pipet.
Ib. Provides adequate sodium thiosulfate for
neutralizing chlorine in sample.
Ic. Return stock sodium thiosulfate solution to
refrigerator.
2a. Use 1 ml pipet.
2b. Provides adequate EDTA chelating agent for
metals in sample.
3a. Protects opening of sample bottle from accidental
contamination.
3-11
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WAFER MONITORING PROCEDURE: Coliform Test by the MPN Method for Drinking Water
3-12
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
9. Pi pet Preparation
4. Sterilize sample bottles
in sterilizing oven.
5. Store sample bottles in
clean, dry place until
used.
1. Inspect pi pets to be pre-
pared for use; discard anc
destroy all having chipped
or cracked tips or tops.
2. Insert plug of non-
absorbent cotton into
mouthpiece of each clean,
dry pi pet.
3. Place a layer of glass
wool or several layers of
paper padding in bottom of
pi pet can.
4. Place 18-24 pipets in each
pi pet can, delivery tip
down.
5. Sterilize cans of pipets
in oven.
6. Store cans in clean, dry
place until used. Mark
cans as 10 ml sterile
pipets.
5a. One hour at 170°C. (See A.2)
la. Cleanliness of pipet must be equivalent to
glassware.
2a. For protection of user when pipetting sample.
2b. Cotton plug must be tight enough to prevent easy
removal, either by the pipetting action or by
handling, and yet loose enough to permit easy
air movement through the plug.
3a. For protection of pipet delivery tips.
4a. Orientation permits removal of sterile pipets
from can without contamination by user.
5a. One hour at 170°C CSee A.2 of procedures).
6a. Laboratory cabinet or drawer recommended.
V.A.9.1-6
(P. 33)
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WATER MONITORING PROCEDURE: Coliform Test by the MPN Method for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
10. Preparation of
Lactose Lauryl
Sulfate Tryptose
Fermentation Broth
(LLSTB)
7. When can of pi pets is
opened for first use,
pass the exposed ends of
the pipets through flame,
slowly.
1. Weigh 53.4 grams of dehy-
drated Lactose Lauryl
Sulfate Tryptose Broth.
Close cover of bottle of
dehydrated medium tightly
after removal.
2. Dissolve in 1 liter dis-
tilled water.
3. Place 20.5 ml of the solu-
tion of prepared LLSTB in
each culture tube.
4. Insert one fermentation
vial into each tube of
medium, open end down.
5. Place tube cap on each
tube of culture medium.
6. Sterilize in autoclave.
7a. Burns off excess cotton sticking out of pi pet
mouthpiece.
7b. Cover kept on can at all times except when samples
are being inoculated.
la. Dehydrated media take moisture out of air; can
become caked.
Ib. Caked media unsatisfactory; should be discarded.
2a. Gentle heat (no boiling) if necessary to complete
dissolving medium. Usually a vigorous agitation
will completely dissolve the medium.
3a. Use 150 x 25 mm tubes.
3b. 25 ml pipet, automatic pipetter, or funnel hose
and pinchcock assembly are acceptable.
3c. Accuracy of delivery: + 0.5 ml.
3d. Approximately 45 tubes will be necessary. This
will suffice for 9 tests based upon procedures
of this WMP (Water Monitoring Procedure).
4a. Tubes and vials previously washed as indicated
(V.6.1-4).
4b. Use 75 x 10 mm tubes.
5a. After all tubes have been filled and have indi-
vidual vial.
6a. Within 1 hour after medium prepared.
6b. Sterilization at 121°C for 15 minutes.
6c. Medium must be removed from autoclave as soon as
possible after pressure has returned to normal.
Use "slow-vent" mode of steam removal.
V.A.9.7
(p. 34)
V.A.lO.Sb
(p. 34)
3-13
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KATER MONITORING PROCEDURE: Coliforro Test by the MPN Method for Drinking Water
3-14
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
11. Preparation of
Brilliant Green
Lactose Bile Broth
(BGLBB)
7. Cool medium to room
temperature.
8. Check pH of finished
medium.
9. If final pH not satis-
factory, discard medium
and prepare new batch
with pH adjustment before
sterilization.
10. Store medium in cool,
dark place.
7a. Medium ready for use when cool and individual
vials are completely filled with fluid. No
bubbles must be present.
8a. Should be 6.7-6.9.
9a. pH value ordinarily drops about 0.2 pH unit.
1. Weigh 40.0 grams of dehy-
drated Brilliant Green
Lactose Bile Broth. Close
cover of bottle of dehy-
drated medium tightly
after removal.
2. Dissolve in 1 liter of
distilled water.
3. Place 10.5 ml of the
solution of prepared
BGLBB in each culture
tube.
lOa. Not in refrigerator. Usually in laboratory
cabinet in darkness.
lOb. May be stored up to 1 week if evaporation not
more than 10% in loose fitting capped tubes.
With screw-capped tubes, it should be held no
longer than 3 months.
la. Dehydrated media takes moisture out of air;
can become caked.
Ib. Caked media unsatisfactory; should be discarded.
2a. Gentle heat (no boiling) if necessary to com-
plete dissolving medium. Usually a vigorous
agitation will completely dissolve the medium.
3a
3b.
3c.
Use 150 x 18 mm tubes.
A 25 ml pi pet, automatic pipetter or funnel
hose and pinchcock assembly are acceptable.
Accuracy of delivery +0.5 ml.
V.A.10.3b
(p. 34)
3d. Approximately 90 tubes will be necessary.
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v-ATER MONITORING PROCEDURE: Coliform Test by the MPN Method for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
12. Final Equipment and
Supply Check
4. Insert one fermentation
vial into each tube of
medium, open end down.
5. Place cap on each tube of
culture medium.
6. Sterilized in autoclave.
7. Cool medium to room
temperature.
8. Check pH of finished
medium.
9. If final pH is not satis-
factory, discard medium
and prepare new batch with
pH adjustment before
sterilization.
10. Store medium in cool,
dark place.
1. Check to be sure that all
equipment and supplies,
solutions, and prepared
media are ready before
starting sample examinatio
4a. Tubes and vials previously washed as indicated
(A.6.1-4).
4b. Use 75 x 10 mm tubes.
5a. After all tubes have been filled and have
individual vial.
6a. Within 1 hour after medium prepared.
6b. Sterilization at 121°C for 15 minutes.
6c. Medium must be removed from autoclave as soon as
possible after pressure has returned to normal.
Use "slow-vent" mode of steam removal.
7a. Medium ready for use when cool and individual
vials are completely filled with fluid. No
bubbles must be present.
8a. Should be 7.1-7.3.
9a. pH value ordinarily drops about 0.2 pH unit.
lOa. Not in refrigerator. Usually in laboratory
cabinet in darkness.
lOb. May be stored up to 1 week if evaporation not more
than 10% in loose-fitting capped tubes. With
screw-capped tubes, it should be held no longer
than 3 months.
la. Check general list of equipment and supplies.
Ib. Each test requires:
5 tubes LLSTB
0-5 tubes of BGLBB
• 1 sample bottle
1-10 ml pipet
3-15
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WATER MONITORING PROCEDURE; Coliform Test by the MPN Method for Drinking Water
3-16
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
2. Make preparations or ad-
justments as necessary
before starting test.
B. First-Day Procedures
1. Equipment
Maintenance
2. Sample Collection
3. Preparation of
Laboratory Data
Sheet
1. Check, record, and adjust
incubator temperature.
2. Add water to pan in incu-
bator as necessary.
1. Collect sample.
2. Record sampling
information.
3. Transport sample to
laboratory.
1. Fill in data sheet to show
sample information.
2. Select sample inoculation
volumes.
la. See A.1.1-6.
la. Locations as selected by requirements.
Ib. Sampling methods as described in Standard Methods
2a. Most organizations have sample tag of some type
which includes such information as date, time,
place of sampling, name of sample collector, and
other information as may be required.
3a. Taken to laboratory without delay.
3b. Samples preferably iced if delay of starting
sample test is greater than one hour. No more
than 30 hours of transportation time is allowed.
la. Needed information should be on sample collection
tag.
Ib. Most data sheets show at least source, date, time
of collection, name of sampler, name of analyst,
laboratory sample number assigned.
2a. For purposes of this VIMP (Water Monitoring
Procedure), sample volumes of 10 ml per tube In a
series of 5 tubes is required.
VII.B.3.1
(p. 35)
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HATER MONITORING PROCEDURE: Coliform Test by the MPN Method for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First-Day Procedures
(Continued)
4. Lab Bench
Disinfection
5. Assembly of
Culture Medium
6. Sample Inoculation
3. Enter information in
laboratory data sheet to
show sample inoculation
volume for 5 tubes.
1. Disinfect laboratory bench;
wipe dry.
1. Place 5 tubes of Lactose
Lauryl Sulfate Tryptose
Broth (LLSTB) in culture
tube rack.
2. Label test set-up.
1. Shake sample vigorously.
2. Deliver 10 ml of sample per
tube of LLSTB.
2b. Workers desiring 100 ml portions for this test
should consult Standard Methods for required
modifications (medium strength, interpretation of
results, etc.)
3a. Recommend showing sample inoculation volumes in
ml or decimal amounts.
Amount
Sample
ml
10
Preservative
LLSTB
24 hr
—
48 hr
Confi
SGLBL
Z4 hr
4^
-
la. Sponge and disinfectant; paper toweling.
2a. First tube or rack can be labeled.
2b. Prevents confusion if a number of tests are
being processed.
la. At least 25 shakes over space of at least 1 foot
in 10 seconds or less.
2a. Use sterile 10 ml pipet.
I.B.6.1
Cp. 28)
3-17
-------
WATER MONITORING PROCEDURE: Coliform Test by the MPN Method for Drinking Water
3-18
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First-Day Procedures
(Continued)
7. Incubation
8. Processing Used
Glassware
9. Lab Bench
Disinfection
1. After completion of sample
inoculation into LLSTB,
shake rack of cultures
gently.
2. Place rack of cultures in
incubator.
1. Drain sample bottle and
pipet into sink.
2. Wash and dry bottle and
pipet.
1. Disinfect laboratory bench
top; wipe dry.
la. Mixes sample with culture medium.
Ib. Avoid Shaking air into fermentation vials.
2a. Twenty-four hours + 2 hours at 35 + 0.5°C.
la. Sterilization unnecessary.
2a. Meets original cleanliness requirements of
glassware.
2b. Glassware ready for reuse.
la. Sponge, disinfectant, paper toweling.
C. Twenty-four Hour
Procedures
1. Equipment
Maintenance
2. Disinfection
1. Check, record, and adjust
incubator temperature.
2. Add water to pan in incu-
bator as necessary.
1. Disinfect laboratory bench
top; wipe dry.
la. See A.1.1-6.
la. See B.4.1
-------
dATER MONITORING PROCEDURE: Coliform Test by the MPN Method for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Twenty-four Hour
Procedures (Continued)
3. Reading and Record-
ing of Results
1. Remove rack of culture
from incubator to lab
bench.
2. Shake culture rack
3. Examine each tube for gas
production and record re-
sults on data sheet.
2a. Hastens release of gas in supersaturated cultures.
2b. Must not shake air into fermentation vials.
3a. If present, gas will be trapped in the fermenta-
tion vial.
3b. Gas in any quantity is a positive test.
3c. Vials with no gas are a negative test.
3d. Each result appears on line corresponding with
the tube label.
3e. All results appear under the "24" of the LLSTB
column.
3f. Plus sign (+) means a gas-positive tube.
3g. Minus sign (-) means a gas-negative tube.
3h. Assume, for instructional purposes, that the
following recordings result:
III.C.3.3
(p. 30)
Obs«.
Ano
Sam
n
to
Jflt
Die
Preservative
LLSTB
H h'r
•f
_
—
—
+
48 hr
— c5K'
D
M
3-19
-------
WATER MONITORING PROCEDURE: Coliform Test by the MPN Method for Drinking Water
3-20
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Twenty-four Hour
Procedures (Continued)
4. Transfers
5. Processing Dis-
carded Cultures
1. Obtain required number of
BGLBB tubes.
2. Transfer each gas-positive
tube of LLSTB to a tube
of BGLBB.
3. Return rack of tubes con-
taining the negative LLSTB
tubes and the freshly
Inoculated BGLBB tubes to
incubator.
1. Sterilize discarded LLSTB
tubes.
2. Remove all labels from
culture tubes.
la. One tube for each LLSTB gas-positive tube.
Ib. In our example test it is necessary to have
two BGLBB tubes.
Ic. Observe BGLBB tubes for sterility (no growth or
turbidity) and completely filled fermentation
vial (no gas in vial).
2a. 3-mm inoculation loop.
2b. Loop flame-sterilized before use and between
successive transfers.
2c. One loopful per transfer.
2d. Place inoculated BGLBB tube into hole of rack
previously occupied by the LLSTB tube from which
the transfer was made.
2e. Place positive LLSTB tube into discard area after
transfer is made. All discard tubes are to be
sterilized prior to cleaning and reuse of caps
and tubes.
2f. Negative (no gas) LLSTB tubes remain untouched in
their rack position.
3a. An additional 24+2 hours at 35° + 0.5°C.
VII.C.4.2
(p. 36)
la. Autoclave: 15 minutes at 121°C.
2a. Best done while still warm after autoclave.
-------
MATER MONITORING PROCEDURE: Coliform Test by the MPN Method for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPErtATIMG GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Twenty-four Hour
Procedures (Continued)
6. Disinfection
3. Empty sterilized cultures
into sink.
4. Wash and dry culture tubes,
fermentation vials, and
tube caps.
1. Disinfect laboratory bench
top; wipe dry.
4a. Meets original cleanliness requirements of
glassware.
4b. Tubes and caps ready for re-use.
la. Sponge and disinfectant; paper toweling.
D. Forty-eight Hour
Procedure
1. Equipment
Maintenance
2. Disinfection
3. Reading and Re-
cording of Results
1. Check, record, and adjust
incubator temperatures.
2. Add water to pan in incu-
bator as necessary.
1. Disinfect lab bench top;
wipe dry.
1. Remove the rack of cultures
from the incubator to lab
bench.
2. Shake culture rack gently.
3. Examine each tube for gas
production and record re-
sults on data sheet.
3a. LLSTB tubes will be recorded under the "48" on
the LLSTB column and the BGLBB tubes under the
"24" column.
3b. Any amount of gas is always considered to be
a "positive" (+) result.
3-21
-------
WATER MONITORING PROCEDURE: Coliform Test by the MPN Method for Drinking Water
3-22
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Forty-eight Hour
Procedures
4. Transfers
1. Discard all of the BGLBB
tubes which have the
positive (+) recordings.
2. Discard all LLSTB tubes
which have the negative
(-) recordings.
3. Re-incubate any BGLBB tube:
which were negative and
assemble for transfer any
positive LLSTB tubes.
4. Label required tube of
sterile BGLBB.
3c. Assume that our "test" never shows the following
recordings:
Amount
Sample
ml
10
Preservative
LLSTB
24 hr
t
-
—
4-
48 hr
+
—
—
Confirmed
SGLBB
H hr
•+-
—
13 hr
la. This will be a total of one tube (see data sheet
recordings in D.3.3c)
2a. This will be a total of 2 tubes (D.3.3c)
2b. LLSTB tubes which show NO GAS production within
48 hours are to be considered as not having
contained coliform bacteria.
3a. There will be one tube of BGLBB which must be
re-incubated for an additional 24 hours at 35° +
0.5°C (D.3.3c).
3b. There will be one positive LLSTB tube (D.3.3c).
4a. This is done so that the re-incubated (24 hour
old) BGLBB tube will not be confused with the
newly inoculated BGLBB tube since both have to
be incubated for 48 hours.
-------
MATER MONITORING PROCEDURE: Coliform Test by the MPN Method for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Forty-eight Hour
Procedures (Continued)
5. Incubation
6. Processing Dis-
carded Tubes of
Media
7. Disinfection
Transfer the positive
LLSTB tube to the sterile
BGLBB tube.
6. After transfer place LLSTB
tube in discard basket.
1. Incubate inoculated BGLBB
tube.
la. (Alternate) If no cultures
for this test procedure
remain to be incubated,
proceed to "Interpretation
of Test Results" and con-
tinue as directed.
1. Sterilize discarded media.
2. Remove all labels from
culture tubes.
3. Empty sterilized cultures
into sink.
4. Wash and dry culture tubes
fermentation vials, and
tube caps.
1. Disinfect laboratory bench
top; wipe dry.
5a. Use 3 mm loop which is flamed prior to entry into
the LLSTB to avoid contamination.
5b. Use one loopful of transfer from the LLSTB to
BGLBB.
la. 35 + 0.5°C for 24 hours.
E. Seventy-two Hour
Procedures
1. Equipment
Maintenance
Check, record, and adjust
incubator temperatures.
3-23
-------
WATER MONITORING PROCEDURE: Coliform Test by the MPN Method for Drinking Water
3-24
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
E. Seventy-two Hour
Procedure (Continued)
2. Disinfection
3. Reading and Record-
ing of Results
4. Processing Dis-
carded Tubes of
Media
2. Add water to pan in incu-
bator as necessary.
1. Disinfect lab bench top;
wipe dry.
1. Remove cultures from incu-
bator to lab bench.
2. Shake cultures gently.
3. Examine each tube for gas
production and record re-
sults on data sheet.
4. Incubate any cultures
which are still negative
if they have not been
incubated a full 48 hours.
1. Sterilize discarded tubes
of media.
2. Remove all labels from
tubes.
3a. In our continuing example, two tubes of BGLBB are
to be examined—one of which will be a "48" entry
and the other of the "24" column entry.
3b. Assume the following recordings will be made:
Amount
Sample
nl
10
Preservative
LLSTB
24 hr
+
—
—
—
+
48 hr
+
—
—
Confirmed
3SL8B
Z< hr
+
—
—
43 hr
—
1
4a. One BGLBB tube will have to be returned to the
incubator since it is only 24 hours old and
still negative.
4b. 35 + 0.5°C for an additional and final 24 hours.
-------
WATER MONITORING PROCEDURE: Coliform Test by the MPN Method for Drinking Water
OPERATING PROCEDURES
STEP SEQUtNCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
E. Seventy-two Hour
Procedures (Continued)
5. Disinfection
3. Empty sterilized tubes
into sink.
1. Disinfect lab bench top;
wipe dry.
F. Ninety-six Hour
Procedures
1. Equipment
Maintenance
2. Disinfection
3. Reading and Record-
ing of Results
1. Check, record, and adjust
incubator temperatures.
2. Add water to pan in incu-
bator as necessary.
1. Disinfect lab bench top;
wipe dry.
1. Remove the BGLBB tube from
incubator to lab bench.
2. Shake culture gently.
3. Examine tube for gas pro-
duction and record results
on data sheet.
3a. In our example assume the final recordings on the
data sheet will be:
..ert.
Amount
Sample
ml
10
Preservative
LL5TB
24 hr
-t-
—
—
—
+
43 hr
+
—
—
Confirmed
ESLSB
24 hr
-h
—
—
43 hr
—
3-25
-------
HATER MONITORING PROCEDURE: Coliform Test by the MPN Method for Drinking Water
3-26
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
F. Ninety-six Hour
Procedures (Continued)
4. Disinfection
5. Processing of
Discarded Tubes
3b. With final recordings completed, one can now
proceed to "Interpretation of Test Results."
3c. Final entries could have been made as early as
the 48-hour procedures up to these 96-hour
procedures.
1. Accomplish as in previous
directives.
1. Accomplish as in previous
directives.
G. Interpretation of
Test Results
1. Determine number of BGLBB
tubes which are positive
in the row of 5 tubes.
2. Write the numbers in the
data sheet.
la. NO consideration of presumptive test (LLSTB) for
interpretation of test results.
Ib. Our example (F.3.3) shows one positive BGLBB tube<
II.6.1
2a.
Observations
ve
•
Confirmed
BGLBB
24 hr
+
—
43 hr
—
—
«*
1
J.
2b. One of the five BGLBB tubes is positive.
3. Select from the proper
table the MPN Index for
the test result.
-------
MATER MONITORING PROCEDURF: Coliform Test by the MPN Method for Drinking Water
OPERATING PROCEDURE1:
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
G. Interpretation of
Test Results
(Continued)
3a. MPN Index for Various Combinations of Positive
and Negative Results When Five 10-ml Portions
Are Used
No. of Tubes
Giving Positive
Reaction out of
5 of 10 ml Each
0
1
2
3
4
5
MPN
Index/
100 ml
<2.2
2.2
5.
9.
16.
.1
.2
4. Record the total coliforms
per 100 ml on the labora-
tory data sheet.
3b. For the example the location of the MPN index is
2.2/100 ml based on the single positive BGLBB
result. The arrow locates the MPN Index.
4a.
J
Results: a.A//00«l
Total collfona MPN
H. Reporting of Results
1. Report results as pre-
scribed by State regula-
tory requirements.
3-27
-------
WATER MONITORING PROCEDURE: Coliform Test by the MPN Method for Drinking Water
TRAINING GUIDE
SECTION TOPIC
I* Introduction
II* Educational Concepts - Mathematics
III* Educational Concepts - Science
IV Educational Concepts - Communications
V* Field and Laboratory Equipment
VI Field and Laboratory Reagents
VII* Field and Laboratory Analyses
VIII Safety
IX Records and Reports
*Training guide materials are presented here under the headings marked *.
These standardized headings are used through this series of procedures.
3-28
-------
WATER MONITORING PROCEDURES: Collform Test by the MPN Method for Drinking Water
INTRODUCTION
Section I
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
B.6.1
These MPN 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 bac-
terial cells, uniformly distributed through the
original sample when the primary inoculation is made.
Test procedures are based on
assumptions:
certain fundamental
a. First, even if only one living cell of the test
organisms 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 cul-
ture medium will produce a result which indicates
presence of the test organism; and
c. Third, unwanted 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.
3-29
-------
WATER MONITORING PROCEDURES: Coliform Test by the MPN Method for Drinking Water
EDUCATIONAL CONCEPTS - MATHEMATICS
Section II
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
G.I
For purely qualitative aspects of testing for indi-
cator organisms, it is convenient to consider the
tests applied to one sample portion, inoculated into
a tube of culture medium, and the follow-up examina-
tions and tests on results of the original inocula-
tion. Results of testing procedures are definite:
positive (presence of the organism-group is demon-
strated) or negative (presence of the organism-group
is not demonstrated).
The combination of positive and negative results is
used in an application of probability mathematics
to secure a single MPN value for the sample.
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 js_ demonstrated
to be present; and
b. The testing procedure must result in one or more
tubes in which the test organism is not demon-
strated to be present.
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.
3-30
-------
MATER MONITORING PROCEDURES: Coliform Test by the MPN Method for Drinking Uater
EDUCATIONAL CONCEPTS - SCIENCE
Section III
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
C.3.3
Interpretation of results on LLSTB:
Development of gas in this medium indicates that the
lactose has been fermented. Fermentation of lactose
with gas production is a basic characteristic of
coliform bacteria. To meet the definition of coli-
forms, gas must be produced from lactose within
48 hours after being placed in the incubator. If a
culture develops gas only after more than 48 hours
incubation, then, by definition, it is not a
coliform.
Meeting previously discussed assumptions (see
I.B.6.1.1) usually makes it necessary to conduct the
tests in a series of stages.
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 in-
cluded 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 includec
in one of the approved media for suppression of
growth of certain non-coliform bacteria. This
additive apparently has no adverse effect on
growth of members of the coliform group in the
concentrations 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.
3-31
-------
WATER MONITORING PROCEDURES: Coliform Test by the MPN Method for Drinking Water
FIELD AND LABORATORY EQUIPMENT
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.I
A.1.1
A.1.2
A.1.3
A.1.5
A.1.6
A.2.1-5
Incubator must be of sufficient size for daily work-
load without causing crowding of tubes to be incu-
bated. Considerations for choice of incubator type
must relate to reliability of operation and not to
cost or attractiveness of equipment.
Incubator should be kept out of drafts or direct
sunlight in order to prevent temperature inside the
incubator from changing outside the temperature
range specified (35° ±0.5°C).
Power supply should be selected so that there won't
be too many pieces of equipment on the same circuit.
Otherwise, circuits will be blown repeatedly.
Mercury bulb thermometer usually used in most incu-
bators. Recording thermometer is acceptable, but,
it should be calibrated against a mercury bulb
thermometer which has been certified by National
Bureau of Standards. The NBS certified thermometer
always should be used with its certificate and
correction chart.
Saturated relative humidity is required in order to
make the incubation more efficient (heat is trans-
ferred to cultures faster than in a dry incubator).
Furthermore, culture medium may evaporate too fast
in a dry incubator.
Allow enough time after each readjustment to permit
the incubator to stabilize before making a new ad-
justment. At least one hour is suggested.
Incubator temperature can be held to much closer
adjustment if operated continuously. Temperature
records should be kept in some form of permanent
record. A temperature record book is suggested with
daily recording of values. If a recording thermom-
eter is used, the charts may be kept as permanent
record; if so, be sure that the charts are properly
labeled to identify the incubator and the period
covered.
Uniform temperature (35°C ^0.5°C) is to be main-
tained on shelves in use.
Since electric sterilizer will be operated inter-
mittently, care should be taken that it is on a
circuit which will not be overloaded when it is
turned on.
Standard Methods for the
Examination of Water and
Nastewater. 14th ed. (1975)
APHA, WPCF, AWWA, p. 880
(Hereafter referred to as:
Std. Meth. 14: (page no.)
Std. Meth. 14:881
3-32
-------
MATER MONITORING PROCEDURES: Cell form Test by the MPN Method for Drinking Water
FIELD AND LABORATORY EQUIPMENT (Continued)
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.2.1-5
(Continued)
A.3.1
A.4.1-2
A time and temperature record is maintained for each
sterilization cycle. Temperature recordings can be
retained for records.
Autoclaves differ greatly in design and in method
of operation. Some are almost like home-style
pressure cookers; others are almost fully automatic.
This is a subject which requires separate instruc-
tion; and should be related to the exact make and
model of equipment you will use in your own
laboratory.
Vertical autoclaves and household pressure cookers
may be used in emergency service if equipped with
pressure gages and thermometers with bulbs positioned
1 inch above the water level. However, they are not
to be considered the equivalent of the general pur-
pose steam sterilizer recommended for permanent
laboratory facilities. Their small size is inade-
quate for large-volume work loads, and they can be
difficult to regulate.
The following requirements must be met regarding
autoclaves of sterilizing units:
a. Reaches sterilization temperature (121°C), main-
tains 121°C during sterilization cycle, and re-
quires no more than 45 minutes for a complete
cycle.
b. Pressure and temperature gages on exhaust side
and an operating safety valve.
c. No air bubbles produced in fermentation vials
during depressurization.
d. Record maintained on time and temperature for each
sterilization cycle.
Distilled water in a bacteriological laboratory must
not contain substances which will prevent any bactff-ia
from growing in culture medium in which the distilled
water is used or will be highly nutritive. There are
>rocedures for testing quality of distilled water;
>ut these should be undertaken only by professional
bacteriologists or in laboratories where this is done
regularly. Use only glass stills or block tin
lined stills.
Std. Meth. 14:881
Std. Meth. 14:645-49
14:888-89
Training Manual (EPA
Current Practices in Water
Microbiology
3-33
-------
WATER MONITORING PROCEDURES: Coliform Test by the MPN Method for Drinking Water
FIELD AND LABORATORY EQUIPMENT (Continued)
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.4.1-2
(Continued)
A.5.1
A.6.1-4a
A.6.1-4b
A.8.1-6
A.9.1-6
3-34
Requirements for distilled water include the following:
Test
PH
Conductivity
Trace metals:
A single metal
Total metals
Test for bactericidal proper-
ties of distilled water
("Standard Methods," 14th
ed., p. 887)
Free Chlorine residual
Standard plate count
Analysis Requirement
4.5-8.5
0.1 megohm as resistivity or <5.0
micromhos/cm at 25°C
Not greater than 0.05 mg/1
Equal to or less than 1.0 mg/1
0.8-3.0
0.0
Less than 10,000/ml
Conducted
Monthly
Monthly
Annually
Annually
Monthly
Monthly
pH Meter: See cited reference
Glassware: See cited reference on pi pets and gradu-
ated cylinders, media utensils, bottles.
Glassware can be checked for bacteriostatic or in-
hibitory residues by a bacteriological test proce-
dure which, like the distilled water suitability
test, should be undertaken only by professional
bacteriologists or in laboratories where this test
is done on a regular basis.
Sample bottles:
Wide-mouthed glass-stoppered bottles suggested, but
other styles acceptable.
If glass-stoppered bottles are used, a strip of paper
should be placed in the neck of the bottle before
placing the stopper in place in preparation for
sterilization. This prevents the glass stopper from
"freezing" in place during sterilization. The paper
strip is discarded at the time of sample collection.
Pi pets:
This procedure is described in terms of reusable
glass pipets. However, single-service prepackaged
glass or plastic pipets may be purchased and used, if
preferred. In case of use of single-service pipets,
they will be sterile when purchased, are used one
time, and discarded immediately after use. Accord-
ingly, in the step-by-step procedures disregard any
instructions about preparation of pipets for reuse in
case of using single-service pipets.
Std. Meth.
Std. Meth.
14:882
14:882-885
Std. Meth. 14:884
14:904
Std. Meth. 14:882-883
-------
WATER MONITORING PROCEDURES: Coliform Test by the MPN Method for Drinking Water
FIELD AND LABORATORY EQUIPMENT (Continued)
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.9.7
Passing the opened can of pi pets through a flame
burns off excess cotton wisps sticking out of the
mouthpiece of the pi pet. If this is not done, it is
almost impossible to control sample measurement
accurately. Some workers may elect to accomplish
this step prior to the sterilization procedure.
A.10.3b
FUNNEL
FUNNEL, HOSE, AND
PINCHCOCK ASSEMBLY
PINCHCOCK
GLASS TUBE
NOTE- UNIT NEED NOT BE
STERILE FOR MEDIUM
DELIVERY ONLY
3-35
-------
WATER MONITORING PROCEDURES: Coliform Test by the MPN Method for Drinking Water
FIELD AND LABORATORY ANALYSES
Section VII
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
B.3.1
There Is no such thing as a "standard" data sheet
for bacteriological tests. A simplified data sheet
is shown below:
Coliform Test
Multiple Dilution Tube (MPN) Method
Sample Type Lab No
Station
Description
Collection Date Time
HM
PM Temp.
AM AH.
Received PM Examined PM. '
Sampler
Amount
Sample
ml
Observations
Preservative
LLSTB
24 hr
48 hr
Confirmed
BGLBB
24 hr
48 hr
*j
s,
10
Results
Total coll form MPN
3-36
-------
WATER MONITORING PROCEDURES: Coliform Test by the MPN Method for Drinking Water
FIELD AND LABORATORY ANALYSES
Section VII
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
C.4.2
Transfers of LLSTB
Transfers can be made, as indicated, with a wire
loop having a diameter of at least 3 mm. An alter-
nate method of transfer authorizes the use of an
"applicator stick" which is a single service hard-
wood transfer device. Its dimensions are 0.2 to 0.3
cm in diameter and 2.5 cm longer than the test tube
used in the analysis. The term single service de-
notes that the stick is pre-sterilized and used for
a single transfer (LLSTB to BG) and then discarded
in the pan containing disinfectant and a new sterile
stick used for the next tube to be transferred. Use
of this stick technique makes the gas burner un-
necessary for the transfer process.
Std. Meth. 14:922
This outline was prepared by: Rocco Russomanno,
Microbiologist, National Training and Operational
Technology Center, MOTD, OWPO, USEPA, Cincinnati,
Ohio 45268
3-37
-------
A PROTOTYPE FOR DEVELOPMENT OF
ROUTINE OPERATIONAL PROCEDURES
for the
COMPLETED TEST FOR THE MPN METHOD FOR DRINKING WATER
as applied In
DRINKING WATER TREATMENT FACILITIES
and In the
DISTRIBUTION SYSTEMS OF DRINKING WATER TREATMENT FACILITIES
National Training and Operational Technology Center
Municipal Operations and Training Division
Office of Water Program Operations
U.S. Environmental Protection Agency
BA.MET.lab.WMP.5a.10.78
4-1
-------
600/8-78-008, May 1978
Manual for the Interim Certification of Laboratories Involved in Analyzing
Public Drinking Water Supplies - Criteria & Procedures
Completed Test for the MPN Method for Drinking Hater
Completed Test
Applied to 10 percent of all positive samples each quarter
Applied to all positive confirmed tubes in each test completed
Positive confirmed tubes streaked on EMB plates for colony isolation
Plates adequately streaked to obtain discrete colonies
Incubated at 35° + 0.5°C for 24 + 2 hours
Typical nucleated colonies, with or without sheen, on EMB plates selected
for completed test identification
If typical colonies absent, atypical colonies selected for completed test
identification
If no colonies or only colorless colonies appear, confirmed test for that
particular tube considered negative
An isolated typical colony or two atypical colonies transferred to lauryl
tryptose broth
Incubated at 35° + 0.5°C; checked for gas within 48 + 3 hours
Cultures producing gas in lauryl tryptose broth within 48 + 3 hours are
considered coliforms.
MINIMUM REQUIREMENTS
4-2
-------
COMPLETED TEST SCHEMATIC
ETC
,
(STREAK
TECHNIQUE )
POSITIVE BGLBB TUBES
FROM CONFIRMED TEST
(POSSIBLES CAN OCCUR IN 48,
72, AND 96 HR STANDARD
TEST TIME INTERVALS)
( INCUBATE 24 ± 2 WRS
AT 35 ± 0.5 C )
PICK APPROPRIATE
COLONIES]
X'
•*<7^A >^ <
f/f ffi CULIURI e«i™«f v
*•// //*» V-ULIUKt j*«
*(///£ #2
^y ^^
CULTURE #1 (24 ± 2 HRS AT 35 ± 0.5 C)
[GENTLY
SHAKE)
GAS -
GAS +
GRAM STAIN
(RE-INCUBATE
TOTAL:
48 ± 3 HRS)
GAS +
GAS -
COLIFORMS
ABSENT
REPEAT FOR
CULTURES 2, 3, ETC)
INTERPRET
RESULTS
RE-EVALUATE, IF NECESSARY, THE
CONFIRMED COLIFORM RESULTS
I
REPORT RESULTS
4-3
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
1. Analysis Objectives:
In drinking water control testing, this test is part of the Standard Test for
Coliforms and is one of the two tests of choice for reporting purposes. The
completed test must be applied in the examination of drinking water to at least
ten (10) percent of all positive samples in each quarter, and, when a specific
sample is being tested, applied to all positive confirmed tubes of that sample.
Repeat samples from the same location that consistently show three or more
positive 10 ml portions should be tested by this procedure.
2. Brief Description of Analysis:
All positive tubes of BGLBB (brilliant green lactose bile broth) from the
confirmed test of the Standard Coliform Test are individually and aseptically
transferred onto EMB Agar by the streaking technique. After incubation for
24 + 2 hours at 35 + 0.5°C, one or more typical isolated colonies are selected
(dark-centered with or without sheen formation) or two or more atypical (if
only these are present) isolated colonies (opaque; un-nucleated; mucoid; pink)
from each plate and transferred to LLSTB (lactose lauryl sulfate tryptose broth)
and a Nutrient Agar Slant (NAS). Thus, each selected pure culture is trans-
ferred to LLSTB and NAS and incubated for 24+2 hours at 35 + 0.5°C. Tubes
are inspected at this time for gas formation in the LLSTB and growth on the
NAS. A Gram Stain is prepared from the NAS at this time and the slant asep-
tically (handled with sterile technique) manipulated and preserved under
refrigeration for possible future need. A positive (gaseous) LLSTB is data
recorded and discarded while a negative (non-gaseous) tube is re-incubated
for an additional 24 hours (total of 48 + 3 hours) when it is again inspected
for gas production.
Coliforms are considered to have populated the original BGLBB tubes if pure
culture gram-negative, non-sporeforming rods, which gaseously fermented
lactose were isolated by this procedure. Any other results are considered
to be the actions of non-coliforms except in the case of lactose fermenters
which are caused by mixed culture (two or more different organisms con-
sisting of gram-positive and gram-negative forms). In this case, the retained
Nutrient Agar Slant is restreaked on EMB and the subsequent procedures repeated
to attempt to isolate the gram-negative pure culture having the coliform
characteristics mentioned. Adjustments, if any, are made to the tube codings
and the MPN re-calculated to give an MPN completed result which is now the
required reportable result.
This procedure conforms to the Standard Total Coliform MPN Tests as described
in Standard Methods for the Examination of Water and Wastewater, 14th ed. (1975),
p. 914
4-4
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
Equipment and Supply Requirements
A. Capital Equipment
1. *Autoclave, providing uniform temperatures up to and including 121°C,
equipped with an accurate thermometer, pressure gauges, saturated
steam power lines and capable of reaching required temperature within
30 minutes
2. Balance, 0.1 g sensitivity at load of 150 g
3. Air *incubator to operate at 35°C + 0.5°C
4. Oven, *hot-air sterilizing or drying, to give uniform temperatures and with
suitable thermometer to register accurately in range of 160-180°C
5. pH meter, accurate to at least 0.1 pH unit, with standard pH reference
solution(s)
6. Water distillation apparatus, (glass or block tin), or source of distilled
water suitable for bacteriological operations^
7. Microscope, compound, oil immersion lens, Abbe condenser
B. Reusable Supplies:
1. Apron or coat suitable for laboratory
2. Baskets, wire,for discarded cultures
3. Hotplate with magnetic whirl feature, if desired
4. Burner, gas, Bunsen burner type
5. Counter, colony, Quebec type, Darkfield Model with guide plate
6. Inoculation loop and needle, 3 mm diameter for loop and both of nichrome
or platinum-indium wire, 26 B&S gauge, in holders
7. Pan, to receive discarded contaminated pipets and glassware (must contain
disinfectant before use)
8. Racks, culture type*, 10x5 openings, to accept tubes at least 25 mm
in diameter
9. Sponge, for cleaning desk top
10. Tubes, culture*, 150 x 18 mm (metal caps for fermentation and screw-cap
for slants)
11. Tubes, fermentation*, 75 x 10 mm vials to be inverted in culture tubes
12. Flasks, Erlenmeyer: 500 ml; 300 ml; 250 ml
13. Graduates: 500 ml; 250 ml
4-5
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
Equipment and Supply Requirements (Continued)
C. Consumable Supplies:
1. Bibulous paper
2. Dishes, petri, 100 x 15 mm, sterile plastic, disposable
3. Disinfectant, for bench tops (Can use household bleach solution prepared
according to instructions on bottle)
4. Distilled water, suitable for bacteriological cultures (note distillation
apparatus required in capital equipment)
5. Eosin methylene blue agar, dehydrated medium (Levine modification)
6. Gram stain solutions, complete set
7. Lactose Lauryl Sulfate Tryptose Broth, dehydrated medium
8. Nutrient Agar, dehydrated medium
9. Slides, microscopic, glass, 1" x 3"
10. Foil, aluminum
11. Matches
12. Wax pencils (recommend soft as equivalent to Blaisdell 169T)
*Iterns marked are needed in quantities or require size or space allowances which
cannot be specified here, as they vary according to the daily analysis schedule.
As a rule-of-thumb, space/size or quantity requirements should be at least 3 times
the normal daily requirements. For further information on specifications for
equipment and supplies, see the Microbiology Section of the current edition of
"Standard Methods for the Examination of Water and Wastewater."
4-6
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE:
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
1. 35°C Incubator
Set-Up, Adjustment
1. Place 35°C incubator in
permanent location.
2. Install thermometer.
3. Install shallow pan of
water in bottom of incu-
bator.
4. Connect incubator to
electric power source.
5. Adjust temperature until
stabilized at required
temperature.
6. Operate bacteriological
incubator continuously.
Aa. All pre-test procedures completed before starting
other first-day procedures.
la. Out of drafts or places where it will be in
sunlight part of day.
Ib. Location convenient to laboratory bench
Ic. Convenient source of electric power.
2a. Thermometer functions at least in 30°-40°C
range and have intervals of 0.5° or less indi-
cated. Meets NBS standards.
2b. Location should be central in incubator.
2c. Mercury bulb thermometer should be fitted with
cork or rubber stopper and mounted in small
bottle filled with liquid (glycerine, water, or
mineral oil).
3a. In most incubators a pan having about 1 square
foot of area, with water about 1 inch deep,
is satisfactory.
3b. Maintains condition of saturated relative
humidity, required in bacteriological incubator.
3c. Requires daily check, with addition of water as
necessary, to keep water in pan at all times.
4a. Many incubators have pilot light to indicate
power turned on.
5a. Manufacturer's instructions for method tempera-
ture adjustment.
5b. Operation must be at 35° + 0.5°C.
5c. Allow about 1 hour between adjustments.
6a. Requires daily check with written temperature
record, with adjustment and water addition as
necessary.
V.A.I
V.A.1.1
(p. 46)
V.A.I.2
(p. 46)
V.A.I.3
(p. 46)
V.A.I.5
(p. 46)
V.A.I.6
(P. 46)
4-7
-------
tJATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
4-8
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
2. Oven, Sterilizer-
Drier, Setup
3. Autoclave Setup
1. Place oven sterilizer in
permanent location.
2. Install thermometer.
3. Connect oven sterilizer to
power source and turn on.
4. Adjust temperature to
stabilize at required
temperature.
5. Operate oven sterilizer
only when needed. Turn
off when not in use.
1. Install and operate auto-
clave according to manu-
facturer's instructions.
la. Convenient to source of electric power usually
on table or bench.
2a. Should indicate the 160° - 180°C range, be
accurate within this interval, and be marked in
1.0 degree intervals.
3a. Usually has pilot light to indicate power on.
4a. Operated as near to 170°C as possible; not lower
than 160° or higher than 180°C.
5a. Turned ON in advance of need to permit reaching
required temperature before introducing material.
5b. Oven used to sterilize or dry glassware, metal
objects.
5c. Oven sterilizer not used with culture media,
solutions, plastics, rubber objects, or with
anything containing or including these.
5d. Paper-wrapped glass pipets, graduates, flasks,
etc. may be sterilized in oven sterilizer.
la. Autoclaves extremely variable in design and
operation; also, potentially dangerous.
Ib. Used to sterilize objects made of, or including
liquids, rubber, culture media.
Ic. Glassware may be autoclave sterilized but must be
dried afterward.
Id. Most plastics not sterilized in autoclave;
plastics usually require chemical sterilizers.
le. Autoclave usually operated at 121°C for 15 min.
If. Sterilized media must be removed from autoclave
as soon as possible after autoclave is reopened.
V.A.2.1-5
(p. 47)
V.A.3.1
(p. 47)
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
Pre-Test Procedures
(Continued)
4. Water Distillation
Equipment
5. pH meter
6. Glassware
1. Install and operate in
accordance with manu-
facturer's instructions.
2. Operate as required to
maintain adequate supplies
of distilled water.
1. Have unit available and
operable.
1. Wash all glassware in hot
detergent solution.
2. Rinse at least once in hot
tap water.
3. Rinse in distilled water,
at least 6 successive
times, and
4. Dry in air or oven.
la. Must produce distilled water meeting quality
requirements for bacteriological tests.
2a. Reserve supplies kept in borosilicate glass
carboys or in plastic carboys made of material
which will not dissolve substances which will
affect growth of bacteria.
2b. Same distillation apparatus used for bacterio-
logical purposes may be used for chemical
reagents.
la. Unit for pH check on finished culture media.
la. Nontoxic detergent.
Ib. Be sure all contents and markings are washed away.
4a. No visible spots or scum; glass should be clean
and sparkling.
4b. Glassware suitable for use in bacteriological
operations.
V.A.4.1-2
(p. 47)
V.A.5.1
(p. 48)
V.A.6.1-4a
(P- 48)
V.A.6.4b
(p. 48)
4-9
-------
WATER MONITORING PROCEDURE; Completed Test for the MPN Method for Drinking Water
4-10
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
7. Preparation of
Lactose Lauryl
Sulfate Tryptose
Fermentation Broth
(LLSTB)
1. Weigh 8.9 grams of dehy-
drated Lactose Lauryl
Sulfate Tryptose Broth.
Close cover of bottle of
dehydrated medium tightly
after removal.
2. Dissolve in 250 ml dis-
tilled water.
3. Place 10.5 ml of the solu-
tion of prepared LLSTB in
each culture tube.
4. Insert one fermentation
vial into each tube of
medium, open end down.
5. Place tube cap on each
tube of culture medium.
6. Sterilize in autoclave.
7. Cool medium to room
temperature.
la. Dehydrated media takes moisture out of air; can
become caked.
Ib. Caked media unsatisfactory; should be discarded.
2a. Use a 500 ml Erlenmeyer flask.
2b. Gentle heat (no boiling) if necessary to com-
plete dissolving medium. Usually a vigorous
agitation will completely dissolve the medium.
3a.
3b.
3c.
3d.
Use 150 x 18 mm tubes.
A 25 ml pipet, automatic pipetter, or funnel,
hose and pinchcock assembly are acceptable.
Accuracy of delivery: ±0.5 ml.
Approximately 23 tubes will be necessary.
V.A.7.3
(p. 48)
4a. Tubes and vials washed as indicated previously.
4b. Use 75 x 10 mm tubes.
5a. After all tubes have been filled and have indi-
vidual vial.
6a. Within 1 hour after medium prepared.
6b. Sterilization at 121°C for 15 minutes.
6c. Medium must be removed from autoclave as soon
as possible after pressure has returned to
normal. Use "slow-vent" mode of steam removal.
7a. Medium ready for use when cool and individual
vials are completely filled with fluid. No
bubbles must be present. Wait for complete
cooling before checking for bubbles.
-------
'.-JATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
8. Preparation of
Eosin Methylene
Blue Agar (EMB
Agar)
8. Check pH of finished
medium.
9. If final pH is not satis-
factory, discard medium
and prepare new batch with
pH adjustment before
sterilization.
10. Store medium in cool dark
place.
1. Weigh 7.5 grams of dehy-
drated eosin methylene
blue agar. Close cover
of bottle of dehydrated
medium tightly after
removal.
2. Dissolve in 200 ml dis-
tilled water.
8a. Should be pH 6.7 - 6.9. It is rare that
deviations occur with this preparation.
9a. pH value ordinarily drops about 0.2 pH unit.
9b. Check for dirty glassware, acid residues in
glassware, etc.
lOa. Not in refrigerator. Usually in laboratory
cabinet in darkness.
lOb. May be stored up to 1 week if evaporation not
more than 10% in loose-fitting capped tubes.
With screw-capped tubes, medium should be held
no longer than 3 months.
la. Use only Levine's modification as this medium
has a number of modifications for differing
purposes.
Ib. Dehydrated media takes moisture out of air; can
become unacceptably caked.
2a. Use a 300 ml Erlenmeyer flask with double layer
foil cap.
2b. Heat to boiling to dissolve completely. Do not
prolong boiling.
2c. Frequent agitation is necessary to prevent burn-
ing of medium.
2d. All of the agar must be in solution. Agar will
be recognized as particulate matter along the
sides of the flask. Gently swirl flask until all
of this material is off of sides and into
medium.
V.A.8.2c
(p. 48)
4-11
-------
HATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
4-12
OPERATING PROCEDURES
STEP SEQUEf.CE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
3. Sterilize medium in
autoclave.
4. Cool medium to 50-60°C and
pour into sterile petri
dishes.
5. Allow dishes to cool to
room temperature and then
dry.
6. Check pH of one of the
plates.
3a. For 15/15 to effect complete sterilization
(15 psi for 15 minutes).
3b. Medium must be removed from autoclave as soon as
possible after pressure has returned to normal.
Use "slow-vent" mode of steam removal.
4a. Can also be poured "hot" from autoclave with
precautions, such as using asbestos glove, for
personal protection.
4b. A flocculant may form after autoclaving. Swirl
flask gently during plate (dish) filling.
4c. About 10-12 mis/plate. About 15 plates will be
required.
4d. Cover plates as they are poured. Do not place
covers on bench where they can become
contaminated.
5a. Agar will solidify and allow plate to be moved
without disturbing medium.
5b. Invert plates (turn upside down) and place in
35° incubator overnight. This will allow plates
to dry and remove excess moisture.
5c. Plates can be used when agar surface is "dry"
(does not have water droplets).
6a. Insert pH meter probes into agar medium using one
of the plates of the batch.
6b. Should read 7.0 - 7.2.
6c. Discard plate after measuring pH. Alternately,
to save medium, one could fill a small clean
receptacle, or, a 60 x 15 mm petri dish for this
check.
6d. Out of range reading denotes unacceptable pro-
cedure, equipment, or materials used (dirty
glassware, poor water supply, overheating, etc.).
Discard plates and rectify problem.
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
9. Prepare Gram-Stain
Solutions
10. Prepare Nutrient
Agar Slants (NAS)
7. Label and date batch of
plates. Store either at
room temperature when use
is made of plates within
several days or in sealed
plastic bags, at 4°C.
1. Prepare solutions
as recommended by
manufacturer.
1. Weigh 2.9 grams of dehy-
drated nutrient agar.
Close cover of bottle of
dehydrated medium tightly
after removal.
2. Dissolve in 125 ml dis-
tilled water.
3. Dispense 6-7 mis of medium
into screw-cap tubes.
4. Place screw caps loosely
on each tube which are
packed loosely in a test-
tube rack, beaker, etc.
7a. Can be kept for one month under refrigeration as
described. Plates may have to be re-dried in the
incubator overnight (inverted) after removal
from refrigerator.
la. Premixed dyes will probably only require
dilution.
Ib. If desirous to prepare dyes from scratch, con-
sult Standard Methods for procedure.
la. Dehydrated media takes moisture out of air; can
become caked.
Ib. Caked media unsatisfactory; should be discarded.
2a. Use a 250 ml Erlenmeyer flask with double layer
foil cap.
2b. Heat to boiling to completely dissolve.
2c. Frequent agitation is necessary to prevent
burning of medium.
2d. All of agar must be in solution. Agar will be
recognized as particulate matter along the sides
of the flask. Gently swirl flask until all of
this material is off of sides and into medium.
3a. Use 150 x 18 mm screw-cap tubes.
3b. A 10 ml pipet, automatic pipetter, or funnel,
hose, and pinchcock assembly are acceptable.
3c. Approximately 25 tubes will be required.
4a. Allows steam to penetrate to medium.
Std. Meth.
14:918-919
V.A.8.2C
(p. 48)
V.A.7.3
(p. 48)
4-13
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
4-14
OPERATING PROCEDURE!
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
5. Sterilize tubes in
autoclave.
6. Tighten caps and slant hot
medium.
5a. For 15/15 to effect complete sterilization (15
psi for 15 minutes). Remove medium as soon as
possible after cycle (slow vent mode) is
completed.
6a. Tight caps will prevent further loosening and
possible contamination.
6b. Necessary to slant while hot so that medium will
not solidify in upright position.
6c. "Slanting" is done to allow a large surface area
for growth of bacteria.
St*NT AREA
6d. Apparatus for tube holding while in the slanted
position can range from expensive "angle" con-
trolled supports to as simple and effective a
method as below:
SLANTED
TUBES
HOSE OD
V.A.3.1
(p. 47)
-------
HATER MONITORING PROCEDURE; Completed Test for the MPN Method for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
IMFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
11. Final Equipment anc
Supply Check
1.
Allow tubes to solidify
before removing from
slanted position and plac-
ing in test tube rack.
Date and label medium as
nutrient agar. Store in
refrigerator.
Check to be sure that all
equipment and supplies,
solutions, and prepared
media are ready before
starting sample
examination.
7a. Solidified tubes can be picked up and will re-
tain "slanted" position of medium. Tubes will
start to harden below 40°C and take on an
"opaque" form as they harden.
8a. Temperature 1-4.4°C.
8b. Can be stored for up to 3 months (if kept in dark
and evaporation is not excessive (less than .25
ml).
la. Check general list of equipment and supplies.
1b. Each test requires:
1 - 5 EMB agar plates
1 - 10 Nutrient agar slants
1-10 LLSTB tubes
Bacteriological loop
Bacteriological needle
1-10 Microbiological slides
Gram stain reagents, set
Since, as shown, the numbers of items can vary
(depending upon the number of confirmed test
positives and subsequent EMB colony forms) this
WMP (Water Monitoring Procedure) will specific-
ally pick a hypothetical situation which will
give the reader a cross-section of conditions
which could occur.
B. Initial Procedures
1. Equipment
Maintenance
1. Check, record, and adjust
incubator temperature.
2. Add water to pan in
incubator as necessary.
la. See A.I.1.1-6.
Ib. Should be in operating condition since MPN test's
earlier phases are in progress (Presumptive and
Confirmed tests).
4-15
-------
MATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
4-16
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Initial Procedures
(Continued)
2. Data Sheet
Inspection
1. Locate data sheet and
verify that the required
sample is being processed.
2. Use active sheet of the
48 hour MPN test (partial
completion of the con-
firmed test) with 48 hour
presumptive tubes and
24 hour confirmed tubes
"saved."
la. A "new" data sheet does not have to be initiated
since the sample is already being processed.
2a. A typical sheet may look like this: (Test
Portion)
VII.B.2
(Suggested
Data Sheet)
(p. 50)
24 hour Column
entry (tubes
processed pre-
viously)
48 hour Column
entry (positive
tube transferred)
\
Confirmed test: positive
tube saved for completed
test. Negative tube is
re-incubated.
Observ<.
Amount '
Sample
ml
10
Preservative
V LLSTB/
\ hr
>
—
—
+
—
48 nr
—
H-
—
*
ConflnMO
sun
Z4Tir
+
_
48 nr
. '
Conoleted
LLSTB
EMB
Z4 48
(Ji
i
2b. Note that 10 ml volumes were used for each of
the 5 tubes for the presumptive test.
2c. Tubes "saved" will be used to initiate the
completed test.
-------
MATER MONITORING PROCEDURE; Completed Test for the MPN Method for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Initial Procedures
(Continued)
3. Lab Bench
Disinfection
4. Continue Standard
MPN Test Procedure
1. Disinfect laboratory
bench; wipe dry.
1. Transfer positive LLSTB
tubes of the presumptive
stage.
la. Sponge and disinfectant; paper toweling.
la. From data sheet (B.2.2.2a), note that one tube
will be transferred from the presumptive stage
to the confirmed stage:
Observations
Amount
Sample
ml
10
Preservative
LLSTB
14 hr
-f-
—
—
•*•
_
1
48 hr
—
4-
/
f-
/
Confirmed
BGLBB
2* hr
4-
__
48 hr
COltD
LI
EMBI
!
i
Transfer this to BGLBB
Ib. Progress of this transfer will be monitored for
possible inclusion to the completed test.
Std. Meth.
14:917
4-17
-------
HATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
4-18
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Initial Procedures
(Continued)
Completed Test Start:
First Day Procedure
5. Select BGLBB
Positives from
Confirmed Test
6. Prepare EMB Agar
Plates
1. Select "positives" from
confirmed "24" hour tubes
for processing.
1. Shake positive BGLBB
tube vigorously.
2. Sterilize a bacteriologi-
cal loop.
la. One positive BGLBB tube is to be processed:
Disregard these negatives
in coliforms absent (see
schematic).
This is the tube to be
processed to EMB.
Negative tube to be re-
incubated as per Con-
firmed test require-
ments (Std. Meth.
14:920).
la. Allows organisms to be suspended in the broth.
2a. Heat in burner to redness all the way to handle:
NOTE
HEAT FULLY ENTIRE
LENGTH OF LOOP
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Initial Procedures
(Continued)
3. Allow loop to cool
(5-10 seconds).
4. Remove cap from BGLBB
tube.
5. Insert loop Into broth to
obtain film transfer.
Cover tube and discard.
6. Streak transfer inocula-
tion from loop to corner
of EMB agar plate.
3a. Avoids possible spattering when loop is inserted
into tube.
5a. "Film" within loop represents transfer volume.
TRANSFER VOLUME
-A
FILM OF
INOCULUM
MUST SHOW
'FILM' WITHIN LOOP
6a. Agar surface must be dry for satisfactory
results.
6b. Streak the inoculation lightly back and forth
over half the agar surface, as in®, avoiding
scratching or breaking the agar surface.
(Over for pictorial representation)
V.B.6.5
(p. 48)
VII.B.6.6
(p. 50)
4-19
-------
HATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
4-20
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Initial Procedures
(Continued)
STREAKED AREA
LOOP
7. Sterilize (flame) loop and
air-cool as before.
8. Streak another segment of
plate to carry portion of
inoculation into another
area of medium.
6c. Use asceptic (sterile) technique to prevent
contamination of medium. Close cover of petri
dish when not streaking.
8a. Turn petri dish about one-quarter-turn in the
holding hand (allows easier streaking).
8b. Streak the loop's tip lightly back and forth over
one-half the agar surface working from area©
into one-half the unstreaked area of the agar.
(Over for pictorial representation)
-------
HATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Initial Procedures
(Continued)
9. Sterilize loop and air-
cool .
10. Streak the remaining un-
streaked area of medium.
8c. Technique allows "dilution" of original heavy
inoculum to occur into an area where less
growth will now result.
lOa. Turn the petri dish one-quarter-turn in the
holding hand.
lOb. Streak the tip lightly back and forth over one-
half the agar surface, working from area ©into
area
lOc. Do not allow any of streaks to touch original
streaking area (separate ©from (P).
lOd. Further "dilution" will now occur to allow "pure"
cultures to grow into colonies.
4-21
-------
U'ATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
4-22
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Initial Procedures
(Continued)
11. Flame sterilize the loop
and set it aside.
12. Invert dish (turn up-side-
down) and identify.
lOe. Close the culture container, and, until the
colonies (bacterial growth forms) are picked,
keep the top and bottom as a unit without
allowing separation to occur.
12a. Use grease pencil (wax pencil) to label bottom of
dish.
12b. A suggested labeling could be:
r312V«-
1
•Lab assigned no. for this sample
•Indicates 1st tube in row of 5
tubes
13. Incubate EMB agar plate.
13a. At 35° + 0.5°C for 24 hours.
13b. Keep in inverted position (avoids water droplets,
if formed, from falling on the medium surface
and ruining the plate).
C. Second-Day Procedures
1. Equipment
Maintenance
2. Lab Bench
Disinfection
3. Data Sheet
Recordings
1. Check, record, and adjust
incubator temperature.
1. Disinfect laboratory
bench, wipe dry.
1. Locate required data
sheet.
2. Remove cultures from in-
cubator and assemble with
data sheet.
la. Sponge and disinfectant; paper toweling.
la. Sample "312" in our example.
2a. 1 EMB plate (24 hours old)
1 BGLBB tube (24 hours old)
1 BGLBB tube (48 + 3 hours old)
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Second-Day Procedures
(Continued)
3. Read BGLBB tubes for gas
and record results.
4. Discard any BGLBB tube
which is negative in
48 hours.
5. Save any 24 hour BGLBB
tube which is positive or
negative.
3a. Any amount of gas is considered positive.
tubes gently before reading.
3b. Assume the following results:
Negative tube
re-incubate
Shake
Ampler
Observations
Amount
Sample
ml
10
Preservative
LLSTB
24 hr
+
_
—
-h
—
i
48 hr
—
-H
*
—
Confirmed
BGLBBT
24 hr/48 hr
•*• /
/I
-^ J
_,**
S^
^
_
w
Conoleted
LLSTB '
EMBI 24 43
GS
;
1
Negative tube-
(discard: coliforms
absent)
(See sche-
matic
diagram)
(P- 3)
5a. None are positive...this possibility would have
made it necessary to streak an EMB agar plate.
5b. There is a negative...reincubate this for an
additional 24 hours.
4-23
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
4-24
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Second-Day Procedures
(Continued)
4. EMB Agar Plate
Inspection
1. Remove cover from EMB
agar plate and inspect
growth.
la. Usual plate growth (colonies) will be as
indicated:
AREA 1
(HEAVY INOCULUM)
AREA 3
(ISOLATED COLONIES)
AREA 2
(MODERATE GROWTH)
APPEARANCE OF STREAK - PLATE
AFTER INCUBATION INTERVAL
OR OCCASIONALLY,
AREA 1
(HEAVY INOCULUM)
AREA 3
(LACK OF COLONY
ISOLATION)
AREA 2
(HEAVY GROWTH)
APPEARANCE OF STREAK - PLATE
AFTER INCUBATION INTERVAL
-------
MATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Second-Day Procedures
(Continued)
Ib. In the case of isolated colonies, one could pro-
ceed to the next step of the completed test
(C.5).
Ic. In the case of a lack of isolated colonies, one
must proceed to re-streak another plate to
attempt isolation of a colony. As follows:
Reisolation Procedure
A. Flame sterilize a loop and air-cool.
B. Immerse the loop into an area which shows a
representative growth mass. Occassionally,
the loop must be touched to two or three
masses to obtain this material.
C. Close cover and discard EMB plate.
D. Streak plate of fresh, dry EMB agar using the
same techniques as previously outlined, except
that it would be wise to allow more streaking
sequences with an increased number of loop
flamings. This would more likely ensure
better isolation:
(p. 45)
RESTREAK(5
FLAME
RESTREAK (7)
FLAME
RESTREAK (3
FLAME
E. Incubate as previously outlined.
ORIGINAL.-
STREAK (7)
FLAME
RESTREAK
FLAME
RESTREAK
4-25
-------
MATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
4-26
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Second-Day Procedures
(Continued)
Id. Recommended technique is to pick pure colony and
with a single transference, inoculate both the
LLSTB and the NAS in a single motion.
le.
in
INOCULATION
2nd
INOCULATION
$„—] U_
OI MORE '
EMB ACAR
LLSTB
NAS... Flame top of tube for about two seconds
prior to entering with sterile (flamed) needle.
Gently swab surface of agar medium. Replace
screw-cap which is held in hand without con-
taminating during procedure.
EMB... Discard plate after inoculation completed
LLSTB... Transfer inoculation directly to LLSTB
tube. Return to colony is not necessary. Flam-
ing of tube top not necessary. Shake needle in
broth for transfer.
Label tubes for identification. Such a labeling
could be as follows:
312
1
TYP
A
3V
1
TYP
A
IQJ
LAB NUMBER
1st TUBE OF 5
TYPICAL COLONY
'A' CULTURE (TO KEEP
TUBES PAIRED)
-------
MATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Second-Day Procedures
(Continued)
If. Incubate tubes at 35° + 0.5°C.
Ig. Indicate the type of EMB plate colonies observed:
Indicates typical colonies
ODSei .u
Amount
Sample
ml
10
Preservative
LLSTB
24 hr
+•
—
_
•h
—
48 hr
—
4-
—
Confirmed *
BGLBB
24 hr
•*-
—
—
48 hr
—
Connie ted
L LLSTB
KMB
TfP
24 48
lib
Ih. Indicate the numbers of pure cultures picked:
Indicates first positive
culture "A"
Observe V
•v
Amount
Sample
ml
10
Preservative
LLSTB
24 hr
+
—
—
+
_
48 hr
—
-H
—
Confirmed
BGLBB
24 hr
-h
—
—
48 hr
—
1
ConpletedV
LLSTB X
EMB
TYP
24 43
GS1
i ^^f**^
L
IK
IB
1C
If more than one pure culture was picked
from the EMB plate derived from the first
tube, indicate here as B, C, etc.
4-27
-------
MATER MONITORING PROCEDURE; Completed Test for the MPN Method for Drinking Water
4-28
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Second-Day Procedures
(Continued)
5. EMB Agar Plate
Colony Transfer
1. Transfer pure cultures to
LLSTB and NAS.
REMOVED
COVER
DISH WITH
EMB MEDIUM
AND COLONIES
II. Atypical Colonies (Usually a Non-Coliform)
These colonies may be opaque, unnucleated,
mucoid, or pink after the prescribed incu-
bation period.
la. Use flamed and air-cooled needle for fishing
(picking).
Ib. Use of colony counter as a magnification aid is
recommended
LENS, MAGNIFIER
ADJUSTING ROD
PETRI DISH
W/0 COVER
DIRECTION OF
LIGHT SOURCE
Ic. Pick one or more typical colonies, or, two or
more atypical colonies and transfer each of them
into their own set of tubes (LLSTB and NAS).
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Second-Day Procedures
(Continued)
Id. Pure growths (colonies) can be regarded as fall-
ing into two groupings:
I. Typical Colonies (Characteristic of Coliforms)
Colonies with dark centers commonly termed
"nucleated" or "fisheye" when viewed from the
bottom of the plate:
©'©•
!r Is etc.
These colonies may or may not have a metallic-
like sheen characteristic on the surface of
the colony.
4-29
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
4-30
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Third-Day Procedures
1. Equipment
Maintenance
2. Lab Bench
Description
3. Data Sheet
Recordings
1. Check, record, and adjust.
incubator temperature.
1. Disinfect laboratory bench.
1. Locate required data
sheet.
2. Remove cultures from in-
cubator and assemble with
data sheet.
3. Read BGLBB tube for gas
and record results.
Tube labeled:
312
3
la. Sponge and disinfectant; paper toweling.
la. Sample "312" is our example.
2a.
1 NAS
1 LLSTB
1 BGLBB
} Sl
I co
[ at
J pr
Since we transferred one colony...
could have been more cultures (i.e.,
least 2 pairs if atypicals were
present only). 24Jiour incubation.
48+3 hours of incubation
See C.3.3b
3a. Any amount of gas is considered positive.
the tube gently before reading.
3b. Assume the following result:
Positive tube (within
48+3 hours)
Shake
Observations
Amount
Sample
ml
10
Preservative
LLSTB
24 nr
t
—
—
-h
—
1
48 nr
—
-*•
—
Confinfed
BGLBBl
24 hr
-T-
—
—
18 hr
\
\
-T-
—
Conoleted
LLSTB
EMB
TIP
24 48
GS
3c. Retain the tube for further processing
)
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
IMFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Third-Day Procedures
(Continued)
4. Check NAS and LLSTB for
growth and gas, respective
ly, and record results.
4. Gram Stain
Preparation
1. Assemble gram staining
materials and culture.
4a. Growth on the NAS is readily visible as an
opaque mass which was not present on the sterile
medium. No recordings are necessary for this
growth—it will be used for a gram stain.
4b. Any amount of gas production in the LLSTB is
considered positive. Shake the tube gently
before reading.
4c. Assume the following result:
*Negative tube (re-incubate for
an additional 24 hours)
Observations
Amount
Sample
ml
10
Preservative
LLSTB
24 hr
-*•
_
—
+
—
48 hr
—
•*•
—
Confirmed
BGLB8
24 hr
+
—
46 hr
*
_
Ctno le ted
\LSTB
EHB
HP
«4 48
'
GS
1
r i
1
IA
i
*Had this tube been positive, the completed test
may have been terminated this third day instead
of tomorrow (fourth day).
la. 1 Bacteriological glass slide
1 Dropper bottle containing ammonium oxalate-
crystal violet dye
1 Dropper bottle containing Lugols solution
(grams modification)
1 Dropper bottle containing safranin dye
1 Dropper bottle containing acetone-alcohol
1 Squeeze bottle containing tap water
Bibulous paper
NAS (culture 1 A) 24 hour culture
4-31
Std. Meth.
14:918-919
III.D.4
(p. 45)
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
4-32
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Third-Day Procedures
(Continued)
2. Prepare glass slide.
3. Obtain culture (NAS) sam-
ple and place on slide.
4. Prepare culture for
staining procedure.
5. Stain culture with re-
agents on the side of the
slide with the culture.
Ib. Twenty-four hour culture is recommended for pre-
paration. Older cultures can give erroneous
results.
2a.
2b.
2c.
Must be clean.
Helpful to clean with alcohol, distilled water,
and lens tissue.
Place a drop of distilled water about 1 inch
from end of slide.
3a. Culture must be obtained with a flamed, air-
cooled needle. NAS then is stored in refriger-
ator for possible need.
3b. Screw cap tube handled asceptically:
* Flamed top of tube
* Sterile needle
* Cap carefully handled and returned to tube
promptly
3c. Only minute amount of culture necessary. Large
amounts can cause staining problems.
3d. Place culture from needle into water droplet
and mix well while extending the droplet size to
about a 1" x 1/2" area.
4a. Allow smear to air-dry completely and then heat
fix by passing slide through the gas flame brief-
ly back-and-forth for a heat exposure of about
two seconds.
5a. Flood the slide with ammonium oxalate-crystal
violet dye.
5b. Allow to cover culture area for 1 minute.
5c. Wash slide gently with tap water.
5d. Apply Lugols-iodine solution to culture area.
5e. Allow to cover culture area for 1 minute.
Std. Meth.
14:918-919
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Third-Day Procedures
(Continued)
6. Examine slide
microscopically.
5f. Wash slide gently with tap water.
5g. Apply acetone-alcohol solution to culture area.
Hold slide and allow solution to flow across
smear until stain is no longer being removed:
DROPPER
BOTTLE
5h.
5k.
51.
6a.
6b.
Do not prolong this alcohol contact period (dis-
colorization step) a? the results may be
erroneous. Some authorities suggest 10-15
seconds maximum.
Wash slide gently with tap water.
Apply Safranin solution (counterstain) for 15
seconds and then wash gently with tap water.
Blot slide gently with bibulous paper using care
not to rub culture area during procedure.
Identify slide to conform to proper culture being
examined. Use of a slide label is convenient
(label 312, 1 A as per our example).
If desired, slide can be retained for later
examination. If the lactose (LLSTB) broth re-
mains negative for the culture (48 + 3 hours),
the slide need not be examined as the culture is
not a coliform.
Become acquainted with microscope from manu-
facturer's literature or individual acquainted
with same.
4-33
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
4-34
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Third-Day Procedures
(Continued)
6c. If examination is desired, place the slide, cul-
ture side up, on the microscope stage of a micro-
scope equipped for oil immersion examination.
6d. Place a drop of a suitable bacteriological
immersion oil on the area to be viewed (culture
smear).
6e. The proper objective is positioned for oil-
immersion (usually labeled oil and having X 97
or X 100 magnification).
See V.D.4.6
for micro-
scope
nomenclature
V.D.4.6.6d
(p. 45)
OIL IMMERSION
OBJECTIVE
TYPICAL OIL
OBJECTIVE
THE OBJECTIVES SCREW INTO
THE TURRET
(TURN AND LOCK FOR SELECTION)
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Third-Day Procedures
(Continued)
5. Gram-Stain
Examination and
Recording
1. Examine stained prepara-
tion for type/types of
bacteria.
2. Record gram-stain data.
6f. Turn Oj| lighting system. Light will be directed
to reflect off the plane side of a mirror through
a condenser assembly and up through the stage
(mirror assembly may be external or internal).
6g. With the illumination system correctly set up,
rack down (or the stage UP on some models) until
the oil-immersion lens touches and disperses the
oil.
6h. Rack down gently with the coarse control (lens
and slide will move towards each other) while
looking down the microscope (into the eyepiece)
until the image begins to come into focus.
6i. Obtain final sharp image using the fine-focus
control.
la. Gram-negative bacteria (typical of coliforms)
will be red or pink colorations.
Ib. Gram-positive bacteria (NOT coliforms) will be
blue-to-purple in color.
Ic. Mixed cultures will show mixtures of the above
and will immediately call for the re-isolation
of pure culture on another EMB agar plate from
the saved nutrient agar slant. Discard the
LLSTB tube as it has no interpretative value
being a mixed culture. Repeat procedures as
before.
Id. If too large of a sample was transferred to the
slide for staining, some areas of matted,
numerous bacterial cells could produce areas
where dyes could not either penetrate or be
washed away. Recommend another smear to be made.
2a. Assume that, for our example, that only gram-
negative organisms were observed during micro-
scopic examination (culture 312 1 A).
V.D.4.6.6g
(p. 45)
4-35
-------
HATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
4-36
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Third-Day Procedures
(Continued)
2b. Enter observation in proper place on data sheet:
Indicates typical culture
for coliforms
2c.
2d.
_„.,,:I vdClOflS
Amount
Sample
ml
10
Preservative
LLSTB
24 hr
t
_
—
+
—
43 hr
—
•t-
—
Confirmed
BGLBB
24 hr
•i-
—
—
43 hr
-»-
_
ConpleXea
LLSTBX
EMB
TYP
24 48W
_
i
TIP
IA
1
Other entries could have been MXD (mixed culture)
or GQ (gram positive).
In the case of a Gfl entry, the culture is not a
coliform and a -(negative) for the completed
test:
Negative tube for
completed test
V
Observations
Amount
Sample
ml
10
Preservative
LLSTB
24 hr
-f-
L_—
-r-
48 hr
—
-*•
—
Confirmed
BGLBB
24 hr
+
—
—
48 hr
-y-
^.
Conoleted V
LLSTB N
FMB
TTP
24 48 GS
_
t
1 |/
/
/
frffi
^-
This tube can be discarded
since it has no further
relevance.
-------
WAFER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Third-Day Procedures
(Continued)
2e. In the case of a MXD (mixed culture) entry, the
following interpretation can be made for the
culture.
* Since it is a mixed culture (gram 9 and Gram 0
organisms growing together), any results for
the LLSTB tube could be in error.
* The NAS (in refrigerator) for the culture is
also mixed growth.
* Reisolation of a pure culture must be made
(NAS + EMB) for valid results.
Discard
this
tube
(...servo I...
Indicates
mixed
growth
New entries for'
this culture
will be made
here as they
are observed
Indicates
fresh EMB
plate was made
from the NAS
4-37
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
4-38
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Third-Day Procedures
(Continued)
6. Process BGLBB
Positive Tube
E. Fourth-Day Procedures
1. Equipment
Maintenance
2. Lab Bench Disin-
fection
3. Data Sheet
Recordings
1. Streak EMB agar plate and
incubate.
1. Check, record, and adjust
incubator.
1. Disinfect laboratory
bench.
1. Locate required data
sheet.
2. Remove cultures from incu-
bator and assemble with
data sheet.
3. Read and record LLSTB
tube.
la. BGLBB tube will be labeled 312.
3
Ib. Method previously described (B. First Day
Procedures).
Ic. Label EMB agar plate.
la. Sponge and disinfectant; paper toweling.
la. Sample "312" is our example.
2a. 1 EMB plate (24 hours old).
1 LLSTB tube (48 hours old).
3a. 48 +_ 3 hours incubation
Tube labeled
312
1A
3c. Assume, for our example, that the tube is
"positive" (contains any amount of gas in
inner vial) and its recording will be:
See data
Sheet
D.3.3.3b &
3c
See D.5.2
-------
HATER K1NITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
E. Fourth-Day Procedures
(Continued)
4. Inspect and process EMB
agar plate.
Positive
LLSTB tube
+ indicates that
coliforms are
positive for
completed test
Observations
Amount
Sample
ml
10
Preservative
LLSTB
24 hr
+•
_
i —
-t-
—
48 hr
—
•f
—
Confirmed
BGLBB
24 hr
-f
—
—
48 hr
-t-
_
CorTBleted
LL\TB
E.MB
ryp
24V48
-V
GS
PTjfP
;
\ :
IR+- i
!
1
Interpretation for First Row
Col i forms present since:
Gram negative non-spore forming rods which
fermented lactose have been demonstrated.
4a. Twenty-four hours incubation for plate labeled:
4b. Assume that the plate shows all colonies which
are Atp (Atypical) and, therefore, two cultures
must be processed:
(Previously
described
in Section
C)
4-39
-------
l-JATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
4-40
OPERATING PROCEDURES
E. Fourth-Day Procedures
(Continued)
F. Fifth-Day Procedures
1. Completed Test
Procedure
Fermentation
STEP SEQUENCE
1. Complete test procedures.
INFORMATION/OPERATING GOALS/SPECIFICATIONS
All colonies
atypical
V-.... .UI.IUII9 r f
JTr.ount
"ml
10
—
P.eser-iative
ILST£
"24 nrl -8 "hr
-t-
—
—
4-
—
—
^-
—
Confir
331!
_
__
........
/
.red
B
-3 nr
-V-
—
C on/1 e ted
yfSTB
r/»f-~+
-t
GS~
TJTf
t
i
IR
3
38
•t-
"A" and *B" indicate
that 2 colonies have
been picked and
processed
la. T
r
o sav
ecord
Ni
i
JCiio
San
n
10
Botl
e repetitive step procedures, th
ings are shown below:
3te: 2 of 5 positive Note: 1
i confirmed test tive in c
, \ test
unt ric;er.itT?^~I Confirmed * Conoleted
pie _ ILSTB > BSLBB LLSTB
1 24 hr <3 hr \24 hrTfS hr" "Fat ~i»' aa
+
—
—
4-
_
—
-*-
—
i non-col iforn
%
—
—
4-
_
^-*
TYF
flTP
|
-
~ *t"
_ _
i
is since Final re
e f
of
oim
TYf
ryf
"cor
inal
5 posi
)leted
IA4-
3
•din
^M
^
gs
-
TRAINING
GUIDE NOTES
(Procedures
as previ-
ously
described)
cultures did not ferment
lactose
here. Note: would have
taken 2 more days to
complete (6 days)
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
F. Fifth Day Procedures
(Continued)
Ib.
Ic.
Had one or both of the cultures checked out as
coliforms, the third row would have been + and,
therefore, resulted in 2 of 5 tubes positive
instead of 1 of 5 in the completed test.
Record essential data on data sheet.
Name/names of
Analyst/s
L
Results Reported:
Total colif^ra "?:;/! 00 ml
Completed"£est results
G. Interpretation of Test
Results
1. Determine number of posi-
tives for the completed
test.
la. Results of confirmed test are not used since
further and more conclusive testing has been
done.
Ib. Our example (F.1.1) shows 1 of 5 positive.
II.G.1.1
(p. 44)
4-41
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
4-42
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
G. Interpretaiton of Test
Results (Continued)
2. Look up and note the MPN
index from the MPN table.
3. Record the calculated
total coliforms per 100 ml
on the data sheet.
2a. For the given example the location of the index
is shown by the arrow.
MPN Index for Various Combinations of
Positive and Negative Results when
Five 10-ml Portions are Used
3a
Std. Meth.
14: 923
No. of Tubes
Giving Positive
Reactions out of
5 of 10 ml Each
0
1
2
3
4
5
MPN
Index/
100 ml
<2.2
2.2-
5.1
9.2
16.
Value is direct index if, as our example, 10 ml
portions were used. If 100 ml portions were
used, the number is 1/10 of the index (or .2
instead of 2.2 for our example).
3b. Record under Completed Test:
1 (
1 : ! 1
I
i |
Results Reported
Total coliform '1PN/100 ml
Confirmed
<
Cor.pl eted .
*ff
*'* \
7
H. Reporting of Results
1. Report results as pre-
scribed under regulatory
requirements.
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method for Drinking Water
TRAINING GUIDE
SECTION TOPIC
I Introduction
II* Educational Concepts - Mathematics
III* Educational Concepts - Science
IV Educational Concepts - Communications
V* Field and Laboratory Equipment
VI Field and Laboratory Reagents
VII* Field and Laboratory Analysis
VIII Safety
IX Records and Reports
*Training Guide materials are presented here under the headings marked*.
These standardized headings are used through this series of procedures.
4-43
-------
MATER MONITORING PROCEDURES: Completed Test for the MPN Method for Drinking Water
EDUCATIONAL CONCEPTS - MATHEMATICS
Section II
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
G.I.I
4-44
For purely qualitative aspects of testing for in-
dicator organisms, it is convenient to consider the
tests applied to one sample portion, inoculated into
a tube of culture medium, and the follow-up exami-
nations and tests on results of the original
inoculation. Results of testing procedures are
definite: positive (presence of the organism -
group demonstrated) or negative (presence of the
organism - group not demonstrated). The combina-
tion of results is used in an application of
probability mathematics to secure a single MPN value
for the sample. The MPN value for a given sample is
obtained through the use of MPN tables. Standard
practice in drinking water tests is to plant 5 tubes
each containing 10 ml of sample (some organizations
utilize 100 ml portions into each of 5 bottles con-
taining increased strength medium).
-------
WATER MONITORING PROCEDURES: Completed Test for the MPN Method for Drinking Water
EDUCATIONAL CONCEPTS - SCIENCE
Section III
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
C.4.1b
D.4
A colony is defined a discrete growth occurring at
least 0.5 cm (approximately .2 inch) from any
other growths. Such growths represent a large
number of developmental successions from an origin-
al viable cell and therefore can be considered a
"pure" culture. All organisms from pure cultures
will exhibit the same characteristics when sub-
jected to standard bacteriological testing.
A gram staining procedure, in general, separates
bacteria into two categories, gram positive (blue
coloration) or gram negative (red coloration). Its
usefullness to the coliform testing procedure is
due to the fact that part of the coliform definition
indicates that "gram negative, non-spore forming
rods" are necessary, and, in addition, no gram
positive organism must be present since some of
these organisms can act synergistically (in con-
junction with other non-coliforms) to produce a
false positive result (gas production in lactose)
which neither could manage independently.
It is desirable to use known pure cultures of both
a gram positive (staphylococcus, bacillus, etc.)
and a gram negative (proteus, enterobacter, etc.) as
controls for the staining procedure.
4-45
-------
WATER MONITORING PROCEDURES: Completed Test for the MPN Method for Drinking Water
FIELD AND LABORATORY EQUIPMENT
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.I
A.1.1
A.1.2
A.1.3
A.1.5
A.1.6
Incubator must be of sufficient size for daily
work load without causing crowding of tubes to be
incubated. Considerations for choice of incubator
type must relate to reliability of operation and
not to cost or attractiveness of equipment.
Incubator should be kept out of drafts or direct
sunlight in order to prevent temperature inside
the incubator from changing outside the tempera-
ture range specified (35° + 0.5°C).
Power supply should be selected so that there will
not be too many pieces of equipment on the same
circuit. Otherwise, circuits will be blown
repeatedly.
Mercury bulb thermometer usually used in most incu-
bators. Recording thermometer is acceptable, but,
it should be calibrated against a mercury bulb
thermometer which has been certified by National
Bureau of Standards. The NBS certified thermometer
always should be used with its certificate and
correction chart.
Saturated relative humidity is required in order to
make the incubation more efficient (heat is trans-
ferred to cultures faster than in a dry incubator).
Furthermore, culture medium may evaporate too fast
in a dry incubator.
Allow enough time after each readjustment to permit
the incubator to stabilize before making a new
adjustment. At least one hour is suggested.
Incubator temperature can be held to much closer
adjustment if operated continuously. Temperature
records should be kept in some form of permanent
record. A temperature record book is suggested with
daily recording of values. If a recording thermom-
eter is used, the charts may be kept as permanent
record; if so, be sure that the charts are properly
labeled to identify the incubator and the period
covered.
Uniform temperature (35°C + 0.5°C) is to be main-
tained on shelves in use.
Standard Methods for the
Examination of Water and
Wastewater, 14th ed. (1975
APHA, WPCF, AWWA, p. 880
(Hereafter referred to as:
Std. Meth. 14: (page no.)
4-46
-------
MATER MONITORING PROCEDURES: Completed Test for the MPN Method for Drinking Water
FIELD AND LABORATORY EQUIPMENT
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.2.1-5
A.3.1
A.4.1-2
Since electric sterilizer will be operated inter-
mittently, care should be taken that it is on a
circuit which will not be overloaded when it is
turned on.
A time and temperature record is maintained for
each sterilization cycle. Temperature recordings
can be retained for records.
Autoclaves differ greatly in design and in method
of operation. Some are almost like home-style
pressure cookers; others are almost fully auto-
matic. This is a subject which requires separate
instruction; and should be related to the exact
make and model of equipment you will use in your
own laboratory.
Vertical autoclaves and household pressure cookers
may be used in emergency service if equipped with
pressure gages and thermometers with bulbs posi-
tioned 1 inch above the water level. However, they
are not to be considered the equivalent of the gen-
eral purpose steam sterilizer recommended for
permanent laboratory facilities. Their small size
is inadequate for large-volume work loads, and
they can be difficult to regulate.
The following requirements must be met regarding
autoclaves or sterilizing units:
a. Reaches sterilization temperature (121°C),
maintains 121°C during sterilization cycle, and
requires no more than 45 minutes for a complete
cycle.
b. Pressure and temperature gages on exhaust side
and an operating safety valve.
c. No air bubbles produced in fermentation vials
during depressurization.
d. Record maintained on time and temperature for
each sterilization cycle.
Distilled water in a bacteriological laboratory
must not contain substances which will prevent any
bacteria from growing in culture medium in which
the distilled water is used or will be highly nu-
tritive. There are procedures for testing quality
of distilled water; but these should be undertaken
only by professional bacteriologists or in labora-
tories where this is done regularly. Use only glass
stills or block tin lined stills.
Std. Meth. 14:881
Std. Meth. 14:881
Std. Meth. 14:645-649
14:888-891
4-47
-------
WATER MONITORING PROCEDURES: Completed Test for the MPN Method for Drinking Water
FIELD AND LABORATORY EQUIPMENT
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.5.1
A.6.1-4a
A.6.1-4b
A.7.3
A.8.2C
B.6.5
pH Meter: See cited reference.
Glassware: See cited reference on pipets and
graduated cylinders, media utensils, bottles.
Glassware can be checked for bacteriostatic or
inhibitory residues by a bacteriological test
procedure which, like the distilled water suit-
ability test, should be undertaken only by pro-
fessional bacteriologists or in laboratories where
this test is done on a regular basis.
Std. Meth. 14:882
Std. Meth. 14:882-885
FUNNEl
FUNNEL. HOSE, AND
PINCHCOCK ASSEMBLY
PINCHCOCK
OLASS TUBE
NOTE: UNIT NEED NOT BE
STERILE FOR MEDIUM
DELIVERY ONLY.
Some workers prefer to utilize a magnetic whirl bar
and hot plate arrangement. This is acceptable and
will require no agitation until the medium is NEAR
BOILING at which time the whirling action should be
terminated and the medium gently swirled by hand and
the flask monitored for boiling.
Alternately, it is authorized to use an "incoulation
stick" for transfers and plate streaking. A pre-
cisely sized and sterilized stick is intended for a
one-time use and, if used, eliminates the need for
a burner during the transfer procedure. Of course,
several will have to be used during the streaking
process since a "sterile" one is required during
the streaking carryover to sterile surfaces.
Also available are resterilized loops used once,
resterilized, and available for future transfers.
Std. Meth.
Std. Meth.
14:917
14:883-884
4-48
-------
WATER MONITORING PROCEDURES: Completed Test for the MPN Method for Drinking Water
FIELD AND LABORATORY EQUIPMENT
Section v
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
D.4.6
D.4.6.6d
D.4.6.6g
Some manufacturers specify that the upper most lens
of the condenser assembly also be coated with a drop
of oil prior to placing the slide on the stage. In
effect, this would be "sandwiching" the slide betweer
two oil interfaces through which the light must pass.
It is extremely important to properly set up the
llumination system for proper results.
Procedures vary according to the type of illumination
provided, the type of diaphragm used, and the con-
trols provided by the particular microscope. Final
results would give, if accomplished correctly:
correct lighting from the light source; centrally
placed optimal lighting; and a sharply focussed
image.
4-49
-------
MATER MONITORING PROCEDURES: Completed Test for the MPN Method for Drinking Water
FIELD AND LABORATORY EQUIPMENT
Section VII
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
B.2
B.6.6
There is no such thing as a "standard data sheet
for bacteriological tests. A simplified sheet is
shown below:
Conform Test
Multiple Dilution Tube (MPN)
Sample Type Lab No
Station
Method
Description
Collection Date
AFT
Received PM
Sampler
Amount
Sample
ml
Time
AM
PH.
Examined
Temp
AM
PM
Observations
Preservative
LLSTB
24 hr
48 hr
Confirmed
BGLBB
24 hr
48 hr
Results Reported
Total coll form MPN/100
Completed
LLSTB
EMB
ml
24 48
Confirmed
ts
Wl
X
1
Completed
4-50
Note: This data sheet could be used only to test
to the confirmed stage and not proceed to the
completed stage so that it can serve for a dual
purpose.
There is no standardized way to accomplish a streak
plate in order to isolate pure cultures. Some
workers prefer to carry the streaks around the plate
several more times with its attendant loop steriliz-
ing sequencing between each of the streakings.
Others prefer to use a specially made petri dish
which features a center partition which "halves" the
dish allowing two separate cultures to be cultivated
These modifications, and others, are not deviations
since the only consideration which matters is that a
pure culture is available for further testing.
-------
WATER MONITORING PROCEDURES: Completed Test for the MPN Method for Drinking Water
This outline was prepared by: Rocco Russomanno,
Microbiologist, National Training and Operational
Technology Center, MOTD, OWPO, USEPA, Cincinnati,
Ohio 45268
4-5]
-------
A PROTOTYPE FOR DEVELOPMENT OF
ROUTINE OPERATIONAL PROCEDURES
for the
TOTAL COLIFORM TEST FOR DRINKING WATER BY THE MEMBRANE FILTER METHOD
as applied in
DRINKING WATER TREATMENT FACILITIES
and in the
DISTRIBUTION SYSTEMS OF DRINKING WATER TREATMENT FACILITIES
National Training and Operational Technology Center
Municipal Operations and Training Division
Office of Water Program Operations
U. S. Environmental Protection Agency
BA.MET.lab.WMP.2a.10.78
5-1
-------
EPA 600/8-78-008, May 1978
Manual for the Interim Certification of Laboratories Involved in Analyzing
Public Drinking Water Supplies - Criteria & Procedures
Total Coliform Test for Drinking Mater by the Membrane Filter Method
The required procedures, which are mandatory, are described in the 13th Edi-
tion of "Standard Methods": single step or enrichment standard total coliform
membrane filter procedure (p. 679-683). Tentative methods are not acceptable.
All other procedures are considered alternative analytical techniques as
described in section 141.27 of the National Interim Primary Drinking Water
Regulations. Application for the use of alternative methods may require accept-
able comparability data.
Membrane Filtration Equipment
Units must be made of stainless steel, glass, or autoclavable plastic. Equip-
ment must not leak and must be uncorroded.
Field equipment is acceptable for coliform detection only when standard labora-
tory MF procedures are followed.
Membrane filter assemblies (wrapped) and sterilized at 121°C/30 minutes.
Membrane Filters and Pads
Membrane filters must be manufactured from cellulose ester materials, white,
grid-marked, 47-mm diameter, 0.45 ym pore size. Another pore size may be used
if the manufacturer gives performance data equal to or better than the 0.45 urn
membrane filter. Membranes and pads must be autoclavable or presterilized
(autoclaved at 121°C for 10 minutes with fast exhaust). Membrane filters used
must be those recommended by the manufacturer for water analysis. The recom-
mendation must be based on data relating to ink toxicity, recovery, retention,
and absence of growth-promoting substances.
Total Coliform Media
M-Endo broth, M-Endo agar, or Les Endo agar used in a single step procedure;
final pH 7.2 + 0.2; total incubation time 22 to 24 hours at 35° + 0.5°C.
In two-step Les M-Endo procedure, MF incubated on lauryl-tryptose-broth-saturated
absorbent pad for 1.5 to 2 hours at 3.5° +_ 0.5°C; then on M-Endo broth or Les
Endo agar for 20 to 22 hours at 35° + 0.5°C.
Reconstituted in laboratory pure water containing 2 percent ethanol (not
denatured).
The membrane filter broth and agar media must be heated in a boiling water bath
until completely dissolved.
Membrane filter (MF) broths must be stored and refrigerated no longer than
96 hours. MF agar media must be stored, refrigerated and used within 2 weeks
if prepared in tight-fitting dishes.
Ampouled media must be stored at 1° to 4.4°C (34° to 40°F); time must be limited
to manufacturer's expiration date.
5-2
-------
MF Culture Dishes
Sterile tight or loose-lid plastic culture dishes or loose-lid glass culture
dishes must be used. For loose-lid culture dishes, relative humidity in the
incubator must be at least 90 percent.
Culture dish containers, if used, must be aluminum or stainless steel; or
dishes may be wrapped in heavy aluminum foil or char-resistant paper.
Open packs of disposable sterile culture dishes must be resealed between
uses.
Stock buffer solution must be prepared according to "Standard Methods" using
laboratory pure water adjusted to pH 7.2. Stock buffer must be autoclaved or
filter-sterilized, labeled, dated, and stored at 1° to 4.4°C. The stored
buffer solution must be free of turbidity.
Rinse and dilution water must be prepared by adding 1.25 ml of stock buffer
solution per liter of laboratory pure water. Final pH must be 7.2 +_ 0.1.
Rinse water volumes of 500 ml to 1,000 ml sterilized at 121°C/45 minutes.
Rinse water in excess of 1,000 ml sterilized at 121°C/time adjusted for volume;
check for sterility.
Filtration assembly sterile at start of each series and must be sterilized
between sample filtration series. A filtration series ends when 30 minutes
or longer elapse between sample filtrations.
At least 2 minutes of UV light or boiling water may be used on membrane filter
assembly to prevent bacterial carry-over between filtrations (optional).
Absorbent pads saturated with medium, excess discarded; or 4.0 ml of agar
medium can be used per culture dish instead of a pad.
Sample shaken vigorously immediately before test.
Test sample portions measured and not less than 100 ml.
Funnel rinsed at least twice with 20- to 30-ml portions of sterile buffered
water.
MF removed with sterile forceps grasping area outside effective filtering area.
MF rolled onto medium pad or agar so air bubbles are not trapped.
Low power magnification device with fluorescent light positioned for maximum
sheen visibility.
Total coliform count calculated in density per 100 ml.
5-3
-------
A start and finish MF control test (rinse water, medium, and supplies) must
be conducted for each filtration series. If sterile controls indicate con-
tamination, all data on samples affected must be rejected and a request made
for immediate resampling of those waters involved in the laboratory error.
The following rules for reporting any problem with MF results must be observed:
- Confluent growth: Growth (with or without discrete sheen colonies) covering
the entire filtration area of the membrane. Results are reported as "con-
fluent growth per 100 ml, with (or without) coliforms," and a new sample
requested.
- TNTC (Too numerous to count): The total number of bacterial colonies on the
membrane is too numerous (usually greater than 200 total colonies), not
sufficiently distinct, or both. An accurate count cannot be made. Results
are reported as "TNTC per 100 ml, with (or without) coliforms," and a new
sample requested.
- Confluent growth and TNTC: A new sample must be requested, and the sample
volumes filtered must be adjusted to apply the MF procedure; otherwise the
MPN procedure must be used.
]MINIMUM REQUIREMENTS except where indicated as OPTIONAL.
5-4
-------
TOTAL COLIFORM TEST
Membrane Filter Method
Flow Sheet
Sample
[m-ENDO Medium]
(100 milliliters)
Colonies counted microscopically
(10 to 15 magnifications)
Total Coliforms
(Colonies with metal lie-
like reflecting sheen
surface)
Count number of colonies
Calculate total coliform/100 milliliters
Record results as total coliforms/100 ml
Non-Coli forms
(Colonies lacking metal lie-
like reflecting sheen
surface)
Total coliforms not present
Report results as prescribed under current
Drinking Water Regulations
en
in
(IMPORTANT: A total of 200 or more colonies of any type
invalidates the sample for counting purposes).
-------
WATER MONITORING PROCEDURE: Total Coliform Test for Drinking Water by
the Membrane Filter Method
1. Analysis Objectives:
In drinking water monitoring, the application of this methodology can be
for any of the following:
a. Test parameter for the conduction of a sanitary survey during an
on-site review of the water source.
b. Special purpose testing such as those taken to determine whether
disinfection practices following pipe placement, replacement, or
repair have been sufficient.
c. Check tests following unsatisfactory coliform results, and,
d. Monitoring potable water supplies.
2. Brief description of analysis:
A standard portion* of 100 milliliters is filtered through a membrane filter
contained within a filtering apparatus. Bacteria in the sample portion are
held on the upper surface of the membrane, while the water passes through
and is discarded.
After several rinses of the funnel of the filtering apparatus with sterile
buffered distilled water, the membrane filter is placed on a paper pad
saturated with a medium called m-ENDO Broth within a petri dish. The closed
and inverted petri dish is now incubated within a high humidity incubator
set at 35°- C +_ 0.5° C for an incubation period of 22-24 hours. On
this medium, coliform bacteria will grow and develop a golden metallic sheen-
like surface on the colonies. Colonies lacking this characteristic reflec-
tive surface are not considered as coliforms. This distinctive surface sheen
may appear at the center, edges, or all-over the colony. At times it can
form as flecks or particles of sheen throughout or partially covering the
colony.
The membrane is inspected with the aid of a microscope or lens having a
magnification of 10X or 15X under reflective lighting from a fluorescent
source. Coliform colonies, if any, are counted and a calculation made in
order to report total coliforms per 100 milliliters.
Analytical Method: Standard Methods for the Examination of Water and
Wastewater, 14th Edition, 1975, Pg. 928 ff.
*Procedures will be hereafter described for monitoring a drinking water
sample and not concerned with other types of samples which require
similar but differing instructions (i.e., more sample portions, more
petri dishes, discrimination of which plates to count, etc.).
5-6
-------
WATER MONITORING PROCEDURE: Total Coliform Test for Drinking Water by
the Membrane Filter Method
Equipment and Supply Requirements
A. Capital Equipment:
1. Autocalve, steam, providing uniform temperatures up to and including
121° C and equipped with an accurate thermometer, pressure gauges,
saturated steam power lines and capable of reaching required temperatures
within 30 minutes. (Alternately, a suitable pressure cooker is acceptable-
see Standard Methods for particulars.)
2. Incubator, air, providing uniform and constant temperature of 35° C
+p.5°C and having an atmosphere of at least 90% relative humidity.
3. Oven, hot-air, providing uniform temperatures within the range of
160 - 180° C.
4. Apparatus, water distillation, distilled water product suitable for
bacteriological operations. (Alternately, a suitable source is
permissible.)
5. Microscope, stereoscopic, 10X to 15X magnification with fluorescent
lighting mandatory. (Alternately, a suitable magnifying lens with
fluorescent lamp is acceptable,)
6. Refrigerator, set for less than 10° C but above the freezing temperature.
7. Vacuum Source, preferably a pump assembly with suitable hoses and shut-off
valve provided. (Alternately, an aspirator or hand pump with the same
provisions are acceptable.)
8. Balance, analytical, sensitivity of 1 mg.
9. Gas Source, suitable for burner. (Alternately, an alcohol lamp can be
used.)
B. Reusable Supplies:
1. Apron, suitable for laboratory operations.
2. Bottle, sample, of sufficient size for standard sample, preferably of
250 ml, wide-mouth, glass stopper, with tag. (Alternately, 120 ml size.)
3. Bottle, squeeze type, containing disinfecting solution.
4. Burner, gas, suitable for laboratory operations with connecting hose.
5. Thermometer, NBS (or NBS calibrated), functions within 20° - 60° C range
with individual markings of 0.2° C or less.
6. Thermometer, NBS (or NBS calibrated), functions within 150° - 190° C
range with individual markings of 1° C.
7. Filtration Unit, MF, a seamless funnel attached to a receptacle bearing
a porous plate (screen, porous disc, etc.) and constructed from stainless
steel, glass, porcelain, plastic, or other suitable material.
8. Hot Plate, controllable heat range up to the 100° C range.
9. Balance, trip, sensitivity of 0.1 gram at a load of 150 grams, with
appropriate weights.
10. Meter, pH, accurate to within 0.1 pH unit, with suitable standard pH
reference solution(s).
11. Can, pipet, non-toxic and sterilizable material (if pre-sterilized
disposable type pi pets are used, this item is unnecessary).
12. Pan, discard, receives contaminated material and pi pets and contains
disinfectant, of sufficient length to receive pipets placed horizontally.
13. Cylinder, graduated, 500 ml, 100 ml and 25 ml size.
5-7
-------
14. Blank, dilution water, 99 ml.
15. Pipets, microbiological, 5.0 ml, with 0.1 ml graduations, sterile cotton
plugged, glass or disposable types (the disposable types are for one time
use and may be glass or Plastic).
16. Pipets, microbiological, 1.0ml, with 0.1 graduations, sterile cotton
plugged, glass or disposable types (the disposable types are for one time
use and may be glass or plastic).
17. Pipets, microbiological, 10 ml, with 1 ml graduations, sterile, cotton
plugged, glass or disposable types (the disposable types are for one time
use and may be glass or plastic).
18. Beaker, 50 ml (for measuring pH).
19. Flask, volumetric, 1 liter capacity (for stock solution of phosphate
buffer).
20. Flask, Erlenmeyer, 500 ml capacity (for holding buffered distilled rinse
water).
21. Flask, sidearm, 1 liter size for reservoir of MF apparatus; proper size
bored, rubber stopper is needed to connect MF filtration flask to flask
and hose required to vacuum source (must be rigid enough to avoid collapse
under vacuum and flexible enough to be controlled by pinch clamp) pinch
clamp - vacuum control.
22. Flask, Erlenmeyer, 50 ml (for preparing m-ENDO medium).
23. Forceps, curved end, round tip.
24. Bottle, small, Methanol or Ethanol volume to cover ends of forceps.
25. Sponge, small, to spread and wipe germicide.
26. Desiccator, media storage, ideally opaque or darkened and containing
desiccating agent to remove moisture.
C. Consumable Supplies:
1. Dish, petri, disposable, tight fitting plastic, 50 x 12 mm, sterile.
2. m-ENDO Broth, medium, dehydrated, total coliform. Distributors, Difco,
BBL, or other equivalent preparation.
3. Pencil, wax, recommended of soft wax equivalent to Blaisdell 169T.
4. Tags, bottle marking.
5. Glass Wool.
6. Cotton, non-absorbent.
7. Paper, Kraft wrapping.
8. Foil, aluminum, heavy duty.
9. Matches or striker.
10. Towels, paper.
11. Detergent, non-toxic, laboratory cleaning.
12. Data Sheet, as required by analyst's agency.
13. Filter, membrane, 47mm, 0.45 ym pore size, white, grid marked, sterile.
14. Pad, absorbent, 48 mm, sterile (usually included with membrane packet).
15. Potassium Dihydrogen Phosphate (KH«PO.), recommended 1/4 Ib.
16. Sodium Thiosulphate (Na2S203SH20)/ *
17. Disinfectant, for bench tops and decontaminating purposes, bleach of
household strength and prepared according to label directions.
18. Sodium Hydroxide (NaOH), IN.
19. Distilled Mater, suitable for bacteriological operations. Obtainable
from distillation apparatus (see Capital Equipment) or suitable source
of supply.
5-8
-------
20. Magnesium Sulfate (MgS04.7H,0).
21. Ethanol, 95%. * *
Item needs In quantities or required size or space allowances cannot be specified,
as they vary according to the daily analysis schedule. As a rule-of-thumb, space/
size or quantity requirements should be at least 3 times the normal daily require-
ments. For further information on specifications for equipment and supplies, see
the Microbiology Section of the current edition of Standard Methods for the
Examination of Water and Wastewater.
5-9
-------
WATER MONITORING PROCEDURE: Total Coliform Test for Drinking Water by the Membrane Filter Method
5-10
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pretest Procedures
1. Incubator Setup,
Adjustment
(35° C + 0.5° C)
1. Place incubator in
permanent location.
2. Provide a saturated
humidity within
incubator.
3. Install thermometer.
4. Connect incubator to
electric power source and
turn ON.
Aa. All pretest procedures completed before starting
other first-day procedures.
la. Floor location for large unit or table or bench
for smaller units.
Ib. Out of drafts or place in which it will be in
direct sunlight part of day.
Ic. Location convenient to laboratory operations.
Id. Convenient source of electric power with a
separate circuit, if possible.
2a. Check manufaturer's handbook for maintenance of
humidification system, if installed.
2b. If humidifier system not installed within
incubator, place beakers or trays containing
distilled water on shelves to provide relative
humidity of at least 90 percent during operating
temperatures.
3a. Functions at least in 30° - 40° C range. Meets
NBS standards. Have 0.2° C increment markings or
less.
3b. Usually a corner location to prevent breakage and
tip immersed in a bottle containing water,
glycerin, etc. for a more stable reading.
3c. If thermometer assembly has been installed by
manufacturer, check for above requirements and
calibrate with NBS thermometer. Calibration may
be possible by removal and testing of installed
unit or by comparsion during incubator operation.
4a. Pilot light should come on.
V.A.1.1
(p. 5-32)
III.A.l.Zb.
(p. 5-31)
V.A.I.3
(P. 5-32)
-------
WATER MONITORING PROCEDURE: Total Coliform Test for Drinking Water by the Membrane Filter Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pretest Procedures
(Continued)
2. Oven Sterilizer-
Drier Setup,
Adjustment
(170° + l.OC)
5. Adjust temperature until
stabilized at required
temperature.
6. Operate incubator
continuously.
1. Place oven sterilizer-
drier in permanent
location.
2. Connect oven/drier to
power source and turn ON.
3. Install thermometer.
5a. Manufacturer's instructions for location and
method of temperature adjustment.
5b. Allow about 1 hour between fine adjustment (less
than 2 degrees) and immediate adjustments can be
made when the calibration is greater than this
amount. Temperature achievement by the setting
knob will be usually indicated by either a light
indication or by an alternate lighting of a "heat-
ON" - "refrigerant-ON" or other arrangement de-
pending upon the incubator type/model.
6a. Operate incubator continuously unless it will be
unused for a relatively long period. (2 weeks or
more).
6b. Daily check of temperature required, preferably
an early morning and late afternoon with a written
record maintained. Adjust temperature if
necessary.
6c. Check at least biweekly the humidity level of
interior of incubator. Add water to humidifier
unit, if applicable, or to trays placed on the
shelves providing humidification by convection.
la. Convenient source of electric power.
2a. Usually an indication is given that power is
applied- such as an indicator light.
3a. Thermometer should read within 160-180° C range,
be accurate within this interval, and be marked
in 1.0° C intervals.
3b. If installed by manufacturer, ascertain if
installation meets the above requirements.
V.A.2
(p. 5-32)
5-11
-------
HATER MONITORING PROCEDURE: Total Coliform Test for Drinking Water by the Membrane Filter Method
5-12
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pretest Procedures
(Continued)
3. Autoclave Setup
4. Water Distillation
Equipment Setup
5. pH Meter Setup
4. Adjust oven temperature
to stabilize at required
sterilizing temperature.
1. Install and operate auto-
clave according to
manufacturer's instruc-
tions.
4a.
4b.
4c.
la.
Ib.
Ic.
Id.
1. Install and operate in
accordance with manu-
facturer's instructions.
2. Operate as required to
maintain adequate supplies
of suitable distilled
water.
1. Setup and operate in
accordance with manu-
facturer's instructions.
170° C is the required temperature. Arbitrarily,
for this publication, a 1 degree leeway is
stipulated.
Manufacturer's instruction for location and
method of temperature adjustment.
Allow about 1 hour between fine adjustments (less
than 2 degrees of desired temperature) and
immediate adjustments can be made when the
calibration is greater than this amount.
Variable in design and operation, and unless
properly operated can be dangerous.
Used to sterilize objects made of or including
liquids, rubber, and some plastics, and, for
glassware, if desired.
Operated for general sterilization at 121° C.
(250° F) for a period of 15 minutes after this
temperature has been attained.
Sterilized media and liquids must be removed as
soon as possible upon completion of sterilization
from the chamber of the autoclave.
la. Must produce water meeting quality requirements
for bacteriological tests.
V.A.4
(p. 5-33)
la. Meter must be accurate to at least 0.1 pH unit.
-------
WATER MONITORING PROCEDURE: Total Coliform Test for Drinking Water by the Memebrane Filter Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pretest Procedures
(Continued)
6. Glassware
Preparation
7. Sample Bottle
Preparation
1. Clean and rinse using a
suitable detergent and
hot water.
2. Use final rinsing of
delonized or distilled
water.
1. Deliver 0.1 ml or 0.2 ml
of 10% sodium thiosulfate
solution to each sample
bottle (0.1 ml to 4 oz. or
120 ml size and 0.2 ml to
6-8 oz. or 250 ml size).
Sodium thiosulfate is
prepared as follows:
* Weigh 10.0 grams of
sodium thiosulfate.
* Dissolve in 50-60 ml of
distilled water.
* Add distilled water to
bring final volume to
100 ml.
* Transfer to labeled
bottle.
2. Place cover on sample
bottle.
3. Place paper or metal foil
cover over bottle cap or
stopper.
la. Nontoxic detergent must be completely removed from
glassware.
2a. 6 to 12 successive rinsings may be required.
2b. Must produce a clean dry glassware which meets
bacteriological requirements for suitability.
la. Bottle meets glassware requirements.
Ib. Use 1 ml pi pet.
Ic. Provides adequate sodium thiosulfate for neutral-
izing chlorine in sample.
Sodium Thiosulfate Preparation
Id. Use of trip balance for weighing acceptable.
le. 100 ml graduated cylinder satisfactory for
volume measurements.
If. Final preparation should be labeled as 10%
Sodium Thiosulfate and stored in refrigerator.
3a. Protects opening of sample bottle from accidental
or natural contamination.
5-13
-------
WATER MONITORING PROCEDURE: Total Coliform Test for Drinking Water by the Membrane Filter Method
5-14
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pretest Procedures
(Continued)
8. Pi pet Preparation
4. Sterilize sample bottle
in autoclave or oven.
5. Store sample bottle in
clean dry place until used
1. Insert a plug of non-
absorbent cotton into
mouthpiece of clean,
dry pi pet.
4a. In oven at 170° C for 1 hour.
15 pounds for 15 minutes.
2. Pass plugged end of pi pet
quickly through burner.
3. Insert a layer of glass
wool or multi-layer of
paper padding in bottom
of pi pet can.
4. Place pi pet in pi pet can
with delivery tip down-
ward and contacting glass
wool or paper. Close can
when full or desirable to
complete preparation.
In autoclave at
la. Pi pets which have chipped or broken tips or tops
should be discarded.
Ib. Cleanliness and suitability of pipets equivalent
to bacteriological suitability of glassware.
Ic. Cotton plug must be tight enough to prevent easy
removal, either by pipeting action or by handling,
and yet be loose enough to permit easy air move-
ment through the plug.
Id. Plug protects user from ingesting sample into
his mouth.
2a. Removes wisps of cotton which interferes with
fingertip control of pipeting action.
3a. Protects tips from damage.
3b. Pipets can be sterilized individually, if desired,
by wrapping in Kraft paper then oven sterilizing.
This technique would make the use of pi pet cans
unnecessary.
4a. Cotton-plugged mouthpiece in pipeting, is finger
control end with the delivery tip on the opposite
end.
4b. Approximately twenty (20) 1 ml pipets or twelve
(12) 10 ml pipets will normally be accommodated
in these cans.
4c. Can must be able to withstand sterilizing condi-
tions. Toxic materials, such as copper, must not
be used. Aluminum is acceptable.
-------
MATER MONITORING PROCEDURE: Total Coliform Test for Drinking Water by the Membrane Filter Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pretest Procedures
(Continued)
9. Blanks, Dilution
Water
5. Sterilize pi pets.
6. Store cans in a clean dry
place until needed.
1. Prepare stock solution of
potassium dihydrogen phos-
phate (KH2P04) by
dissolving 34.0 grams of
this chemical in 500 ml of
distilled water and adjust
ting its pH to 7.2 with
IN NaOH. Dilute to 1
liter in volumetric flask.
2. Prepare stock solution of
magnesium sulfate
(MgS04.7H20) by dissolving
50 grams of this chemical
in 500-600 mis of distille
water and, after complete
dissolving, bring the
final volume to 1 liter in
a volumetric flask.
5a. At least 1 hour in oven at 170° C, or
5b. In autoclave at 15 pounds steam pressure for 15
minutes. Cans removed quickly from autoclave
after sterilizing with aid of asbestos gloves and
opened quickly and slightly to allow residual
steam to escape for a few seconds.
la. Distilled water may be measured in 500 ml gradu-
ated cylinder.
Ib. Label to show contents, identity of preparer,
and date of preparation.
le. Stored in refrigerator.
Id. Discarded if mold or turbidity appear.
5-15
-------
WATER MONITORING PROCEDURE: Total Coliform Test for Drinking Water by the Membrane Filter Method
5-16
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pretest Procedures
(Continued)
3. Prepare working solution
of dilution water by add-
ing 1.25 ml of the
potassium dihydrogen
phosphate stock solution
and 5 ml of the magnesium
sulfate stock solution to
each liter of distilled
water to be used in the
preparation of dilution
water.
4. Deliver enough working
solution to each dilution
water bottle so that after
sterilization the bottle
will contain 99 + 2 ml of
dilution water.
5. Place caps on bottles
loosely.
6. Sterilize in autoclave.
7. Remove from autoclave,
tighten bottle caps; cool
to room temperature.
8. Store in cool place.
3a. A 10 ml or 5 ml pipet is satisfactory for
delivery of both of these stock solutions
provided that it has graduation marks to
deliver the proper amount. Use separate pipets
for each solution to prevent contamination.
4a. Recommended that dilution water bottles have a
marking at the desired 99 ml quantity. Amount
to be delivered to bottle before sterilization
cannot be stated exactly as evaporation is
different with differing conditions and auto-
claves. Ordinarily about 102 ml will be
required.
6a. 15 minutes at 121° C.
8a. Dilution bottles ready for use. May be stored
indefinitely.
8b. Some evaporation loss may occur in time and in
these cases, sterile similarly prepared water
may be added. This is why a calibrated marked
bottle is desirable.
-------
WATER MONITORING PROCEDURE: Total Coliform Test for Drinking Water by the Membrane Filter Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pretest Procedures
(Continued)
10. Preparation of
Total Coliform
Medium
1. Retrieve and inspect
dehydrated m-ENDO broth
medium.
2. Weigh 1 gram of dehydrated
m-ENDO broth.
3. Place powder in a clean,
dry 50 ml Erlenmeyer flask
4. Prepare an alcohol-water
solution as follows:
a. Place 0.4 ml of
ethanol in a clean,
dry 25 ml graduate.
b. Add distilled water to
the graduate to the
20 ml mark.
5. Add a small amount of the
ethanol-water solution to
the powder in the flask
(about 5 ml). Swirl flask
to mix powder and then add
the remainder of the water
la. Best stored in desiccator which prevents moisture
from damaging medium.
Ib. Powder must be light pink without signs of
hardening or color change to blood red hue.
2a. Sufficient for 20 ml of medium which prepares
10 petri dishes.
2b. Analytical balance having a sensitivity of 1 mg
will be required.
2c. More than 2 grams being weighed can be done on
less sensitive balance. This would provide more
plates, but, of course, some medium can be
discarded.
4a. Graduate need not be sterile. No acceptable
substitutes for ethanol. Use 1 ml pi pet
graduated in 0.1 ml increments.
4b. A squeeze bottle addition to the graduate makes
control of the distilled water addition easier.
VI.A.10.1
(p. 5-35)
[I.A.10.2
[p. 5-30)
VI .A.10.4
(p. 5-35)
5a. Small addition of water makes it easier to
remove powder from walls of flask.
5-17
-------
WATER MONITORING PROCEDURE: Total Coliform Test for Drinking Water by the Membrane Filter Method
5-18
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pretest Procedures
(Continued)
11. Preparation of
m-ENDO Plate
6. Cover top of flask with
aluminum foil.
7. Heat flask on a hot plate
set to HIGH heat range.
8. Remove at first sign of
boi 1 i ng.
1. Remove a sterile petri
dish from its container.
Loosen its cap without
removal.
2. Remove a sterile absorptior
pad from its container and
place in dish. Replace
cap which is still loosely
fitting.
3. Transfer approximately
2 ml of the m-ENDO broth
to the absorption pad
within the dish.
4. Gently tip the opened
petri dish until a droplet
of medium forms on the
inner lower edge.
6a. Some laboratories use a cap to cover a screw-cap
flask. If this is to be practiced, make sure
that the cap is LOOSE when heating to relieve
pressure built up during heating.
7a. Constant stirring is necessary to prevent
charring or burning of medium.
8a. Prolonged heating reduces selectivity of
medium.
8b. Do not autoclave this medium.
8c. Medium ready for use. Can be stored in
refrigerator for up to 96 hours before
discarding.
la. Usually in a sleeve of pre-.sterilized plastic
one-time-use dishes.
Ib. Laboratory resuable sterilized glass dishes can
alternately be used.
2a. Usually packaged with membrane filters, or,
alternately, can be separately laboratory
sterilized.
3b. Use a forceps which has been standing in a flask
of alcohol and then passed quickly through a
flame to remove residual alcohol.
3a. Plate can be stored in refrigerator for up to
96 hours before discarding,or used immediately.
4a. A 2 ml broth addition is usually an execessive
amount.
4b. Hold petri dish cover in other hand. Do not
allow it to become contaminated.
-------
WATER MONITORING PROCEDURE: Total Coliform Test for Drinking Water by the Membrane Filter Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pretest Procedures
(Continued)
5. Gently shake out large
droplet to waste. Replace
cover tightly.
5a. Plate is ready for use in analysis.
5b. Keep plate from excessive exposure to light,
particularly sunlight.
5c. It has been found that this procedure will in-
variably give an optimum amount of medium,
whereas, trying to measure precisely the same
amount for each plate will give less than optimum
amounts more frequently.
5d. If plate is to be used within the hour, simply
cover with a paper towel on the bench. If a
greater time is expected, place in refrigerator
until used.
B. First Day Procedure
1. Equipment
Maintenance
2. Sample Collection
and Handling
1. Check, record, and adjust,
if necessary, the 35° C
incubator.
2. Check, record (if done)
and adjust (if necessary)
the refrigerator.
1. Collect sample, use a
grab, direct filling, or
suitable device collection
technique.
2. Apply label to bottle
and enter required
information.
la. Representative of water supply system.
Ib. Leave sufficient air space in bottle to allow
shaking of sample (at least 2.5 cm or 1 inch).
Ic. Do not rinse bottle before collecting sample as
this would cause loss of dechlorinating agent.
Id. Exercise care to prevent contamination of samples
2a. Enter required information as per agency
requirements. A minimum useful amount of entries
include:
* name of sampler (complete name, not initials)
* location/code of collection site
* time of collection
* chlorine residual (water before sampling)
* date of collection.
5-19
-------
WATER MONITORING PROCEDURE: Total Coliform Test for Drinking Water by the Membrane Filter Method
5-20
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First Day Procedure
(Continued)
3. Preanalysis
Preparation
4. Equipment and
Material Assembly
3. Place bottle in closed ice
chest.
4. Transport to laboratory
and dispose of sample in
accordance with laboratory
policies.
1. Prepare laboratory data
sheet.
2. Disinfect laboratory
bench; wipe dry.
1. Assemble required
equipment and material.
3a. Use ice in chest if possible, although there is
no strict requirement for this procedure. How-
ever, protect the bottle from detrimental
conditions such as direct sunlight, extreme heat
or cold, etc.
4a. An undesirable, but acceptable practice, is to
retain the bottle for up to 30 hours prior to
analysis before discarding as an unacceptable
sample. Sample should be analyzed as soon as
possible.
la. No standard data sheet. Use form recommended by
1aboratory/Agency.
Ib. Some of required information will be on sample
label.
2a. Use sponge and disinfectant; paper toweling.
la. Filtration funnel assembly, sterile.
Ib. Side arm suction flask, 1 liter size.
Ic. Hose, suction w/clamp.
Id. Vacuum source, operational.
le. Sheet, data.
If. Prepared m-ENDO dish.
Ig. Membrane filter, sterile.
Ih. Buffered distilled rinse water (about 100 ml per
test).
11. Forceps and disinfectant container (methanol).
Ij. Pencil, marking.
Ik. Sample bottle.
11. Graduate, sterile, 100 ml, foil hood protected.
1m. Burner, gas, w/hose joined to gas source.
-------
WATER MONITORING PROCEDURE: Total Collform Test by the Membrane Filter Method
-------
HATER MONITORING PROCEDURE: Total Coliform Test for Drinking Water by the Membrane Filter Method
5-22
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First Day Procedure
(Continued)
2. Assemble the units of the
filtration apparatus.
3. Test the filtration
apparatus for operation.
Assemble the units of the
filtration unit assembly:
Unwrap sterile funnel
base from wrapping and
place on base unit.
2a. Suction side of apparatus may, depending on
choice of equipment, consist of sidearm suction
flask, suction line, pinch clamp, and suction
device. Such an arrangement is shown below:
hose
pinch clamp
stopcock
sidearm flask
suction main line
3a. Check suction units for cleanliness and operation
Open suction line by turning on stopcock and
removing pinch clamp and check for suction at
neck of sidearm flask by placing palm of hand
over neck of flask and noting presence of suction
Replace and close pinch clamp and note if
suction is cut off from the flask. NOTE: This
test is made without the filtration funnel
assembly being installed.
4a. The filtration unit assembly consists of a funnel
and a base which should be clean, sterile, and in
operational status.
4b. Manufacturers usually provide kits for
maintenance of units.
4c. Do not contaminate working areas of funnel
assembly (screen, inner area of funnel, funnel
lip, etc.).
4d. Stopper may be retained on base of filtration
unit throughout the usage and sterilization of
the base.
V.B.4.2
(p. 5-33)
-------
MATER MONITORING PROCEDURE; Total Collform Test for Drinking Water by the Membrane Filter Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First Day Procedure
(Continued)
4e. Units of filtration assembly are sterilized by
steam after wrapping in Kraft paper or aluminum
foil.
funnel
base
filtration
funnel
assembly
stopper
5. Sample Filtration
5. Light burner.
6. Label m-ENDO plate with
necessary identification
markings.
1. Place membrane filter
(MF) on base of funnel
unit and centered evenly
on the screen assembly.
5a. Some laboratories use an alcohol lamp.
6a. Conforms to data sheet.
la. Funnel top removed carefully to avoid contami-
nation. Do not place on contaminated surface.
Best to hold in hand while using forceps in
other.
5-23
-------
WATER MONITORING PROCEDURE: Total Coliform Test for Drinking Water by the Membrane Filter Method
5-24
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First Day Procedure
(Continued)
Ib. MF placed grid or inked side UP.. MF handled
with flamed forceps and only on the membranes
outer 3/16 inch of its circumference.
2. Deliver a small volume of
sterile buffered distilled
rinse water inside the
funnel.
3. Deliver standard sample
volume to funnel by using
a sterile graduate.
Ic. Replace funnel top. Avoid over-tightening which
can damage the MF or cause leakage.
2a. Use approximately 10 ml of water.
2b. Observe funnel for leakage. If any, disassemble
unit and repeat from Step 1 after inspecting
base of funnel for possible debris or damage.
Persistent leakage will necessitate
maintenance or replacement of funnel unit.
3a. Thoroughly shake sample bottle prior to filling
graduate. A minimum requirement would be 25
complete up-and-down (or back-and-forth) movements
of about 0.3 m (1 foot) in 7 seconds.
3b. Sterile graduate is prepared by oven sterili-
zation with an aluminum foil cap.
-------
WATER MONITORING PROCEDURE: Total Coliform Test for Drinking Water by the Membrane Filter Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First Day Procedure
(Continued)
3c. Volume in graduate is measured by sighting if the
meniscus lower curve being even with the 100 ml
mark.
meniscus line
glass graduate
4. Gently pour standard
sample (100 ml) into
funnel.
Graduate marked TC (to
contain): Rinse graduate
several times with sterile
water and pour each
rinsing individually into
funnel.
Graduate marked TD (to
deliver): Rinsing not
necessary, but, allow at
least 5 seconds drainage
time and then gently tap
off last drop into funnel.
eye level
(bottom of meniscus line
touching 100 ml line
in a parallel plane)
4a. Avoid splashing. Pour slowly and close to top
of funnel without touching sides.
4b. Allow a 5 second drainage period before shaking
off the last drop.
5-25
-------
MATER MONITORING PROCEDURE: Total Coliform Test for Drinking Water by the Membrane Filter Method
5-26
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First Day Procedure
(Continued)
6. Membrane Plating
6. Open vacuum control valve
and remove pinch clamp
to allow vacuum to filter
sample through MF.
7. Rinse funnel three times
with sterile buffered
distilled water.
8. Replace pinch clamp on
suction hose.
1. Disconnect funnel locking
device and lift funnel
from base to expose MF.
2. Remove membrane from
funnel base.
6a. Vacuum must not be allowed to enter system prior
or during Step 5 as this would suck sample pre-
maturely and bacterial dispersion will not occur
over membrane.
6b. Allow complete passage of sample through MF.
7a. Rinsings remove all of residual sample droplets
from sides of funnel.
7b. Allow complete flushing of each rinse through
membrane before applying next rinse.
7c. Use about 25 ml for each rinse and pass around
funnel to rinse complete circumference
(circular motion of hand around funnel) of
funnel. Do not touch inside area of funnel.
8a. Interrupts vacuum delivery to flask.
8b. Will not allow MF to be lifted from base without
possible damage due to strong suction being
continued.
8c. Some laboratories may elect to use control valve
for this operation and not use pinch clamp.
la. Best to hold funnel in one hand while using
forceps with other. Some laboratories may
elect to either:
* Use a germicidal cabinet to hold funnel
* Use a funnel holding device
But, in any event, DO NOT place funnel where it
can become contaminated if it is to be used for
another sample.
2a. Again, handle membrane carefully with flamed
forceps (quickly flamed after removing from
alcohol immersion jar - NOT HEATED) and only
on outer 3/16 inch of membrane.
V.B.6.1
(p. 5-33)
-------
WATER MONITORING PROCEDURE: Total Coliform Test for Drinking Water by the Membrane Filter Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First Day Procedure
(Continued)
4.
Replace funnel on base if
it is held in other hand,
or, replace when con-
venient if held in holding
device or UV light box.
Remove cover from m-ENDO
dish. Do not allow it to
become contaminated.
Can either be held in the
hand or placed on the lab
bench.
2b. Break residual vacuum in flask by gently lifting
edge of MF before removing.
3a. Funnel unit is now ready to receive the next
sample as the three rinses have been found to
be sufficient to cleanse the funnel of bacteria.
which can influence this test.
cover
base with
m-ENDO
medium
5-27
-------
WATER MONITORING PROCEDURE: Total Coliform Test for Drinking Water by the Membrane Filter Method
5-28
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First Day Procedure
(Continued)
7. Incubation Procedure
5. Place MF over the m-ENDO
medium. Use a rolling
action to eliminate air
pockets. Close the dish
tightly when membrane
shows elimination of air
pockets.
1. Invert petri dish (turn
upside down). The bottom
or plate base will now be
on top and the MF will be
upside down.
2. Place dish in the in-
verted position within
the 35C C incubator.
5a.
5b.
5c.
5d.
2a.
2b.
2c.
Grid or inked side surface contains the bacteria
and must not be placed next to the m-ENDO*
If air pockets persist (indicated by white areas
with the pink colored m-ENDO liquid) pick up the
MF by its edge and re-roll. Persistent clear or
white areas usually indicate that there is too
little broth on the pad. Add a drop of m-ENDO
to the pad if necessary while holding up a corner
of the MF. Do not place broth over the membrane.
Do not run forceps or any object over the MF as
it is very delicate and damage can result in
poor plate results.
Some amount of air spots is tolerable if they
are outside the working area of where the
bacteria were plated. About 3/16 inch is
acceptable.
Plate is inverted to prevent droplets from
"falling down" on MF destroying the colonial
growth of the bacteria.
Do not crowd plates. If a number of them have to
be stacked, place them no more than three high
with an unused area around them equal to the size
of a petri dish.
Allow an incubation period of 22-24 hours. Be
sure time of plating is indicated on data sheet.
-------
WATER MONITORING PROCEDURE: Total Coliform Test for Drinking Water by the Membrane Filter Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Second Day Procedure
1. Counting Procedure
Remove petri dish from
incubator with careful
handling to avoid jarring
of plate. Turn plate over
where cap is now on top.
Remove cover to facilitate
counting and place plate
bottom on stage of micro-
scope or under lens of
magnifier.
Adjust cool, white,
fluorescent light source
to give maximum sheen
development to colonies,
if any.
Microscopically scan
membrane with a back-and-
forth movement over the
grids and count all
colonies having sheen.
la. Incubation period has been within the 22-24 hour
period. No deviations are permitted.
Ib. Rough handling can cause spattering of droplets
within plate and possibly causing difficulty in
counting.
2a. MF colonies are best counted with a magnification
of 10 to 15 diameters. A wide fteld dissecting
microscope is preferred but a magnifier is
acceptable.
3a. A nearly vertical light adjustment is usually
optimum.
3b. Do not use any other lighting source than the
cool, white, fluorescent bulb.
3c. Sheen is characteristic of the coliform group of
bacteria and is a golden, metal lie-like re-
flective property on the surface of the colonies.
4a.
The dashed circle indicates the effective
filtering area. The dashed back-and-forth
line indicates the colony counting pathway.
5-29
-------
WATER MONITORING PROCEDURE: Total Coliform Test for Drinking Water by the Membrane Filter Method
5-30
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Second Day Procedure
(Continued)
2. Recording Results
Observation of numerous
colonies on the MF, even
if they are not sheen con-
taining colonies, will re-
quire counting since there
is a 200 count maximum
allowable colony count.
1. Since 100 ml is the
standard sample volume,
the number of coliforms
counted will be the
count/100 mis and this
value is recorded on the
data sheet.
4b. Sheen development may be in a variety of forms -
solid sheet, pin points, flecks, etc. Any amount
of sheen is enough to consider the colony as
being presumptively a coliform.
4c. Colonies are raised, usually circular, growths of
original bacteria which was planted on the
membrane and are considered to be the result of
a single organism multiplying many times to
produce a visible colony.
5a. The 200 colonies or more of all types (which
includes coliforms) is an amount of growth
which produces interferences with validity of
results.
5b. A resampling should be requested in this case,
and if persistent, treatment of the source must
be instituted before laboratory results can be
meaningful. It may be useful to run two 50 ml
plates or four 25 ml plates and add the coliform
colonies from the series to reduce the back-
ground colonies produced on the larger volumes.
Thus, individual acceptable plates are obtained
and the standard volume is still being processed.
la. If any coliforms are present on the MF, each must
be verified as being a coliform bacteria.
Ib. In this case, close the dish and initiate the
verification procedure as soon as possible.
Ic. Turn in data sheet if no coliforms are present
and take organizational policy steps if any
coliforms are presumptively present.
V.C.I.Ac
(p. 5-34)
V.C.2.1a
(p. 5-34)
-------
WATER MONITORING PROCEDURE; Total Coliform Test for Drinking Water by
the Membrane Filter Method
SECTION TOPIC
I Introduction
II* Educational Concepts - Mathematics
III* Educational Concepts - Science
IV Educational Concepts - Communications
V* Field and Laboratory Equipment
VI* Field and Laboratory Reagents
VII Field and Laboratory Analyses
VIII Safety
IX Records and Reports
*Training guide materials are presented here under the headings marked*.
These standardized headings are used through this series of procedures.
5-31
-------
WATER MONITORING PROCEDURES:
Total Coliform Test for Drinking Water by
the Membrane Filter Method
Educational Concepts - Mathematics
Section II
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.10.2
Since 48 grams of m-ENDO broth powdered medium and
20 mis of 95% Ethanol are required to prepare 1
liter (1000 mis) of m-ENDO broth, it is possible
to calculate weights and volumes to prepare any
requirement based upon the number of plates
desired. Calculations are based upon knowing the
above figures and the requirement of 2.0 ml of
broth for each pad saturation.
For rapid calculations the following two formulas
can be used:
1. No. of plates desired x 0.096 = grams m-ENDO.
2. No. of plates desired x 0.04 = ml Ethanol.
EXAMPLE: If 47 plates of m-ENDO are required:
1. 47 x 0.096 = 4.512 or 4.5 grams m-ENDO.
2. 47 x 0.04 = 1.88 or 1.9 mis Ethanol.
NOTE: Due to the practical and technical diffi-
culties involved in weighing very small portions
as, for instance, 0.096 grams of m-ENDO for one
plate requirement, it would be wise to prepare
at least 10 plates (0.96 or 1.0 gram m-ENDO and
0.4 ml Ethanol) as a minimum requirement.
5-32
-------
MATER MONITORING PROCEDURES: Total Coll form Test for Drinking Water by
the Membrane Filter Method
Educational Concepts - Science
Section III
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.1.2b
A relative humidity of over 90 percent is necessary
in order to obtain bacterial growth on the membrane
filter (colony) which has not been inhibited by a
lack of this moisture. Inhibited colonies will in-
variably be smaller and less apt to give the typical
sheen characteristic of a frank coliform.
An accurate solid heat sink incubator is acceptable
for use. This is constructed of a solid metallic
block having slots for insertion of the petri dishes.
Since there are no provisions for a high humidity
chamber in this type of incubator, it is important
to only use the types of petri dishes having a tight
attachment of cover-to-base thus preventing loss of
moisture during the incubation period.
A closed container, such as a plastic vegetable
crisper, may be placed within the incubator and
have within the container a saturated humid atmos-
phere. A convenient way of accomplishing this is to
wet a few paper towels and place within the crisper
or box.
Std. Meth. 14:937
5-33
-------
WATER MONITORING PROCEDURES: Total Collforn Test for Drinking Water by
the Membrane Filter Method
Field and Laboratory Equipment
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.I.I
A.I.3
A. 2
Incubator should be kept out of drafts or direct
sunlight in order to prevent temperature inside the
incubator from changing outside the temperature
range specified (35* C + 0.5° C).
Power supply should be selected so that there will
not be too many pieces of equipment on the same
circuit. Otherwise circuits will be blown repeat-
edly.
Mercury bulb thermometer usually used in most in-
cubators and a recording thermometer are acceptable.
Thermometers must be calibrated against a mercury
bulb thermometer which is (or calibrated against)
a National Bureau of Standards issue and used with
the certificate and correction chart.
Sterilizing ovens should be of sufficient size to
prevent crowding of materials to be sterilized. The
information below summarizes the use of the oven.
MATERIAL
STERILIZED CONDITIONS REMARKS
Standard Methods for the
Examination of Mater and
Mastewater 14th Ed. (1975)
APHA, WPCF, AWWA, p. 880 ff.
(Hereafter referred to as:
Std. Meth. 14:(Page No.)
Std. Meth. 14:881
:885
Glassware
Glassware
170° C for at
least 60 min
160° C for at
least 60 min
If Internal oven
characteristics are
unknown
If oven temperature
uniform throughout
chamber
Glassware within 170° C for at
metal container least 120 min
Other material
170° C for at
least 60 min
Material must be
capable of with-
standing sterilizinc
conditions
Alternately, a gas sterilizing unit can be used in
place of the hot-air oven. Refer to Standard Methods
and manufacturer's catalogs for details of such a
unit (ethylene oxide gas).
Conflicting temperature/time relationships appear in
differing references, but, the over-riding consider-
ation is how this time/temperature relationship works
in your hands, with your equipment, and considering
the results of sterility testing.
5-34
-------
WATER MONITORING PROCEDURES:
Total Coliform Test for Drinking Water by
the Membrane Filter Method
Field and Laboratory Equipment
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.4
B.4.2
B.6.1
Distilled water must not contain substances pre-
venting bacterial growth or be highly nutritive.
There are required procedures for testing distilled
water and should be undertaken only by professional
bacteriologists or in laboratories where this is
done regularly. Alternately, a source of deionized
water which meets all requirements as imposed on
distilled water is suitable for use in bacteriologicc1
operations.
Diagram and equipment listing describe the type of
units most commonly used in the membrane filter
procedure. They are by no means the only acceptable
arrangement which can be utilized. Different modes
of obtaining a vacuum or its control are available.
Also, various vacuum flask arrangements are accept-
able as well as the types of vacuum controls. To
preclude numerous examples, the one described will
be sufficient to give technological procedures which
are acceptable and the reader can refer to Standard
Methods and manufacturer's catalogs for further
information regarding system components or field
units which are acceptable.
A germicidal cabinet is an enclosed unit which con-
tains an active germicidal lamp (UV) which produces
a 99.9% bacterial kill in 2 minutes. It is impor-
tant not to have UV leakage from cabinet which can
be detrimental to the analyst's eyes. A funnel
holding device is designed to hold the funnel and
prevent its contamination.
EXAMPLE
Std. Meth. 14:887
Std. Meth. 14:933
iplit-ring
holding device
funnel in holder
NQ
5-35
-------
WATER MONITORING PROCEDURES: Total Coliform Test for Drinking Mater by
the Membrane Filter Method
Field and Laboratory Equipment
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
B.6.3
C.1.4C
C.2.1a
Funnel units are considered to be acceptable for use
for the next sample unless an interval of 30 minutes
or longer elapses before the next sample is run. In
this case the unit must be resterilized.
Occasionally colonial growth will be observed to be
irregular such as the following:
Std. Meth. 14:932
B
Usually, as in A and B, the colonies are readily
discernible as being multiple colonies - 2 for A
and 3 for B. In the case of C and D, however, this
separation is not readily apparent and the judgment,
based on experience, of the analyst becomes importanl
In the case of D the long strand growth may be causec
by a particle of debris which allowed channeled
growth of one or more bacteria.
The verification test is accomplished by picking the
presumptive sheened coliform colony with a sterile
needle and passing it through a series of broth
media to observe for another coliform characteristic-
gas formation in a selective medium. Refer to
Standard Methods for a detailed performance of this
verification test.
Std. Meth. 14:920
:931
5-36
-------
WATER MONITORING PROCEDURES: Total Coliform Test for Drinking Water by
the Membrane Filter Method
Field and Laboratory Reagents
Section VI
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.10.1
A.10.4
Procedures are given for m-ENDO broth medium
preparation which is, however, not the only
acceptable method available. Other acceptable
m-ENDO medium preparations include:
A. m-ENDO Agar Medium
This includes the addition of the complex
carbohydrate agar in order to solidify the
medium. In this preparation the absorption
pad is not required for analysis.
B. Pre-prepared Ampouled m-ENDO Medium
A complete prepared medium enclosed in a glass
tube. Contains enough medium for a single
medium for a single test and has the advantages
of a larger shelf life and convenience of use.
Is somewhat more costly than laboratory pre-
paration.
Ethanol is added to distilled water in a 2% dilution
for the m-ENDO medium. The amounts, of course,
would be different depending on the petri dish re-
quirements. The table below gives some useful in-
formation as reference:
Std. Meth. 14:895
No. of Plates
m-ENDO Re-
quired
Alcohol-Mater
Required
Ethanol
mis
m-ENDO Broth
Powder gm.
500
250
50
25
10
5
1 liter
(1000 ml)
500
100
50
20
10
20
TO
2
1
0.4
0.2
48
24
4.8
2.4
.96 or 1.0
.48 or T
Some laboratories prepare a large amount of the 2%
solution and, when tightly stoppered, can be used
for extended periods.
This outline was prepared by: Rocco Russomanno,
Microbiologist, National Training and Operational
Technology Center, MOTD, OWPO, USEPA, Cincinnati,
Ohio 45268.
5-37
-------
A PROTOTYPE FOR DEVELOPMENT OF
ROUTINE OPERATIONAL PROCEDURES
for the
VERIFIED MEMBRANE FILTER TEST FOR DRINKING WATER
as applied In
DRINKING WATER TREATMENT FACILITIES
and In the
DISTRIBUTION SYSTEMS OF DRINKING WATER TREATMENT FACILITIES
National Training and Operational Technology Center
Municipal Operations and Training Division
Office of Water Program Operations
U.S. Environmental Protection Agency
BA.MET.lab.WMP.8a.10.78
6-1
-------
EPA 600/8-78-008, May 1978
Manual for the Interim Certification of Laboratories Involved in Analyzing
Public Drinking Water Supplies - Criteria & Procedures
Verified MF Test for Drinking Water1
All typical coliform (sheen) colonies or at least five randomly selected sheen
or borderline sheen colonies must be verified from each membrane containing
five or more such colonies.
Needle must be sterile before selecting colonies.
Counts must be adjusted based on verification.
The verification procedure must be conducted by transferring growth from
colonies into lauryl tryptose broth (LIB) tubes and then transferring growth
from gas-positive LIB cultures to brilliant green'lactose bile (BGLB) tubes.
Colonies must not be transferred exclusively to BGLB because of the lower
recovery of stressed coliforms in this more selective medium. However, colonies
may be transferred to LIB and BGLB simultaneously. Negative LIB tubes must be
reincubated a second day and confirmed if gas is produced.
It is desirable to verify all sheen and borderline sheen colonies (optional).
If there is more than one analyst in laboratory, at least once per month each
analyst should count the sheen colonies on a membrane from a polluted water
source. Colonies on the membrane should be verified and the analysts' counts
compared to the verified count. (Optional)
Sheen colonies in mixed confluent growth reported and verified (optional).
]MINIMUM REQUIREMENTS except where indicated as OPTIONAL.
6-2
-------
Verified Membrane Filter Test Flow Sheet
Pure culture
m-ENDO test with colonies
Pure culture #2
t t
LLSTB LLSTB
ETC.
ETC
J..
Incubate 24 - 2 hn. at 35°C - 0.5°C
Gai
I
Gas(-
BGLBB
Reincubate for an additional 24hrs.
(48 + 3 hrs.) at 35°C + 0.5°C
Incubate for 24 hr*.
at 35°C ± 0.5°
1
Gas (+)
Coliform
verified
eulate percent
I
1
Gas(+)
Coliform
verified
1
verified
± 2 hr,. \ T
\ Gas (+) Gas(-)
\_J
1
Gas {-)
1
Reincubate for an
additional 24 hrs.
(48 1 3 hrs) at
35°C ±0.5eC
|
1
Gas(-)
Coliform
absent
Coliform
absent
gas(+) - Tube containing
at least one bubble
of gas
gas(— ) - Complete absence
of gas in tube.
LLSTB - Lactose lauryl sulfate
tryptose broth
BGLBB - Brilliant green lactose
bile broth
— Record in
OC records
adjust count reported,
I if necessary
Report results as prescribed
under regulatory requirements
6-3
-------
WATER MONITORING PROCEDURE: Verified Membrane Filter Test for Drinking Water
1. Analysis Objectives:
a. In water treatment plant quality control, the objective of the test fs to
determine if the effluent quality is in compliance with bacteriological
requirements as prescribed in the Federal Drinking Water Standards.
b. In distribution network and individual consumer tapping locations, the test
determines compliance with bacteriological requirements with the above
mentioned standards.
c. Supplies conclusive determination of coliforms or lack of coliforms of
mixed growth appearing fn m-ENDO medium with apparent coltform-like
colony(s) within mixed culture.
d. As part of a required Analytical Quality Control Program the test has
applications for the following:
1) m-Endo medium quality check
2) laboratory personnel comparative check for new or established workers
3} supporting evidence of colony interpretations for legal or routine
operations.
2. Brief Description of Analysis:
Discrete colony/s, whose characteristics are recorded, are aseptically (sterile
technique) picked from a suitably incubated m-ENDO plate membrane filter and
transferred to an LLSTB fermentation tube which are incubated at 35°C +_ 0.5°C.
After 24 hours and again at 48 hours (if previously negative), the LLSTB tube
cultures are examined and results recorded for gas production. Cultures show-
ing gas production are transferred to BGLBB fermentation tubes and incubated at
55° +_ 0.5°C. BGLBB tubes are examined, usually at 24 hour periods, for gas up
to 48 hours + 3 hours and those showing gas are considered a positive
coliform bacteria.
Based on the number of positives originally counted, it may be necessary
to adjust the count reported for drinking water quality.
Results are entered in a quality control record book for necessary data per-
taining to laboratory certification requirements or possible legal data
requirements.
This procedure conforms to the Standard Total Coliform MPN Test as described in
Standard Methods for the Examination of Water and Wastewater, 14th Edition
(1975), p. 931.
6-4
-------
WATER MONITORING PROCEDURE: Verified Membrane Filter Test for Drinking Water
Equipment and Supply Requirements
A. Capital Equipment:
Autoclave, providing uniform temperatures.up to and including 121°C, equipped
with an accurate thermometer, pressure gages, saturated steam power lines and
capable of reaching required temperature within 30 minutes
Balance, 0.1 g sensitivity at load of 150 g
Microscope, stereoscope viewing, 5-15 x, fluorescent, vertical Ifght source
Air incubator to operate at 35°C +_ 0.5°C
pH meter, accurate to at least 0.1 pH unit, with standard pH reference
solution(s)
Water distillation apparatus (glass or block tin), or source of distilled
water suitable for bacteriological operations.
B. Reusable Supplies:
Apron or coat suitable for laboratory
Baskets, wire for discarded cultures
Bottle, squeeze type, with disinfecting solution
Burner, gas, Bunsen Burner type
Metal caps* to fit 25 x 150 mm and 18 x 150 mm culture tubes
Pan, to receive discarded contaminated glassware (must contain disinfectant
before use)
Inoculation needle and loop, 3 mm diameter loop, of nichrome of platinum-
iridium wire, 26 B&S gage, in holder
Racks, culture type*, having at least 5 openings capable of accepting tubes
at least 20 mm in diameter
Sponge, for cleaning desk top
Tubes, culture*, 150 x 25 mm and 150 x 18 mm
Tubes, fermentation*, 75 x 10 mm vials to be inverted in culture tubes
C. Consumable (must be replaced when stocks get low):
Distilled water, suitable for bacteriological cultures (note distillation
apparatus required in capital equipment)
6-5
-------
WATER MONITORING PROCEDURE: Verified Membrane Filter Test for Drinking Water
Equipment and Supply Requirements (Continued)
BGLBB (Brilliant Green Lactose Bile Broth), dehydrated (recommend purchase
of 1/4-lb. units)
LLSTB (Lactose Lauryl Sulfate Tryptose Broth), dehydrated (recommend purchase
of 1-lb. units)
Disinfectant, for bench tops (Use household bleach solution prepared according
to instructions on bottle.)
Wax pencils (recommend soft wax equivalent to Blaisdell 169T)
Data sheets (Verified Test)
*Iterns marked are needed in quantities or require size or space allowances which
cannot be specified here, as they vary according to the daily analysis schedule.
As a rule-of-thumb, space/size or quantity requirements should be at least 3
times the normal daily requirements. For further information on specifications
for equipment and supplies, see the Microbiology Section of the current edition
of "Standard Methods for the Examination of Water and Wastewater."
6-6
-------
WATER MONITORING PROCEDURE: Verified Membrane Filter Test for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCF
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
1. 35°C Incubator
Set-up, Adjustment
1. Place 35°C incubator in
permanent location.
2. Install thermometer.
3. Install shallow pan of
water in bottom of
incubator.
4. Connect incubator to
electric power source.
5. Adjust temperature until
stabilized at required
temperature.
6. Operate bacteriological
incubator continuously.
Aa. All pre-test procedures completed before starting
other first-day procedures.
la. Out of drafts or places where it will be in di-
rect sunlight part of the day.
Ib. Location convenient to laboratory bench.
Ic. Convenient source of electric power.
2a. Thermometer functions at least in 30°-40°C range
and have intervals of 0.5° or less indicated.
Meets NBS standards.
2b. Location should be central in incubator.
2c. Mercury bulb thermometer should be fitted with
cork or rubber stopper and mounted in small
bottle filled with liquid (glycerine, water, or
mineral oil).
3a. In most laboratory incubators a pan having about
1 square foot area, with water about 1 inch deep,
is satisfactory.
3b. Maintains condition of saturated relative humid-
ity, required in bacteriological incubator.
3c. Requires daily check, with addition of water as
necessary, to keep water in pan at all times.
4a. Many incubators have pilot light to indicate
power turned on.
5a. Manufacturer's instructions for method of
temperature adjustment.
5b. Operation must be at 35 i0.5°C.
5c. Allow about 1 hour between adjustments.
6a. Requires daily check with written temperature
record, with adjustment and water addition as
necessary.
V.A.I
(p. 26)
V.A.I.2
(p. 26)
V.A.I.2
(p. 26)
V.A.I.3
(p. 26)
V.A.I.5
(p. 26)
V.A.I.6
(p. 26)
6-7
-------
WATER MONITORING PROCEDURE: Verified Membrane Filter Test for Drinking Water
6-8
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
2. Autoclave Set-Up
3. Water Distillation
Equipment
4. pH Meter
5. Glassware
1. Install and operate auto-
clave according to manu-
facturer's instructions.
1. Install and operate in
accordance with manu-
facturer's instructions.
2. Operate continuously or
intermittently as required
to maintain adequate
supplies of distilled
water.
1. Have unit available and
operate in accordance with
procedures described in
other lab procedures.
1. Wash all glassware in hot
detergent solution;
2. Rinse at least once in hot
tap water;
la. Autoclaves extremely variable in design and
operation; also, potentially dangerous.
Ib. Used to sterilize objects made of, or including
liquids, rubber, culture media.
Ic. Glassware may be autoclave sterilized but must be
dried afterward.
Id. Most plastics not sterilized in autoclave;
plastics usually require chemical sterilizers.
le. Autoclave usually operated at 121°C for 15 min.
If. Sterilized media must be removed from autoclave
as soon as possible after autoclave is reopened.
la. Must produce distilled water meeting quality
requirements for bacteriological tests.
2a. Reserve supplies kept in borosilicate glass car-
boys or in plastic carboys made of material
which will not dissolve substances which will
affect growth of bacteria.
2b. Same distillation apparatus used for bacterio-
logical purposes may be used for chemical
reagents.
la. Unit for pH check on finished culture media.
Ib. Used in preparation of stock solution of
potassium dihydrogen phosphate.
la. Nontoxic detergent.
Ib. Be sure all contents and markings are washed
away.
V.A.2.1
(p. 26)
V.A.3.1-2
(P. 27)
V.A.4.1
(p. 28)
V.A.5.1-4
(p. 28)
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WATER MONITORING PROCEDURE: Verified Membrane Filter Test for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
6. Preparation of
Lactose Lauryl
Sulfate Tryptose
Fermentation Broth
(LLSTB)
3. Rinse in distilled water,
at least 6 successive
times, and,
4. Dry in air.
1. Wei gh 17.8 grams of dehy-
drated Lactose Lauryl
Sulfate Tryptose Broth.
Close cover of bottle of
dehydrated medium tightly
after removal.
2. Dissolve in 500 mis dis-
tilled water.
3. Place 10.5 ml of the solu-
tion of prepared LLSTB in
each culture tube.
4. Insert one fermentation
vial into each tube of
medium, open end down.
5. Place tube cap on each
tube of culture medium.
6. Sterilize in autoclave.
4a. No visible spots or scum; glass should be clean,
and sparkling.
4b. Glassware suitable for use in bacteriological
operations.
la. Dehydrated media take moisture out of air; can
become caked.
Ib. Caked media unsatisfactory; should be discarded.
2a. Gentle heat (no boiling) if necessary to complete
dissolving medium. Usually a vigorous agitation
will completely dissolve the medium.
3a. Use 150 x 18 mm tubes.
3b. 25 ml pi pet, automatic pipetter, or funnel hose
and pinchcock assembly are acceptable.
3c. Accuracy of delivery: +0.5 ml.
3d. Approximately 45 tubes will be necessary.
4a. Tubes and vials washed as previously indicated.
4b. Use 75 x 10 mm tubes.
5a. After all tubes have been filled and have indi-
vidual vial.
6a. Within 1 hour after medium prepared.
6b. Sterilization at 121°C for 12 minutes.
6c. Medium must be removed from autoclave as soon as
possible after pressure has returned to normal.
Use "slow-vent" mode of steam removal.
/.A.5.4.4b
[p. 28)
...
[p. 28)
6-9
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WATER MONITORING PROCEDURE; Verified Membrane Filter Test for Drinking Water
6-10
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
7. Cool medium to room
temperature.
8. Check pH of finished
medium.
9. If final pH not satis-
factory, discard medium
and prepare new batch
with pH adjustment before
sterilization.
10. Store medium in cool, dark
place.
7. Preparation of
Brilliant Green
Lactose Bile Broth
(BGLBB)
7a. Medium ready for use when cool and individual
vials are completely filled with fluid. No
bubbles must be present.
8a. Should be 6.7-6.9.
9a. pH value ordinarily drops about 0.2 pH unit.
1. Weigh 40.0 grams of dehy-
drated Brilliant Green
Lactose Bile Broth. Close
cover of bottle of dehy-
drated medium tightly .
after removal.
2. Dissolve in 1 liter of
distilled water.
3. Place 10.5 ml of the solu-
tion of prepared BGLBB in
each culture tube.
lOa. Not in refrigerator. Usually in laboratory
cabinet in darkness.
lOb. May be stored up to 1 week if evaporation not
more than 10% in loose fitting capped tubes.
With screw-capped tubes, it should be held no
longer than 3 months.
la. Dehydrated media takes moisture out of air; can
become caked.
Ib. Caked media unsatisfactory; should be discarded.
2a. Gentle heat (no boiling) if necessary to com-
plete dissolving medium. Usually a vigorous
agitation will completely dissolve the medium.
3a. Use 150 x 18 mm tubes.
3b. A 25 ml pi pet, automatic pipetter or funnel
hose and pinchcock assembly are acceptable.
V.A.6.3b
(p. 28)
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WATER MONITORING PROCEDURE: Verified Membrane Filter Test for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedure
(Continued)
4. Insert one fermentation
vial into each tube of
medium, open-end-down.
5. Place cap on each tube of
culture medium.
6. Sterilized in autoclave.
7. Cool medium to room
temperature.
8. Check pH of finished
medium.
9. If final pH is not satis-
factory, discard medium
and prepare new batch with
pH adjustment before
sterilization.
10. Store medium in cool, dark
place.
4a. Tubes and vials washed as previously indicated.
4b. Use 75 x 10 mm tubes.
5a. After all tubes have been filled and have
individual vial.
6a. Within 1 hour after medium prepared.
6b. Sterilization at 121°C for 12 minutes.
6c. Medium must be removed from autoclave as soon as
possible after pressure has returned to normal.
Use "slow-vent" mode of steam removal.
7a. Medium ready for use when cool and individual
vials are completely filled with fluid. No
bubbles must be present.
8a. Should be 7.1-7.3.
9a. pH value ordinarily drops about 0.2 pH unit.
lOa. Not in refrigerator. Usually in laboratory
cabinet in darkness.
lOb. May be stored up to 1 week if evaporation not
more than 10% in loose-fitting capped tubes.
With screw-capped tubes, it should be held no
longer than 3 months.
6-11
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HATER MONITORING PROCEDURE; Verified Membrane Filter Test for Drinking Water
6-12
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedure
(Continued)
8. Final Equipment anc
Supply Check
Check to be sure that all
equipment and supplies,
solutions, and prepared
media are ready before
starting test procedures.
la. Check general list of equipment and supplies.
Ib. Each test requires:
tubes LLSTB
tubes of BGLBB
inoculation needle
inoculation loop
stereoscopic microscope (5-15x magnifica-
tion)
burner
35° incubator
1-5
0-5
1
1
1
1
1
Other:
Sponge
Disinfectant
Wax pencil
Data sheet
Since, as shown, the numbers of items can vary
(dependency upon the number of colonies picked
from the m-ENDO plate for this procedure a
hypothetical situation will be generated which
will give the reader a cross section of condi-
tions apt to occur.
B. First-Day Procedures
1. Equipment
Maintenance
1. Check, record and adjust
incubator temperature.
2. Add water to pass in
incubator as necessary.
la. See previous information.
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MATER MONITORING PROCEDURE; Verified Membrane Filter Test for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First-Day Procedures
(Continued)
2. Unite Sample and
Paperwork
1.
Locate m-ENDO plate and
corresponding data sheet
of sample to be subjected
to verification test.
la. For purposes of this training exercise, our
sample will be numbered "435" (data sheet and
plate reflect this identification) and have the
following characteristics:
3. Prepare Data Sheet
for Test
1.
Indicate on data sheet the
required information.
Data Sheet
As required by agency
or reporting facility
m-ENDO Plate
Count was made within
1 hour of initiating this
procedure
7 colonies on filter:
2 frank sheen
5 borderline sheen
Ib. Colonies to be subjected to verification test are
usually predetermined during colony counting of
m-ENDO plates since:
A. All sheen colonies are tube verified, or,
at least five sheen or borderline sheen
colonies must be verified from each membrane
containing five or more such colonies.
B. It is optimally desirable to verify all sheen
and borderline sheen colonies.
la. On "suggested" data sheet indicate:
A. Sample number and date
B. Culture number
C. Type of Colony
D. Analyst and start-of-test time.
(See data sheet on following page.)
VII.B.3
(p. 29)
6-13
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HATER MONITORING PROCEDURE: Verified Membrane Filter Test for Drinking Water
6-14
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First-Day Procedures
(Continued)
4. Prepare Laboratory
Test Area
5. Inoculation and
Incubation of LLSTB
Tubes
Data Sheet
Verified Membrane Filter Tests
Sample No.
and Date
1. Disinfect bench top; wipe
dry.
2. Assemble the following:
7 LLSTB tubes
1 Inoculation needle
Sample 435 MF plate
1 Burner (light with
match or striker)
1 Microscope with
fluorescent light,
5-15x magnification
1. Starting with 435-1, which
is a typical colony, focus
at 15x the colony to be
cultured.
Culture
No.
ias~l
13S-J
135-1
Type MF
Colony
rvp
TYP
ATVP
ATYP
LLSTB
BGL
IEHB
_Z4 hr I 48 hr L 24 hrl 43 hr I Aaar
Indicates "sheen"
colony
Gram
Stain
Indicates colonies
•with "borderline"
sheen development
Assighed test
numbers this
column (7 columns)
la. Sponge and disinfectant; paper toweling.
la. Remove cover from dish before placing it on
microscope stage.
Ib. Typical colony is characterized by metallic sheen,
Any amount of sheen is considered positive.
III.B.5
(P. 25)
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WATER MONITORING PROCEDURE: Verified Membrane Filter Test for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First-Day Procedures
(Continued)
2. Pick the colony with a
sterile needle.
Ic. Have fluorescent light positioned vertically or
near vertical to the plate.
Id. Focus microscope to give'the sharpest image of
the colony to be cultured.
2a. Sterilize needle to red hot stage along the
entire length of the needle in a burner flame.
Allow flame to heat part of the holder for
several seconds.
Holder Needle
Heated Zone
2b. Allow needle assembly to cool for about 5
seconds.
2c. With microscopic viewing, pick colony by allow-
ing needle to penetrate growth mass. Withdraw
needle from colony and pass away from scope.
Do not touch needle at any time to anything but
the colonial culture.
2d. Pick, if possible, only pure cultures for
testing. A pure culture is one which shows a
"separation" from the nearest neighboring colony
of at least 0.5 cm:
Needle
Colony #1 Colony #2
Separation
6-15
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WATER MONITORING PROCEDURE: Verified Membrane Filter Test for Drinking Water
6-16
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First-Day Procedures
(Continued)
3. Transfer culture to LLSTB.
2e. If necessary to pick "touching" colonies, culture
from the extreme end of the desired colony pro-
viding the greatest distance from the other
colony:
3a.
3b.
Side View
Top View
Membrane Filter
2f. If necessary to pick a sheen colony within a
mass of background growth, simply touch the
needle to the colony without regard to main-
taining purity.
Do not allow needle to touch anything as this
could cause contamination to be transferred.
Insert needle into LLSTB tube and "macerate"
culture at glass-broth junction:
Needle
Rotate and
Press to Wall
Mixing Zone
LLSTB
Wall of Tube
3c. Sterilize needle and return to storage location.
-------
WATER MONITORING PROCEDURE; Verified Membrane Filter Test for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First-Day Procedures
(Continued)
4. Identify LLSTB tube.
5. Repeat procedures for
other colonies to be
cultured.
6. Incubate all tubes at
358C + 0.5°C.
7. Disinfect lab test area;
wipe dry.
4a. Wax marking pencil: "435-1"
5a. For 435-2; 435-3; 435-4; 435-5; 435-6; and 435-7
6a. For 24 hours + 2 hours before inspection.
7a. Sponge and disinfectant; paper toweling.
C. Second-Day Procedures
1. Pre-Test
Preparations
2. Read and Record
LLSTB Results
1. Accomplish equipment
maintenance.
2. Complete bench
disinfection.
3. Assemble data sheet and
test components.
1. Inspect the seven LLSTB
tubes for gas formation
and record results.
la. As previously described.
2a. As previously described.
3a. Data sheet for this procedure.
3b. Components include: (#435 cultures)
7 LLSTB tubes from 35°C incubator.
la. Gently shake rack of tubes to allow possible
supersaturation of gases to exit from solution.
Ib. After several minutes, inspect for gas: any
amount of gas in inner vial is considered
positive.
6-17
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WATER MONITORING PROCEDURE; Verified Membrane Filter Test for Drinking Water
6-18
OPERATING PROCEDURES
C. Second- Day Procedures
(Continued)
3. Prepare for and
Complete BGLBB
Transfers
STEP SEQUENCE
-
1. Assemble LLSTB tubes for
transfers.
2. Assemble materials re-
quired for transfers.
3. Transfer one loopful of
culture from each LLSTB
tube to a BGLBB tube.
INFORMATION/OPERATING GOALS/ SPECIFICATIONS
Ic. Assume the following results:
Data She
Verified Membrane
.• No.
ate
Culture
No.
*t3S-\
HZ5-3L
V3S--J
fSS-'L
V3f-f
¥3S-k
•us-1
Type HF
Colony
TXP
TVP
/mp
ATVP
*ryf»
AT|fP
ATVP
LLSTB
'2* hr
\
4-
+
+
—
+
— «J
48 hr
H-
Enter results in
this column
la. Five tubes will be required for transfers (all
of the + tubes); two tubes will be re- incubated
as they are negatives (435-5 and 435-7).
2a. Required:
1 Bacteriological loop
1
5
Burner
BGLBB
sterile tubes
Matches or striker
Wax pencil
3a. Only a sterile
ture from LLSTB
air-cooled loop
for
loop must be used to obtain cul-
tube. Flamed (to redness) and
must have film of inoculation
proper transfer:
(See drawing on
next page.)
TRAINING
GUIDE NOTES
See Flow
Sheet of
Test Pro-
cedures
V.C.3.2
(p. 28)
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WATER MONITORING PROCEDURE; Verified Membrane Filter Test for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Second-Day Procedure
(Continued)
TRANSFER VOLUME
-A
FILM OF
INOCULUM
MUST SHOW
'FILM' WITHIN LOOP
4. Label each BGLBB tube,
then incubate five
cultures.
5. Disinfect lab test area;
wipe dry.
3b. Transfer all positive LLSTB cultures to BGLBB
tubes. Treat each tube aseptically (sterile
technique) to avoid cross-contamination.
4a. Wax pencil used for marking.
4b. Label each BGLBB tube to correspond to the LLSTB
tube from which culture was obtained.
4c. Incubation at 35°C + 0.5°C for 24 hours (first
inspection).
5a. Sponge and disinfectant; paper toweling.
D. Third-Day Procedures
1. Pre-Test
Preparations
1. Accomplish equipment
maintenance.
2. Complete bench
disinfection.
3. Assemble data sheet and
test components.
la. As previously described.
2a. As previously described.
3a. Data sheet for this procedure (#435 cultures),
3b. Components included:
2 LLSTB cultures (48 + 3 hours incubation)
5 BGLBB cultures (24 + 2 hours incubation)
6-19
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HATER MONITORING PROCEDURE; Verified Membrane Filter Test for Drinking Water
6-20
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Third-Day Procedures
(Continued)
2. Read and Record
LLSTB Results
1. Inspect the 2 LLSTB
cultures for gas produc-
tion and record results.
3. Perform Transfer to
Confirming Medium
4. Read and Record
BGLBB Results
1. Transfer culture 435-5
(LLSTB tube) to BGLBB.
1. Inspect the 5 BGLBB tubes
for gas formation, process
tubes, and record results.
la
Ib
Gently shake rack of tubes to allow possible
super-saturation of gases to exit from solution.
After several minutes, inspect for gas; any
amount of gas in inner vial is considered
positive.
Ic. Assume the following:
Data :
Verified Membra
ture
o.
Type NF
Colony
TvP
ATVP
flTYF
LLSTB
24 hr
48 hr
Culture 435-5 + in LLSTB...
,must be transferred to
BGLBB
Culture 435-7 - in LLSTB...
-coliform negative; discard
tube
la. Loop transfer method as described previously.
Ib. Label inoculated BGLBB tube with wax pencil as
435-5.
Ic. Incubate this tube at 35°C + 0.5°C.
la. All tubes have been incubated for 24+2 hours.
Ib. Gently shake rack of tubes to allow possible
super-saturation of gases to exit from solution.
Ic. After several minutes, inspect tubes for gas;
any amount of gas in inner vial is considered
positive.
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WATER MONITORING PROCEDURE: Verified Membrane Filter Test for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Third-Day Procedures
(Continued)
2. Disinfect lab test area;
wipe dry.
Culture
No.
Type MF
Colony
LLSTB
TVP
VJ5--V
rT/P
BGLBB
24 hr
Id. Assume, for our continuing example, the follow-
ing results:
Verified
+ BGLBB; verified for
coli forms—di scard
'tubes, unless further
required
- BGLBB; must be
"re-incubated
See Flow
Sheet
III.D.4.1
(p. 25)
le. Incubate culture 435-6 at 35°C + 0.5°C.
Mote to summarize:
We have processed 7 cultures and at the present
state we have the following status:
Verified as Coliforms
435-1; 435-2; 435-3; 435-4
Verification Negative (Not Coliforms)
435-7
Testing Incomplete (Pending)
435-5 (BGLBB tube in process)
435-6 (BGLBB still incubating)
2a. As described previously.
6-21
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MATER MONITORING PROCEDURE: Verified Membrane Filter Test for Drinking Water
6-22
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
E. Fourth-Day Procedures
1. Continue Testing
Procedure
2. Analyze Verifica-
tion Test Results
1. Continue test procedures
as previously outlined
for 435-5 and 435-6.
1. Calculate the percent
verified for the sample.
2. Record this percent
verified on the data
sheet.
la. Assume the following results:
Verified Membrane Filter Te»
BGLBB +; coliform
verified
BGLBB -; coliform
absent
*Note that this particular result could have gone
to a fifth day of inspection had it been nega-
tive today.
la. From the data sheet:
5 of 7 were + for coliforms; therefore:
x 100 = 71.4%
2a.
Remarks i
Analyst
7^ «6v2s.^7?
aSS
-------
WATER MONITORING PROCEDURE: Verified Membrane Filter Test for Drinking Water
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
E. Fourth-Day Procedures
(Continued)
3. Perform Data
Adjustments
Necessary
1. Adjust total coliform
counts of original sample,
la. Examples:
Suppose sample 435 had 12 colonies which could
have been reportable (combination of frank sheen
and borderline sheen colonies).
71.4% of 12 = 8.56 or 9 coliform's reported value
The same rationale is followed for other hypo-
thetical values such as given below:
Sample MF
Colonies
11
9
7
Calculated
Value
7.85
6.42
4.99
Reported
Value
8
*7
5
*As a factor of safety, report the "higher" value
of fractional portions. Note: This procedure
is not spelled out in any document, it is
author's opinion.
6-23
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WATER MONITORING PROCEDURE: Verified Membrane Filter Test for Drinking Water
TRAINING GUIDE
SECTION TOPIC
I Introduction
II Educational Concepts - Mathematics
III* Educational Concepts - Science
IV Educational Concepts - Communications
V* Field and Laboratory Equipment
VI Field and Laboratory Reagents
VII Field and Laboratory Analysis
VIII Safety
IX Records and Reports
Training Guide materials are presented here under the headings marked*. These
standardized headings are used through this series of procedures.
6-24
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WATER MONITORING PROCEDURES: Verified Membrane Filter Test for Drinking Water
EDUCATIONAL CONCEPTS - SCIENCE
Section III
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
B.5
It is an acceptable procedure to also inoculate a
BGLBB tube along with the LLSTB tube from the cul-
ture derived from the colony to be verified. This
procedure can save from 1 to 4 days in the test
procedure:
(Simultaneous transfer)
LLSTB BGLBB
(35'C)
Test Results
+....any amount
ol gas formation
—....lack of gas
production
Conclusions
Coliforms
Present
LLSTB +
and
BGLBB +
Coliforms
Absent
LLSTB +
and
BGLBB -
or
LLSTB -
and
BGLBB -
Remarks
Impossible to have LLSTB -
and BGLBB +. If this occurs,
inoculate the LLSTB with the
culture from the BGLBB and
observe for + within 48 hrs.
D.4.1
[f requirements call for the continuation of testing
to the gram staining procedure, it will be necessary
to further process the BGLBB tube and not discard it
at the positive stage.
Std. Meth. 14:918-19
6-25
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EFFLUENT MONITORING PROCEDURE: Verified Membrane Filter Test for Drinking Water
FIELD AND LABORATORY EQUIPMENT
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.I
A. 1.1
A.1.2
A.1.3
A.1.5
A.1.6
A.2.1
6-26
Incubator must be of sufficient size for daily work-
load without causing crowding of tubes to be incu-
bated. Considerations for choice of incubator type
must relate to reliability of operation and not to
cost or attractiveness of equipment.
Incubator should be kept out of drafts or direct
sunlight in order to prevent temperature inside the
incubator from changing outside the temperature
range specified (35° + 0.5°C).
Power supply should be selected so that there won't
be too many pieces of equipment on the same circuit.
Otherwise, circuits will be blown repeatedly.
Mercury bulb thermometer usually used in most incu-
bators. Recording thermometer is acceptable, but,
it should be calibrated against a mercury bulb
thermometer which has been certified by National
Bureau of Standards. The NBS certified thermometer
always should be used with fts certificate and
correction chart.
Saturated relative humidity is required in order to
make the incubation more efficient (heat is trans-
ferred to cultures faster than in a dry incubator).
Furthermore, culture medium may evaporate too fast
in a dry incubator.
Allow enough time after each readjustment to permit
the incubator to stabilize before making a new ad-
justment. At least one hour is suggested.
Incubator temperature can be held to much closer
adjustment if operated continuously. Temperature
records should be kept in some form of permanent
record. A temperature record book is suggested with
daily recording of values. If a recording thermom-
eter is used, the charts may be kept as permanent
record; if so, be sure that the charts are properly
labeled to identify the incubator and the period
covered.
Uniform temperature (35°C + 0.5°C) is to be main-
tained on shelves in use.
Autoclaves differ greatly in design and in method
of operation. Some are almost like home-style
pressure cookers; others are almost fully automatic.
This is a subject which requires separate instruc-
tion; and should be related to the exact make and
model of equipment you will use in your own
laboratory.
Std. Beth. 14:880
Std. Meth. 14:881
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WATER MONITORING PROCEDURES: Verified Membrane Filter Test for Drinking Water
FIELD AND LABORATORY EQUIPMENT
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.2.1 (Cont'd.)
A.3.1-2
Vertical autoclaves and household pressure cookers
may be used in emergency service if equipped with
pressure gages and thermometers with bulbs posi-
tioned 1 inch above the water level. However, they
are not to be considered the equivalent of the gen-
eral purpose steam sterilizer recommended for
permanent laboratory facilities. Their small size
is inadequate for large-volume workloads, and they
can be difficult to regulate.
The following requirements must be met regarding
autoclaves of sterilizing units:
a. Reaches sterilization temperature (121°C), main-
tains 121°C during sterilization cycle, and re-
quires no more than 45 minutes for complete
cycle.
b. Pressure and temperature gages on exhaust side
and an operating safety valve.
c. No air bubbles produced in fermentation vials
during depressurization.
d. Record maintained on time and temperature for
each sterilization cycle.
Distilled water in bacteriological laboratory must Std. Meth.
not contain substances which will prevent any bac-
teria from growing in culture medium in which the
distilled water is used or will be highly nutritive.
There are procedures for testing quality of dis-
tilled water; but these should be undertaken only by
professional bacteriologists or in laboratories where
this is done regularly. Use only glass stills or
block tin lined stills.
Requirements for distilled water include the following:
Test Analysis Requirement
PH 4.5-875 '
Conductivity 0.1 megohm as resistivity or
<5.0 micromhos/cm at 25°C
Trace metals:
A single metal
Total metals
14:645-49
14:888-89
Test for bactericidal proper-
ties of distilled water
(Std. Meth. 14:880)
•ree chlorine residual
Standard plate count
Not greater than 0.5 mg/1
Equal or less than 1.0 mg/1
0.8-3.0
0.0
Less than 10,000/ml
Conducted
Monthly
Monthly
Annually
Annually
Monthly
Monthly
6-27
-------
WATER MONITORING PROCEDURES: Verified Membrane Filter Test for Drinking Water
FIELD AND LABORATORY EQUIPMENT
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.4.1
A.5.1-4
A.5.4.4b
A.6.3b
C.3.2
pH Meter: See cited reference.
Glassware: See cited reference on pipets and gradu-
ated cylinders, media utensils, bottles.
Glassware can be checked for bacteriostatic or in-
hibitory residues by a bacteriological test proce-
dure which, like the distilled water suitability
test, should be undertaken only by professional
bacteriologists or in laboratories where this test
is done on a regular basis.
Std. Meth. 14:882
Std. Meth. 14:882-85
FUNNEL
FUNNEL, HOSE, AND
PINCHCOCK ASSEMBLY
PINCHCOCK
GLASS TUBE
NOTE UNIT NEED NOT BE
STERILE FOR MEDIUM
DELIVERY ONLY.
Alternately, it is authorized to use in "inoculation
stick" for transfers and even for colony picking. A
precisely sized and sterilized stick is intended for
a one-time use and, if used, eliminates the need for
a burner during the transfer or colony picking pro-
cedure. Of course, individual ones will be required
for each tube as colony to be processed to maintain
purity of culturing. Discard into a germicidal
solution prior to discarding.
Also available are re-sterilizable loops used once,
re-sterilized, and available for future transfers.
Std. Meth. 14:917
14:883-84
6-28
-------
EFFLUENT MONITORING PROCEDURE: Verified Membrane Filter Test for Drinking Water
FIELD AND LABORATORY ANALYSIS
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
B.3
There is no such thing as a "standard" data sheet
for bacteriological tests. A suggested one is
shown below and will be used for this training
exercise:
Data Sheet
Verified Membrane Filter Tests
Sample No.
and Date
Culture
No.
Type MF
Colony
L
24 hr
STB
46 hr
BG
24 hr
LBB
46 hr
EMB
Aqar
Gram
Stain
Remarks 1
Analyst
Note: Some data sheets are made to include the
verified test with the standard coliform MF test
results on the same sheet. Whatever approach is
made, however, it is important to have a place to
indicate the reportable MF coliform count based on
the results of the verified test which could cause
a count adjustment.
This outline was prepared by: Rocco Russomanno,
Microbiologist, National Training and Operational
Technology Center, MOTD, OWPO, USEPA, Cincinati,
Ohio 45268
6-29
-------
A PROTOTYPE FOR DEVELOPMENT OF
ROUTINE OPERATIONAL PROCEDURES
for the
STANDARD PLATE COUNT
as applied in
WATER TREATMENT FACILITIES
WASTEWATER TREATMENT FACILITIES
and in the
MONITORING OF EFFLUENT WASTEWATERS
National Training and Operational Technology Center
Municipal Operations and Training Division
Office of Water Program Operations
U. S. Environmental Protection Agency
BA.MET.lab.WMP.3.5.78
7-1
-------
STANDARD PLATE COUNT
FLOW SHEET
BLANK
CONTROL
VOLUMES
DELIVERED
(0.1 ML) ( 1 ML]
OPTIONAL, OR
WHEN REQUIRED
MEDIUM
CONTROL
OPTIONAL TESTS
PIPET
CONTROL
DISH
CONTROL
ROOM
CONTROL
INCUBATE DISHES
AT 35 °C ± 0.5°C
FOR 48+3 MRS
SELECT PLATE/PLATES
WITH COLONIES NUMBERING
30 - 300
CALCULATE COUNT/ML
REPORT COUNTS AS REQUIRED
BY REGULATORY AGENCY OR
STORE IN OFFICAL RECORDS
7-3
-------
WATER MONITORING PROCEDURE: Standard Plate Count
1. Applicability of Test:
a. In water treatment plant, reservoir, and distribution line quality
control, the objective of the test is to:
1) provide a method of monitoring for changes in the bacteriological
quality of finished water in storage reservoirs and distribution
systems,
2) indirectly limit the occurrence and magnitude of Pseudomonas,
Flavobacteriurn and other secondary pathogenic invaders that
could pose a health risk in the hospital environment,
3) reduce problems in the detection of low densities of total
coliforms due to interference by non-coliform bacteria,
4) monitor the effectiveness of chlorine throughout the
distribution network and provide a warning of filter effluent
quality deterioration and the occurrence of coliform break-
through, and
5) indicate the existence of sediment accumulation in the
distribution network that provides a protective habitat for
the general bacterial population.
Reference (1-5 above): Is the Total Count Necessary, Geldreich, E.,
AWWA Technology Conference Proceedings,
Cincinnati, Ohio, December 3-4, 1973.
b. Although not currently a test requirement, strong recommendations have
been made for its future inclusion or use:
Academy of Science
National Interim Primary Drinking Water Regulations
Office of Water Supply, U. S. Environmental Protection Agency
(Dec. 1975).
c. Knowledge of test procedure is required for conducting the "Suitability
of Distilled Water Test" which is a required bacteriological test
procedure.
d. Establishment of "base line" general bacteriological data in conjunction
with the coliform analysis regarding proposed regulations concerning
modification of existing disinfection practices.
2. Brief Description of Analysis:
A selected aliquot of water sample or its dilution is measured into a petri
dish and a liquified, temperature controlled agar medium is added. An even
distribution of organisms is accomplished by plate rotation and then the
plate is allowed to harden prior to plate inversion and incubation at 35° +_
0.5° C for 48+3 hours. Plate(s) having proper range(s) of colonies are
counted and caTculation(s) made to determine the count/ml.
7-4
-------
WATER MONITORING PROCEDURE: Standard Plate Count
3. Applicability of this Procedure:
a. The range of total count concentrations:
These ranges of total count
If the sample volumes used are organisms covered are
1 ml, 0.1 ml and 0.01 ml 30 to 30,000/ml
b. Pretreatment of samples in accordance with Standard Methods, 14th Ed.
(p 904).
This procedure conforms to the Standard Plate Count as described in Standard
Methods for the Examination of Water and Wastewater. 14th Ed. (1975), p. 908.
7-5
-------
WATER MONITORING PROCEDURE: Standard Plate Count
Equipment and Supply Requirements
A. Capital Equipment:
1. Autoclave*, providing uniform temperatures up to and including 121° C,
equipped with an accurate thermometer, pressure gauges, saturated
steam power lines and capable of reaching required temperature within
30 minutes.
2. Balance, 0.1 g sensitivity at load of 150 g.
3. Incubator*, air, to operate at 35° C + 0.5° C.
4. Oven*, hot-air sterilizing, to give uniform temperatures and with
suitable thermometer to register accurately in range of 160-180° C.
5. pH Meter, accurate to at least 0.1 pH unit, with standard pH reference
solution(s).
6. Water Distillation Apparatus*, glass or block tin, or source of distilled
water suitable for bacteriological operations.
7. Incubator*, water, to operate at 45° +_ 1° C.
8. Refrigerator*, to operate at 4° C.
9. Thermometer, mercury bulb, certified NBS or calibrated against a certified
NBS thermometer 0.5° intervals and have 160-180° C as part of range.
B. Reusable Supplies:
1. Apron or coat suitable for laboratory.
• 2. Baskets, wire for discarded cultures.
3. Bottles, dilution*, 6 oz. screw caps, with 99 ml volume level etched on
one side.
4. Bottles, sample*, preferred characteristics being 250 ml (6-8 oz.),
wide mouth, glass stopper.
5. Bottle, squeeze type, with disinfecting solution.
6. Burner, gas, Bunsen burner type.
7. Cans, pipet, aluminum or steel; not copper (If plastic, or other type of
prepackaged disposable pi pets are used, this item is unnecessary.)
8. Counter, colony, Quebec type, Darkfield model with guide plate, hand talley.
9. Cylinder, graduated, 100 ml.
10. Cylinder, graduated, 500 ml.
11. Dish*, petri, sterile, 100 mm diameter, >_ 15 mm in height, with glass or
porous tops preferred (presterilized, sterile one-time-use plastic tubes
may be used).
12. Flask*, Erlenmeyer, 250 ml capacity.
13. Flask, Volumetric, 1 liter.
14. Pan, to receive discarded contaminated pi pets and glassware (must contain
disinfectant before use).
15. Pipets*, 1 ml, having 0.1 ml increments, sterile, cotton plugged, glass or
disposable plastic, TD type (NOT a "blowout" type).
16. Pipets, 5 ml, having 1 ml increments (have several on hand).
17. Sponge, for cleaning desk top.
18. Thermometer, mercury bulb, certified NBS or calibrated against a certified
NBS thermometer 0.5° intervals and have 30-40° C as part of range.
7-6
-------
WATER MONITORING PROCEDURE: Standard Plate Count
Equipment and Supply Requirements (Continued)
C. Supplies Used Up In the Analysis (must be replaced when stocks get low):
1. Cotton, nonabsorbent.
2. Disinfectant, for bench tops. (Use household bleach solution prepared
according to Instructions on bottle.)
3. Distilled water, suitable for bacteriological cultures (note distillation
apparatus required In capital equipment).
4. EDTA (ethylene dinitrilotetraacetic add).
5. Foil, aluminum.
6. Paper, Kraft.
7. Magnesium Sulfate (MgS04-7H20) (recommend purchase of 1/4 Ib. units).
8. Pencil, wax, (recommend soft wax equivalent to Blaisdell 169T).
9. Potassium Di hydrogen Phosphate (KH.POJ (recommend purchase of 1/4 Ib. units).
10. Sheet, Data, SPC.
11. Sodium Hydroxide (NaOH).
12. Sodium Thiosulfate
13. Tryptose Glucose Yeast Agar, dehydrated medium (recommend purchase of
1/4 Ib. unit).
*Iterns marked are needed 1n quantities or require size or space allowances
which cannot be specified here, as they vary according to the dally analysis
schedule. As a rule-of-thumb, space/size or quantity requirements should be
at least 3 times the normal dally requirements. For further Information on
specifications for equipment of supplies, see the Microbiology Section of
the current edition of Standard Methods for the Examination of Hater and
Wastewater.
7-7
-------
MATER MONITORING PROCEDURE: standard Plate Count
7-8
OPERATING PROCEDURES
STEP SEQUENCE
INFORMAT ION/OPERAT ING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
1. 35° C incubator
set-up, adjust-
ment
1. Place 35° C Incubator In
permanent location.
2. Install thermometer.
3. Install shallow pan of
water in bottom of incu-
bator.
4. Connect incubator to
electric power source.
5. Adjust temperature until
stabilized at required
temperature.
6. Operate bacteriological
incubator continuously.
Aa. All pretest procedures completed before starting
other first-day procedures.
la. Out of drafts or places where it will be in direct
sunlight part of day.
Ib. Location convenient to laboratory bench.
Ic. Convenient source of electric power.
2a. Thermometer functions at least in 30° - 40° C
range and has intervals of 0.5° or less indicated^
Meets NBS standards.
2b. Location should be central in incubator.
2c. Mercury bulb thermometer should be fitted with
cork or rubber stopper and mounted in small
bottle filled with liquid (glycerine, water, or
mineral oil).
3a. In most laboratory incubators a pan having about
1 square foot of area, with water about 1 inch
deep, is satisfactory.
3b. Maintains condition of saturated relative humidity*
required in bacteriological incubator.
3c. Requires daily check, with addition of water as
necessary, to keep water in pan at all times.
4a. Many incubators have pilot light to indicate
power turned ON.
5a. Manufacturer's instructions for method of
temperature adjustment.
5b. Operation must be at 35 +, 0.5° C.
5c. Allow about 1 hour between adjustments.
6a. Requires daily check with written temperature
record, with adjustment and water addition as
necessary.
V.A.I
(p. 7-45)
V.A.1.1
(p. 7-45)
V.A.I.2
(p. 7-45)
V-A.l.3
(p. 7-45)
V.A.I.5
(p. 7-45)
V.A.I.6
(p. 7-45)
-------
WATER MONITORING PROCEDURE: Standard Plate Count
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
2. Water bath Incuba-
tor setup, adjust-
ment
1. Place water bath Incubator
In permanent location.
2. Put water In water bath.
3. Install thermometer.
4. Connect water bath Incu-
bator to electric power
source and turn ON.
5. Adjust temperature until
stabilized at required
temperature.
6. Operate water bath incu-
bator continuously.
la. On bench or table surface.
Ib. Out of drafts or place in which it will be in
direct sunlight part of day.
Ic. Location convenient to laboratory bench.
Id. Convenient source of electric power.
2a. Distilled or deionized water preferred, tap water
accepted.
2b. Water must be deep enough that when a flask of
medium is placed in the water bath the water is as
high as the medium inside the flask. Yet it must
not be so deep as to let the flask float or reach
the cap or closure.
3a. Functions at least in 40° - 50° C range. Meets
NBS standards. Have at least 0.5° C increment
markings.
3b. Most water baths provide for corner location of
thermometer (for protection from breakage).
4a. Pilot light should come on.
5a. Manufacturer's Instructions for location and
method of temperature adjustment.
5b. Operation must be at 45° + 1.0° C.
5c. Allow about 1 hour between adjustments.
6a. Requires daily check with written temperature
record, with adjustment as necessary.
6b. Requires daily check of water level and addition
of more as needed.
6c. With tap water in water bath, may require periodic
scum removal from inner walls.
7-9
-------
WATER MONITORING PROCEDURE: Standard Plate Count
7-10
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
3. Oven, sterilizer
setup
4. Autoclave setup
1. Place oven sterilizer in
permanent location.
2. Install thermometer.
3. Connect oven sterilizer to
power source and turn on.
4. Adjust temperature to
stabilize at required
temperature.
5. Operate oven sterilizer
only when needed. Turn
off when not in use.
1. Install and operate auto-
clave according to manu-
facturer's instructions.
la. Convenient to source of electric power; usually
on table or bench.
2a. Should indicate the 160° - 180° C range, be
accurate within this interval, and be marked in
1.0 degree intervals. Thermometer bulb is within
a cylinder filled with a fine sand and positioned
on the center shelf of chamber.
3a. Usually has pilot light to indicate power on.
4a. Operated as near to 170° C as possible; not lower
than 160 nor higher than 180° C. Check to verify
that the 170° C temperature is reached and is
maintained within +. 10° for a 2 hour period.
5a. Turned ON in advance of need to permit reaching
required temperature before introducing material
to be sterilized.
5b. Oven sterilizer used to sterilize dry glassware,
metal objects.
5c. Oven sterilizer NOT used with culture media,
solution, plastics, rubber objects, or with
anything containing or including these.
5d. Paper-wrapped glass pi pets may be sterilized in
oven sterilizer.
la. Autoclaves extremely variable in design and
operation; also, potentially dangerous.
Ib. Used to sterilize objects made of, or including
liquids, rubber, culture media.
V.A.3.1-5
(p. 7-46)
V.A.4.1
(p. 7-46)
-------
WATER MONITORING PROCEDURE: Standard Plate Count
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
5. Water distillation
equipment setup
6. pH meter
operation
7. Glassware
preparation
1. Install and operate in
accordance with manu-
facturer's instructions.
2. Operate continuously or
intermittently as required
to maintain adequate
supplies of distilled
water.
1. Have unit available and
operational.
1. Wash all glassware in hot
detergent solution.
2. Rinse at least once in
hot tap water.
3. Rinse in distilled water,
at least 6 successive
times.
Ic. Glassware may be autoclave sterilized but must be
dried afterward.
Id. Most plastics NOT sterilized in autoclave;
plastics usually require chemical sterilizers.
le. Autoclave usually operated at 121° C for 15 min.
If. Sterilized media must be removed from autoclave
as soon as possible after autoclave is reopened.
la. Must produce distilled water meeting quality
requirements for bacteriological tests.
2a. Reserve supplies kept in borosilicate glass
carboys or in plastic carboys made of material
which will not dissolve substances which will
affect growth of bacteria.
2b. Same distillation apparatus used for bacterio-
logical purposes may be used for chemical
reagents.
la. Unit for pH check on finished culture media.
Ib. Used in preparation of stock solution of
potassium dihydrogen phosphate.
la. Nontoxic detergent.
Ib. Be sure all contents and markings are washed
away.
V.A.5.1-2
(P. 7-47)
V.A.6.1
(P. 7-47)
V.A.7.1-4a
(P. 7-47)
7-11
-------
WATER MONITORING PROCEDURE: Standard Plate Count
7-12
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
8. Chemical solutions
preparation for
sample bottles
4. Dry in air.
Sodium Thiosulfate
1. Weigh 10.0 grams of sodium
thiosulfate
2. Dissolve completely in
50-60 ml distilled water.
3. Add distilled water to
bring final volume to
100 ml.
4. Transfer to labeled bottle
EDTA
*
5. Weigh 15.0 grams of EDTA.
6. Dissolve completely in 50-
60 ml distilled water.
7. Add distilled water to
bring the final volume to
100 ml.
8. Transfer to labeled
clean bottle.
4a. No visible spots or scum; glass should be clean,
and sparkling.
4b. Glassware suitable for use in bacteriological
operations.
la. Used for dechlorination of samples. Not required
(but not detrimental to) for unchlorinated samples,
Ib. Use of trip balance accepted.
2a. 100 ml graduated cylinder satisfactory.
4a. Labeled as 10% sodium thiosulfate; dated;
preparer's name; and stored in refrigerator.
5a. Used for water samples high in copper, zinc, or
heavy metals. Normally not necessary for most
treated water supplies.
5b. Use of trip balance accepted.
6a. A 100 ml graduated cylinder is satisfactory.
8a. Labeled as 15% ethylene dinitrilotetraacetic
acid (EDTA); dated; preparer's name; and stored
in refrigerator.
V.A.7.1.4b
(p. 7-47)
-------
WATER MONITORING PROCEDURE: Standard Plate Count
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedure
(Continued)
9. Sample bottle
preparation
10. Pipet preparation
1. Deliver 0.1 ml or .2 ml
of 10% sodium thiosulfate
solution to each sample
bottle. (.1 ml to 4 ounce
or 120 ml size and .2 ml
to 6-8 ounce or 250 ml
size).
2. Deliver .3 ml or .6 ml of
15% EDTA solution to each
sample bottle (.3 ml to
4 ounce or 120 ml size and
.6 ml to 6-8 ounce or
250 ml size).
3. Place cover on sample
bottle.
4. Place paper or metal foil
cover over bottle cap or
stopper.
5. Sterilize sample bottles
in sterilizing oven.
6. Store sample bottles in
clean, dry place until
used.
1. Inspect pipets to be pre-
pared for use; discard and
destroy all having chipped
or cracked tips or tops.
la. Use 1 ml pipet.
Ib. Provides adequate sodium thiosulfate for
neutralizing chlorine in sample.
Ic. Return stock sodium thiosulfate solution to
refrigerator.
2a. Use 1 ml pipet.
2b. Provides adequate EDTA chelating agent for metals
in sample. Not necessary for sample which does
not contain copper, zinc, or heavy metals.
2c. Return stock solution of EDTA to refrigerator.
V.A.9.1-6
(p. 7-48)
4a. Protects opening of sample bottle from accidental
contamination.
5a. One hour at 170° C. (See A.3).
la. Cleanliness of pipet must be equivalent to
glassware.
7-13
-------
WATER MONITORING PROCEDURE: Standard Plate Count
7-14
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedure
(Continued)
11. Plate count agar
preparation
(tryptose glucose
yeast agar)
2. Insert plug of non-
absorbent cotton Into
mouthpiece of each clean,
dry plpet.
3. Place a layer of glass
wool or several layers
of paper padding In bottom
of plpet can.
4. Place 18-24 pi pets In each
plpet can, delivery tip
down.
5. Sterilize cans of pi pets
In oven.
6. Store cans In clean, dry
place until used. Mark
cans as 1 ml sterile
pipets.
7. When can of pi pets Is
opened for first use, pass
the exposed ends of the
pipets through flame,
slowly.
1. Weigh 2.4 grams of de-
hydrated plate count
agar. Cover bottle of
mediurn tightly after
removal.
2a.
2b.
For protection of user when pipetting sample.
Cotton plug must be tight enough to prevent easy
removal, either by the pipetting action or by
handling, and yet loose enough to permit easy
air movement through the plug.
3a. For protection of pipet delivery tips within can.
4a. Orientation permits removal of sterile pipets from
can without contamination by user.
5a. 1 hour at 170° C. (See A.3 of procedures).
6a. Laboratory cabinet or drawer recommended.
7a. Burns off excess cotton sticking out of pipet
mouthpiece.
7b. Cover kept on can at all times except when
samples are being inoculated.
la. Dehydrated media can take moisture out of air
(hygroscopic).
Ib. Discard caked media; use only dry powder.
V.A.10.1-6
(p. 7-48)
V.A.10.7
(p. 7-48)
-------
WATER MONITORING PROCEDURE: Standard Plate Count
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedure
(Continued)
2. Dissolve in TOO ml of
distilled water.
3. Close neck of flask with
a plug of non-absorbent
cotton. Cover with a
cap of aluminum foil.
4. Sterilize in autoclave.
5. Pour a small amount of
medium (3-6 mis) into a
small petri dish or clean
container.
6. Cool hot liquid medium to:
A. Room temperature and
place in storage area.
2a. Distilled water meets suitability requirements.
2b. Use 250 ml Erlenmeyer flask with foil cap.
2c. Use flowing steam (100° C) or boiling water to
dissolve. Some autoclaves can be adjusted to give
flowing steam mode. See manufacturer's instruc-
tion manual.
2d. Do not prolong boiling or exposure to steam.
Agitate frequently when boiling is used to
prevent burning. Boil for 1 minute.
2e. Expose to boiling or steam only until agar in
medium has dissolved.
3a. Cotton tight enough to support weight of flask
but not too tightly packed to resist easy re-
moval .
4a. Within 1 hour after medium prepared.
4b. Sterilization at 121° C for 15 minutes.
4c. Medium must be removed from autoclave as soon as
possible after pressure has returned to normal.
Use "slow-vent" (liquid cool) mode of steam
removal.
4d. Total elapsed time from time of placement in
autoclave to removal after sterilization, must
not exceed 45 minutes.
5a. Use sterile (aseptic) handling techniques to
prevent medium contamination. Recap flask
quickly.
6.A.a. If medium is to be used for future tests.
6.A.b. If more than three hours will elapse before
test procedure.
6.A.c. place in refrigerator (4°).
V.A.11.2b
(p. 7-48)
V.A.11.2b
(p. 7-48)
V.A.11.6.A.C
(p. 7-49J
7-15
-------
WATER MONITORING PROCEDURE; Standard Plate Count
7-16
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedure
(Continued)
12. Dilution water
blanks preparation
B. 44-46° C in water bath.
7. Check pH of prepared
medium in dish (See 11.5),
1. Prepare stock solution of
potassium dihydrogen phos-
phate (KH2P04); dissolve
34.0 grams of the KH2P04
in 500 ml distilled water.
Adjust to pH 7.2 with
IN NaOH, and dilute to 1
liter with distilled water
2. Prepare stock solution of
magnesium sulfate (MgSO..
7H20) by dissolving 50
grams of this chemical in
500-600 mis of distilled
water and, after complete
dissolving, bring the
final volume to 1 liter in
a volumetric flask.
6.B.a. Hold for no longer than 3 hours.
6.B.b. If not used within the above time interval,
cool to room temperature and place in
refrigerator. (See A.11.6.A.c.)
7a. Should be pH 7 +_0.1. Probe(s) of pH meter can
be inserted in medium.
7b. Record pH in Quality Control records.
7c. Discard plate after pH check.
7d. Medium will usually be within pH requirements. If
not, reject prepared medium and check procedures,
glassware, etc. for abnormalities. Prepare a new
lot of Standard Plate Count medium when the
cause has been found (glassware with acid residue,
poor water supply, etc.).
la. Distilled water may be measured in 500 ml
graduated cylinder.
Ib. Finished solution labeled "Stock KH,PO. for
Dilution Water." * 4
Ic. Stored in refrigerator.
Id. Discard stock solution and prepare new solution
if mold appears.
V.A.12.1.1d
(p. 7-49)
-------
WATER MONITORING PROCEDURE: Standard Plate Count
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedure
(Continued)
13. Petri dish
preparation
Prepare working solution
of dilution water by add-
ing 1.25 ml
KH2P04
and 5 ml of the magnesium
sulfate stock solution to
each liter of distilled
water to be made up as
dilution water.
4. Deliver enough working
solution to each dilution
water bottle so that after
sterilization the bottles
will contain 99 +_2 ml of
dilution water.
5. Place caps on dilution
bottles loosely.
6. Sterilize in autoclave.
7. Promptly remove from auto-
clave, tighten bottle caps
cool to room temperature.
8. Store in cool place.
1. Clean and dry dishes;
sterilize.
3a. 5 ml pi pet satisfactory for 1 liter amounts of
dilution water. 10 ml pi pet better when several
liters are being made.
3b. 1-liter graduated cylinder satisfactory for
measurement of distilled water.
3c. Use separate pipets for each solution to prevent
contamination.
4a. 100 ml graduated cylinder ordinarily satisfactory,
Pipetting machine desirable but not mandatory.
Amount cannot be stated exactly, as sterilization
evaporation differs from one autoclave to another,
Commonly, about 102 mis are required.
6a. 15 minutes at 121° C.
steam evacuation.
Use "slow-vent" mode of
8a. Dilution water ready for use. May be stored
indefinitely in screw-capped bottles.
la. If petri dishes are glassware, they meet re-
quirements as previously described. Sterilize
at 15/15 in the autoclave or 170° for 1 hr. in
the oven. Glass dishes may be sterilized and
stored in stainless steel or aluminum cans or
wrapped in Kraft paper before sterilizing.
V.A.12.4
(p. 7-49)
V.A.12.5
(p. 7-49)
V.A.12.6
(p. 7-49)
V.A.12.8
(p. 7-49)
7-17
-------
WATER MONITORING PROCEDURE; Standard Plate Count
7-18
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedure
(Continued)
14. Final equipment
and supply check
1. Check readiness of
equipment and supplies
before starting sample
examinations.
lb. If petri dishes are plastic and presterilized
as purchased, they are used directly as taken
from the packaging for single use only.
la. Check general list of equipment and supplies.
B. First Day Procedures
1. Equipment
maintenance
2. Sample collection
1. Check, record, and adjust
incubator temperatures.
1. Collect sample.
2. Record the on-site
sampling informations.
la. Location as determined by requirement.
lb. Sampling methods as described in Standard Methods.
3. Transport sample to
laboratory.
2a. Most organizations have sample tags which at
least include:
Date of Sampling
Time of Sampling
Sample Locaton
Collector's Name.
3a. Ideally under refrigeration (below 10° C) or in
iced condition.
3b. If unrefrigerated, the maximum time allowable
between collection and examination is 8 hours
(NOTE: The maximum transit time is 6 hours).
3c. If extended holding time is unavoidable, maintain
temperature below 10° C and do not exceed 30 hours
between collection and examination.
-------
WATER MONITORING PROCEDURE: Standard Plate Count
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First Day Procedures
(Continued)
3. Preparation of
data sheet
1. Fill in data sheet to
show sample information.
2. Select sample inoculation
volumes.
3. Enter information in
laboratory data sheet
for sample volumes.
la. Required information should be on sample tag.
Ib. Most data sheets record:
Information as in B.2.2a
Name of Analyst
Laboratory sample Identification
Time of Start of Analysis.
2a. For purposes of this WMP (Water Monitoring
Procedure), sample volumes of 1.0 ml; 0.1 ml,
and 0.01 ml are required.
2b. Above volumes are recommended for drinking water
samples.
2c. Samples other than drinking water may require
higher dilutions (Ex. 0.1; 0.01; 0.001).
3a. Show sample inoculation volumes in mi Hi liters
(mis) or decimal amounts (1; 1/10; 1/100)
(See VII.B.3).
ianple
tolume
o.l
48 hr
VII.B.3
(p. 7-53)
7-19
-------
WATER MONITORING PROCEDURE: Standard Plate Count
7-20
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First Day Procedures
(Continued)
4. SPC medium
preparation
5. Laboratory bench
disinfection
6. Assembly of test
related materials
1. Prepare or have available
sufficient SPC medium.
If medium is taken from
refrigerator: (A.11.6.A)
A. Melt medium.
B. Cool to 44-46° C.
If from water bath:
(A.11.6.B)
C. Check that medium has
not been incubated
excessively.
1. Disinfect laboratory
bench; wipe dry.
l.A.a. Melt by exposure to flowing steam or by
placing flask in boiling water.
l.A.b. Heated only until fully liquified. DO NOT
RESTERILIZE.
l.A.c. Only one remelting is authorized; discard if
not used.
l.B.a. Place in water bath until at temperature
(15 to 20 minutes for the 100 ml preparation).
l.B.b. Medium must not be held in incubator for over
3 hours.
l.C.a. Medium must not be held in incubator for
over 3 hours.
'a. Sponge and disinfectant; paper toweling.
B6. Consult general list of equipment and supplies.
-------
WATER MONITORING PROCEDURE: Standard Plate Count
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First Day Procedures
(Continued)
1. Label 8 sterile petri
dishes for SPC Test.
la. Three sample volumes run In duplicate.
Ib. Label with wax pencil as follows:
ASSIGNED
LABORATORY
NUMBER
SAMPLE VOLUME
2. Select and label a
a representative 99 ml
dilution blank.
Ic. Does not include "optional tests."
2a. Representative of a "batch" of sterilized bottles
which were prepared and sterilized together.
2b. Select one which has meniscus above calibration
mark, or, if all bottles are below mark:
Transfer, by using aseptic (sterile) techniques,
"batch" water from one bottle to another to
above calibration mark (approx. 1/8 inch above).
2c. Label bottle with assigned sample number and
0.01 dilution volume:
V.B.6.1C
(p. 7-51)
MENISCUS
7-21
-------
WATER MONITORING PROCEDURE: Standard Plate Count
7-22
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First Day Procedures
(Continued)
7. Sample inoculations,
dilutions, and
pouring
1. Agitate sample bottle.
2. Fill 1 ml pipet with
sample from sample bottle,
3. Place 1 ml volume in petri
dishes labeled 1.0 ml
(two dishes).
la. At least 25 shakes over space of at least 1 foot
in 7 seconds or less.
2a, Do not place pipet tip more than 1 inch below
surface of sample.
2b. Do not wet cotton plug.
2c. Line sample water line with "0" marking on pipet.
2d. Touch off any droplets along inside of bottle
before removing pipet. Do not touch outside of
sample bottle.
2e. Close sample bottle; retain on bench for lateruse.
3a. Do not perform test in direct sunlight.
3b, Allow dishes to be opened only enough to insert
pipet easily.
COTTON PLUG
TOUCH BOTTOM DISH
I.B.7.1
(p. 7-40
-------
HATER MONITORING PROCEDURE: Standard Plate Count
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First Day Procedures
(Continued)
4. Place 0.1 ml volume in
petri dishes labeled 0.1
ml (two dishes).
5. Pour the four dishes (two
1.0 ml and two 0.1 ml).
3c
Pipet 1.0 ml volume into dishes. When volume
has been delivered, touch off once any remaining
droplet against a dry spot on the dish bottom and
withdraw pi pet.
If pipet becomes contaminated touching bench;
touching outside of petri dish; touching hand;
etc.; discard and replace with a sterile one;
repeat step.
3e. Close petri dishes immediately.
3d
4a.
4b.
5a.
5b.
5c.
5d.
5e.
Repeat steps 3b - 3e using the same sterile pipet
but using a 0.1 ml volume and not retouching the
plate.
Any convenient 0.1 ml volume located between the
0 and 1.0 ml graduations is acceptable.
0.1 ml O.lml 0.1 ml
0
•
Ol
0
•
Wl
o
•
-p»
o
CO
Use SPC medium from either B.4.1A-B or B.4.1C.
Quickly pour 10-12 ml of the melted (44-46° C)
SPC into each of the four dishes.
Avoid splashing of medium.
Open cover of dish only enough to allow ease of
pouring of medium.
Gently "swirl" each of the four dishes to obtain
distribution of bacteria within the medium. Keep
plates on bench while moving.
7-23
-------
WATER MONITORING PROCEDURE; Standard Plate Count
7-24
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First Day Procedures
(Continued)
6. Prepare blank control
plate.
7. Prepare 0.01 (or 10"2)
dilution.
5f. An acceptable "swirling" sequence could be as
fol1ows:
III.B.7.5.
(p. 7-42)
5f
1 234
Do not allow motions to splash medium on cover of
dish. If this occurs re-inoculate another dish.
5g. Allow plates to remain on bench to "harden"
without disturbing.
6a. Use previously labeled sterile petri dish "blank
control" (316 BC).
6b. Using a sterile 1 ml pipet, remove water from the
dilution blank after shaking as previously
described, until the meniscus is even with the
etched marking on the blank (See B.5.2.2a-c) and
place this water into the blank control place.
Close blank and retain for further requirement.
6c. Using techniques as in B.5.5a-g, prepare blank
control plate.
7a. Shake sample bottle using agitation method
previously described.
7b. Using a sterile 1 ml pipet, add 1 ml from the
sample bottle to the 99 ml dilution blank which
was prepared in B.6.6b. Discard pipet.
7c. Shake the dilution blank. Proper agitation
previously described.
-------
WATER MONITORING PROCEDURE: Standard Plate Count
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First Day Procedures
(Continued)
8. Incubation of
plates
8. Place 0.01 ml volume in
dishes labeled 0.01 ml
(two dishes).
9. Pour the two 0.01 dishes.
10. Pour the MC (medium
control) plate.
1. Collect all of the
hardened plates.
2. Place inverted'plates in
35° C incubator.
8a. Using a 1.0 ml delivery volume with a sterile
pi pet, follow the previous steps to place 1 ml
volume in dishes from the dilution blank. Touch-
off droplet against a dry spot. Close petri
dishes. (Steps B.7.3.b-e).
9a. Use previously learned steps for pouring
(B.7.5.5.a-g).
lOa. Use previously labeled sterile dish (316 MC).
lOb. Pour 10-12 ml of SPC medium into dish. Close
dish immediately.
lOc. Allow to harden. "Swirling" is not necessary as
MC plate does not use sample.
la. Eight plates should be ready for further process-
ing (6 test plates; 2 control plates).
Ib. Allow no more than 20 minutes to elapse from
beginning test to collecting these plates.
Ic. Hardened plates can be inverted (turned over)
without flowing from fixed position.
2a. Plates inverted to prevent condensation droplets
from spreading on and causing irregular growth
to occur on surface of medium.
2b. Do not crowd dishes in incubator. No rrore than
four high and no touching of stacks with other
stacks or top or sides of incubator.
2c. Incubate for 48+3 hours.
7-25
-------
WATER MONITORING PROCEDURE; Standard Plate Count
7-26
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Third Day
Procedures
1. SPC colony
counting
1. Retrieve all plates from
incubator (8).
2. Count control plates.
la. Bring to lab bench where colony counting is done.
Ib. Assemble plates by sample volume
2 - 1 ml
2 - 0.1 ml
2 - 0.01 ml
1 - MC
1 - BC
2a.
2b.
Both MC and BC plates should show no bacterial
growth. Occasional growth, such as less than
2 colonies, can be tolerated as chance
contaminants.
View plates with the Quebec Colony Counter.
Good results are achieved (author method) by
removing dish cover and placing dish on guide
plate and then adjusting magnifier lens.
(P. 7-42)
V.C.2.2b
(p. 7-51)
LENS, MAGNIflER
ADJUSTING ROD
- DIRECTION OF
LIGHT SOURCE
DISH WITH
SPC MEDIUM
2c. If control plates are acceptable, proceed to the
plate counts. If control plates are unacceptable,
review procedures and/or discard contaminated
materials and abort test until fresh materials
are prepared for a fresh sampling.
-------
HATER MONITORING PROCEDURE: standard Plate Count
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Third Day
Procedures
(Continued)
3. Count test plates.
4, Record counts on data
sheet.
3a.
3b.
Scan the 3 pairs of plates for the ones which fall
between 30-300 colonies/plate.
With practice these can be easily ascertained,
but, with the new worker counts would have to be
made when uncertain until this skill is acquired.
Example:
1.0 ml
VOLUME
0.1 ml
VOLUME
0.01
VOLUME
TOO NUMEROUS
ACCEPTABLE
TOO LOW
4a. Since a number of situations can be found related
to counts, the possibilities are shown below:
Plates having 30-300 colonies
Sample
Volume
/
M
».«
T*f
•TAS
«/«
.fife
3
7
_
48
NOTE: TN indicates Too Numerous ( >300)
counts of 0.1 volume will be used for
calculations.
7-27
-------
WATER MONITORING PROCEDURE: Standard Plate Count
7-28
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Third Day
Procedures
(Continued)
4b.
No plate with 30-300 colonies
Sample
Volume
/
6.1
sue
AM
•?7
5i
A
4f
48 h
—
NOTE: Counts of 0.1 volume will be used for
calculations since they are CLOSER to
the limits.
4c. All plates with fewer than 30 colonies
Sample
tolune
/
a/
0*1
5
£
9»
/
0
0
48 hr
.
_
NOTE; Counts of 1 ml volume will be used for
calculations since they are the LOWEST
dilution plated (largest sample volume).
-------
WATER MONITORING PROCEDURE: Standard Plate Count
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Third Day
Procedure
(Continued)
4d.
Plate with no colonies
iaiqile
Volume
0.1
DkDl
4e.
NOTE; Counts of 1 ml volume (LARGEST VOLUME)
will be used for calculations.
All plates greater than 300 colonies
Sanple
Volume
001
NOTE: Counts of 0.01 volume (SMALLEST VOLUME)
will be used for calculations.
7-29
-------
MATER MONITORING PROCEDURE: Standard Plate Count
7-30
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Third Day
Procedure
(Continued)
4f. Crowded plates using Quebec Counter to
estimate Case #1:
Less than 10 colonies per square cm
A. There are less than 10 colonies per square cm
when the number of colonies is less than 10 as
the dish is viewed in the counter and one
representative square is counted:
PETRI DISH
VIEWING
WINDOW
W/GRIDS
NOTE: 7 COLONIES IN CENTER SQUARE
-------
WATER MONITORING PROCEDURE: Standard Plate Count
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Third Day
Procedure
(Continued)
B. Count 13 squares which have representative
colonial distribution. Seven (7) consecutive
horizontal and six (6) consecutive vertical
SOUMI
HOIIZONIAL
:©
C. Sum Squares: 47 + 42 = 89 colonies.
NOTE: When counting vertical and horizontal
squares, do not count a square more
than one time.
7-31
-------
WATER MONITORING PROCEDURE: Standard Plate Count
7-32
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Third Day
Procedure
(Continued)
D. Assuming that the 0.01 ml dilutions had this
situation, the entries would appear as follows
Sample
Volume
ft/
41
•As per example
Hypothetical ly derived
value from second plate
using same rationale.
Case #2
I More than 10 colonies per square cm
A. See C1.4.4f Case #1 for counting squares.
B. Count 4 representative squares. For example:
12; 17; 13; 20.
C. Average the count per sq. cm.
12 + 17 + 13 + 20 = 62
62 T 4 = 15.5 or 16.
-------
WATER MONITORING PROCEDURE: standard Plate Count
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Third Day
Procedure
(Continued)
2. SPC calculations
1. Calculate count per ml.
D. Assuming that the 0.01 ml dilutions had this
situation, the entries would appear as follows:
la.
iample
Volume
•As per example
Hypothetically derived
value from second plate
using same rationale.
Plates having 30-300 colonies
(See C.1.4.4a for example)
Formula:
Sum of colonies _
Sum of Volumes Tested, ml
Given Example:
45 + 58
.1 + .1
Count/ml
= 515 Count/ml
7-33
-------
WATER MONITORING PROCEDURE: Standard Plate Count
7-34
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Third Day
Procedure
(Continued)
A special case exists when more than one dilution
contains 30-300 colonies. Suppose, for example,
that the following counts were recorded:
Volu
a/
ifi-
The following calculation is necessary:
1777 Count/ml
180 + 145 * 35_+ 31
"TT+.1 + .01 + .01
Ib.
No plates with 30-300 colonies
(See C.1.4.4b for example)
* "
-r = 250 Count/ml
The counts of 27 and 23 were used since they
were closer to the plate range of 30-300.
-------
MATER MONITORING PROCEDURE: Standard Plate Count
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Third Day
Procedure
(Continued}
1C.
Id.
le.
All plates with fewer than 30 colonies
(See example C.1.4.4c)
y-J-y = ^| = 6.5 or 7 Count/ml
Plate with no colonies
(See example C.1.4.4d)
In order to use, for calculation purposes, a
series of plates, none of which have colonies,
assign a count of one (1) to each of the
largest sample volume and calculate the
count:
2.
2
1 Count/ml
However, a count derived from this reasoning
must be preceded by a less than (<) value.
Therefore, the recorded count would be < 1
Count/ml.
All plates greater than 300 colonies
(See example C.1.4.4e)
385 + 360
.01 + .01
745
= 37,250 Count/ml
7-35
-------
WATER MONITORING PROCEDURE: Standard Plate Count
7-36
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Third Day
Procedure
(Continued)
If. Crowded Plates
(See C.1.4.4f Case #1 and Case #2)
A. Case #1:
Multiply sum of 13 squares by 5:
Plate #1 89 (No. of colonies previously
calculated) x 5 = 445.
Plate #2 95 x 5 = 475.
445 + 475
= 460 estimated count
B. Case #2:
Multiply the Count/sq cm (previously found
to be 16 and 28) by 65 (No. of sq. cm. of
petri dTsh): ~~
Plate #1 16 x 65 = 1040
Plate #2 28 x 65 = 1820
Multiply by the reciprocal of the dilution
to determine the count per ml.
Let us assume that the dilution was 0.01 ml
for the plates being counted:
Plate #1 1040 x 100 = 104,000
Plate #2 1820 x 100 = 182,000
104.000 * 182.000
? = 143,000 Count/ml Estimated
II.C.I.4.4f
Case #2
(p. 7-41)
-------
WATER MONITORING PROCEDURE: standard Plate Count
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Third Day
Procedure
(Continued)
2. Record QC information and
Count/ml on data sheet.
2a. See data sheet (VII.B.4):
1
1
Quality Con
Medium Cent
P1pet Contr
Room Contra
Blank Contr
I ~
'
i 1
trol Information:
rol
fll
1
•ol
Reported Values
SPC/ml
2b. With the usual conditions of good control and
asceptic (sterile) handling techniques the QC
information will be acceptable:
Quality Control Information:
Medium Control
P1pet Control
Room Control
Blank Control
7-37
-------
MATER MONITORING PROCEDURE; Standard Plate Count
7-38
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Third Day
Procedure
(Continued)
2c. Record values to be reported:
Reported Values
SPC/ml
For the examples given note the following:
Calculated Count
per ml
515
1777
250
7
< 1
37,250
460
143,000
Reported Count
per ml (2 significant figures)
520 SPC See C. 2.1. la
1800 SPC See C. 2.1. la
250 SPC See C.Z.l.lb
7 SPC (See C.2.1.1c
< 1 SPC (See C. 2.1. Id
37,000 SPC (See C.2.1.1e
460 Estimated Plate Count (See C.2.1
Case #1)
140,000 Estimated Plate Count (See C.2.1
Case #2)
.If
.If
-------
WATER MONITORING PROCEDURE: Standard Plate Count
TRAINING GUIDE
SECTION TOPIC
I* Introduction
II* Educational Concepts - Mathematics
III* Educational Concepts - Science
IV Educational Concepts - Conmunications
V* Field & Laboratory Equipment
VI Field & Laboratory Reagents
VII* Field & Laboratory Analyses
VIII Safety
IX Records and Reports
*Training guide materials are presented here under the headings marked*.
These standardized headings are used through this series of procedures.
7-39
-------
WATER MONITORING PROCEDURES: Standard Plate Count
INTRODUCTION
Section I
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
B.7.1
The SPC method 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.
SPC procedures are based on certain fundamental
assumptions:
A. First, all of the living bacterial organisms
will remain viable if they are capable of
growth under the conditions cf the test; and;
B. Second, resultant growth of the organisms will
produce, within 48 hours at 35° C, a visible
colony under appropriate magnification.
7-40
-------
WATER MONITORING PROCEDURES: Standard Plate Count
Educational Concepts - Mathematics
Section II
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
C.1.4.4f
Case n
A reciprocal Is the fractional "Inversion" of a
value (I.e., reciprocal of 4 Is ]_; of 1 Is 4;
etc.) 4 4
Sample Volumes Also
Used Written As
1.0
0.1
0.01
1
1/10
1/100
Reciprocal
1
10
100
7-41
-------
WATER MONITORING PROCEDURES: Standard Plate Count
Educational Concepts - Science
Section
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
B.7.5.5f
C.1.2.2a
A gentle "swirling" action allows the bacteria
within the sample volume to mix with the SPC
medium and be distributed evenly throughout the
area of the petrl dish bottom. Keeping the
plate on the surface of the bench allows an
easier management of the manipulation. Since
the type of sample being considered in this WMP
does not allow the novice to this procedure to
"see" the sample being ultimately mixed and
distributed, a "practice" plate can be made by
using an opaque fluid (milk, cream, etc.) and
seeing the gradual and complete mixing occurring
as the sequential swirling is accomplished.
Bacterial colonies are visible growths which have
resulted from the multiplication of a simple
organism which was trapped within the gelled
agar-nutrient material. Colonial shapes are
usually easily discernable forms which the
beginner must learn to recognize and differentiate
from debris which in this medium (SPC) is usually
insoluble phosphate or undissolved particles.
Colonies are more uniform in shape than the debris
particles:
COLONIES
DEBRIS
(USUALLY
SMALLER)
A special type of colonial growth may sometimes
be encountered which requires special counting
rules. This growth 1s called a "Spreader" and
constitutes an irregular and possibly extensive
growth area which may or may not have originated
from a single organism. Spreaders are usually
top surface growth which therefore can grow with
less restrictions than 1s the case within the
agar mass where constraints to movement produce
a small confined growth area. The presence of
surface moisture as well as certain genera of
bacteria can cause extensive growth areas.
7-42
-------
WATER MONITORING PROCEDURES: Standard Plate Count
Educational Concepts - Science
Section III
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
C.1.2.2a
(Continued)
Plates containing spreading colonies must be so
reported on the data sheet. If spreaders exceed
one-half of the total plate area, the plate 1s not
used. Report as: No results. Colonies can be
counted on representative portions of plates 1f
spreading colonies constitute less than one-half
of the total plate area, and the colonies are
well-distributed.
A. Count each chain of colonies as a single
colony.
B. Count each spreader colony that develops as a
film of growth between the agar and the petrl
dish bottom as one colony.
C. Count the growth that develops In a film of
water at the edge or over the surface of the
agar as one colony.
D. Report as: Estimated Standard Plate Count/ml.
If spreading colonies (spreaders) are encountered
on the plates/s selected, count colonies on
representative portions only when
A. Colonies are well distributed In spreader-free
areas, and
B. The area covered by the spreader/s does not
exceed one-half the plate area.
When spreading colonies must be counted, count
each unit of the following types as one:
A. The first 1s a chain of colonies
that appears to be caused by dis-
integration of a bacterial clump
as the agar and sample were mixed.
Count each such chain as a single
colony, do not count each
Individual colony 1n the chain.
.«•* Spreader
7-43
-------
r
WATER MONITORING PROCEDURES: Standard Plate Count
Educational Concepts - Science
Section in
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
C.1.2.2a
(Continued)
B. The second type of spreader
develops as a film of growth
between the agar and the
bottom of the petH dish.
C. The third type forms 1n
a film of water at the
edge or over the surface
of the agar.
Spreader
lop
bottom
lop
bollom
If plates prepared from the sample have excessive
spreader growth, report as "Spreaders" (Spr).
When plates are uncountable because of missed
dilution, accidental dropping, and contamination,
or the control plates Indicate that the medium
or other material or labware was contaminated,
report as "Laboratory Accident" (LA).
7-44
-------
WATER MONITORING PROCEDURES: Standard Plate Count
Field and Laboratory Equipment
Section
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.I
A.1.1
A.1.2
A.I.3
A.I.5
A.I.6
Incubator must be of sufficient size for daily
work load without causing crowding of plates to
be incubated. Considerations for choice of
incubator type must relate to reliability of
operation and not to cost or attractiveness of
equipment.
Incubator should be kept out of drafts or direct
sunlight in order to prevent temperature inside
the incubator from changing outside the tempera-
ture range specified (35° ^0.5°).
Power supply should be selected so that there
will not be too many pieces of equipment on the
same circuit. Otherwise, circuits will be blown
repeatedly.
Mercury bulb thermometer usually used in most
incubators. Recording thermometer is acceptable,
but, it should be calibrated against a mercury
bulb thermometer which has been certified by
National Bureau of Standards. The NBS certified
thermometer always should be used with its
certificate and correction chart.
Saturated relative humidity is required in order
to make the incubation more efficient (heat is
transferred to cultures faster than in a dry
incubator). Furthermore, culture medium may
evaporate too fast in a dry incubator.
Allow enough time after each readjustment to per-
mit the incubator to stabilize before making a
new adjustment. Al least one hour is suggested.
Incubator temperature can be held to much closer
adjustment if operated continuously. Temperature
records should be kept in some form of permanent
record. A temperature record book is suggested
with daily recording of values. If a recording
thermometer is used, the charts may be kept as
permanent record; if so, be sure that the charts
are properly labeled to identify the incubator
and the period covered.
Uniform temperature (35° C +0.5) is to be
maintained on shelves in use.
Standard Methods for the
Examination of water and
Wastewater, 14th ed. 1975
APHA, WPCF, AWWA, p. 880
(Hereafter referred to as
Std. Meth. 14: (page no.)
7-45
-------
MATER MONITORING PROCEDURES: Standard Plate Count
Field and Laboratory Equipment
Section v
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.3.1-5
A.4.1
Since electric sterilizer will be operated Inter-
mittently, care should be taken that It 1s on a
circuit which will not be overloaded when It Is
turned on.
A time and temperature record Is maintained for
each sterilization cycle. Temperature recordings
can be retained for records.
Autoclaves differ greatly in design and in method
of operation. Some are almost like home-style
pressure cookers; others are almost fully auto-
matic. This Is a subject which requires separate
instruction; and should be related to the exact
make and model of equipment you will use in your
own laboratory.
Vertical autoclaves and household pressure
cookers may be used in emergency service if
equipped with pressure gages and thermometers
with bulbs positioned 1 inch above the water
level. However, they are not to be considered
the equivalent of the general purpose steam
sterilizer recommended for permanent laboratory
facilities. Their small size is Inadequate for
large-volume workloads, and they can be diffi-
cult to regulate.
The following requirements must be met regarding
autoclaves or sterilizing units:
A. Reaches sterilization temperature (121° C),
maintains 121° C during sterilization cycle,
and requires no more than 45 min. for a
complete cycle.
B. Pressure and temperature gages on exhaust side
and an operating safety valve.
C. No air bubbles produced in fermentation vials
during depressurization.
D. Record maintained on time and temperature for
each sterilization cycle.
Std. Meth. 14:881
Std. Meth. 14:881
7-46
-------
WATER MONITORING PROCEDURES: Standard Plate Count
Field and Laboratory Equipment
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.5.1-2
Distilled water in a bacteriological laboratory
must not contain substances which will prevent
any bacteria from growing in culture medium in
which the distilled water is used or will be
highly nutritive. There are procedures for
testing quality of distilled water; but these
should be undertaken only by professional
bacteriologists or in laboratories where this is
done regularly. Use only glass stills or block
tin lined stills.
Requirements for distilled water include the
following:
Std. Meth.
14:645-49
14:888-891
Test
PH
Conductivity
Trace Metals:
A single metal
Total metals
Test for bactericidal properties
of distilled water ("Standard
Methods," Nth Ed. p. 887)
Free chlorine residual
Standard plate count
Analysis Requirement
5.5 - 7.5
0.1 megohm as resistivity or
< 5.0 micromhos/cm at 25° C
Not greater than 0.05 mg/1
Equal to or less than 1.0 mg/1
0.8 - 3.0
0.0
Less than 10,000/ml
Conducted
Monthly
Monthly
Annually
Annually
Monthly
Monthly
A.6.1
A.7.1-4a
A.7.1-4b
pH Meter: See cited reference
Glassware: See cited reference on pi pets and
graduated cylinders, media utensils, bottles.
Glassware can be checked for bacteriostatic or
inhibitory residues by a bacteriological test
procedure which, like the distilled water suit-
ability test, should be undertaken only by
professional bacteriologists or in laboratories
where this test is done on a regular basis.
Std. Meth. 14:882
Std. Meth. 14:882-885
Std. Meth. 14:885
7-47
-------
WATER MONITORING PROCEDURES: Standard Plate Count
Field and Laboratory Equipment
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.9.1-6
A.10.1-6
A.10.7
A.ll.Zb
Sample bottles:
Wide-mouthed glass-stoppered bottles suggested,
but other styles acceptable.
If glass-stoppered bottles are used, a strip of
paper should be placed in the neck of the bottle
before placing the stopper in place in preparatior
for sterilization. This prevents the glass stop-
per from "freezing" in place during sterilization.
The paper strip is discarded at the time of
sample collection.
Pi pets:
This procedure is described in terms of reusable
glass pi pets. However, single-service pre-
packaged glass or plastic pi pets may be purchased
and used, if preferred. In case of use of
single-service pipets, they will be sterile when
purchased, are used one time, and discarded
immediately after use. Accordingly, in the
step-by-step procedures disregard any instruc-
tions about preparation of pipets for reuse in
case of using single-service pipets.
Passing the opened can of pipets through a flame
burns off excess cotton wisps sticking out of the
mouthpiece of the pipet. If this is not done, it
is almost impossible to control sample measurement
accurately. Some workers may elect to accomplish
this step prior to the sterilization procedure.
Alternate medium containers which can be
utilized are:
1. Flasks with screw caps
2. Tubes of at least 50 ml capacity with 15-20
ml of medium/tube
3. Dilution bottles
It is preferable to use a container which has all
of the medium for a single test since the medium
control test will have greater test result
assurance.
Std. Meth. 14:884
14:904
Std. Meth. 14:882-883
7-48
-------
WATER MONITOP.ING PROCEDURES: Standard Plate Count
Field and Laboratory Equipment
Section
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.11.6.A.C
A.12.1.Id
A.12.4
A.12.5
A.12.6
A.12.8
Recommended Time Limits for Holding Prepared
Media at 4°T
Agar or broth in loose-cap tubes One Meek
Agar or broth in screw-cap tubes,
tightly closed Three Months
Large volumes of Agar in screw-
cap flasks or bottles, tightly
closed Three Months
See cited reference. In time, this solution will
become moId-infested. At this time it must be
discarded and a new stock solution prepared.
Dilution water preparation:
Measurement of dilution water into bottle with a
100 ml graduated cylinder is time-consuming,
but effective. An automatic pipetting machine
can be considered a luxury, but is a real time-
saver.
If caps are not placed on bottles of dilution
water loosely, they may crack in autoclave;
furthermore, steam will not be able to get in
contact with the material being sterilized. After
sterilization, tightening caps on bottles of dis-
tilled water will permit them to be kept for long
periods.
Always pack material loosely and away from walls
in autoclave when preparing to sterilize. Steam
must flow freely around materials being steri-
lized.
If water should evaporate noticeably or become
contaminated by microbial growth, the bottle of
distilled water should be discarded.
Std. Meth. 14:892
7-49
-------
UATER MONITORING PROCEDURES: Standard Plate Count
Field and Laboratory Equipment
Section
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
B.S.lc
Optional Tests are not run as routine procedures
due to the fact that inordinate materials and
time are required for their performance. They
should, however, be occasionally run to estab-
lish that all materials and conditions are
satisfactory. Possibly every 25th SPC test
(author criteria) can be done in total and proper
Quality Control records maintained to document
satisfactory results. Indicated below are the
full complement of control tests:
10 ML
POUR
10 ML
POUR
IPIPET 2 ML AND
POUR 8 ML OF
MEDIUM)
INCUBATE
48 HR AT
31°C
o
OPEN AFTER
HARD FOR 15
SEC INC,
48HR AT 35°C
o
INC, 48 HR
35°C
o
PIPETED
DILUTION
WATER,
10 ML
MEDIUM ]
INC,
48 HR 35°C
OBSERVE PLATES (SHOULD NOT CONTAIN MORE THAN OCCASIONAL
COLONIES OR < 3 COLONIES)
PIPET
MEDIUM
DILUTION
BLANK
MC - MEDIUM CONTROL
RC - ROOM CONTROL
PC - PIPET CONTROL
BC - BLANK CONTROL
7-50
-------
MATER MONITORING PROCEDURES: Standard Plate Count
Field and Laboratory Equipment
Section
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
B.6.1C
(Continued)
The majority of control tests will fall Into
the following patterns from which decisions can
be made as to the status of materials:
TEST RESULTS
MC
+
-
-
-
RC
+
-
-
+
PC
+
+
-
-
BC
+
-
+
-
REMARKS
Medium possibly contaminated; petri dishes
possibly contaminated; both of above
possibly contaminated.
P1pet contaminated.
Blank contaminated.
Room atmosphere contaminated.
C.2.2b
- .... No Growth, sterile plate
+ .... >_3 colonies; indicates contamination
NOTE: Although 48 hours Incubation time is
stipulated, the plates should be examined at
24 hours since gross contamination can be
observed at this Interval and a + can therefore
be found earlier.
A number of alternately acceptable counters are
available, and, if they can be shown to be
equivalent to the discussed method of counting
(manual), they would be acceptable for use.
Included among these counters are electrohlc-
asslst devices which registers each colony with
a sensing probe and have an automatic tabulation.
Recently a fully-automatic counter was made
available which scans and registers all particles
(colonies) above a preset threshold-size.
7-51
-------
WATER MONITORING PROCEDURES: Standard Plate Count
Field and Laboratory Analyses
Section
VII
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
B.3
There is no such thing as a "Standard" data
sheet for bacteriological tests. Entries for the
SPC may be an integral part of a multi purpose
data sheet or be used only for the specific test.
A simplified data sheet is presented below:
STANDARD PLATE COUNT
Sample Type Lab. No.
Station
Description
Collection Date Time ARM
Received
APM Examined APM
Sampler Name
Analyst Name
Remarks
iample
l/olume
48 hr count
72 hr count
Count per ml
Quality Control Information:
tedium Cont
P1pet Contr
loom Contra
Blank Contr
rol
Reported Values
*>! -rpr/n
ol
1
7-52
-------
WATER MONITORING PROCEDURES: Standard Plate Count
Section
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
This outline was prepared by: Rocco Russomanno,
M1crob1olog1st, National Training and Operational
Technology Center, MOID, OWPO, USEPA, Cincinnati,
Ohio 45268.
7-53
-------
RESIDUAL CHLORINE AND TURBIDITY
I. INTRODUCTION
The Interim Primary Drinking Water Regulations (Federal Register, December 24,
1975) permits the options of substitution of up to 75 percent of the bac-
teriological samples with residual chlorine determinations. Any community
or non-community water system may avail themselves of this option with
approval from the State based upon results of sanitary surveys. Residual
chlorine determinations must be carried out at the frequency of at least
four for each substituted microbiological sample.
Since many potable water plants carry out their own microbiological deter-
minations, it will be necessary that these laboratories be certified for
the bacteriological parameters. Residual chlorine determinations may be
carried out by any person acceptable to the State and the analytical
method and techniques used must be evaluated in some manner to assure that
reliable information is obtained.
Since the presence of high turbidity can interfere with the disinfection
capability of chlorine, a maximum allowable limit has been set for turbidity
as follows:
A. One turbidity unit (TU) as determined by a monthly average except
that five or fewer turbidity units may be allowed if the supplier
of water can demonstrate to the State that the higher turbidity
does not
1. Interfere with disinfection,
2. Prevent maintenance of residual of disinfectant throughout
distribution system, or,
3. Interfere with microbiological determinations.
B. Five turbidity units based on an average of two consecutive days.
The Criteria and Procedures Document for Water Supply Laboratory Certifi-
cation suggests that some quality control guidelines be instituted for
the residual chlorine and turbidity measurements at the State level for
the purpose of ensuring data validity for these critical measurements.
In response to public comments regarding the proposed Primary Regulations
(Federal Register, December 24, 1975) it is stated that operators per-
forming residual chlorine and turbidity analyses " be certified,
approved, or at least minimally trained to perform the analytical tasks
before a State could accept their analytical determinations "
CH.TURB. 3.9.77 8-1
-------
II. RESIDUAL CHLORINE
Since residual chlorine analysis would be carried out in "field" conditions
or in the small laboratories of treatment plants, perhaps by unskilled
operators, it is necessary to keep the analytical method as simple as
possible. For a number of years, operators had utilized the orthotolidine
technique in a kit form to determine the chlorine residual. Recent
studies and regulatory guidelines have dictated against this test procedure.
The acceptable test procedure is now the DPD Test (13th Ed., Standard Methods
for the Examination of Water and Wastewater. pgs. 129-132), for which kits
are available from at least two companies and which meet requirements for
accuracy and reliability. These kits are capable of measuring both free
and combined chlorine of which only the free chlorine is measured to meet
compliance requirements. Kit procedures call for a premeasured single
powder or tablet reagent added to the test cell with the sample and a
resultant color development measures by comparison the standardized colors
within one minute. Standard Methods includes cautions regarding temperature
and pH control regarding this test parameter and this test procedure, the
DPD Test, is least effected by temperature and the pH is adjusted by the
added reagents. The only interfering substance, oxidized manganese, can
be determined in a preliminary step and compensated for in the final test
value.
III. TURBIDITY
Turbidity has long been used in the water supply industry for indicating proper
operational techniques. Turbidity should be clearly understood to be an ex-
pression of the optical property of a sample which causes light to be scattered
and absorbed rather than transmitted in straight lines through the sample.
The standard method for the determination of turbidity has been based on the
Jackson candle turbidimeter. However, the lowest turbidity value which can
be measured directly on the Jackson turbidimeter is 25 units which is well
above the monitoring level. Because of these low level requirements, the
nephelometric method was chosen and procedures are given in Standard Methods
(13th Ed., 1971).
IV. NEPHELOMETRIC MEASUREMENTS FOR COMPLIANCE MONITORING
The subjectivity and apparatus deficiencies involved in visual methods of
measuring turbidity make each unsuitable as a standard method.
Since turbidity is an expression of the optical property of scattering or
absorbing light, it was natural that optical instruments with photometers
would be developed for this measurement.
The type of equipment specified for compliance monitoring^ ' ' utilizes
nephelometry.
A. Basic Principle^''
The intensity of light scattered by the sample is compared (under defined
conditions) with the intensity of light scattered by a standard reference
solution (formazin). The greater the intensity of scattered light, the
greater the turbidity. Readings are made and reported in NTUs (Nephelometric
Turbidity Units).
8-2
-------
B. Schematic
Lamp
Turbidity Particles
Scatter Light
Sample Cell
(Top View)
Figure 2 NEPHELOMETER
(90° Scatter)
Light passes through a polarizing lens and on to the sample in a cell.
Suspended particles (turbidity) in the sample scatter the light.
Photocell(s) detect light scattered by the particles at a 90° angle to the
path of the incident light. This light energy is converted to an electric
signal for the meter to measure.
1. Direction of Entry of Incident Light to Cell
a. The lamp might be positioned as shown in the schematic so the
beam enters a sample horizontally.
b. Another instrument design has the light beam entering the sample
(in a flat-bottom cell) in a vertical direction with the photocell
positioned accordingly at a 90° angle to the path of incident light.
2. Number of Photocells
The schematic shows the photocell(s) at one 90° angle to the path of
the incident light. An instrument might utilize more than one photo-
cell position, with each final position being at a 90° angle to the
sample liquid.
3. Meter Systems
a. The meter might measure the signal from the scattered light in-
tensity only.
b. The meter might measure the signal from a ratio of the scattered
light versus light transmitted directly through the sample to a
photocel1.
8-3
-------
4. Meter Scales and Calibration
a. The meter may already be calibrated in NTUs. In this case,
at least one standard is run in each instrument range to be
used in order to check the accuracy of the calibration scales.
b. If a pre-calibrated scale is not supplied, a calibration curve
is prepared for each range of the instrument by using appropriate
dilutions of the standard turbidity suspension.
C. EPA Specifications for Instrument Design^ '
Even when the same suspension is used for calibration of different
nephelometers, differences in physical design of the turbidimeters will
cause differences in measured values for the turbidity of the same sample.
To minimize such differences, the following design variables have been
specified by the U. S. Environmental Protection Agency.
1. Defined Specifications
a. Light Source
Tungsten lamp operated at not less than 85% of rated voltage
and at not more than rated voltage.
b. Distance Traveled by Light
The total of the distance traversed by the incident light plus
scattered light within the sample tube should not exceed 10 cm.
c. Angle of Light Acceptance of the Detector
Detector centered at 90° to the incident light path and not to
exceed ± 30° from 90°.
(Ninety degree scatter is specified because the amount of scatter
varies with size of particles at different scatter angles).
d. Applicable Range
The maximum turbidity to be measured is 40 units. Several ranges
will be necessary to obtain adequate coverage. Use dilution for
samples if their turbidity exceeds 40 units.
2. Other EPA Design Specifications
a. Stray Light
Minimal stray light should reach the photocell(s) in the absence
of turbidity.
8-4
-------
3.
Some causes of stray light reaching the photocell(s) are:
•
1) Scratches or imperfections in glass cell windows.
2) Dirt, film or condensation on the glass.
3) Light leakages in the instrument system.
A schematic of these causes is shown in Figure 3.
Meter
Photoc*ll(i)
Light Leakage
from Lens System
Lamp
\
Light Leakage from
Transmitted Light
CJ
\
I
f A
A?
IX
\ \
i \ " ^ \ -
>
r
/ j
Light Scatter by glass tube
(Top View)
Figure 3 NEPHELOMETER
SOURCES OF STRAY LIGHT
Stray light error can be as much as 0.5 NTU. Remedies are
close inspection of sample cells for imperfections and dirt,
and good design which can minimize the effect of stray light
by controlling the angle at which it reaches the sample.
b. Drift
The turbidimeter should be free from significant drift after a
short warm-up period. This is imperative if the analyst is
relying on a manufacturer's solid scattering standard for setting
overall instrument sensitivity for all ranges.
c. Sensitivity
In waters having turbidities less than one unit, the instrument
should detect turbidity differences of 0.02 unit or less.
Several ranges will be necessary to obtain sufficient sensitivity
for low turbidities.
Examples of instruments meeting the specifications listed in 1 and 2
above include:
a. Hach Turbidimeter Model 2100 and 2100A.
b. Hydroflow Instruments DRT 100, 200, and 1000.
8-5
-------
4. Other turbidimeters meeting the listed specifications are also
acceptable.
D. Sources of Error
1. Sample Cells
a. Discard scratched or etched cells.
b. Do not touch cells where light strikes them in instrument.
/0\
c. Keep cells scrupulously clean, inside and out.1 '
1) Use detergent solution.
2) Organic solvents may also be used.
3) Use deionized water rinses.
4) Rinse and dry with alcohol or acetone.
2. Standardizing Suspensions^ '
a. Use turbidity - free water for preparations. Filter distilled
water through a 0.45ym pore size membrane filter if such filtered
water shows a lower turbidity than the distilled water.
b. Prepare a new stock suspension of Formazin each month.
c. Prepare a new standard suspension and dilutions of Formazin
each week.
3. Sample Interferences
a. Positive
1) Finely divided air bubbles
b. Negative
1) Floating debris
2) Coarse sediments (settle)
3) Colored dissolved substances
(absorb light)
8-6
-------
E. Reporting Results*7'
NTU RECORD TO NEAREST
0.0-1.0 0.05
1-10 0.1
10-40 1
40-100 5
100-400 10
400-1000 50
>1000 100
F. Precision and Accuracy* '
1. In a single laboratory (EMSL), using surface water samples at
levels of 26, 41, 75 and 180 NTU, the standard diviations were
+0.60, ±0.94, +1.2 and +4.7 units, respectively.
2. Accuracy data is not available at this time.
V. STANDARD SUSPENSIONS AND RELATED UNITS(9)
One of the critical problems in measuring turbidity has been to find a
material which can be made into a reproducible suspension with uniform sized
particles. Various materials have been used.
A. Natural Materials
1. Diatomaceous earth
2. Fuller's earth
3. Kaolin
4. Naturally turbid waters.
Such suspensions are not suitable as reproducible standards because
there is no way to control the size of the suspended particles.
B. Other materials
1. Ground glass
2. Microorganisms
3. Barium Sulfate
4. Lates spheres
Suspensions of these also proved inadequate.
8-7
-------
C. Formazin
1. A polymer formed by reatu-ig hydrazine sulfate and hexamethy 1 enete-
tramine sulfate.
2. It is more reproducible than previously used standards. Accuracy
of + one percent for replicate solutions has been reported.
3. In 1958, the Association of Analytical Chemists initiated a standard-
ized system of turbidity measurements for the brewing industry by:
a. Defining a standard formula for making stock Formazin solutions
and
b, Designating a unit of measurement based on Formazin, i.e., the
Formazin Turbidity Unit (FTU).
4. During the 1960's Formazin was increasingly used for water quality
turbidity testing. It is the currently recognized standard for
compliance turbidity measurements.
D. Units
1. At first results were translated into Jackson Turbidity Units (JTU).
However, the JTU was derived from a visual measurement using con-
centrations (mg/liter) of silica suspensions prepared by Jackson.
They have no direct relationship to the intensity of light scattered
at 90 degrees in a nephelometer.
2. For a few years, results of nephelometric measurements using specified
Formazin standards were reported directly as Turbidity Units (TUs).
3. Currently, the unit used is named according to the instrument used for
measuring turbidity. Specified Formazin standards are used to calibrate
the instrument and results are reported as Nephelometric Turbidity
Units (NTUs).
VI. SUMMARY
The importance of residual chlorine determination can be seen in its possible
effect on the health of the consumers. The Criteria and Procedures for
Laboratory Certification suggests that some form of quality assurance should
be instituted on a state level to assure valid data for both the chlorine and
turbidity measurements. The comments on the public responses to the proposed
Interim Primary Regulations also suggests some form of quality assurance on
the state level to be instituted. Consequently, the Regional Certification team
should point out to the principal laboratories the importance of some kind of
effort being instituted. States might wish to offer some kind of formal
training effort as part of the approval mechanism for the operators doing
the chlorine and/or turbidity measurements.
8-8
-------
A PROTOTYPE FOR DEVELOPMENT OF
ROUTINE OPERATIONAL PROCEDURES
for the
COLIFORM TEST BY THE MULTIPLE DILUTION TUBE METHOD (MPN)
as applied in
WATER TREATMENT FACILITIES
WASTEWATER TREATMENT FACILITIES
and in the
MONITORING OF EFFLUENT WASTEWATERS
National Training and Operational Technology Center
Municipal Operations and Training Division
Office of Water Program Operations
U. S. Environmental Protection Agency
BA.MET.lab.WMP.4.5.78
9-1
-------
COLIFORM TEST
MULTIPLE DILUTION TUBE
IMPNI METHOD SLOW SHEET
S TUBES OF EACH OF EACH OF A
DECIMAL SERIES OF 3 OR MORE
SAMPLE VOLUMES |EACH SUCCESSIVE
VOLUME IS 1/10 THE PREVIOUS
VOLUME)
INCUBATE~z4 ± 2 HOURS A~35° c ± o.s° c
I
INCUBATE FOR 24 HOURS ± 2 HOURS
AT 35° C ± 0 5° C
I
GAS |*|
COLIFORMS
PRESENT
GAS |-|
I
REINCUBATE FOR AN ADDITIONAL 24
HOURS |48 i 3 HOURS) AT 35'
C * 0.5° C
GASJ-J_
~REINCUBATfFOH AN ADDITIONAL
24 HOURS (48 1 3 HOURS) AT
35" C + 0 5° C
GAS |+]
COLIFORMS
PRESENT
GAS |-|
COLIFORMS
ABSENT
GAS |-|
COLIFORMS
ABSENT
GAS |+| TUBE CONTAINING AT
LEAST ONE BUBBLE OF GAS
GAS )-) COMPLETE ABSENCE OF
GAS IN TUBE
LLSTB - LACTOSE LAURTL
SULFATE TRYPTOSE BROTH
BGLBB - BRILIANT GREEN
LACTOSE BILE BROTH
CODIFY RESULTS BASED UPON
POSITIVE AND NEGATIVES IN
EACH ROW
DETERMINE MPN INDEX
APPLY CORRECTION FACTOR
AS REQUIRED
RECORD RESULTS AS
COLIFORMS/100 ml
REPORT RESULTS AS PRESCRIBED
UNDER REGULATORY REQUIREMENTS
9-3
-------
WATER MONITORING PROCEDURE: Coliform Test by the Multiple
Dilution Tube (MPN) Method
1. Analysis Objectives:
In water treatment plant quality control, the objective of the test may be
one or both of the following:
a. To determine whether water treatment plant influent quality meets
requirements set by law or regulatory authority.
b. To determine water body quality as pertaining to upstream flow in
a sanitary survey to locate source of excessive counts.
2. Brief Description of Analysis:
Three or more decimal series dilutions of a sample (For example: Five
fermentation tubes with 10 ml portions, another five tubes with 1 ml
portions, etc.) are inoculated into lactose lauryl sulfate tyrptose
broth (LLSTB) and incubated at 35° C + 0.5° C. After 24 hours and
again at 48 hours, the LLSTB tube cultures are examined and results
recorded for gas production. Cultures showing gas production are
transferred at each examination interval to BGLBB fermentation tubes
and incubated at 35° C + 0.5° C. BGLBB tubes are examined at 24 and
48 hour intervals for presence of gas and those showing gas are con-
sidered gas (+) and containing coliforms while those completely without
gas as gas (-) or not containing coliforms.
At the end of the overall incubation period, individual tubes are
summarized as positive or negative and these results coded to represent
rows of the inoculation series. A Table of Most Probable Numbers (MPN)
is used with properly selected codes to determine the MPN Index. This
Index is corrected, if necessary, to agree with the actual sample
volumes indicated (the Table is based on 10 ml; 1 ml; and 0.1 ml volumes
for the series). The final results are recorded and reported as the
coliforms per 100 ml of sample.
3. Applicability of this Procedure:
a. Range of Coliform Concentration
If these dilutions are used These ranges of Coliforms are covered
10; 1; 0.1; 0.01 > 2 to > 24,000
1; 0.1; 0.01; 0.001 20 to > 240,000
0.1; 0.01; 0.001; 0.0001 200 to > 2,400,000
etc. etc.
b. Pretreatment of Samples
In accordance with Standard Methods, 14th ed. (p. 904).
This procedure conforms to the Standard Total Coliform MPN Tests as described in
Standard Methods for the Examination of Water and Wastewater, 14th ed., (1975),
p. 916 ff.
9-4
-------
WATER MONITORING PROCEDURE: Coliform Test by the Multiple
Dilution Tube (MPN) Method
Equipment and Supply Requirements
A. Capital Equipment:
1. Autoclave, providing uniform temperatures up to and including 121° C,
equipped with an accurate thermometer, pressure gauges, saturated
steam power lines and capable of reaching required temperature within
30 minutes
2. Balance, 0.1 g sensitivity at load of 150 g
3. Air Incubator to operator at 35° C + 0.5° C
4. Oven, *hot-air sterilizing, to give uniform temperatures and with
suitable thermometer to register accurately in range of 160-180° C
5. pH Meter, accurate to at least 0.1 pH unit, with standard pH reference
solutions(s)
6. Water distillation apparatus, (glass or block tin), or source of
distilled water suitable for bacteriological operations
B. Reusable Supplies:
1. Apron or coat suitable for laboratory
2. Baskets, wire for discarded cultures
3. Bottles, dilution*, 6-oz. screw caps, with 99 ml volume level etched
on one side
4. Bottles, sample*, preferred characteristics being 250 ml (6-8 oz.),
wide mouth, glass stopper
5. Burner, gas, Bunsen burner type
6. Cans, pipet, aluminum or steel; not copper (If plastic, or other type
of prepackaged disposable pi pets are used, this item is unnecessary.)
7. Metal caps* to fit 18 and 25 mm culture tubes
8. Pan, to receive discarded contaminated pi pets and glassware (must
contain disinfectant before use)
9. Inoculation loop, 3 mm diameter loop of nichrome or platinum-iridium
wire, 26 B&S gauge, in holder
10. Pipets*, 1 ml, with 0.1 ml graduations, Mohr type preferred, sterile,
cotton plugged, glass or disposable plastic
11. Pipets*, 10 ml, with 1.0 ml graduations, Mohr type preferred, sterile,
cotton plugged, glass or disposable plastic
12. Racks, culture type*, 10 x 5 openings, to accept tubes at least 25 mm
in diameter
13. Sponge, for cleaning desk top
14. Tubes, culture*, 150 x 25 mm
15. Tubes, culture*, 150 x 18 mm
16. Tubes, fermentation*, 75 x 10 mm vials to be inverted in culture tubes
C. Consumable Supplies:
1. Distilled water, suitable for bacteriological cultures (note distillation
apparatus required in capital equipment)
2. BGLBB (Brilliant Green Lactose Bile Broth), dehydrated (recommend purchase
of 1/4 Ib. units)
3. Lactose Lauryl Sulfate Tryptose Broth, dehydrated (recommend purchase of
1 Ib. units)
4. Postasium Dihydrogen Phosphate (KH9PO.) (recommend purchase of 1/4 Ib.
units) *
9-5
-------
WATER MONITORING PROCEDURE: Coliform Test by the Multiple
Dilution Tube (MPN) Method
C. Consumable Supplies (Continued):
5. Disinfectant, for bench tops. (Use household bleach solution prepared
according to instructions on bottle|
6. Wax pencils (recommend soft wax equivalent to Blaisdell 169T)
7. EDTA (ethylene dinitrilotetraacetic acid)
8. Sodium thiosulfate (Na^SpO-.S H.O)
*Items marked are needed in quantities or require size or space allowances which
cannot be specified here, as they vary according to the daily analysis schedule.
As a rule-of-thumb, space/size or quantity requirements should be at least 3 times
the normal daily requirements. For further information on specifications for
equipment and supplies, see the Microbiology Section of the current edition of
"Standard Methods for the Examination of Water and Wastewater."
9-6
-------
WATER MONITORING PROCEDURE:
Coliform Test by the Multiple
Dilution Tube (MPN) Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOAl S/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
1. 35° C Incubator
Set-up, Adjustment
1. Place 35° C Incubator In
permanent location.
2. Install thermometer.
3. Install shallow pan of
water 1n bottom of incu-
bator.
4. Connect Incubator to
electric power source.
5. Adjust temperature until
stabilized at required
temperature.
6. Operate bacteriological
incubator continuously.
Aa. All pretest procedures completed before starting
other first-day procedures.
la. Out of drafts or places where it will be in
sunlight part of day.
Ib. Location convenient to laboratory bench.
Ic. Convenient source of electric power.
2a. Thermometer functions at least in 30°-40° C
range and has intervals of 0.5° or less indi-
cated. Meets NBS standards.
2b. Location should be central in incubator.
2c. Mercury bulb thermometer should be fitted with
cork or rubber stopper and mounted in small
bottle filled with liquid (glycerine, water, or
mineral oil).
3a. In most laboratory incubators a pan having about
1 square foot of area, with water about 1 inch
deep, is satisfactory.
3b. Maintains condition of saturated relative humidity
required in bacteriological incubator.
3c. Requires daily check, with addition of water as
necessary, to keep water in pan at all times.
4a. Many incubators have pilot light to indicate
power turned on.
5a. Manufacturer's instructions for method
temperature adjustment.
5b. Operation must be at 35° + 0.5° C.
5c. Allow about 1 hour between adjustments.
6a. Requires daily check with written temperature
record, with adjustment and water addition as
necessary.
V.A.I
(P- 9-42)
V.A.1.1
(p. 9-42)
V.A.I.2
(p. 9-42)
V.A.I.3
(P- 9-42)
V.A.I.5
(p. 9-42)
V.A.I.6
(P. 9-42)
9-7
-------
WATER MONITOR1NG PROCEDURE:
Coliform Test by the Multiple
Dilution Tube (MPN) Mehtod
9-8
OPERATING PROCEDURES
A. Pre-Test Procedures
(Continued)
2. Oven, Sterilizer
Set-up
3. Autoclave Set-up
STEP SEQUENCE
1. Place oven sterilizer in
permanent location.
2. Install thermometer.
3. Connect oven sterilizer to
power source and turn on.
4. Adjust temperature to
stabilize at required
temperature.
5. Operate oven sterilizer
only when needed. Turn
off when not in use.
1. Install and operate auto-
clave according to manu-
facturer's instructions.
INFORMATION/OPERATING GOALS/SPECIFICATIONS
la. Convenient to source of electric power usually
on table or bench.
2a. Should indicate the 160° - 180° C range, be
accurate within this interval, and be marked in
1.0 degree intervals.
3a. Usually has pilot light to indicate power on.
4a. Operated as near to 170° C as possible; not lower
than 160° or higher than 180° C.
5a. Turned ON in advance of need to permit reaching
required temperature before introducing material
to be sterilized.
5b. Oven sterilizer used to sterilize dry glassware,
metal objects.
5c. Oven sterilizer not used with culture media,
solution, plastics, rubber objects, or with
anything containing or including these.
5d. Paper-wrapped glass pi pets may be sterilized in
oven sterilizer.
la. Autoclaves extremely variable in design and
operation; also, potentially dangerous.
Ib. Used to sterilize objects made of, or including
liquids, rubber, culture media.
Ic. Glassware may be autoclave sterilized but must be
dried afterward.
Id. Most plastics not sterilized in autoclave;
plastics usually require chemical sterilizers.
le. Autoclave usually operated at 121° C for 15 min.
If. Sterilized media must be removed from autoclave
as soon as possible after autoclave is reopened.
TRAINING
GUIDE NOTES
V.A.2.1-5
(p. 9-43)
V.A.3.1
(p. 9-43)
-------
WATER MONITORING PROCEDURE:
Coliform Test by the Multiple
Dilution Tube (MPN) Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
4. Water Distillation
Equipment
5. pH Meter
6. Glassware
1. Install and operate in
accordance with manu-
facturer's instructions.
2. Operate continuously or
intermittently as required
to maintain adequate
supplies of distilled
water.
1. Have unit available and
operate in accordance with
procedures described in
other lab procedures.
1. Wash all glassware in hot
detergent solution.
2. Rinse at least once in
hot tap water.
3. Rinse in distilled water,
at least 6 successive
times and,
4. Dry in air.
la. Must produce distilled water meeting quality
requirements for bacteriological tests.
2a. Reserve supplies kept in borosilicate glass
carboys or in plastic carboys made of material
which will not dissolve substances which will
affect growth of bacteria.
2b. Same distillation apparatus used for bacterio-
logical purposes may be used for chemical
reagents.
la. Unit for pH check on finished culture media.
Ib. Used in preparation of stock solution of
potassium dihydrogen phosphate.
la. Nontoxic detergent
Ib. Be sure all contents and markings are washed away.
4a. No visible spots or scum; glass should be clean.
and soarklinq.
4b. Glassware suitable for use in bacteriological
operations.
V.A.4.1-2
(p. 9-4.3)
V.A.5.1
(p. 9-4.4)
V.A.6.1-4a
(p. 9-44)
V.A.6.1-4b
(p. 9-44)
9-9
-------
WATER MONITORING PROCEDURE:
Coliform Test by the Multiple
Dilution Tube (MPN) Method
9-10
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
7. Sodium Thiosulfate
Solution
8. Ethylenedinitrilote
traacetic Acid
(EDTA) Solution
The following special conditions may apply to the
sample to be analyzed:
If the sample is chlorinated influent which contains copper, zinc, or heavy
metals, do operating procedures A.7, A.8 and A.9 completely.
If the sample is unchlorinated influent which contains copper, zinc, or heavy
metals, eliminate steps A.7 and A.9.1.
If the sample is chlorinated influent which does not contain copper, zinc, or
heavy metals, eliminate steps A.8 and A.9.2.
If the sample is unchlorinated and contains no copper, zinc, or heavy metals,
eliminate steps A.7, A.8, A.9.1 and A.9.2.
1. Weigh 10.0 grams of sodium
thiosulfate.
2. Dissolve in 50-60 ml dis-
tilled water.
3. Add distilled water to
bring final volume to
100 ml.
4. Transfer to labeled bottle
1. Weigh 15.0 grams of EDTA.
2. Dissolve in 50-60 ml dis-
tilled water.
la. Used for dechlorination of samples.
Ib. Use of trip balance accepted.
2a. 100 ml graduated cylinder satisfactory.
la. Labeled as 10% sodium thiosulfate and stored in
refrigerator.
la. Used for water samples high in copper or zinc or
wastewater samples high in heavy metals.
Ib. Use of trip balance accepted.
2a. A 100 ml graduated cylinder is satisfactory.
-------
UATER MONITORING PROCEDURE:
Coliform Test by the Multiple
Dilution Tube (MPN) Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
9. Sample Bottle
Preparation
3. Add distilled water to
bring final volume to
100 ml.
4. Transfer to labeled clean
bottle.
1. Deliver 0.1 ml or .2 ml
of 10% sodium thiosulfate
solution to each sample
bottle. (.1 ml to 4 ounce
or 120 ml size and .2 ml
to 6-8 ounce or 250 ml
size).
2. Deliver .3 ml or .6 ml of
15% EDTA solution to each
sample bottle (.3 ml to
4 ounce or 120 ml size and
.6 ml to 6-8 ounce or
250 ml size).
3. Place cover on sample
bottle.
4. Place paper or metal foil
cover over bottle cap or
stopper.
5. Sterilize sample bottles in
sterilizing oven.
6. Store sample bottles in
clean, dry place until
used.
4a. The bottle should be labeled as 15% Ethylene-
dinitrilotetraacetic acid (EDTA) and stored
in refrigerator.
la. Use 1 ml pi pet.
Ib. Provides adequate sodium thiosulfate for
neutralizing chlorine in sample.
Ic. Return stock sodium thiosulfate solution to
refrigerator.
2a. Use 1 ml pi pet.
2b. Provides adequate EDTA chelating agent for metals
in sample.
2c. Return stock solution of EDTA to refrigerator.
V.A.9.1-6
(p. 9-44)
4a. Protects opening of sample bottle from accidental
contamination.
5a. One hour at 170° C. (See A.2)
9-11
-------
WATER MONITORING PROCEDURE.:
Coliform Test by the Multiple
Dilution Tube (MPN) Method
9-12
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
10. Pipet Preparation
1. Inspect the 10 ml and 1 ml
pi pets to be prepared for
use; discard and destroy
all having chipped or
cracked tips.
2. Insert plug of non-
absorbent cotton into
mouthpiece of each clean,
dry pi pet.
3. Place a layer of glass wool
or several layers of paper
padding in bottom of pi pet
can.
4. Place 12-24 pipets of the
same size in each pi pet can
delivery tip down. Mark
cans as either 10 ml or
1 ml.
5. Sterilize cans of
pipets in oven.
6. Store cans in clean, dry
place until used.
7. When can of pipets is
opened for first use, pass
the exposed ends of the
pipets through flame,
slowly.
la. Cleanliness of pipet must be equivalent to
glassware.
2a. For protection of user when pipetting sample.
2b. Cotton plug must be tight enough to prevent easy
removal, either by the pipetting action or by
handling, and yet loose enough to permit easy
air movement through the plug.
3a. For protection of pipet delivery tips.
4a. Orientation permits removal of sterile pipets
from can without contamination by user.
5a. 1 hour at 170° C. (See A.2 of procedures)
6a. Laboratory cabinet or drawer recommended.
7a. Burns off excess cotton sticking out of pi pet
mouthpiece.
7b. Cover kept on can at all times except when
samples are being inoculated.
V.A.10.1-6
(p. 9-44)
V.A.10.7
(p. 9-44)
-------
kATER MONITORING PROCEDURE:
Collform Test by the Multiple
Dilution Tube (MPN) Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPE-WING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
11. Dilution Water
Blanks
1. Prepare stock solution of
potassium dihydrogen phos-
phate (KH2P04); dissolve
34.0 grams of the KH2P04
in 500 ml distilled water.
Adjust to pH 7.2 with
IN NaOH, and dilute to 1
liter with distilled water
2. Prepare stock solution of
magnesium sulfate (MgSO..
7H20) by dissolving 50 *
grams of this chemical in
500-600 mis of distilled
water and, after complete
dissolving, bring the
final volume to 1 liter in
a volumetric flask.
3. Prepare working solution
of dilution water by add-
ing 1.25 ml KH2PH4
and 5 ml of the magnesium
sulfate stock solution to
each liter of distilled
water to be made up as
dilution water.
4. Deliver enough working
solution to each dilution
water bottle so that after
sterilization the bottles
will contain 99 + 2 ml of
dilution water.
la. Distilled water may be measured in 500 ml
graduated cylinder.
Ib. Finished solution labeled "Stock KH-PO. for
Dilution Water." * 4
Ic. Stored in refrigerator.
Id. Discard stock solution and prepare new solution
if mold appears.
V.A.11.1.Id
(p. 9-44)
3a. 5 ml pi pet satisfactory for 1 liter amounts of
dilution water. 10 ml pi pet better when several
liters are being made.
3b. 1-liter graduated cylinder satisfactory for
measurement of distilled water.
3c. Use separate pi pets for each solution to prevent
contamination.
4a. 100 ml graduated cylinder ordinarily satisfactory.
Pipetting machine desirable but not mandatory.
4b. Amount cannot be stated exactly, as sterilization
evaporation differs from one autoclave to another.
Commonly, about 102 mis are required.
V.A.11.4
(p. 9-45)
9-13
-------
MATER MONITORING PROCEDURE:
Coliform Test by the Multiple
Dilution Tube (MPN) Method
9-14
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
12. Preparation of
Lactose Lauryl
Sulfate Tryptose
Fermentation Broth
(LLSTB)
5. Place caps on dilution
bottles loosely.
6. Sterilize in autoclave.
7. Promptly remove from auto-
clave, tighten bottle caps,
cool to room temperature.
8. Store in cool place.
Single-Strength Medium
1. Weigh 35.6 grams of dehy-
drated Lactose Lauryl
Sulfate Tryptose Broth.
Close cover of bottle of
dehydrated medium tightly
after removal.
2. Dissolve in 1 liter dis-
tilled water.
3. Place 10.5 ml of the solu-
tion of prepared LLSTB in
each culture tube.
6a. 15 minutes at 121° C.
steam evacuation.
Use "slow-vent" mode of
8a. Dilution water ready for use. May be stored
indefinitely in screw-capped bottles.
la. Dehydrated media takes moisture out of air;
can become caked.
Ib. Caked media unsatisfactory; should be discarded.
2a. Gentle heat (no boiling) if necessary to com-
plete dissolving medium. Usually a vigorous
agitation will completely dissolve the medium.
3a. Use 150 x 18 mm tubes.
3b. A 25 ml pi pet, automatic pipetter, or funnel hose
and pinchcock assembly are acceptable.
3c. Accuracy of delivery: + 0.5 ml.
3d. Approximately 90 tubes will be necessary. This
will suffice for 6 tests based upon procedures
of this WMP (Water Monitoring Procedure).
V.A.11.5
(p. 9-45)
V.A.11.6
(p. 9-45)
V.A.11.8
(p. 9-45)
V.A.12.3b
(p. 9-45)
-------
U'ATER MONITORING PROCEDURE:
Collform Test by the Multiple
Dilution Tube (MPN) Method
OPERATING PROCEDURES
STEP SLQUENCE
INFORMAT10N/OPCRA1ING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
4. Insert one fermentation
vial into each tube of
medium, open end down.
5. Place tube cap on each
tube of culture medium.
6. Sterilize in autoclave.
7. Cool medium to room tem-
perature.
8. Check pH of finished
medium.
9. If final pH is not satis-
factory, discard medium
and prepare new batch with
pH adjustment before
sterilization.
10. Store medium in cool dark
place.
4a. Tubes and vials previously washed as indicated
(A.6.1-4.)
4b. Use 75 x 10 mm tubes.
5a. After all tubes have been filled and have
individual vial.
6a. Within 1 hour after medium is prepared.
6b. Sterilization at 121° C for 15 minutes.
6c. Medium must be removed from autoclave as soon
as possible after pressure has returned to
normal. Use "slow-vent" mode of steam removal.
7a. Medium ready for use when cool and individual
vials are completely filled with fluid. No
bubbles must be present.
8a. Should be pH 6.7 - 6.9.
9a. pH value ordinarily drops about 0.2 pH unit.
lOa. Not in refrigerator. Usually in laboratory
cabinet in darkness.
lOb. May be stored up to 1 week if evaporation is not
more than 10% in loose-fitting capped tubes.
With screw-capped tubes should be held no
longer than 3 months.
9-15
-------
WATER MONITORING PROCEDURE:
Coliform Test by the Multiple
Dilution Tube (MPN) Method
9-16
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
Increased Strength Medium
11. Weigh 53.4 grams of dehyd-
rated Lactose Lauryl Sul-
fate Tryptose Broth.
Close cover of bottle of
dehydrated medium tightly
after removal.
12. Dissolve in 1 liter dis-
tilled water.
13. Place 20.5 ml of the
solution of prepared LLSTB
in each culture tube.
13. Preparation of
Brilliant Green
Lactose Bile Broth
(BGLBB)
14. Continue step sequence as
in 12.4-10 to complete
preparation of increased
strength LLSTB.
1. Weigh 40.0 grams of de-
hydrated Brilliant Green
Lactose Bile Broth. Close
cover of bottle of de-
hydrated medium tightly
after removal.
2. Dissolve in 1 liter dis-
tilled water.
lla. Dehydrated media takes moisture out of the air;
can become caked.
lib. Caked media unsatisfactory; should be discarded.
12a. Gentle heat (no boiling) if necessary to complete
dissolving medium. Usually a vigorous agitation
will completely dissolve the medium.
13a. Use 150 x 25 mm tubes.
13b. 25 ml pi pets, automatic pipetter, or funnel hose
and pinchcock assembly are acceptable.
13c. Accuracy of delivery: + 0.5 ml.
13d. Approximately 45 tubes will be necessary. This
will suffice for 9 tests based upon procedures
of this WMP.
V.A.12.3b
(p. 9-45)
la. Dehydrated media takes moisture out of the air:
can become caked.
Ib. Caked media unsatisfactory; should be discarded.
2a. Gentle heat (no boiling) if necessary to complete
dissolving medium. Usually a vigorous agitation
will completely dissolve the media.
-------
WATER MONITORING PROCEDURE:
Coliform Test by the Multiple
Dilution Tube (MPN) Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
3. Place 10.5 ml of the solu-
tion of prepared BGLBB in
each culture tube.
4. Insert one fermentation
vial into each tube of
medium, open end down.
5. Place cap on each tube of
culture medium.
6. Sterilize in autoclave.
7. Cool medium to room
temperature.
8. Check pH of finished
medium.
9. If final pH not satis-
factory, discard medium and
prepare new batch with pH
adjustment before
sterilization.
3a. Use 150 x 18 mm tubes
3b. A 25 ml pipet, automatic pipetter or funnel hose
and pinchcock assembly are acceptable.
3c. Accuracy of delivery ± 0.5 ml.
3d. Approximately 90 tubes will be necessary.
4a. Tubes and vials previously washed as indicated
(A.6.1-4).
4b. Use 75 x 10 mm tubes.
5a. After all tubes have been filled and have in-
dividual vial.
6a. Within 1 hour after medium prepared.
6b. Sterilization at 121° C for 15 minutes.
6c. Medium must be removed from autoclave as soon as
possible after pressure has returned to normal.
Use "slow-vent" mode of steam removal.
7a. Medium ready for use when cool and individual
vials are completely filled with fluid. No
bubbles must be present.
8a. Should be 7.1 - 7.3.
9a. pH value ordinarily drops about 0.2 pH unit.
V.A.12.3b
(p. 9-45)
9-17
-------
WATER MONITORING PROCEDURE:
Coliform Test by the Multiple
Dilution Tube (MPN) Method
9-18
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
10. Store medium in cool dark
place.
14. Final Equipment
and Supply Check
1. Check to be sure that all
equipment and supplies,
solutions, and prepared
media are ready before
starting sample examina-
tion.
2. Make preparations or ad-
justments as necessary be-
fore starting test.
lOa. NOT in refrigerator. Usually in laboratory
cabinet in darkness.
lOb. May be stored up to 1 week if evaporation not
more than 10% in loose-fitting capped tubes.
With screw-capped tubes should be held no
longer than 3 months.
la. Check general list of equipment and supplies.
Ib. Each test requires (with 4 sample volumes per
test):
5 tubes 1.5X LLSTB (150 x 25 rim tubes)
15 tubes IX LLSTB (150 x 18mm tubes)
10-15 tubes BGLBB
1 sample bottle, sterile
1 10 ml pipet, sterile
2 1 ml pi pets
1 99 ml sterile dilution blank.
B. First-day Procedures
1. Equipment
Maintenance
2. Sample Collection
1. Check, record, and adjust
incubator temperature.
2. Add water to pan in
incubator as necessary.
1. Collect sample.
2. Record sampling informa-
tion.
3. Transport sample to
laboratory.
la. See A.1.1-6
2a. Most plants have sample tag of some type which
includes such Information as date, time, place
of sampling, name of sample collector, and other
information as may be required.
3a. Taken to laboratory without delay.
3b. Samples iced if delay of starting sample test
is greater than one hour. No more than 6 hours
of transportation time is allowed.
-------
WATER MONITORING PROCEDURE:
Coliform Test by the Multiple
Dilution Tube (MPN) Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First-day Procedures
(Continued)
3. Preparation of
Laboratory Data
Sheet.
1. Fill in data sheet to
show sample information.
2. Select sample inoculation
volumes.
la. Needed information should be on sample collection
tag.
Ib. Most data sheets show at least source, date,
time of collection, name of sampler, name of
analyst, laboratory sample number assigned.
2a. According to coliform density range predicted for
the sample.
2b. For conforms per 100 ml in the range
VII.B.3.1
(p. 9-46)
VII.B.3.2
(p. 9-46)
from
20 -
200 -
2,000 -
20,000 -
to inoculate 5 tubes each of ml
16,000 10.0, 1.0,
0.1, 0.01
160,000 1.0, 0.1, 0.01, 0.001
1,600,000 0.1, 0.01, 0.001, 0.0001
16,000,000 .01, .001, .0001, .00001
160,000,000 .001,.0001,.00001, .000001
2c. For chlorinated Influents, 1.0, 0.1, 0.01, and
0.001 ml sample portions are recommended.
2d. For raw (untreated) sewage, use sample portions
of 0.0001, 0.00001, 0.000001, and 0.0000001 ml.
2e. For other waters, other combinations of sample
volumes may be required, particularly 1n
environmental waters receiving raw or Incompletely
treated sewage. It may be necessary to conduct
exploratory tests.
2f. For purposes of this UMP, the selected volumes
will be:
10.0; 1.0; 0.1; and 0.01
9-19
-------
WATER MONITORING PROCEDURE: Coliform Test by the Multiple
— Dilution Tube (MPN) Method
9-20
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First-day Procedures
(Continued)
4. Lab Bench
Disinfection
Enter information in
laboratory data sheet to
show sample inoculation
volume for each series
(row) of 5 tubes.
1. Disinfect laboratory
bench; wipe dry.
3a. Recommend showing sample inoculation volumes in
ml or decimal amounts.
. ieci
\
\
\
\
Received PM. Examined
PH
Observations
Amount
Sample
ml
10
1
.1
.01
—
.
Presumtive
LLSTB
24 hr
48 hr
Confirmed
BGLBB
24 hr
48 hr
I
la. Sponge and disinfenctant; paper toweling.
-------
WATER MONITORING PROCEDURE:
Coliform Test by the Multiple
Dilution Tube (MPN) Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First-day Procedures
(Continued)
5. Assembly and Label-
ing of Culture
Medium
6. Sample Inoculations
1. Place 5 tubes of Lactose
Lauryl Sulfate Tryptose
Broth (LLSTB) in each of
4 rows in culture tube
rack. (20 total tubes)
2. Label tubes of culture
medium to show sample
number, sample volume,
and position of tube in
the series of 5 tubes per
sample volume.
Row 1
Shake sample vigorously.
2. Deliver 10 ml of sample
into each of tubes in Row
1.
Row 2
3. Deliver 1 ml of sample
into each of tubes of Row
2.
Row 3
4. Deliver 0.1 ml of sample
into each of tubes of Row
3.
la. First row of 5 tubes to contain 1.5X LLSTB
(increased strength broth) and the next 3 rows
to contain the single strength medium.
2a. Use labeling code which allows instructor to
follow manipulation of tubes by trainee through-
out procedure.
2b. Label every tube. Only the experienced worker
should take short-cuts in labeling.
2c. Use wax pencil. Soft wax equivalent to
Blaisdell 169T is suggested.
la. At least 25 shakes over space of at least 1 foot
in 10 seconds or less.
2a. Use the same originally sterile 10 ml pi pet for
each of the 5 tubes.
2b. Discard pi pet into discard tray.
3a. Use the same originally sterile 1 ml pi pet for
each of the 5 tubes.
3b. Do not contaminate (bench-top, hands, etc.) pi pet
as it will be needed further.
4a. Use the 1 ml pi pet (as used for Row 2) to deliver
0.1 ml into each of the 5 tubes.
4b. Do not contaminate (bench-top, hands, etc.) pipet
as it will be needed further.
VII.B.5.2
(p. 9-47)
VII.B.6
I.B.6.1.1
(P. 9-48)
(p. 9-36)
9-21
-------
WATER MONITORING PROCEDURE:
Coliform Test by the Multiple
Dilution Tube (MPN) Method
9-22
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First-day Procedures
(Continued)
Row 4
5. Deliver 1.0 ml of sample
into 99 ml dilution blank.
5a. Water within 99 ml blank must have meniscus in
line with etched bottle marking before sample
del i very.
tfX/hi
OUTER
GLASS
WALL
CORRECT —
EYE —
LEVEL
CURVED
MENISCUS
LINE
ETCHED
LINE
e
6. Shake dilution blank
vigorously.
7. Deliver 1 ml of dilution
blank water into each of
tubes of Row 4.
LEVEL TABLE
5b. Discard 1 ml pipet into discard tray.
6a. As previously described.
7a. Use a sterile 1 ml pipet.
7b. Discard pipet into discard tray.
-------
UATER MONITORING PROCEDURE: Coliform Test by the Multiple
————— Dilution Tube (MPN) Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First-day Procedures
(Continued)
7. Incubation
8. Processing Used
Glassware
9. Lab Bench
Disinfection
1. After completion of sample
inoculation into LLSTB,
shake rack of cultures
gently.
2. Place rack(s) of cultures
in incubator.
1. Drain sample bottles,
dilution bottles, and
pi pets into sink.
2. Wash and dry bottles,
pi pets.
1. Disinfect laboratory
bench top; wipe dry.
la. Mixes sample with culture medium.
Ib. Avoid shaking air into fermentation vials.
2a. 24 hours + 2 hours at 35 + 0.5° C.
la. Sterilization unnecessary.
2a. Meets original cleanliness requirements of
glassware.
2b. Glassware ready for reuse.
la. Sponge, disinfectant, paper toweling.
C. 24-Hour Procedures
1. Equipment
Maintenance
2. Disinfection
3. Reading and Record-
ing of Results
1. Check, record, and adjust
incubator temperature.
2. Add water to pan in
incubator as necessary.
1. Disinfect laboratory
bench top; wipe dry.
1. Remove rack(s) of cultures
from incubator to lab
bench.
la. See A.1.1-6.
la. See B.4.1.
9-23
-------
WATER MONITORING PROCEDURE;
Coliform Test by the Multiple
Dilution Tube (MPN) Method
9-24
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. 24-Hour Procedures
(Continued)
2. Shake culture rack gentl
3. Examine each tube for gas
production and record
results on data sheet.
2a. Hastens release of gas in supersaturated cultures
2b. Must not shake air into fermentation vials.
3a. If present, gas will be trapped in the fermenta-
tion vial.
3b. Gas in any quantity is a positive test.
3c. Vials with no gas are a negative test.
3d. Each result appears on line corresponding with
the tube label.
3e. All results appear under the "24" of the LLSTB
column.
3f. Plus sign (+) means a gas-positive tube.
3g. Minus sign (-) means a gas-negative tube.
3h. Assume, for instruction purposes, that
the following recordings result:
III.C.3.3
(P. 9-41)
JJbservatu
Amount
Sample
ml
10
1
.1
0
Presumtlve
LLSTB
24 hr
+
4-
+
4.
4.
+
+
+
_
4-
_
+
+
•
-
-
—
_
_
48 hr
_
-------
WATER MONITORING PROCEDURE:
Coliform Test by the Multiple
Dilution Tube (MPN) Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. 24-Hour Procedures
(Continued)
4. Transfers
5. Processing Dis-
carded Cultures
1. Label and assemble tubes
of BGLBB.
2. Transfer each gas-positive
tube of LLSTB to a labeled
tube of BGLBB.
3. Return rack of tubes con-
taining the negative LLSTB
tubes and the freshly
Inoculated BGLBB tubes to
the 35° C Incubator.
1. Sterilize discarded LLSTB
tubes.
2. Remove all labels from
culture tubes.
la.
Ib.
Ic.
2a
One tube of each LLSTB gas-positive tube.
Each BGLBB tube label corresponds with label on
gas-positive LLSTB tube.
Labeled BGLBB tubes assembled in a culture tube
rack in same relative position as gas-positive
LLSTB tubes in their rack. In our example there
will be eleven tubes of BGLBB required.
Label on inoculated tube of BGLBB is the same as
the label on the tube of LLSTB from which the
transfer is made.
2b. 3 mm inoculation loop.
2c. Loop flame-sterilized before use and between
successive transfers.
2d. One loopful per transfer.
2e. Place inoculated BGLBB tube into hole of rack
previously occupied by the LLSTB tube from
which the transfer was made.
2f. Place positive LLSTB tube into discard area after
transfer is made. All discard tubes are to be
sterilized prior to cleaning and reuse of caps
and tubes.
3a. An additional 24 + 2 hours at 35° +_ 0.5° C.
VII.C.4.2
(p. 9-50)
la. Autoclave: 15 minutes at 121° C.
2a. Best done while still warm after autoclave.
9-25
-------
WATER MONITORING PROCEDURE; Coliform Test by the Multiple
Dilution Tube (MPN) Method
9-26
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. 24-Hour Procedures
(Continued)
6. Disinfection
3. Empty sterilized cultures
into sink.
4. Mash and dry culture tubes
fermentation vials, and
tube caps.
1. Disinfect laboratory bench
top; wipe dry.
4a. Meets original cleanliness requirements of
glassware.
4b. Tubes and caps ready for reuse.
la. Sponge and disinfectant; paper toweling.
D. 48-Hour Procedures
1. Equipment
Maintenance
2. Disinfection
3. Reading and
Recording of
Results
1. Check, record, and adjust
1ncubator temperatures.
2. Add water to pan in
Incubator as necessary.
1. Disinfect lab bench top;
wipe dry.
1. Remove the rack of cultures
from the incubator to lab
bench.
2. Shake culture rack gentl
Examine each tube for gas
production and record
results on data sheet.
3a. LLSTB tubes will be recorded under the "48" on
the LLSTB column and the BGLBB tubes under the
"24" column.
3b. Any amount of gas is always considered to be a
"positive" result.
-------
WATER MONITORING PROCEDURE: Coliform Test by the Multiple
Dilution Tube (MPN) Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. 48-Hour Procedures
(Continued)
3c. Assume that our "test" now shows the following
recordings:
..nervations
Amount
Sample
ml
10
1
.1
.1)
—
Presuntlve
LLSTB
24 hr
+
+
+
+
+
+
+
X
_
+
-
4-
_
+
_
-
_
_
_
.
48 hr
+
—
_
4-
—
_
—
—
.
—
Confirmed
BGLBB
24 hr
+
+
+
+
+
+
+
4.
+
+
I
48
4. Transfers
1. Discard all of the BGLBB
tubes which have the
positive recordings.
2. Discard all LLSTB
tubes which have the
negative recordings.
la. This will be a total of ten tubes (See data sheet
recordings in D.3.3.C).
2a. This will be a total of seven tubes (D.3.3.c).
2b. LLSTB tubes which show no gas production within
48 hours are to be considered as not having
contained coliform bacteria.
9-27
-------
WATER MONITORING PROCEDURE:
Coliform Test b
Dilution Tube
the Multiple
N) Method
9-28
OPERATING PROCEDURES
STtP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. 48-Hour Procedures
(Continued}
5. Processing
Discarded Tubes of
Media
3. Re-incubate any BGLBB tube;
which were negative and
assemble for transfer any
positive LLSTB tubes.
4. Label required tubes of
sterile BGLBB tubes.
5. Transfer each of the two
gas-positive LLSTB tubes tc
its corresponding tube of
BGLBB.
6. After each transfer, place
LLSTB tubes in discard
basket.
7. Place inoculated BGLBB
tubes in the 35° + 0.5° C
incubator.
7a.(Alternate) If no cultures
for this test procedure
remain to be incubated,
proceed to Interpretation
of Test Results and
continue as directed.
1. Sterilize discarded media.
2. Remove all labels from
culture tubes.
3. Empty sterilized cultures
into sink.
3a. There will be one tube of BGLBB which must be
re-incubated for an additional 24 hours at 35° +
0.5° C.
3b. There will be two positive LLSTB tubes.
4a. Two tubes of BGLBB should be labeled to
correspond to the two markings of the positive
LLSTB tubes.
5a. Use 3 mm loop which is flamed prior to entry
into the LLSTB to avoid contamination or cross-
contamination.
5b. Use one loopful of transfer from the LLSTB to
BGLBB.
6a. Contaminated tubes are to be sterilized prior to
cleaning operation.
-------
HATER MONITORING PROCEDURE:
Coliform Test by the Multiple
Dilution Tube (MPN) Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. 48-Hour Procedures
(Continued)
6. Disinfection
4. Wash and dry culture tubes,
fermentation vials, and
tube caps.
1. Disinfect laboratory bench
top; wipe dry.
E. 72-Hour Procedures
1. Equipment
Maintenance
2. Disinfection
3. Reading and
Recording of
Results
1. Check, record, and adjust
incubator temperatures.
2. Add water to pan in
incubator as necessary.
1. Disinfect lab bench top;
wipe dry.
1. Remove cultures from
Incubator to lab bench.
2. Shake cultures gently.
9-29
-------
WATER MONITORING PROCEDURE:
Coliform Test by the Multiple
Dilution Tube (MPN) Method
9-30
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
E. 72-Hour Procedures
(Continued)
3. Examine each tube for gas
production and record
results on data sheet.
4. Incubate any cultures
which are still negative
if they have not been
incubated a full 48 hours.
3a. In our continuing example, 3 tubes of BGLBB are
to be examined - one of which will be a "48"
entry and the other two of the "24" column entry.
Assume the following recordings:
jnint
Obset ..tior.s
Amount
Sample
ml
0
1
.1
.01
-
Presumfive
LLSTB
24 hr
+
+
4-
4-
J
4.
+
+
_
4-
+
_
+
_
_
_
_
_
43 hr
+
_
_
+
-
_
—
_
Confirmed
BGLBB
24 hr
4-
+
+
+
+
4.
+
+
4-
+
+
+
"
48 hr
+
4a.
Since both "24 hour" recordings of the BGLBB have
become positive, NO further culturings are
necessary and one could proceed with the
Interpretation of Test Results instead of the
96 hour procedure.
-------
WATER MONITORING PROCEDURE:
Coliform Test by the Multiple
Dilution Tube (MPN) Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
4. Processing
Discarded Tubes
of Media
5. Disinfection
1. Sterilize discarded tubes
of media.
2. Remove all labels from
tubes.
3. Empty sterilized tubes
into sink.
1. Disinfect lab bench top;
wipe dry.
F. Interpretation of
Test Results
Determine number of BGLBB
tubes which are positive
for each group of five
tubes of equal sample
volumes.
la. NO consideration of Presumptive Test (LLSTB)
for interpretation of test results.
Ib. Our example (E.3.3) shows
5 positive 1st row
5 positive 2nd row
3 positive 3rd row
0 positive 4th row
II.F.1-2
(p. 9-37)
9-31
-------
WATER MONITORING PROCEDURE:
Col i form Test by the Multiple
Dilution Tube (MPN) Method
9-32
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
F. Interpretation of
Test Results
(Continued)
2. Write the numbers in the
data sheet.
3. Select the 3-digit code
which applies to the
number of gas-positive
tubes of BGLBB.
2a.
•M. Examined
•vatIons
Confi rmed
BGLBB
24 hr
48 hr
3a. In a test involving 4 sample volumes this will
be based on rows 1, 3, 3, or on rows 2, 3, 4;
and
3b. If all tubes are positive in rows 1 and 2, then
the 3-digit code is based on rows 2, 3, 4.
3c. In all other cases the 3-digit code is based on
rows 1, 2, 3.
II.F.3
(p. 9-37)
-------
WATER MONITORING PROCEDURE:
Coliform Test by the Multiple
Dilution Tube (MPN) Method
OPERATING PROCEDURES
F. Interpretation of
Test Results
(Continued)
*
STEP SEQUENCE
4. Look up and record on the
data sheet the MPN Index.
5. Divide the MPN Index by
the number of mis of sample
represented by the middle
digit of the MPN Code.
The number obtained is the
MPN (Most Probable Number)
per 100 ml of original
sample.
INFORMATION/OPERATING GOALS/SPECIFICATIONS
4a. For the given example the location of the MPN
index is shown by the arrow based on the 5-3-0
code.
Table of Most Probable Numbers (MPN)
No. of Tubes Giving Positive
Reaction out of
5 of 10
ml Each
5
5
5
5
5
5
5
5a. Calculates
5 of 1
ml Each
1
1
1
2
2
2
3
to be 790.
5 of 0.1
ml Each
0
1
2
0
1
2
0
MPN
Index
per
100 ml
33
46
63
49
70
94
79
TRAINING
GUIDE NOTES
II. F. 4
(p. 9-38)
II. F. 5
(p. 9-38)
9-33
-------
WATER MONITORING PROCEDURE:
Coliform Test by the Multiple
Dilution Tube (MPN) Method
9-34
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
F. Interpretation of
Test Results
(Continued)
6. Record the calculated
Total Coliforms per 100 ml
on the laboratory data
sheet.
RESULTS: Coliform MPN
790
II.F.6
(p. 9-38)
G. Reporting of Results
1. Report results as
prescribed under
regulatory requirements.
-------
WATER MONITORING PROCEDURE: Coliform Test by the Multiple
Dilution Tube (MPN) Method
TRAINING GUIDE
SECTION TOPIC
I* Introduction
II* Educational Concepts - Mathematics
III* Educational Concepts - Science
IV Educational Concepts - Communications
V* Field & Laboratory Equipment
VI Field & Laboratory Reagents
VII* Field & Laboratory Analyses
VIII Safety
IX Records and Reports
*Training guide materials are presented here under the headings marked*.
These standardized headings are used through this series of procedures.
9-35
-------
WATER MONITORING PROCEDURES:
Coliform Test by the Multiple
Dilution Tube (MPN) Method
INTRODUCTION
Section I
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
B. 6.1.1
These MPN 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.
Test procedures are based on certain fundamental
assumptions:
a. First, even if only one living cell of the
test organisms 1s 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, unwanted 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.
9-36
-------
WATER MONITORING PROCEDURES: Coliform Test by the Multiple
Dilution Tube (MPN) Method
EDUCATIONAL CONCEPTS - MATHEMATICS
Section II
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
F.l-2
F.3
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 me'dium, 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).
The combination of positive and negative results
is used in an application of probability mathe-
matics to secure a single MPN value for the sample.
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 ii demon-
strated 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.
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.
Standard practice in water tests made by most or-
ganizations is to plant five tubes in each of a
series of sample increments, in sample volumes
decreasing at decimal intervals.
As an example, assume that all tubes were positive
for a sample portion of 10 ml, all five tubes were
positive on the portions of 1 ml, three of the
five 0.1 ml portions were positive, and none of the
five 0.01 ml portions were positive.
The numbers, on the above example, would be
5-5-3-0.
1. Pursuing the above example, the code would be
5-3-0.
2. Selection of codes is sometimes complicated.
For further.information study training guide
notes and cited references.
Std. Meth. 14:923 ff
9-37
-------
WATER MONITORING PROCEDURES: Coliform Test by the Multiple
Dilution Tube (MPN) Method
EDUCATIONAL CONCEPTS - MATHEMATICS
Section II
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
F.4
F.5
F.6
1. Appears on MPN Table (attached to this Section)
2. Pursuing the given example, the MPN Index for
MPN Code 5-3-0 would be 79.
1. As indicated above, the middle digit 1s 3; and
it represents a sample portion of 0.1 ml. An
MPN Index of 79 divided by 0.1 1s 790.
The Coliforms per 100 ml would be recorded as 790.
9-38
-------
HATER MONITORING PROCEDURES: Col1 form Test by the Multiple
Dilution Tube (MPN) Method
EDUCATIONAL CONCEPTS - MATHEMATICS
Section II
TRAINING GUIDE NOTE
Table of Most Probable Numbers (MPN)
No. of Tubes Giving Positive
Reaction out of
5 of 10
ml Each
0
0
0
0
1
1
1
1
1
2
2
2
2
2
2
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5 of 1
ml Each
0
0
1
2
0
0
1
1
2
0
0
1
1
2
3
0
0
1
1
2
2
3
0
0
1
1
1
2
2
3
3
4
0
0
0
5 of 0.1
ml Each
0
1
0
0
0
1
0
1
0
0
1
0
1
0
0
0
1
0
1
0
1
0
0
1
0
1
2
0
1
0
1
0
0
1
2
MPN
Index
nov*
per
100 ml
<2
2
2
4
2
4
4
6
6
5
7
7
9
9
12
8
11
11
14
14
17
17
13
17
17
21
26
22
26
27
33
34
23
31
43
REFERENCES/RESOURCES
9-39
-------
MATER MONITORING PROCEDURES: Coliform Test by the Multiple
Dilution Tube (MPN) Method
EDUCATIONAL CONCEPTS - MATHEMATICS
Section II
TRAINING GUIDE NOTE
Table of Most Probable Numbers (MPN)
No. of Tubes Giving Positive
Reaction out of
5 of 10
ml Each
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5 of 1
ml Each
1
1
1
2
2
2
3
3
3
3
4
4
4
4
4
5
5
5
5
5
5
5 of 0.1
ml Each
0
1
2
0
1
2
0
1
2
3
0
1
2
3
4
0
1
2
3
4
5
MPN
Index
per
100 ml
33
46
63
49
70
94
79
110
140
180
130
170
220
280
350
240
350
540
920
1600
52400
REFERENCES/RESOURCES
9-40
-------
WATER MONITORING PROCEDURES: Coll form Test by the Multiple
———————— Dilution Tube (MPN) Method
EDUCATIONAL CONCEPTS - SCIENCE
Section III
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
C.3.3
Interpretation of results on LLSTB:
Development of gas in this medium indicates that
the lactose has been fermented. Fermentation of
lactose with gas production is a basic characteris-
tic of coliform bacteria. To meet the definition
of coliforms, gas must be produced from lactose
within 48 hours after being placed in the incubator
If a culture develops gas only after more than 48
hours incubation, then, by definition, it is not a
coliform.
Meeting previously discussed assumptions (See
I.B.6.1.1) usually makes it necessary to conduct
the tests in a series of stages.
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 ex-
traneous bacteria, but this feature never should
be included if it also inhibits growth of any
cells of the group for which the test is de-
signed. 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 pro-
duction 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 non-
col i form bacteria. This additive apparently has
no adverse affect on growth of members of the
coliform group in the concentrations 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 fur-
ther 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.
9-41
-------
WATER MONITORING PROCEDURES:
Coliform Test by the Multiple
Dilution Tube (MPN) Method
FIELD AND LABORATORY EQUIPMENT
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.I
A.1.1
A.1.2
A.1.3
A.1.5
A.1.6
Incubator must be of sufficient size for daily
work load without causing crowding of tubes to be
incubated. Considerations for choice of incubator
type must relate to reliability of operation and
not to cost or attractiveness of equipment.
Incubator should be kept out of drafts or direct
sunlight in order to prevent temperature inside
the incubator from changing outside the tempera-
ture range specified (35° +. 0.5°).
Power supply should be selected so that there will
not be too many pieces of equipment on the same
circuit. Otherwise, circuits will be blown
repeatedly.
Mercury bulb thermometer usually used in most incu-
bators. Recording thermometer is acceptable, but,
it should be calibrated against a mercury bulb
thermometer which has been certified by National
Bureau of Standards. The NBS certified thermo-
meter always should be used with its certificate
and correction chart.
Saturated relative humidity is required in order
to make the incubation more efficient (heat is
transferred to cultures faster than in a dry incu-
bator). Furthermore, culture medium may evaporate
too fast in a dry incubator.
Allow enough time after each readjustment to per-
mit the incubator to stabilize before making a
new adjustment. At least one hour is suggested.
Incubator temperature can be held to much closer
adjustment if operated continuously. Temperature
records should be kept in some form of permanent
record. A temperature record book is suggested
with daily recording of values. If a recording
thermometer is used, the charts may be kept as
permanent record; if so, be sure that the charts
are properly labeled to identify the incubator and
the period covered.
Uniform temperature (35° C + 0.5) is to be main-
tained on shelves in use.
Standard Methods for the
Examination of Water and
Wastewater. 14th ed. (1975
APHA, WPCF, AWWA, p. 880
(Hereafter referred to as:
Std. Meth. 14: (page no.)
9-42
-------
MATER MONITORING PROCEDURES: Coliform Test by the Multiple
———————— Dilution Tube (MPN) Method
FIELD AND LABORATORY EQUIPMENT
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.2.1-5
A.3.1
A.4.1-2
Since electric sterilizer will be operated inter-
mittently, care should be taken that it is on a
circuit which will not be overloaded when it is
turned on.
A time and temperature record is maintained for
each sterilization cycle. Temperature recordings
can be retained for records.
Autocalves differ greatly in design and in method
of operation. Some are almost like home-style
pressure cookers; others are almost fully auto-
matic. This is a subject which requires separate
instruction; and should be related to the exact
make and model of equipment you will use in your
own laboratory.
Vertical autoclaves and household pressure cookers
may be used in emergency service if equipped with
pressure gages and thermometers with bulbs
positioned 1 inch above the water level. However,
they are not to be considered the equivalent of
the general purpose steam sterilizer recommended
for permanent laboratory facilities. Their small
size is inadequate for large-volume work loads, and
they can be difficult to regulate.
The following requirements must be met regarding
autoclaves or sterilizing units:
a. Reaches sterilization temperature (121°C),
maintains 121°C during sterilization cycle, and
requires no more than 45 min. for a complete
cycle.
b. Pressure and temperature gages on exhaust side
and an operating safety valve.
c. No air bubbles produced in fermentation vials
during depressurization.
d. Record maintained on time and temperature for
each sterilization cycle.
Distilled water in a bacteriological laboratory
must not contain substances which will prevent
any bacteria from growing in culture medium in
which the distilled water is used or will be highly
nutritive. There are procedures for testing quality
of distilled water; but these should be undertaken
only by professional bacteriologists or in labora-
tories where this is done regularly. Use only glass
stills or block tin lined stills.
Std. Meth. 14:881
Std. Meth. 14:881
St. Meth.
14:645-49
14:888-891
9-43
-------
MATER MONITORING PROCEDURES: Coliform Test by the Multiple
Dilution Tube (MPN) Method
FIELD AND LABORATORY EQUIPMENT
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.5.1
A.6.1-43
A.6.1-4b
A. 9.1-6
A.10.1-6
A.10.7
A.11.1.Id
pH Meter: See cited reference.
Glassware: See cited reference on pi pets and
graduated cylinders, media utensils, bottles.
Glassware can be checked for bacteriostatic or
inhibitory residues by a bacteriological test
procedure which, like the distilled water suit-
ability test, should be undertaken only by
professional bacteriologists or in laboratories
where this test is done on a regular basis.
Sample bottles:
Wide-mouthed, glass-stoppered bottles suggested,
but other styles acceptable.
If glass-stoppered bottles are used, a strip of
paper should be placed in the neck of the bottle
before placing the stopper in place in preparation
for sterilization. This prevents the glass stopper
from "freezing" in place during sterilization. The
paper strip is discarded at the time of sample
collection.
Pi pets:
This procedure is described in terms of reusable
glass pi pets. However, single-service prepackaged
glass or plastic pi pets may be purchased and used,
if preferred. In case of use of single-service
pipets, they will be sterile when purchased, are
used one time, and discarded Immediately after use.
Accordingly, in the step-by-step procedures dis-
regard any instructions about preparation of pipets
for reuse in case of using single-service pipets.
Passing the opened can of pipets through a flame
burns off excess cotton wisps sticking out of the
mouthpiece of the pi pet. If this is not done, it
is almost impossible to control sample measurement
accurately. Some workers may elect to accomplish
this step prior to the sterilization procedure.
See cited reference. In time, this solution will
become mold-infested. At this time it should be
discarded and a new stock solution prepared.
Std. Meth. 14:882
Std. Meth. 14:882-885
Std. Meth. 14:884
14:904
Std. Meth. 14:882-883
Std. Meth. 14:892
9-44
-------
WATER MONITORING PROCEDURES:
Coliform Test by the Multiple
Dilution Tube (MPN) Method
FIELD AND LABORATORY EQUIPMENT
Section v
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.11.4
A.11.5
A.11.6
A.11.8
A.12.35
Dilution water preparation:
Measurement of dilution water into bottle with a
100 ml graduated cylinder is time-consuming, but
effective. An automatic pipetting machine can be
considered a luxury, but is a real time-saver.
If caps are not placed on bottles of dilution water
loosely, they may crack in autoclave; furthermore,
steam will not be able to get in contact with the
material being sterilized. After sterilization,
tightening caps on bottles of distilled water will
permit them to be kept for long periods.
Always pack material loosely and away from walls
in autocalve when preparing to sterilize. Steam
must flow freely around materials being sterilized.
If water should evaporate noticeably or become
contaminated by microbial growth, the bottle of
distilled water should be discarded.
Funnel
Funnel, Hose, and
Pinchcock Assembly
Pinchcock
Hose
Glass Tube
NOTE: Unit need not be
sterile for medium
delivery only.
9-45
-------
WATER MONITORING PROCEDURES:
CoHform Test by the Multiple
Dilution Tube (MPN) Method
FIELD AND LABORATORY ANALYSES
Section
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
B.3.1
B.3.2
There is no such thing as a "standard" data sheet
for bacteriological tests. A simplified data sheet
is shown below.
In this procedure, it is recommended that the worker
learn to select a series of 4 sample volumes in de-
creasing amounts as indicated.
It is possible to use as few as three sample
volumes, but often the worker will fail to get a
measurable result. On the other hand, one could
have 5, 6, or even more sample volumes in de-
creasing amounts.
M
Sample T
Station
COL I FORM TEST
ultiple Dilution Tube (MPN) Method
voe Lab. No.
Description
Collection Date
Time
APM
Received PH. Examined
pH
' TefflP^
PM.
Observations
Amount
Sampl e
ml
Presumtive
LLSTB
24 hr
48 hr
Conf 1 rmed
BGLBB
24 hr
48 hr
RESULTS: Conform MPN
9-46
-------
WATER MONITORING PROCEDURES:
Coliform Test by the Multiple
Dilution Tube (NPN) Method
FIELD AND LABORATORY ANALYSES
Section vil
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
B.5.2
Suggested labeling code for tubes:
1. Every tube shows the laboratory bench number
(323 in example shown below).
2. Below the laboratory bench number on each tube
will be found a code symbol which represents
the sample volume and the tube of each series
of five. Thus:
Sample volume, ml Tubes are labeled
10.0
1.0
0.1
0.01
0.001
0.0001
0.00001
0.000001
0.0000001
A B C D
a, b, c, d,
«L» k» £»
-------
WATER MONITORING PROCEDURES:
Coliform Test by the Multiple
Dilution Tube (MPN) Method
FIELD AND LABORATORY ANALYSES
Section VII
B.6.
TRAINING GUIDE NOTE
Multiple dilution tube tests for quantitative
determinations apply a Most Probable Number (MPN)
technique. In this procedure one or more measured
portions of each of a series of decreasing sample
volumes is inoculated into the first-stage culture
medium. Through decreasing the sample increments,
eventually a volume is reached where only one cell
is introduced into some tubes. Each of the several
tubes of sample-inoculated first-stage medium is
tested independently, according to the principles
described.
Another way to represent sample dilution and inocu-
lation is shown below. Sample dilutions are made a:
needed during the inoculation procedure; they are
not made up before starting to inoculate tubes of
culture medium. Bacteria shall not be suspended
in any dilution water for more than 30 minutes at
room temperature.
Table of sample portions
To get Deliver From
(ml) (ml) (sample preparations)
1.0 1.0 original sample
0.1 (1:10) 0.1 original sample
0.01 (1:100) 1.0 1:100 dilution
0.001 (1:1000) 0.1 1:100 dilution
0.0001 (1:10000) 1.0 1 : 10000 dilution
0.00001 (1:100000) 0.1 1 : 10000 dilution
0.000001 (1:1000000) 1.0 1:1000000 dilution
Dilutions of original samples
Deliver to
To get 99-ml blank From
1:100 1 ml Original sample
1:10000 1 ml 1:100 dilution
1:1000000 1ml 1 :1 0000 dilution
REFERENCES/RESOURCES
9-48
-------
WATER MONITORING PROCEDURES: Coliform Test by the Multiple
Dilution Tube (MPN) Method
FIELD AND LABORATORY ANALYSES
Section
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
DILUTION RATIOS.
1100
1.10000
10 ml DELIVERY VOLUME 1 ml 01 ml 1 nl 01 ml
TUBES
ACTUAL VOLUME
OF SAMPLE IN TUBE
1 nl
PETRI DISHES OR CULTURE TUBES
01 ml 0 01 ml 0 001 ml
0001 nl
0 00001 nl
9-49
-------
UATER MONITORING PROCEDURES:
Conform Test by the Multiple
Dilution Tube (MPN) Method
FIELD & LABORATORY ANALYSES
Section VII
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
C.4.2
9-50
Transfers of LLSTB
Transfers can be made, as indicated, with a wire
loop having a diameter of at least 3 mm. An
alternate method of transfer authorizes the use of
an "applicator stick" which is a single service
hardwood transfer device. Its dimensions are 0.2
to 0.3 cm in diameter and 2.5 cm longer than the
test tube used in the analysis. The term single
service denotes that the stick is pre-sterilized
and used for a single transfer (LLSTB to BGLBB) and
then discarded in the pan containing disinfectant
and a new sterile stick used for the next tube to
be transferred. Use of this stick technique makes
the gas burner unnecessary for the transfer process
Std. Meth. 14:922
This outline was prepared by: Rocco Russomanno,
Microbiologist, National Training and Operational
Technology Center, MOTD, OWPO, USEPA, Cincinnati,
Ohio 45268.
-------
A PROTOTYPE FOR DEVELOPMENT OF
ROUTINE OPERATIONAL PROCEDURES
for the
COMPLETED TEST FOR THE MPN METHOD
as applied in
WATER TREATMENT FACILITIES
WASTEWATER TREATMENT FACILITIES
and in the
MONITORING OF EFFLUENT WASTEWATERS
National Training and Operational Technology Center
Municipal Operations and Training Division
Office of Water Program Operations
U.S. Environmental Protection Agency
BA.MET.lab.WMP.6.5.78
10-1
-------
COMPLETED TEST SCHEMATIC
* «
V\ ETC
(STREAK
TECHNIQUE J
POSITIVE BGLBB TUBES
FROM CONFIRMED TEST
(POSSIBLES CAN OCCUR IN 48,
72, AND 96 HR STANDARD
TEST TIME INTERVALS)
EMB AGAR PLATES
X
(INCUBATE 24 ± 2 HRS
xxx AT 35 ± 0.5 C)
^
(PICK APPROPRIATE
COLONIES)
CULTURE
#3
ETC
CULTURE #
I
1 (24 ± 2 MRS AT 35 ± 0.5 C)
I
1
[ GENTLY
SHAKE)
f
GAS -
I
GAS +
GRAM STAIN
r
(RE-INCUBATE
TOTAL:
48 ± 3 HRSJ
GAS -
CONFORMS
ABSENT
[ REPEAT FOR
CULTURES 2, 3, ETC)
INTERPRET
RESULTS
RE-EVALUATE, IF NECESSARY, THE
CONFIRMED COLIFORM RESULTS
10-3
REPORT RESULTS
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method
1. Analysis Objectives:
In control testing, this test is part of the Standard Test for Coliforms and
is one of the two tests of choice for reporting purposes. The completed test
should be applied to such a proportion of tests as to establish beyond reason-
able doubt the value of the confirmed test in determining the sanitary quality
of water, and, as a rule of thumb, applied to at least ten (10) percent of all
positive samples. When a specific sample is being tested, the completed test
is applied to all positive confirmed tubes of that sample.
2. Brief Description of Analysis:
All positive tubes of BGLBB (Brilliant green lactose bile broth) from the
confirmed test of the Standard Coliform MPN Test are individually and ascep-
tically transferred onto EMB Agar by the streaking technique. After incubation
for 24 + 2 hours at 35 + 0.5°C, one or more typical isolated colonies (dark-
centered with or without sheen) or two or more atypical colonies (opaque; un-
nucleated; mucoid; or pink) if only these are present, are selected from each
plate and transferred to LLSTB (lactose lauryl sulfate tryptose broth) and a
nutrient agar slant (NAS). Each pure culture is incubated for 24+2 hours
at 35 +_ 0.5°C on these media and then inspected for gas formation Tj-LSTB) and
growth (NAS). A gram stain is prepared from each NAS at this time with the
slant asceptically (sterile technique) manipulated and then preserved under
refrigeration for possible future need. A positive (gaseous) LLSTB is data
recorded and discarded while a negative (non-gaseous) tube is re-incubated for
an additional 24 hours (total of 48 +_ 3 hours) when it is again inspected for
gas production. Coliforms are considered to have populated the original BGLBB
tubes if pure culture, gram-negative, non-spore forming rods which gaseously
fermented lactose were isolated by this procedure. Any other results are
considered to be the actions of non-coliforms except in the case of lactose
fermentaters which are caused by mixed culture (two or more different organisms
consisting of gram-positive and gram-negative forms). In this case, the re-
tained nutrient agar slant is restreaked on EMB and the subsequent procedures
repeated to attempt to isolate a pure culture with the coliform characteristics.
Adjustments, if any, are made to the tube codings and the MPN re-calculated to
give a MPN completed result which is now the required reportable result.
This procedure conforms to the Standard Total Coliform MPN Test as described in
Standard Methods for the Examination of Water and Wastewater, 14th Edition
(1975), p. 916 ff.
10-4
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method
Equipment and Supply Requirements
A. Capital Equipment:
1. *Incubator, air, to operate at 35° * 0.5°C
2. *0ven, hot air, sterilizing-drying, to give uniform temperatures and with
suitable thermometer to register accurately in range of 160-180°C
3. *Autoclave, providing uniform temperatures up to and including 121°C,
equipped with an accurate thermometer, pressure gauges, saturated steam
power lines and capable of reaching required temperature within 30 minutes
4. Balance, 0.1 g sensitivity at load of 150 g
5. pH Meter, accurate to at least 0.1 pH unit, with standard pH reference
solution(s)
6. Mater distillation apparatus, (glass or block tin), or source of distilled
water suitable for bacteriological operations^.
7. Microscope, compound, oil immersion lens, Abbe condenser
B. Reusable Supplies:
1. Apron or coat suitable for laboratory
2. Baskets, wire for discarded cultures
3. Tubes, culture*, 150 x 18 mm (metal caps for fermentation and screw-cap
for slants)
4. Tubes, fermentation*, 75 x 10 mm vials to be inverted in culture tubes
5. Inoculation loop and needle, 3 mm diameter for loop and both of nichrome or
platinum-iridium wire, 26 B&S gauge, in holders
6. Hotplate with magnetic whirl feature, if desired
7. Burner, gas, Bunsen burner type
8. Sponge, for cleaning desk top
9. Counter, colony, Quebec type, Darkfield Model with guide plate
10. Racks, culture type*, 10 x 5 openings, to accept tubes at least 25 mm in
diameter
11. Pan, to receive discarded contaminated pipets and glassware (must contain
disinfectant before use)
12. *Flasks, Erlenmeyer, 500 ml; 300 ml; 250 ml
13. Cylinder, 500 ml; 250 ml
C. Consumable Supplies:
1. Bibulous paper
2. Dishes, petri, 100 x 15 mm sterile plastic, disposable
3. Disinfectant, for bench tops. (Can use household bleach solution prepared
according to instructions on bottle.)
4. Distilled water, suitable for bacteriological cultures (Note distillation
apparatus required in capital equipment.)
5. Eosin methylene blue agar, dehydrated (Levine modification)
6. Gram stain solutions, complete set
7. Lactose Lauryl Sulfate Tyrptose Broth, dehydrated
8. Nutrient agar, dehydrated
9. Slides, microscopic, glass 1" x 3"
10-5
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method
10. Foil, aluminum
11. Matches or striker
12. Wax pencils (recommend soft as equivalent to Blaisdell 169T)
*I terns marked are needed In quantities or require size or space allowances which
cannot be specified here, as they vary according to the daily analysis schedule.
As a rule-of-thumb, space/size or quantity requirements should be at least 3 times
the normal daily requirements. For further information on specifications for
equipment and supplies, see the Microbiology Section of the current edition of
"Standard Methods for the Examination of Water and Wastewater."
10-6
-------
WATER MONITORING PROCEDURE; Completed Test for the MPN Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
1. 35°C Incubator
Set-up, Adjustment
1. Place 35°C incubator in
permanent location.
2. Install thermometer.
3. Install shallow pan of
water in bottom of
incubator.
4. Connect incubator to
electric power source.
5. Adjust temperature until
stabilized at required
temperature.
6. Operate bacteriological
incubator continuously.
Aa. All pre-test procedures completed before starting
other first-day proceudres.
la. Out of drafts or places where it will be in
sunlight part of day.
Ib. Location convenient to laboratory bench
Ic. Convenient source of electric power.
2a. Thermometer functions at least in 30°-40°C range
and have intervals of 0.5° or less indicated.
Meets NBS standards.
2b. Location should be central in incubator.
2c. Mercury bulb thermometer should be fitted with
cork or rubber stopper and mounted in small
bottle filled with liquid (glycerine, water, or
mineral oil).
3a. In most incubators a pan having about 1 square
foot of area, with water about 1 inch deep, is
satisfactory.
3b. Maintains condition of saturated relative
humidity, required in bacteriological incubator.
3c. Requires daily check, with addition of water as
necessary, to keep water in pan at all times.
4a. Many incubators have pilot light to indicate power
turned on.
5a. Manufacturer's instructions for method temperature
adjustment.
5b. Operation must be at 35° + 0.5°C.
5c. Allow about 1 hour between adjustments.
6a. Requires daily check with written temperature
record, with adjustment and water addition as
necessary.
V.A.I
A.A.1.1
(p. 48)
V.A.I.2
(p. 48)
V.A.I.3
(p. 48)
V.A.1.5
(p. 48)
V.A.I.6
(p. 48)
10-7
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method
10-8
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
2. Oven, Sterilizer-
Dryer Set-up
3. Autoclave Set-up
1. Place oven sterilizer in
permanent location.
2. Install thermometer.
3. Connect oven sterilizer to
power source and turn on.
4. Adjust temperature to
stabilize at required
temperature.
5. Operate oven sterilizer
only When needed. Turn off
when not in use.
1. Install and operate auto-
clave according to manu-
facturer's instructions.
la. Convenient to source of electric power usually on
table or bench.
2a. Should indicate the 160°-180° range, be accurate
within this interval, and be marked in 1.0 degree
intervals.
3a. Usually has pilot light to indicate power on.
4a. Operated as near to 170°C as possible; not lower
than 160° or higher than 180°C.
5a. Unless materials are "heat shocked" adversely,
oven is turned ON in advance of need to permit
reaching required temperature before introducing
material.
5b. Oven used to sterilize or dry glassware, metal
objects.
5c. Oven sterilizer not used with culture media,
solutions, plastics, rubber objects, or with
anything containing or including these.
5d. Paper-wrapped glass pipets, graduates, flasks,
etc. may be sterilized in oven sterilizer.
la. Autoclaves extremely variable in design and
operation; also, potentially dangerous.
Ib. Used to sterilize objects made of, or including
liquids, rubber, culture media.
Ic. Glassware may be autoclave sterilized but must be
dried afterward.
Id. Most plastics not sterilized in autoclave;
plastics usually require chemical sterilizers.
le. Autoclave usually operated at 121°C for 15minutes,
If. Sterilized media must be removed from autoclave
as soon as possible after autoclave is reopened.
V.A.2.1-5
(p. 49)
V.A.3.1
(p. 49)
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
4. Water Distillation
Equipment
5. pH Meter
6. Glassware
Preparation of
Lactose Lauryl
Sulfate Tryptose
Fermentation Broth
(LLSTB)
1. Install and operate in
accordance with manu-
facturer's instructions.
2. Operate as required to
maintain adequate supplies
of distilled water.
1. Have unit available and
operable.
1. Wash all glassware in hot
detergent solution.
2. Rinse at least once in hot
tap water.
3. Rinse in distilled water,
at least 6 successive
times and,
4. Dry in air or oven.
Weigh 8.9 grams of dehy-
drated Lactose Lauryl
Sulfate Tryptose Broth.
Close cover of bottle of
dehydrated medium tightly
after removal.
la. Must produce distilled water meeting quality
requirements for bacteriological tests.
2a. Reserve supplies kept in borosilicate glass
carboys or in plastic carboys made of material
which will not dissolve substances which will
affect growth of bacteria.
2b. Same distillation apparatus used for bacterio-
logical purposes may be used for chemical
reagents.
la. Unit for pH check on finished culture media.
la. Nontoxic detergent.
Ib. Be sure all contents and markings are washed away.
4a. No visible spots or scum; glass should be clean
and sparkling.
4b. Glassware suitable for use in bacteriological
operations.
la. Dehydrated media takes moisture out of air; can
become caked.
Ib. Caked media unsatisfactory; should be discarded.
V. A. 4. 1-2
(p. 49)
V.A.5.1
(p. 50)
V.A.6.1-4a
(p. 50)
V.A.6.4b
Cp. 50)
10-9
-------
MATER MONITORING PROCEDURE: Completed Test for the MPN Method
10-10
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
2. Dissolve in 250 ml dis-
tilled water.
3. Place 10.5 ml of the solu-
tion of prepared LLSTB in
each culture tube.
4. Insert one fermentation
vial into each tube of
medium, open end down.
5. Place tube cap on each
tube of culture medium.
6. Sterilize in autoclave.
7. Cool medium to room
temperature.
8. Check pH of finished
medium.
9. If final pH not satis-
factory, discard medium
and prepare new batch with
pH adjustment before
sterilization.
2a. Use a 500 ml Erlenmeyer flask.
2b. Gentle heat Cno boiling) if necessary to com-
plete dissolving medium. Usually a vigorous
agitation will completely dissolve the medium.
3a. Use 150 x 18 mm tubes.
3b. A 25 ml pipet, automatic pipetter, or funnel, hose
and pinchcock assembly are acceptable.
3c. Accuracy of delivery: +_ 0.5 ml.
3d. Approximately 23 tubes will be necessary.
4a. Tubes and vials washed as indicated previously.
4b. Use 75 x 10 mm tubes.
5a. After all tubes have been filled and have
individual vial.
6a. Within 1 hour after medium prepared.
6b. Sterilization at 121°C for 15 minutes.
6c. Medium must be removed from autoclave as soon as
possible after pressure has returned to normal.
Use "slow-vent" mode of steam removal.
7a. Medium ready for use when cool and individual vials
are completely filled with fluid. No bubbles must
be present. Wait for complete cooling before
checking for bubbles.
8a. Should be pH 6.7-6.9. It is rare that deviations
occur with this preparation.
9a. pH value ordinarily drops about 0.2 pH unit.
9b. Check for dirty glassware, acid residues in
glassware, etc.
V.A.7.3
[p. 50)
-------
HATER MONITORING PROCEDURE: Completed Test for the MPN Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
8. Preparation of
Eosin Methylene
Blue Agar
(EMB Agar)
10. Store medium in cool, dark
place.
1. Weigh 7.5 grams of de-
hydrated eosin methylene
blue agar. Close cover
of bottle of dehydrated
medium tightly after
removal.
2. Dissolve in 200 ml dis-
tilled water.
3. Sterilize medium in
autoclave.
4. Cool medium to 50-60°C and
pour into sterile petri
dishes.
lOa. Not in refrigerator. Usually in laboratory
cabinet in darkness.
lOb. May be stored up to 1 week if evaporation not
more than 10% in loose-fitting capped tubes.
With screw-capped tubes, it should be held no
longer than 3 months.
la. Use only Levine's Modification as this medium has
a number of modifications for differing purposes.
Ib. Dehydrated media takes moisture out of air; can
become unacceptably caked.
2a. Use a 300 ml Erlenmeyer flask with double layer
foil cap.
2b. Heat to boiling to dissolve completely. Do not
prolong boiling.
2c. Frequent agitation is necessary to prevent
burning of medium.
2d. All of the agar must be in solution. Agar will
be recognized as particulate matter along the
sides of the flask. Gently swirl flask until all
of this material is off of sides and into medium.
3a. For 15/15 to effect complete sterilization
(15 psi for 15 minutes).
3b. Medium must be removed from autoclave as soon as
possible after pressure has returned to normal.
Use "slow-vent" mode of steam removal.
4a. Can also be poured "hot" from autoclave with
precautions, such as using asbestos glove, for
personal protection.
V.A.8.2C
(p. 50)
10-11
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method
10-12
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Proceudres
(Continued)
5. Allow dishes to cool to
room temperature and
then dry.
6. Check pH of one of the
plates.
7. Label and date batch of
plates. Store either at
room temperature when use
Is made of plates within
several days or In sealed
plastic bags, at 4°C.
4b. A flocculant may form after autoclavlng. Swirl
flask gently during plate (dish) filling.
4c. About 10-12 mis/plate. About 15 plates will be
required.
4d. Cover plates as they are poured. Do not place
covers on bench where they can become contaminated
5a. Agar will solidify and allow plate to be moved
without disturbing medium.
5b. Invert plates (turn upside down) and place in
35° incubator overnight. This will allow plates
to dry and remove excess moisture.
5c. Plates can be used when agar surface 1s "dry"
(does not have water droplets).
6a. Insert pH meter probes into the agar medium
using one of the plates of the batch.
6b. Should read 7.0-7.2.
6c. Discard plate after measuring pH. Alternately,
to save medium, one could fill a small clean
receptacle, or, a 60 x 15 mm petri dish for this
check.
6d. Out of range reading denotes unacceptable pro-
cedure, equipment or materials used (dirty
glassware, poor water supply, overheating, etc.).
Discard plates and rectify problem.
7a. Can be kept for one month under refrigeration as
described. Plates may have to be re-dried in the
incubator overnight (inverted) after removal
from refrigerator.
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
9. Prepare Gram-
Stain Solutions
10. Prepare Nutrient
Agar Slants (NAS)
1. Prepare solutions as
recommended by
manufacturer.
2. Place in dropper bottles
for use.
1. Weigh 2.9 grams of dehy-
drated Nutrient Agar.
Close cover of bottle of
dehydrated medium tightly
after removal.
2. Dissolve in 125 ml dis-
tilled water.
3. Dispense 6-7 mis of medium
into screw-cap tubes.
4. Place screw caps loosely
on each tube which are
packed loosely in a test
tube rack, beaker, etc.
5. Sterilize tubes in
autoclave.
la. Premixed dyes will probably only require dilution.
Ib. If desirous to prepare dyes from scratch, consult
Standard Methods for procedure.
la. Dehydrated media takes moisture out of air; can
become caked.
Ib. Caked media unsatisfactory; should be discarded.
2a. Use a 250 ml Erlenmeyer flask with double layer
foil cap.
2b. Heat to boiling to completely dissolve.
2c. Frequent agitation is necessary to prevent
burning of medium.
2d. All of agar must be in solution. Agar will be
recognized as particulate matter along the sides
of the flask. Gently swirl flask until all of
this material is off of sides and into medium.
3a. Use 150 x 18 mm screw-cap tubes.
3b. A 10 ml pipet, automatic pipetter, or funnel,
hose, and pinchcock assembly are acceptable.
3c. Approximately 25 tubes will be required.
4a. Allows steam to penetrate to medium.
Std. Meth.
14:918-919
See V.A.8.2c
(p. 50)
See V.A.7.3
(p. 50)
5a. For 15/15 to effect complete sterilization (15 psi See V.A.3.1
for 15 minutes). Remove medium as soon as p. 49)
possible after cycle (slow vent mode) is completed.
10-13
-------
MATER MONITORING PROCEDURE: Completed Test for the MPN Method
10-14
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
6. Tighten caps and slant hot
medium.
7. Allow tubes to solidify
before removing from
slanted position and
placing in test tube rack.
8. Date and label medium as
Nutrient Agar. Store in
refrigerator.
6a,
6b.
6c.
6d.
Tight caps will prevent further loosening and
possible contamination.
Necessary to slant while hot so that medium will
not solidify in upright position.
"Slanting" is done to allow a large surface area
for growth of bacteria.
SLANT AREA
MEDIUM
Apparatus for tube holding while in the slanted
position can range from expensive "angle" con-
trolled supports to as simple and effective a
method as below:
SLANTED
TUBES
1"
- HOSE OD
7a. Solidified tubes can be picked up and will retain
"slanted" position of medium. Tubes will start to
harden below 40°C and take on an "opaque" form as
they harden.
8a. Temperature 1-4.4°C.
8b. Can be stored for up to 3 months (if kept in dark
and evaporation is not excessive (less than .25
ml).
-------
MATER MONITORING PROCEDURE; Completed Test for the MPN Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pre-Test Procedures
(Continued)
11. Final Equipment
and Supply Check
1. Check to be sure that all
equipment and supplies,
solutions, and prepared
media are ready before
starting sample examina-
tion.
la. Check general list of equipment and supplies.
Ib. Each test requires:
1-15 EMB agar plates
1-20 Nutrient Agar Slants
1-20 LLSTB tubes
1 Bacteriological loop
1 Bacteriological needle
1-20 Microbiological slides
Gram stain reagents, set
Since, as shown, the numbers of items can vary
(depending upon the number of confirmed test
positives and subsequent EMB colony forms) this
WMP (Water Monitoring Procedure) will specifically
pick a hypothetical situation which will give the
reader a cross-section of conditions which could
occur.
B. Initial Procedures
1. Equipment
Maintenance
2. Data Sheet
Inspection
1. Check, record, and adjust
incubator temperature.
2. Add water to pan in incu-
bator as necessary.
1. Locate data sheet and
verify that the required
sample is being processed.
la. See A.1.1-6.
Ib. Should be in operating condition since MPN test's
earlier phases are in progress (presumptive and
confirmed tests).
la. A "new" data sheet does not have to be initiated
since the sample is already being processed.
VII.B.2
(Suggested
Data Sheet)
(P. 52)
10-15
-------
MATER MONITORING PROCEDURE: Completed Test for the MPN Method
10-16
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Initial Procedures
(Continued)
3. Lab Bench
Disinfection
Use active sheet of the
48 hour MPN test (partial
completion of the con-
firmed test) with 48 hour
presumptive tubes and 24
hour confirmed tubes
"saved."
2a. For our hypothetical test, the data sheet
shows as follows:
1. Disinfect laboratory
bench; wipe dry.
24 hour column
entry (tubes
processed
previously)
48 hour column
entry (tubes saved)
2b. Tubes "saved" will be used to initiate the com-
pleted test or to proceed to the confirmed test.
la. Sponge and disinfectant; paper toweling.
-------
WATER MONITORING PROCEDURE; Completed Test for the MPN Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Initial Procedures
(Continued)
4. Continue Standard
MPN Test Procedure
1. Transfer positive LLSTB
tubes of the presumptive
stage.
la. From data sheet (B.2.2.2a), note that one tube
will be transferred from the presumptive stage
to the confirmed stage:
Std. Meth.
14:917
'Amount
Sample
' ml
10
I
O.I
_.
Preservative
LLSTB
24 hr
+
•h
4-
—
+
_
•+•
I __
43 hr
—
—
+
4
r
Confirmed
SSL8B
24 hr
•*'
+-
4-
•V
48 hr
— |
,
!-—!/-— !
; —
: — I
I —
-/ -
"7
—
Conoieted
LLSTB
EM3I 24 48
i
~sr
—
i
r
—
i •
1 :
1
Transfer this to BGLBB
Ib. Progress of this transfer will be monitored for
possible inclusion to the comleted test.
10-17
-------
HATER MONITORING PROCEDURE: Completed Test for the MPN Method
10-18
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Initial Procedures
(Continued)
Confirmed Test Start
First Day Procedures
5. Select BGLBB
Positives from
Confirmed Test
1. Select "positives" from
confirmed "24" hour tubes
for processing.
la. Four positive BGLBB tubes are to be processed:
This positive trans-
ferred to confirmed
test
Amoun
Process these 4 tubes
to EMB
Discard these negatives...
coliforms absent (see
schematic)
Negative tube to be
re-incubated as per
confirmed test re-
quirements (Std. Meth
14:920)
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Initial Procedures
(Continued)
6. Prepare EMB Agar
Plates
1. Shake all positive BGLBB
tubes vigorously after
labeling each tube.
2. Sterilize a bacteriologic-
al loop.
3. Allow loop to cool
(5-10 seconds).
4. Remove cap from the first
positive BGLBB tube (10/1).
5. Insert loop into broth to
obtain film transfer.
Cover tube and discard.
la. Labeling avoids correlation errors in tube plate
matching. Mark tubes 10/1; 10/2; 10/3; and 1/1
for the four positive tubes in order (labeled
according to inoculation volume/number in row).
Ib. Shaking allows organisms to be suspended in the
broth.
2a. Heat in burner to redness all the way to handle:
NOTE:
HEAT FUUY ENTIRE
LENGTH OF LOOP
3a. Avoids possible spattering when loop is inserted
into tube.
5a. "Film" within loop represents transfer volume.
TRANSFER VOLUME
A
LOOP
MUST SHOW
'FILM' WITHIN LOOP
FILM OF
INOCULUM
V.B.6.5
(p. 50)
10-19
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method
10-20
OPERATING PROCEDURES
B. Initial Procedures
(Continued)
STEP SEQUENCE
6. Streak transfer inocula-
tion from loop to corner
of EMB agar plate.
7. Sterilize (flame) loop
and air-cool as before.
INFORMATION/OPERATING GOALS/SPECIFICATIONS
6a. Agar surface must be dry for satisfactory results.
6b. Streak the inoculation lightly back and forth
over half the agar surface, as in®, avoiding
scratching or breaking the agar surface.
SUEAKED AREA
LOOP
,.LAIf
6c. Use asceptic (sterile) technique to prevent
contamination of medium. Close cover of petri
dish when not streaking.
TRAINING
GUIDE NOTES
VII.B.6.6
(p. 52)
-------
MATER MONITORING PROCEDURE: Completed Test for the MPN Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Initial Procedures
(Continued)
Streak another segment of
plate to carry portion of
inoculation into another
area of medium.
8a.
8b.
Turn Petri dish about one-quarter turn in the
holding hand (allows easier streaking).
Streak the loops, tip lightly back and forth over
one-half the agar surface, working from area (D
into one-half the unstreaked area of the agar.
9. Sterilize loop and air
cool.
0. Streak the remaining un-
streaked area of medium.
8c. Technique allows "dilution" of original heavy
Inoculum to occur into an area where less growth
will now result.
lOa. Turn the Petri dish one-quarter turn 1n the
holding hand.
lOb. Streak the tip lightly back and forth over one-
half the agar surface, working from area 2
into area
10-21
-------
HATER MONITORING PROCEDURE: Completed Test for the MPN Method
10-22
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Initial Procedures
(Continued)
11. Flame sterilize the loop
and set it aside.
12. Invert dish (turn up-side-
down) and identify.
13. Incubate EMB agar plate.
14. Streak, label, and incu-
bate EMB plates from the
other three positive
BGLBB tubes (10/2; 10/3;
and 1/1).
lOc. Do not allow any of streaks of one group to touch
a more concentrated area than the area streaking
from (in effect, separate 3 from 1 or over-growth
may occur.)
lOd. Close the culture container, and, until the
colonies (bacterial growth forms) are picked,
keep the top and bottom as a unit without allow-
ing separation to occur.
12a. Use grease pencil (wax pencil) to label bottom of
dish.
12b. For this positive BGLBB tube label as follows:
Lab number for this
particular sample
-Indicates 1st tube of row
which received 10 ml sample
inoculation
13a. At 35° + 0.5°C for 24 hours.
13b. Keep in inverted position (avoids water droplets,
if formed, from falling on the medium surface and
ruining the plate).
14a. Use techniques for streaking as previously
described.
14b. Labeled plates will read:
14c. Incubate as previously described.
-------
WATER MONITORING PROCEDURE; Completed Test for the MPN Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Second-Day Procedure
1. Equipment
Maintenance
2. Lab Bench
Disinfection
3. Data Sheet
Recordings
1. Check, record, and adjust
incubator temperature.
1. Disinfect laboratory
bench, wipe dry.
1. Locate required data
sheet.
2. Remove cultures from incu-
bator and assemble with
data sheet.
3. Read BGLBB tubes for gas
and record results.
la. Sponge and disinfectant; paper toweling.
la. Sample "217" in our example.
2a. 4 EMB plates (24 hours old)
1 BGLBB tube (24 hours old)
1 BGLBB tube (48 + 3 hours old)
3a. Any amount of gas is considered positive.
tubes gently before reading.
3b. Assume the following results:
See B.2.2a
Shake
Se-
It
10
\
0.1
ole
1 _
_ _LLS
_2« _nr
-H
4-
_
—
_±_
4-
_
7 _
_
—
TB
_
__
.Jr~
_
_
_
—
BSLB
V"
-1-
_*J
—
- i - 1
-•
_
b
>
/
S
-*
— ^
.
/
1
1
H"
—
-
'
Negative tube
r(re-incubate)
^Negative tube
'(coliforms a sent
See
Schematic
^i^grain
J. 3)
10-23
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method
10-24
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Second-Day Procedure
(Continued)
4. EMB Agar Plate
Inspection
4. Discard any BGLBB tubes
which are negative in
48 hours.
5. Save any 24 hour BGLBB
tube which is positive
or negative.
Remove cover from one of
the four EMB agar plates
and inspect growth (most
convenient to open plate
10/1).
4a. There is one such tube (1 ml sample volume;
3rd tube of row).
4b. Coliforms absent in this tube.
5a. None are positive. This possibility would have
made it necessary to streak an EMB agar plate.
5b. There is a negative. Re-incubate this for an
additional 24 hours (1 ml sample volume; 2nd
tube of row).
la. Usual plate growth (colonies) will be as
indicated:
AREA 1
(HEAVY INOCULUM)
AREA 3
(ISOLATED COLONIES)
AREA 1
(MODERATE GROWTH)
APPEARANCE OF STREAK - PLATE
AFTER INCUBATION INTERVAL
OR OCCASIONALLY,
AREA 1
(HEAVY INOCULUM)
AREA 3
(LACK OF COLONY
ISOLATION)
AREA 2
(HEAVY GROWTH)
APPEARANCE OF STREAK - PIATE
AFTER INCUBATION INTERVAL
-------
MATER MONITORING PROCEDURE; Completed Test for the MPN Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Second-Day Procedure
(Continued)
Ib. In the case of isolated colonies, one could pro-
ceed to the next step of the completed test
(C.5).
Ic. In the case of a lack of isolated colonies, one
must proceed to re-streak another plate to
attempt isolation of a colony. (As follows):
Re-isolation Procedure
A. Flame sterilize a loop and air cool
B. Immerse the loop into an area which shows a
representative growth mass. Occasionally,
the loop must be touched to two or three
masses to obtain this material.
C. Close cover and discard EMB plate.
D. Streak plate of fresh, sterile, dry EMB agar
using the same technique as previously
outlined except that it would be wise to allow
more streaking sequences with an increased
number of loop flamings. This would more
likely ensure better isolation:
III.C.4.1
(p. 47)
RESTREAK (5
FLAME
RESTREAK
FLAME
RESTREAK (3
^-*
FLAME
ORIGINAL ^_
STREAK (T)
FLAME
RESTREAK (2
FLAME
RESTREAK (T
E. Incubate as previously outlined..
10-25
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method
10-26
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Second-Day Procedure
(Continued)
Id. Pure growths (colonies) can be regarded as fall-
ing into two groupings:
I. Typical Colonies (characteristic of coliforms)
Colonies with dark centers commonly termed
"nucleated" or "fisheye" when viewed from
the bottom of the plate:
0:
EFC.
These colonies may or may not have a metal!ic-
like sheen characteristic on the surface of
the colony.
II. Atypical Colonies (usually a non-coliform)
These colonies may be opaque, unnucleated,
mucoid, or pink after the prescribed incu-
bation period.
-------
HATER MONITORING PROCEDURE: Completed Test for the MPN Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Second-Day Procedure
(Continued)
5. EMB Agar Plate
Colony Transfer
1. Transfer pure cultures to
LLSTB and NAS.
la. Use flamed and air cooled needle for fishing
(picking).
Ib. Use of colony counter as a magnification aid
is recommended:
LENS, MAGNIFIER
ADJUSTING ROD
- DIRECTION OF
LIGHT SOURCE
DISH WITH
EMB MEDIUM
AND COLONIES
Ic. Pick one or more typical colonies, or, two or
more atypical colonies and transfer each of them
into their own set of tubes (LLSTB and NAS)
(See
schematic
of test)
10-27
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method
10-28
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Second-Day Procedure
(Continued)
Id. Recommended technique is to pick a pure colony
and, with a single transference, inoculate both
the LLSTB and the NAS in a single motion.
1st
INOCULATION
2nd
INOCULATION
STERILE NEEDLE
.5 cm J U—
OR MORE '
EMB AGAR
NAS
LLSTB
NAS...Flame top of tube for about two seconds
prior to entering with needle. Gently
swab surface of agar medium. Replace
screw-cap which is held in hand without
contaminating during procedure.
• EMB...Discard plate after inoculations.
LLSTB...Transfer inoculum directly to LLSTB
tube. (Return to colony is not
necessary.) Flaming of tube top not
necessary. Shake needle in broth for
transfer.
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Second-Day Procedure
(Continued)
le. Label tubes for identification. Such a labeling
could be as follows:
a
217
1
TYP.
A
1
217
1
TYP
A
IOJ
"217" Lab number
•10/1" 1st tube of 5 of 10 ml.
sample volume
"TYP." Typical colony "A" culture
(to keep lubes paired)
If. Incubate tubes at 35° + 0.5°C.
Ig. Indicate the necessary information regarding the
step just completed on the data sheet:
Amount
Sample
ml
10
Preservative
LLSTB
24 hr
4-
•h
4.
—
—
48 hr
—
—
Confirmed
BGLBB
24 hr
+
-f-
4-
48 hr
jt
/
/
Completed
LLSTD
Culture 1
/6/I
EHB
T/P
v
\
>
24
\
48
GS
1st tube of row
of 10 ml sample
volume
EMB culture was
"typical" (See
C4.1.1d for
definition)
NOTE: Only one colony will be processed for this
first tube of the first row since the
colony is typical (one or more could have
been picked).
10-29
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method
10-30
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Second-Day Procedure
(Continued)
Ih. Utilizing the same procedures as in a - g of this
section, process the next culture (10/2 EMB agar
plate). Assuming an atypical colony formation on
the plate, we will process three cultures (2 or
more are required for this condition) and record
them as follows:
Observations
Amount
Sample
ml
10
Preservative
LLSTB
24 hr
4-
-t-
+
—
—
•h
—
48 hr
—
—
+
Confirmed
BGLBB
24 hr
-»•
•+•
+
j/
48 hr
/
/
Completed
LLSTB
Culture 1
/O/l
n/*A
UfiLB-
1*1* C
\
\
\
EMB
TYP
»T»P
»TfP
nyf
a
*e>
48
i***^
GS
f^
t.
t
-•C
C
:Atypical
•colonies
on EMB
Three cultures
processed
Letter designations will keep
the three cultures separated
(Note: the same "A"; "B" and
"C" will appear on the LLSTB
tubes and NAS tubes.)
li. Process the two remaining cultures and assume the
following recordings for all four cultures:
(See data sheet on following page.)
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method
OPERATING PROCEDURES
C. Second-Day Procedure
(Continued)
D. Third-Day Procedure
1. Equipment
Mai ntenance
2. Lab Bench Disin-
fection
3. Test Observations,
Recordings, and
Processing
STEP SEQUENCE
1. Check, record, and adjust
incubator temperature.
1. Disinfect laboratory bench
DATA SHEET ENTRIES
1. Locate required data
sheet.
2. Remove cultures from incu-
bator and assemble with
data sheet.
INFORMATION/OPERATING GOALS/SPECIFICATIONS
Observations ,,.,uiybt
Preservative
,. LLSTB
24 hr
4-
4-
1-
-
.4.
—
+
—
—
—
—
—
••
—
48 hr
—
—
-T
—
_
—
_.
—
—
—
Confirmed
BGLBB
24 hr
•v-
+
•r-
•4-
—
—
48 hr
..
Completed
LLSTB
du' ture I
ffifl
!*/*• 4
ikfa. fl
lft/7 A
10
in
i.
? ft
3 C,
r
FHR
TfP
!»TYP
AT7P
KT1P
AW
^TYP
TYP
?4
4ft
IK
Completed
LLSTB
Culture 1
FMR
TT
7?
la. Sponge and disinfectant; paper toweling.
la. Sample "217" is our example.
2a 8 LLSTB} 24 hours 1ncubat1on
1 BGLBB 48 +_ 3 hours of incubation
TRAINING
GUIDE NOTES
See
C.3.3b
10-31
-------
HATER MONITORING PROCEDURE: Completed Test for the MPN Method
10-32
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Third-Day Procedure
(Continued)
3. Read BGLBB tube for gas
and record results.
21 7
3a. Tube labeled ^2 after we find it is positive
(any amount of gas considered positive).
Observations
Ana.
Amount
Sample
ml
10
I
•
Preservative
LLSTB
24 hr
4-
4-
4.
—
—
4-
—
4-
—
—
—
48 hr
—
_
4-
—
—
—
—
Confirmed
BGLBB
24 hr
4-
4-
4-
4-
—
—
48 hr
•v-
— ^
Completed
LLSTB
Cultured
16/1
|/|/
10
(0
llj
Afi
a R
3 P
h A
\ R
I«J3 C.
.A
X
\
N
FMB
ryp
KTff
ATYP
KT1P
KTjr
ffftf
ATYP
^TYP
74
48
f,S
Cultu
3b.
3c.
Positive BGLBB^twithin
48+3 hours)
Tube would have been discarded if it were nega-
tive (coliforms absent) and a negative (-) in
this case, assigning a tube number would have
been unnecessary.
Process this (+) BGLBB tube to an EMB agar
streak plate as outlined previously and then
discard the (+) tube.
See B.6
-------
MATER MONITORING PROCEDURE: Completed Test for the MPN Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Third-Day Procedure
(Continued)
4. Check NAS and LLSTB tubes
for growth and gas, re-
spectively and record
results.
4a. Eight pairs of tubes to be read and recorded.
Assume the following:
.attons
.
onftrmed
B6LBB
24 hr
+
4-
+
+
—
_ _
48 hr
—
Cc
Culture 1
/all
loj: ft
io,~ .«_
10'.: '-
IC/- f:
10 L- ;--
lOl- c
///
mplet
LLSTB
-w
Tff
rtr>?
hT- 'J
h"if
ftT/P
fiT?'?
RTYP
T/P
?d
~W'
4-
-»•
Jr
--
^^ .
__,
-*•
48 GS
^X
r
Culture 1
^
^
Ihb
X
Re-incubate
negatives
for an addi-
tional 24
hours.
4b,
Growth on the MAS is readily visible as an
opaque mass which was not present on the sterile
medium. No recordings are necessary for this
growth—it will be used for a GRAM STAIN. It
is quite rare that no growth will occur on this
medium. (If this rarity occurs, restreak the
NAS tube from its companion LLSTB tube.)
10-33
-------
WATER MONITORING PROCEDURE; Completed Test for the MPN Method
10-34
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Third-Day Procedure
(Continued)
GRAM STAINING
5. Assemble Gram staining
materials and cultures.
6. Prepare glass slides.
Place NAS culture on slide
(use 10/1 culture first
then repeat procedure
with 10/2 A, 10/2 B, etc.,
each on a different slide)
5a. 8 bacteriological glass slides
6a.
6b.
6c.
7a.
7b.
7c.
7d.
1
dropper bottle containing ammonium oxalate
crystal violet dye
dropper bottle containing Lugols solution
(Grams modification)
dropper bottle containing safranin dye
dropper bottle containing acetone-alcohol
squeeze bottle containing tap water
bibulous paper
NAS cultures...(24 hour cultures)
10/1
10/2 A
10/2 B
10/2 C
10/3 A
10/3 B
10/3 C
1/1
Std. Meth.
14:918-19
III.D.3.5
(p. 47)
VII.D.3.5
(p. 53)
Must be clean.
Helpful to clean with alcohol, distilled water,
and lens tissue.
Place a drop of distilled water about 1 inch from
end of slide.
Screw-cap tube handled asceptically (sterile
technique):
* flame top of tube
* needle flamed to sterilize
* cap handled carefully and returned promptly
to tube
NAS stored in refrigerator for possible need.
Only minute amount of culture necessary. Large
amounts can cause staining problems.
Place culture from needle with water droplet on
slide and mix well while extending the droplet
size to about a 1" x 1/2" area.
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Third-Day Procedure
(Continued)
8. Prepare culture for
staining procedure.
9. Stain culture with reagents
on the side of the slide
with the culture.
8a.
9a.
9b.
9c.
9d.
9e.
9f.
9g.
9h.
9k.
Allow smear to air-dry completely and then heat
fix by by-passing slide (culture on upper side)
through the gas flame briefly back-and-forth
for a heat exposure of about two seconds.
Flood the slide with ammonium oxalate-crystal
violet dye.
Allow to cover culture area for 1 minute.
Wash slide gently with tap water.
Apply Lugolsriodine solution to culture area.
Allow to cover culture area for 1 minute.
Wash slide gently with tap water.
Apply acetone-alcohol solution to culture area.
Hold slide and allow solution to flow across
smear until stain is no longer being removed:
Dropper
Bottle
Do not prolong this alcohol contact period
(decolorization step) as the results may be
erroneous. Some authorities suggest 10-15
seconds maximum.
Wash slide gently with tap water.
Apply Safranin solution (counter-stain) for
15 seconds and then wash gently with tap water.
Blot slide gently with bibulous paper using care
not to rub culture area during procedure.
(Continued on next page)
Std. Meth.
14:918-19
10-35
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method
10-36
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Third-Day Procedure
(Continued)
91. Identify slide to conform
being examined. Use of a
venient (label "217 10/1"
10. Repeat step sequences 7-9
for cultures 10/2 A; 10/2
B; 10/3 A; and 1/1.
11. Examine slides
microscopically.
to proper culture
slide label is con-
as per our example),
lla. If desired, slides can be retained for later
examination. If the lactose (LLSTB) broth re-
mains negative for the culture (48 + 3 hours),
the slide need not be examined as the culture
Is NOT a collform.
lib. Become acquainted with microscope from
manufacturer's literature or individual
acquainted with same.
lie. If examination 1s desired, place the slide,
culture side up, on the microscope stage of a
microscope equipped for oil Immersion examination
lid. Place a drop of a suitable bacteriological
immersion oil on the area to be viewed (culture
smear).
lie. The proper objective is positioned for oil
immersion (usually labeled "oil" and having
x 97 or x 100 magnification).
V.D.S.ll.lld
(P. 51)
OIL IMMERSION
OBJECTIVE
TYPICAL OIL
OBJECTIVE
THE OBJECTIVES SCREW INTO
THE TURRET.
(TURN AND LOCK FOR SELECTION)
-------
HATER MONITORING PROCEDURE; Completed Test for the MPN Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Third-Day Procedure
(Continued)
llf.
llg.
llh.
Hi,
EXAMINATION AND RECORDING
OF STAINS
12. Examine stained preparatior
for bacteria.
12a.
12b.
12c.
12d.
Turn ON lighting system. Light will be directed
to reflect off the plane side of a mirror through
a condenser assembly and up through the stage.
(Mirror assembly may be external or internal.)
With the illumination system correctly set up,
rack down (or the stage up on some models),
until the oil-immersion lens just touches and
disperses the oil.
Rack down gently with the coarse control (lens
and slide will move toward each other) while
looking down the microscope (into the eyepiece)
until the image begins to come into focus.
Obtain final sharp image using the fine-focus
control.
Gram-negative bacteria (typical of coliforms)
will be red or pink colorations.
Gram-positive bacteria (NOT coliforms) will be
blue-to-purple in color.
Mixed cultures will show mixtures of the above
and will immediately call for the re-isolation
of pure culture on another EMB agar plate from
the saved nutrient agar slant. Discard the
LLSTB tube as it has no interpretative value
being a mixed culture. Repeat procedures as
before.
If two large of a sample was transferred to the
slide for staining, some areas of matted,
numerous bacterial cells could produce areas
where dyes could not either penetrate or be
washed away. Recommend another smear to be
made.
12e. Examine each of the stains prepared.
.
(P. 51)
10-37
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method
10-38
OPERATING PROCEDURES
D. Third-Day Procedure
(Continued)
STEP SEQUENCE
13. Record gram stain data.
INFORMATION/OPERATING GOALS/SPECIFICATIONS
13a. Assume, for our examples, that only Gram
negative (GN) organisms were observed during
microscopic examination.
13b. Enter observations in proper place on data sheet:
"USTB*"
-Bhr
Culture
10/1
ltt!3. A
lf'3 B
li5/A t.
W/3 ft
ic '3 b
1 .• ' T 1
?,'!
l cm
TYP
0TVP
fffHf
MVP
ATVP
ATr'P
ATVf
ryp
24 4
4*
-|-
f
-
.4.
-'
—
f
Sample Confirmed
Volume Test
| Observations
Lc
ti
C(
Anunt
f\
10
ictose nega-_ .-«<
'ves, not
)li forms
1
*Definite col if
1, 2, and 3 of
r
1 ~ "
•
Confirmed
-r
-t-
•\-
>^
** •*••"'***
+
t>-^.'
1 BS
GM
frV
vK
L.»%i
Sf
fiii
61
5^
u
•
Completed
Test
- -,
*
*
*
*
Completed ^s
LI.S1B
Culture I EHB
Ut'i rvf
Itf/i ft ATV?
lj5j;!_^L ftfTVP
w.'s. '.A-VP
j'-j.Ji A ftT'i?
.'•? ;'i B ATVr
1 r"' "• ft /-TV**
.'-': TJT
4-
-t-
-t-
i-
—
4-
48
US
&vj
'«. * '
<*»s
Vi
Oti
ftN
ftKi
TRAINING
GUIDE NOTES
>
•
•
«*
8
\
t-
j
k
+•
+
t
^
f
\
1
orms at this stage. Note that rows
10 ml and row 1 of 1 ml inocula-
10 ml vol-
umes for 5
•tubes of
completed
test row
/I ml vol-
I ume in 1st
{ tube of
1 compl eted
Vtest row
tion volume all have at least 1 coliform repre-
sented by culturing.
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method
OPERATING PROCEDURES
D. Third-Day Procedure
(Continued)
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
13c. Other entries could have
or GP (Gram Positive).
13d. In
the
been MXD
case of a GP entry, the
col i form and
the specific
no further action
(mixed culture)
culture
need
be
is not a
taken for
culture.
Multiple
Sample Type
Station
Lab. No
Conform Test
Dilution Tube (HPN)
TRAINING
GUIDE NOTES
Method
Results Reported:
Total collform HPN/109 ml
Description
Collection Date
Time
AM
Received PH Examined
Sampler
Amount
Sample
ml
10
L
AH
PM
AH
PH
Temp.
Observations
Preservative
LLSTB
44 hr
actos
48 hr
i
J\
/ *
f
e nee
Confirmed
BGLBB
Z4 hr
•if
•if
+•
"^^
^>^
jative
48 hr
^^>^
><^
S
Confirmed
Completed
Analyst
Completed
Culture f
LLSTB
EHB
24
48
65
Completed
LLSTB
Culture 1
lt>f\
10/1 A
10 1 A. B
If,
l°l
J4
a C.
3 /»
,3 B
jfja C
EMB
TVP
rryp
HJH
iTYf
WP
24
^.
4.
•(•
—
^.
—
—
40
Not coli forms
(1) 10
GN
(2) 10
GN
(3) 10
GN
ml/first row Positive
Conf i rmed
Test...
lactose fermenter (culture 10/1)
ml /second
row Positive Confirmed Test...
lactose fermenter (cultures 10/2 A and 10/2
ml /third row Positive
Confirmed
lactose fermenter (culture 10/3
Note: Culture 10/3 B and
processed since a
needed
from
these
B)
Test. . .
A)
10/3 C need no longer
positive
is
be
no longer
respective cultures.
Had
GS
&|4
&H
&K
ftP
Sh
nu
&u
3
S
*,
41
tt
01
1
LJ
-v-
^.
f
_.
_
f
0)
(2)
(3)
(4)
*.
(Continued)
10-39
-------
HATER MONITORING PROCEDURE: Completed Test for the MPN Method
10-40
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Third-Day Procedure
(Continued)
culture 10/3 A been a negative for coliforms,
however, the other two cultures must be pro-
cessed to determine coll form content:
10/3 B Mixed culture EMB-^{L£STB etc>
10/3 C Negative LLSTB...hold another 24
hours for possible fermentation (+)
(4) 1 ml/first row Positive Confirmed Test...
GN lactose fermenter (culture 1/1)
-------
UATER MONITORING PROCEDURE: Completed Test for the MPN Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
E. Fourth-Day Procedure
1. Equipment
Maintenance
2. Lab Bench
Disinfection
3. Data Sheet
Recordings
4. EMB Agar Plate
Processing (Culture
217 1/2)
1. Check, record, and adjust
incubator.
1. Disinfect laboratory bench
1. Locate required data sheet.
2. Remove cultures from incu-
bator and assemble with
data sheet.
1. Transfer two of the atypi-
cal cultures to HAS and
LLSTB.
la. Sponge and disinfectant; paper toweling.
la. Sample "217" is our example.
2a. 1 EMB agar plate (#217 1/2), 24 hours old.
2b. Record necessary data regarding colony
characteristics on EMB agar plate:
..tiler _ _ Observations _
Amount
SMRple
ml
/o
1
p
-
-
Confirmed
BGLBB
24 hr
i-
-r
4-
"><
+
—
j
/
J
48 hr
^>-~r~
.'•-<'
jh
f
r-
-
-
-
s+
Completed
USTB
Culture 1
10/1
/•3,U t-.
10'; 3
It';. C.
1 •••':- k
\t'z fc
io73 :.
i'r
^|/X /i
»•/ i b
EHB
T/°
fSTjf
(•-••\:
ft";?
KTit-
AT;'?
TV.'
£T'ff
rvp
24
f
A.
t
.w
-
-
4-
w
J;?.
vN
•^H
ft'i
•'•iM
^S
;Vi
•
-f
-f-
_
4-
Culture Two Atypical cultures
217 1/2 to be processed to
NAS and LLSTB
la. As per data sheet (1/2 A and 1/2 B).
Ib. Use procedures outlined in C.5.1.
Ic. Incubate cultures at 35°C + 0.5°C.
See "Data
Sheet"
D.3.3.3a
10-41
-------
WATER MONITORING PROCEDURE; Completed Test for the MPN Method
10-42
OPERATING PROCEDURES
F. Fifth/Sixth Day
Procedures
1. Completed Test
Termination
STEP SEQUENCE
1. Completed test terminated
with processing of 1/2 A
and 1/2 B.
INFORMATION/OPERATING GOALS/SPECIFICATIONS
la. Gi
ii
Ib. LI
lc. As
d<
Id. Ha
fc
be
pc
a
•am stains made in accorc
istructions. Recordings
.STB observations and rec
>sume the following infor
ita sheet regarding these
R«
Code for rows— ««,
Name of Analyst-—^,
Amount
Sample
ml
10
1
'
- •
Confirmed
BGLOB
24 hr
4-
4.
4-
p>*
s
lance with previous
made.
:ordings made.
•mat ion added to the
» two cultures:
suits Reported:
Total conform MPN/IOO ail
I Confirmed
f
Com
3-
AnTiTsTa.tf.
pie ted 1
A,^
Completed
LLSTB
Culture 1
/a/i
10 'a f-
[013 B
tola, o
16\± f-
10'Z B
it. 13 . A
p-l/a. B
EHB
TYP
ftrvP
ftTVP
ftTYP
ff'fP
AT^p
TVP
24
^.
•V
•t
__
-t
—
.
-i
—
48
-
-.
GS
^rM
IfiN
frN
gM
fiN
^kl
AH
£N
r-'W
SN
Completed Test Result
rh
+
—
_
-r-
«y
Coli forms NOT present
(lactose not fermented)
d one or both cultures checked out as coli-
irms, the 2nd tube of the 2nd row would have
•en (+) and, therefore, resulted in 2 of 5 tubes
sitive (instead of 1 of 5 for this row), giving
3-2-0 code.
TRAINING
GUIDE NOTES
la of 5 (+)
V2nd tube of
Vow is (-),
therefore 1
of 5 (+)
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method
OPERATING PROCEDURES
F. Fifth/Sixth Day
Procedures (Continued!
2. Interpretation of
Test Results
STEP SEQUENCE
1. Determine number of posi-
tives for the completed
test.
2. Look up and note the MPN
Index from the MPN table.
INFORMATION/OPERATING GOALS/SPECIFICATIONS
la. Results of confirmed test are not used since
further and more conclusive testing has been
done (completed test).
Ib. Our example (F.l.l.lc) shows:
Sample Volume
10
1
1/10
No. Positives
3
1
0
2a. For the given example (3-1-0) a typical table of
MPN's will show an index of 11 as noted by arrow
below:
MPN INDEX 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
Positive Reaction out of
5 of 10
ml each
0
0
0
k^O^x'
3
3
3
3
\^
5 of 1
ml each
0
0
1
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method
10-44
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
F. Fifth/Sixth Day
Procedures (Continued]
3. Record the calculated
total coliform/100 ml com-
pleted test count on the
data sheet.
3a. Value is direct index if, as our example, 10 ml
portions were used in the first row. Other
volumes used would have necessitated adjusting
the index value.
3b. Record as follows:
II.F.2.3.3a
Results Reported:
Total collform MPN/100 ml
Code for 3 rows
Count/100 ml
G. Reporting of Results
1- Report results as pre-
scribed under regulatory
requirements.
-------
WATER MONITORING PROCEDURE: Completed Test for the MPN Method
TRAINING GUIDE
SECTION TOPIC
I Introduction
II* Educational Concepts - Mathematics
III* Educational Concepts - Science
IV Educational Concepts - Communications
V* Field and Laboratory Equipment
VI Field and Laboratory Reagents
VII* Field and Laboratory Analysis
VIII Safety
IX Records and Reports
*Training Guide materials are presented here under the headings marked *.
These standardized headings are used through this series of procedures.
10-45
-------
WATER MONITORING PROCEDURES: Completed Test for the MPN Method
EDUCATIONAL CONCEPTS - MATHEMATICS
Section II
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
F.2.1
F.2.3.3a
For purely qualitative aspects of testing for indi-
cator organisms, it is convenient to consider the
tests applied to one sample portion, inoculated into
a tube of culture medium, and the follow-up examina-
tions and tests on results of the original inocula-
tion. Results of testing procedures are definite:
positive (presence of the organism/group demon-
strated) or negative (presence of the organism/group
not demonstrated).The combination of results is
used in an application of probability mathematics to
secure a single MPN value for the sample. The MPN
value for a given sample is obtained through the
use of MPN tables. Standard practice in environ-
mental water testing is to plant 3 rows of tubes
(15 tubes - 3 rows of 5 tubes each) with each row
containing equal increments of sample/tube and
usually having a tenfold sample dilution factor be-
tween rows.
When the series of decimal dilutions is other than
10, 1.0 and 0.1 ml, use the following formula:
MPN index
(from table) x
10
Largest quantity tested
= MPN/100 ml
Example: From a sample of water, 5 out of five
0.01-ml portions, 2 out of five 0.001-ml portions,
and 0 out of five 0.0001-ml portions, gave positive
reactions.
From the code 5-2-0 in the MPN table, the MPN index
is 49.
49 10
(from table) x OT = 49>000
MPN/100 ml = 49,000
10-46
-------
WATER MONITORING PROCEDURES: Completed Test for the MPN Method
EDUCATIONAL CONCEPTS - SCIENCE
Section III
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
C.4.1
D.3.5
A colony is defined as a discrete growth occurring
at least 0.5 cm (approximately .2 inch) from any
other growths. Such growths represent a large
number of developmental successions from an original
viable cell and therefore can be considered a "pure"
culture. All organisms from pure cultures will ex-
hibit the same characteristics when subjected to
standard bacteriological testing.
A gram staining procedure, in general, separates
bacteria into two categories, gram positive (blue
coloration) or gram negative (red coloration).
Its usefulness to the coliform testing procedure is
due to the fact that part of the coliform definition
indicates that "gram negative, non-spore forming
rods" are necessary, and, in addition, no gram
positive organism must be present since some of these
organisms can act "synergistically" (in conjunction
with other non-coliforms) to produce a false
positive result (gas production in lactose) which
neither could manage independently.
It is desirable to use known pure cultures of both
a gram positive (staphylococcus, bacillus, etc.)
and a gram negative (proteus, enterobacter, etc.) as
controls for the staining procedure. A 24 hour
culture is recommended for stained preparations since
older cultures can give erroneous results.
10-47
-------
WATER MONITORING PROCEDURES: Completed Test for the MPN Method
FIELD AND LABORATORY EQUIPMENT
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.I
A.1.1
A.1.2
A.1.3
A.1.5
A.1.6
Incubator must be of sufficient size for daily work
load without causing crowding of tubes to be incu-
bated. Considerations for choice of incubator
type must relate to reliability of operation and
not to cost or attractiveness of equipment.
Incubator should be kept out of drafts or direct
sunlight in order to prevent temperature inside
the incubator from changing outside the tempera-
ture range specified (35° + 0.5°).
Power supply should be selected so that there will
not be too many pieces of equipment on the same
circuit. Otherwise, circuits will be blown
repeatedly.
Mercury bulb thermometer usually used in most incu-
bators. Recording thermometer is acceptable, but,
it should be calibrated against a mercury bulb
thermometer which has been certified by National
Bureau of Standards. The NBS certified thermometer
always should be used with its certificate and
correction chart.
Saturated relative humidity is required in order to
make the incubation more efficient (heat is trans-
ferred to cultures faster than in a dry incubator).
Furthermore, culture medium may evaporate too fast
in a dry incubator.
Allow enough time after each readjustment to permit
the incubator to stabilize before making a new
adjustment. At least one hour is suggested.
Incubator temperature can be held to much closer
adjustment if operated continuously. Temperature
records should be kept in some form of permanent
record. A temperature record book is suggested
with daily recording of values. If a recording
thermometer is used, the charts may be kept as
permanent record; if so, be sure that the charts
are properly labeled to identify the incubator and
the period covered.
Uniform temperature (35°C t 0.5°) is to be main-
tained on shelves in use.
Standard Methods for the
Examination of Mater and
Hastewater. 14th ed. (1975
APHA, WPCF, AWWA, p. 880
(Hereafter referred to as:
Std. Meth. 14: (page no.)
10-48
-------
WATER MONITORING PROCEDURES: Completed Test for the MPN Method
FIELD AND LABORATORY EQUIPMENT
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.2.1-5
A.3.1
A.4.1-2
Since electric sterilizer will be operated inter-
mittently, care should be taken that it is on a
circuit which will not be overloaded when it is
turned on.
A time and temperature record is maintained for each
sterilization cycle. Temperature recordings can be
retained for records.
Autoclaves differ greatly in design and in method
of operation. Some are almost like home-style
pressure cookers; others are almost fully automatic.
This is a subject which requires separate instruc-
tion; and should be related to the exact make and
model of equipment you will use in your own
laboratory.
Vertical autoclaves and household pressure cookers
may be used in emergency service if equipped with
pressure gages and thermometers with bulbs posi-
tioned 1 inch above the water level. However, they
are not to be considered the equivalent of the
general purpose steam sterilizer recommended for
permanent laboratory facilities. Their small size
is inadequate for large-volume work loads, and they
can be difficult to regulate.
The following requirements must be met regarding
autoclaves or sterilizing units:
. Reaches sterilization temperature (121°C),
maintains 121°C during sterilization cycle, and
requires no more than 45 minutes for a complete
cycle.
, Pressure and temperature gages on exhaust side and
an operating safety valve.
No air bubbles produced in fermentation vials
during depressurization.
Record maintained on time and temperature for
each sterilization cycle.
Distilled water in bacteriological laboratory must
not contain substances which will prevent any
bacteria from growing in culture medium in which the
distilled water is used or will be highly nutritive.
There are procedures for testing quality of dis-
tilled water; but these should be undertaken only by
professional bacteriologists or in laboratories
where this is done regularly. Use only glass stills
or block tin lined stills.
Std, Meth. 14:881
Std. Meth. 14:881
Std. Meth.
14:645-49
14:888-91
10-49
-------
MATER MONITORING PROCEDURES: Completed Test for the MPN Method
FIELD AND LABORATORY EQUIPMENT
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.5.1
A.6.1-4a
A.6.1-4b
A.7.3
A.8.2c
B.6.5
pH Meter: See cited reference.
Glassware: See cited reference on pi pets and
graduated cylinders, media utensils, bottles.
Glassware can be checked for bacteriostatic or in-
hibitory residues by a bacteriological test proce-
dure which, like the distilled water suitability
test, should be undertaken only by professional
bacteriologists or in laboratories where this test
is done on a regular basis.
Std. Meth. 14:882
Std. Meth. 14:882-85
Std. Meth. 14:885
FUNNEL
FUNNEL. HOSE, AND
PINCHCOCK ASSEMBLY
PINCHCOCK
CLASS TUBE
NOTE UNIT NEED NOT BE
STERILE FOR MEDIUM
DELIVERY ONLY
Some workers prefer to utilize a magnetic swirl bar
and hot plate arrangement. This is acceptable and
will require no agitation until the medium is
near boiling at which time the swirling action should
be terminated and the medium gently swirled by hand
and the flask monitored for boiling.
Alternately, it is authorized to use an "inoculation
stick" for transfers and plate streaking. A pre-
cisely sized and sterilized stick is intended for a
one-time use and, if used, eliminates the need for
a burner during the transfer procedure. Of course,
several will have to be used during the streaking
process since a "sterile" one is required during the
streaking carry-over to sterile surfaces.
Also available are re-sterilizable loops used once,
resterilized, and available for future transfers.
Std. Meth. 14:917
Std. Meth. 14:883-84
10-50
-------
WATER MONITORING PROCEDURES: Completed Test for the MPN Method
FIELD AND LABORATORY EQUIPMENT
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
D.3.11
D.3.11.lid
D.S.ll.llg
Some manufacterurs specify that the upper most lens
of the condenser assembly also be coated with a drop
of oil prior to placing the slide on the stage. In
effect, this would be "sandwiching" the slide between
two oil Interfaces through which the light must pass,
It Is extremely important to properly set up the
illumination system for proper results. Procedures
vary according to the type of illumination provided,
ihe type of diaphragm used, and the controls provided
by the particular microscope. Final results would
give, 1f accomplished correctly: correct lighting
From the light source; centrally placed optimal
lighting; and a sharply focused image.
10-51
-------
EFFLUENT MONITORING PROCEDURE: Completed Test for the MPN Method
FIELD AND LABORATORY ANALYSIS
Section VII
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
B.2
There is no such thing as a "standard" data sheet
for bacteriological tests. A suggested data sheet
is shown below:
B.6.6
10-52
Sample
Station
Type
Conform Test
Multiple Dilution Tube (MPN) Method
Lab. No. Results Reported.
Description
Collection Date
Tire
AM
Received PM Examined
Sampler
Amount
Sample
ml
Preservative
LLSTB
24 hr
40 hr
AM
PM
"AM
PM
Observations
Confined
BGIBB
24 hr
48 hr
Temp.
Completed
LLSTB
Culture 1
EM
24
48
OS
Total collform MPN/100 ml
Confirmed
Completed
Analyst
Completed
LLSTB
Culture 1
1
EMB
a
48
Note: This data sheet could also be used exclusive-
ly for the confirmed test and not for only the
completed test stage.
There is no standardized way to accomplish a streak
plate in order to isolate pure cultures. Some work-
ers prefer to carry the streaks around the plate
several more times with its attendant loop steriliz-
ing sequencing between each of the streakings,
Others prefer to use a specially made petri dish
which features a center partition which "halves" the
dish allowing two separate cultures to be cultivated.
These modifications, and others, are not deviations
since the only consideration which matters is that a
pure culture is available for further testing.
(iS
3
M
«J
s
1
I
-------
WATER MONITORING PROCEDURES: Completed Test for the MPN Method
FIELD AND LABORATORY ANALYSIS
Section VII
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
D.3.5
You will note that three cultures (10/2 C; 10/3 B;
and 10/3 C) are being processed for gram staining
even though their 24 hour LLSTB results are negative,
This is done for the following reasons:
1. Growth used after 24 hours may give erroneous
staining patterns.
2. Staining is quickly accomplished and is pre-
ferable over restreaking a MAS and waiting
an additional day for culturing.
3. Microscopic examination need not be done after
staining and can wait for the 48 hour fermen-
tation tube results...if positive, proceed
microscopically; if negative, coliforms
absent and discard stained slide.
This outline was prepared by: Rocco Russomanno,
Microbiologist, National Training and Operational
Technology Center, MOTD, OWPO, USEPA, Cincinnati,
Ohio 45268
10-53
-------
A PROTOTYPE FOR THE DEVELOPMENT OF
ROUTINE OPERATIONAL PROCEDURES
for the
TOTAL COLIFORM TEST BY THE MEMBRANE FILTER METHOD
as applied In
WATER TREATMENT FACILITIES
WASTEWATER TREATMENT FACILITIES
and In the
MONITORING OF EFFLUENT WASTEWATERS
National Training and Operational Technology Center
Municipal Operations and Training Division
Office of Water Program Operations
U.S. Environmental Protection Agency
BA.MET.lab.WMP.9.5.78
11-1
-------
Sample
I (m-ENDO Medium)
I (40ml) * I (8ml)'
I (.8ml)4
I
(.08ml)*
Incubate 35°C - 0.5°C for 23 hr. - 1hr.
Select plate/5 within
countable plate range
Total Coliforms
(Colonies with metallic- like
reflecting sheen surface)
Count number of colonies
I
Calculate total coliform /100 mis
I
Record results as total
coliforms/100ml
Non-Coliforms
(Colonies lacking metallic-like
reflecting sheen surface)
I
Total Coliforms not present
•Note: Since sample volumes necessarily change dependent upon the
existing water quality, these have been arbitrarily selected
to give a cross-section of laboratory procedural methodology
11-3
-------
WATER MONITORING PROCEDURE: Total Coliform Test by the Membrane Filter Method
1. Analysis Objectives
In environmental water monitoring, the application of this methodology can
be for any of the following:
a. Test parameter for the conduction of a sanitary survey during an on-sfte
review of the water source.
b. Monitoring of influent waters of treatment plant.
c. Test parameter for special purpose waters (recreational, boating,
controlled loading, etc.).
2. Brief Description of Analysis:
Multiple portions of a representative sample are filtered through membrane
filters contained within a filtering apparatus. Bacteria in the sample
portions are held on the upper surface (gridded) of each of the membranes,
while the sample water passes through and is discarded. After several rinses
of the funnel of the filtering apparatus with sterile buffered distilled
water, each membrane filter is placed on a paper pad (absorption pad) saturated
with a medium called m-ENDO Broth within a petri dish. The closed end inverted
dishes are incubated within a high humidity incubator set at 35°C + 0.5°C for
an incubation period of 22-24 hours.
On this medium, coliform bacteria will grow and develop a golden metallic
sheen-like surface on the colonies. Colonies lacking this characteristic
reflective surface are not considered as coliforms. This disttnctive surface
sheen may appear at the center, edges or all-over the colony. At times it
can form as flecks or particles of sheen throughout or partially covering the
colony.
The membranes are inspected with the aid of a microscope or lens having a
magnification of lOx to 15x under reflective lighting from a fluorescent
source. Coliform colonies, if any, from suitable membrane/s are counted
and a calculation made to determine total coliforms per 100 millimeters.
3. Applicability of this Procedure:
a. The range of total coliform concentrations:
If the sample volumes These ranges of total
used are coliforms covered are
40 ml to .08 ml 50 to 100,000/100 ml
b. Pretreatment of samples in accordance with Standard Methods, 14th Ed.
(P- 904-907)
This procedure conforms to the Standard Total Coliform MPN Tests as described
in Standard Methods for the Examination of Water and Wastewater, 14th ed (1975)
p. 914 '"
11-4
-------
WATER MONITORING PROCEDURE: Total Coliform Test by the Membrane Filter Method
Equipment and Supply Requirements
A. Capital Equipment:
1. Autoclave, steam, providing uniform temperatures up to and including
121°C and equipped with an accurate thermometer, pressure gauges,
saturated steam power lines and capable of reaching required temperatures
within 30 minutes. (Alternately, a suitable pressure cooker is acceptable--
see Standard Methods for particulars.)
2. Incubator, air, providing uniform and constant temperature of 35°C.
+0.5°C and having an atmosphere of at least 90% relative humidity.
3. Oven, hot-air, providing uniform temperatures within the range of 160-180°C
4. Apparatus, water distillation, distilled water product suitable for
bacteriological operations (alternately, a suitable source is permissible).
5. Microscope, stereoscopic, 10X to 15X magnification with fluorescent
lighting mandatory. (Alternately, a suitable magnifying lens with
fluorescent lamp is acceptable.)
6. Refrigerator, set for less than 10°C but above the freezing temperature.
7. Vacuum source, preferably a pump assembly with suitable hoses and shut-off
valve provided. (Alternately, an aspirator or hand pump with the same
provisions are acceptable.)
8. Balance, analytical, sensitivity of 1 mg.
9. Gas source, suitable for burner. (Alternately, an alcohol lamp can be
used.)
B. Reusable Supplies:
1. Apron, suitable for laboratory operations.
2. Bottle, sample, of sufficient size for standard sample, preferably of
250 ml, wide-mouth, glass stopper, with tag. (Alternately, 120 ml size)
3. Bottle, squeeze type, containing disinfecting solution.
4. Burner, gas, suitable for laboratory operations with connecting hose.
5. Thermometer, NBS (or NBS calibrated), functions within 20°-60°C range
with individual markings of 1°C.
6. Thermometer, NBS (or NBS calibrated), functions within 150°-190°C range
with individual markings of 1°C.
7. Filtration Unit, MF, a seamless funnel attached to a receptacle bearing
a porous plate (screen, porous disc, etc.) and constructed from stainless
steel, glass, porcelain, plastic, or other suitable material.
8. Hot plate, controllable heat range up to the 100°C range.
9. Balance, trip, sensitivity of 0.1 gram at a load of 150 grams, with
appropriate weights.
10. Meter, pH, accurate to within 0.1 pH unit, with suitable standard pH
reference solution(s).
11. Can, pi pet, non-toxic and sterilizable material (if pre-sterilized
disposable type pi pets are used, this item is unnecessary).
12. Pan, discard, receives contaminated material and pi pets and contains
disinfectant. Should be of sufficient length to receive pipets placed
horizontally.
13. Cylinder, graduated, 500 ml, 100 ml, 50 ml, and 25 ml size.
(The 50 ml size is covered with a "cap" of foil or Kraft paper and
then sterilized.)
11-5
-------
WATER MONITORING PROCEDURE: Total Coliform Test by the Membrane Filter Method
Equipment and Supply Requirements (Continued)
14. Blank, dilution water, 99 ml.
15. Pipets, microbiological, 50. ml, with 0.1 ml graduations, sterile cotton
plugged, glass or disposable types (the disposable types are for one time
use and may be glass or plastic).
16. Pipets, microbiological, 1.0 ml, with 0.1 graduations, sterile cotton
plugged, glass or disposable types (the disposable types are for one time
use and may be glass or plastic).
17. Pipets, microbiological, 10ml, with 1 ml graduations, sterile, cotton
plugged, glass or disposable types (the disposable types are for one-time
use and may be glass or plastic).
18. Beaker, 50 ml (for measuring pH).
19. Flask, volumetric, 1 liter capacity (for stock solution of phosphate
buffer).
20. Flask, Erlenmeyer, 500 ml capacity (for holding buffered distilled rinse
water).
21. Flask, sidearm, 1 liter size (for reservoir of MF apparatus; proper size
bored, rubber stopper is needed to connect MF filtration flask to flask
and hose required to vacuum source (must be rigid enough to avoid collapse
under vacuum and flexible enough to be controlled by pinch clamp) pinch
clamp - vacuum control.
22. Flask, Erlenmeyer, 50 ml (for preparing m-ENDO medium).
23. Forceps, curved end, round tip.
24. Bottle, small, Methanol or Ethanol volume to cover ends of forceps.
25. Sponge, small, to spread and wipe germicide.
26. Desiccator, media storage, ideally opaque or darkened and containing
desiccating agent to remove moisture.
C. Consumable Supplies:
1. Dish, petri, disposable, tight fitting plastic, 50 x 12 mm, sterile.
2. m-ENDO Broth, medium, dehydrated, total coliform. Distributors, Difco,
BBL, or other equivalent preparation.
3. Pencil, wax, recommended of soft wax equivalent to Blaisdell 169T.
4. Tags, bottle marking.
5. Glass Wool.
6. Cotton, non-absorbent.
7. Paper, Kraft wrapping.
8. Foil, aluminum, heavy duty.
9. Matches or striker.
10. Towels, paper.
11. Detergent, non-toxic, laboratory cleaning.
12. Data Sheet, as required by analyst's agency.
13. Filter, membrane, 47mm, 0.45 pm pore size, white, grid marked, sterile.
14. Pad, absorbent, 48 mm, sterile (usually included with membrane packet).
15. Potassium Dihydrogen Phosphate (KH2P04), recommended 1/4 Ib.
16. Sodium Thiosulphate (Na2S203SH20).
17. Disinfectant, for bench tops and decontaminating purposes, bleach of
household strength and prepared according to label directions.
18. Sodium Hydroxide (NaOH), IN.
19. Distilled water, suitable for bacteriological operations. Obtainable
from distillation apparatus (see Capital Equipment) or suitable source
of supply.
11-6
-------
WATER MONITORING PROCEDURE: Total Coliform Test by the Membrane Filter Method
Equipment and Supply Requirements (Continued)
20. Magnesium Sulfate (MgSO.-7H?0).
21. Ethanol, 95%. H
Item needs in quantities or required size or space allowances cannot be specified,
as they vary according to the daily analysis schedule. As a rule-of-thumb, space/
size or quantity requirements should be at least 3 times the normal daily require-
ments. For further information on specifications for equipment and supplies, see
the Microbiology Section of the current edition of Standard Methods for the
Examination of Mater and Wastewater.
11-7
-------
WATER MONITORING PROCEDURE: Total Coll form Test by the Membrane Filter Method
11-8
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pretest Procedures
1. Incubator Setup,
Adjustment
(35°C + 0.5°C)
1. Place Incubator In perma-
nent location.
2. Provide a saturated
humidity within
incubator.
3. Install thermometer.
4. Connect incubator to
electric power source and
turn ON.
Aa. All pretest procedures completed before starting
other first-day procedures.
la. Floor location for large unit or table or bench
for smaller units.
Ib. Out of drafts or place in which it will be in
direct sunlight part of day.
Ic. Location convenient to laboratory operations.
Id. Convenient source of electric power with a
separate circuit, if possible.
2a. Check manufacturer's handbook for maintenance of
humidification system, if installed.
2b. If humidifier system not installed within incu-
bator, place beakers or trays containing dis-
tilled water on shelves to provide relative
humidity of at least 90 percent during operating
temperatures.
3a. Functions at least in 30°-40°C range. Meets NBS
standards. Have 0.2°C increment markings or lessj
3b. Usually a corner location to prevent breakage and
tip immersed in a bottle containing water,
glycerin, etc. for a more stable reading.
3c. If thermometer assembly has been installed by
manufacturer, check for above requirements and
calibrate with NBS thermometer. Calibration may
be possible by removal and testing of installed
unit or by comparison during incubator operation.
4a. Pilot light should come on.
V.A.1.1
III.A.l.Zb
V.A.I.3
-------
HATER MONITORING PROCEDURE: Total Coliform Test by the Membrane Filter Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pretest Procedures
(Continued)
2. Oven Sterilizer-
Drier Setup,
Adjustment
(170° + 1.0°C)
5. Adjust temperature until
stabilized at required
temperature.
6. Operate incubator
continuously.
1. Place oven sterilizer-
drier in permanent
location.
2. Connect oven/drier to
power source and turn ON.
3. Install thermometer.
5a. Manufacturer's instructions for location and
method of temperature adjustment.
5b. Allow about 1 hour between fine adjustment (less
than 2 degrees) and immediate adjustments can be
made when the calibration is greater than this
amount. Temperature achievement by the setting
knob will be usually indicated by either a light
indication or by an alternate lighting of a
"heat-ON" - "refrigerant-ON" or other arrangement
depending upon the incubator type/model.
6a. Operate incubator continuously unless it will be
unused for a relatively long period. (2 weeks or
more).
6b. Daily check of temperature required, preferably
an early morning and late afternoon with a
written record maintained. Adjust temperature if
necessary.
6c. Check at least biweekly the humidity level of
interior of incubator. Add water to humidifier
unit, if applicable, or to trays placed on the
shelves providing humidification by convection.
la. Convenient source of electric power.
2a. Usually an indication is given that power is
applied—such as an indicator light.
3a. If installed by manufacturer, ascertain if
installation meets the above requirements.
V.A.2
(p. 38)
11-9
-------
HATER MONITORING PROCEDURE: Total Coliform Test by the Membrane Filter Method
11-10
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pretest Procedures
(Continued)
3. Autoclave Setup
4. Water Distillation
Equipment Setup
5. pH Meter Setup
4. Adjust oven temperature
to stabilize at required
sterilizing temperature.
1.
Install and operate auto-
clave according to manu-
facturer's instructions.
1.
Install and operate in
accordance with manu-
facturer's instructions.
2. Operate as required to
maintain adequate supplies
of suitable distilled
water.
1. Setup and operate in
accordance with manu-
facturer's instructions.
4a. 170°C is the required temperature. Arbitrarily,
for this publication, a 1 degree leeway is
stipulated.
4b. Manufacturer's instruction for location and
method of temperature adjustment.
4c. Allow about 1 hour between fine adjustments (less
than 2 degrees of desired temperature) and
immediate adjustments can be made when the
calibration is greater than this amount.
la. Variable in design and operation, and unless
properly operated can be dangerous.
Ib. Used to sterilize objects made of or including
liquids, rubber, and some plastics, and, for
glassware, if desired.
Ic. Operated for general sterilization at 121°C.
(250°F) for a period of 15 minutes after this
temperature has been attained.
Id. Sterilized media and liquids must be removed as
soon as possible upon completion of sterilization
from the chamber of the autoclave.
la. Must produce water meeting quality requirements
for bacteriological tests.
V.A.4
(p. 39)
la. Meter must be accurate to at least 0.1 pH unit.
-------
WATER MONITORING PROCEDURE; Total Coliform Test by the Membrane Filter Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pretest Procedures
(Continued)
6. Glassware
Preparation
7. Sample Bottle
Preparation
1. Clean and rinse using a
suitable detergent and
hot water.
2. Use final rinsings of
deionized or distilled
water.
1. Deliver 0.1 ml or 0.2 ml
of 10% sodium thiosulfate
solution to each sample
bottle (0.1 ml to 4 oz. or
120 ml size and 0.2 ml to
6-8 oz. or 250 ml size).
Sodium thiosulfate is
prepared as follows:
*Weigh 10.0 grams of
sodium thiosulfate.
*Dissolve in 50-60 ml of
distilled water.
*Add distilled water to
bring final volume to
100 ml.
*Transfer to labeled
bottle.
2. Place cover on sample
bottle.
3. Place paper or metal foil
cover over bottle cap or
stopper.
la. flontoxic detergent must be completely removed
from glassware.
2a. Six to twelve successive rinsings may be required
2b. Must produce a clean dry glassware which meets
bacteriological requirements for suitability.
la. Bottle meets glassware requirements.
Ib. Use 1 ml pipet.
Ic. Provides adequate sodium thiosulfate for neutral-
izing chlorine in sample. Note: If the sample
does not contain chlorine, it is not necessary
to add the sodium thiosulfate.
Sodium Thiosulfate Preparation
Id. Use of trip balance for weighing acceptable.
le. 100 ml graduated cylinder satisfactory for volume
measurements.
If. Final preparation should be labeled as 10%
Sodium Thiosulfate and stored in refrigerator.
3a. Protects opening of sample bottle from accidental
or natural contamination.
11-11
-------
WATER MONITORING PROCEDURE: Total Coliform Test by the Membrane Filter Method
11-12
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pretest Procedures
(Continued)
8. Pi pet Preparation
4. Sterilize sample bottle
in autoclave or oven.
5. Store sample bottle in
clean dry place until used
1. Insert a plug of non-
absorbent cotton into
mouthpiece of clean, dry
pi pet.
2. Pass plugged end of pi pet
quickly through burner.
3. Insert a layer of glass
wool or multi-layer of
paper padding in bottom
of pipet can.
4. Place pi pet in pi pet can
with delivery tip down-
ward and contacting glass
wool or paper. Close can
when full or desirable to
complete preparation.
4a. In oven at 170°C for 1 hour.
15 pounds for 15 minutes.
In autoclave at
la. Pi pets which have chipped or broken tips or tops
should be discarded.
Ib. Cleanliness and suitability of pi pets equivalent
to bacteriological suitability of glassware.
Ic. Cotton plug must be tight enough to prevent easy
removal, either by pipeting action or by handling
and yet be loose enough to permit easy air move-
ment through the plug.
Id. Plug protects user from ingesting sample into his
mouth.
2a. Removes wisps of cotton which interferes with
fingertip control of pipeting action.
3a. Protects tips from damage.
3b. Pipets can be sterilized individually, if desired
by wrapping in Kraft paper, then oven sterilizing.
This technique would make the use of pi pet cans
unnecessary.
4a. Cotton-plugged mouthpiece in pipeting is finger
control end with the delivery tip on the opposite
end.
4b. Approximately twenty (20) 1 ml pipets or twelve
(12) 10 ml pipets will normally be accommodated
in these cans.
4c. Can must be able to withstand sterilizing condi-
tions. Toxic materials, such as copper, must not
be used. Aluminum, stainless steel, or glass
(Pyrex) are acceptable.
-------
WATER MONITORING PROCEDURE: Total Coliform Test by the Membrane Filter Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pretest Procedures
(Continued)
9. Blanks, Dilution
Water
5. Sterilize pi pets.
6. Store cans in a clean dry
place until needed.
1. Prepare stock solution of
potassium dihydrogen phos-
phate (KH2P04) by dissolv-
ing 34.0 grams of this
chemical in 500 ml of dis-
tilled water and adjusting
its pH to 7.2 with IN NaOH
Dilute to 1 liter in
volumetric flask.
2. Prepare stock solution of
magnesium sulfate
(MgS04-7H20) by dissolving
50 grams of this chemical
in 500-600 ml of distilled
water and, after complete
dissolving, bring the
final volume to 1 liter in
a volumetric flask.
5a. At least 1 hour in oven at 170°C, or
5b. In autoclave at 15 pounds steam pressure for 15
minutes. Cans removed quickly from autoclave
after sterilizing with aid of asbestos gloves and
opened quickly and slightly to allow residual
steam to escape for a few seconds.
la. Distilled water may be measured in 500 ml gradu-
ated cylinder.
Ib. Label to show contents, identity of preparer, and
date of preparation.
Ic. Stored in refrigerator.
Id. Discarded if mold or turbidity appear.
11-13
-------
HATER MONITORING PROCEDURE: Total Coliform Test by the Membrane Filter Method
11-14
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pretest Procedures
(Continued)
3. Prepare working solution
of dilution water by add-
ing 1.25 ml of the
potassium dihydrogen
phosphate stock solution
and 5 ml of the magnesium
sulfate stock solution to
each liter of distilled
water to be used in the
preparation of dilution
water.
4. Deliver enough working
solution to each dilution
water bottle so that after
sterilization the bottle
will contain 99 +_ 2 ml of
dilution water.
5. Place caps on bottles
loosely.
6. Sterilize in autoclave.
7. Remove from autoclave,
tighten bottle caps; cool
to room temperature.
8. Store in cool place.
3a. A 10 ml or 5 ml pi pet is satisfactory for
delivery of both of these stock solutions pro-
vided that it has graduation marks to deliver
the proper amount. Use separate pi pets for each
solution to prevent contamination.
4a. Recommended that dilution water bottles have a
marking at the desired 99 ml quantity. Amount
to be delivered to bottle before sterilization
cannot be stated exactly as evaporation is
different with differing conditions and auto-
claves. Ordinarily about 102 ml will be required
6a. 15 minutes at 121°C.
8a. Dilution bottles ready for use. May be stored
indefinitely.
8b. Some evaporation loss may occur in time and in
these cases, sterile similarly prepared water
may be added. This is why a calibrated marked
bottle is desirable.
-------
WATER MONITORING PROCEDURE: Total Coliform Test by the Membrane Filter Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pretest Procedures
(Continued)
10. Preparation of
Total Coliform
Medium
1. Retrieve and inspect
dehydrated m-ENDO broth
medium.
2. Weigh 1 gram of dehydrated
m-ENDO broth.
3. Place powder in a clean,
dry 50 ml Erlenmeyer flask
4. Prepare an alcohol-water
solution as follows:
a.
b.
Place 0.4 ml of
ethanol in a clean,
25 ml graduate.
dry
Add distilled water to
the graduate to the
20 ml mark.
Add a small amount of the
ethanol-water solution to
the powder in the flask
(about 5 ml). Swirl flask
to mix powder and then add
the remainder of the water
la. Best stored in desiccator which prevents moisture
from damaging medium.
Ib. Powder must be light pink without signs of
hardening or color change to blood red hue.
2a. Sufficient for 20 ml of medium which prepares
10 petri dishes.
2b. Analytical balance having a sensitivity of 1 mg
will be required.
2c. More than 2 grams being weighed can be done on
less sensitive balance. This would provide more
plates, but, of course, some medium can be
discarded.
4a. Graduate need not be sterile. No acceptable
substitutes for ethanol. Use 1 ml pipet
graduated in 0.1 ml increments.
4b. A Squeeze bottle addition to the graduate makes
control of the distilled water addition easier.
5a. Small addition of water makes it easier to re-
move powder from walls of flask.
VI.A.10.1
(P. 41)
II.A.10.2
(p. 36)
VI.A.10.4
(p. 41)
11-15
-------
WATER MONITORING PROCF.DURE; Total Coliform Test by the Membrane Filter Method
11-16
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pretest Procedures
(Continued)
11. Preparation of
m-ENDO Plate
6. Cover top of flask with
aluminum foil.
7. Heat flask on a hot plate
set to high heat range.
8. Remove at first sign of
boiling.
1. Remove a sterile petri
dish from its container.
Loosen its cover without
removal.
2. Remove a sterile absorp-
tion pad from its container
and place in dish. Replace
cover which is still kept
loosely fitting.
3. Transfer approximately 2 m
of the m-ENDO broth to the
absorption pad within the
dish.
4. Gently tip the opened
petri dish until a droplet
of medium forms on the
inner lower edge.
6a. Some laboratories use a screw-cap to cover the
flask. If this is to be practiced, make sure
that the cap is LOOSE when heating to relieve
pressure built up during heating.
7a. Constant stirring is necessary to prevent
charring or burning of medium.
8a. Prolonged heating reduces selectivity of medium.
8b. Do not autoclave this medium.
8c. Medium ready for use. Can be stored in refrig-
erator for up to 96 hours before discarding.
la. Usually in a sleeve of pre-sterilized plastic
one-time-use dishes.
Ib. Laboratory reusable sterilized glass dishes can
alternately be used.
2a. Usually packaged with membrane filters, or,
alternately, can be separately laboratory
sterilized.
2b. Use a forceps which has been standing in a flask
of alcohol and then passed quickly through a
flame to remove residual alcohol to handle the
pad.
3a. Plate can be stored in refrigerator for up to
96 hours before discarding or used immediately.
4a. A 2 ml broth addition is usually an excessive
amount.
4b. Hold petri dish cover in other hand. Do not
allow it to become contaminated.
-------
EFFLUENT MONITORING PROCEDURE: Total Coliform Test by the Membrane Filter Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Pretest Procedures
(Continued)
5. Gently shake out large
droplet to waste. Replace
cover tightly.
5a. Plate is ready for use in analysis.
5b. Keep plate from excessive exposure to light,
particularly sunlight.
5c. It has been found that this procedure will in-
variably give an optimum amount of medium,
whereas, trying to measure precisely the same
amount for each plate will give deviations from
optimum amounts more frequently.
5d. If plate is to be used within the hour simply
cover with a paper towel on the bench. If a
greater time is expected, place in refrigerator
until used.
B. First Day Procedure
1. Equipment
Maintenance
2. Sample Collection
and Handling
1. Check, record, and adjust,
if necessary, the 35°C
incubator.
2. Check, record (if done)
and adjust (if necessary)
the refrigerator.
1. Collect sample, use a
grab, direct filling, or
suitable device collection
technique.
2. Apply label to bottle and
enter required information
la. Representative of water supply system.
Ib. Leave sufficient air space in bottle to allow
shaking of sample (at least 2.5 cm or 1 inch).
Ic. Do not rinse bottle before collecting sample as
this would cause loss of dechlorinating agent.
Id. Exercise care to prevent contamination of samples,
2a. Enter required information as per agency re-
quirements. A minimum useful amount of entries
include:
*name of sampler (complete name, not initials)
*location/code of collection site
*time of collection
*chlorine residual (water before sampling)
*date of collection.
11-17
-------
EFFLUENT MONITORING PROCEDURE: Total Coliform Test by the Membrane Filter Method
11-18
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First Day Procedure
(Continued)
3.
Preanalysis
Preparation
4.
Equipment and
Material Prepara-
tion/Assembly
3. Place bottle in closed ice
chest.
4. Transport to laboratory
and dispose of sample in
accordance with laboratory
policies.
1. Prepare laboratory data
sheet.
2. Disinfect laboratory
bench; wipe dry.
1. Assemble required
equipment and material.
4a. Sample should be analyzed as soon as possible.
Immediate analysis is best but up to 6 hours
holding time is acceptable.
la. No standard data sheet. Use form recommended by
1aboratory/agency.
Ib. Some of required information will be on sample
label.
2a. Use sponge and disinfectant; paper toweling.
la. Filtration funnel assembly, sterile.
Ib. Side arm suction flask, 1 liter size.
Ic. Hose, suction w/clamp.
Id. Vacuum source, operational.
le. Sheet, data.
If. Prepared m-ENDO dishes. (4 required)
Ig. Membrane filters, sterile.
Ih. 99-ml buffered, distilled water blank.
li. Forceps and disinfectant container (methanol).
Ij. Pencil, marking.
Ik. Sample bottle.
11. Graduate, sterile, 50 ml, foil hood protected.
1m. Burner, gas, w/hose joined to gas source.
* pipets, 10 ml, 1 ml sterile, '(not shown)
* buffered distilled rinse water (about 250 ml
per analysis).
-------
WATER MONITORING PROCEDURE; Total Collform Test for Drinking Water by the Membrane Filter Method
-------
WATER MONITORING PROCEDURE: Total Coliform Test by the Membrane Filter Method
11-20
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING COALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First Day Procedure
(Continued)
2. Assemble the units of the
filtration apparatus.
3. Test the filtration
apparatus for operation.
Assemble the units of the
filtration unit assembly:
Unwrap sterile funnel base
from wrapping and place on
base unit.
2a.
Suction side of apparatus may, depending on
choice of equipment, consist of sidearm suction
flask, suction line, pinch clamp, and suction
device. Such an arrangement is shown below:
V.B.4.2
(p. 39)
hose
pinch clamp
stopcock
sidearm flask
suction main line
3a. Check suction units for cleanliness and operation
Open suction line by turning on stopcock and re-
moving pinch clamp and check for suction at neck
of sidearm flask by placing palm of hand over
neck of flask and noting presence of suction.
Replace and close pinch clamp and note if suction
is cut off from the flask. NOTE: This test is
made without the filtration funnel assembly being
installed.
4a. The filtration unit assembly consists of a funnel
and a base which should be clean, sterile, and in
operational status.
4b. Manufacturers usually provide kits for mainte-
nance of units.
4c. Do not contaminate working areas of funnel
assembly (screen, inner area of funnel, funnel
lip, etc.).
4d. Stopper may be retained on base of filtration
unit throughout the usage and sterilization of
the base.
-------
HATER MONITORING PROCEDURE: Total Coliform Test by the Membrane Filter Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First Day Procedure
(Continued)
4e. Units of filtration assembly are sterilized by
steam after wrapping in Kraft paper or aluminum
foil.
funnel
base
filtration
funnel
assembly
stopper
5. Sample Filtrations
A. 40 ml Volume
5. Light burner.
6. Label m-ENDO plate with
necessary identification
markings.
1. Place membrane filter (MF)
on base of funnel unit and
centered evenly on the
screen assembly.
5a. Some laboratories use an alcohol lamp.
6a. Conforms to data sheet. (See flow sheet.)
la. Funnel top removed carefully to avoid contami-
nation. Do not place on contaminated surface.
Best to hold in hand while using forceps in other
11-21
-------
WATER MONITORING PROCEDURE: Total Coliform Test by the Membrane Filter Method
11-22
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First Day Procedure
(Continued)
2. Deliver a small volume of
sterile buffered distilled
rinse water inside the
funnel.
3. Deliver sample volume to
funnel by using a sterile
graduate.
Ib. MF placed grid or inked side UP. MF handled
with flamed forceps and only on the membranes
outer 3/16 inch of its circumference.
Ic. Replace funnel top. Avoid over-tightening which
can damage the MF or cause leakage.
2a.
2b.
3a
3b.
Use approximately 10 ml of water.
Observe funnel for leakage. If any, disassemble
unit and repeat from Step 1 after inspecting
base of funnel for possible debris or damage.
Persistent leakage will necessitate maintenance
or replacement of funnel unit.
Thoroughly shake sample bottle prior to filling
graduate to 40 ml mark. A minimum requirement
would be 25 complete up-and-down (or back-and-
forth) movements of about 0.3 ml (1 foot) in
7 seconds.
Sterile graduate is prepared by oven steriliza-
tion with an aluminum foil cap.
See Flow
Sheet
(P. 3)
-------
HATER MONITORING PROCEDURE:'Total Coliform Test by the Membrane Filter Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First Day Procedure
(Continued)
4. Gently pour sample (40 ml)
into funnel.
3c. Volume in graduate is measured by sighting if the
meniscus lower curve being even with the 40 ml
mark.
meniscus line
4a,
4b,
4c.
4d.
-40
30
glass graduate
eye level
(bottom of meniscus line
touching 40 ml line
in a parallel plane)
Avoid splashing. Pour slowly and close to top
of funnel without touching sides.
Allow a 5 second drainage period before shaking
off the last drop.
Graduate marked TC (to contain): Rinse graduate
several times with sterile water and pour each
rinsing individually into funnel.
Graduate marked TD (to deliver): Rinsing not
necessary, but, allow at least 5 seconds drainage
time and then gently tap off last drop into
funnel.
11-23
-------
UATER MONITORING PROCEDURE; Total Coliform Test by the Membrane Filter Method
11-24
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First Day Procedure
(Continued)
5. Open vacuum control valve
and remove pinch clamp to
allow vacuum to filter
sample through MF.
6. Rinse funnel three times
with sterile buffered
distilled water.
7. Replace pinch clamp on
suction hose.
8. Disconnect funnel locking
device and lift funnel
from base to expose MF.
9. Remove membrane from
funnel base.
5a. Vacuum must not be allowed to enter system prior
or during the previous step as this would suck
sample prematurely and bacterial dispersion will
not occur over membrane.
5b. Allow complete passage of sample through MF.
Ga
6b.
6c.
7a.
7b.
7c.
Rinsings remove all of residual sample droplets
from sides of funnel.
Allow complete flushing of each rinse through
membrane before applying next rinse.
Use about 25 ml for each rinse and pass around
funnel to rinse complete circumference (circular
motion of hand around funnel) of funnel. Do not
touch inside area of funnel.
Interrupts vacuum delivery to flask.
Will not allow MF to be lifted from base without
possible damage due to strong suction being
continued.
Some laboratories may elect to use control valve
for this operation and not use pinch clamp.
8a. Best to hold funnel in one hand while using
forceps with other. Some .laboratories may elect
to either:
*Use a germicidal cabinet to hold funnel.
*Use a funnel holding device.
But, in any event, DO NOT place funnel where it
can become contaminated if it is to be used for
another sample as is this analysis.
9a. Again, handle membrane carefully with flamed
forceps (quickly flamed after removing from
alcohol immersion jar - NOT HEATED) and only on
outer 3/16 inch of membrane.
V.B.6.1
(p. 39)
-------
WATER MONITORING PROCEDURE; Total Coliform Test by the Membrane Filter Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First Day Procedure
(Continued)
10. Replace funnel on base if
it is held in other hand,
or, replace when conven-
ient if held in holding
device or UV light box.
11. Remove cover from m-ENDO
dish.
9b. Break residual vacuum in flask by gently lifting
edge of MF before removing.
lOa. Funnel unit is now ready to receive the next
sample as the three rinses have been found to
be sufficient to cleanse the funnel of bacteria,
which can influence this test (carry-over or loss
of bacteria).
lla. Do not allow it to become contaminated. Can
either be held in the hand or placed on the lab
bench.
V.B.6.3
(p. 40)
cover
base with
m-ENDO
medium
11-25
-------
WATER MONITORING PROCEDURE: Total Coliform Test by the Membrane Filter Method
11-26
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First Day Procedure
(Continued)
12. Place MF over the m-ENDO
medium. Close the dish
tightly when membrane
shows elimination of air
pockets.
12a.
12b.
12c,
12d.
13. Invert petri dish (turn
upside down). The bottom
or plate base will now be
on top and the MF will be
upside down. Label dish.
14. Place dish in the inverted
position within the 35°C
incubator.
Grid or inked side surface contains the bacteria
and must not be placed next to the m-ENDO.
If air pockets persist (indicated by white areas
with pink colored m-ENDO liquid) pick up the
MF by its edge and re-roll. Persistent clear or
white areas usually indicate that there is too
little broth on the pad. Add a drop of m-ENDO
to the pad if necessary while holding up a corner
of the MF. Do not place broth over the membrane.
Use a rolling action to eliminate air pockets.
Do not run forceps or any object over the MF as
it is very delicate and damage can result in poor
plate results.
Some amount of air spots is tolerable if they
are outside the working area of where the
bacteria were plated. About 3/16 inch is
acceptable.
13a. Use wax marker or a label used by facility.
13b. Indicate time of plating and sample number.
14a. Plate is inverted to prevent droplets from
"falling down" on MF destroying the colonial
growth of the bacteria.
14b. Do not crowd plates. If a number of them have to
be stacked, place them no more than three high
with an unused area around them equal to the size
of a petri dish.
14c. Allow an incubation period of 22-24 hours. Be
sure time of plating is indicated on data sheet.
-------
WATER MONITORING PROCEDURE; Total Coliform Test by the Membrane Filter Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First Day Procedure
(Continued)
B. 8 ml Sample
Volume
1. Place membrane filter (MF)
on base of funnel unit
and centered evenly on the
screen assembly.
2. Deliver a small volume of
sterile buffered distilled
rinse water inside the
funnel.
3. Deliver sample volume to
funnel by using a sterile
pi pet.
4. Gently pi pet the 8 ml into
the funnel.
5. Open vacuum control valve
and remove pinch clamp to
allow vacuum to filter
sample through MF.
6. Rinse funnel three times
with sterile buffered
distil led'water.
7. Replace pinch clamp on
suction hose.
la. As previously described.
2a. As previously described.
3a. Thoroughly shake sample bottle as described
previously.
3b. Fill pi pet to about the 10 ml mark and apply
finger pressure to hold this amount within pipet.
3c. Allow volume to fall to exactly the 8 ml gradua-
tion. Hold and maintain this volume by finger
pressure.
4a. As previously described in B.5A.4a-b.
6a. As described previously.
7a. As described previously.
11-27
-------
WATER MONITORING PROCEDURE; Total Coliform Test by the Membrane Filter Method
11-28
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First Day Procedures
(Continued)
C. 0.8 ml Sample
Volume
8. Disconnect funnel locking
device and lift funnel
from base to expose MF.
9. Remove membrane from
funnel base.
10. Replace funnel on base if
it is held in other hand,
or, replace when con-
venient if held in holding
device or UV light box.
11. Remove cover from m-ENDO
dish.
12. Place MF over the m-ENDO
medium. Close the dish
tightly when membrane
shows elimination of air
pockets.
13. Invert petri dish.
14. Place dish in the inverted
position within the 35°C
incubator.
1. Accomplish complete fil-
tration procedure as
described 'previously
(steps 1-14) for other
volumes.
8a. As described previously.
9a. As described previously.
lOa. As described previously.
lla. As described previously.
12a. As described previously.
14a. As described previously.
la. All items of step sequence and of this column
are identical except for the means of obtaining
the sample volume which is as follows:
A. Use a 1 ml pi pet. Fill pi pet to zero mark and
apply finger pressure to hold this amount
within pi pet. Drop level to 0.2 ml mark.
B. Gently pi pet the & ml sample volume into the
funnel. Pi pets may be of two general types:
(Continued)
-------
WATER MONITORING PROCEDURE: Total Coliform Test by the Membrane Filter Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. First Day Procedures
(Continued)
D. 0.08 ml Sample
Volume
1. Accomplish complete fil-
tration procedures as
described previously
(steps 1-14) for other
volumes.
a. TD (To Deliver)...This type allows proper
volume delivery when the liquid level
either drops to a marked line value or
falls to the delivery tip with full finger
release.
b. TC (To Contain)...This type will have
proper volume delivery of the full pipet
contents only when the last remaining
volume is forcibly ejected from the
by blowing.
la. All items of step sequence and of this column
are identical except for the means of obtaining
the sample volume which is as follows:
A. Required materials:
a. Pipet, sterile, 1 ml
b. Pipet, sterile, 10 ml
c. 99-ml dilution blank water level adjusted,
if necessary, by use of sterile pipet or
sterile pipet and dilution water.
B. Add 1 ml of well shaken sample water to the
99-ml blank and shake this well to distribute
sample.
C. Water the 10 ml pipet, obtain water from the
blank and pipet 8 ml to the funnel.
C. Second Day Procedure
1. Colony Counting
Procedure
Remove petri dishes from
incubator with careful
handling to avoid jarring
of plates. Turn plates
over where cap is now on
top.
la. Incubation period has been within the 22-24 hour
period. No deviations are permitted.
Ib. Rough handling can cause spattering of droplets
within plate and possibly causing difficulty in
counting.
11-29
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WATER MONITORING PROCEDURE; Total Coliform Test by the Membrane Filter Method
11-30
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Second Day Procedure
(Continued)
Observe visually which
plates are within the
countable plate range (20-
80 colonies). Select those
within this range for
counting.
3. Count selected plates for
total collform colonies
with microscopic aid. Ad-
just light source to give
maximum sheen development
to colonies, if any.
2a. This ability comes with experience, but plates
which are overcrowded or those with less than 20
are readily apparent.
2b. Observation of numerous colonies on the MF, even
if they are not sheen containing colonies, will
require counting since there is a 200 count maxi-
mum allowable colony count. The 200 colonies or
more of all types (which includes conforms) Is ar
amount of growth which produces Interferences witt
validity of results.
2c. It 1s necessary only to record counts within the
given range, but, If not possible, we will cover
exceptions later with examples.
3a. Binocular wide field dissecting microscope with
10-15x magnification preferred, but magnifying
lens acceptable.
3b. Cool, white, fluorescent lighting system neces-
sary. A near vertical light adjustment 1s
usually optimum.
3c. Sheen Is characteristic of the collform group of
bacteria and 1s a golden, metalllc-like reflective
property on the surface of the colonies. Sheen
can completely or partially cover the colony. It
can also appear as flecks. ANY AMOUNT OF SHEEN
1s considered positive.
-------
MATER MONITORING PROCEDURE; Total Coliform Test by the Membrane Filter Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Second Day Procedure
(Continued)
4. Select colony count/counts
to use. Utilize formula
to calculate count/100 ml.
3d. Microscopically scan membrane with a back-and-
forth movement over the grids and count all
colonies having sheen.
3e.
\
= = = = »
The dashed circle indicates the effective filter-
ing area. The dashed back-and-forth line indi-
cates the colony counting pathway.
Colonies are raised, usually circular, growths of
original bacteria which was planted on the mem-
brane and are considered to be the result of a
single organism multiplying many times to produce
a visible colony.
V.C.l.Se
(p. 40)
4a. Formula
Total Col 1 forms/100 ml = 100 x
Example #1
mis Filtered No. Colonies
40
Colony Count
No. of mis Filtered
TC/100 ml
TNTC (Indicates too
numerous to count)
8 TNTC
0.8 TNTC
0.08 35 43,750
Use: 35 colonies with 0.08 ml sample volume which
calculates to 44,000/100 ml (Use two significant
figures).
-------
WATER MONITORING PROCEDURE; Total Coliform Test by the Membrane Filter Method
11-32
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Second Day Procedure
(Continued)
Example #2
mis Filtered
40
B
0.8
0.08
No. Colonies^
70)
22 f
below 20
below 20
TC/100 ml
Combined
calculation
40 ml + 8 ml = 48 ml
70 colonies + 22 colonies = 92 colonies
TC/100 ml = 100 x
190/100 ml
Use: 190/100 ml as count based on combined values
of the plates.
Example #3
mis Filtered No. Colonies
40
8
0.8
0.08
Use:
TNTC
TNTC
270 (80 conforms &
190 background
colonies)
75
TC/100 ml
>200 (greater
than 200)
Limit sur-
passed—not
used
94,000
75 colonies from the 0.08 volume to give a
count of 94,000/100 mis
-------
MATER MONITORING PROCEDURE; Total Coliform Test by the Membrane Filter Method
OPERATING PROCEDURES
C. Second Day Procedure
(Continued)
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
Example #4
mis Filtered No. Colonies TC/100 ml
40 TNTC
8 TNTC
0.8 150
0.08 90 110,000
Rationale: Use that count which is closer to the
maximum 80 count.
Example #5
mis Filtered No. Colonies TC/100 ml
40 17 43
8 3
0.8 0
0.08 0
Rationale: Use that count which is closer to the
minimum 20 count.
Example #6
mis Filtered No. Colonies TC/100 ml
40 0 <3 (Less than 3)
80
0.8 0
0.08 0
Rationale: Assume that the largest volume delivered
has one colony. Use this in calculations
and call the result < (less than).
TRAINING
GUIDE NOTES
11-33
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WATER MONITORING PROCEDURE; Total Coliform Test by the Membrane Filter Method
11-34
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Second Day Procedure
(Continued)
2. Recording Data
3. Disposition of
Counted Plates
1. Record counts as values
per 100 ml.
1. Process to verification
test, if necessary.
2. Dispose of plates in
approved manner.
la. Record and process data as required by organiza-
tion/agency.
la. Some, all, or none of the plates may be processed
to this test dependent upon requirements of or-
gani zation/agency.
2a. Any LIVE organisms are to be considered as
POTENTIALLY DANGEROUS to humans.
2b. Usual disposition is by autoclaving (steam
sterilizing) in a metal container, then discard-
ing in waste recepticles (Note: Red dye can
still cause staining so handle to preclude
contamination.
V.C.3.1a
-------
WATER MONITORING PROCEDURE: Total Coliform Test by the Membrane Filter Method
SECTION TOPIC
I Introduction
II* Educational Concepts - Mathematics
III* Educational Concepts - Science
IV Educational Concepts - Communications
V* Field and Laboratory Equipment
VI* Field and Laboratory Reagents
VII Field and Laboratory Analyses
VIII Safety
IX Records and Reports
*Training guide materials are presented here under the headings marked *.
These standardized headings are used through this series of procedures.
11-35
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WATER MONITORING PROCEDURES: Total Coliform Test by the Membrane Filter Method
EDUCATIONAL CONCEPTS - MATHEMATICS
Section H
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.10.2
Since 48 grams of m-ENDO broth powdered medium and
20 ml of 95% Ethanol are required to prepare 1 liter
(1000 ml) of m-ENDO broth, it is possible to calcu-
late weights and volumes to prepare any requirement
based upon the number of plates desired. Calcula-
tions are based upon knowing the above figures and
the requirement of 2.0 ml of broth for each pad
saturation.
For rapid calculations the following two formulas
can be used:
1. No. of plates desired x 0.096 = grams m-ENDO
2. No. of plates desired x 0.04 = ml Ethanol.
EXAMPLE: If 47 plates of m-ENDO are required:
1. 47 x 0.096 = 4.512 or 4.5 grams m-ENDO.
2. 47 x 0.04 = 1.88 or 1.9 ml Ethanol.
94 ml
total
volume
NOTE: Due to the practical and technical difficul-
ties involved in weighing very small portions as, for
instance, 0.096 grams of m-ENDO for one plate re-
quirement, it would be wise to prepare at least 10
plates (0.96 or 1.0 gram m-ENDO and 0.4 ml Ethanol)
as a minimum requirement.
11-36
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MATER MONITORING PROCEDURES: Total Coliform Test by the Membrane Filter Method
EDUCATIONAL CONCEPTS - SCIENCE
Section III
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.1.2b
A relative humidity of over 90 percent is necessary
in order to obtain bacterial growth on the membrane
filter (colony) which has not been inhibited by a
lack of this moisture. Inhibited colonies will in-
variably be smaller and less apt to give the typical
sheen characteristic of a frank coliform.
A closed container, such as a plastic vegetable
crisper, may be placed within the incubator and have
within the container a saturated humid atmosphere.
A convenient way of accomplishing this is to wet a
few paper towels and place within the crisper or box.
An accurate solid heat sink incubator is acceptable
for use. This is constructed of a solid metallic
block having slots for insertion of the petri dishes.
Since there are no provisions for a high humidity
chamber in this type of incubator, it is important
to only use the types of petri dishes having a tight
attachment of cover-to-base thus preventing loss of
moisture during the incubation period.
Std. Meth. 14:937
11-37
-------
HATER MONITORING PROCEDURES: Total Coliform Test by the Membrane Filter Method
FIELD AND LABORATORY EQUIPMENT
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A. 1.1
A.1.3
A.2
Incubator should be kept out of drafts or direct
sunlight in order to prevent temperature inside the
incubator from changing outside the temperature
range specified (35°C + 0.5°C).
Power supply should be selected so that there will
not be too many pieces of equipment on the same
circuit. Otherwise circuits will be blown repeat-
edly.
Mercury bulb thermometer usually used in most in-
cubators and a recording thermometer are acceptable.
Thermometers must be calibrated against a mercury
bulb thermometer which is (or calibrated against) a
National Bureau of Standards issue and used with
the certificate and correction chart.
Sterilizing ovens should be of sufficient size to
prevent crowding of materials to be sterilized. The
information below summarizes the use of the oven.
Standard Methods for the
Examination of Water and
Wastewater. 14th Ed. TT975)
APHA, WPCF, AWWA, p. 880 ff.
(Hereafter referred to as:
Std. Meth. 14:(Page No.)
Std. Meth. 14:881
:885
MATERIAL
STERILIZED
ilassware
ilassware
CONDITIONS
170°C for at
least 60 min.
160°C for at
least 60 min.
Glassware within 170°C for at
netal container least 120 min.
Other material
170°C for at
least 60 min.
REMARKS
If internal oven,
characteristics are
unknown
If oven temperature
uniform throughout
chamber
Material must be
capable of with-
standing sterilizing
conditions
Alternately, a gas sterilizing unit can be used in
place of the hot-air oven. Refer to Standard Methods
and manufacturer's catalogs for details of such a
unit (ethylene oxide gas).
ionflicting temperature/time relationships appear in
differing references, but, the over-riding consider-
ation is how this time/temperature relationship works
in your hands, with your equipment, and considering
the results of sterility testing.
11-38
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MATER MONITORING PROCEDURES: Total Coliform Test by the Membrane Filter Method
FIELD AND LABORATORY EQUIPMENT
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.4
B.4.2
B.6.1
Distilled water must not contain substances pre-
venting bacterial growth or be highly nutritive.
There are required procedures for testing distilled
water and should be undertaken only by professional
bacteriologists or in laboratories where this is
done regularly. Alternately, a source of deionized
water which meets all requirements as imposed on
distilled water is suitable for use in bacterio-
logical operations.
Diagram and equipment listing describe the type of
units most commonly used in the membrane filter
procedure. They are by no means the only acceptable
arrangement which can be utilized. Different modes
of obtaining a vacuum or its control are available.
Also, various vacuum flask arrangements are accept-
able as well as the types of vacuum controls. To
preclude numerous examples, the one described will
be sufficient to give technological procedures which
are acceptable and the reader can refer to Standard
Methods and Manufacturer's catalogs for further
information regarding system components or field
units which are acceptable.
A germicidal cabinet is an enclosed unit which con-
tains an active germicidal lamp (UV) which produces
a 99.9% bacterial kill in 2 minutes. It is important
not to have UV leakage from cabinet which can be
detrimental to the analyst's eyes. A funnel holding
device is designed to hold the funnel and prevent its
contamination.
EXAMPLE
Std. Meth. 14:887
split-ring
holding device
funnel in holder
N0
Std. Meth. 14:933
11-39
-------
WATER MONITORING PROCEDURES: Total Coliform Test by the Membrane Filter Method
FIELD AND LABORATORY EQUIPMENT
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
B.6.3
C.1.3e
C.S.la
Funnel units are considered to be acceptable for use
for the next sample unless an interval of 30 minutes
or longer elapses before the next sample is run. In
this case the unit must be resterilized.
Occasionally colonial growth will be observed to be
irregular such as the following:
Std. Meth. 14:932
B
Usually, as in A and B, the colonies are readily
discernible as being multiple colonies - 2 for A and
3 for B. In the case of C and D, however, this
separation is not readily apparent and the judgment,
based on experience, of the analyst becomes impor-
tant. In the case of D the long strand growth may be
caused by a particle of debris which allowed chan-
neled growth of one or more bacteria.
The verification test is accomplished by picking the
presumptive sheened coliform colony with a sterile
needle and passing it through a series of broth media
to observe for another coliform characteristic-gas
Formation in a selective medium. Refer to Standard
Methods for a detailed performance of this verifica-
tion test.
Std. Meth. 14:920
:931
11-40
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WATER MONITORING PROCEDURES: Total Coliform Test by the Membrane Filter Method
FIELD AND LABORATORY REAGENTS
Section VI
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.10.1
A.10.4
Procedures are given for m-ENDO broth medium pre-
paration which is, however, not the only acceptable
method available. Other acceptable m-ENDO medium
preparations include:
A. m-ENDO Agar Medium
This includes the addition of the complex carbo-
hydrate agar whose purpose is to solidify the
medium. In this preparation the absorption pad
is not required for the analysis.
B. Pre-prepared Ampouled m-ENDO Medium
A complete prepared medium which is enclosed in a
glass tube. Contains enough medium for a single
test and has the advantages of a longer shelf life
and convenience of use. Is somewhat more costly
than laboratory preparation, especially when many
plates are to be processed.
thanol is added to distilled water in a 2% dilution
'or the m-ENDO medium. The amounts, of course, would
e different depending on the petri dish requirements.
he table below gives some useful information as
eference:
Std. Meth. 14:895
o. of Plates
-ENDO Re-
uired
500
250
50
25
10
5
Alcohol-Water
Required, mis
1 liter
(1000 ml)
500
100
50
20
10
Ethanol
mis
20
10
2
1
0.4
0.2
m-ENDO Broth
Powder, qm
48
24
4.8
2.4
.96 or 1.0
.48 or .5
ome laboratories prepare a large amount of the 2%
olution and, when tightly stoppered, can be used
or extended periods.
'his outline was prepared by: Rocco Russomanno,
licrobiologist, National Training and Operational
Technology Center, MOTD, OWPO, USEPA, Cincinnati,
Ohio 45268
11-41
-------
LABORATORY SAFETY PRACTICES
I INTRODUCTION
A Safe Use. Handling and Storage of Chemicals
1 Chemicals in any form can be safely
stored, handled, and used if their
hazardous physical and chemical
properties are fully understood and the
necessary precautions, including the
use of proper safeguards and personal
protective equipment are observed.
2 The management of every unit within a
manufacturing establishment must give
wholehearted support to a well integrated
safety policy.
B General Rules for Laboratory Safety
1 Supervisory personnel should think
"safety. " Their attitude toward fire
and safety standard practices is reflected
in the behavior of their entire staff.
2 A safety program is only as strong as
the worker's will to do the correct
things at the right time.
3 The fundamental weakness of most
safety programs lies in too much lip
service to safety rules and not enough
action in putting them into practice.
4 Safety practices should be practical and
enforceable.
5 Accident prevention is based on certain
common standards of education, training
of personnel and provision of safeguards
against accidents.
II LABORATORY DESIGN AND EQUIPMENT
A Type of Construction
1 Fire-resistant or noncombustible
2 Multiple story buildings should have
adequate means of exit.
3 Stairways enclosed with brick or
concrete walls
4 Laboratories should have adequate exit
doors to permit quick, safe escape in
an emergency and to protect the
occupants from fires or accidents in
adjoining rooms. Each room should be
checked to make sure there is no
chance of a person being trapped by
fire, explosions, or release of dangerous
gases.
5 Laboratory rooms in which most of the
work is carried out with flammable
liquids or gases should be provided
with explosion-venting windows.
B Arrangement of Furniture and Equipment
1 Furniture should be arranged for
maximum utilization of available space
and should provide working conditions
that are efficient and safe.
2 Aisles between benches should be at
least 4 feet wide to provide adequate
room for passage of personnel and
equipment.
3 Desks should be isolated from benches
or adequately protected.
4 Every laboratory should have an eye-
wash station and a safety shower.
C Hoods and Ventilation
1 Adequate hood facilities should be
installed where work with highly toxic
or highly flammable materials are used.
2 Hoods should be ventilated separately
and the exhaust should be terminated
at a safe distance from the building.
3 Make-up air should be supplied to
rooms or to hoods to replace the
quantity of air exhausted through the
hoods.
PC.SA. lab. 1. 11.77
12-1
-------
Laboratory Safety Practices
4 Hood ventilation systems are best
designed to have an air flow of not less
than 60 linear feet per minute across
.the face of the hood, with all doors open
and 150. if toxic materials are involved.
5 Exhaust fans should be spark-proof if
exhausting flammable vapors and
corrosive resistant if handling corrosive
fumes.
6 Controls for all services should be
located at the front of the hood and
should be operable when the hood door
is closed.
7 All laboratory rooms should have the
air changed continuously at a rate
depending on the materials being
handled.
D Electrical Services
1 Electrical outlets should be placed
outside of hoods to afford easy access
and thus protect them from spills and
corrosion by gases.
2 Noninterchangeable plugs should be
provided for multiple electrical services.
3 Adequate outlets should be provided and
should be of the three-pole type to
provide for adequate grounding.
E Storage
1 Laboratories should provide for adequate
storage space for mechanical equipment
and glassware which will be used
regularly.
2 Flammable solvents should not be stored
in glass bottles over one liter in size.
Large quantities should be stored in
metal safety cans. Quantities requiring
containers larger than one gallon should
be stored outside the laboratory.
3 Explosion proof refrigerators should be
used for the storage of highly volatile
and flammable solvents.
4 Cylinders of compressed or liquified
gases should not be stored in the
laboratory.
F Housekeeping
1 Housekeeping plays an important role
in reducing the frequency of laboratory
accidents. Rooms should be kept in a
neat orderly condition. Floors, shelves,
and tables should be kept free from
dirt and from all apparatus and chemi-
cals not in use.
2 A cluttered laboratory is a dangerous
place to work. Maintenance of a clean
and orderly work space is indicative of
interest, personal pride, and safety-
mindedness.
3 Passageways should be kept clear to all
building exits and stairways.
4 Metal containers should be provided for
the disposal of broken glassware and
should be properly labeled.
5 Separate approved waste disposal cans,
should be provided for the disposal of
waste chemicals.
6 Flammable liquids not miscible with
water and corr.osive materials, or
compounds which are likely to give off
toxic vapors should never be poured
into the sink.
G Fire Protection
1 Laboratory personnel should be
adequately trained regarding pertinent
fire hazards associated with their work.
2 Personnel should know rules of fire
prevention and methods of combating
fires.
3 Fire extinguishers (CO. type) should
be provided at convenient locations and
personnel should be instructed in their
use.'
4 Automatic sprinkler systems are
effective for the control of fires in
chemical laboratories.
12-2
-------
Laboratory Safety Practices
H Alarms
1 An approved fire alarm system should
be provided.
2 Wherever a hazard of accidental release
of toxic gases exists, a gas alarm
system to warn occupants to evacuate
the building should be provided.
3 Gas masks of oxygen or compressed air
type should be located near exits and
selected personnel trained to use them.
in HANDLING GLASSWARE
A Receiving, Inspection and Storage
1 Packages containing glassware should
be opened and inspected for cracked or
nicked pieces, pieces with flaws that
may become cracked in use. and badly
shaped pieces.
2 ' Glassware should be stored on well-
lighted stockroom shelves designed and
having a coping of sufficient height
around the edges to prevent the pieces
from falling off.
B Laboratory Practice
1 Select glassware that is designed for the
type of work planned.
2 To cut glass tubing or a rod. make a
straight clean cut with a cutter or file
at the point where the piece is to be
severed. Place a towel over the piece
to protect the hands and fingers, then
break away from the body.
3 Large size tubing is cut by means of a
heated nichrome wire looped around the
piece at the point of severance.
4 When it is necessary to insert a piece
of glass tubing or a rod through a
perforated rubber or cork stopper.
select the correct bore so that the
insertion can be made without excessive
strain.
Use electric mantels for heating
distillation apparatus, etc.
To remove glass splinters, use a
whisk broom and a dustpan. Very
small pieces can be picked up with a
large piece of wet cotton.
IV GASES AND FLAMMABLE SOLVENTS
A Gas Cylinders
1 Large cylinders must be securely
fastened so that they cannot be dis-
lodged or tipped in any direction.
2 Connections, gauges, regulators or
fittings used with other cylinders must
not be interchanged with oxygen
cylinder fittings because of the possi-
bility of fire or explosion from a
reaction between oxygen and residual
oil in the fitting.
3 Return empty cylinders promptly with
protective caps replaced.
B Flammable Solvents
1 Store in designated areas well
ventilated.
2 Flash point of a liquid is the temperature
at which it gives off vapor sufficient to
form an ignitible mixture with the air
near the surface of the liquid or within
the vessel used.
3 Ignition temperature of a substance is
the minimum temperature required to
initiate or cause self-sustained com-
bustion independently of the heating or
heated element.
4 Explosive or flammable limits. For
most flammable liquids, gases and
solids there is a minimum concentration
of vapor in air or oxygen below which
propagation of flame does not occur on
contact with a source of ignition.
There is also a maximum proportion of
vapor or gas in air above which
12-3
-------
Laboratory Safety Practices
propagation of flame does not occur.
These limit mixtures of vapor or gas
with air, which if ignited will just
propagate flame, are known as the
"lower and higher explosive or flammable
limits."
5 Explosive Range. The difference
between the lower and higher explosive
or flammable limits, expressed in
'terms of percentage of vapor or gas in
air by volume is known as the "explosive
range."
6 Vapor Density is the relative density
of the vapor as compared with air.
7 Underwriter's Laboratories Classification
is a standard classification for grading
the relative hazard of the various
flammable liquids. This classification
is based on the following scale:
Ether Class 100
Gasoline Class 90 - 100
Alcohol (ethyl) Class 60 - 70
Kerosene Class 30 - 40
Paraffin Oil Class 10- 20
8 Extinguishing agents
V CHEMICA L HA ZA RDS
A Acids and Alkalies
1 Some of the most hazardous chemicals
are the "strong" or "mineral" acids
such as hydrochloric, hydrofluoric,
sulfuric and nitric.
2 Organic acids are less hazardous
because of their comparatively low
ionization potentials. However, such
acids as phenol (carbolic acid),
hydrocyanic and oxalic are extremely
hazardous because of their toxic
properties.
3 Classification of acids
B Oxidizing Materials
1 Such oxidizing agents as chlorates,
peroxides, perchlorates and perchloric
acid, in contact with organic matter
can cause explosions and fire.
2 They are exothermic and decompose
rapidly, liberating oxygen which reacts
with organic compounds.
3 Typical hazardous oxidizing agents are:
Chlorine Dioxide
Sodium Chlorate
Potassium Chromate
Chromium Trioxide
Perchloric Acid
C Explosive Power
1 Many chemicals are explosive or form
compounds that are explosive and
should be treated accordingly.
2 A few of the more common examples
of this class of hazardous materials are:
Acetylides
Silver Fulminate
Peroxides
Peracetic Acid
Nitroglycerine
Picric Acid
Chlorine and Ethylene
Sodium Metal
Calcium Carbide
D Toxicity
1 Laboratory chemicals improperly
stored or handled can cause injury to
personnel by virtue of their toxicity.
2 Types of exposure. There are four
types of exposure to chemicals:
a Contact with the skin and eyes
b Inhalation
c Swallowing
d Injection
12-4
-------
Laboratory Safety Practices
VI PRECAUTIONARY MEASURES
A Clothing and Personal Protective Equipment
1 'Chemical laboratories should have
special protective clothing and equipment
readily available for emergency use and
for secondary protection of personnel
working with hazardous materials.
2 Equipment should be provided for adequate:
a Eye protection
b Body protection
c Respiratory protection
d Foot protection
e Hand protection
B Bodily Injury
1 Burns, eye injuries, and poisoning are
the injuries with which laboratory
people must be most concerned.
2 First emphasis in the laboratory
should be on preventing accidents.
This means observing all recognized
safe practices using necessary personal
protective equipment and exercising
proper control over poisonous sub-
stances at the source of exposure.
3 So that a physician can be summoned
promptly, every laboratory should have
posted the names, telephone numbers,
and addresses of doctors to be called
in an emergency requiring medical care.
REFERENCES
Guide for Safety in the Chemical Laboratory,
the General Safety Committee of the
Manufacturing Chemists Association, Inc..
VanNostrand. New York (1954).
This outline was prepared by Paul F. Hallbach,
Chemist, National Training and Operational
Technology Center. MOTD. OWPO. USEPA,
Cincinnati, Ohio 45268
Descriptors: Safety, Laboratory. Practices
Safety. Laboratory Design Chemical Storage,
Gas Cylinders, Flammable Solvents
12-5
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|