ENVIRONMENTAL PROTECTION AGENCY
STANDARD METHODS
USED BY
REGION IX
MICROBIOLOGY LABORATORY
Laboratory Support Branch
620 Central Avenue, Bldg. 1
Alameda, CA 94501 November, 1973
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11775
Region IX
Activities of the Microbiology Section
The Microbiology Laboratory has established tests which
it can perform. These include: indicator organisms assays
(total and fecal coliform, fecal streptococci) by multiple
tube dilution and membrane filter techniques; plate counts
at 20° and 35°C; pathogen isolation (Salmonella), serology,
and fluorescent-antibody scanning. Methodology is attached.
Upon request the Section can adapt existing techniques
and develop special ones for recovering such organisms as:
anaerobic bacteria; photosynthetic bacteria; Pseudomonas sp.;
sulfur oxidizers; Klebsiella; and other specific groups.
Staff from the Section will also offer technical advice
and consultation for review of grants, permits, standards
and research proposals, etc. Lectures and training courses
in laboratory and field techniques and sample collection have
been given by the Section in the past and can be developed or
modified upon request.
Kathleen G. Shimmin
Chief, Microbiology Section
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TABLE OF CONTENTS
PAGE
Sample Collection and Chain-of-Custody 1
Determination of Coliform Organisms by
Membrane Filter Procedure
Total Coliforms 4
Fecal Coliforms 7
Determination of Fecal Streptococcus Organisms
by Membrane Filter Procedure using:
M-Enterococcus Agar 9
KF Streptococcus Agar 11
Most Probable Number (MPN) Test for the
Detection and Enumeration of Coliform and
Fecal Coliform Organisms 12
Most Probable Number (MPN) Test for the
Detection and Enumeration of Fecal
Streptococcus Organisms 16
Estimation of Bacterial Density 18
Gram's Stain Technique 23
IMViC Procedures 24
Procedure for Salmonella Isolation 26
Fluorescent Antibody (FA) Salmonella Screening 31
Serological Grouping of Salmonella 32
Diagram of Slide Agglutination 34
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TABLES
PAGE
*407(1):
*407(2) :
I.
II.
MPN Index and 95% Confidence Limits
for Various Combinations of Positive
and Negative Results when Five 10-ml
portions are used
MPN Index and 95% Confidence Limits
for Various Combinations of Positive
and Negative Results when Five 10-ml
Portions, Five 1-ml Portions, and Five
0.1-ml Portions are used.
Incubation Time and Colonial
Appearance for Various Organisms in
Selected Media
Salmonelleae - Parameters and
Biochemical Reactions
20
21
28
29
*Refers to Section Numbers in Standard Methods, 13th Edition,
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STANDARD PROCEDURES USED BY REGION IX
LABORATORY FOR BACTERIOLOGICAL EXAMINATION
OF TOTAL AND FECAL COLIFORMS
1. Sample Collection and Chain-of-Custody
a. Procedure for collection of samples shall be
done in accordance with methods as outlined in
Standard Methods for the Examination of Water
and Wastewater; 13th Edition.
b. Samples should be properly iced immediately after
collection (ice bucket or chest).
c. Chain-of-custody should be as follows:
1. Two people are required; one as sampler,
one as witness.
2. Label on sample container should read: (See
Attachment - page 3)
Sample Number (Use a self-sticking
Sample Description label which will not
Time and Date Collected deteriorate in ice chest)
Collected by
Witnessed by
3. If samples change hands during transport back
to laboratory, label or tag should be attached
to ice bucket or chest, signed by person and
witness to whom custody was given. (See Attachment)
4. Upon delivery to laboratory the technican should
make out a receipt stating time and condition
samples were received.
5. Before processing, log in time received, time
collected, time processed, and processor. This
can be done in same log book as results are
recorded.
2. Processing
a. Do at least duplicate replicate plates for each
dilution.
b. For unknown water, do at least four dilutions with at least
two replicates for each dilution. (A minimum of eight plates
per medium per sample.)
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3 . Time lapse between sample collection and processing is
as follows:
a. Seawater 4 hours for Total Coliforms
2 hours for Fecal Coliforms
b. Freshwater 6 hours for Total Coliforms
3-4 hours for Fecal Coliforms
c. Shellfish 6 hours for Total and Fecal
12 hours maximum
Examination of shellfish for total and fecal coliforms
should be done according to the procedures recommended
in most recent edition of American Public Health Association,
Recommended Procedures for the Examination of Seawater and
Shellfish.
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ATTACHMENT
SAMPLE BOTTLE LABEL
ENVIRONMENTAL PROTECTION AGENCY
EAH.'LE UO.
PRINT NAME £f!D TITLE OnCHJCtor,
GRAIN OF CUSTODY LABEL
'i:
1-1 J
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I hereby certify that I received this sample and disposed of it as
not'.'d fic'ow
RECEIVED FriOM
DISPOSITION OF SAMPLE
DATE RECEIVED TIME RECEIVED
SIGNATURE
I hovby certify that I obtained this sample ard dispatched it as
shov.-n below.
PATE OBTAINED " IME OBTAINED 1 SOURCE
1
DATE DISPATCHED TIME DISPATCHED MElHOD OF SHIPMENT
SENT TO
SIGNATURE
CHAIN OF CUSTODY TAG
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Standard Method used
by
Region IX, Laboratory
THE DETERMINATION OF COLIFORM ORGANISMS
BY THE MEMBRANE FILTER PROCEDURE
M-ENDO - Total Coliforms
I. Preparation of Medium
1. Boil 500 ml. distilled water.
2. Add 1.5% Bacto-Agar (7.5 g) and stir to dissolve.
3. Add 24 g. Difco M-Endo medium and 10 ml of 95% ethanol.
Bring to boil to dissolve (keep stirring).
DO NOT CONTINUE TO BOIL the medium once boiling point
reached.
4. Cool medium slightly and distribute about 5 ml per
petri dish (sterile, disposable, 50 x 12 mm).
5. Allow the plates to harden. Pack the plates in an
inverted position in a basket and cover with brown
paper. Place the basket in the refrigerator.
In the dark and under cool conditions the prepared
medium can be stored for short periods of time. The
prepared plates should be used within a 24 hour period
Under no circumstances will the plates be stored for
periods of 72 hours or longer. Results from such
plates are questionable.
II. Testing Procedures
1. All filtrations should be carried out according to the
protocol outlined in Section 408, page 678 of Standard
Methods, 13th Edition, 1971.
2. Sample volumes to be filtered should be chosen so that
at least one membrane filter contains between 20-80
coliform colonies, and not to exceed 200 colonies of
all types on the filter.
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In the absence of previous bacterial data the
following are recommended volumes:
a. Treated water supplies minimum of 50 ml,
100 ml recommended.
b. Untreated water supplies — 5 - 50 ml.
c. Unpolluted surface water — 1, 4, 15 and 60 ml
(covers range 33-8000) .
d. Polluted surface water 0.02, 0.08, 0.15 and
0.5 ml (covers range
of 4000 - 40,000).
e. Sewage 0.0003, 0.001, 0.003
and 0.01 ml (covers
range of 200,000 -
27,000,000 cells/lOOml).
3. The plates containing the membranes are placed in
a 35°C incubator in an inverted position.
4. After a period of 24 - 2 hours the plates are removed
from the incubator. Coliform colonies are dark red
and have a green metallic surface sheen. Non-coliform
colonies range from colorless to pink, however, the
metallic sheen is absent. Such type colonies should
not be included in the coliform count.
5. The characteristic metallic sheen colonies are counted
with the aid of a wide-field binocular microscope
using 10 or 15 x magnification. For illumination,
use a light source directly over the membrane filter
so that an image of the light source is reflected
off the colony surface into the microscope lens system.
(Suitable for this purpose is a Stereozoom Microscope
[A/0 or B&L] with a fluorescent illuminator).
6. Select the membranes that have between 20 to 80
coliform colonies and compute the density per 100 ml.
The actual colony counts and the calculated density
per 100 ml are entered on the data sheet.
No of coliform colonies counted x 100 = No f colonies
Sample volume filtered xn ml r 100 ml
7. The plates are then placed in metal containers and
sterilized in the autoclave. At no time will any
culture medium containing bacteria be disposed of
first without adequate sterilization.
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8- Calculations
a. For routine work, at least 3 dilutions of the
sample are made. However, when testing water
where no previous information is available then
it may be necessary to use as many as 4 or 5
dilutions.
b. Select the membrane that has between 20-80
coliform colonies and compute the density
per 100 ml.
c. If several sample volumes have coliform colonies
in the range 20-80, then average the counts per
100 ml.
d. If all membranes have counts outside the range
20-80, the following procedure should be used:
1. Low counts (below 20 colonies). Calculate
the density if 20 colonies were to have
been present. Report on this calculation
on the data sheet preceded by "<" ("less
than").
2t High counts (above 80 colonies). Calculate
the density if 80 colonies were to have
been present. Report this calculation on
the data sheet preceded by ">" ("greater
than").
It must be realized that data reported with
"less than" and "greater than" have limited use
when strict interpretation of data is required.
It does provide some idea as to the relative
coliform density of the sample. Most important
of all, it shows the need for repeat sampling and
adjustment of sample filtration volume so that
a membrane with the desired range 20-80 may
be obtained.
In order to obtain at least one membrane having
an acceptable number of colonies, the range of
filtration volumes should vary by a factor of
4 or less. Different factors apply to fecal
coliform and fecal streptococcus.
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REGION IX
r
THE DETERMINATION OF FECAL COLIFORM ORGANISMS
BY THE MEMBRANE FILTER TECHNIQUE
I. Medium Preparation
1. Rehydrate Bacto-M-FC Broth Base by adding 3.7g in
100 ml distilled water.
2. Add one ml of a rosolic acid solution prepared by
dissolving one gram rosolic acid in 100 ml 0.2N NaOh.
3. Add 1.5% Bacto Agar.
4. Dissolve ingredients in a boiling water bath or in
"Instatherm" apparatus.
5. Bring to a boil and pour approximately 5 ml into
sterile, disposable 50 x 12mm petri dishes. Final
reaction of the medium is pH 7.4.
6. Allow the medium to harden.
7. Pack the plates in baskets in the inverted position
and place in the refrigerator.
8. Prepared medium "shelf life" is 5-7 days if stored in
the refrigerator away from light.
9. The rosolic acid is stable indefinitely in the dry
state. In aqueous solution it is stable for two
weeks under refrigeration. After this period
degradation is evidenced by the change of color
(Red to Brown).
II. Testing Procedures
1. All filtrations should be carried out according to
the protocol outlined in Section 408, page 678 of
Standard Methods, 13th Edition, 1971.
2. Sample volumes to be filtered should be chosen so
that at least one membrane filter contains between
20-60 fecal coliform colonies.
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3. The plates containing the membranes are placed in
water-proof plastic bags (Whirl-Pak Bags) and
submerged in a 44.5 ± 0.2°C water bath for 24 hours.
(Plates should not be held no longer than 20 minutes
at ambient temperature after filtration. Immediate
introduction of plates into the 44.5°C water bath
is recommended).
4. After incubation, colony counts are made using a
wide-field binocular microscope - lOx magnification.
Fecal coliform colonies are deep blue in color and
may vary from one to three mm in diameter. Non-
fecal coliform colonies will appear as pink or
colorless type colonies and should not be included
in the fecal coliform count.
5. The colony counts are entered on the data sheet and
reported per 100 ml.
No. of fecal coliform colonies counted x ]_QQ = NO . of colonies
Sample volume filtered in ml' por 100 ml.
6. Calculations and selection of sample filtration
volumes follow the same, theory as that discussed in
the Method, Total Coliform Determination
by Membrane Filter Procedure . Exceptions are that
the desired range of fecal coliform colonies on the
membrane is 20-60, and that fecal coliform counts
should be based on filtration volumes varying by a
factor of 3 or less.
7. The plates are than placed in metal containers and
sterilized in autoclave. At no time will any culture
medium containing bacteria be disposed of first without
adequate sterilization.
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THE DETERMINATION OF FECAL STREPTOCOCCI
BY THE MEMBRANE FILTER PROCEDURE
I. Preparation of Medium
1. Weigh out 21 grams of M-Enterococcus Agar and place
in one liter flask.
2. Add 500 ml cold distilled water and bring solution
to a boil using "Instatherm" or boiling water bath
and remove as soon as agar is in solution.
(Do not overheat.)
3, Final pH should be: 7.2
4. Pour approximately 5 ml of the medium into sterile,
disposable, 50 x 12 mm petri dishes.
5. Allow the medium to harden. Packthe plates in an
inverted position in a basket and cover with brown
paper. The prepared plates can be stored in the
refrigerator up to four weeks.
6. The filtration of the sample should be carried out
according to the methods outlined in the section on
Filtering Techniques.
7. Sample volume to be filtered should be chosen so that
at least one membrane filter contains between 20-100
fecal streptococcus colonies.
8. The plates containing the membranes are placed in a
35°C incubator in an inverted position.
9. After a period of 48 hours the plates are removed
from the incubator. With a wide-field binocular
microscope, using 10 or 15x magnification, count
all pink, red and purplish-red colonies. Colonies
other than these are not fecal streptococci and should
not be included in the count.
10. The colony counts are entered on the data sheet and
reported per 100 ml.
No. of fecal streptococci counted „ inn - M~ f •<= n j- ^ 4
— . jT —, x 1UU = No . of fecal streptococci
Sample volume filtered in ml inn i
r per 100 ml
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10
11. Calculation and selection of sample filtration
volumes follow the same principle as that
discussed in Method of .Coliform Determination By-
Membrane Filter Procedure, except that the desired
range of colonies on the membrane is between 20-100
and the counts should be based on filtration volumes
varying by a factor of 5 or less.
12. The plates are than placed in metal containers and
sterilized in the autoclave and discarded.
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11
Standard Methods used by Region IX for the Determination of
Fecal Streptococcus by Membrane Filter using Bacto-KF
Streptococcus Agar
I. Preparation of Medium
a. Weigh out 76.4 grams KF Streptococcus agar and
place in flask.
b. Add 1000 ml cold distilled water and heat to boiling
to dissolve completely.
c. Dispense in 100 ml amounts or multiples thereof
into flasks and sterilize for 10 minutes at 15 Ib
pressure (121°C) .
d. Cool to 60°C and add one ml Bacto TTC Solution 1%.
(Triphenyltetrazolium Chloride 1%) per 100 ml sterile
medium.
e. Mix to obtain uniform distibution of the TTC.
f. Final pH 7.2.
g. Pour approximately 5 ml of medium into sterile
disposable, 50x12 mm petri dishes.
h. Incubate inoculated plates for 48 hours at 35°c.
i. Using a dissecting microscope with a magnification
of 15 diameters count all colonies showing red or
pink center as streptococcus.
j. Colony counts are entered on data sheet and reported
per 100 ml.
k. Calculation: f
Number of fecal streptococcus counted n nri f°a , /n nn i
Sample volume filtered in ml X 10° = streptococcus/lQQm}
1. Desired range of colonies on membrane is 20-100 and
counts should be based on filtration volumes varying
by a factor of 5 or less.
m. Plates are then placed in metal containers and
sterilized in an autoclave. (121°C, 15 Ib pressure
for 1/2 hour) .
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12
THE MOST PROBABLE NUMBER (MPN) TEST
FOR THE DETECTION AND ENUMERATION OF
COLIFORM AND FECAL COLIFORM ORGANISMS
I. Presumptive Test
A. Preparation of medium
1. For 1 ml of sample inoculation: weigh out
35.6g lauryl tryptose broth and add to one
liter of distilled water. (For 10 ml of
sample inoculation 53.4g per one liter of
distilled water).
2. Dissolve ingredients and dispense ten ml of
mixture 1 above into test tubes. Dispense
20 ml of 53.4g per one liter mixture into
large .test tubes.
3. Insert one Durham gas tube, inverted position,
into each tube containing the broth. Place auto-
clavable plastic Kaputs on tubes. The double-
strength tubes may be coded by closing them with
Kaputs of a different color.
4. Sterilize in the autoclave for 12 minutes at
12 Ibs. pressure.
B. Procedure
1. The five tube, multiple fermentation technique
will be used.
2. Inoculate a series of five tubes in each dilution,
In all such analyses, at least 3 dilutions must
be used. (Region IX routinely uses 5 dilutions.)
3. The portions of the water sample used for
inoculating the fermentation tubes will vary
with the type water being tested. In general,
decimal multiples and sub-multiples of one ml
will be used.
4. Incubate the fermentation tubes at 35.0 ± 0.5°C.
Examine each tube at the end of 24 ± 2 hours
and, if no gas had yet been produced, examine
again at the end of48 hours ± three hours.
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13
Record the presence or absence of gas
formation at each examination of the tubes.
The smallest type bubble in the gas tube
should be recorded as a positive tube, even
though this may appear as trapped air in the
tube and not really gas production from the
fermentation process.
II. Confirmed Test
A. Preparation of medium
1. Weigh out 40.Og of Bacto-Brilliant Green Bile
2% and add to one liter of distilled water.
2. Dissolve ingredients and dispense ten ml into
each test tube.
3. In the inverted position, insert a Durham gas
vial into each test tube. Use Kaput closures
to cover the tubes.
4. Sterilize in the autoclave for 12 minutes at
12 Ibs. pressure. Final reaction of the medium
is pH 7.2.
B. Procedure
*1. Using a 24 gauge wire loop, with a loop of 3 mm
in diameter, transfer one loopful of the positive,
lauryl tryptose broth into a tube of brilliant
green bile broth.
(If active fermentation appears in the primary
fermentation tube before the expiration of the
24 hour period of incubation, it is preferable
to transfer to the confirmatory brilliant green
bile broth without waiting for the full 24"hour
period to elapse.)
2. Incubate the inoculated brilliant green lactose
bile broth tube for 48 ± 3 hours at 35° ± 0.5°C.
3. The formation of gas in any amount in the Durham
tube, constitutes a positive Confirmed Test.
Record all positives and negatives on the data
sheet.
*As an alternative to transfering with a wire loop, sterilized
hardwood applicator sticks (sterilized in dry heat, 1-1/2
hours, 170°C, stored in gloss tubes or syringe-sterilization
bags) may be used. Each stick is used once and then dis-
carded into a disinfectant-filled discard container.
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14
III. Fecal Coliform (MPN)
A. Preparation of medium
1. Weigh out 37.0g of Bacto E.G. medium and
add to one liter of cold distilled water.
2. Dissolve the ingredients and dispense into
test tubes in ten ml amounts.
3. Insert a Durham gas vial into each tube.
(In the inverted position.) Use Kaput closures
for these tubes.
4. Sterilize in the autoclave for 12 minutes at
12 Ibs. pressure. Final reaction of the medium
is pH 6.9.
B. Procedure
*1. Using a 24 gauge wire loop, with a loop of 3 mm
in diameter, transfer one loopful of the positive,
lauryl tryptose broth into a tube of E.G. medium.
2. The inoculated tube must be put into a 44.5 -,
0.2°C water bath not later than 20 minutes after
initial inoculation.
3. The formation of any amount of gas in the vial
at the end of 24 hours constitutes a positive
test. At the end of 24 hours the positive
and negative results are entered on the data
sheet. Readings after 24 hours are invalid.
IV. Computing of MPN
1. The number of positive findings of coliform group
organisms (Presumptive, Confirmed and Fecal) resulting
from multiple-portion, decimal dilution inoculations
should be computed and recorded in terms of the
"most probable number" (MPN).
2. See MPN and 95% Confidence Limits for Various
Combination of Positive Results in Standard Methods,
13th Edition, 1971.
*The single-use, sterile, hardwood applicators may be used
as an alternative to transferring with a wire loop.
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15
V. Schematic Diagram Illustrating Steps in MPN Procedures.
Sample
Lauryl Tryptose Broth
Incubate in air incubator at 35°C ± 0.5°C for 24 hours
No gas produced
Return to incubator
Production of gas
Positive Presumptive Test
for Coliform Group
*-
No gas:
coliform
group absent
J,
+gas, Positive
Presumptive Test
for Coliform Group
i ..
4-
V
1
Brilliant Green Bile Broth
Incubate in 35 ± 0.5°C in incubator
for 24 hours
E.G.
Incubate in 44.5 ± 0.2°C
water bath for 24 hours.
No gas produced
return to incubator
for another 24 hrs.
+Gas
Positive Confirmed
Test for Coliform
Group
Mo gas =
Fecal coliform
group absent
+ gas v
Fecal coli-
form group
present
No gas produced
Coliform group
absent
+Gas:
Positive Confirmed
Test for Coliform
Group
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16
THE MOST PROBABLE NUMBER (MPN) TEST FOR THE DETECTION
AND ENUMERATION OF FECAL STREPTOCOCCI ORGANISMS
I. Presumptive Test
A. Preparation of medium
1. For 1 ml of sample inoculation: weigh out 34.7 grams
Azide Dextrose Broth and add to one liter (1000 ml)
of distilled water (for 10 ml of sample inoculation
prepare double-strength medium).
2. Dissolve ingredients and dispense 10 ml of mixture
into test tubes for 1 ml sample inoculation. Dis-
pense 10 ml of double strength medium into large
(18 x 150 mm) test tube for a 10 ml sample inoculum.
3. Place plastic or metal caps on tubes.
4. Sterilize in the autoclave for 15 minutes at 15
pounds pressure (121°C). Final reaction of medium
is pH 7.2 25°C.
B. Procedure
1. The five-tube, multiple fermentation technique will
be used.
2. Inoculate a series of five tubes in each dilution.
In all such analyses, at least 3 dilutions must be
used (EPA, Region IX routinely uses 5 dilutions).
3. The dilutions of the water sample used for inoculat-
ing the fermentation tubes will vary with the type
of water being analyzed. Decimal multiples and
decimal dilutions of one ml are used.
4. Incubate the inoculated tubes at 35.0 ± 0.5°C.
Examine each tube for the presence of turbidity at
end of 24 ± two hours. If no definite turbidity is
present, reincubate and read again at end of 48 ±
three hours.
II. Confirmed Test
A. Preparation of Medium
1. Weigh out 35.8 grams EVA Broth (Ethyl Violet Azide)
and add to 1 liter (1000 ml) distilled water.
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17
2. Dissolve and dispense 10 ml portions into test tubes,
3. Sterilize in autoclave for 15 minutes at 15 pounds
pressure (121°C). Final reaction of medium pH 7.0
at 25°C.
B. Procedure
1. Transfer 3 loopfuls of growth or use a wooden appli-
cator to transfer growth from each azide dextrose
broth tube to a tube containing 10 ml ethyl violet
azide broth.
2. Do not discard positive tubes (presumptive). Hold
in the incubator.
3. Incubate the inoculated tubes for 24 hours at 35 i
0.5°C. The presence of fecal streptococci is indi-
cated by formation of a purple button at the bottom
of the tube or by a very dense turbidity.
4. Record all positive tubes. Discard those tubes.
5. If no growth (purple button or heavy tubidity)
appears in ethyl violet azide in 24 hours, reinocu-
late the tubes with an additional 3 loopfuls (or use
wooden applicator) from the original positive azide
broth cultures and reincubate for another 24 hours.
6. Record results.
C. Computing and Recording MPN
Same as with computation of total and fecal coliform
organisms.
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18
STANDARD METHODS, APHA, 13th EDITION
407 D. ESTIMATION OF BACTERIAL DENSITY
1. Precision of Fermentation Tube Test
It is desirable to bear in mind that unless a large
number of portions of sample are examined, the precision
of the fermentation tube test is rather low. For example,
even when the sample contains 1 coliform organism per
milliliter, about 37% of 1-ml tubes may be expected to
yield negative results because of irregular distribution
of the bacteria in the sample. When five tubes, each
with 1 ml of sample are employed under these conditions,
a completely negative result may be expected less than
1% of the time.
Even when five fermentation tubes are employed, the precision
of the results obtained is not of a high order. Consequently,
great caution must be exercised when interpreting, in
terms of sanitary significance, the coliform results
obtained from the use of a few tubes with each dilution
of sample, especially when the number of samples from
a given sampling point is limited.
2. Computing and Recording of MPN
The number of positive findings of coliform group organisms
(either presumptive, confirmed or completed) resulting
from multiple-portion decimal-dilution plantings should
be computed as the combination of positives and recorded
in terms of the Most Probable Number (MPN). The MPN,
for a variety of planting series and results, is given
in Tables 407(1) through (6).* Included in these tables
are the 95% confidence limits for each MPN value
determined.
The quantities indicated at the heads of the columns
relate more specifically to finished waters. The values
may be used in computing the MPN in larger or smaller
portion plantings in the following manner: If, instead
of portions of 10, 1.0 and 0.1 ml, a combination of
portions of 100, 10 and 1 ml is used, the MPN is recorded
as 0.1 times the value given in the applicable table.
*Since Region IX routinely uses the five tube MPN procedure,
only Table 407(1), and 407(2) from Standard Methods 13th Ed.
are included in this manual.
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19
If, on the other hand, a combination of corresponding
portions at 1.0, 0.1 and 0.01 ml is planted, record
10 times the value shown in the table; if a combination
of portions of 0.1, 0.01 and 0.001 ml is planted,
record 100 times the value shown in the table; and
so on for other combinations.
When more than three dilutions are employed in a
decimal series of dilutions, the results from only
three of these are used in computing the MPN. To
select the three dilutions to be employed in determining
the MPN index, taking the system of five tubes of each
dilution as an example, the highest dilution which
gives positive results in all five portions tested
(no lower dilution giving any negative results) and
the next succeeding higher dilutions should be chosen.
The results at these three volumes should then be used
in computing the MPN index. In the examples given
below, the significant dilution results are shown in
boldface. The number in the numerator represents
positive tubes; that in the denominator, the total
tubes planted; the combination of positives simply
represents the total number of positive tubes per
dilution:
Example 1 ml
0.1 ml 0.01 ml 0.001 ml
Combination
of positives
(a)
(b)
(c)
5/5
5/5
0/5
5/5
4/5
1/5
2/5
2/5
0/5
0/5
0/5
0/5
5-2-0
5-4-2
0-1-0
In c, the first three dilutions should be taken, so as to
throw the positive result in the middle dilution.
When a case such as shown below in line d arises, where a
positive occurs in a dilution higher than the three chosen
according to the rule, it should be incorporated in the
result for the highest chosen dilution, as in e:
Example 1 ml
O.lml
0.01ml
~n
.001 ml
Combination
of pOSitiVes
(d)
(e)
5/5
5/5
3/5
3/5
1/5
2/5
1/5 -]
0/5 j
5-3-2
When it is desired to summarize with a single MPN value
the results from a series of samples, the geometric mean, the
arithmetic mean, or the median may be used.
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20
TABLE 407(1): MPN INDEX AND 95% CONFIDENCE LIMITS 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
per 100 ml
<2.2
2.2
5.1
9.2
16.
>16.
95% Confidence
Limits
Lower
0
0.1
0.5
1.6
3.3
8.0
Upper
6.0
12.6
19.2
29.4
52.9
Infinite
Formula* for MFN Calculation:
MPN/100 ml = Numbfir Positive Tubes x 100
?
Knuscrip I o in x (>nl s triple
negative tubes) in all tubes)
*From Thomas, H.A. Jr. 19^2. Bacterial densities from
fermentation tube tests. JAW/A 3-'4:572.
-------
21
TABLE 407 (2): MPN INDEX AND 95% CONFIDENCE LIMITS FOR VARIOUS COMBINATIONS
OF POSITIVE AND NEGATIVE RESULTS WHEN FIVE 10-ML PORTIONS, FIVE 1-ML
PORTIONS AND FIVE 0.1-ML PORTION ARE USED
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
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
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
MPN
Index
per
100 ml
< 2
2
2
4
2
4
4
6
6
5
7
7
9
9
12
8
11
11
14
95% Confidence
Limits
Lower
< 0.5
< 0.5
<0.5
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
1
1
2
2
3
1
2
2
4
14 | 4
17
17
13
17
17
21
26
22
5
5
3
5
5
7
9
7
Upper
7
7
11
7
11
11
15
15
13
17
17
21
21
28
19
25
25
34
34
46
46
31
46
46
63
78
67
Continued on page 22
-------
22
(Cont'd. from page 21)
ABLE 407(2): MPN INDEX AND 95% CONFIDENCE LIMITS FOR VARIOUS COMBINATIONS
F POSITIVE AND NEGATIVE RESULTS WHEN FIVE 10-ML PORTIONS, FIVE 1-ML
PORTIONS AND FIVE 0.1-ML PORTION ARE USED
No. of Tubes Giving
Positive Reaction out of
: of 10
il Each
4
4
4
4
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
*-f
5
5
5 of 1
ml Each
2
3
3
4
0
0
0
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
1
0
1
0
0
1
2
0
1
2
0
•I
2
0
1
2
3
0
1
2
3
4
0
1
2
3
4
5
MPN
Index
per
100 ml
26
27
33
34
23
31
43
33
46
63
49
70
94
79
110
140
180
130
170
220
230
350
240
350
540
920
1600
>2400
95% Confidence
Limits
Lower
9
9
11
12
7
11
15
11
16
21
17
23
28
25
31
37
44
35
43
57
90
120
68
120
180
300
640
Upper
78
80
93
93
70
89
110
93
120
150
130
170
220
190
250
340
500
300
490
700
850
1,000
750
1,000
1,400
3,200
5,800
-------
23
GRAMS STAIN
1. Using a clean slide, make a thin smear of the culture
on the slide.
2. Air dry
3. Heat fix by passing slide briefly through flame.
4. Add gentian violet dye - let stand one minute. Rinse
with tap water. Rinse with Gram's iodine.
5. Add Gram's iodine - let stand one minute. Drain slide,
6. Add decolorizer - let stand 10-15 seconds.
Rinse with water. Rinse with Safranin.
7. Add Safranin dye to counterstain - one minute. Rinse
with tap water. Blot dry with paper towel.
View slide under microscope.
Gram-positive organisms appear blue-violet; gram-
negative bacteria retain only the counterstain,
appearing red when Safranin is counterstain.
-------
24
IMViC PROCEDURES
(Standard Methods, 13th Ed.)
Be sure to use a pure culture for all IMViC inoculations.
Indole production from tryptophane broth
1. Prepare Medium:
Add 10.0 g. tryptone (or trypticase) to 1 liter distilled
water. Distribute in 5 ml portions into screw-capped
test tubes. Autoclave at 120°C for 15 minutes.
2. Prepare reagent:
Dissolve 5 g. paradimethylaminobenzaldehyde in 75 ml
isoamyl (or normal amyl) alcohol, ACS grade. Add 25 ml
concentrated hydrochloric acid (perform this operation
under a hood if possible or else in a room with good
ventilation). The reagent should turn yellow. Final
pH should be less than 6.0.
3. Procedure:
Inoculate 5 ml medium. Incubate at 35 ± 0.5°C for
24 ± 2 hours. Add 0.2-0.3 ml reagent and shake tube.
Let stand for 10 minutes.
4. Results:
Dark red color in surface layer = (+) for indole.
Yellow (Color of reagent) color in surface layer =
(-) for indole. Orange color in surface layer =
(±) reaction.
Methyl Red Test
1. Prepare Medium:
Add 17 g. MR-VP Broth Base (or equivalent) to 1 liter
distilled water. Heat slightly to dissolve. Distribute
10-ml portions into screw-capped test tubes. Autoclave
at 121°C for 12-15 minutes.
2. Prepare reagent:
Dissolve 0.1 g. methyl red dye in 300 ml 95% ethyl alcohol
Dilute to 500 ml total volume with distilled water.
3. Procedure:
Inoculate 10-ml medium. Incubate at 35°C for 5 days.
-------
25
After incubation, pipette 5 ml culture into a clean test tube.
Add 5 drops (about 0.25 ml) methyl red reagent.
4. Results:
Distinct red color = (+) methyl red. Distinct yellow
color = (-) methyl red. Mixed shade = questionable results.
Voges-Proskauer Test
1. Prepare medium:
The same tube of MR-VP broth inoculated for the methyl
red tests may be used for the Voges-Proskauer test as well.
2. Prepare reagents:
Naphthol solution: Dissolve 5 g. purified naphthol
(melting point 92.5 or higher) in 100 ml absolute ethyl alcohol.
Solution should be prepared fresh each day.
Potassium Hydroxide solution: Dissolved 40 g. KOH in
100 ml distilled water.
3. Procedure:
Inoculate 5 ml medium. Incubate at 35 + 0.5°C for 48
hours. Pipette 1 ml culture into a clean test tube. Add to '
this 0.6 ml naphthol solution and 0.2 ml KOH solution.
4. Results:
Pink to crimson color develops in 2-4 hours = (+) for V-P.
Sodium Citrate Test
1. Prepare mediums
Add 24.2 g. Simmons Citrate Agar to 1 liter distilled
water. Mix thoroughly. Heat with frequent agitation until
medium" boils for one minute. Distribute into screw-capped test
tubes. Autoclave at 121°C for 15 minutes. Cool tubes in
slanted position.
2. Procedure:
Inoculate the medium with a straight needle, using both
a stab and a streak. Incubate 48 hours at 35 + 0.5°C.
3. Results:
Growth on the medium with a blue color (usually) = (+)
for citrate utilization. No growth = (-) test.
-------
26
PROCEDURE FOR SALMONELLA ISOLATION
(for shellfish or sediment modifications see note following
Serological Grouping)
Water Samples, Filtration
1. Set up filter system. Millipore assembly may be used.
Substitute filter pad for actual filter grid. On top of pad
(with funnel in place) add one inch of diatomaceous earth
(Celite or equivalent). Add sterile buffered water to saturate
Celite column.
2. Filter one liter sample.
3. Add filter pad plus diatomaceous earth to enrichment broth.
Repeat filtration procedure for each flask of enrichment broth
(a total of six filtrations for two broths and three tempera-
tures) .
Enrichment
1. Two enrichment broths (tetrathionate and selenite) should
be used at each of three different temperatures (37°, 41.5°,
43°C) . In case of limited laboratory capability the 43°C incuba-
tion may be omitted.
2. Incubate 18-24 hours at appropriate temperature.
Isolation
1. From the enrichment flask make streak plates and plates for
impression smears onto Brilliant Green and XLD agars (reincubate
enrichment broth). Incubate plates at same temperature as for
flasks.
2. Time of incubation for streak plates is indicated in Table 1.
Impression smears should be made on slides after 2-4 hours
incubation.
3. Smears should be stained and examined as indicated in
fluorescent-antibody staining directions. If no fluorescing cells
(3+, 4+) are found, sample may be said to contain no Salmonellae.
If fluorescing cells are present, continue isolation procedure
(steps 4-5); streaking from enrichment broths may be repeated
after 2 and 3 days incubation (or up to the point at which no
Salmonella-like colonies are recovered).
4. From each medium select isolated colonies of Salmonella-like
appearance (consult Table 1) and restreak until pure cultures
are obtained. Incubate at 37°C.
5. Inoculate isolates into differential media in order indicated.
Differential Tests
1. Using a single colony, inoculate 1/2 into a Triple Sugar Iron
slant and the other 1/2 into urea agar. Incubate at 37°C (times
are indicated in Table 1).
2. Those isolates with Salmonella-like reactions in both TSI and
urea should be inoculated into the following: Brilliant Green
-------
27
and XLD agar plates (streak); SIM (stab); Lysine Decarboxylase;
Nutrient Agar. Use material from the TSI slant for these
inoculations. Incubate at 37°C for times indicated in Table 1.
Serological Grouping
1. Isolates displaying a Salmonella-like pattern in differential
media may be grouped according to their somatic antigens. This
agglutination reaction may be lost when cultures are not freshly
isolated.
2. To prime the isolate for the agglutination test, inoculate
it into a Brain Heart Infusion slant (or broth). Incubate
24 hours at 37°C.
3. Repeat step 2 at least once or twice, ending up with a fresh
BHI slant.
4. Perform slide agglutination tests as indicated in directions
accompanying Difco Salmonella 0 Antisera. Perform the agglu-
tination test for each of the sets of antisera (Sets A-l, A,
B, C, D, E, F, G).
5. Final typing for 0 and H antigens may be done at a convenient
typing center (California State Public Health, Berkeley).
MODIFICATIONS FOR SEDIMENT OR SHELLFISH SAMPLES
Sediment
1. Omit filtration procedure.
2. Inoculate sediment directly into enrichment broth.
For oily sample, use 1 gm. For other material, use 10 gin.
3. Proceed as directed through outline.
Shellfish
1. Omit filtration procedure.
2. As directed in Recommended Procedures for the Examination of
Sea Water and Shellfish (1970) , weigh 100 gm (approximately) of~~
shellfish meat and liquor and add an equal weight of buffered
dilution water. Grind in a blender about 2 minutes. Pipet 20 ml
into each enrichment flask.
3. Proceed as directed through outline.
-------
28
Table 1: Incubation Time and Colonial Appearance for Various
Organisms in Selected Media
Medium
Brilliant Green
Incubation
(Hours)
48
Colonial Appearance
Salmonella Shigella Proteus Coliforms
pinkish
with red
background
green
green
XLD
Lysine
24
Decarboxylase
Indole
Motility
Urease
TSI-slant
-butt
-H2S
24
24
24
24
18-4
red with red yellow yellow
black ctr.
+(purple) -(yellow) - +,-
Al
AG
Al
A
A
AG
A
AG
A = acid production (indicator turns yellow)
Al = alkaline reaction (indicator turns red)
G = gas formation (bubbles appear in agar)
Rapid Procedure - In addition to and supplement of Table 1:
Omit Indole, Motility, Urease, TSI, lysine decarboxylase
Inoculate Improved Enterotube. Instructions in use of
Enterotube accompany package.
The Enterotube is prepared by Roche Diagnostics, Division of
Hoffmann - La Roche, Inc., Nutley, New Jersey, 07110.
Table II shows parameters and biochemical reactions for
Salmonelleae (Salmonella, Arizona, Citrobacter).
-------
29.
TABLE II SALMONELLEAE
Parameters and Biochemical Reactions
TEST OR SUBSTRATE
SALMONELLEAE
Salmonella
Arizona
Citrobacter
Indol
Methyl Red
Voges - Proskauer
Simmons' Citrate
Hydrogen Sulfide (TSI)
Urease
KCN
Motility
Gelatin (22°C)
Lysine Decarboxylase
Arginine Dihydrolase
Ornithine Decarboxylase
Phenylalanine Deaminase
Malonate
Gas From Glucose
Lactose
Sucrose
Mannitol
Dulcitol
Salicin
( + ) or +
+ or (+)
+ or -
w
d
d
d
d
d
d
(Continued next page)
-------
(Continued)
30. -
TABLE II SALMONELLEAE
Parameters and Biochemical Reactions
TEST OR SUBSTRATE
Adonitol
Inositol
Sorbitol
Arabinose
Raf f inose
Rhamnose
SALMONELLEAE
Salmonella
d
+
+ (D
-
+
Arizona
-
-
+
+
-
+
Citrobacter
-
+
+
d
+
(1) S_. typhi, S. cholerae-suis, S. enteritidis bioser. Paratyphi A
and Pullorum, and a few otheFs ordinarily do not ferment
dulcitol promptly. s. cholerae~suis does not ferment arabinose,
+, 90 percent or more positive in 1 or 2 days. -, 90 percent or
more negative, d, different biochemical types [+,(+),-].
(+), delayed positive. + or -, majority of cultures positive.
- or +, majority negative, w, weakly positive reaction.
Compiled by Difco Laboratories, Detroit, Michigan
-------
* 31
Standard Method used
by
Region IX Laboratory
FA - SALMONELLA SCREENING
1. From colony or agar slant, make light saline suspension.
(Use fresh agar slant culture and suspend in a small amount of
solution made by mixing 0.85 g NaCl to 100ml distilled water.
2. Prepare smears of this suspension on clear glass FA slides
(1.0 to 1.1 mm thick).
3. Air dry the smears - then fix for 2 minutes in Kirkpatrick's
Fixative. Rinse briefly in 95% ethanol. Allow to dry.
Do not blot.
4. Cover the fixed smears with one drop of Salmonella
polyvalent OH conjugate. (use 1:8 dilution of the conjugate)
*5. Place slides in a moist chamber to prevent evaporation of
the staining reagent. After 30 minutes, wash away excess
reagent by dipping slide into buffered saline (pH 7.5 - 8.0).
6. Place slide in second bath of buffered saline for 10 minutes.
7. Remove slides, rinse in distilled H-p) and allow to drain
dry.
8. Place a small drop of mounting fluid on the smear and cover
with a No. 1 coverslip. Examine under fluorescence scope,
using UG-1 (2 mm) primary filter and GG-9 (1 mm) ocular
filter. A combination of BG-12 (3mm) and OG-1 (1 mm) will
also give satisfactory results.
Kirkpatrick's Fixative
60 ml absolute ethanol
30 ml chloroform
10 ml formalin
^Buffered Saline: Bacto-FA Buffer Dried, prepared by Difco
Laboratories is recommended. Instructions
for preparation accompany the package.
F. Brezenski
Region II
-------
32
TECHNIQUES USED BY REGION IX FOR SEROLOGICAL GROUPING OF SALMONELLA
1. After completion of Differential Test, choose an isolated
colony from Brilliant Green Agar and/or XLD plates which
were streaked from Triple Sugar Iron slant (to obtain well
isolated colonies it may be necessary to re-streak several
times).
2. From Brilliant Green Agar and/or XLD plates, pick well isolated
Salmonella-like colony. Inoculate it into Brain Heart
Infusion broth. Incubate 24 hours at 37°C.
3. Repeat priming of isolate by transferring a loopful of
Brain Heart Infusion (BHI) broth culture into a fresh tube
of BHI broth. After 3-4 transfers, inoculate onto BHI
slant. Make two slants (always keep one for stock).
4. Perform slide agglutination test (see also page 27 Salmonella
Procedures, Serological Grouping, Step 4).
a. Prepare a dense suspension of organism from fresh 18-
hour BHI slant in 0.5 ml of 0.85% sodium chloride
solution. Suspension should be homogeneous and at
least as concentrated as that of Bacto McFarland Barium
Sulfate Standard #10 (which corresponds to 3xl09 cells/ml).
b. Using alcohol-cleaned slide mark slide into 4 sections
1 cm square. Using wax penciL mark heavily (form
continuous lines) to avoid spilling from one section
to another.
c. Place one drop ( use capillary pipet with rubber bulb)
of 0.05 ml of the Bacto-Salmonella 0 Antiserum Poly
within one square.
d. To the square next to antiserum, place one drop of
0.85% sodium chloride solution (This serves as negative
control) .
e. Using a clean inoculating loop, transfer a loopful
(0.05 ml) of bacterial suspension in 0.85% sodium
chloride prepared in step a and gently mix to emulsify
thoroughly-
f. Transfer another loopful of bacterial suspension to
section containing antiserum.
g. Gently rock the slide 1-2 minutes watching for agglutination,
(Using a small inverted fluorescent lamp aids in
detecting agglutination process.)
-------
33
Positive agglutination is rapid. Delayed agglutination
(over 2 minutes) or partial agglutination should be
considered negative.
5. If culture reacts with Bacto-Salmonella 0 Antiserum Poly
(step g) but does not react with the specific Salmonella 0
Antiserum groups, it should be checked with Bacto-Salmonella
Vi Antiserum by same method as described above. If the
culture does not agglutinate with Salmonella Vi antiserum,
the culture may be regarded as not of the Salmonella genus.
If the culture does react with the Vi antiserum, proceed
as follows:
a. Heat the culture suspension in a boiling water bath for
10 minutes, cool.
b. After cooling the heated culture should be re-tested
with the desired individual Salmonella O antiserum
groups and the Salmonella Vi antiserum.
6. If the organism does not react with the Vi antiserum after
heating, it is ready to be confirmed by a Public Health
Laboratory or typing center.
a. Send a pure culture slant.
b. List all parameters which were performed and results
obtained. Send along with culture slant.
c. It is preferable to deliver culture to Public Health
Laboratory; however, if this cannot be done, the culture
(in a screw-capped tube) should be carefully packed
inside a metal screw cap tube and then into a mailing
tube properly labelled and sent as registered mail.
Attachment: Diagram of slide agglutination
-------
34
Diagram of slide agglutination
Frosted
Culture
#
"0"
Antiseri:
A-l
(1)
n 0)
(2)
(4)
(Example)
Wa5c pencil enclosure
.Sl,ide__Se_ction Drop to contain
#1
#2
#3
Antiserum alone
Antiserum + 0.85% NaCl
Bacterial Suspension in
0.85% NaCl
#4
Bacterial Suspension in
0.85% NaCl + Antiserum
-------
in advance. Medium for fecal coliforms (mFC)
may be stored for 5 to 7 days.
2. Membrane-filter technique
a) Advantages; confirmed results within 24 hours;
test may be done entirely in field (media is
not autoclaved); permanent record of results;
less space necessary than for tubes.
b) Limitations; suitable filtration volume must
be selected; high turbidity interferes; large
numbers of non-coliform inhibit appearance of
coliforms; colonies must be recognized and
counted; some percentage of counts should be
verified by tube testing.
3. Multiple-tube technique
a) Advantages: media may (must) be prepared in
advance; positive test is easy to read; less
interference from turbidity and large numbers
of non-coliforms (than is the case with membrane
filter technique).
b) Limitations; media cannot be prepared in field/-
confirmed results require a minimum of 48 hours
and a maximum of 96 hours incubation; a large
amount of storage and incubator space is required
(as compared to that necessary for membrane
filters) .
-15-
------- |