9221 MULTIPLE-TUBE FERMENTATION TECHNIQUE FOR MEMBERS OF THE

COLIFORM GROUP*

9221 A. Introduction

The coliform group consists of several genera of bacteria

belonging to the family Enterobacteriaceae. The historical definition of this group has been based on the method used for

detection, lactose fermentation, rather than on the tenets of

systematic bacteriology. Accordingly, when the fermentation

technique is used, this group is defined as all facultative anaerobic, gram-negative, non-spore-forming, rod-shaped bacteria

that ferment lactose with gas and acid formation within 48 h at

35°C.

The standard test for the coliform group may be carried out by

the multiple-tube fermentation technique or presence–absence

procedure (through the presumptive-confirmed phases or completed test) described herein, the membrane filter (MF) technique

(Section 9222) or the enzymatic substrate coliform test (Section

9223). Each technique is applicable within the limitations specified and with due consideration of the purpose of the examination. Production of valid results requires strict adherence to

quality control procedures. Quality control guidelines are outlined in Section 9020.

When multiple tubes are used in the fermentation technique,

coliform density can be estimated by using a most probable

number (MPN) table. This number, based on certain probability

formulas, is an estimate of the mean density of coliforms in the

sample. Results of coliform testing, together with other information obtained from engineering or sanitary surveys, provide the

best assessment of water treatment effectiveness and the sanitary

quality of source water.

The precision of the fermentation test in estimating coliform

density depends on the number of tubes used. The most satisfactory information will be obtained when the largest sample

inoculum examined shows acid and/or gas in some or all of the

tubes and the smallest sample inoculum shows no acid and/or

gas in any or a majority of the tubes. Bacterial density can be

estimated by the formula given or from the table using the

number of positive tubes in the multiple dilutions (9221C.2). The

number of sample portions selected will be governed by the

desired precision of the result. The MPN tables are based on the

assumption of a Poisson distribution (random dispersion). However, if the sample is not adequately shaken before the portions

are removed or if bacterial cells clump, the MPN value will be

an underestimate of actual bacterial density.

1. Water of Drinking Water Quality

When analyzing drinking water to determine if the quality

meets U.S. Environmental Protection Agency (EPA) standards,

use the fermentation technique with 10 replicate tubes each

containing 10 mL, 5 replicate tubes each containing 20 mL, or a

single bottle containing a 100-mL sample portion. When examining drinking water via the fermentation technique, process all

tubes or bottles demonstrating growth, with or without a positive

acid or gas reaction, to the confirmed phase (9221B.3). Drinking

water samples that are positive for total coliforms also must be

tested for thermotolerant (fecal) coliforms (9221E) or Escherichia coli (9221F).

For routine examinations of public water supplies, the objective of the total coliform test is to determine the efficiency of

treatment plant operations and the integrity of the distribution

system. It is also used as a screen for the presence of fecal

contamination. Some coliform occurrences in a distribution system may be attributed to growth or survival of coliforms in

bacterial biofilms in the mains, rather than treatment failure at

the plant or well source, or outside contamination of the distribution system. Because it is difficult to distinguish between

coliforms entering the distribution system and coliforms already

present in the pipe biofilm and sediments, assume that all coliforms originate from a source outside the distribution system.

2. Water of Other than Drinking Water Quality

When examining nonpotable waters, inoculate a series of

tubes with appropriate decimal dilutions of the water (multiples

of 10 mL), based on the probable coliform density. Use the

presumptive– confirmed phases of the multiple-tube procedure.

Use the more labor-intensive completed test (9221B.4) as a

quality control measure on at least 10% of coliform-positive

nonpotable water samples on a seasonal basis. The objective of

the examination of nonpotable water, generally, is to estimate the

bacterial density, determine a source of pollution, enforce water

quality standards, or trace the survival of microorganisms. The

multiple-tube fermentation technique may be used to obtain

statistically valid MPN estimates of coliform density. Examine a

sufficient number of water samples to yield representative results

for the sampling station. Generally, the geometric mean or

median value of the results of a number of samples will yield a

value in which the effect of sample-to-sample variation is minimized.

3. Other Samples

The multiple-tube fermentation technique is applicable to the

analysis of salt or brackish waters as well as muds, sediments,

and sludges. Collect samples as directed in Sections 9060A and

B, using sample containers specified in Section 9030B.19. Follow the precautions given above on portion sizes and numbers of

tubes per dilution.

* Approved by Standard Methods Committee, 2006.

Joint Task Group: Ellen B. Braun-Howland (chair), Paul S. Berger, Robert J.

Blodgett, Clifford H. Johnson, Shundar Lin, Mark C. Meckes, Eugene W. Rice.

1
To prepare solid or semisolid samples, weigh the sample and add

diluent to make a 10–1 dilution. For example, place 30 g sample in

sterile blender jar, add 270 mL sterile phosphate buffered or 0.1%

peptone dilution water, and blend for 1 to 2 min at high speed

(8000 rpm). Prepare the appropriate decimal dilutions of the homogenized slurry as quickly as possible to minimize settling.

9221 B. Standard Total Coliform Fermentation Technique

1. Samples

Collect samples as directed in Sections 9060A and B, using

sample containers specified in Section 9030B.19.

2. Presumptive Phase

Use lauryl tryptose broth in the presumptive portion of the

multiple-tube test. If the medium has been refrigerated after

sterilization, incubate overnight at room temperature (20°C)

before use. Discard tubes showing growth and/or bubbles.

a. Reagents and culture medium: If possible, use a commercially available medium.

Lauryl tryptose broth:

Tryptose ................................................................................... 20.0 g

Lactose....................................................................................... 5.0 g

Dipotassium hydrogen phosphate, K2HPO4 ............................. 2.75 g

Potassium dihydrogen phosphate, KH2PO4.............................. 2.75 g

Sodium chloride, NaCl.............................................................. 5.0 g

Sodium lauryl sulfate ................................................................ 0.1 g

Reagent-grade water.................................................................. 1 L

Add dehydrated ingredients to water, mix thoroughly, and heat

to dissolve. Before sterilization, dispense—in fermentation tubes

with an inverted vial (Durham tube)—sufficient medium to

cover the inverted vial at least one-half to two-thirds after

sterilization. Alternatively, omit the inverted vial and add

0.01 g/L bromcresol purple to lauryl tryptose broth to determine

acid production, an indicator of a positive result in this part of the

coliform test. Close tubes with metal or heat-resistant plastic

caps.

Make lauryl tryptose broth of such strength that adding

100-mL, 20-mL, or 10-mL portions of sample to the medium

will not reduce ingredient concentrations below those of the

standard medium. Prepare in accordance with Table 9221:I.

Autoclave medium at 121o

C for 12 to 15 min. Ensure that

inverted vials, if used, are free of air bubbles. Medium pH should

be 6.8  0.2 after sterilization.

b. Procedure:

1) Arrange fermentation tubes in rows of five or ten tubes each

in a test tube rack. The number of rows and the sample volumes

selected depend on the quality and character of the water to be

examined. For potable water, use five 20-mL portions, ten

10-mL portions, or a single bottle of 100-mL portion; for nonpotable water, use five tubes per dilution (of 10, 1, 0.1 mL, etc.).

When making dilutions and measuring diluted sample volumes, follow the precautions given in Section 9215B.2. Use

Figure 9215:1 as a guide to preparing dilutions. Shake sample

and dilutions vigorously about 25 times. Inoculate each tube in

a set of five with replicate sample volumes in increasing decimal

dilutions, if decimal quantities of the sample are used. Mix test

portions in the medium by gentle agitation.

2) Incubate inoculated tubes or bottles at 35  0.5°C. After 24

 2 h swirl each tube or bottle gently and examine it for growth,

gas, and/or acidic reaction (shades of yellow color) and, if no gas

or acidic reaction is evident, re-incubate and re-examine at the

end of 48  3 h. Record presence or absence of growth, gas,

and/or acid production. If the inner vial is omitted, growth with

acidity (yellow color) signifies a positive presumptive reaction.

c. Interpretation: Production of an acidic reaction and/or gas

in the tubes or bottles within 48  3 h constitutes a positive

presumptive reaction. Submit tubes or bottles with a positive

presumptive reaction to the confirmed phase (9221B.3).

The absence of acidic reaction and/or gas formation at the end

of 48  3 h of incubation constitutes a negative test. Submit

drinking water samples demonstrating growth without a positive

gas or acid reaction to the confirmed phase (9221B.3). An

arbitrary 48-h limit for observation doubtless excludes occasional members of the coliform group that grow very slowly (see

Section 9212).

3. Confirmed Phase

a. Culture medium: Use brilliant green lactose bile broth

fermentation tubes for the confirmed phase. If possible, use a

commercially available medium.

Brilliant green lactose bile broth:

Peptone ................................................................................ 10.0 g

Lactose................................................................................. 10.0 g

Oxgall .................................................................................. 20.0 g

Brilliant green........................................................................ 0.0133 g

Reagent-grade water.............................................................. 1 L

Add dehydrated ingredients to water, mix thoroughly, and heat

to dissolve. Before sterilization, dispense—in fermentation tubes

with an inverted vial—sufficient medium to cover the inverted

vial at least one-half to two-thirds after sterilization. Close tubes

TABLE 9221:I. PREPARATION OF LAURYL TRYPTOSE BROTH

Inoculum

mL

Amount of

Medium in

Tube

mL

Volume of

Medium 

Inoculum

mL

Dehydrated Lauryl

Tryptose Broth

Required

g/L

1 10 or more 11 or more 35.6

10 10 20 71.2

10 20 30 53.4

20 10 30 106.8

100 50 150 106.8

100 35 135 137.1

100 20 120 213.6

MULTIPLE TUBE FERMENTATION TECHNIQUE (9221)/Standard Total Coliform Fermentation Technique

2
with metal or heat-resistant plastic caps. Autoclave medium at

121o

C for 12 to 15 min. Ensure that inverted vials are free of air

bubbles. Medium pH should be 7.2  0.2 after sterilization.

b. Procedure: Submit all presumptive tubes or bottles showing

growth, any amount of gas, or acidic reaction within 24  2 h of

incubation to the confirmed phase. If additional presumptive

tubes or bottles show active fermentation or acidic reaction at the

end of a 48  3 h incubation period, submit these to the

confirmed phase. Simultaneous inoculation into brilliant green

lactose bile broth for total coliforms and EC broth for thermotolerant (fecal) coliforms (see 9221E) or EC-MUG broth for

Escherichia coli (see 9221F) may be used. To confirm presumptive coliform colonies growing on a solid medium using fermentation media, see Section 9222B.4f.

Gently shake or rotate presumptive tubes or bottles showing

gas or acidic growth to resuspend the organisms. With a sterile

loop 3.0 to 3.5 mm in diameter, transfer one or more loopfuls of

culture to a fermentation tube containing brilliant green lactose

bile broth. Alternatively, insert a sterile wooden applicator at

least 2.5 cm into the culture, promptly remove, and plunge

applicator to bottom of fermentation tube containing brilliant

green lactose bile broth. Remove and discard applicator. Repeat

for all other positive presumptive tubes.

Incubate the inoculated brilliant green lactose bile broth tubes

at 35  0.5°C. Formation of gas in any amount in the inverted

vial of the brilliant green lactose bile broth fermentation tube at

any time within 48  3 h constitutes a positive confirmed phase.

To estimate the coliform density, calculate the MPN value from

the number of positive brilliant green lactose bile tubes (as

described in 9221C).

c. Alternative procedure: Use this alternative only for polluted

water or wastewater known to produce positive results consistently.

If all presumptive tubes are positive in two or more consecutive dilutions within 24 h, submit to the confirmed phase only the

tubes of the highest dilution (smallest sample inoculum) in

which all tubes are positive and any positive tubes in still higher

dilutions. Submit to the confirmed phase all tubes in which gas

or acidic growth is produced in 24 to 48 h.

4. Completed Phase

To verify the presence of coliform bacteria and to provide

quality control data for nonpotable water sample analysis, use

the completed test on 10% (or a set percentage) of positive

confirmed tubes (see Figure 9221:1). Use the completed test on

at least one positive sample per quarter. If no positive sample

occurs within a quarter, a QC check must be performed using a

known positive sample. Simultaneous inoculation into brilliant

green lactose bile broth for total coliforms and EC broth for

thermotolerant (fecal) coliforms (9221E) or EC MUG broth for

Escherichia coli (9221F) may be used. Positive results from

incubation in EC and/or EC-MUG broths at elevated temperature

(44.5  0.2o

C) can be considered as a completed test. Parallel

Figure 9221:1. Schematic outline of presumptive, confirmed, and completed phases for total coliform detection.

MULTIPLE TUBE FERMENTATION TECHNIQUE (9221)/Standard Total Coliform Fermentation Technique

3
positive brilliant green lactose bile broth cultures with negative

EC or EC-MUG broth cultures indicate the presence of nonfecal

coliforms. Parallel positive EC or EC-MUG tubes and negative

brilliant green lactose bile broth cultures are recorded as positive

for fecal coliforms or E. coli, respectively. Alternatively, the

completed test for positive total coliforms may be performed as

follows.

a. Culture media and reagents: If possible, use a dehydrated,

commercially available medium.

1) LES Endo agar: See Section 9222B.2a. Use 100-  15-mm

petri plates.

2) MacConkey agar:

Peptone .................................................................................. 17 g

Proteose peptone...................................................................... 3 g

Lactose................................................................................... 10 g

Bile salts .................................................................................. 1.5 g

Sodium chloride, NaCl............................................................ 5 g

Agar ....................................................................................... 13.5 g

Neutral red............................................................................... 0.03 g

Crystal violet ........................................................................... 0.001 g

Reagent-grade water ............................................................... 1 L

Add ingredients to water, mix thoroughly, and heat to boiling

to dissolve. Sterilize by autoclaving for 15 min at 121°C. Temper

agar after sterilization and pour into petri plates (100  15 mm).

Medium pH should be 7.1  0.2 after sterilization.

3) Nutrient agar:

Peptone ........................................................................................ 5.0 g

Beef extract.................................................................................. 3.0 g

Agar ........................................................................................... 15.0 g

Reagent-grade water.................................................................... 1 L

Add ingredients to water, mix thoroughly, and heat to dissolve. Before sterilization, dispense in screw-capped tubes. Autoclave at 121°C for 15 min. Medium pH should be 6.8  0.2

after sterilization. After sterilization, immediately place tubes in

an inclined position so the agar will solidify with a sloped

surface. Tighten screw caps after cooling and store in a protected, cool storage area.

4) Gram-stain reagents: Reagents are commercially available

as prepared solutions.

a) Ammonium oxalate-crystal violet (Hucker’s): Dissolve 2 g

crystal violet (90% dye content) in 20 mL 95% ethyl alcohol.

Dissolve 0.8 g (NH4)2C2O4  H2O in 80 mL reagent-grade water.

Mix the two solutions and age for 24 h before use. Filter through

paper into a staining bottle.

b) Lugol’s solution, Gram’s modification: Grind 1 g iodine

crystals and 2 g KI in a mortar. Add reagent-grade water a few

milliliters at a time, and grind thoroughly after each addition

until solution is complete. Rinse solution into an amber glass

bottle with the remaining water, using a total of 300 mL.

c) Counterstain: Dissolve 2.5 g safranin dye in 100 mL 95%

ethyl alcohol. Add 10 mL to 100 mL reagent-grade water.

CAUTION: Flammable.

d) Acetone alcohol: Mix equal volumes of ethyl alcohol (95%)

with acetone. CAUTION: Flammable.

b. Procedure:

1) Using aseptic technique, streak one LES Endo agar (Section

9222B.2a.) or MacConkey agar plate from each tube of brilliant

green lactose bile broth showing gas, as soon as possible after the

observation of gas. Streak plates in a manner to ensure the

presence of some discrete colonies separated by at least 0.5 cm.

To obtain a high proportion of successful isolations if coliform

organisms are present, the following approach may be used:

(a) Use a sterile 3-mm-diam loop or an inoculating needle

slightly curved at the tip; (b) tap and incline the fermentation

tube to avoid picking up any membrane or scum on the needle;

(c) insert the end of the loop or needle into the liquid in the tube

to a depth of approximately 0.5 cm; and (d) streak a plate for

isolation with the curved section of the needle in contact with the

agar to avoid a scratched or torn surface. Flame the loop between

the second and third quadrants to improve colony isolation.

Incubate plates, inverted, at 35  0.5°C for 24  2 h.

2) The colonies developing on LES Endo agar are defined as

typical (pink to dark red with a green metallic surface sheen) or

atypical (pink, red, white, or colorless colonies without sheen)

after 24 h incubation. Typical lactose-fermenting colonies developing on MacConkey agar are red and may be surrounded by

an opaque zone of precipitated bile. From each plate pick one or

more typical, well-isolated coliform colonies or, if no typical

colonies are present, pick two or more colonies considered most

likely to be organisms of the coliform group. Transfer growth

from each isolate to a single-strength lauryl tryptose broth fermentation tube and onto a nutrient agar slant.

If needed, use a colony magnifying device to provide optimum

magnification when colonies are picked from the LES Endo or

MacConkey agar plates. When transferring colonies, choose

well-isolated ones and barely touch the surface of the colony

with a flame-sterilized, air-cooled transfer needle to minimize

the danger of transferring a mixed culture.

Incubate secondary broth tubes (lauryl tryptose broth with

inverted fermentation vials ) at 35  0.5°C for 24  2 h; if gas

is not produced within 24  2 h, reincubate and examine again

at 48  3 h. Microscopically examine Gram-stained preparations

from those 24-h nutrient agar slant cultures corresponding to the

secondary tubes that show gas.

3) Gram-stain technique—The Gram stain may be omitted

from the completed test for potable water samples only because

the occurrences of gram-positive bacteria and spore-forming

organisms surviving this selective screening procedure are infrequent in drinking water.

Various modifications of the Gram stain technique exist. Use

the following modification by Hucker for staining smears of pure

cultures; include a gram-positive and a gram-negative culture as

controls.

Prepare separate light emulsions of the test bacterial growth

and positive and negative control cultures on the same slide,

using drops of distilled water on the slide. Air-dry, fix by passing

slide through a flame, and stain for 1 min with ammonium

oxalate-crystal violet solution. Rinse slide in tap water, and drain

off excess; apply Lugol’s solution for 1 min.

Rinse stained slide in tap water. Decolorize for approximately

15 to 30 s with acetone alcohol by holding slide between the

fingers and letting acetone alcohol flow across the stained smear

until the solvent flows colorlessly from the slide. Do not overdecolorize. Counterstain with safranin for 15 s, rinse with tap

water, blot dry with absorbent paper or air dry, and examine

microscopically. Gram-positive organisms are blue; gramnegative organisms are red. Results are acceptable only when

controls have given proper reactions.

MULTIPLE TUBE FERMENTATION TECHNIQUE (9221)/Standard Total Coliform Fermentation Technique

4
c. Interpretation: Formation of gas in the secondary tube of

lauryl tryptose broth within 48  3 h and demonstration of

gram-negative, nonspore-forming, rod-shaped bacteria from the

agar culture constitute a positive result for the completed test,

demonstrating the presence of a member of the coliform group.

5. Bibliography

MEYER, E.M. 1918. An aerobic spore-forming bacillus giving gas in

lactose broth isolated in routine water examination. J. Bacteriol.

3:9.

HUCKER, G.J. & H.J. CONN. 1923. Methods of Gram Staining. N.Y. State

Agr. Exp. Sta. Tech. Bull. No. 93.

NORTON, J.F. & J.J. WEIGHT. 1924. Aerobic spore-forming lactose fermenting organisms and their significance in water analysis. Amer. J.

Pub. Health 14:1019.

HUCKER, G.J. & H.J. CONN. 1927. Further Studies on the Methods of

Gram Staining. N.Y. State Agr. Exp. Sta. Tech. Bull. No. 128.

PORTER, R., C.S. MCCLESKEY & M. LEVINE. 1937. The facultative sporulating bacteria producing gas from lactose. J. Bacteriol. 33:163.

COWLES, P.B. 1939. A modified fermentation tube. J. Bacteriol. 38:677.

SHERMAN, V.B.D. 1967. A Guide to the Identification of the Genera of

Bacteria. Williams & Wilkins, Baltimore, Md.

GELDREICH, E.E. 1975. Handbook for Evaluating Water Bacteriological

Laboratories, 2nd ed. EPA-670/9-75-006, U.S. Environmental Protection Agency, Cincinnati, Ohio.

EVANS, T.M., C.E. WAARVICK, R.J. SEIDLER & M.W. LECHEVALLIER.

1981. Failure of the most-probable number technique to detect

coliforms in drinking water and raw water supplies. Appl. Environ.

Microbiol. 41:130.

SEIDLER, R.J., T.M. EVANS, J.R. KAUFMAN, C.E. WAARVICK & M.W.

LECHEVALLIER. 1981. Limitations of standard coliform enumeration

techniques. J. Amer. Water Works Assoc. 73:538.

GERHARDS, P., ed. 1981. Manual of Methods for General Bacteriology.

American Soc. Microbiology, Washington, D.C.

KRIEG, N.R. & J.G. HOLT, eds. 1984. Bergey’s Manual of Systematic

Bacteriology, Vol 1. Williams & Wilkins, Baltimore, Md.

GREENBERG, A.E. & D.A. HUNT, eds. 1985. Laboratory Procedures for

the Examination of Seawater and Shellfish, 5th ed. American Public

Health Assoc., Washington, D.C.

U.S. ENVIRONMENTAL PROTECTION AGENCY. 1989. National primary drinking water regulations: analytical techniques; coliform bacteria; final

rule. Federal Register 54(135):29998 (July 17, 1989).

9221 C. Estimation of Bacterial Density

1. Precision of the Multiple-Tube Fermentation Test

Unless many sample portions are examined, the precision of the

Multiple-Tube Fermentation Test is rather low. Consequently, use

caution when interpreting the sanitary significance of any single

coliform result. When several samples from a given sampling point

are estimated separately and the results combined in their geometric

mean, the precision is greatly improved. Although most probable

number (MPN) tables and calculations are described for use in the

coliform test, they also can be used to determine the MPN of any

other organisms, provided that suitable test media are available.

2. Table Reading and Recording of Most Probable Number

(MPN)

Record coliform concentration as MPN/100 mL. MPN values

for a variety of positive and negative tube combinations are

given in Tables 9221:II, III, and IV. The sample volumes indicated in Tables 9221:II and III are chosen especially for examining drinking waters. Table 9221:IV illustrates MPN values for

combinations of positive and negative results when five 10-mL,

five 1.0-mL, and five 0.1-mL sample portion volumes of nonpotable water are tested. If the sample portion volumes used are

those found in the tables, report the value corresponding to the

number of positive and negative results in the series as MPN/

100 mL. When the series of decimal dilutions is different from

that in Table 9221:IV, select the MPN value from Table 9221:IV

for the combination of positive results and calculate according to

the following formula:

MPN/100 mL  (Table MPN/100 mL)  10/V

TABLE 9221:II. MPN INDEX AND 95% CONFIDENCE LIMITS FOR ALL

COMBINATIONS OF POSITIVE AND NEGATIVE RESULTS WHEN FIVE 20-ML

PORTIONS ARE USED

No. of Tubes Giving

Positive Reaction Out

of 5 (20 mL Each)

MPN Index/

100 mL

95% Confidence

Limits (Exact)

Lower Upper

0 1.1 — 3.5

1 1.1 0.051 5.4

2 2.6 0.40 8.4

3 4.6 1.0 13

4 8.0 2.1 23

5 8.0 3.4 —

TABLE 9221:III. MPN INDEX AND 95% CONFIDENCE LIMITS FOR ALL

COMBINATIONS OF POSITIVE AND NEGATIVE RESULTS WHEN TEN 10-ML

PORTIONS ARE USED

No. of Tubes Giving

Positive Reaction Out of

10 (10 mL Each)

MPN Index/

100 mL

95% Confidence

Limits (Exact)

Lower Upper

0 1.1 — 3.4

1 1.1 0.051 5.9

2 2.2 0.37 8.2

3 3.6 0.91 9.7

4 5.1 1.6 13

5 6.9 2.5 15

6 9.2 3.3 19

7 12 4.8 24

8 16 5.8 34

9 23 8.1 53

10 23 13 —

MULTIPLE-TUBE FERMENTATION TECHNIQUE (9221)/Estimation of Bacterial Density

5
where:

V  volume of sample portion at the lowest selected dilution.

When more than three dilutions are used in a decimal series1

of dilutions, use the following guidelines to select the three most

appropriate dilutions and refer to Table 9221:IV. Several illustrative examples (A through G) of combinations of positives are

shown in Table 9221:V. First, remove the highest dilution

(smallest sample volume) if it has all negative tubes and at least

one remaining dilution has a negative tube. Next, remove the

lowest dilution (largest sample volume) if it has all positive tubes

and at least one remaining dilution has a positive tube. AccordTABLE 9221:IV. MPN INDEX AND 95% CONFIDENCE LIMITS FOR VARIOUS COMBINATIONS OF POSITIVE RESULTS WHEN FIVE TUBES ARE USED PER

DILUTION (10 ML, 1.0 ML, 0.1 ML)*

Combination of

Positives MPN Index/100 mL

Confidence

Limits Combination of

Positives MPN Index/100 mL

Confidence Limits

Low High Low High

0-0-0 1.8 — 6.8 4-0-3 25 9.8 70

0-0-1 1.8 0.090 6.8 4-1-0 17 6.0 40

0-1-0 1.8 0.090 6.9 4-1-1 21 6.8 42

0-1-1 3.6 0.70 10 4-1-2 26 9.8 70

0-2-0 3.7 0.70 10 4-1-3 31 10 70

0-2-1 5.5 1.8 15 4-2-0 22 6.8 50

0-3-0 5.6 1.8 15 4-2-1 26 9.8 70

1-0-0 2.0 0.10 10 4-2-2 32 10 70

1-0-1 4.0 0.70 10 4-2-3 38 14 100

1-0-2 6.0 1.8 15 4-3-0 27 9.9 70

1-1-0 4.0 0.71 12 4-3-1 33 10 70

1-1-1 6.1 1.8 15 4-3-2 39 14 100

1-1-2 8.1 3.4 22 4-4-0 34 14 100

1-2-0 6.1 1.8 15 4-4-1 40 14 100

1-2-1 8.2 3.4 22 4-4-2 47 15 120

1-3-0 8.3 3.4 22 4-5-0 41 14 100

1-3-1 10 3.5 22 4-5-1 48 15 120

1-4-0 10 3.5 22 5-0-0 23 6.8 70

2-0-0 4.5 0.79 15 5-0-1 31 10 70

2-0-1 6.8 1.8 15 5-0-2 43 14 100

2-0-2 9.1 3.4 22 5-0-3 58 22 150

2-1-0 6.8 1.8 17 5-1-0 33 10 100

2-1-1 9.2 3.4 22 5-1-1 46 14 120

2-1-2 12 4.1 26 5-1-2 63 22 150

2-2-0 9.3 3.4 22 5-1-3 84 34 220

2-2-1 12 4.1 26 5-2-0 49 15 150

2-2-2 14 5.9 36 5-2-1 70 22 170

2-3-0 12 4.1 26 5-2-2 94 34 230

2-3-1 14 5.9 36 5-2-3 120 36 250

2-4-0 15 5.9 36 5-2-4 150 58 400

3-0-0 7.8 2.1 22 5-3-0 79 22 220

3-0-1 11 3.5 23 5-3-1 110 34 250

3-0-2 13 5.6 35 5-3-2 140 52 400

3-1-0 11 3.5 26 5-3-3 170 70 400

3-1-1 14 5.6 36 5-3-4 210 70 400

3-1-2 17 6.0 36 5-4-0 130 36 400

3-2-0 14 5.7 36 5-4-1 170 58 400

3-2-1 17 6.8 40 5-4-2 220 70 440

3-2-2 20 6.8 40 5-4-3 280 100 710

3-3-0 17 6.8 40 5-4-4 350 100 710

3-3-1 21 6.8 40 5-4-5 430 150 1100

3-3-2 24 9.8 70 5-5-0 240 70 710

3-4-0 21 6.8 40 5-5-1 350 100 1100

3-4-1 24 9.8 70 5-5-2 540 150 1700

3-5-0 25 9.8 70 5-5-3 920 220 2600

4-0-0 13 4.1 35 5-5-4 1600 400 4600

4-0-1 17 5.9 36 5-5-5 1600 700 —

4-0-2 21 6.8 40

* Results to two significant figures.

MULTIPLE-TUBE FERMENTATION TECHNIQUE (9221)/Estimation of Bacterial Density

6
ing to these guidelines, the three dilutions in Example A are

selected by removal of the highest (0.001-mL) and the lowest

(10-mL) dilutions.

If the lowest dilution does not have all positive tubes, and

several of the highest dilutions have all negative tubes, then

remove the highest negative dilutions (Example B).

More than three dilutions may remain after removal of the

lowest dilution with all positive tubes and high dilutions with all

negative tubes. In this case, if the highest dilution with all

positive tubes is within two dilutions of the highest dilution with

any positive tubes, then use the highest dilution with any positive

tubes and the two immediately lower dilutions. In Example C,

the highest dilution with all positive tubes is 0.1 mL, which is

within two dilutions of 0.001 mL, which has one positive tube.

In Example D, the highest dilution with all positive tubes is

0.01 mL, which is within two decimal dilutions of 0.001 mL, to

yield a combination of 4-5-1.

If, after removal of the lowest dilution with all positive tubes,

no dilution with all positive reactions remains, then select the

lowest two dilutions and assign the sum of any remaining dilutions to the third dilution. In Example E, the highest dilution with

all positive tubes contains 10 mL; this dilution was removed in

the second step. Four dilutions, none of which have all positive

tubes, remain. Under these circumstances, select the two lowest

remaining dilutions corresponding to 1 and 0.1 mL sample. For

the third dilution, add the number of positive tubes in all higher

dilutions (0.01 and 0.001 mL sample), to yield a final combination of 4-4-1.

If no dilution has all positive tubes (Example F), select the

lowest two dilutions, corresponding to 10 and 1 mL sample. For

the third dilution, add the number of positive tubes in the

remaining dilutions (0.1, 0.01, and 0.001 mL sample), to yield a

final combination of 4-3-2. If the third dilution is assigned more

than five positive tubes, then the selected combination will not be

in Table 9221:IV.

If the three dilutions selected are not found in Table 9221:IV,

then something in the serial dilution was unusual. In this case,

the usual methods for calculating the MPN, presented here, may

not apply. If a new sample cannot be collected and an MPN

value is still desired, use the highest dilution with at least one

positive tube and the two dilutions immediately lower as the

three selected dilutions. In Example G, the first selection, 4-3-6

(the outcome from the highest three dilutions), is not in Table

9221:IV because 6 is greater than 5. The second selection,

according to the above guidelines, would be 3-2-1. If this second

set of selected dilutions is not in Table 9221:IV, then use the

following formula to calculate the MPN:

 230.3

zs

log101  xszs



js

K

njzj

where:

j  a dilution,

s  the highest dilution with at least one positive tube,

xs  the number of positive tubes in the sth dilution,

nj  the number of tubes in the jth dilution,

zj  the amount of the original sample inoculated into each tube in

the jth dilution,

zs  the amount of the original sample inoculated into each tube of

the sth dilution, and

K  the number of dilutions.

For example, in the series x-x-3-0-0, where the third dilution

level (zs) equals 0.1 mL, xszs  0.3, and  nj

zj  0.555. Thus, the

calculated MPN  780/100 mL.

This formula also applies to serial dilutions having all positive

tubes in a single dilution, and can serve as an approximation for

outcomes like 5-5-5-0-0-0, where five tubes are used per dilution, by using just the last four dilutions.

Table 9221:IV shows all but the improbable positive tube

combinations for a three-dilution series. In testing 10 samples,

there is a 99% chance of finding all the results among these 95

outcomes. If untabulated combinations occur with a frequency

greater than 1%, it indicates that the technique is faulty or that

the statistical assumptions underlying the MPN estimate are not

being fulfilled (e.g., growth inhibition at low dilutions).

The MPN for combinations not appearing in the table, or for

other combinations of tubes or dilutions, may be estimated as

follows: First, select the lowest dilution that does not have all

positive results. Second, select the highest dilution with at least

one positive result. Finally, select all the dilutions between them.

For example, from (10/10, 10/10, 4/10, 1/10, 0/10) use only (–,

–, 4/10, 1/10, –), corresponding to 4/10 @ 0.1 mL sample/tube

and 1/10 @ 0.01 mL sample/tube. Likewise, from (10/10, 10/10,

10/10, 0/10, 0/10), select only (–, –, 10/10, 0/10, –), corresponding to 10/10 @ 0.1 mL sample/tube and 0/10 @ 0.01 mL

TABLE 9221:V. EXAMPLES FOR CHOICE OF THREE COMBINATIONS OF POSITIVES FROM FIVE DILUTIONS

Example

Volume

mL Combination of

Positives

MPN Index

10 1 0.1 0.01 0.001 No./100 mL

A 5 51 0 0 x-5-1-0-x 330

B 45 1 0 0 4-5-1-x-x 48

C 52 52 1 x-x-5-2-1 7000

D 45 45 1 x-x-4-5-1 4800

E 5 44 0 1 x-4-4-1-x 400

F 43 0 1 1 4-3-2-x-x 39

G 43 32 1 x-x-3-2-1 1700

MULTIPLE-TUBE FERMENTATION TECHNIQUE (9221)/Estimation of Bacterial Density

7
sample/tube. Use only the selected dilutions in the following

formula of Thomas:1

MPN/100 mL (approx.)  100  P/(N  T)1/2

where:

P  number of positive results,

N  volume of sample in all the negative portions combined, mL, and

T  total volume of sample in the selected dilutions, mL.

That is, N  (nj

–xj)zj, P  xj, and T  njzj, where the

summations are over the dilutions selected, and xj  the number

of positive tubes in the jth dilution.

In the first example above,

MPN/100 mL (approx.)  100  5/(0.69  1.1)1/2

500/0.87 570/100 mL

In the second example above,

MPN/100 mL (approx.)  100  10/ (0.1  1.1)1/2

1000/0.332 3000/100 mL

The two examples compare well with the true MPNs, 590/

100 mL and 2400/100 mL, respectively. The second example is

a special case for which an exact solution can be calculated

directly for the two selected dilutions.

When it is desired to summarize the results from several

samples with a single MPN value, use the geometric mean or the

median. The geometric mean is calculated by averaging the

logarithmic values; for example, the geometric mean of A, B, and

C is 10L where:

L  (log10 A log10 B log10 C)/3

Mean values are reported as the antilog of L.

3. Reference

1. THOMAS, H.A., JR. 1942. Bacterial densities from fermentation tube

tests. J. Amer. Water Works Assoc. 34:572.

4. Bibliography

MCCRADY, M.H. 1915. The numerical interpretation of fermentation

tube results. J. Infect. Dis. 12:183.

MCCRADY, M.H. 1918. Tables for rapid interpretation of fermentationtube results. Pub. Health J. 9:201.

HOSKINS, J.K. 1933. The most probable numbers of B. coli in water

analysis. J. Amer. Water Works Assoc. 25:867.

HOSKINS, J.K. 1934. Most Probable Numbers for evaluation of coliaerogenes tests by fermentation tube method. Pub. Health Rep.

49:393.

HALVORSON, H.O. & N.R. ZIEGLER. 1933–35. Application of statistics to

problems in bacteriology. J. Bacteriol. 25:101; 26:331, 559;

29:609.

EISENHART, C. & P.W. WILSON. 1943. Statistical methods and control in

bacteriology. Bacteriol. Rev. 7:57.

COCHRAN, W.G. 1950. Estimation of bacterial densities by means of the

“Most Probable Number.” Biometrics 6:105.

WOODWARD, R.L. 1957. How probable is the Most Probable Number?

J. Amer. Water Works Assoc. 49:1060.

DEMAN, J.C. 1983. MPN tables, corrected. Eur. J. Appl. Biotechnol.

17:301.

GARTHRIGHT, W.E. 1998. Appendix 2. Most probable number from serial

dilutions. FDA Bacteriological Analytical Manual, 8th ed., Rev. A.

AOAC International, Gaithersburg, Md.

BLODGETT, R.J. & W.E. GARTHRIGHT. 1998. Several MPN models for

serial dilutions with suppressed growth at low dilutions. Food

Microbiol. 15:91.

BLODGETT, R.J. 2002. Measuring improbability of outcomes from a serial

dilution test. Commun. Statist. Theory Meth. 31:2209.

GARTHRIGHT, W.E. & R.J. BLODGETT. 2003. FDA’s Preferred MPN

methods for standard, large, or unusual tests, with a spreadsheet.

Food Microbiol. 20:439.

BLODGETT, R.J. 2006. Appendix 2. Most probable number from

serial dilutions. FDA Bacteriological Analytical Manual. (http://

www.fda.gov/Food/ScienceResearch/LaboratoryMethods/

BacteriologicalAnalyticalManualBAM/ucm109656.htm. Accessed November 2011.

9221 D. Presence–Absence (P–A) Coliform Test

The presence–absence (P–A) test for the coliform group is a

simple modification of the multiple-tube procedure. Simplification— by use of one large test portion (100 mL) in a single

culture bottle to obtain qualitative information on the presence or

absence of coliforms—is justified on the theory that no coliforms

should be present in 100 mL of a drinking water sample. Another

advantage includes the possibility of examining a larger number

of samples per unit of time. Comparative studies with the membrane filter procedure indicate that the P–A test may maximize

coliform detection in samples containing many organisms that

could overgrow coliform colonies and cause problems in detection.

The P–A test is intended for use on routine samples collected

from distribution systems or water treatment plants.

1. Samples

Collect samples as directed in Sections 9060A and B, using

sample containers specified in Section 9030B.19.

2. Presumptive Phase

a. Culture medium:

P–A broth: This medium is commercially available in dehydrated and in sterile concentrated form.

MULTIPLE-TUBE FERMENTATION TECHNIQUE (9221)/Presence–Absence Coliform Test

8
Beef extract............................................................................ 3.0 g

Peptone .................................................................................. 5.0 g

Lactose................................................................................... 7.46 g

Tryptose ................................................................................. 9.83 g

Dipotassium hydrogen phosphate, K2HPO4 ......................... 1.35 g

Potassium dihydrogen phosphate, KH2PO4.......................... 1.35 g

Sodium chloride, NaCl.......................................................... 2.46 g

Sodium lauryl sulfate ............................................................ 0.05 g

Bromcresol purple ................................................................. 0.0085 g

Reagent-grade water.............................................................. 1 L

Make this formulation triple strength (3) when examining

100-mL samples. Dissolve the P–A broth medium in water

without heating, using a stirring device. Dispense 50 mL prepared medium into screw-capped 250-mL milk dilution bottles

or equivalent containers. A fermentation tube insert is not necessary. Autoclave for 12 min at 121°C, with the total time in the

autoclave limited to 30 min or less. Medium pH should be 6.8 

0.2 after sterilization. When the P–A medium is sterilized via

filtration, a 6 strength medium may be used. Aseptically dispense 20 mL of the 6 medium into a sterile 250-mL dilution

bottle or equivalent container.

b. Procedure: Shake sample vigorously for 5 s (approximately

25 times) and inoculate 100 mL into a P–A culture bottle. Mix

thoroughly by inverting bottle once or twice to achieve even

distribution of the medium throughout the sample. Incubate at

35  0.5°C and inspect after 24  2 and 48  3 h for acid

reactions.

c. Interpretation: A distinct yellow color forms in the medium

when acidic conditions exist following lactose fermentation. If

gas also is being produced, gently shaking the bottle will result

in a foaming reaction. Any amount of gas and/or acid constitutes

a positive presumptive test requiring confirmation. Simultaneous

inoculation of EC broth or EC MUG and then brilliant green

lactose bile broth is permitted.

3. Confirmed Phase

The confirmed phase is outlined in Figure 9221:1.

a. Culture medium: Use brilliant green lactose bile (BGLB)

fermentation tubes (see 9221B.3a).

b. Procedure: Use a sterile wire loop or wooden applicator to

transfer all cultures that show acid reaction or acid and gas

reaction to BGLB broth for incubation at 35  0.5°C (see

9221B.3).

c. Interpretation: Gas production in BGLB broth culture

within 48  3 h confirms the presence of coliform bacteria.

Report result as P–A test positive or negative for total coliforms

in 100 mL of sample. Drinking water samples that are positive

for total coliforms also must be tested for thermotolerant (fecal)

coliforms (9221E) or E. coli (9221F).

4. Completed Phase

The completed phase, required for nonpotable water sample

analysis, is outlined in 9221B.4 and Figure 9221:1.

5. Bibliography

WEISS, J.E. & C.A. HUNTER. 1939. Simplified bacteriological examination of water. J. Amer. Water Works Assoc. 31:707.

CLARK, J.A. 1969. The detection of various bacteria indicative of water

pollution by a presence–absence (P–A) procedure. Can. J. Microbiol. 15:771.

CLARK, J.A. & L.T. VLASSOFF. 1973. Relationships among pollution

indicator bacteria isolated from raw water and distribution systems

by the presence–absence (P–A) test. Health Lab. Sci. 10:163.

CLARK, J.A. 1980. The influence of increasing numbers of nonindicator

organisms upon the detection of indicator organisms by the membrane filter and presence–absence tests. Can. J. Microbiol. 26:827.

CLARK, J.A., C.A. BURGER & L.E. SABATINOS. 1982. Characterization of

indicator bacteria in municipal raw water, drinking water, and new

main water samples. Can. J. Microbiol. 28:1002.

JACOBS, N.J., W.L. ZEIGLER, F.C. REED, T.A. STUKEL & E.W. RICE. 1986.

Comparison of membrane filter, multiple-fermentation-tube, and

presence–absence techniques for detecting total coliforms in small

community water systems. Appl. Environ. Microbiol. 51:1007.

RICE, E.W., E.E. GELDREICH & E.J. READ. 1989. The presence–absence

coliform test for monitoring drinking water quality. Pub. Health

Rep. 104:54.

9221 E. Fecal Coliform Procedure

Thermotolerant coliforms (those that ferment lactose to produce gas at 44.5°C) were traditionally called fecal coliforms, but

they also have been documented in organically rich waters or

tropical climates in the absence of recent fecal contamination.

So, testing for E. coli—a specific indicator of fecal contamination—is recommended.

Nevertheless, current regulations may require that thermotolerant (formerly fecal) coliforms be identified and enumerated. In

the multiple-tube fermentation technique, this group of organisms is identified by their ability to ferment lactose to produce

gas at 44.5°C.

A test for thermotolerant coliforms can be performed using

one of the multiple-tube procedures described here or the membrane filter methods described in Sections 9222D and E.

1. Thermotolerant Coliform Test (EC Medium)

The thermotolerant coliform test using EC medium is applicable to investigations of drinking water, stream pollution, raw

water sources, wastewater treatment systems, bathing waters,

seawaters, and general water-quality monitoring. Do not use EC

medium for the direct isolation of thermotolerant coliforms from

water. Prior enrichment in a presumptive medium is required for

optimum recovery of thermotolerant coliforms. For testing of

presumptive coliform colonies growing on solid media, refer to

Section 9222G.2.

a. EC medium: Preferably use a dehydrated, commercially

available medium.

Tryptose or trypticase.................................................................20.0 g

MULTIPLE-TUBE FERMENTATION TECHNIQUE (9221)/Fecal Coliform Procedure

9
Lactose......................................................................................... 5.0 g

Bile salts mixture or bile salts No. 3 ......................................... 1.5 g

Dipotassium hydrogen phosphate, K2HPO4 ............................... 4.0 g

Potassium dihydrogen phosphate, KH2PO4................................ 1.5 g

Sodium chloride, NaCl................................................................ 5.0 g

Reagent-grade water.................................................................... 1 L

Add dehydrated ingredients to water, mix thoroughly, and

heat to dissolve. Before sterilization, dispense sufficient medium, in fermentation tubes with an inverted vial, to cover the

inverted vial at least one-half to two-thirds after sterilization.

Close tubes with metal or heat-resistant plastic caps. Autoclave medium at 121o

C for 12 to 15 min. Ensure that inverted

vials are free of air bubbles. Medium pH should be 6.9  0.2

after sterilization.

b. Procedure:

1) Gently shake or rotate fermentation tubes or bottles showing gas, growth, or acidity. Using a sterile 3- or 3.5-mm-diam

loop or sterile wooden applicator stick, transfer growth from

each presumptive or confirmed fermentation tube or bottle to EC

broth (see Section 9221B.3).

2) Place all EC tubes in a water bath within 30 min after

inoculation. Incubate inoculated EC broth tubes in a water bath

at 44.5  0.2°C for 24  2 h. Maintain a sufficient water depth

in the water bath incubator to immerse tubes to the upper level

of the medium.

c. Interpretation: Gas production and growth in an EC broth

culture within 24  2 h or less is considered a positive thermotolerant coliform reaction. Failure to produce gas (with little or

no growth) constitutes a negative reaction. If multiple tubes are

used, calculate the MPN of thermotolerant coliforms from the

number of positive EC broth tubes (as described in 9221C).

When using only one tube for subculturing from a single presumptive bottle, report as presence or absence of thermotolerant

coliforms. If heavy growth occurs with no gas production, subject the culture to a thermotolerant coliform or E. coli test using

a different medium.

2. Thermotolerant Coliform Direct Test (A-1 Medium)

a. A-1 medium: This medium may be used for the direct

isolation of thermotolerant coliforms from source water, treated

wastewater and seawater, but not drinking water. Unlike EC

medium, A-1 medium does not require prior enrichment in a

presumptive medium for optimum recovery of thermotolerant

(fecal) coliforms. Preferably use a dehydrated, commercially

available medium.

Lactose..................................................................................... 5.0 g

Tryptone................................................................................. 20.0 g

Sodium chloride, NaCl............................................................ 5.0 g

Salicin ...................................................................................... 0.5 g

Polyethylene glycol p-isooctylphenyl ether*.......................... 1.0 mL

Reagent-grade water................................................................ 1 L

Heat to dissolve solid ingredients, add polyethylene glycol

p-isooctylphenyl ether, and adjust to pH 6.9  0.1. For 10-mL

samples, prepare double-strength medium so the final concentration of ingredients after sample addition is correct. Before

sterilization dispense, in fermentation tubes with an inverted

vial, sufficient medium to cover the inverted vial at least one-half

to two-thirds after sterilization. Close with metal or heat-resistant

plastic caps. Sterilize by autoclaving at 121°C for 10 min. Ensure

that inverted vials are free of air bubbles. Store in the dark at

room temperature for not longer than 7 d. Ignore formation of

precipitate during storage.

b. Procedure: Inoculate tubes of A-1 broth as directed in

9221B.2b). Incubate for 3 h at 35  0.5°C. Transfer tubes to a

water bath at 44.5  0.2°C and incubate for another 21  2 h.

c. Interpretation: Gas production in any A-1 broth culture

within 24 h or less is a positive reaction indicating the presence

of thermotolerant coliforms. Calculate the MPN of thermotolerant coliforms from the number of positive A-1 broth tubes (as

described in 9221C).

3. Bibliography

PERRY, C.A. & A.A. HAJNA. 1933. A modified Eijkman medium.

J. Bacteriol. 26:419.

PERRY, C.A. & A.A. HAJNA. 1944. Further evaluation of EC medium for

the isolation of coliform bacteria and Escherichia coli. Amer. J.

Pub. Health 34:735.

GELDREICH, E.E., H.F. CLARK, P.W. KABLER, C.B. HUFF & R.H. BORDNER.

1958. The coliform group. II. Reactions in EC medium at 45°C.

Appl. Microbiol. 6:347.

GELDREICH, E.E., R.H. BORDNER, C.B. HUFF, H.F. CLARK & P.W. KABLER.

1962. Type distribution of coliform bacteria in the feces of warmblooded animals. J. Water Pollut. Control Fed. 34:295.

GELDREICH, E.E. 1966. Sanitary significance of fecal coliforms in the

environment. FWPCA Publ. WP-20-3 (Nov.). U.S. Dep. Interior,

Washington, D.C.

ANDREWS, W.H. & M.W. PRESNELL. 1972. Rapid recovery of Escherichia

coli from estuarine water. Appl. Microbiol. 23:521.

OLSON, B.H. 1978. Enhanced accuracy of coliform testing in seawater by

a modification of the most-probable-number method. Appl. Microbiol. 36:438.

STANDRIDGE, J.H. & J.J. DELFINO. 1981. A-1 Medium: Alternative technique for fecal coliform organism enumeration in chlorinated

wastewaters. Appl. Environ. Microbiol. 42:918.

9221 F. Escherichia coli Procedure Using Fluorogenic Substrate

Escherichia coli is a member of the indigenous fecal flora

of warm-blooded animals. The presence of E. coli in water is

considered a specific indicator of fecal contamination and the

possible presence of enteric pathogens. Tests for E. coli are applicable to the analysis of drinking water, surface water, groundwater,

and wastewater. Testing for E. coli can be performed using the

multiple-tube procedure described here, by the membrane filter

method described in Section 9222G, or by chromogenic enzyme

* Triton X-100, Rohm and Haas Co., or equivalent.

MULTIPLE-TUBE FERMENTATION TECHNIQUE (9221)/Escherichia coli Procedure Using Fluorogenic Substrate

10
substrate tests described in Section 9223. Other E. coli procedures

are presented in 9221G.

For the E. coli test using EC-MUG medium, E. coli is defined

as the species of coliform bacteria possessing the enzyme

-glucuronidase and capable of cleaving the fluorogenic substrate 4-methylumbelliferyl--D-glucuronide (MUG) with the

corresponding release of the fluorogen within 24  2 h or less

when grown in EC-MUG medium at 44.5°C.

1. Escherichia coli Test (EC-MUG Medium)

Use EC-MUG medium to test for E. coli in a total coliformpositive culture. The procedure also can be used to confirm

presumptive total coliform colonies growing on solid media,

described in Section 9222B.4f.

a. EC-MUG medium:

Tryptose or trypticase.......................................................... 20.0 g

Lactose................................................................................... 5.0 g

Bile salts mixture or bile salts No. 3 ................................... 1.5 g

Dipotassium hydrogen phosphate, K2HPO4 ......................... 4.0 g

Potassium dihydrogen phosphate, KH2PO4...........................1.5 g

Sodium chloride, NaCl.......................................................... 5.0 g

4-Methylumbelliferyl--D-glucuronide (MUG).................... 0.05 g

Reagent-grade water.............................................................. 1 L

Add dehydrated ingredients to water, mix thoroughly, and heat

to dissolve. Before sterilization, dispense in tubes that do not

fluoresce under long-wavelength (366 nm) ultraviolet (UV) light.

An inverted tube is not necessary. Close tubes with metal or

heat-resistant plastic caps. Medium pH should be 6.9  0.2 after

sterilization for 15 min at 121°C.

b. Procedure:

1) Gently shake or rotate fermentation tubes or bottles showing growth, gas, or acidity. Using a sterile 3- or 3.5-mm-diam

metal loop or sterile wooden applicator stick, transfer growth

from the fermentation tube or bottle to EC-MUG broth.

2) Place all EC-MUG tubes in water bath within 30 min after

inoculation. Incubate inoculated EC-MUG tubes for 24  2 h in

a water bath maintained at 44.5  0.2°C. Maintain a sufficient

water depth in the water-bath incubator to immerse tubes to the

upper level of medium.

c. Interpretation: Examine all tubes exhibiting growth for

fluorescence using a 6W, 365 to 366 nm long-wavelength UV

lamp. The presence of bright blue fluorescence is considered a

positive result for E. coli. Growth in the absence of bright blue

fluorescence is considered a negative result. To aid in the interpretation of results and to avoid confusion of weak autofluorescence of the medium or glass tubes as a positive response,

include in the assay a positive control consisting of a known

E. coli (MUG-positive) culture, a negative control consisting of

a thermotolerant Klebsiella pneumoniae (MUG-negative) culture, and an uninoculated medium control. If multiple tubes are

used, calculate the MPN for E. coli from the number of positive

EC-MUG broth tubes as described in Section 9221C. When

using only one tube, or subculturing from a single presumptive

bottle or colony, report as presence or absence of E. coli.

2. Bibliography

FENG, P.C.S. & P.A. HARTMAN. 1982. Fluorogenic assays for immediate

confirmation of Escherichia coli. Appl. Environ. Microbiol.

43:1320.

HARTMAN, P.A. 1989. The MUG (glucuronidase) test for E. coli in food

and water. In A. Balows et al., eds., Rapid Methods and Automation

in Microbiology and Immunology. Proc. 5th Intl. Symp. on Rapid

Methods and Automation in Microbiology & Immunology, Florence, Italy, Nov. 4 – 6, 1987.

SHADIX, L.C. & E.W. RICE. 1991. Evaluation of -glucuronidase assay

for the detection of Escherichia coli from environmental waters.

Can. J. Microbiol. 37:908.

9221 G. Other Escherichia coli Procedures (PROPOSED)

For the E. coli test using the GAD reagent, E. coli is defined

as the species of coliform bacteria possessing the enzyme glutamate decarboxylase (GAD), and capable of producing an alkaline reaction within 4 h in a reagent containing glutamic acid

and a lytic agent. The procedure is used to test for E. coli after

prior enrichment in a medium used for the identification of

coliform bacteria. The procedure is particularly useful for determining the presence of MUG-negative strains of E. coli, some of

which are pathogenic (see also Section 9260F).

1. Escherichia coli Test (GAD Procedure)

Use the GAD procedure to test for E. coli in a total coliformpositive culture

a. GAD reagent:

L-Glutamic acid ................................................................. 1.0 g

Sodium chloride, NaCl.................................................... 90.0 g

Bromcresol green .............................................................. 0.05 g

Polyethylene glycol octylphenyl ether* ........................... 3.0 mL

Reagent-grade water ......................................................... 1 L

Add ingredients to water and mix thoroughly until all ingredients are dissolved. pH should be 3.4  0.2. The reagent is

stable for 2 months when stored at 5°C. It can be filter-sterilized

(0.2-m filter) and treated as a sterile solution.

b. Procedure:

1) Gently shake or rotate presumptive tubes or bottles showing

growth, gas, or acidity. Using a graduated pipet, transfer 5 mL broth

from the fermentation tube or bottle to 15-mL centrifuge tube.

2) Concentrate the bacterial cells from the broth by centrifugation at 2500 to 3000  g for 10 min. Discard supernatant and

resuspend cells in 5 mL phosphate buffer. Reconcentrate cells by

centrifugation (2500 to 3000  g, 10 min). Discard supernatant

* Triton X-100, Union Carbide Co., or equivalent.

MULTIPLE-TUBE FERMENTATION TECHNIQUE (9221)/Other Escherichia coli Procedures

11
and add 1.0 mL GAD reagent. Vigorously swirl tube to resuspend cells in GAD reagent.

3) Incubate tubes at 35°C and observe after 1 h. Tubes may be

incubated for a maximum of 4 h.

c. Interpretation: Examine all tubes for a distinct color change

from yellow to blue. The presence of a blue color is considered

a positive result for E. coli. To assist in interpretation of results,

incorporate in the assay a positive control consisting of a known

E. coli (GAD-positive) culture, a negative control consisting of

a known total coliform organism [e.g., Enterobacter cloacae

(GAD-negative)] and an uninoculated GAD reagent control. If

multiple tubes are used, calculate the MPN for E. coli from the

number of positive GAD tubes as described in Section 9221C.

When using only one tube or a single presumptive bottle, report

as presence or absence of E. coli.

2. Escherichia coli Test (Indole Production)

For the purposes of this test, E. coli is defined as the species

of coliform bacteria that can produce indole within 24  2 h

when grown in tryptone water at 44.5  0.2°C. However, it

should be noted that Klebsiella oxytoca is indole positive. Use

tryptone water and Kovacs’ reagent to test for E. coli in a total

coliform-positive culture.

a. Reagents:

1) Tryptone water:

Tryptone .........................................................................................20 g

Sodium chloride, NaCl ....................................................................5 g

Reagent-grade water........................................................................1 L

Add ingredients to water and mix thoroughly until dissolved.

Adjust pH to 7.5. Dispense 5-mL portions into tubes, cap, and

sterilize for 10 min at 115°C.

2) Kovacs’ reagent:

p-Dimethylaminobenzaldehyde.................................................. 5 g

Amyl alcohol (analytical grade)............................................... 75 mL

Hydrochloric acid, conc............................................................ 25 mL

Dissolve aldehyde in alcohol. Cautiously add acid to aldehyde-alcohol mixture and swirl to mix. Store in the dark at 4°C.

CAUTION: Reagent is corrosive and flammable. This reagent

should be pale yellow to light brown in color. Use of low-quality

amyl alcohol may produce a dark-colored reagent; do not use

such a reagent.

b. Procedure: Gently shake or rotate presumptive tubes or

bottles showing growth, gas, or acidity. Using a sterile 3- or

3.5-mm-diam metal loop or sterile wooden applicator stick,

transfer growth from presumptive fermentation tube or bottle to

a tube containing 5 mL tryptone water. Incubate inoculated

tryptone water tubes in a water bath or incubator maintained at

44.5°C for 24  2 h. After incubation, add 0.2 to 0.3 mL

Kovacs’ reagent to each tube of tryptone water.

c. Interpretation: Examine all tubes for the appearance of a

deep red color in the upper layer. The presence of a red color is

considered a positive result for E. coli. To assist in interpretation

of results, incorporate into the assay a positive control consisting

of a known E. coli (indole-positive) culture, a negative control

consisting of a known total coliform organism [e.g., Enterobacter cloacae (indole-negative)] and an uninoculated reagent control. If multiple tubes are used, calculate the MPN for E. coli

from the number of indole-positive tubes as described in Section

9221C. When using only one tube or a single presumptive bottle,

report as presence or absence of E. coli.

3. Bibliography

FIEDLER, J. & J. REISKE. 1990. Glutaminsauredecarboxylase-schnelltest

zur identifikation von Escherichia coli. Z. Ges. Hyg. Grenzgeb.

36:620.

RICE, E.W., C.H. JOHNSON, M.E. DUNNIGAN & D.J. REASONER. 1993.

Rapid glutamate decarboxylase assay for detection of Escherichia

coli. Appl. Environ. Microbiol. 59:4347. Errata. 1995. Appl. Environ. Microbiol. 61:847.

STANDING COMMITTEE OF ANALYSTS. 1994. Report on Public Health and

Medical Subjects No. 71, Methods for the Examination of Waters

and Associated Materials, The Microbiology of Water, Part

1—Drinking Water. HMSO Books, London, U.K.

RICE, E.W., C.H. JOHNSON & D.J. REASONER. 1996. Detection of Escherichia coli O157:H7 in water from coliform enrichment cultures.

Lett. Appl. Microbiol. 23:179.

MULTIPLE-TUBE FERMENTATION TECHNIQUE (9221)/Other Escherichia coli Procedures

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