Multiple-Tube Fermentation Technique: Coliforms

Soil Microbiology



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 aerobic and facultative anaerobic, Gram-negative, nonspore-forming, rod-shaped bacteria that ferment lactose with gas and acid formation within 48h at 35oC.

The standard test for the coliform group may be carried out either by the multiple-tube fermentation technique (through the presumptive-confirmed phases or completed test), by the membrane filter (MF) technique, or by the proposed chromogenic substrate coliform test. Each technique is applicable within the limitations specified and with due consideration of the purpose of the examination.

When multiple tubes are used in the fermentation technique, results of the examination of replicate tubes and dilutions are reported in terms of the Most Probable Number (MPN) of organisms present. This number, based on certain probability formulas, is an estimate of the mean density of of coliforms in the sample. Coliform density provides the best assessment of water treatment effectiveness and the sanitary quality of untreated water.

The precision of each test depends on the number of tubes used. The most satisfactory information will be obtained when the largest sample inoculum shows no gas in all 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 multiple dilutions. The number of sample portions selected will be governed by the desired precision of the result. 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 clumping of bacterial cells occurs, the MPN value will be an underestimate of the actual bacterial density.

Standard Total Coliform Fermentation Technique

1. Presumptive Phase:

Use lauryl tryptose broth in the presumptive portion of the multiple-tube test.
a. Reagents and culture medium: Add dehydrated products to water, mix thoroughly, and heat to dissolve. pH should be 6.8 +/- 0.2 after sterilization. Before sterilization, dispense sufficient medium, in fermentation tubes with an inverted vial, to cover inverted vial at least one-half to two-thirds after sterilization. Alternatively, omit inverted vial and add 0.01 g/L bromcresol purple to presumptive medium to determine acid production, the indicator of a positive result in this part of the coliform test. Close tubes with metal or heat-resistant plastic caps.
b. Procedure:
1.Arrange the fermentation tubes in rows of five tubes each in a test tube rack. The number of five-tube rows and the sample volumes selected depend upon 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.).
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 +/- 0.5oC. After 24 +/- 2h swirl each tube or bottle gently and examine it for heavy growth, gas, and acidic reaction (shades of yellow color) and, if no gas or acidic growth has formed, reincubated and reexamine at the end of 48 +/- 3h. Record Presence or absence of heavy growth, gas, and acid production. If the inner vial is ommited, growth with acidity signifies a positive presuptive reaction.
c. Interpretation: Production of gas or acidic growth in the tubes or bottles within 48 +/- 3h constitutes a positive presumptive reaction. Submit tubes with a positive presumptive reaction to the confirmed phase.
The absence of acidic growth or gas formation at the end of 48 +/- 3h of incubation constitutes a negative test. An arbitrary 48-h limit for observation doubtless excludes occasional members of the coliform group that grow very slowly.

The Presumptive Phase. The three MPN tubes on the left are positive (yellow) and the two on the right are negative (blue-green). The medium is lauryl tryptose broth.

2. Confirmed Phase

a. Culture Medium: Use brilliant green lactose bile broth fermentation tubes for the confirmed phase.
Add dehydrated ingredients to water, mix thoroughly, and heat to dissolve. pH should be 7.2 +/- 0.2 after sterilization. Before sterilization, dispense, in fermentation tubes with an inverted vial, sufficient medium to cover inverted vial at least one-half to two-thirds after sterilization. Close tubes with metal or heat-resistant plastic caps.
b. Procedure: Submit all primary tubes or bottles showing heavy growth, any amount of gas, or acidic growth within 24h of incubation to the confirmed phase. If active fermentation or acidic growth appears in the primary tube earlier than 24h, transfer to the confirmatory medium, preferably without waiting for the full 24h period to elapse. If additional primary tubes or bottles show active fermentation or acidic growth at the end of a 48h incubation period, submit these to the confirmed phase.
Gently shake or rotate primary tubes or bottles showing gas or acidic growth to resuspend the organisms. With a sterile metal loop 3 mm in diameter, transfer one loopful of culture to a 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 tube for 48 +/- 3 h at 35 +/- 0.5oC.
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. Calculate the MPN value from the number of positive brilliant green lactose bile tubes.
c. Alternative procedure: Use this alternative only for polluted water or wastewater known to produce 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 only after 48 h.

3. Completed Phase

To establish the presence of coliform bacteria and to provide quality control data, use the completed test on at least 10% of positive confirmed tubes. Double confirmation into brilliant green lactose bile broth for total coliforms and EC broth for fecal coliforms may be used. Consider positive EC broth elevated temperature (44.5oC) results as a positive completed test response. Parallel positive brilliant green lactose bile broth culture with negative EC broth cultures indicate the presence of nonfecal coliforms and must be submitted to the completed test procedures to validate the presence of coliforms.
a. Culture media and reagents: Add ingredients to water, mix thoroughly, and heat to dissolve. pH should be 6.8 +/- 0.2 after sterilization. Before sterilization, dispense in screw-capped tubes. After sterilization, immediately place tubes in an inclined position so that the agar will solidify with a sloped surface. Tighten screw caps after cooling and store in a protected, cool storage area.
b. Procedure:
1. Using aseptic technique, streak one LES Endo agar 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 insure presence of some discrete colonies separated by at least 0.5 cm. Observe the following precautions when streaking plates to obtain a high proportion of successful isolations if coliform organisms are present: (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 end of loop or needle into the liquid in the tube to a depth of approximately 0.5cm; and (d) streak plate for isolation with curved section of the needle in contact with the agar to avoid a scratched or torn surface. Flame the loop between second and third quadrants to improve colony isolation.
Incubate plates (inverted) at 35 +/- 0.5oC for 24 +/- 2h.
2. The colonies developing on LES Endo agar are defined as typical ( pink to dark red with a green metallic surface sheen); atypical (pink, red, white, or colorless colonies without sheen) after 24 h incubation; or negative (all others). 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 consist of organisms of the coliform group, and transfer growth from each isolate to a single-strength lauryl tryptose broth fermentation tube and onto a nutrient agar slant. (The latter is unnecessary for drinking water samples.)

4. Estimation of Bacterial Density

a. Precision of Fermentation Tube Test: Unless a large number of sample portions is examined, the precision of the fermentation tube test is rather low. For example, even when the sample contains 1 coliform organism/mL, about 37% of 1-mL tubes may be expected to yield negative results because of random distribution of the bacteria in the sample. When five tubes, each with 1 mL sample, are used under these conditions, a completely negative result may be expected less than 1% of the time.
Even when five fermentation tubes are used, the precision of the results obtained is not of a high order. Consequently, exercise great caution when interpreting the sanitary significance of coliform results obtained from the use of a few tubes with each sample dilution, especially when the number of samples from a given sampling point is limited.
b. Computing and Recording of MPN: To calculate coliform density, compute in terms of the Most Probable Number. The MPN values are given in Table 1 (as an example). Included in this table is the 95% confidence limits for each MPN value determined. If the sample volumes used are those found in the tables, report the value corresponding to the number of positive and negative results in the series as the MPN/100 mL or report as total or fecal coliform presence or absence.
When the series of decimal dilutions is different from that in the table, select the MPN value from the table for the combination of positive tubes and calculate according to the following formula:
MPN value(from table) x (10/largest volume tested) = MPN/100mL
# of tubes giving positive reaction out of 10 of 10mL each MPN Index/100mL 95% Confidence Limits (approximate)
Lower Upper
0 less than 1.1 0 3.0
1 1.1 0.03 5.9
2 2.2 0.26 8.1
3 3.6 0.69 10.6
4 5.1 1.3 13.4
5 6.9 2.1 16.8
6 9.2 3.1 21.1
7 12.0 4.3 27.1
8 16.1 5.9 36.8
9 23.0 8.1 59.5
10 greater than 23.0 13.5 infinite

When more than three dilutions are used in a decimal series of dilutions, use the results from only three of these in computing the MPN. To select the three dilutions to be used in determining the MPN index, choose the highest dilution that gives positive results in all five portions tested (no lower dilution giving any negative results) and the two next succeeding higher dilutions. Use the results at these three volumes 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 MPN index/100 mL
a 5/5 5/5 2/5 0/5 5-2-0 5000
b 5/5 4/5 2/5 0/5 5-4-2 2200
c 0/5 1/5 0/5 0/5 0-1-0 20

In c, select the first three dilutions so as to include the positive result in the middle dilution.

5. Development of a Microtiter MPN procedure

A most-probable-number microtitration technique for isolating fecal coliforms from soil was developed. A correlation coefficient of 0.86, with a 95% confidence interval of 0.76 to 0.92, was obtained when this technique was compared with the standard elevated temperature fecal coliform most-probable-number procedure. From Microtechnique for Isolating Fecal Coliforms in Soil. Peter Hartel and Charles Hagedorn. Applied and Environmental Microbiology, Aug 1983, p 518-520.

The microtiter plate containing lauryl tryptose broth. The dilution series was 1:2, and the first four rows are positive (yellow). In the fifth row, 4 of 8 wells are positive and 4 are negative (blue-green).

A 48-prong multipoint inoculator was used to inoculate 48 wells onto a plate of Eosin Methylene Blue Agar (EMB). Note the greenish metallic sheen on the colonies that is typical of E.coli.

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