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9221 MULTIPLE-TUBE FERMENTATION TECHNIQUE FOR MEMBERS OF THE COLIFORM GROUP*

 

* Approved by Standard Methods Committee, 1994.

 

9221 B. Standard Total Coliform Fermentation Technique

 

1. 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 (20ºC) before use. Discard tubes showing growth and/or bubbles.

    a. Reagents and culture medium:

    1) Lauryl trytose 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. 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.

    Make lauryl tryptose broth of such strength that adding 100-mL, 20-mL, or 10-mL portions of sample to medium will not reduce ingredient concentrations below those of the standard medium. Prepare in accordance with Table 9221:I.

    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 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.).

    In 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 ± 0.5C. After 24 ± 2 h swirl each tube or bottle gently and examine if for growth, gas, and acidic reaction (shades of yellow color) and, if no gas or acidic reaction is evident, reincubate and reexamine at the end of 48 ± 3 h. Record presence or absence of growth, gas, and acid production. If the inner vial is omitted, growth with acidity signifies a positive presumptive reaction.

    c. Interpretation: Production of an acidic reaction or gas in the tubes or bottles within 48 ± 3 h constitutes a positive presumptive reaction. Submit tubes with a positive presumptive reaction to the confirmed phase (9221B.2).

 

 

 

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

 

    The absence of acidic reaction 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.2). An arbitrary 48-h limit for observation doubtless excludes occasional members of the coliform group that grow very slowly (see Section 9212).

 

 

 

 

2. Confirmed Phase

 

    a. Culture medium: Use brilliant green lactose bile broth fermentation tubes for the confirmed phase.

    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. 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 presumptive tubes or bottles showing growth, any amount of gas, or acidic reaction with 24 ± 2 h of incubation to the confirmed phase. If active fermentation or acidic reaction appears in the presumptive tube earlier than 24 ± 2 h, transfer to the confirmatory medium; preferably examine tubes at 18 ± 1 h. 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.

    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 or 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 tube 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 (e.g., 6 ± 1 h, 24 ± 2 h) within 48 ± 3 h constitutes a positive confirmed phase. Calculate the MPN value from the number of positive brilliant green lactose bile tubes as described in Section 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 only after 48 h.

 

 

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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 (see Figure 9221:1). Simultaneous inoculation into brilliant green lactose bile broth for total coliforms and EC broth for fecal coliforms (see Section 9221E below) or EC-MUG broth for Escherichia coli may be used. Consider positive EC and EC-MUG broths elevated temperature (44.5°C) results as a positive completed test response. Parallel positive brilliant green lactose bile broth cultures with negative EC of EC-MUG broth cultures indicate the presence of nonfecal coliforms.

    a. Culture media and reagents:

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

    4) Gram-stain reagents:

    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.

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

    b. Procedure:

    1) Using aseptic technique, streak one LES Endo agar (Section 9222B.2) 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 0.5 cm; 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 loop between 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 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.)

    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 inserted) 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 culture; include a gram-positive and 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 and 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 over-decolorize. 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; gram-negative organisms are red. Results are acceptable only when controls have given proper reactions.

    c. Interpretation: Formation of gas in the secondary tube of lauryl trypotose 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 coiform group.

 

 

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4. 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 & W.M. 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 and 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).

 

 

©Standard Methods for the Examination of Water and Wastewater. 20th Ed. American Public Health Association, American Water Works Association, Water Environment Federation.