SOP: Determination of Coliform Count in Water (Presumptive, Confirmed and Completed Tests)

GIFSQ — Global Institute for Food Safety & Quality

Principle

Escherichia coli is one of the few bacteria able to ferment lactose with the production of acid and gas. If acid and gas are produced in lactose broth inoculated with the water being tested, this is presumptive evidence of sewage pollution — but several non-intestinal bacteria also give this result, so a presumptive test must be confirmed. The coliform count therefore proceeds in three stages: the presumptive test (gas production in lactose broth), the confirmed test (typical colonies on eosin-methylene blue agar and gas in brilliant green lactose bile broth), and the completed test (pure culture of Gram-negative rods producing gas in lactose broth), carried out when the confirmatory test does not give clear-cut results.

Apparatus required

• Five large test tubes containing 10 ml of double-strength lactose broth with inverted Durham tubes
• Two test tubes containing 10 ml of single-strength lactose broth with inverted Durham tubes
• Pour plates of sterile eosin-methylene blue agar (EMB agar, Levine’s)
• Tubes of brilliant green lactose bile broth (2%) with inverted Durham tubes
• Nutrient agar slants
• Tubes of single-strength lactose broth with Durham tube
• Sterile 1 ml and 10 ml pipettes (the book prints “1.1 ml and 10 ml”, carried as printed)
• Straight inoculating needle
• Incubator set at 37°C
• Microscope with Gram-staining materials

Reagents

a. Lactose broth — take 3 g of beef extract, 5 g of peptone and 1,000 ml of distilled water; heat slowly in a water bath with stirring until the solid materials dissolve; add 5 g of lactose and make up the volume to 1,000 ml with distilled water; adjust the pH so that after sterilization it is between 6.8 and 7.0; transfer to fermentation tubes containing Durham tubes and sterilize at 15 psig for 15 min; remove from the autoclave and cool rapidly. Repeat this procedure on three successive days; the total time of exposure to heat in the autoclave should not exceed 60 min. For the double-strength broth, use double the quantities of the ingredients for the same quantity of water and transfer 10 ml into each tube.

b. Eosin-methylene blue (EMB) agar — mix 10 g of peptone, 2 g of dipotassium phosphate and 15 g of agar with 1,000 ml of distilled water; note the volume by marking with a glass marking pencil; heat on a water bath until dissolved and make up the loss due to evaporation with distilled water; transfer aliquots (100 or 200 ml) into flasks or bottles and sterilize in the autoclave at 15 psig for 15 min. Just before use, add to each 100 ml of the melted agar: 5 ml of 20% sterile lactose solution, 2 ml of 2% aqueous eosin yellowish solution, and 1.3 ml of 0.5% aqueous methylene blue solution (the book prints “0.5°%%”, carried here as 0.5%). Alternatively, all ingredients may be added to the stock agar at the time of preparation and sterilized together; decolourization occurs during autoclaving, but the colour reappears on cooling.

c. Brilliant green lactose bile broth — prepare solution A by dissolving 10 g each of peptone and lactose in 500 ml of water; dissolve 20 g of dehydrated ox-gall in 200 ml of distilled water and adjust the pH of the solution between 7 and 7.5 (solution B); mix solutions A and B and make up with distilled water to approximately 975 ml; adjust the pH to 7.4; add 13 ml of 0.1% solution of brilliant green in distilled water and make up to 1,000 ml; filter through cotton, distribute in 5 ml quantities in fermentation tubes (6 × 5/8 in.) and sterilize at 10 psig for 15 min. The pH should be not less than 7.1 and not more than 7.4.

d. Nutrient agar slants — dissolve 3 g of yeast extract or meat extract and 5 g of peptone in 1,000 ml of distilled water on a water bath and cool; adjust the pH to 7.4 using phenol red as the indicator; weigh 25 g of agar (washed, shredded or powdered) — if not shredded, cut it into pieces, tie in a muslin bag, wash in running water for 15 min, squeeze out the excess water and add to the yeast peptone mixture; autoclave at 15 psig for 20 min and immediately filter through paper pulp in a Buchner funnel or through a wad of cotton; test the pH of the filtrate at 50°C and adjust to 7 if necessary; dispense into tubes and allow to solidify as slants, then plug the tubes and autoclave at 15 psig for 20 min (final pH at room temperature should be 7.2).

Sample preparation

Use the water sample as collected; no preparation beyond shaking is needed.

Procedure

Presumptive test:

Using a 10 ml pipette, transfer 10 ml portions of the water being tested to each of the tubes containing double-strength lactose broth

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Into the two single-strength lactose broth tubes, place 0.1 ml and 1 ml of water respectively

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Incubate at 37°C and examine after 24 and 48 hr

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Mark a tube positive when gas fills 10% or more of its Durham tube

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Record the pattern of positive tubes for the Results interpretation

Confirmed test:

Select a positive presumptive tube — preferably the highest dilution showing gas (0.1 or 1 ml if these are positive), and prefer a tube showing gas in 24 hr over one showing gas only after 48 hr

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Flame a straight inoculating needle, insert it into the positive tube holding the tube in a slanted position to avoid picking up any scum or surface membrane, and streak on the EMB agar plate

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Flame the inoculating needle again and streak a second time to ensure a distribution giving discrete colonies

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Inoculate a loopful from the same tube, or another positive tube of the series, into a tube of the brilliant green lactose bile broth

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Incubate at 37°C and examine at the end of 24 hr

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Examine the plate and the broth culture and record the colony types observed: typical E. coli colonies have dark to black centres, button-like in appearance, and are often surrounded by a greenish metallic sheen

Completed test (made when the positive confirmatory test does not give clear-cut results):

Transfer a well-isolated, typical or atypical E. coli colony from the EMB agar plate to a nutrient agar slant and a tube of lactose broth

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Incubate both for 24 hr at 37°C

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Make a stained mount of the nutrient agar culture: a completed test shows a pure culture of Gram-negative short rods, and gas is produced in the lactose broth tube

Results

Presumptive test: record the results indicating the probable degree of pollution presumed to be present. Gas in all the tubes, including those receiving 1 and 0.1 ml of water, indicates gross contamination; a sample showing only one positive 10 ml tube should be classed as questionable.

Confirmed test: gas in the brilliant green lactose bile broth is taken as confirmatory evidence that E. coli is present. Report the colonies on the plate as typical E. coli colonies present, and report also any atypical colonies — these may be Aerobacter aerogenes or intermediate coliforms.

Precautions

• Several non-intestinal bacteria ferment lactose with acid and gas, so never report a presumptive test as confirmed.
• For the confirmed test, select the positive tube at the highest dilution showing gas, and prefer a 24 hr positive over a 48 hr positive.
• When inserting the needle into the positive tube, hold the tube slanted so that scum or surface membrane is not picked up.
• Atypical colonies (Aerobacter aerogenes or intermediate coliforms) must be reported as such, not as E. coli.

References

Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.

Manual of Methods for the Examination of Water, Sewage and Industrial Wastes, Special Report No. 47, Indian Council of Medical Research, New Delhi (1963) — cited in the book.

Standard Methods for the Examination of Water and Wastewater, 13th edn., American Public Health Association, Washington, D.C. (1971) — cited in the book.

Taylor, E.W., The Examination of Water and Water Supplies, 7th edn., J. & A. Churchill Ltd., London (1958) — cited in the book.