SOP: Determination of Faecal Coliform Count by the Membrane Filter Technique

GIFSQ — Global Institute for Food Safety & Quality

Principle

If the total coliform count is made by the membrane filter technique, the faecal coliform count may be made by the same procedure using an enriched lactose medium (M-FC medium) and incubating the dishes at 44.5 ± 0.2°C (the book prints “44.5 + 0.2° C”, carried as printed). The elevated incubation temperature, together with the rosolic acid salt in the medium, suppresses the growth of non-faecal coliforms, so the faecal coliform colonies develop a blue colour while non-faecal colonies remain grey to cream.

Apparatus required

• Filtration assembly as for the total coliform membrane filter procedure (funnel, receptacle with porous plate, Buchner flask, vacuum source with trap), sterilized as before
• Filter membranes and absorbent paper pads as for the total coliform procedure
• Sterile forceps
• Tight-fitting 60 × 15 mm plastic dishes — sterilize by immersion in 70% alcohol for 30 min or by ultra-violet radiation; if sterilized using alcohol, wipe with a sterile towel and close employing sterile techniques
• Sterile pipettes
• Water-proof bags for protecting the dishes
• Water bath maintained at 44.5 ± 0.2°C (the book prints “44.5 + 0.2° C”, carried as printed)
• Low-power (10–15 magnifications) binocular wide-field dissecting microscope

Reagents

a. M-FC medium — weigh 10 g of tryptose, 5 g of proteose peptone No. 3 or polypeptone, 3 g of yeast extract, 5 g of sodium chloride, 12.5 g of lactose, 1.5 g of bile salt No. 3 or bile salts mixture and 0.1 g of aniline blue (the book prints “0.1’g of aniline|blue”, carried here as 0.1 g); dissolve in 1 litre of water containing 10 ml of 1% rosolic acid in 0.2 N sodium hydroxide; heat the medium to the boiling point and cool quickly to 45°C — do not autoclave, as the rosolic acid decomposes; the final pH should be 7.4. Store the finished medium at 2–10°C and discard the unused medium after 4 days. The stock solution may be stored at 2–10°C for 2 weeks; discard earlier if the colour changes from dark red to muddy brown.

Sample preparation

Use the water sample as collected. The volume of water sample chosen should yield a countable membrane; in addition, select two additional quantities representing one-tenth and ten times this quantity respectively. Sample quantities yielding between 20 and 60 faecal coliform colonies give the greatest accuracy of density determination.

Procedure

Prepare the assembly and filter the sample as in the total coliform membrane filter procedure

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Place a sterile absorbent pad in each culture dish and pipette approximately 2 ml of M-FC medium onto it; carefully remove the surplus liquid from the dish

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Place the filter membrane through which the water has been filtered on the pad impregnated with the medium, and cover the dish

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Place the dishes in water-proof bags for protection and submerge the bags below the water level in the bath maintained at 44.5 ± 0.2°C (the book prints “44.5 + 0.2° C”, carried as printed)

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Incubate for 24 hr — begin the incubation no later than 30 min after filtration

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Examine the membranes: faecal coliform colonies appear blue, while non-faecal colonies appear grey to cream; the background colour of the filter membrane varies from a yellowish cream to faint blue depending upon the age of the rosolic acid salt reagent

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Count the colonies with the aid of the low-power (10–15 magnifications) binocular wide-field dissecting microscope — the number of colonies should be in the range of 20–60 because of the larger colony growth on M-FC medium

Calculation

Calculate as in the total coliform membrane filter count: faecal coliforms per 100 ml = (colonies counted × 100) ÷ (ml of sample filtered).

• Where, faecal coliforms per 100 ml = faecal coliform density in the sample;
• colonies counted = number of blue colonies on the membrane;
• ml of sample filtered = volume of water passed through the membrane.

Report as faecal coliforms per 100 ml.

Precautions

• Start the incubation within 30 min of filtration.
• Choose the sample volumes so that the membranes yield 20–60 faecal coliform colonies, with additional membranes at one-tenth and ten times the chosen volume.
• Usually few non-faecal coliform colonies are observed on M-FC medium because of the elevated incubation temperature and the addition of the rosolic acid salt reagent to the medium — do not mistake grey to cream colonies for faecal coliforms.
• The background colour of the membrane varies with the age of the rosolic acid salt reagent; discard the medium if the stock changes from dark red to muddy brown.

References

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

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