SOP: Determination of Coliform Count by the Membrane Filter Technique

GIFSQ — Global Institute for Food Safety & Quality

Principle

A measured volume of the sample is filtered through a thin bacteria-retaining membrane (the book prints “150 um”, carried as printed); the bacterial cells are trapped on the surface. The membrane is then transferred onto a thin absorbent pad saturated with a selective medium — a modified Endo medium for coliform organisms — and incubated so that the trapped bacteria grow into countable colonies. The method is more direct and quicker than the traditional multiple-tube tests (giving results in 18–24 hr) and permits the use of larger volumes of sample, which renders the results more representative.

Apparatus required

• Seamless funnel fastening to a receptacle bearing a porous plate for supporting the filter membrane, with a convenient locking device so that the membrane is securely held without mechanical damage and all the fluid passes through it during filtration
• Filtration (Buchner) flask with a rubber stopper, and a trap between the flask and the vacuum source
• Filter membranes providing full bacterial retention, stability in use, and free from chemicals inimical to the growth of organisms
• Absorbent paper pads and high-grade filter paper free from sulphites or other substances inimical to the growth of the organisms, cut into circles of 4.8 cm diameter and of thickness sufficient to absorb 1.8 to 2.2 ml of nutrient
• Sterile forceps
• Sterile Petri dishes, 10 cm in diameter and 60 × 15 mm flat dishes
• Sterile pipettes
• Incubator set at 37°C
• Low-power lens (10–15 diameters) for counting by reflected light

Sterilization: wrap the two parts of the filter assembly separately in heavy wrapping paper and sterilize by boiling, autoclaving or ultra-violet radiation. For the membranes, remove the paper separators (not the absorbent paper pads), place 10 to 12 pads in 10 cm diameter Petri dishes or wrap in paper and sterilize by autoclaving at 15 psig for 10 min. Sterilize the filter paper circles along with the membranes.

Reagents

a. Lauryl tryptose broth (enrichment medium) — dissolve 20 g of tryptose, 5 g of lactose, 2.75 g each of dipotassium hydrogen phosphate (K₂HPO₄) and potassium dihydrogen phosphate (KH₂PO₄), 5 g of sodium chloride and 0.1 g of sodium lauryl sulphate in 1,000 ml of water (the book’s printed formulae for the phosphates are garbled, carried here in standard form); adjust the pH so as to be approximately 6.8 after sterilization; transfer 10 ml to each tube and sterilize at 10 psig for 15 min.

b. Modified Endo medium — weigh 10 g of tryptose or polypeptone, 5 g of thiopeptone or thiotone, 5 g of cositone or trypticase, 1.5 g of yeast extract, 12.5 g of lactose (the book prints “12.5 of lactose”, carried here as grams), 5 g of sodium chloride, 4.375 g of dipotassium hydrogen phosphate (K₂HPO₄), 1.375 g of potassium dihydrogen phosphate (KH₂PO₄), 0.05 g of sodium lauryl sulphate, 0.1 g of sodium desoxycholate, 2.1 g of sodium sulphite and 1.05 g of basic fuchsin; dissolve in 1 litre of water containing 20 ml of 95% ethanol (the book’s printed phosphate formulae are garbled, carried here in standard form); heat the medium to boiling point, promptly remove from the heat and cool to below 45°C — do not sterilize by autoclaving; the final pH should be between 7.1 and 7.3. Store the finished medium in the dark at 2–10°C and discard the unused medium after 96 hr.

c. LES Endo agar (agar-based alternative to the liquid medium) — weigh 1.2 g of yeast extract, 3.7 g of casitone or trypticase, 3.7 g of thiopeptone or thiotone, 7.5 g of tryptose, 9.4 g of lactose, 3.3 g of dipotassium hydrogen phosphate (K₂HPO₄), 1.0 g of potassium dihydrogen phosphate (KH₂PO₄), 3.7 g of sodium chloride, 0.1 g of sodium desoxycholate, 0.05 g of sodium lauryl sulphate, 1.6 g of sodium sulphite, 0.8 g of basic fuchsin and 15 g of agar; rehydrate in 1 litre of distilled water containing 20 ml of 95% ethanol; bring to a boil and cool to 45–50°C; pipette 4 ml into each 6 cm diameter Petri dish (for dishes of other size, add sufficient volume to give an equivalent depth). Store in the dark at 2–10°C; the plates may be stored up to 2 weeks; do not expose the plates to direct sunlight.

Sample preparation

Use the water sample as collected; choose a volume that will yield a countable membrane.

Procedure

Sterilize the filtration assembly before use; attach the filter holder to the rubber stopper, insert in the vacuum flask and connect the flask to the vacuum source

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Using sterile forceps, transfer a sterile filter pad to the platform base of the filter unit; place the filter with the ruled side up; place the matched funnel unit over the filter disc, making sure it is clamped firmly by the scissors-type clamp

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Pour the sample into the funnel and filter by maintaining a slight vacuum; rinse the funnel with two 30 ml portions of sterile water

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Add 1.5–2 ml of lauryl tryptose broth to a sterile absorbent pad in a sterile culture plate to saturate the pad, and carefully remove any surplus liquid

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Aseptically remove the filter membrane from the filtration unit and place it on the filter paper soaked with the medium, excluding any air between the two discs

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Incubate the filter membrane in the enrichment medium for 2 hr at 37°C

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Place a fresh sterile absorbent pad in a sterile Petri dish (60 × 15 mm, flat) and pipette onto it 2 ml of modified Endo medium

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Transfer the filter disc to the surface of the fresh pad enriched with Endo medium with the help of a sterile forceps, taking care to exclude air

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Invert the dish and incubate at 37°C for 24 hr; alternatively, where the agar-based medium (LES Endo agar) is used instead of liquid medium, remove the filter membrane from the enrichment pad and roll it onto the surface of the agar, taking care to prevent entrapment of air, invert the dish and incubate as before

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Make a direct estimation of the number of coliform organisms by counting every dark colony that shows a greenish metallic surface sheen, under a low-power lens (10–15 diameters) by reflected light

Calculation

Total coliform colonies per 100 ml = (coliform colonies counted × 100) ÷ (ml of sample filtered).

• Where, total coliform colonies per 100 ml = coliform density in the sample;
• coliform colonies counted = number of dark colonies with greenish metallic lustre on the membrane;
• ml of sample filtered = volume of water passed through the membrane.

Precautions

• The success of the method depends on the use of effective differential or selective media that allow easy identification of the colonies.
• Exclude air between the membrane and the pad or agar surface when transferring the filter disc; trapped air prevents contact with the medium.
• Wash the funnel through with two separate 30 ml portions of sterile water after filtration.
• Count under low power (10–15 diameters) by reflected light only.
• Through slight variation in technique and sampling equipment, the same method has wide potential application in foods, industrial bacteriology and air contamination.

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.