SOP: Determination of Plate Count in Water

GIFSQ — Global Institute for Food Safety & Quality

Principle

A measured amount of the water is mixed with a sterile, solidifiable culture medium (nutrient agar) and incubated for a fixed time so that the bacteria present grow into visible colonies, which are then counted. The plate count measures the general bacterial purity of the water and is used for treatment purposes.

Apparatus required

• Nutrient agar sterilized in 15 ml quantities in plugged tubes
• Sterilized Petri dishes (about 10 or 12 cm in diameter)
• Dilution bottles each containing 90 ml of quarter-strength Ringer’s solution, or tubes each containing 9 ml of sterile quarter-strength Ringer’s solution
• Sterilized 1 ml and 10 ml pipettes
• Boiling water bath (for melting the agar)
• Incubator set at 37°C
• Magnifying glass or a colony counter

Reagents

a. Nutrient agar — 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; add 15 ml to each tube, plug the tubes and autoclave at 15 psig for 20 min (final pH at room temperature should be 7.2). When required for use, melt the tubes in boiling water and cool to 42–45°C. Unless used within one week of preparation, store in the refrigerator to retain the moisture in the medium.

b. Quarter-strength Ringer’s solution — prepare the full-strength solution from 9 g of sodium chloride, 0.42 g of potassium chloride, 0.48 g of calcium chloride and 0.2 g of sodium bicarbonate in 1,000 ml of distilled water (the book prints “0.48 ¢” and “0.2 ¢”, carried here as grams); dilute 250 ml of this solution with 750 ml of distilled water and sterilize by autoclaving at 15 psig for 20 min. Dispense 90 ml into each dilution bottle or 9 ml into each dilution tube.

Sample preparation

Shake the sample vigorously twenty-five times to distribute the bacteria fairly evenly throughout the sample, then remove the stopper and flame the mouth of the bottle.

Procedure

Melt the required number of nutrient agar tubes in boiling water until the medium completely melts, then cool to 45–50°C and keep the medium liquid at this temperature

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Choose the amount of sample according to the anticipated purity: where the water is expected to be unpolluted, withdraw 2 ml with a sterile pipette and introduce it into a sterile Petri dish; where the water is expected to be polluted, prepare decimal dilutions by adding 1 ml of the well-shaken sample to 9 ml of quarter-strength Ringer’s solution and continuing in the same way, using a fresh sterilized pipette for each dilution and ensuring thorough mixing

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Inoculate at least two Petri dishes with 1 ml each of the different dilutions

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Pour at least 10 ml of the melted nutrient agar, at 42–45°C, over the water in the Petri dishes

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Blend the sample into the medium with quick back-and-forth and circular swirling, keeping the dish level on the table

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After mixing, leave the plates on the table until the medium solidifies, then place them inverted in the incubator

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Incubate at 37°C for 24 ± 3 hr (the book prints “24 + 3 hr”, carried here as 24 ± 3 hr)

Results and interpretation

Remove the plate from the incubator and count the colonies with the aid of a magnifying glass; a suitable colony counter may be used. The best plates to count are those with 30–300 colonies — count all the colonies in these. In plates with fewer than 30 colonies, count the plate inoculated with the undiluted water. Treat each spreader as one colony, and reject any plate on which spreaders cover half of the plate or more. Where all the plate counts for the dilutions inoculated are over 300, a fair approximation may be obtained by counting the colonies in a few sectors and calculating the total.

The bacterial colonies growing on nutrient agar are largely derived from soil, dust, sewage and other extraneous materials that have gained access to the water, so the number indicates the “cleanness” or “dirtiness” of the sample. A variety of colonies of different sizes and shapes, presenting a disagreeable appearance and an unpleasant odour, is suggestive of undesirable pollution. Surface waters generally show a high plate count, especially after rainfall following a period of dry weather; high counts may also be obtained with waters from newly sunk wells, decreasing gradually with continuous pumping. A rise in the plate count of a deep well or treated water may afford an early indication of contamination. A correctly filtered and chlorinated water supply should give a total count of no more than 10 per ml.

Calculation

Multiply the observed count by the dilution factor and record the result to only two significant figures. Report the result as “plate count per ml at 37°C in 24 hr.”

• Where, plate count per ml = observed colony count × dilution factor.

Precautions

• Use a fresh sterilized pipette for each dilution and mix each dilution thoroughly.
• Record load of spreaders honestly: count each spreader as one colony, and discard any plate on which spreaders cover half the plate or more.
• Use the plates with 30–300 colonies for counting; avoid estimating from overcrowded plates except by sector counting where no better plate exists.
• Judge a rise against the normal plate count of that particular water; a rise in the count of a deep well or treated water may indicate contamination or falling purification efficiency.

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.