SOP: Direct Microscopic Count of Bacteria in Tomato Products

GIFSQ — Global Institute for Food Safety & Quality

Principle

High bacterial counts indicate secondary spoilage that may have occurred in the factory through breakdown or careless cleaning methods. The rod-shaped bacteria are counted directly under the microscope on the same mount as that prepared for yeasts and spores, and the count per unit volume is computed from the known area examined and the chamber depth. Bacterial counts have no direct relationship with the mould count.

Apparatus required

• Blood counting cell (chamber depth 1/10 mm), preferably ruled in the Thoma or the old Neubauer system; clean so as to give good Newton’s rings
• Cover glass for the counting cell
• Compound microscope giving a magnification of about 500 diameters
• Graduated cylinder
• Beaker

Sample preparation

Prepare the diluted sample and mount it exactly as for the direct microscopic count of yeasts and spores (fill a graduated cylinder with water to the 20-ml mark, add the sample to the 30-ml mark, shake vigorously for 15–20 sec, stir, stand 3–5 sec, mount a drop on the central disc and cover immediately).

Procedure

Allow the sample to stand for not less than 15 min after mounting before counting

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If the counting cell used is ruled by the Thoma or old Neubauer system, count and record the number of rod-shaped bacteria having a length more than 1.5 times their width, in an area comprising five small-sized squares

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Count the number in five such areas, preferably one from near each corner of the ruled portion of the slide and one from near the centre (each of these groups has an area of 0.0125 sq mm, so the total area examined is 0.0625 sq mm)

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If the counting cell used has a chamber depth of 1/10 mm but is ruled by some other system, count the bacteria in five squares each with an area of 0.0125 sq mm

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Keep the bacteria under observation long enough to establish their true character, since they sometimes exhibit slight motion and may present momentarily an end view instead of a side view

Calculation

Multiply the total number of rod-shaped bacteria counted in the areas examined by 4,80,000; this gives the number of rod-shaped bacteria per ml (when a dilution of one part of sample with two parts of water is used).

Where:

• If a dilution of one part of sample with three parts of water is used instead of one part of sample with two parts of water, the total count obtained must be multiplied by 14,40,000
• Count only rod-shaped bacteria having a length more than 1.5 times their width

Precautions

• Allow the slide to stand for at least 15 min after mounting before beginning the bacterial count
• Do not attempt to count the micrococci, as they are likely to be confused with other bodies
• Keep the organisms under observation long enough to confirm their rod shape, since slight motion can give a momentary end view

References

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

Official Methods of Analysis, 11th edn., Association of Official Analytical Chemists, Washington (1970) — direct microscopic count, cited in the book.