SOP: Counting of Spores

GIFSQ — Global Institute for Food Safety & Quality

Principle

The spore suspension for thermal death time determination must contain more than 10 million spores per ml (a suspension below 100,000 spores per ml is generally useless). The count is obtained either by direct microscopic counting or by subculturing, the heated dilutions being plated or tubed so that only activated spores grow.

Apparatus required

• Microscope with oil immersion objective
• Counting cell 0.02 mm thick, with a black background
• Dilution tubes with sterile water
• Water bath at 100 °C (for activation)
• Petri dishes with agar plates (for acidifying aerobes)

Reagents

a. Liver broth — prepare liver broth, add an equal quantity of ground tomatoes and 10 g of soil per 100 g; add a few pieces of liver to each tube; adjust the pH to 5; sterilize, cool and inoculate each tube with 1 ml of dilution.

Procedure — Direct counting

Count the cells by direct observation under the microscope against a black background, using the oil immersion objective and a cell 0.02 mm thick

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Note that living spores cannot be distinguished from dead spores by this method; if the suspension has been prepared as described, the results are similar to the culture method

Procedure — Counting by subculturing

Prepare successive decimal dilutions in sterile water; heat to destroy vegetative forms and to activate the spores

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Prepare the liver broth as above; inoculate each tube with 1 ml of dilution; create anaerobic conditions either by exhausting the air or by sealing the surface; use two to five tubes for each dilution

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From the positive tubes, count the number of cells

Procedure — Counting of acidifying aerobes on agar plates

Agar plates are specially suitable for counting acidifying aerobes (B. stearothermophilus, B. coagulans). Prepare a 1:100 dilution of the suspension and distribute 2 ml per tube; submit the tubes in pairs to increasingly long periods of heating (for example 5 to 60 min) at 100 °C, which destroys the vegetative forms and activates the spores

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Inoculate on agar plates and, after incubation, count the colonies; record the highest number — it corresponds to the degree of heating most favourable to activation

Precautions

• Usually an actual heating period of 3–5 min at 100 °C is sufficient to destroy vegetative forms in the dilutions and to activate the spores of most aerobic or anaerobic species mentioned; for thermophilic aerobes the optimal activation time must be determined.
• For obligate anaerobic species, use the surface culture technique of Miller et al.

References

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

Miller, N.J., O.W. Garett & P.S. Prickett, Food Res., 4, 447 (1939) — cited in the book.