SOP: Cleaning of Spore Suspensions

GIFSQ — Global Institute for Food Safety & Quality

Principle

Harvested spore suspensions contain vegetative cells and extraneous material that must be removed before thermal death time determinations, because solutes and debris influence heat resistance. Vegetative cells are lysed by lysozyme or aeration, and the solutes are removed by repeated centrifugal washing in sterile distilled water.

Apparatus required

• Centrifuge
• Refrigerator (4 °C)
• Mixer or shaker (for breaking up spore clusters)

Reagents

a. Lysozyme solution — 0.25–0.3 mg of lysozyme per ml of suspension.

b. Sterile distilled water — for repeated washing.

Procedure — Removal of vegetative cells

To the spore suspension add 0.25–0.3 mg of lysozyme per ml; adjust the pH to 11 — lysis occurs almost immediately

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For spore suspensions of obligate anaerobes, lysis of the vegetative cells may instead be brought about by aeration for 15 to 20 min

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In some cases, where sporulation builds alongside lytic systems, store the spore suspension at 4 °C for 10 to 15 days, during which lysis of the vegetative cells occurs

Procedure — Removal of solutes by washing

Centrifuge the spore suspension at 1,000 g for 5 min (or at approximately 600 g for 30 min); discard the supernatant

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Re-suspend the residue in sterile distilled water and repeat the centrifuging process several times; wash spores grown in meat broth at least 5 times

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After the final washing, suspend the spores in sterile distilled water and shake the container to break up spore clusters; use a mixer if necessary and strain as before

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Store in a refrigerator (4 °C) till required

Precautions

• The presence of a few vegetative cells is not objectionable in heat resistance work, but solutes are — wash thoroughly.
• Break up all spore clusters before counting.

References

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