SOP: Spore Staining of Bacteria

GIFSQ — Global Institute for Food Safety & Quality

Principle

Certain bacteria form endospores (spores) within the cell; these spores are remarkably resistant to conditions that quickly kill vegetative cells. They resist the usual stains and appear as relatively transparent spots in the cell, and in old cultures the spore’s cell wall often breaks down, freeing the spore.” A special staining procedure is therefore needed to stain the spores properly; spore size, shape and position help identify Bacillus and Clostridium species.

Apparatus required

• Glass slides
• Small test tubes
• Boiling water bath (for Dorner’s method)
• Inoculating loop
• Microscope with oil immersion objective

Reagents

a. Ziehl’s carbol fuchsin: solution A — mix 0.3 g of basic fuchsin with 10 ml of ethyl alcohol (95%); solution B — mix 5 g of phenol with 95 ml of distilled water. Mix solutions A and B.

b. Dorner’s nigrosin solution: add 10 g of nigrosin to 100 ml of distilled water and dissolve by heating for 30 min on a boiling water bath; replace the water lost by evaporation and add 0.5 ml of formalin; filter twice through double filter paper.

c. Malachite green: dissolve 5 g of malachite green in 100 ml of distilled water.

d. Safranin: prepare a 2.5% solution of safranin O in 10 ml of ethyl alcohol (95%) and 100 ml of distilled water.

Procedure — Dorner’s method

Make a heavy suspension of the organism in 2 or 3 drops of distilled water in a small test tube

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Add an equal quantity of carbol fuchsin and let the mixture stand in boiling water for 10 min or longer

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On a slide, mix 1 loopful of the stained preparation with 1 loopful of the nigrosin solution

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Smear as thin as possible and dry rapidly — dry it quickly — nigrosin films that dry slowly crack when they are thick

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Examine thin portions of the preparation with the oil immersion objective: the background appears greyish, the cells colourless, and the spores a brilliant red; note the presence of free spores in the field

Procedure — Wirtz’s method (modified)

Make a smear of the culture on the slide and fix it by passing over the flame 5 or 6 times

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Flood the smear with malachite green and bring to the steaming point; let it remain for 2 or 3 min and wash in tap water

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Apply the safranin solution and let it remain for 30 sec

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Wash, dry, and examine under the oil immersion objective: the spore stains green, the remainder of the cell a light red

Precautions

• In Dorner’s method, smear as thin as possible and dry rapidly — thick nigrosin films crack on slow drying.
• Keep the malachite green at the steaming point for the full 2–3 min.
• Examine the thin portions of the preparation; thick areas obscure the spores.

References

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