SOP: Negative Staining of Bacteria

GIFSQ — Global Institute for Food Safety & Quality

Principle

In negative staining the dye colours the background, not the cells: the organisms appear light against a dark field. Because the cells themselves are not stained, they are not distorted as in ordinary staining, so negative staining is a rapid method for studying the organisms and for measuring their true size.

Apparatus required

• Glass slides
• Inoculating loop
• Microscope with oil immersion objective

Reagents

a. 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.

Procedure

Place 2 loopfuls of the suspension on a slide and add 1 loopful of the nigrosin solution

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Mix thoroughly and spread the material to make a thin, even film

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Air dry — do not fix with heat

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Examine the preparation under the oil immersion objective of the microscope

Note — source flag: the printed sentence describing how to spread the film is partly illegible in the book extract; only the legible instructions (mix, spread to a thin film, air dry, no heat fixation) are carried.

Precautions

• Never fix a negative-stained preparation with heat.
• Spread the material to a thin, even film and air dry without heat fixation.

References

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