SOP: Monochrome Staining of Bacteria

GIFSQ — Global Institute for Food Safety & Quality

Principle

Certain dyes bind to bacterial cell walls and raise the contrast of the cells under the microscope. In monochrome (simple) staining a single dye is applied to a fixed smear, so the shape and size of the organisms — the morphology — can be observed and the nature of the staining noted.

Apparatus required

• Glass slides, 3 × 1 in.
• Wire loop (sterilizable in a flame)
• Bunsen burner or spirit lamp
• Filter paper
• Microscope with oil immersion lens

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. Loeffler’s methylene blue: dissolve 0.3 g of methylene blue in 30 ml of ethyl alcohol.

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

Procedure

Clean the glass slides thoroughly and wipe them with a dry cloth — on a clean grease-free slide, a drop of water spreads as a thin film

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Heat a wire loop to redness in the flame, let it cool, and transfer a loopful of the bacterial suspension from the broth to the centre of the slide (if the preparation is from a solid medium, touch the sterile loop lightly on the colony and transfer it to a drop of water placed on the slide); reflame the loop and cool it

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Spread the drop into a thin film on the slide and let it air dry

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Fix the dry smear by passing the slide rapidly through the low flame of a Bunsen burner or spirit lamp — apply only enough heat that the slide can be tolerated on the back of one’s palm

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Cover the smear with several drops of any one of the dyes: allow 15–20 sec in the case of carbol fuchsin, or 3–5 min in the case of methylene blue

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Wash carefully with water, press dry with filter paper, and examine under the microscope using the oil immersion lens; note the shape of the cells and the nature of the staining

Precautions

• The slide must be grease-free.
• Let the smear air dry completely before fixing; apply only enough heat that the slide can be tolerated on the back of one’s palm.
• Wash the dye off carefully with water.

References

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