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Operation method

Gram staining test for bacteria

Principle

G + bacteria cell wall peptidoglycan content, dense structure, less lipid, ethanol is not easy to penetrate the decolorization; G - bacteria cell wall peptidoglycan content is less, loose structure, more lipids, ethanol is easy to dissolve lipids and penetrate the decolorization. G + bacteria contain a large number of magnesium salts of ribonucleic acid, can be combined with crystal violet, iodine, so that it is not easy to decolorize ethanol; G - bacteria iso-electric point is lower than the G - bacteria under the same pH conditions than G - bacteria have more negative charges. -G+ bacteria isoelectric point is lower than G-bacteria, under the same PH conditions than G-bacteria with more negative charge, easy to positively charged alkaline dyes (crystal violet), so it is not easy to be decolorized by ethanol.

Move

1. Preparation of specimen sheets

(1) Take a clean slide and divide it into 2 areas with a marker and mark them;

(2) Add 1 drop of saline to each of the two areas.

(3) Pick a small amount of bacteria with a sterilized inoculation ring, mix and spread in a saline, forming a thin film of bacteria.

(4) Dry naturally at room temperature.

(5) Fix the specimen by flaming 2 to 3 times and cool naturally.

2. Gram stain

(1) Initial staining: add drops of crystal violet to the bacterial membrane to the extent of covering the membrane, about 1 minute, then rinse the stain with a fine stream of water.

(2) Enzyme staining: add several drops of Lugol's iodine solution, rinse gently after about 1 minute.

(3) Decolorization: add 3-4 drops of 95% alcohol, gently shake the slide for a few seconds, tilt the slide to discard the alcohol, and so on for several times, the flowing alcohol is almost colorless or slightly mauve, and finally rinse.

(4) Re-staining: add a few drops of diluted red dye solution, rinse after about 1 minute.

After staining, dry the slide with absorbent paper, add a few drops of cedar oil and examine it microscopically.

3. Results: Staphylococcus albicans was stained purple for Gram-positive (G+) bacteria, E. coli was stained red for Gram-negative (G-) bacteria.

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