How to Gram-stain a specimen

How to Gram-stain a specimen.

Observing Microorganisms

1. Prepare the specimen using the heat fixation process .

2. Place a drop of crystal violet stain on the specimen.

3. Apply iodine on the specimen using an eyedropper. The iodine helps

the crystal violet stain adhere to the specimen. Iodine is a mordant

which is a chemical that fixes the stain to the specimen.

4. Wash the specimen with ethanol or alcohol-acetone solution, then

wash with water.

5. Wash the specimen to remove excess iodine. The specimen appears

purple in colour.

6. Apply the safranin stain to the specimen using an eyedropper.

7. Wash the specimen.

8. Use a paper towel and blot the specimen until the specimen is dry.

9. the specimen is ready to be viewed under the microscope. Gram-positive bacteria

appear purple and gram-negative bacteria appear pink.

Here is how to apply the Zichi-Neelsen acid-fast stain to a specimen.

1. Prepare the specimen (see “Smear” earlier in this chapter).

2. Apply the red dye carbon-fuchsin stain generously using an eyedropper.

3. Let the specimen sit for a few minutes.

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4. Warm the specimen over steaming water. The heat will cause the stain to

penetrate the cell wall.

5. Wash the specimen with an alcohol-acid or acid-alcohol decolorizing solution

consisting of 3 percent hydrochloric acid and 93 percent ethanol. The

hydrochloric acid will remove the color from non-acid-fast cells and the

background. Acid-fast cells will stay red because the acid cannot penetrate the cell

wall.

6. Apply methylene blue stain on the specimen using an eyedropper.