L.17 GRAM STAINING

OBJECTIVES:

- Differenciate yogurt bacteria.
- Relate the staining procedure with the structure of the cells.

MATERIALS:

- 1 Slide
- 1 Cover slip
- Tongs
- Needle
- Gram stain: crystal violet, iodine and safranin.
- Descolorize reagent: ethanol 96%
- Microscope
- Yogurt

PROCEDURE:

- Prokariotic cell observations: GRAM STAINING

1. Prepare a heat-fixed sample of the bacteria to be stained.
2. Cover the smear with crystal violet for an exposure of 1 min.
3. Rinse with distilled water.
4. Apply Iodine solution for 1 min.
5. Rinse the sample with distilled water.
6. Decolorize using ethanol. Drop by drop until the purple stops flowing. Wash immediately with distilled water.
7. Cover the sample with the safrain stain for an exposure time of 45 seconds.
8. Rinse the sample with distilled water.
9. Gently dry the slide with paper.

=> Gram staining is a method of differentiating bacterial species into two large groups: Gram positive and Gram negative. This differentiation is based by the chemical and physical properties of their cell walls by detecting a peptidoglycan, which is present in a thick layer in gram-positive bacteria. The result is:

> Gram-negative: stain pink or reddish color.
> Gram-positive: stain purple color. 

Gram stain:
- Complete the table that you have below:

	GRAM POSITIVE	GRAM NEGATIVE
Crystal violet: Color?	Violet	Violet
Iodine	Contrast	Contrast
Ethanol: Decolorize?	No,  too thick retaining the dye	Yes, open the pores and the Coloring goes.
Safranin: Color?	No	Reddish