L14: Gram staining

Introduction: 

Gram staining is a method of differentiating bacterial species into two large groups:  gram postive 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

Objectives:

- Differentiate yogurt bacteria
- Relate the stanning procedure with the structure of the cells.

Materials:

- Toothpick
- Slide
- Cover Slip
- Tongs
- Needle
- Gram stain: crystal violet, iodine and safranin.
- Decolorize reagent: ethanol 96%
- Microscope
- Yogurt

Procedure:

- Prepare a heat-fixed sample of the bacteria to be stained.
- Cover the smear with crystal violet for an exposure of 1 min.
- Rinse with destilled water.
- Apply iodine solution for 1 min.
- Rinse the sample with distilled water.
- Decolarize using ethanol. Drop by drop until the purple stops flowing, "Wash immediately with distilled water"
- Cover the sample with the safranin stain for an exposure time of 45 seconds.
- Rinse the sample with tap water.
- Gently dry the slide with paper (only the under part of the slide)

Results:

	GRAM +	GRAM -
Crystal Violet (color?)	PURPLE	PURPLE
Iodine (changes?)	YES	YES
Ethanol (decolorize?)	NO	YES
Safranin (color?)	NO	REDDISH

400x 

400x