STAINING

Coloring organism with a dye

Microorganisms must be FIXED to microscopic slide first

Kills microbe

Attaches organism to the slide

Preserves various parts with minimal distortion

Steps to fix:

SMEAR is placed on slide and allowed to dry

Passed through Bunsen burner several times

Stain then applied and washed off

STAINING

Stains are salts composed of positive and negative ions (one is colored and is called a chromophore)

Basic dyes-color is positive ion

Acidic dyes- color is negative ion

Bacteria are slightly negatively charged at pH 7.0, so basic dye is attracted more to them. Includes:

crystal violet

methylene blue

malachite green

safranin

STAINING

Acidic dyes not attracted to bacteria because stain’s negative ion is repelled

Used to stain background instead (some bacteria are colorless). This is called negative staining

Great when fixing is not possible because only background is stained

STAINING

TYPES OF STAINING:

Simple

Differential

Gram Stain

Acid Fast

Special

Negative staining for capsules

Endospore Staining

Flagella Staining

Simple

Sometimes an additive called a mordant. It increases uptake of the dye.

Differential

Gram Stain (developed in 1884)

Procedure:

1. apply heat. this fixes smear; slide is covered with purple dye (primary stain)

2. dye is washed off and the mordant, iodine, is placed and washed off

3. slide washed with alcohol (decoloring agent)

4. slide is re-stained with safranin (red dye is a counterstain)

Differential

Acid Fast

Binds strongly to waxy material in cell walls

Procedure:

1. red dye carbolfuchsin is applied to a fixed smear and slide is heated

2. slide is cooled and washed with water and then acid-alcohol

3. If colorless, slide is counterstained with methylene blue

STAINING

Special Stains

Negative Staining for capsules

Organism is placed in solution with India ink or nigrosin and the contrastained with safranin

STAINING

Endospore (spore staining)

Most common stain is Schaeffer-Fulton endospore stain

Malachite green (primary stain) is applied to heat-fixed smear and heated to steaming for 5 min

Washed with water

Next safranin (counterstain)

…