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)
…