Phenotype Staining

Simple staining
It is a simple stain which uses methylene blue, crystal violet or safranin. Simple staining is sufficient determine the bacterial shape and the arrangement characteristics. An air-dried smear is stained, rinsed, dried and examined using oil immersion lens of the microscope.

Differential staining
Gram stain
• Gram positive bacteria have thick cell walls which have low lipid content. This result the cell wall to becomes less permeable during the treatment with decolourizer.
• Gram negative bacteria have thin cell walls which have higher lipid content. This result the cell wall to be soluble and thus dissolves in decolourizer.


Procedures for gram staining
Fixation-----> crystal violet-----> iodine treatment-----> decolourization----->counter stain safranin

1. Heat fix the smear
2. Cover the smear with crystal violet and rinse
3. Cover the smear with iodine and rinse
4. Apply alcohol decolourizer
5. Cover the smear with safranin and rinse

It is used for Mycobacteria because they have a thick and waxy coat. Acid-fast stain uses dyes (carbon fuchsin) with heat to allow the dye to go into the bacterium. Once acid-fast bacteria are stained, they are resistant to decolourization. This is because of its thick and waxy coat.
When non acid-fast bacteria are decolourized, they take up the counterstain, which is the methylene blue.

Result: Acid-fast – pink
Non Acid-fast - blue


Special staining
Capsular stain
Capsules do not have the same affinity for dyes as other cell components. The result of negative staining is the capsule will halo around the cell against a dark background.

Endospore stain
Endospore has a special resistant. Dormant structure formed within a cell that protects the bacteria during adverse environmental conditions.

Flagella stain
The flagella are too small to be seen under microscope. In order to see it, we have to increase the thickness of flagella by piling the stain on it.

References:
http://www.ruf.rice.edu/~bioslabs/studies/invertebrates/kingdoms.html
http://biology.northwestcollege.edu/biology/b1010lab/bactypes.htm
http://www.lima.ohio-state.edu/biology/biodiv/webbact.htm