Gram Stain
Determines Gram + or Gram -
Results
Red/Pink: Gram - (will keep secondary color: Saffranium)
Purple: Gram + (will keep primary color: Crystal Violet)
Gram - bacteria has lipid outer cell wall and thin peptidoglycan wall that dissolves in ethanol which doesn’t hold the Crystal Violet.
Gram + is able to retain the Crystal Violet stain.
Procedure
1) Heat fix slide with bacteria
2) Smear with Crystal Violet stain: 1 minute
3) Rinse with distilled water
4) Smear with Iodine stain: 1 minute
5) Rinse with distilled water
6) Decolorize: Run with 95% ethanol for 3 seconds
7) Rinse with distilled water
8) Counterstain with Safranin stain for 1 minute
9) Blot with bibulous paper
Acid - Fast Stain
Presence of Mycolic acid in cell wall
Results
Redish Pink/Purple: Acid fast + (will keep primary color: Kinyoun C.)
Blue: Acid fast - (will keep secondary color: Methylene Blue)
Mycolic acid is a waxy substance = Higher affinity for primary stain and resistance to decolorization. Phenol in Kinyoun stain helps the stain penetrate and become trapped inside the waxy substance.
Procedure
1) Heat fix slide with bacteria
2) Smear with Kinyoun Carbolfuchsin stain: 5 mins
3) Rinse with distilled water
4) Decolorize with acid-alcohol
5) Rinse with distilled water
6) Counterstain with Methylene blue: 1 min
7) Rinse with distilled water
8) Blot with bibulous paper
Capsule Stain
Presence of capsule
Results
Halo: Capsule +
No Halo: Capsule -
Capsules are Mucoid Polysaccharides or Polypeptides. It repels the stain giving it a halo look.
Procedure
1) Put drop of Congo Red stain on one end of the slide
2) Put bacteria on the drop
3) Do smear (like a blood smear)
4) Air dry
5) Smear with Maneval’s stain: 1 min
6) Rinse with distilled water
7) Blot with bibulous paper
Positive
Endospore Stain
Presence of Keratin protein
Results
Red bacteria with Green spore: Endospore +
Only Red bacteria: Endospore -
Endospore is dormant form of bacteria. It helps them to survive harsh conditions.
Keratin protein (outer covering of Spore) is resistant to heat and stainning so extreme measures must be taken.
- Terminal - Spore located at the ends of bacteria
- Central - Spore located in the middle of the bacteria
- Subterminal - Spores located between middle and ends of bacteria
Procedure
1) Heat fix slide with bacteria
2) Put a strip of bibulous paper over slide
3) Smear with Malachite Green stain regularly so it doesn’t dry out.
4) Steam: 7-10 min
5) Rinse with distilled water
6) Counterstain with Safranin stain: 1 min
7) Rinse with distilled water
8) Blot with bibulous paper
Simple Stain
aka
Postive Stain
Determines shape of bacteria
Chromogen: Capable of producing color/pigment
Chromophore: Is responsible for the color. (Gives the color)
Auxochrome: - charged particle (ouside of the cell)
Basic stain: Alkaline is + charged particle that attracts Auxochrome
Basic Stain (+) & Auxochrome (-) = Color
Procedure
1) Heat fix slide with bacteria
2) Smear with Methylene Blue: 1 min
3) Rinse with distilled water
4) Blot with bibulous paper
Negative Stain
Determine morphology without heat fixing
Stains background
Chromogen (-) & Acidic stain (-) = Uncolored
It is repelled.
Procedure
1) Put a drop of Nigrosin Black stain at the end of slide
2) Put bacteria on the drop
3) Smear across slide (like a blood smear)
4) Air dry