STAINING TECHNIQUES

ANTHONY’S CAPSULE STAINING TECHNIQUE

Procedure: 

1. Take a clean grease free glass slide.
2. Prepared a smear of bacterial culture at centre of the clean slide.
3. Allow the smear to air dry. DO NOT HEAT FIX (to avoid destroying or distorting the capsule or causing shrinkage).
4. Cover the slide with 1% crystal violet for 2 minutes.
5. Rinse gently with a 20% solution of copper sulfate.
6. Air dry the slide. DO NOT BLOT. (Blotting will remove the un-heat-fixed bacteria from the slide and/or cause disruption of the capsule.)
7. Examine the slide under an oil immersion lens.

Bacterial cells will appear purplish while the capsules will appear transparent in the light purplish background.

SCHAEFFER-FULTON METHOD FOR STAINING ENDOSPORES

• Malachite green stain  (0.5% (wt/vol) aqueous solution)

          0.5 g of malachite green in 100 ml of distilled water

• Decolorizing agent: Tap water

• Safranin counterstain: 2.5 g of safranin in100 ml of 95% ethanol

Procedure: 

1. Take a clean grease free glass slide.
2. Prepared a smear of bacterial culture on the slide. 
3. Heat fixed bacterial slide
4. Placed on steam/water bath after placing a paper towel on the slide
5. Flood the filter paper covered slide placed in steam bath with the primary stain malachite green for five minutes. Add drops of malachite stain if it dries. (Steaming allows entry of malachite stain by increasing porosity of bacterial cell wall and membrane).
6. Remove the slide from the steaming apparatus and rinse the slide with water until water runs clear.
7. Flood slide with the counterstain safranin for 1 minute, then rinse.
8. View specimen under oil immersion objective of compound light microscope.

Endospores will appear green, having retained the primary stain, malachite green. Vegetative cells (bacteria are in the active, metabolizing state) will appear pink, having retained the counterstain, safranin

NEGATIVE STAINING TECHNIQUE

Procedure: 

1. Take a clean grease free glass slide.
2. Add a drop of nigrosin stain or India ink on the slide.
3. Add bacterial culture on the slide and spread well in drop stain.
4. Smear the stain containing bacterial culture using another clean slide horizontally at 45˚C.
5. Allow the smear to air dry.
6. Examine the slide under an oil immersion lens.

Bacterial cells will appear clear/unstained on dark background.