1. Negative Stain and Acid Fast Stain
Mic 224 Lab 5
Negative Stain and Acid Fast Stain

2. Negative Stain
The negative stain is particularly useful for determining cell size and arrangement. It can also be used to stain cells that are too delicate to be heat-fixed
We use nigrosin or india ink as our negative stain.
India Ink and Nigrosin are acidic stains. This means that the stain readily gives up a hydrogen ion and becomes negatively charged.  Since the surface of most bacterial cells is negatively charged, the cell surface repels the stain. The glass of the slide will stain, but the bacterial cells will not. The bacteria will show up as clear spots against a dark background.
Capsules (pathogenic agent of infection) that surround bacteria stand out clearly in a negative stain
Bacteria are not heat fixed and are therefore live throughout this procedure
Heat fixing can distort cell morphology

3. Procedure
Place a single drop of india ink on a clean microscope slide, adjacent to the frosted edge.
Using a flamed loop and sterile technique, remove some organism from your stock culture of Bacillus and mix it into the drop of ink. Be sure there are no large clumps of organism, but try to avoid spreading the drop.
Place the end of another clean microscope slide at an angle to the end of the slide containing the organism and spread the drop out into a film. This is done by contacting the drop of india ink with the clean microscope slide and using the capillary action of the dye/microscope slide to spread the india ink across the smear
Allow the film to air dry.
Observe the slide under the microscope, using proper microscope technique.
SEE IMAGE ON PAGE 33 OF LAB MANUAL

4. Results

5. Acid Fast Stain
Acid fast organisms comprise two species: Nocardia and Mycobacterium
These organisms have a high content of lipids and waxes (mycolic acid) in their cell walls making them difficult to stain
Examples: Mycobacterium leprae, and Mycobacterium tuberculosis

6. Kinyoun’s Acid Fast Method
1.  Prepare a mixed smear of Mycobacterium and S. aureus. Heat fix an air dried smear.
2.  Place the slide smear side up on the slide rack and cover the smear with filter paper
3. Flood slides with Kinyoun’s carbolfuchsin (primary stain) reagent and allow to stain for 6 minutes at room temperature. This method uses a high concentration of carbolfuchsin that contains the chemical phenol that is the mordant
4. Rinse with water and tilt slide to drain and discard filter paper into biohazard bag.
5. Decolorize with 2% acid-alcohol for 10 seconds and rinse again with water.
6. Flood slide with methylene blue counterstain and allow to stain for 2 minutes.
7. Rinse with distilled water and allow to air dry.
8. Examine under high magnification, and confirm acid-fast structures under oil immersion (100X). 

7. Results
Mycobacterium smegmatis (pink) and S. aureus (blue)