researchers need to infect animals, isolate the strain, and ensure the
resulting strain has “regained” its virulence. The derived culture
should be used immediately as a seed lot. This way, the researchers
will have access to a virulent strain without the need of animal work.
2 Materials
2.1 Preparation
of Bacterial Smears
1. Mycobacterial cells to stain.
2. Sterile water or broth.
3. Glass slides.
4. Bacteriological loop.
5. Methanol.
2.2 Gram Staining
1. Glass slides.
2. Gram stain reagents: including Gram crystal violet, decolorizer,
Gram staining iodine and safranin (Gram staining kit
from VWR).
3. Basin or sink.
4. Clean running water.
5. Microscope with 100Â oil immersion lens.
6. Immersion oil.
7. Culture of mycobacteria.
8. Sterile saline, water, or broth.
9. Methanol.
10. Bibulous paper.
2.3 Kinyoun Staining
(Cold Acid-Fast
Staining)
1. Kinyoun carbol fuchsin stain (see Note 1): In 83.3 mL of
deionized (DI) water, dissolve 3.33 g basic fuchsin, 6.67 g
phenol, and 16.7 mL ethanol.
2. Decolorizer (3% acid alcohol) (see Note 1): Combine 3.0 mL of
hydrochloric acid with 97 mL of ethanol.
3. Brilliant green counterstain (see Note 1): Dissolve 1.0 g of
brilliant green in 100 mL of DI water.
4. Methylene blue counterstain (see Note 1): Dissolve 0.3 g of
methylene blue in 100 mL of DI water.
2.4 Ziehl-Neelsen
Staining
1. Carbol fuchsin stain (see Note 1): Dissolve in 90 mL of DI
water 0.3 g basic fuchsin, 5.0 g of phenol, and 10 mL of
ethanol.
2. Decolorizer (3% acid alcohol) (see Subheading 2.3, item 2).
3. Carbol fuchsin counterstain (methylene blue) (see Subheading
2.3, item 4).
24
Elizabeth Wallace et al.