7. Squeeze Gram decolorizer over the slide for a few seconds.
Slides with a thick film of cells will need to decolorize longer
than those with only a few cells. Quickly rinse the decolorizer
off with clean water.
8. Add Gram safranin to cover slide. Wait 60 s and rinse gently.
9. Dry the slide with bibulous paper.
10. Examine under a microscope with a 100Â oil immersion objective without a phase filter. Mycobacteria are generally weakly
Gram positive (see Note 20).
3.3 Kinyoun Staining
1. Prepare the slide for staining as indicated in Subheading 3.1.
2. Flood the slide with Kinyoun carbol fuchsin for 4 min.
3. Rinse slide with a gentle stream of tap water to remove fuchsin.
4. Flood the slide with decolorizer for 3–5 s.
5. Rinse slide with a gentle stream of tap water.
6. Counterstain with brilliant green or methylene blue for 30 s.
7. Rinse slide with a gentle stream of tap water.
8. Air-dry the slide.
9. Examine the slide on an appropriate light microscope, using a
100Â oil immersion objective without a phase filter (see Note
21) (see Fig. 1).
3.4 Ziehl-Neelsen
Staining (See Fig. 6)
[16]
1. Prepare the slide for staining as indicated in Subheading 3.1.
2. Cover the smear with a piece of bibulous paper.
3. Flood with carbol fuchsin.
4. Heat the slide for 5 min while keeping the bibulous paper moist
with the carbol fuchsin. Heat can be achieved with a Bunsen
burner or a hot electric plate at 65–75
C (recommended).
During the process, avoid drying by addition of more stain as
needed.
5. Let the slide cooldown.
6. Remove the bibulous paper and gently rinse the slide with
distilled water to remove excess carbol fuchsin.
7. Decolorize by pouring acid alcohol onto the slide until the
runoff is clear.
8. Gently rinse the slide with distilled water to remove
decolorizer.
9. Counterstain with methylene blue for 1 min.
10. Rinse the slide with distilled water.
11. Blot (do not rub) the slide dry in a tablet of bibulous paper.
12. When the slide is dry, observe the bacteria under a microscope
with oil immersion.
32
Elizabeth Wallace et al.
Slides with a thick film of cells will need to decolorize longer
than those with only a few cells. Quickly rinse the decolorizer
off with clean water.
8. Add Gram safranin to cover slide. Wait 60 s and rinse gently.
9. Dry the slide with bibulous paper.
10. Examine under a microscope with a 100Â oil immersion objective without a phase filter. Mycobacteria are generally weakly
Gram positive (see Note 20).
3.3 Kinyoun Staining
1. Prepare the slide for staining as indicated in Subheading 3.1.
2. Flood the slide with Kinyoun carbol fuchsin for 4 min.
3. Rinse slide with a gentle stream of tap water to remove fuchsin.
4. Flood the slide with decolorizer for 3–5 s.
5. Rinse slide with a gentle stream of tap water.
6. Counterstain with brilliant green or methylene blue for 30 s.
7. Rinse slide with a gentle stream of tap water.
8. Air-dry the slide.
9. Examine the slide on an appropriate light microscope, using a
100Â oil immersion objective without a phase filter (see Note
21) (see Fig. 1).
3.4 Ziehl-Neelsen
Staining (See Fig. 6)
[16]
1. Prepare the slide for staining as indicated in Subheading 3.1.
2. Cover the smear with a piece of bibulous paper.
3. Flood with carbol fuchsin.
4. Heat the slide for 5 min while keeping the bibulous paper moist
with the carbol fuchsin. Heat can be achieved with a Bunsen
burner or a hot electric plate at 65–75
C (recommended).
During the process, avoid drying by addition of more stain as
needed.
5. Let the slide cooldown.
6. Remove the bibulous paper and gently rinse the slide with
distilled water to remove excess carbol fuchsin.
7. Decolorize by pouring acid alcohol onto the slide until the
runoff is clear.
8. Gently rinse the slide with distilled water to remove
decolorizer.
9. Counterstain with methylene blue for 1 min.
10. Rinse the slide with distilled water.
11. Blot (do not rub) the slide dry in a tablet of bibulous paper.
12. When the slide is dry, observe the bacteria under a microscope
with oil immersion.
32
Elizabeth Wallace et al.