3.5 Auramine/
Rhodamine Staining
1. Place slides on a staining rack so they are at least 1 cm apart, and
flood with auramine/rhodamine stain and let stand for 20 min.
2. Rinse the stain away with chlorine-free water and tilt slide to
drain.
3. Flood the slide with 0.5% acid alcohol and let stand for 2 min.
4. Wash off the acid alcohol with distilled water.
5. Flood slides with 0.5% potassium permanganate for 1–2 min
(see Note 22).
6. Wash off the stain with distilled water.
7. Allow slides to air-dry in the slide rack. DO NOT BLOT!
8. Protect smears from light and examine immediately using the
fluorescent microscope. If unable to read right away, place
slides in dark box.
3.6 Fite-Faraco
Staining
and Enumeration
of M. leprae
Suspension (See
Fig. 7)
1. Make dilutions of M. leprae suspension (1:100, 1:1000, and
1:10,000) using sterile broth (7H9, RPMI-10, or
RPMI 1640).
2. Add 10 μL of serum–phenol solution to each circle of the
counting slide, then add 10 μL of the M. leprae suspension to
each circle of the slide (see Note 23).
Fig. 6 Ziehl-Neelsen staining of purified M. leprae harvested from an infected armadillo spleen, with no or little
tissue contamination (panels a, b), or from liver with about 1% tissue contamination (panels c, d)
Culturing Mycobacteria
33
Rhodamine Staining
1. Place slides on a staining rack so they are at least 1 cm apart, and
flood with auramine/rhodamine stain and let stand for 20 min.
2. Rinse the stain away with chlorine-free water and tilt slide to
drain.
3. Flood the slide with 0.5% acid alcohol and let stand for 2 min.
4. Wash off the acid alcohol with distilled water.
5. Flood slides with 0.5% potassium permanganate for 1–2 min
(see Note 22).
6. Wash off the stain with distilled water.
7. Allow slides to air-dry in the slide rack. DO NOT BLOT!
8. Protect smears from light and examine immediately using the
fluorescent microscope. If unable to read right away, place
slides in dark box.
3.6 Fite-Faraco
Staining
and Enumeration
of M. leprae
Suspension (See
Fig. 7)
1. Make dilutions of M. leprae suspension (1:100, 1:1000, and
1:10,000) using sterile broth (7H9, RPMI-10, or
RPMI 1640).
2. Add 10 μL of serum–phenol solution to each circle of the
counting slide, then add 10 μL of the M. leprae suspension to
each circle of the slide (see Note 23).
Fig. 6 Ziehl-Neelsen staining of purified M. leprae harvested from an infected armadillo spleen, with no or little
tissue contamination (panels a, b), or from liver with about 1% tissue contamination (panels c, d)
Culturing Mycobacteria
33