2.5 Auramine/
Rhodamine Staining
(See Note 1)
1. Auramine/rhodamine stain: In 75 mL of glycerol, dissolve
1.5 g of auramine O and 0.75 g of rhodamine B. Then add
10 mL of heated phenol crystals and 50 mL of DI water. Filter
through glass wool to clarify.
2. Decolorizer (0.5% acid alcohol): Combine 0.5 mL of HCl in
100 mL 70% ethanol.
3. 0.5% potassium permanganate counterstain solution. Dissolve
0.5 g of KMn0 4 in 100 mL of DI water.
2.6 Fite-Faraco
Staining (See Note 1)
1. Serum phenol: Prepare a solution of 5% phenol w/v in DI
water + 2% v/v fetal bovine serum (FBS).
2. 37% v/v formaldehyde.
3. Gelatin phenol: In 90 mL DI water, add 0.5 g gelatin and
10 mL 5% w/v crystal phenol (see Note 2).
4. Carbol fuchsin staining solution (see Subheading 2.4, item 1).
5. Acid alcohol: Combine 946.25 mL of DI water, 2.375 mL
ethanol, and 50 mL H 2 SO 4 (36 N).
6. Methylene blue or brilliant green counterstaining solution (see
Subheading 2.3, items 3 and 4).
7. Reich counting slides.
8. Boiling water bath with flat glass cover (large inverted Petri
dish) or hot plate at 100
C.
9. Microscope with known 100Â field constant (see Note 3).
10. 100Â objective immersion oil.
11. Platinum tip spreading tool.
12. M. leprae suspension.
2.7 Small-Scale
Growth
1. ADC enrichment (see Note 1): Aseptically, add 5.0 g bovine
albumin, 2.0 g dextrose, and 3.0 mg beef catalase to 100 mL of
DI water.
2. OADC enrichment (see Note 1): To 100 mL of DI water, add
5.0 g bovine albumin, 2.0 g dextrose, 0.85 g sodium chloride,
4.0 mg beef catalase, and 50.0 mg oleic acid.
3. Middlebrook 7H9 broth + ADC: To 900 mL of DI water, add
2.5 g disodium phosphate, 1.0 g monopotassium phosphate,
0.5 g L-glutamic acid, 0.5 g ammonium sulfate, 0.1 g sodium
citrate, 50.0 mg magnesium sulfate, 40.0 mg ferric ammonium
citrate, 1.0 mg zinc sulfate, 1.0 mg copper sulfate, 1.0 mg
pyridoxine, 0.5 mg calcium chloride, 0.5 mg biotin, and
2.0 mL glycerol (see Note 1). Heat and stir to dissolve. Autoclave at 121
C for 15 min. Once medium is at room temperature, add aseptically 100 mL of ADC enrichment.
Culturing Mycobacteria
25
Rhodamine Staining
(See Note 1)
1. Auramine/rhodamine stain: In 75 mL of glycerol, dissolve
1.5 g of auramine O and 0.75 g of rhodamine B. Then add
10 mL of heated phenol crystals and 50 mL of DI water. Filter
through glass wool to clarify.
2. Decolorizer (0.5% acid alcohol): Combine 0.5 mL of HCl in
100 mL 70% ethanol.
3. 0.5% potassium permanganate counterstain solution. Dissolve
0.5 g of KMn0 4 in 100 mL of DI water.
2.6 Fite-Faraco
Staining (See Note 1)
1. Serum phenol: Prepare a solution of 5% phenol w/v in DI
water + 2% v/v fetal bovine serum (FBS).
2. 37% v/v formaldehyde.
3. Gelatin phenol: In 90 mL DI water, add 0.5 g gelatin and
10 mL 5% w/v crystal phenol (see Note 2).
4. Carbol fuchsin staining solution (see Subheading 2.4, item 1).
5. Acid alcohol: Combine 946.25 mL of DI water, 2.375 mL
ethanol, and 50 mL H 2 SO 4 (36 N).
6. Methylene blue or brilliant green counterstaining solution (see
Subheading 2.3, items 3 and 4).
7. Reich counting slides.
8. Boiling water bath with flat glass cover (large inverted Petri
dish) or hot plate at 100
C.
9. Microscope with known 100Â field constant (see Note 3).
10. 100Â objective immersion oil.
11. Platinum tip spreading tool.
12. M. leprae suspension.
2.7 Small-Scale
Growth
1. ADC enrichment (see Note 1): Aseptically, add 5.0 g bovine
albumin, 2.0 g dextrose, and 3.0 mg beef catalase to 100 mL of
DI water.
2. OADC enrichment (see Note 1): To 100 mL of DI water, add
5.0 g bovine albumin, 2.0 g dextrose, 0.85 g sodium chloride,
4.0 mg beef catalase, and 50.0 mg oleic acid.
3. Middlebrook 7H9 broth + ADC: To 900 mL of DI water, add
2.5 g disodium phosphate, 1.0 g monopotassium phosphate,
0.5 g L-glutamic acid, 0.5 g ammonium sulfate, 0.1 g sodium
citrate, 50.0 mg magnesium sulfate, 40.0 mg ferric ammonium
citrate, 1.0 mg zinc sulfate, 1.0 mg copper sulfate, 1.0 mg
pyridoxine, 0.5 mg calcium chloride, 0.5 mg biotin, and
2.0 mL glycerol (see Note 1). Heat and stir to dissolve. Autoclave at 121
C for 15 min. Once medium is at room temperature, add aseptically 100 mL of ADC enrichment.
Culturing Mycobacteria
25