159temperature (see Note 2) and add 100 mL of Albumin Dextrose
Complex (ADC) (see Subheading 2.1, item 7).- Middlebrook 7H11 Agar: Add 21 g of powder 7H11 medium
(Becton Dickinson, UK) and 5 mL of glycerol to 900 mL of
distilled water followed by autoclaving at 121 °C for 15 min.
After sterilization, cool the agar to 55 °C (see Note 2) and add
100 mL of Oleic Albumin Dextrose Complex (OADC, Becton
Dickinson, UK) (see Subheading 2.1, item 8) immediately
before pouring to 90 mm sterile petri dishes. - Kirchner liquid medium: add 17.4 g of powder Kirchner liquid
medium (MAST) and 20 mL of glycerol to 1 L of distilled
water, autoclave at 121 °C for 15 min. After sterilization, cool
the medium to room temperature and add 100 mL of sterile
inactivated horse serum (Oxoid, UK) before use. - Löwenstein-Jensen slopes: In 600 mL purified water, add
monopotassium phosphate 2.4 g, potato flour 30 g, magne-
sium sulfate 0.24 g, asparagine 3.6 g, sodium citrate 0.6 g, and
Malachite Green 0.4 g and glycerol 12 mL. After sterilization
by autoclaving at 121 °C for 15 min, add fresh 1000 mL whole
egg. Dispense the complete medium into sterile screw capped
tubes and arrange in a slant position in a suitable rack. These
slopes are obtained from Becton Dickinson and stored at
2–8 °C in the dark (see Note 3). - Blood agar: Add 39 g of powder Columbia blood agar base
(Oxoid, UK) to 1 L of distilled water. Sterilize by autoclaving
at 121 °C for 15 min. Cool to 50 °C and add 5% sterile defi-
brinated blood (Oxoid) immediately before pouring to 90 mm
sterile petri dishes. - Sabouraud dextrose agar: Add 65 g of Sabouraud dextrose
agar (Oxoid) to 1 L of distilled water. Sterilize by autoclaving
at 121 °C for 15 min. Mix well and pour into 90 mm sterile
petri dishes. - Albumin Dextrose Complex (ADC): add 10 g of albumin frac-
tion V, 4 g of d-glucose, and 1.7 g NaCI to 200 mL sterilized
distilled water. Stir to completely solubilize the albumin.
Sterilize using 0.2 μm fliter and store at 4 °C. - Oleic Albumin Dextrose Complex (OADC): obtain from
Becton Dickinson, UK. OADC contains 5% of albumin frac-
tion V, 2% of glucose, 0.85% of NaCI, 0.05% of oleic acid, and
0.004% catalase. - Phosphate Buffered Saline: NaCl 0.138 M, KCl 0.0027 M,
pH 7.4. - Selectatab (Mast Diagnostica GmbH): each tablet contains poly-
myof polymyxin B 200,000 unit/L, carbenicillin 100 mg/L,
trimethoprim 10 mg/L, and amphotericin B 10 mg/L.
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