3-2-1: Conventional Culture Method

3-2-3-1-Conventional Culture Method (Marks 1972): The infected lesions of the tuberculin positive reactors were collected and placed in to a sterile mortar containing sterile sand. The fat was removed and the suspected parts were cut into minute pieces. Then 2 ml of distilled water added to the mixture, homogenized and crushed well until the suspension was obtained. Then 2 ml of H2So4 acid 4% added to the mixture then incubated the mixture at 37 0C for 1/2 h. The mixture was diluted with 16 ml of distilled water and centrifuged at 3000rpm for 1/3h. The supernatant fluid was discarded into 5% phenol solution and the sediment was cultured into Lowenstein – Jensen media, 2 glycerated and 2 pyruvated. Then the slants incubated at 37 0C
One of each slant was wrapped well in aluminum foil. The slants were incubated at 370C and when the unwrapped media showed growth, the aluminum foil was removed from the other media and the color of the growth in the both media was compared. The growth in the wrapped slants was directed to light for 1h and examined in the second day for any change in color.

The isolates produced yellow or orange pigment when their cultures exposure to the light, but not produced any pigments when kept in the dark were considered (photo chromogenic). The isolates produced pigments in slants incubated in the dark and the intensity of the colour increase on exposure to light were considered (scoto chromogenes). While the isolates produced white or pale yellow pigments and on exposure to light the color not intensified were considered (non-photo chromogenic).
3-2-4-4- Growth at different temperature degrees (Kubica, 1973): Tested isolates inoculated on Lowenstein – Jensen slants were and incubated at different degrees of temperatures (280C, 450C and 52°C) then examined after 7 days for growth.
3-2-4-5 – Biochemical
A positive test is shown by change in the colour of tween 80 from straw yellow to pink.
D- Catalase test (Kubica, 1973)
Procedure:
Spot test: Add 2 drops from freshly mixed Tween peroxide solution to a colony of mycobacterial and shown for 4 to 5 second of bubbles. Appearance of bubbles is a positive test while lack of any bubbles is a negative test for catalase.
Semi – quantative test: Inoculated 0.1 ml of a 7 day liquid culture of the test organism on to the surface of a tube of Lowenstein Jensen medium then incubated at 370C for 2 weeks. Caps on the culture tubes must be loss to permit adequate exchange of air. Add 1 ml of freshly prepared Tween – peroxide solution (1:1 mixture of 1% Tween 80 and 30% H2O2) and leave upright for 5 minutes. Measure the height of the column of bubbles above the surface of the culture medium.

Test for heat – stable catalase pH7, 68 0C for 20