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Also, add sterile calf serum 100 ml
Mix the above carefully and distribute, under sterile conditions, in 10 ml amounts.
Check sterility by overnight incubation at 37
o
C and store in the cold.
5. CULTURE BY SOLID CULTURE METHODS
5.1CSF and pericardial fluid
Smear:
1. Label a clean dry slide with the lab number and place the slide and the
sample container inside the cabinet
2. Mix well and aseptically remove one loopful of the fluid and place in the
centre of the slide; close the container and allow the drop to air-dry
3. Place one more drop of the CSF on the same spot and let dry.
4. Place the third drop after processing the sample as below:
Culture:
Culture of CSF is done in two steps:
1. Direct inoculation in media 2. Inoculation after decontamination
Direct
1. Place one loopful of CSF on to one slope each of LJ and LJ-P
2. Add 0.2 ml of CSF in to one bottle containing SK medium
3.
Decontamination
1. Add 1ml of 5% H
2
SO
4
to CSF
2. Mix well and let stand for 15 minutes
3. Fill the container with sterile distilled water and centrifuge at 3000 x g for
15 minutes
4. Aspirate the supernatant carefully without disturbing the deposit or discard
carefully in to a disinfectant bin containing 5% phenol or any other
mycobactericidal solution
5. Inoculate one slope each of LJ and LJ-P with one loopful of deposit for
each slope
6. Transfer the remaining deposit in to one bottle of SK
7.
8. Incubate both set A and B at 37
o
C
5.2 BAL
1. Make a direct smear
2. Process using 5% H
2
SO
4
as in CSF
3.
Inoculate two slopes each of
LJ and LJ-P with one loopful of deposit using
5mm twisted wireloop
4. Transfer the remaining deposit in to one bottle of SK
5. Incubate the slopes and SK medium at
37
o
C
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