163- Then perform the culture in a tenfold serial dilution by adding
0.5 mL of the mixed cell suspension to 4.5 mL of culture fil-
trates (see Note 15). - Incubate the cultures at 37 °C without disturbance.
- At 10-day intervals over a 2-month period of incubation, the
broth cultures are examined for visible turbidity. - Plate M. tuberculosis in turbid tubes on 7H11 agar plates to
confirm colonial morphology (see Note 16). - Estimate the MPN of viable bacilli from the patterns of posi-
tive and negative tubes [ 15 ]. - Plate the cultures from turbid tubes on blood agar medium
(Oxoid) and Sabouraud dextrose agar (Oxoid) to check the
sterility of the culture free from bacterial and fungal contami-
nation (see Note 17). - Student’s t-test is used to determine the difference of CFU
counts and broth counts between the experimental groups. - The difference between the relapse rates is determined by Chi-
square test and Fisher’s exact test. P-values <0.05 are consid-
ered significant.
4 Notes
- M. tuberculosis forms clumps when grown in 7H9 medium.
The addition of Tween 80 is to prevent the formation of
clumps. The reconstituted medium needs to be mixed well till
the powder is completely dissolved before sterilization. - ADC and OADC are heat sensitive and need to be added in
the medium after cooling to 55 °C. - In order to obtain consistent results, this slopes will be obtain
from Becton Dickinson. On receipt, store slope tubes in the
dark at 2–8 °C. Avoid freezing and overheating and minimize
exposure to light. Media stored according to the labeled
instruction may be inoculated up to the recommended incuba-
tion times before the expiration date. - In the in vitro model, OD value of 1–1.5 will be achieved at
15–20 days of incubation. It is extremely important that the
bacteria are in the stage to actively produce RPF but there is
still sufficient nutrient available in the medium for subsequence
period of incubation. - For M. tuberculosis to grow in 7H9 medium, an inoculation of
log-phase culture to the medium is essential. In the hypoxia
model, log-phase growth is defined from 7 to 10 days of
growth which represents about 2 × 107 CFU/mL.
3.7 Statistical
Analysis
A Method to Evaluate Persistent Mycobacterium tuberculosis In Vitro and in the...