1.0 INTRODUCTION:
Tuberculosis
(TB) is caused by a set of closely related mycobacterial strains such as Mycobacterium tuberculosis, M. bovis, M. africanum, and others,
known collectively as the M. tuberculosis complex. M. tuberculosis is carried in airborne
particles, called droplet nuclei, of 1– 5 microns in diameter. Infectious
droplet nuclei are generated when persons who have pulmonary or laryngeal TB
disease cough, sneeze, shout or sing. Depending on the environment, these tiny
particles can remain suspended in the air for several hours. M. tuberculosis is
transmitted through the air, not by surface contact. Transmission occurs when a
person inhales droplet nuclei containing M. tuberculosis, and the droplet
nuclei traverse the mouth or nasal passages, upper respiratory tract, and
bronchi to reach the alveoli of the lungs.
2.0 METHODOLOGY:
·
M. tuberculosis H37Rv
ATCC 27294 will be grown for 15 days in Middlebrook 7H9 broth supplemented
with OADC enrichment (oleic acid, albumin, dextrose, and catalase); 0.5%
glycerol as a carbon source; and 0.5% Tween 80 to prevent clumping.
·
Suspensions will be prepared,
and the turbidity will be adjusted to a McFarland no. 1 standard.
·
Stock solutions of the test
sample will be prepared in DMSO, and further dilute to obtain final
concentrations ranging from 0.98 to 250μg/mL in Middlebrook 7H9 broth
supplemented with oleic acid, albumin, dextrose, and catalase.
·
Isoniazid, rifampicin,
streptomycin, and ethambutol will be solubilized according to the
manufacturers’ recommendations and used as positive control drugs.
· After further dilutions to
reach the final bacterial suspension concentration (5×105 UFC/mL),
100 μL of the inoculum will be added to each well of a 96-well microtiter
plate containing the test sample.
· The plates will be incubated
for 7 days at 37°C, and after incubation, 30μL of 0.1mg/mL resazurin will
be added.
·
The assays will be set up in
duplicate.
·
After 24h, the wells will be
ready for color change and fluorescence in a spectrafluor Plus microfluorimeter
(excitation/emission with 530/590 nm filters, respectively).
·
The MIC (minimum inhibitory
concentration) will be defined as the lowest concentration resulting in a 90%
growth inhibition of M. tuberculosis.
·
The MIC values of Isoniazid,
rifampicin, streptomycin, and ethambutol will be determined in a single plate as
standards.
·
ENDPOINT PARAMETER
·
MIC value
3.0 REFERENCE(S):
3.1 Gemechu, A., Giday, M., Worku, A. et
al. In vitro Anti-mycobacterial activity of selected medicinal plants
against Mycobacterium tuberculosis and Mycobacterium
bovis Strains. BMC Complement Altern Med 13, 291 (2013). https://doi.org/10.1186/1472-6882-13-291.
3.2 Araujo, R.C., Neves, F.A., Formagio,
A.S. et al. Evaluation of the anti-mycobacterium
tuberculosis activity and in vivo acute toxicity of Annona
sylvatic . BMC Complement Altern Med 14, 209 (2014). https://doi.org/10.1186/1472-6882-14-209.
END OF DOCUMENTS
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