1. Bacteriological diagnosis of TB

2. Microscopic examination
It is based on a smear of a clinical sample and identifies mycobacteria highlighting their acid-alcohol resistance property
Smear is sterile prepared (bacteriological loop) is air dry (at room temperature or a heating platinum) and not dry up in flames
Fixing: Hold the smear with forceps and fix the smear by passing it through a flame (smear side up) three or four times.

3. ZIEHL-NEELSEN staining
The reference staining used to highlight BAAR (Acid-Alcohol Resitant Bacillus)
Can be made with fluorescent substances: the auramine-rhodamina
Fuxin hot staining can be made to allow dye entry within mycobacteria, and thus are colored in red

4. ZIEHL-NEELSEN staining
Discoloration using acid-alcohol (70 ° alcohol and sulfuric acid) – until macroscopic disappearance of red coloration
All structures are discolored, except mycobacteria that stay stained in red
For contrast – recoloration using methylene blue

5. Microscopic examination
The exam is done with the optical microscope with immersion objective 100x
Mycobacteria appear as thin rods, red, slightly incurbate, isolated or grouped in pairs or groups, on a blue background
BAAR are counted on 100 microscopic fields
The results are expressed quantitatively depending on the density of bacilli on the slide
Sensitivity is relatively small and requires the presence of 10,000 bacteria/ml for a positive result

6. The semi-quantitative expression of the results of sputum microscopy for the presence BAAR- immersion objective 1000X

7. Culture of mycobacteria
The reference method for diagnosing TB
Allows identification of mycobacterial strain and then testing its sensitivity to anti-TB drugs
Contaminated clinical samples (sputum) should be decontaminated with usual antiseptic and homogenized
Centrifuged and then neutralized with a weak acid
The product thus prepared or directly if the sample is sterile, is inoculated on culture media.

8. LÖWENSTEIN JENSEN medium
Culture on solid media is the standard reference for the isolation of MTB
MTB growth period is 4-6 weeks
Culture on liquid media with radioactive or fluorescent detection allows their detection in 1-2 weeks, but is more expensive and less available

9. LÖWENSTEIN JENSEN MEDIUM

10. Culture of mycobacteria
MTB colonies on solid media are round, pale yellow, cauliflower, rough surface, isolated or confluent according to the initial inoculum density of bacilli
Species identification is made by biochemical tests
Expression results are semiquantitatively according to the density of colonies

11. REFERENCES
Tuberculosis - course for students, 2004 – Institute of Pneumology “Marius Nasta” - National Tuberculosis Control Programme