2 Microscopic examination It is based on a smear of a clinical sample and identifies mycobacteria highlighting their acid-alcohol resistance propertySmear is sterile prepared (bacteriological loop) is air dry (at room temperature or a heating platinum) and not dry up in flamesFixing: 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-rhodaminaFuxin 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 colorationAll structures are discolored, except mycobacteria that stay stained in redFor contrast – recoloration using methylene blue
5 Microscopic examination The exam is done with the optical microscope with immersion objective 100xMycobacteria appear as thin rods, red, slightly incurbate, isolated or grouped in pairs or groups, on a blue backgroundBAAR are counted on 100 microscopic fieldsThe results are expressed quantitatively depending on the density of bacilli on the slideSensitivity 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 TBAllows identification of mycobacterial strain and then testing its sensitivity to anti-TB drugsContaminated clinical samples (sputum) should be decontaminated with usual antiseptic and homogenizedCentrifuged and then neutralized with a weak acidThe 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 MTBMTB growth period is 4-6 weeksCulture on liquid media with radioactive or fluorescent detection allows their detection in 1-2 weeks, but is more expensive and less available
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 bacilliSpecies identification is made by biochemical testsExpression results are semiquantitatively according to the density of colonies
11 REFERENCESTuberculosis - course for students, 2004 – Institute of Pneumology “Marius Nasta” - National Tuberculosis Control Programme