Bacteriological diagnosis of TB

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. The reference staining used to highlightBAAR (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 ZIEHL-NEELSEN staining

4. 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 ZIEHL-NEELSEN staining

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 strainand 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. 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 Culture of mycobacteria

11. REFERENCES • http://images.google.ro/imgres?imgurl=http://telmeds.org/AVIM/Abacterio/mycobacterias%2520y%2520nocardia/El_medio_de_cultivo_se_llama_Lowenstein_Jensen.JPG&imgrefurl=http://telmeds.org/AVIM/Abacterio/mycobacterias%2520y%2520nocardia/mycobacterias_nocardia.htm&h=600&w=900&sz=171&hl=ro&start=15&um=1&tbnid=4P7BwIflgWEkmM:&tbnh=97&tbnw=146&prev=/images%3Fq%3DLowenstein%2BJENSEN%26svnum%3D10%26um%3D1%26hl%3Dro%26lr%3Dlang_ro%26sa%3DG • Tuberculosis - course for students, 2004 – Institute of Pneumology “Marius Nasta” - National Tuberculosis Control Programme • http://faculty.plattsburgh.edu/jose.deondarza/Bio406/Handouts/notes_09.htm http://images.google.ro/imgres?imgurl=http://telmeds.org/A