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Bacteriological diagnosis of TB. Microscopic examination It is based on a smear of a clinical sample and identifies mycobacteria highlighting their acid-alcohol.

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Presentation on theme: "Bacteriological diagnosis of TB. Microscopic examination It is based on a smear of a clinical sample and identifies mycobacteria highlighting their acid-alcohol."— Presentation transcript:

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 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 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. 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 highlightBAAR (Acid-Alcohol Resitant Bacillus) The reference staining used to highlight BAAR (Acid-Alcohol Resitant Bacillus) Can be made with fluorescent substances: the auramine-rhodamina 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 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 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 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 BAAR are counted on 100 microscopic fields The results are expressed quantitatively depending on the density of bacilli on the slide 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 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 The reference method for diagnosing TB Allows identification of mycobacterial strain and then testing its sensitivity to anti-TB drugs 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 Contaminated clinical samples (sputum) should be decontaminated with usual antiseptic and homogenized Centrifuged and then neutralized with a weak acid Centrifuged and then neutralized with a weak acid The product thus prepared or directly if the sample is sterile, is inoculated on 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 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 Species identification is made by biochemical tests Expression results are semiquantitatively according to the density of colonies Expression results are semiquantitatively according to the density of colonies

11 REFERENCES http://images.google.ro/imgres?imgurl=http://telmeds.org/AVIM/Abac terio/mycobacterias%2520y%2520nocardia/El_medio_de_cultivo_se_ll ama_Lowenstein_Jensen.JPG&imgrefurl=http://telmeds.org/AVIM/A bacterio/mycobacterias%2520y%2520nocardia/mycobacterias_nocardi a.htm&h=600&w=900&sz=171&hl=ro&start=15&um=1&tbnid=4P7B wIflgWEkmM:&tbnh=97&tbnw=146&prev=/images%3Fq%3DLowen stein%2BJENSEN%26svnum%3D10%26um%3D1%26hl%3Dro%26l r%3Dlang_ro%26sa%3DG http://images.google.ro/imgres?imgurl=http://telmeds.org/AVIM/Abac terio/mycobacterias%2520y%2520nocardia/El_medio_de_cultivo_se_ll ama_Lowenstein_Jensen.JPG&imgrefurl=http://telmeds.org/AVIM/A bacterio/mycobacterias%2520y%2520nocardia/mycobacterias_nocardi a.htm&h=600&w=900&sz=171&hl=ro&start=15&um=1&tbnid=4P7B wIflgWEkmM:&tbnh=97&tbnw=146&prev=/images%3Fq%3DLowen stein%2BJENSEN%26svnum%3D10%26um%3D1%26hl%3Dro%26l r%3Dlang_ro%26sa%3DG Tuberculosis - course for students, 2004 – Institute of Pneumology “Marius Nasta” - National Tuberculosis Control Programme 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://faculty.plattsburgh.edu/jose.deondarza/Bio406/Handouts/notes_ 09.htm http://images.google.ro/imgres?imgurl=http://telmeds.org/A


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