Bacteriological diagnosis of Bacteriological diagnosis of TBTB

Microscopic Microscopic examinationexamination

It is based on a smear of a It is based on a smear of a clinical sample and identifies clinical sample and identifies mycobacteria highlighting their mycobacteria highlighting their acid-alcohol resistance propertyacid-alcohol resistance property

Smear is sterile prepared Smear is sterile prepared (bacteriological loop) is air dry (bacteriological loop) is air dry (at room temperature or a (at room temperature or a heating platinum) and not dry heating platinum) and not dry up in flamesup in flames

Fixing: Hold the smear with Fixing: Hold the smear with forceps and fix the smear by forceps and fix the smear by passing it through a flame passing it through a flame (smear side up) three or four (smear side up) three or four times.times.

ZIEHL-NEELSEN stainingZIEHL-NEELSEN staining The reference staining The reference staining

used to highlightused to highlight BAAR (Acid-Alcohol BAAR (Acid-Alcohol Resitant Bacillus)Resitant Bacillus)

Can be made with Can be made with fluorescent fluorescent substances: the substances: the auramine-rhodaminaauramine-rhodamina

Fuxin hot staining can Fuxin hot staining can be made to allow dye be made to allow dye entry within entry within mycobacteria, and mycobacteria, and thus are colored in thus are colored in redred

ZIEHL-NEELSEN stainingZIEHL-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

Microscopic Microscopic examinationexamination

The exam is done with the optical The exam is done with the optical microscope with immersion objective 100xmicroscope with immersion objective 100x

Mycobacteria appear as thin rods, red, Mycobacteria appear as thin rods, red, slightly incurbate, isolated or grouped in slightly incurbate, isolated or grouped in pairs or groups, on a blue backgroundpairs or groups, on a blue background

BAAR are counted on 100 microscopic fieldsBAAR are counted on 100 microscopic fields The results are expressed quantitatively The results are expressed quantitatively

depending on the density of bacilli on the depending on the density of bacilli on the slideslide

Sensitivity is relatively small and requires Sensitivity is relatively small and requires the presence of 10,000 bacteria/ml for a the presence of 10,000 bacteria/ml for a positive resultpositive result

The semi-quantitative expression of the results of The semi-quantitative expression of the results of sputum microscopy for the presence BAAR- sputum microscopy for the presence BAAR-

immersion objective 1000Ximmersion objective 1000X

Culture of mycobacteriaCulture of mycobacteria The reference method for diagnosing TBThe reference method for diagnosing TB Allows identification of mycobacterial Allows identification of mycobacterial

strainstrainand then testing its sensitivity to anti-TB and then testing its sensitivity to anti-TB drugsdrugs

Contaminated clinical samples (sputum) Contaminated clinical samples (sputum) should be decontaminated with usual should be decontaminated with usual antiseptic and homogenizedantiseptic and homogenized

Centrifuged and then neutralized with a Centrifuged and then neutralized with a weak acidweak acid

The product thus prepared or directly if The product thus prepared or directly if the sample is sterile, is inoculated onthe sample is sterile, is inoculated on culture media.

LLÖÖWENSTEIN JENSEN WENSTEIN JENSEN mediummedium

Culture on solid media is the Culture on solid media is the standard reference for the isolation standard reference for the isolation of MTBof MTB

MTB growth period is 4-6 weeksMTB growth period is 4-6 weeks Culture on liquid media with Culture on liquid media with

radioactive or fluorescent detection radioactive or fluorescent detection allows their detection in 1-2 weeks, allows their detection in 1-2 weeks, but is more expensive and less but is more expensive and less availableavailable

LLÖÖWENSTEIN JENSEN WENSTEIN JENSEN MEDIUMMEDIUM

Culture of mycobacteriaCulture of mycobacteria MTB colonies on solid MTB colonies on solid

media are round, pale media are round, pale yellow, cauliflower, yellow, cauliflower, rough surface, isolated rough surface, isolated or confluent according or confluent according to the initial inoculum to the initial inoculum density of bacillidensity of bacilli

Species identification Species identification is made by is made by biochemical testsbiochemical tests

Expression results are Expression results are semiquantitatively semiquantitatively according to the according to the density of coloniesdensity of colonies

REFERENCESREFERENCES

http://images.google.ro/imgres?imgurl=http://telmeds.org/AVIM/http://images.google.ro/imgres?imgurl=http://telmeds.org/AVIM/Abacterio/mycobacterias%2520y%2520nocardia/Abacterio/mycobacterias%2520y%2520nocardia/El_medio_de_cultivo_se_llama_Lowenstein_Jensen.JPG&imgrefurl=El_medio_de_cultivo_se_llama_Lowenstein_Jensen.JPG&imgrefurl=http://telmeds.org/AVIM/Abacterio/mycobacterias%2520yhttp://telmeds.org/AVIM/Abacterio/mycobacterias%2520y%2520nocardia/%2520nocardia/mycobacterias_nocardia.htm&h=600&w=900&sz=171&hl=ro&start=mycobacterias_nocardia.htm&h=600&w=900&sz=171&hl=ro&start=15&um=1&tbnid=4P7BwIflgWEkmM:&tbnh=97&tbnw=146&prev=/15&um=1&tbnid=4P7BwIflgWEkmM:&tbnh=97&tbnw=146&prev=/images%3Fq%3DLowenstein%2BJENSEN%26svnum%3D10%26umimages%3Fq%3DLowenstein%2BJENSEN%26svnum%3D10%26um%3D1%26hl%3Dro%26lr%3Dlang_ro%26sa%3DG%3D1%26hl%3Dro%26lr%3Dlang_ro%26sa%3DG

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

http://faculty.plattsburgh.edu/jose.deondarza/Bio406/Handouts/http://faculty.plattsburgh.edu/jose.deondarza/Bio406/Handouts/notes_09.htmnotes_09.htm

http://images.google.ro/imgres?imgurl=http://telmeds.org/A