Mycobacteria
General characteristic of Mycobacterium:1- Slender rods bacteria slightly curved rod with rounded ends although sometime granular or filamentous forms may be seen.
2- Aerobic bacteria.
3- Difficultly stained by gram stain so are stained by Ziehl-Neelsen technique.
4- Non motile
5- Non capsulated
6-Non spore forming
7- These bacteria destroyed by pasteurization process also by to ultraviolet light, autoclaving methods, phenol, alcohol, formaldehyde, microwave.
Pathogenicity:
This genus Mycobacterium contains many species, the major medically important pathogens are:M. tuberculosis, cause tuberculosis in human.
M. bovis, cause T.B in human and cattle.
M. africanum, cause T.B in human and monkey.
M. leprae cause leprosy.
Mycobacteria other than tuberculosis (MOTT) such as
M. avium
M. intracellulare cause disease in person with chronic lung disease, leukemia and AIDS.
Identification of Mycobacteria:
1- Sputum testing.
The specimen is treated with 3% KOH or NaOH for liquefaction.
The specimen is centrifuged at high speed to concentrate the mycobacterium at the bottom of centrifuge tube.
Stained by Ziehl-Neelsen (acid-fast stain) technique.
Acid-fast stain is used in the diagnosis of disease caused by acid-fast species such as: tuberculosis & leprosy diseases, these bacteria contain unusual complex lipid (mycolic acid) of the cell wall. These lipids give the cell waxy properties and make it difficult to get stain inside the cell
Procedure:
1- Fix the slide with mycobacterium bacteria.
2- Cover the smear with basic carbol fuchsin (red) & steam over boiling water bath for 5 min, adding additional stain, if necessary to prevent smear drying.
3- Allow slide to cool & wash with water.
4- Decolorizing by adding acid-alcohol (95% ethyl alcohol containing 3% Hcl) drop by drop to the slide until the primary stain fall from the smear.
5- Wash with water.
6- Counter stain with methylene blue for 30 sec.
7- Wash with water & dry.
Result:
Acid fast bacteria appear as red
Non-acid fast bacteria appear as blue
2- Culture:
Lowenstein-Jensen medium: special selective media contain:1. HYPERLINK "http://en.wikipedia.org/wiki/Malachite_green" \o "Malachite green" Malachite green which inhibit all bacteria except Mycobacteria species.
2. HYPERLINK "http://en.wikipedia.org/wiki/Glycerol" \o "Glycerol" Glycerol which inhibit the growth of M. bovis and allow the growth of
M. tuberculosis
3. HYPERLINK "http://en.wikipedia.org/wiki/Asparagine" \o "Asparagine" Asparagine
5. HYPERLINK "http://en.wikipedia.org/wiki/Coagulation" \o "Coagulation" Coagulated HYPERLINK "http://en.wikipedia.org/wiki/Egg_(biology)" \o "Egg (biology)" eggs.
6. HYPERLINK "http://en.wikipedia.org/wiki/Potassium_dihydrogen_phosphate" \o "Potassium dihydrogen phosphate" Potassium dihydrogen phosphate.
7. HYPERLINK "http://en.wikipedia.org/wiki/Magnesium_sulfate" \o "Magnesium sulfate" Magnesium sulfate.
8. HYPERLINK "http://en.wikipedia.org/wiki/Sodium_citrate" \o "Sodium citrate" Sodium citrate.
*The medium appears green, opaque and opalescent.
*Slow growing (2-3 weeks) at 37oC.
Colony on Lowenstein-Jensen medium appears as brown, granular colonies sometimes called (rough, tough, and buff).
3- Chest x-ray: Abnormalities on chest radiographs may be suggestive of, but are never alone diagnostic of, TB.
4- Tuberculin skin test (Mantoux skin test): A standard dose is 5 tuberculin units TU (0.1 ml)is injected intradermally (between the layers of dermis) and read 48 to 72 hours later. This intradermal injection is termed the Mantoux technique. A person who has been exposed to the bacteria is expected to mount an immune response in the skin containing the bacterial proteins.
The reaction is read by measuring the diameter of HYPERLINK "http://en.wikipedia.org/wiki/Induration" \o "Induration" induration(palpable raised, hardened area) across the forearm in millimeters. If there is no induration, the result should be recorded as "0 mm". HYPERLINK "http://en.wikipedia.org/wiki/Erythema" \o "Erythema" Erythema (redness) should not be measured.
If a person has had a history of a positive tuberculin skin test, or had a recent tuberculin skin test (within one year), another skin test should be used.