MYCOBACTERIUMLab#10Basmah Almaarik
MYCOBACTERIUM It has many species one of them is
Mycobacterium tuberculosis that cause TB.
Some other common species seen include: M. avium AIDS patients. M. kansasiiM. fortuitum
MYCOBACTERIUM Specimen seen in lab are mainly
sputum.
Bone biopsy, CSF, urine and other.
MYCOBACTERIUM MORPHOLOGY Non spore forming, non capsulated
bacilli.
It has a fatty cell wall so it dose not stain well with gram stain. The gram stain can not penetrate this layer.
Acid Fast Bacilli cause it can not be decolorized with acid.
MYCOBACTERIUM STAINING1. Ziehl-Neelsen stain.
2. Kinyoun stain (cold method)
3. Fluorochrome stain.
MYCOBACTERIUM STAINING Ziehl-Neelsen technique:
CarbolfuchsinAcid alcohol 3%Methylene blue
Once Carbolfuchsin go inside the bacterial cell it can not be decolorized by acid because of the fatty
cell wall
ZIEHL-NEELSEN TECHNIQUE
1. Do a regular smear.
2. Heat fix slide.
3. Cover slide with carbolfuchsin allow heated Stain to stay for 5 min
Need longer fixation time because of the thick fatty cell wall
a) We apply heat under slide until vapor just begins to rise (this will allow penetration of
stain inside the cells .)
b) Or we better use heated carbolfuschin and if it dray we apply more of the stain (this to avoid fire hazard)
ZIEHL-NEELSEN TECHNIQUE
4. Wash with water.Now the carbolfoschin and the fatty layer of
cell wall make a complex that is red and cannot be decolorized
5. Cover slide with acid alcohol 3-5 mint tell all the red color is gone.
(take care cause acid alcohol is flammable)6. Wash slide well with water.7. Cover slide with methylene blue for 3-
5 mint8. Wash and examin.
ZIEHL-NEELSEN TECHNIQUE
1 2 3
4 5 6
ZIEHL-NEELSEN TECHNIQUE
7
Red bacilli against a blue background
Results
Very slender bacilli, arranged in single, or in pairs or in long parallel bundles
making cord formation
MODIFIFIED ZIEHL-NEELSEN TECHNIQUE
Acid Fast Bacilli
Non- Acid Fast Bacilli
Carbolfuschin
Decolorizer
Methylin Blue
KINYOUN STAIN (COLD METHOD) Same steps as Zhiehl-Neelsen but with
few changes: No heating Concentrated carbolfuschin. Concentrated decolorizing agent.
Same results (red bacilli- blue back ground)
FLUOROCHROME STAIN When exposed to UV light it will
fluorescent. These slides are examined using
fluorescent microscope. If Bacilli are present it will fluorescence
against a dark background. This method is used for screening if
positive it is confirmed by Common fluorochrome stain:
Auramine-Rhodamin stain Acridine orange stain
FLUOROCHROME STAIN
1 .Heat fix slide2 .Flood with
auramine-phenol stain 10 m
3 .Wash with water
4 .Decolorize 1% acid alcohol 5 m
5 .Wash with water
6 .Cover with potassium
permanganate 10 sec
FLUOROCHROME STAIN
7 .Rinse thoroughly with water
Examine at x25 or X40
Bacilli will appear yellow orange against
a black background.
Always include a positive
control smear with each batch to
make sure stain is ok
EXAMINING THE SLIDE
Fluorochrome Stine are examined at X20 or
X40 objective
Ziehl-Neelsen and Kinoyoun are examined in oil
immersion X100 objective
MYCOBACTERIUM CULTURE The media used is Lowenstein Jensen (LJ) It is enriched medium Mycobacterium grow aerobically. 35-37 ˚C. Slow grower. M.tuberculosis raised , dry cream (buff)
colonies. Visible colonies appear after 2-3 weeks of
incubation. But culture should be incubated up to 6 weeks
before discarding.
LOWENSTEIN JENSEN Media contain:
Glycerol (enhances the growth of Mycobacterium tuberculosis)
Antibiotic (Low levels of penicillin and nalidixic acid are also present in LJ medium to inhibit growth of gram positive and gram negative bacteria)
Malachite green (inhibits most other bacteria)
Egg yolk
LOWENSTEIN JENSENColi flower
appearance colonies, easily removed but
very difficult to emulsify, non
pigmented
RAPID METHODS FOR DETECTIONBACTEC™ 12B SYSTEM
Fluid medium containing palmotic acid carbon 14 labelled (C14)