Lab10 Basmah Almaarik It has many species one

Lab#10 Basmah Almaarik

� It has many species one of them is Mycobacterium tuberculosis that cause TB. � Some �M. other common species seen include: avium AIDS patients. �M. kansasii �M. fortuitum

� Specimen � Bone seen in lab are mainly sputum. biopsy, CSF, urine and other.

� 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.

1. Ziehl-Neelsen stain. 2. Kinyoun stain (cold method) 3. Fluorochrome stain.

� Ziehl-Neelsen technique: �Carbolfuchsin �Acid alcohol 3% �Methylene blue Once Carbolfuchsin go inside the bacterial cell it can not be decolorized by acid because of the fatty cell wall

1. 2. Do a regular smear. Heat fix slide. Cover slide with carbol fuchsin allow heated Stain to stay for 5 min 3. 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)

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. 7. 8. Wash slide well with water. Cover slide with methylene blue for 3 -5 mint Wash and examin.

1 2 3 4 5 6

Results Red bacilli against a blue background 7 Very slender bacilli, arranged in single, or in pairs or in long parallel bundles making cord formation

Acid Fast Bacilli Non- Acid Fast Bacilli Carbolfuschin Decolorizer Methylin Blue

� Same steps as Zhiehl-Neelsen but with few changes: No heating Concentrated carbolfuschin. Concentrated decolorizing agent. � Same results (red bacilli- blue back ground)

� 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

Always include a positive control smear with each batch to make sure stain is ok Examine at x 25 or X 40 Bacilli will appear yellow orange against a black background.

Ziehl-Neelsen and Kinoyoun are examined in oil immersion X 100 objective Fluorochrome Stine are examined at X 20 or X 40 objective

� 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.

� 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

Coli flower appearance colonies, easily removed but very difficult to emulsify, non pigmented

Fluid medium containing palmotic acid carbon 14 labelled (C 14)