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)