Download MYCOBACTERIUM

Lab#10
Basmah Almaarik
 It
has many species one of them is
Mycobacterium tuberculosis that cause TB.
 Some



other common species seen include:
M. 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
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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.
Red bacilli against a blue background
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 x25 or X40
Bacilli will appear
yellow orange against a
black background.
Ziehl-Neelsen and Kinoyoun are
examined in oil immersion X100
objective
Fluorochrome Stine are
examined at X20 or X40
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
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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
(C14)