Download Zoonotic disease unit

Family : mycobacteriaceae
Genus : mycobacterium
Species : 1. mycobacterium tuberculosis in human causes tuberculosis .
2. mycobacterium leprae in human causes Leprosy .
3. mycobacterium bovis produce disease in cattle & other animals,spread to
Human by raw milk ingestion. Disease in human similar to that caused by TB.
4. mycobacterium avium also known as mycobacterium avium complex (MAC)
May develop into pulmonary infection.
Morphology & staining :
The mycobacteria are thin straight rods or slightly curved rods of varying lengths & sometimes
branching filamentous forms occure . The mycobacteria don’t take up the dyes of the gram stain
because the cell wall are rich in lipid that called mycolic acid . mycobacteria cells are not easily
decolourised even by acid- alcohol so it have acid-fast stain (Ziehl-Neelsen). The cells take red color
(carbol fuchsin) by using mild heat or heatless method by addition of a wetting agent (Turgitol)&
back ground is blue (methylene blue).
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Natural habitat :
The source of pathogenic mycobacteria is usually infected animals. M. bovis is excreted in
respiratory discharge, faeces, milk,urine & semen. M. avium & M.paratuberculosis are shed in
faeces . M. tuberculosis mainly in respiratory discharge .
Cultural characteristics :
The mycobacteria are aerobic & oxidative & non motile bacteria .They don’t have capsules, and most
don’t form spore . Many Mycobacterium species adapt readily to growth on very
simple substrates, using ammonia or amino acids as nitrogen sources and glycerol as a
carbon source in the presence of mineral salts. Optimum growth temperatures vary widely
according to the species and range from 25 °C to over 50 °C.
Some species can be very difficult to culture (they are fastidious) .
Decontaminating agents such as : oxalic acid 5% , NaoH 2-4% can be used on specimens before
culture .
Media for mycobacteria :
1. The egg-based Lowenstein- Jensen medium
2. Stone brinks media
3. Middle brook broth & media
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They are most commonly used for culture of Mycobacterium, notably Mycobacterium
tuberculosis. Composition of these media are includ e :
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Malachite green
Glycerol
Asparagine
Potato starch
Coagulated eggs
Mineral salt solution
 Potassium dihydrogen phosphate
 Magnesium sulfate
 Sodium citrate
These media are prepared as a solid slants in screw capped bottles. Malachite green dye is commonly
used as the selective agent
Low levels of penicillin and nalidixic acid are also present in LJ medium to inhibit growth of gram
positive and gram negative bacteria, in order to limit growth to Mycobacteria species only. Presence of
malachite green in the medium inhibits most other bacteria . Presence of glycerol enhances the growth
of Mycobacterium tuberculosis & M. avium & many of the typical mycobacteria but glycerol is inhibitory to
M. bovis while sodium pyruvate enhances its growth .
The medium appears green, opaque and opalescent.
Colony morphology on media :
M. tuberculosis on glycerol- containing media giving the characteristic dry, rough, tough&
buff colonies, hard to break up, is known as Eugonic . the growth of M. avium in the same
media also called Eugonic .
M. bovis has sparse thin growth on glycerol- containing media that is called dysgenic.
(white,small,moist,flat colonies ) .
M. bovis grow well on pyruvate containing media without glycerol .
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Pigment production :
*M. that produce yellowish- orange pigments are called chromogenic .
*The term photo chromogenic is applied to those M. that produce pigment only if exposed to light.
*Scoto chromogenic M. produce pigment when incubated either in the light or in the dark.
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Niacin test: All species of mycobacterium produces Niacin (nicotinic acid) and the Mycobacterium tuberculosis accumulates the
most. A positive niacin test provides preliminary evidence that an organism that exhibits a buff-colored, slow-growing rough
colony may be M. tuberculosis.
Nitrate Reduction test: Mycobacterium tuberculosis is a strongly nitrate positive organism. This test is valuable for the
identification of M. tuberculosis, M. kansasii, M. szulgai and M. fortuitum.
Rapid growers such as M. fortuitum can be tested within 2 weeks, but slow growers should be tested after 3-4 weeks of luxuriant
growth. Both chemical procedure and commercially available nitrate strips are available .
Catalase test: Semiquantitative catalase test is used for the identification of Mycobacteria. Catalase is an enzyme which splits
Hydrogen peroxide to water and oxygen and positive catalase test is indicated by formation of gas bubbles. Most species of
Mycobacteria, except strains of M. tuberculosis complex and M. gastri, produce catalase enzyme.
Tween 80 hydrolysis test: Tween 80 hydrolysis test is used to separate the species of Photochromogens, scotochromogens and
nonchromogens. Non pathogenic slow growing scotochromogens and nonchromogens produce a lipase that is able to hydrolyze
Tween 80 into oleic acid and polyoxyethylated sorbitol, where as pathogenic species do not.
Tellurite reduction test: The ability of mycobacterial species to reduce tellurite in 3 to 4 days is used to distinguish members
of M. avium complex from most other non-chromogenic species. All rapid growers reduce tellurite in 3 days.
Arylsulfatase test: Arylsulfatase enzyme is present in most mycobacteria. The rate by which arylsulfatase enzyme breaks down
phenolphthalein disulfate into phenolphthalein (which forms a red color in the presence of sodium bicarbonate) and other salts is
used to differentiate certain strains of Mycobacteria. 3 day arylsulfatase test is used to identify rapid growers such as M. fortuitum
and M. chelonae. Slow growing M. marinum and M. szulgai .
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Growth type
Colony form on media with glycerol
M. Tb
Eugenic
Rough,tough&buff ,
dry&hard tobreak up
Growth time
Glycerol for growth
Na pyruvate for growth
Niacin production
Urease
Nitrate reduction
Pyrazin amidase
Growth on TCH
3-8 week
+ enhanced
- Inhibited
+
+
+
+
+
M. bovis
Dysgenic
Small,moistsheen,breakup easily
3-8 week
- Inhibited
+ enhanced
+
-
Dr. Ban sahib Abed Al-Nabi
Zoonotic disease unit
Post graduate lecture
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M. avium
Eugenic
White,stickey,
breakup
easily
2-6 week
+ enhanced
+ enhanced
+
+
-