ACID FAST BACTERIA

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ALT CULTURE CLUB TOPIC

ACID FAST BACILLI

ISOLATION AND IDENTIFICATION

Acid Fast Bacilli are those that do not stain readily but once stained will resist decolorization with acid alcohol.  They occur as single bacilli or in small irregular clumps, sometimes beaded or pleomorphic in stained smears.  The group includes numerous organism pathogenic for man, the most important of which is Mycobacterium tuberculosis.

The presence of acid fast bacilli (AFB) does not identify M. tuberculosis since the stain will not differentiate pathogenic from non-pathogenic strains.  Direct microscopic examination often makes possible and early presumptive diagnosis and gives information as to the numbers of acid fast bacilli as well as the presence of other agents and evidence of disease.  Concentrate or direct smear of sputum, spinal or other body fluids including catheterized urine, and homogenized tissue material or impression slides are satisfactory specimens for smear.  The presence of non-pathogenic acid fast bacilli in gastric content or in voided urine make direct smears difficult to interpret.

Generally, acid fast bacilli from sputum nearly always represent tubercle bacilli, but this is not true for smears from gastric washings or urine specimens.  Smegma bacillus are occasionally encountered in urine.  Acid fast organisms have been found in tap and "distilled" water.  Cultural methods should be employed to demonstrate the presence of tubercle bacilli since this technique is recommended for diagnosis of tuberculosis.

The tubercle bacillus is capable of attacking and altering nearly all tissue of the body.  Thus any available secretion, excretion, body fluid or tissue from a tuberculosis patient may become a specimen for laboratory test.  Many media have been described for the cultivation of M. tuberculosis.  The composition of these media vary widely although most of them use coagulated egg as a base.  Media with selective agents are generally employed for the primary growth of these organism from pathological material.  After a concentration and digestion procedure to destroy other common bacteria, the selected culture media (Lowenstein, Middlebrook and Dubos) are inoculated and incubated at 35 C to 37 C and room temperature, for 6 to 8 weeks with examination for growth at irregular intervals (reading in 2-3 days to weekly).  Colonial characteristics and growth rate of M. tuberculosis and other acid fast organisms when grown on solid egg media such as ATS (American Trudeau Society), Lowenstein or Petragnani, are such as to often make it possible to recognize non-pathogenic acid fast organisms.

Colonies of human tubercle bacilli are 8-12 mm in diameter following an incubation period (depending on concentration numbers of AFB, if many, growth as early as 3 ? to 4 weeks).  They are granular, rough, dry, worty, friable and easily detached from the surface of the medium but emulsify with difficulty.  Bovine tubercle bacilli require a much longer incubation period, 3 to 6 weeks, and form small, pale, smooth, pyrimidal colonies which adhere to the surface of the medium and emulsify readily.  Avian types develop hemispherical colonies, pale yellow or grey in color depending on the media used, after 2 - 3 weeks incubation and slightly larger than colonies of bovine types.  Acid fast saprophytes generally form pigmented colonies after several days incubation and will grow well at room temperature.  Such colonies are soft, creamy, and usually smooth.

TYPES

M. tuberculosis variants:

1.  Human
2.  Bovine
3.  Avian

Mycobacterium Other Than Tuberculosis (MOTT)

1.  Photochromogens (Group I)
2.  Scotochromogens (Group II)
3.  Non-Photochromogens (Group III)
4.  Rapid Growers (Group IV)

BIOCHEMICAL TESTS

Catalase:
M. tb and variants   (+)
MOTT (+++)

Auramine disc:

M.tb and variants  (+)

Groups I, II, III  (+/-)

Rapid Growers (-)

Niacin:

M.tb var. human (+)

M.tb var. bovine (-)

M.tb var. avian (-)

MOTT (-)

ANIMAL PATHOGENICITY

Mouse:  M.tb var. human, bovine, avian (+); MOTT - Photochromogen (+); Scotochromogen (-); Non-Photochromogen (+, some -); Rapid Growers (-, some +) 

Guinea pig: M.tb (+); M.tb var. avian (-); MOTT (-)

Rabbit:  M.tb var. human, bovine, avian (+); MOTT (-)

Fowl: M.tb var. human, bovine (-); M.tb var. avian (+); MOTT (-)

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