Morphology

It is a slender or slightly curved bacillus, in direct smears

measuring about 2.5 to 3.5 by 0.3 µm although both shorter

and longer forms may be seen. In cultures, short forms are

found especially on solid media, but longer forms may be

found in liquid media. The bacillus may occur singly, or in

pairs, or in larger or smaller masses.

The tubercle bacillus (TB or Koch’s Bacillus) does not

stain easily by the ordinary dyes, but it stains well with a

strong dye with a mordant, such as carbol fuchsin when the

stain is hot but it takes a longer time when the stain is cold.

When once it has been stained it resists decolorization

with 20% H2SO4 or HNO3 (Nitric acid); it also resists

decolorization with alcohol, and so it is both acid and

alcohol fast. The method generally used for staining is

Ziehl-Neelsen’s method or one of its modifications. The

bacilli may stain evenly, or they may show beading or

barred staining or sometimes may have terminal granules.

The bacilli are gram-positive but by this method they are

stained only with great difficulty and Gram’s method is of

no use for their identification. A more recent method is the

use of auramine-phenol fluorescent stain.

Cultures

The tubercle bacillus will not grow on ordinary media.

Primary cultures are usually made on some form of egg

844 Concise Book of Medical Laboratory Technology: Methods and Interpretations

medium of which Lowenstein-Jensen (LJ) medium is

probably the most widely used. Dorset’s egg medium

or Petragnani’s media may also be used. After the

primary culture has been established, it is possible to

make subcultures on media without egg, such as Dubos,

Proskauer and Beck, Kirschner and Youman’s media.

All the varieties, viz. hominis, bovis and avium may be

found in man but in India, the bovine seems to be rare in

man, and the avian is not a common finding.

Cultural Characteristics

M. tuberculosis is aerobic, and the optimum temperature

for the human and bovine varieties is 37oC, and for

the avian 40–44oC. It grows slowly. It does not grow on

ordinary media but for the primary isolation requires an

enriched medium, a medium including eggs is used most

commonly. Glycerol stimulates the growth of the human

and avian varieties but not the bovine. On LJ medium,

the general appearance of the growth is dry, irregular,

tough and tenacious, a buff to light orange in color, but

if the surface of the medium is moist the appearance of

the colony is smoother. The growth is said to be eugonic

that is growing well. The bovine variety is dysgonic that

is growing with difficulty and the colonies are smaller,

discrete, rather smooth, slightly moist, and grayish yellow

in color. The avian grows rapidly, the colonies are moister,

more luxuriant and individual colonies have a smooth

shiny surface, yellowish to faint pink in color.

Laboratory Diagnosis

In many specimens the finding of acid-fast bacilli typical

in shape and staining, is accepted as sufficient for calling

them tubercle bacilli, but it must always be remembered

that there are many other acid-fast bacilli, which may be

found particularly in stomach-wash, urine, feces, and even

in sputum. Also when bacilli are few in number, they may

not be found in direct smear. Concentration methods such

as sodium hydroxide and trisodium phosphate may help

but on the whole considerable labor is involved without a

great increase in positive findings. Therefore, cultures are

being increasingly used even if 3 to 4 weeks elapse before a

positive result is given, and most workers report a negative

only after 6 to 8 weeks.

The specimens most commonly examined for tubercle

bacilli are sputum, stomach wash, laryngeal swabs,

urine, CSF, pleural or peritoneal fluid, pus and tissue. The

specimens are collected in clean, sterile vessels, and most

are treated with 4% NaOH or 6% H2SO4 (v/v) before the

culture is made. The treatment homogenices the specimen

and also destroys organisms other than mycobacteria.

Concentration Methods

Except in very severe cases, the organism is not always

present in large numbers and for this reason, techniques

have been devised for concentrating the organisms present

to facilitate their detection and isolation. Concentration

serves two other purposes.

1. The mucoid material is broken down and the sample

homogenized.