Tuberculosis

Tuberculosis is an infectious
bacterial disease affecting
primarily the lungs, but any other
organs may be affected. The
disease can be transmitted from
cattle to humans through the
consumption of raw milk or
airborne spread and is of major
public health significance in the
world.
 Causative Agent
Mycobacterium spp. produce the classic disease
known as tuberculosis: M. tuberculosis, M bovis, M.
avium, M. africanum,...

Organisms are non motile, non spore forming,

slightly curved or straight rods that resist staining
with basic dyes due to high lipid content (wax) in
the cell wall. The cells usually appear weakly
positive on Gram stain. If special methods are used
to stain the organisms, the stain is not easily
removed, even with acidified organic solvents.
Therefore, the mycobacteria are called acid-fast
bacilli.
Cell wall of Gram’s positive bacteria VS Cell wall of Mycobacteria
 Sample Type
- Sputum
- L.N
- Pleural effusions
- Urine
- Stool
- CSF
- Aspiration ( gastric – cold abscess)
- Milk
 Treatment of Sample
Petroff’s Method

Use of 4 % NaOH to be mixed with the

sample for decontamination. Spin the
mixture and then decant the supernatant
and use the sediment in culture,
preparation of a stained film and any
other test.
 Ziehl – Neelsen’s Stain
Acid – fast Stain
Stained smears help in the early diagnosis of
mycobacterial infections. This is important because
most mycobacteria are slow growers and cultures may
take weeks to appear on laboratory media. Staining
can also be used to confirm the acid-fast properties of
cultures isolated in the laboratory.

TB can resist the decolorization with acid-alcohol, so

called Acid – fast bacilli. (Fastness = Resistance).

ZN stain is a differential stain that differentiates between

acid – fast (Red) and non acid – fast bacilli (Blue).
 Ziehl-Neelsen Technique
Flood the slide with STRONG Carbol Fuchsin and HEAT for 5
minutes
AVOID BOILING

Acid fast Non Acid fast

bacilli bacteria
Let the slide cool for a minute

Decolorize using Acid Alcohol (15-20 seconds)

AVOID OVER-DECOLORIZATION

Flood the slide with Methylene blue and leave to act for
30 sec.

Blot dry, add drop of cedar wood oil, then examine under oil immersion
lens.
Acid fast Bacilli
Single or in Cords
Page 55

Bacterial film stained with ZN stain showing red acid

fast bacilli in-between blue non-acid fast organisms
 Culture of Mycobacterium spp.
The following solid media are recommended:
1) Solid egg-based:
A- Lowenstien-Jensen (LJ) slants
B- Dorset’s egg media.

2) Solid agar-based:
A- Middlebrook 7H-9
B- Middlebrook 7H-10
C- Middlebrook 7H-11

3) Media for M. paratuberculosis:

Herrold’s egg – yolk medium contains mycobactin J
 Supplements may be added
Sodium Pyruvate:
Enhances the growth of M. bovis

Glycerol:
Enhances the growth of M. tuberculosis and M. avium.

Mycobactin J:
Essential supplement for the growth of M.
paratuberculosis, the causative agent of Johne’s
disease.
Lowenstien-Jensen
Dorset’s egg
 Biochemical Identification
Nitrate Reduction Test
Nitrate -ve Nitrate +ve

M. bovis M. tuberculosis