This document provides information on Mycobacterium tuberculosis. It discusses that M. tuberculosis is the primary cause of human tuberculosis, along with M. bovis. M. tuberculosis is an acid-fast, aerobic bacterium that grows slowly and forms characteristic colonies on culture media. It discusses the morphology, cultural characteristics, resistance properties, host range, antigenic properties, and biochemical reactions of M. tuberculosis that aid in its identification. Typing methods like phage typing, bacteriocin typing, IS6110 RFLP typing, and spoligotyping are also summarized for distinguishing strains of M. tuberculosis.

1. MYCOBACTERIUM
TUBERCULOSIS
Anushi Jain
Roll No. : 08
Paper II
Msc II

2. INTRODUCTION
• Mycobacteria are slender
rods.
• They sometimes show
branching, filamentous
forms resembling fungal
mycelium.
• They are aerobic, non-
motile, non capsulated and
non-sporing , usually
having slow growth.
• They are acid fast bacilli.
• Mycobacteria can be
classified as
Mycobacterium I or
Mycobacterium II.

3. • The genus Mycobacteria contains three groups : obligate
parasites, opportunistic pathogens and saprophytes.
• Obligate Parasites : this includes :-
1. Mycobacterium tuberculosis complex : M. tuberculosis,
M. bovis, M. africanum, M. microti, M. canetti, M. caprae,
M. pinnipeddi .
2. Mycobacterium leprae
• Opportunistic pathogens : These are the non-
tuberculous mycobacteria (NTM). They are categorized
as photochromogens, scotochromogens, non-
photochromogens and rapid growers.
• Saprophytes : Saprophytic mycobacterium are isolated
from many sources. These includes M. phlei (from grass)
and M. smegmatis (from smegma).

4. MYCOBACTERIUM TUBERCULOSIS
• M. tuberculosis and M. bovis are
the human tuberculosis causing
mycobacteria.
• M. tuberculosis was discovered in
1882 by Robert Koch.
MORPHOLOGY
• M. tuberculosis is a straight or
slightly curved rod whereas M.
bovis is straight and short.
• 3 x 0.3 μm in size.
• They can occur singly, in pairs or as
small clumps.
• They are gram positive.
• They are filamentous, club shaped
and branching.
• They are acid and alcohol fast.

5. CULTURAL CHARACTERISTICS
• The bacilli have slow growth, with a generation time of
14-15 hours in vitro.
• the colonies maybe appear after 2weeks and may take
upto 8 weeks.
• The optimum temperature is 37°c and the growth does
not occur below 25°c or above 40°c.
• Optimum pH is 6.4-7.0
• M. tuberculosis is eugonic whereas M. bovis is dysgonic.
• The addition of 0.5% glycerol improves the growth of M.
tuberculosis but have no effect on or even impair the
growth of M. bovis whereas sodium pyruvate helps the
growth of both types.

6. • Several media, both solid and
liquid, can be used for growth of
tubercle bacilli.
Solid Media :
• The most widely used solid media
is Lowenstein-Jensen (LJ).
• On solid media, M. tuberculosis
forms dry, rough, raised, irregular
colonies with a wrinkled surface.
• The colonies are creamy white,
becoming yellowish or buff-
coloured on further incubation.
• Other media that can use are
Petragnini media, Dorset media,
Tarshis medium, Loeffler media or
Pawlowsky media.

7. Liquid Media
• The most liquid media
used are Dubos’s,
Middlebrooks's,
Proskauer and Beck’s,
Sula’s and Sauton’s.
• In liquid media, the
growth begins at the
bottom, creeps up and
forms prominent pellicle
at the surface.

8. RESISTANCE
• Mycobacteria are not completely heat resistance, being
killed at 60°c for 10-15 minutes.
• Cultures may be killed by exposure to direct sunlight for
two hours, but the bacteria survive in sputum for 20-30
hours and may remain viable in droplet nuclei for 8-10
days.
• They are resistance to chemical disinfectants like 5%
phenol, 15% Sulphuric acid, 3% nitric acid, 5% oxalic acid
and 4% sodium hydroxide.
• They are sensitive to formaldehyde and glutaraldehyde.
• They can be destroyed by 80% ethanol in 2 minutes and
by iodine in 5 minutes.

9. Host Range
• M. tuberculosis causes natural infection in humans, other
primates, dogs and other animals in close contact with
humans.
• It is highly infectious for guinea pigs ands hamsters.
• M. bovis is more pathogenic for animals.
• It causes tuberculosis in cattle, carnivores, dogs and cats,
swine, parrots and other birds of prey.
• It is moderately pathogenic for rats.
• ‘African strains’ or M. africanum was isolated from some
parts of South Africa, showing intermediate properties
between human and bovine types.
• ‘Asian Type’ of tubercle bacilli was isolated from South
India, where of low virulence for guinea pigs.

10. ANTIGENIC PROPERTIES
Many antigens have been identified in the mycobacterial cell
wall.
They constituents of lipids, proteins, polysaccharides.
1. Lipids :
• The cell wall is rich in mycolic acid, which plays an important
role in pathogenesis.
• Mycolic acid is also responsible for the acid fastness of the
bacteria and cellular tissue reactions of the body.
2. Proteins :
• They are responsible for the tuberculin reaction.
• They induce delayed type hypersensitivity.
3. Polysaccharides :
• Their role in pathogenesis is not certain.
• They induce delayed type hypersensitivity.

11. BIOCHEMICAL REACTIONS
NIACIN TEST :
• Niacin is detected by
addition of 10% cyanogen
bromide and 4% aniline in
96% ethanol to a suspension
of the culture.
• Positive reaction – Canary
yellow colour.
• M. tuberculosis – Positive.
• M. bovis – Negative.

12. Aryl Sulphatase Test
• The bacilli is grown in a
medium containing
0.001M tripotassium
phenolphthalein
disulphate.
• 2N NaOH is added, drop
by drop.
• Positive reaction – Pink
colour.
• Only positive for atypical
mycobacteria.

13. Catalase-Peroxidase Test
• Equal volumes of H2O2 and 0.2%
catechol in distilled water are
added to 5ml of test culture and
allowed to stand.
• Effervescence – Catalase
positive.
• Browning – Peroxidase positive.
• Atypical mycobacteria – Catalase
positive and Peroxidase
negative.
• M. tuberculosis – Catalase
negative and Peroxidase
positive.

14. Nitrate Reduction Test
• Sulphanilamide and n-
naphthyl-ethylene is
added to bacterial
suspension in nitrate
medium.
• Positive reaction – Red
colour.
• M. tuberculosis –
Positive.
• M. bovis – Negative.

15. Amidase Test
• Five amides are tested,
namely, acetamide,
benzamide, carbamide,
nicotinamide and
pyrazinamide.
• A 0.00165M solution of
amide is incubated with
bacillary suspension at
37°c and 0.1ml of
MnSO4.4 H2O, 1.0ml of
phenol solution and
0.5ml of hypochlorite
solution are added.
• Positive reaction – blue
colour.

16. Pyrazinamide Test
• Enzyme pyrazinamidase
hydrolyses pyrazinamide to
ammonia and pyrazionic
acid which is detected by
adding ferric ammonium
sulphate.
• Positive reaction – Pink
colour band.
• M. tuberculosis – Positive.
• M. bovis – Negative.

17. Neutral Test
• Virulent strains of tubercle
bacilli can bind to neutral
red in alkaline buffer
solution, virulent strains
cannot bind.
Tween 80 Hydrolysis test
• Tween 80 is added to the
medium.
• Positive reaction – Colour
change from yellow to red
at pH 7.
Inhibition by thiophene-2
carboxylic acid (T2H) test
can also performed.

18. TYPING METHODS
Phenotypic
• Bacteriophage Typing :
Tubercle bacilli has been
classified into four
phages type : A, B, C and
a intermediate between
A and B, designated I.
• Bacteriocin Typing : M.
tuberculosis can also be
typed by means of
bacteriocin produced by
rapidly growing
mycobacteria.
Molecular Typing
• IS6110 RFLP Typing :
Restriction endonuclease
treatment yields nucleic
acid fragments of varying
lengths, the pattern of
which are strain specific.
• Spoligotyping (spacer
oligotyping) : This is
based on polymorphism
in the direct repeat (DR)
locus.

19. THANK YOU