Mycobacterium tuberculosis

OPTOM FASLU MUHAMMED

Morphology• It is a straight or slighty curved rod.• 3 X 0.3 pm in size.• Occuring singly, in pairs or small clumps.• The size depents on condition of growth• Long filamentous , club-shaped and branching

forms may some times be seen• Tubercle bacilli – gram +ve

• Tubercle bacilli resist decolourisation by 20 % sulphuric acid & absolute alcohol for 10 minutes ( acid & alcohol fast ).

• Acid fastness has been ascribed variously to the presence in the bacillus of an unsaponifiable wax (myocoloic acid) or to a semipermiable membrane around the cell.

• It is related to the integrity of the cell and and appears to be a property of the lipid-rich waxy cell wall.

• Staining may be uniform or granular.• Beaded or barred forms are frequently seen in

M tuberculosis, but M bovis stains more uniformly.

Cultural charecteristics• The bacilli grow slowly,• The generation time in vitro being 14-15 hrs.• Colonies appears in about 2 weeks & may

some times take up to 8 weeks.• Optimum temp = 37 ᴼC.• Growth does not occure below 25 ᴼC or above

40 ᴼC.• Optimum pH is 6.4 to 7.0 .• M tuberculosis is an oligate aerobe.

Mycobacteria colonies

• The solid media contain egg (Lowenstein-Jensen,Petragnini,Dorset),blood (Tarshis), serum (Loeffler) or potato ( Pawlowsky).

• The solid medium most widely employed for routine culture is the Lowenstein-jensen(LJ) medium without starch, as reommended by the International Union Against Tuberculosis (IUAT).

• On solid media, M tuberculosis forms dry, rough, raised, irregular colonies with wrinkled surface.

• They are creamy white, becoming yellowish or buff coloured on further incubation.

• They are tenacious and not easily emulsified.• M bovis colonies , in comparison are flat,

smooth, moist, white and break up easily when touched.

• In liquid media without dispersing agents the growth begins at the bottom , creeps up the slides and form a prominent surface pellicle which may extend along the sides above the medium.

BIOCHEMICAL REACTIONS

Niacin test• 10% cyanogen bromide and 4% aniline in 96 %

ethanol are added to a suspension of the culture,

• A canary yellow color indicates a + ve reaction.• This test + ve with the human type and – ve

with the bovine type of bacilli.

Aryl sulphatase test• This test +ve only with a typical mycobacteria .• The bacilli are grown in a medium containing

0.001 M tripotassium phenolphthalein disulphate.

• 2 N , NaOH is added drop by drop to the culture.

• A pink colour indicates +ve reaction.

Neutral red test• Virulent stains of tubercle bacilli are able to

bind neutral red in alkaline buffer solution, while avirulent stains are unable to do so.

Catalase-peroxidase test• A mixture of equal volumes of 30 vol. • H₂O₂ and 0.2 % catechol in distilled water is

added to 5 ml of the test culture and allowed to stand forfew minutes.

• Effervescence indicates peroxidase activity.

Amidase tests• A 0.00165 M soltion of amide is incubated

with the bacillary suspension at 37 ᴼC and 0.1 ml of MnSO₄ . 4 H₂O , 0.1 mi of phenol solution and 0.5 ml of hypochloride solution are added

• The tubes are placed in boiling water for 20 minutes.

• A blue colour indicates a +ve test.

Nitrate reduction test• This is +ve with M tuberculosis and –ve with

m bovis.

Antigenic properties• Infection by bacilli , delayed hyper sensitivity is

devoleped to the bacillary protein (tuberculin)

BACTERIOCINS• M tuberculosis is divisible into two types by

mean of bacteriocins produced by rapidly growing mycobacteria.

MOLECULAR TYPING• Restriction fragment length polymorphism

(RFLP) enables strain typing for epidemiological purposes.