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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.