Lab Identification Of Mycobacterium Tuberclosis 17.4.2014

ObjectivesAt the end of the practical demonstration, student should be able to:Identify mycobacteria on the basis of their:• Acid fast staining.• Morphology as seen on microscopy• Culture medium• Differentiate between gram staining and acid fast staining• Clinical features of tuberclosis

Mycobacterium

Differences and similarities between Actinomyces,Nocardia and Mycobacteria

GenusGenus SporeSpore Aerobic Aerobic GrowthGrowth

ExotoxinExotoxin Facultative Facultative IntracellularIntracellular

IC IC HostsHosts

Acid Acid FastFast

Branching Branching rodsrods

ActinomycesActinomyces __ __ __ __ __ __ ++

NocardiaNocardia __ ++ __ __ ++ ++ ++

MycobacteriumMycobacterium __ ++ __ ++ ++ ++ __

Mycobacterium TUBERCLOSIS MORPHOLOGY

•Are slender bacilli exhibit filamentous form resembling fungal mycelium(myces meaning fungus)•Stained by hot carbol-fuchsin and resist declorization by dilute mineral acids this why refered to acid –fast bacilli• NONMOTILLE•NONSPORING•NONCAPSULATED BACILLUS

Labrotary diagnosis of mycobacteria

1- Detection Microscopy – carbolfuchsin acid fast test (Z.N stain)Morning sputum collection for 3 days Culture-A- solid egg-based LJ(lowenstien jensn medium B- solid agar –based or broth –based Middlebrook medium

2- Identification •Morphological properties•Biochemical reaction•Analysis of cell wall lipids•Nucleic acid probe 3- Skin test Mantoux test

IDENTIFICATION OF M. tuberculosis

• • Growth rate slow• • Growth temperature 35°-37°C only• • No pigmentation• • Niacin positive• • Nitrate positive• • Catalase negative at 68°C• • growth on LJ medium

CULTURE OF MYCOBACTERIA TB•Lowenstien-jensn medium•Middlebrook broth media•Tubercle bacilli are aerobes•Grow slowly12-24 h and rquire 8 weeks for incubation•Enreched media ; containing egg potatoes , serum and meat.•NICAIN +

MORPHOLOGYMICROSCOPY

The term acid fastness refers to resistance showed by these bacteria to decolorization by acid alcohol(95%ethyl alcohol and 3% Hcl).At this strength acid alcohol decolorizes all bacteria except for mycobacteria.

Colonies on LJ medium take 18-24 days to appear .They appear as dry,rough,creamy or buff colored colonies.

Skin test• Mantoux test•Intradermal test 0.1 ml of PPD•Result : positive reaction local edema after 48-72 h of 10 mm diameter •Reagents• old tuberculin test –koch test•PPD Purified Protien derivative

Clinical Disease and symptoms•Primary infection •Active tuberclosis(chronic caseous granuloma)•Miliary tuberclosis•Symptoms :•chronic cough - productive cough • fever (sweating)•Weight loss

Treatment and VacinationComined antituberclous drugs:• Isonazid• Rifampicin• Ethambutol • Streptomycin

Vaccination:• BCG (Live attenuated vaccine)

Thank you for your attention

Dr.Mashair Abdelrahman