1. Lab Identification Of Mycobacterium Tuberclosis 17.4.2014

2. Objectives At 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

3. Mycobacterium Strict Pathogens M.Tuberculosis complex M.Tuberculosis M.Bovis M.Africanum M.micoti Animal Pathogens Lepra bacilli Atypical Myco- bacteria Saprophytic Mycobacteri a

4. Differences and similarities between Actinomyces,Nocardia and Mycobacteria GenusSpore Aerobic Growth Exotoxin Facultative Intracellular IC Hosts Acid Fast Branching rods Actinomyces______+ Nocardia_+__+++ Mycobacterium_+_+++_

5. 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

6. 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

7. 2- Identification Morphological properties Biochemical reaction Analysis of cell wall lipids Nucleic acid probe 3- Skin test Mantoux test

8. 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

9. 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 +

10. MORPHOLOGY MICROSCOPY

12. 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.

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

14. 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

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

16. Treatment and Vacination Comined antituberclous drugs: Isonazid Rifampicin Ethambutol Streptomycin Vaccination: BCG (Live attenuated vaccine)

17. Thank you for your attention Dr.Mashair Abdelrahman