1. Experiment seven Mycobacterium tuberculosis

2. Pathogenesis primary infection primary infection 1) lung infection secondary infection secondary infection 2) Out lung infection

5. Who is at risk: Primary infection: children Primary infection: children Secondary infection: age>25 Secondary infection: age>25

6. M. tuberculosis General Features

9. Clinical syndromes a. fatigue, weakness, weight loss and fever a. fatigue, weakness, weight loss and fever b. pulmonary involvement: chronic cough,spit blood b. pulmonary involvement: chronic cough,spit blood c. meningitis or urinary tract involvement c. meningitis or urinary tract involvement d. bloodstream dissemination: miliary tuberculosis with lesions in many organs and a high mortality rate. d. bloodstream dissemination: miliary tuberculosis with lesions in many organs and a high mortality rate.

10. MYCOBACTERIUM TUBERCULOSIS Can infect (disseminate) and cause disease in many different body locations such as: 1.Meninges 2.Brain 3.Bone 4.Kidney 5.Essentially any organ (lung primary target)

11. Diagnosis The steps to diagnose TB infection and disease include: A medical evaluation that includes history and risk assessment A medical evaluation that includes history and risk assessment The tuberculin skin test The tuberculin skin test A chest x-ray A chest x-ray A bacteriological examination A bacteriological examination

12. Treatment for Tuberculosis Treated with a combination of multiple drugs for a long period of time: rifampin, isoniazid (INH), pyrazinamide, ethambutol, and streptomycin. Treated with a combination of multiple drugs for a long period of time: rifampin, isoniazid (INH), pyrazinamide, ethambutol, and streptomycin. Emergence of multi-drug resistant M. tuberculosis strains.

13. Principle While the majority of bacterial organisms are stainable by either simple or Gram-staining procedures, a few genera, particularly the members of the genus Mycobacterium like M tuberculosis, are resistant and can only be visualized by the acid fast method. The stain is of diagnostic value in identifying these organisms. While the majority of bacterial organisms are stainable by either simple or Gram-staining procedures, a few genera, particularly the members of the genus Mycobacterium like M tuberculosis, are resistant and can only be visualized by the acid fast method. The stain is of diagnostic value in identifying these organisms.

14. Principle The characteristic difference between mycobacteria and other organisms is the presence of a thick waxy (lipoidal) wall that makes penetration by stains extremely difficult. Once the stain has penetrated, however, it cannot be readily removed even with the vigorous use of acid alcohol as a decolorizing agent. Because of this property, these organisms are called acid-fast, while all other microorganism, which are easily decolorized by acid alcohol, are non-acid- fast.The acid-fast stain use three different reagents as follows: The characteristic difference between mycobacteria and other organisms is the presence of a thick waxy (lipoidal) wall that makes penetration by stains extremely difficult. Once the stain has penetrated, however, it cannot be readily removed even with the vigorous use of acid alcohol as a decolorizing agent. Because of this property, these organisms are called acid-fast, while all other microorganism, which are easily decolorized by acid alcohol, are non-acid- fast.The acid-fast stain use three different reagents as follows:

15. Acid-fast stain 1.Prepare a Smear Smear Dry Fixed 2.Acid-fast stain

16. Process of Acid-fast stain 9% carbolfuchsin acid-alcohol (Primary staining) (Decoloration) Methylene blue Methylene blue (Conterstain) (Conterstain) Washing 5min 5min5min 1-2min Washing dry the slide with bibulous papers Observation with the oil immersion lens Results :acid-fast bacteria:red color non-acid-fast:blue color

17. Mycobacterium tuberculosis:red colour Shape:silm rod,o.4×3μm Background and the other bacteria:blue colour

18. Corynebacterium diphtheriae

20. Gray-black colonies on tellurite 亚碲酸盐 medium

21. Gram stain PROCEDURE: PROCEDURE: –Smear: size of a dime to form a thin film –Dry : air dry –Fix: through the warm air above the flame two or three times.

22. NEISSER’S STAIN Ⅰ and Ⅱ NEISSER’S STAIN Ⅲ washing 2min Blot dry with bibulous papers Observation with the oil immersion lens 30s Process of Neisser’sStain

23. Metachromatic granules