Acid fast bacillus - Myobecterium Tuberculaes

Microbiology,
Acid Fast Bacillus - Specialized staining technique for myobecterium tuberculae and other bacterias having complex cell wall.

1. Mycobecterium

2. Presented By : Presented To :

3. Acid Fast

4. History of Acid Fast Staining In 1882 ROBERT KOCH reported the discovery of the tubercle bacillus and described the appearance of the bacilli resulting from a complex staining procedure.  During  the same time period several other researchers (Ehrlich, Ziehl, Rindfleisch, and Neelsen), intending to improve on Koch’s method, introduced modifications to the reagents and the procedure.  

5. Franz Ziehl was the first to use carbolic acid (phenol) as the mordant.  Friedrich Neelsen kept Ziehl’s mordant, but changed the primary stain to the basic fuchsin . This method became known as the Ziehl-Neelsen method in the early to mid 1890s. 

6. Acid fast  organisms like Mycobacterium contain large amounts of lipid substances within their cell walls called mycolic   acids . These  acids  resist  staining  by ordinary methods such as a Gram  stain . It can also be used to  stain a few other bacteria, such as Nocardia .

7. LIPID RICH CELL WALL

8. The stains used are the red colored Carbol fuchsin that stains the bacteria and a counter stain like Methylene blue or Malachite green.

9. Procedure ACID FAST STAINING Methylene Blue Carbon Fuschin

10. Basic Requirements Carbolfuchsin (Red) Acid Alcohol Counterstain with Methylene Blue Acid - Fast Cells – Red Non Acid – Fast – Blue

11. Acid-fast Stain Reaction Explained Primary stain:   The hot pink appearance of Acid-fast cells is caused by Carbol fuchsin , the primary (first) stain, which is driven into acid-fast cells using the heat from a water bath. Carbol Fuchsin is a lipid soluble, phenolic compound, which is able to penetrate the cell wall

12. Secondary stain  ( counterstain ): The methylene blue counterstain imparts Blue color to the colorless nonacid-fast bacteria, but doesn't change the color of acid-fast cells.​

13. Application of Primary Stain 1. Carbol fuchsin primary stain of acid-fat stain; 2. Carbol fuchsin being applied to slide that had been prepared with acid-fast controls and an unknown bacteria. Blotting paper has been put on top of the slide. Then the blotting paper is saturated with stain and heated over water bath; 3. Clothes pins are useful for handling the slide; 4. Blotting paper is discarded and slide is rinsed. 

14. Application of Decolorizer 1. Acid alcohol decolorizer for acid-fast stain; 2. Drizzle decolorizer down slide for 10 - 15 seconds, while watching to see that stain is removed from negative control; 3. Rinse

15. Application of Counterstain : 1. Secondary stain ( counterstain ), crystal violet; 2 . Crystal violet is applied to slide and left for one minute; 3 . Rinse; 4. Stained acid fast slide ,

16. Observation Myobacteriu m

17. Mycobacterium Kingdom: Bacteria Phylum: Actinobacteria Class: Actinobacteria Order: Actinomycetales Suborder: Corynebacterineae Family: Mycobacteriaceae Genus: Mycobacterium Species: M.tuberculosis

18. MYCOBACTERIUM-INTRODUCTION Mycobacteria are  aerobic, Rod shaped  and nonmotile bacteria (except for Mycobacterium marinum , which shows motile within macrophages Cell wall –rich in lipids Very slow growing (15-20 Hr)  They do not have capsules, and most do not form  endospores . are characteristically acid fast.

19. Cell wall The distinguishing characteristic of all  Mycobacterium  species is that the cell wall is thicker than in many other bacteria, being hydrophobic, waxy, and rich in  mycolic acids or mycolates . contains a polypeptide layer, a peptidoglycan layer, and free lipids. There are porins in the membrane to facilitate transport. Beneath the cell wall, there are layers of arabinogalactan and peptidoglycan that lie just above the plasma membrane.

20. Mycobacterial  cell wall: 1-outer lipids, 2-mycolic acid, 3-polysaccharides ( arabinogalactan ), 4-peptidoglycan, 5-plasma membrane, 6-lipoarabinomannan (LAM), 7-phosphatidylinositol mannoside , 8-cell wall skeleton`

21. The high concentration of lipids in the cell wall of  Mycobacterium tuberculosis have been associated with these properties of the bacterium: Impermeability to stains and dyes  Resistance to many antibiotics  Resistance to killing by acidic and alkaline compounds  Resistance to osmotic lysis via complement deposition  Resistance to lethal oxidations and survival inside of macrophages

22. Mycobacterium tuberculosis complex M. tuberculosis M. bovis ( subsp.   bovis   and   caprae ) vaccine strain  M. bovis   BCG ( Bacille Calmette-Guérin ) M. africanum M. canettii M. microti M. pinnipedii These species are, with the exception of  M. bovis   BCG, considered to cause tuberculosis (TB) in humans and animals. Despite their close genetic similarity, these organisms differ considerably with regard to epidemiology, pathogenicity and their host spectrum.

23. Habitate Mycobacteria are widespread organisms, typically living in water (including tap water treated with chlorine) and food sources. Some, however, including the tuberculosis and the leprosy organisms, appear to be obligate parasites and are not found as free-living members of the genus.

24. Myobacterium Division A natural division occurs between  slowly – and  rapidly–growing  species. Mycobacteria that form colonies clearly visible to the naked eye within seven days on subculture are termed rapid growers, - while those requiring longer periods are termed slow growers

25. Two media are used to grow MTB 1- Middlebrook's medium   -which is an agar based medium and 2- Lowenstein-Jensen medium  which is an egg based medium.

26. “Tuberculosis is defined as an infectious disease caused by a bacterium; that most commonly affects the lungs.” It can also be a crippling and deadly disease, and is on the rise in both developed and developing worlds.  Globally, it is the leading cause of deaths resulting from a single infectious disease.  Currently, it kills “three million people” a year and could claim up to 30 million lives if not controlled. What is Tuberculosis?

27. Types of Tuberculosis Mycobacterium which is carried by humans. Mycobacterium T.B. can present it self in the human body in different forms effecting any where from “the intestines, bones, joints, skin, and the genito urinary , lymphatic, and nervous systems.”

28. The primary stage of the disease may be symptom-free, or the individual may experience a flu-like illness . This is called the “inactive stage.” Within the active stage of the disease, there might be a slight fever, night sweats, weight loss, fatigue. The symptoms may vary depending on what type of tuberculosis you contract. Symptoms of Tuberculosis

29. PULMONARY TUBERCULOSIS

30. INTRODUCTION Pulmonary Tuberculosis (TB) is an infectious disease that mainly affect the lungs parenchyma. TB is a contagious bacterial (M. tuberculosis) infection that mainly affects the lungs parenchyma, but may spread to other organs. TB is an ancient disease. Signs of skeletal TB (pott disease) were evident in Europe from Neolithic times, ancient Egypt, and in the pre-Columbian New World. Physicians in ancient Greece called this illness as “phthisis” reflecting its wasting character.

31. TB has remained an enemy of human society for all age. TB is not only a problem for the person suffering from it or their families but a public health problem of the entire world.

32. TB spread from person to person by airborne transmission. Infected person release droplet nuclei (1-5 micro meter in diameter) through, Talking Coughing Sneezing Laughing Singing If not treated properly, TB can be fatal.

33. Clinical presentation of Pulmonary TB Chronic cough Weight loss Pyrexia of unknown origin Unresolved pneumonia Chest pain fatigue

34. SIGNIFICANT LAB TEST Tuberculin skin test: Injecting a small amount of protein from tuberculosis bacteria between the derived layer of the skin (usually forearm). Sputum examination and Cultures; Is examined under a microscope to look for tuberculosis bacteria and used to grow the bacteria in a culture.

35. Interferon-gamma Blood test ; A simple blood is mixed with synthetic proteins similar to those produced by the tuberculosis bacteria. If people are infected with tuberculosis bacteria, their white blood cells produce certain substances (interferons) in response to the synthetic proteins. SIGNIFICANT LAB TEST

36. IMAGING CONSIDERATION Chest CT Scan Chest X-ray

37. Treatment Anti TB drugs: Duration 9 months: Isoniazid along with pyridoxine (vit B6) Rifampicin Ethambutol Pyrazinamide