Afb stain by manoj

Afb stain by manoj

1. ZIEHL-NEELSEN STAIN Manoj Mehta MSc. Clinical microbiology

2. What is staining? Colored compound is used to develop a contrast between the specimen and the background. This process of imparting colour to the cell  is known as staining . What is the difference between stain and dye? Dyes are the textile colouring agents that have been prepared with lesser specifications and they may contain  the impurities . Stains are the biological colouring agents that are more pure and prepared with greater care and specification   Dye is crude and stain is purified form.  

3. Why stain cells ? Cells are stain to reveal the size and shape of microorganisms. Cells are stained is to enhance visualization of the cell or certain cellular components under a microscope.  Cells may also be stained to highlight metabolic processes . To denotiate between live and dead cells in a sample .  Cells may also be enumerated by staining cells to differetermine biomass in an environment of interest.  Cells are stained to demonstrate the presence of  internal and external structures . Cells are stained to distinguish between different types of organisms .    

4. STAINING METHODS IN MICROBIOLOGY Simple Stains: only one dye is employed for the colouration of bacterial smear Ex. Methylene blue Negative Stains: Negative :staining is a technique by which bacterial cells are not stained, but are made visible against dark background.  Ex. Eosin,Nigrosin,India Ink etc Differential Stains: Differential Stains use two or more stains and allow the cells to be categorized into various groups or types. Other staining techniques allow the observation of cell morphology, or shape, but differential staining usually provides more information about the characteristics of the cell wall (Thickness ). Ex. Gram Staini , Acid Fast Stain

5. ACID-FAST STAIN Ziehl – Neelsen stain, also known as the acid-fast stain Ziehl – Neelsen stain was first described by two German doctors ; Franz Ziehl (1859 to 1926), a bacteriologist and Friedrich Neelsen (1854 to 1894) a pathologist . Principle : When the smear is stained with carbol fuchsin , is lipid-soluble and contains phenol, which helps the stain penetrate the cell wall. This is further assisted by the addition of heat. The smear is then rinsed with a very strong decolorizer , which strips the stain from all non-acid-fast cells but does not permeate the cell wall of acid-fast organisms . The decolorized non-acid-fast cells then take up the counterstain . . Only decolorized cells absorb the counter stain and take its color and appears blue while acid-fast cells retain the red color. Ex. Mycobacteria, Actinomyces in tissue sections, cultures of Nocardia , oocysts of Cryptosporidium, Isospora and bacterial spores

6. Chemicals used in AFB stain Primary stain : Strong carbol fuchsin (consists of basic fuchsin and carbolic acid phenol) Decolourizer : 20% sulfuric acid (H2SO4) or acid alcohol Counterstain : methylene blue or malachite green AFB : Once bacteria are stained with strong carbol fuchsin solution with the help of heat, those which are not decolorized by acid (20% H2So4), are called acid fast bacilli (AFB) . AAFB :those which are not decolorized by acid and alcohol solution (3%acid/alcohol) are called acid alcohol fast bacilli AAFB

7. Modifications in the percentage of sulfuric acid 5% H2SO4 for M.leprae 1% H2SO4 for Actinomyces in tissue 0.5 % H2SO4 for cultures of Nocardia 0.25-0.5%for spores and for oocysts of Cryptosporidium and Isospora

8. Sample Collection Sputum -3 consecutive samples . Early morning complete sputum. 1 st spot sputum, 2 nd early morning sputum and 3 rd spot sputum (DOTS protocol) Urine -3 or more consecutive samples or 24 h pooled urine.

9. Preparation of smear Thin smear on a clean and grease free slides DOTS protocol: 3 sputum samples , 1 st spot sample, 2 nd early morning sample 3 rd spot sample Other specimen included pus, CSF, Urine, endometrial biopsy etc Smear should be oval, 2.5 cm length and 1.5 cm width in and thin enough to see the printed matters through it .

10. Materials required : Carbol fuchsin stain (filtered) Acid alcohol, 3% v/v Malachite green 5 g/l (0.5% w/v) or Methylene blue, 5g/l

11. Acid Fast Reagents Carbolfuchsin (red ) Primary stain Lipid soluble; phenol Acid alcohol Decolorizer Removes carbol fuchsin that has not bound to a mycolic acid. Methylene blue Counterstain Cannot penetrate mycolic acid; provides contrast to non acid fast cells.

12. Fixation Fixation may kill some bacilli makes smear stick to slide by heat or alcohol do not overheat safe smear on may kill some bacilli makes smear stick to slide by heat or alcohol do not overheat safe smear may kill some bacilli makes smear stick to slide by heat or alcohol do not overheat safe smear ? Cell wall structure of Acid Fast Bacilli

13. Ziehl-Neelsen Staining procedure Spread the sputum evenly over the central area of the slide using a continuous rotational movement the recommended size of the smear is about  20 mm by 10 mm Place slides on dryer with smeared surface upwards, and air dry for about 30 minute Heat fix dried smear Cover the smear will carbol fuchsin stain Heat the smear until vapour just begins to rise (i.e. about 60 degree Celsius ). Do not overheat. Allow the heated stain to remain on the slide for  5 minutes . Wash off the stain with clean water .

14. Cover the smear with  3% v/v acid alcohol for 2-5 minutes  ( or 20% sulfuric acid)  or until the smear is sufficiently decolorized, i.e. pale pink . Wash well with clean water Cover the stain with  malachite green stain for 1-2 minutes Wash off stain with clean water Wipe the back of the slide clean, and place it in a draining rack for smear to air dry (do not blot dry). Examine the smear microscopically, using the 100x oil immersion objective and scan the smear systematically.

15. Aseptic transfer

16. Overview of a bacterial staining procedure

17. Aseptic transfer and the Bunsen burner flame reductant flame oxidant flame inner cone outer cone

18. sample 1 sample 2 Preparation of the heat-fixed smear

19. Results of AFB Stain AFB : Rod-shaped bacilli meas. 2-4 x 0.2-0.5 μ m . Pink red coloured , slightly curved bacilli in singles or in small clumps seen against a blue background of epithelial cells and pus cells. , often beaded due to uneven staining. Cells :   Green Background material : Green

20. Reporting on AFB Microscopy > 10 AFB/high power field            –> (+++) 1-10 AFB/high power field             –> ( ++) 10-100 AFB/100 high power field –> (+) 1-9 AFB/100 high power fields       –> exact number However if no AFB is seen, write the result as ‘no AFB seen’ and never write negative.

21. Advantages of sputum microscopy More reliable than x-ray for the diagnosis of infectious TB Simple to perform Easy to read Minimal infrastructure required Inexpensive Quick Only tool to monitor and declare patients as “cured ’’

22. AFB MICROSCOPY Advantages ( continue) 1. Rapid 2. High specificity -All mycobacterium are acid fast 3. Accurate diagnosis - Using simple and available equipment Disadvantage 1. Low sensitivity; Reported sensitivity ranging 25 to 65% when compared to culture (>10,000 bacilli/ml of sputum) 2. Species differentiation impossible. False positive; Saprophytic mycobacteria

23. false-positive results : Re-use of containers or positive slides; C ontaminated stain prepared with water containing environmental mycobacteria; use of scratched slides ; AFB floated off one slide and became attached to another during the staining procedure because there was no space between adjacent slides; I nadequate decolourization ; lack of experience , confusion with artefacts (especially if stains are not or poorlyfiltered ); M icroscope (lamp) in poor condition or poorly adjusted : interpreting glitter as AFB; P oor quality of staining solutions

24. false-negative results Poor quality of specimen; N ot taking proper portion of s pecimen for smear preparation; E xcessive decolourization ; P oorly prepared staining solution; T oo little time staining with carbol fuchsin ; O ver-staining with methylene blue; O verheating during fixing

25. Limitations :   Microscopy for acid-fast bacilli (AFB) cannot distinguish Mycobacterium tuberculosis  from NTM, Viable from non-viable organisms, Drug-susceptible from drug-resistant strains.

26. List of Acid Fast organisms (Other than Mycobacteria Nocardia spp : Partial Acid Fast Rhodococcus spp : Partial Acid Fast Legionella micdadei : Partially acid fast in tissue Cyst of  Cryptosporidium : Acid Fast Cyst of  Isospora : Acid Fast

27. Alternate stain for AFB Fluorescent dye ( Auramine O and Rhodamine B) Good for labs with high workload. Auramine O- Bright yellow Auramine O-Rhodamine B- Yellow orange.