Ziehl-Neelsen n stain / Acid fast stain

Acid Fast stain/Z N stain

1. Z N STAIN dr. yogita

2. TOPICS HISTORY PRINCIPLE REAGENTS PROCEDURE NAMES OF ACID FAST ORGANISMS IMPORTANCE OF Z N STAIN DIFFERENT MODIFICATION OF REAGENTS ERRORS

3. It was first discovered by Earlich in 1881 and modified by Zeihl & Neelsen. It is a differential stain used mainly to detect mycobacteria. ACID FAST means bacteria which protect the primary dye to be washed of from the action of acid alcohol decolorizer.

4. Principle: Acid fastness has been ascribed to the high content and variety of lipid , fatty acid and higher alcohols found in tubercle bacilli. A lipid particular to acid fast bacilli is a long chain fatty acid (mycolic acid ), Acid fast is not a property of lipid alone but depends also on integrity of the cell wall .

5. Z N stain is a modification of Ehrlich’s original method for differential staining of acid fast bacilli by use of aniline gentian violet followed by strong nitric acid. The ordinary aniline dye solution do not readily penetrate the acid fast bacilli. So by use of powerful staining solution that contain phenol and application of heat , the dye can be made to penetrate the bacillus. Phenol will solubilise the cell wall and heat will increase the stain penetration. Once stained the tubercle bacilli will withstand the action of powerful decolorizing agents for considerable period of time , retains the primary stain when every thing else has been decolorized.

6. Z N REAGENTS : Primary dye : carbol fuchsin(basic fuchsin with phenol) Decolourizer : sulfuric acid , alcohol , acid alcohol Counter stain : methylene blue , malachite green The dye is basic and its combination with a mineral acid used as decolourizer produces a reddish pink compound that is readily dissolved out of all structures except acid fast bacteria. To provide contrast to this red stain acid fast bacteria, counter stain is used which also help to see other cells , non acid fast bacteria also.

7. CARBOL FUCHSIN Basic fuchsin (powder) 5gm Phenol(crystalline) 25 gm Alcohol(95 % or 100%) 50ml Distilled water 500ml Dissolve the fuchsin in phenol by placing them in a 1 liter flask over a boiling water bath for 5 min , shaking the contents from time to time . When solution is complete, add alcohol and then distilled water .Mix thoroughly . Filter the mixture before use.

8. DECOLOURIZER SULPHURIC ACID (20%) Concentrated sulfuric acid 250ml Distilled water 1 liter Pour the water into a large flask and place the flask in 5-8 cm of cold water in the sink. Add the acid in 50ml lots over 10 min , Pouring slowly down the side of the flask into water . Mix gently . Mixture will become hot.

9. ALCOHOL 95% Ethanol 95 ml Methylated spirit 100ml ACID ALCOHOL Concentrated hydrochloric acid 75ml Methylated sprit 2425ml

10. COUNTERSTAIN: METHYLENE BLUE Methylene blue 0.5 gm Glacial acetic acid 0.5 ml Distilled water 100 ml

11. PROCEDURE PREPARATION OF SMEAR Cover a heat fixed , dried smear with a small rectangle of filter paper. Apply 5-7 drops of carbol fuchsin stain to thoroughly moistened filter paper. The slide is intermittently heated from underneath using a spirit lamp until fumes arise(kept for 5min). Care must be taken not to boil the solution or drying of the slide. Slide washed in water and decolourized by 20% H2SO4 until the slide is almost colourless or pale pink. The smear is then washed and counterstained with methylene blue solution for 2 minutes. The slide is washed again and dried.

12. STRUCTURES THAT ARE ACID FAST All Mycobacteria - M. tuberculosis, M. leprae and atypical Mycobacterium Actinomyces – Nocardia ,Rhodococus Head of sperm Bacterial spores Cysts of some coccidian parasites: Cryptosporidium parvum Isospora belli Cyclospora cayetanensis A few other parasites: Taenia saginata eggs Hydatid cysts, especially their hooklets stain irregularly with ZNstain

13. ACID FAST BACILLI

14. CYCLOSPORA

15. ISOSPORA

16. IMPORTANCE OF Z N STAIN FOR M. TB BACILLI Acid fast staining reaction of mycobacteria, along with their characteristic size and shape, is a valuable aid in the early detection of infection and in the monitoring of therapy for mycobacterium disease,. The presence of acid fast bacilli in the sputum, combine with a history of cough, weight loss and chest radiographic evidence of pulmonary infiltrate, is the presumptive evidence of active tuberculosis .

17. METHOD FOR REPORTING NUMBERS OF ACID FAST BACILLI OBSERVED IN STAINED SMEARS NUMBER OF BACILLI OBSERVED GRADE - 1-2/300 FIELDS + OR - 1-9/100 FIELDS 1+ 1-9/10 FIELDS 2+ 1-9/FIELD 3+ MORE THAN 9/FIELD 4+

18. Acid fast smears are also useful in following response to treatment . After antimicrobial drugs are started, culture become negative before the smear do , suggesting that organisms are not capable of replicating but capable of binding the stain.

19. With continued treatment , more organisms are killed and fewer shed , so assessing the number of organisms in sputum during treatment can provide an early objective measure of response. If the number of organisms fail to decrease after therapy is started , the possibility of drug resistance must be considered and additional cultures and susceptibility studies should be obtained. ADV : Rapid ,easy DISADV : At least 10000 bacilli/ml should be present

20. Grading of Lepra bacilli No. of bacilli Grading No bacilli /100 fields 0 1-10 / 100 fields +1 1-10/ 10 fields +2 1-10 / field +3 10-100/ field [phone] / field +5 > 1000 /field +6

21. DIFFERENT MODIFICATIONS OF REAGENTS TO IDENTIFY THE ACID FAST ORGANISMS 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% H2SO4 for spores and for oocysts of Cryptosporidium and Isospora 0.5% acetic acid ---- Brucella

22. Use of acid-alcohol as decolourizer Instead of using 20% sulfuric acid as decolourizer, 3% HCl in 95% alcohol may be used. This also differentiates tubercle bacilli from saprophytic mycobacteria . It is especially used in diagnosis of renal tuberculosis.

23. Use of alcohol as secondary decolourizer: After primary decolourization with sulfuric acid, the smear may be treated with 95% alcohol as secondary decolourizer. M. tuberculosis is both acid fast and alcohol fast , while saprophytic mycobacteria are only acid fast .

24. ERRORS: Causes of false positive smears: Food particles Precipitated stains Saprophytic AFB Spores Fibers and pollens

25. Causes of false negative smears: Inadequate specimen inadequate storage of specimens Failure to select suitable material to prepare smear Inadequate preparation of smears Inadequate examination Mistakes in labelling, false recording

26. REFERENCES ANANTHANARAYAN & PANIKER MACKIE & MCCARTNEY KONEMAN

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