Modified Ziehl-Neelsen Stain

Modified Ziehl-Neelsen Stain

1. MODIFIED Z N STAIN

2. Classic ZN stain Cold ZN stain: Kinyoun’s Method Gabett’s Method

3. ZN stain for spores: Muller’s method Dorner’s method Schaffer fulton stain Muller chermock tergitol method

4. For tissue sections: Ellis and Zabrowarny stain Fite faroco stain Wade fite stain Modified bleach ZN method Cooper’s modifications

5. KINYOUN STAIN Kinyoun stain  is a method of staining  acid-fast  microorganisms, specifically  Mycobacterium  and  Nocardia and cryptosporidium oocyst . The procedure for Kinyoun staining is similar to the  Ziehl-Neelsen stain , but does not involve heating the slides being stained. The Kinyoun staining method uses  carbol fuchsin  as a primary stain, followed by decolorization with an acid-alcohol solution and  methylene blue  as a counter stain. Kinyoun carbol fuschsin has a greater concentration of  phenol  and basic  fuchsin  and does not require heating in order to stain properly. When viewed under a microscope, a Kinyoun stained slide will show acid-fast organisms as red and nonacid-fast organisms as blue.

6. Procedure Flood slides with Kinyoun  carbol fuchsin  for 5 minutes. Rinse gently with water until the water flows off clear. Flood slides with acid-alcohol (3% HCl in ethanol) for 3~5 seconds. Rinse gently with water until the water flows off clear. Flood slides with methylene blue for 3 minutes. Rinse gently with water until the water flows off clear. Allow slides to air dry before viewing.

7. Modifications: A solution of 1% sulfuric acid can be substituted in place of 3% HCl solution. The sulfuric acid solution does not decolorize as strongly as the hydrochloric acid. This makes it useful for staining organisms that are weakly acid fast, such as  Nocardia .  Brilliant Green  may be substituted for Methylene Blue as a counter stain, resulting in non-acid fast organisms appearing green rather than blue. Another alternative is 20% sulphuric acid instead of HCl.

8. In Gabett’s stain: It is a two step method. First step is primary staining. Second step is a combination step of discoloration and counterstaining.

9. CS method was done by using 2 different time duration, that is, with duration of primary stain (carbolfuchsin) kept for 10 min (CS 10 min) and primary stain kept for 20 min (CS 20 min) simultaneously using Gabbett’s methylene blue modification. But with CS 20 only the results are same as that with classical ZN stain. The 2 step cold acid fast stain, is an improved alternative method over the traditional ZN hot stain, due to its sensitivity, simplicity and rapidity. CS 20 method was found to be equally sensitive in detecting AFB as the traditional ZN method .

10. 1% carbol-fuchsin stain (SAME AS CLASSICAL ZN STAIN ) 10 g --- basic fuchsin, 100 ml--- methylated spirit 50 g --- phenol 1,000 ml---- distilled water Gabbett’s methylene blue( DIFFERENT FROM CLASSICAL Z N STAIN ) 1 g --- methylene blue 20 ml ---- sulfuric acid, 30 ml --- absolute alcohol 50 ml ---- distilled water Decolourization and counterstaining are done in one step.

11. ZN stain for spores Muller’s method Dorner’s method Schaffer fulton stain Muller chermock tergitol method

12. ZN stain for spores Muller’s method Dorner’s method Schaffer fulton stain Muller chermock tergitol method

13. MULLER’S METHOD Endospores  are surrounded by a highly resistant spore coat. The spore coat is highly resistant to excessive heat, freezing as well as chemical agents. More importantly, spores are resistant to commonly employed staining techniques. Therefore alternative staining methods are required. Moeller staining involves the use of a steamed dye  reagent  in order to increase the stainability of the spores;  Carbol fuchsin  is the primary stain used in this method. Endospores are stained red, while the counter stain,  Methylene blue  stains the vegetative bacteria blue.

14. Carbol fuchsin is applied to a heat-fixed slide. The slide is then heated over a Bunsen burner, or suspended over a hot water bath, covered with a paper towel, and steamed for 3 minutes. The slide is rinsed with acidified ethanol, and counter-stained with  Methylene blue . An improved method involves the addition of the surfactant Tergitol 7 to the carbol fuchsin stain, and the omission of the steaming step.

15. DORNER’S STAIN Carbolfuchsin stain 0.3 g ---- basic fuchsin 10 ml ---- ethanol(95% ) 5 ml ---- phenol 95 ml ---- distilled water Decolorizing solvent (acid-alcohol) 97 ml of ethanol, 95% (vol/vol) 3 ml of hydrochloric acid (concentrated) Counter stain (Nigrosin solution) 10 g of nigrosin 100 ml of distilled water

16. Dorner method for staining endospores Air dry or heat fix and cover with a square of blotting paper. Saturate with carbol fuchsin and steam for 5 to 10 minutes.  Alternatively, the slides may be steamed over a container of boiling water Remove the blotting paper and decolorize the film with acid-alcohol for 1 minute; rinse with tap water and blot dry. Dry a thin even film of saturated aqueous nigrosin on the slide.   Vegetative cells are colorless, endospores are red, and the background is black.

17. Variation on the Dorner method Mix an aqueous suspension of bacteria with an equal volume of carbol fuchsin in a test tube. Immerse the tube in a boiling water bath for 10 minutes. Mix a loopful of 7% nigrosin on a glass slide with one loopful of the boiled carbol fuchsin-organism suspension and air dry to a thin film. Examine the slide under the oil immersion lens (1,000X) for the presence of endospores.    

18. SCHAEFFER FULTON STAIN Malachite green stain  (0.5%) 0.5 g of malachite green 100 ml of distilled water Decolorizing agent Tap water Safranin counterstain Stock solution (2.5%)     2.5 g of safranin O     100 ml of 95% ethanol Working solution      10 ml of stock solution    90 ml of distilled water

19. Schaeffer-Fulton method Air dry and heat fix the glass slide and cover with a square of blotting paper. Saturate with malachite green stain solution and steam for 5 minutes.  Alternatively, the slide may be steamed over a container of boiling water. Wash the slide in tap water. Counterstain with safranin for 30 seconds.  Wash with tap water; blot dry. Examine the slide under the oil immersion lens (1,000X) for the presence of endospores.  Endospores are bright green and vegetative cells are brownish red to pink. 

20. Muller Chermock Tergitol Method Cover smear with carbolfuchsin containing 1 drop Tergitol no.7 to 25 ml of stain. Allow to stain for 2 min without heat. Decolorize with acid alcohol. Counter stain with methylene blue dilute 1:20 for 5-15 sec.

21. STAIN FOR TISSUE SECTIONS Fite-Faraco Staining Fite stain --- For lepra bacilli Wade Fite staining Ellis and Zabrowarny stain(no Phenol/carbolic acid)

22. FITE FARACO STAIN Xylene and peanut oil xylene ---- 2 parts peanut oil -- 1 part  carbol fuchsin (1%)                            sulphuric acid 10%           distilled water --- 90.0 ml conc.sulphuric acid ----- 10.0  ml acetified methylene blue      methylene blue ----- 0.25gm           distilled water ------ 99.0 ml glacial acetic acid ----- 1.0 ml

23. Principle Mycobacterial cell walls contain a waxy substance composed of mycolic acids. These are ß- hydroxyl carboxylic acids with chain lengths of up to 90 carbon atoms. The property of acid fastness is related to the carbon chain length of the mycolic acid found in any particular species. The leprosy bacillus is much less acid and alcohol fast than the tubercle bacillus, therefore alcohol is removed from the hydrating and dehydrating steps and 10% sulphuric acid is used as a decolourizer in place of acid / alcohol solution. The sections are also deparaffinised using peanut oil/ xylene mixture, this helps to protect the more delicate waxy coat of the organisms.

24. Procedure Deparaffinise with two parts xylene and then with one part vegetable oil for 15 mins. Blot dry and wash in water. Repeat if any xylene-oil remain on the section. Filter on carbol fuchsin solution, DO NOT HEAT, for 20 mins. Wash in running tap water. Decolourize in 10.0% sulphuric acid for 2 mins.

25. Wash well in running tap water, rinse distilled water. Counterstain in 0.25% methylene blue for 20 seconds. Wash and blot dry. Clear in xylene. Repeat the blotting- xylene treatment until section is clear.

26. Results   Leprosy bacilli ……………magenta Nuclei, background…………………………blue Red blood cells……………………………… pale pink       

27. FITE'S ACID FAST STAIN - LEPROSY PURPOSE: To demonstrate mycobacterium leprae (leprosy), which are acid fast organisms. PRINCIPLE: This technique combines peanut oil with the deparaffinizing solvent (xylene), minimizing the exposure of the bacteria's cell wall to organic solvents, thus protecting the precarious acid-fastness of organism.

28. RESULTS: Acid-fast bacilli red Background blue NOTE: Mineral oil may be substituted for peanut oil.

29. WADE FITE STAIN Ziehl's Carbol fuchsin 1% aqueous hydrochloric acid Methylene blue, 0.1% aqueous Dewaxing Mixture Rectified turpentine ---- 2 volume Liquid paraffin  ------ 1 volume M. leprae (and other mycobacteria) - red Nuclei - grey/blue

30. ELLIS AND ZABROWARNY STAIN (NO PHENOL OR CARBOLIC ACID IS USED) To develop a method for staining acid fast bacilli which excluded highly toxic phenol from the staining solution. A lipophilic agent, a liquid organic detergent, LOC High Studs, distributed by Amway, was substituted. LOC High Suds is considerably cheaper than phenol. The acid fast bacilli stained red; nuclei, cytoplasm, and cytoplasm elements stained blue on a clear background. These results compare very favorably with acid fast bacilli stained by the traditional method. Detergents are efficient lipophilic agents and safer to handle than phenol.

31. Staining solutions Solution A Basic fuchsin ---- 1 g Absolute ethyl alcohol ---- 10 ml Solution B L.O.C. High Suds ---- 0.6 ml Distilled water --- 100 ml 3% hydrochloric acid in 95% ethyl alcohol Absolute ethyl alcohol ---- 95ml Distilled water ---- 2 ml Concentrated hydrochloric acid ----3 ml 0.25% methylene blue in 1% acetic acid Methylene blue ----- 0.25 g Distilled water ----- 99 ml Acetic acid ----- 1 ml

32. Place the staining solution in a coplin jar and pre-heat to 60˚C for 10 mins Deparaffinise sections, bring to water and stain in the pre-heated solution for 15 mins Place the coplin jar containing the slides into running cold tap water for 2 mins before removing the slides from the coplin jar. Remove the slides and wash in running water for 1 min

33. Decolourize with 3% hydrochloric acid in 95% ethyl alcohol until no more colour runs from the slide Wash briefly in water. Counterstain with 0.25% methylene blue for 15 to 30 sec.

34. MODIFIED BLEACH METHOD By Ziehl–Neelsen (ZN) method has many advantages when it comes to speed and feasibility though it has a low sensitivity. If the sensitivity could be improved, it has the potential to become an even more valuable tool for detection of AFB. The modified bleach method was more sensitive and safer than routine ZN staining. As the background was clear, the bacilli were easily visible and the screening time can be shorten. The classic cytomorphological pattern of tuberculosis of epithelioid granulomas, langhans giant cells and caseous necrosis can be seen better with it. PROCEDURE 2 ml of 5% NaCl is used mixture was incubated at room temperature for 15 min by shaking at regular intervals. centrifugation at 300 RPM for 15 min after addition of 2 ml of distilled water. The supernatant was discarded and the sediment was transferred to a clean sterile slide . ZN stain done.

35. COOPER MODIFICATION Cooper carbol fuchsin Carbol fuchsin ---- 100ml Sodium chloride ---- 3 ml Cooper brilliant green A. Brilliant green ---- 1 gm Distilled water ----100ml B. Sodium hydroxide --0.1 ml Distilled water ---- 10 ml Mix 100ml sol.A and 1ml sol. B

36. Stain with cooper carbol fuchsin 3-5 min, gently heating slide. Decolorize with 5% nitric acid in 95% alcohol. Counter stain with cooper brilliant green 30sec.

37. SUMMARY Various method of modification of Z N stain are helpful by their modification to see less acid fast structure , acid fast bacilli in tissue section and also spores. It also causes less damage to this structure. It also increases the sensitivity of stain.

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