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

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.

16. 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

17. 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.


19. 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.

21. 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

22. 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.


25. 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.

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

27. 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

28. • 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.

29. 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.

30. 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.

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

33. 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.

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

36. 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

38. 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.

39. • 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

40. • 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

41. • 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.

43. 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.

45. 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

46. • 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.

47. • 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.

48. THANK YOU