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
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.
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
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
0 -
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 +4
100-1000 / 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.
