Afb staning

 For Smear preparation
 Sputum container of 50 ml capacity to collect sputum
 Wire loop with an inner diameter of 3mm or applicator
stick to spread sputum on the slide
 New glass slide (no grease and no scratches on the slide)
 Diamond marker/ marking pencil to enter identification
number on the slide
 Forceps to hold slide with smear
 Bunsen burner or spirit lamp to sterilize wire loop, fix
smeared slide and flame the same during staining
 Metal waste bin/can with disinfectant to discard infected
material
 Alcohol-sand flask to remove and disinfect excess sputum
on the wire loop

Wire loop
New glass slide
Sputum container Forceps
Alcohol
spirit lamp

 Balance
 Staining rack to hold smeared slides to
stain
 1% Carbol fuchsin to stain acid-fast bacilli
 25% Sulphuric acid to decolorize stained
smears
 0.1% Methylene blue to counter stain
decolorized material in the smear
 Water to rinse/ wash the staining solution
 Slide rack to place stained slides to air dry

Carbol fuchsin Sulphuric acid Methylene blue

 Microscope
 Immersion oil for 100X objective
 Tissue paper to absorb immersion oil on
stained slides before storing.
 Slide box to store stained slides for quality
assurance (Blinded rechecking)
 70% ethanol to clean lenses (frequent use
is not recommended)
 Clean, dry lens tissue to clean microscope
lenses

Microscope
Tissue paper
Immersion oil
Slide box

 TB laboratory register
 Laboratory Request/Report form
 Red pen to write positive result

 Three specimens should be collected for the
laboratory diagnosis of tuberculosis as
follows:
 One spot specimen when the patient first
visits the health facility
 One early morning specimen (preferably
the next day)
 One spot specimen when the early morning
specimen is being submitted for examination

 An essential prerequisite for the safe collection and
transport of satisfactory specimens is the use of a
proper container. The following specifications are
recommended to facilitate the choice of a proper
container for sputum specimen collection:
 Wide-mouthed so that patient can expectorate
easily inside the container without contaminating the
outside
 Approximately 50 ml in capacity
 Made of transparent material to ease observation of
specimen volume and quality without opening the
container
 Screw-capped to obtain a water tight seal & to
reduce the risk of leakage during transport
 Easily labeled to allow permanent identification
 Rigid, to avoid breakage during transit

 The patient is given a container on his first visit to
health facility. He/she should be instructed, with
demonstration by actual actions, to
 Inhale deeply 2- 3 times
 Cough out deep from the chest during exhalation
 Open the container and spit the sputum into the
container
 Avoid saliva or nasal secretions
 Close the container
 Instruct the patient to collect an early morning
specimen in a similar manner and bring it to the
laboratory. On the second attendance, a third
specimen (spot) is collected

 Physical appearance of sputum sample
should be thick, purulent or and/or
bloodstained.
 Sufficient quantity of at least 3-5 ml.
 Contains no food particles or other
remnants

 Label a new clean, unscratched slide at one
end with the laboratory serial number. Label
frosted slides with pencil and non-frosted
slides with diamond pencil.

 Use muco-purulent, opaque, grayish or
yellowish portion for smear preparation.
Transfer an appropriate portion of the
specimen to the slide by using an applicator
stick or wire

 Smear the specimen over an area of
approximately 1cm x 2cm to 2cm x 3cm.
Make it thin enough to be able to read
through it. Use a new slide for each
specimen

 Allow smears to air-dry for 15 minutes. Do
not use heat for drying.

 Pass slide through a flame three or four
times with the smear uppermost. Do not
overheat. Allow to cool before staining.

 Discard the applicator stick in disinfectant
and use a new one for each specimen.
Remove particles of adherent sputum from
wire loop by moving it up and down through
a sand alcohol bottle containing 70% alcohol.
Flame wire loop thoroughly prior to re-use.

 AFB resists decolourization by weak acid and
retains the primary stain. The counter stain
is used to stain other materials and the
background which gives a contrast
background for the easy visibility of the Acid
Fast Bacilli. After the smear is stained with
Ziehl Neelsen technique, AFB appears red
against a blue background.

 1. Place the numbered
slides on a staining rack
with the smeared side
facing up (individual or
in batch of maximum 12
slides). Ensure to leave
a finger thickness gap
between slides to
prevent the transfer of
material from one
smear to another and
the solution running off
from slides.

 2. Flood the entire
surface of slide
with Ziehl-Neelsen
1% Carbol fuchsin
solution

 3. Heat each slide
slowly until steam
arises. Maintain
steaming for 5
minutes by using
intermittent heat.
(In no case must
the stain solution
boil or dry during
staining process.)

 4. Rinse each slide
individually in a
gentle flow of
water until all free
stain is washed
away. Drain off
excess rinse water
by tilting the slide.

 5. Flood the slide
with the
decolorizing
solution (25%
Sulphuric acid) for
3 minutes. (If the
slide is under
decolorized after 3
minutes, further
decolorize for 1
minute.)

 6. Rinse the slide
thoroughly with
water. Drain off
excess water from
the slide

 7. Wipe off back of
slide with cotton
soaked in
decolorizer to
clean the dried
stains.

 8. Rinse the slide
again with water
and drain off
excess water from
the slide by tilting
the slide

 Flood the slide
with 0.1%
Methylene blue
and counter-stain
for 60 seconds

 Rinse the slide
thoroughly with
water and drain
off any excess
stain from the
slide by placing
the slide under
gentle stream of
running water

 Stand the rinsed
slide on the slide
holding block and
allow the smear to
air dry.
 (Do not heat or use
blotting paper to
dry the smear. Do
not place the slide
under direct
sunlight to dry the
smear.)

 To obtain excellence in
smear examination, a
good microscope and a
comfortable work area are
required. Reading of
smears must be systematic
and standardized to
ensure that a
representative area of the
smear is examined. To
ensure that an area is
covered only once, the
smear should be examined
in an orderly manner and
the following procedure is
recommended:

 Focus the stained
slide under low
power objective
lens (5x or 10x) and,
observe the staining
quality and
evenness of smear.
Select the field
where the smear is
evenly distributed
and WBCs & mucus
can be seen.

 Apply a drop of
immersion oil on the
stained smear and focus
the smear using 100x
objective lens. Do not
touch the smear with
the dropper. Doing so
may contaminate the
applicator whereby may
spread AFB from one
slide to the next
resulting in false-
positive results.

 Always examine
ZN-stained smears
with a 100x
objective lens
under oil
immersion.

 Include a known positive slide and a known
negative slide each day. The positive control
ensures the staining capability of the solutions
and of the staining procedure. The negative
control confirms that acid-fast contaminants are
not present in the stains or other solutions.
 Make a series of systematic examination over
the length of the smear. After examining a
microscopic field, move the slide longitudinally
so that the neighboring field to the right can be
examined. Search each field thoroughly

 Examine a minimum of 100 fields before the
smear is reported as negative. For skilled
microscopists this will take approximately five
minutes. In a smear of 2.0 cm x 1.0 cm size, the
number of microscopic fields in one length of the
slide corresponds to around 100. If the smear is
moderately or heavily positive, fewer fields may
be examined and a report of “positive” may be
made even though the entire smear has not been
examined.
 Before examining the next slide, wipe the
immersion lens with a piece of lens tissue.

 Unexpected objects may be seen when using
the microscope. If these objects move only when
the slide is moved, they may be materials
occasionally found in the specimen or object,
precipitated stains, contaminants from the stains
or contaminants in the immersion oil.
 Artifacts that move only when the eyepiece is
rotated are in the eyepiece or on its lenses.
Artifacts may also be caused by material on the
condenser, lenses, mirror or light source.
 Keep all the slides for quality control according
to established procedures

No of acid-fast bacilli Fields Report
No AFB In 100 immersion fields Negative
1-9 AFB In 100 immersion fields Positive scanty
Record exact figure
10 to 99 AFB In 100 immersion fields 1+
1 to 10 AFB Per field (examine 50
fields
2+
More than 10 AFB Per field (examine 50
fields
3+

 Connected with the specimen
 Inadequate specimen volume and/or poor
quality.
 Carelessness in marking the container.
Marking should be done on the body of the
container and not on its lid.

 Insufficient or poorly lit work surface.
 Mixing-up of slides.
 Preparing too many slides at once. A
maximum of 12 is recommended per batch.
 Re-use of slides that had been positive
earlier. These should be discarded.
 Specimen contamination due to careless
use of loops/wooden applicators.

 Using scratched slides on which deposits of
stain may look like bacilli.
 Using unfiltered fuchsin, which may
contain crystals.
 Carelessness in heating the fuchsin,
allowing it to dry and crystallize on the
smear.
 Inadequate decolorizing of the smear,
which may leave red stain on saprophytic
bacilli which then appear to be acid-fast.

 Failure to check the slides and renumber
them, if the number is obscured during
staining. This may lead to substitutions.
 Failure to clean the immersion lens with
lens tissue after each examination, especially
after a smear was found to be positive.
 Carry-over of bacilli from a positive smear
due to immersion oil dropper touching the
smear. The oil drop should be allowed to fall
without any contact between the dropper
and slide.
 Erroneous recording of the results.

Problem Possible causes Solution
The field is dim Condenser may be too low
Condenser iris may be closed
Raise the condenser.
Open the diaphragm
Dark shadows in the field which
move when eye piece is moved
Eyepiece may be dirty.
Eyepiece or objective may be
contaminated with fungi.
Surface of eyepiece may be
scratched.
Clean the eye piece
Eye piece or objective may
need cleaning
A new eye piece may be needed
The image is not clear The smeared portion of the
slide may be upside down
There may be an air bubble in
the oil
The oil may be of poor quality
There may be dirt on the lens
Turn the slide over
Move the 100x lens from side to
side
Use only good quality immersion
oil.
Clean the lens
The image through low power is
not clear
There may be oil on the lens
There may be dust on the upper
surface of the lens
The lens may be broken
Clean the lens
Clean the lens
A new lens may be needed

‘‘NEVER TRUNED BEHIND
BE SUCCESS IN YOUR LIFE’’
Thank you

Afb staning

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