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AFB Microscopy Module


Purpose To provide participants with an overview of the principles and

steps in AFB microscopy

Learning At the end of the module, the participant will be able to:
Objectives Explain collection of appropriate sputum specimens
Explain processing and registration of sputum specimens
Describe sputum smearing and staining
Describe the features of properly prepared and well
stained smears (ZN, auramine staining)
Describe smear examination and define grading of smears
(ZN, auramine staining)

Content outline Collection of sputum specimens

Quality of sputum specimens
Processing of sputum specimens
Registration of sputum specimens
Sputum smearing and staining
Smear examination
Recording and reporting

Appendices 1. Laboratory register for sputum smear microscopy

2. Request and report form for sputum smear examination


AFB Microscopy Module




For diagnostic purposes, one to three sputum specimens must be collected and examined.
The national policy for declaring a smear-positive TB case and for exclusion of a smear-
positive case should be followed.
- Strategy 1: two positive sputa to declare a smear-positive case, three negative
sputa to exclude smear-positive TB
- Strategy 2: one positive sputum to declare a smear-positive case, two negative
sputa to exclude smear-positive TB

For out-patients, the first specimen should be collected at the time symptoms
suspected of TB are identified, this is commonly called a spot specimen.
For hospitalized patients early morning specimens on consecutive days may be
For patients on treatment, follow-up sputum specimens should be collected at
intervals specified by the NTP. Early morning sputum is recommended.

The patient should be given clear instructions on how to collect the sputum. This includes
- the importance of sputum examination for diagnosis of TB,
- the importance of producing real sputum and not saliva,
- how to produce good sputum (i.e. by repeatedly deeply inhaling and exhaling
followed by cough from as deep inside the chest as possible; if the condition of
the patient allows, fast walking/running around the health facility may help to
initiate a good cough),
- how to open and close the sputum container,
- how to avoid contamination of the outside of the container by carefully spitting
into the container and properly closing the container,
- how to safely deliver the sputum container to the laboratory or assigned person.


A good specimen should be 3-5 ml. It is usually thick and mucoid, but may be fluid and
contain pieces of purulent material. The color varies from opaque white to green; when
blood is present, it may be reddish to brown. Blood-streaked sputum is suitable; however
a specimen that is very bloody should not be examined. The patient should instead be
referred to a medical person for immediate management before sputum smear
Clear saliva is not acceptable and should only be tested if the patient cannot produce
sputum even after carefully following the instructions. This may be more common when
collecting follow-up specimens. Although not the optimal specimen, saliva can produce
positive results.

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The specimens should be processed as soon as possible, although technically speaking

the time between collection and staining matters little, and older specimens should never
be rejected. Once a fixed smear has been prepared and is kept out of reach of insects such
as flies, further processing can wait if necessary, for example until after the weekend.
However, for the benefit of the patient results should be available as soon as possible.

If the health facility does not perform smear microscopy, there are a number of options:
- refer the patient to the nearest facility with smear microscopy,
- collect the specimens at the health facility and send these by public transport or an
assigned person to the nearest smear microscopy facility,
- collect the specimens at the health facility, or at assigned points in the
communities, have the smears prepared and fixed by a trained person, then send
the slides by public transport or an assigned person to the nearest smear
microscopy facility.

Each option has advantages and disadvantages.

Referral of the patient will more easily allow for collecting a repeat sample if the quality
of a specimen is unsatisfactory. However, it may be too expensive for the patient to go to
another site; patients may not go, not be diagnosed or diagnosis may be delayed.
Referral of the specimens requires careful packing, clear labeling and completed request
forms for examination. Transport may be irregular, especially in remote areas, which may
cause delays in diagnosis. This also applies to sending slides.
Besides the advantage for the patient of sending sputum specimens or fixed slides, fixed
slides are much less infectious than sputum specimens. However, preparing slides
requires some equipment and training for the peripheral health facilities.


All specimens requested for AFB microscopy have to be registered in the TB laboratory
register; one line for the diagnostic smears of one patient and one line for each follow-up
(one or two smears). The sputum container should be labeled on the side, and not on the
lid, with the laboratory number and serial number. Also the slides should be labeled with
the laboratory line serial number and with the column number or code.


Preparing the slides

To prepare slides for smearing and staining:
- use only new, clean, grease-free, unscratched slides, free of fingerprints.
Degreasing is not needed, as this is done at the factory,
- write the laboratory line serial and column number/code on the slide using a
pencil for frosted slides or a diamond pencil for unfrosted slides. Ensure that the
number on each slide is the same as that on the sputum container.

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- place each slide on its container to avoid mixing up slides with the wrong

Sputum smearing
To make good quality sputum smears:
- smears should be prepared in a clean area, best covered with waste paper,
- place the slide on the smearing area, and open the corresponding container (and
only this one),
- use an application stick (e.g. of bamboo), or a wire loop, to select and pick up the
thick particles of sputum. If yellowish particles are not present (e.g. follow-up
sputum), pick up a whitish particle or the most mucoid part of the sample,
- prepare the smear in an oval shape in the center of the slide; 2-3 cm in length and
1-2 cm in width. This will allow for reading 100-150 fields (2 by 1 cm smear or 3
by 2 cm smear) in one length,
- spread the sputum on the slide by firmly pressing the stick/wire loop
perpendicular to the slide in small concentric circles. Continue spreading the
smear until an even result with fine streaks and no thick parts has been obtained.
Proper spreading is very important; the smear should be neither too thick nor too
thin. The thickness is correct if the letters can be read when the smear is held 4-5
cm over a piece of printed paper,
- place the stick into a discard container and use a new stick for each specimen,
- if a wire loop is used, dip this in a sand-alcohol bottle. Move the wire loop up and
down to remove excess sputum. Heat the wire loop in a flame until red-hot, and
allow to cool before reusing,
- allow the smear to air-dry at room temperature; do not dry in direct sunlight or
over a flame,
- after it is completely dry, use forceps to pass the slide, holding it upwards, over a
flame 2-3 times for 2-3 seconds each. Alternatively cover the smear with alcohol
and let it evaporate.

Ziehl-Neelsen (ZN) staining

A well stained smear by ZN shows strong red AFB, against a weak, blue, background.
The background should not show much remaining red and there should not be any red
artifacts. The technique proposed here using 1% carbol fuchsin as stain (=strong red) and
0.1% methylene blue (=weak blue) as counter stain was recommended by a panel of
experts in November 2007.
Contact with carbol fuchsin should be at least 10 minutes and not more than 15 minutes.
The slide should be completely destained, and the destaining steps can be repeated to
ensure complete absence of red color from the background. If the smear is too thick
destaining will be difficult or incomplete, even if repeated. The commonly used
destaining reagents are 3% hydrochloric acid in alcohol or 20-25% sulfuric acid in water.

Procedure for ZN staining of slides:

- arrange slides in serial order on staining bridge, with smear side up, while taking
care that they do not touch each other,

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- apply filtered 1% carbol fuchsin on the slides (best to filter through a funnel and
paper while pouring the staining solution on the smears),
- gently heat until steaming,
- keep the carbol fuchsin on the slide for at least 10 minutes, rinse with water and
- apply decolorizing solution for 3-5 minutes, then rinse with water and drain,
- repeat the decolorizing step if too much red remains, rinse with water and drain,
- apply methylene blue 0.1% counter stain for 1 minute (no more than 2 minutes),
rinse with water and drain,
- air dry on slide rack.

Auramine (Fluorochrome) staining for Fluorescent Microscope

In a well stained auramine smear AFB appear as yellowish fluorescent bacilli against a
dark background. The background should not show much remaining fluorescence, but
some fluorescing artifacts will often be present. Contact with auramine should be at least
15 minutes and not more than 30 minutes. Complete destaining with 0.5% acid alcohol is
necessary and may be repeated if required. The commonly used counter stain reagent is
0.5% potassium permanganate. If the slides cannot be examined immediately after
staining, keep them in the dark, closed slide box, to prevent rapid fading.

Procedure for auramine staining of slides:

- arrange slides in serial order on staining bridge, with smear side up, while taking
care that they do not touch each other,
- apply filtered 0.1% auramine on the slides,
- keep the auramine on the slides for at least 15 minutes, rinse with water and drain,
- apply decolorizing solution for 1-3 minutes, rinse with water and drain,
- apply counter stain for a maximum of 1 minute, rinse with water and drain,
- air dry on slide rack.


ZN Smears
Examine 1 length of the smear (2cm) or 100 fields with bright field microscope, using
1000x magnification. If less than 10 AFB are found in 100 fields, the number of AFB
should be counted. For high positives, examination of only 20-30 fields is sufficient.

Auramine (Fluorochrome) smears

Examine one length of an auramine slide with a fluorescent microscope (FM), using 200-
250x magnification, to cover 30 fields in one length, equivalent to 300 fields at 1000x
magnification (high-power fields or HPF). Alternatively 400x magnification can be used,
covering 40 fields at this magnification per length, corresponding to 200 HPF. Because
fluorescent artifacts may be present in the smear it is important that the examiner applies
strict criteria for proper cell morphology to avoid misreading and reporting false
positives. While AFB has a yellowish fluorescence, artifacts may be green and some are
not quite fluorescent. At least some AFB should have the typical curved-rod shape.
Confirmation can be done by changing to a higher power objective. If < 30 AFB at 200-

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250 x magnification, or < 20 AFB at 400x magnification, are found, the exact number of
AFB should be counted. For high positives, examination of less than 1 length may be

Grading of the smears is presented in the following table (HPF= high power field, or the
view field size at 1000x magnification)

scale 1000 x magnification, 200-250x 400x magnification,
100 HPF per length of magnification, 40 fields= 200 HPF
the smear 30 fields= 300 HPF per length of the
per length of the smear
Negative Zero AFB/1 length Zero AFB/1 length Zero AFB/1 length
Scanty 1-9 AFB/1 length 1-29 AFB/1 length 1-19 AFB/1 length
1+ 10-99 AFB/1 length 30-299 AFB/1 length 20-199 AFB/1
2+ 1-10 AFB/1 HPF on 10-100 AFB/1 field 5-50 AFB/1 field on
average on average average
3+ >10 AFB/1 HPF on >100 AFB/1 field on >50 AFB/1 field on
average average average


The results should be registered in the TB Laboratory Register (Appendix 1), be recorded
on the sputum examination request form (Appendix 2) and be forwarded to the person
requesting the sputum examination(s).

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Appendix 1
Laboratory register for sputum smear microscopy


Lab Date Name Sex Age Name of Address Reason for Result of specimen Signatur Remarks
serial M/F treatment new examination * e
No. unit patients diagnosi follow 1 2 3
s** up

* Check the appropriate category from the Request for Sputum Smear Examination
** Add the TB treatment registration number for all cases put on treatment at the center; or the name of the center where they were sent to take
their treatment
*** Write the TB treatment registration number

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Appendix 2

Request and reporting form for sputum smear examination

Treatment Unit_______________________________________ Date_______

Patients name:__________________________________________________

Age________ Sex (M/F)________

Address (precise) ___________________________________________


Reason for examination: diagnosis follow-up

If follow-up examination Month: __
Registration number: __________
Signature of person requesting examination

Results (to be completed in the laboratory)

Laboratory serial No._________ Technique used : Ziehl-Neelsen / Fluorochrome

Result (check one)

Date Specimen Appearance* neg 1-9 + ++ +++
* visual appearance of sputum (blood-stained, purulent, mucous, mucopurulent, salivary

Date_____________ Examined by ______________________________


The completed form (with results) should be sent promptly to the treatment unit