Direct sputum

smear microscopy
TB LABORATORY HANDBOOK
2021

DSSM remains to be an indispensable laboratory test for TB due

to its low cost, procedure simplicity, and high specificity. This
allows the detection of acid-fast bacilli, such as Mycobacterium
tuberculosis, in good quality smears directly prepared from sputum
specimens collected from presumptive TB, or people with TB
undergoing treatment. It is highly possible that individuals whose
specimens test AFB-positive by smear microscopy are ten times
more infectious than those with negative results. Thus, smear
microscopy plays a vital role in both bacteriological diagnosis of
TB, and monitoring of anti-TB treatment effectiveness.
Direct sputum smear microscopy
TB LABORATORY HANDBOOK

© National Tuberculosis Reference Laboratory

Research Institute for Tropical Medicine - Department of Health
2021

All rights reserved.

You may copy, redistribute, and adapt this work in part or in full for non-profit
purposes, provided that the work is appropriately cited, and credit is given to the
National Tuberculosis Reference Laboratory of the Research Institute for Tropical
Medicine - Department of Health.

NATIONAL TUBERCULOSIS REFERENCE LABORATORY

Research Institute for Tropical Medicine - Department of Health
9002 Research Drive, Filinvest City, Alabang
Muntinlupa City, Philippines 1781
+63-2-8807-2631/32/37 loc 101
labnetwork.ntrl@gmail.com
www.ritm.gov.ph
 Contents

Contents

Purpose.....................................................................................................................iv

Acknowledgements...................................................................................................iv

Acronyms...................................................................................................................v

Biosafety....................................................................................................................1

Specimen receiving....................................................................................................4

Smear preparation......................................................................................................5

Staining of smears.....................................................................................................7

Examination of smears...............................................................................................8

Recording and reporting...........................................................................................10

Decontamination and disposal.................................................................................11

Quality assurance.....................................................................................................12

References...............................................................................................................15

iii
 Direct sputum smear microscopy

Purpose
This Direct sputum smear microscopy: TB laboratory handbook was developed
by NTRL to serve as a quick reference for personnel working in smear microscopy
laboratories within the national TB laboratory network. Using this document as their
guide, along with other materials such as training manuals, smear microscopy laboratory
personnel are expected to draft their own guidelines, standard operating procedures,
and/or work instructions.

Acknowledgements
This document was developed by the NTRL led by Ramon P. Basilio, MD (OIC, Head)
through a technical writing group composed of the following personnel:

Ma. Cecilia Vanessa M. Serrano, RMT, MPH (Head, LNSS)

Earl Christian P. Mantes, RMT (QMS Thematic Lead, LNSS)
Raiza Carmella C. Adao, RMT (Staff, PSQMS)

The technical writing group acknowledges the contributions of the following technical
reviewers:

Marienella P. Galit (Quality Manager, NTRL)

Lorenzo T. Reyes, RMT, RN (Laboratory Manager, NTRL)
Catherine Ann PL. Sacopon, RMT, MPH, CBO (Biosafety Officer, NTRL)
Ryan V. Castro, RMT (Head, LDS)
Michellin Roxanne S. Baje, RMT (Head, LSS - Ancillary Unit)
Roeus Vincent Arjay G. Reyes, RMT (Head, LSS - Routine Unit)
Ralph Jason G. Esteban, RMT (Quality Officer, LSS)

NTRL is also grateful for the support extended by the following in this endeavor:

Amado O. Tandoc, III, MD, FPSP (Chief, LRD)

Celia C. Carlos, MD, CESO III (Director IV, RITM)

iv
 Acronyms

Acronyms
AFB Acid-fast bacilli
BSC Biosafety cabinet
BSL2+ Enhanced biosafety level two
COVID-19 Coronavirus disease - 2019
DSSM Direct sputum smear microscopy
EQA External quality assessment
HPF High power field
ID Identification
IUATLD International Union Against Tuberculosis and Lung Disease
LDS Learning and Development Section
LED-FM Light-emitting diode fluorescence microscopy
LIS Laboratory information system
LNSS Laboratory Network Strengthening Section
LPF Low power field
LRD Laboratory Research Division
LRF Laboratory request form
LSS Laboratory Services Section
NTRL National Tuberculosis Reference Laboratory
OIC Officer-in-charge
OIF Oil immersion field
PPE Personal protective equipment
PSQMS Program Support and Quality Management Section
QA Quality assurance
QC Quality control
QMS Quality management system
RITM Research Institute for Tropical Medicine
TAT Turnaround time
TB Tuberculosis
WHO World Health Organization
ZN Ziehl-Neelsen

v
 Biosafety

Biosafety
As a smear microscopy laboratory different requirements as to facility,
personnel, am I at risk of getting infected practices and procedures, and PPE.
with TB?
Yes, when you inhale infectious aerosols Note: If DSSM is performed in the same
generated when doing mechanical laboratory as with TB culture, DST and/or
procedures with the specimen like shaking LPA, then all BSL2+ practices must be strictly
and stirring. However, DSSM is relatively observed.
a low-risk procedure provided that the
minimum biosafety requirements for a
TB smear microscopy laboratory, and its
personnel are met. Facility
How do I keep myself safe? How should a TB smear microscopy
laboratory be set up?
To ensure your safety when working
inside a TB smear microscopy laboratory, The laboratory should have adequate
you must observe the minimum biosafety ventilation, and must be divided into
requirements at all times. There are clean and dirty zones (see figure 1):

RECORDING &
STAINING MICROSCOPY
REPORTING

DOOR
PREPARATION
SMEAR

HANDWASH
OPEN WINDOW

PPE
RECEIVING
SPECIMEN

SPECIMEN REAGENTS &

STORAGE SUPPLIES STORAGE

Figure 1: TB smear microscopy laboratory set-up showing separation of areas as to clean (right of
dotted line) and dirty (left of dotted line), and unidirectional airflow from clean to dirty areas (depicted
by arrow)

1
 Direct sputum smear microscopy

• The clean zone is dedicated for How do I prevent generating infectious

microscopy, record keeping, and aerosols?
storage areas. Laboratory furniture,
especially the bench/table used to • Avoid shaking/stirring specimens.
hold the microscope, should be sturdy.
• If specimens have been disturbed, let
• The dirty zone is where specimen them stand for at least ten minutes
receiving, and smear preparation and before opening them.
staining are performed. The smear
preparation area should be well-lit. • Make sure smears are completely air
Meanwhile, the staining sink must dried before heat fixing.
be deep enough to contain water
• Be careful when staining smears to
splashes. Water supply should be
prevent splashes.
dependable, and of good quality.

How do I achieve adequate ventilation? What other practices should I always do?

Windows may be opened, and fans may be • Restrict laboratory access to

installed such that there is unidirectional authorized personnel only (e.g. by
airflow from clean to dirty areas. This posting the international biohazard
means that the air should flow from the sign on the laboratory entry door; see
laboratory personnel, across the specimen figure 2)
receiving / smear preparation / staining
areas, and outside of the laboratory (see • Washing your hands thoroughly
figure 1). with soap and water before entering,
and exiting the laboratory.
Do I need a BSC?
• Proper cleaning and disinfection of
A BSC is not required for a laboratory laboratory benchtops for specimen
where only DSSM is performed. Adequate receiving and processing (see section
ventilation and unidirectional airflow, in on Decontamination and disposal),
addition to the observance of all other and correct arrangement of
minimum biosafety requirements for a TB materials on the clean, working, and
smear microscopy laboratory, will suffice. dirty zones of the benchtop.

Practices and procedures

How should I work inside a TB smear
microscopy laboratory?

Treat all specimens as potentially

infectious, always handle them with
caution. Also, be careful when receiving
and processing specimens in order to
Figure 2: International biohazard symbol
avoid generating infectious aerosols.
2
 Biosafety

• Storing flammable reagents in a What should I not do?

cool, well-ventilated area.
• Do not put anything inside your mouth
• Making sure that appropriate (e.g. mouth-pipetting, licking labels
disinfectants are readily available and envelopes).
for decontamination of surfaces
and materials (see section on • Do not eat, drink, smoke, apply
Decontamination and disposal). cosmetics, handle contact lenses,
and use personal gadgets inside the
• Proper decontamination of all laboratory.
laboratory wastes before disposal
following established healthcare waste • Do not enter the laboratory without
management regulations (see section appropriate PPE, and do not wear
on Decontamination and disposal). PPE outside the laboratory.

How do I arrange materials on the PPE

laboratory benchtop during specimen
receiving / processing?
Divide the benchtop into clean, working • Laboratory coat or gown –
and dirty zones:
• Clean: where all clean materials to be
used are placed. It should be on one
side of the working zone, and opposite
the side where the dirty zone is.
• Working: where you will be working.
It should be in the middle. Place • Footwear – closed footwear should
an absorbent liner soaked with
tuberculocidal solution on the working
zone. This is where specimens must • Medical mask – a regular 3-ply
be put on at all times.
• Dirty: where you will place a discard
pan lined with infectious waste bag
containing tuberculocidal solution. All
laboratory wastes generated during
specimen receiving / processing
should be disposed of here.
What should I wear at the minimum inside
a TB smear microscopy laboratory?
ESSENTIAL; must be worn at all times
inside the laboratory
• Gloves – ESSENTIAL; must be
worn when performing laboratory
procedures that involve contact with
the specimen.
be worn inside the laboratory
medical mask is sufficient to be worn
inside the laboratory
How about other types of PPE like the N95
respirator?
Other types of PPE are not required,
but may be worn based on local risk
assessment (e.g. when there is community
transmission of Covid-19; refer to the
latest biosafety guidelines disseminated by
NTRL).

3
 Direct sputum smear microscopy
Specimen receiving
What kind of sputum specimens can be
accepted for AFB smear microscopy?
Sputum specimens that are purulent,
mucoid, blood-streaked or salivary
can be accepted. Also, all of the following
acceptance criteria should be met:
• Completely and correctly labelled
specimen
• Specimen accompanied by a to resolve the issue within the specimen’s
completely and correctly filled out LRF
• Specimen with volume of at least 3-5
mL
What kind of specimens should be
rejected?
• Specimen with no label
• Specimen with no LRF
• Improperly collected / stored /
transported specimen (e.g. preserved
with formalin, collected on tissue
paper)
• Specimen with damaged, leaking or
broken specimen container/tube
• Grossly bloody specimen
• Specimen with insufficient volume
• Specimen more than 7 days old
What should I do when rejecting a
specimen?
Indicate the reason for rejection on the
LRF, and send it back to the sending
facility. Inform them that the specimen
was rejected, and advise for recollection if
possible.
4
What if there are problems with the
specimen?
In instances when there are issues with the
specimen (e.g. problem with the label and/
or LRF), store the specimen temporarily
inside a refrigerator at 2-8oC, taking into
consideration its viability period (i.e.
up to seven days from collection date).
Coordinate with the sending health facility
viability period so the specimen can be
processed accordingly. If the issue was
not resolved within the specimen’s viability
period, inform the sending facility that the
specimen will be disposed of, and advise
for recollection if possible.
What do I need to prepare before
receiving specimens?
Equipment
• Laboratory benchtop for specimen
receiving
• Refrigerator
Materials
• Absorbent liner soaked with
disinfectant solution
• Discard pan lined with infectious waste
bag containing disinfectant solution
• Laboratory register
• Paper towel / tissue paper
• Pen and/or marker
• Specimen package for receiving
Media and reagents
• 70% alcohol
• Freshly prepared tuberculocidal
solution
 Smear preparation

How do I receive specimens? 7. If there are issues, coordinate with the

1. Check for completeness of information
in the accompanying LRF and
specimen receiving form (paper-
based and/or electronic copies).
sending health facility.
8. If the specimen is to be rejected,
contact the referring health facility,
indicate reason on the LRF.

2. Prepare and disinfect the laboratory a. For paper-based LRF, send back
benchtop (see section on the document to the referring
Decontamination and disposal). health facility.

3. Arrange materials on the benchtop: b. For electronic LRF, encode

rejection information in the LIS.
a. Clean: Pen and/or marker, paper
towel / tissue paper 9. If the specimen is to be accepted,
assign a laboratory ID number. Label
b. Working: Absorbent liner, the specimen with it.
specimen package
10. Proceed to smear preparation.
c. Dirty: Discard pan, disinfectants Otherwise, keep the specimen in a
refrigerator at 2-8oC until it can be
4. Disinfect transport boxes, and allow processed, taking into consideration
them to stand for at least ten minutes its viability period. Clean up and
before unpacking them. disinfect the laboratory benchtop.
5. Assess the specimen against the 11. At the recording and reporting area,
acceptance and rejection criteria. log the accepted specimen into the
Record specimen volume and laboratory register. If the request was
consistency on the LRF. sent electronically via the LIS, accept
the request on the LIS.
6. Check that the information in the LRF
matches the label on the specimen.

Smear preparation
What do I need to prepare before Materials
preparing smears? • Alcohol lamp
Equipment • Discard pan lined with infectious waste
bag containing disinfectant solution
• Laboratory benchtop for smear
preparation • Labelled specimens
• Refrigerator • Pencil
• Ruler
• Slide tray
5
 Direct sputum smear microscopy
Supplies
• 70% alcohol
• Applicator sticks
• Denatured alcohol
• Glass slides with frosted end
• Match/lighter
• Paper towel / tissue paper
• Tuberculocidal solution
How do I prepare smears?
1. Prepare and disinfect the laboratory
benchtop (see section on
Decontamination and disposal).
2. Arrange materials:
a. Clean: pencil, ruler, empty slide
tray, unused applicator sticks,
unused glass slides, paper towel /
tissue paper, match/lighter
b. Working: specimens, slide tray
with smears, glass slides with
smears
c. Dirty: discard pan, alcohol lamp,
disinfectants
3. Using a pencil, label the glass slide
with the laboratory ID number.
6
4. Using an applicator stick, fish out a
purulent / blood-stained portion of the
sputum sample.
5. Make a 3cm x 2cm oval-shaped
smear by spreading the sample on the
glass slide in small, repetitive, coil-like
patterns (see figure 3). Dispose of the
applicator stick.
6. Air dry the smear completely, away
from direct sunlight.
7. Heat-fix the smear by passing the
bottom of the slide over a lit alcohol
lamp 2-3 times for 2-3 seconds each
time. Do not overheat.
8. Set smears aside on a slide tray for
staining.
9. Clean up and disinfect the laboratory
benchtop.
3 CM
2 CM
Figure 3: A correctly prepared smear showing
repetitive coiling method of spreading.
 Staining of smears

Staining of smears
What do I need to prepare before
Note: For brightfield microscopy, use Ziehl-
staining smears? Neelsen staining kit and tap water. For LED-FM,
use fluorescence staining kit and distilled water.
Equipment
• Staining sink

• Timer
How do I stain smears?

Materials
Note: The timing of staining explained here is
• Alcohol lamp (for Ziehl-Neelsen) for staining solutions prepared in-house. If you
are using commercially prepared staining kits,
• Forceps follow the instructions on the package insert.

• Heat-fixed smears
1. On the staining bridge, arrange heat-
• Positive & negative control slides
fixed smears and control slides about
• Slide tray a finger width apart.

• Squeeze bottles 2. Flood the entire slide with the primary

stain:
• Staining bridge
Reagents Ziehl-Neelsen Fluorescence
1% Carbol-fuchsin 0.1% Auramine
• 70% alcohol
Using an alcohol
• Denatured alcohol lamp, apply heat on
the bottom of the
• Tap or distilled water slide until steam is Do not heat.
produced. Do not
• Tuberculocidal solution boil, nor allow the
slide to dry up.
• Ziehl-Neelsen or fluorescence staining Leave for 10 minutes Leave for 20 minutes
kit

Supplies 3. Using forceps, tilt the slide to drain off

stain, wash under a gentle stream of
• Match/lighter (for Ziehl-Neelsen)
water, then tilt again to drain off excess
• Paper towel / tissue paper water. Do not splash adjacent slides.

• Pipettes

7
 Direct sputum smear microscopy

4. Flood the entire slide with the

decolorizer: Note: Potassium permanganate is a strong
quenching agent. Avoid prolonged contact
Ziehl-Neelsen Fluorescence time of the smear with this reagent since it may
reduce fluorescence.
3% Acid alcohol 0.5% Acid alcohol
Leave for at least 3
Leave for 1-2
minutes, or until no
minutes
more color comes off

5. Wash the slide (see step 3).

6. Flood the entire slide with the

counterstain / quenching agent:
Figure 4: Smear after staining (ZN/FM) with
Ziehl-Neelsen Fluorescence
Methylene blue as counterstain (blue in color).
0.5% Potassium
0.1% Alkaline
permanganate, or
methylene blue
0.3% Methylene blue
Leave for 30-60 Leave for 60
seconds seconds

7. Wash the slide (see step 3).

8. Air dry the smears on a slide tray Figure 5: Smear after staining (FM) with
away from direct sunlight. Do not blot Potassium permanganate as quenching agent
smears, nor examine them wet. (pale gold in color).

Examination of smears
What do I need to prepare before Reagents
examining smears? • Immersion oil (for brightfield
Equipment microscopy)
• 70% alcohol
• Brightfield microscope or LED-FM unit
• 95% alcohol (for cleaning of eyepice
• Tally counter
and objective lens)
Materials Supplies
• Microscopy worksheet and/or result • Lens tissue (for cleaning of eyepiece
form and objective lens)
• Pens (black/blue and red) • Tissue paper / paper towel
• Slide box
• Stained smears
8
 Examination of smears

How do I examine smears? 7. If no AFB are seen in one length,

examine another horizontal length of
Brightfield microscopy the slide.
1. Clean and disinfect the laboratory
benchtop (see section on 8. Record observation on the microscopy
Decontamination and disposal). Wipe worksheet and/or result form (see
the microscope with alcohol. Prepare section on Recording and reporting).
the microscopy worksheet, and pens.
9. Leave the slide face down on a
2. Start examination with the control tissue paper / paper towel to dry off
slides, then patient smears. If control immersion oil. Store dried off slides
fails, investigate and take appropriate chronologically in a slide box, and
action (see section on Quality keep them for EQA.
assurance).
10. Clean up and disinfect the microscope,
3. Apply a drop of immersion oil onto the and laboratory benchtop.
smear without allowing the applicator
to touch the slide.

4. Using the scanner objective, focus on

purulent/mucoid material that contains
inflammatory cells. Avoid areas that
are too thick/thin, or contain epithelial
cells only.

5. Switch to the oil immersion objective, Figure 7: Appearance of AFB (in red) under the
and focus until cells appear sharp brightfield microscope (ZN-stained). (Image
without allowing the lens to touch the adapted from TB Microscopy. Tokyo: RIT.)
slide. LED-FM
6. Start examining the horizontal center 1. Clean and disinfect the laboratory
of the smear from end to end. Look for benchtop (see section on
AFB (slender, red rods that may occur Decontamination and disposal). Wipe
in singles, fragments, V-shaped forms, the microscope with alcohol. Prepare
or clumps; see figures 6 & 7). the microscopy worksheet, and pens.

2. Examine smears on the same day

A of staining. If they cannot be read
B immediately, keep them in the dark
after staining.

Figure 6: (A) Proper method of scanning a Note: Restain smears if they were not examined
smear (may be left to right, or right to left);
within 24 hours from the time they were originally
(B) Scanning a second row (either above or
below A) if no AFB are seen in A is required for stained.
brightfield microscopy.

9
 Direct sputum smear microscopy

3. Start examination with the control 7. Record observation on the microscopy

slides, then patient smears. If control worksheet and/or result form (see
fails, investigate and take appropriate section on Recording and reporting).
action (see section on Quality
assurance). 8. Store slides chronologically in a slide
box, and keep them for EQA.
4. Using the low power objective, focus
the smear. 9. Clean up and disinfect the laboratory
benchtop, and microscope.
5. Examine the entire horizontal center of
the smear. Look for AFB which appear
as long, slender, slightly curved, bright
yellow to greenish rods occurring in
singles, small groups or large clumps.
AFB may be uniformly stained or with
varying intensity, may contain one or
more gaps, or may give a granular
appearance.

6. Use the high power objective when Figure 8: Appearance of AFB (in bright yellow)
confirming suspicious objects, and under the LED-FM (Auramine-stained). (Photo
presence of scanty bacilli. adapted from TB Microscopy. Tokyo: RIT.)

Recording and reporting

Table 1: WHO / IUATLD grading scale

Brightfield Microscopy LED-FM

Grading Reading (OIF / 100x) Reading (LPF / 20x) Reading (HPF / 40x)
0 No AFB seen in 2 lengths No AFB seen in one length
1-4 AFB seen in 1 1-2 AFB seen in 1
For confirmation*
length length
5-49 AFB seen in 1 3-24 AFB seen in 1
+n 1-9 AFB seen in 1 length
length length
10-99 AFB seen in 1 3-24 AFB seen per 1-6 AFB seen per
1+
length LPF HPF
1-10 AFB per field in at 25-250 AFB seen 7-60 AFB seen per
2+
least 50 OIFs per LPF HPF
>10 AFB per field in at >250 AFB seen per >60 AFB seen per
3+
least 20 OIFs LPF HPF

Note: *Confirmation to be done by another microscopist, or by preparing and reading another

smear.

10
 Decontamination and disposal

What do I need to prepare before 4. Prepare a laboratory results releasing

recording and reporting?
Materials
• Black/blue and red pens
• Laboratory register
• Laboratory result form
form, and send it together with the
result form to the requesting facility.
If the request was received through
an LIS, encode the result via the LIS,
and have it verified by a senior staff /
supervisor so it can be forwarded to
the requesting facility.

• Laboratory results releasing form 5. Result should be released within

• Laboratory report form three working days from specimen
collection date.
• Microscopy worksheet
How do I report smear microscopy
How do I record and release DSSM laboratory data?
results?
1. During the reporting period, fill up a
1. Use the WHO/IUATLD grading scale laboratory reporting form using data
(see table 1). from the laboratory register (for further
instructions, refer to the TB laboratory
2. Record the result on the microscopy recording and reporting guidebook).
worksheet, laboratory result form, and
laboratory register. Use red ink for 2. A senior staff / supervisor should verify
positive results, and black for negative the report. If the head of laboratory is
ones (for further instructions, refer available, they should also sign the
to the TB laboratory recording and report.
reporting guidebook).
3. Submit the report to the provincial/
3. A senior staff / supervisor should verify city health office. For electronic-based
the result. If the head of laboratory is reporting, encode the report in the
available, they must also sign the LIS. Have it verified by a senior staff
result. / supervisor so it can be forwarded to
the provincial/city health office.

Decontamination and disposal

it is not required for a laboratory
Decontamination performing DSSM only. Chemical
How do I decontaminate smear microscopy decontamination may be used as an
laboratory wastes? alternative.

• Autoclaving: Used laboratory • Chemical decontamination: Use

materials, and wastes for disposal of properly prepared solutions of
are autoclaved at 121oC, 15psi for appropriate disinfectants, taking into
at least 30 minutes. Although this is consideration their prescribed contact
the best decontamination method, times (see table 2).

11
 Direct sputum smear microscopy

Table 2: Different disinfectants that can be used in a TB smear microscopy laboratory

Disinfectant Preparation Contact time Use

Tuberculocidal; for decontamination
Phenolic 5% in water At least 10 of laboratory benchtops before
compound (freshly prepared) minutes and after work, and wastes prior to
disposal.
1:100 and 1:10 1:100 dilution is used for general
Sodium
in water (freshly surface disinfection; 1:10 dilution
hypochlorite At least 10
prepared; stored may be used as a substitute
(household minutes
in dark, well- for phenolic compound; NOTE:
bleach)
ventilated area) Corrosive to metals
Alcohol Non-tuberculocidal; for routine
(ethanol, 70% in water N/A disinfection of surfaces and
isopropanol) materials

How do I disinfect laboratory benchtops of following established healthcare

and surfaces?
Wipe with phenolic compound or sodium
hypochlorite solution, and observe contact
time (see table 2). Afterwards, rinse
disinfectant residues by wiping twice with
alcohol solution.
Disposal
How do I dispose of laboratory wastes?
• For sharp items such as glass slides,
discard them into a rigid container
filled with tuberculocidal solution.
Label it with “Biohazardous Sharps
Waste”. Once it is three quarters full, Note: Never wash and reuse used sputum cups.
tightly close the container, and dispose
waste management regulations in your
facility/area. Do not fill the container to
or over capacity.
• For all other laboratory wastes,
discard them into an infectious
waste bag (e.g. yellow bag). Label it
with “Infectious Waste”. Submerge
wastes completely in tuberculocidal
solution (for sputum cups, fill them
with tuberculocidal solution). Dispose
of following established healthcare
waste management regulations in
your facility/area.

Quality assurance
How do I ensure the quality of my AFB Quality control
smear microscopy results? What should I observe and do inside the
Observe the three components of quality laboratory to ensure quality?
assurance: quality control, external • Personnel performing DSSM should
quality assessment, and quality be adequately trained.
improvement (for further information,
refer to the Quality assurance on DSSM • Microscope should be properly
manual). maintained.
12

• Staining kits should be correctly 3. Gently mix the liquefied sputum while
stored in a cool, dry place, away from
direct sunlight. Perform QC testing
when opening a new batch of staining
kit by staining and reading positive 4. Label slide with the positive control
and negative control slides. Never use
expired staining kits.
• Process only specimens that meet all
of the acceptance criteria, and none
of the rejection criteria (see section
on Specimen receiving).
• Perform QC testing for every batch of
patient smears being stained (at least
once in a day).
• Record all specimens received and
their results in the laboratory register.
Release results within the desired
TAT (see section on Recording and
reporting).
• All results and reports should be
verified by a senior staff / supervisor.
• Collect and analyze significant data
quarterly, taking note of the following:
• Total number of presumptive TB
cases examined;
• Total number of smears prepared;
• Total number of cases that tested
positive; and
• Positivity rate
How to prepare positive control slides?
1. Use a known low positive (1+) sputum.
2. Allow sputum to stand overnight until
liquefied.
Quality assurance
it is still capped. Let stand for 10
minutes before opening.
batch number, smear preparation
date, and staff initials.
5. Prepare as many smears as possible.
Completely air dry, then heat fix the
smears.
6. Randomly pick around 10% of the total
number of positive smears prepared,
stain them, and examine under the
microscope to check number of AFB.
a. If sampled smears yield 1+
results, keep all prepared smears
in a slide box, and label it with
“Positive Control Smears”.
b. Otherwise, dispose of the entire
batch of prepared smears.
How do I prepare negative control slides?
1. Use egg white diluted to 5% in water,
and mix with a little amount of known
negative sputum/saliva.
2. Label slide with the negative control
batch number, smear preparation
date, and staff initials.
3. Prepare as many smears as possible.
Completely air dry, then heat fix them.
4. Keep all prepared smears in a slide
box, and label it with “Negative Control
Smears”.
13
 Direct sputum smear microscopy

How do I perform QC testing of staining 4. Record results in a QC Logbook. Take

kit?
1. Upon opening a new batch of staining
kit, pick two positive, and two negative
control slides.
2. Stain the positive controls once, and
the negative controls thrice.
3. Examine control slides for number
of AFB, intensity of AFB color,
decolorization of background, and
presence of crystals and artifacts (see
table 3 for the expected results).
Table 3: Expected QC testing results
appropriate action for unacceptable
results as follows:
a. Check the smear preparation and
staining technique. Check also
the stains used. Resolve issue
identified.
b. If still uncertain, stain another
batch of control slides using the
same batch of staining kit while
ensuring correct technique. If the
QC results are still unacceptable,
discard batch of staining kit used,
and perform QC on new batch of
reagents.

QC Area Acceptable Unacceptable

Number of AFB
Intensity of AFB color
Positive Control: 1+;
Negative Control: 0
Ziehl-Neelsen: strong red;
LED-FM: bright to greenish
yellow
Positive Control: +n/0;
Negative Control: +
Pale

Decolorization Complete Incomplete

Crystals & Artifacts Absent Present

How do I perform QC testing during a. Check staining technique by

staining of patient smears?
1. Before staining a batch of patient
smears, pick one positive, and one
negative control slide.
2. Stain both controls together with the
patient smears.
3. Examine the control smears according
to the four QC areas (see table 3).
4. Record results in a QC Logbook.
Investigate problems, if present, and
take appropriate action:
14
staining the same batch of
control slides using the same
batch of staining kit. If QC result
is acceptable, discard previously
stained patient smears, and
prepare new ones from stored
sputum. Observe correct staining
technique.
b. Check staining kit by staining
the same batch of control slides
using a different batch of staining
kit. If QC result is acceptable,
discard the previously used batch
 References

of staining kit, and previously EQA

stained patient smears. Prepare
new smears from stored sputum, How is EQA done?
and stain them using the batch of In addition to internal QC, your smear
staining kit that passed QC. microscopy laboratory should regularly
participate in the EQA program
c. Check control slides by staining
implemented by the NTRL. For blinded
a different batch of control slides
rechecking, keep all read smears properly
using the same batch of staining
and chronologically in slide boxes until
kit. If QC result is acceptable,
slides are selected by the QA Center.
discard the previously used batch
of control slides, and previously Quality improvement
stained patient smears. Prepare
new smears from stored sputum, How do I improve the quality of AFB smear
and stain them together with microscopy testing in our laboratory?
the batch of control slides that
passed QC. Consider the outputs of your data analysis,
and EQA / on-site supervision visit
feedback given by senior staff / supervisor,
and/or quality controller / coordinator.
Implement corrective actions to address
areas for improvement.

References
Fujiki, A. (2002). TB Microscopy. Tokyo: Research Institute of Tuberculosis.

Fujiki, A. (2007). TB Microscopy for National Tuberculosis Program. Tokyo: Research Institute of
Tuberculosis.

Global Laboratory Initiative. (2014). Mycobacteriology Laboratory Manual. Geneva: Global

Laboratory Initiative.

Lumb, R., Van Deun, A., Bastian, I., & Fitz-Gerald, M. (2013). Laboratory Diagnosis of Tuberculosis
by Sputum Microscopy. Adelaide: SA Pathology.

National Tuberculosis Reference Laboratory. (2019). Manual on the Collection, Storage & Transport
of Specimens for TB Testing, 2nd Ed. Muntinlupa City: NTRL-RITM.

Philippine National Tuberculosis Programme. (2004). Quality Assurance for Sputum Smear
Microscopy. Manila: Department of Health.

Singhal, R., & Myneedu, V. P. (2015). Microscopy as a diagnostic tool in pulmonary tuberculosis.
International Journal of Mycobacteriology, 4(1), 1-6.

World Health Organization. (2012). Tuberculosis Laboratory Biosafety Manual. Geneva: WHO
Press.

15