Laboratory Diagnosis

The handbook
Laboratory Diagnosis
of Tuberculosis by Sputum
Microscopy
Pacific Island Countries

The handbook
Microscopy
Richard Lumb
Ivan Bastian
Acknowledgments
This edition of The Handbook, for Pacific Island Countries, was produced
with financial assistance from the South Australian TB Services within the Royal Adelaide
Hospital Department of Thoracic Medicine, and the Australian Tuberculosis and
Chest Association.
The authors wish to thank the following for their valuable input and support:
• Dr Armand van Deun

Bacteriology Consultant
Union
Antwerp, Belgium
• Mr David Dawson
TB Laboratory Consultant
Brisbane, Australia
• Mr John Elliot
Pacific Paramedical Training Centre
Wellington, New Zealand
• Ms Linda Kuo
Department of Health Services
Richmond, California, USA
• Ms Kay Withnall
Laboratory Consultant
Darwin, Australia
• Prof Barrie Vernon-Roberts
IMVS
LABORATORY DIAGNOSIS OF TUBERCULOSIS

BY SPUTUM MICROSCOPY Page 1
The handbook
Microscopy
Published by:
Institute of Medical and Veterinary Science
Frome Road
Adelaide
South Australia 5000
www.imvs.sa.gov.au
All rights reserved. No part of this book may be reproduced or

transmitted in any form or by any means without the written permission
of the publisher. The authors assert their moral rights in the work.
ISBN 0-9750285-2-9
Production
Project Editor
Mark Fitz-Gerald
Technical Editors
Richard Lumb, Ivan Bastian
Manuscript Editors
Mark Fitz-Gerald, Richard Lumb
Illustrations
Kerry Reid
Design
Sue Dyer Design
© 2005 Institute of Medical and Veterinary Science

Page 2 BY SPUTUM MICROSCOPY
Contents
Page Page
Foreword 4 7 Summary 26
False-negatives 26
Introduction 6 – Consequences
– Prevention
1 Symbols and warnings 7 False-positives 26
– Consequences
2 Sputum collection 8 – Prevention
Spot-morning-spot 8
Hospital patients 8 8 Appendices 27
Safe collection 8 Specimen containers 27
Pre-collection patient advice 9 Documentation 28
How to collect a specimen 10 Laboratory layout 30
Specimen quality 10 Safety 31
Rejection criteria 10 Ziehl-Neelsen reagent preparation 34
Registration 11 Quality Control 37
Storage and transport 12 The microscope 38
Trouble shooting
3 Smear preparation 14 – Staining 45
What you need 14 – Microscopy 47
Making a smear 15 Patient information 52
4 Staining 18
What you need 18
The Ziehl-Neelsen method 20
5 Examination 22
Reading smears 22
Appearance of acid fast bacilli 24
6 Reporting 25
How to report 25

Foreword
Tuberculosis is a treatable disease that

affects many individuals and almost all
communities. The well-being and
productivity of a country may be affected
severely by this insidious disease.
The World Health Organization developed the Directly Observed
Treatment, Short course (DOTS) strategy for tuberculosis (TB) control
which has been adopted by many National Tuberculosis Programmes
(NTPs). The DOTS strategy recommends direct smear microscopy as
the most effective tool for the diagnosis of TB and for monitoring patient
response to treatment. An effective TB control programme therefore
depends upon laboratories providing accurate, reliable and timely
detection of infectious cases.
In 2004, in collaboration with Indonesian colleagues, The Handbook

was developed and translated into Bahasa. 15,000 copies were printed
and distributed throughout the country. It has subsequently received
widespread acceptance and generated interest in other countries.
This version has been revised for Pacific Island Countries many
of which are isolated and have relatively high rates of TB in widely-
dispersed communities. These conditions present unique challenges for
TB control and the provision of laboratory services. The Handbook
is intended to assist the NTPs of Pacific Island Countries to achieve
excellence in sputum smear microscopy.
MR RICHARD LUMB
DR IVAN BASTIAN

Introduction
The purpose of this Handbook is to teach technicians how to safely collect, process
and examine sputum specimens for the laboratory diagnosis of tuberculosis (TB).
It should be used together with ‘Quality Assurance of Sputum Microscopy in DOTS
Programmes – Guidelines for Pacific Island Countries’ (WHO).
Sputum microscopy
Sputum smear microscopy is one of the most efficient tools for identifying people
with infectious TB.
Smear-positive patients are up to ten times more likely to be infectious than are
smear-negative patients.
The purpose of sputum microscopy is to:

• Diagnose people with infectious TB
• Determine the infectivity of the disease
• Monitor the progress of treatment
• Confirm that cure has been achieved
Consistent and accurate laboratory practice helps to save lives and improves public health.
Risk of Infection
Risk of infection for laboratory technicians is very low during smear preparation.
A higher risk of infection exists when talking to TB patients before specimens are collected.
In contrast, doctors and nurses working in TB wards and clinics have a much higher risk
of becoming infected with TB.
Personal safety
When performed correctly sputum examination will not place laboratory technicians
at increased risk of developing TB.

Page 6 Introduction BY SPUTUM MICROSCOPY
Symbols and warnings 1
Failure to follow these instructions may harm your health or cause immediate
damage to equipment
Failure to follow these instructions may affect test outcomes, or cause equipment
damage over time
Correct – the preferred way to do something
✗ Do not do this
You should wear gloves for this procedure
You should wear a gown for this procedure
Wash your hands

Always wash your hands after preparing sputum smears
and before leaving the laboratory
This substance is toxic
This substance is corrosive

8

BY SPUTUM MICROSCOPY Symbols and warnings Page 7
2 Sputum collection
Spot-morning-spot
Three sputum specimens are recommended for the laboratory diagnosis of TB. For the
convenience of the patient collect specimens using the spot-morning-spot principle.
Spot Day – 1
• Collect the first specimen when the patient presents to the clinic.
• Give the patient a labelled sputum container for the next morning’s sputum collection.
Morning Day – 2
• Patient collects early morning sputum and brings it to the clinic.
Spot Day – 2
• Collect the third specimen when the patient returns to the clinic with the morning
specimen.
Hospital patients
If the patient is in hospital, collect a sputum specimen each morning on three
consecutive days.
Safe collection
Transmission of TB occurs because infectious droplets are released into the air
when a diseased patient coughs.
Collect specimens outside so that infectious droplets are diluted in an open,

well-ventilated area
To reduce the possibility of laboratory staff becoming infected:
Tell the patient to cover their mouth when coughing.
Collect sputum outside the laboratory, preferably outside the building

well away from other people.
✗ Do not collect sputum specimens in:

• Laboratories
• Toilet cubicles
• Waiting rooms
• Reception rooms
• Any poorly ventilated area

Page 8 Sputum collection BY SPUTUM MICROSCOPY
Sputum collection 2
Pre-collection patient advice
N AT I O N A L T U B E R C U L O S I S C O N T R O L P R O G R A M M E

R E Q U E S T F O R S P U T U M E X A M I N AT I O N
Treatment unit Date
Contact person Phone

Diagnosis
Patient name
or
✗
Date of birth Age Sex M F
Other names
Home address Follow up

Island
Examination for Diagnosis Follow-up

Patient Identification Number
RESULTS (Laboratory to complete)
Laboratory Register Number

Appearance Results (check one)
Blood Muco
Saliva
Collection Date Specimen Stained Purulent neg 1-9 + ++ +++
1
Examined by (signature) Date • Clearly label the

S E N D C O M P L E T E D F O R M A N D R E S U LT S
TO T H E T R E AT M E N T U N I T P R O M P T LY sputum container with
the patient’s name and
• Check the Specimen Request Form date of collection
• Fill in any missing details • Label the container,
• Tick Diagnosis or Follow-up never the lid
If dentures are present,

remove them and rinse
mouth with water.
Tell the patient the best
specimen comes from
the lungs.
Saliva or nasal secretions
are unsuitable.

BY SPUTUM MICROSCOPY Sputum collection Page 9
2 Sputum collection
How to collect a specimen

Patient Information
page 52
✗
Do not stand in front of the patient during collection
Instruct the patient to:
1. Inhale deeply 2 to 3 times, breathe out hard each time
2. Cough deeply from the chest
3. Place the open container close to the mouth to collect the sputum
4. Screw the lid on tightly
Several attempts may be necessary to obtain a good quality specimen.
If the specimen is poor:
1. Keep the most mucoid-purulent sample
2. Discard all the other samples
Specimen quality
✗
Good quality specimen Good quality specimen Blood stained Poor quality specimens
Mucoid Purulent are thin and watery
or composed largely
of bubbles
Rejection criteria
• Broken or leaking specimen containers
• Specimen in inappropriate/non-sterile container
• Specimen container details do not match the Specimen Request Form
• The specimen has been collected into a fixative (e.g. formalin)
• The specimen has been collected into a tissue

Sputum collection 2
Registration
Register the specimen before processing
3
Laboratory Register
4
757/1 Laboratory Register
Reason for Microscopy

Lab Name of examination* Signature Remarks
Sex Complete address Results
Register Date Name in full Age referring Health
M/F (for new patients)
No. Centre Diagnosis Follow-up 1 2 3
Treatment unit Date
Patient name
Other names * If sputum is for diagnosis, put a tick () mark in the space under “Diagnosis”
Home address
Island
2 If sputum is for follow-up of patients on treatment, write Patient’s Identity Number (PIN) in the space under “Follow-up”.
Collection Date
1
Blood Muco
Specimen Stained Purulent
Saliva
neg 1-9 + ++ +++
5
2
Examined by (signature) Date
TO T H E T R E AT M E N T U N I T P R O M P T LY
Specimen Request Form
1. Check patient details on container match the Specimen Request Form

2. Transfer patient details from the Specimen Request Form to the Laboratory Register
3. Write the Laboratory Register Number (LRN) on the side of the specimen container
4. Write the LRN on the Specimen Request Form
5. For Follow-up patients write the Patient Identity Number (PIN) in the Remarks Column
of the Laboratory Register
For each patient, use the same LRN and the numbers 1, 2, 3 to identify the:
• Spot (1)
• Morning (2)
• Spot (3) specimens.
Saliva specimens must be reported on the Specimen Request Form.

2 Sputum collection Specimen storage and transport
In remote settings, sputum specimens need to be transported to a microscopy laboratory.
Storage
To preserve specimen quality:
• Prior to dispatch store specimens in a refrigerator
(do not freeze), or keep as cool as possible
• Protect from sunlight
What you need

• Strong specimen containers (see page 27)
Specimen storage and transport
• Permanent marker to write details on the side of the container

• Plastic, ziplock bag for each specimen
• Transport box
• Master list of specimens
• Sputum request forms
Specimen containers must be strong enough to withstand transportation effects
Approved secondary packaging (transport box) must:

• Be leak proof and strong
• Contain absorbent material, bench roll etc
• Be kept out of sunlight
• Keep request forms separate from sputum specimens
Packing checklist
Is the sputum container clearly labelled with
• Patient name
• Date of collection
• Spot (1) Morning (2) Spot (3)
Always label the container never the lid (see page 9)
• Are Request Forms completed correctly?

• Are Request Forms packed separately from specimens?
Prepare a Master list that contains the details for each specimen being transported
Ensure the Master list contains the name and address of the laboratory
sending the specimens
Check that the number of specimens equals that on the Master list
Transport
• Check your local regulations for transport by air
• Dispatch stored specimens twice weekly

Specimen storage and transport Sputum collection 2
Packing specimens
Put several layers of absorbent paper in the bottom of the shipper.
3
RE
Treatment QUEST
unit FOR SPUTUM EXAMIN AT I O N
Date
Patient name
R unit
Treatment EQUEST FOR SPUTUM EXAMIN AT I O N
Date
Other names
Patient name
Home address
Treatment unit Date
Date of birth Age Island Sex M F
Other names
Patient name
Home address

Island
Other names
Home address
1
Island

2 5
4
7
6 Keep upright
Absorbent paper 8
1. Check each specimen container is labelled with

– Patient name
– Date
– Spot (1), Morning (2), Spot (3)
2. Seal each container in a separate bio-hazard bag
3. Cross check specimens against Request Form
4. Put sealed bio-hazard bags into the shipper
5. Put Request Forms and Master list into a separate sealed bag
6. Pack shipper to prevent movement Keep cool
7. Add sealed bag containing Request Forms last Store upright
8. Seal and address the shipper Deliver urgently

3 Smear preparation What you need
Sputum smears must be prepared promptly after collection.
To effectively prepare smears, you will need:
A solid bench with a non-absorbent surface that can be disinfected
Handle slides by
edges only
Discard bucket
containing disinfectant Alcohol/sand trap for
New, clean glass slides (e.g. 5% Phenol) cleaning loops
Bamboo/wooden applicator sticks or wire loop
Spirit lamp
Slide rack for drying smears
Never reuse sputum smear slides
Applicator stick
Bamboo/wooden applicator sticks are better because they:
• Separate purulent material from saliva faster
• Pick up more sputum than wire loops
• Are easier to handle
• Are faster to use
Wire loops
Some technicians prefer wire loops because they can be reused however they:
• Are more time consuming
• Collect a smaller sample volume
• Are less efficient

Page 14 Smear preparation BY SPUTUM MICROSCOPY
Making a smear Smear preparation 3
Never put more than one sputum specimen on each slide
Add Laboratory Code

1
Number (LCN) if
applicable
LRN Spot
Write the LRN and the 1, 2, or 3 identifier on the frosted end For clear slides use
of each slide using a lead pencil a diamond pen
Do not mix purulent/bloodstained portions with saliva/mucous
A
Select only purulent or
bloodstained portions of
Sputum specimen with purulent portions within saliva sputum
More acid fast bacilli

(AFB) will be found A B
in the purulent portions
of a specimen.
Ziehl-Neelsen stained smear – purulent Ziehl-Neelsen stained smear – saliva

BY SPUTUM MICROSCOPY Smear preparation Page 15
3 Smear preparation Making a smear
3 4
1cm
2cm
✗
Discard the applicator
stick into discard
Smear the specimen in the centre of the slide, covering bucket after use, do not
2cm by 1cm flame, do not reuse
Always clean and sterilise a wire loop between each specimen
5 6
Retain all specimens until
results are reported
To clean a wire loop

• Insert loop in sand trap and rotate
• Flame the loop to red-hot and allow to cool
Wash your hands after smear preparation

Page 16 Smear preparation BY SPUTUM MICROSCOPY
Making a smear Smear preparation 3
7 8
Dry smears on a slide rack, out of direct sunlight
When dry, heat fix the

9 smears:
• ensure the smear is
facing upwards
• pass 3 times through
the flame of a spirit
lamp.

Correctly prepared smear
Overheating will
damage the bacilli
Stained smears resulting from poor smear

preparation
Too thick Too thin Not centred and Multiples

too small
✗ ✗ ✗ ✗
BY SPUTUM MICROSCOPY Smear preparation Page 17
4 Staining What you need
To stain smears using the Ziehl-Neelsen method you will need:
Slide rack to support slides over sink or bucket
Forceps
Spirit burner
Slide rack for drying stained slides Water
Timer

Page 18 Staining BY SPUTUM MICROSCOPY
What you need Staining 4
Carbol fuchsin 0.3 %
Methylene blue 0.3 %
3% HCI in ethanol
or 25% H2S04
You will require 2 – 3 volumes of decolouriser for each

volume of stain.

BY SPUTUM MICROSCOPY Staining Page 19
4 Staining The Ziehl-Neelsen method
1 2
Place the slides smear upwards, in LRN order, Cover each slide completely with carbol fuchsin
on a staining rack over the sink or basin, about
a finger-width apart
Ensure the slides are level
4
Leave the heated stain on the slides –
minimum 5 minutes
3
A longer time is OK provided the stain
does not dry on the slide
• Heat each slide from below until steam rises, always

keep the flame moving
• Stop heating when steam rises
Do not boil
5
Do not splash adjacent slides ✗
• Gently rinse each slide with a stream of water

• Tilt each slide to drain off excess water

Page 20 Staining BY SPUTUM MICROSCOPY
The Ziehl-Neelsen method Staining 4
7 8
Add decolourising solution to the slide and leave • Gently rinse each slide with water
for 3 minutes • Do not splash other slides
9 10
Cover each slide with methylene blue for • Gently rinse each slide with water
30 seconds only • Do not splash other slides
11
12
Do not examine slides until they have dried
• Air dry away from direct sunlight

• Do not dry slides using blotting paper
A correctly stained smear

BY SPUTUM MICROSCOPY Staining Page 21
5 Examination Reading smears
AFB’s are stained red. Some males are red/green colour-blind, hence all male technicians
should be tested for colour blindness.
Smears must be consistently and systematically examined to ensure a

representative area of the smear is reported.
1 2
Use the 10X objective lens to find a suitable purulent
area to examine:
• It should have more inflammatory cells than
epithelial cells
• For uniformly poor quality specimens, find an area
relatively free of epithelial cells
• Check the smear is facing upwards

• Apply one drop of immersion oil
• The drop must fall freely onto the smear so that the oil
applicator does not become contaminated with TB
Inflammatory cells (high power) – look for areas like this
organisms
Never allow the oil applicator to

touch the slide
3
Carefully rotate the 100X oil objective lens
over the slide
Avoid areas containing epithelial cells (low power)

Page 22 Examination BY SPUTUM MICROSCOPY
Reading smears Examination 5
4 5
Carefully adjust the fine focus until cells are sharp
Never allow the lens to touch the

glass slide
6
Direction of traversing the stained slide
Examine at least 100 high power fields

before recording a negative result
You should take approximately 5 minutes
to read a negative smear
Gently wipe the oil from the slide with the edge
of a clean piece of tissue paper
Avoid contamination, always use a

clean piece of toilet paper
8

Store the slides in LRN order as they will be
Wipe the lens gently with tissue paper to remove needed for External Quality Assessment (EQA)
immersion oil after you have finished examining
a batch of slides ✗ Do not write the result on the slide
✗ There is no need to treat slides with xylol

BY SPUTUM MICROSCOPY Examination Page 23
5 Examination Appearance of acid fast bacilli (AFB)
• Viewed with an oil immersion lens, AFB are red, slender rods, sometimes with one
or more granules
• Tubercle bacilli may occur singly, as V-shaped forms, or as clumps of bacilli
Typical morphological characteristics of Mycobacterium tuberculosis
single bacilli V-shaped forms
clumps of bacilli
Where possible all new positive smears should be reviewed

by another technician

Page 24 Examination BY SPUTUM MICROSCOPY
How to report Reporting 6
The number of AFB What you see What to report
found indicates how
infectious the patient is, No AFB in 100 fields Negative for AFB
hence it is important 1 – 9 AFB in 100 fields Report what you see
to record exactly 10 – 99 AFB in 100 fields 1+
what you see.
1 – 10 AFB per field, check 50 fields 2+
More than 10 AFB per field, check 20 fields 3+
1 R E Q U E S T F O R S P U T U M E X A M I N AT I O N
757/1
Treatment unit
Contact person
Date
Phone
4 2 Patient name
Date of birth
Other names
Age Sex M F
Home address
Record Results Island
Laboratory Register
757/1
Register Date Name in full Age referring Health Laboratory Register Number
Blood Muco
Saliva
1
Examined by (signature)
Date Date
Sign 3
* If sputum is for diagnosis, put a tick () mark in the space under “Diagnosis”
If sputum is for follow-up of patients on treatment, write Patient’s Identity Number (PIN) in the space under “Follow-up”.
Laboratory Register 5 Return to doctor or clinic
1. Check the smear LRN matches the Specimen Request Form

2. Write the results on the patient’s Specimen Request Form
3. Date and sign the Specimen Request Form
4. Record the results in the Laboratory Register immediately after reading
the smear. Use red pen for Positive results
5. Return the completed Specimen Request Form to the doctor or clinic
Do not give results to the patients as lost reports may delay treatment
Do not write the results on the slide as they are needed for QC checking

BY SPUTUM MICROSCOPY Reporting Page 25
7 Summary
Be consistent and accurate in all your work, lives depend on you
False-negatives – consequences
False-negative means • Patients with TB may not be treated resulting in on-going disease, disease
reported negative but truly transmission, or death
smear-positive • Intensive phase treatment may not be completed, resulting in inadequate treatment and
potential drug resistance
Prevention
• Label sputum containers, slides and laboratory forms accurately
• The specimen must contain sputum not saliva
• Select purulent material to make the smear
• Smear preparation – centred, not too thick or thin, 2cm x 1cm in size
• Heat carbol fuchsin until steaming
• Do not boil during fixation
• Stain with carbol fuchsin – minimum 5 minutes
• Do not overheat the carbol fuchsin
• Decolourise until no more carbol fuchsin is released, maximum 3 minutes
• Counterstain – maximum 30 seconds
• Keep the microscope well maintained and the lenses clean
• Perform regular QC on stains and reagents
• Check the slide LRN matches the Specimen Request Form before recording the result
Don’t rush – examine a smear for 5 minutes minimum before

recording a negative
False-positives – consequences
False-positive means • Patients are treated unnecessarily
reported positive but truly • Treatment may be continued longer than necessary
smear-negative • Medications will be wasted
Prevention
• Ensure technicians can reliably recognise acid-fast bacilli
• Label sputum containers, slides and laboratory forms accurately
• Always use new unscratched slides
• Use bamboo/wooden sticks once only
• Do not allow carbol fuchsin to dry on the smear
• Decolourise adequately
• The oil applicator must not touch the slide
• Keep the microscope well maintained, the lenses clean, store appropriately
• Perform regular QC of stains and reagents
• Check the slide LRN matches the Specimen Request Form before recording the result

Page 26 Summary BY SPUTUM MICROSCOPY
Specimen containers Appendices 8
Ideal specimen container

Multi-thread screw cap
Wide-mouth
Clear break-resistant

plastic
Can be written on with a
permanent marker pen
Single-use
Clean
Unacceptable containers
✗ ✗ ✗ ✗
Opaque Extra insert lid Too small Coloured

Too small Screw cap inadequate Screw cap inadequate Screw cap inadequate

BY SPUTUM MICROSCOPY Specimen containers Page 27
8 Appendices Documentation

This example shows the type of information required on a Specimen Request Form.
Treatment unit Date
Patient name
Other names
Home address
Island

Blood Muco
Saliva
1
Examined by (signature) Date

Page 28 Specimen Request Form BY SPUTUM MICROSCOPY
Documentation Appendices 8
Laboratory Register
This example shows the type of information required on a Laboratory Register
Laboratory Register

Register Date Name in full Age referring Health
* If sputum is for diagnosis, put a tick () mark in the space under “Diagnosis”
If sputum is for follow-up of patients on treatment, write Patient’s Identity Number (PIN) in the space under “Follow-up”.
The Laboratory Register Numbering System

The LRN begins at number 1 at the start of each year.
It increases by one with each patient, until the end of the year.
Do not return to LRN Number 1 at the end of each day, week or month

BY SPUTUM MICROSCOPY Laboratory Register Page 29
8 Appendices Laboratory layout
Biological safety cabinets (BSC)

• Preparing sputum smears does not require a BSC
• Only laboratories performing culture and drug susceptibility testing need
a functioning BSC
Never use a clean air cabinet as it can blow TB organisms

into the laboratory
The basic requirements for a sputum microscopy laboratory include:

• A table/bench to prepare smears
• Sink or plastic basin to stain smears
• A table to examine smears
• A table for paperwork
• Basin for hand washing
• Adequate lighting
• Non-skid flooring
• An area for receiving specimens
• Good ventilation
Window
Microscopy bench Records bench
Waste bin
Window opens to exterior
Sink
Storage
cupboards
Smear
preparation
area
Hand
washing
basin
Bench for incoming
specimens Gown rack
Sliding window to
receive specimens

Page 30 Laboratory layout BY SPUTUM MICROSCOPY
Safety Appendices 8
Transmission of TB occurs through micro-aerosols called infectious droplets that
contain tubercle bacilli. Good laboratory methods aim to minimise formation
of droplet nuclei.
Assume all specimens are potentially infectious
General laboratory safety

• Never smoke, eat or drink in the laboratory
• Do not wear gowns/coats or gloves outside the laboratory
• A surgical mask provides no worthwhile protection for technicians
• Wash hands regularly, especially after removing gloves, and always before
leaving the laboratory
• Do not allow unauthorised people within the laboratory area
• Do not perform work for which you are not trained
• Always follow the safety procedures
Gowns
The laboratory should provide a supply of clean gowns
• Hang your gown in a designated area when not in use
• Change gowns on a regular basis
• Replace immediately if contaminated
Gloves
• Disposable gloves should be worn when handling specimens
• Do not wear gloves for microscopy, report writing or during staining
If gloves are not available, you can still prepare sputum smears but you must
wash your hands after processing is completed
Disinfection methods
Surfaces Spills Prepare
Phenol 5% Every 2 days
Alcohol 80% v/v ✗ Weekly
Hypochlorite 5%* ✗ Every 2 days
*Corrosive to metal
How to clean up spills

1. Put on a gown and gloves
2. Place a paper towel or cloth over the spill area and liberally apply phenol solution
3. Leave covered – minimum 15 minutes
4. Clean up the contaminated material and put into the waste container
5. Clean with a final wash using 80% v/v alcohol
6. Wash your hands after the clean up is complete

BY SPUTUM MICROSCOPY Safety Page 31
8 Appendices Safety
Waste disposal
Locate the burning drum away from people in an open area as the
fumes are toxic
1 2
Add phenol to a lined bucket Tighten caps, add specimen
containers and contents of the
discard bucket to the phenol bucket
3
Burn bucket
contents
weekly
4
When cool bury burning
drum contents at least
1.5 metres deep

Page 32 Safety BY SPUTUM MICROSCOPY
Safety Appendices 8
Ergonomics
✗
Good posture – supporting your feet

straightens your back Poor posture – feet unsupported
✗
Good posture – raise the microscope

to help straighten your back and keep Poor posture – microscope too low
your feet flat on the floor and feet not flat

BY SPUTUM MICROSCOPY Safety Page 33
8 Appendices Ziehl-Neelsen reagent preparation
Record each batch of prepared reagent in a Reagent Preparation Workbook including:

• Reagent name
• Signature of the technician who prepared it
• The date of preparation
• The results of QC testing
Reagent Preparation Workbook
Date Reagent QC Results Initials

Positive Negative
25/11/03 Carbol fuchsin 0.3% RL
25/11/03 3% HCl/alcohol RL
Ready to use reagents must also be QC tested, and the results recorded in the
Reagent Preparation Workbook.

Page 34 Ziehl-Neelsen reagent preparation BY SPUTUM MICROSCOPY
Ziehl-Neelsen reagent preparation Appendices 8
Wash your hands after preparing reagents
Carbol fuchsin – 0.3%
Phenol crystals and vapour are corrosive, toxic and may cause burns
8 Use care, prepare in a well ventilated area
Grade
Basic fuchsin powder 3.0g Certified
Ethanol (or methanol) 100ml Technical
Phenol crystals* 50g *use colourless not tinted crystals Analytical
Distilled water 900ml
Preparation
1. Add 100ml of alcohol (or methanol) to a one litre glass flask
2. Add 50g of phenol crystals and dissolve
3. Add 3.0g of basic fuchsin powder
4. Mix well with gentle heating until dissolved
5. Add distilled water to make one litre
6. Label the bottle – “0.3% carbol fuchsin”, date and initial
7. Store in a dark bottle in a cupboard at room temperature (expiry 6 months)
8. Prior to use filter stock solution into the staining bottle
Label
0.3% carbol fuchsin
Date
Initial
Filter stock solution before use

BY SPUTUM MICROSCOPY Ziehl-Neelsen reagent preparation Page 35
8 Appendices Ziehl-Neelsen reagent preparation
Decolourising solution
Always add the acid to ethanol or water. Solutions will generate heat.
3% HCI ethanol Grade

Concentrated hydrochloric acid (HCl) 30ml Technical
95% ethanol 970ml Technical
25% H2SO4
Concentrated sulphuric acid (H2SO4) 250ml Technical
Preparation
1. Carefully add the HCl to the ethanol or the H2SO4 to the water
2. Label the bottle “3% HCl in ethanol” or “25% H2SO4”, date and initial
Counterstain
Methylene blue chloride 3.0g

Preparation
1. Dissolve the methylene blue chloride in distilled water
2. Label the bottle – “0.3% methylene blue”, date and initial
4. Prior to use filter stock solution into the staining bottle
Label
0.3% Methylene blue
Date
Initial
Filter stock solution before use

Page 36 Ziehl-Neelsen reagent preparation BY SPUTUM MICROSCOPY
Quality Control Appendices 8
Preparing positive control slides
1. Obtain a 2+ or 3+ smear-positive specimen
2. Label slides as positive controls (include preparation date)
3. Prepare 50 smears and air dry
4. Heat fix and store in dark, dry place
5. Use within 12 months of preparation
Preparing negative control slides

Prepare smears from any smear-negative sputum (not saliva) specimen.
Quality Control procedure

Each week
Stain and examine one smear-positive and one smear-negative control slide on the first
staining run of each week:
• Record the result in the Laboratory Register
• Store the control slides in sequence with completed slides
When using new reagents

• Stain and examine one smear-positive and one smear-negative using new reagents
• Record the result in the Reagent Preparation Workbook
Unacceptable results
• Positive control has poor staining or no visible AFB
• Negative control has AFB or AFB-like objects present
• Background is not properly decolourised
• Stain deposit is present on the QC slides
Report all unacceptable results to the laboratory supervisor immediately

BY SPUTUM MICROSCOPY Ziehl-Neelsen reagent preparation Page 37
8 Appendices The microscope
The microscopy area should be:

• Free from dust
• On a sturdy level platform
• Away from centrifuges and refrigerators
• Away from water, sinks or chemicals to avoid splashes or spills
• Ergonomically correct work position (see page 33)
Binocular eye pieces Power On/Off
Diopter ring adjustment
Nose piece Voltage

regulator
Lenses
Stage
Condenser diaphragm Coarse

focus
Centering screws
Field diaphragm
Fine
focus
Stage X Stage Y
movement movement

Page 38 The microscope BY SPUTUM MICROSCOPY
The microscope Appendices 8
Setting up the microscope
For binocular microscopes with pre-centred and fixed condensers:
1. Set the variable voltage regulator to minimum
2. Turn the power on
3. Slowly adjust until the desired light intensity is reached
4. Place a stained slide onto the stage
5. Rotate the nose-piece to the 10X objective
6. Bring the smear into focus with the coarse and fine adjustment knobs
Always use the focusing adjustment knobs to lower the stage

away from the lens
7. Adjust the interpupillary distance until the right and left images merge
8. Focus the image with the right eye by looking into the right eye-piece and adjusting
with the fine focus knob
9. Focus the image with the left eye by looking into the left eye piece and turning the
dioptre ring
10. Open the condenser iris diaphragm so that the field is evenly lit
11. Place one drop of immersion oil onto the smear and rotate the 100X objective
into place

BY SPUTUM MICROSCOPY The microscope Page 39
12. Focus using the fine adjustment knob

13. Use the variable voltage regulator to achieve a comfortable illumination
14. Once the smear has been read, rotate the 100X objective away, locate the 10X objective
over the slide, and then remove the slide
15. When finished, reset the voltage regulator to a minimum, and turn the power off
16. At the end of each day, use lens paper, muslin cloth, or fine tissue paper to carefully
remove immersion oil from the 100X lens, cover the microscope, or put it in the
microscope box and return to the humidity controlled cupboard
Do’s and Don’ts

• The 100X objective is the only lens requiring immersion oil
• Keep immersion oil away from other lenses
• Immersion oil must have medium viscosity and a refractive index (RI) greater than
1.5. Any synthetic, non-drying oil with an RI > 1.5 is suitable (refer to
manufacturer’s instructions).
• Do not use cedar wood oil as it leaves a sticky residue on the lens
• Do not use liquid paraffin because it has a low refractive index resulting in
an inferior image
Immersion oil – a simple test
Good immersion oil Poor immersion oil
✗
Glass rod ‘disappears’ Glass rod still visible

RI > 1.5 below the surface RI < 1.5

Maintenance
Xylol produces irritant fumes
Cleaning lenses
Some cleaning agents will damage lenses over time
Cleaning Agent Long term use Infrequent use

Manufacturer’s recommendation
Ethyl ether/alcohol (80/20)
Alcohol ✗
Benzene/petrol ✗
Acetone/ketones ✗
Xylol ✗ ✗
• When ever possible use the cleaning fluid recommended by the manufacturer
• Use a minimum amount of cleaning fluid, never dip a lens into cleaning fluid
• Lens paper is best for cleaning optical surfaces as it does not scratch the lens
• Alternatives are muslin cloth, silk, or fine quality toilet paper
• Do not use ordinary paper or cotton wool to clean lenses
• Keep the microscope covered when not in use
• Keep the eye-pieces in place
• Fungus or dust may enter through holes where objectives in the nose-piece are missing
✗
Cover holes from missing objectives

• If the image appears hazy with black dots, check for dust or dirt on the lenses
(eye-pieces, objectives, condenser and illuminator lens). If:
– the black dot moves when the eye-piece is rotated, then the dust is on the eye-piece
– the black dot moves when the slide is moved, then it is on the slide
– these two are ruled out, then assume the dust is on the objective (if inside the
objective, it appears as dots; if on the outside, then as a hazy image)
• Dust can be removed using a camel-hair/artist brush or by blowing over the lens
with an air brush
A simple air brush made using a pasteur pipette and

rubber bulb
Light source
• Never touch the glass bulb surface as skin oils will burn, reducing light intensity
• Use lens paper to hold the bulb when inserting into the microscope
Use a tissue, do not touch the globe with your fingers
Mechanical parts
• Never disassemble the microscope
• Stiffness of movement may be due to an accumulation of dust in the sliding channel,
or in the rack and pinion
• Remove the dust with an air brush or artist brush, clean with a solvent such as petrol,
then polish and apply high-quality silicone grease to lubricate the moving parts
• Stiffness may be due to bending of some part. The up and down movement of the
mechanical stage will loosen over time. Both problems need to be assessed by
a service engineer

Fungal growth
Fungus growing inside the eyepiece tube
Fungus growing inside the microscope head piece
• Fungus grows on the lenses, the eye-piece tube and prisms causing the microscope
image to become hazy and unclear
• To check for fungus turn the microscope on:
– Rotate the 10x objective into the light path
– Take out both eyepieces, look down the eyepiece tubes for fungus
• To prevent fungal growth, the microscope should be kept in a warm cupboard.
– A cupboard with a tightly fitting door, heated by a light globe (maximum 40W),
located at the top of the cupboard near to the microscope head
– Always leave the cupboard light on, even when the microscope
is not in the cupboard
– Check the temperature inside the cupboard is at least 5°C warmer than room
temperature
– Microscopes must be kept in the cupboard even if the laboratory is air-conditioned

Warming box for microscope storage
• Where power is unreliable, store the microscope in its box (or tight fitting cover) with
silica gel, salt, or rice (~100 grams) placed in an open pot on the microscope stage.
– Replace the salt when it begins to look wet
– Replace the rice when it is no longer dry and crisp
– Silica gel changes colour from blue to pink when it is unable to absorb
any more moisture
– Dry gel by placing in a hot air oven or heating in a saucepan until the
blue colour reappears
Placement of desiccating agent
• If proper storage not available, keep the microscope in the shade and with
good air circulation.

Trouble-shooting Staining Appendices 8
Well stained slides
Problem Cause Remedy

Smear too pink Insufficient decolourisation Decolourise for longer
Acid/alcohol concentration For commercial reagents, check
less than 3% with NTP
For in-house reagents, recheck
stain preparation and QC results
Acid/alcohol reagent has expired Replace reagent
or been stored in direct sunlight Store stain bottle in the dark
Carbol fuchsin (CF) has dried Check smears are level over sink
on smear Add sufficient CF
Smear too thick Prepare new smear
When correctly stained this slide

looks like A above

BY SPUTUM MICROSCOPY Trouble-shooting Staining Page 45
8 Appendices Trouble-shooting Staining

Pale acid-fast bacilli CF concentration < 0.3% For commercial reagents,
check with NTP
For in-house reagents, recheck stain
preparation and QC results
CF insufficiently heated Heat CF to steaming
CF staining time less than 5 minutes Stain for a minimum of 5 minutes
Smear overheated during preparation Pass over flame 3 times, 1-2
or staining seconds each time
Stop heating when CF steams
CF reagent has expired or stored Replace reagent
in direct sunlight Store stain bottle in the dark
Over decolourised with 3% Decolourise for 3 minutes
HCl/ethanol maximum

Counterstain too dark Excessive staining time Do not exceed 30 seconds
Inadequate washing step after Extend washing step
counterstaining
Methylene blue concentration For commercial reagents, check
too strong with NTP
For in-house reagents, recheck stain
preparation and QC results
Smear too thick Prepare new smear

Deposit on slide Stains not filtered Filter stains prior to use
Soot deposit on underside of smear Clean with a moist tissue

Page 46 Trouble-shooting Staining BY SPUTUM MICROSCOPY
Trouble-shooting Microscopy Appendices 8
Light flickers or does Loose plug or connection Check wall sockets, transformer,
not turn on power supply
Loose light bulb Reinstall the bulb – Do not touch
bulb with fingers
Dirty bulb contacts Replace bulb
Erratic voltage supply Use a voltage stabiliser
Faulty on-off switch Replace the switch
Fuse blown or transformer blown Replace the fuse
Discoloured bulb/burnt out Replace the bulb – Do not touch
bulb with fingers

Uneven illumination Field of view partially blocked Rotate the nose-piece until it
clicks into position
Iris diaphragm is almost closed Recalibrate microscope
or condenser is not aligned
Dirty lenses Gently wipe the lenses with lens
paper/soft cloth. If the trouble
persists clean with lens paper
soaked in the recommended lens
cleaning fluid
Heavy fungal growth on lenses Clean the lens using lens cleaning
fluid as recommended by the
manufacturer

Excessive image contrast Iris diaphragm is almost closed Open diaphragm

BY SPUTUM MICROSCOPY Trouble-shooting Microscopy Page 47
8 Appendices Trouble-shooting Microscopy

Unclear image with glare Iris diaphragm too far open Close the iris diaphragm to make
the opening smaller

Specimen focused at Slide upside down Turn it over
10x but not at higher
magnification

Specimen goes out of focus Slide is not flat on the stage Clean the stage and underside
more than usual at high of slide
magnification

Mechanical stage cannot Lock set too low Adjust to proper height and
be raised lock

Mechanical stage is loose Poor tension adjustment on the Adjust tension with tension
or stiff mechanical stage adjustment device
Solidified lubricants Microscope requires service

Page 48 Trouble-shooting Microscopy BY SPUTUM MICROSCOPY
Good

Oil immersion objective does Is oil being used? Apply immersion oil
not give a clear image
Light source collector lens dirty Clean using lens paper and
cleaning fluid
Poor quality immersion oil Use quality immersion oil
(low refractive index) (see page 40)
Surface of the lens is dirty Clean lens with lens paper

If oil/fungus inside the
objective, replace lens
Water on slide Air dry slides
Bubbles in immersion oil Remove oil from slide and
carefully reapply oil
Oil inside lens Clean or replace lens

8 Appendices Trouble-shooting Microscopy
Good

Dust/dirt visible in the field Dust on the collector lens of the Clean all surfaces
of view light source
Dust on the top-most lens of the Clean all surfaces
condenser
Dust on the eye-piece Clean all surfaces

Cracked objective lens Lens has been dropped Replace lens
Lens forced into slide or stage Replace lens

Page 50 Trouble-shooting Microscopy BY SPUTUM MICROSCOPY
Regular or semi regular The glass slide is scratched Learn to recognise glass artefacts
crescent shapes that maybe
confused for AFBs

Headaches/incomplete Eye-pieces are not matched Use matched eye-pieces
binocular vision
Improper adjustment of interpupillary Adjust the interpupillary distance
distance
Dioptre adjustment was not done Adjust dioptre settings

Fuse blows frequently Fuse incorrectly rated Replace with correctly rated fuse
Unstable line voltage Use voltage protection device

8 Appendices Patient information
• Your doctor/nurse has sent you to the laboratory because they suspect that you
may have the symptoms of tuberculosis (TB).
• To diagnose TB three sputum specimens are needed and they will be collected:
1. At first presentation
2. Next morning before breakfast
3. When you return to the laboratory tomorrow
• Good quality specimens from the lungs are required not saliva or nasal secretions.
• Rinse your mouth out with water if you have recently eaten, or if you have dentures
Please cover your

(remove them first).
• To obtain a good quality sputum specimen:
mouth when coughing! 1. Inhale deeply 2-3 times, breathe out hard each time
2. Cough deeply from the chest
3. Place the open container close to your mouth to collect the specimen
4. Screw the lid on tightly when done
• You may be asked to repeat the collection to obtain a good quality specimen
It is important that you provide three, good quality specimens
✗
The specimens must be from the lungs, not saliva

or nasal secretions.
If you have recently

eaten, or have dentures
(remove them) then
rinse your mouth out
with water.

Page 52 Patient information BY SPUTUM MICROSCOPY
Institute of Medical and Veterinary Science
Quality Pathology supporting Training and Research

Laboratory Diagnosis

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Laboratory Diagnosis

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The handbook

Pacific Island Countries

• Dr Armand van Deun

LABORATORY DIAGNOSIS OF TUBERCULOSIS

All rights reserved. No part of this book may be reproduced or

© 2005 Institute of Medical and Veterinary Science

LABORATORY DIAGNOSIS OF TUBERCULOSIS

LABORATORY DIAGNOSIS OF TUBERCULOSIS

Tuberculosis is a treatable disease that

In 2004, in collaboration with Indonesian colleagues, The Handbook

LABORATORY DIAGNOSIS OF TUBERCULOSIS

The purpose of sputum microscopy is to:

LABORATORY DIAGNOSIS OF TUBERCULOSIS

Correct – the preferred way to do something

You should wear gloves for this procedure

You should wear a gown for this procedure

Wash your hands

This substance is toxic

This substance is corrosive

LABORATORY DIAGNOSIS OF TUBERCULOSIS

Collect specimens outside so that infectious droplets are diluted in an open,

To reduce the possibility of laboratory staff becoming infected:

Tell the patient to cover their mouth when coughing.

Collect sputum outside the laboratory, preferably outside the building

✗ Do not collect sputum specimens in:

LABORATORY DIAGNOSIS OF TUBERCULOSIS

Treatment unit Date

Contact person Phone

Home address Follow up

Examination for Diagnosis Follow-up

RESULTS (Laboratory to complete)

Laboratory Register Number

Examined by (signature) Date • Clearly label the

If dentures are present,

LABORATORY DIAGNOSIS OF TUBERCULOSIS

How to collect a specimen

LABORATORY DIAGNOSIS OF TUBERCULOSIS

Reason for Microscopy

Treatment unit Date

Contact person Phone

Date of birth Age Sex M F

Examination for Diagnosis Follow-up

Patient Identification Number

RESULTS (Laboratory to complete)

Laboratory Register Number

Appearance Results (check one)

Examined by (signature) Date

Specimen Request Form

1. Check patient details on container match the Specimen Request Form

LABORATORY DIAGNOSIS OF TUBERCULOSIS

In remote settings, sputum specimens need to be transported to a microscopy laboratory.

What you need

• Permanent marker to write details on the side of the container

Specimen containers must be strong enough to withstand transportation effects

Approved secondary packaging (transport box) must:

Always label the container never the lid (see page 9)

• Are Request Forms completed correctly?

LABORATORY DIAGNOSIS OF TUBERCULOSIS

Contact person Phone

Examination for Diagnosis Follow-up

1. Check each specimen container is labelled with

LABORATORY DIAGNOSIS OF TUBERCULOSIS

Sputum smears must be prepared promptly after collection.