Lab Diagnosis of TB by Sputum Microscopy

Laboratory Diagnosisof Tuberculosis by SputumMicroscopy

The

hand

book

Paci

fic Is

land

Cou

ntrie

s

Richard LumbIvan Bastian

AcknowledgmentsThis edition of The Handbook, for Pacific Island Countries, was produced with financial assistance from the South Australian TB Services within the Royal AdelaideHospital 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 DeunBacteriology ConsultantUnionAntwerp, Belgium

• Mr David DawsonTB Laboratory ConsultantBrisbane, Australia

• Mr John ElliotPacific Paramedical Training CentreWellington, New Zealand

• Ms Linda KuoDepartment of Health ServicesRichmond, California, USA

• Ms Kay WithnallLaboratory ConsultantDarwin, Australia

• Prof Barrie Vernon-RobertsIMVS

Laboratory Diagnosis of Tuberculosis by SputumMicroscopy

The

hand

book

Page 1LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

Published by:Institute of Medical and Veterinary ScienceFrome Road Adelaide South Australia 5000www.imvs.sa.gov.au

All rights reserved. No part of this book may be reproduced ortransmitted in any form or by any means without the written permissionof the publisher. The authors assert their moral rights in the work.

ISBN 0-9750285-2-9

ProductionProject EditorMark Fitz-Gerald

Technical EditorsRichard Lumb, Ivan Bastian

Manuscript EditorsMark Fitz-Gerald, Richard Lumb

IllustrationsKerry Reid

DesignSue Dyer Design

© 2005 Institute of Medical and Veterinary Science

Laboratory Diagnosis of Tuberculosis by SputumMicroscopy

The

hand

book

Page 2 LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

7 SummaryFalse-negatives – Consequences– PreventionFalse-positives – Consequences– Prevention

8 AppendicesSpecimen containers DocumentationLaboratory layoutSafetyZiehl-Neelsen reagent preparationQuality Control The microscopeTrouble shooting – Staining– MicroscopyPatient information

Contents

Foreword

Introduction

1 Symbols and warnings

2 Sputum collectionSpot-morning-spotHospital patientsSafe collectionPre-collection patient adviceHow to collect a specimenSpecimen qualityRejection criteriaRegistrationStorage and transport

3 Smear preparationWhat you needMaking a smear

4 StainingWhat you need The Ziehl-Neelsen method

5 ExaminationReading smearsAppearance of acid fast bacilli

6 ReportingHow to report

4

6

7

88889

1010101112

141415

181820

222224

2525

2626

26

2727283031343738

454752

Page Page


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 ObservedTreatment, Short course (DOTS) strategy for tuberculosis (TB) controlwhich 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 patientresponse to treatment. An effective TB control programme thereforedepends upon laboratories providing accurate, reliable and timelydetection of infectious cases.

In 2004, in collaboration with Indonesian colleagues, The Handbookwas developed and translated into Bahasa. 15,000 copies were printedand distributed throughout the country. It has subsequently receivedwidespread 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 forTB control and the provision of laboratory services. The Handbookis intended to assist the NTPs of Pacific Island Countries to achieveexcellence in sputum smear microscopy.

MR RICHARD LUMB

DR IVAN BASTIAN


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 DOTSProgrammes – Guidelines for Pacific Island Countries’ (WHO).

Sputum microscopySputum 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 InfectionRisk 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 safetyWhen performed correctly sputum examination will not place laboratory technicians at increased risk of developing TB.

Introduction

Page 6 Introduction LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

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 handsAlways wash your hands after preparing sputum smears and before leaving the laboratory

This substance is toxic

This substance is corrosive

Symbols and warnings 1

�

✗

8

Symbols and warnings Page 7LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

2 Sputum collection

Spot-morning-spotThree sputum specimens are recommended for the laboratory diagnosis of TB. For theconvenience 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 patientsIf the patient is in hospital, collect a sputum specimen each morning on three consecutive days.

Safe collectionTransmission 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 collectionLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

Sputum collection 2Pre-collection patient advice

If dentures are present,remove them and rinsemouth with water.

• Check the Specimen Request Form• Fill in any missing details • Tick Diagnosis or Follow-up

Tell the patient the bestspecimen comes from the lungs.Saliva or nasal secretionsare unsuitable.

• Clearly label the sputum container with the patient’s name and date of collection

• Label the container, never the lid

��

�

✗

✗

NATIONAL TUBERCULOSIS CONTROL PROGRAMME

REQUEST FOR SPUTUM EXAMINATION

Treatment unit Date

Contact person Phone

Patient name

Date of birth Age Sex � M � F

Other names

Home address

Island

Examination for � Diagnosis � Follow-up

Patient Identification Number

RESULTS (Laboratory to complete)

Laboratory Register Number

Collection Date Specimen

Examined by (signature) Date

SEND COMPLETED FORM AND RESULTS

TO THE TREATMENT UNIT PROMPTLY

Appearance Results (check one)

BloodStained

MucoPurulent Saliva neg 1-9 + ++ +++

1

2

3

DiagnosisorFollow up

�

Sputum collection Page 9LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

How to collect a specimen

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 time2. Cough deeply from the chest3. Place the open container close to the mouth to collect the sputum4. 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 specimenMucoid

Good quality specimenPurulent

Blood stained Poor quality specimens are thin and watery or composed largely of bubbles

� � � ✗

2 Sputum collection

✗

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


Patient Informationpage 52



Treatment unit Date


Patient name


Other names

Home address

Island










BloodStained


1

2

3

Name in full

Laboratory Register

LabRegister

No.Date Age

SexM/F

Complete address(for new patients)

Name ofreferring Health

Centre

Reason forexamination*

MicroscopyResults

Signature Remarks

Diagnosis Follow-up 1 2 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

RegistrationRegister the specimen before processing

Sputum collection 2

1. Check patient details on container match the Specimen Request Form2. 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 container4. Write the LRN on the Specimen Request Form5. 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

4

Specimen Request Form

1

3

757/1

5


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

StorageTo 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)• 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 checklistIs 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

2 Sputum collection Specimen storage and transportSp

ecim

en s

tora

ge a

nd tr

ansp

ort


5


Sputum collection 2Specimen storage and transport



Treatment unit Date


Patient name


Other names

Home address

Island





Treatment unit Date


Patient name


Other names

Home address

Island



2

3

6

4

��

1



Treatment unit Date


Patient name


Other names

Home address

Island



8

7

Keep upright

Absorbent paper

Packing specimensPut several layers of absorbent paper in the bottom of the shipper.

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 bag3. Cross check specimens against Request Form4. Put sealed bio-hazard bags into the shipper5. Put Request Forms and Master list into a separate sealed bag6. Pack shipper to prevent movement7. Add sealed bag containing Request Forms last8. Seal and address the shipper

Keep coolStore uprightDeliver urgently

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

3 Smear preparation What you need

Never reuse sputum smear slides

Applicator stickBamboo/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 loopsSome technicians prefer wire loops because they can be reused however they:• Are more time consuming• Collect a smaller sample volume • Are less efficient

New, clean glass slides

Discard bucket containing disinfectant (e.g. 5% Phenol)

Bamboo/wooden applicator sticks or wire loop

Alcohol/sand trap forcleaning loops

Spirit lamp

Slide rack for drying smears

Page 14 Smear preparationLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

Handle slides by edges only

Sputum specimen with purulent portions within saliva

Making a smear

Never put more than one sputum specimen on each slide

Write the LRN and the 1, 2, or 3 identifier on the frosted end of each slide using a lead pencil

For clear slides use a diamond pen

Do not mix purulent/bloodstained portions with saliva/mucous

Select only purulent orbloodstained portions ofsputum

Ziehl-Neelsen stained smear – purulent

1

2

Ziehl-Neelsen stained smear – saliva

Smear preparation 3

B

A

A BMore acid fast bacilli

(AFB) will be found in the purulent portions

of a specimen.

Smear preparation Page 15LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

Add Laboratory CodeNumber (LCN) ifapplicable

LRN Spot

Smear the specimen in the centre of the slide, covering2cm by 1cm

�

✗

Discard the applicatorstick into discard bucket after use, do notflame, do not reuse

To clean a wire loop• Insert loop in sand trap and rotate• Flame the loop to red-hot and allow to cool

Always clean and sterilise a wire loop between each specimen

Retain all specimens untilresults are reported

3 4

65

1cm

2cm

Making a smear3 Smear preparation

Wash your hands after smear preparation

Page 16 Smear preparationLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

Dry smears on a slide rack, out of direct sunlight

When dry, heat fix thesmears:• ensure the smear is

facing upwards • pass 3 times through

the flame of a spirit lamp.

Overheating willdamage the bacilliCorrectly prepared smear

�

Stained smears resulting from poor smear preparation

Too thick

✗

Too thin

✗

Not centred and too small

✗

Multiples

✗

87

9

Making a smear Smear preparation 3

Smear preparation Page 17LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

To stain smears using the Ziehl-Neelsen method you will need:

Slide rack to support slides over sink or bucket

Forceps

Slide rack for drying stained slides

Spirit burner

Water

Timer

What you needStaining 4

Page 18 StainingLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

Staining

Carbol fuchsin 0.3 %

3% HCI in ethanol or 25% H2S04

You will require 2 – 3 volumes of decolouriser for eachvolume of stain.

Methylene blue 0.3 %

What you need 4

Staining Page 19LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

Staining 4

Cover each slide completely with carbol fuchsin

21

• Heat each slide from below until steam rises, always keep the flame moving

• Stop heating when steam rises

Do not boil

3

Do not splash adjacent slides

6

• Gently rinse each slide with a stream of water• Tilt each slide to drain off excess water

5

4

Place the slides smear upwards, in LRN order, on a staining rack over the sink or basin, about a finger-width apart

Ensure the slides are level

Leave the heated stain on the slides – minimum 5 minutes

A longer time is OK provided the stain does not dry on the slide

✗

The Ziehl-Neelsen method

Page 20 StainingLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

Staining 47

Add decolourising solution to the slide and leave for 3 minutes

9

Cover each slide with methylene blue for 30 seconds only

11

• Air dry away from direct sunlight• Do not dry slides using blotting paper

10

• Gently rinse each slide with water• Do not splash other slides • Tilt each slide to drain off excess water

12Do not examine slides until they have dried

A correctly stained smear

8

• Gently rinse each slide with water• Do not splash other slides • Tilt each slide to drain off excess water

The Ziehl-Neelsen method

Staining Page 21LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

AFB’s are stained red. Some males are red/green colour-blind, hence all male techniciansshould be tested for colour blindness.

Smears must be consistently and systematically examined to ensure a representative area of the smear is reported.

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

Inflammatory cells (high power) – look for areas like this

2

• 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 organisms

Never allow the oil applicator to touch the slide

3

Carefully rotate the 100X oil objective lens over the slide

Reading smears

Avoid areas containing epithelial cells (low power)

Examination5

✗

�

Page 22 ExaminationLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

1

5Examination

4

Carefully adjust the fine focus until cells are sharp

Never allow the lens to touch the glass slide

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

6

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

Store the slides in LRN order as they will be needed for External Quality Assessment (EQA)

Do not write the result on the slide

There is no need to treat slides with xylol

Wipe the lens gently with tissue paper to remove immersion oil after you have finished examining a batch of slides

Reading smears

5

7

8

✗✗

�

Examination Page 23LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

• 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

Where possible all new positive smears should be reviewed by another technician

single bacilli V-shaped forms

clumps of bacilli

Examination5 Appearance of acid fast bacilli (AFB)

Page 24 ExaminationLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY



Treatment unit Date


Patient name


Other names

Home address

Island










BloodStained


1

2

3

What you see What to report

No AFB in 100 fields Negative for AFB

1 – 9 AFB in 100 fields Report what you see

10 – 99 AFB in 100 fields 1+

1 – 10 AFB per field, check 50 fields 2+

More than 10 AFB per field, check 20 fields 3+

Reporting 6How to report

1. Check the smear LRN matches the Specimen Request Form2. Write the results on the patient’s Specimen Request Form3. Date and sign the Specimen Request Form4. Record the results in the Laboratory Register immediately after reading

the smear. Use red pen for Positive results5. Return the completed Specimen Request Form to the doctor or clinic

Do not give results to the patients as lost reports may delay treatmentDo not write the results on the slide as they are needed for QC checking

Name in full

Laboratory Register

LabRegister

No.Date Age

SexM/F



Centre


MicroscopyResults

Signature Remarks



Date Sign

757/1

757/1

1

3

4

5

Record Results

Return to doctor or clinic

2

Laboratory Register

Specimen Request Form

Reporting Page 25LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

The number of AFB found indicates how

infectious the patient is,hence it is important

to record exactly what you see.

Be consistent and accurate in all your work, lives depend on you

False-negatives – consequences• Patients with TB may not be treated resulting in on-going disease, disease

transmission, or death• 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• Patients are treated unnecessarily• Treatment may be continued longer than necessary• 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

7 Summary

False-negative meansreported negative but truly

smear-positive

False-positive meansreported positive but truly

smear-negative

Page 26 SummaryLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

Ideal specimen container

Specimen containers Appendices 8

Unacceptable containers

✗ ✗ ✗ ✗

�Clear break-resistant plastic

�Wide-mouth �Multi-thread screw cap

�Single-use

�Clean

�Can be written on with a permanent marker pen

OpaqueToo small

Extra insert lidScrew cap inadequate

Too smallScrew cap inadequate

ColouredScrew cap inadequate

Specimen containers Page 27LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

Specimen Request FormThis example shows the type of information required on a Specimen Request Form.

8 Appendices Documentation



Treatment unit Date


Patient name


Other names

Home address

Island










BloodStained


1

2

3

Page 28 Specimen Request FormLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

Documentation Appendices 8Laboratory RegisterThis example shows the type of information required on a Laboratory Register

Name in full

Laboratory Register

LabRegister

No.Date Age

SexM/F



Centre


MicroscopyResults

Signature Remarks



The Laboratory Register Numbering SystemThe 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

Laboratory Register Page 29LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

The basic requirements for a sputum microscopy laboratory include:

8

• 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

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

Microscopy bench Records bench

Bench for incomingspecimens

Sliding window toreceive specimens

Win

dow

open

s to

exte

rior

Storagecupboards

Window

Smearpreparation

area

Sink

Handwashingbasin

Waste bin

Gown rack

Page 30 Laboratory layoutLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

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

GownsThe 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 mustwash 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 spills1. Put on a gown and gloves2. Place a paper towel or cloth over the spill area and liberally apply phenol solution3. Leave covered – minimum 15 minutes4. Clean up the contaminated material and put into the waste container5. Clean with a final wash using 80% v/v alcohol6. Wash your hands after the clean up is complete

Safety Appendices 8

Safety Page 31LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

Waste disposal

Locate the burning drum away from people in an open area as the fumes are toxic

8 Appendices Safety

1

Add phenol to a lined bucket

Burn bucket contents weekly

3

2

Tighten caps, add specimencontainers and contents of the discard bucket to the phenol bucket

4

When cool bury burning drum contents at least1.5 metres deep

Page 32 SafetyLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

8

Poor posture – feet unsupportedGood posture – supporting your feetstraightens your back

Poor posture – microscope too lowand feet not flat

Good posture – raise the microscopeto help straighten your back and keepyour feet flat on the floor

✗

✗

�

�

Safety Appendices

Ergonomics

Safety Page 33LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

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

8 Appendices Ziehl-Neelsen reagent preparation

Ready to use reagents must also be QC tested, and the results recorded in the 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

Page 34 Ziehl-Neelsen reagent preparationLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

Wash your hands after preparing reagents

Carbol fuchsin – 0.3%

Phenol crystals and vapour are corrosive, toxic and may cause burns 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

Preparation1. Add 100ml of alcohol (or methanol) to a one litre glass flask 2. Add 50g of phenol crystals and dissolve3. Add 3.0g of basic fuchsin powder4. Mix well with gentle heating until dissolved5. Add distilled water to make one litre6. Label the bottle – “0.3% carbol fuchsin”, date and initial7. 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

Ziehl-Neelsen reagent preparation Appendices 8

Label0.3% carbol fuchsinDateInitial

Filter stock solution before use

Ziehl-Neelsen reagent preparation Page 35LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

8

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


Preparation1. Carefully add the HCl to the ethanol or the H2SO4 to the water2. Label the bottle “3% HCl in ethanol” or “25% H2SO4”, date and initial3. Store in a dark bottle in a cupboard at room temperature (expiry 12 months)

Counterstain

Methylene blue chloride 3.0g


Preparation1. Dissolve the methylene blue chloride in distilled water2. Label the bottle – “0.3% methylene blue”, date and initial3. Store in a dark bottle in a cupboard at room temperature (expiry 12 months)4. Prior to use filter stock solution into the staining bottle

8 Appendices Ziehl-Neelsen reagent preparation

Filter stock solution before use

Label0.3% Methylene blueDateInitial

Page 36 Ziehl-Neelsen reagent preparationLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

Preparing positive control slides1. Obtain a 2+ or 3+ smear-positive specimen2. Label slides as positive controls (include preparation date)3. Prepare 50 smears and air dry4. Heat fix and store in dark, dry place5. Use within 12 months of preparation

Preparing negative control slidesPrepare smears from any smear-negative sputum (not saliva) specimen.

Quality Control procedureEach weekStain and examine one smear-positive and one smear-negative control slide on the firststaining 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

Quality Control Appendices 8

Ziehl-Neelsen reagent preparation Page 37LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

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)

8 Appendices The microscope

Binocular eye pieces

Diopter ring adjustment

Nose piece Voltageregulator

Coarsefocus

Finefocus

Lenses

Stage

Condenser diaphragm

Field diaphragm

Centering screws

Power On/Off

Stage Ymovement

Stage Xmovement

Page 38 The microscopeLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

Setting up the microscopeFor binocular microscopes with pre-centred and fixed condensers:1. Set the variable voltage regulator to minimum2. Turn the power on3. Slowly adjust until the desired light intensity is reached4. Place a stained slide onto the stage5. Rotate the nose-piece to the 10X objective6. 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

The microscope Appendices 8

11. Place one drop of immersion oil onto the smear and rotate the 100X objective into place

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

The microscope Page 39LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

12. Focus using the fine adjustment knob13. Use the variable voltage regulator to achieve a comfortable illumination14. Once the smear has been read, rotate the 100X objective away, locate the 10X objective

over the slide, and then remove the slide15. When finished, reset the voltage regulator to a minimum, and turn the power off16. 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


Glass rod ‘disappears’RI > 1.5

Glass rod still visiblebelow the surface RI < 1.5

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Good immersion oil Poor immersion oil


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

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• 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 andrubber 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

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

Use a tissue, do not touch the globe with your fingers


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



• 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

Warming box for microscope storage

Placement of desiccating agent

• If proper storage not available, keep the microscope in the shade and with good air circulation.


Trouble-shooting Staining Appendices 8Well stained slides

Problem Cause RemedySmear 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 reagentor been stored in direct sunlight Store stain bottle in the dark

Carbol fuchsin (CF) has dried Check smears are level over sinkon smear Add sufficient CF

Smear too thick Prepare new smear

When correctly stained this slide looks like A above

A

Trouble-shooting Staining Page 45LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

8 Appendices Trouble-shooting Staining

Problem Cause RemedyPale acid-fast bacilli CF concentration <0.3% For commercial reagents,

check with NTP For in-house reagents, recheck stainpreparation 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, 1-2or staining seconds each time

Stop heating when CF steams

CF reagent has expired or stored Replace reagentin direct sunlight Store stain bottle in the dark

Over decolourised with 3% Decolourise for 3 minutes HCl/ethanol maximum

Problem Cause RemedyCounterstain too dark Excessive staining time Do not exceed 30 seconds

Inadequate washing step after Extend washing stepcounterstaining

Methylene blue concentration For commercial reagents, check too strong with NTP

For in-house reagents, recheck stainpreparation and QC results

Smear too thick Prepare new smear

Problem Cause RemedyDeposit 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 StainingLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

Problem Cause RemedyLight 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 bulbErratic voltage supply Use a voltage stabiliserFaulty on-off switch Replace the switchFuse blown or transformer blown Replace the fuseDiscoloured bulb/burnt out Replace the bulb – Do not touch

bulb with fingers

Problem Cause RemedyUneven illumination Field of view partially blocked Rotate the nose-piece until it

clicks into positionIris diaphragm is almost closed Recalibrate microscopeor condenser is not alignedDirty 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 cleaningfluid as recommended by the manufacturer

Trouble-shooting Microscopy Appendices 8

Problem Cause RemedyExcessive image contrast Iris diaphragm is almost closed Open diaphragm

Trouble-shooting Microscopy Page 47LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

Problem Cause RemedyUnclear image with glare Iris diaphragm too far open Close the iris diaphragm to make

the opening smaller

Problem Cause RemedySpecimen focused at Slide upside down Turn it over10x but not at higher magnification

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

Problem Cause RemedyMechanical stage is loose Poor tension adjustment on the Adjust tension with tension or stiff mechanical stage adjustment device

Solidified lubricants Microscope requires service

Problem Cause RemedyMechanical stage cannot Lock set too low Adjust to proper height and be raised lock

8 Appendices Trouble-shooting Microscopy

Page 48 Trouble-shooting MicroscopyLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

Problem Cause RemedyOil immersion objective does Is oil being used? Apply immersion oilnot 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 paperIf oil/fungus inside the objective, replace lens

Water on slide Air dry slidesBubbles in immersion oil Remove oil from slide and

carefully reapply oilOil inside lens Clean or replace lens



Good�

Problem Cause RemedyCracked objective lens Lens has been dropped Replace lens

Lens forced into slide or stage Replace lens

8 Appendices Trouble-shooting Microscopy

Problem Cause RemedyDust/dirt visible in the field Dust on the collector lens of the Clean all surfacesof view light source

Dust on the top-most lens of the Clean all surfaces condenserDust on the eye-piece Clean all surfaces

Page 50 Trouble-shooting MicroscopyLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

Good�

Problem Cause RemedyHeadaches/incomplete Eye-pieces are not matched Use matched eye-piecesbinocular vision

Improper adjustment of interpupillary Adjust the interpupillary distancedistanceDioptre adjustment was not done Adjust dioptre settings

Problem Cause RemedyFuse blows frequently Fuse incorrectly rated Replace with correctly rated fuse

Unstable line voltage Use voltage protection device

Problem Cause RemedyRegular or semi regular The glass slide is scratched Learn to recognise glass artefactscrescent shapes that maybeconfused for AFBs



8 Appendices Patient information

Page 52 Patient informationLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY

• 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 presentation2. Next morning before breakfast3. 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 (remove them first).

• To obtain a good quality sputum specimen:1. Inhale deeply 2-3 times, breathe out hard each time2. Cough deeply from the chest3. Place the open container close to your mouth to collect the specimen4. 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

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The specimens must be from the lungs, not saliva or nasal secretions.

If you have recentlyeaten, or have dentures(remove them) thenrinse your mouth outwith water.

Please cover your mouth when coughing!

Institute of Medical and Veterinary ScienceQuality Pathology supporting Training and Research

Lab Diagnosis of TB by Sputum Microscopy

Documents

lter stock

filter stock

black dot

pacic island

variable voltage

good quality

acid fast

specimen request