Basic Malaria Microscopy - Part 1 Learner's Guide, 2nd Edition, WHO, 2010

7/30/2019 Basic Malaria Microscopy - Part 1 Learner's Guide, 2nd Edition, WHO, 2010

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MALARIAMICROSCOPY

Basic

Part I. Learners guide

Second edition


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MALARIAMICROSCOPY

Basic

Part I. Learners guide

Second edition


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World Health Organization 2010

All rights reserved. Publications of the World Health Organization can be obtained from WHO Press, World Health Organization, 20 AvenueAppia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: [email protected]). Requests for permis-sion to reproduce or translate WHO publications whether for sale or for noncommercial distribution should be addressed to WHO Press,at the above address (fax: +41 22 791 4806; e-mail: [email protected]).

The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoeveron the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or con-cerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may notyet be full agreement.

The mention of specific companies or of certain manufacturers products does not imply that they are endorsed or recommended by theWorld Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names ofproprietary products are distinguished by initial capital letters.

All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication.However, the published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for theinterpretation and use of the material lies with the reader. In no event shall the World Health Organization be liable for damages arisingfrom its use.

Design and layout by WHO GraphicsPrinted in Switzerland

WHO Library Cataloguing-in-Publication Data

Basic malaria microscopy 2nd edition.

Contents: - Part 1: Learners guide - Part 2: Tutors guide.

1.Malaria - laboratory manuals. 2.Malaria - diagnosis. 3.Microscopy - laboratory manuals. 4.Teaching materials.

ISBN 978 92 4 154782 6 (Part 1) (NLM Classification: W 25)


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Preface 1Introduction 3

Learning unit 1

Malaria, the disease 7

Learning unit 2

Cleaning and storing microscope slides 13

Learning unit 3

Keeping records 19

Learning unit 4

Preparing blood films 21

Learning unit 5

Staining with Giemsa stain 29

Learning unit 6

The microscope 37

Learning unit 7

Examining blood films 45

Learning unit 8

Examining blood films for malaria parasites 51

Learning unit 9

Routine slide examination 69

Learning unit 10

Supervision in malaria microscopy 77

Contents


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Preface to the second edition

An norma WHO consuaon on quay assurance or maara mcroscopy,ed n Kuaa Lumpur, Maaysa, n 2004 recommended a e 1991 edon oWHOs Basc maara mcroscopy1 be revsed. hs second edon s e resu oa recommendaon.

Few rea canges n e mcroscopy o maara parases ave occurred snce 1991,bu muc as canged n e way maara s dagnosed and reaed. here s beerundersandng n remoe communes a maara s a medca emergency andrequres rapd dagnoss and reamen. As par o efors n many counres o

expand access o reamen, mcroscopy servces are beng renewed and upgraded.Parasoogca conirmaon o a dagnoss o maara w srengen e surve-ance o maara and mprove conro o e dsease.

Mcroscopss are va o maara programmes, and er dagnosc and ecncasks are reed on n bo curave servces and dsease surveance. hus, ran-ng n maara mcroscopy mus be sound and mus reac odays g sandards.Wen mcroscopss are raned and abe o make quay-assured dagnoses omaara, communes a rsk ave greaer conidence n er servces, and bopaens and prescrbers benei.

he ranng package presened ere as been adjused o mee e canged cond-ons. he ranng manua s dvded n wo pars: a earners gude (Par I) and auors gude (Par II). he package ncudes a CD-ROM, prepared by e UnedSaes Ceners or Dsease Conro and Prevenon, wc conans mcropoo-graps o e dferen maara parase speces and ecnca normaon n Pow-erPon orma, wc can be sown durng ranng sessons and reerred o by eparcpans. Empass s paced on eacng and earnng, ncudng monorngand evauang ndvduas and e group durng ranng.

he Basc Maara Mcroscopy programme connues o use e compeence-basedconcep o acevng se arges o compeence. Aemps ave been made o nd-

cae e approprae sandards a w quay a parcpan or graduaon andor progress beween earnng uns. he eves o compeence o be aaned a eend o s ranng course are e mnmum eves deined n e WHOMaaramcroscopy quaty assurance manua2. For exampe, Reacng 80% accuracy ndagnosng maara parases (assessed agans a sandard se o mcroscopy sdes)s consdered acevabe by every parcpan. I s recognzed, owever, a someprogrammes may no ye be abe o reac suc sandards and nay mus seer own. he course organzers soud ndcae e sandards ey expec ran-

1 WHO. Basic malaria microscopy: Part I Learners guide; Part II Tutors guide. Geneva, World Health Organization, 19912 WHO. Malaria microscopy quality assurance manual. Manila, Western Pacific Regional Office, 2009.


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BasicMALARIA MICROSCOPY

ees o reac. As e ranees, once ey ave graduaed, w be makng decsonsa deermne e managemen o a poenay aa dsease, a g sandard ocompeence mus be ensured.

hs second edon o e Basc Maara Mcroscopy package s a sand-aoneproduc, provdng a a s needed o conduc a compee ranng course. I sconans e beauu and accurae waer-coour usraons prepared or e irsedon o e manua by e ae Yap Loy Fong. Experence as sown a coourdrawngs are bes n ranng new recrus o recognze parase sages and spe-ces, because snge pane pcures ep sudens o exrapoae rom wa ey seeunder e mcroscope, ocussed a a number o oca panes, o a compee vewo e parase. Laer, ey can move rom drawngs and use mcropoograps,wc w ave an addona, posve mpac. hus, e ranng course s urersrengened copes o e o e Benc ads or maara mcroscopy1 are asomade avaabe o ranees.

he ex or s edon was exensvey revsed by Jon Sorey, on e bass o re-vews by Proessor Amed A. Abde-Hameed Adee, Dr Hoda Aa, Dr A. Bejaev,Dr Davd Be, Dr Andrea Bosman, Ms Leg Dn, Dr Jon Frean, Dr M.A. Ka-a, Dr D. Karkowsk, Dr Ken Ley, Dr Ear Long, Dr Majed A Zedja and Dr R.Veayudan. In addon, Donao Esparar, Ronad Espna, Serwn Ga, ZenadaGrad, Fesa Gubaa, Jon Fe Poro and Arene Lea Sanago esed and made

vauabe commens on e new keys o ck and n ims n e Learners gude.

hs projec was coordnaed or e WHO Goba Maara Programme by eWHO Wesern Pacic Regona Oce and receved inanca suppor rom Au-sAd and e Russan Federaon, or wc graeu acknowedgemen s made.

1 WHO. Bench aids for malaria microscopy. Geneva, World Health Organization, 2009.


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Introduction

The Learners guide

hs andbook (Par I o e Basc Maara Mcroscopy ranng modues) w as-ss parcpans durng ranng n e mcroscopc dagnoss o uman maara.Desgned as e oundaon or orma ranng o 45 weeks duraon, e gude sdesned or parcpans w ony eemenary knowedge o scence.

On compeng e ranng, ese personne w be responsbe or dagnosngmaara n bood ims rom suspeced cases n er communes, and mporan

reamen decsons w be based on er compeence n ensurng unsupervseddagnoss o maara. In order o gan e conidence o e pubc and e easysem, e quay o ranng o ese personne mus be o e ges possbesandard and demonsraed o be so.

he course as a compeence-based srucure, n wc e essena ecnca n-ormaon or e acquson o sks and ow o nsrucons are gven n an eas-y undersood orma. he ranng s mosy ands-on. By e end o e course,e ranees mus be demonsraed o ave acqured a g eve o compeence.Compeence-based ranng s a poweru, we-esed way o acqurng sks es-sena or pubc ea servces and ea care.

In addon o ranng ea workers n basc maara mcroscopy, e moduescan be used or rereser ranng or esabsed saf conducng sandard Gem-sa-based maara mcroscopy. As ese personne w aready ave a irm back-ground and work experence, ey soud be abe o reac e course objecveswn 1112 workng days. Dsrc and provnca ospa aboraory ecncansamar w e aboraory procedures coud benei rom a sorened course; a-oug maara mcroscopy s par o er day roune, rereser ranng coursesare beneica o ensure accuracy.

he gude s dvded no earnng uns. he noes and nsrucons n eac unare sucen o mnmze exensve noe-akng by e parcpans so a eycan parcpae uy n presenaons and dscussons. A page or noes s provdeda e end o eac un.

Sandard operang procedures are ouned and oowed wen approprae, soa, once ranng s compeed, e gude w connue o be a reerence. hs sparcuary useu or peope workng n soaed areas, were g sandards muss be ensured.

Beore movng rom one earnng un o e nex, ranees mus reac a desg-naed eve o compeence n eac denied sk. Faure o do so ndcaes a apersons sks are nsucen, and e ranng mus be repeaed un masery s

demonsraed.


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Note: Levels of accuracy are based on minimum grades of competence, as defined in the WHO

manual for malaria microscopy quality assurance.1 The levels are usually set at 8095%. For ex-

ample, a microscopist working at peripheral level should be able to detect the presence of a parasite

accurately in 90% of slides (after review of a standard set of slides for accreditation) and to identify the

species of plasmodium accurately in 80% of slides. These figures may appear too high to some andtoo low to others; the levels will be decided by the course organizers. When a patients life is at risk,

the highest level of accuracy must be achieved. With this kind of training and the amount of practice

time provided, participants should be able to reach the level of accuracy selected for the course. This

approach keeps errors in microscopy to the lowest possible level and helps reduce severe malaria

morbidity and mortality in communities.

Competence-based training is well described

by the last line of the Chinese proverb below.

Facilitators and trainees follow this

strategy throughout the course.

Hear and forget.

See and remember.

Do and understand.

Course objectives

Overall objectiveshe overa objecves descrbe broady wa e earner w be abe o do by e

end o ranng. Parcpans w be abe o:

organze and run a sma maara mcroscopy aboraory; andO

accuraey dagnose, w Gemsa mcroscopy and nernaonay recognzedOsandard operang procedures, maara necons n paens.

Specific objectiveshe specic objecves cover e knowedge, sks and audes a parcpansw acqure and er aby o use em. hey aso usrae e sep-wse ap-proac used or acevng eac objecve. Parcpans soud be aware o wa sexpeced o em rom e very begnnng o ranng.

Ater compeng ranng, ranees w ave successuy acqured e sks andcompeence o:

descrbe e mporance o maara as a poenay e-reaenng dsease, nOwc eary, accurae dagnoss and reamen are essena or paen recoveryand survva;

descrbe our common cnca sgns and sympoms o maara n paens;O

record on e correc aboraory or survey orms reevan paen deas orOsubsequen normaon and paen oow-up;

demonsrae er aby o prepare sdes or bood imng correcy;O

1 WHO. Malaria microscopy quality assurance manual. Manila, Western Pacific Regional Office, 2009.


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Introduction

adequaey prepare a se number o ck and n bood ims;O

demonsrae e correc pracce and precauons or prevenng ransmsson oOboodborne paogens wen andng bood;

demonsrae e correc Gemsa sanng procedures or sanng ck and nO

bood ims or maara mcroscopy;

demonsrae and descrbe e meods used o manan mcroscopes nOworkng order;

demonsrae and use e correc procedures or examnng saned ck andOn bood ims or maara parases;

demonsrae er aby o deny correcy e componens o normaObood;

recognze and deny maara parases presen n bood ims; deny eOsage(s) o pasmoda, e presence o ndvdua speces or mxed necons oPasmodum acparum, P. vvax,P. maarae and P. ovae; and esabs edensy o maara parases n e im;

record e resus o e mcroscopy examnaon on e correc orm;O

norm ose responsbe o e indngs n a mey manner;O

demonsrae er undersandng o e requremen o observe paenOconidenay and eca ssues;

oow e correc procedures o e naona programme, subm repors,Osores sdes or auds and prepare requess or suppes, o ensure e smoounconng o e mcroscopy acy;

use e andbook as a resource o eac ea workers ow o make ck andO

n bood ims, as par o e ranser o sks and eam deveopmen; andorganze, oowng e poces and requremens o e naona maara conroOprogramme, e coaboraon necessary or reguar supervson o e work oe aboraory.

Note: Any mention of stain in the text refers to Giemsa stain, unless otherwise stated.

The training programme

A course uor carres ou e ranng, asssed by a eam o acaors. he cass

s dvded no sma groups conssng o ree o ive parcpans eac, and oneacaor s assgned o eac group. Usng e Learners gude, parcpans areguded roug eac un. he acaor ensures a eac ranee receves appro-prae gudance and reaces e requred eves. hus, earners receve ndvduaaenon rom an experenced acaor, wo monors er progress o ensurea eac as reaced e requred sandard beore proceedng o e nex un andgves addona uon wen requred.

he essons conss o 1520-mn presenaons oowed by acves suc as dem-onsraons, sma group dscussons and roe-pay. Mos o e ranng consss opracca sessons. Reguar pracce eps earners o acqure e sks and know-

edge needed or ecen Gemsa maara mcroscopy. he meabe provdes or as


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muc pracce as possbe, o ep ranees gan pracca experence n a aspecso maara mcroscopy. Fedwork s par o pracce, and an addona objecves o provde e nerpersona experence o workng w suspeced maara pa-ens n a rea-e seng. hs can revea oer probems a can arse n everyday

suaons.

Forma evauaons are conduced reguary o assess acevemen and provde n-ormaon a an ndvdua and a coecve eve.

Evaluaton of the learner: hese evauaons can ncude mupe-coce quesons,spo ess, parcpan presenaons and examnaon o known sdes. he aers a reguar exercse as earners progress roug ranng and eps acaors ogauge ndvdua acevemen. I s useu or denyng areas n wc a earneras probems, gvng an opporuny o address and correc ose probems.

Evaluaton of the tranng by the learner: By means o a quesonnare, e uor

asks e earners ow ey nk e ranng as eped em and ow mg bemproved. hese reguar evauaon sessons aso aow parcpans o commen one eacng, e eacng sandards, e quay o maeras used and oer cond-ons o ranng. Tranees make er commens anonymousy, aowng beneicamodicaons.

he uor and acaors w nroduce you o e course and e maeras o beused, ncudng s andbook. Soud you ave dcuy durng any par o eranng, do no esae o conac your acaor or urer ep.

Please read Learning unit 1 in preparation

for the start of the course.


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Learning unit 1

Malaria, the disease

Learning objectives

By e end o s un, you w be abe o:

describeO why malaria is an important public health problem in

many parts of the world;

describeO four common symptoms of malaria;

describeO why some people have malaria parasites in their blood

but have no clinical symptoms;

explainO how malaria parasites produce disease in people;

explainO how some species of femaleAnophelesmosquito

transmit malaria; and

explainO why accurate diagnosis of malaria depends on correct

microscopic identification.

The importance of malaria

Maara s a serous pubc ea probem n many pars o e word. Aacks oe dsease can be severe and can ead qucky o dea unreaed. Communesw g eves o maara ave many croncay members, resung n absen-eesm rom work and scoo. Repeaed aacks no ony resu n eavy spendngon reamen bu aso afec educaon, e amoun o ood e amy can grow ande money a amy earns. Maara s a serous rsk o pregnan women and nansand s a common cause o mscarrage. In areas o g ransmsson, maara sresponsbe or underweg nans a br and anaema n e moer (irs preg-nances are parcuary a rsk). Lack o knowedge abou maara, povery andcronc dsease ogeer orm a vcous crce, wc s dcu o break.

Maara s caused by a sma vng organsm, caed a parase, wc necs a per-sons red bood ces. I s ransmed rom one person o anoer by e be o e-maeAnopees mosquoes. he parase mus go roug a compex cyce n boe mosquo and n umans beore ransmsson can ake pace. In e mosquo

vecor, e cyce ass or 13 weeks, dependng on a number o acors, suc as emaara parase speces, e amben emperaure and e reave umdy.

In e pas ew years, aemps ave been made o conro e dsease roug euse o nseccde-reaed nes, promp dagnoss and approprae reamen. hese

ave resued n sgnican reducons n moray and morbdy n some coun-res. In oer paces, e dsease connues o be e prmary cause o ness anddeas.


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Clinical signs and symptoms of malaria

he dsease s reasonaby easy o recognze n peope wo ave no ad maarabeore, or ave ad ew aacks. he common sympoms o maara are: g ever,eadace, severe cs or rgor, prouse sweang and genera body pans. Somepaens may ave vomng, coug or darroea. In perssen and recurren nec-ons, anaema may be presen.

As smar cnca sgns are seen n oer common dseases, urer nvesgaonsare necessary beore a reabe dagnoss o maara can be made. he cnca pre-senaon o maara s even ess cear n paens wo ave ad a number o maaraaacks, as ey generay sow no cear sgns or sympoms. Care mus aso be akeno esabs weer e paen as aken anmaara medcnes beore gong oospa, as s can mody e cnca presenaon. Prevous reamen w an-maara medcnes, by reducng e parase densy o very ow eves, may makemcroscopc dagnoss more dcu. Knowng wc reamen was receved smporan n order o avod an overdose o anmaara medcnes, wc can bedangerous, especay e paen was unconscous wen admed o ospa.

Diagnosing malaria: What is the best way?

In mos vage, dsrc and provnca maara aboraores, e mos reabe me-od or dagnosng maara s mcroscopc examnaon o a paens saned boodim by a raned mcroscops. Maara mcroscopy s a sked exercse requrnggrea care a eac sep o e sandard operang procedures and precse vsua and

dferena sks.

Note: Your tutor or facilitator will explain the meanings of visual and differential skills and other

words in this text that may not be familiar. Most will be easy to understand after such explanations.

Maara s caused by a parase n e bood; e parases are very sma (mcro-scopc) and can be seen ony under a mcroscope w g magnicaon. Beoree parases can be seen, owever, a bood im mus be made, dred, saned andexamned under e mcroscope. Wen e mcroscops sees saned parases, edagnoss o maara s conirmed. Mcroscopss wo use e sks earn durng

s ranng can deny e sages and speces o maara parase and e densyo e necon. Usng s normaon, e pyscan or ea-care worker canrea e paen w e mos approprae anmaara agens, n e bes possbeway.

Suspected malaria is an emergency, and the person must

see a health worker quickly. Examination of patients

blood films ensures a quick diagnosis and helps them

receive the correct treatment early. Failure to do

this can put patients at great risk.


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Wen you read e descrpons o e dferen seps or maara mcroscopy, eymay seem very dcu o earn. hs s no e case. Learnng uns 210, wcoow, w, w your acaors ep, ake you roug eac sep and esabse sandards you need o do suc work. By e me you ave reaced e end

o un 10, you w ave aceved e mporan sks o maara mcroscopy. Inrecognon o s, you w be awarded a cericae or accredaon o compe-ence, dependng on e poces o your counry. hs w be expaned durng ecourse.

A patient in whom malaria is wrongly diagnosed might

not be further investigated. This may result in

another serious illness being missed.

The malaria parasite life cycle

A descrpon o e maara parase e cyce s ncuded ere or your neresand normaon, o sow you ow compex s and ow dcu can be o con-ro maara (Fgure 1). Uness nsruced o do so by your acaor, you need nomemorze e varous porons o e cyce, bu you can reer o e ex and edagram a e end o s un.

Malaria in the mosquito vector

The sexual cyclehe sexua cyce o maara parases begns wen a parcuar speces o emae

Anopees mosquo eeds on an neced person. Mae maara parases (mcrog-ameocyes) n e neced persons bood, sucked no e mosquos somac,produce our o eg lagea. Eaclageum separaes rom e paren body andswms roug e coaguang bood n e mosquos somac; wen inds aemae maara parase (macrogametocyte), eners and erzes . Ater erza-on, a zygoe s ormed, wc raves o e wa o e mosquos somac, were squeezes beween e ces o e somac wa, sees under e ouer nngand encyss. In s oocyst, e maara parases mupy un e oocys conansmany ousands o new parases. Evenuay, e oocys rupures and reeasese spnde-saped sporozotes, wc make er way o e mosquos savarygands. he me needed or compeon o e parase e cyce n e mosquo,a s, beween e me e emae mosquo ngess an neced bood mea ande me se can ransm maara, vares accordng o e speces and e ambenemperaure and umdy, bu s usuay 721 days.

Malaria in humansThe phase in the liver

Wen e neced emae Anopees mosquo bes a uman beng, sporozotesare nroduced w e sava a e mosquo uses as an ancoaguan. hs

ancoaguan prevens e bood rom cong n e mosquos very sma, ube-

Learning Unit 1. Malaria, the disease


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ke proboscs or mou pars. Once nsde e uman beng, e sporozoes movequcky o e ver, were ey ry o nvade ver ces.

In neced ver ces, a snge parase dvdes and generaes many ousands onew parases over 721 days. he enarged ver ce, caed a ver sczont, inayburss, reeasng ousands omerozotes no e boodsream, wc qucky ad-ere o and ener red bood ces. On enerng a red bood ce, e maara parasesars o grow, usng e conens o e ce as ood, and becomes a tropozote.

hs bre descrpon o wa appens n e ver pase appes o wo o e ma-ara parase speces a afec umans, P. acparum and P. maarae. he oerwo speces, P. vvaxand P. ovae, ave a sgy dferen cyce, as a number oe parases a orgnay ener e ver ces do no mmedaey become versczons bu ener a knd o seepng pase. Caed ypnozoes, ese dormanparases are responsbe or e reapses a occur a nervas ater e irs ma-ara aack.

The blood phase

he blood pase s te focus of ts course, and you wll be famlar wt everyaspect of t by te end of te tranng.

Rg now, a s a you need o know abou maara parases and er e cyce.Your uor w descrbe oer aspecs o e subjec as you progress roug ecourse. he dagram beow ceary sows e e cyce and ransmsson o e par-ase. You w ind ou more abou e appearance o maara parases n n andck bood ims n earnng uns 7 and 8.

For clarity, read the Introduction and Learning unit 1 again.

When you have done that, read Learning unit 2

in preparation for the next session.


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Learning Unit 1. Malaria, the disease

Figure 1. The malaria parasite life cycleFigure reproduced, with minor amendments, from Bruce-Chwatts essential malariology, London, Arnold, 1993, with the

permission of H.M. Gilles and D.A. Warrell, eds.


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Notes


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Learning unit 2

Cleaning and storing microscope slides

Learning objectives


describeO one standard operating procedure and explain its

importance for malaria microscopy;

selectO from previously used slides those suitable for making blood

films and demonstrate why other slides are not suitable; and

demonstrateO the two correct ways of washing, drying, wrapping

and storing microscope slides for making blood films.

Working in a laboratory

I s s e irs me you ave worked n a aboraory, you may ee srange or ner-vous. Dont worry. Once you are amar w e way aboraores uncon, youw ee as oug you ave worked n one a your e. Some smpe rues o oown e aboraory w ep you see n qucky:

Basic rules for the laboratory:

Do not touch, open or smell bottles, jars and containers orO

chemicals unless you have been instructed to do so or unless you

know what you are doing and know what is in the bottle.

Clean up when you have finished your work: do not leave dirtyO

glassware or slides for others to wash.

Do not eat or drink in the laboratory: eat and drink in designatedO

areas.

Do not smoke.O

Use the correct precautions when handling biological specimens,O

chemicals and sharps, such as needles and lancets.

Take appropriate care when handling liquids that may be corrosiveO

or acidic or have strong fumes.

Wear protective gloves when handling materials contaminatedO

with or holding blood.

Discard contaminated materials into the designated receptacles; ifO

you are not sure, ask someone who will know.

As soon as a job is finished, wash your hands with soap andO

water.


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Standard operating procedures

Sandard operang procedures are used wdey by cncans and n aboraores.hey ave been descrbed as a se o wren nsrucons a documen e cor-rec way o carryng ou a roune or repeve acvy. hs s a smpisc dei-non, bu e acaor w expan ow sandard operang procedures appy oyour work. Remember, as you proceed roug eac earnng un, a eac acv-y oows a seres o seps, and eac sep mus be aceved o a desgnaed sandardor eve. Wen e sep s correcy oowed and e sandard reaced, your workproduc (n s un, ceaned and wrapped sdes) w be sasacory. Devangrom e nsrucons gven n an esabsed sandard operang procedure wresu n a produc o poorer quay or reaby.

he bass o s ranng programme s observance o e recommended seps. Indong so, you w reac e arge eves o compeence a quay you o pracsemaara mcroscopy.

he irs esson o be earn s a you mus oow e nsrucons gven by youracaor, as se or e w be usng sandard operang procedures a resu nreabe maara mcroscopy.

Cleaning slides

Correc ceanng o sdes s your irs acvy. Lke mos o e acves covered ne uns, s que smpe. Devaon rom e procedures w gve poor resus.

Poorly cleaned slides result in substandard blood films,

poor-quality staining and imprecise microscopy and

diagnosis. This places patients at risk. To avoid

this, ensure that slides are well selected and

properly cleaned, wrapped and stored.

Slides for malaria microscopyhe gass sdes used n mcroscopy, oten caed mcro-sdes, are usuay sup-ped n boxes o 50 or 72. hey may be descrbed on e abe as wased or pre-ceaned.

For maara mcroscopy, preer pan gass sdes o superor quay, w groundedges and a rosed end. he rosed end soud be used o abe e sde. he gassused n superor quay sdes does no og or become opaque n ropca cond-ons. Poorer quay gass sdes are ceaper bu deerorae qucky n a o, umdcmae; wasng does no remove e opaque paces, and e sdes are useess orprecse mcroscopy. Aoug e sdes are descrbed as wased or pre-ceaned,s does no mean a ey can be used drecy rom e box.Mcro-sldes mustbe washed, dred and wrapped before beng used for blood ilms.


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Sdes a are sgy scraced and consdered unsuabe or bood ims can beanded over o oer secons o e aboraory servce or roune use.

Washing and preparing slides

Two ways o wasng and preparng sdes or bood ims are descrbed beow.Your acaor w ake you roug ese seps, descrbng and demonsrange wo meods.

For hospital laboratories

In ospas, paens usuay come sngy, and ere s me o cean sdes as eyare needed. I s sucen o open one box o new sdes a a me.

You will need:

one box o new superor quay mcro-sdes;O

one medum-szed pasc bow or basn;O

good-quay qud or powder deergen;O

a wasng co or sot sponge;O

cean, n-ree coon cos (e knd used o dry crockery or gassware);O

mey acooO 1;

a wde-mou jar w a screw-ing op, o od acoo and sdes; andO

a suppy o cean waer.O

The method:

Separae new sdes one rom e oer and soak n deergen souon or 48 ,1.conveneny overng.

Ater soakng, cean eac sde on bo sdes by rubbng e wo suraces n e2.wasng co or sponge beween e oreinger and umb.

Rnse e sdes ndvduay n cean waer o was of e deergen.3.

Dran excess waer rom e sdes, beore pacng em n e jar o acoo w4.e d irmy screwed on. Keep ou o drec sung.

Wen requred, remove a sde and dry orougy w a cean, n-ree co-5.on co. Aways ande sdes by e edges.

he sde s ready or use; does no need wrappng.6.

For national malaria control programmes

In suc programmes, maara mcroscopy acves vary rom a mcroscopsworkng aone n a remoe aboraory w ew aces, o arge epdemoogcasurveys, sudes o monor drug ressance and oer acves n e ied. To en-sure a saf ave e correc maeras, ceaned, wrapped sdes, sans and oersuppes are oten prepared and provded rom a cenra ocaon. In some ruraareas, owever, aboraory saf mus cean er own sdes or even recyce em,

1 Methanol (methyl alcohol) is highly toxic and flammable; it can cause blindness and even death if swallowed in anyquantity. When not in use, it should be stored in a locked cupboard.

Learning Unit 2. Cleaning and storing microscope slides


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due o sorages o suppes.1 In ese suaons, a good suppy o sdes s needed,wc mus be ready, ceaned and wrapped beoreand. hs ncreases ecencysgnicany, as ensures e avaaby o e arge number o sdes requred oracves ar rom e aboraory. Ceanng s bes done n a sma group.

You will need:

new superor quay mcro-sdes or recyced, used sdes;O

wo medum-szed pasc bows or basns;O

good-quay qud or powder deergen;O

a wasng co or sot sponge;O

a suppy o cean, n-ree coon cos (e knd used o dry crockery andOgassware);

a suppy o cean waer;O

sees o cean paper cu o abou 11 cm x 15 cm;O

empy sde boxes o e ype n wc new sdes are supped;O

rubber bands or cear adesve ape; andO

a warm cupboard or a desccaor w acvaed sca ge.O

The method:

Trea new sdes n e same way as descrbed above n seps 14, bu en dry1.em (do no sore em n acoo).

Soak used, dry sdes n warm waer and deergen or a mnmum o 68 .2.

Ater soakng, cean eac sde ndvduay by e meod descrbed n sep3.

2 above, un a races o e od bood im and mmerson o are removed.hs may requre more an one soakng, dependng on e sae o e sdes;ence e second bow.

Wen e sdes are cean, rnse em n cean waer o remove a races o4.deergen.

Dry eac sde w a coon co. Cpped or scraced sdes are unsu-5.abe or aemaoogy and soud be dscarded; ey may be used or medcaenomoogy.

Wrap e dred sdes n packs o 10 n e peces o paper. Turn down e ends6.o e wrapper and secure em w cear adesve ape, and pace e packs

no e cardboard boxes, ready or use.As eac box ods abou 10 o ese packs, s easy o cacuae e number o7.sdes avaabe or use or dspac.

Sore boxed cean sdes n eer a sma, warm cupboard or a desccaor o en-8.sure a ey reman dry un requred. Sdes sored a room emperaure ag umdy w sck ogeer ater a ew weeks and canno be used unessey are rewased and dred.

For quay conro, abe eac box w e dae o ceanng and e name o9.e person responsbe or ceanng.

1 Use of new slides is recommended, but many programmes are unable to afford them, and slides must be selected

from recycled ones.


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Learning Unit 2. Cleaning and storing microscope slides

In warm, damp climates, fungus grows quickly on glass

slides, microscope lenses and prisms. Unless this is

prevented by storage in a dry environment, heavy fungal

growth may make it impossible to use a simple slide

or a more complex microscope. In either case,

you will be unable to do your job effectively.

The facilitator will describe warm cupboards

and other ways of protecting glassware

and microscopes from fungus.

Read Learning unit 3 in preparationfor the next session.


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Notes


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Learning unit 3

Keeping accurate records

Learning objectives

By e end o s Learnng un, you w be abe o:

identifyO the correct record form(s) and register(s) for entering

information on patients;

demonstrateO preparation of accurate, error-free records on the

appropriate form;

selectO the correct copy of each record form, or completed

summary, for dispatch to the supervisor;

describeO examples of the possible consequences of mixing up

patients records; and

explainO why a patients details are confidential and must not be

shared with unauthorized persons.

I s mporan o ensure a paens deas can be raced easy by recordng aapproprae normaon wen ey aend e cnc or wen nervewed a ome.Mos normaon s sored n a compuer daabank and requres specay desgnedorms, wc usuay cover:

e regon, provnce, dsrc or zone n wc e work was done;O

e own, vage or ocay n wc e paen ves;O

e sree and ouse number a wc e paen can be conaced;O

e paens name, sex and age;O

e paens number, wc may aso be e bood im number;O

oer deas, suc as sympoms, body emperaure and weg;Oe resus o e bood im examnaon, suc as posve or negave or maaraOparases, speces and sages seen and weer P. acparum gameocyes wereobserved;

any anmaara reamen receved beore mcroscopy examnaon; andO

oer commens, observaons or nsrucons o e cncan.O

Even you do no ave a compuer, e essena deas mus be recorded n a dayregser. Your acaor w provde exampes o e orms curreny n use andadvse you ow o compee em correcy. You w aso pracse compeng emunder norma workng condons n e aboraory or e ied.


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Remember:

A patients details are confidential. It is unethical to discuss information

in a patients records with unauthorized persons. Patient records

should be stored securely, safe from unauthorized access.

Read Learning unit 4 in preparation

for the next session.

Notes


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Learning unit 4

Preparing blood films

Learning objectives


explainO why blood must always be regarded as potentially

contaminated;

nameO four diseases found in infected blood;

demonstrateO the normal precautions used when handling blood;

demonstrateO the action to take when blood contaminates

something accidentally;

listO the materials required for making thick and thin blood films;

demonstrateO the correct method for preparing a thick and a thin

blood film on the same slide, for malaria microscopy;*

demonstrateO the correct way of labelling a blood film;

separateO thick and thin blood films of acceptable quality from

unacceptable ones, giving reasons for the selection; and

describeO and identify common mistakes and faults in makingthick and thin blood films and the causes.

* A minimum of 80% of blood films must be prepared to a satisfactory standard or meet

the satisfactory level decided for your course.

Accdena conamnaon w a paens bood presens poena rsks o easaf and paens or a number o dseases. he rsks are kep o exremey ow ev-es e oowng precauons are aken:

Wear proecve goves wen akng bood sampes or andng bood.O

Avod geng bood, ncudng dry bood rom ims, on your ingers or ands.O

Cover cus or abrasons on your ands w a waerproo dressng.O

Avod accdenay prckng yourse wen andng sarp nsrumens aOave been n conac w bood.

horougy was your ands w soap and waer as soon as you ins a job.O

I you ge bood on your skn, qucky wpe of w a coon swab dampenedOw acoo; en, was e afeced area w soap and waer as soon aspossbe.


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Bood-conamnaed maeras suc as ances, broken sdes and coon swabsOmus be dscarded n a sarps bn. I a sarps bn s no avaabe, oowyour programmes esabsed pracce and saey dspose o e maeras byncneraon.

Some people carry a disease in their blood even when they

do not appear to be ill. Diseases in the blood are

not easily detected, and the tests to demonstrate them

are sometimes complicated and expensive.

Hepatitis, HIV/AIDS, malaria and syphilis are the

commonest, but others, such as leptospirosis, may be

seasonal and common in certain areas.

Ensure, when handling blood, that you practise

the correct preventive measures.

Kinds of blood lm

In maara mcroscopy, wo knds o bood im are used: ck and n.

The thick filmA ck im s aways used o searc or maara parases. he im consss omany ayers o red and we bood ces. Durng sanng, e aemogobn n ered ces dssoves (deaemogobnzaon), so a arge amouns o bood can be

examned qucky and easy. Maara parases, wen presen, are more concen-raed an n a n im and are easer o see and deny.

The thin filmhe n im s used o conirm e maara parase speces, wen s canno bedone n e ck im. I s used o searc or parases ony n excepona sua-ons. A we-prepared n im consss o a snge ayer o red and we boodces spread over ess an a e sde. he rosed end o e sde s used orabeng. Use o e n im as a abe s no onger recommended. I sdes w arosed end are no avaabe, en deas can be wren on e n im w a sot

ead penc. Do no ck e end o e penc durng use.

Preparation of a thin and a thick blood lm on thesame slide

You will need:

proecve quay aex goves wou acum powder (wo o ree pars perOperson per exercse);

ceaned, wrapped sdes (more an are needed);O

sere ances (one per paen, pus 10%);O


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Learning Unit 4. Preparing blood lms

70% eano;O

absorben coon woo;O

a sarps conaner;O

a sde box or ray or dryng sdes orzonay and proecng em rom lesOand dus;

our or ive cean, n-ree coon cos;O

record orms or a regser;O

bapon nk-pen or e record orms or regser; andO

an HB ead penc o wre on e n im and sma sarpener.O

The method:

Ater recordng e paens deas on e orm

or regser, wearng proecve aex goves, ode paens et and, pam acng upwards, andseec e rd inger rom e umb, caed erng inger. For nans, e bg oe can be used,no e ee. Never use e umb, or eer c-dren or adus.

Cean e inger w coon woo dampened wacoo. Use irm srokes o remove dr and osrom e ba o e inger.

Dry e inger w a cean coon co, usngirm srokes o smuae bood crcuaon.

Usng a sere ance and a quck rong acon,puncure e ba o e inger or oe.

Appy gene pressure o e inger or oe and ex-press e irs drop o bood; wpe away w drycoon woo, makng sure a no coon srandsreman a mg aer be mxed w e bood.

Workng qucky and andng e sdes ony by

e edges, coec e bood as oows:Appy gene pressure o e inger and coec asnge sma drop o bood abou s szeton emdde o e sde. hs s or e n im.

Appy urer gene pressure o express morebood, and coec wo or ree arger drops on esde, abou 1 cm away rom e drop nended ore n im. Wpe e remanng bood of einger w coon woo.


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he tn ilm: Usng anoer cean sde as aspreader and w e sde w e bood res-ng on a la, irm surace, ouc e sma drop obood w e edge o e spreader, aowng e

bood o run rg aong e edge.

Frmy pus e spreader aong e sde, keep-ng a an ange o 45o. he edge o e spreadermus reman n even conac w e surace oe oer sde we e bood s beng spread.

he tck ilm: Handng e sdes by e edg-es or a corner, make e bood im by usng ecorner o e spreader o jon e drops o bood,and spread em o make an even, ck im. Dono sr e bood. A crcuar or recanguar imcan be made by ree o sx quck srokes w ecorner o e spreader.

he crcuar ck im soud be abou 1 cm ndameer.

he ck im soud be dred eve and be pro-eced rom dus, les, sung and exremeea.

Under norma condons, e n im dres

qucky. In e pas, e paens deas, sdenumber and dae used o be recorded w a sotead penc on e cker par o e n im.Preeraby sdes w a rosed end soud be usedand e rosed end used as e abe. Usng en im as a abe s no onger recommended.

Avod oucng wrng nsrumens o e boodim. Do no use a bapon or ge pen o abesdes, as e nk w spread wen e im sixed.

Wen e ck im s compeey dry, wrap esde n e paens record orm and qucky or-ward o e aboraory. Sdes a are no o beprocessed mmedaey can be sored n a descca-or beore sanng

Sdes a are correcy made eave e bood one spreader. he spreader sde can be used ormakng ck and n ims rom e nex pa-en, we anoer cean sde rom e pack sused as e res spreader.


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Common faults in preparing blood lms

Faus commony seen n bood ims may afec e abeng, e sanng or eexamnaon se and, ereore, e oucome or e paen.

Poorly positioned blood filmsI ims are no correcy sed on e sde, ey may be mpossbe o examne.Pars o e ck im can be rubbed of by e edges o e sanng roug, dryngrack or sde rame.

hs n im s oo arge; e ck im s wrongy posoned and w be dcuo examne under e o mmerson objecve.

Too much bloodSaned ck ims made w oo muc bood w ave a very bue background.here w be oo many we bood ces per ied, wc may obscure any parasesa are presen. In n ims a are oo ck, e red ces w be on op o oneanoer, makng mpossbe o see parases ceary.

Too little bloodWen ere s oo e bood n e ims, ere are no enoug we bood cesn e ck im ied or sucen bood or a sandard examnaon. he n imw usuay be useess or speces dagnoss.

Greasy slidesBood ims made on a greasy sde w spread uneveny, and pars o e ck imw loa of durng sanng. Examnaon o bo ck and n ims w be d-icu because o e pacy dsrbuon o bood.


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Edge of the spreader slide chippedWen e edge o e spreader sde s cpped, n ims spread uneveny, aresreaky and ave many as. Cpped spreaders can aso afec e way e ckim spreads.

Oer probems w e preparaon, coecon or sorage o unsaned boodims can ncude e oowng:

Fes, ans, cockroaces and oer nsecs ea e we or dryng bood and damageO

e ims. Sdes soud be covered durng dryng and ten stored overngt n anartgt box or desccator carged wt sca ge.

Use o scraced sdes or bood ims makes mcroscopc examnaon o eOims dcu. Scratced or cpped sdes soud not be used or makng bood

ims. hey soud be dscarded.

Uneven dryng o ck ims eads o varaon n e quay o a im, makngOsandard mcroscopc examnaon dcu. Bood ims must be dred on a lat,orzonta surace.

Auoixaon o ck ims akes pace wen sdes ave been sored or ooOong a g amben emperaure and umdy wou sanng. hs can

appen wen sdes mus be sored wou sanng, suc as sdes o knownparasoogy coeced or eacng or sde banks durng proonged ied surveys.Autoixed sdes stan poory, but autoixaton can be deayed by keepng te sdesn a desccator carged wt sca ge. Avod pacng newy coected sdes n drectsungt or on te loor o a vece over a ot exaust ppe durng transport. hck

ims can be deaemogobnzed by mmersng tem n cean, preeraby bufered(pH 7.2), water or about 5 mn, torougy dryng tem and storng tem n adesccator.

hck ims a are ncompeey dred beore ey are sacked ron o back andOsored n used cardboard sde boxes w sck o one anoer. Sdes must bedred competey beore tey are packed or storage or transport.




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Notes


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Notes


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Learning unit 5

Staining blood films with Giemsa stain

Learning objectives


demonstrateO correct operation of the analytical balance;*

make upO the buffered water used to dilute Giemsa stain;

demonstrateO correct use of the colour comparator or pH meter;*

make upO the 2% correcting fluids used to adjust the pH ofbuffered water;

explainO why pH 7.2 buffered water is best for good Giemsa

staining;

demonstrateO two correct methods of fixing thin blood films;

explainO when the rapid and slow Giemsa staining methods are

used for malaria microscopy;

demonstratO e mastery of the rapid and slow Giemsa staining

methods;

describeO

the correct ways of handling and storing Giemsa stain;and

demonstrateO the correct drying and storing of stained slides.

* This objective applies only where this type of equipment is used.

Buffered water

On propery saned bood ims, maara parases can be seen ceary under emcroscope. Beore sanng bood ims, prepare e bufered waer used o duee san.

Using buffered water at the correct pH

helps to ensure good staining.

pH expresses e acdy or akany o a lud. I s based on a scae o near 0 (veryacd) o 14 (very akane). Lquds a are neer acd nor akane are descrbedas neura, a pH 7.0. he pH o a qud can be measured w a pH meer or w acoour ndcaor, suc as e Lovbond comparaor. Paper ndcaor srps can asobe used, bu ey are rapdy afeced by g umdy and become unreabe.

In s un, you w use e pH meer or comparaor recommended n your na-ona maara conro programme.


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Waer can be made more acd or more akane by e addon o ceran sas,caed bufer sas. hese are sored separaey un combned n e correc pro-porons n a ixed voume o waer o gve e requred pH. Bufer sas are wegedon a baance. I s mporan o ensure a ey are sored correcy and canno

absorb mosure rom e ar; oerwse, ey w no work.

Formuaed abes (bufer abes) are commercay avaabe, wc gve a spe-cic pH wen mxed n a ixed amoun o waer (usuay 1 re). Bufer abes dono need o be weged and are useu n paces w med aces. hey mus,owever, be kep n an arg ube under dry condons; oerwse, ey rapdyabsorb mosure and mus en be dscarded. Some workers consder a e re-sus o sanng are neror wen bufer abes are used, bu ere s no evdenceo suppor s percepon.

To prepare buffered water

You will need:

an anayca baance accurae o 0.01 g (a wo-pan rp baance s dea); varoussnge-pan, eecrcay operaed baances are avaabe a are easy o use andsuabe;

ier papers, 11 cm n dameer;O

one gass conca lask, 1 re capacy;O

one gass beaker, 250 m capacy;O

wooden spauas (wooden ongue depressors are ready avaabe);O

dsed or deonzed waer, 1 re;O

poassum dydrogen pospae (anydrous) (KH2PO4); andO

dsodum ydrogen pospae (anydrous) (Na2HPO4).O

The method:

I you are usng a radona, wo-pan anayca baance, oow a e seps rom1 o 10. I you are usng an eecrc baance, oow e acaors nsrucons; youw probaby sar a sep 5.

Make sure a e poner o e baance s se a zero by adjusng e baanc-1.ng screw on e rg arm.

Pace a ier paper n eac pan; se e baance o zero, s me by movng e2.gram weg aong e gram scae arm.

Move e gram weg a urer 0.7 g aong e scae arm, ready or wegng3.e poassum dydrogen pospae.

Usng a wooden spaua, pace some o e KH2PO4 on e ier paper n e4.et-and pan.

Transer e weged KH2PO4 o e gass beaker, add abou 150 m o waer,5.and sr w a cean spaua un e sa dssoves.

Pace a res ier paper n e et-and pan.6.

Rese e baance as beore, bu s me adjus e gram weg o 1 g or e7.

Na2HPO4.


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Learning Unit 5. Staining blood lms witth Giemsa stain

Usng a cean, dry spaua, add e Na2HPO4 o e rg-and pan, baancng8.e weg as descrbed n sep 4 above.

Add e Na2HPO4 o e souon n e beaker and sr as n sep 5.9.

Wen e sas ave dssoved, add e souon o e conca lask and op up10.

o e 1 re mark w waer.

he bufered waer s now ready or adjusmen o pH 7.2 ater e correcng ludas been made up.

To make up the 2% correcting fluids

You will need:

an anayca baance accurae o 0.01 g or beer (a wo-pan rp baance s dea, oruse an eecrcay operaed one-pan baance);

ier papers, 11 cm n dameer;O

wo gass-soppered boes, eac o 100 or 150 m capacy;O

poassum dydrogen pospae (anydrous) (KH2PO4) ;O

dsodum ydrogen pospae (anydrous) (Na2HPO4);O

dsed or deonzed waer, abou 200 m;O

wooden spauas;O

wo beakers o 250 m capacy;O

one measurng cynder o 100 m capacy; andO

abes.O

The method:

Foow seps 1 and 2 o e meod or makng bufered waer, en move e1.gram weg a urer 2 g aong e scae arm.

Weg 2 g o Na2HPO4 and add o 100 m o waer n e beaker; sr w e2.spaua un e sas ave dssoved.

Pour e souon no one o e gass boes and abe e boe 2%3.Na2HPO4.

Repea seps 1 o 3 above, ony s me use 2 g o KH2PO4; abe e boe as4.suc.

Sore n a coo pace away rom sung.5.

To check and adjust the pH of buffered waterCeck e pH o bufered waer rouney beore use. To adjus e pH, add smaquanes o e correcng luds o e bufer: 2% Na2HPO4 e pH s beow 7.2(oo acd) or 2% KH2PO4 e pH s above 7.2 (oo akane). Adjusmens can bemade as ouned beow:

Remember: There are many kinds of pH meter available.

You will learn to operate the kind used in your country.


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You will need:

bufered waer n a conca lask;O

e wo boes o correcng luds;O

a pH meer or a pH coour ndcaor;O

wo pH coour ndcaor gass ces;O

one boe o bromo-ymo-bue ndcaor; andO

one measurng ppee, capacy 1 m.O

The method:

Pour some o e bufered waer o be esed no eac o e pH coour ndca-1.or gass ces up o e 10 m mark.

Pace one ce n e et-and comparmen o e pH coour ndcaor, as e2.conro ce.

Ppee 0.5 m o bromo-ymo-bue ndcaor no e oer ce, mx, and pace3.e ce n e rg-and comparmen.

Hodng e pH coour ndcaor owards a ceary , we background, urn4.e dsc un s coour maces a n e rg-and ce.

Adjus e pH o e waer n e conca lask by addng drops o e reevan5.correcng lud: Na2HPO4 o make akane, KH2PO4 o make acd.

Giemsa stain

Gemsa san s an acoo-based Romanowsky san. I s purcased ready o use or

s made up a regona cenres by sked ecncans and en dsrbued roug-ou e aboraory and maara conro programme nework. Gemsa san s amxure o eosn, wc sans parase croman and sppng sades o red orpnk, and meyene bue, wc sans parase cyopasm bue. We-ce nucesan bue o amos back, dependng on e ype o we ce. hs s expaned na aer earnng un.

Some mporan ngs o remember w regard o e sock souon o GemsaOsan are:

Keep e boe gy soppered o avod evaporaon and oxdaon o e sanOby g umdy.

Sore n a dark gass boe n a coo, dry, sady pace, away rom drecOsung.

For day requremens, measure sma amouns o san no a gy sopperedOboe (abou 25 m), so a e sock souon s ess key o be conamnaed.

Do no add waer o e sock souon; even e smaes amoun w cause eOsan o deerorae, makng sanng progressvey nefecve.

Do no sake e boe o san beore use. Sakng re-suspends precpaes,Owc see on ims durng sanng and obscure mporan deas durngmcroscopy.

Do no reurn unused san o e sock boe or o e boe used n your dayO

roune. Once san s ou o e boe, mus be used qucky or dscarded.


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Staining blood filmshere are wo meods o sanng w Gemsa san: e rapd (10%) meod ande sow (3%) meod. he rapd meod s used n oupaen cncs and busyaboraores were a quck dagnoss s an essena par o paen care. he sow

meod s used or sanng arger numbers o sdes, suc as ose coeced durngcross-secona or epdemoogca surveys and ied researc.

The rapid (10%) method

hs s e commones meod or sanng 115 sdes a a me. I s used n abo-raores were a quck resu o deermne a paens maara saus s requred.he meod s ecen, bu more san s used. he need or speed jusies e ad-dona cos.

You will need:

Gemsa san, decaned rom e sock souon no a 25-m boe;Omeano;O 1

absorben coon woo;O

es ubes o 5 m capacy;O

dsed or deonzed waer bufered o pH 7.2;O

a Paseur ppee w a rubber ea;O

a curved pasc sanng ray, pae or rack;O

a sde-dryng rack;O

a mng cock; andO

a sma eecrc ar-drer.Ohck bood ims mus be compeey dry beore beng saned. hey can be dredqucky w warm ar rom a sma ar-drer or by careu warmng over a amp ora g bub. Avod overeang sdes as ey can ea ix and en san poory.

The method:

Fx e n im by dabbng w a pad o coon woo dampened w me-1.ano or by brely dppng e im no meano. Avod conac beween eck im and meano, as meano and s vapours qucky ix e ck im,and does no san we.

Usng a es ube or a sma conaner o od e prepared san, make up a 10%2.souon o Gemsa n e bufered waer by mxng ree drops o Gemsa rome sock souon, usng e Paseur ppee, w 1 m o bufered waer. Eacsde needs approxmaey 3 m o san o cover .

Dependng on weer you are usng a sanng ray, pae or rack, pace e3.sdes o be saned ace down on e curved sanng ray or ace upwards one pae or rack.

1 Methanol (methyl alcohol) is highly toxic and flammable; it can cause blindness and even death if swallowed in

any quantity. When not in use, it should be stored in a locked cupboard.



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Pour e san geny under e sanng ray un eac sde s covered w4.san, or geny pour e san ono e sdes yng ace upwards on e pae orrack.

San e ims or 810 mn. Experence w e san you are usng w ep5.

you o decde e exac me needed or good sanng.Geny was e san rom e sde by addng drops o cean waer. Do no6.pour e san drecy of e sdes, or e meac-green surace scum wsck o e im, spong or mcroscopy.

Wen e san as been wased away, pace e sdes n e dryng rack, im7.sde downwards, o dran and dry. Ensure a ck ims do no scrape eedge o e rack.

The slow (3%) method

hs meod s ess approprae wen a quck resu s needed bu s exceen or

sanng arge numbers (20 or more) o sdes. I s dea or sanng bood imsrom surveys or researc work or baces o sdes or eacng. I perorms beswen sdes ave dred overng. he meod s economca because muc esssan s used (3% raer an 10%).

You will need:

Gemsa san;O

meano;O 1

absorben coon woo;O

sanng rougs o od 20 sdes paced back o back;O

waer bufered o pH 7.2;Oa measurng cynder, capacy 100500 m;O

a measurng cynder, capacy 1025 m;O

a lask or beaker (capacy w depend on e amoun o san o be made up);O

a mng cock; andO

a sde-dryng rack.O

The method:

Fx eac n im by dabbng geny w a pad o coon woo dampened1.w meano or by dppng n a conaner o meano or a ew seconds.

Avod conac beween e ck im and meano, as meano and s va-pours qucky ix e ck im, and does no san we.

Pace e sdes back o back n a sanng roug, makng sure a e ck2.ims are ogeer a one end o e roug.

Prepare a 3% souon o Gemsa san by addng 3 m o Gemsa sock souon3.o 97 m o waer bufered o pH 7.2, or mupes o s.

Pour e san no e roug. Do no pour drecy ono e ck ims, as4.ey may loa of e sdes.

1 Methanol (methyl alcohol) is highly toxic and flammable; it can cause blindness and even death if swallowed inany quantity. When not in use, it should be stored in a locked cupboard.


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San or 4560 mn; experence w ndcae e correc me.5.

Geny pour cean waer no e roug o loa of e rdescen scum. To6.avod dsurbng e ck ims, pour e waer no e n im end. A esssasacory way o lusng sdes s o mmerse e woe roug n a basn

ied w cean waer and make sure o avod e rdescen scum wen re-movng e roug rom e basn.

Geny pour of e remanng san and rnse w cean waer.7.

Careuy remove e sdes, one by one, pacng em im sde down n e8.dryng rack o dry. Make sure a e ck ims do no ouc e edge o erack.

During staining with Giemsa stain (3% or 10%), the

surface is covered with a metallic green scum.

Avoid getting it onto blood films duringrinsing as it can impair examination.

Care of glassware and measuring equipment

Measurng cynders, ppees, sanng rougs and beakers mus be cean and drybeore use. Sanng bood ims w dry uenss gves unsasacory resus.

he equpmen used or Gemsa sanng soud be rnsed mmedaey ater use

n cean waer o remove as muc o e san as possbe. I soud en be soakedor a we n a deergen souon beore wasng. Wasng uenss w a mddeergen s sasacory, provded ey are rnsed orougy n cean waer beoredryng. Any deergen a s et on gass and pasc-ware can aer e pH o ewaer and e san, resung n poor sanng wen e equpmen s nex used.





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Notes


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Learning unit 6

The microscope

Learning objectives


demonstrateO the correct set-up and use of a binocular

microscope with artificial and with natural light;

demonstrateO the correct use of the x10 paired oculars and x100

oil immersion objective;*operateO the mechanical stage correctly;

nameO correctly 10 component parts of the microscope;

describeO the correct way in which to maintain a microscope in

good working order;

describeO two ways of storing a microscope correctly; and

demonstrateO the correct way of packing a microscope for long-

distance transport.

* Or x7 ocuars ey are used n e programme

For efficient malaria microscopy, learn

to use the microscope correctly; know

its limitations and how to keep it in

good working condition.

Monocuar mcroscopes ave a snge eyepece (ocuar). hey are mos useu wenno power suppy s avaabe. Dayg provdes a brg mcroscopc ied or mon-ocuar mcroscopes. Bnocuar mcroscopes, w wo eyepeces, ave repaced

monocuar ones, as ey are more comorabe o use, bu dayg provdes poorumnaon or ese mcroscopes.

he mcroscope you w use durng ranng and back a your ome base s caed acompound bnocuar mcroscope. Opma maara mcroscopy s done w mcro-scopes ied w x10 pared eyepeces and an x100 o mmerson objecve.1

To ensure e g sandards o umnaon requred or roune bnocuar ma-ara mcroscopy, s essena o ave a good, reabe source o arica g. I aconsan suppy o eecrcy s no avaabe, a generaor can be used. Deverng

1 Some programmes prefer 7-paired oculars but they are not easy to obtain. The 7 ocular covers more blood perfield and is therefore considered by some workers to be more sensitive.


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even sma generaors and ue o remoe cncs can be dcu, owever, and grunnng coss make s meod unaccepabe. Ceaper, easer sources o aricag or mcroscopy are g-emng dodes (LED), a orm o eecroumnes-cence a can be derved rom sma, ow-voage baeres. he baeres can be

carged by a sma soar pane mouned on a poe or e roo o e aboraory. Arange o ese producs s avaabe on e marke. Mos are afordabe, easy o useand requre mnma manenance. Your acaor w dscuss s subjec urer,dependng on ow mporan s o you and e programme.

The LED light illustrated here can run for a minimum of 200 hours on four standard 1.5-volt batteries.

1

2

3

4

5

6

7

8

9

10

11

12

Parts of the compound binocular microscope

he man pars o a ypca compound bnocuar mcroscope are sown above.

1 and 2. Main tube and body tubeCoecvey caed e mcroscope ead, e man ube and body ube are desgnedo sope owards e user and are caed an ncned ead. Posed gass prsms


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Learning Unit 6.The microscope

nsde e body ube o e ncned ead bend e g so a e mage reacese users eyes roug e pared ocuars.

3. Revolving nosepiece

hree or our objecve enses o dferen magnicaons screw no e nosepece.he nosepece revoves o pace a dferen objecve over e specmen, n ne we eyepeces, wc ncreases or decreases magnicaon o e specmen.

4. Objective lensesA e pars o e mcroscope are mporan, bu e objecve enses mus bereaed w parcuar care. An objecve consss o wo or more enses kep npace by a speca gue or cemen. Sovens suc as acoo, xyo and aceone candssove e cemen odng e ens n pace and soud no be used o cean eobjecves or any oer par o e mcroscope.

An objecve s reerred o by s magnyng power, wc s usuay marked on esde o e body. Eac mcroscope usuay as a x10, a x40 and a x100 objecve. hex100 s caed e o mmerson objecve and can be denied by a dsncveback, red or we rng.

Wen you examne an objecve ens, you w noce a e sze o e ron ensdecreases w e magnyng power. he workng dsance beween e ron ensand e ocused specmen on e sage canges w e magnicaon. hus, eger e objecves magnyng power, e sorer e workng dsance. Caremus ereore be aken no o damage e specmen w e objecve ens.

Aoug ere may be sma varaons accordng o e manuacurer, e work-ng dsance or eac objecve s approxmaey:

x10 15.98 mm

x40 4.31 mm

x100 1.81 mm (o mmerson)

he mcroscope mus be used w care, as specmens, sdes and even e ob-jecve ens can easy be damaged by roug manpuaon or wen objecves arecanged.

5. Mechanical stagehe mecanca sage ods e sde secure we aowng specmens o be movedsmooy. A scae ied o wo sdes sows e specmens poson and subsequenmovemen durng examnaon. hs scae s caed e Verner scae. You w uses scae o race porons o e bood im a soud be re-examned or sowno oers. In modern bnocuar mcroscopes, e sage moves wen e specmen socused. In oder mcroscopes, e body and ube move durng ocusng.

21 Y 51 Y 211 Y

Objectives, showing working distance between

front lens and specimen


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6. Substage condenser, iris diaphragm and filter holderhe subsage condenser consss o a number o enses a cenre e g rome source or mrror ono a cenra spo on e mcroscopc ied. he subsage con-denser can be rased or owered o gve maxmum or mnmum umnaon.

Insde e condenser s e rs dapragm, wc s used o conro e amoun og passng roug e condenser. I consss o a number o n, nerockngmea eaves, wc are adjused by movng a sma ever.

Benea e rs dapragm s e ier oder, n wc a rosed bue-gass ier spaced wen eecrcy s e g source. hs makes e mcroscopc ied appearwe raer an yeow.

he procedure or seng e correc umnaon o e mcroscope, .e. Kerumnaon, s mporan or opmum resouon and conras, ensurng an even-y umnaed ied, removng gare and reducng eang o e specmen, as de-

scrbed n e encosed CD-ROM.

7. IlluminatorModern mcroscopes ave a ixed umnaor, n wc a bu-n prsm mrrorbrngs g o e mcroscopc ied. Oers ave a removabe umnaor, wccan be repaced by a mrror wen eecrcy s no avaabe.

he subsage mrror s used o drec g rom e g source o e mcroscopeied. I as wo sdes: one pane (la) and e oer concave. he la surace s usedw e subsage condenser. he concave sde s used wou e subsage con-

denser, as e curved surace se acs as a condenser.

8. Base or footTo avod movemen or wobbng, e sod base, or oo, o e mcroscope musres on a irm, la surace. he sape o e oo may vary. Mos ave a readedoe n e undersde o e base o receve a securng screw a keeps e mcro-scope rgd n e box durng ranspor.

9. Ocular, or eyepiecehe op o e man ube o modern mcroscopes s ied w a bnocuar ead, .e

w wo ocuars, one or eac eye. Monocuar mcroscopes are sedom used odayn naona maara conro programmes.

he ocuar is no e upper end o e man ube, and e mcroscops ooksroug wen usng e mcroscope. he magnyng power o eac ocuar smarked on . he magnyng power s e number o mes by wc w mag-ny e mage produced by e objecve. For exampe, w ocuars o x10 andan o mmerson objecve o x100, e oa magnicaon o e specmen woudbe 10 x 100 = 1000 dameers. he magnicaon s acuay a e more, bu 1000dameers s accurae enoug or our purposes.

Ocuars are avaabe n a range o powers, rom x5 o x25 or even x30. In ma-

ara mcroscopy, a range o x6 o x10 s used rouney. One arge programme asused x5 ocuars or many years. Today, x10 s probaby e mos commony used.Programmes are srongy advsed o use ocuars beween x7 and x10 or rounemaara mcroscopy.


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Ocuars ied o bnocuar mcroscopes are caed pared ocuars. he mark-ng x10P on e rm o a x10 ocuar ndcaes a s one o a pared se oeyepeces.

10. Arm or limbhe arm orms a rgd suppor or e man ube and sage o e mcroscope. I srobus and can be used as a ande or carryng e mcroscope. Wen carryng amcroscope n s way, aways suppor e base o e mcroscope w e oerand.

11 and 12. Coarse and fine adjustmentshe wo adjusmen sysems, coarse and ine, are used o ocus on e specmenbeng examned. he coarse adjusmen s used or rapd, reavey arge vercaocusng movemens, we e ine adjusmen s or e more precse ocusng

requred w ger-powered objecves. In modern mcroscopes, e coarse andine adjusmens rase and ower e mecanca sage. In oder mcroscopes, eman ube s rased o ocus.

Usuay, a specmen s irs examned w e coarse adjusmen and en exam-ned n dea w e ine adjusmen.

he coarse adjusmen s used dfereny wen e o mmerson objecve s used,as w be expaned n a aer earnng un.

Use of the microscopeIn e pracca sessons, you w use and become amar w a e eaures oe mcroscope. Eary on, you w see e mage o e specmen becomng argeras e magnicaon s ncreased. hs akes pace wen you cange objecves.You w aso examne everyday objecs and see ow dferen ey ook under emcroscope. hese exercses are desgned o ep you earn o adjus e umna-on correcy and o use e subsage condenser and rs dapragm. You w asopracse usng e mecanca sage and Verner scae.

The light source

A good source o arica g s needed o examne specmens propery. Lga s eer oo brg or oo dm w nerere w maara mcroscopy.

Wen e o mmerson objecve o a bnocuar mcroscope s used rouney, eec-rc g rom a mans suppy or a generaor soud be used. Baery-operaed LEDg sources are a useu aernave wen eecrc g s no avaabe and soudbe dreced owards e mrror. Arica LED g raves roug e mrror ona pa rom e source as oows:

source mirror substage condenser and diaphragm specimen objective oculars

Wen arica g s used, a rosed bue ier mus be paced beween e source

and e subsage condenser. he la sde o e mrror s used.



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Dayg soud be used ony n an emergency. Wen dayg s e g source,e concave mrror soud be used wou e subsage condenser. I s danger-ous o pon e mrror drecy a e sun wen obanng umnaon, as serousdamage can be caused o e eyes.

Obtaining even illumination

Usng x10 pared ocuars and an x10 objecve:

Pace e sde on e mecanca sage, w e specmen over e cenra1.openng n e sage.

Focus on e specmen usng e coarse adjusmen.2.

Make sure a e rs dapragm s wde open, and rase e subsage con-3.denser un e mcroscopc ied s brges.

Remove e eyepeces and, ookng down e ube, adjus e mrror ( s be-4.

ng used) un e objecve ens s uy umnaed.Repace e eyepeces. Use e ine adjusmen o sarpen e ocus on e5.specmen.

Remove e eyepeces agan, and sowy cose e rs dapragm un e aper-6.ure o e objecve s wo-rds vsbe. he specmen w appear cearer, wmaxmum resouon.

Repace e eyepeces, and revove e nosepece o seec e objecve you wan7.o use. Eac me you cange e objecve, you mus reocus.

I e nensy o e g rom e subsage amp s consan, e umnaon8.can be adjused by ncreasng or decreasng e aperure o e rs dapragm.

In some mcroscopes, s possbe o adjus e nensy o e g rom esubsage amp.

Using the oil immersion objective

Wen preparng e mcroscope or o mmerson mcroscopy:

Arrange e umnaon as descrbed above, en observe e nex seps rom1.e sde o e mcroscope.

Usng e coarse adjusmen, rack e sage down, away rom e objecve2.ens.

Pace e sde on e mcroscope sage, w e bood im uppermos.3.

Makng sure a ere w be sucen space beween e sage and e x1004.objecve, revove e nosepece un e x100 objecve s over e specmen.

Pace one or wo drops o mmerson o on e area o e bood im o be5.examned.

Usng e coarse adjusmen, move e sage un e objecve ens s n conac6.w e mmerson o. Rase e sage sgy, makng sure a e ens ando reman n conac.

Lookng down e eyepeces, ocus on e specmen w e ine adjusmen.7.

Make sure a e ens does no ouc e sde. Correc e umnaon byadjusng e rs dapragm.


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Immerson o s used beween e mcroscope sde and e objecve ens o re-duce scaerng o ransmed g. he o mus reproduce e opca propereso e gass used or e enses and mus ereore ave a reracve ndex o 1.515,wc s approxmaey 1.5 mes e reracve ndex o waer.

Commercay avaabe mmerson os can be ceaned of e objecve ens w asot coon co. Do no use s co o cean oer enses. Immerson o on boodims can be geny wased away w e soven recommended by e manuac-urers, or e sdes can be paced ace down or a we on cean, we absorbenssue paper a soaks up e o. Some workers wpe e o of ims w absor-ben ssue, bu s meod s roug and s no recommended. Anoer meod so ro examned sdes n we ssue paper (oe paper w do), w one ayer ossue paper beween eac sde. Ater a ew days, wen e paper as absorbed eo, e sdes can be removed rom e paper. Cooured ssue soud no be usedas s oten acdc and w de-san bood ims.

Care of the microscope

Provded norma care and common sense are exercsed, your mcroscope w re-man n good condon or many years.

Removing dust and greaseDurng e day, wen e mcroscope s no n use, soud be kep covered wa cean co or pasc cover o proec e enses rom seng dus. Overng, or e mcroscope w reman unused or a ong me, soud be paced nsde s

box, w e door gy cosed. To proec e objecve enses, e x10 objecvesoud be roaed o ne up w e ocuar.

O rom eyeases, aca skn and ingers s easy deposed on enses and ocuarsdurng use. hese pars soud be ceaned careuy w ens ssue or a sot coonco.

O mmerson objecves mus be ceaned mmedaey ater use. I no, e ow cken and arden over me, and e objecve w become useess. To avodurer ranser o os, never use conamnaed cos o cean oer objecves,ocuars or e mrror.

Preventing fungal growthIn warm, umd cmaes, unga grows are easy esabsed on enses andprsms. Funga grow causes probems and can become so bad a a mcroscopecanno be used. In suc cases, e afeced suraces mg ave o be ceaned andreposeda job usuay done by e manuacurer, wc akes me and can beexpensve.

Fungus canno grow on gass suraces wen e amospere s dry. hereore, Os mporan o sore e mcroscope n dry condons wen no n use. One oe oowng meods soud be used.

Keep e mcroscope n a warm cupboard, wc as a gy ing door andOwo or more, consany burnng 25-wa bubs, dependng on e sze o e



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cupboard. he emperaure nsde e cupboard soud be a consan 3035 C,w ow umdy.

Keep a enses and prsm eads n an arg box or desccaor conanngOacve sca ge, wc s a desccan and absorbs waer vapour rom e ar.

Se-ndcang sca ge s bue wen acve and becomes pnk as absorbswaer vapour. Wen s brg pnk, can be reacvaed by eang; s readyo use agan (ater coong) wen as become brg bue.

I possbe, keep e mcroscope n a connuousy ar-condoned room. RoomsOa are ar-condoned ony durng e workng day are no suabe.

Transporting the microscopeWen ransporng e mcroscope beween aboraores or o e ied, s m-poran o ensure a s propery secured nsde s box. he bes way o do ss by screwng e securng devce roug e oe n e boom o e box noe base or oo o e mcroscope. Wen s s done correcy, e mcroscoperemans rgd n s box on even e rouges road.



Notes


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Learning unit 7

Examining blood films

Learning objectives

By oowng eac sep n s Learnng un, you w be abe o:

listO the components of normal blood;

demonstrateO each method used for examining a thick blood film

and a thin blood film for malaria parasites;

recognizeO and classify the normal components of blood;

nameO correctly the main parts of a white blood cell; and

recognizeO common contaminants of blood films.

Note: The standards of accuracy required are not listed for these learning objectives. The tutor will

designate the levels of accuracy expected for the competence and the methods for assessment during

this course.

Components of normal blood

Wen bood s aken drecy rom a ven andcoeced n a es-ube, s a red qud. Atersandng or 520 mn, e bood separaesno wo ayers, as n e dagram. he serumayer s a pae-yeow lud; e bood co s asem-sod subsance a becomes dark-red oramos back. he co conans red bood ces,we bood ces and paees. hese compo-nens are very sma and can be seen ony we ad o a mcroscope, on a bood im maderom resy aken bood and dred and sanedbeore examnaon.

Quanes o bood arger an a obaned rom a inger-prck are usuay akendrecy no a ube reaed w an ancoaguan, wc prevens e bood romcong. Coed bood canno be used n many aboraory procedures. Fms madew ancoaguaed bood mus be anded careuy, as aderes poory o esde. Some ancoaguans cange e pH sgy, wc can afec e quay o

Tfsvn

Cmppe dmpu dpoubjojohsfe cmppe dfmmt-

xijuf cmppe dfmmt-qmbufmfut


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sanng. I ancoaguaed bood s et sandng on e benc or et n e rerg-eraor or more an 1 or 2 ours, e morpoogy o componens ke we boodces, paees and parases can cange and make dagnoss dcu.

What do normal blood components look like?he norma appearance o e varous componens o a ck and a n bood ims aways sgy dferen, and s mporan o be abe recognze em.

Blood in Giemsa-stained thin filmshn ims examned w e x100 o mmerson objecve and e x10 ocuarconan: red bood ces (or eryrocyes), we bood ces (or eukocyes) andpaees (or rombocyes).

Red blood cells

he sape o e red bood ce, or eryrocye, s descrbed as a bconcave dsc. Is e commones ce n n bood ims. here are abou 5 000 000 n eac m-crore () o bood. W Gemsa sanng, e red ce appears as a pae-greyso g-pnk dsc measurng abou 7.5 m n dameer.

Sfe cmppe dfmm ps fszuispdzuf

he red ce does no ave a nuceus. Some ces may appear arger an norma redbood ces (normocyes) and n some rare cases may even appear ova.

White blood cells

he number o we bood ces, or eukocyes, per mcrore o bood s normay60008000, muc ewer an red ces. he number can vary wdey under cerancondons and n some ndvduas. here are severa ypes o eukocye. As eacsans dfereny, ey are easy o dsngus w pracce. he pars o a ypcawe bood ce are sown n e usraon.

A white blood cell

Cytoplasmand granules

Cell membrane

Nucleus

Eac eukocye as a nuceus surrounded by cyopasm; somemes, e cyopasms granuar. Some eukocyes ave a muobed nuceus, as sown n e us-raon above. Leukocyes are dvded no wo groups, poymorponucear andmononucear eukocyes.


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Learning Unit 7. Examining blood lms

Polymorphonuclear leukocytes

Neutrophls make up 65% o e oa we ce coun n a eay person. heyave we-deined granues n e cyopasm, and er nuce san a deep purpe.Wen a maara parase s presen, neurops may conan maara pgmen,

wc s a by-produc o parase meabosm and s a a remans o parasesa ave been pagocyosed (engued or eaen) by e neurop. Maara pg-men can be goden brown o amos back. I does no ake up Gemsa san.

Eosnophls make up 14% o e oa we ce coun n a eay person. hegranues are a dsncve pnks (eosn) coour and are a good ndcaor o san-ng quay. he number o eosnops can ncrease dramacay n dseases keasma, emnass and oer necons and aerges. Wen e percenage oeosnops s 8% or ger (eosnopa), e ac mus be recorded and reporedo e cncan.

Basophls are rare, makng up ess an 1% o e oa. hey are seen as arge bueor mauve granues n e cyopasm ater Gemsa sanng.

Mononuclear leukocytes

Monocytes are e arges o e we bood ces, measurng 1218 m n dam-eer. he nuceus s arge and kdney or bean saped; e cyopasm may conan aew granues a san pnks or red. Monocyes make up 210% o e oa wece coun, and, ke neurops, ey acvey pagocyose maara parases.

Lymphocytes occur as wo ypes, arge and sma, and ogeer ey comprse 2045% o e oa we ce coun. he nuceus o a arge ympocye s round and

deep-mauve. he arge area o cyopasm sans a cear waer-bue and may conana ew mauve-sanng granues. Sma ympocyes are sgy arger an a nor-ma red bood ce. here s e cyopasm surroundng e nuceus, wc sansdark-bue and somemes amos back.

Platelets

Paees are sma, rreguary saped bodes, wou a nuceus bu w ine redgranues on a bues background. Lke eosnops, paees can be used as sens-ve ndcaors o e quay o sanng. Numberng abou 100 000 per mcroreo bood, ey usuay occur n groups o 510 bu orm arger cumps wen a boodim s poory made. Inexperenced mcroscopss may conuse em w maara

parases.

Blood in Giemsa-stained thick filmsWen a saned ck bood im s examned under a x100 o mmerson objecveand x10 pared ocuars, e vewer w see e remans o red bood ces, webood ces and paees. he we bood ces and paees ook muc e same asn n ims, excep a e cyopasm around e nuce s no vsbe.

A ck bood im consss o deaemogobnzed red bood ces, ayer on ayer na ck mass. Wen a ck im s saned, e waer n e san acs on e unpre-

served red ces, and e aemogobn n e ces dssoves no e waer. hs pro-cess s caed deaemogobnzaon. I can be observed wen an unsaned ckim s paced n a Per ds o cean waer. As soon as e sde eners e waer, e


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red aemogobn sars o low ou, eavng e ck im pae and opaque ater aew mnues. hs akes pace durng sanng, and a a remans wen sanngs compee are e remnans o red bood ces, saned we ces and paees.

he usraons beow w ep you o deny e red ces and e knds o weces a are presen, as we as paees. Un you gan experence n er den-icaon, conirm eac cassicaon w your acaor. You w noce a eseusraons, and mos o ose n s andbook, are coour drawngs. hs s be-cause, n e begnnng, s no easy o recognze saned bood eemens undere mcroscope. Cooured drawngs make easer o do so. As you gan exper-ence, you w probaby progress rom ceckng agans e drawngs o ceckngagans e mcropoograps (poograps aken down e mcroscope) sownn e Benc ads.

In e exercses a oow, you w aso become amar w areacs and boodconamnans. hese can make dagnoss o maara parases dcu, bu you wind ess dcuy as you gan n experence. Areacs and bood conamnans areaso dea w n Learnng un 8.

How blood elements appear in thick and in thin blood films

LEUKOCYTESThin Film

Thin Film

Thick Film

Thick FilmERYTHROCYTES

N = Neutrophil, E= Eosinophil, M = Monocyte, L = Lymphocyte, P = Platelets

NC = Normocyte, MC = Microcyte, PM = Polychromatic macrocyte, PC = Poikilocyte,

PB = Punctate basophilia, CR = Cabots ring, HJ = Howell-Jolly bodies, RC = Reticular clouds

and chromatoid bodies in severe anaema

N

E

P

ML

NE

N

P

L

L

M

NC

MC

PM

PCPB

CR

HJ

RC


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Learning Unit 7. Examining blood lms



Artefacts and contaminants that can cause confusion

BLOOD ELEMENTS

BACTERIA

SPORES

VEGETABLE CELLS

VARIOUS SOURCES

FUNGUS

Hyphae and spores

Crystalline pits indevitrified slide

Herring-bone scratchesin glass slide

Giemsa stain crystalsDust particles

Clouds and chromatoid debris derived fromimmature erythrocytes in severe anaemia

Isolated groups ofeosinophilic granules

Blood platelets.Lymphocyte for

comparison of size


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Notes


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Learning unit 8

Examining blood films for malaria

parasites

Learning objectives

By oowng e seps n s Learnng un and meeng eac o e sandardsesabsed or your course, you w be abe o:

nameO the parts of a malaria parasite correctly;

distinguishO malaria parasites in thin and in thick films, identifying

trophozoite, schizont and gametocyte stages;

identifyO , in thin and in thick films, the four human species of

malaria parasite, P. falciparum, P. vivax, P. malariaeand P. ovale;

describe and demonstrateO , in thick and thin blood films, the

main morphological differences between the four species of

human malaria parasite;

demonstrateO common contaminants seen in blood films that are

often mistaken for blood components or malaria parasites;

recognizeO and name other blood parasites common to humans inyour area; and

describeO ways of preventing some artefacts from contaminating

blood films.

Accuracy connues o be an mporan eemen o your work. To aceve andmanan e se eves, you w ave o examne a range o bood ims or pracceand o gan experence. In addon o e advce avaabe rom your acaor, sLearnng un conans an exensve range o dagrams, and e mcropoograpsn e Benc ads can be consued. hese w ep you work on your own, beore

conirmng your ina dagnoss w e acaor.

Aoug e eve o accuracy expeced o ranees aendng s course were seseparaey1 and are no descrbed ere, e eve w be 90% or dsngusngmaara parases n n and ck ims and denyng ropozoe, sczon andgameocye sages and 80% or denyng e our uman speces o maara para-se. he eve aaned by ranees w be assessed on e bass o er examnaono a sandard se o sdes provded by e uor. We ese eves may seem g,ey are a e ow end o e range. As your experence, knowedge and sks m-prove, your accuracy w ncrease sgnicany. W s approac and connu-ous pracce, you w no ind dcu o aceve an accuracy o 80% or some

acves. Many parcpans w consseny reac ger eves o compeence.

1 WHO. Malaria microscopy quality assurance manual. Manila, Western Pacific Regional Office, 2009.


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Recognition of a malaria parasite

Gemsa san coours eac par o a maara parase dfereny. W good san-ng, s easy o dsngus e pars sown n e dagram.

s of a malaria parasite in a red blood cell

Cytoplasm (blue)matin (red)

uole (clear)

Host redblood cell

Pigment (golden brown to black)

Stippling (pink)

Bufered waer a a sabe pH 7.2 s essena or good sanng o parases and wece eemens. Wou a sabe pH, e sanng w vary wdey and, dependng on

e pH, can be smar o ose n e dagram beow.Effect of pH on parasite morphology

Maara parases pass roug a seres o deveopmena sages, durng wc ersape vares wdey. Nevereess, e same pars o e parase san e samecoour a eac sage.

ChromatnO , par o e parase nuceus, usuay round, sans brg red.

CytoplasmO sans bue; e one o bue may dfer beween speces and ssomemes a dferenang caracersc.

PgmentO s a granuar by-produc o parase grow. I does no ake up

san bu vares n coour rom goden-brown o back. he coour and szeo pgmen granues vares accordng o e speces and, w coour, s otencaracersc.

StpplngO , spos, dos or cets are descrpons o e efec a e paraseas on e os ce, wc s empaszed by good sanng. he bes known andeases o demonsrae s Scufner sppng, e mass o pnk dos a appearso i some P. vvax-paraszed red bood ces. In P. ovae necons, e amos

pH 6.4 6.8 7.2 7.6


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Learning Unit 8. Examining blood lms for malaria parasites

mauve sppng, wc can even obscure e parase se, s caed Jamesdos, aoug mos workers connue o use e erm Scufner sppng odescrbe . Oer dos or cets, suc as Maurer cets, seen n some paraszedces n n ims oP. acparum, are ess easy o demonsrae and depend on

e quay o sanng.

Stages of the malaria parasite

Durng ese pracca exercses, you w irs earn o recognze maara parasesand er sages n n bood ims. hs s an exacng ask, and you w be en-couraged o pracse and work on your own as muc as you can.

To ep you roug ese exercses, use e n and ck im key on pages 5556and e dagnosc ads n Fgures 3, 4 and 5 and Paes 48 o s andbook. Asyou become more amar w wa you see, you w ind a e poomcro-

graps n e Benc ads w be o ncreasng ep. Un you are more experenced,connue

Basic Malaria Microscopy - Part 1 Learner's Guide, 2nd Edition, WHO, 2010

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