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·LABORATORY TECHNIQUE
FOR THE
STUDY OF MALARIA
Ву
PERCY G SHUTE
MOE FRES
and
MARJORIE E MARYON
FRES
Malaria Reference Laboratory Florion Surrey
Foreword by
SIR GORDON COVELL
BD DPI1
Adruser on Afolaria to the Ministry of Health
SITH 25 ILLUSTRATIONS
ANTONS
J&A CHURCHILL I TE
304 GLOLCESTER PLACE. TOVO 11
1960

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I&A CHURCHILL LTD 1960

·FOREWORD The treatment of neurosyphilis by malarsatherapy introduced in Austria by Wagner Jauregg in 1917 was first used in Great Britain In 1922 in the following year on the advice of Lt. Col S P James Adviser of Tropical Diseases to the Ministry of Health it was decided to provide facilities for the routine treatment of neuro syphletic patients by malariatherapy throughout the United hingdom From 1923 to 1925 batches of mosquitoes caught as adults from buildings in the low lysng areas of hent and Essex Mere fed on patients suffering from induced malarta in vanous hospitals mostly in the London area The mosquitoes were then taken to the Ministry of Health in Whitchali ndividuals from each batch being dissected from time to time until sposozostes were found in the salvary glands Requests from hospuals were then met by taking infected mosquitoes to them and allow ng them to feed on the patient requiring malanatherapy With the increasing demand for infective material this make shirt arrangement proved unsausfactory and a was then decided to estabish a special malana laboratory in the grounds of a mensal hospital The Manor Hospital at Epsom which had been used for the treatment of tropical discases during Warld War I was first considered as a location but since this was now occupied exclusively by mental defectises another hospital Horton situated in the same area was eventually selected A small villa in its grounds containing 12 beds was taken over a treatment ccnire and two rooms in it were fitted up for laboratory work Mr P G Shute who had been 33MICS s assistant since 1918 and had previously worked in the laboratory of the Manor Hospital under James and Ronald Ross has been in charge of the llorton Laboratory stace its inception Tor the last 21 years he has been assisted by Miss 11 Maryon who has been co author with him of a number of sciencific Popert In order to fulfil the functions of the laboratory it has been necessary not only to infect mosquitoes but to deep them ativt for several weeks beyond the time shot the salsary glands ·FOREWORD become infected so that they may be transported to hospitals in any part of the country and used to infect patients requiring malariatherapy It has also been necessary to breed and maintain large numbers of mosquitoes in the insectaria so that they may be available at short notice when patients undergoing malariatherapy are found to have gametocytes in their blood The practice of malariatherapy has rendered it possible to study in detail the natural course of primary attacks of malaria in non immune subjects and of relapses and a wealth of clinical material has become available for research in chemotherapy parasitology pathology and immunology Advances have also been made in the study of mosquito ecology and of malars carrying properties of different species of mosquitoes from many parts of the world These activities have involved the use of a large number of techniques both for the establishment and maintenance of laboratory colonies of mosquitoes and the study of the malaria parasite in all its phases in the mosquito and the human host In some cases new techniques have been evolved while in others some modification of an existing technique has led to improve ment in the results obtained It is the purpose of this book to describe the techniques which have been adopted for routine use in the laboratory The authors do not claim that all these are original but merely that they or the modifications introduced by them have proved useful in their hands They are aware that other methods are used by other workers which may be of equal value or even superior to those here described If they have not been mentioned it is either because the authors have not tried them or having tried them have not found them entirely satisfactory I am confident that the techniques described in this book representing as they do the results of 35 years of experience of laboratory and field work will be of great practical value to students of malarıology and to all those engaged in the study of insect borne diseases GORDON COVELL ·PRFFACE Tus book has been written as the result of numerous requests from workers in various parts of the world many of whom have been given facilities for study at various periods since our laboratory was established 33 years ago During this period more than 13 000 patients have been infected with one or other of the four species of human Plasmodia involving the use of some 100 000 mosquitoes most of which were bred in the laboratory The monkey Plasmodium P knowles, was also used to infect a limited number of patients during the period 1934-35 Pertinent references are cited at the end of ench section and the titles of a number of other papers published from this laboratory which contain details of special techniques are listed at the end of the book PGS MM ·ACKNOWLEDGEMENTS It is difficult to express adequately our thanks and appreciation to Sir Gordon Covell for the valuable help and advice he has given us while writing this book We are grateful too to Sir Richard Christophers for his help and advice with the chapter on sectioning mosquitoes We wish to express our thanks to Mrs E Jones for the typing of the manuscript and for her valuable help in collecting material from our record books Finally we are greatly indebted to the Wellcome Trust for financial help and to Colonel Bozman and Dr Duggan of the Wellcome Museum for kindly preparing most of the photographs ·CONTFNTS Y FOREWORD by Sir Gordon Covell PREFACE ti SECTION 1-PREPARATION AND STAINING OF BLOOD FILAIS Techniques for thin and thick blood films with modifi cations of the Romanowsky stains 1 15 16 17 21 25 SECTION 2-MISCELLANEOUS LABORATORI PRO CEDURES Preparing blood films for demonstrating exflagellating male gametocytes Preparing specimens of ookinetes or vermicules Technique for dissecting the salivary glands and stomach of a mosquito To male permanent and stained preparations of sporo- 201te and infected mid guts Sectioning and staining secuons of mosquitoes Preparing specimens of sporozoiles from rupturing oocysts Species diagnosis of human malarial oocysts l'osition of malaria! oocysts on the stomachs of mosquitoes Rosss Black Spores Technique of counting sporozoites and inducing malana by injecting them intravenously Culture of the malaria parasite Preparing sections and smears of post mortem and biopsy material 30 33 39 41 43 47 48 ·ACKNOWLEDGEMENTS It is difficult to express adequately our thanks and appreciation to Sir Gordon Covell for the valuable help and advice he has given us while writing this book We are grateful too to Sir Rickard Christophers for his help and advice with the chapter on sectioning mosquitoes We wish to express our thanks to Mrs E Jones for the typing of the manuscript and for her valuable help in collecting material from our record books Finally we are greatly indebted to the Wellcome Trust for financial help and to Colonel Bozman and Dr Duggan of the Wellcome Museum for kindly preparing most of the photographs ·CONTENTS FOREWORD by Sir Gordon Covell V PREFACE 11 SFCTION ) - PREPARATION AND STAINING OF BLOOD FILMIS Techniques for thin and thick blood films with modifi cations of the Romanowsky stains 1 15 16 17 21 25 SECTION 2-MISCELLANEOUS LABORATORY PRO CEDURES Preparing blood films for demonstrating exflagellating male gametocytes Preparing specimens of Ookinetes or sermicules Technique for dissecting the salıvary glan and stomach of a mosquito To make permanent and stained preparations of sporozoites and infected mid guts Sectioning and staining sections of mosquitoes Preparing specimens of sporozoites from rupturing oocysts Species diagnosis of human malarial pocysts Position of malarial oocysts on the stomachs of mosquitoes Rosss Black Spores Technique of counting sporozoites and inducing malaria by injecting them intravenously Culture of the malaria parasite Prepunng sections and smears of post mortem and biopsy material 30 33 39 41 47 48

·r
CONTENTS
SECTION 3 ESTABLISHING AND MAINTAINING
COLONIES OF ANOPHELES IN THE LABORATOR
General Principles
S3
Laboratory rearing of Anopheles stephensı
55
Laboratory rearing of Anopheles maculipennis arropan us 61
Methods of obtaining eggs from individual mosquitoes 64
Laboratory infections of Anopheles with malaria 66
SECTION PRACTICAL RINTS
74-80
SELECTED BIBLIOGRAPHY
80
INDEX
85

·SECTION 1 PREPARATION AND STAINING OF BLOOD FIL 115 Glass slides used for making blood films Slides known to the trade as half white are satisfactory 76 mm x 25 mm (3 in x 1 in) x 08-1 mm thick with ground smooth edges It is essential that the slides used for both thin and thick films should be grease free scratch free clean and dry If a slide 15 greasy a thin film will not spread evenly and a dried blob of blood will peel off a thick film during the process of staining We find that with new slides all that is required is to wipe them with a clean cloth and pass them through a mixture (eqval parts) of ether and methyl alcohol For used slides we find that soaking them in 25 lysol for a few days removes the old film They are then thoroughly rinsed in hot water to remove all races of lysol then soaked for a day or two in 1 / tecpol again thorouehly rinsed in hot water and dried with a clean grease frec cloth Some slides retain their transparency for a long time but some become rogey and mottled with brownish paiches which cannot be remored because the discoloration is in the glass and not on the surface these should be discarded THY BLOOD FILMS Technique of mahing thin films Aſier cleaning the pad of the finges of lobe of the car the skin should be wiped dry Puncture the skin with a straight tnangular surgical needle and wipe off the first drop of blood A bead of blood for a thin film should be no larger ihan the head of a pin If a slide is used for spreading the film soc that it is a thin one and has a smooth edge Contact of the side with the bead of blood should be at an angle of 45 degrees and the film should be made by pushing the spreader away from the blood The aim in making a thin film should be to have the corpuscles only onc decp the cells should almost touch one another but not overlap Good then films cannot be made of a thick slide is used for spreading the film of the drop of blood is top large or of the blood has been ·2 PREPARATIOY AND STAINING OF BLOOD FILMS spread 100 rapidly The sude used as a spreader should have a smooth edge otherwise gaps will occur in the film Leishman stain METHODS OF PREPARATION There are three methods of preparing the stain (1) 05gm of dry crystals are placed in a mortar small quan filies of methyl alcohol are added and pounded with the pestle and the supernatant fluid is poured into a clean dry boule of hard glass with a ground glass stopper Then more alcohol is added by degrees until all the crystals are dissolved and finally made up to 100 ml Stain prepared in this way is ready for use within 24 hours Letshman stain should nerer be filtered (2) 100 ml of methyl alcohol is poured into the bottle and 0-15 gm of crystals added Several times throughout the day the bottle containing the stain is rocked to prevent the dye settling and slicking to the bottom of the container The stars is ready for use within 3 or 4 days (3) 0 15 gm of crystals are reduced to pouder with pestle and mortar Small quantities of methyl alcohol are added and the mixture is surred with the passle further methyl alcohol being added until she 100 ml is used when all the dye will have been absorbed The stain is ready for use within an hour It will be found that a small putty like residue remains but this can be Temoved and discarded of the abole methods of preparing Leishman s stain we prefer No 3 For demonstrating the suppling in host cells of all species of mammalian malana parasites Leishman s stain is excellent pro viding the correct techmque is employed (James! >> Unfor tunately however there is considerable variation in the quality of different brands of Leishman stain and therefore samples of each should be tried unul de dest is found for staming malanta parasites Leishman saja should aluays be prepared in the laboratory and never purchased a solution The dye keeps indefimtely and we are still getting satisfactory results with stain prepared from dry crystals purchased over 20 years ago We are aware that in the tropics Leishman stain is not now extensively used at least not in malaria suneys There are several reasons for thus The prepared stain does not keep ery long at hsgh tem peratures At 27 C 1 deteriorates in a week or to Howeves ·TECHNIQUE FOR STAINING WITH LEISHMAN 3 at 16-17 C it retains its full staining properties up to one year some technical knowledge is required to obtain the best results and it is not as suitable as Gremsa for field surveys After many years of trials and experiments with this stain we believe we have cvolved a technique which gives consistent results and that for staining malaria parasites and the stippling in the host cells in thin films Leishman is the best of all the Romanowsky modifica tions Our technique of staining is as follows On a shelf on the right hand side over the laboratory sink we keep a 5 litre aspirator of distilled water On the same shelf we kecp a jar containing two standard tubes of phenol red pH 70 and pH 72 a cordite tube marked at 5 ml onc 50 ml drop bottle containing a filtered saturated solution of lithium carbonate and another containing 0-02 / phenol red Before beginning the days work we test the pH or the distilled water in the aspirator by running of 5 ml of water and adding 0 5 ml of phenol red if the displed water is acid a few drops of lithum carbonate are added until the pH registers 72 Some is then poured into a 152 mm (6 in ) diameter petri dish exposed to the air and rested on the laboratory bench for 15-20 minutes If at the end of this time the pH 15 neutral the water is ready for use but if it is acid more lithium carbonate is added to the distilled water in the aspirator and the procedure repeated (As a rough guide when 10 drops of lithium carbonate are added to 5 litres of distilled water the pH is neutral) Sometimes depending the amount of Co, in the atmosphere it may be necessary for the distilled water in the aspirator to have a pH of eren 80 if it is to have a pH of 7-7 2 after being exposed to the air for 15 minutes which is the optimum time for starting a film if the distilled water used for diluting the stain becomes acıd within a few minutes of exposure to the atmosphere on the laboratory bench the films when flushed off at the end of the staining period will be pink Such films seldom show stippling of the host cells and moreover the cytoplasm of the parasite is poorly coloured on the other hand the water is too alkalınc (over 76) at the end of the staining period the film will be blue with consequent poor staining of the chromatin The stippling will cither be absent only faintly seen To the naked eye a washed film should be of a faint greyish bronze colour A satisfactory test is to look at the granules of an cosinophile leucocyte If the staining solution is neutral at the (imc of washing off the film the cosinophilc granules will be a pale ·4 PREPARATION AND STAINING OF BLOOD FILMS greyish colour If the solution has become acid the granules will be bright pink and if it has become alkaline the granules will be deep blue For hæmatological work eg differential leucocyte counts workers often prefer a pink coloured film But for optimum demonstration of malaria parasites the red blood cor puscles should be inconspicuous by comparison The parasites are then prominently displayed morphologically against the greyesh bronze coloured host cells The importance of the pH of the distilled water for staining with Leishman cannot be over emphasised This is illustrated by Field and Shute Some workers use buffered distilled water but we prefer the water to be adjusted to pH 72 with lithium carbonate only But it is not sufficient to adjust the destilled water to pH 7 2 without taking other factors into consideration For example in a laboratory where there are gas fires or bunsen burners exposed distilled water adjusted to pH 72 usually becomes acıd within a few minutes This also occurs of all the laboratory windows are closed day and night We have tested this on many occasions by staining one film in the laboratory and another in the open air For staining blood films we use a room where there are no coal or gas fires or bunsen burners For routine work where films are not wanted for future reference or class purposes a buffered water at pH 7 2 will save a great deal of detailed work Tablets of buffer salts sufficient for a litre of water are now available However it is our experience that films fade much more rapidly when the water is buffered and also the colouring is not quite so vivid METHOD OF STAINING Put 6 or 7 drops of stain on the film and allow it to act for 15 seconds not more This is sufficient to fix the film and if the stain is left longer than 15 seconds before diluting it especially in hot weather evaporation brings about precipitation and spouls the film Add 12 drops of distilled water (pH 7 2) and thoroughly mix the stain by gently rocking the The diluted stain is allowed to act for 15-20 minutes when it is flushed off by a stream of distilled water The flushing should occupy only a few seconds otherwise there is a risk of washing out the steppling especially the Maurer s dots of P falciparum infections In very hot weather it is advisable to fix the film with one or two drops of methyl alcohol and after it has dried stain as above ·TECHNIQUE FOR STANAG HITU LEISIVAN 5 For routine ekamation 1 slightly acid or slightly alkaline Leishman solution will not present malaria parasites from being recognisech But species diagnosis is more difficult especially with young trophozoites and in the absence of stippling Another important reason for the starning solution to be neutral at the end of the slauring period is that it helps to present fading for films which are to be kept for class and demonstration purposes 11 15 cssential that the stagin solution should be neutral at the end of the staining period There are a number of so-called neutral mounting mediums on the market and probably all arc satis factory under optimum conditions yet when the question as to which is satisfactory and which is unsafısfactory is discussed among a group of workers there is often much disagreement The real reason for the disagreement as we beliese often over looked because it may have nothing to do with the mounung medium but may be due to the film itself If the film is pink (acid) « will fade within a few weeks first the blue of the cyro plasm and then the pink of the chromatin If the film is blush (alkaline) then the chromatın ifade first followed later by fading of the cytoplasm It is quite useless to altempt to presene films of this kind whatcier mountant is used on the other hand if the disted stain is neutral at the end of the staining period and the film is then mounted with a neutral medium it will often deep for years ir This laboratory we have blood films which were stained with Leishman and mounted in Euparal sert over 30 years apo (Shute) Some of these specimens are still shoun at 1arious laboratory meetings and if fading has occurred is imperceptible The only precaution ne sake ss to keep the films away from heat and overlong exposure to strong light In our cxperience Leishman stais is unsatisfactory for thick films either when the drop of blood is first deh zmoglobinised dried and stained or when Leishman is first dituted and the film stained and dchemoglobirised at the same time as is usually pracused with Gimsa stain Although Leishman is ctcellent for staining all mammalian malang parasites in thin films and for Staining sporozoites from the salvary glands 1 series o other useful purpose is a poor stan compared with Gems for the parassles of avian malaria thick films sections smears of bone marrow post mortem material and crocrythrocytic rarasites ·4 PREPARATION AND STAINING OF BLOOD FILMS greyish colour If the solution has become acid the granules will be bright pink and if it has become alkaline the granules will be deep blue For hæmatological work eg differential leucocyte counts workers often prefer a pink-coloured film But for optimum demonstration of malaria parasites the red blood cor puscles should be inconspicuous by comparison The parasites are then prominently displayed morphologically against the greyish bronze-coloured host cells The importance of the pH of the distilled water for staining with Leishman cannot be over-emphasised This is illustrated by Field and Shutes Some workers use buffered distilled water but we prefer the water to be adjusted to pH 7 2 with lithium carbonate only But It is not sufficient to adjust the distilled water to pH 7 2 without taking other factors into consideration For example in a laboratory where there are gas fires or bunsen burners exposed distilled water adjusted to pH 7 2 usually becomes acid within a few minutes This also occurs if all the laboratory windows are closed day and night we have tested this on many occasions by Staining one film in the laboratory and another in the open air For staining blood films we use a room where there are no coal or gas fires or bunsen burners For routine work where films are not wanted for future reference or class purposes a buffered water at pH 7 2 will save a great deal of detailed work Tablets of buffer salts sufficient for a litre of water are now available However It is our experience that films fade much more rapidly when the water is buffered and also the colouring is not quite so vivid METHOD OF STAINING Put 6 or 7 drops of stain on the film and allow it to act for 15 seconds not more This is sufficient to fix the film and if the stain is left longer than 15 seconds before diluting it especially in hot weather evaporation brings about precipitation and spoils the film Add 12 drops of distilled water (pH 7 2) and thoroughly mix the stain by gently rocking the slide The diluted stain is allowed to act for 15-20 minutes when it is flushed off by a stream of distilled water The flushing should occupy only a few seconds otherwise there is a risk of washing out the stippling especially the Maurer s dots of P falciparum infections In very hot weather It is advisable to fix the film with one or two drops of methyl alcohol and after it has dried stain as above ·TECHNIQUE FOR STAINING WITH LEISHMAN $ For routine examination 2 slightly acid or slighily alkaline Leishman solution will not prevent malaria parasites from bung recognised but species diagnosis is more difficult especially with young trophozoites and in the absence of stippling Another important reason for the staining solution to be neutral at the end of the staring period is that it helps to prevent fading For films which are to be kept for class and demonstration purposes it is essenual that the staining solution should be neutral at the end of the staining period There are a number of so called neutral mounting mediums on the market and probably all are saus factory under optimum conditions Yet when the question as to which is satisfactory and which is unsatisfactory is discussed among a group of workers there is often much desagreement The real reason for the disagreement as we believe often over looked because it may have nothing to do with the mounting medium but may be due to the film self If the film is pink (acid) it will fade within a few weeks first the blue of the cyto plasm and then the pink of the chromanın 1ht fun 15 blursa (alkalıne) then the chromatin will fade first followed later by fading of the cytoplasm It is quite useless to attempt to preserve films of this kind whatever mountant is used on the other hand if the diluted stanı us neutral at the end of the stassing period and the film is ther: mounted with a neutral medium it will often keep for years in tias laboratory we have blood films which were stained with Leishman and mounted in Euparal vert over 30 years ago (Shute") Some of these specimens are still shown at varsous laboratory meetings and is fadmg has occurred its imperceptible The only precaution we take is to keep the films away from hear and Overlong exposure to strong ught In our experience Lesshman stain is unsatisfactory fos thick films either when the drop of blood is first dehæmoglobinised dried and stained or when Leishman is first diluted and the film stained and dehemoglobsrised at the same time as is usually practised with Glemsa stassi Although Lershmaan is excellent for staining all mammalian malarsa parasites in thin films and for staining sporozoites from the salivary glands it serves no other useful purpose it is a poor stain compared with Gietisa for the parasites of avian malaria thick films sections smears of bone marrow post mortens material and eroerythrocytic parasttes ·6 PREPARATION AND STAINING OF BLOOD FILMS 30 gm 08 gm Giemsa stalo Unlike Leishman stain Gremsa keeps indefinitely in all climates It is easily prepared from Gremsa powder or from the two azures separately (1) Giemsa powder 38 gm Glycerol (pure) 250 ml Methyl alcohol (certified purity) 250 ml (2) Azur 11 eosin Azur U Glycerol (pure) 250 ml Methyl alcohol (certified purity) 250 ml We prepare the stain by mixing the alcohol and glycerol and then adding small quantities at a time to the powder in the mortar and grinding until all the colour is absorbed As with Leshman crystals so it is with Gremsa ponder some brands are better than others In our experience with several brands better results are obtained when the stain is prepared from the two azures instead of from the powder already mixed by the manu facturers but why this is so we do not know It is best not to filter Gremsa stain for about a week after it is made up A putty like residue always remains however thoroughly the dye is powdered in the mortar By leaving the mixture for about a week after its preparation before filtering the maximum amount of the dye is absorbed by the glycerol and alcohol The prepared stain should be kept in a botile made of hard glass with a close fitting ground glass stopper and kept away from strong light After the films have been fixed for half a minute in methyl alcohol or one minute in ethyl alcohol they are ready for staining The dilution of 5-7 ml stain to 100 mi distilled water gives excellent results Better results are obtained when the water is neutral rather than slightly alkaline if the water is even very slightly alkaline the red corpuscles stain greyish blue but when this happens it can be corrected by drying the film after staining and then soaking it for a few minutes in normal salıne buffered 10 neutral The stippling of all the four species of human malaria parasites can be demonstrated with Giemsa stain but it is not so vividly displayed compared with the best that Leishman can do Although we are able consistently to produce Ziemann s dots in P malaria with Leishman stain we haie seldom succeeded with ·7 IMPORTANCE OF PH WHEN STAINING Glemsa even after prolonged staining He find that for thin films staining with Gremsa for 30 minutes gives best results (twice as long as with Leishmas) When films which are not wanted for future reference or for Teaching purposes are to be stained in large numbers for routine examination distilled water buffered to p/ 70 can be used The buffer salts required are the alkaline and acid phosphate potas sium dihydrogen phosphates and disodium hydrogen phosphate A buffer solution which gives a pH of 7 2 xs Potassium d hydrogen phosphate kH, PO. D sodium hydrogen phosphate Na, HPO Distilled water 1000 m2 01 gm 10 gm The solution should be tested after its preparation and if necessary a uttle more of one or the other salts added Once a cortect pH is obtained the solution remains stable almost indefinitely Tablets of these buffer salts can be purchased either for 103 mi or 1000 ml of water Thin films stained for from 20-30 minutes give opumum results Thss procedure stains not only the parasite but also the stippling However sf a dragnosis of malaria only is required the parasites are recognisable after staining for only 5 minutes After the ${3:38g period is completed the film is Qushed with a stream of dystalica water This should not be prolonged beyond a few seconds because of the parasites are young trophozoites of P fokuparum the colouring of Maurer s dots may be washed out If the film is too blue we find it best to dry it and then soak it for a few minutes in normal salspe buffered to neutral We recommend driuting Gremsa stain with distilled water which has a pH of 70 battered if for routine work but for specy mens which it is intended to keep for future reference or for class work the distilled water should be brought to a pH of 70 with an alkalsne reagent such as lithium carbonate Glemsa çalıne for thin mms Pampana was the first to show that Glemsa diluted with saline gave better results than Gemsa diluted with distilled water for thick films but he made no mention of thin films Later Field ? based his raped method for thick films on Pampana s findings but he tos did not menuen thin films It was qune by accident that on one occasion we used saline instead of distilled water when ·8 PREPARATION AND STAINING OF BLOOD FILMS making up Glemsa to stain some thin films When later we examined the films (Pinar) we were surprised to see a type of staining quite unlike our routine Gremsa stained specimens The uninfected red cells were semi transparent the leucocytes and the malaria parasites were deeply and beautifully coloured and the stippling was so prominent that the granules could be counted Later we were able to trace the cause of this unusual picture and so we decided to carry out a series of tests with both thick and thin films (Shute) For staining thin films following fixation with methyl alcohol in the usual way Giemsa stain is diluted at the rate of 5-7 ml of stain to 100 ml of salıne (085 per cent) The parasites are nicely stained aſter about 30 minutes but best results are obtained when films are stained for one hour There are a number of features about this method of staining all of which appear to be advantageous (1) The length of time thin films are left to stain seems to be quite immaterial (beyond the minimum of 30 minutes to 1 hour) Beyond that time no changes occur and even when the films are left in the stain for a week the picture is the same as when the films are stained for an hour (2) The pH is not very important at least providing the water is not acid For example tap water is just as satisfactory as distilled water and films stained with tap water saline and flushed with tap water are as clean and as well stained as those with distilled water The tap water we have tried was the local Epsom main supply By the courtesy of the Epsom Borou n Engineer we are able to give the analysis pH 72 Hardness Total 255 Free carbon dioxide 18 Total solids 370 Alkalınıty as calcium carbonate 245 A further series of tests were carried out using a local supply from the nearby town of Leatherhead The analysis of this water is pH 91 Hardness Total 94 Free carbon diorde NII Total solids 153 Alkalinity as calcium carbonate 46 ·TECHNIQUE OF STAINING WITH Gigysa SALINE Films stained with Gremsa and Legshman diluted with this water are quite useless the red cells stare deep blue the parasites are only faintly coloured and the whole film is covered with fine granule deposits Yet when this water is made saline the staining results with Gremsa are quite good partscularly when the pH is around 7-74 For staining films in bulk this method is excellent in fact we have used the same solution of stain on as many as five occasions om the same day staining a hundred films with 100 ml of stain The only disadvantage is that all our efforts to demonstrate Maurer s dots in P falciparum have failed even after prolonged staining and when as a control these dots have been beautifully demonstrated by Leishmans stain We have no explanation to offer why this should be so However it is interesting to remember that Maurer s dots have never as far as we are aware been demonstrated in thick films by any method of staining and we are therefore inclined to doubt whether Maurer s dots of P falciparum and Schuffner s dots of Pear are produced by the same mechanisms (Shuie and Afaryon) The reasons wisy saline Gremsa gives so much better stasning results than Giemsa diluted with distilled water we do not know It has been suggested by Sur Rickard Christophers that salınc is a better protein solvent than distilled water and that it probably removes something from the film which remains after treatment with water THICK BLOOD FILMS Technque of preparation There is a tendency to make thick films too thick The drop of blood should not be more than twice or at most three times the size required for a thun film The blood should be spread to cover about three times sis original area and this is best done with the corner of another clean slide A simple test to estimate the correct thickness of a film is to place the blood over a piece of newspaper with small print or over the face of a watch The small print or figures round the deal of the watch should just be visible if sot then the drop of blood is too thick if seen too clearly st us too then Some workers advocate a very thick film that has about 20 leucocytes per micro field (2 min 03 mmersion lens with a x5 or 6 ocular) This represents 20 to 10 times the ·10 PREPARATION AND STAINING OF BLOOD FILMS thickness of a thin film We deplore this advice because (1) scanty parasites especially P falciparum rings could easily be hidden beneath a leucocyte (11) the drop of blood is likely to peel from the slide during staining and (111) more intensive and prolonged search of each micro field becomes necessary On the other hand with an average of about 10 leucocytes per micro field the above disadvantages are not encountered After the film is prepared it must be put aside to dry in a horizontal position and covered to protect it from dust and flies It is often stated that as soon as a thick film is dry it is ready for staining This is not our experience and we find that some heat is required to ensure that the drop of blood will adhere to the slide during the staining process If a thick film is to be stained within an hour of taking it we put it on the top of the microscope Jamp where the temperature is 50-60 C and leave it for 7 minutes Too much heat will of course fix the film and prevent the hemoglobin from dissolving during staining Thick films which have been kept overnight in the incubator at blood heat usually adhere to the slide (Shute) Technique of staining thick films There are four recognised methods of staining thick films all of which we have used in this laboratory There is also a fifth method JSB (Singh and Bhattacharjı') which many workers have found useful but which we have not This may be because we have not mastered the technique but since many workers have obtained satisfactory results we are giving the formula METHOD 1 Staining thick films with Gremsa stain diluted with distilled water The dilution of 5-7 ml of stain to 100 ml of distilled water adjusted to neutral is used The blood films are covered with the stain without previous dehemoglobinisation and the stain is allowed to act for from 20-30 minutes The films are then gently flushed with distilled water care being taken not to let the stream of water come into direct contact with the blood otherwise there is a risk that the films will be washed from the slide After they have dried following staining it is best to soak them for a few minutes in saline to remove the surface residual stain and the envelopes of the lysed cells These stain greyish blue and so reduce visual contrast and make examination tedious ·THICK FILM STAINING TECHNIQUE METHOD 2 Giensa stain diluted with saline buffered to pH 72 3-7 ml stain to 100 mi saline (085 sodium chloride) Stara for 30 minutes to I hour Pampanat was the first to draw attention to the use of an isotonic solution for diluting Giemsa stain for thick films He ponied out that leucocytes and protozoa were undama ed and that red cells lysed more readily than when distilled water was used The technique is the same as that used for ordinary Gsemsa statning The stairs is diluted and thoroughly mued and poured into the vessel containing the slides We use glass containers with Ogrooves which hold 100 ml if thin slides are used each groove will hold two and these are placed back to back thus each container will hold 20 slides The films are starned for 30 minutes and the stain is then gently flushed off in the usual way In P trar and P orale the stippling is vuidly coloured but as with all other techniques of staining thick films of P falciparum Maurer s dots are not seen (Shute and Maryon) METHOD 3 Field s stan's This consists of two solutions 08 gm Solution A Methylene blue (med nal) Ature? D sod um hydrogen phosphate (anhydrous) Posassrum dihydrogen phosphate Dist Hled water 05 em 50 gm 625 gm *DX0 ml Sant B Eosin D sou um hydrogen phosphate (arhydraus) Potass um d hyd o en phosphate stilled water log 50 gm 6 25 pm 500 ml The phosphate salts are dissolved and then the stain is added Leave for 23 hours and then filter if the anhydrous salt ss not available sodium phosphate cryst BP (Na HPO, 12H,0) 126 grammes can be used ·12 PREPARATION AND STAINING OF BLOOD FILMS Method of staining On the bench by the sink place 3 wide mouth staining Jars 101 mm x 38 mm (4 in x plin) Number 1 Solution A Nurnber 2 Buffer (without stain) Nurnber 3 Solution B (a) Dip the slide for one second in Solution A (6) Wash off the stain from the back of the slide with distilled water (c) Dip the slide in the buffer solution until most of the excess blue colouring has left the film (dl) Dip the slide for one second in Solution B (e) Wash gently for a few seconds with distilled water to remove excess eosin Place slide film downwards in rack to dry The chief advantage of this stam oler ordinary Glemsa diluted with distilled water is that the background of the film is quite clean the parasites and leucocytes are sharply defined and the leucocytes are not distorted A disadvantage is that by this technique films cannot be stained in bulk and though useful for the clinical laboratory it is not suitable for field survey work when large numbers of films have to be stained However as a method of staining individual thick films it is a great improvement on the ordinary Giemsa method Because thick films cannot be stained in bulk by Field s method for field surveys it is better to stain with Glemsa diluted with saline with the latter method the background is quite clean and the parasites and leucocytes are more vividly coloured than they are with Field s stain METHOD 4 Field s method and countersiaınıng with Leishman (Shutelt) It is only for convenience that we are describing this as a separate method the only ifference from Method 3 bein, that after the film has dried it is again stared with Leishman exactly as though it were a thin film Very beautiful results are obtained and for special purposes the extra time and labour required are well worth while The background of the film remains quile clean the parasites are intensely coloured and in pintar and ·THICK FILM STAINING TECHNIQUE Poale the stippling is stained as vividly as in an ordinary thin film All our attempts to demonstrate Maurer > dots by this technique have failed We are therefore incited to the belief that It is not possible to demonstrate Maurer s dots 10 other than thin films and only in these when the trophozonte has been attached to the cell for some tiene probably over 12 hours METHOD 5 The J.S B stan! There are two solutions Solution 1 Ale licinal methylene blue Potassium dichromate Sulphuric acid (1 by volume) Potassium hydroxide (1) Water 01 gm 0 gm 06 ml Omi 100 ml Solutt # 2 Ecs 1 water soluble) Tap wa er 10 gm 500 mi Method of staining thick films Imrerse slide in solution I for 10 s conds 2 Wash in jar containing acidulated water (pH 6 2-66) for 2 seconds 3 Stain with solution 2 for one second 4 Wash in same jar (2) for S seconds 5 Immerse in solution 1 again for 10 seconds 6 Wash as above for 2 seconds or ut smear gives a pink background (2) 7 Dry JSB method of combined staining i For the thin smear by dipping that part of the slide in a jar containing methyl alcohol for a second or two 2 Dry thoroughly preferably by waving the slide is the air 3 Immerse the whole slide in solution i for 30 seconds 4. Wash in a jar containing audulated tap water (pH 6 2-6 6) With tap water in Delhi (pH 76 srdıcator' bromo thymol blue) approumately 50 mg of sodium dihydrogen phosphate or 5 drops of 5 acetic or citric acid solutions for each 100 ml of water arc necessary

·14
PREPARATION AND STAINING OF BLOOD FILMS
S Stain with solution 2 for one second
6 Wash in same jar (4) for 4 seconds
7 Immerse in solution 1 again for 30 seconds (3)
8 Wash as above for 10 seconds or until the smear gives a pink
background (4)
9 Dry
REFERENCES
1 JAMES S P (1979) Trans R Soc frop Med Hug 23 269
2 -(1930) Trans R Soc frop Med Hys 23 437
3 FIELD J W SHUTE P G (1956) The microscopic dia, nosis of human
malaria Part 11 Plate 38 p 193 Government Press Kuala Lumpur
No 24
4 SHUTE P G (1947) Trans R Soc trop Afed Hış 41 13
S PAMPANA E J (1938) Rn di Malariologia 17 300
6 FIELD J W (1941) Inst Med Research Bull No 2 Federation of
Malav States 16
7 -(1941) Trans R Soc trop Med Hig 35 35
8 Shute P G (1955) Nature 175 727
9 SHUTE P G MARYON M (1957) Trans R Soc trop Med Hyg 51 998
10 SHUTE P G (1946) Public Health Bulletin 5 32
11 SINGH J BHATTACHARJI L M (1944) Ind Med Ga 79 107
12 SHUTE P G MARYOW M (1955) Trans R Soc trop Med Hug 49 306
13 FIELD J W (1948) The microscopic diagnosis of malaria Part 1
Studies from the Inst of Med Research No 23 Government Press
Kuala Lumpur Federation of Malaya
14 SHUTE P G (1954) Trans R Soc trop Afed Hyg 39 7

·SECTION 2 MISCELLANEOUS LABORATORY PROCEDURES PREPARING BLOOD FILMS FOR DEMONSTRATING EXFLAGELLATING NIALE CAMETOCYTES 2 Filter paper MATERIALS REQUIRED | Nests of petr dishes 88 mm (3) in ) diameter 3 Triangular pieces of 6 3 mm (in) glass rod or tubing 4 Spint lamp 5 Glass slides Prepare five or sıx petri dishes by inserting a layer of filter paper accurately cut to fit in the lid and in the bottom of cach (James') Lay a triangular piece of glass rod or tubing in each dish and moisten the filter paper with as much hor water as it will absorb but not more The filter paper should be uetted to saturation at least one hour before the dishes are to be us d Take the dishes and the slides to the bed of the patient accompanied by an assistant who will hold a separate dish ready for each blood him as it is made Clean and prick the finger make a blm slightly thicker than an ordinary thin film and at once breathe on it and quickly lay it on the glass rod in the dish from which the assistant has momentarily removed the lid The led is quickly replaced and more specimans are prepared sn the same way The dishes must be kept quite level and taken to the laboratory without delay (unless the temperature in the room where the specimens are prepared is 23 C or higher) If it is required to see the early changes in the process of exflagellation remove a slide from a dish at 5 minute intervals At 25 C cxflagellation is completed in 12-15 minutes for prilar and P o ale and in from 15-30 minutes for P falciparum After semoving the slide from the prin dish quickly wipe the under side of the slide on ite back of the hard This enabi sone to make sure that the blood film has not dried If it is still moist pass the slide once only through the frame of a spirit lamp to dry the film quickly For special cytological studies instead of passing 15 ·16 MISCELLANEOUS LABORATORY PROCEDURES the film through the flame of a spirit lamp expose it to osmic acid vapour for a few seconds If the film is dry when taken out of the petri dish this is either because the atmosphere in the dish is not sufficiently moist or because the film dried before it was put into the dish Such films should be discarded Atmospheric temperature is very important Below 15 C exflagellation does not occur at 16-20 C it may take as long as half an hour whereas at 25 C it occurs in 12-15 minutes after 20 minutes free flagella and often fertilisation of the female parasite can be seen REFERENCE 1 JAMES S P (1934) Trans R Soc trop Med Hyg 28 104 PREPARING SPECIMENS Or OOKINETES OR VERMICULES Feed some anopheles on a patient whose blood contains numerous gametocytes the males having been tested for ripeness (exflagellation) Select and transfer to a very small cage mosqui toes which have gorged themselves with blood and keep them in a room where the temperature is 24 -27 C and the humidity is relatively high (80 per cent) At intervals up to 24 hours remote one or two mosquitoes and proceed as follows Anästhetise the mosquito and remove its legs and wings (stunning the mosquito in the test tube will nearly always rupture the stomach and spoil the specimen because the blood will stick to the inside of the test tube) Dissect the mid gut in saline or Locke s fluid by nicking the chitin on both sides of the 5th or 6th abdominal segment so as to draw out the mid gut containing the blood clot without tearing the wall of the stomach Cut away the malpighian tubules bit the clot on to the point of a dissecting needle and transfer it to a drop of Locke s fluid previously placed on another clean slide about one inch from one end of it with the aid of the dissectin microscope against a white background tear the clot so that the blood becomes mixed with the Locke s fluid The released blood should be confined to as small a space as possible so as to present any part of it from drying when the blood and the fluid are thoroughly mixed make thin films on clean slides in the same way as if the material were a drop of finger blood After the films have dried stain them in the same way as a blood film with either Glemsa or Leishman ·TECHNIQUE FOR DISSECTING MOSQUITOES 17 Search for ookinetes with the 2 mm (it in otammersion lens The amount of saline used should be about the same volume as the blood clot Some dilution but not too much is necessary b cause the blood being semi digested and coagulated contains much debris and fundiluted examination would be difficult (James') It should be noted that ookinetes wat form in the gut of anı blood sucking arthropod The very large culiline Theobaldia annulara takes at least twice the quantity of blood that is imbibed by most anophelines and we have often prepared as many as 16 blood stars from the diluted stomach content of a 7 annulata which had fed on a gametocyte carrier 24 hours tarker for demonstration purposes REFERENCE 1 Immes S P (1934) T ans sac top Med H18 28 107 TECHNIQUE FOR DISSECTING THE SALIVARI GLANDS AND STOVACIL OF A WOSQUITO MATERIALS PEQUIRED I Ordinary glass slides 75 mm x 25 mm (3 in x jin) 2 19 mm (in) No I square cover slips 3 Two bayonet shaped dissecting needles 4 Dissecting mucroscope 5 Ordinary microscope with mechamcal stage removed 6 Test tubes 127 mm x 127 mm (5 in x 'in) 7 Watch glass containing normal saline 8 Chloroíorm or ether Transfer the mosquito to be dissected to a narrow test tube and when the insect is at the bottom rap the end of the tube sharply against the palm of the hand five or six times This is sufficient to stun the mosquito which is then removed to n glass slide Holding the insect by one wing remove the legs one at a time and afterwards pull off one wint Return the mosquito 10 the slide and with a dissecting needle cut off the remaining wing The insect is nowd void of wings and legs and ready for dissection (Shule) Place the insect in the centre of a dry slide with the anterior end pointing to the right and with a needle in the right hand cut off the head sharply and cleanly Place a drop of sahne (the size ·18 MISCELLANEOUS LABORATORY PROCEDURES of a pin s head) quite close to but not touching the anterior end of the thorax The point of the dissecting needle held in the left hand is gently but firmly planted on the thorar just below th parts where the glands fie With a needle in the right hand bring gentle pressure to bear on the thorax a little above the left hand needle Very little pressure is needed to express the glands from the thorax As soon as this occurs touch the bead of salıne with NIE Y Law mid gut Fig 1 The dissection of a mosquito the point of the right hand needle and bring it into contact with the glands Next remove the mosquito and transfer it to the watch glass containing the salıne to awant the dissection of the Transfer the slide containing the salivary glands to the platform of the microscope and bring the specimen into focus with the low power (16 mm -- in lens) Reduced light should always be used and the glands should be in the centre of the slide When the glands are in focus and in the centre of the microscopic field drop a 19 mm (8 in) square coverslip on to them at an angle so that one corner of the Loverslip is over the glands and the opposite ·TECHNIQUE FOR DISSECTING MOSQUITOES 19 comer protruding beyond the slide The coverslip should be dropped sufficiently heavily on to the glands to rupture the cells without desplacing them The object of applying the coverslıp at an angle is to prevent the saline from becoming too widely distributed and so make the examination easy and quick as the glands and their contents are concentrated in a very small area Focus the specimen with the 4 mm (din) lens and cramine for sporozoites Instead of stunning the mosquito the insect can be anæsthetised with ether or chloroform by dropping two or three drops on to the plug of cotton wool covering the mouth of the test tube This is preferable of the mosquito has blood in the stomach or contains ripe eggs Care should be taken not to use too much chloroform or ether otherwise some of it may wet the mosquito and cause partial fixation and so make dissection difficult To dissect the mid gut If the glands have been dissected with care the remainder of the insect will be undamaged Lift it from the watch glass con taining the salıne on to the centre of a glass slide The insect should be lying on a drop of saline with the thorax pointing to the left Steady the thorax on the slide by transfixing it with the point of the left hand needle With the right hand needle nick the chitin above and below on either side of the 6th or 7th abdominal segment Gently pull the detached segments with the right hand needle until the mid gut and the attached malpighian tubules come into view Sever the alimentary canal sufficiently far forward to bring away a portion of the fore gut if the correct quantity of salıne has been used the malpighian tubules will tend to float backwards leaving the whole of the mid gut clear Anchor the gut by placing the point of the left hand needle on the fore gut and with the right hand needle cut through the alimentary canal at the junction of the mid and hind gut severing the base of the malpighian tubules at the same time Remove the hind gut and malpighian tubules so that only the mid gut (with the attached ta, of esophagus) remains on the slide Let one edge of the coverslip rest on the slide but not in contact with the stomach and the opposite edge rest on the point of the dissecting needle which is then gradually lowered till the coverslıp comes to rest on the stomach If the stomach is lying on too small a drop of saline the weight of the coverslip will rupture the stomach cells ·20 MISCELLANEOUS LABORATORY PROCEDURES and this will spoil the preparation and make examination for oocysts difficult On the other hand if too much saline is used the stomach will not flatten sufficiently in this case some of the excess saline can be removed by holding a strip of filter paper against the edge of the coverslip To examine for oocysts Place the slide containing the specimen on the platform of the microscope and examine with the 16 mm (3 in ) lens with reduced light (it is best not to use the mechanical stage but to manipulate the specimen with the index finger and thumb of the left hand) If large oocysts are present they are easily seen with the low power of the microscope Failing this examine with the 4 mm (tin) lens and if no oocysts are seen eramine with the 2 mm Gin) oil immersion lens for this it may be necessary to use the mechanical stage of the microscope Mosquitoes which are infected and kept at 25 C show oocysts under the following magnifications 3-4 days 2 mm ( in ) lens x No 6 eyepiece 5-7 days 4 mm (in) lens x No 6 cycpiece 16 mm (fin) lens x No 6 eyepiece It is often easier to detect the very young oocysts 3-4 days old than those which are about half grown in the very young oocysts the pigment is concentrated within a very small sphere whereas in those which are half grown it is distributed over a much larger area of the cyst and much of it is obscured by the dividing nucle: There should be no difficulty in detecting large oocysts because their sporoblast or sporozoite contents are prominent Reduced light should be used throughout When large numbers of mosquitoes are to be dissected it is a good plan to have on the laboratory bench a large sheet of white paper and a piece of black paper about 152 mm (6 in ) in diameter covered by a piece of plate glass For removing the legs and wings of a mosquito a white background is helpful but for seeing clearly the dissected tissue (stomach and glands) a black background is necessary There should also be a black platform on the dissectin microscope for the same reason As regards the type of dissecting microscope the simple model with a x 10 cyepiece and arm rests appears to us to be quite adequate it leaves plenty of room for manipulating the needles and the magnification is sufficient for the tissues to be seen clearly With this type of microscope 8-11 days ·PERMANENT PREPARATIONS OF GUT AND GLANDS 21 *c have frequently dissected 200 mosquitoes in a day without undu fatigue He cannot see any advantage to using binocular microscopes for these dissections and after ertensive trials we consider that they slow up the work and increase fatigue How ever the simple monocular microscope mentioned above is only 157-203 mm (6-8 in ) high and when large numbers of mos quitoes are 10 be dissected it is advisable to rest the instrument on a block of wood or other material about 101 mm (4 in) thick to prevent having to stretch the neck at an awkward angle for long periods It is also an advantage when carrying out many dissections to have two small petri dishes each containing a wet plug of cotton wool one for the right hand needle and one for the Ielt Following a dissection the head and proboscis of the mosquito usually adhere to the right hand needle and the reo skeleton of the abdomen to the left hand needle The wet plugs of cotton wool enable one quickly to wipe the needles The dissecting Needles With practice almost any type of needle can be used but one which is well designed facilitates speedy and clean dissections The pattern wc prefer is made of stainless steel bayonet shaped with a sharp Lutting edge and a rubber covered hand! This is important because of the blade becomes rusty the dissected tissue will stick to it The edge of the needle is sharp making it easy to sever the tissue while the rubber holder ersures a firm grip REFERENCE I SHUTE P G (1940) Ri di Atalarial 19 16 TO MAKE PERMANENT AND STAINED PREPARATION OF SPOROZOITES AND INFECTED MID GUTS MATERIALS REQUIRED (For SPOROZOSTES) 1 Leishman or Giemsa stain 2 Euparal I No 1 coverslips 19 mm in diameter Before staining make a circle surrounding the specimen with a blue grcase pencii on the reverse side of the shde. The makes it casy to locate the specimen when the time comes to examine it This type of d ssecting needls dess ned to our specfication is fanu a tured by Bard & Tatlak Ltd Chadwell Hicath Esset and is catalogued The Shute D s.cctun Needle

·22
MISCELLANEOUS LABORATORY PROCEDURES
Fig 2 Equipment and arrangement for mass
dissections of mosquitoes
Fly 3 Pair of dissecting needles and petri dish
contatning a wad of wet cotton wool
Fig 4

·PERMANENT PREPARATIONS OF GUT AND GLANDS 23 Sporozoites stain equally well with either Leishman or Gremsa The technique is the same as for blood films but it is important that the pH of the distilled water be adjusted to neutral or pH 71 72 otherwise the nucleus and the cytoplasm will not be prominently displayed If the specimen is worth keeping mount it in Euparal vert with 2 19 mm ( in ) drameter No 1 coverslıp MATERIALS REQUIRED (FOR MID GUTS) 1 Sabine 2 Bles fixatıc 90 ml 70/ alcohol 7 ml formalın 3 ml acetic acid (glacial) 3 MAYER S hemalum (Pantin? 1948) in 1000 ml distilled water dissolve i gramme hæmatoxylın 02 gramme sodium iodate 50 grammes potassium alum Shake frequently until the solution is blue violet (some hours) add 50 grammes chloral hydrate 1 gramme citric acid crystals The solution now turns red violet Keep in a hard glass bottle Mayer shemalum is a progressive self differentiating stain nuclei blue cytoplasm pink 4 Carbol xylol 30/ phenol dissolved in xylol 5 Canada balsam 6 Coverslips No 1 19 mm (in) diameter Dissect the gut in normal saline in the centre of a clean slide as already described Then proceed as follows See that the gut is floating on the saline if it is sticking to the slide it will rupture when the coverslip is applied With a dissect ing needle gently lower a 19 mm (in) diameter coverslip on to the specimen to flatten it if it is dropped on heavily the gut will rupture and be spoilt With practice the right quantity of saline Tequired to flatten the tissue will quickly be learnt if there is too much salınc the gut will not be evenly flattened and if there is not enough it will rupture Without touching the coverslıp place one drop of Bles fixative on the slide so that it will run under the coverslıp mir with the salıne and so be in the process of fixation Leave for 2 or 3 minutes and then add another drop of firate After 5 minutes the specimen will be partially fixed and it is then 1 ·24 MISCELLANEOUS LABORATORY PROCEDURES transferred to a petri dish containing fresh Bles to complete fixation Care must be taken not to disturb the coverslip Leave for half an hour or longer and then remove the coverslıp with the aid of dissecting needles The petri dish containing the specimen should be resting on a black surface so that the gut can be seen clearly Hold the needle in the left hand so that it is just touching the edge of the coverslip Hold the right hand needle at an angle of 45 and very gently insert its point between the coverslıp and the slide The needle in the left hand must prevent the coverslip from slıdıng while the right hand needle gently raises the coverslip In most cases the gut will be found adhering to the latter If the gut adheres to the slide the coverslıp can be discarded If it adheres to the coverslıp continue lifting the latter by keeping the left hand needle steady until the coverslıp turns over with the tissue uppermost When fixation is complete the gut will become opaque Transfer the specimen to 50 / alcohol for 15-20 minutes and then for about the same time immerse it in slightly alkalıne distilled water To stain the gut In our experience the most satisfactory stain is Mayer s hemalum If the stain is allowed to act for about half a minute or less the oocysts are nicely stained and stand out prominently against the surrounding tissue cells Most other hæmatoxylins we have tried have not given satisfactory results because the depth of staining of both tissue and oocysts has been about equal With Mayer s hæmalum and also with Weigert s hæmatorylın there is better differentration between oocysts and the surrounding tissue cells We prefer Mayer s hämalum to Weigert s hæma toxylın because it keeps better and it is not necessary to prepare two solutions on every occasion After staining the specimen is blued in the usual manner and taken through graded alcohols a few minutes in each being suffi cient To ensure complete dehydration the specimen is placed in carbol xylol for some minutes and then given two or three changes of xylol every trace of carbol xylol must be removed otherwise fading soon occurs If the gut is on the slide it is mounted in Canada balsam in the usual way If however it is on the coverslıp it should be mounted as follows A drop of balsam is placed in the centre of a clean slide and the

·PREPARING AND STAINING SECTIONS OF MOSQUITOES 25
coverslıp applied but with the gut on the upper surface Then a
further drop of balsam us placed on the gut and another clean
coverslıp applied The reason for this is that the oocysts on the
upper surface of the gut are stained but not those on the under
surface
REFERENCE
I PANTIN CF A (1948) Notes on microscopical technique for zoolo ists
Univers ty Press Cambridge
SECTIONING AND STAINING SECTIONS OF MOSQUITOES
MATERIALS AND SOLUTIONS REQUIRED (PANTIN')
Fixatives
Industrial methylated spirit 74 OP
Benzene
Methyl salicylate
Methyl benzoate + 1, cellodin
Methyl benzoate + 3 celloidin
FS Longitud nal sect on of infected mosquito The gut 13 studded
with ook ysts and some are free in the lumen

·F, 6 Longitudinal section of a maxillary palp of A stephenst
showin, masses of Pinar sporozoites in the lumen
TEERIT
Fig 7 Transverse section of mid out of A stephenst
showing large numbers of mature P mar oocysts
Specimen shows the remains of a blood meal taken 36
hours before dissection

·PREPARATION OF SECTIONS OF MOSQUITOES To section whole mosquitoes Anesthetise the insect in a 127 mm x 127 mm (5 18 x 11 test tube and remove its wings and legs Hold the mosquito by one wing and remove the legs one at a time and then one wing Lay de insect on a slide and detach the remaining ring with a dissecting needle (there is no need to puncture the mosquito to enable the fitative and other funds to penetrate) FIXATIVES We find that the two following fitaires give satisfactory results and do not interfere with subsequent staining or cause brittleness t ce am fi volite 00! absolute al ohol tindustrial methylated spurt OP) 30 ml chosoft 10 ml aceta, aced (glacra) Fix for 1 ? hours no longer 2 les / ratic 90 m3 0 alcohol 7 ml foras ) mi acetic acid (glacial) Iis for 12 24 hours A Transfer to 90 alcohol (ino changes) and leave for 12 hours ar overnight 2 Absolute alcohol or 74 OP Two chan_cs at one hourly intervals C Methyl benzoate + 1 celioidin (12-24 hours) (Pantin') Ir necessary objects can be kept in C indefinitely A came? har brush is useful for transferring the specimen from one solution to another Ta embed the species Dip & square covership m melied wax and after it has cookd transfer to a metri dish and add a large drop of 3 telosdus 5 meshyl benzoate solution on the centre of the waxed covership The celloidin blob should be large enough for the tissue to rest que flat and well within the cellordin With a small camel hair brush carefully fift the mosquito from the cellodin in me thy benzoate solution and transfer it to the centre of the drop Again with a smal camel hus brush or create the mosquito so that it is 117 the position required for sectioning and then food the covership ·28 MISCELLANEOUS LABORATORY PROCEDURES with benzene to gel the cello.dis This usually takes from 1-2 hours After the war on the coverslıp has dissolsed the spera men will leave the coverslıp After a further change an benzer embed the specimen in paraffin wax (56-60°C) two changes at one hourly intervals Block out in the usual way If when cutting the sections they fail to leave the knife as a ribbon remove the block and dip it in Overheated wax at a lower temperature €5 45 C But this is not usually necessary when the temperature of tre room is 21 -24 C To prepare sections of the dissected gut Carnoy or Bles are quite satisfactory for fixation If the mosquito had a blood meal 36-48 hours previously and has not completely digested at the gut keeps its shape and the dark colour of the blood makes it easy to follow the specimen through all the various solutions and also for embedding and sectioning If Carnay is used 30 minutes is sufficient ir Bles is used 2-4 hours are necessary The procedure is then the same as for sectioning whole mosquitoes However considerable stretching of the issue occurs during the act of dissecting the gut When there fore a gut is to be sectioned it should after dissection be floated in saline for about 5 minutes to allow the tissue to contract back to normal It can then be transferred to the chosen fixative Demonstration of cokinetes in transit To foltou up the various developmental processes specimens should be prepared at 4 hourly intervals beginning 12 hours after the infectie blood meal and continuing for 48 hours For this study we prefer transverse se loss of the whole mosquito since owing to the stretching of the w3l after a blood meat there is always a risk or damage to the gut during the process of dissection with consequent loss of part of the clot Some of the pokinetes formed close to the gut wall will pass through or between the cells and become pocysts in less than 24 hours though still showing a single nucleus Olhers may be seen at night angles to the axis of the gut in the act of penetralias the wall Marked displacement of the endothekal celis may be seen at the point of penetration Ooksnetes formed in the central zone of the clot do not reach and penetrate the wall for several hours after those which happes to be near st at the time of formation Many of these become ·STAINING SECTIONS OF MOSQUITOES phagocytosed before reaching the sali Qokinetes may be found in the lumen of the gut as late as 48 hours after the infective feed and on many occasions we have seen them in the feces of mos qustocs especially in specimens collected 36 to 48 hours after the infectrve feed These findings may in part explain why when more than one species of Anopheles are fed on the same human host the number of oocysts seen in the smaller species ofien cxlceds that found in the larger We prefer Carnoys fixative whether for the whole mosquito or the dissected gut Sections may be stated either with hema toxylın or with Gsemsa For demonstration of the nucles in very young oocysts the modification of Gemsa recommended by Short and cooper' for staming exo en throcylic parasites is to be preferred Salmary glands Sections of the salivary glands can be prepared in the same way as for the dissected gul However these are best displayed by cutting longitudinal sections of the whole mosquito An important advantage of this technique is that complete dehydration and clearing is obtained without the use of xylol also the time required for the issue to remain in absolute alcohol is quite short by comparison wnh other techniques and so there is no risk of the tissue becoming brstile Staining For stalning sections of mosquitoes with malarial oocysts well matured Ehrlich s hämatorylin is excellent I Stain with undiluted Ehrlichs hematorylin for 30 minutes for longer) 2 Wash 1 water (a few minutes) 3 Soak int HC) in 70 alcohol (a few seconds) * Wash is running water for 5-10 minutes and then leave for a further half hour in vates which has been made alkaline by adding a few drops of a saturated solution of lithium carbonate 5 Upgrade in alcohol 50 75 and 90 a few minuies in cach 6 Counterstain in 0 0 alcoholic erythrosine 15 minutes of longer 7 Absolute alcohol (industrial 54511 740P) for 15 minutes 8 Carhol vylol foc 15 minutes ·30 MISCELLANEOUS LABORATORY PROCEDURES 9 Xylol (tuo changes until clear) All traces of phenol must be washed out by xylol otherwise rapid fadmg occurs 10 Mount in Canada balsam The very thin coating of celloidin which covers the selfods seems not to interfere with the stainıng If however it is con sidered advisable to dissolve the celloidın before staming this can be accomplished by passing the slide through a solution of equal parts of ether and alcohol This of course should be done alter passing the specimen through absolute alcohol and after the wax has been dissolved wsth xylol Nerther alcohol alone nor ether alone dissolves the gelied ceiloidin it must be a mixture of the two To demonstrate sporozoites in sections stain with Gremsa the technique of Short and Cooper for staining exo erythrocytic parasites in the liver gives good results if it is intended to stara sections with Gremsa the fixative must not contain formala Although tissues treated with fixatives containing formalin do not stain satisfactorily with any of the Romanowsky formulatsons those fixed with Carnoy stain well with all the hæmatouins we have tried and also with the Romanousky formulations It sometimes happens that when a mosquito as put into fatne at fasts to sink to the bottom of the fluid Usually the reason is that there is an air bubble at the top of the underneath part of the thorax Unless this is dispersed the insect should be discarded because the fixative and cleaning solutions will not penetrate REFERENCES I PANTIN CFA (1948) Notes on microscopical technique for zoologists University Press Camondge 2 SHORTT H'E COOPER W (1948) runs R Sor Trap Med Byg 4 4'7 PREPARING SPECIMENS OF SPOROZOTTES FROM RUPTURING OCISTS Although the salivary glands of a mosquito may be heavily infected specimens prepared from them seldom show more than a few sporozoites per micro field As the result of dissecting and crushing the glands beneath a coverstop the fluid containing sporo zortes is usually distributed over a fairly large area of the slide however the specimen is dissected dry and a small bead of salıne is immediately placed on the glands which are then crushed with ·DEMONSTRATION OF SPOROZOITES FROM OOCYSTS 31 only the corner of the coverslip the sporozoites are concentrated in a very small area By this method however only one specimen is obtained from each set of glands If numbers of specimens are required these can be prepared from mature and rupturing oocysts The technique is as follows Feed a batch of mosquitoes on a good gametocyte carrier and incubate them at about 25 -26 C On the 4th and Sth day after feeding dissect a few and examine the stomachs for oocysts If the number of oocysts per gut averages less than about 10 the batch should be discarded as unsuitable If the polysts are numerous incubate the mosquitoes at 25 C until the 7th day after feeding and then remove them to a room where the tempera ture is 16 C During this period they should be fed on 10 glucose soaked in cotton wool Reducing the temperature to 16 C slows down but does not stop the development of oocysts It must be remembered that the number of oocysts per gut varies enormously within a given batch of mosqutfoes even when they have all fed at the same time on a patient in whose blood gameto cytes were very numerous The following figures illustrate this point Twenty eight A maculipennis atropart us were fed on a single occasion on a patient whose blood contained 850 exflagellating male and 1 920 female P max parasites Re ill of d ssections Scr al number Oocysts Serial number Oocysts 113 45 6 1 18 19 0 21 8 9 10 t1 12 12 14 is 16 0 9 IS 26 31 J6 65 68 77 86 88 0 97 113 129 130 149 15 184 274 238 36 4'5 738 23 4 25 7 On the 10th-13th day after infecting the mosquitoes dissect one or two and cramine the gut with the 16 mm ( in ) lens without ·32 MISCELLANEOUS LABORATORY PROCEDURES applying a coverslıp to ascertain approximately the number of oocysts If they are sufficiently numerous agair examine the preparation this time with a 4 mm (td in ) lens still asthout applying a coverslıp if some oocysts are ripe and rupturing free sporozoites will be seen moving about in the fluid Hayang obtained a specimen in this stage of development quickly prepare a dozen slides as follows In the centre of each slide put a bead of saline no larger than the head of a pin With the aid of the dissecting needles manauvre the mid gut on to the tsp of one of them and gently pass of 3 or 4 times through the bead of salme in most cases it will be found that the mid gut tissue will adhere to the point of the needle and the procedure is then repeated unul all the dozen slides have been prepared if the gut leaves the point of the needle while preparing one specimen it can easily be picked up agaia It is best to do this under the dissecting microscope against a dark background It is also a good plan to add a small bead of fresh blood to about 5 ml of the sale which is used in the dissections The red cells make it easy to focus the specimen whereas the sporozoites being refractile make focusing difficult even with reduced light After the salıne on the slide has dried and before applying the stain encircle the specimen with a grease pencil or diamond marker on the reverse side of the slide Unless the drop of blood is added the specimen bemg semi transparent is not clearly visible even after staining From a few mosquitoes with mature and rupturing cocysts many specimens of sporozostes can be prepared providing the oocysts rupture without artificial pressure The sporozoites are as perfectly formed as they are when prepared from the salivary glands They are of course much more numerous and as we have shown (Shute') they are Infective It may be thought that a more simple method would be to crush the stomach beneath a coverslip and then to make several specs mens from the emulsion But oocysts do not all develop at the same rate so that crushing the stomach in this way would produce an abnormal picture Some immature cocysts would be ruptured and the specimen would show a number of stumpy and immature sporozoites REFERENCE } SHUTE P G (1943) Jour 1 op Afed' Hig 46 57 ·SPECIES DIAGNOSIS OF OOCYSTS 33 SPECIES DIAGNOSIS OF HUMAN MALARIAL OOCYSTS BY THE ARRANGEMENT AND DEGREE OF COARSENESS OF THE PIGVENT It is generally agreed that there are no discernible morphological differences between the sporozoites of the four species of human Plasmodia This however is not the case with oocysts The number of grains of pigment in an oocyst varies from species to species as also do the colour of the pigment and the pattern the pigment forms in the cyst (see Figs 8-11) The pattern varies from day to day during the growth of the oocysts an obser vation which shows the great importance of a knowledge of this changing character in species diagnosis In addition to being able to identify the species concerned one can also estimate from the character of the pigment the number of days before the oocysts would have ruptured taking into consideration of course the prevailing atmospheric temperatures that temperature plays a vital part in the time required for the parasite to complete its development in the insect host is too well known to need further emphasis It is however not so fully realised how much variation occurs in the time factor between different species at a given temperature At a constant temperature of 25 C the parasite of P inar completes its cycle in 9 days P falciparum in 11-12 days P oi ale in 14-15 days and P malaria in 15-21 days It is interesting to note that the two less common species take the longest time to complete their development in the mosquito There is no evidence to suggest that there are any differences in the arrangement and degree of coarseness of the pigment of a particular species of parasite in different species of Anopheles The descriptions given by us are from studies carried out with infections seen in Anopheles maculipennis atroparrus and A Stephens (Shute and Maryon!) it should be remembered that although quinine does not sterilise the gametocytes of P falcı arum some of the synthetic drugs do so either partially or completely Mepacrine may cause the famclocytes of P falciparum to lose their pigme without sterilising them Oocysts produced from these pigmentless gametocytes do not of course conform to pattern Therefore in areas where malaria control includes mass drug therapy other than quinine atypical oocysts may occasionally be seen

·34
MISCELLANEOUS LABORATORY PROCEDURES
Pilar
3
$
6.
oo
000000
000000
"OOOOO
00000
8
om
NIMBERS DENOTE AGE IN DAYS
Fig & The pigments greenish brown but some of the granules ere
darker than o hers and may be dark brown The granules are
uneven in size and share but are mostly ta short rods The number
of granules is between 0 and 100 The completion of the cycle takes
only 9 days ał a constant temperature of 25 C The pigment is fine
and because the grans are so numerous does not form any patien
in very young boysts (3 dave) When the cyst has increased in sic
and is 4 and 5 days old there is a tendeny to a pitern with the
pigment in chain formation often 15 three hnes and resemblin
Prince of Wales feathers By the sixth day of growth multiple
division of the nucle will have obscured much of the pigment and by
the seventh day only a few grains can be seen On the cighth day it 15
extremely diffi uit 10 see any pigmert

·ARRANGEMENT OF PIGMENT IN OOCYSTS
35
P orale
1000000 100000000
0.0000
7
O
nh
000
0000000
NA BEAJ BENJIE ACE N DAYS
SoH
Fig 9 The pgment is very dark brown and the granules thou h
not so large as n Plospa uni are larger than those of Prnar
They compare ſa ourably in size and colour with P mala ir There
are between 0 and 60 fra ns 19 an oocyst The completion of the
clc takes 15 days at SC A peculiar f ature of the poment is its
tendency to form chains wh ch cross cach other at right angl s in
the centre of the cyst Ths is seldom of cice seen in any other
species and it is a good dia nost s character It occurs 10 oocysts
from the fourth day of growth and is most pla nly seen on the sth
to cghth day From the eleventh day onwards much of the pgment
18 obscure but some can usually be sen

·36
MISCELLANEOUS LABORATORY PROCEDURES
Pfalciparum
3
5
0700%
0000
GOO00000
0000
or
O
9
С
NUMBERS DENOTE AGE IN DAYS
Fig 11 The pigment is almost bla k and the granules are relatively
farge The number of gran
between 10 and 20 The
completion of the cycle takes 10 days at 25 C The pigment $ 1ery
coarse and forms a pottery This is usually a double sersecucle
around the persphery of the cyst but it may be a small cucke in the
centre of the cyst or even a double straight chan This is the usual
picture 13 pocysts aged 3-7 days By the eighth day much of the
pigment becomes obscured but a few grains can usua}ly be seemt
Even on the dirth day when there is sporoblast formation odu SRINI
of Pigtuent are usually seen

·ARRANGEMENT OF PIGMENT IN OOCYSTS
37
P malaria
re
OOOO
ooooo
O ()
U
Soy
NUMBERS DENOTE AE N DAYS
It !! The pgment is try dark brown and the granules are larger
than those of P tirar but small r than ih se of p ful parim
There are seldom more than 30 granules and these vary cons derably
in size Dutin the first 7 8 days of growth the piment is stri
buted over the syst but from about the nnth day onwards it is
clumped and is invariably s tuated in a mass at the peniphery of the
004 yst en in young cocysts a decree of clumpin, may be seen
and give the appearance of scry large lumps of grient which are
In fact accumulated granules The clumping of the pgment in a
smalt arsa at the penchery of the cyst is of much da nostc alue

·TABLE 1 Species Colour of Pigment and degree of coarseness Day by day arrangement of the pigment Number Number of Measurement of grains days for of oocysts in of completion # (smallest Pigment of cycle and largest) Plitar 50-100 9 10-46 P olale 50-60 15 9-37 Greenish 3 days Without pattern brown 4-5 days In grains often arcan,ed in Prince of Wales feather formation Fine 6-7 days Much of pigment obscured by dividing nucler 8-9 days Seldor seen Dark brown 3-4-5-6-days Concentrated at the periphery of the cyst in to near semicircles or double lines often crossing at night angles black in two rows of dots 7--8-9-10 days Comparable to carlier stages but more clearly defined Medium 11-12-13-14 days Much of the pigment obscured and in some none visible Blackish 3-4-5-6-7 days Tendency to concentrate at the periphery of the cyst often in double rows but may also be in two straight chains Very coarse 8-9 days Much of the pigment obscured but some grains usually visible 10 days Seldorn scen Very dark 3-4 5 days D stributed throughout the cyst but some brown to clumping occurs near black 6-9 days Increased clumping Medium 10 days and beyond Markedly clumped and s tuated at the periphery of the cyt In mature oocysts pigment may or may not be es ble P falcipa um 10-20 10 8-60 P maları About 30 Minimum 15 Max mum 21 BEEFRENSE ·MISCELLANEOUS LAIORATORY PROCEDURES 39 POSITION OF MALARIAL QOCYSTS OY THE STOWACJIS OF MOSQUITOES The distribution of oocysts on the gut wall varies in different species of mosquitoes It is dependent at least to some extent on whether the secretion of the salıvarv glands of a species of mos qu to agglutinales the blood as do all the 4 stuculipernis complex Fig 12 stephen I Docysts distributed o er the whole of the gut or whether as with A Stephens A gambırand many other species the saltsary gland secretion acts as an anticoagulant (Shute') Usually in d maculipennis most of the oocysts are confined to the posterior two thirds of the gut (Fig 13) If however im mediately after taking a blood meal the insect is placed head downwards and kept these for 48 hours (until the oolinetes have penctrated the gut wall) the picture is reversed most of the 00cysts being confined to the anterior half of the stomach (Shute" (Fig 14 ) In those species of mosquitoes whose salivary glands do not ·Fig 13 A maculipennis Oocysts confined to the posterior tho thurds of the gut Fig 14 Most of the ooLysts confined to the anterior portion of the gut as the result of keepin, the insect head downwards for 48 hours after the infective feed ·Ross s BLACK SPORES agglutinate their blood meals the oocysts are distributed unevenly throughout the stomach In heavy infections some cysts are often seen at the posterior end of the anterior portion of the organ (Fig 12 ) However in all species of mosquitoes oocysts are more numer ous on the lower half of the gut This suggests that gravity is the deciding factor (Huff) It is not we believe due to any differ ences in the varying degree of thickness of the gut wall or to the presence of a peritrophic membrane as has sometimes been sug gested (Buxton) REFERENCES 1 SHUTE P G (1935) Jou 1 op Med 38 2? - (1919) Trans R Soc trop Afed Hvg 42 374 3 HUFF CO (1934) Am J Hyg 19 123 4 BUXTON P A (1931) 7 ans R Soc i op Arel Hg 24 477 ROSS S BLACK SPORES In the past 35 years we have dissected many thousands of anopheles mosquitoes The great majority were found to have cocysts on the stomach wall or sporozoites in the salivary glands but in about 10, no evidence of infection was detected During the whole period these negative mosquitoes must have amounted to several thousands yet we have never found Ross s black spores in negative mosquitoes dissected up to and including the seventh day after the infective feed The spores have been found on numerous occasions in both stomachs and glands of old mosquitoes in which only very scanty sporozoites were detected Our observations lead us to believe that Ross s black spores are the result of malaria infection and that if on dissecting a mosquito neither oocysts nor sporozoites are found but the spores of Ross are present either on the stomach in the thoracic muscles or in the salivary glands it is evidence of a previous infection When these spores are found on the stomach they may be round and in a solid clump or a bunch of small globular like bodies often banana shaped and light chocolate to nearly black in colour But when they are present in the thoracic muscles or the salivary glands they are always banana shaped and of a deep chocolate colour In our experience too the percentage of a batch inſected with these spores is higher at particular seasons of the year in o maculipennis the highest percentage occurs in September and ·42 MISCELLANEOUS LABORATORY PROCEDURES October (James) at least under laboratory conditions We have found them in mosquitoes infected with all four species of human malaria as well as in culicines infected with avan malaria On many occasions only a small percentage of a batch of A macul penns has become infected when ſed on one of our patients suffering from Pinar and we think it is significant that where Ross s black spores have been found in such circumstances it was only in those showing either oocysts or sporozoites Fig 15 Ross s Black Sports As regards their origin it would seem that they are chitinised parasites although some workers believe they are chitinised iracher (Bruce Mayne ) If however they are chitinised para sites ie oocysts and sporozoites then it is possible that the organisms descnbed by Mayne may be other than the true spores of Ross When mosquitoes are infected with these spores they can oftes be seen at the time of dissection with the dissecting microscope Permanent preparations of infected tissue can be prepared by fiung with Bles flud between the slide and the coverslip dchy drating clearing and mounting in Canada balsam It is best not to stain the specimen ·INTRAVENOUS INJECTION OF SPOROZOITES 43 REFERENCES 1 JAMES S P (19 8) Trans R S trop Afed Hug 21 258 2 MAYNE B (1999) Ind Jour Ved Res 17 109 TECHNIQUE FOR COUNTING SPOROZOITES AND INDUCIVC MALARIA BY INJFCTING THEM INTRAVENOUSLY The transmission of malaria by injecting sporozoites intra venously was first practised by us in 1926 (James Nicol and Shute) During the past 30 years hundreds of patients have been infected by this technique and also from sporozoites from rupturing oocysts which have not come into contact with the sylivary glands (Shute) In 1926 on arriving at the ward of a certain hospital with a jar containing mosquitoes we were called to the telephone before the mosquitoes had ſed on the patients On returning to the ward about half an hour later it was found that an enterprising nurse had put the jar of mosquitoes on the radiator to keep them warm All were either dead or moribund and the problem arose how to infect the patients It was decided to dissect the salivary glands of some of the moribund mosquitoes in Locke s fluid and inject them intra Yenously This was carried out and all the patients developed malaria within the usual incubation period Because it was found to be completely successful this method of inducing Infection has been used extensively and sometimes exclusively by other research workers on simian and avran malaria It is interesting to record that none of our patients has failed to develop malaria with any of the human Plasmodia following injections of sporozoites from ripe oocysts or from the salivary glands and nonc has had a protracted incubation period The procedure is quite safe The slides needles coverslips and Locke s fluid are all sterilised but the mosquito is not because of the risk of killing the sporozoites However on many occasions years ago when we were trying to culture sporozoites by dissecting the infected salıvary glands and mixing them with blood the culture often remained free of bacterial contamination for several days even thou h no special precautions were taken to sterilise the mosquitoes before dissections It seemed as though the secretion of the salivary glands contained some antibiotic properties Duncan' has demonstrated that a bactericidal principle occurs in both the gut contents and the frees of many ·MISCELLANEOUS LABORATORY PROCEDURES Insects including mosquitoes it seems likely that this applies 4180 to the secretions of the salivary glands of anopheles Inoculation of sporozoite suspensions can be useful in several ways in malaria therapy the injection of the salivary glands of one inſected mosquito has always proved to be sufficient to induct infection For mass infections the number of sporozoites injected from the salivary glands of 100 infected mosquitoes is at least the equivalent of the butes of a thousand We have proved this on several occasions in the following way A mosquito was ſed on a patient and allowed to gorge itsell with blood Within half an hour after feeding its salnary glands were dissected crushed beneath a coverslıp and a suspenston prepared The sporozoites were then stained and counted if the mosquito was heavily infected (hundreds of oocysts) the number of sporozoites counted often exceeded 200 000 The volunteer who was infected at Horton with Pinan to demonstrate pre erythrocytic parasites (Short et al ') was given an injection containing the salivary glands of 200 mosquitoes and a subsequent count of sporozoites proved that at least 15 000 000 were injected All the mosquitoes had fed on the solunteer a few hours earlier thus proving that each mosquito after taking a blood meal still retained about 150 000 sporozoites in its salivary glands This enormous dose of 15 000 000 sporozoites must have contributed to the numerical prevalence of pre erythrocytic forms whers were subsequently found in sections of the hver Each mosquito to be dissected is collected in a rimless 127 mm x 127 mit (5 in x 1 in) test (ube The head is cleanly severed and the salvary glands are dissected by the technique described in Section 2 As soon as the glands are dissected a drop of Locke s fluid is brought into contact with them The specimen is then brought into focus with the 16 mm (s in ) lens and a square coverslıp is applied If the covership is dropped on the glands fairly heavily and providing there is not too much Locke s fluid the glands will rupture and release the sporozoites into the fluid Examination with the 4 mm (10) lens by reduced light will quickly reveal the presence or absence of sporozoites ll the sporo zoites are to be injected the coserslip is carefully hifted from the slide without dragging it and a further drop of Locke s fluid is quickly added The fluid is then drawn up in a l mi or 2 ml 2!! glass Syringe and injected into the animal with as little delay as possible if it is considered advisable to suspend the sporozoites ·TECIINIQUE FOR COUNTING SPOROZOITES in serum this should be from the animal to be infected or at least from one of the same species It is however better to dissect the glands in Locke s fluid than in serum because the sporozoites are then more easily sucked up into the syringe If a heavy infection is required ie the glands of hundreds of mosquitoes the materiał can be pipetted off into narrow tubes until the dissections are completed This calls for a combined operation with 3 or 4 workers dissecting as rapidly as possible with roving technicius equipped with a syringe collecting the sus pensions as they become available Because very little is known about the longevity of sporozoites in either serum or Locke s flud it is best to inject the sporozoites within an hour of dissec non We do not know the exact chemical composition of the salivary cands of mosquitoes and although we have had one or Iwo patients who have developed fever and parasitomia induced by 13ecting sporozostes which have been in Lockes fluid for 24 hours before being injected we still do not know the percentage of sporozoites which survived for this in th of time All ne do know is that in this particular case a sufficient number survived to induce infection Our technique for countsng sporozoites is as follows A 19 mm (in) square coverslip is fired to a clean side witt Canada balsam The salivary glands of a mosquito are dissected in Lockes fluid care being taken to obtain all sit lobes These are then macerated beneath a coverslıp which has been applied so that one corner protrudes over the slide The coverslip is then carefully 1: Ned by kolding its corner and raising ut so that the Muld and the sporozoites which st contains is concentrated in a small arca somewhere near the centre of the slide The covership is then washed 3 or 4 times with a few drops of Locke s fluid and this is added to the Nuid on the slide The Bud is then 13.cn up into a l m hypodermie syringe and ſuiher drops of Lockes Suid are added 10 ensure that most of the sporozoites are remorcu This is continued until the quantity in the syrin C is exactly im! The contents of the syringe are then discharged into small flat bottomed watch glass and mured thoroughly by drawing the malerral in and out of the syringe a few umes Prick the pad of the middle finger of the len hand and with the point of the needle not a speck of blood and add to ine fluid this gnts a smable background on which so ſosus when counting sporozoites but only a few hundred cells are required for the purpose ·46 MISCELLANEOUS LABORATORY PROCEDURES Draw up 0 05 ml of the fluid and blow it on to the square fixed coverslip without spilling any over the slide With a needle distribute the fluid evenly over the coverslıp and cover with a petri dish until it is quite dry Fix with methyl alcohol for a few minutes then dry and stain for half an hour with weak Gıemsa (one part of stain to 20 parts of distilled water) To count the sporozoites An Fhrlich eyepiece or its equivalent and a 2 mm (in) oil immersion lens are essential Focus on to the specimen (the scanty red cells present will be helpful here) and move along until the extreme right or left edge of the coverslip is reached Count the number of sporozoites in a horizontal strip and repeat twice at different parts of the coverslıp and then strike an average we prefer counting one strip across the top of the coverslıp one across the bottom and one across the centre The number of microscopic fields from top to bottom of the coverslip is pre determined by smearing a drop of blood over a coverslip of the size to be used and staining it in the ordinary way the cor puscles enabling one to estimate the exact number of fields It is best to make all counts with the draw tube of the microscope closed When the average number of sporozoites per strip is known the number of sporozoites in the fluid is calculated If for example there are 100 microscope fields from top to bottom of the coverslıp and if the average number of sporozoites per horizontal strip is 100 then the number of sporozoites on the whole of the coverslip is 100 x 100 = 10 000 "If the quantity of flud on the coverslip represents th of the whole then the total number of sporozoites equals 200 000 If then the whole of the fluid remaining in the syringe is injected into the patient the total number of sporozoites injected will be 200 000 minus the 10 000 in the 0 05 ml used on the coverslip REFERENCES 1 JAMES S P NICOL W D SHUTE PC (1997) Trans R Sor trop ved Hig 21 233 2 SHUTE P G (1943) Jour trup Afed Hyg 46 57 3 DUNCAN J T (1976) Parasit 18 238 4 SHORTT HE GARNHAM PCC COVELL G SHUTE P G (1943) BMI 347 ·CULTURE OF MALARIA PARASITE 47 CULTURE OF THE MALARIA PARASITE As an aid to the diagnosis of malaria no useſul purpose is served by culture methods for there is as yet no practical technique whereby sub cultures increase appreciably the number of parasites following segmentation Furthermore as in three of the four species of human Plasmodium the complete erythrocytic cycle occurs in the peripheral circulation specimens from cultures of these three species would produce nothing which differs from a peripheral blood film However cultures of Pfalciparum trophozoites are of great value for teaching purposes because it is seldom that growing forms beyond the ring stage are seen in the peripheral circulation when they do occur the patient is often gravely ill and drug therapy is a matter of urgency From our cxperience with P falciparum in malaria therapy in primary cases up to about the seventh day of erythrocytic parasitemiz although fever is nearly always quotidian or frequently inter mittent the patient is not unduly distressed and ricors seldom fever occur However beyond the seventh day of fever parasites often occur in the peripheral circulation in enormous numbers often a thousand parasites per twenty five micro fields ic ten per cent of the red cells infected in these cases parasites beyond the ring stage including schizonts are quite common and these parasites are of course heavily pigmented There is a striking difference in the pattern of the pigment of P falciparum compared with the same stage of growth in the other three species Instead of a number of separate grains as in Pinar P orale and p malaria in P fahiparum the pigment is in one two or at most three clumps Our reason for stressing the above description is that students of malariology should learn to recognise developin P Silciparuni trophozoites in peripheral blood smcars because when they occur in numbers in patients undergoing a primary attack the prognosis may be grave Many workers on malaria who have lived in the fropics for years have told us that they haıc recognised and appreciated the significance of descloping tropozoites of P falciparunt for the first time when paying a visit to our laboratory for teaching purposes smears of P falciparunt parasites can be prepared from cultures which will demonstrate the characteristic I ment as it occurs in trophozoites and schizonts in the internal ·48 MISCELLANEOUS LABORATORY PROCEDURES organs the placenta and occasionally in the periphera] circulation An alternative to culture smears for obtaining trophozoites and schizonts of P falciparum is to make smears of parasitized placental blood in pregnant women living in holo or hyperen demic areas the placenta is frequently very heavily infected with all stages of the asexual cycle even when parasites are scanty in the peripheral blood Frequently also a large percentage of the leucocytes including polymorphonuclears contains masses of pigment the result of phagocytosis Technique for culturing to maturity a single generation of P folcrparum parasites Select a patient in whose blood there are numerous ring stares of the parasite eg at least five parasites per field of a thin film 1 Tale 20 ml of Venous blood and esther defibrinate it or add 160 units of heparin 2 Add 0 2 ml of a 50% solution of sterile dextrose and mit thoroughly with the blood 3 Pipette off into suitable culture tubes to form a column of about 4 c mm and incubate at 37 -40°C The parasites live and grow at the top of the column of sedimented red cells and most of the parasites beneath this layer die within a few hours 7 At intervals up to 36-48 hours of incubation withdraw a small quantity of blood witha pipette from the toplayer of the sedimented cells for examination The culture tubes must never be tilted 5 When drawing off some of the top layer of blood for pre paring thin films include a little of the serum Because the asexual cycle of P faliparum is completed in about 48 hours it may be thought that parasites should be cultured for this length of time in order to obtain fully segmenting parasites This is not so and 24-36 hours is about optimum The reason is that the parasites may be several hours old at the time the blood 25 taken and before the culture is prepared for class purposes films should be made at frequent intervals so that all stages of growth of the parasite are available for study PREJ ARING SECTIONS AND STEARS OF POST MORTEM AND PIOPSY MATERIAL Malaria parasites are very difficult to stain satisfactorily in tissue sections and direct smears give better results Even in smears of ·PREPARATION OF Post MORTEM MATERIAL 49 post mortem material the parasites do not stain wohl unless they are prepared within about three hours after death It is seldom if ever that a patient dies from an uncomplicated attack of malaria caused by any species other than P falciparum When death is directly due to falciparum malaria parasite density is generally high and parasites are distributed throughout the vascular organs The brain and spleen are nearly always heavily infected and to a lesser extent the heart kidney lungs and bone marrow As pointed out by Field and Shutel parasites are recognised from tissue smears prepared shortly after death but in preparations made several hours after death they are usually unrecognisable and heavy growths of saprophytic bacteria obscure the microscopic picture In smears of this kind the parasites look more like large cocci due presumably to contrac tions and degeneration Therefore post mortem tissue should be prepared as soon after death as possible The brain and spleco are the two organs most likely to reveal large numbers of parasites malarial pigment and phagocytosis Where a post mortem is not permissible for religious or traditional reasons some spleen material may be obtained with a syringe and a large cxploring needle Brain tissue may be obtained by passing a needle attached to a syringe through the orbital plate of the frontal bone and applying suction The best method we have seen was that demonstrated by Dionisi in Rome many years ago Instead of a syrin e he used a cobbler s needle The point of the instrument was skilfully inserted through the conjunctiva under the eyelid and then forced upwards through the orbital plate into the brain the instrument was then resolved two or three times and this filled the eye of the needle with grey matter if this operation is performed carefully there is no disfigurement In autopsics performed within 24 hours or longer after death most of the parasites have degenerated almost beyond recognition However the pigment remains and this offers the best guide for diagnosis Some reasons why smears are preferable to sections are (1) A really thin smear is thinner than a thin section (2) The parasite stains better (3) In brain smears the capillaries are well preserved and can be traced for a lonver distance than is usually possible in sections ·SO MISCELLANEOUS LABORATORY PROCEDURES Technique of preparing and staining smears of brain tissues A small piece of brain no larger than the head of a pin is placed in the centre of a dry clean slide A second slide is applied in such a manner as to squash the piece of issue and drag it over the surface of the first slide exerting continuous pressure in this way two very thin smears are made After the smears are quite dry they are fired for 5-10 minutes in methyl alcohol (longer than for an ordinary blood film) and are then stained with Gemsa diluted with distilled water 7 ml -100 ml with a pH of 7-7 2 for 30 minutes Flush with distilled water and dry After the films are dry soak them for 5 minutes in normal salıne This removes much of the methylene blue from the tissue cells without decolorising the cytoplasm of the parasites and gives a much clearer picture After treating the films with salıne wash again with distilled water to remove all particles of salt Dry and examine To prepare and stain smears of splenic material It is difficult to make very thin films of pulpy splenic tissue and we find it a good plan to break down a small piece in a few drops of salıne From this material satisfactory thin films can be prepared After the films have dried they are fired for several minutes with methyl alcohol and stained with Gremsa It is our experience that neither Leishman the hæmatorylıns nor the new rapid methods now used for staining thick blood films are as satisfac tory as Gremsa for staining parasites in tissue smears Of course the pigment is prominent whatever stain is used but so it is when the film is examined without staining The technique described above for making and staining smears of brain and spleen is also applicable to other organs heart iner kidney and bone marrow If it is desired to remove the pigment before staining this can be done after thorough firation by soaking the film for at leasi one hour in 1/ HCI in acetone In addition to dissolving the malarial pigment this also dissolses extraneous hæmoglobin without interfering with the cytologic picture The treated films must be washed in running water for at least 15 minutes to remove every trace of acid before staining ·PREPARATION OF TISSUE FOR SECTIONING 51 Firation and preparation of malaria tissue for staining and sectioning The fixatiye generally used is 10% formal salınc This is quite satisfactory for subsequent staining with any of the hematoxylins Unfortunately however sections of formalin fired material cannot be stained satisfactorily with Gremsa If therefore sections are to be stained with Giemsa a fixative not containing formalın must be used Seclions of tissue fixed in Carnoy stain beautifully with Gremsa as described by Shortt and Cooper" for demonstrating eroerythrocytic malaria parasites in the liver Their technique is as follows MATERIALS REQUIRED Glemsa stain Acetone and vylol solutions 10, -15 solution of colophonium resin in acetone Prepare stain as follows 10 ml Gemsa stain 10 ml methyl alcohol 10 ml acetone 70 ml distilled water pl 72 After sections are quite dry they should be placed between two pieces of filter paper and rolled flat with a squeegee (1) Dissolve wax with xylol () Downgrade through alcohols to water (3) Stain sections for at least 1 hour (4) Wash in tap water for several minutes (5) Immerse slide in jar containing colophonium resin acetone solution (The time required for optimum differentiation varies from a few seconds to a minuts or cien longer depending on the depth of staining) When the sections are taken out of the staining jar they are a bluish mauv ecolour But after a few seconds in the colophonium resin accione solution much of the methylene blue is extracted and they are then a violer pink With practice the exact time to check the decolorising process can be judged with the naked ce but to beginners new to this technique the specimen should be examined under the low power of the microscope How eller before doing this the specimen should be batbed in 70 Slika AIR ·MISCELLANEOUS LABORATORY PROCEDURES acetone xylol solution otherwise a resinous film will form on the section When the desired degree of differentiation has been obtained the sections are passed through three grades of acetone xylol solutions a few minutes in each (1) 70% acetons-30% xylo! (2) 50% acetone--50 / xylol (3) 25*, acetonem 75% xylol This is followed by two changes in xylol and the sections are then mounted in Euparal sert Specimens starned by this technique and mounted yn Euparat (not Canada balsam) do not fade for several years The disadvantage of Carnay as a fixate is that it causes lysis of the red cells As far as we are aware there is no fixatre for tissue which fulfits all the requirements for subsequent satisfactory staining by both Gremsa and the hemato tylins Of all the hematoxylıns we have tried we prefer Ehrlichs (undiluted) prepared in the laboratory and thoroughly matured Sections are stained for one hour or longer and then bathed for a minute or two in al solution of HCI in 70, alcohol Providing the laboratory tap sater is slightly alkaline washing the seccions for halt an hour is sufficient to blue hem An alternative us to soak them in distilled water resdered alkaline by adding a few drops of a saturated solution of lithium carbonate After upgrading in alcohol the sections are counterstained in a 001 solution of erythrosie in absolute alcohol for half an hour This is preferable to counterstaining with a stronger eosin solution for a shorter period They are then passed through 30, phenol xylol cleared 38 xylol (two changes to remove every trace of phenol otherwise fading quickly occurs) and finally mounted in ncutral balsam REFERENCES 1 FIELD W SHITE P G (1956) The microscopie diagnosis of human malarsa Part II Goscrament Press Kuala Lumpur Federat on or Malaya 2 SHORT'HE COOPER W (1947) Tross R Soe top Med Hag 41 427 ·SFCTION 3 ESTABLISHING AND MAINTAINING COLONIES OF ANOPHELES IN THE LABORATOR) GENERAL PRINCIPLES WALCE it is comparatively easy to raise a single generation of mosquitoes from eges laid by females collected in the field it is often extremely difficult and with many species quite impossible to maintain succes ive generations continuously in the laboratory One of the earliest records of success we have been able to trace is by Major Harold RAMC' who succeeded in raising several generations of Anopheles maculipennis at the School Hygiene at Aldershot The gravid adults were collected from Sandwich Rent At the time he was carrying out his researches it was not known that there are several varieties within the species A maculipennis but from personal observations we now know that alroparrus is the predominant variety present in Sandwich and it is obvious that Harold s original work was with this form it is Interesting to record that at one time when he lost his strain he tried to replace it with specimens of A maculipennis collected in the Aldershot area He failed but met with success when he again started with the Sandwich strain The predominant variety in the Altershot district is messere and the difference between (arolds failure and success is accounted for by the difference between the two variclics in their ability to copulate in confined spaces in the laboratory afroparius will mate in cages one foot cubed while messere will not do so in relatively large rooms Harold s success in breeding several generations of A maruli penis in the laboratory was subsequently repeated by many other workers with A maculipennis atropart us In the tropics Moores appears to have been the first to breed Successfic generations of Anopheles gamoice in Africa and at the present time many species of Anopheles are being maintained as laboratory colonies in various parts of the world These workers have coninbuted to our knowledge of mosquito bionomics and it is difficult to see how the present intensive researches on insect cales could be carried out if it were still not possible to breed and

·Fig 16 Sution pump used for collecting pupt
Fig 17 Small bottles conta ning 10' glucose with protruding
plug of cotton wool as used in store cises to provide food for both
male and females

·LABORATORY REARING DE ASTEPHE SI $7 Trays for sand 914 4 mm x 1292 2 mm x 101 6 mm (3RX 4ft x 415 ) Long sleeved glove Dimethyl phthalate Hy 51 FA 18 Stock cage conta nin, mosquitoes showing an antsthclised guinea po Cages--cubical-457 2 mm (18 in ) The sides back and front are covered with mosquito netling stretched on a wooden frame The top and bottom are made of three plywood The bottom hair of the front of the cage is divided into two cach with a netted slcose to enable the anesthetiscd rabbit to be inserted and also for the introduction of mosquitoes and pupe jars Parallel with and about 254 mm (1 in) away from the outside of the cage a plastic covered wiC is fixed on which damp clothes can be tung to increase humidity A wooden shell is fired inside the cage for the cge liyin dish and on this shell (na clips are screwed about 1143 mm 141 in) apart to hold the boities con taining the glucose cotton wool The colony of A Stephens was established in 1947 from cers sent Hy Lt Col Jaswant Singh Director Malaria Institute of india ·S8 LABORATORY REARING OF MOSQUITOES The insectarium used for the breeding of this species is an ex army Nissen hut druided into two parts cach with two double windows The room with a south-east aspect is used for the bred ing of the larvæ because it gives the best possible condmons of natural lighting The adjoining room with a north west aspect is used for housing the adult mosquitoes Both rooms are lined with plaster boards and have concrete floors Heating is supplied by non radiant heat the heat being enclosed in tubular heaters which are in two pairs in each room producing 360 watts per tube The heaters are connected to two thermostats per room and are kept at 250-27°C The tubular heaters are protected by guards made of perforated metal The light in the room is suppls by standard daylight tube lighting and each room contains a portable lamp readily adjustable to any position These lamps are of low voltage to ensure perfect safety from shock risks All electrical apparatus is properly earthed in view of the damp conditions Both the room for the breeding of the larvæ and the room for the housing of the adults contain a sink hot and cold water taps and a draining board for cleaning the breeding bowls etc The bowls containing the eggs and larvae of the colony are accommodated on three tables about 2 m (7 ft) long two under the windows and one opposite the window in the adjoining room the cages containing the adult insects are placed on similar tables The humidity is maintained by a constant drip of hot water from a tap in cach room and also there are several trays half filled with saturated sand ballast it is important that the surface of the ceilings and walls be not freshly painted whitewash or an untreated surface is considered satisfactory and is to be preferred If paint has to be used the adult mosquitoes should not be housed in the insectary for at least a month after the painting as completed The lasya Success or failure in the rearing of a mosquito colony depends a great d-al on the technique used for raising the larvt When the A stephens: colony was first started the method used for breeding the larvæ was that described by Shute for the reasing of A atraparcus Although this was fastly satisfactory the method used by the East Africa Malaria Unit Aman for the breeding of A gambla is far superior (Shute GT) This tech nique is ideal for the breeding of A Stephens larvæ and hundreds ·LABORATORY REARING OF A STEPHENSI 59 of thousands of insects can be obtained with the minimum of elfort TICINIQUE White polythene bowls 3048 mm diameter x 127 mm deep (12 mm x 3 mm) are used as rearing pans Fresh distilled water or filtered rain water is added to the bowls to a depth of 331 mm (11 in) and cvaporation is made good by replenishing the water cach day The dishes containing the stephensi cegs are taken from the cages which house the adults and are allowed to hatch Forty cight hours later 100-150 larvz are put into bowls containing the distilled water where they are kept until they pupate The pupe are then transferred to a glass jar which is placed in the cage in which the adults will emerge Larvæ should not be overcrowded in the rearing bowls Overcrowding is just as deletenous to the second and third instar as it is to the fourth instar larve and an increase in the quantity of food provided cannot compensate for such Overcrowding The farvæ are fed on dried yeast tablets finely ground with a postle and mortar in a proportion of 1 gramme of yeast guer 100 ml of water On hatching 2 ml of the yeast water mixture 15 pipetted into the egg laying dish On the day when the larvt are separated 5 ml of the mixture is added to cach dish and this procedure is repeated daily until the pupe develop feeding should ther cease The yeast water mixture should be prepared immediately before use as it ramdly deteriorates when left standing The yeast tablets should be hert either in a desiccator or in a dry glass jar with an airtight hd Fuery baich of 100 150 Jasse has its appropriate bon) and position on the table according to age and as soon as pupa tion is complete the bowl is cleaned ready for new larut and the next boni containing larve about to pupaic is moved into position The advantage of this method is that all the lant develop at about the same rate of growth and puration is reached within about 48 hours of each other The pupe are collected by means of a vacuum pump which is connected to an intakchottle This mechanical collecior enables large numbers of pups to be collected in a short time The rupt are transferred from the intaks toitle into plass jars and placed in the breedin. CHCS ·LABORATORY REARING OF MOSQUITOES The adults The majority of A stephensı adults emerge in 30-40 hours after pupation when the temperatures 25 -27 C A stephens will matein a cage of any size but the one described above is the most suitable The adults for stock purposes are kept soom facing north west where a temperature of 18 C is maintained and at this temperature the adults are given a blood meal three times per Week Etther a human arm or a rabbit is used for ctus purpose The rabbit us anxsibetised by Nembutal injected sntravenously intramuscularly or interperitoneally The makers recommend gramme (1 ml) for each 5 lb weight but it must be remembered that animals have a Sarymg tolerance for the barbiturates some requiring a higher dose than others Nembut3l should be given slowly when administered intravenously For intramuscular ne interperitoneal injections the same dose is usually employed anesthesia is complete in the majority of cases in from 10-15 minutes and will last for a period of 1-2 hours for intramuscular injections we inject the Nembutal between the scapulæ Anæsthetised rabbits are used in preference to guinea pres for feedmg & stephensı because a luigh death rate in the insects occurs in a few hours after they feed on guinea pig blood the death rate is presumably due to the inability of this species of Anopheles to digest the blood meal We do not share the larr of any part of the animal though no doubt this would be an advantage of only a few were available When there are several hundreds of mosquitoes to be fed the anesthetised animals are placed in the cage on their backs and the mosquttoes feed from the naked parts legs parts of the head the ears and around the anus Neither anophelines nor culicines are able to feed through the very harry or furry parts of an animal although repeated efforts are made to do so Nembutal fulfils a very useful role in the insectary With care even very small animals such as 3 or 4 week old chicks can be anästhetised and immobilised for from 1-4 hours sufficient time to allow fundreds of mosqutoes to feed When adults are collected by hand from a cage for the purpose of sn fecting them with malária or when collecting infectite mosquitoes many will bite if the hand remains in the cage for only a few seconds We find it satisfactory 10 smeas the hand and ass with dimethyl phihalale or alternatively to wear a long sleeved glove ·LAHORATORY REARING OF A ATROPARIUS 61 REFERENCES 1 SHUTE P G (1936) Jou trop Afet Hyr 39 233 2 SIUTE G T (1956) Ann tror Afet Pa asi 5D 99 THE LABORATORI REARIX OF AYOPHELES MACULIPENVIS ATROPARIIS EQUIPMENT REQUIRED The apparatus is almost the same as is used for the breeding of the Astephensi colony creept in the requirements for the breeding of larva Unglazed carthenware pans 457 2 mm (18 in ) diameter x 1143 mm (44 in deep Unglazed carthenware pans 1270 mm (5 in ) diameter x 381 mm (fin) deep for egg layang Ras water Jug and funnel for filtering the water Filter made of fine mesh gauze Sods or grass Farcx (Glaxo Lid London) Bemar (Vitamins Ltd London) Dog biscuit (Enrw histles Lid Liverpool) Glass jar with screw lop for keeping Farer Glass jar with screw top and wite cloth cenite for Bemar and dog biscuit mitlure Wire cloch--mild steel 60 x 60 mesh x 37s W (Greenings Warrington) Closc mesh soye or Vories hand grinding mill (Bryan Corcoran london) Small fly flo pump for acrating saler (Medealf Bros Florence Works įshington N)) Rabbits or guinc4 pios The colony was started in 1934 from the cers of a single female caught at Grain in the county of Kent for 9 cars prior to this colony being established the adults of 4 maculiye nu var atropursus used in the laboratón acre caucht under natural conditions from an area fordering on the Thames Estuary The insectarium used for the breeding of this species 113 equipped to that strea y described for the breeding of 4 The rooms where the larst are bed and the adults ·62 LABORATORY REARING OF MOSQUITOES situated on the ground floor of the Malarsa Therapy Vaut They are of the same brick and concrete construction as the rest of the building The farya The method used for breeding airoparius lant is based on the work by Shutel Three to four hundred lart are put into 27 earthenware pan containing filtered rain water with a small sod Fig 19 Breeding pan showing the water being aerated by a pump of grass placed in the middle The mud part of the grass sod is beneficial to the growth of the larve and it is quite unneces sary to renew the grass sod or change the water except when fresh bowls are prepared for newly hatched larvæ These pans should be thoroughly scoured with boiling water before preparing a new breeding ground 3f the breeding pans have concave bottoms (usual in earthen ware vessels) they will not fie flat on the laboratory bench When grass sods are added to the basins sooner or later ants will be introduced inevitably nests will be made on the bench under the hollows of the pans and the insectanum will soon become infested The pans should rest on bricks or bartens and so be raised above the level of the bench by a few inches Ants can be ·LABORATORY REARING OF A ATROPARIUS 63 dog kept under control and prevented from entering under doors and through crevices by sprinkling a mixture of equal parts of sifted icing sugar and borar around the rooms Ants are partial to sugar and the borax acts as a poison Rain water is stored in a large tank covered with a metal lid to prevent the breeding of all aquatic insects The larve are fed at intervals throughout the day with either Farer or a mixture of 10 / Bemar and 90 dog biscuit Farex is an infant food (carbohydrate 730 Protein 140 fat 30 mineral salts 40/ moisture 60 ) It spreads evenly over the surface of the water and floats for a considerable time Bemax is a rich natural vitamin protein mineral supplement (protein 27/ carbohydrates 50, approximately fibre not more than 3/ calorific value 104 per oz) and the dog biscuit All in One kennel meal Ten per cent of the Bemar is added to 90 biscuit the mixture is passed through a close mesh sieve or a Vortet hand grinding mill and is placed in a glass jar with a screw top in the top of the jar is fired a very fine wire cloth (60 x 60 mesh x 37 swg) and by using this sprinkler the mix fure spreads evenly over the surface of the water when not in use the wire mesh top of the jar should always be covered by an in Verted petri dish so as to prevent the food from getting damp Should this occur the food will not pass through the fine wire cloth and therefore it should be thoroughly dried and the wire cloth scrubbed and dried also the quantity of food used and the fre quency of application depends on the number of larut in the pans One should aim at supplying the maximum amount of food that the lart will consumc but no more than this excess of food will foul the water and cause a high death rate among the larvt It is best to feed with small quantities at a time and at frequent intervals However should a pellicle form it can be broken up by aerating the water with the Pły flo pump When breeding Anopheles in the laboratory it is very important to ascertain whether the larse are exclusively surface feeders (as maculipennis atroparrus appears to be) or whether like A ganha and a slephensi feeding takes place both on the surface and at the bottom of the breeding pans Larse which feed only on the surface must be given food which has the consistency of a powder and which floats on the surface Lane particles of food are rejected by the youn larse and these parlıcles cientually sink to the hoitom of the breeding pan ·62 LABORATORY REARING OF MOSQUITOES situated on the ground floor of the Malaria Therapy Unst They ate of the same brick and concrete construction as the rest of the building The Barra The method used for breeding atroparius farve is based on the work by Shutei Three to four hundred larvæ are put into an earthenware pan containing filtered rain water with a small sod Fig 19 Breeding pan showing the water being acrated by a pump of grass placed in the middle The mud part of the grass sod is beneficial to the growth of the farve and it is quite unneces sary to renew the grass sod or change the water except when fresh bowls are prepared for newly hatched lanıt These pans should be thoroughly scoured with boiling water before preparan a new breeding ground If the breeding pans have concase bottoms (usual in earthen ware vessels) they wsll not lie flat on the laboratory bench When grass sods are added to the basins sooner or later ants will be introduced Inevitably nests will be made on the bench under the hollows of the pans and the insectarium will soon become infested The pans should rest on bricks or baffens and so be raised above the level of the bench by a few inches Ants can be ·LABORATORY REARING OF A ATROPARIUS 63 kept under control and prevented from entering under doors and through crevices by sprinkling a mixture of equal parts of sifted icing sugar and borat around the rooms Ants are partial to sugar and the borax auts as a poison Rain water is stored in a large tank covered with a metal lid to prevent the breeding of all aquatic insects The larva are led at intervals throughout the day with either Farex or a mixture of 10 / Bemax and 90 / dog biscuit Farex is an infant food (carbohydrate 73 0% protein 140 fat 30 mineral salts 40/ moisture 60 ) It spreads evenly over the surface of the water and floats for a considerable time Bemax 1$ a rich natural vitamin protein mineral supplement (protein 27/ carbohydrates 50 approximately fibre not more than 3/ calonitic value 104 per oz) and the dog biscuit All in One kennel meal Ten per cent of the Bemar is added to 90 dog biscuit the mixture is passed through a close mesh sieve or a Vortex hand grinding mill and is placed in a glass jar with a Surew top in the top of the jar is fixed a very fine wire cloth (60 x 60 mesh x 37 sw g) and by using this sprinkler the mix ture spreads evenly over the surface of the water when not in use the wire mesh top of the jar should always be covered by an in verted petri dish so as to prevent the food from getting damp Should this occur the food will not pass through the fine wire cloth and therefore it should be thoroughly dried and the wire cloth scrubbed and dried also the quantity of food us d and the fre quency of application depends on the number of larvæ in the pans One should aim at supplying the maximum amount of food that the larve will consume but no more than this excess of food will foul the water and cause a high death rate among the larvæ It is best to feed with smalt quantities at a time and at frequent intervals However should a pellicle form it can be broken up by aerating the water with the Hy no pump When breeding Anopheles in the laboratory it is very important to ascertain whether the larvæ are exclusively surface feeders (as niacuupennis airoparius appears to be) or whether like A gambia and A stephenst feeding takes place both on the surface and at the bottom of the breeding pans Laryt which feed only on the surface must be given which has the consistency of a powder and which floats on the surface Large particles of rood are rejected by the young larve and these particles eventually sink to the bottom of the breedin, pan

·04
LABORATORY REARING OF MOSQUITOES
and cause the water to become sour Therefore it is essential
when dealing with larvæ which feed on the surface of the water
that the food should be of a dust like consistency
REFERENCE
1 SHUTE P G (1936) Jour trop Med Hyg 39 233
METHODS OF OBTAINING EGGS FROY I\DIVIDUAL
AIOSQUITOES
MATERIALS REQUIRED
Wooden rack 457 mm x 76 mm (18 in x 3 in) Near the
top on each side there is a narrow bar which keeps the cylinders
in position One of the bars is halved both sections being
hinged to the frame This bar is lifted before removing the
cylinders from the rack
Cylinders (glass) 101 mm x 444 mm (4 in deep x 11 in
diameter)
Petri dishes 38 mm (1) in diameter)
White card This is cut into pieces 63 5 mm x 63 5 mm
(24 in x 21 in ) and inserted inside the cylinders as a resting place
for the mosquitoes without coming into contact with the water in
the petri dish
Fig 20 Racks and cylinder jars for obtaining eggs
from individual mosquitoes

·Fig 21 The same w th cross has raised for cemo 1
cylinders after eg, layas
Agta mosqu to al resin one of the syl nders
is

·66 LABORATORY REARING OF MOSQLITOES METHODI If more than one batch of eggs are wanted from an individual mosquito these may be obtained in the following way A mosquito which is known to be fertilised is given a blood meal and transferred to a glass cylinder which is then put back into the rack over the pein dish of water Eggs are usually land 2-3 days after the insect has taken a blood meal most often af night After a batch of egts is laid the cylinder contaming the mosquito is caretully lifted from the rack The mosquito is then given a blood meal after which it is again put into the glass cylinder and returned to the rack over a petri dish containing fresh water where it remams until a further batch of eggs is fald som lines but not often two blood meals may be necessary to bring the varies to maturity By this method we have succeeded in obtaining 17 batches of ebes from an individual mosquito and some individuals have surved for over 80 days (Shute) Method 2 A mosquito with ripe ovaries will nearly always lay its eggs of the following procedure is adopted Collect a gravid adult in a 127 mm x 127 mm (5 in ) test tube shake it to the bottom of the tube and rap the bottom end of the tube sharply 7 or 8 times on the palm of the hand 10 stun the insect (Shute i When it is immobilised remove it from the test tube carefully remove a wing and transfer it to a pein dish of water Within a few mgutes the mosquito will begin to lay its eggs and providing it remains alne laying wilt connue unti all its eggs are lard Care must be taken not to kill the mos quito it should only be rendered sufficiently immobile to enable ut to be handled for the purpose of removing a wing to present it escaping REFERENCES 1 SHUTE PO (1936) AAR trop Med Parasa 30 31 2 -(1933) ANN lip lied Parasır 27 409 LABORATORŁ INFECTIONS OF ANOPHELES W777 MALARI There are four obvious reasons for infecting mosquitoes in the laboratory

·INFECTION OF ANOPHELES WITH MALARIA
67
(1) For providing infective material for therapy purposes
(2) For provding infective material for hegry infections of
sporozoites in the study of the pre and exo-erythrocytic cycle of
the par3site
(3) For comparative studies of carrier species of anopheles in
1 given area
(4) For ascertaining the infectivity to anopheles of the
inhabitants in an endemic area
Very little apparatus is required for investigating malaria
infections in mosquitoes (Sinton Shute)
19791
wyn
F: 3 Type of c2.c used for separating -from unred
mosquitoes and for stor ng infcied mosquitoes
APPARATUS REQUIRED
(!) A colony of anopheles mosquitoes
(") Sustable pars for feedin, purposes
(3) Test tubes 127 mm x 127 mm (5 in x n )
(4) Cages 3048 mm x 263 2 mm (1210 x8 in) The sides
back and front are covered with #hite mosquito netuing stretched
on a wooden frame with a false ceiling about 127 mm (11)
below the true ceiling to present the mosquitoes hiding in the
corners The front of the cage is provided with a netted sleeve to
enable the antsthetised animal to be inserted and for the introduc
uon of mosquitoes We consider this size cage to be safer for the

·68 LABORATORY REARING OF MOSQUITOES housing of the infected insects than the one described for housing the uninfected mosquitoes and makes it much easier to collect thern The technique for reasons (1) and (2) is approximately the same but for (3) and (4) some modifications are required and these will therefore be described separately For the provision of heavily infected mosquitoes the three essentials are (1) A suitable animal in whose blood there are numerous mature gametocytes which have previously been tested for ripeness as described in Section 2 (2) Very heavy infections re several hundreds of oocysts per gut (3) Over 90% of a batch infected as the result of a single feed These conditions are only obtained when the blood film shows 200 or more exfiagellating male gametocytes per c mm (approx mately 5–10 males per 100 leucocytes in an animal showing about 6 000 leucocytes per c mm) The feeding jars The most suitable jars we have found are made of thick glass cylindrical with a heavy flanged mouth and measuring 889 mm (3' in ) in diameter and height (James' 2) In the bottom of each jar is placed a closely fitting disc of dry filter paper to absorb any moisture which may accumulate during the process of feedin especially if the patient is sweating Over the mouth of each jar is tied with tape a piece of mosquito netting which has an opening in the centre a little larger than the diameter of the test tube used for collecting mosquitoes The insects are captured in a test tube and transferred through the opening into the jar When about 50 mosquitoes have been inserted into the jar the top is covered with another layer of netting which is tied in position with a second piece of tape With some species of mosquitoes such as the comparatively sluggish A maculipennis ten or even twenty specimens are easily collected in a single tube But with some other species such as a stephensı or A gambia it is difficult to collect more than two or three in a tube at a time As the test tube containing the mosquitoes has to be inserted several times into the hole through the netting covering the top of the jar the second piece of netting without a hole in the centre is used as a plug to prevent escape until all the mosquitoes have been bottled ·INFECTION OF ANOPHELES WITH MALARIA 69 then the top layer of netting is tied down We prefer white netting both for our cages and for the tops of the jars Also we use an all purpose netting which has 130 holes to 6 4 sq cm (square inch) This is suitable for dealing with relatively large mosquitoes such as A maculipennis and also for the small species such as A stephens and A gambic We prefer test tubes for collecting mosquitoes from the cages rather than the sucking tube used by many workers in the field When mosquitoes are being infected for use after the 2 1 Fig 24 Jars used for feeding mosquitoes on humans completion of the sporogonic cycle (sporozoites in the salivary glands) every care must be taken not to injure them at any stage and especially at the time of infecting them If the sucking tube is used many of the mosquitoes are damaged and many lose one or more legs and also the wings are often damaged To infect a batch of mosquitoes from a human gametocyte carrier we proceed as follows The patient lies on his back and is made comfortable so that he will remain quite still the leg is bared from the thich to the knee and 4 jars as described above are pressed firmly against the sain The person responsible for feeding the mosquitoes should also make himself comfortable so that he can press the jars avainst the skin and remain quite steady for at least 20 minutes ·72 LABORATORY REARING OF MOSQUITOES is best not to provide water for egg laying because they drown very easily However great care must be taken to ensure & constant high humidity (70-80%) otherwise there will inevitably be a high death rate If more than one species of parasite or more than one strann of parasite is being maintained the ideal is to have a separate room for each This we would suggest is essential where more than one strain of the same species is maintained especially if there are no morphological characters to differentate between the parasites of strains either in the cocysts in the mosquito or in the parasites in the peripheral blood of the vertebrate host It is a good plan to paint the woodwork of the cages with a distinctive colour for each species of parasite The technique of infecting large batches of mosquitoes from monkeys birds and other animals is the same as that already described except that instead of collecting the mosquitoes into jars and feeding them by hand the animal is either anæsthetised or other vise immobilised so that it cannot move about inside the cage Whan investigating the comparative susceptibility to infection between several local species of Anopheles all should be fed on the same patient at the same tsme This may be difficult and call for much patience because it necessitates the availability of large numbers of Anopheles at the time when gametocytes are present in the peripheral blood As the normal death rate among mosquito colonies is usually high not less than 50 mosquitoes of each species should be fed Normally 50-70% will surve for about 7-8 days which means that 20-30 are avarlable for dis section on or about the seventh day (Shute and Maryon" In many experiments of this kind which we have conducted it has been found that the seventh day is about optimum for dissection At this stage of growth with reduced light oocysts are readily detected with the 16 mm (sp) lens and No 6 eyepsoce 11 20-30 mosquitoes of each species are dissected a fairly accurate comparison of suscepubility to infection of species is arrived at One of the most striking and perhaps surprising findings in this laboratory has been the inconsistency between gametocyte density and mosquito infectivity For example on many occasions we have found that mosquitoes have become infected when gametocytes have been difficult to hnd in this or even in thick films and indeed on some occasions when none has been found ·INFECTION OF ANOPELES WITH MALARIA 73 even after a prolonged search This has caused us to consider the problem from the epidemiological angle and to consider how much importance can be attached to gametocyte surveys is the field During surveys an individual blood film is examined for one supposes 2-3 minutes If a workes spends 4 hours at his microscope he will examine 80-100 films From these he may find one or more gametocytes in 5-10, of the population This would suggest that about 10 in every 100 individuals are infecine and from our experience this could be inaccurate it seems to us that gametocyte surveys based on blood flm examinations would not gue such accurate information as sample feedings of a carrier species If in surveys of this kind about so mosquitoes were fed on a percenta e of the population according to age groups and the surviving insects dissected on about the Sesenth day a far more accurate picture of the percentage of the population who are at a given time capable of infecting mosqustocs would be arrived at REFERENCES Syvyo JA (1938) Public Malik Reps x No 8+ I 2 SHUTE PG (1951) Bir tel 8ill $ 56 3 JAMES S P (19) League of Narons Reports CH /Malars/57(1) 1 4 -(1997) League of Aaron R po u C* /Malarsa173 $ - (1931) TO# R S T op vted His 24 $37 6 SHUTE P G (1945) Tans R S op sy lig 38 493 7 SHUTE P MARYON AT (1957) Tans R Sp Mtel Ny 31 403 ·SECTION 4 PRACTICAL HINTS When preparing blood films for teaching purposes no depar ture from the usual routine is necessary but because the specimens will be required to last for an indefinite period precautions must be taken to delay fading Adjust the distilled water so that it is neutral or very slightly alkaline after it has been exposed to the atmosphere of the laboratory for 15-20 minutes If possible films should be stained on the day they are taken but in any case not later than 48 hours afterwards Unmounted films probably keep slightly better than those which are mounted in a neutral mountant but if used repeatedly unmounted films soon get scratched or damaged Never use Canada balsam for mounting films stained with Lershman ar Giemsa stain Euparal vert is excellent and specimens mounted in this medrum often fade very little after 20 years or more If films are not mounted the immersion oil should be thoroughly dissolved with neutral xylene before wiping the film To put xylene on the oil and then to wipe the film before the oil is thoroughly dissolved quickly ruins the film Xylene is sometimes strongly acid especially when it is old or when the bottle is only half full for long periods Only No 1 coverslips should be used for mounting films which are subsequently to be examined with an jon lens Films when ild be ark and if possible in a dry calcium chloride) and } 1a des ·PRACTICAL HINTS 75 Thick films unstained for a week or more should be soaked in normal saline before being stained especially in the tropics The length of time a film should be soaked in saline depends on its age and thickness The aim should be to continue soaking the film in saline until most but not all of the hæmoglobin is removed The film should then be stained while wet Very thick films even thou, h they may have been taken only a few hours previously should be soaked before staining All stains and especially the Romanowsky modifications Leishman and Glemsa should be prepared in the laboratory and not purchased in solution Stock solutions of Giemsa and Leishman stain should be kept away from strong light It is a good plan to cover the vessels containing the prepared stains with brown paper held in position with rubber bands Because heat also plays a part in stain deterioration stock solutions should be kept at temperatures below 16 C For diluting Glemsa measure by millilitre and not by drops This prevents conſuston concerning the number of drops to a millilitre Thick and thin films will stain perfectly up to at least 3 years of kept in a desiccator which is stored in a domestic refrigerator Thick and thin films which are to be stored in a desiccator for subsequent staining should be prepared from freshly drawn blood and on no account shoull these films be fixed prior to storing Films prepared from defibrinated or citrated blood do not stain well even after a few days Films from heparinised blood stain satisfactorily several weeks after they are prepared A more even thick film is obtained of the blood is distributed on the slide to form a square instead of a circle and the staining i$ more uniform When thick and thin films are made on the same slide great care must be taken to present any alcohol from touching the thick ·76 PRACTICAL HINTS film If this happens the latter will not dehemoglobiise The thick film should be made about 127 mm (} in ) from one end of the slide the thin film shovid be spread in the opposite direction A gap of about 127 mm (fin) being left between the two It frequently happens in the tropics that absolute alcohol is tampered with by irresponsible employees It is therefore advis able to test the alcohol before use when preparing fixatres etc The best method is to use a hygrometer where this is not available a good indicator is copper sulphate Industrial spin 34 OP 15 99 per cent alcohol and is much cheaper than ethyl alcohol The relatively new method of producing mineral free water by passing it through a demoniser has much to recommend it Once the apparatus is available the cost of maintaining mineral free water is cheaper than producing distilled water and what is more important it is the equal of treble distilled water This deionised water is ercellent for rearing mosquito larve by the method described in this book for rearing A stephens. It is also ercellent for diluting alcohol and all water solent stains If distilled water is boiled in a soft glass flask it becomes strongly alkalınc Always test the pH of distilled water which has been boiled before using it for diluting Romanowsky stains If the distilled water is too alkalıne never add acid for adjusting Instead add distilled water which has become acid through exposure to CO Perfect thin films cannot be made with thick sides or even with thin ones which have chipped edges Always keep a few extra thin glass slides with clean bevelled edges for preparing thin films Instead of coating a paraffin block with a low melting point war to ensure a nbbon raise the temperature of the room where the sections are to be cut When sections separate from cach other as they leave the knife it is often due to the temperature of the room being too low Although this can be corrected by coating the paraffin block with a low melting point war thishas the dis advantage of causing the sections to become widely spaced when they are heated for flattening on the shde ·PRACTICAL HINTS 77 Mosquitoes which are to be killed and fired for sectioning should not be led on sugar solutions for at least 3 days before killing them when the diverticulum is distended with fluid it presses against the mid gut and causes distortion When only a thin film is available for examination and para sites are not found easily with the 2 mm ( in ) oil immersion lens change to a 375 mm fluorite lens Although magnification is thereby reduced by nearly one half the smallest P falciparum ring can be seen quite clearly The 3 75 lens is also useful when searching for scanty gametocytes with the modern high grade binocular microscopes a 3 75 lens is adequate for all routine blood film examinations of both thick and thin films and is a great time saver Alternatively a 4 mm (fin) dry lens with a No 6 or No 10 eyepiece can be used It is necessary however to cover the specimen with a film of oil and apply a No I coverslip Stomachs and sections of mosquitoes showing oocysts stained with hematoxyhin and mounted in a neutral medrum usually do not fade perceptibly over several years If fading has occurred however valuable specimens can be restored by the following technique (1) To remove the coverslıp soak the slide in xylof [ the specimen was mounted in Canada balsam or in Eupara! essence if it was mounted in Euparal This usually takes several days (2) Absolute alcohol (two changes) at half hourly intervals (3) One per cent HCl in 70 alcohol and leave until all the stain is removed (usually 1-2 hours) (4) Running water for at least one hour to remove all traces of HCI (5) Stain for about 10 seconds with Mayer s hemalum (Pantin s) (6) Blue in alkaline water (7) Upgrade in alcohols to absolute (74 OP Industrial Spirit) a few minutes in cach (8) Carbol xylol in 30 phenol in vylol (30 minutes) (9) Two or three changes in vylol to remove all traces of phenol (10) Mount in a neutral medium ·78 PRACTICAL HINTS If difficulty is experienced in starting a laboratory colony of a Species of mosquito whose habits are unknown the first essential is to ascertain whether copulation occurs in the labora tory Remove one or two females from the cage after they haie been in contact with the males for a few days and dissect out the spermotheca For this dissection use the white background of the dissecting microscope because then the dark brown spermotheca will show up clearly Dissect the spermotheca in saline transfer the specimen to the microscope and apply a coverslip under pressure sufficient to rupture the sac Examine with the 4 mm (fin) lens If positive the sperms will be seen swarming from the ruptured sac Even in an unruptured spermotheca the sperms can often be scen and there is usually a rotary antr-clockwise movement of the sperms within the sac To observe the process of egg hatching lift an egg from the surface of the water with a tria gular prece of filter paper sclecting one that has been laid about 36 hours Transfer it to a cavity slide containing 10 / formalın and apply a covership in a few minutes the larva well emerge and the formalin will kill it it can then be mounted as a fluid specimen or the formalın can be pipetted off and a different mountant used When injecting animals with Nembutal wipe the needle to prevent the drug leaking This will prevent sores developing al the seat of injection The points of dissecting needles soon become damaged unless special care is taken to protect them even sucking them in a cork can be harmful keep the needles blades downwards in a test tube which has a cotton wool plug at the bottom and a stopper at the top If ordinary steel needles are used they should be Hashed in hot water at the end of the days work to remove all traces of salt and then thoroughly dried to prevent rust forming Slide dryer slab When large numbers of slides are to be stained in bulk they can be set up to dry in the following way A wooden slab preferably of teak of made to the following measurements will carry half a gross of slides 240 mm x 240 mm x 18 mm (916 in x 9 p s8 in) The slab is divided into nine lines of grooves each 24 m apart ·PRACTICAL HINTS 79 The grooves are 2 m wide and 3 mm deep By having the grooves twice the thickness of the slide it is ensured that the slide will rest at a slightly slanting angle If the slides are placed in the grooves with the blood film underneath they will remain dust free while drying If the lines are too close to each other or if the grooves are too wide the slides when at rest will touch each Other and so present them drying Plastic material prepared to Fig 25 Slide dryer slab to hold half a gross of sl des the above measurements would probably be as satisfactory as teak and of course would be much lighter Hardening and brittleness of tissue can be avoided if after the tissue has been in alcohol for not more than 2 hours it is then transferred to methyl benzoate for 24 hours or longer Transfer to benzene two changes 1 hour in cach and then into paraffin in the usual way Many substitutes for cedar oil immersion lenses are being used citensively these days Anisolus satisfactory and has the advantage ·80 SELECTED BIBLIOGRAPHY that it is not sticky or greasy It does not harden on the slide and a drop of Anisol disappears from the slide in about 20 minutes In the humid tropics immersion oils quickly take up water vapour Also the most breath of the microscopist can cause water vapour to reach the film and make examination tedious and difficult SELECTED BIBLIOGRAPHY Set out in chronological ord's JAMES S P (1923) The parasites of inoculated malaria in England Trans R Snc trop Vet Hug 17 340 - (1924) Observations on malaria made during treatment of general paralysis Trans R Soc trop Ved Hy18 33 -(1974) Naturally infected malaria in cases of general paralysis Trans R Soe trop Met Hug 18 2 -(1974) Rosss Black Spores in Anopheles macul pennts fed on BT malaria in England Trans Soc inep Med Hug 18 241 -(1925) Contribution to discussion of Professor Warrin ton Yorkes paper Further observations on malaria made durin, treatment of general paralysis Trans R Soc trop Afed Hjg 19 125 - (1926) Epidemiological results of a laboratory study of induced mataria in England Trans R Soc trop Med His zó 3 -(1927) History of a group of anopheles mosquitoes infected with Plasma dium crear bhandi ad Gebiet d Auslandskunk 26 270 -(1927) Problems of malaria prophylaxis Brit Med 2,340 JAMES S P NICOL X D SHUTE P G (1927) Habits of anopheles in relation to their role in the spread of malaria Trans FEAT 7th Congress held in India 11 712 -(1977) Experiments on the treatment of malaria in En land Trans FEAT117th Congress held in India 11 788 JAMES S P SHUTE P G (1930) Infection of En lish Anopheles maculpennis with P falciparum Trans R Sor tap Med Hyg 24 369 JAMES S P NICOL, W D SHUTE PĆ (1931) Prevention of malaria with plasmoquine The Lancet 2 341 JATE S P (1931) The use of plasmoquine in the prevention of malaria! infections Proc R Acai Sai Amsterdam 34 14 JAMES S P (1931) Chemoprophylaxis in malaria Bull of Intern Hig Pub 23 7175 JAMES S P Nirou WD SHUTE P G (1932) A study of induced malignant tertian malaria Proc R Soc Aled 25 1153 ---(1937) Plasmodium ouale Stephens passage of the parasite throu n mosquitoes and successful transmission by their bites Annette Parasit 26 139 JAMES S P (1932) Cycle of Plasmodium orale Stephens in man and Anopheles maculipennis Trans R Soc trap Med Hug 26 3 - (1939) Synthetic antimalarial remedies and quinine Trans R Sor 1 mp Aled His 26 105 ·SELECTED BIBLIOGRAPHY 81 JAMES S P NICOL WD SHUTE P G (1933) Plasm drum orale Stephens 1927 Pa asi 25 87 JAMES S P (1933) (mmun ty in malaria Trans R Soc frp Wed Hs 26 sas -(1933) Remarks preceding a demonstration relatin, to the practie of malanatherapy at Hartan Bu I ten Du 9 230 - (1933) The therapeutics of malaria League of Nat ns Qua iprly Bullelin 11 (1) 183 - (193) The direct effect of atebrn on the parasite of benign tertian and quartan malaria Trans R S trop Med Hyg 28 3 JAMES S P NICOL W D SHUTE PG (1939) The specifi status of Plasmo d um orale Stephens Ante frop Med 15 187 JAMES S P (1935) Le paludisme par Plasmodium o ale Ri di Malarul SHUTE P G (1935) The value of the external sp ne of the harpa o in ident fyn sarieties of Anopheles na ulipennis Pa asi 27 361 (1933) A note on the habits of Anopheles maculipenn s as observed dur ng a recent tour of Roumania and Bessarabia with particular referen e to Hosp cal Socola Report to the League of Nations Commiss on Genc a Junc JAMES S P (1935) Dscuss on on the chemotherapy of malaria Matu e 136 743 - (1936) Consecutive reports on the antimalarial chemotherapeutic tests carried out at the Devon Mental Hospital 194 1935 - (1936) Speces and races of malaria paras tes 3rd International Congress on Malaria Madrid. October JAMES S P Nicou WD SHUTE P G (1936) Clin cal and paras tological observations on induced malatia Proc R Sor tfel 29 21 SINUTE P G (1937) A technique for the inoculation of known numbers of sporozo tes as an ard to malaria rescarch Ann 1 op Afed Pa as / 31 85 -(1997) The morphology of the external spines of the harpa ocs of a sin le race of macul pennis var ar opart us and its value for d agnostic purposes Lea wc of Mat ons C/H/Malart3/ H May 13th SHUTE P G BADEVSAJ G (1937) A study of the numer cal prevalence of asexual parasites in primary malaria Ash Roum de Pathul Erpe 10 453 SHUTE PO UNGUREANU E (1938) Comparative stud es of the engs of An ph les marulipenis var atropa 11 in ihe field and in the laboratory Rio Malu tot 17 358 SHUTE P G (1938) The position of human malaria oocysts on the stomach of An ph kes macul pennis Ach Rum Pathol Exper 11 331 SINTON ) A SKUTE P G (1938) A report on the longer ty of mosquitoes in relation to the transmission of malara an nature Ph Health Reports 85 SINTON JAHUTTO EL SHUTE PG (1938) Failure to transmit an nfection of Pleinkudium ai nomoly to man by blood inoculation and mosquito bies # 1 op ved HIT 43 175 ---- (1938) Unsuccessful attempts at causal prophylaxis with certunan malignant tertian maldria Trans R Some IP ved Hve J 19 SINT J A (1913) A report on the di trebution and pro sion of infective ·82 SELECTED BIBLIOGRAPHY material for the practice of malariatherapy in En land and Wales Public Health Reports 84 SINTON J Å SHUTE P G (1919) Spirochæjal infections of mosquiides Jour trop Med Hyg 42 125 SHUTE P G UNGUREANU E (1939) Preliminary report on the longevity of the races of Anopheles maculipennis League of Nations CH /Afalania/273 SHUTE P G (1939) Protracted incubation periods in indigenous cases of malaria in En_land Jour trop Med Hyg42 201 HUTTON EL SHUTE P G (1939) The risk of transmitting malaria by blood transfusion Jcur trop Ved flyg 12 309 SINTON JA HUTTON EL SHUTE P G (1939) Some successful trials of proseptasıne as a causal prophylactic against infection with Plasmodium falciparunt Ann trop Med Parasu 33 37 -- (1949) Studies of infections with Plastodium ouale 1 Natural resistance to ouale infections Trans R Soc trop Med Hyg 32 751 -----(1939) Studies of infections with Plasmodium orale 2 Acquired resistance to orole infections Trans R Soe trop Med Hy8 3 47 SHUTE P G (1940) Failure to infect English specimens of Anopheles macuh pennis var aire panus with certain strains of Plasmodium falciparum of tropical origin Jur trop Med Hyg 43 175 - (1941) A species of mosquito infesting deep shelters in London The Lancet 2 6 MURRAY ELLIOTT J SHUTE P G (1942) A case of P falcipa um malara The Lancet 2 365 WHELEN M SHUTE P G (1943) Thiobismol in therapeuta malaria Jour frop Med Hyg 46 1 SHUTE P G (1943) Mosquito problems in static water tanks Jour R San Insı 63 117 - (1944) War and malaria Kings College Hosp Ga else 341 (1944) Indigenous malaria and mosquito control in En land after the war Jour R San Inst 64 85 - (1944) Relapse of quartan fever after 12 and 21 years The Lancer 2 146 -- (1945) Malaria in England Public Health 58 67 -- (1945) Mosquitoes in static water tanks The Lancet 2 155 --(1945) Clinical observations on malaria Post Grat Med J ur 21 38 (1946) The maintenance of a strain of Plasmodium iner through man and mosquito over a period of 21 years Public Health Bull 4 110 - (1946) Congenital malaria Brit Ved Jour 1 930 - (1946) Latency and long term relapses in ben, a tertian malarra Trons R S trop Ved His 40 189 UNGUREANU E SHUTE PG (1947) The value of the wing scales as an aid to the taxonomy of adult Anopheles maculipennis Pro R Ěn Sx London 16 7-8 79 SHUTE PO MARYON M (1947) A study of a strains of Plasmodium falcı parum indigenous to the Bet, an Congo Ann Sor Belge Ated rp 27 39) SHUTE P G (1947) A case of malari (quartan) malaria brought about as the result of whole blood transfusion Trans R Soc trop Wed Hyg 40 37) SHUTE P G (1947) Indigenous malaria in Great Britain Afonthly Bulletin Ministry of Health 6 116 ·SELECTED BIBLIOGRAPHY 83 -- (1947) A case of latent P oral esth a spontaneous recovery Trans R Soc! Wed His 40 373 (1948) Discuss on on the Exo erythrocytic parasites of Plasand une anom 1 Ton R Spe 1 op Wed H 41 714 SHUTE P G MarYOV Mt (1948) The gametocytocidal action of paludrınc upon infections of Plasmodium falc po um Pó o 38 64 Suite P G (1948) The history of a group of Anoph les maculipennur var of openw (3 600) infected with paar malaria 1 Athly Bullern Ministry of 11 alth 7162 --- (1949) A review of und genous malarsa si Great Britain after the war of 1939-45 compared with the correspondin, rer od after the 1954-18 war (With some observat ons on the xt olo yi Vonikly B lletin Mursistry of Health & 2 COVELL, G Nicol WD SKUTE PG MARYON M (1949) Paludisme (proguanil) in prophylaxis and treatment of malana nfections caused by a West African stra RotP faleipa um Bu 11 jou 1 88 --(1949) Studies on a West African strain of Plasmod um fol parum The efficacy of paludenc (pro uznil) as a prophylactic agent Trans R Sciop ved His 4341 (1949) Studies on a West African strain of Plasmodium falc pa un The sficacy of paludame (pro, 1991) as a therapeut agent Tom RS. WoH 4465 Secure P G (1949) Mosquitoes in London Worthly Bull im Atimisery of Health & 28 SHIRTT HE HAMILTON FAIRLEY N COVELL G SHUTE PG GARNHAM PCC (1949) The pre-erythrocytic sta e of Plasmos um falcipa um Mo Jour 2 1006 JAMES S P Nice WD SHUTE PG (1949) O ale maları3 Malars ology Mark FB) SKUTE P G (19501 Reply to Professor Swellen, rebels paper The malaria er dem of 1943 46 in the provine of North Holland Tan R Soc IM HE 43 467 Shure PG (1951) Twenty five years of malarsaiherapy Discore ) March 76 SHUTE PG MARYON M (1951) A study of gastrocytes in a West Afnan stran of P falcipa um 7 ans 8 Soc 1 op 11 Hyg 44 4.5 Siront He Hautltoy FAIRLEY N COVELL,G SKUTE PG CAR HAY PCC (1951) The pre-crythrocytic stage of Plasmod um falc pa um Tant | Sec I op al d H 4 405 MAITINLY PI ROZENBOOY EL LAVE HANGT AL DALYMOND F# CHEMISTOPITER SR Suure PG (1951) The Culere pent complex TON En! So London, 102, 331 SHUTE P G (1951) Sub-ten an malana on tropical aerodroms 8 Med IN 2 1 8D SUUTE P GMAXYON A (1959) Studies in the transm ss on of Pla nwum malor by anotheis mosqu toes Pa ar 1 41 2) SUTI P G (1999) Rssly to Dr Lansers pax Sorx obsesyal ons on sub-testian malaria. 81YJTW 2.43 -195 ) Returks on daraç m 7 ans R Some trop Vaug 46 0 (1972) Atalanal Feser Therapy The Lon « , 333 ·84 SELECTED BIBLIOGRAPHY - (1953) Mosquitoes and the floods Brit Afed Jour 1 616 (1953) Contribution to the Culer pipients complex Trans 19th Int Congress Ent Amsterdam 2 289 COVELL G SHUTE P G MARYON M (1953) Pyrimethamine (daraprım) as a prophylactic agent against a West African strain of P falciparum Brit Aled Jour 1 1081 ---(1953) Pyrimethamine (daraprim) in the treatment of the ar mataria Bit Aled Jour 2 258 SHUTE PG MARYON M (1954) The effect of pyrımethamine upon the oocysts and gametocytes of P tar and P falciparum malaria parasites Trans R Soc trop Afed Hig 48 50 SHUTE P G (1954) Indigenous P irlar malaria in London belicved to have been transmitted by A plumbeus Afonthly Bulletin Ministry of Health 13 48 COVELL G SHUTE PC (1954) Memorandum on the control of mosquito nuisances in Great Britain 4th Edition Ministry of Health Memo/238/ Med SHUTE P G MARYOV M (1954) A contribution to the problem of strains of human Plasmodium RR di Malarsol 33 147 ---(1954) Observations on P otale 5th Inter Con.sess trop Med. & Malaria Instanbul 4 SHUTE P G (1954) Preparation of blood flms in suspected malaria The Lancer 2 499 GAIRDNER G SHUTE P G (1955) Nephrosis treated by malaria Results in 65 cases The Lancer 2 9-16 SHUTE P G (1955) The discovery of the third cyle in the life history of the human malaria parasite Aings College Hosp Ga este 34 234 SHUTE P G MARYON M (1955) Transmission of Plasmodium malanır by laboratory bred A maculipennis atropartus Mergen Ann trop Med Parası 49 451 (1956) is the malaria parasite within or upon the red blood corpuscle With particular reference to the stippling and other morphological changes observed in the host cell Trans Soc top Med mig 40 139 (1957) A fatal case of P falciparum malaria Trans R Sor trop Vred Hug 51 298 SHUTE PC (1957) Brief review of results obtained from thirty years of malariatherapy Congress National des Sciences Medicales Communi cations des invites estranxes Bucharest 419 - (1957) Malaria in Roumania The Lancet 1 1198 -- (1957) Thirty years of malariatherapy Suur trop Med Hug 61 57 ·INDEX Adult mo qu toc care of 60 cms sta n } $ 6 7 8 9 10 11 Ang the lion of labo atory an mis 12 16 1 ) 9 30 46 50 51 55 60 78 sal c stan 7 8 9 11 An ol 80 Im Ipe ar al op $ !!ts o techn que 74 80 Laboratory breed ng of 61 A paisip 1 I feci o of mosq tocs 66 laboratory breed ng of 55 Intra eno ject of spo ozo tes A oph i ne mosquitoes 43-45 breed ng and colo z ng of $3 [ bo tory onfect on wth malan JSB sta 10 13 66 method of Ohta ning o a from ind Labo atory colo ics of mosq 1ocs 55 dual adults 64 61 prepa alio for secl ont x 25 Labo atory tech 19c cst ng faded sectos of 77 m scellan ou h ne on 74 80 La of mo quitoes Borsy mat ! ra gand ca e of 58 6 prep ning seet ons nd smer or Ieshni st1 2 3 4 5 6 7 91 48 21 23 so Black po es of Ross 41 Blood smears Mla 4 paras les demo trai on or 75 culture of 47 Nagellion in 15 Maurer s dots prepar rior Icach ng pu poses d motrat on of 4 7 9 11 13 74 Md gut th in preparalon and sta ng or ds ect on of 17 19 CU mnat on ro oocyst on 0 tam nal on or 14 poto of oocysts on 40 th k prepration id lain & of Stjin ng faded spe me sor 77 1 14 Mosqu locs I bo to y olo or cm mat on of 9 10 55 61 Breed 3 of mo qu tóc: $$ start ng nd maint na ce of 53 Cult of malr pa as tes 47 Needlc d sect 6 or of 1 Descator for sto ing blood fim 75 Nemb: 1 pt lisd water Islhn que fo admin strat lo $ bst tute fo76 I borat ty nm 60 import re of pH of 3 4791) 23 DosysІ Dying or 1bfo 78 79 taminat nr 0 polo on m gut of 40 Fraat 74 prepar ! on of roto o les from 30 Enllato demon trai on of IS peces dros of 33 ng d maki & roman ni pre Tulus 30 of 13 Ook nets Gameta_y16 male per to is how on d mot it on offellt n 15 cios, 16 85 ·86 INDEX Smears of sple! Star Spore bp Ookinetes (conid ) preparation of s cions showing 28 Ova of mosquitoes method of obtaining from Individual adult insects 64 mounting of 78 observation of hatching of 78 Pampanas modification of Gremsa stain 7 Paraffin block treatment of 76 Pigment in oocyst as an aid to species diagnosis 33 Post mortem material preparation of section and smcars of 48 Pupa of mo care of 59 Sporozowy count intrave prepar 30 starnung para? Stomach dissecti traming Ross s black spores 41 faded si Stain Felds Gemsa 16 Gems! JSB 1 Le shma 21 23 Staining biood fil oocysts Se tions 1 Sporozat Technique Practi al Saline use of wth Glemsa stain 7 8 9 11 12 Salivary gland dissection of 17 examination for sporozoites 19 Schuffner s dots 9 Sections of biopsy mater al preparation and staining of 48 of mosquitoes preparation and stain ing of 5 21 of post mortem material preparation and sta ming or 48 Si des glass 1 slab fo dry ng 78 79 specifi ation and treatment of 1 Smears of branssue Peparation and staining of so 31 Th ohald a blood m Tissue for Preparati Vermicules? Pepa atus Preparat Zemanasa demonstra SMS I EDICAL COLLE LIBRARI, JAIPI

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