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A method of preparing and staining thick blood films for the diagnosis of malaria
275 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE. Vol. XXVI. No. 3. November, 1932.
A METHOD
OF P R E P A R I N G AND
STAINING
THICK
BLOOD
F I L M S FOR T H E D I A G N O S I S OF MALARIA. BY R I C H A R D GREEN, M.D., B.S., D.T.M.*
(From the Institute for Medical Research, Kuala Lumpur, F.M.S.)
1.
INTRODUCTION.
The search for malarial parasites in blood is undoubtedly made easier by the use of thick films which have been properly prepared and stained. It may be said that, when the diagnosis of malarial infection rather than the definition of the actual parasite species becomes all-important, and where large numbers of blood films are to be examined efficiently for malarial parasites, the use of thick films becomes the method of necessity rather than the method of choice. It is a common experience to find, however, that among those concerned with the diagnosis of malaria, the use of thick films is often abandoned after a few trials on account of the technical difficulties involved, or because of the poor results obtained. The method to be described is, strictly speaking, not an original one, but various steps, that have been recommended by text books or by individuals, have been tried from time to time; some processes have been modified or retained, others have been rejected. From tiffs series of trials a simple method has been evolved, which has afforded uniformly good results over a long period. Details are given as follows :-* The writer is indebted to Dr. A. N ~ w KINGSBURY,Director, Institute for Medical Research, for permission to publish this paper ; and to Dr. W. FLETCHER and Dr. H. O. HOPKINS for suggestions regarding the staining of thick films.
276
PREPARING AND STAINING TIIICK BLOOD FILMS FOR DIAGNOSIS OF MALARIA. 2.
TECHNIQUE OF PREPARING AND STAINING T H I C K FILMS.
(a) Preparation. 1. Making the Film. The finger of the patient is cleaned and dried thoroughly. It is then pricked with a triangular pointed needle and a larger quantity of blood than is used for thin films is gently squeezed out. The finger being dry, this blood takes the form of a globule. The pricked surface is held facing upwards and the lower surface of a microscope slide is rubbed carefully over the globule of blood to form a circle about half an inch in diameter. The circular motion of the slide is continued until the blood is evenly distributed in the form of a disc. At first the beginner must be careful not to make the film too thick ; nor must the slide touch the skin of the finger. The slide is then turned rapidly so that the blood faces upwards; it is tilted, if necessary, to secure even distribution of the blood ; then placed on a level surface to dry. It must be protected from dust. The process of making the film should occupy a few seconds only.
2. Drying the Film. After preliminary drying on a level surface the films must continue to dry for some hours before staining, otherwise they may become detached from the slide in the staining trough.* If a desiccator or an incubator is available', the period of drying may be reduced to two or three hours. A makeshift drying chamber may be devised by inserting a lighted electric lamp at the end of flexible wire into a drawer. A thermometer is placed in the drawer and, by moving the drawer in or out, the temperature within may be regulated to about 37 ° C. A period of two or three hours in this form of drying chamber is generally sufficient. It is inadvisable to keep thick films unstained for more than twenty-four hours because, after this, they do not stain so well ; also, moulds and bacteria are liable to develop in t h e m , Finally, during drying, the film must be protected from insects, such as ants and cockroaches. The latter often eat thick films after they have been stained.
(b) Staining. The following apparatus and solutions are required.
Apparatus.-(i) (ii) (iii) (iv)
Measuring cylinders, 50 c.cm. or 100 c.cm. Staining troughs to take slides on edge. Dropping bottles. Troughs or beakers for washing slides.
* The period required for drying varies With the local condition~ of temperature and humidity, but is soon ascertained by experience.
RICHARD GREEN.
277
Solutiom.--(a) Giemsa's stain. (b) Buffered distilled water or buffered tap water. The full complement of apparatus as detailed above is desirable. It is best to stain the slides on edge, but these, supported on glass rods, may be placed film downwards in a shallow staining trough. If the diluted stain is poured on to slides allowed to remain film upwards, the particles of stain precipitated on the film may be difficult to remove later. Alternatively, the slides, separated suitably, may be made into bundles and immersed in stain which is contained in a glass beaker or porcelain cup.
(a) Giemsa's Stain. Geimsa's stain is often so badly made up that notes on its preparation are given below : The formula of Giemsa's stain for 500 c.cm. is as follows :-Azur II-eosin 3.0 gram Azur II 0.8 gram Glycerine 250 c.cm. Methyl alcohol 250 c.cm. The azur II-eosin and azur II are generally combined in the form of powder. To make 500 c.cm. of stain proceed as follows : Weigh out 3.8 grams of the combined Giemsa's stain powder and place it in a glass or glazed mortar. Measure 250 c.cm. of pure glycerine and pour it from time to time over the powder, using the pestle to assist in mixing and dissolving. Then add intermittently a total quantity of 250 c.cm. methyl alcohol (acetone free) stirring rapidly. This latter part must be done fairly quickly, otherwise the alcohol will evaporate. When the powder is dissolved sufficiently, pour the contents of the mortar into a glass-stoppered bottle, wash out the mortar with what remains of the 250 c.cm. methyl alcohol and put these washings into the bottle. The bottle should be shaken frequently during the next twenty-four hours, and the stain should be ready for use in forty-eight hours. When required, small quantities of the stain are filtered into dropping bottles. During filtration, the funnel is covered over with half a petri dish to prevent evaporation of the methyl alcohol. Prepared in this way, the stain has been found to retain its properties for twelve months or more in the tropical climate of Malaya. In many cases it will be sufficient to prepare 50 c.cm. of the stain only. With this volume of course, one-tenth of the quantities given above are used. If soloids (Burroughs Wellcome) are used, one soloid of Giemsa's stain (indexed as eosin-azur) is dissolved in 5 c.cm. of a mixture of equal parts of glycerine and methyl alcohol. Soloids, however, are sometimes difficult to dissolve completely. They should be ground to a fine powder before use. The glycerine used is of importance in making good stain. It should be as pure as possible (re-distilled) and obtained from a reliable drug house, because F
978
PREPARINGAND STAINING THICK BLOOD FILMS FOR DIAGNOSIS OF MALARIA.
most brands of glycerine become acid in reaction soon after the bottle is opened. The acidity varies in degree. To counteract this, buffered solution is used when the slides are stained. Leishman's stain may also be used for thick films. It gives almost as good results as Giemsa's stain, but needs to be prepared more frequently. For the method to be described, however, Leishman's stain, in its usual formula of 0"15 grams to 100 c.cm. methyl alcohol, is unsuitable; and the stain should be made up in concentrations five times the normal; that is, 0.75 grams Leishman's powder to 100 c.cm. methyl alcohol.
(b) Buffered Distilled Water or Buffered Tap Water. If the water used for diluting the Giemsa's stain is buffered with phosphates, acidity or alkalinity, which may have developed in the water, is counteracted. Any change in reaction which may have occurred in the solvents of the Giemsa's stain is also corrected. The use of buffered solution ensures that the stain acts in a medium which is neither acid nor alkaline, and that the red and blue elements in the stain will be taken up in the correct degree by the material to be stained. Distilled water generally becomes acid in reaction soon after preparation and needs to be adjusted by some means before use. Tap water, provided that it has a low content of organic matter and a low degree of hardness may be buffered and used as a diluent for staining thin and thick blood films. Experiments were carried out by the writer with artificially prepared samples of hard water. Water of a degree of total and permanent hardness approximating to that of the Thames was used. This water, when buffered with phosphates, was found to be unsuitable for use with Leishman's and Giemsa's stains. The samples were supplied by courtesy of Mr. R. W. BLAIR, of this Institute. Alternatively, rain water may be used. The writer has used buffered tap water from local sources for all thin and thick film work over a period of years, because it has been found to give as good results as the more expensive and less easily obtained distilled water. There may be additional factors apart from high organic content and high degree of hardness which may render tap waters unsuitable for making up buffered solution used in staining blood films. It is probable, however, that in many parts of the tropics, tap water may be used instead of distilled water for this purpose. Buffered water is easy to prepare, costs little, and will " keep " for a considerable period. A simple form of this solution, suggested by COENAES~ is made up as follows : To one litre of water add one gram of potassium dihydrogen phosphate (KH~PO~) and two grams of di-sodium monohydrogen phosphate (NaIHPO4, 12H~O). Shake and dissolve. This has a pH value of about 7.0 and possesses ¢~ COENAES, E . S .
v. Geneesk., Lxxxl, 1934.
(1927).
Giemsa Staining of Blood Preparations.
Nederl. Tijdschr,
RICHARD GREEN.
~79
considerable latitude in taking up acid or alkali. This solution may be used for both thin and thick films. If used for Leishman-stained thin films, after the preliminary fixation and dilution, five mirmtes' staining is then given where leucocytes are to be differentiated, but ten minutes' when search is to be made for malarial parasites.
(c) Method of Staining. The slides bearing the thick films are placed on their edge in the staining trough. These troughs are generally made with a capacity of 50 or 100 c.cm. The stain is prepared by adding one drop of Giemsa's stain to every 3 c.cm. of buffered solution. Thus, if the capacity of the trough is 100 c.cm., about thirty-three drops of Giemsa's stain are added to 100 c.cm. of buffered solution. This is mixed thoroughly and poured gently into the trough until the films are immersed. The slides remain in this diluted stain for one hour. During that period the water removes the h~emoglobin from the red cells, while the stain acts on the malarial parasites, the leucocytes and the " ghosts " of the red cells. The slides are then removed, dipped gently five or six times in water contained in a trough or beaker, for the purpose of washing and of differentiating the red and blue elements. Washing for longer periods is unnecessary. Ordinary tap water or rain water is usually satisfactory for this purpose, but buffered solution may have to be used in certain localities. Finally, the slides are tilted almost vertically to drain the water off and are dried as rapidly as possible. For rapid drying an electric fan is useful, if available. Blotting paper should not be used. If the films are allowed to dry too slowly, this often results in overdifferentiation and lack of contrast in the stained material. APPmmlX. Notes on the Examination of Thick Films.
Good microscopy is essential. More intense illumination is required for thick films than for thin ones. Search for parasites is made with an oil immersion objective and a low power eye-piece. When found, confirmation is made with an eyepiece of higher power. The parasites stain as in thin films, though frequently looking somewhat smaller, and are seen against a background of destroyed red cell envelopes. This background is more or less even in appearance, purplish-red in colour, and the field is seen to contain numbers of leucocytes, some of which can be readily classified if desired.* At the edge of the film where it is thinner, parasites may often be seen within a red ceU which still has its envelope intact. Occasionally, conditions are such that parasiteinfected corpuscles resist being " dehmmoglobinized," while the surrounding uninfected ones are seen to have lost their h~emoglobin. SCHOFFNF.R'Sdots in benign tertian infections * The percentage of eosinophils may be determined rapidly in a well stained thick film. Incidentally, microfilari~e may be readily detected in thick films, using the lower power dry objectives of the microscope. For more accurate differential diagnosis of microfilarim, the Giemsa's stain may be removed with acid alcohol and the film restained with weak hmmatoxylin
9~80
PREPARING A N D STAINING THICK B L O O D FILMS FOR DIAGNOSIS OF MALARIA.
sometimes become apparent with good staining. These are seen best at the edge of the film. Parasites in thick films retain practically the same shape as in thin films. The ring forms, however, may be seen tilted and on edge, while the more advanced forms, such as schizonts and gametocytes, often appear smaller because they are not flattened as in thin films. Crescents may sometimes become abnormal in appearance in thick films, but this will be referred to later.
1. Rules in Diagnosis. Rule 1. Before making a diagnosis resolve what is thought to be a malarial parasite into its components of (i) chromatin (red), (ii) protoplasm (blue) and (iii) pigment (if present). Pigment is not always present but unless (i) and (ii) are clearly defined the diagnosis remains negative. This rule is important because contaminating cocci of various kinds take up the red element of the stain and look like dots of chromatin material. Occasionally these become associated with a blue-staining piece of fibrin or a red cell envelope. It is unlikely that this will happen more than once or twice in the same film, which leads to a further rule as follows : Rule 2 . - - D o not diagnose malarial infection on the finding of what appears to be one parasite only. The film should be searched until the diagnosis is confirmed again and again. These rules apply particularly to films in which the parasites are very scanty. 2.
The Distinction Between the Different Parasite Species.
Without illustration it is diffficult to describe the specific charac:ers of the different species of malarial parasites as encountered in thick films. Sometimes the distinction is difficult to make but, in the majority of films, this can be done fairly easily after the observer has had some experience of the method. The following general observations are not complete but should help in differential diagnosis : - -
(a) Subtertian Malaria. In infections with P. falciparum the asexual cycle is rarely seen, in its entirety, in the peripheral blood. Schizogony seems to take place mainly in the capillaries of the internal organs, while the peripheral blood usually shows small ring forms only. In thick films, therefore, the main feature of a subtertian malarial infection is generally the presence of numbers of small ring forms. It is seldom that one finds schizonts except in severe and very heavy infections. T h e additional finding of crescents will denote present or recent infection with P. falciparum and will assist in confirming the diagnosis Crescents are generally the most easily recognised parasite in thick films. As a rule, they retain their crescentic outline but occasionally, while the thick film is drying in a moist atmosphere, conditions in the midgut of the mosquito are imitated, with the result that the female crescents become circular and extrude some of their chromatin in the form of a polar body, while the male crescents go through the process of exflaggelation. When this occurs, these female crescents which have become circular are apt to be mistaken for quartan gametocytes.
(b) Benign Tertian Malaria. Larger ring forms are usually seen, b u t the main feature is polymorphism ; that is, parasites in all stages will be found, mostly in the form of growing trophozoites. One may at least, after a little practice, diagnose benign tertian malaria with certainty, for these large diffuse-looking parasites with their fine greenish-yellow pigment are sufficiently eharactertistic. On occasions when the specific diagnosis is in doubt, if two or three thick films are taken from a benign tertian case at short intervals, polymorphism will always be found.
(c) Quartan Malarm. T h e quartan parasite also is usually found in all stages in thick films, but when compared with the benign tertian parasite, it is smaller, more compact, generally circular in
RICHARD GREEN.
281
outline, and the protoplasm, particularly of the larger forms, becomes more densely stained. T h e pigment is coarse and of a distinct greenish colour. This becomes apparent soon after the ring forms start to grow, whereas in the benign tertian parasite, pigment production occurs comparatively late.
(d) Mixed Infections. T h e diagnosis, in thick films, of the presence of more than one species of malarial parasite is often difficult. No help may be obtained, as in thin films, from alterations which may have occurred in the size and appearance of the infected erythrocytes ; and it is necessary, in thick films, to study the morphology of the parasites more closely. With increasing experience, the diagnosis of mixed infections becomes less difficult ; but, in doubtful cases, a series of thin films should be obtained from the patient. On the other hand, in thick films, on account of the greater concentration of the blood in each microscope field, mixed infections are, as a rule, less likely to be overlooked.
3. Artefacts in Thick Films. Artefacts are more likely to give rise to error in thick films than in thin films. Thick films taken from dirty fingers on old, scratched slides and kept too long before staining are more likely to contain artefacts. These are seldom seen in well prepared and stained films. Protozoal contamination of the water used in staining sometimes gives rise to difficulties in interpretation. More common artefacts which may, in part, simulate the appearance of malarial parasites are as follows : Certain elongate mould spores may be mistaken for crescents. Crescentic shaped scratches on the slide sometimes retain the stain and may also be mistaken for crescents. Blood platelets joined together sometimes have a crescentic outline. In size and in reaction to staining, the chromatin of ring forms may be imitated by cocci from the skin of the patient. These cocci generally take up the red element of the stain, and pieces of fibrin, epithelium, or other blue-staining matter by being closely juxtaposed to this red-staining dot, may help to produce an artefact resembling a malarial parasite. T h e application of the two rules given above should, however, prevent a wrong diagnosis from being made.
4. General. (a) Cleaning the Slides.--Special mention is made of the necessity of cleaning thoroughly the slides used for thick film work. A common fault in cleaning slides is that they are allowed to remain stuck together during immersion in cleaning fluids. Careless mechanical cleansing later may not rub off all the old film, which is then colourless and not easily seen by the unaided eye. If cleaning is not done properly, parasites may remain on the slide, and, although the next film taken on the same slide is really negative for malaria parasites, these old underlying parasites will restain quite well and give rise to a positive diagnosis. (b) Cedar Wood Oil, when left on thick films, causes them to fade rapidly. If required for further examination, the cedar wood oil should be tilted off at once and the remainder dissolved out with xylol or chloroform. T h e use of these solvents, however, sometimes results in minute refractile granules appearing in the thicker part of the film. T o remove these, the film should be washed very rapidly with alcohol and dried immediately. SUMMARY. i. A m e t h o d o f p r e p a r i n g a n d s t a i n i n g t h i c k b l o o d f i l m s is d e s c r i b e d . In this method, various steps usually recommended are omitted. For example, t h e s p r e a d i n g o f e a c h film w i t h a n e e d l e a n d p r e l i m i n a r y " d e - h ~ e m o g l o b i n i z a t i o n " w i t h s p e c i a l fluids a r e b o t h r e n d e r e d u n n e c e s s a r y . B u f f e r i n g o f t h e water used for diluting the stain counteracts acidity and alkalinity which may
289.
PI~J~PARINGAND STAINING THICK BLOOD FILMS FOR DIAGNOSIS OF MALARIA.
have developed in the stain or in the diluent, and helps to ensure constant results. Water buffered with phosphates is regarded as simpler to prepare and as having more reserve than water which has been adjusted to a certain pH value by the addition of acid or alkali in the presence of an indicator. It is pointed out that, in some localities, it may be unnecessary to use distilled water with Leishman's or Giemsa's stains, and that tap water may be used. ii. In an appendix, notes are given on the examination of thick blood films for malarial parasites, on differential diagnosis between parasite species, and on common artefacts which may appear in thick films.
A METHOD
OF P R E P A R I N G AND
STAINING
THICK
BLOOD
F I L M S FOR T H E D I A G N O S I S OF MALARIA. BY R I C H A R D GREEN, M.D., B.S., D.T.M.*
(From the Institute for Medical Research, Kuala Lumpur, F.M.S.)
1.
INTRODUCTION.
The search for malarial parasites in blood is undoubtedly made easier by the use of thick films which have been properly prepared and stained. It may be said that, when the diagnosis of malarial infection rather than the definition of the actual parasite species becomes all-important, and where large numbers of blood films are to be examined efficiently for malarial parasites, the use of thick films becomes the method of necessity rather than the method of choice. It is a common experience to find, however, that among those concerned with the diagnosis of malaria, the use of thick films is often abandoned after a few trials on account of the technical difficulties involved, or because of the poor results obtained. The method to be described is, strictly speaking, not an original one, but various steps, that have been recommended by text books or by individuals, have been tried from time to time; some processes have been modified or retained, others have been rejected. From tiffs series of trials a simple method has been evolved, which has afforded uniformly good results over a long period. Details are given as follows :-* The writer is indebted to Dr. A. N ~ w KINGSBURY,Director, Institute for Medical Research, for permission to publish this paper ; and to Dr. W. FLETCHER and Dr. H. O. HOPKINS for suggestions regarding the staining of thick films.
276
PREPARING AND STAINING TIIICK BLOOD FILMS FOR DIAGNOSIS OF MALARIA. 2.
TECHNIQUE OF PREPARING AND STAINING T H I C K FILMS.
(a) Preparation. 1. Making the Film. The finger of the patient is cleaned and dried thoroughly. It is then pricked with a triangular pointed needle and a larger quantity of blood than is used for thin films is gently squeezed out. The finger being dry, this blood takes the form of a globule. The pricked surface is held facing upwards and the lower surface of a microscope slide is rubbed carefully over the globule of blood to form a circle about half an inch in diameter. The circular motion of the slide is continued until the blood is evenly distributed in the form of a disc. At first the beginner must be careful not to make the film too thick ; nor must the slide touch the skin of the finger. The slide is then turned rapidly so that the blood faces upwards; it is tilted, if necessary, to secure even distribution of the blood ; then placed on a level surface to dry. It must be protected from dust. The process of making the film should occupy a few seconds only.
2. Drying the Film. After preliminary drying on a level surface the films must continue to dry for some hours before staining, otherwise they may become detached from the slide in the staining trough.* If a desiccator or an incubator is available', the period of drying may be reduced to two or three hours. A makeshift drying chamber may be devised by inserting a lighted electric lamp at the end of flexible wire into a drawer. A thermometer is placed in the drawer and, by moving the drawer in or out, the temperature within may be regulated to about 37 ° C. A period of two or three hours in this form of drying chamber is generally sufficient. It is inadvisable to keep thick films unstained for more than twenty-four hours because, after this, they do not stain so well ; also, moulds and bacteria are liable to develop in t h e m , Finally, during drying, the film must be protected from insects, such as ants and cockroaches. The latter often eat thick films after they have been stained.
(b) Staining. The following apparatus and solutions are required.
Apparatus.-(i) (ii) (iii) (iv)
Measuring cylinders, 50 c.cm. or 100 c.cm. Staining troughs to take slides on edge. Dropping bottles. Troughs or beakers for washing slides.
* The period required for drying varies With the local condition~ of temperature and humidity, but is soon ascertained by experience.
RICHARD GREEN.
277
Solutiom.--(a) Giemsa's stain. (b) Buffered distilled water or buffered tap water. The full complement of apparatus as detailed above is desirable. It is best to stain the slides on edge, but these, supported on glass rods, may be placed film downwards in a shallow staining trough. If the diluted stain is poured on to slides allowed to remain film upwards, the particles of stain precipitated on the film may be difficult to remove later. Alternatively, the slides, separated suitably, may be made into bundles and immersed in stain which is contained in a glass beaker or porcelain cup.
(a) Giemsa's Stain. Geimsa's stain is often so badly made up that notes on its preparation are given below : The formula of Giemsa's stain for 500 c.cm. is as follows :-Azur II-eosin 3.0 gram Azur II 0.8 gram Glycerine 250 c.cm. Methyl alcohol 250 c.cm. The azur II-eosin and azur II are generally combined in the form of powder. To make 500 c.cm. of stain proceed as follows : Weigh out 3.8 grams of the combined Giemsa's stain powder and place it in a glass or glazed mortar. Measure 250 c.cm. of pure glycerine and pour it from time to time over the powder, using the pestle to assist in mixing and dissolving. Then add intermittently a total quantity of 250 c.cm. methyl alcohol (acetone free) stirring rapidly. This latter part must be done fairly quickly, otherwise the alcohol will evaporate. When the powder is dissolved sufficiently, pour the contents of the mortar into a glass-stoppered bottle, wash out the mortar with what remains of the 250 c.cm. methyl alcohol and put these washings into the bottle. The bottle should be shaken frequently during the next twenty-four hours, and the stain should be ready for use in forty-eight hours. When required, small quantities of the stain are filtered into dropping bottles. During filtration, the funnel is covered over with half a petri dish to prevent evaporation of the methyl alcohol. Prepared in this way, the stain has been found to retain its properties for twelve months or more in the tropical climate of Malaya. In many cases it will be sufficient to prepare 50 c.cm. of the stain only. With this volume of course, one-tenth of the quantities given above are used. If soloids (Burroughs Wellcome) are used, one soloid of Giemsa's stain (indexed as eosin-azur) is dissolved in 5 c.cm. of a mixture of equal parts of glycerine and methyl alcohol. Soloids, however, are sometimes difficult to dissolve completely. They should be ground to a fine powder before use. The glycerine used is of importance in making good stain. It should be as pure as possible (re-distilled) and obtained from a reliable drug house, because F
978
PREPARINGAND STAINING THICK BLOOD FILMS FOR DIAGNOSIS OF MALARIA.
most brands of glycerine become acid in reaction soon after the bottle is opened. The acidity varies in degree. To counteract this, buffered solution is used when the slides are stained. Leishman's stain may also be used for thick films. It gives almost as good results as Giemsa's stain, but needs to be prepared more frequently. For the method to be described, however, Leishman's stain, in its usual formula of 0"15 grams to 100 c.cm. methyl alcohol, is unsuitable; and the stain should be made up in concentrations five times the normal; that is, 0.75 grams Leishman's powder to 100 c.cm. methyl alcohol.
(b) Buffered Distilled Water or Buffered Tap Water. If the water used for diluting the Giemsa's stain is buffered with phosphates, acidity or alkalinity, which may have developed in the water, is counteracted. Any change in reaction which may have occurred in the solvents of the Giemsa's stain is also corrected. The use of buffered solution ensures that the stain acts in a medium which is neither acid nor alkaline, and that the red and blue elements in the stain will be taken up in the correct degree by the material to be stained. Distilled water generally becomes acid in reaction soon after preparation and needs to be adjusted by some means before use. Tap water, provided that it has a low content of organic matter and a low degree of hardness may be buffered and used as a diluent for staining thin and thick blood films. Experiments were carried out by the writer with artificially prepared samples of hard water. Water of a degree of total and permanent hardness approximating to that of the Thames was used. This water, when buffered with phosphates, was found to be unsuitable for use with Leishman's and Giemsa's stains. The samples were supplied by courtesy of Mr. R. W. BLAIR, of this Institute. Alternatively, rain water may be used. The writer has used buffered tap water from local sources for all thin and thick film work over a period of years, because it has been found to give as good results as the more expensive and less easily obtained distilled water. There may be additional factors apart from high organic content and high degree of hardness which may render tap waters unsuitable for making up buffered solution used in staining blood films. It is probable, however, that in many parts of the tropics, tap water may be used instead of distilled water for this purpose. Buffered water is easy to prepare, costs little, and will " keep " for a considerable period. A simple form of this solution, suggested by COENAES~ is made up as follows : To one litre of water add one gram of potassium dihydrogen phosphate (KH~PO~) and two grams of di-sodium monohydrogen phosphate (NaIHPO4, 12H~O). Shake and dissolve. This has a pH value of about 7.0 and possesses ¢~ COENAES, E . S .
v. Geneesk., Lxxxl, 1934.
(1927).
Giemsa Staining of Blood Preparations.
Nederl. Tijdschr,
RICHARD GREEN.
~79
considerable latitude in taking up acid or alkali. This solution may be used for both thin and thick films. If used for Leishman-stained thin films, after the preliminary fixation and dilution, five mirmtes' staining is then given where leucocytes are to be differentiated, but ten minutes' when search is to be made for malarial parasites.
(c) Method of Staining. The slides bearing the thick films are placed on their edge in the staining trough. These troughs are generally made with a capacity of 50 or 100 c.cm. The stain is prepared by adding one drop of Giemsa's stain to every 3 c.cm. of buffered solution. Thus, if the capacity of the trough is 100 c.cm., about thirty-three drops of Giemsa's stain are added to 100 c.cm. of buffered solution. This is mixed thoroughly and poured gently into the trough until the films are immersed. The slides remain in this diluted stain for one hour. During that period the water removes the h~emoglobin from the red cells, while the stain acts on the malarial parasites, the leucocytes and the " ghosts " of the red cells. The slides are then removed, dipped gently five or six times in water contained in a trough or beaker, for the purpose of washing and of differentiating the red and blue elements. Washing for longer periods is unnecessary. Ordinary tap water or rain water is usually satisfactory for this purpose, but buffered solution may have to be used in certain localities. Finally, the slides are tilted almost vertically to drain the water off and are dried as rapidly as possible. For rapid drying an electric fan is useful, if available. Blotting paper should not be used. If the films are allowed to dry too slowly, this often results in overdifferentiation and lack of contrast in the stained material. APPmmlX. Notes on the Examination of Thick Films.
Good microscopy is essential. More intense illumination is required for thick films than for thin ones. Search for parasites is made with an oil immersion objective and a low power eye-piece. When found, confirmation is made with an eyepiece of higher power. The parasites stain as in thin films, though frequently looking somewhat smaller, and are seen against a background of destroyed red cell envelopes. This background is more or less even in appearance, purplish-red in colour, and the field is seen to contain numbers of leucocytes, some of which can be readily classified if desired.* At the edge of the film where it is thinner, parasites may often be seen within a red ceU which still has its envelope intact. Occasionally, conditions are such that parasiteinfected corpuscles resist being " dehmmoglobinized," while the surrounding uninfected ones are seen to have lost their h~emoglobin. SCHOFFNF.R'Sdots in benign tertian infections * The percentage of eosinophils may be determined rapidly in a well stained thick film. Incidentally, microfilari~e may be readily detected in thick films, using the lower power dry objectives of the microscope. For more accurate differential diagnosis of microfilarim, the Giemsa's stain may be removed with acid alcohol and the film restained with weak hmmatoxylin
9~80
PREPARING A N D STAINING THICK B L O O D FILMS FOR DIAGNOSIS OF MALARIA.
sometimes become apparent with good staining. These are seen best at the edge of the film. Parasites in thick films retain practically the same shape as in thin films. The ring forms, however, may be seen tilted and on edge, while the more advanced forms, such as schizonts and gametocytes, often appear smaller because they are not flattened as in thin films. Crescents may sometimes become abnormal in appearance in thick films, but this will be referred to later.
1. Rules in Diagnosis. Rule 1. Before making a diagnosis resolve what is thought to be a malarial parasite into its components of (i) chromatin (red), (ii) protoplasm (blue) and (iii) pigment (if present). Pigment is not always present but unless (i) and (ii) are clearly defined the diagnosis remains negative. This rule is important because contaminating cocci of various kinds take up the red element of the stain and look like dots of chromatin material. Occasionally these become associated with a blue-staining piece of fibrin or a red cell envelope. It is unlikely that this will happen more than once or twice in the same film, which leads to a further rule as follows : Rule 2 . - - D o not diagnose malarial infection on the finding of what appears to be one parasite only. The film should be searched until the diagnosis is confirmed again and again. These rules apply particularly to films in which the parasites are very scanty. 2.
The Distinction Between the Different Parasite Species.
Without illustration it is diffficult to describe the specific charac:ers of the different species of malarial parasites as encountered in thick films. Sometimes the distinction is difficult to make but, in the majority of films, this can be done fairly easily after the observer has had some experience of the method. The following general observations are not complete but should help in differential diagnosis : - -
(a) Subtertian Malaria. In infections with P. falciparum the asexual cycle is rarely seen, in its entirety, in the peripheral blood. Schizogony seems to take place mainly in the capillaries of the internal organs, while the peripheral blood usually shows small ring forms only. In thick films, therefore, the main feature of a subtertian malarial infection is generally the presence of numbers of small ring forms. It is seldom that one finds schizonts except in severe and very heavy infections. T h e additional finding of crescents will denote present or recent infection with P. falciparum and will assist in confirming the diagnosis Crescents are generally the most easily recognised parasite in thick films. As a rule, they retain their crescentic outline but occasionally, while the thick film is drying in a moist atmosphere, conditions in the midgut of the mosquito are imitated, with the result that the female crescents become circular and extrude some of their chromatin in the form of a polar body, while the male crescents go through the process of exflaggelation. When this occurs, these female crescents which have become circular are apt to be mistaken for quartan gametocytes.
(b) Benign Tertian Malaria. Larger ring forms are usually seen, b u t the main feature is polymorphism ; that is, parasites in all stages will be found, mostly in the form of growing trophozoites. One may at least, after a little practice, diagnose benign tertian malaria with certainty, for these large diffuse-looking parasites with their fine greenish-yellow pigment are sufficiently eharactertistic. On occasions when the specific diagnosis is in doubt, if two or three thick films are taken from a benign tertian case at short intervals, polymorphism will always be found.
(c) Quartan Malarm. T h e quartan parasite also is usually found in all stages in thick films, but when compared with the benign tertian parasite, it is smaller, more compact, generally circular in
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outline, and the protoplasm, particularly of the larger forms, becomes more densely stained. T h e pigment is coarse and of a distinct greenish colour. This becomes apparent soon after the ring forms start to grow, whereas in the benign tertian parasite, pigment production occurs comparatively late.
(d) Mixed Infections. T h e diagnosis, in thick films, of the presence of more than one species of malarial parasite is often difficult. No help may be obtained, as in thin films, from alterations which may have occurred in the size and appearance of the infected erythrocytes ; and it is necessary, in thick films, to study the morphology of the parasites more closely. With increasing experience, the diagnosis of mixed infections becomes less difficult ; but, in doubtful cases, a series of thin films should be obtained from the patient. On the other hand, in thick films, on account of the greater concentration of the blood in each microscope field, mixed infections are, as a rule, less likely to be overlooked.
3. Artefacts in Thick Films. Artefacts are more likely to give rise to error in thick films than in thin films. Thick films taken from dirty fingers on old, scratched slides and kept too long before staining are more likely to contain artefacts. These are seldom seen in well prepared and stained films. Protozoal contamination of the water used in staining sometimes gives rise to difficulties in interpretation. More common artefacts which may, in part, simulate the appearance of malarial parasites are as follows : Certain elongate mould spores may be mistaken for crescents. Crescentic shaped scratches on the slide sometimes retain the stain and may also be mistaken for crescents. Blood platelets joined together sometimes have a crescentic outline. In size and in reaction to staining, the chromatin of ring forms may be imitated by cocci from the skin of the patient. These cocci generally take up the red element of the stain, and pieces of fibrin, epithelium, or other blue-staining matter by being closely juxtaposed to this red-staining dot, may help to produce an artefact resembling a malarial parasite. T h e application of the two rules given above should, however, prevent a wrong diagnosis from being made.
4. General. (a) Cleaning the Slides.--Special mention is made of the necessity of cleaning thoroughly the slides used for thick film work. A common fault in cleaning slides is that they are allowed to remain stuck together during immersion in cleaning fluids. Careless mechanical cleansing later may not rub off all the old film, which is then colourless and not easily seen by the unaided eye. If cleaning is not done properly, parasites may remain on the slide, and, although the next film taken on the same slide is really negative for malaria parasites, these old underlying parasites will restain quite well and give rise to a positive diagnosis. (b) Cedar Wood Oil, when left on thick films, causes them to fade rapidly. If required for further examination, the cedar wood oil should be tilted off at once and the remainder dissolved out with xylol or chloroform. T h e use of these solvents, however, sometimes results in minute refractile granules appearing in the thicker part of the film. T o remove these, the film should be washed very rapidly with alcohol and dried immediately. SUMMARY. i. A m e t h o d o f p r e p a r i n g a n d s t a i n i n g t h i c k b l o o d f i l m s is d e s c r i b e d . In this method, various steps usually recommended are omitted. For example, t h e s p r e a d i n g o f e a c h film w i t h a n e e d l e a n d p r e l i m i n a r y " d e - h ~ e m o g l o b i n i z a t i o n " w i t h s p e c i a l fluids a r e b o t h r e n d e r e d u n n e c e s s a r y . B u f f e r i n g o f t h e water used for diluting the stain counteracts acidity and alkalinity which may
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PI~J~PARINGAND STAINING THICK BLOOD FILMS FOR DIAGNOSIS OF MALARIA.
have developed in the stain or in the diluent, and helps to ensure constant results. Water buffered with phosphates is regarded as simpler to prepare and as having more reserve than water which has been adjusted to a certain pH value by the addition of acid or alkali in the presence of an indicator. It is pointed out that, in some localities, it may be unnecessary to use distilled water with Leishman's or Giemsa's stains, and that tap water may be used. ii. In an appendix, notes are given on the examination of thick blood films for malarial parasites, on differential diagnosis between parasite species, and on common artefacts which may appear in thick films.