The morphology of malarial parasites in thick blood films. Part I. The thick-film morphology of Plasmodium vivax

467 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE.

Vol. XXXII. No. 4. January, 1939.

COMMUNICATIONS. THE

MORPHOLOGY OF MALARIAL PARASITES IN THICK BLOOD FILMS. P A R T I. THE THICK-FILM MORPHOLOGY OF PLASMODIUM FIVAX. BY

J. w. FIELD AND

H. LE FLEMING, From the Institute for Medical Research, Federated Malay States. FOREWORD.

Thick-film methods suitable for the demonstration of malarial parasites in the blood have been known for many years. First suggested by Ross in 1903 and simplified by KOCH during the German Trypanosomiasis Expedition to East Africa in 1907, they are now fully established as a valuable aid to diagnosis. The thick-film process depends on the fact that it is possible, by dissolving out the haemoglobin, to render thick opaque smears of blood, dried on glass slides, sufficiently transparent for examination by transmitted light. Cellular structures, other than the red cells, are preserved well enough to be recognized, concentrated to an extent depending on the thickness of the film. The advantages of concentration are obvious but some sacrifice of the integrity of cytoplasmic outline must be accepted, for leucocytes and parasites seldom pass unscathed through the process of haemolysis which is an essential step in the staining technique. Descriptions of the morphology of malarial parasites are usually based on appearances in thin films fixed with methyl alcohol and stained by one of the Romanowsky methods. These appearances are not fully preserved in thick films. Thick-film staining entails important departures from thin-film technique. The host red cells are destroyed and the parasites, exposed to the disruptive effects of a hypotonic solution of stain, are modified in contour, colour and size. They tend to appear smaller and to be less regular in outline. They are seen moreover in an unfamiliar setting; no longer, as in fixed thin films, neatly framed by their host cells, but stripped and distorted on a mottled blue-grey ground of red cell residues. Some of the difficulties of the thick-film diagnosis of malaria arise from these facts. This paper and subsequent papers on Plasmodiumfalciparum and P. malariae attempt to describe the changed morphology.

468

PLASMODIUM

VIVAX.

TECHNICAL METHODS.

The water colour drawings which illustrate this series of papers were made directly from the microscope by one of us (H. LE V.) with no preconceived ideas of parasite appearances. * They attempt to reproduce what was seen without diagrammatic simplification. Thick and thin blood films, taken at the same time from patients with acute untreated malaria and stained by a Giemsa technique, were the sources of illustrative material. A standard Leitz binocular microscope with a 1/12 inch fluorite objective, × 5 eyepieces, and a " S p e n c e r " concentrated filament half-watt lamp with a Coming " Daylite " filter, were used throughout. Descriptions are based on I0 years' experience of thick-film methods developed in the Malaria Research Division of this Institute by our colleague, D r . R. GREEN. W e may perhaps emphasize that the form, colour and setting of parasites in thick blood films, lysed and Romanowsky-stained, varies with the method of staining and are particularly related to the quality of the film background. Morphological description must therefore be limited to definite staining conditions. The following account is based solely on, and has reference only to, the appearance of parasites in thick films prepared and stained by the methods detailed on pages 475 to 477. T H I C K - F I L M APPEARANCES OF

P. ?)ivax.

Young Trophozoites. The most distinctive feature of young P. vivax trophozoites in thick films is the variation of cytoplasmic outline and arrangement. Haemolysis seems to modify the clear-cut ring forms seen in fixed thin films in two ways ; by favouring the collapse of the parasite vacuole and by distorting its outline, often with a loss of cytoplasmic continuity. The signet-ring formation familiar in thin films is only occasionally retained and ring forms tend to be broken or collapsed. The cytoplasm is often contracted into one or two fairly homogeneous masses arranged mutually and, in relation to the nuclear chromatin, in fairly characteristic patterns depending on the manner and plane of collapse. To facilitate description we may perhaps coin the following terms for the common appearances :-(a) Interrupted rings. (b) Exclamation marks. (c) " Swallow " forms. Interrupted rings are still recognizably " ring " forms. The cytoplasm is broken into a series of fragments along the line of the original contour. The chromafin is a single circular bead usually placed somewhere on this line. *" In the representation of colour each artist will render his own interpretation which will differ, however slightly from that of other artists. In the making of the coloured plate of blood cells there are three separate artists concerned, the one who makes the original painting, the lithographer and the printer. Thus each deviation from the original is liable not only to perpetuation but to a three-fold exaggeration and it is almost impossible to ensure that the final result is an exact representation of the original." This reminder of the difficulties of the exact reproduction of colour (NAPIER and NEN-GUPTA, 1938) prompts the comment that despite the excellence of the plates there are slight departures from the original appearance seen under the microscope. (1) Sehiiffner's dots shown in the thin-film field are too red and, in the cell containing the schizont, somewhat too solid looking. (2) the background of the thick-film field is too uniform ; a vague mottling, normally faint but sometimes as distinctive as the cloud patterns of a mackerel sky, is more

usual

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469

F I E L D A N D H . LE F L E M I N G .

The exclamation mark has a single, compact, often elongated wisp of cytoplasm, either straight or on the curve like a " question-mark," extending from a single bead of chromatin. Chromatin and cytoplasm usually appear continuous but may be separated by a considerable gap. In the swallow form the cytoplasm is "disposed more or less symmetrically as two " wings " which extend at a wide angle from the central chromatin bead. These cytoplasmic patterns of the young P. vivax trophozoite in thick blood films are illustrated in Fig. 1 and in Plate I (6-18). Departure from thin-film appearances occurs only in the cytoplasm. The nuclear chromatin is usually a single circular" bead." Occasionally the chromatin is broken into two, rarely into three, smaller " beads." The familiar staining reactions of chromatin and cytoplasm seen in Romanowsky-stained thin films, the bright maroon of the chromatin and the pale blue of the cytoplasm, are unchanged. The parasites are isolated on a blue-grey mottled background derived from the remains of the lysed red cells. The host cells usually disappear completely Exclamation marks. Swallow forms. Unbroken rings. Broken rings.

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leaving no trace either of their original contour or of stippling, but occasionally, particularly at the thinner edge of the film, infected cells persist with Schiiffner's dots vaguely seen as a stippled zone around the parasites. Advanced Trophozoites. With advancing growth the contour of the parasite becomes less definite. There is a tendency to what may be termed " cytoplasmic scatter." The relatively compact cytoplasm of the early forms has dispersed into tenuous pale-blue wisps and strands scattered around the chromatin " bead." Sometimes the cytoplasm is broken into a cluster of isolated fragments suggesting early segmentation. Cytoplasmic " scatter" .and fragmentation with the

470

PLASMODIUM V1V.JX.

chromatin as yet undivided we have noted only with P. vivax and believe to be characteristic of this species. The appearance is illustrated in Fig. 2.

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FIo. 2 . - - C o n t o u r drawings o f P . vivax trophozoites with the cytoplasm broken into a cluster of isolated fragments ; a c o m m o n a n d characterlO,u istic formation of advanced trophozoites in , I thick-blood films.

The chromatin mass tends to be larger and less consistently circular in form. Though clearly defined as a rule, it is sometimes absent, having apparently disappeared during staining. When seen isolated from the cytoplasm the colour of the chromatin is bright maroon-red as in the younger forms. PLATE I. A.

B.

C.

P. vivax IN Trophozoites in Thin 6-11, 13-18 : 10, 13, 17 : 6, 14, 15 : 8, 9 : 12, 19 :

GIEMSA-STAINED THIN AND THICK BLOOD FILMS. (1-5) and Thick (6-19) Blood Films. young trophozoites. " broken ring " forms. " exclamation mark " forms. " swallow " forms. advanced trophozoites with early pigment. Note the delicacy and dispersion of the cytoplasm (12, 19) and the fragmented cytoplasm and isolated pigment (12). This breaking up of the cytoplasm into a cluster of separate fragments while the chromatin is still undivided is very characteristic of P. vivax in thick films. Schizonts in Thin (20-24) and Thick (25-32) Blood Films. 25, 26, 29, 30 : early schizonts. 27, 28, 31 : advanced schizonts. 32 : mature schizont. Note the prominence of the pigment when isolated (26), the persistence of Schtiffner's dots (28, 30) and the change in colour of the dividing chromatin (27, 28, 31). Individual segments of the advanced schizont sometimes tend to be somewhat pale and distended (28). Gametocytes in Thin (33-37) and Thick (38-47) Blood Films. T h e drawings illustrate a range of appearances. No attempt has been made to differentiate sex; the distinction in thick films between male and female is not well defined. 39, 43, 45 : gametocytes relatively undamaged. 38, 40, 44 : gametocyte slightly damaged b u t with cytoplasm, chromatin and pigment still clearly distinguishable. 41, 42, 46, 47 : gametocytes showing extensive damage ; pigment and chromatin alone visible (46, 47), pigment and cytoplasm (41). 45: a very distinctive form with an excessively large and vividly stained mass of chromatin and a p r o m i n e n t halo of pigment.

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MORPHOLOGY

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THIN

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J. w .

FIELD AND H. LE FLEMING.

471

Pigment is often visible at this stage as a yellow haze tinging the offshoots of cytoplasm or as small scattered granules dispersed throughout the parasite. Sometimes the pigment is isolated against the blue-grey of the film background. Pigment tends to be more clearly defined than in fixed thin films, being unobscured by Schiiffner's stippling and thrown into sharper relief by the thinning and dispersion of the cytoplasm.

Early Schizonts. The cytoplasm is now more abundant, sometimes dispersed in delicate palely stained wisps, sometimes more deeply stained and more compact but still with great variety of form. This tendency to cytoplasmic delicacy and tenuity is a fairly constant feature of P. vivax in thick-blood films and a useful aid to species diagnosis. The nuclear chromatin is divided and appears in compact rounded or ovoid masses enveloped in blue-staining cytoplasm. The colour of the divided chromatin is no longer the bright maroon of the trophozoite stage but reddish purple. This change in colour is probably more apparent than real. The ehromatin of the young forms is usually seen isolated, with its true colour unobscured whereas the divided chromatin is often enveloped in a covering of basophilic cytoplasm. The pigment is increased in amount and, as a rule, more prominent than in thin films. It appears as a yellowish-brown haze in the cytoplasm or in tiny rodlets which vary in colour from bright yellow, when seen isolated on the film background, to dark brown when visible through a haze of blue cytoplasm. Sometimes pigment and chromatin alone remain, the cytoplasm having disappeared during staining. Advanced Schizonts. The parasite is now less tenuous in texture and more vividly stained. It is irregularly round or oval in shape often with a scalloped contour from the projection of individual segments. The cytoplasm is more abundant and though occasionally fragmented and partly lost, the scattering seen in the earlier forms is less marked. Division of the cytoplasm is advanced, the individual segments having either separated completely or become delineated by a light peripheral zone of demarcation. The dim outline of the host cell is sometimes visible-more often than with the younger forms--as a faintly stippled zone around the parasite. The nuclear chromatin is divided at this stage into segments arranged with a fair uniformity throughout the parasite though occasionally concentrated to one side. The chromatin segments are rounded, oval or sometimes irregular and vividly contrasted against the pale blue of the cytoplasm which forms their immediate background. They are usually seen through a covering of basophilie cytoplasm which modifies their colour to a deep rich purple.

472

PLASMODIUM VIVAX.

The pigment is concentrated into one or two central, occasionally peripheral, collections of rodlets varying in colour from bright yellow-green to dark-brown according to the depth of cytoplasm through which they are seen. Mature Schizonts. With advancing maturity the parasite seems better able to resist the influences which damage the younger forms and fully segmented parasites differ little in appearance from those seen in thin films. The mature schizont is a rounded or oval cluster of deeply stained fully differentiated segments which are either contiguous, with a little pale staining intersegmental residue, or dispersed and cleanly separated. T h e pigment is usually concentrated into a single dense mass, usually near the centre, but occasionally at the periphery or even outside the merozoite cluster. From concentration it is now darker in colour, sometimes almost black with a hazy yellow fringe. The degree of concentration varies. Although commonly seen as a single fairly dense homogeneous mass the pigment may also appear as a compact collection of a few large but discrete granules. Rarely the pigment is scattered among the merozoites, possibly from dispersion during the staining of the film. Gametocytes. The gametocytes of P. vivax are not as a rule difficult to identify in thick films, but show much greater morphological variation than in fixed thin films. This variation seems to depend on the degree of disruption of cytoplasm during lysis of the blood and occurs no matter how careful the staining or constant the technique. A single film may show gametocytes with cytoplasm intact and contours undamaged alongside others with nothing remaining but a blob of ehromatin and a halo of pigment. PLATE II. SPLIT ~/~ICROSCOPIC FIELD ILLUSTRATING CHARACTERISTIC DIFFERENCES IN THE APPEARANCE OF P. "oi',uax IN GIEMSA-STAINED THIN AND THICK BLOOD FILMS.

T h i n film ; i. a " ring " form. ii. a female gametocyte. iii. an early schizont. T h e thick film illustrates various stages of development : i. young trophozoites w i t h a characteristic range of cytoplasmic patterns. ii. advanced trophozoites including one example of cytoplasmic scatter, a distinctive appearance which is sometimes seen at this stage. iii. two young schizonts, one with two and one with four chromatin masses. iv. a gametocyte. T h e changed appearance of the leucocytes in the thick film is shown. T h e cytoplasm and granules of the neutrophils is tattered and partly lost though the nuclei are well preserved. T h e eosinophil is less damaged ; eosinophilic granules are often retained[intact. A group of platelets is shown in the top corner of the half field.

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GIEMSA-STAINED

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THICK

BLOOD

FILMS.

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The undamaged gametocyte does not differ much in appearance from that seen in fixed thin films except that it is unobscured by Schiiffner's dots, tends to be smaller and paler, and seldom has so definite a peripheral contour. It appears as a large round or oval parasite with fairly homogeneous cytoplasm varying in colour from pale blue to bluish-purple. With partial disruption, the cytoplasm shows great variation in amount and arrangement. Common appearances are illustrated in the accompanying diagram (Fig. 3). T h e chromatin is undivided and often more clearly defined than in thin films; a single deeply stained reddish-purple mass without characteristic relationship to the cytoplasm but tending to a peripheral position. Sometimes the chromatin is diffuse, pale staining and difficult to define and occasionally it may not be seen at all, the fact that the parasite is a gametocyte being suggested only by its advanced development, the absence of evidence of segmentation, and the quantity and distribution of the pigment. A very distinctive form of • ° 2,

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FIG. 3 . - - C o m m o n arrangements of the chromatin, c y t o p l a s m a n d p i g m e n t o f P. vivax g a m e t o c y t e s in G i e m s a - s t a i n e d t h i c k - b l o o d films. i~i

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and (8) Cytoplasm as a single mass of irregular contour. and (8) Cytoplasm completely lost. (5) (6) and (7) Cytoplasm broken : chrornatin and, part of pigment isolated.

unknown significance is illustrated in Plate I (45). T h e chromatin mass is excessively large and vividly stained with the pigment scattered in a small circular area around it or to one side, and with cytoplasm limited in amount and often absent• The pigment of the gametocyte is often of itself sufficiently distinctive for identification of species and phase. It is never concentrated as in schizonts of similar size but scattered in yellowish-brown rodlets throughout the cytoplasm with a tendency to a circular distribution around the periphery. Sometimes pigment and chromatin or pigment and cytoplasm alone remain, either cytoplasm or chromatin having been lost during staining•

474

PLASMODIUM V1FAX.

Young gametocytes are not easily distinguished from advanced trophozoites with undivided chromatin. That a young parasite is an early gametocyte is suggested by the more compact form of the cytoplasm; but, until the parasite has attained a size at which the chromatin of the asexual form would normally have divided, definite differentiation may be impossible. Nor does it seem possible to identify the sex of P. v i v a x gametocytes in thick films with certainty. Some gametocytes have sharply defined chromatin with compact blue cytoplasm and are presumably females. Others with illdefined chromatin and pale, tattered cytoplasm may possibly be males. But no constant association of diffuse chromatin with cytoplasmic pallor, or of compact chromatin with deep blue cytoplasm has been established. There is variation also in the coarseness and arrangement of the pigment. How far this variation is related to sex we have been unable to determine. ExflageUation of the male gametocyte is not seen in thick blood films prepared and stained by the methods described in this paper. OBSERVATIONS ON THE STIPPLING OF INFECTED CELLS IN THICK FILMS OF P. vivax.

Thick-blood films prepared and stained by the methods we employ seldom show Schfiffner's stippling around the parasites. Infected cells usually disappear during lysis of the blood though occasionally they are still to be seen at the edge of the film particularly when the parasites are advanced in growth. With the persistence of infected cells Schiiffner's dots are seen over and around the parasites as a stippled zone limited by the outline of the host cells. The stippling tends to be paler than in Giemsa-stained thin films and the dots fewer in number and hazier in form. Occasionally the original contour of infected cells is suggested by a vague halo free from stippling. Schiiffner's dots are readily demonstrated in thick films by modifying the technique of staining. Prolonged staining, or the use of more concentrated stain even though the time of staining is shortened proportionately, accentuates stippling. Prolonged drying of films before staining d8 hours or more--often has the same effect. But modifications of technique designed to emphasize stippling are not free from drawbacks. The persistence of Schiiffner's dots in a thick film usually implies a darkening and " bluing " of the background. With darkening of the ground there is a loss of contrast and an obscuring of the definition of the finer details of parasite structure on which the diagnosis of species and phase is largely based. OBSERVATIONS ON THE BACKGROUNDOF GIEMSA-STAINED THICK BLOOD FILMS. Thin-blood films, stained by the Romanowskyprocess, have a clearunstained background. Bloodcells are seen against the luminous white of the glass slide. Malarial parasites lie contrasted against the pale flesh tint of their host cells.

j. w. FIELD AND H. LE FLEMING.

475

Thick-blood films, however, when lysed and stained, have a " ground " of red cell residues. It is against this stained " ground " that parasites and other cellular structures are viewed. Thick-film staining thus has a limiting intensity beyond which there is no gain in the definition of parasites and cells but actually a loss from the deepening of the ground on which they lie. Staining is probably optimal when the parasite-background contrast is maximal. Well-stained thick films of normal blood have a clear mottled blue-grey ground, pale and almost luminous, on which the leucocytes stand out vividly purple. Nuclear contour is well preserved but the cytoplasm is ragged and fragmentary. Neutrophilic granules tend to disappear; eosinophilic granules are more resistant to the destructive efforts of lysis and are usually retained. The blood platelets, isolated or in clusters, sometimes in large clusters filling the microscopic field, are easily recognized by their distinctive texture and hazy outline. No other cellular elements are normally present, but in severe anaemia lysis tends to be less complete and the background is often clouded with the nuclear and reticular debris of immature red cells. When heavily stained the general background of thick blood films is darkened, with special emphasis on basophilic structures. Instead of a pale smooth ground of faintly mottled blue-grey there is a darkened ground flecked with blue-staining residues against which the delicate arrangements and colour distinctions of the cytoplasm, chromatin and pigment of malarial parasites can be defined only with difficulty. T h e advantages of intensive staining are that cytoplasmic contour is better preserved and Schiiffner's dots often retained. Some workers prefer the deeply stained film with the outlines and stippling of infected cells retained at the cost of precise colour distinctions ; others sacrifice these advantages for the more delicate appreciation of the internal detail of parasites which the lighter ground affords. Our experience suggests that, on the whole, species diagnosis is easier in films that are relatively lightly stained. ADDENDA. A.

T H E PREPARATION AND STAINING OF T H I C K BLOOD FILMS.

The thick-film methods used in this laboratory are based on a simplified technique suggested by K o c h (1907) and modified by GREEN (1931) tO suit local conditions. The following technical details are drawn mostly from GREEN'S account.

Preparation. The pulp of a finger, cleaned with alcohol and thoroughly dried with a clean fibre-free tow'el, is pricked with a triangular cutting needle and a fairly large globule of blood gently squeezed out. The under surface of a glass slide, held between finger and thumb, is lowered on to the globule and the blood rapidly spread as a smear the size of a sixpence by a circular movement of the slide in the plane of its surface, making contact so far as possible only with the D

476

PLASMODIUM

VIVAX.

blood, not with the surface of the finger. T h e slide is then turned film uppermost, tilted a little if necessary to ensure an even spread of the blood and placed on a level surface to dry. T h e more quickly the film is m a d e the better, as a rule, it will be. Delay favours the formation of fibrin which clouds the background of the stained f i l m ; prolonged tilting in various directions to get an even thickness of film m a y cause agglutination of the red cells with a patchy distribution.

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4.--Usual size and arrangement of single and multiple films on the slide. a. Ordinary thick fihns in duplicate from a single case. The second film is a spare for use should the first be damaged. b. Five films, 511-515, on one slide. Our usual method of arranging thick films for routine survey work. c. Five films, A5 to 9. "A" is the index letter of the series ; the number following is the day of the month. Our usual arrangement of films from which parasite counts may be required. The first film from each case is an ordinary thick film ; the second a fowl-cell film. FIG.

T h e best thickness of smear is soon ascertained with a little practice. T h e t e r m " thick drop " is misleading. A drop of blood, unless spread as a smear, cannot be made sufficiently transparent b y removing the haemoglobin for examination by transmitted light. T h e thickness of film at which we aim is such that a microscopic field, u n d e r a 1/12th oil i m m e r s i o n lens and a x 5 eyepiece, contains f r o m ten to thirty leucocytes. T h e stained film is of such density that large print can dimly be seen through it. T h i s simple " finger-slide " method of making thick films we use only when the necessary e q u i p m e n t for the more elaborate " pipette " m e t h o d is not at hand. Routine thick films are made with a capillary pipette. P i p e t t e - m a d e

j. w.

FIELD AND H. LE FLEMING.

477

thick films are more uniform and can be arranged five or more to one slide with an economy of time and material. A short column of from ¼ to ½ inch of blood is drawn into a capillary pipette and spread rapidly on a slide as a uniform smear. Capillary tubes ordinarily supplied for vaccine lymph are used in an ingenious pipette described by CHRISTOPHERS, SINTON and COVELL (1936). A brief note on this pipette is given on page 479. The usual size and arrangement of single and multiple films on the slide are shown in the accompanying diagram. Drying.--While still wet the films are placed in a horizontal tray which is covered with a strip of stiff cardboard as a protection from dust--a precaution which we believe to be important. Preliminary drying under local climatic conditions appears complete in a quarter of an hour or so but the films cannot be stained satisfactorily for about 8 hours. They may be kept unstained for as long as 48 hours, but thereafter deteriorate rapidly. There is a partial autofixation of the red cells; the removal of haemoglobin is incomplete and the background of the stained film becomes more and more opaque to transmitted light. Even at 48 hours the background stains very deeply, though as a small compensation, Schiiffner's stippling is often preserved. Our films are air dried until they can be taken from hospital or field to the laboratory, and then stored overnight in an incubator at 37 ° C. for staining the next morning.

Staining. The routine stain is Giemsa's buffered with a phosphate diluent.

Equipment required :-i. Giemsa's stain in a drop bottle. ii. Phosphate buffer. iii. Measuring jar of 100 c.c. capacity. iv. Staining trough of 50 or 100 c.c. grooved to take slides on edge. v. Clean water in a bowl or other wide mouthed vessel. A timing clock and, in a humid climate, an electric fan are useful accessories.

Technique of staining :-i. ii. iii. iv. v. vi. vii.

Arrange the films, which have been stored in an incubator at 37 ° C. overnight, side edge down in a staining trough. Prepare sufficient stain to fill the trough by adding one drop of Giemsa for every 3 c.c. of the buffered diluent. Pour the diluted stain into the staining trough. Leave for 1 hour ; lysis and staining are simultaneous. Allow a little water to trickle from a tap into the trough to float off the scum which might otherwise deposit on the slides as they are removed. Remove the slides one by one and differentiate by waving very gently for about 3 seconds in clean tap water. Dry rapidly, if possible in the draught of a fan.

PLASMODIUM V1VAX.

478 B.

THE PREPARATION AND STAINING OF THIN BLOOD FILMS.

Thin films are prepared by conventional methods. The end edge of a glass slide, or of a rectangular coverslip, is dipped into a smaIl droplet of bIood as it exudes freshly from the pulp of a finger pricked with a triangular cutting needle, placed at about 45 ° on a second slide and rapidly pushed along with the blood drawn as a film behind the moving edge. We prefer a cover-slip spreader because of its truer edge and smoother passage along the slide.

Fixing and Staining. Films are fixed in methyl alcohol and stained with buffered Giemsa. The concentration of stain is adjusted to the time of staining on the assumption that intensive staining followed by thorough differentiation in water gives optimal contrast and best demonstrates the stippling of infected cells. Equipment required : ~ i. Giemsa's stain in a drop bottle. ii. Methyl alcohol in a drop bottle. iii. Phosphate buffer. iv. A burette or measuring jar calibrated in cubic centimetres. v. A small beaker. vi. A staining rack over a sink. vii. A brisk stream of clean water from a tap or other source. Technique of Staining :-i. Arrange the slides on a staining rack. ii. Prepare diluted stain by adding three drops of Giemsa's stain for every 1 c.c. of the phosphate buffer. (The total quantity of diluted stain required for a number of films is prepared at one time. Each slide requires about 3 c.c. ; if, for instance, 25 thin films are to be stained, 75 c.c. of the diluted Giemsa is prepared in a small beaker by adding 225 drops of Giemsa's stain to 75 c.c. of the diluent.) iii. Fix the films for about 10 seconds with methyl alcohol. iv. Pour off the alcohol and immediately add diluted Giemsa to cover the slide. v. Stain for 30 minutes. vi. Flush off the stain under a brisk stream of tap water and differentiate under this stream for 20 seconds. vii. Dry rapidly in air. C.

THE PREPARATION OF GIEMSA'S STAIN.

The method of preparing Giemsa's stain is that described by GREEN (1931) from whose account the following details are taken. The stain usually used is that supplied by British Drug Houses or by G. T. Gurr, of London.

J. W . FIELD AND H. LE FLEMING.

479

To make 500 c.c. of stain, " weigh out 3.8 grammes of the combined Giemsa's stain powder and place it in a glass mortar. Measure 250 c.c. of pure glycerine (Merck, or British Drug Houses, A.R.) and pour it from time to time over the powder using a glass pestle to assist in mixing and dissolving. Then pour intermittently a total quantity of 250 c.c. of methyl alcohol (Merck or British Drug Houses, A.R.) stirring rapidly. The later part must be done fairly quickly otherwise the alcohol will evaporate. When the stain is dissolved sufficiently pour the contents of the mortar into a bottle, wash out the mortar with what remains of the 250 c.c. methyl alcohol and put these washings into the bottle. The bottle should be shaken frequently during the next 24 hours, and the stain should be ready for use in 48 hours. When required, small quantities of the stain are filtered into dropping bottles. During filtration, the funnel is covered with a lid to prevent evaporation of the methyl alcohol." D.

PREPARATION OF PHOSPHATE BUFFER FOR GIEMSA'S STAIN.

T h e phosphate solution we use to dilute and buffer the stain is made b y dissolving 2.0 grammes of disodium hydrogen phosphate (Na2HPO412H20) and 0.4 gramme of potassium dihydrogen phosphate (KH2PO4) in 1 litre of distilled water. T h e solution should be of p H 7.2. T h e p H remains stable for some weeks but is tested once at the time of preparation and adjusted if necessary. This preliminary testing we find to be necessary because of occasional errors in the p H of the final solution due possibly to variation in different batches of the salts or in the p H of the distilled water. T h e p H indicator is b r o m o - t h y m o l blue. One c.cm. of a 1 per cent. solution of the indicator is added to 9 c.c. of the buffer solution in a test tube and matched against a set of b r o m o - t h y m o l blue colour standards. E.

THE CAPILLARY PIPETTE USED FOR PREPARING THICK BLOOD FILMS.

CHRISTOPHERS, SINTON and COVELL (1936) have described an ingenious pipette for making thick-blood films with the capillary tubes ordinarily supplied for vaccine lymph. ~* T h e advantages of a pipette technique are : - (a) that there is full control over the thickness of the films. (b) that several films can be made on a single slide with an economy of time and material. (c) that the m e t h o d can be readily adapted to the S i n t o n - D r e y e r fowl-cell technique for counting parasites. T h e special advantages of the pipette suggested by CHRISTOPHERS and his associates are : - (a) that the main body of the pipette can be used indefinitely with little or no attention. (b) that control over the inflow of blood into the pipette is arranged by a capillary valve. (¢) that, as the capillary tubes only are changed after each film is made, the e q u i p m e n t required for making large numbers of films takes up Capillary tubes supplied for vaccine lymph are obtained from W. J. Hancher, 34, Bridge Road West, London, S.W.11, at a cost of 6s. 6d. per 1,000, or through Messrs. Baird & Tatlock. Size 90 mm. x 1-5 mm.

480

PLASMODIUM VIVAX.

very little space. A vessel of 2½ inch diameter, the size of an ordinary cigarette tin, holds enough tubes for nearly a thousand films. This pipette, slightly modified by one of our assistants, Mr. LAI S~E FOON, is prepared as follows : - 1. A short piece of glass tubing about 2½ inches long and from ¼ to [ inch bore is heated a little over ½ inch from one end and drawn out to form a slight constriction (Fig. 5, a). 2. Two solid rubber corks to fit the tubing are drilled from end to end with a red hot needle (Fig. 5, b).

a

l

b

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c

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FIc. 5.--Preparation of capillary pipette. (Modifiedfrom CHRISTOPHERS,SINTONand COVELL.)

3.

A short piece of capillary tube with one end drawn out to a fine bore is fitted into one of the corks and the latter inserted into the end of the glass tubing distant from the constriction till almost flush with the rim. The drawn out fine end of the capillary tube is now within the outer tube. This acts as an air valve. The second rubber cork is inserted into the other end of the outer tubing until somewhat compressed by the constriction (Fig. 5, c). 4. A rubber teat is attached to the outer tube at the air-valve end ; at the other end a lymph capillary tube is inserted until it engages firmly in the rubber cork where squeezed and narrowed by the constriction of the outer tube. Lymph tubes vary somewhat in bore. With the original pipette of CHRISTOPHERS, SINTON and COVELL the rubber cork sometimes failed to grip the smaller tubes firmly enough to prevent an air-leak and poor control over the column of blood. To obviate this difficulty, Mr. LAI SRE FOON'S modification is the introduction of the constriction in the outer tube. REFERENCES. CHRISTOPHERS, S. R., SINTON, J. A. ~ COVELI., G. (1936). Hlth Bull. No. 14. Malaria Bureau No. (q., pp. 97/8, 3rd. Ed. Revised by J. A..qinton. Delhi : Manager of

Publications.

GREEN, R. (1931). Bull. Inst. reed. Res. F.M.S., No. 1. NAPIER, L. E. & SEN GUPTA, P . C .

(1938).

Indian reed. Gaz., 73, 3.