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A note on the shute technique for staining malaria parasites with Leishman's stain and on the stippling in infected red blood corpuscles which it reveals
269 TRANSACTIONS TROPICAL
OF THE ROYAL SOCIETY OF MEDICINE AND HYGIENE.
Vol. XXIII.
No. 3. November,
A NOTE MALARIA
ON
THE
PARASITES
STIPPLING
IN
1929.
SHUTE
TECHNIQUE
WITH
LEISHMAN’S
INFECTED WHICH
RED IT
FOR STAIN
BLOOD
STAINING AND
ON
THE
CORPUSCLES
REVEALS. BY
S. P. JAMES, Adviser
on
Tropical
(Received
M.D., Diseases
30th
D.P.H.,
I.M.S.
(Red.).
to
Ministry
of
the
August,
Health.
1929).
For finding and identifying malaria parasites in thin blood films, Leishman’s stain (which requires no preliminary fixation of the blood) is in general use ; but it happens frequently that the results obtained with it are not at all satisfactory. Mr. P. G. SHUTE, Senior Laboratory Assistant in the Malaria Laboratory of the Ministry of Health, by investigating systematically each factor which seemed to have an influence on the staining process, has worked out a technique which enables excellent results to be obtained without fail. An important outcome of the use of this technique in our laboratory has been the demonstration of a hitherto unnamed type of stippling in red blood corpuscles infected with quartan parasites (Plasmodium malaria), as well as the demonstration of Schiiffner’s dots and Maurer’s spots in red blood corpuscles infected with younger stages of P. vivax and P. falciparum respectively, than the stages with which those characteristic phenomena are usually associated. For these reasons, and particularly because constant and consistent results are obtained by using Leishman’s stain in the way described below, the technique is of great assistance in the correct identification of the species of parasite present in blood films. The importance of this matter will be appreciated by those who are aware of the frequency with which errors in identification have been made even by
370
NOTE ON THE SHUTE TECHNIQUE FOR STAINING MALARIA PARASITES.
experienced workers, in the past, and are still being made today wherever microscopic examination of blood films is practised. ~ DESCRIPTION OF THE TECHNIQUE. New microscope glass slides should be used. T h e slides with which the best results are obtained are those known in the trade as " half white " ; the results m a y not be so good when slides known as " white " or as " French special " are used. T h e slides should be dipped in " nitric alcohol " (30 parts nitric acid, 70 parts absolute alcohol), and, after being wiped dry, should be kept in absolute alcohol. On no account should " methylated spirit " be used as a substitute for absolute alcohol in any part of the procedure of taking and staining blood films. T h e slides, after removal from absolute alcohol, should be rubbed dry with a clean soft cloth. T h e stain is made with pure methyl alcohol (" free from acetone ") and crystals of Leishman's stain. T h e reaction of the methyl alcohol must be tested before making up the stain. This is done with the aid of an outfit for determining the hydrogen ion concentration, the indicator used being phenol red, and the range of the standard tubes being from p H 6.6 to p H 8.0 (Baird and Tatlock's outfit No. P.2759). Into the test-tube made of cordite glass which is supplied with the outfit, pipette 5 c.cm. of the methyl alcohol to be tested. With a separate pipette add 0.5 c.cm. of a .01 per cent. solution of phenol red. Shake and, after a m o m e n t or two, compare the tint with that in the standard tubes provided. Every brand of methyl alcohol which we have tested in this way gives a slightly acid reaction. After adding the indicator to 5 c.cm. of the brand which we use, the tint corresponds nearly always with that of the standard tube marked 6-8, b u t sometimes with that marked 6.6. It is our practice to discard supplies of methyl alcohol which are as acid as is indicated by the tube marked 6.6, and we have had to do so even with some supplies which makers p u t up in hermetically sealed tubes " for use in microscopic staining." T o make up the stain, rinse a glass stoppered bottle # thoroughly with some of the methyl alcohol that will be used for the stain, and then p u t in 0.15 g. of Leishman's crystals (usually called " Leishman's powder "). Add 100 c.cm. of the methyl alcohol. Shake f r o m time to time during the next 24 hours, after which period nearly all the crystals will be dissolved, and the stain will be ready Interesting examples in the literature of more than twenty-five years ago will be found in LEISHMAN'Sfirst article on the application of Romanowsky's stain for the microscopic diagnosis of malaria (Brit. Med. Jl., 16th March, 1901, page 635), and in STEPHENS'and CHRISTOPHERS'first article on the malarial infection of native children in British Central Africa (Reports to the Malaria Committee of the Royal Society, Third Series, 1900, page 4). It is obvious from the descriptions and illustrations in those articles that LEISHMANidentified and described as a quartan parasite what was really a malignant tertian parasite, and that NTEPHENS a n d CHRISTOPHERSseem to have made precisely the reverse error. Examples in modem times are too numerous to cite. t The bottle should be of hard glass (green glass).
S. P. JAMES.
271
for use. For several reasons it is unwise to make the solution in a pestle and mortar, or to filter it, as is usually recommended in text-books. Next deal with the distilled water which will be used in the staining process and for washing the stained slides. We work with a 1-1itre flask of distilled water which has been treated as follows : Shake up the water in the flask and wash out a 5 c.cm. pipette with water from it ; test 5 c.cm. of the water in the same way as was described for testing the methyl alcohol. Probably the water will be found to be at least as acid as is indicated by the standard tube marked 6.6. Add to the water three or four drops of a saturated filtered solution of lithium carbonate, shake the flask to ensure thorough mixing, and repeat the test. Continue the procedure of adding one or two drops of the lithium carbonate solution and of testing until the water in the flask becomes exactly of an alkalinity indicated by observing that, after adding 0.5 c.cm. of the phenol red solution to 5 c.cm. of the water, the resulting colour matches the colour of the solution in the standard tube marked 7.2. This is the degree of alkalinity that must be reached when the methyl alcohol is of an acidity represented by the tube marked 6"8. To stain a blood film, drop four drops of the staining solution on the film, rock for ten seconds, add twelve drops of the distilled water, and mix thoroughly by tilting and rocking. We do not use a glass rod for mixing the water and the stain on the slide, because a rod often carries specks of dust or of cotton fibre which are transferred to the slide ; but a good deal of practice in tilting and rocking the slide is required in order to obtain quick and complete mixing of the water and the stain without spilling some of it off the slide, or on the fingers, and without allowing any of the stain to dry on the film before the water has reached it. Nor do we employ the usual practice of making a barrier with a wax pencil across the proximal end of the slide, because, when this is done, particles of methylene blue from the pencil invariably become mixed with the stain and alter the result. By using four drops of stain and twelve drops of distilled water, the stain is diluted three times which, we think, gives the best results. Four drops of stain, carefully applied, are quite sufficient to cover a film and, when twelve drops of water are added, the amount of fluid on the slide is easy to manipulate so that none spills off the slide or reaches the fingers. We time the ten seconds during which the stain alone is on the film by a watch with a large second hand. The length of time during which the stain is allowed to act on the film is very important. When dealing with a film about which we do not know the species of malaria parasite present, thirty minutes is the correct period during which the stain should be allowed to act ; for although the older stages of the parasites present will be too deeply stained, that period is necessary for bringing out clearly the stippling in red blood corpuscles infected with quartan parasites and Schiiffner's dots and Maurer's spots in the youngest ring or marginal stages of corpuscles infected with P. vivax or P. falciparum. When we know what species of parasite is present in the films, the period
579'
NOTE O N THE SHUTE TECHNIQUE FOR STAINING MALARIA PARASITES.
of staining should be varied according to whether it is desired particularly to study the morphology of the parasites or the stippling and other changes in the red blood corpuscles containing them. The parasites stain much more quickly than the changes indicated by stippling or dotting of the red blood corpuscles ; and Schiiffner's dots stain more quickly than Maurer's spots or the stippling characteristic of a quartan infection. After staining for ten minutes Schiiffner's dots, characteristic of infection with P. vivax, are vividly stained in all corpuscles containing quarter to half-grown and later stages of the parasite, but in that period no stippling can be seen either in corpuscles infected with the youngest forms of this parasite or in any corpuscles infected with P. malari~e. Stippling of corpuscles infected with the latter parasite is best studied in films stained for 45 minutes, though it is well seen in most (though not in all) infected corpuscles after 30 minutes staining. For routine identification work 30 minutes is the best time. * On the termination of staining, the stain must not be poured off the slide before beginning to wash the film ; a good stream of distilled water must be applied at once so that all stain and deposit is flushed off in the first moment. Washin[~ in the stream of distilled water should be continued for fifteen seconds by the watch. I am afraid that the technique, and my description of it, may be criticised on the ground of meticulolls accuracy. My excuse is that during a recent tour of investigation in Africa I was so much impressed by the reports of difficulties and failures with Leishman's stain which were brought to my notice, as well as with errors resulting from working with poor stains, that I hardly think it possible to exaggerate the importance of the details to which I have drawn attention above. NOMENCLATURE AND DESCRIPTION OF THE DOTS AND SPOTS. Hitherto, observers who have described characters for distinguishing between the " ring forms " of the three species of the malaria parasite, have been embarrassed by the fact that they could never be sure that the blood films which they examined for this purpose (being obtained from patients infected in nature) contained only one species of parasite. The unrecognised presence of two species of the parasite was obviously the cause of the errors in identification and description to which reference was made in the footnote on page 270. In our laboratory we are in a more favourable position because our patients are intentionally infected with what has been proved by repeated passage to be a pure strain of only one species of the parasite. Thus both the youngest and the oldest ring forms which appear in the peripheral circulation are known to belong to the single species with which the patient was infected. Our benign tertian strain has not changed its characters since it was obtained in 1925, and our quartan strain has remained equally pure since it was obtained from Dr. W. KIRSCHBAUM, The slide must be covered by a Petri dish during the process of staining.
273
S. P. JAMES.
of the Friedrichsberg Mental Hospital, Hamburg, a year ago. We have studied ring forms of the malignant tertian parasite particularly in cases of primary infection intentionally induced by blood inoculation, and afterwards watched for a long period to see that no other type of fever or parasite appeared, but we have not maintained a strain of that species continuously. We have compared the findings in blood films of our cases of each type of malaria with those in blood films from natives of East Africa stained by the same technique, and have found that among the latter, mixed infections (particularly with quartan and subtertian) are frequent. I do not propose, in this article, to describe ail the points by which, using .the staining procedure described, one is able to recognise and distinguish from one another the ring forms of the three species of parasite ; what I wish to do is to draw attention to the phenomenon of stippling in corpuscles infected with quartan parasites, to note the character of the stippling in some uninfected red corpuscles which is demonstrable in films of malarial blood stained in this way, and to correct an error of nomenclature relative to Maurer’s spots which has crept into English text-books and articles. STIPPLING
OF
RED
CELLS
IN
QUARTAN
MALARIA.
The illustration below shows Schiiffner’s dots in corpuscles infected with a ring form of P. vivax, the quartan stippling or dotting in corpuscles infected with a ring form of P. malank, and Manrer’s spots in corpuscles infected with a ring form of P. falciparum. It is reproduced from a water colour drawing kindly
made for me by Mr. Jobling.* It will be seen that in corpuscles containing ring forms of approximately the same size : (1) Schtiffner’s dots are relatively large and round, numerous and very distinct ; their colour shades from deep mauve through violet to pale rose ; (2) the quartan stippling consists of numerous separate dots and points, smaller than Schtiffner’s dots and not so definitely round nor so distinct ; their colour is the same as that of Schiiffner’s dots, but more of them are pale rose. In corpuscles containing quartan parasites which nearly fill the corpuscle, the dots are nearly as large as Schtiffner’s dots, but they are fewer in number, and they stain faintly pale rose. Their reaction to staining * I have to thank Dr. C. M. WENYON, Director in Chief, Wellcome Bureau of Scientific Research, for kindly permitting Mr. Jobling’s services to be lent to me for this purpose, as well as for valuable suggestions kindly made while the paper was passing through the press. The coloured drawings, with others, will be published as a plate in The Kenya and East African Rledical Journal.
~74[
NOTE ON TnE SHUTE TECHNIQUE FOR STAINING MALARIA PARASITES.
is evidently quite different from that of Schiiflher's dots because, while Schiiffner's dots can be demonstrated readily in films stained for only five minutes in the manner described, the stippling of cells containing quartan parasites is not demonstrable until the film has been stained for at least twenty minutes, and is best brought out by staining the films from thirty to forty-five minutes. This is why we recommend thirty minutes as the period of staining for routine identification work. It will be remembered that MAURER,to obtain his" third and fourth degrees of staining," which showed the stippling of infected corpuscles best, recommended that films should be stained for one hour. Evidently in modern practice much is sacrificed to the demand for quick results ; for example, in the ninth edition of Manson's Tropical Diseases, p. 859, five minutes is the period recommended for staining with Leishman's stain. (3) Maurer's spots are few in number, and larger and of more irregular shape than the dots in either of the other types ; their number can always be counted, and some of them are tiny rings, loops or streaks ; their colour is deep mauve to violet, and the corpuscle itself is pale blue-grey, and often outlined sharply by the rose stain. PROPOSED
NAME FOR THE
QUARTAN S T I P P L I N G .
As names have been given to the stippling of corpuscles infected with P. vivax (Schfiffner's dots) and to the stippling of corpuscles infected with P. falciparum (Maurer's spots) it will probably be thought desirable to name the stippling associated with the presence of P. malarice. It is not easy to ascertain from the literature who has the right to priority of observation and description in this matter, particularly because most, if not all, observers who have mentioned stippling in corpuscles which they described as containing a quartan parasite, were working with films from cases with a mixed infection. SCHOFFNER in his classical monograph, Beitrag zur Kenntniss der Malaria, published in 1899, states that in quartan infections the blood corpuscles retain their normal staining properties, but that " only very occasionally there is a delicate blue line or loop in the interior of the red cell, which cannot possibly, however, be confused with the dots described " (that is, with Schiiffner's dots). His illustration of these lines or loops shows that he was not describing (and apparently had not seen) the type of quartan stippling with which we are concerned at present. ZIEMANN, whose observations were made in Togoland in 1900, noted SCHi)FFNER'S observations on the staining characters of corpuscles infected with quartan parasites and reported that, for his part, he had sometimes seen, when employing an alkaline Romanowsky stain, an exceedingly delicate Schiiffner dotting in those corpuscles. MAURER,in his articles published in 1900 and 1902, does not mention any change in corpuscles infected with quartan parasites. STEPHENSand CHRISTOPHERS,in their article on the malarial infection of native children, published in 1900, give a drawing (Plate I, drawing No. 8) which might serve to illustrate the stippling of a blood corpuscle containing a nearly full-grown quartan parasite, but they described the parasite as a female
S. P. JAMES.
275
gametocyte of P. j’alciparum. CRAIG, in his textbook published in 1909, states that the form of degeneration represented by the occurrence of Schiiffner’s granules or dots is probably never present in quartan infections, “ although in a very few such infections I have observed a stippling indistinguishable from that produced by Schiiffner’s dots in invaded red blood corpuscles. However, such an appearance occurs so rarely that it is of no practical importance.” WENYON, in describing quartan infections on page 942 of Vol. II of his textbook PTotozoology, says : “ Very rarely dots resembling those seen in the case of P. vivax have been noted.” He has kindly informed me that this statement is based on his own experience and that for many years he has taught that occasionally such dots are seen in quartan infections.* Dr. MARY LAWSON, in an article published in 1916, gave a photograph of “ two young quartan parasites in a red corpuscle showing Maurer’s rings and dots in a red cell showing (Fig. SO),” and another of “ a quartan microgametocyte Schiiffner’s granules (Fig. Sl).” Unfortunately, her paper contains no statement of the grounds on which the identification of these parasites as quartan was made, and I do not see how it would be possible to agree with her opinion that quartan parasites sometimes cause the destructive changes in red blood corpuscles which are demonstrable as Maurer’s spots, loops and rings ; it seems probable that, as has happened to many observers, she was dealing with a mixed infection. The same criticism is applicable to papers by several other workers who have described stippling of different types in red blood corpuscles said to contain quartan parasites. There are, however, descriptions in another category to be considered, namely, those in which parasites have been classed and named as a new species chiefly because, while resembling the quartan parasite in most particulars, they were present in red cells showing stippling said to be like Schiiffner’s dots. The parasite described by STEPHENS in 1922, and again by STEPHENS and OWEN in 1927, under the name Plasmodium ovale, is in this category. From an examination of the coloured plate published with the description of P. ovale it seems probable that in this instance the stain, or the distilled water, used for the staining process happened accidentally to possess the reaction which brings out stippling in corpuscles infected with quartan parasites. Probably this is an example in which the characteristic stippling of corpuscles infected with quartan parasites was observed, although, in my opinion, it was wrong to describe the stippling as “ Schiiffner’s dots.” The coloured plate of P. ovale shows that the dots are less numerous and smaller than Schiiffner’s dots, and it is mentioned in the description that they do not stain so deeply as Schiiffner’s dots usually do, * In this connection I hope it will be clearly understood that we do not consider our finding of stippling in quartan infections to be a new observation. I may be permitted to say, however, that, by the use of the technique described, it has been possible to give precision to previous observations on the subject, and to point out that, in several important respects, the stippling described does not, in fact, resemble that observed in vivax infections.
276
NOTE O N THE SHUTE TECHNIQUE FOR STAINING MALARIA PARASITES.
although the films were stained for one hour. The fact that the stippling was seen only after long staining is in favour of the view that P. ovale is a quartan parasite seen in cells stippled in the manner described in this paper. Having regard to the various points mentioned in the above summary, I consider that ZIEMANNhas the right of priority of observation and of description in the matter of-stippling of red corpuscles infected with quartan parasites ; his observations were made earlier than those of any of the other workers quoted, and his description of them, though meagre, was correct. He noted that, in red corpuscles containing quartan parasites forty-eight hours old, the stippling could be brought out by using Brug's staining method as well as by using an alkaline Romanowsky stain. For these reasons I suggest that the characteristic dotting of red corpuscles invaded by quartan parasites should be named " Ziemann's stippling." I choose the word stippling because it is advisable that the nomenclature should indicate the character of the stained markings; Schfiffner's are dots, Maurer's are spots, and Ziemann's are a mixture of points, dots and spots. STIPPLING OF UNINFECTED CORPUSCLES. P Stippled red corpuscles not containing parasites are common in films from cases of subtertian malaria stained by the Shute technique, and are also seen (less frequently) in films from cases of quartan malaria. The dots and points are numerous, and most of them are small and of a pale rose colour. They are quite different from the basophilic dots with which some other red blood corpuscles in films from cases of malaria are often studded. MAURER,in 1902, described and illustrated this type of stippling of uninfected corpuscles. PROPOSED CORRECTION OF NOMENCLATURE. The error of nomenclature which I desire to correct is that in English textbooks, and articles " Maurer's dots " are wrongly named " Stephens' and Christophers' dots." I am the more anxious to rectify this error because I was myself among those who made it (on page 24 of my book, Malaria at Home and Abroad). I refrain from naming all the other English writers who, in textbooks and articles, have made the same mistake. It arose after the publication by STEPHENS and CHRISTOPHERSof their " Note on the Changes in the Red Cell produced by the Malignant Tertian Parasite " (Brit. Med. ~l., 28th March, 1903, page 730), in which they said that, as far as they were aware, they were the first to point out the changes in red cells invaded by malignant tertian parasites. The article from which they quoted a sentence to support this belief was their first report to the Malaria Committee of the Royal Society on The Malarial and Blachwater Fevers of British Central Africa. This report was published in 1900. But, evidently, they were not aware of the publication, early in 1899, of SCH~)EENER'S article Beitrag zur Kenntniss der Malaria, in which, as well as describing the dots associated with P. vivax infections Which now bear his name,
277
S. P. JAMES.
he described (and illustrated accurately and precisely in a coloured plate) the markings associated with P. falciparum infections which were afterwards named Maurer’s dots. This article by SCH~FFNER was published in a special volume (Festschrift XUT Feier Leipzig) as the 64th
des 100 Jahrigen
Bestchens
der Medicinischen
Klinik
2;u
volume of the Deutsche Archiv fiir Klinische Medicin. The copy which I have consulted is stamped with the date stamp of the Royal Medical and Chirugical Society, 24th May, 1899, so it must have been received in London some time before this date. On the other hand, the manuscript of STEPHENS and CHRISTOPHER& article was not received by the Malaria Committee of the Royal Society until the 10th December, 1899, and the article was not published until some time in 1900. Therefore, it seems clear that SCH~FFNER was the first to describe and to point out in a published article these malignant tertian markings, as well as the benign tertian markings with which his name is always associated. MAURER, in his articles published in 1900 and in 1902, fully acknowledged SCH~~FFNER’S priority of observation and description as regards both kinds of stippling, and he described his own contributions as a confirmation and extension of SCH~~FFNER’S results by using the Romanowsky stain instead of the Mannaberg and hzmotoxylin stains which SCH~FFNER used. On this subject Professor E. P. SNIJDERS, of the Institute for Tropical Hygiene, Amsterdam, has written to me as follows : “ SCH~FFNER first detected the ‘ dots ’ of benign tertian malaria and the ‘ spots ’ of malignant tertian malaria (perniciosa). Afterwards, independently of each other, RUGE and MAURER (both in 1900), by the application of the Romanowsky stain, confirmed his findings with regard to the ‘ dots,’ and MAURER confirmed his findings with regard to the SCH~~FFNER and MAURER were ‘ spots ’ (Flecken), and laid full stress on them. great friends, and I think you will act in accordance with SCH~~FFNER’S views if you will state that SCH~FFNER was the first to describe the subtertian ‘ spotting ’ (Fleckung) as well as the benign tertian ‘ dotting ’ (Ti@felung), that MAURER acknowledged this fully, and that it was ever a great joy to SCH~~FFNER that the ‘ spots ’ bear the name of his friend MAURER in recognition of the latter’s great contributions to the knowledge of staining methods for malaria.” REFERENCES. ARGUTINSKY,
P. (1903). Zur Kenntniss des Tropicaparasiten ; Die Tiipfelung der Wirts-
zellen der Halbmonde. Cent.f. Bakt., Abt. 1, Orig., xxxiv. (2), 144. CRAIG, C. F. (1909). Malarial Fevers. J. and A. Churchill, London and New York, 1909. -. (1915). New Varieties and Species of Malaria Plasmodia. JZ. Pamsit., 1, (2), 85. LAWSON, M. R. (1916). Distortion of the Malarial Parasite. An Interpretation of “ Plasmodinm Tenue ” (Stephens). yl. Experim. Med., xxiv, (3), 291. LEISHMAN, W. B. (1901). The Application of Romanowsky’s Stain in Malaria. Brit. Med. Jl., i, 635 ; 16th March. MAIJRER, G. (1900). Die Ttipfelung der Wirtszelle des Tertianaparasiten. Cent. f. Bakt., Abt. 1, xxviii, 114. ---. (1901). Die Malariaparasiten. Mtinch. Med. Woch., xlviii, 337; 26th Feb. -. (1902). Die Malaria Perniciosa. Cent. f. Bakt., Abt. 1, Orig., xxxii, 695.
278
NOTE ON THE SHUTE TECHNIQUEFOR STAININGMALARIAPARASITES•
MACFIE, J. W. S•, and INGRAM, A. (1917). Observations on Malaria in the Gold Coast Colony, West Africa. Ann. Trop. Med. ~.~ Parasit., xi, (No. 1), 1. RUGE, REINHOLD• (1900). Ein Beitrag zur Chromatinf/irbung der Malariaparasiten. Zeitsehr. f. Hyg. u. Infektionskr., xxxiii, 178. SCH/.~IFFNER. (1899). Beitrag zur Kenntniss der Malaria. Deut. Arch. f. Klin. Med,, lxiv, 428. STEPHENS, J. W. W., and CHRISTOPHERS,S. R. (1900). Th e Malarial and Blackwater Fevers
of British Central Africa. Reports to the Malaria Committee of the Royal Society. Series 1, p. 12. Series, p. 4". (1900). T h e Malarial Infection of Native Children. Ibid. T h i r d (1903). Note on the Changes in the Red Cell produced by the Malignant ~Fertian Parasite. Brit. Med. Jl., 730 ; 28th March. STEPHENS, J. W . W . (1922). A New Malaria Parasite of Man. Ann. Trop. Med. Parasit., xvi, (4), 383. STEPHENS,J• W. W., and OWEN, D. U. (1927). Plasmodium ovale. Ann. Trop. Med. Parasit., xxi, (2), 293. THOMSON, J• G. (1928)• Stippling of the Red Cells in Malaria• Proc. Roy. Soc. Med. (See. Trop. Dzs. ~ Parasit•), xxi, 464 ; 3rd January• • (1924). Researches on Blackwater Fever in Southern Rhodesia. Lond. Sch. Trop. Med. Res. Mem. Set., vi. THOMSON, J. G., and ROBERTSON,ANDREW. (1929). T h e Malarial Parasites of Man. Protozoology, p. 14. London : Balli&re, Tindall & Cox. WEIqYON, C . M . (1928). Protozoology, ii, 942. London : Balli~re, Tindall & Cox. ZIEMANN, HANS. (1898). Neue Untersuchungen fiber die Malaria und den malariaerregem nahestehende Blutparasiten. Deut. Med. Woch., 123 ; 24th Feb. • (1915). Ueber eigenartige Ma]ariaparasitenformen. Cent. f. Bakt., Abt. 1, Orig. lxxvi, (5), 384. (1906)• In Mense's Handbueh der Tropenkrankheiten, iii, 1st edition. Leipsic (1918). Ibid., v, 2nd edition, pp. 30, 33 and 37.
OF THE ROYAL SOCIETY OF MEDICINE AND HYGIENE.
Vol. XXIII.
No. 3. November,
A NOTE MALARIA
ON
THE
PARASITES
STIPPLING
IN
1929.
SHUTE
TECHNIQUE
WITH
LEISHMAN’S
INFECTED WHICH
RED IT
FOR STAIN
BLOOD
STAINING AND
ON
THE
CORPUSCLES
REVEALS. BY
S. P. JAMES, Adviser
on
Tropical
(Received
M.D., Diseases
30th
D.P.H.,
I.M.S.
(Red.).
to
Ministry
of
the
August,
Health.
1929).
For finding and identifying malaria parasites in thin blood films, Leishman’s stain (which requires no preliminary fixation of the blood) is in general use ; but it happens frequently that the results obtained with it are not at all satisfactory. Mr. P. G. SHUTE, Senior Laboratory Assistant in the Malaria Laboratory of the Ministry of Health, by investigating systematically each factor which seemed to have an influence on the staining process, has worked out a technique which enables excellent results to be obtained without fail. An important outcome of the use of this technique in our laboratory has been the demonstration of a hitherto unnamed type of stippling in red blood corpuscles infected with quartan parasites (Plasmodium malaria), as well as the demonstration of Schiiffner’s dots and Maurer’s spots in red blood corpuscles infected with younger stages of P. vivax and P. falciparum respectively, than the stages with which those characteristic phenomena are usually associated. For these reasons, and particularly because constant and consistent results are obtained by using Leishman’s stain in the way described below, the technique is of great assistance in the correct identification of the species of parasite present in blood films. The importance of this matter will be appreciated by those who are aware of the frequency with which errors in identification have been made even by
370
NOTE ON THE SHUTE TECHNIQUE FOR STAINING MALARIA PARASITES.
experienced workers, in the past, and are still being made today wherever microscopic examination of blood films is practised. ~ DESCRIPTION OF THE TECHNIQUE. New microscope glass slides should be used. T h e slides with which the best results are obtained are those known in the trade as " half white " ; the results m a y not be so good when slides known as " white " or as " French special " are used. T h e slides should be dipped in " nitric alcohol " (30 parts nitric acid, 70 parts absolute alcohol), and, after being wiped dry, should be kept in absolute alcohol. On no account should " methylated spirit " be used as a substitute for absolute alcohol in any part of the procedure of taking and staining blood films. T h e slides, after removal from absolute alcohol, should be rubbed dry with a clean soft cloth. T h e stain is made with pure methyl alcohol (" free from acetone ") and crystals of Leishman's stain. T h e reaction of the methyl alcohol must be tested before making up the stain. This is done with the aid of an outfit for determining the hydrogen ion concentration, the indicator used being phenol red, and the range of the standard tubes being from p H 6.6 to p H 8.0 (Baird and Tatlock's outfit No. P.2759). Into the test-tube made of cordite glass which is supplied with the outfit, pipette 5 c.cm. of the methyl alcohol to be tested. With a separate pipette add 0.5 c.cm. of a .01 per cent. solution of phenol red. Shake and, after a m o m e n t or two, compare the tint with that in the standard tubes provided. Every brand of methyl alcohol which we have tested in this way gives a slightly acid reaction. After adding the indicator to 5 c.cm. of the brand which we use, the tint corresponds nearly always with that of the standard tube marked 6-8, b u t sometimes with that marked 6.6. It is our practice to discard supplies of methyl alcohol which are as acid as is indicated by the tube marked 6.6, and we have had to do so even with some supplies which makers p u t up in hermetically sealed tubes " for use in microscopic staining." T o make up the stain, rinse a glass stoppered bottle # thoroughly with some of the methyl alcohol that will be used for the stain, and then p u t in 0.15 g. of Leishman's crystals (usually called " Leishman's powder "). Add 100 c.cm. of the methyl alcohol. Shake f r o m time to time during the next 24 hours, after which period nearly all the crystals will be dissolved, and the stain will be ready Interesting examples in the literature of more than twenty-five years ago will be found in LEISHMAN'Sfirst article on the application of Romanowsky's stain for the microscopic diagnosis of malaria (Brit. Med. Jl., 16th March, 1901, page 635), and in STEPHENS'and CHRISTOPHERS'first article on the malarial infection of native children in British Central Africa (Reports to the Malaria Committee of the Royal Society, Third Series, 1900, page 4). It is obvious from the descriptions and illustrations in those articles that LEISHMANidentified and described as a quartan parasite what was really a malignant tertian parasite, and that NTEPHENS a n d CHRISTOPHERSseem to have made precisely the reverse error. Examples in modem times are too numerous to cite. t The bottle should be of hard glass (green glass).
S. P. JAMES.
271
for use. For several reasons it is unwise to make the solution in a pestle and mortar, or to filter it, as is usually recommended in text-books. Next deal with the distilled water which will be used in the staining process and for washing the stained slides. We work with a 1-1itre flask of distilled water which has been treated as follows : Shake up the water in the flask and wash out a 5 c.cm. pipette with water from it ; test 5 c.cm. of the water in the same way as was described for testing the methyl alcohol. Probably the water will be found to be at least as acid as is indicated by the standard tube marked 6.6. Add to the water three or four drops of a saturated filtered solution of lithium carbonate, shake the flask to ensure thorough mixing, and repeat the test. Continue the procedure of adding one or two drops of the lithium carbonate solution and of testing until the water in the flask becomes exactly of an alkalinity indicated by observing that, after adding 0.5 c.cm. of the phenol red solution to 5 c.cm. of the water, the resulting colour matches the colour of the solution in the standard tube marked 7.2. This is the degree of alkalinity that must be reached when the methyl alcohol is of an acidity represented by the tube marked 6"8. To stain a blood film, drop four drops of the staining solution on the film, rock for ten seconds, add twelve drops of the distilled water, and mix thoroughly by tilting and rocking. We do not use a glass rod for mixing the water and the stain on the slide, because a rod often carries specks of dust or of cotton fibre which are transferred to the slide ; but a good deal of practice in tilting and rocking the slide is required in order to obtain quick and complete mixing of the water and the stain without spilling some of it off the slide, or on the fingers, and without allowing any of the stain to dry on the film before the water has reached it. Nor do we employ the usual practice of making a barrier with a wax pencil across the proximal end of the slide, because, when this is done, particles of methylene blue from the pencil invariably become mixed with the stain and alter the result. By using four drops of stain and twelve drops of distilled water, the stain is diluted three times which, we think, gives the best results. Four drops of stain, carefully applied, are quite sufficient to cover a film and, when twelve drops of water are added, the amount of fluid on the slide is easy to manipulate so that none spills off the slide or reaches the fingers. We time the ten seconds during which the stain alone is on the film by a watch with a large second hand. The length of time during which the stain is allowed to act on the film is very important. When dealing with a film about which we do not know the species of malaria parasite present, thirty minutes is the correct period during which the stain should be allowed to act ; for although the older stages of the parasites present will be too deeply stained, that period is necessary for bringing out clearly the stippling in red blood corpuscles infected with quartan parasites and Schiiffner's dots and Maurer's spots in the youngest ring or marginal stages of corpuscles infected with P. vivax or P. falciparum. When we know what species of parasite is present in the films, the period
579'
NOTE O N THE SHUTE TECHNIQUE FOR STAINING MALARIA PARASITES.
of staining should be varied according to whether it is desired particularly to study the morphology of the parasites or the stippling and other changes in the red blood corpuscles containing them. The parasites stain much more quickly than the changes indicated by stippling or dotting of the red blood corpuscles ; and Schiiffner's dots stain more quickly than Maurer's spots or the stippling characteristic of a quartan infection. After staining for ten minutes Schiiffner's dots, characteristic of infection with P. vivax, are vividly stained in all corpuscles containing quarter to half-grown and later stages of the parasite, but in that period no stippling can be seen either in corpuscles infected with the youngest forms of this parasite or in any corpuscles infected with P. malari~e. Stippling of corpuscles infected with the latter parasite is best studied in films stained for 45 minutes, though it is well seen in most (though not in all) infected corpuscles after 30 minutes staining. For routine identification work 30 minutes is the best time. * On the termination of staining, the stain must not be poured off the slide before beginning to wash the film ; a good stream of distilled water must be applied at once so that all stain and deposit is flushed off in the first moment. Washin[~ in the stream of distilled water should be continued for fifteen seconds by the watch. I am afraid that the technique, and my description of it, may be criticised on the ground of meticulolls accuracy. My excuse is that during a recent tour of investigation in Africa I was so much impressed by the reports of difficulties and failures with Leishman's stain which were brought to my notice, as well as with errors resulting from working with poor stains, that I hardly think it possible to exaggerate the importance of the details to which I have drawn attention above. NOMENCLATURE AND DESCRIPTION OF THE DOTS AND SPOTS. Hitherto, observers who have described characters for distinguishing between the " ring forms " of the three species of the malaria parasite, have been embarrassed by the fact that they could never be sure that the blood films which they examined for this purpose (being obtained from patients infected in nature) contained only one species of parasite. The unrecognised presence of two species of the parasite was obviously the cause of the errors in identification and description to which reference was made in the footnote on page 270. In our laboratory we are in a more favourable position because our patients are intentionally infected with what has been proved by repeated passage to be a pure strain of only one species of the parasite. Thus both the youngest and the oldest ring forms which appear in the peripheral circulation are known to belong to the single species with which the patient was infected. Our benign tertian strain has not changed its characters since it was obtained in 1925, and our quartan strain has remained equally pure since it was obtained from Dr. W. KIRSCHBAUM, The slide must be covered by a Petri dish during the process of staining.
273
S. P. JAMES.
of the Friedrichsberg Mental Hospital, Hamburg, a year ago. We have studied ring forms of the malignant tertian parasite particularly in cases of primary infection intentionally induced by blood inoculation, and afterwards watched for a long period to see that no other type of fever or parasite appeared, but we have not maintained a strain of that species continuously. We have compared the findings in blood films of our cases of each type of malaria with those in blood films from natives of East Africa stained by the same technique, and have found that among the latter, mixed infections (particularly with quartan and subtertian) are frequent. I do not propose, in this article, to describe ail the points by which, using .the staining procedure described, one is able to recognise and distinguish from one another the ring forms of the three species of parasite ; what I wish to do is to draw attention to the phenomenon of stippling in corpuscles infected with quartan parasites, to note the character of the stippling in some uninfected red corpuscles which is demonstrable in films of malarial blood stained in this way, and to correct an error of nomenclature relative to Maurer’s spots which has crept into English text-books and articles. STIPPLING
OF
RED
CELLS
IN
QUARTAN
MALARIA.
The illustration below shows Schiiffner’s dots in corpuscles infected with a ring form of P. vivax, the quartan stippling or dotting in corpuscles infected with a ring form of P. malank, and Manrer’s spots in corpuscles infected with a ring form of P. falciparum. It is reproduced from a water colour drawing kindly
made for me by Mr. Jobling.* It will be seen that in corpuscles containing ring forms of approximately the same size : (1) Schtiffner’s dots are relatively large and round, numerous and very distinct ; their colour shades from deep mauve through violet to pale rose ; (2) the quartan stippling consists of numerous separate dots and points, smaller than Schtiffner’s dots and not so definitely round nor so distinct ; their colour is the same as that of Schiiffner’s dots, but more of them are pale rose. In corpuscles containing quartan parasites which nearly fill the corpuscle, the dots are nearly as large as Schtiffner’s dots, but they are fewer in number, and they stain faintly pale rose. Their reaction to staining * I have to thank Dr. C. M. WENYON, Director in Chief, Wellcome Bureau of Scientific Research, for kindly permitting Mr. Jobling’s services to be lent to me for this purpose, as well as for valuable suggestions kindly made while the paper was passing through the press. The coloured drawings, with others, will be published as a plate in The Kenya and East African Rledical Journal.
~74[
NOTE ON TnE SHUTE TECHNIQUE FOR STAINING MALARIA PARASITES.
is evidently quite different from that of Schiiflher's dots because, while Schiiffner's dots can be demonstrated readily in films stained for only five minutes in the manner described, the stippling of cells containing quartan parasites is not demonstrable until the film has been stained for at least twenty minutes, and is best brought out by staining the films from thirty to forty-five minutes. This is why we recommend thirty minutes as the period of staining for routine identification work. It will be remembered that MAURER,to obtain his" third and fourth degrees of staining," which showed the stippling of infected corpuscles best, recommended that films should be stained for one hour. Evidently in modern practice much is sacrificed to the demand for quick results ; for example, in the ninth edition of Manson's Tropical Diseases, p. 859, five minutes is the period recommended for staining with Leishman's stain. (3) Maurer's spots are few in number, and larger and of more irregular shape than the dots in either of the other types ; their number can always be counted, and some of them are tiny rings, loops or streaks ; their colour is deep mauve to violet, and the corpuscle itself is pale blue-grey, and often outlined sharply by the rose stain. PROPOSED
NAME FOR THE
QUARTAN S T I P P L I N G .
As names have been given to the stippling of corpuscles infected with P. vivax (Schfiffner's dots) and to the stippling of corpuscles infected with P. falciparum (Maurer's spots) it will probably be thought desirable to name the stippling associated with the presence of P. malarice. It is not easy to ascertain from the literature who has the right to priority of observation and description in this matter, particularly because most, if not all, observers who have mentioned stippling in corpuscles which they described as containing a quartan parasite, were working with films from cases with a mixed infection. SCHOFFNER in his classical monograph, Beitrag zur Kenntniss der Malaria, published in 1899, states that in quartan infections the blood corpuscles retain their normal staining properties, but that " only very occasionally there is a delicate blue line or loop in the interior of the red cell, which cannot possibly, however, be confused with the dots described " (that is, with Schiiffner's dots). His illustration of these lines or loops shows that he was not describing (and apparently had not seen) the type of quartan stippling with which we are concerned at present. ZIEMANN, whose observations were made in Togoland in 1900, noted SCHi)FFNER'S observations on the staining characters of corpuscles infected with quartan parasites and reported that, for his part, he had sometimes seen, when employing an alkaline Romanowsky stain, an exceedingly delicate Schiiffner dotting in those corpuscles. MAURER,in his articles published in 1900 and 1902, does not mention any change in corpuscles infected with quartan parasites. STEPHENSand CHRISTOPHERS,in their article on the malarial infection of native children, published in 1900, give a drawing (Plate I, drawing No. 8) which might serve to illustrate the stippling of a blood corpuscle containing a nearly full-grown quartan parasite, but they described the parasite as a female
S. P. JAMES.
275
gametocyte of P. j’alciparum. CRAIG, in his textbook published in 1909, states that the form of degeneration represented by the occurrence of Schiiffner’s granules or dots is probably never present in quartan infections, “ although in a very few such infections I have observed a stippling indistinguishable from that produced by Schiiffner’s dots in invaded red blood corpuscles. However, such an appearance occurs so rarely that it is of no practical importance.” WENYON, in describing quartan infections on page 942 of Vol. II of his textbook PTotozoology, says : “ Very rarely dots resembling those seen in the case of P. vivax have been noted.” He has kindly informed me that this statement is based on his own experience and that for many years he has taught that occasionally such dots are seen in quartan infections.* Dr. MARY LAWSON, in an article published in 1916, gave a photograph of “ two young quartan parasites in a red corpuscle showing Maurer’s rings and dots in a red cell showing (Fig. SO),” and another of “ a quartan microgametocyte Schiiffner’s granules (Fig. Sl).” Unfortunately, her paper contains no statement of the grounds on which the identification of these parasites as quartan was made, and I do not see how it would be possible to agree with her opinion that quartan parasites sometimes cause the destructive changes in red blood corpuscles which are demonstrable as Maurer’s spots, loops and rings ; it seems probable that, as has happened to many observers, she was dealing with a mixed infection. The same criticism is applicable to papers by several other workers who have described stippling of different types in red blood corpuscles said to contain quartan parasites. There are, however, descriptions in another category to be considered, namely, those in which parasites have been classed and named as a new species chiefly because, while resembling the quartan parasite in most particulars, they were present in red cells showing stippling said to be like Schiiffner’s dots. The parasite described by STEPHENS in 1922, and again by STEPHENS and OWEN in 1927, under the name Plasmodium ovale, is in this category. From an examination of the coloured plate published with the description of P. ovale it seems probable that in this instance the stain, or the distilled water, used for the staining process happened accidentally to possess the reaction which brings out stippling in corpuscles infected with quartan parasites. Probably this is an example in which the characteristic stippling of corpuscles infected with quartan parasites was observed, although, in my opinion, it was wrong to describe the stippling as “ Schiiffner’s dots.” The coloured plate of P. ovale shows that the dots are less numerous and smaller than Schiiffner’s dots, and it is mentioned in the description that they do not stain so deeply as Schiiffner’s dots usually do, * In this connection I hope it will be clearly understood that we do not consider our finding of stippling in quartan infections to be a new observation. I may be permitted to say, however, that, by the use of the technique described, it has been possible to give precision to previous observations on the subject, and to point out that, in several important respects, the stippling described does not, in fact, resemble that observed in vivax infections.
276
NOTE O N THE SHUTE TECHNIQUE FOR STAINING MALARIA PARASITES.
although the films were stained for one hour. The fact that the stippling was seen only after long staining is in favour of the view that P. ovale is a quartan parasite seen in cells stippled in the manner described in this paper. Having regard to the various points mentioned in the above summary, I consider that ZIEMANNhas the right of priority of observation and of description in the matter of-stippling of red corpuscles infected with quartan parasites ; his observations were made earlier than those of any of the other workers quoted, and his description of them, though meagre, was correct. He noted that, in red corpuscles containing quartan parasites forty-eight hours old, the stippling could be brought out by using Brug's staining method as well as by using an alkaline Romanowsky stain. For these reasons I suggest that the characteristic dotting of red corpuscles invaded by quartan parasites should be named " Ziemann's stippling." I choose the word stippling because it is advisable that the nomenclature should indicate the character of the stained markings; Schfiffner's are dots, Maurer's are spots, and Ziemann's are a mixture of points, dots and spots. STIPPLING OF UNINFECTED CORPUSCLES. P Stippled red corpuscles not containing parasites are common in films from cases of subtertian malaria stained by the Shute technique, and are also seen (less frequently) in films from cases of quartan malaria. The dots and points are numerous, and most of them are small and of a pale rose colour. They are quite different from the basophilic dots with which some other red blood corpuscles in films from cases of malaria are often studded. MAURER,in 1902, described and illustrated this type of stippling of uninfected corpuscles. PROPOSED CORRECTION OF NOMENCLATURE. The error of nomenclature which I desire to correct is that in English textbooks, and articles " Maurer's dots " are wrongly named " Stephens' and Christophers' dots." I am the more anxious to rectify this error because I was myself among those who made it (on page 24 of my book, Malaria at Home and Abroad). I refrain from naming all the other English writers who, in textbooks and articles, have made the same mistake. It arose after the publication by STEPHENS and CHRISTOPHERSof their " Note on the Changes in the Red Cell produced by the Malignant Tertian Parasite " (Brit. Med. ~l., 28th March, 1903, page 730), in which they said that, as far as they were aware, they were the first to point out the changes in red cells invaded by malignant tertian parasites. The article from which they quoted a sentence to support this belief was their first report to the Malaria Committee of the Royal Society on The Malarial and Blachwater Fevers of British Central Africa. This report was published in 1900. But, evidently, they were not aware of the publication, early in 1899, of SCH~)EENER'S article Beitrag zur Kenntniss der Malaria, in which, as well as describing the dots associated with P. vivax infections Which now bear his name,
277
S. P. JAMES.
he described (and illustrated accurately and precisely in a coloured plate) the markings associated with P. falciparum infections which were afterwards named Maurer’s dots. This article by SCH~FFNER was published in a special volume (Festschrift XUT Feier Leipzig) as the 64th
des 100 Jahrigen
Bestchens
der Medicinischen
Klinik
2;u
volume of the Deutsche Archiv fiir Klinische Medicin. The copy which I have consulted is stamped with the date stamp of the Royal Medical and Chirugical Society, 24th May, 1899, so it must have been received in London some time before this date. On the other hand, the manuscript of STEPHENS and CHRISTOPHER& article was not received by the Malaria Committee of the Royal Society until the 10th December, 1899, and the article was not published until some time in 1900. Therefore, it seems clear that SCH~FFNER was the first to describe and to point out in a published article these malignant tertian markings, as well as the benign tertian markings with which his name is always associated. MAURER, in his articles published in 1900 and in 1902, fully acknowledged SCH~~FFNER’S priority of observation and description as regards both kinds of stippling, and he described his own contributions as a confirmation and extension of SCH~~FFNER’S results by using the Romanowsky stain instead of the Mannaberg and hzmotoxylin stains which SCH~FFNER used. On this subject Professor E. P. SNIJDERS, of the Institute for Tropical Hygiene, Amsterdam, has written to me as follows : “ SCH~FFNER first detected the ‘ dots ’ of benign tertian malaria and the ‘ spots ’ of malignant tertian malaria (perniciosa). Afterwards, independently of each other, RUGE and MAURER (both in 1900), by the application of the Romanowsky stain, confirmed his findings with regard to the ‘ dots,’ and MAURER confirmed his findings with regard to the SCH~~FFNER and MAURER were ‘ spots ’ (Flecken), and laid full stress on them. great friends, and I think you will act in accordance with SCH~~FFNER’S views if you will state that SCH~FFNER was the first to describe the subtertian ‘ spotting ’ (Fleckung) as well as the benign tertian ‘ dotting ’ (Ti@felung), that MAURER acknowledged this fully, and that it was ever a great joy to SCH~~FFNER that the ‘ spots ’ bear the name of his friend MAURER in recognition of the latter’s great contributions to the knowledge of staining methods for malaria.” REFERENCES. ARGUTINSKY,
P. (1903). Zur Kenntniss des Tropicaparasiten ; Die Tiipfelung der Wirts-
zellen der Halbmonde. Cent.f. Bakt., Abt. 1, Orig., xxxiv. (2), 144. CRAIG, C. F. (1909). Malarial Fevers. J. and A. Churchill, London and New York, 1909. -. (1915). New Varieties and Species of Malaria Plasmodia. JZ. Pamsit., 1, (2), 85. LAWSON, M. R. (1916). Distortion of the Malarial Parasite. An Interpretation of “ Plasmodinm Tenue ” (Stephens). yl. Experim. Med., xxiv, (3), 291. LEISHMAN, W. B. (1901). The Application of Romanowsky’s Stain in Malaria. Brit. Med. Jl., i, 635 ; 16th March. MAIJRER, G. (1900). Die Ttipfelung der Wirtszelle des Tertianaparasiten. Cent. f. Bakt., Abt. 1, xxviii, 114. ---. (1901). Die Malariaparasiten. Mtinch. Med. Woch., xlviii, 337; 26th Feb. -. (1902). Die Malaria Perniciosa. Cent. f. Bakt., Abt. 1, Orig., xxxii, 695.
278
NOTE ON THE SHUTE TECHNIQUEFOR STAININGMALARIAPARASITES•
MACFIE, J. W. S•, and INGRAM, A. (1917). Observations on Malaria in the Gold Coast Colony, West Africa. Ann. Trop. Med. ~.~ Parasit., xi, (No. 1), 1. RUGE, REINHOLD• (1900). Ein Beitrag zur Chromatinf/irbung der Malariaparasiten. Zeitsehr. f. Hyg. u. Infektionskr., xxxiii, 178. SCH/.~IFFNER. (1899). Beitrag zur Kenntniss der Malaria. Deut. Arch. f. Klin. Med,, lxiv, 428. STEPHENS, J. W. W., and CHRISTOPHERS,S. R. (1900). Th e Malarial and Blackwater Fevers
of British Central Africa. Reports to the Malaria Committee of the Royal Society. Series 1, p. 12. Series, p. 4". (1900). T h e Malarial Infection of Native Children. Ibid. T h i r d (1903). Note on the Changes in the Red Cell produced by the Malignant ~Fertian Parasite. Brit. Med. Jl., 730 ; 28th March. STEPHENS, J. W . W . (1922). A New Malaria Parasite of Man. Ann. Trop. Med. Parasit., xvi, (4), 383. STEPHENS,J• W. W., and OWEN, D. U. (1927). Plasmodium ovale. Ann. Trop. Med. Parasit., xxi, (2), 293. THOMSON, J• G. (1928)• Stippling of the Red Cells in Malaria• Proc. Roy. Soc. Med. (See. Trop. Dzs. ~ Parasit•), xxi, 464 ; 3rd January• • (1924). Researches on Blackwater Fever in Southern Rhodesia. Lond. Sch. Trop. Med. Res. Mem. Set., vi. THOMSON, J. G., and ROBERTSON,ANDREW. (1929). T h e Malarial Parasites of Man. Protozoology, p. 14. London : Balli&re, Tindall & Cox. WEIqYON, C . M . (1928). Protozoology, ii, 942. London : Balli~re, Tindall & Cox. ZIEMANN, HANS. (1898). Neue Untersuchungen fiber die Malaria und den malariaerregem nahestehende Blutparasiten. Deut. Med. Woch., 123 ; 24th Feb. • (1915). Ueber eigenartige Ma]ariaparasitenformen. Cent. f. Bakt., Abt. 1, Orig. lxxvi, (5), 384. (1906)• In Mense's Handbueh der Tropenkrankheiten, iii, 1st edition. Leipsic (1918). Ibid., v, 2nd edition, pp. 30, 33 and 37.