Comprising Notes on Recent Researches, FormulÆ of some Culture Media, and Brief Notes on General Protozoological Technique.

BY

H.B. FANTHAM, M.A., D.Sc.

I.—NOTES ON RECENT RESEARCHES.

Since the foregoing section on Protozoology was sent to press, certain interesting observations and results have been published. Brief notes on such, and some references thereto, are now added.

It is necessary, however, to remark that sometimes it is impossible to give a precise or rigid definition to a genus of Protozoa, owing to differences of opinion, to differences regarding nomenclature or to incompleteness of knowledge. Such a lack of definition, while inconvenient for the time being, is not unhopeful, as it directs attention to the necessity for further work, which is inevitable in such a relatively new and wide subject as protozoology. Thus, it may be noted in illustration that Minchin, in 1912, in his text-book regarding the genus Entamoeba writes: “The entozoic amoebÆ are commonly placed in a distinct genus, Entamoeba, distinguished from the free-living forms by little, however, except their habitat and the general (but not invariable) absence of a contractile vacuole.”

Differences between Entamoeba histolytica and E. coli.—In continuation of the remarks on pp.34 and 40, it may be added that Lugol’s solution (iodine in aqueous potassium iodide solution) in fresh specimens shows by brownish staining the presence of glycogen in the vacuoles of Entamoeba coli. Such a reaction is rarely or never given by E. histolytica.

PhagedÆnic AmoebÆ.—Carini and others record cases in which the skin around an operation wound in connection with liver abscess became gangrenous. AmoebÆ, possibly Entamoeba histolytica, were found therein and may have been responsible for the gangreno-phagedÆnic action.

Endamoeba gingivalis (see pp.43, 44).—Smith and Barrett,1248 after analysing the early literature, state (June, 1915) that Endamoeba gingivalis, Gros, 1849, is the correct name for the following organisms: E. buccalis, Prowazek, 1904 (see p.43); Amoeba gingivalis, Gros, 1849; Amoeba buccalis, Steinberg, 1862, and Amoeba dentalis, Grassi, 1879. They conclude that E. gingivalis is the causal agent of pyorrhoea alveolaris, and that this disease responds to treatment with emetine.

Entamoeba kartulisi (see p.44), synonym E. maxillaris, Kartulis, is considered to be E. gingivalis.

Smith and Barrett adopt the generic name Endamoeba, Leidy, 1879 (see footnote on p.31, also p.34). Leidy worked on Endamoeba blattÆ.

Craigia and Craigiasis (see p.45).—Barlow1249 (May, 1915) found Craigia (Paramoeba) hominis in cases of chronic diarrhoea and mild dysentery in Honduras. He also described a new species of Craigia under the name of C. migrans. Fifty-six cases were studied, five of which were due to Craigia hominis, the remainder to C. migrans. In C. migrans, each flagellate, on attaining full development, becomes an amoeba without dividing. Each amoeba encysts and produces a number of flagellates which are somewhat like cercomonads. On the other hand, in C. hominis the flagellate form produces, by longitudinal fission, several generations of flagellates before entering upon the amoebic stage. The cysts of C. migrans contain fewer “swarmers” (flagellulÆ) than those of C. hominis, but the “swarmers” are somewhat larger, namely, 5µ instead of 3µ in diameter. Further, there is no accessory nuclear body in C. migrans, but its flagellum stains more deeply than that of C. hominis and has a peculiar banded appearance.

Human Trichomoniasis (see pp.52–56).—Lynch1250 (April and May, 1915), working in Charleston, seems to favour the view that the trichomonads found in the vagina, urethra, mouth, lungs and alimentary tract are one and the same organism, and that these flagellates may further excite already existing inflammatory conditions. He gives detailed histories of cases of (a) infection of the vagina and gums, and (b) intestinal infection manifested as intermittent attacks of diarrhoea. The flagellates were found in catarrhal vaginal discharge, in blood-stained scrapings from the gums (together with Endamoeba buccalis), and in stools after a purge of magnesium sulphate. The parasites were tetratrichomonads (see footnote, p.53), that is, each possessed four flagella anteriorly as well as an undulating membrane. Lynch successfully infected rabbits from the cases and from cultures of the parasite. Encysted trichomonads were seen in a patient’s stools, in rabbits infected therefrom and in cultures. The culture medium used was bouillon acidified with about 0·05 per cent. acetic acid and the cultures were maintained at 30°C.

Trichomonads occur in the digestive tracts, for example, the cÆca of rats and mice (fig.422). In man allied flagellates can occur in similar situations, as well as in other parts of the intestine.

Other trichomonad-like organisms have been recently described from the fÆces of man, more particularly from cases of chronic dysentery in the tropics. Derrieu and Raynaud1251 (July, 1914), working in Algeria, found a flagellate possessing five free flagella anteriorly and an undulating membrane apparently lateral. They named the parasite Hexamastix ardin-delteili, but the generic name Hexamastix is pre-occupied. Chatterjee1252 (January, 1915), working in India, found probably the same flagellate and called it Pentatrichomonas bengalensis.

Chilomastix (Tetramitus) mesnili (see p.57).—Alexeieff1253 (1914) now places the parasite originally called Macrostoma mesnili, by Wenyon (1910), in the genus Chilomastix, Alexeieff. The differential characters of the genera Tetramitus and Chilomastix are not especially well marked. According to Alexeieff, Tetramitus is characterized by four unequal flagella (which he figures anteriorly), a ventral cytostome in the form of a linear cleft and a pulsatile vacuole in front of the anterior nucleus. Chilomastix, according to the same author, has three forwardly directed flagella and a fourth backwardly directed one in the cytostome, which is well developed (fig.423). Some authors consider that the fourth flagellum forms the edge of an undulating membrane in the cytostome.

Diagrams of Chilomastix mesnili are given in fig.423.

Giardia (Lamblia) intestinalis (see p.57).—Alexeieff1254 (1914) considers that Lamblia intestinalis, Lambl, should be placed in the genus Giardia, Kunstler, 1882. Bipartition occurs in the encysted state. The axostyles persist in the quadrinucleate cyst.

Cercomonas hominis (see p.61).—This parasite is considered by some authors to be of a doubtful nature, as it is thought to have been mistaken for deformed or incompletely observed Trichomonas or Chilomastix or even Lamblia.

Wenyon1255 (1910) described Cercomonas longicauda from cultures of human fÆces. It is considered that the genus is very confused, and the author points out that the tail flagellum has been overlooked. He considers that the genus Cercomonas should include flagellates with an anterior blunt end from which arises a single long flagellum, and a posterior tapering end also with a flagellum, which can be traced over the surface of the body towards the insertion of the anterior flagellum.

Another species, Cercomonas parva, has been found in cultures of human fÆces by Hartmann and Chagas1256 (1910). It has a somewhat different structure.

Further researches are necessary on the organisms variously referred to the genus Cercomonas.

Transmissive Phase of Trypanosomes in Vertebrates.—In addition to the general remarks on the morphology of trypanosomes set forth on pp.70 to 72, it may be noted that Woodcock1257 (November, 1914) states that, in certain cases, there is a definite transmissive phase of a trypanosome in its vertebrate host. He quotes the work of Minchin and himself on T. noctuÆ of the little owl, in which the transmissive form is spindle-shaped and occurs in the bird’s peripheral blood during the early summer months (see p.69). A similar phase occurs in T. fringillarum, and Robertson1258 has found that the short, stumpy form of T. gambiense is its transmissive phase in vertebrates.

Trypanosoma lewisi (see p.88).—Brown (1914–15) has published some interesting results on the potential pathogenicity of T. lewisi.

Blepharoplastless Trypanosomes (see p.101).—Laveran1259 (April, 1915) suggested a practical use of strains of blepharoplastless trypanosomes produced by the action of drugs. He finds that tryposafrol will also produce such strains, and remarks on blepharoplastless strains of T. evansi and T. brucei, which in the former case can undergo 450 passages without reversion, and in the latter 273 passages. He states that if it is desired to inoculate surra or nagana to CapridÆ or BovidÆ in order to produce immunity, use should be made of the blepharoplastless races of the respective trypanosomes, which races are a little less virulent than the corresponding normal ones. Also, the immunity which follows from an infection due to blepharoplastless T. evansi or T. brucei is only a little less complete than that following infections from either of the respective normal strains.

The Experimental Introduction of certain Insect Flagellates into various Vertebrates, and its bearing on the Evolution of Leishmaniasis.—In continuation of the remarks on pp.103, 104, and 112, further researches have been conducted on the introduction into vertebrates of flagellates normally parasitic in insects. The vertebrates became infected by inoculation with the flagellates or by being fed on insects containing the protozoa. Fantham and Porter1260 (June, 1915) published the following results. Flagellates from sanguivorous and non-sanguivorous insects were used, and cold-blooded as well as warm-blooded vertebrates as hosts. The introduced protozoa were pathogenic to the mammals, but not markedly so to the cold-blooded vertebrates. Herpetomonas jaculum, H. stratiomyiÆ, H. pediculi, and Crithidia gerridis (parasitic in certain waterbugs) proved pathogenic to mice. A puppy was infected by way of the digestive tract with H. ctenocephali. Frogs became infected with H. jaculum and with C. gerridis, toads and grass snakes with H. jaculum, lizards with C. gerridis, and sticklebacks with H. jaculum. Second and third passages of some of the parasites were obtained. The protozoa, whether Herpetomonas or Crithidia, were present in the vertebrate hosts in either the non-flagellate or the flagellate form, or usually both. They were more abundant in the internal organs of the hosts, more particularly in the liver, spleen and bone-marrow. In all experiments in which C. gerridis was used the parasite invariably retained the crithidial facies in the vertebrate host. No transition to a trypanosome was ever seen. Infections in adult animals were not so heavy as in the young ones, and the parasites were more virulent in young hosts, as is the case with Mediterranean kala-azar in children.

The mode of infection of the vertebrate in Nature seems to be contaminative, either by its food or through an already existing abrasion or puncture on the surface of its body. Cases in which the flagellate-infected insects have been allowed to suck the blood of vertebrates have proved negative up to the present. In areas where leishmaniases are endemic, an examination should be made of all insects and other invertebrates likely to come into contact with men or dogs, or rats and mice (see below), in order to ascertain if these invertebrates harbour herpetomonads. Preventive measures should be directed against such invertebrates, especially arthropods. Further, it is likely that certain vertebrates, such as reptiles and amphibia (especially those that are insectivorous), may serve as reservoirs of leishmaniases, or, as they should preferably be termed, herpetomoniases. From such reservoirs the herpetomonads may reach man by the agency of ectoparasites or flies, especially such as are sanguivorous.

That vertebrates in Nature can harbour herpetomonads in their blood has been shown by the work of Dutton and Todd (1903) on the herpetomonads of Gambian mice, while the recently published investigations of Fantham and Porter1261 (June, 1915) on natural herpetomonads in the blood of mice in England have shown that these rodents may be a natural reservoir of herpetomoniasis. The origin of the infection of mice is to be sought in a flagellate of an ectoparasite of the mouse, very probably Herpetomonas pattoni parasitic in various fleas, which protozoÖn can adapt itself to life in the blood of mice. Herpetomonads were also found naturally in the blood of birds by Sergent (1907). Recently, Fantham and Porter have successfully infected birds with herpetomonads experimentally.

The significance of insect flagellates in relation to the evolution of disease has recently been set forth by Fantham1262 (June, 1915). The deductions to be made from the occurrence of a herpetomonad stage in Leishmania, especially in L. tropica, in man himself, and of flagellate stages of the so-called Histoplasma capsulatum in man are fully discussed and correlated. It is pointed out that flagellosis of plants (see p.104) may possibly be connected with leishmaniasis. The evolution of Leishmania from flagellates of invertebrates is thus traced and the way again indicated for preventive measures against leishmaniasis, as first set forth by Dodds Price and Rogers.

Franchini and Mantovani (March, 1915) have successfully infected rats and mice by inoculation or by feeding with Herpetomonas muscÆ domesticÆ obtained from flies and from cultures.

It is of great interest to note that the recent observations of Ed. and Et. Sergent, Lemaire and Senevet1263 (1914) have demonstrated the presence of a herpetomonad flagellate in cultures of the blood and organs of geckos obtained from areas in Algeria in which Oriental sore, due to L. tropica, is prevalent. Phlebotomus flies, which may harbour a natural herpetomonad, feed on the geckos and on men. Hence animals like geckos may possibly act as reservoirs of leishmaniasis. Lindsay1264 (1914) writes that the parasite of dermo-mucosal leishmaniasis in Paraguay is believed by native sufferers to be conserved in rattlesnakes, and spread by ticks or flies (Simulium) feeding on the reptiles and transferring the parasite to man.

The Transmission of SpirochÆta duttoni (see p.116).—It is probable that Ornithodorus savignyi acts as the transmitting agent of S. duttoni in places like Somaliland (Drake-Brockman, 1915).1265

SpirochÆta bronchialis (see p.122).—The morphology and life-history of S. bronchialis have been investigated by Fantham1266 (July, 1915). From researches conducted in the Anglo-Egyptian Sudan, he found that S. bronchialis is an organism presenting marked polymorphism, a feature that has only been determined by the examination of numerous preparations from the deeper bronchial regions of various patients.

S. bronchialis varies in length from 5µ to 27µ, and its breadth is about 0·2µ to 0·6µ. These variations are due to the processes of growth and division. Many of the parasites measure either 14µ to 16µ long, or 7µ, to 9µ, the latter resulting from transverse division of the former. The ends show much variation in form, but approach the acuminate type on the whole. The discrepancies in dimensions given by the very few previous workers on the subject are probably the result of the measurement of a limited number of parasites. All such sizes can be found on some occasion during the progress of the disease, when a larger number of spirochÆtes is examined.

The movements of S. bronchialis are active, but of relatively short duration, when it is removed from the body. The number of coils of the spirochÆte is rather an index of its rapidity of motion than a fixed characteristic of the species.

The motile phase of S. bronchialis is succeeded by one of granule formation, the granules or coccoid bodies serving as a resting stage from which new spirochÆtes are produced. The formation of coccoid bodies and reproduction of spirochÆtes from them can be observed in life.

S. bronchialis is a species distinct from the spirochÆtes occurring in the mouth. It differs from them in morphology, pathogenicity and in staining reactions. It is not a developmental form of any bacterium, and is an entity in itself.

The passage from man to man is effected most probably by means of spirochÆtes, and especially coccoid bodies, that leave the body in the spray with expired air and by way of the nasal secretions. Owing to the fragility and short life of S. bronchialis extracorporeally, the resistant coccoid bodies in air, in dried sputum and dust, and possibly also on the bodies of flies and other insects, are probably instrumental in inducing attacks of bronchial spirochÆtosis in human beings, especially those having a lowered bodily resistance, such as occurs after a chill. Lurie (December, 1915), has described a case from Serbia.

The SpirochÆtes of the Human Mouth (see p.122).—Two species of spirochÆtes were recorded as occurring in the human mouth about forty or fifty years ago. These are SpirochÆta buccalis, Steinberg (often ascribed to Cohn, 1875), and S. dentium, Miller (often attributed to Koch, 1877).

The most recent work on S. dentium and S. buccalis is that of Fantham1267 (July, 1915), who observed the parasites ascribed to Cohn and to Koch, these being the two common spirochÆtes seen in the mouths of natives of the Sudan and of Europeans in England, as well as the forms described and cultivated by recent investigators. Some of the mouth spirochÆtes are not very active, but there is marked corkscrew and boring movement, and they are flexible. Tangles or tomenta of these mouth spirochÆtes are common. Internal structure is seen with some difficulty, but in some specimens it can be determined, and chromatin granules are then seen. MÜhlens (1907) figured stained specimens of S. buccalis and S. dentium, in which chromatin-coloured granules were distributed along the bodies of the organisms.

S. dentium has tapering ends, and varies in length from 4µ to 10µ. S. dentium is rather like Treponema pallidum, and has been placed by some workers—for example, Dobell—in the genus Treponema. It has already been mentioned, on p.128, that Noguchi cultivated three species of Treponema from the human mouth—namely, T. macrodentium, T. microdentium, and T. mucosum, but they cannot be easily distinguished morphologically, and so may appear to be biological varieties of S. dentium.

S. buccalis has somewhat rounded or bluntly acuminate ends and varies in length from 9µ to 22µ. A slight membrane or crest may sometimes be observed. S. buccalis was found to be the predominant spirochÆte in the mouths of eight natives examined by Fantham in the Anglo-Egyptian Sudan.

S. buccalis and S. dentium take up stains well and with relative ease. Intracellular stages of the parasites are uncommon. Multiplication by binary fission has also been observed. Coccoid bodies or granule stages of the mouth spirochÆtes are formed, but appear to be relatively few in number.

J.G. and D. Thomson1268 (1914) have written an interesting paper on various spirochÆtes occurring in the alimentary tract of man and of some of the lower animals. They have also given a useful list of references, and the work of some of the earlier authors is discussed in the paper.

With regard to the general morphology of spirochÆtes, it may be noted that the so-called axial fibre of Zuelzer is acknowledged to be homologous with the membrane or crista of molluscan spirochÆtes.

Coccidia in Cattle.—Regarding the remarks on coccidiosis or “red dysentery” in cattle on p.147, it may be added that Schultz1269 (July, 1915) has found the malady among cattle in the Philippine Islands. He states that some irregular or atypical cases of apparent rinderpest are really due to coccidia. As has been pointed out by Montgomery, rinderpest can be transmitted by blood inoculation, while coccidiosis cannot be so transmitted, but may be diagnosed by the microscope. These differences should be remembered as the two diseases are often found to be associated and are difficult to separate clinically. Coccidia have also been found in Australian cattle.

The HÆmosporidia.—It is likely that this order (see p.151) may be soon abolished. Mesnil1270 (April, 1915) considers that the grouping of the three families, PlasmodiidÆ (or HÆmamoebidÆ), HÆmogregarinidÆ and PiroplasmidÆ in the order HÆmosporidia is no longer possible, because of the coccidian nature of the HÆmogregarines (see p.154). The Coccidia are divisible into the Adeleidea and the Eimeridea (see p.141). The HÆmogregarinidÆ are allied to the former, and the PlasmodiidÆ to the latter. The PiroplasmidÆ, until more is known of their life-cycle in the invertebrate host, cannot be more definitely placed.

The Leucocytozoa of Birds.—Regarding the statement, on p.153, that Laveran and FranÇa consider that avian leucocytozoa may inhabit red blood cells, it may be added that FranÇa1271 (April, 1915) remarks that the action of the parasites on the red cells is very rapid and very intense. The host cells become so altered that it is difficult to recognize their true nature. He used very young birds in his researches. Two shapes of host cell are considered, namely, those with fusiform prolongations, and those which are rounded and without such prolongations (see p.153). The movements and form of the Leucocytozoa determine the shape of the host cell, as was pointed out by Fantham1272 in 1910.

Schizogony of these parasites has been seen by FranÇa (1915) and by Coles (1914), in addition to Fantham (1910), and to Moldovan (1913), mentioned on p.153. Schizogony may also take place in the lungs of the host. The genus LeucocytozoÖn, established by Ziemann in 1898, belongs to the family HÆmamoebidÆ.

II.—FORMULÆ OF SOME CULTURE MEDIA.

(1) Culture Media for growing AmoebÆ.—There has been much discussion as to whether the true parasitic EntamoebÆ or EndamoebÆ can be grown on culture media (see p.42). Undoubtedly certain free-living amoebÆ can be so grown, and it is considered that some of the earlier researches on the so-called artificial growth of the dysenteric amoebÆ were really due to contaminations with free-living forms. The following media are worthy of note:—

Musgrave and Clegg in 1904 devised a culture medium for amoebÆ. The organisms grown by them were probably not dysenteric amoebÆ, as was thought, but free-living forms. Phillips1273 (1915) gives a slightly modified formula of Musgrave and Clegg’s medium, thus:—

Without clarifying, sterilize at 7 kilograms pressure per square centimetre for about three-quarters of an hour. It should be neutral to phenolphthalein.

Anna W. Williams1274 (1911) described a medium consisting of fresh tissue spread on agar plates for the culture of amoebÆ. There are three stages in the procedure: (1) obtaining living amoebÆ free from other living organisms; (2) obtaining sterile tissue; and (3) making successive transplants of amoebÆ and tissue, and showing that every transplant is free from other living organisms. Each step requires many controls. The essentials of the method may now be given. Remove aseptically and rapidly the tissue required, such as brain, liver, kidney, or spleen, from a freshly killed animal (guinea-pig, rabbit, or dog). Put each tissue on a separate agar plate. Cut the selected tissue into tiny pieces, and spread them over freshly made agar plates. Place these plates in a thermostat at 36°C. for twenty-four hours to insure sterility. Add the broken up tissue to the amoebÆ, free from bacteria, and maintain the cultures in thermostats, some at 36°C., and some at 20°C. to 24°C. Emulsions of liver and brain in sterile neutral glycerine may also be used. The freshly removed tissue serves as food for the amoebÆ.

The cultural amoebÆ mentioned on p.42 were grown on such media or modifications thereof. One modified medium actually used was brain tissue, to which blood was added from day to day, and an easily assimilable bacterium (one of the influenza group of bacilli) was present, which did not overgrow the medium at a temperature of 38°C. Different conditions of food and of temperature produced morphological variations in the cultural amoebÆ.

Couret and J. Walker1275 (1913) state that they have cultivated five varieties of intestinal amoebÆ, the associated bacteria having been previously separated. They used a medium consisting of agar to which sterile autolysed tissue had been added. The sterile tissue, such as brain or liver, was kept in a sterile thermostat at a temperature of 40°C. for ten to twenty days. The surface of the agar should be broken up before use, and the medium must not be too acid (not over 1·5 per cent.). They consider that autolysed tissue is necessary for the growth of EntamoebÆ, and that naturally associated bacteria aid growth by autolysing the tissues.

(2) Culture Media for the growth of Protozoa parasitic in the Blood.—MacNeal and Novy,1276 in 1903, used a mixture of blood and agar for the cultivation of trypanosomes such as T. lewisi and T. brucei. They employed varying proportions of the blood and agar, a medium consisting of two parts of defibrinated rabbit’s blood mixed with one part of agar being useful. The trypanosomes grew in the water of condensation. Some of the authors’ earlier formulÆ contained different proportions of blood and agar with a little peptone, while one of these media contained meat extract, agar, peptone, salt and sodium carbonate. The temperature, like the proportion of blood and agar, varied with the trypanosome investigated, but the optimum was 25°C.

Mathis1277 (1906) somewhat simplified the technique of Novy and MacNeal. He collected the blood of a suitable animal, such as rabbit, cow or dog, strict asepsis not being essential. The blood was defibrinated in the ordinary way. One part of blood was added to two parts of agar at 50°C. The mixture was sterilized several times by heating to 75°C. or 100 ° C. Slopes were made and the water of condensation was inoculated with a little blood containing the trypanosomes. Blood may be obtained from a superficial vein or from the heart.

Novy-MacNeal-Nicolle or N.N.N. Medium.—In 1908 C. Nicolle1278 brought forward a modification of the Novy-MacNeal (N.N.) medium. The formula is as follows:—

Apparently pure sodium chloride can be substituted equally well for sea salt. The mixture is placed in tubes and sterilized in an autoclave. To each tube one-third of its volume of rabbit blood, taken by aseptic puncture of the heart, is added. The salt agar is kept liquid at 45°C. to 50°C. and the blood is added to the mixture. The culture medium so prepared is maintained for five days at 37°C., and then for a few days at room temperature. This medium was devised for the cultivation of Leishmania (see p.106), but trypanosomes may also be grown thereon. Subsequently, Nicolle recommended the use of citrated rat’s blood heated to 45°C. for half an hour, instead of defibrinated rabbit’s blood. On such a medium, J.G. Thomson and Sinton1279 (1912) succeeded in growing Trypanosoma gambiense and T. rhodesiense (see pp.76, 83).

Noguchi’s media for the cultivation of SpirochÆtes and Treponemata are described on pp.123, 125. Hata’s modification is discussed on p.126.

Bass’s glucose-blood medium for the cultivation of malarial parasites is described on pp.170–172. It has also been used successfully for the cultivation of Piroplasma or Babesia (see p.172).

III.—BRIEF NOTES ON GENERAL PROTOZOOLOGICAL TECHNIQUE.

The object of this book is to give accounts of the structure and life-histories of the numerous parasitic organisms that affect man more particularly. It is, therefore, inappropriate to devote much space to a consideration of technique, regarding which many volumes have already been written. Methods of procedure are largely matters of opinion, and the technique that gives brilliant results when used by one investigator may be a complete failure in the hands of another. In the present appendix, brief notes regarding certain relatively simple methods only can be given, because the number of fixatives in use is very great; there are also large numbers of stains as well as many modifications of them, while the methods of applying both fixatives and stains are, perhaps, still more numerous. There are so many, in fact, that confusion frequently arises from the multiplicity of choice presented to the worker. Those desiring more information on the subject of technique are advised to consult the treatises of Bolles Lee1280 and of Langeron.1281

Fresh Material.

(a) Simple Examination.

Fluid Substances, such as Blood and Sputum.—A small quantity of the substance to be examined is taken on a sterile platinum loop and transferred to a perfectly clean glass slide. A clean cover-slip is gently lowered on to the drop, air bubbles being avoided. The preparation is luted with vaseline or paraffin and examined first with a low power and then with a high power objective. The light is cut down by partly closing the diaphragm of the substage of the microscope.

Skin Ulcers and Similar Sores.—Scrapings are made from the edge of the sore, mixed with sterile physiological salt solution, and prepared and examined as above.

FÆces.—A small portion of fÆces, or flakes of mucus (which may be blood-stained) from the same, is removed on a sterile platinum loop, spread out thinly after dilution, if necessary, with physiological salt solution on a slide, covered and examined as before.

Alternatively, hanging drop preparations of blood, ulcerative tissue, or fÆces, appropriately diluted if necessary with sodium citrate or physiological salt solution, may be made on a cover-slip, which is inverted over a slide with a well in it. The cover-slip is then luted and examined.

For the elucidation of the developmental processes of such organisms as trypanosomes, spirochÆtes and piroplasms, fresh preparations may be often kept under observation longer by the use of a thermostat, maintained at or near blood heat, in which the microscope is inserted.

(b) Intra vitam Staining of fresh Preparations.

Intra vitam staining is of service on some occasions, more particularly for the study of the nucleus and other chromatoid substances of the living organism. Two methods are in common use. In the first case, the stain, employed usually in very dilute solution, is mixed with the medium containing the organism. The latter takes up some of the stain, the amount of coloration depending on the organism concerned and on the stain employed.

The commoner intra vitam stains are pure, medicinal (zinc-free) methylene blue and neutral red, used in aqueous solutions. A solution of methylene blue of 1 per 1,000 of water may be tried, while neutral red in the proportion of 1 per 3,000 parts of water has proved of service.

The second method of vital colouring consists in placing a drop of 1 per cent. solution of methylene blue on a slide or cover-slip, slightly spreading it, and allowing it to dry. The living organism is then placed in a drop of saline on the prepared slide or cover-slip, which is then mounted and examined under the microscope. Progressive staining of the organism occurs and its internal structure can be seen. A similar procedure may be followed for neutral red. Intra vitam staining is useful for relatively large and easily deformed protozoa such as ciliates, as well as for amoebÆ and flagellata of the gut.

When examining very actively motile organisms, it is sometimes useful to endeavour to restrict their movements by adding a little gum or gelatine to the medium.

(c) Examination by aid of the Paraboloid Condenser.

The use of one of the dark-ground illuminators (so-called ultra-microscopes) is of service for the detection of minute living organisms or of organisms present in small numbers only. The forms of paraboloid condenser manufactured by the firms of Zeiss and Leitz can be recommended. For details of their methods of employment, reference should be made to the leaflets of the firms supplying the said instruments. By the use of the paraboloid condenser, the finer details of certain stages of life-cycles, such as the formation of granules in spirochÆtes and treponemata, can be observed more readily than by using the ordinary substage of the microscope. The use of the paraboloid condenser for the detection of small numbers of living organisms renders it of value for rapid diagnostic purposes.

Stained Material.

Fuller accounts of the technique of fixed and stained material will be found in Bolles Lee and in Langeron, already mentioned.

Thin Films.—For the examination of blood-inhabiting Protozoa, it is necessary to make first thin films or smears of blood. There are many ways of doing this, and opinions differ as to their respective merits. A simple method is to take a straight surgical needle about 2in. long, the eye of which has been removed, and a clean glass slide. The patient’s skin is pricked, and when the bead of blood reaches the size of a small pin’s head, the slide is applied to the surface of the blood, about 1/3in. from the far (left-hand) end of the slide. The shaft of the needle is laid across the drop of blood, which spreads between the slide and the needle. The latter is drawn evenly along the slide towards the right. The film is dried by waving it in the air. The film should possess a straight edge parallel with that of the slide and should be as uniform and thin as possible. Another glass slide may be used as a spreader, or a cover-slip or thin glass rod may be employed.

Thick Films.—These are of service in detecting malarial parasites or trypanosomes, especially when the parasites are few. The method of Ross, or a modification thereof, has been much used. A small drop of fresh blood is spread evenly and quickly with a needle-point over a square area somewhat less than that of an ordinary square cover-glass. The blood is allowed to dry. The film is then carefully dehÆmoglobinized in water in which there is a trace of acetic acid. The dehÆmoglobinizing fluid is then carefully drained off and the film again dried. It is fixed in absolute alcohol and stained with Romanowsky’s solution. A cubic millimetre of blood divided into quarters may be thus dehÆmoglobinized and stained. The parasites in such a cubic millimetre of blood may be counted. Such a procedure was followed by R. Ross and D. Thomson,1282 in determining the periodic variation of the numbers of trypanosomes in the blood of a patient, as mentioned and figured on pp.78 and 79.

For cytological details of various Protozoa, thin film preparations on cover-slips or slides are often useful. Cover-slip preparations are preferable, unless the organisms under investigation are extremely scanty. The medium containing the organisms, such as blood, lymph, intestinal contents, sputum, scrapings of ulcers, and urine, is spread thinly, either alone or diluted with a little physiological salt solution, on the cover-slip. Fixation while still wet is necessary. Various methods are employed.

Fixatives.—A useful procedure is to fix the wet film by exposure to 4 per cent. osmic acid vapour for ten to thirty seconds, then place in absolute alcohol for five minutes to harden. Grade down from absolute alcohol through 90 per cent., 70 per cent., 50 per cent., and 30 per cent. alcohols to water. Stain wet with a suitable stain such as hÆmatoxylin, and gradually dehydrate by grading through the necessary strengths of alcohol, clear in xylol or other oily clearing medium and mount in Canada balsam.

Other fixatives may be employed, such as are also useful for fixing pieces of tissue for sectioning. Films or smears on cover-slips while still wet are floated on the surface of the fixative in a watch glass. Some good fixatives of wide application are:—

Schaudinn’s Fluid.—This consists of a mixture of

Two modifications of Schaudinn’s formula may be found useful. A saturated solution of corrosive sublimate in physiological salt solution may be substituted for the aqueous one, and the addition of a few drops of glacial acetic acid to either of the preceding mixtures may be made.

Some workers prefer to use hot fixatives, raised to a temperature of about 50°C.

Fixation by corrosive sublimate solutions must be followed by thorough removal of the mercury salt by washing repeatedly in 30 per cent. alcohol or with iodine-alcohol.

Bouin’s Fluid, or modifications thereof, is also very useful for wet fixation. Bouin’s picro-formol solution consists of:—

The best-known modification is one due to Duboscq and Brasil, and often known as Bouin-Duboscq Fluid. Its formula is as follows:—

Thorough washing of the smear or cover-slip preparation with 70 per cent. alcohol until the yellow colour disappears is necessary to remove excess of fixative.

Other fixatives, which may be of use, more especially for fixing small pieces of tissue for sectioning, are the solutions of Flemming (chromo-aceto-osmic acids) and of Zenker (sublimate-bichromate-acetic, with sodium sulphate).

Regarding the time of fixation, there is much difference of opinion. Usually, exposure to or contact with the fixative for five minutes is sufficient in the case of films or smears. Material for sections should be cut into small cubic pieces, of a thickness of about 5mm. ( 1/5in.). One or two hours should be sufficient time for the fixation of such pieces of tissue, though some, as Langeron, prefer a longer time of fixation. On the other hand, Gustav Mann1283 recommends a short fixation period. The excess of fixative should be thoroughly washed out of the tissue in the manner appropriate to the particular fixative used. If it is desired to keep the tissue for some time before sectioning and staining, it should be transferred to 70 per cent. alcohol.

When fluid fixatives are employed, large quantities of the fixing media are necessary. The volume of the fixative should be at least ten to twenty times that of the object, and the latter should be suspended in the middle of the fixative. The tissue should be fixed as soon as possible after the death of the host.

For sectioning tissue parasitized by Protozoa, embedding in paraffin is generally recommended. Microtome sections should not, if possible, exceed 5µ in thickness. Details of special procedures must be sought in larger works.

Staining.—Here, as with fixatives, much choice is presented. The various modifications of the Romanowsky stain have aided greatly in the detection of various Protozoa parasitic in the blood. Such stains, however, leave something to be desired in the revealing of finer cytological details. Other stains, more especially the hÆmatoxylins, must be employed for cytological purposes.

FormulÆ of some of the principal Romanowsky and hÆmatoxylin stains may now be given.

The underlying principle of the Romanowsky Stain is the reaction between alkaline methylene blue and eosin, forming the so-called eosinate of methylene blue which stains chromatin purplish-red. A solution of medicinal methylene blue after having been subjected to the action of an alkali, such as sodium carbonate, becomes partly converted into certain derivatives, the chief of which are methylene azure and methylene violet. These substances are also present in matured polychrome methylene blue.

The formula of a slightly modified Romanowsky Stain which gives excellent results is given below:—

Two stock solutions are required—

Keep in a warm incubator for two or three days, until the solution is distinctly purple in colour. It improves with age.

This solution must be kept in the dark, in dark-tinted (amber-coloured) bottles, as unfortunately it is decolorized by light.

Before use each stock solution must be diluted. Thus, make up 5c.c. of each stock solution to 100c.c. by adding distilled water. For staining, 1 volume of solution A is added to 2 or 3 volumes of solution B. Mix thoroughly by shaking, pour the mixture over the film, previously fixed in absolute alcohol, and stain for ten to fifteen minutes. Wash carefully in running water, then dry. The cytoplasm of a protozoan parasite will be stained blue, the chromatin purplish-red and vacuoles or very tenuous protoplasm will remain colourless.

The exact proportions of solutions A and B, which must be mixed together, should be determined by experiment. Freshly mixed stain must be used on each occasion.

Leishman’s Stain is the precipitate resulting from the interaction of alkaline methylene blue and eosin. The washed and dried precipitate is collected and dissolved in pure methyl alcohol, which acts as a fixative; 0·015grm. of Leishman powder may be dissolved in 10c.c. of methyl alcohol for staining films. The film is covered with the solution for one minute, twice the volume of water is then added and mixed with the stain on the slide. The staining is then continued for five to ten minutes, and the film is finally washed with water.

Giemsa’s Stain.—This should be procured ready made. Azure II is a mixture of methylene azure and methylene blue. (Methylene azure is sometimes known as Giemsa’s Azure I.) The formula given by Giemsa himself in 1912 is:—

The film is first fixed in absolute alcohol. The proportion of stain usually used is one drop of stain to 1c.c. of water. Stain for about ten minutes and then wash in water.

The details of the application of the Giemsa stain to films fixed wet and to sections must be sought in larger works on technique. These works should also be consulted for information regarding the use of Pappenheim’s Panchrome mixture.

There are numerous formulae of stains containing ripened HÆmatoxylin or its essential principle, HÆmatein. A mordant is necessary, one of the alums being usually employed. The mordant may be included as an ingredient in the staining mixture, or it may be used separately as in the case of the so-called iron-hÆmatoxylins, wherein ferric ammonium alum is used separately and is followed by staining with hÆmatoxylin or hÆmatein. A few of these stains of general application may now be mentioned.

Delafield’s (or Grenachier’s) HÆmatoxylin.

Mix these ingredients, and leave exposed to light and air for three to four days. Filter and add—

Allow the mixture to stand until the colour is sufficiently deep, then filter and place in a stoppered bottle. The solution should be allowed to ripen for at least two months before use. Dilute aqueous solutions of the stain are of service for films and for sections. A trace of acetic acid may be added at the moment of use, for sharp differentiation.

Ehrlich’s acid hÆmatoxylin, Mayer’s hÆmalum, and Mayer’s glychÆmalum are also useful. Their formulÆ will be found in larger works.

The chief Iron-HÆmatoxylin Stain is that devised by Heidenhain. Unfortunately the procedure involved is a long one, and various modifications have been made to obviate this disadvantage. HÆmatein may be used instead of ripened hÆmatoxylin.

One efficacious modification of Heidenhain’s stain is that of Rosenbusch. The smear or tissue, after fixation, must be graded downwards through the alcohols to water. Mordant for one and a half hours in a 3 1/2 per cent. aqueous solution of ferric ammonium sulphate. Stain for about three minutes in 1 per cent. solution of ripe hÆmatoxylin or hÆmatein in absolute or 96 per cent. alcohol, to which a drop of saturated aqueous solution of lithium carbonate, sufficient to produce a wine-red colour, has been added. Differentiate under the microscope with a very dilute solution of the ferric ammonium sulphate. Wash, gradually dehydrate, clear and mount in balsam. It must be remarked that iron-hÆmatoxylin is a regressive stain, hence great care must be exercised in differentiating with the iron alum.

Gentian Violet.—A 1 per cent. alcoholic solution of gentian violet, or of methyl violet, or of crystal violet, will be found useful for staining spirochÆtes.

Methyl Green.—This substance is considered to be a chromatin stain, for either fresh or perhaps recently fixed tissues. A concentrated aqueous solution contains about 1 per cent. of the stain. This should be added to a 1 per cent. solution of acetic acid. It may be used for demonstrating the nuclei of ciliates.

In conclusion it is essential to remember that the actual magnification of figures of Protozoa should be given, and not merely the combination of objective and ocular that has been used, for unless the tube-length and distance of the drawing board from the ocular be also given, it is not possible to compute the magnification from such information. Drawings should always be made with the aid of a camera lucida, drawing prism or other form of projection apparatus.