modification of the Romanowsky stain (see Plate I., fig. 1). This stain, which may be obtained ready prepared from Griibler, of Leipzig, under the name of “Giemsa’sche Lösung für die Romanowsky Färbung,” is made as follows: Azur II.-eosin compound, 3 grms. and Azur II. 0·8 grm. are mixed and dried thoroughly in the desiccator over sulphuric acid; this mixture is then very finely pulverized, passed through a fine-meshed silk sieve and dissolved at 60° C. in Merck’s glycerin, 250 grms., the mixture being well shaken; 250 grms. of methyl-alcohol (Kahlbaum I.), which has been previously heated to 60° C, is then added. The whole, after being well shaken, is allowed to stand for twenty-four hours and filtered. The solution, now ready for use, should be kept in a yellow glass bottle. To 1 c.c. of ammonia-free distilled water add 1 drop of this stain. Stain for from a quarter to three-quarters of an hour. Wash in running water, blot, dry, and mount in Canada balsam. Longer exposure to the action of a more dilute Giemsa fluid often gives excellent results.
The stained organisms may be seen as delicate, reddish, regular spirals with pointed extremities. They usually measure from 4 to 14μ in length, though they may reach 18 or 22μ; the breadth is about 0·25μ. In a section of the liver from a case of congenital syphilis an enormous number of these spirochaetes may be found.
Stain by Levaditi’s method as follows: Fix fragments of tissue not more than 1 mm. thick in 10% formol solution for twenty-four hours. Rinse in distilled water and harden in 96% alcohol for twenty-four hours. Then wash in distilled water for some minutes, i.e. until the pieces fall to the bottom of the vessel, and transfer to a 1·5–3% solution of nitrate of silver (3% is preferable when the tissues have been obtained from the living patient). This impregnation should be carried on at a temperature of 38° C. for from three to five days, according to the nature of the tissue. "Reduce" the silver in the following solution: Pyrogallic acid, 2–4%, Formol, 5 c.c, Aq. dest., 100 c.c. Allow this solution to act on the tissues for from twenty-four to forty-eight hours at room temperature. Again wash in distilled water, dehydrate with alcohol, clear with xylol and cedar-oil, and embed in paraffin. The sections should not be more than 5μ thick. In a section so stained the spirochaetes are seen as dark spirals standing out against a pale yellow background. On staining with a weak counterstain many of the spirals may be seen actually within the liver cells.
This organism may be found in the lung, spleen and other visceral organs, and even in the heart of a patient suffering from syphilis. It has also been found in syphilitic lesions produced experimentally in the higher apes, especially the chimpanzee. As a result of these observations it is now generally accepted as being the primary cause of syphilitic lesions in the human subject. It is certainly present in the lesions usually met with in cases of primary and secondary syphilis of the human subject, and by its action on the blood and tissues of the body produces an antigen, a specific (?) substance, the presence of which has been utilized by Wassermann in the diagnosis of syphilis. He uses the method of deviation of complement by the antigen substances contained in the syphilitic fluid blood or cerebro spinal fluid—by which the lytic action of a haemolysing fluid is prevented.
Kála-ázar.—The non-malarial remittent fever, met with in China, known as dum-dum fever in India and as kála-ázar in Assam, is associated with peculiar parasitic bodies described by Donovan and Leishman (Herpetomonas Donovani) (? Helcosoma tropicum, Wright). This fever is characterized by its great chronicity, associated with very profound, and ultimately fatal, bloodlessness, in which there is not only a fall in the number of red blood corpuscles, but a marked diminution in the number of white blood corpuscles. Ulceration of the skin and mucous membrane, especially of the lower parts of the small intestine and of the first part of the colon is often present, this being accompanied by dropsy and by distinct enlargement of the liver and spleen. Leonard Rogers, who has given an excellent account of this condition, points out that there is a marked increase in the number of cells in the bone-marrow.
The Leishman-Donovan bodies have been found in large numbers, especially in the spleen (see Plate I., fig. 7); they may also be found in the ulcerating surfaces and wherever the cellular proliferation is marked. These organisms may be found in sections, or they may be demonstrated in film preparations made from the material scraped from the freshly-cut surface of the spleen.
The films are best stained by Leishman’s method: Solution A.—Medicinal methylene-blue (Grübler), 1 part; distilled water, 100 parts; sodium carbonate, 1·5 parts. This mixture is heated to 65° C. for twelve hours and then allowed to stand at room temperature for ten days. Solution B.—Eosin extra B.A. (Grübler), 1 part; distilled water, 1000 parts. Mix equal parts of solutions A and B in a large open vessel and allow to stand for from six to twelve hours, stirring from time to time with a glass rod. Filter, and wash the precipitate which remains on the paper with a large volume oi: distilled water until the washings are colourless or only tinged a pale blue. Collect the insoluble residue, dry and pulverize.
Make a 0·15% solution of the powder (which may also be obtained from Grübler & Co., Leipzig) in absolute methyl alcohol (Merck’s “for analysis”), and transfer to a clean, dry, well stoppered bottle. Pour three or four drops of this stain on to the prepared film (blood, bone, marrow, &c.) and run from side to side. After about half a minute add six or eight drops of distilled water, and mix thoroughly by moving the slide or cover-glass. Allow the stain to act for five minutes longer or, if the film be thick, for ten. Wash with distilled water, leaving a drop or two on the glass for about a minute. Examine at once or after drying without heat and mounting in xylol balsam.
These peculiar parasitic bodies appear as deeply stained points, rounded, oval or cockle-shaped, lying free or grouped in the large endothelial cells of the spleen. Examined under a magnification of 1000 diameters they are found to measure from 3·5 to, 2·5μ, or even less, in diameter. Their protoplasm is stained, somewhat unequally, light blue; and from this light blue background two very deeply stained violet corpuscles of unequal size stand out prominently; the smaller of these is more deeply stained than the larger, is thinner, somewhat more elongated or rod-shaped, and parallel or running at right angles to the large corpuscle or obliquely from it. The larger corpuscle is rounded or oval, conical, or sometimes almost dumb-bell shaped. These bodies may appear to touch one another, though usually they are disconnected. Most of these Donovan-Leishman bodies are embedded in the protoplasm of the large endothelial or mononuclear splenic cells, of similar cells in the bone marrow, or of certain lymphatic glands. They may also be seen lying in the protoplasm of the endothelial cells lining the capillary vessels and lymphatics. They are considered by Leishman and Leonard Rogers to be organisms in an intermediate stage of development of either a Trypanosome or some form of Herpetomonas. Rogers, who succeeded in cultivating them outside the body, described changes which he considers are associated with this latter germ. Patton goes further than this, and states that the Leishmania donovani Lav. et Mesn. taken up by the bed bug closely resembles in its life cycle that of the Herpetomonas of the common housefly. It is thought that the Leishman-Donovan bodies are the tissue parasite stage, and that the herpetomonas stage is probably to be sought for in the blood of the patient.
Tsetse-Fly Disease (Trypanosomiasis).—The interesting observations carried out by Sir David Bruce have invested the tsetse fly with an entirely new significance and importance. In 1895 Bruce first observed that in the tsetse disease—n’gana—there may be found a flagellated haematozoon closely resembling the Trypanosoma Evansii found in Surra. This, like the Surra organism, is very similar in appearance to, but considerably smaller than, the haematozoon often found in the blood of the healthy rat. It has, however, as a rule a single flagellum only. A small quantity of blood, taken from an affected buffalo, wildebeest, koodoo, bushbuck or hyaena—in all of which animals it was found by Bruce—when inoculated into a horse, mule, donkey, cow, dog, cat, rabbit, guinea-pig, rat or mouse, produces a similar disease, the organisms being found sometimes in enormous numbers in the blood of the inoculated animal, especially in the dog and in the rat. He then found that the tsetse-fly can produce, the disease in a healthy animal only when it has first charged itself with blood from a diseased animal, and he produced evidence that Glossina morsitans is not capable of producing the disease except by carrying the parasites from one animal to another in the blood that it takes through its proboscis into its stomach. The parasites taken in along with such blood may remain in the stomach and alive for a period of 118 hours, but shortly after that the stomach is found to be empty, and the parasites contained in the excrement no longer retain their vitality. The mode of multiplication of these organisms has been studied by Rose-Bradford and Plimmer, who maintain that the multiplication takes place principally in the spleen and lymphatic glands. The tsetse-fly parasite, however, is still imperfectly understood, though much attention is now being paid to its life-history and development.
