Many of our rats died.

Plain evidences of sickness coupled with heavy blood infection were almost sure to give a fatal result. We made no pathological study of the internal organs.

STAINING.

In the beginning we attempted to stain the parasites with the ordinary aniline dyes. We used eosin, methylene blue, hemalum, carbofuchsin, and carbo-thionin, but all were very disappointing. We then turned to the Romanowsky double stain of methylene blue and and eosin, Goldhorn's polychrome methylene blue and Jenner's stain, all of which give most beautiful effects indeed; perhaps the best is Romanowsky's stain. Some modification of this stain is the one which many of the investigators have employed within the last few years for trypanosomes. In the hands of the various experimenters this stain seems to give somewhat different effects. The two points on which they all agree are that the nucleus takes a beautiful rose red and that the protoplasm takes a blue color. Rabinowitsch and Kempner state, and it is clearly shown in their figures, that in the hind part of the adult parasite there is an oval or round spot of a homogeneous structure which takes the stain uniformly and intensely red. In the front end of the trypanosome is a larger body with a net-like structure which stains rose red. In their hands the protoplasm of the body and the free outer border of the undulating membrane and the flagellum stained quite uniformly blue. The web of the membrane remained unstained.

Wasielewski and Senn state that the protoplasm is almost homogeneous, stains a light blue, and has a fine granular structure. In it are one to three clear, oval areas, generally in front of the centrosome, which can be considered as vacuoles. With the Romanowsky stain they succeeded in giving a red tint, not only to the centrosome and nucleus, but to the free border of the undulating membrane, to the web of the undulating membrane, to the flagellum, and to the entire border of the body of the parasite. They state, however, that the tint of the centrosome, flagellum, undulating membrane, and "periplast" was a bluish red.

Laveran and Mesnil picture the centrosome and body of the parasite as blue, and the nucleus, flagellum, and undulating membrane as red. With Romanowsky's stain we gave a blue color to the body of the trypanosome. The nucleus took a rose red, and the centrosome was deep red. The flagellum and border of the undulating membrane were red, and the web of the undulating membrane was faintly red. Wright's modification of Romanowsky's stain.-Wright (7), in speaking of Romanowsky's stain, which came out in 1891 as a differential stain for the chromatin and cytoplasm of the malarial parasite, refers to the difficulties and uncertainties attending the preparation of the stain until it became finally modified by Leishman (8), whose

method Wright has further simplified. Wright, in his directions for preparing the stain, says to add 1 per cent methylene blue to a onehalf per cent solution of sodium bicarbonate and steam the mixture in an Arnold steam sterilizer for one hour, which renders the methylene blue polychromatic. When cold he adds eosin until the color changes from blue to purple and a metallic scum forms on the surface and a black precipitate appears in suspension. The precipitate is collected on a filter, dried, and dissolved in methyl alcohol. Wright gives the following summary for using the stain:

1. Make films of blood, spread thinly, and allow them to dry in the air. 2. Cover the preparation with the alcoholic solution of the dye for one minute. 3. Add to the alcoholic solution of the dye on the preparation sufficient water, drop by drop, until the mixture becomes semitranslucent and a yellowish metallic scum forms on the surface. Allow this mixture to remain on the preparation for two or three minutes.

4. Wash in water, preferably in distilled water, until the film has a yellowish or pinkish tint in its thinner or better spread portions.

5. Dry between filter paper and mount in balsam.

Goldhorn's stain (9).-Dry the film and fix in pure methyl alcohol fifteen seconds, wash in running water, stain in 0.1 per cent aqueous solution of eosin for thirty seconds, wash in running water, stain one minute in Goldhorn's polychrome methylene blue, wash in water, dry in air, mount in balsam.

The polychrome methylene blue is made as follows:

1. Dissolve 2 grams of methylene blue and 4 grams of lithium carbonate in 300 c. c. of warm water.

2. Heat in porcelain dish in a boiling water bath fifteen minutes. 3. Pour into a glass-stoppered bottle and set aside for several days. 4. Render only slightly alkaline by adding 4 to 5 per cent acetic acid solution.

With this stain we have obtained beautiful preparations showing the chromatin of the ring form of æstivo-autumnal malaria and the chromatin of the tertian parasite. We have also well-stained preparations of blood platelets. It is a good stain for the nuclei of animal parasites. It shows the chromatin of the segmenting bodies of malaria, the chromatin of the crescents, the eosinphilic and neutrophilic granules and nuclei of leucocytes. Mast cells and myelocytes are well stained.

Jenner's stain.-This stain is not so good for trypanosomes as the other two. Equal parts of 1 per cent aqueous solutions of eosin and methylene blue are mixed and set aside for twenty-four hours. The mixture is filtered, the precipitate is washed with water and dried and then dissolved in methyl alcohol. In using this stain no previous fixing is necessary. After staining one to three minutes the specimen is thoroughly washed until the corpuscles appear pink. Dry in the air and mount in balsam.

The stain can be bought ready for use from dealers, or a powder

can be gotten from Grübler, which is to be dissolved in methyl alcohol.

We have gone at some length into these stains, for without some one of them the investigator will attain little in the way of getting instructive preparations of trypanosomes and because their more general use may bring out some points still unmentioned in the structure of animal parasites generally. Since a thorough understanding of the stains is necessary to fine work, the reader is referred to the original articles mentioned in the bibliography.

ACTIVE IMMUNITY.

With very few exceptions a single infection with trypanosomes renders the rats free from parasites thereafter. Rabinowitsch and Kempner had no second infections following the injection of heavily infected trypanosome blood into the peritoneal cavity of rats, which, after artificial inoculation, had become spontaneously free of parasites. Laveran and Mesnil, in their series of thirty, found two susceptible to a second infection. One of our rats infected by feeding and another infected by intrastomachal injection proved susceptible to a second infection by intraperitoneal inoculation in two and five months, respectively, after they had become free of parasites. Their second infections lasted only three days.

PASSIVE IMMUNITY.

From our knowledge of the antitoxins of the bacterial diseases, we are led naturally to the investigation of the protective property of the serum of immunized rats, and it is found that there is produced a specific immune serum. The serum of rats which have been immunized by one or more inoculations of trypanosome blood does give protection to other rats within certain limits. Laveran and Mesnil found that their most active serum came from a rat which had been given 13 inoculations.

If we add in vitro 1 c. c. of immune serum to 1 c. c. of trypanosome blood and inject the mixture into a fresh rat, no infection will follow. We also separated the two injections in time to see whether immune serum would prevent infection if injected before the trypanosome blood; likewise, whether the immune serum would prevent infection if it were injected after the injection of infected blood. Our results were somewhat variable, but in general they corresponded to the limits of time which have been set by Rabinowitsch and Kempner and confirmed by Laveran and Mesnil, namely, that 1 c. c. of immune serum injected into a fresh rat twenty-four hours before or twenty-four hours after the injection of trypanosomes will prevent infection. They found that emulsions of the spleen, bone marrow, liver, or brain conferred no passive immunity.

The limits of the preventive and curative power of the immune

serum, although quite narrow, are, however, as wide as we would expect.

Five pregnant rats were inoculated, with a view to finding whether, their young would acquire an immunity by placental transmission. Although the mothers bore a very heavy infection, we could never, demonstrate any parasites in the fetuses nor did the young show any evidences of an increased resistance to subsequent infection. Laveran and Mesnil mention one immune rat which had two litters; the first litter was immune, the second susceptible. In this connection we may mention that no case of placental transmission of malaria has been reported in which the possibility of postnatal infection has been excluded.

SUSCEPTIBILITY OF ANIMALS TO TRYPANOSOMA LEWISI.

The wild rat and the sewer rat are the only animals in which there is a spontaneous conveyance to each other. The white and spotted rats are susceptible by inoculation only. Young rats are more susceptible than old ones. Rabinowitsch and Kempner report the failure to infect pregnant rats. We inoculated five pregnant females, and all bore heavy infections. No one has found that the white rats harbored trypanosomes spontaneously in their blood. No investigators have succeeded in infecting other animals with rat trypanosomes except Laveran and Mesnil, who infected guinea pigs. We tried in vain to infect guinea pigs, rabbits, white mice, cats, a dog, a goat, and a horse by intraperitoneal inoculation. In one guinea pig we found six parasites in the blood twenty-four hours after inoculation, but subsequent examinations of the blood showed an absence of all parasites, so that this can not be considered a case of infection. Laveran and Mesnil, after intraperitoneal injection of a guinea pig with 1 c. c. of blood rich in trypanosomes, found multiplication forms in the peritoneum two to five days after injection. They had numerous failures in bringing about a blood infection, but some guinea pigs showed parasites in the blood in the proportions of 1:20 and 1:50 of the red blood corpuscles. The infection was of short duration.

When attempting to infect the various animals other than rats we injected large amounts of heavily infected rat blood. In some cases we injected a rat's entire blood. White rats and wild rats are so susceptible that only one to three drops of infected blood, mixed with a little sterile salt solution or bouillon and injected intraperitoneally, will cause a marked infection.

AUTO-AGGLUTINATION.

We have brought forward the use of this term to signify the agglutination of a rat's own trypanosomes while still circulating in his own blood. If daily examinations are made of the blood of an infected rat,

he parasites will be seen to show agglutination during the period of their lecline in numbers and disappearance from the blood. When autogglutination is well advanced we see very few parasites occurring ingly. They are collected into masses; but these masses in turn how a tendency to collect close together, which must necessarily leave ertain drops of blood almost free from all parasites while other drops vill show typical fields of auto-agglutination. On this account a single hanging drop or a single stained preparation taken from a rat s not sufficient to give a correct idea of the condition of the parasites In the blood. As many as eight to ten slides may be made in which will be found only a few scattered parasites, and the next slide will show pictures such as are seen in Plate II, figure 8, and Plate IV, figures 13 and 14. Figure 13 represents a single focus of agglutination, and in figure 14 there are three such foci near to each other. A close examination of these agglutinations will show that they have nothing to do with the rosettes of multiplication. They are found in the blood after the period of multiplication has passed, and they are made up of adult parasites instead of young forms. We consider these agglutinations as an evidence of the presence of agglutinin in the rat's blood and as an omen of an impending rapid disappearance of the parasites from the blood. We have in the laboratory in several instances seen the agglutination of the parasites in a rat's blood for a few successive days before a sudden disappearance of all parasites over night. We conclude from this observation that agglutination is a step toward dissolution, and that it foretells the disappearance of the trypanosomes from the blood.

From a daily study of the blood of numerous cases in which agglutinations of parasites were present we were able to prophesy that active immunity was near at hand.

It is not every case of infection which shows auto-agglutination. We would explain its absence on the ground of an insufficient generation of agglutinin. As will be referred to later, there is a difference in the agglutinating power of the immune sera of different rats. As soon as a rat loses his parasites he is considered an immune and his serum is admitted to have agglutinating power on fresh parasites to which it may be added. The agglutinating power of the immune serum is, moreover, attributed to the agglutinin which it contains. Now, we think that the production of this agglutinin is a gradual process which is begun before a rat loses his parasites, and we think that just previous to the disappearance of all parasites from a rat's blood there is a short period during which he may generate sufficient agglutinin to agglutinate the parasites circulating in his own blood. We would offer as a possible explanation of our own cases that perhaps our trypanosomes were more virulent than those used by other observers. This view seems to be supported by the shorter time which elapsed between inoculation and heavy blood infection, the