556 TRYPANOSOMES AND TRYPANOSOMIASES 



concentrate them by preliminary centrifuging. Frequently, however, 

 it is only necessary to obtain a drop of blood, for instance, from 

 the ear of a larger animal or from the tip of the tail of a rat, or mouse, 

 allow it to fall on a clean slide and cover it with a cover-glass. This 

 simple preparation should be examined at once with the microscope. 

 The first search can be made with a low-power lens, 16 mm. or two- 

 thirds inch focus. With this magnification a peculiar agitation among 

 the red blood corpuscles can frequently be seen in a part of the field. 

 If this spot is placed in the centre the very characteristic micro- 

 organisms can generally be seen either with a high-power dry or 

 with an oil-immersion lens. In fresh preparations trypanosomes easily 

 betray their presence even to the tyro by their shooting, darting, or 

 spiral motions in the blood, and, in some trypanosome infections, as, for 

 instance, in dourine in horses, in the juice expressed from the patchy 

 infiltrations of the skin or the inguinal glands, and in sleeping sickness in 

 man in the centrifuged cerebrospinal fluid. If the morphology of the 

 pathogenic trypanosomes is to be studied in detail, dry stained prepar- 

 ations are necessary. The most useful stains are generally a com- 

 bination of eosin and methylene blue as found in the stains of Roman- 

 owski, Leishman, and Wright. The last named, in particular, is 

 very satisfactory, because its use is very simple. It is only necessary 

 to make a blood smear on a cover-glass or slide; the smear is allowed 

 to become air dry, and Wright's stain is poured on at once. The 

 stain, dissolved in methyl alcohol also fixes the preparation. The 

 undiluted stain is left on for one minute, then enough distilled water 

 is added to the fluid to cause it to show a dark precipitate, while at 

 the same time the pink of the eosin shows. This dilute stain is left 

 on for two minutes, then the preparation is washed in distilled water 

 for thirty to sixty seconds, finally it is dried with filter paper and 

 examined with an oil-immersion lens. 



Artificial Culture Media. The artificial cultivation of trypanosomes 

 was first successfully carried out by Novy and McNeal, whose culture 

 medium consists of ordinary agar distributed to tubes and kept 

 melted at 50 C. Twice the volume of aseptically collected defi- 

 brinated rabbit's blood is then added to each tube. When the agar- 

 defibrinated-blood mixture has solidified in a slanting position the 

 condensed water is inoculated with the blood of an infected animal 

 or from a previous culture. This comparatively simple culture 

 medium has enabled Novy and his assistants to obtain much infor- 

 mation about the morphology and multiplication of the trypanosomes. 



The rat trypanosome, Trypanosoma Lewisii, is very easy to cultivate; 

 the pathogenic trypanosomes are more difficult. Novy, McNeal, and 

 Hare, however, also succeeded in obtaining Trypanosoma Brucei and 

 Trypanosoma Evansii in artificial culture, while Laveran and Mesnil 

 cultivated Trypanosoma Brucei, Trypanosoma dimorphon, and 

 Trypanosoma gambiense. 



Novy and Knapp, while working with the flagellates found in the 



