Behrens, C.A.

Travelbee, H.C.

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297The Erdmann New Culture Medium for Protozoa.C. A. Behrens and H. C. Travelbee—Purdue University.It is a well-known fact that the first culture in vitro of a pathogenictrypanosome (Tryponosoma Brucei) was obtained by Novy and Mac-NeaP in 1903. The medium used was a meat extract agar plus twoparts of defibrinated rabbit's blood. Of fifty animals tested only 4, or8% positive cultures resulted. In 1905 Smedley', using a similar medium,found that three out of ten attempts, or 30Vc, were successful.Because of the inconsistent results we deemed it advisable to at-tempt an improvement of the medium. The first attempts along theselines were in 1909''. The media with their per cent, of positive growthsare as follows:1 Novy MacNeal blood agar 25%lA Novy MacNeal blood 0%2 Bean and pea extract blood agar 53%2A Bean and pea extract blood 0%3 Nicolle blood agar 48%3A Nicolle blood 0%4 Dialyzed meat extract blood agar 80%4A Dialyzed meat extract blood 0%5 Dialyzed meat extract dilute serum agar 100%)5A Dialyzed meat extract dilute serum 0%6 Dialyzed meat extract inactivated serum agar 100%6A Dialyzed meat extract inactivated serum 0%7 Dialyzed meat extract dilute red blood cells agar 38%-7A Dialyzed meat extract dilute red blood cells 0%8 Dialyzed meat extract Ascitic fluid agar 0%8A Dialyzed meat extract Ascitic fluid 0%9 Veal extract blood minus white blood cells agar 100%9A Veal extract blood minus white blood cells 0%iJour. Amer. Med. Assn., 1903, 41, p. 1266: Jour. Infect. Dis., 1904, 1, p. 1.= Jour. Hyg., 1905, 5, p. 38.3 Jour. Infec. Dis., 1914, 15, 1, p. 4.298The above table indicates that successful cultures ranging h-om25 to 100 per cent, are obtained when the solid type of medium is em-ployed and that in every case where the liquid medium is used negativeresults occurred. In the successful cultures growth always resulted inthe water of condensation after a period of incubation from one to fourweeks at a temperature ranging from 25° to 28° C.We therefore naturally were very much interested when in 1914Rh. Erdmann^ announced a new liquid culture medium for TrypanosomaBrucei. Erdmann states that by using the plasma of the host as themedium she grew Trypanosoma Brucei in hanging-drop cultures andkept them in normal condition for an indefinite period. The techniqueemployed in brief was as follows : The plasma was obtained by themethod of Harrison'', Burrows', and Walton'. "The blood from the in-fected rat was taken and put into a small drop of plasma on a cover-glass and then this was further diluted with plasma in order to reducethe number of blood corpuscles in the hanging-drop which was takenfrom this." The cover glass with hanging-drop was either placed on adepression or regular slide and sealed. Precautions to secure asepticconditions were taken.We attempted to follow the technique thus outlined as nearly aspossible. These cultures showed no signs of bacterial contamination atthe end of forty-five days. In only a few instances were actively motilesurvivals in evidence for more than five days when kept at 10°C. Inpreparations incubated at 20 °C, or above no survivals were observedafter forty-eight hours.In the course of an extensive series of attempts using heterologousand homologous sera under various conditions we found it impossible atany time to obtain a second generation by the Erdmann method. Thehomologous sera used were rat and guinea pig. The heterologous serawere human, horse, beef, sheep, pig, rabbit and chicken. These serawere used in a dilute one to one, inactivated, and normal form and thepreparations were incubated at temperatures of 10, 15, 20, 25, 28, 30,35, Pj1V2, and 40 °C. Ascitic fluid was also used without success.It is true that trypanosomes will multiply and remain actively> Soc. Exp. Biol, and Med., 1914, XII, p. .57.'' Proc. Soc. Exp. Biol, and Med., 1907, IV, p. 10: Jour. Exp. Zool., 1010, IX, p. 787."Jour. Amer. Med. Assn., 1910, LV ; Jour. Exp. Zool., 1911, X, p. 63.^ Proc. R. S. L., Ser. B., 87, p. 452.299motile when first placed in a medium such as described by Erdmann.We have especially noticed this in connection with our work with solidmedia. Good survival forms of other pathogenic trypanosomes as thosecausing human sleeping sickness, dourine, and mal de caderas wereobserved as late as the twenty-eighth day, but in no case did theseforms result in positive growth or second generation when transplantedto similar medium under similar conditions.In summing up our work we can positively say that at no time,under no conditions were we able to obtain a positive culture using theErdmann cultural medium. As a matter of fact the easily cultivatedtrypanosome of Lewis would not develop successfully on this medium.

The Erdmann New Culture Medium for Protozoa

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The Erdmann New Culture Medium for Protozoa

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