Etiology of Yellow Fever. Vi. Cultivation, Morphology, Virulence, and Biological Properties of Leptospira Icteroides. by Hideyo Noguchi, M.d

As the nature of the causative agent of yellow fever was unknown, it was necessary at the beginning of these experiments to formulate a spedal method of cultivation. The methods employed have been similar to those recommended 1 for the cultivation of Leptospira icterohcemorrhagice (Inada and Ido). Instead of the serum and citrate plasma of the rabbit or other animal, serum and plasma from nonimmune persons were used during the early stage of the cultivation" experiments. The principal medium consisted of a mixture of 1 part of the serum and 3 parts of Ringer solution, used in a combination of the liquid form and a form made semisolid by adding melted neutral agar (0.3 per cent), the liquid half (8 cc.) of the medium being superimposed on the semisolid half (8 cc.) in a tall culture tube such as that used in the cultivation of spirochetes. * The first step in the inoculation of the medium was to mix about 0.5 to 1 cc. of the citrate blood, drawn from the median basilic vein of the patient, with the lower or semisolid portion of the medium, while the latter was still in the fluid state (42°C.), and allow the mixture to solidify by cooling. The serum-Ringer dilution was t h e n poured on the semisolid portion and about 0.5 to 1 cc. more of the same blood introduced. A layer of paraffin oil was finally added to