A study of the growth requirements of Spirochœta obermeieri resulted in the perfection of a method which enabled us (1) to cultivate the organisms consistently from the blood of infected mice and rats, (2) to maintain the viability of cultures for periods of at least 3 to 7 weeks, and (3) to carry them on in successive subcultures by transplanting at intervals of 2 to 4 weeks. This method is essentially the same as the Noguchi technique for the cultivation of the Leptospira group, but emphasizes control of the physicochemical factors that act to limit and prevent growth and prescribes the conditions necessary to counteract the injurious influences. The main facts may be briefly summarized as follows:
(a) Ascitic fluid, horse or rabbit serum may be used as culture fluids.
(b) These fluids become progressively more alkaline on exposure to air.
(c) Uniformly successful results depend chiefly on the proper adjustment and stabilization of the reaction.
(d) A balanced reaction can be secured by adding 1.0 per cent peptone broth or egg albumin as buffer, and covering the culture with a layer of oil.
(e) The reaction limits for growth and survival are between pH 6.8 and 8.2, with the optimum at pH 7.2 to 7.4.
(f) Spirochœta obermeieri is a strict aerobe, consequently in order to permit adequate aeration, the oil layer should not exceed 1.5 cm. in height.