1. The presence of suitable animal or human serum is essential for the cultivation of Leptospira icterohæmorrhagiæ.
2. The nutrient value of serum is considerably reduced by heating to 60°C. for 30 minutes and is destroyed by boiling (100°C). Filtration through a Berkefeld filter does not diminish the nutrient value of the serum.
3. The cultural value of different animal sera varies considerably. It is entirely absent from the sera of the rat and the pig. The sera of the rabbit, horse, and goat are better suited for the growth of the organism than those of the guinea pig, sheep, donkey, or calf. Human serum is suitable, but not ascitic fluid.
4. Fresh or heated emulsions of the liver, kidney, heart muscle, or testicle of the normal guinea pig or rabbit have no cultural value for the organism. The same may be said of both the white and yolk of the hen's egg.
5. A luxuriant growth takes place in a medium of Ringer's solution to which more than 10 per cent of normal rabbit serum is added. There is only moderate growth with 5 per cent of serum, and none when less than 2 per cent is present. The use of an undiluted serum offers no advantage over a diluted one, provided the latter contains at least 10 per cent of serum. In the case of certain animal sera dilution seems to make them more suitable for cultivation purposes, owing perhaps to its reduction of their inherent alkalinity.
6. The tonicity of the culture medium has but little influence upon the growth and morphology of the organism. A medium containing distilled water as diluent or one containing 8 per cent sodium chloride seems to give identical results. The viability of the organism was greatest in a medium in which Ringer's solution or isotonic salt solution was used as diluent.
7. The reaction of the medium is an important factor in the cultivation of the organism, which thrives most vigorously in a medium of which the reaction is slightly alkaline, not exceeding that of the serum. If the reaction is neutral, the growth is meager, and the culture is short lived. When the reaction of a medium becomes alkaline by the addition of a small amount of sodium hydroxide, or faintly acid by the addition of a little hydrochloric acid, no growth can take place.
8. Leptospira icterohæmorrhagiæ is an obligatory aerobe. Any hindrance to the access of oxygen constitutes an unfavorable factor in obtaining a culture.
9. The addition of carbohydrates to media has no perceptible effect upon the growth or morphology of the organism. The reaction of the media is not modified by their presence.
10. Leptospira icterohæmorrhagiæ grows at any temperature between 37° and 10°C., the optimum zone being 30–37°C. Growth proceeds more rapidly at 37°C. than at 30° or at 25°, but the cultures remain viable much longer at the latter temperatures. No growth takes place at 42°C.
11. Three different media are described for the cultivation of freshly isolated strains. After prolonged cultivation on these media a strain may be readily cultivated in a serum diluted with Ringer's or isotonic salt solution.