1. Leptospira icterohæmorrhagiæ is unable to grow in the urine, either with or without the addition of suitable culture ingredients, the acidity of the urine being detrimental to the growth. It survives less than 24 hours, unless the urine is neutralized or slightly alkalized, when the period of survival is somewhat longer. If suitable nutrient ingredients are added to the neutralized or slightly alkalized urine, the organism is able to grow for about 10 days, after which multiplication ceases.
2. Feces from normal or jaundiced persons destroy Leptospira icterohæmorrhagiæ within 24 hours when a rich culture is added and the mixture allowed to stand at 26°C. The addition of blood serum and corpuscles does not prevent the destruction of the organism. Autoclaved specimens and filtrates of unheated feces do not constitute a suitable medium in which to keep the organism alive for any length of time, but the addition of blood corpuscles and serum in adequate quantities renders them fairly satisfactory as media. Under natural conditions Leptospira icterohæmorrhagiæ cast off in the feces cannot survive more than 24 hours.
3. Polluted water, sewage, and soil will not serve to keep Leptospira icterohæmorrhagiæ alive for more than 3 days at the most. When deprived by filtration or autoclaving of their bacteria they become indifferent diluents and may be used to make up a culture medium when mixed with serum and citrate plasma of a suitable animal. Sterilized soil with a neutral reaction, when added to a culture, has an unfavorable effect upon the growth of the organism.
4. Most of the aerobic bacteria found in feces, sewage, soil, and tap water inhibit the growth of Leptospira icterohæmorrhagiæ when inoculated into the same medium. Bacillus fæcalis alkaligenes and many strains of non-hemolytic streptococci caused the least interference, although growth was never so vigorous or lasting in the media in which they were present as in the control media. Certain pathogenic bacteria (Bacillus typhosus, Bacillus paratyphosus, Bacillus dysenteriæ, pneumococcus) are antagonistic to the growth of the spirochete.
5. Leptospira icterohæmorrhagiæ is highly sensitive to the destructive action of bile, bile salts, and sodium oleate, but resists the action of saponin. In this last respect it differs from many so called spirochetes. The destructive action of these agents is counteracted by blood serum.
6. The larvae and adults of the Culex mosquito, the larvæ of the house-fly and bluebottle fly, wood ticks (Dermacentor andersoni), and leeches failed to become carriers of the spirochetes when fed on infected guinea pigs or their organs; that is, they cannot play the part of an intermediary host of Leptospira icterohæmorrhagiæ.