THE ANTISTREPTOMYCIN ACTIVITY OF LIPOSITOL

In an earlier report' it was shown that the antibacterial action of streptomycin on Eberthella typhosa and Staphylococcus azueus was greatly decreased when the bacteria were grown in Difco brain-heart infusion.ln a continuation of this work it was found that a methanol extract of the brainheart infusion contained the streptomycin-inhibiting factor and that Difco heart infusion had no activity.In order to study the nature of the factor, Difco Casamino acids medium was used as the base medium.It was first determined that the vitamin content of Casamino acids medium and even added vitamin did not carry the factor.Comparison of peptone, neopeptone, proteose peptone, peptone 3, tryptose, tryptone, trypticase, phytone, and Liver Fraction L indicated that phytone (a plant peptone) was the most active.In order to save time a shorter assay method was devised at this point.The modified procedure consisted of adding 2.5 ml. of double strength base medium to a solution of the material to be assayed in 2 ml. of water.The final solution was sterilized and cooled.Streptomycin (0.5 ml.) of the desired concentration and 1 standard drop of a 24 hour broth culture of the test organism were then added.The total volume was 5 ml. in all tests and the amount of streptomycin added in the tests with E. typhosa was 200 units per ml., or a total of 1000 units, and with S. aureus 50 units per ml., or a total of 250 units.Incubation was at 37' for 15 hours.The presence or absence of turbidity was used as the measure for the presence of the active factor.Final checks were made by plating 1 ml. of the solution after the 15 hour incubation interval.Several different concentrations of the assay material were tested in order to determine the minimum amount that gave effective inhibition of streptomycin.The original brain-heart infusion was active at a level of 25 to 50 mg.A special Difco brain infusion2 gave inhibition at a much lower level.At this point a systematic fractionation of the active substance from brain was undertaken.As was expected, aqueous extracts were ac-