The biotin requirement of Micrococcus lysodeikticus.

The response of M. lyodeiktiwu to biotin or other nutrients was determined by turbidimetric measurement of growth. Five ml of test medium contained in aluminum-capped test tubes were used in each assay. Additions to 4 ml of basal medium varied the composition of the nutrient material. The basal medium (no. 1) (table 1) was stored under toluene in the refrigerator and aliquots withdrawn aseptically when needed. Inoculation of the assay tubes was made as follows: A 24 hour agar slant culture of the organism was washed into 10 ml of sterile normal saline, centrifuged, and again washed with saline. After centrifuging, the cells were resuspended in 10 ml saline and diluted 1-10. A single drop of this suspension added from a standardized pipette served to inoculate each tube. The tubes were incubated at 37 C for 72 hours. Since incubation without shaking results in only slight growth, the cultures were agitated by means of a reciprocal shaker operating at approximately 125 three inch strokes per minute. Solutions of crystalline -biotin were used in the experiments. L-Aspartic acid was neutralized before adding to the medium. Sodium oleate solutions were made up as follows: 100 mg of oleic acid were dissolved in 4 ml of 95 per cent ethyl alcohol, titrated with 0.9 N NaOH, and made to 5 ml with alcohol. The solution was diluted 1-100 with distilled water for use as a stock solution. Growth experiments indicated