Isolation of rhizobia from preserved nodules.

During 1951 and 1952 various leguminous plants from the grounds, gardens, and greenhouses of Vassar College were collected and examined for the presence of root nodules. On the day of collection nodules were plated with medium # 79 (Fred and Waksman, 1928). Nodules from soybeans, clover, peas, beans, alfalfa, lespedeza, hairy vetch and birdsfoot trefoil contained viable rhizobia. On the same day, the remaining nodules were washed with running water; excess root tissues and stems were removed; and nodules were drained on filter paper. Each set of nodules was divided into three parts. Two groups were placed in sterile test tubes or jars, and covered with sterile mineral oil. One jar or test tube was then stored in the refrigerator and the other left at room temperature. The third group was placed in sterile test tubes or jars and stored in a deep freeze cabinet. In 1953, 1 to 2 years after collection, samples of all nodules were examined for the presence of viable rhizobia. Mineral oil was removed from nodules by washing with 70 per cent alcohol. Half of all nodules were washed with 1:1000 mercuric chloride for 30 seconds, followed by washing in 70 per cent alcohol for 30 seconds, and finally rinsed with sterile water three times. The remaining portion of all the nodules was washed with sterile water three times. Washing with mercuric chloride reduced the number of contaminants appearing in plates, but did not reduce the number of colonies of rhizobia. After washing, the nodules were placed in Petri dishes containing sterile water. The nodules were crushed and a crude dilution series was prepared by transferring two loops of the mixture of crushed nodule and water from the first Petri dish to a second. A third dilution was prepared in the same manner. Six media were used for the isolations: # 79, #79 plus 1: 80,000 crystal violet, # 79 plus 1:15,000 rose