A broth dilution method of assaying streptothricin and streptomycin.

The agar dilution (Waksman, 1943) and the agar diffusion (Foster and Woodruff, 1943b) methods of assay of streptothricin and streptomycin are the procedures most commonly employed and have led to what Waksman calls "dilution units" (Waksman, 1943) and "diffusion units" (Schatz, Bugie, and Waksman, 1944). However, in addition, Waksman speaks of "Escherichia coli units," or "Bacillus subtilis units," or "Staphylococcus aureus units," and the picture is further complicated by the possibility of attempting to compare Escherichia coli dilution units and Staphylococcus aureus diffusion units, etc. To assign more than one activity value to a given substance leads to confusion. Foster and Woodruff's (1943b) choice of an arbitrary unit based on Bacillus subtilis inhibition in a cup plate test has value as a reference unit and we have used it as such in the determination of the toxicity of streptothricin (Rake, Hamre, Kavanagh, Koerber, and Donovick, 1945). No absolute value can be given to such a unit in terms of antibacterial activity because this activity varies from day to day with the conditions of the test (Foster and Woodruff, 1943a; Waksman and Reilly, 1944). Since crystalline preparations of streptothricin and streptomycin have now been described (Fried and Wintersteiner, 1945), it should be possible in the near future to define the unit of each of these substances in absolute terms, i.e., a fixed number of units per milligram of crystalline material. For the present, however, the use of an arbitrary reference unit would appear to be most valuable. This entails the use of a reference standard solution to be tested together with samples of unknown potency each day. Foster and Woodruff (1943b) stated that assay values obtained by the agar diffusion method, using a reference standard, for replicate streptothricin samples showed a variability between 5 and 15 per cent on a given day, whereas on different days this variability might be up to 15 to 25 per cent. Using the same method of assay, Waksman and Reilly (1944) found that a given concentration of streptothricin or streptomycin caused zones of inhibition the diameters of which varied with the period of incubation. Secondary and tertiary zones were formed in streptomycin plates. Hence this assay method required the choice of a standard period of incubation as well as a standard preparation for comparison. It has been found expedient in this laboratory to employ a method of assay for streptomycin and streptothricin which is a modification of a procedure which has been used here in the assay of penicillin. The test organism was chosen by plating out a number of cultures on beef extract agar containing varying amounts of