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In developing the microbiological assay for nystatin1 in feeds, reported by Pagano, studies were made of the following factors: effect of heat and of pH on nystatin stability; effect of solvents on nystatin recovery; effect of feed extractives on Candida tropicalis; recovery of nystatin at different concentrations in feed; and preparation of a nystatin-free feed. Heat and pH must be rigidly controlled to keep nystatin loss at a minimum. Best recoveries were obtained with a hexane wash and methanol extraction.

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lntroductionIn estimating t h e concentration of a n antibiotic in solution by o n e of several analytical methods (chemical or biological), it is customary to prepare preselected dilutions of t h e sample calculated to produce varying inhibitory v a l u e s i n t h e range of t h e linear portion of the standard doseresponse curve. Usually t h e readings for two or three d o s e s of the unknown a r e graphed and through t h e s e points a curve is drawn t h a t c a n b e compared with that of a standard.T h e a n a l y s t is a t a disadvantage b e c a u s e h e must e s t i m a t e t h e concentrations of antibiotic that when diluted and a s s a y e d would yield r e s p o n s e s on t h e linear portion of t h e dose-response curve. A second disadvantage is that a curve produced by graphing t h e v a l u e s for s e v e r a l d o s e s of t h e unknown solution g i v e s only a n approximation of i t s shape, s i n c e a11 other points a r e determined by interpolation or extrapolation.A more satisfactory technique would permit t h e a n a l y s t t o obtain all points on the dose-response curve. Although t h i s is ideal, i t h a s heretofore been impractical. B y a continuous dilution d e v i c e for t h e Auto-Analyzer instrumental system w e a r e now a b l e to produce full descriptions of dose-response curves. Methods and R e s u l t s T h e schematic diagram shown i n F I G U R E 1 s h o w s a v e s s e l containing a solution of antibiotic agitated continuously by a magnetic stirrer. TWO polyethylene t u b e s of e q u a l diameter a r e inserted into t h e solution;as one tube withdraws a quantity of sample the other simultaneously res t o r e s a n equal quantity of diluent. S i n c e t h e solution in t h e v e s s e l is maintained a t a constant volume while i t is simultaneously being sampled and diluted, t h e concentration of s o l u t e s in solution will d e c r e a s e logarithmically a s a linear function of time. A similar application of t h i s principle w a s recently described by Menzies' for preparing dilutions of s o i l s u s p e n s i o n s for making bacterial counts. Menzies also presented a review of t h e mathematics.It occurred to u s t h a t t h e technique of continuous dilution could b e coupled t o t h e AutoAnalyzer microbiological a s s a y for quantitation of antimicrobial agents. To t e s t t h i s hypothesis we examined t h e activity of three polyene antifungal agents, fungichromin, amphotericin A, and 644