292 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS N-O-D-GLUCOPYRANOSYL-5-ARALKYLIDENERHODANINES N-(2,3,4,6-Tetra-0-acetyl-/•-D-glucopyranosyl)-benzylidenerhodanine (2.56 g, 0.0046 mole) was suspended in 700 ml of methanol, and 1.9 M hydrochloric acid (10.5 ml) was added. The reaction mixture was stirred at room temperature for 3 d. The reaction solution was filtered and evaporated to a semisolid material at a temperature under 30 ø in vacuo. The semisolid was crystallized from ethanol and water, yielding 1.7 g (96.5%) mp 104-110 ø, [cr]•) 2ø = --78.0 ø (C = 0.8, pyridine) (lit. mp 104-110 ø) (24). The same method was applied for the preparation of the other glucosylrhodanine derivatives. TEST ORGANISMS One Gram-positive and two Gram-negative bacteria, one yeast and one mold were selected for the test. These were, respectively, Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 11229), Pseudomonas aeruginosa (ATCC 15442), Candida albicans (ATCC 10259), and Aspergillus niger(ATCC 1015). PREPARATION OF INOCULUM Portions of four or five discrete colonies representative of the organisms to be tested were inoculated into 10.0 ml of a suitable broth medium and marked accordingly. Bacterial cultures were incubated at 37 ø, while fungal cultures were incubated at 25 ø for 18-24 hr. AGAR PLATE STREAK METHOD FOR QUALITATIVE EVALUATION Trypticase Soy Agar (BBL) was used for bacteria and Sabouraud Maltose Agar (BBL) for the yeast and mold. ^ 10-cm round plastic plate was used for the preparation of the medium. Each plate was divided into four areas by marking on the bottom of the plate. The agar plate was streaked by the appropriate inoculating loop. Three thin 1oopfuls of inocula were applied and covered the agar plate completely. Approxi- mately 10-15 mg of compound to be tested was placed on the corresponding area of each inoculated plate. The plates inoculated with S. aureus, E. co/i, and P. aeruginosa were incubated at 37 ø for 24-48 hr, and those inoculated with C. albicans and A. niger were incubated at 25 ø for 48-72 hr. After incubation, if a zone of inhibition was visible around the compound, the susceptibility of the organism to the compound was considered to be positive. The degree of inhibition of the compound was decided by the size of the zone. The results of the qualitative evaluation of the compounds are shown in Tables I and II. BROTH DILUTION METHOD FOR THE DETERMINATION OF THE MINIMAL INHIBITORY CONCENTRATION (MIC) Quantitative evaluation was made for the compounds which showed some inhibitory activity in the preliminary agar screening test against any of the five organisms. The broth dilution method was chosen for the determination of the MIC. A 10 -s molar stock solution was prepared by dissolving or suspending the required amount of

ANTIMICROBIAL ACTIVITY OF N-GLUCOSYLRHODANINES 293 Table I Agar Plate Screening Test of Antimicrobial Activity of Glucosylthioureas Compounds tested S. E. P. C. A. R aureus coli aeruginosa albicans niger --N(CH,)• + .... --NH(CH0•CH• + + - _ + + --NHCH2•OCH 3 + - _ + + --NHCH2CH•-• + .... --NHCH2- • - + + - _ --N(%H0• ..... --N N--CH3 ..... --N O ..... --N•/S --NHCH•CH•H2 + + .... --NHN(CH3)2 + - - + - --N• + - + -- - --NH2 + + - -- + + Rhodanine + + + + + + + + + + + + + + + Very active + + moderately active + slightly active - no activity. compound in sterilized distilled water. The desired number of sterile test tubes were aseptically filled with 9.0 ml of Trypticase Soy Broth (BBL). One ml of the stock solution was added to the first tube and mixed thoroughly. With a sterile pipette, 1.0 ml was transferred from the first tube to the second tube, 1.0 ml was transferred with a separate pipette to the third tube. This process was continued through the penultimate tube, from which 1.0 ml was removed and discarded. Eventually, a serial dilution of 10 -2 , 10 -3 , 10 -4 , 10 -5 , and 10 -6 molar concentrations was made. The sixth tube without any antimicrobial agent served as a growth control, and also a blank tube was prepared, separately. Except for the blank tube, the inoculum was prepared by adding 0.05 ml of 24 hours-old broth culture without any dilution. The tubes were incubated for 24-48 hr at 37 ø for bacteria and 48-72 hr at 25 ø for the yeast and mold. The lowest concentration of antimicrobial compound resulting in complete inhibition of visible growth represented the MIC a very faint haziness or a small clump of possible growth