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6 Effect of horse blood on antibacterial activity of SMX in MUELLER-HINTON agar media (Eiken, Lot No. : TY009L, TA000U, TA000L and TG000T) in vitro Effect of inoculum size on in vitro antibacterial activity of TMPand SMX
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8 Staphylococcus aureus FDA 209-P JC-1 Escherichia coli NIHJ JC-2 2) DARRELL, J. H.; L. P. GARROD & P. M. WAT- ERWORTH : J. Clin. Path. 21 : 202 `209, ) HARPER, G. J. & W. C. CAWSTON : J. Path. Bact. 57 : 59, ) WALKER, N. ; R. PHILIP, M. W. SMYTH, & J. W. MCLEOD : J. Path. Bact. 59 : 631, ) WATERWORTH, P. M. : Postgraduate Med. J. Suppl. 45 : 21 `27, ) BUSHBY, S. R. M. & G. H. HITCHINGS : Brit. J. Pharm. Chemother. 33 : 72 `90, ) WOODS, D. D. : J. Gen. Microbiol. 29 : 687, 1962
9 VOL. 21 NO. 2 CHEMOTHERAPY 75 METHODS OF SENSITIVITY TESTING FOR SULFAMETHOXAZOLE-TRIMETHOPRIM COMBINATION PRODUCT Ad Hoc Committee of the Japan Society of Chemotherapy for the Evaluation of Sensitivity Testing Methods for Sulfamethoxazole and Trimethoprim* *Members of this committee are : KEIMEI MASHIMO, YASUMICHI KATO and OSAMU YAJIMA (Hokkaido University) YASUSHI UEDA, FUMIO MATSUMOTO and ATSUSHI SAITO (Jikei University School of Medicine) KIHACHIRO SHIMIZU and OTOHIKO KUNII (University of Tokyo) RYOCHI FUJII and MASATOSHI KONNO (Teikyo University) SHUNJI ISHIYAMA, ISSEI NAKAYAMA, HIDEO IWAMOTO and SIGETOMI IWAI (Nihon University) MASAAKI OHKOSHI and YORIO NAIDE (School of Medicine. Keio University) SHOGO KUWAHARA and SACHIKO GOTO (Toho University) SHozo NAKAZAWA (Kyoto College of Pharmacy) KENZO SHIOTA and Fumio MIKI (Osaka Municipal University) HIROSHI OKUBO and YURUKO OKAMOTO (Kansai Medical School) SHIGEMI AWATAGUCHI (Tanabe Seiyaku) KEN KATAGIRI and MIKAO MAYAMA (Shionogi Seiyaku) An ad hoc committee has been organized by members of the Japan Society for Chemotherapy with an aim to establish acceptable methods for determination of minimal inhibitory concentrations (MICs) of sulfamethoxazole (SMX) - trimethoprim (TMP) combination product. The following summary outlines the conclusion derived from investigations by this committee. 1. Sensitivities shall be assayed by the agar plate dilution method and activities shall be expressed in terms of MIC (the minimal concentration at which bacterial growth is completely inhibited). 2. Culture medium for inoculation MUELLER-HINTON broth (Difco) shall be used without modifying its ph (7. 4 } 0. 2). For those test organisms which require specific nutrients, appropriate media shall be selected. 3. Dilution of culture broth Sterilized physiological saline shall be used in general as a diluting medium but other appropriate material may be employed for specific organisms. 4. Culture medium for sensitivity testing MUELLER-HINTON agar (Eiken) or Medium for Sensitivity Disc Testing (Nissan) shall be employed enriched with 7.5% hemolyzed horse blood (ph 7.4). 5. Method of inoculating test organisms Culture medium for inoculation purposes shall be incubated at 37 Ž for 18 `20 hours. The culture broth thus obtained shall be diluted 100-fold for gram positive cocci, or 1000-fold for gram negative rods to make inoculating materials for sensitivity testings. A loopful of this culture broth will be streaked over 2 cm on a sensitivity plate. 6. Judgement After incubating the plate at 37 Ž for 18 `20 hours, the minimal concentration at which the bacterial growth is completely inhibited shall be examined macroscopically to determine MIC. The growth of a single colony, or of a very thin bacterial film shall be judged as bacterial growth (in case the bacterial growth is insufficient after the aforementioned conditions, the judgement shall be made after 48 hours' incubation). 7. Concentrations of drugs Two-fold dilutions shall be made in the following order : 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78,
10 76 CHEMOTHERAPY MAR and mcg/ml. When employing concentrations in excess of 100 mcg/m I, concentrations shall be 200, 400 and 800 mcg/ml. 8. Preparation of standard solution of TMP and SMX Ten mg of TMP are dissolved in a small volume of 4/100 N HCI, to which water is added to make a total volume of 10 ml. Ten mg of SMX is dissolved in a small volume of 1/8 N NaOH, to which water is added to make a total volume of 10 ml. The following alternative may be acceptable for TMP : Ten mg of TMP is dissolved in 0.5 ml of dimethylformamide or 0.5 ml propyleneglycol, to which water is added to make a total of 10 ml. 9. Preparation of plate for sensitivity testing Hemolyzed horse blood shall be added when the temperature of culture medium is cooled down to approximately Ž. Drugs in prescribed quantities are poured into the plate and the culture medium in 9-fold volume is added while the plate is gently shaken. 10. Test organisms (clinical isolates and standard bacterial strains) Test organisms must be fresh within 2-3 culture generations after the clinical isolation. As reference standard of organisms, the following 2 strains as recommended by the Japan Society for Chemotherapy shall be employed ; Staphylococcus aureus FDA 209-P JC-1 and Escherichia coli NIHJ JC-2. The MICs of these standard strains as assayed by the foregoing procedures are as follows :
VOL. 21 NO. 2 CHEMOTHERAPY 137
VOL. 21 NO. 2 CHEMOTHERAPY 137 Minimal inhibitory concentration (mcg/m1) of TM? and SMX in MUELLER-HINTON agar with 7.5% lysed horse blood, Diagnostic sensitivity test medium with 7. 596 lysed horse blood
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