Effects of incubation temperature, inoculum size, and medium on agreement of macro- and microdilution broth susceptibility test results for yeasts

Abstract

We examined the effects of temperature and inoculum on the agreement of macro-and microdilution broth Drugs. SCH 39304 (Schering-Plough Research Corp., Bloomfield, N.J.), fluconazole (Pfizer Research, Groton, Conn.), amphotericin B (E. R. Squibb & Sons, Princeton, N.J.), and ketoconazole (Janssen Pharmaceutical, Inc., Piscataway, N.J.) were used to prepare 1-mg/ml stock solutions in 2% dimethyl sulfoxide (Sigma Chemical Co., St. Louis, Mo.) for the triazoles and 10% dimethyl sulfoxide for the other drugs. Stock solutions of flucytosine (Hoffmann-La Roche Inc., Nutley, N.J.) were prepared at a concentration of 10 mg/ml in 10% dimethyl sulfoxide. All drugs were stored at -700C.Media and buffers. Unbuffered synthetic amino acid media for fungi (SAAMF) were prepared as previously described (2, 6), except that solutions of amino acids (Irvine Scientific Sales Co., Inc., Santa Ana, Calif.) were used instead of powdered amino acids and cystine was replaced with equimolar amounts of cysteine (7). Yeast nitrogen broth (Difco), RPMI 1640 (Sigma), and high-resolution broth (kindly supplied by Peter Troke, Pfizer Inc., Sandwich, England) were prepared as specified by the manufacturers, sterilized by filtration, and stored at 4°C. On the day of use, morpholinepropanesulfonic acid (MOPS) buffer (Sigma) was added to each medium to a final concentration of 0. 165 M, the pH was adjusted to 7.0 with 10 M sodium hydroxide or 6 M hydrochloric acid, and the medium was resterilized by filtration.Susceptibility testing. Macrodilution broth assays were performed as previously described (8,9). Briefly, twofold dilutions of the antifungal agents were prepared by standard methods (10) in media to 10 times the final concentrations, as listed below. Yeast inocula were prepared by suspending 1542

Improved medium for fluconazole susceptibility testing of Candida albicans

“…At least for the most common organism, C. albicans, some of the parameters affecting test results, such as inoculum size, medium composition, pH, incubation time, and incubation temperature, have been tentatively standardized after the comparative work of many different groups (3,5,6,9,10,(14)(15)(16). We therefore used as the starting point in our antifungal susceptibility tests inocula of ca.…”

In vitro studies of a new antifungal triazole, D0870, against Candida albicans, Cryptococcus neoformans, and other pathogenic yeasts

“…In this report, we have analyzed the effect of changing each of several test conditions that influence test results with other antifungal agents (2,5,10,11,18,19,29 Yeasts. A group of seven yeast isolates (one each of C. albicans, Candida lusitaniae, Candida tropicalis, Candida parapsilosis, T. glabrata, C. krusei, and C. neofornans)was used for the initial studies.…”

Effects of temperature on anti-Candida activities of antifungal antibiotics

“…However, Block et al (2) noted a considerable increase in the susceptibility of Cryptococcus neoformans to flucytosine at 37°C compared with that at 32°C, while precisely the opposite effect of temperature was noted by Johnson et al, working with nystatin (14), and Galgiani et al, working with flucytosine (11), both in tests with Candida spp. An incubation temperature of 35°C has emerged in some recent reports as preferable both to 37°C (5) and to 30°C (23,25) for optimal reader agreement with visual MIC endpoints. Temperature was unequivocally a less significant factor influencing interlaboratory agreement between MICs than was the composition of the test medium (23).…”