Nystatin, amphotericin B, ketoconazole and fluconazole (Sigma, St. Louis, MO, USA) were dissolved in dimethylsulphoxide (DMSO). Caspofungin (Merck and Company Inc, Waterhouse Station, NJ, USA) was dissolved in sterile distilled water. These anti-Candida agents were prepared initially as 10,000 µg/ml and stored at -20°C prior to each experiment as previously described4 ,5 . It was thereafter suspended/diluted in the following medium during the exposure period of yeasts: RPMI (Roswell Park Memorial Institute) 1640 medium buffered with 0.165 M MOPS (3-(N-morpholino) propanesulfonic acid) containing L-glutamine and lacking sodium bicarbonate (Sigma, USA), dissolved in 1 liter of sterile distilled water and adjusted to a pH of 7.2 and sterilized filter.
Lacking sodium bicarbonate
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Lacking sodium bicarbonate is a laboratory product that serves as a buffer solution. It is used to maintain a specific pH level in various experiments and applications.
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4 protocols using lacking sodium bicarbonate
1
Preparation of Antifungal Agents for Candida Experiments
2
Antifungal Agents Preparation Protocol
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Antifungal agents was prepared as done previously in similar studies.9 (link) Briefly, nystatin, amphotericin B, ketoconazole and fluconazole (Sigma, St. Louis, MO, USA) were dissolved in dimethylsulphoxide (DMSO). Caspofungin (Merck and Company Inc, Waterhouse Station, NJ, USA) was dissolved in sterile distilled water. These antimycotic agents were prepared initially as 10,000 μg/mL and stored at −20 °C prior to each experiment as previously described.9 (link) It was thereafter suspended/diluted in the following medium during the exposure period (1 h) of yeasts. RPMI 1640 medium buffered with 0.165 M MOPS (3-(N-morpholino) propanesulfonic acid) containing l -glutamine and lacking sodium bicarbonate (Sigma, USA), was dissolved in 1 l of sterile distilled water and adjusted to a pH of 7.2 and filter sterilized.
3
Antifungal Agent Preparation and Dilution
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Anti-fungal agents were prepared as in previous similar studies [14 ,15 (link)]. Briefly, nystatin, amphotericin B, ketoconazole and fluconazole (Sigma, St. Louis, Mo., USA) were dissolved in dimethylsulphoxide. Initially, 10,000 µg/ml of each anti-mycotic agent was prepared and stored at −20°C as previously described [14 ,15 (link)]. For 1 h of exposure to the yeasts, each agent was suspended/diluted in RPMI 1640 medium buffered with 0.165 M MOPS [3-(N-morpholino)propanesulphonic acid] containing L -glutamine and lacking sodium bicarbonate (Sigma), which was dissolved in 1 litre of sterile distilled water and adjusted to a pH of 7.2 and then filter sterilized. Chlorhexidine gluconate (0.2%; GlaxoSmithKline, Brentford, UK) was dissolved in sterile phosphate-buffered saline (PBS) at a pH of 7.2 and diluted to obtain a concentration of 0.005% prior to each experiment as previously described [16 (link),17 (link)].
4
Nystatin Dosing and Preparation
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As described previously [12 (link),13 (link),14 (link),15 (link)], nystatin (Sigma, St. Louis, Mo., USA) was dissolved in dimethyl sulfoxide (DMSO) and absolute ethanol (3:2 ratio), respectively, and was initially prepared as a 10,000-µg solution and stored at −20°C before use. It was thereafter suspended in the following medium during the period of exposure to yeasts (1 h): Rosewell Park Memorial Institute (RPMI) 1640 medium buffered with 0.165 M morpholinopropanesulfonic acid (MOPS) containing L -glutamine and lacking sodium bicarbonate (Sigma) in 1 liter of sterile distilled water adjusted to a pH of 7.2 and filter sterilized [12 (link),13 (link),14 (link),15 (link)]. This liquid (RPMI) was stored at 2–8°C. The stock solution was used to obtain the drug concentrations [i.e. 2× the minimum inhibitory concentration (MIC)] used in the experiments.
Since nystatin was dissolved in DMSO and absolute ethanol, equivalent amounts of the latter chemicals were tested initially as was done in previous studies using the same isolates to ascertain whether they had an effect on the isolates tested. Minute amounts of the chemicals used in this study did not have any effect on yeast survival/growth when compared to the controls.
Since nystatin was dissolved in DMSO and absolute ethanol, equivalent amounts of the latter chemicals were tested initially as was done in previous studies using the same isolates to ascertain whether they had an effect on the isolates tested. Minute amounts of the chemicals used in this study did not have any effect on yeast survival/growth when compared to the controls.
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