Strains were preserved in storage media (10% glycerol, 1% NaCl, and 1% Tween 20) at −80 °C. They were revitalized and routinely grown on yeast peptone dextrose (YPD) agar plates (2% Bacto Peptone, 1% yeast extract, 2% dextrose, and 2% Bacto agar) at 35 °C, regularly sub-cultured before each experiment. Throughout the assays we also used YPD broth (2% Bacto Peptone, 1% yeast extract, 2% dextrose), Sabouraud dextrose agar (3% Sabouraud dextrose from Sigma-Aldrich, 2% Bacto agar), and RPMI (1.04% RPMI-1640 from Sigma-Aldrich, 3.453% morpholinepropanesulfonic acid from Sigma-Aldrich, pH adjusted to pH 7 with 10 M NaOH solution) (Adams et al., 1998 ).
Sabouraud dextrose
Manufactured by Merck Group
Sourced in Germany
Sabouraud dextrose is a type of culture media used for the isolation and cultivation of fungi, particularly those that cause fungal infections. It provides the necessary nutrients and growth conditions for the development of fungal colonies, enabling their identification and analysis.
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Lab products found in correlation
Morpholinepropanesulfonic acid, by Merck Group (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Mueller hinton, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Hexane, by Merck Group (1 mentions)
Anhydrous sodium sulfate, by Merck Group (1 mentions)
Ciprofloxacin, by HiMedia (1 mentions)
Fluconazole, by HiMedia (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Chloramphenicol, by Merck Group (1 mentions)
Mueller hinton agar (mha), by Merck Group (1 mentions)
Vitek 2, by bioMérieux (1 mentions)
Glycerol, by Fagron (1 mentions)
Sabouraud dextrose agar, by Merck Group (1 mentions)
8 protocols using sabouraud dextrose
1
Preserving and Culturing Fungal Strains
2
Antimicrobial Assays and Reagents
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Hexane, anhydrous sodium sulfate, dimethylsulfoxide (DMSO), trypticase soy, Mueller–Hinton and Sabouraud dextrose were purchased from Sigma-Aldrich (Steinheim, Germany). Fluconazole and ciprofloxacin were purchased from Hi Media (Mumbai, India). Sodium chloride (NaCl) was purchased from local markets.
3
Immunosuppression and Radiation Exposure in Candida albicans Murine Study
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C. albicans (ATCC: 10231) was used through all stages of the study. Male inbred BALB/c mice, at 6–8 weeks of age, were purchased from Pasteur Institute of Iran and kept in a standard animal housing facility with adequate pellet and water for animal consumption. The animal study was approved by the Institutional Ethics Committee. Dexamethasone (Sigma: D1756) was used for immunosuppression. Chlorpromazine was used as a sedating agent for better restraining of the animals during manipulations. Sabouraud Dextrose (SD) (Merck: 105438), SD agar or SD broth mediums were used as culture media with chloramphenicol (Merck: 220551) as an antibiotic to prevent from undesired microbial contamination of the cultures. The radiation generator was a standard jammer with 900 MW output and 3 pieces omnidirectional antennas. All culture and isolation media and related reagents were from the university resources.
4
Candida albicans ATCC 10231 Cultivation
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C. albicans ATCC 10231 strain was obtained from Laboratory Gen Lab from Peru S.A.C. Sabouraud dextrose, Mueller Hinton agar, and sodium sulfate (Na2SO4) anhydrous (Merck, Darmstadt, Germany) were used, which were prepared according to the same manufacturer’s standards.
5
Identification of Candida kefyr in COWE Samples
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The presence of Candida kefyr in the COWE samples was determined by inoculating 10 μL of the solution over the Sabouraud dextrose (Merck, Pro-Analise, BR) agar surface. The solution was streaked in all directions, in a loop. After 48 hours of incubation at room temperature, the colonies were analyzed and the microculture technique was carried out to identify yeast genus and observe the pseudohyphe formation at 400x magnification. The Vitek 2 (Biomérieux, Brazil AS) automated system was used to confirm the yeast species. The sample received the identification number of 6057315063047100. Briefly, this system performs micro-organisms identification by continuously monitoring the growth and activity of the micro-organisms within the wells of the charts. The optical transmission system uses visible light to directly measure the growth of microorganisms at 15 minute intervals based on an initial reading prior to a significant microbial growth. The process was repeated 3 times. Auxanogram (C and N medium; raffinose, xylose, melybiose, and inositol) and zymogram (sucrose, lactose, galactose, trealose, maltose, and dextrose) were also performed to identify the yeast on the COWE solution. After 24–48 hours of incubation at 35–37 °C, the presence of gas on tubes or cloudy halo formation on carbohydrates, the peptone or potassium nitrate sites in the agar were observed.
6
Cultivation and standardization of Candida species
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Strains of C. albicans (ATCC 90028) and C. glabrata (ATCC 2001) were cultivated aerobically on Sabouraud Dextrose (Merck KGaA, Germany) agar at 37 °C. Cell suspensions were grown in RPMI 1640 (Inlab diagnóstica, Brazil) during 24 h at 37 °C. Before experiments, cells were centrifuged (5000 g for 5 min), washed twice with sterile saline, and suspended in RPMI 1640 medium. Suspensions were standardized at OD600 = 1.0 (1 × 106 cells/mL) (LGL Scientific 0741/16, Brazil), based in experiments described previously with some adaptations [22 (link)].
7
Topical Clotrimazole Formulation Development
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Clotrimazol was purchased from Pol-Aura (Poznan, Poland). Carbopol® EZ-3 was purchased from Lubrizol Corp. (Wickliffe, OH, USA). Glycerol was purchased from Fagron (Kracow, Poland). Ethanol 96%, isopropanol and sodium chloride were purchased from Avantor Performance (Gliwice, Poland). Phosphate buffer concentrate was purchased from Chempur (Piekary Śląskie, Poland). Resin was purchased from Phrozen Aqua-Blue (Hsinchu City, Taiwan). Triizopropanolamine was purchased from Sigma-Aldrich (St. Luis, MI, USA). Sabouraud dextrose and yeast culture broth were purchased from Merck KGaA (Darmstadt, Germany).
8
Fungal Cultures and Identification
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Human pathogenic fungal cultures i.e., Candida albicans ATCC 10231 and Candida galeberata ATCC 20001 obtained from Oxoid, UK, and grown onto sabouraud dextrose agar (SDA) (Merck, Germany) at 28 °C for 48 h. Multi-cellular and dimorphic plant-associated fungal isolates i.e. Trichoderma longibrachiatum( MW564026), Purpureocillium lilacinum (MW566158), Aspergillus terreus (MW570851), Aspergillus flavus, Paecilomyces variotii, and Fusarium oxysporum, and two yeast species Meyerozyma guilliermondi (MW564205) and Saccharomyces cerevisiae were grown onto sabouraud dextrose and czapek’s dox agar (Merck, Germany) for 2–7 days at room temperature (Colozza et al., 2012 (link); Elefanti et al., 2013 (link)). Fungal strains were identified at the molecular level by the ITS gene sequencing and were submitted to the gene bank providing the accession numbers.
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