C. albicans was a kind gift from Dr. Khaled Alobaid (Mycology Reference Laboratory at Mubarak Al-Kabeer Hospital, Kuwait). Single colonies of the clinical isolate (141/11/20) were inoculated into yeast extract peptone dextrose (YPD) broth and incubated at 30 °C for 48 h, with shaking at 200 rpm. YPD broth was made by dissolving 10 g yeast extract (Oxoid, Cambridge, UK), 20 g mycological peptone (Oxoid), and 20 g D-glucose (Fisher Scientific, Loughborough, UK) in 1 L distilled water and then autoclaving the mixture. The cultures were then centrifuged three times at 6000 rpm in an Eppendorf 5415 R centrifuge (Eppendorf®, Hamburg, Germany), resuspended in PBS, and adjusted to a final concentration of 1 × 108/mL. Heat-killing was then done by incubating cells at 100 °C for 1 h on a static heat block. Killing was confirmed by plating out cells on YPD agar and monitoring growth for up to 72 h. YPD agar was prepared like that used for the YPD broth described above but also included 20 g technical agar (Oxoid) for every 1 L preparation.
Mycological peptone
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Mycological peptone is a nutrient source used in microbiology and mycology applications. It is a powder that provides essential nutrients for the growth and cultivation of various microorganisms, including fungi and bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Yeast extract, by Thermo Fisher Scientific (6 mentions)
Centrifuge 5415r, by Eppendorf (1 mentions)
Technical agar, by Thermo Fisher Scientific (1 mentions)
D glucose, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Tyrosol, by Merck Group (1 mentions)
3 n morpholino propanesulfonic acid, by Merck Group (1 mentions)
Absolute ethanol, by Avantor (1 mentions)
Low molecular weight chitosan, by Merck Group (1 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Isopropanol, by Merck Group (1 mentions)
Acetone, by Merck Group (1 mentions)
Ammonium carbonate, by Merck Group (1 mentions)
Acetic acid, by Merck Group (1 mentions)
Lp0021, by Thermo Fisher Scientific (1 mentions)
Bacto malt extract, by BD (1 mentions)
Yeast extract, by BD (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
12 protocols using mycological peptone
1
Preparation of Heat-Killed Candida albicans
2
Cultivation of Candida and Saccharomyces
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Candida auris and other yeast strains used in this study are listed in Table S1. Candida albicans SC5314, and Saccharomyces cerevisiae BY4741 and BY4743 were used as control organisms. Yeast cells were grown at 30 °C on YPD plates (1% yeast extract, 2% mycological peptone, 2% glucose, 2% agar; Oxoid, Basingstoke, UK) or shaking at 200 rpm in YPD broth (same as plates, but without agar).
3
Characterization of C. auris Strain
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C. auris isolate 12 (NCPF 8973), derived from the South Asian/Indian lineage, was obtained from the National Mycology Reference Laboratory (United Kingdom) (21 (link)). The strain was maintained on yeast extract–peptone–dextrose (YPD) solid medium [10 g/L of yeast extract (Alfa Aesar, USA), 20 g/L of mycological peptone (Oxoid, United Kingdom), 20 g/L of dextrose, and 20 g/L of agar (VWR International LLC, Hungary), pH 5.6]. Culturing and biofilm formation were performed in RPMI-1640 (with L-glutamine and without bicarbonate, pH 7.0, and with 3-(N-morpholino) propanesulfonic acid; Merck Ltd, Budapest, Hungary). Farnesol (Merck Ltd.) was obtained as a 3-M stock solution, which was diluted to 30 mM in 100% methanol. The working concentration of farnesol (75 µM) was prepared in the RPMI-1640 medium. Drug-free RPMI-1640 controls were supplemented with 1% (vol/vol) methanol. Tyrosol [2-(4-hydroxyphenyl) ethanol] (Merck Ltd.) was prepared as a 0.1-M stock solution in sterile physiological saline. The working concentration of Tyrosol (15 mM) was prepared in RPMI-1640.
4
Hydrogel Biomaterials Characterization
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Sodium alginate from brown algae (1% viscosity 35 mPa s; mannuronic/guluronic ratio 70/30) was kindly donated by Dompè S.p.A (L’Aquila, Italy), whereas pectin amid CF 025 D (amidated low methoxyl grade, degree of esterification 23–28%, degree of amidation 22–25%, molecular weight 120 kDa) was kindly donated by Herbstreith & Fox (Werder/Havel, Germany). Chitosan low molecular weight (50,000–190,000 Da,1% viscosity in acetic acid 20–80 mPa s; 75–85% deacetylated) was purchased from Sigma Aldrich (Milan, Italy). Sodium bicarbonate, ammonium carbonate, sodium chloride, as well as acetic acid, isopropanol, and acetone were acquired from Sigma Aldrich (Milan, Italy). Absolute ethanol was obtained from VWR Chemicals (VWR International, Radnor, PA, USA). Mycological peptone was purchased from Oxoid Ltd., Basingstoke, Hants, UK) and fetal bovine serum that was qualified and heat-inactivated was obtained from Gibco (Thermo Fischer Scientific, São Paulo, Brazil). All other chemicals used in this work were of commercial analytical grade.
5
Osmotic stress response in Candida albicans
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A single C. albicans colony was cultured overnight at 30 °C in shaking incubator in 5 mL CAI4-CIp10 in YPD-T, pH 7.4 (Tris buffered YPD containing 2% w/v glucose, 2% w/v mycological peptone (Oxoid, Hampshire, United Kingdom), 1% w/v yeast extract (Oxoid), 100 mM Tris-HCl, pH 7.4)28 (link). The following day 500 µL culture was inoculated into 50 mL YPD-T and allowed to grow overnight. Fresh medium was then inoculated with culture to OD600 = 0.2 and the cells were grown to OD600 = 0.8. At this point the culture was divided into two, diluted with medium to OD600 of 0.2 and NaCl added to one flask (final concentration 1M), the other serving as the control. The triplicate analysis (three high salt, three controls) were harvested after 1 h of further growth and the cell pellets were flash frozen in liquid nitrogen. Three biological replicates per treatment group were produced in this way.
6
Preparing and Quantifying Candida albicans
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Candida albicans strain SC5314 (Gillum et al. 1984 (link)) was prepared by plating 2–10 µl of frozen glycerol stock on YPD plates [1% w/v yeast extract (Oxoid LP0021, Basingstoke, UK), 2% w/v mycological peptone (Oxoid LP0040), 2% w/vd -glucose, and 2% w/v agar No. 2 (Oxoid LP0012)] and incubating at 30°C for 48 h. A single colony was transferred from the Petri dish into NGY broth (0.1% yeast extract (Oxoid LP0021), 0.1% neopeptone (Difco, Franklin Lakes, NJ, USA), and 0.4% w/v d -glucose; MacCallum et al. 2006 (link)) and incubated at 30°C, with shaking at 200 rpm, overnight. The concentration of C. albicans cells in suspension (cells/ml) was estimated by counting using a haemocytometer. Yeast growth was assessed by measuring optical density of the cultures at a wavelength of 600 nm using a spectrophotometer. For determination of C. albicans CFUs in samples, cells were plated on Sabouraud dextrose agar (SDA; 4% w/v d -glucose, 1% w/v mycological peptone, and 2% w/v agar No. 2, pH 5.6).
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Candida albicans strain SC5314 (Gillum et al. 1984 (link)) was prepared by plating 2–10 µl of frozen glycerol stock on YPD plates [1% w/v yeast extract (Oxoid LP0021, Basingstoke, UK), 2% w/v mycological peptone (Oxoid LP0040), 2% w/v
7
Non-Isocaloric Experimental Diets
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We designed five non‐isocaloric experimental diets that vary in protein: carbohydrate (P:C) ratios. The ratios were 3:1, 1:1, 1:3, 1:5 and 1:8, and the corresponding total calories in each diet were 194, 110, 65, 108 and 102 kcal respectively (Table S1 ). In these diets, we used mycological peptone (Oxoid Ltd.) as the protein source and Bacto malt extract (BD) as the carbohydrate source. Solid media were made with 1.5% w/v agar (GELITA Pty Ltd.). The precise amounts (g/L) of peptone, malt extract and agar were calculated based on each ingredient's percentage of protein and carbohydrate.
8
Cultivating Candida albicans for Experiments
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All fungal strains used in this study are shown in Table 5 and were cultured from glycerol stocks (−70°C) and maintained on YPD agar plates containing 2% (wt/vol) glucose, 2% (wt/vol) mycological peptone, 1% (wt/vol) yeast extract, and 2% (wt/vol) agar (all from Oxoid, Cambridge, UK). For C. albicans strains, unless stated otherwise, a single colony was grown in YPD medium (as described above but without the agar) and grown overnight at 30°C with shaking at 200 rpm. To induce hypha formation, cells were grown in RPMI 1640 modified medium (Sigma-Aldrich) containing 10% heat-inactivated fetal calf serum (FCS) and incubated at 37°C for 2 to 4 h. For mouse studies, C. albicans SC5314 was grown in NGY medium containing 0.1% Neopeptone (BD, Wokingham, UK), 0.4% glucose, and 0.1% yeast extract (BD) at 30°C with constant rotation at 200 rpm.
9
Yeast Extract Peptone Dextrose (YEPD) Broth and Agar Preparation
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The yeast extract peptone dextrose (YEPD) [1% (w/v) yeast extract, 2% (w/v) mycological peptone, and 2% (w/v) D-glucose] broth was prepared by dissolving 4 g of yeast extract (Oxoid) and 8 g of mycological peptone (Oxoid) in Milli-Q water up to a total volume of 380 mL. After autoclaving, 20 mL of filter-sterilised 40% (w/v) D-glucose (ChemSupply) was added. For YEPD plates, 8 g of agar no. 2 (Oxoid) was added to the solution before autoclaving.
10
Culturing Candida albicans and Escherichia coli
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The C. albicans strains used in this study are listed in Table 2 . Yeast was grown in YPD medium (1% [wt/vol] yeast extract [Oxoid], 2% [wt/vol] mycological peptone [Oxoid], 2% [wt/vol] glucose) at 30°C. Uridine prototrophs were selected on synthetic defined (SD) medium (0.67% [wt/vol] yeast nitrogen base [YNB; Difco], 2% [wt/vol] glucose). Hyphae were induced in 20% (vol/vol) fetal bovine serum (FBS; Sigma) with 2% glucose (wt/vol) at 37°C. E. coli strains were grown in Luria-Bertani (LB) medium (1% [wt/vol] tryptone, 0.5% [wt/vol] yeast extract, 0.5% [wt/vol] NaCl) at 37°C.
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