P. thymicola IBT 5891 was obtained from the IBT culture collection (Kgs. Lyngby, Denmark). A. nidulans FGSC A4 and A. nidulans FGSC A1145 was obtained from Fungal Genetics Stock Center (http://www.fgsc.net/ ). P. thymicola was maintained on PDA (potato dextrose agar, BD) 5 days for sporulation or liquid PDB medium (PDA medium without agar) and SDA (Sabouraud Agar, BD) for gene over-expression, genomic DNA and RNA extraction. A. nidulans was maintained on CD agar for sporulation, or liquid CD-ST medium for gene over-expression, genomic DNA and RNA extraction (http://www.fgsc.net/ ). P. thymicola were maintained on SDA at 24°C, 7 days for the production of penigequinolone A/B.
Sabouraud agar
Manufactured by BD
Sourced in United States, Germany, France
Sabouraud agar is a laboratory culture medium used for the cultivation and isolation of fungi, particularly those that cause mycotic infections. It provides the necessary nutrients and growth conditions for the growth of a wide range of fungal species.
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Lab products found in correlation
Macconkey agar, by BD (3 mentions)
Potato dextrose agar (pda), by BD (2 mentions)
High pure pcr template preparation kit, by Roche (1 mentions)
Lightcycler 480 sybr green 1 master, by Roche (1 mentions)
Gentamycin, by BD (1 mentions)
Chloramphenicol, by BD (1 mentions)
Sabouraud broth, by Thermo Fisher Scientific (1 mentions)
Api yeast identification system, by bioMérieux (1 mentions)
Thoma cell counting chamber, by Marienfeld (1 mentions)
Schaedler agar, by bioMérieux (1 mentions)
Mcconkey agar, by bioMérieux (1 mentions)
Columbia agar, by BD (1 mentions)
Microflex maldi tof mass spectrometer, by Bruker (1 mentions)
Chocolate agar, by bioMérieux (1 mentions)
Sheep blood agar, by BD (1 mentions)
Brain heart infusion broth, by BD (1 mentions)
Sterile container, by Bandelin (1 mentions)
Maldi tof mass spectrometer, by Bruker (1 mentions)
Mueller hinton agar plate, by Thermo Fisher Scientific (1 mentions)
Tryptic soy broth (tsb), by Merck Group (1 mentions)
19 protocols using sabouraud agar
1
Cultivation of Penicillium and Aspergillus
2
Molecular Detection of GBS and E. coli
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For the molecular detection of GBS and E. coli, DNA extraction from the two Copan swabs of each subject was carried out at ITM by thawing the swabs at room temperature for 30 minutes. After adding 1200 μL of diluted PBS, each swab was gently vortexed for 15 seconds, and 1 mL of each swab suspension was pooled into a final volume of 2 mL. An aliquot of 250 μL was extracted using the Abbott m24sp automated extraction platform (Abbott, Maidenhead, UK), according to the manufacturer’s instructions, and 200 μl of eluted DNA—to be used in the quantitative PCR (qPCR) assays—was stored at– 80°C.
For the construction of qPCR standard curves, DNA was extracted from overnight cultures of S. agalactiae LMG 14694T on TSA + 5% sheep blood, E. coli ATCC 25922 grown on TSA + 5% sheep blood, and C. albicans ATCC 90028 grown on Sabouraud agar (all BD). All growth was harvested from the plate and resuspended in 1 ml of saline. DNA of this suspension was extracted using the High Pure PCR Template Preparation Kit (Roche Applied Science, Basel, Switzerland) according to the manufacturer’s instructions.
For capsular genotyping of GBS, 1 ml of inoculated Lim Broth medium (seeMicrobiological culturing ) was used for DNA extraction using the High Pure PCR Template Preparation Kit (Roche), according to the the manufacturer’s instructions.
For the construction of qPCR standard curves, DNA was extracted from overnight cultures of S. agalactiae LMG 14694T on TSA + 5% sheep blood, E. coli ATCC 25922 grown on TSA + 5% sheep blood, and C. albicans ATCC 90028 grown on Sabouraud agar (all BD). All growth was harvested from the plate and resuspended in 1 ml of saline. DNA of this suspension was extracted using the High Pure PCR Template Preparation Kit (Roche Applied Science, Basel, Switzerland) according to the manufacturer’s instructions.
For capsular genotyping of GBS, 1 ml of inoculated Lim Broth medium (see
3
Quantifying C. albicans in Vaginal DNA
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To detect C. albicans in vaginal DNA extracts, a C. albicans specific qPCR was carried out, using primers targeting the ITS-1 gene (adapted from [25 (link)]). The qPCR reactions were performed in a final volume of 10 μl, containing 5 μl of LightCycler 480® SYBR Green I Master (Roche), 0.3 μM of both forward primer CA_FW (5’-CAACGAACTGAACTGGCAGA-3’) and reverse primer CA_RV (5’- CATTACGCTGCGATGGAT -3’) (Eurogentec) and 2 μl of DNA extract or 2 μl of HPLC water (as negative template control). Cycling conditions were as follows: 50°C for 2 min, 95°C for 10 min; 40 cycles of 95°C for 15 s and 60°C for 1 min. A standard series (using C. albicans ATCC 90028 grown on Sabouraud agar (BD)), was constructed as described for S. agalactiae.
4
Standardized C. albicans Growth and Identification
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Yeasts were grown aerobically at 37°C on Sabouraud agar with 0.4 g/l chloramphenicol and 0.04 g/l gentamycin (BD Diagnostics, Franklin Lakes, NJ, USA). All in vitro investigations were conducted on a third subculture of C. albicans ATCC 10231 (Oxoid; Thermo Fisher Scientific, Inc., Waltham, MA, USA) suspended in Sabouraud broth (cat. no. CM147; Oxoid™; Thermo Fisher Scientific, Inc.) or in distilled water. The suspension was adjusted to 1–20×106 blastoconidia per ml by dilution, following a blastoconidia count using a Thoma cell counting chamber (Marienfeld™, Lauda-Königshofen, Germany). Wild strains were isolated by swabbing from dentures and identified on the basis of their colony aspect on CHROMagar™ medium (BD Diagnostics), by chlamydoconidia formation on BT™ Rice Extract agar (BD Diagnostics) and by the API yeast identification system (bioMérieux, Marcy-l'Etoile, France).
5
Comprehensive Microbial Isolation and Identification
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All samples (ESwabs) were streaked and incubated for 24h and 48h at 36°C (6% CO2) on various media. Five media were used for aerobic isolation: Columbia-Agar (with 5% sheep blood) (BD Diagnostic, Heidelberg, Germany), Chocolate-Agar (BioMérieux, Nürtingen, Germany), McConkey- Agar (BioMérieux), Burkholderia cepacia-Spezial-Agar (7 days, 36°C) (BD), Sabouraud- Agar (7 days, 36°C) (BD). Additionally, two media were used for anaerobic isolation (36°C): Schaedler-Agar (BioMérieux) and Kanamycin-Vancomycin-Agar (BD). Anaerobic cultures were processed within an hour after receiving the sample. Once colonies were isolated, they were identified with a Microflex MALDI-TOF mass spectrometer (Bruker Daltonik, Bremen, Germany) [28 (link)].
6
Cultivation of Penicillium and Aspergillus
7
Microorganism Identification from Stent Surfaces
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The prepared stent was put into a sterile container (Bandelin, Germany) and completely covered with 5–10 mL of Brain Heart Infusion Broth (Becton Dickinson, Franklin Lakes, NJ, USA). To disrupt the advice microorganism on the inner surface of the stent, the specimen was vortexed for 30 s and subsequently exposed to low-frequency (40 kHz) ultrasound for 15 min [26 (link)]. Thereafter, the container was vortexed again for 30 s. Aliquots of the sonication fluid were cultivated on conventional solid media, 5% (v/v) sheep blood agar, MacConkey agar and Sabouraud agar (Becton Dickinson). The plates were incubated at 37 °C in an aerobic atmosphere overnight. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometer (Bruker Daltonics, Billerica, MA, USA was used for genus and species of microrganism [27 (link)].
8
Preparation of Microbial Suspensions for Testing
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Bacteria and yeasts deep frozen for storage were grown on Mueller–Hinton agar plates (Oxoid, Hampshire, UK) and subcultivated overnight in tryptic soy broth (Merck) at 37 °C. Subsequently, they were washed twice in 0.9% saline before use. Strains used were Staphylococcus aureus ATCC 25923 and 6538, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 11229, and Candida albicans CBS 5982 (60% pseudohyphae and 40% blastoconidia). Aspergillus fumigatus ATCC 204305 was grown on Sabouraud agar (Becton & Dickinson, Heidelberg, Germany) for 72 h. Suspensions of conidia were gained by harvesting them from the agar plates with 5.0 ml of 0.9% saline plus 0.01% Tween 20, followed by 10-µm filtration (CellTrics; Partec GmbH, Görlitz, Germany) to gain a pure conidia suspension without hyphae and three washing steps in phosphate-buffered saline (Lackner et al. 2015 (link)).
9
Microbial Assessment of Air Quality
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Three different media were used to grow a wide spectrum of microorganisms: Columbia 5% Sheep Blood Agar (CAB, Becton Dickinson), Tryptic Soy Agar (TSA) (BIOMÉRIEUX), and Sabouraud Agar (SAB, Becton Dickinson). A microbiological assessment of the air in the control and experimental rooms was performed twice (before and after the experiment), and was based on three replicates of each medium at three measurement points. The TSA plates were incubated for 72 h at 37° C and a further 14 days at 28 °C. Based on the plate analysis of the number of colony-forming units (CFU/m3), the number of microorganisms in 1 m3 of air was determined.
10
Morphological Comparison of Mold Species
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To assess morphological differences between M. capitatus and M. clavatus that could be useful for identification, all isolates were streaked onto the following seven different agars and incubated for 1 to 5 days at 35°C: Columbia blood agar, chocolate agar, Sabouraud agar, or Difco malt extract agar (all Becton Dickinson, Heidelberg, Germany); ChromAgar Orientation (CHROMagar, Paris, France), inhibitory mold agar (in house); and ChromID CPS agar (bioMérieux).
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