Sabouraud agar

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Sabouraud agar is a growth medium used for the isolation and cultivation of fungi, including yeasts and molds. It provides the necessary nutrients and growth conditions for the optimal growth of fungal species.

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38 protocols using sabouraud agar

Surface and Air Microbial Pollution Assessment

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The CFU of bacteria and fungi on the surfaces were tacked with Count-Tact Applicator (pressure 500 g, 10 s). Rodac contact plates were used for investigation of surface microbial pollution on the surface of 25 cm². The plates contained trypticase soy agar (TSA) for bacteria (Oxoid, UK) and Sabouraud agar (Oxoid, UK) for fungi.
Air samples were taken using Microbial Air Monitoring System MAS-100 (Merck, Germany) based on impaction systems, with sampling times of 10 min equivalent to circulation of 1 m³ of air. Standard Petri dishes were used for growing microorganisms: with blood agar (COS-Columbia Agar + 5% sheep blood, bioMérieux, France) for bacteria and Sabouraud agar (Oxoid, UK) for fungi. During the experiment, the MAS-100 was placed in the middle of seats in such a way that air flowing from the air conditioning system was directed at the MAS-100 head and Petri dishes, with all doors and windows closed.

Bukłaha A., Wieczorek A., Kruszewska E., Majewski P., Iwaniuk D., Sacha P., Tryniszewska E, & Wieczorek P. (2022). Air Disinfection—From Medical Areas to Vehicle. Frontiers in Public Health, 10, 820816.

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Generating Kinase Knockout Mutants in Aspergillus fumigatus

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A. fumigatus MFIG001 was used as the parental isolate to generate kinase knockout mutants [20 (link)]. Knockout cassettes were generated using the protocol from Zhao et al 2019 [25 (link)]. Briefly, 1kb flanking regions of the gene of interest were PCR amplified and fused to a hygromycin resistance cassette via additional fusion PCR. A. fumigatus MFIG001 was cultured in Sabouraud agar (Oxoid, UK) overnight at 37 °Celsius at 120 rpm, followed by protoplasting for 3 hours in Sabouraud agar + VinoTaste Pro solution (freshly filtered in 0.6M KCl, Citric Acid). Protoplasts were filtered through Miracloth, washed twice in 0.6M KCl and resuspended in 0.6 M KCl + 200 mM CaCl2. Fusion PCR product was added to 1×10⁵ protoplasts, followed by addition of PEG. This was incubated on ice for 30 minutes. 600 μL of PEG was added and the mixture was then incubated at room temperature for 10 minutes. Transformation mixture was plated on selective medium (YPS + 100 mg/L hygromycin). Transformants were twice purified on Sabouraud agar + 100 mg/L hygromycin and PCR validated.

van Rhijn N., Zhao C., Al-Furaji N., Storer I., Valero C., Gago S., Chown H., Baldin C., Fortune-Grant R., Shuraym H.B., Ivanova L., Kniemeyer O., Krüger T., Bignell E., Goldman G., Amich J., Delneri D., Bowyer P., Brakhage A., Haas H, & Bromley M. (2023). Functional analysis of the Aspergillus fumigatus kinome reveals a DYRK kinase involved in septal plugging is a novel antifungal drug target. Research Square.

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Antimicrobial Activity of Metal Complexes

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The ~2 × 10⁵ cfu/ml of bacterial or fungal culture were grown for 24 h at 37 °C with 5% CO₂ in stationary culture in Tryptic Soy Broth (Oxoid, USA) or Sabouraud Broth (Oxoid, USA), respectively, in the presence of metal complexes at non-cytotoxic concentrations: 60 µM for [Co(iaa)₂(H₂O)₂]·H₂O, [Co(1-allim)₆](NO₃)₂ and 30 µM for [Cu(iaa)₂(H₂O)], [Cu(1-allim)₄(NO₃)₂]. The Co(II) and Cu(II) ions alone and free ligands (1-allylimidazole and imidazole-4-acetate anion) at 60 µM have been used as controls. Additionally, the microorganisms viability was expressed as colony-forming units (CFU/ml) on Tryptic Soy Agar (Oxoid, USA) or Sabouraud Agar (Oxoid, USA). All samples were measured in three independent experiments.

Gałczyńska K., Ciepluch K., Madej Ł., Kurdziel K., Maciejewska B., Drulis-Kawa Z., Węgierek-Ciuk A., Lankoff A, & Arabski M. (2019). Selective cytotoxicity and antifungal properties of copper(II) and cobalt(II) complexes with imidazole-4-acetate anion or 1-allylimidazole. Scientific Reports, 9, 9777.

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Anti-biofilm Potential of L. multifida Essential Oil

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The antibiofilm potential of L. multifida essential oil was assessed on yeasts and filamentous fungi. Dermatophyte species included both collection strains such as Microsporum gypseum CECT 2908, Trichophyton mentagrophytes var. interdigitale CECT 2958, and T. rubrum CECT 2794, and clinical strains, namely Epidermophytom floccosum FF9, M. canis FF1, and T. mentagrophytes FF7, whereas C. albicans ATCC 10231 was the representative yeast strain tested. Strains were cultured in potato dextrose agar (Oxoid Limited, Hampshire, UK) or Sabouraud agar (Oxoid Limited) at 37 °C for 48 h (C. albicans) or 30 °C for 7 days (dermatophytes).

Alves-Silva J., Zuzarte M., Cavaleiro C, & Salgueiro L. (2023). Antibiofilm Effect of Lavandula multifida Essential Oil: A New Approach for Chronic Infections. Pharmaceutics, 15(8), 2142.

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Characterizing C. tropicalis Isolates

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A total of 68 C. tropicalis isolates were obtained from 3 different general hospitals during the period of August 2013–July 2014 in Beijing, China. These strains belong to the archive collection of the Chinese Centre for Control and Prevention. The origins of these strains were diverse, including 31 from sputum, 10 from urea, 7 from feces, 4 from vaginal secretion, 3 from blood, 3 from drainage, 2 from throat swab, 1 from prostatic secretion, 1 from sanies, and 6 from other unknown. All isolates were identified by internal transcribed spacer (ITS) sequencing and AUX 20C (BioMérieux, Lyon, SA, France) in our lab. The universal primers ITS1 and ITS4 were used to amplify and sequence the ITS fragment in both directions.^{[8 (link)]} The strains were stored at 280°C in brain-heart infusion (Oxoid, Basingstoke, UK). The isolates were maintained on Sabouraud agar (Oxoid) during the study. In vitro enzymatic activities (aspartyl protease, phospholipase, and hemolytic activities), adhesion, and biofilm formation were analyzed for all C. tropicalis isolates.

Zhang L.J., Yu S.B., Li W.G., Zhang W.Z., Wu Y, & Lu J.X. (2019). Polymorphism analysis of virulence-related genes among Candida tropicalis isolates. Chinese Medical Journal, 132(4), 446-453.

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Antibacterial Activity of Pyrazoles

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Pyrazoles’ antibacterial activity was evaluated by using a panel of clinically isolated Gram-positive and Gram-negative bacteria (Staphylococcus aureus DH-432, Streptococcus pyogenes DH-3467, Enterococcus faecalis DH-5478, Pseudomonus aeruginosa DH-5698, Acinetobacter baumannii DH-243, and Escherichia coli DH-5987). The bacterial isolates were obtained from different clinical samples collected by the Central Laboratories, EL-Demerdash Hospital, Ain-Shams University, Cairo, Egypt. The CV026, a Chromobacterium violaceum mutant deficient in production of Nhexanoyl-L-homoserine lactone (C6HSL), which can produce purple pigmentation on an external application of C6HSL, was used as a reporter organism to test for QSI. Microbiological media, tryptic soy (TS) broth or agar, and sabouraud agar were obtained from Oxoid (Hampshire, UK). Dimethyl sulfoxide (DMSO) was used as a solvent for the synthesized derivatives, and the DMSO and other chemicals were of analytical grade.

Ragab A., Fouad S.A., Ammar Y.A., Aboul-Magd D.S, & Abusaif M.S. (2023). Antibiofilm and Anti-Quorum-Sensing Activities of Novel Pyrazole and Pyrazolo[1,5-a]pyrimidine Derivatives as Carbonic Anhydrase I and II Inhibitors: Design, Synthesis, Radiosterilization, and Molecular Docking Studies. Antibiotics, 12(1), 128.

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Antimicrobial Efficacy of Plant Extracts

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The antimicrobial activities of the plant extracts (CFP, CFE and CFM) were determined using the agar well diffusion method. Nutrient agar and Sabouraud agar (Oxoid Limited, United Kingdom) were used. Molten sterile agar (20 mL) was aseptically inoculated with 0.1 mL of a 24 h suspension of the test organisms containing approximately 10⁶ CFU/mL and rolled in the palm to mix thoroughly. The seeded agar was then aseptically transferred into sterile Petri dishes. After the agar was set, cork borer No. 5 (diameter 10 mm) was flamed and used to bore four (4) wells equidistant from each other. The wells were labelled and filled with 0.2 mL of 4 different concentrations (20, 10, 5 and 2.5 mg/mL) of the plant extract. The diameter zones of growth inhibition (the area around the well where no visible microbial growth was observed) were measured using a millimetre rule after 24 h of incubation at 37 °C for bacteria and 72 h at 28 °C for fungus. Ciprofloxacin in concentrations of 4, 2, 1 and 0.5 μg/mL, and ketoconazole in concentrations of 64, 32, 16 and 8 μg/mL were used as standard drugs. Tween 80 (Carl Roth GmbH, Karlsruhe, Germany) was tested to rule out any antimicrobial activity or contamination [20 ]. The above procedure was carried out in three replicates.

Ankomah A.D., Boakye Y.D., Agana T.A., Adu F, & Agyare C. (2021). Antimicrobial properties of Cnestis ferruginea Vahl ex DC. Heliyon, 7(11), e08266.

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Microbiological Analysis of Water and Swab Samples

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The water samples were well mixed and using disposable sterile loops (2 mm inside diameter) each of the samples was inoculated onto MacConkey agar (MA) and blood agar (BA) (Oxoid Ltd., Basingstoke, UK). The swabs were also inoculated (directly) onto MA and BA. The plates were incubated at 37°C for 24 hours. The number of colonies seen was counted using a colony counter and recorded as colony forming unit per gram (cfu/g) for swabs and colony forming unit per millilitre (cfu/mL) for water samples. Bacteria colonies were further subcultured onto MA and BA, to obtain pure cultures and primary identification done using colonial morphology. Further identification was done based on a number of procedures including microscopy, Gram staining, oxidation-fermentation tests, and other biochemical tests such as urease test, catalase test, citrate utilization test, indole test, and triple sugar iron test [20 –23 ].
Similarly, the water samples and swabs were inoculated on Sabouraud agar (Oxoid Ltd., Basingstoke, UK) for fungal growth and subsequent identification using standard protocol [19 ].

Tetteh-Quarcoo P.B., Anim-Baidoo I., Attah S.K., Abdul-Latif Baako B., Opintan J.A., Minamor A.A., Abdul-Rahman M, & Ayeh-Kumi P.F. (2016). Microbial Content of “Bowl Water” Used for Communal Handwashing in Preschools within Accra Metropolis, Ghana. International Journal of Microbiology, 2016, 2617473.

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Automated Microbial Detection in Contrast Media

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For microbiological analysis 1 ml of the contrast medium/ saline specimen was inoculated into BacT/Alert bottles iAST and iNST (bioMérieux, Nuertingen, Germany) for automated microbial detection based on the colorimetric detection of CO₂ produced by growing microorganisms in Casein Soja-Pepton bouillon. The aerobic bottle was incubated at 22±1°C for seven days and the anaerobic bottle at 32±1°C also for seven days based on the method described in the European Pharmacopoeia for sterility testing [10 ]. Automatic readings were performed every 10 minutes. In case of positivity, aliquots of the bouillon were plated for culturing on solid media (Columbia blood agar, Chocolate agar, MacConkey agar, Universal BEER agar, Schaedler anaerobe agar, Sabouraud agar, all from Oxoid, Wesel, Germany). The time from start of incubation of the bottles to the signal of the system of positivity (time to positivity) was calculated. Identification of microorganisms was performed with the mass spectrometry VITEK MS, or the semi-automated platforms VITEK 2 (bioMérieux, Nuertingen, Germany) and WalkAway (Beckman Coulter, Krefeld, Germany).

Goebel J., Steinmann J., Heintschel von Heinegg E., Hestermann T, & Nassenstein K. (2019). Bacterial contamination of automated MRI contrast injectors in clinical routine. GMS Hygiene and Infection Control, 14, Doc05.

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Antimicrobial Activity Assessment of Essential Oils

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One hundred microliters of bacterial suspension after incubation were spread on the Mueller–Hinton agar (MHA, Oxoid) for the agar disc diffusion method. The filter paper discs (6 mm diameter) were infused with 15 µL of the EO, tested, and placed on the inoculated MHA. The MHA was kept at 4 °C for 2 h and then at 37 °C for 24 h aerobically. For yeasts, 100 µL of the microbial suspension was spread onto Sabouraud agar (Oxoid) and cultivated at 37 °C for 24 h. After the incubation period, the diameter of the inhibition zones was measured (mm). The growth inhibition was compared with standard drugs. Tests were performed in three replications.

Semerdjieva I., Zheljazkov V.D., Radoukova T., Dincheva I., Piperkova N., Maneva V., Astatkie T, & Kačániová M. (2021). Biological Activity of Essential Oils of Four Juniper Species and Their Potential as Biopesticides. Molecules, 26(21), 6358.

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