Brilliance candida agar, by Thermo Fisher Scientific - Product details

1

Candida spp. Isolation and Identification Protocol

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CAT tubes were vortexed for 10 s to release all cells from swabs. Swabs were subsequently pressed against the tube wall to release all free liquid and then disposed of. One millilter of sample was transferred to a 2 mL microfuge tube and frozen at −80 °C until DNA extraction. The remaining sample (approximately 900 μL) was incubated at 37 °C for 24 h to enrich for low cell titres of Candida spp. Following incubation, two 10 μL loops of sample were plated onto Candida Brilliance agar (Oxoid, Thebarton, South Australia, Australia) and incubated at 37 °C for 72 h.
Positive cultures on Candida Brilliance agar (Oxoid) were classified as follows: Green colonies = C. albicans; pink/yellow/beige/brown colonies = non-albicans Candida spp. All positive cultures were re-plated for purity and following incubation, pure cultures were re-suspended in 2 mL of Sabaraud-Dextrose broth (Oxoid) and frozen at −80 °C.

Payne M.S., Ireland D.J., Watts R., Nathan E.A., Furfaro L.L., Kemp M.W., Keelan J.A, & Newnham J.P. (2016). Ureaplasma parvum genotype, combined vaginal colonisation with Candida albicans, and spontaneous preterm birth in an Australian cohort of pregnant women. BMC Pregnancy and Childbirth, 16, 312.

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2

Identification and Typing of Candida Isolates

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Candida isolates from clinical, patient and staff screening and environmental swabs were plated on Sabouraud dextrose agar plates and identified using Chromogenic ager (Brilliance Candida Agar, Thermo Scientific, Basingstoke, UK). Non-C.albicans isolates including C. auris were speciated by Matrix Assisted Laser Desorption Ionization-Time of Flight mass spectrometry (MALDI-TOF; Bruker, Bremen, Germany) using the Biotyper v3.1 software (Bruker Ltd, Coventry, UK). Antifungal susceptibility testing was done by microbroth dilution (Sensititre YeastOne; Trek Diagnostic Systems Ltd, East Grinstead, UK).
Typing of Candida isolates from representative isolates of a number of global outbreaks was done by AFLP analysis as previously described [7 (link), 8 (link)]. Briefly, genomic DNA was extracted from 48 h liquid cultures using the MasterPure yeast DNA purification kit (Epicentre Biotechnologies, Cambridge, United Kingdom) with an additional bead beating step included. Extracted gDNA was quantified using a Qubit 2.0 fluorometer and dsDNA BR (double-stranded DNA, broad range) assay kit (Life Technologies, Carlsbad, CA, USA).

Schelenz S., Hagen F., Rhodes J.L., Abdolrasouli A., Chowdhary A., Hall A., Ryan L., Shackleton J., Trimlett R., Meis J.F., Armstrong-James D, & Fisher M.C. (2016). First hospital outbreak of the globally emerging Candida auris in a European hospital. Antimicrobial Resistance and Infection Control, 5, 35.

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3

Microorganism Screening Protocol

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The microorganisms were cultured on nalidixic acid/cetrimide agar (Sigma-Aldrich
Chemie GmbH, Switzerland) for the first screening of Pseudomonasspecies, while Mannital Salt agar (Acumedia Manufacturers, USA) was used for the
first screening of Staphylococcus species and Brilliance
Candida Agar (Thermo Fisher Scientific Inc.) was used for the
first screening of Candida species. The nalidixic acid/cetrimide
agar and Mannital Salt agar plates were incubated at 37ºC under aerobic conditions
for 2 days, while Candida-GS agar plates were incubated at 30ºC for
3 days.

PAN J., ZHAO J, & JIANG N. (2014). Oral cavity infection: an adverse effect after the treatment of oral cancer in aged individuals. Journal of Applied Oral Science, 22(4), 261-267.

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4

Candida auris Identification and Antifungal Susceptibility

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Culture-based approaches remain the mainstay of the laboratory diagnosis of C. auris. Candida isolates from clinical swabs were plated on BD Sabouraud Agar with gentamicin and chloramphenicol agar plates (Becton Dickinson GmbH, Heidelberg, Germany) and identified using chromogenic agar with five days at 37 °C implemented for the incubation protocol (Brilliance Candida Agar, Thermo Scientific, Basingstoke, UK). Non-C.albicans isolates including C. auris were identified to the species level by MALDI-TOF (Bruker, Bremen, Germany) using the Biotyper v4.1.100 software. MIC determination was conducted by microbroth dilution according to the standard EUCAST with the commercial method MICRONAUT-AM antifungal agents MIC (Bruker, Bremen, Germany).

Corcione S., Montrucchio G., Shbaklo N., De Benedetto I., Sales G., Cedrone M., Vita D., Costa C., Zozzoli S., Zaccaria T., Silvestre C., Cavallo R., Brazzi L, & De Rosa F.G. (2022). First Cases of Candida auris in a Referral Intensive Care Unit in Piedmont Region, Italy. Microorganisms, 10(8), 1521.

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5

Detecting Candida albicans in blood

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C.albicans in blood cultures was detected as described previously22 (link). Briefly, fetal blood was inoculated into culture vials and incubated at 37 °C for 72 hours. Every day, 1 ml sample was removed and subcultured on sheep blood agar at 37 °C for 48 hours. C.albicans colonial morphology was confirmed by growth on Brilliance Candida Agar (Oxoid, Adelaide, Australia) as previously reported22 (link).

Nikiforou M., Jacobs E.M., Kemp M.W., Hornef M.W., Payne M.S., Saito M., Newnham J.P., Janssen L.E., Jobe A.H., Kallapur S.G., Kramer B.W, & Wolfs T.G. (2016). Intra-amniotic Candida albicans infection induces mucosal injury and inflammation in the ovine fetal intestine. Scientific Reports, 6, 29806.

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6

Standardized Candida albicans Inoculum Preparation

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A single Western Australian clinical isolate of C. albicans was cultured on Difco Sabaraud-Dextrose agar (Becton Dickinson & Co., Franklin Lakes, NJ.) at 37°C for 48 h and single colonies were inoculated into sterile 1 × phosphate buffered saline (Sigma-Aldrich, St. Loius, MO.). C. albicans colony morphology was confirmed by growth on Brilliance Candida Agar (Oxoid). Inoculums were quantified using a plate dilution series as per standard microbiological methods and recorded as CFU/mL. Quantified inoculums (10⁷ CFU in 2 mL 1 × phosphate buffered saline) were stored at −80°C prior to use.

Payne M.S., Kemp M.W., Kallapur S.G., Kannan P.S., Saito M., Miura Y., Newnham J.P., Stock S., Ireland D.J., Kramer B.W, & Jobe A.H. (2014). Intrauterine Candida albicans infection elicits severe inflammation in fetal sheep. Pediatric research, 75(6), 716-722.

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Standardized Candida albicans Inoculum Preparation

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A single Western Australian clinical isolate of C. albicans was cultured on Difco Sabaraud-Dextrose agar (Becton Dickinson & Co., Franklin Lakes, NJ.) at 37°C for 48 h and single colonies were inoculated into sterile 1 × phosphate buffered saline (Sigma-Aldrich, St. Loius, MO.). C. albicans colony morphology was confirmed by growth on Brilliance Candida Agar (Oxoid). Inoculums were quantified using a plate dilution series as per standard microbiological methods and recorded as CFU/mL. Quantified inoculums (10⁷ CFU in 2 mL 1 × phosphate buffered saline) were stored at −80°C prior to use.

Payne M.S., Kemp M.W., Kallapur S.G., Kannan P.S., Saito M., Miura Y., Newnham J.P., Stock S., Ireland D.J., Kramer B.W, & Jobe A.H. (2014). Intrauterine Candida albicans infection elicits severe inflammation in fetal sheep. Pediatric research, 75(6), 716-722.

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8

Candida albicans Isolation and Characterization

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A Western Australian clinical isolate of C. albicans (19) was cultured on Difco Sabaraud-Dextrose agar (Becton Dickinson, Franklin Lakes, NJ) at 37 °C for 48 h and single colonies were inoculated into sterile phosphate-buffered saline (PBS) (Sigma-Aldrich, St. Loius, MO) (18) . C. albicans colonial morphology was confirmed by growth on Brilliance Candida Agar (Oxoid, Adelaide, Australia). Inoculums were quantified using a plate dilution series as per standard microbiological methods and recorded as CFU/ml. Quantified inoculums (10 7 CFU in 2 ml PBS) were stored at -80 °C prior to use. For amniotic fluid culture, 100 µl of fresh amniotic fluid was inoculated onto Difco Sabaraud-Dextrose agar and evenly distributed across the plate with a sterile spreader. Incubation conditions were as described above. For blood culture, 2 ml fetal blood samples were inoculated into BACTEC Peds Plus culture vials (Becton Dickinson, Franklin Lakes, NJ) and incubated aerobically at 37 °C for 72 h. Every 24 h, a 1 ml sample was aseptically removed and 100 µl of this was subcultured on 5% sheep blood agar (Pathwest Laboratories, Perth, Western Australia) at 37 °C for 48 h. For both amniotic fluid and blood cultures, C. albicans colonial morphology was confirmed as described above. RNA extracted from fetal lung was screened using a real-time PCR assay targeting the RNase P RNA gene of C. albicans (19) .

Maneenil G., Payne M.S., Senthamarai Kannan P., Kallapur S.G., Kramer B.W., Newnham J.P., Miura Y., Jobe A.H, & Kemp M.W. (2015). Fluconazole treatment of intrauterine Candida albicans infection in fetal sheep. Pediatric research, 77(6).

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9

Quantitative PCR for Candida albicans

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RNA extracted from fetal lung, skin, chorioamnion and spleen was screened using a real-time PCR assay targeting the RNase P RNA gene of C. albicans ^{29 (link)}. RNA-based reactions were performed using an EXPRESS One-Step SuperScript qRT-PCR Kit (Life Technologies) with 0.5 μM each primer, 0.2 μM probe, 400 ng template RNA and water to a final volume of 20 μL. To enable quantitation of C. albicans within each sample, a standard curve of pure C. albicans (study isolate) RNA was included in each assay at a final concentration of 40, 4 and 0.4 ng per 20 uL reaction. Reaction cycling conditions were as follows: 15 min reverse transcription at 50°C and an initial denaturation at 95°C for 20 s, followed by 40 cycles of 95°C for 3 s and 60°C for 30 s. All reactions were performed in 96 well plates on a ViiA7 real-time PCR thermocycler (Life Technologies). The presence of viable C. albicans in amniotic fluid samples was determined using a Sabaraud-Dextrose agar plate dilution series as described above. Three single colonies from positive plates were subsequently inoculated onto Candida Brilliance agar (Oxoid, Basingstoke, UK) for confirmation of isolate identification.

Payne M.S., Kemp M.W., Kallapur S.G., Kannan P.S., Saito M., Miura Y., Newnham J.P., Stock S., Ireland D.J., Kramer B.W, & Jobe A.H. (2014). Intrauterine Candida albicans infection elicits severe inflammation in fetal sheep. Pediatric research, 75(6), 716-722.

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10

Quantitative PCR for Candida albicans

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RNA extracted from fetal lung, skin, chorioamnion and spleen was screened using a real-time PCR assay targeting the RNase P RNA gene of C. albicans ^{29 (link)}. RNA-based reactions were performed using an EXPRESS One-Step SuperScript qRT-PCR Kit (Life Technologies) with 0.5 μM each primer, 0.2 μM probe, 400 ng template RNA and water to a final volume of 20 μL. To enable quantitation of C. albicans within each sample, a standard curve of pure C. albicans (study isolate) RNA was included in each assay at a final concentration of 40, 4 and 0.4 ng per 20 uL reaction. Reaction cycling conditions were as follows: 15 min reverse transcription at 50°C and an initial denaturation at 95°C for 20 s, followed by 40 cycles of 95°C for 3 s and 60°C for 30 s. All reactions were performed in 96 well plates on a ViiA7 real-time PCR thermocycler (Life Technologies). The presence of viable C. albicans in amniotic fluid samples was determined using a Sabaraud-Dextrose agar plate dilution series as described above. Three single colonies from positive plates were subsequently inoculated onto Candida Brilliance agar (Oxoid, Basingstoke, UK) for confirmation of isolate identification.

Payne M.S., Kemp M.W., Kallapur S.G., Kannan P.S., Saito M., Miura Y., Newnham J.P., Stock S., Ireland D.J., Kramer B.W, & Jobe A.H. (2014). Intrauterine Candida albicans infection elicits severe inflammation in fetal sheep. Pediatric research, 75(6), 716-722.

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Brilliance candida agar

Lab products found in correlation

10 protocols using brilliance candida agar

Candida spp. Isolation and Identification Protocol

Identification and Typing of Candida Isolates

Microorganism Screening Protocol

Candida auris Identification and Antifungal Susceptibility

Detecting Candida albicans in blood

Standardized Candida albicans Inoculum Preparation

Standardized Candida albicans Inoculum Preparation

Candida albicans Isolation and Characterization

Quantitative PCR for Candida albicans

Quantitative PCR for Candida albicans

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