Blood cultures from 217 patients were processed according to routine standard operating procedures in each of the referring laboratories. When available, multiple isolates were collected from some of these patients within the observation period of 30 days. All clinical isolates obtained during this period were independently identified by the use of Colorex Candida chromogenic plates (E&O Laboratories, Bonnybridge, UK), as confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry at the Public Health England Southwest Laboratory (Bristol), and were stored in Microbank vials (Pro-Lab Diagnostics, Bromborough, UK) at −80°C until further use. These isolates were subcultured on Sabouraud's dextrose agar (Sigma-Aldrich, Poole, UK). Plates were incubated at 30°C for 48 h, and maintained at 4°C.
Sabouraud s dextrose agar
Manufactured by Merck Group
Sourced in Germany
Sabouraud's dextrose agar is a culture medium used for the isolation and cultivation of fungi. It is composed of dextrose, peptone, and agar, and provides a nutrient-rich environment suitable for the growth of a variety of fungal species.
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Lab products found in correlation
Candida medium, by CHROMagar (2 mentions)
Microbank vial, by Pro-Lab Diagnostics (1 mentions)
Tryptic soy broth (tsb), by Merck Group (1 mentions)
Id 32, by bioMérieux (1 mentions)
Glycerol, by Merck Group (1 mentions)
α tocopherol, by Merck Group (1 mentions)
Milli q system, by Merck Group (1 mentions)
Acetonitrile, by Avantor (1 mentions)
Cholesterol, by Avanti Polar Lipids (1 mentions)
Soybean phosphatidylcholine, by Avanti Polar Lipids (1 mentions)
Nutrient broth, by Merck Group (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Mueller hinton agar (mha), by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Gentamicin, by Merck Group (1 mentions)
Cornmeal agar, by BD (1 mentions)
Corn meal agar, by Merck Group (1 mentions)
Chloramphenicol, by Merck Group (1 mentions)
8 protocols using sabouraud s dextrose agar
1
Candida Isolate Collection and Identification
2
Isolation and Identification of Candida Strains
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Fifty clinical isolates of Candida (16 strains of C. albicans and 34 strains of C. glabrata) were isolated from stool, urine, blood, wound, catheters, sputum and throat swabs. They were identified by ID32 (bioMerieux) test in the Department of Microbiology of the Wrocław Medical University. All strains were preserved in −80 °C in Tryptic Soy broth (Sigma-Aldrich), supplemented with 10 % glycerol (Sigma-Aldrich) and subcultured onto Sabouraud’s dextrose agar (Sigma-Aldrich) for 24 h to ensure viability and purity prior testing.
3
Quantification of Voriconazole and Metabolite
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Cholesterol and soybean phosphatidylcholine (PC) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Voriconazole (VOR) was purchased from Hangzhou Dayangchem Co., Limited (Hangzhou, China). and voriconazole-N-oxide (VNO) was purchased from Genone Biotechnologies (Rio de Janeiro, RJ, Brazil) Alpha-tocopherol and Sabouraud’s dextrose agar were purchased from Sigma Aldrich (St. Louis, MO, USA). Acetonitrile was purchased from J. T. Baker (Phillipsburg, NJ, USA). Water was purified using a Milli-Q system (Millipore, Billerica, MA, USA) with a 0.22 µm pore end-filter. All other chemicals and reagents were of analytical grade or superior.
4
Amaranthus cruentus Linn Hemagglutinin Purification
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Mueller Hinton agar, Nutrient broth, Luria Bertani broth, Gentamicin, Sabouraud's Dextrose Agar, DMEM, Streptomycin, Dimethyl sulphoxide (DMSO), Crystal Violet, were of analysis grade commercially available from Sigma Chemical Co., St. Louis, MO, USA. A voucher specimen was put into Kerala University Botanical Herbarium with voucher No. KUBH 7501. Amaranthus cruentus Linn seeds were weighed and homogenised using mortar and pestle in 5mM PBS (PH 7.4); centrifuged, filtered and the supernatant was collected. The supernatant was subjected for ammonium sulphate (0-60%) precipitation and kept for half an hour at 4ºC. After the period, the centrifuged precipitate so obtained was dissolved in minimum volume of buffer (1mL PBS in each tube). Dialysis was done against 1mM PBS at 4ºC with a three times buffer change with constant stirring in a magnetic stirrer. The dialysed sample was subjected to gel filtration using Sephadex G-75 1.5 x 75 cm column equilibrated with 5mM PBS. Loaded the column with 1mL sample and allowed to pass into the gel matrix. Elution was done with PBS and 2mL fractions were collected. Absorbance of the eluted samples were measured at 280nm with PBS as blank. An elution profile is plotted with OD at 280nm against number of fractions. Each fraction was tested for heamagglutination.
5
Vulvovaginitis Infection Susceptibility
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During a cross-sectional study, from Feb 2018 to Jul 2018, 150 patients with vvulvovaginitis symptoms such as itching, burning sensation, pain, inflammation, and excessive and stinging discharge were enrolled in this study. Laboratory test for determining the presence of infection and evaluation the in vitro susceptibility to three anti-fungal agents was done in Shahroud University of Medical Sciences, Iran.
In the first step, demographic data of the patients were obtained through questionnaires, and then the collection of posterior vaginal fornix or vaginal wall specimens by the senior clinicians was done for laboratory testing. Patients who were previously treated with anti-fungal drugs within two months ago were excluded from the study. Samples were prepared by KOH 10%, cultured on Sabouraud’s dextrose agar (Merck, Germany), and kept at 37 °C for one week. Isolates were identified based on production chlamydoconidia in cornmeal agar (Becton, France) and colony color on chromogenic CHROMagar Candida medium (CHROMagar, Paris, France). In addition, for confirmation of species identification isolates were subjected to the polymerase chain reaction– restriction fragment length polymorphism (PCRRFLP) technique (11 (link)).
In the first step, demographic data of the patients were obtained through questionnaires, and then the collection of posterior vaginal fornix or vaginal wall specimens by the senior clinicians was done for laboratory testing. Patients who were previously treated with anti-fungal drugs within two months ago were excluded from the study. Samples were prepared by KOH 10%, cultured on Sabouraud’s dextrose agar (Merck, Germany), and kept at 37 °C for one week. Isolates were identified based on production chlamydoconidia in cornmeal agar (Becton, France) and colony color on chromogenic CHROMagar Candida medium (CHROMagar, Paris, France). In addition, for confirmation of species identification isolates were subjected to the polymerase chain reaction– restriction fragment length polymorphism (PCRRFLP) technique (11 (link)).
6
Oral Candidiasis in Hematologic Malignancies
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Fifty swabs were collected from the mouths of patients with hematologic malignancies undergoing chemotherapy from the oncology units of the teaching hospitals (Afzalipour, Shahid-Bahonar and Shafa Hospitals) in Kerman, Iran, from March 2017 to February 2018. Patients were from Kerman, Sistan-Baluchestan and Hormozgan provinces of Iran. Fifty samples including 13 patients with acute lymphoid leukemia (ALL); 13 patients, acute myeloid leukemia (AML); 13 patients, chronic lymphoid leukemia (CLL); 5 patients, chronic myeloid leukemia (CML); 5 patients, Hodgkin's lymphoma (HL); 4 patients and non-Hodgkin's lymphoma (NHL); 10 patients.
Samples were collected and transferred to the medical mycology laboratory of Kerman University of Medical Sciences. Mouth swabs were subjected to direct examination with 20% KOH and cultured on Sabouraud's dextrose agar (Merck, Germany) containing chloramphenicol (0.5 μg/mL) (Merck KGa A, Darmstadt, Germany). All isolates were presumptively identified by phenotypic methods such as the color of colonies on CHROMagar Candida medium (CHROMagar, India, Cat no: 212961), germ-tube formation in serum and production of chlamydoconidia in corn meal agar (Merck, Germany) with 1% Tween 80.
Samples were collected and transferred to the medical mycology laboratory of Kerman University of Medical Sciences. Mouth swabs were subjected to direct examination with 20% KOH and cultured on Sabouraud's dextrose agar (Merck, Germany) containing chloramphenicol (0.5 μg/mL) (Merck KGa A, Darmstadt, Germany). All isolates were presumptively identified by phenotypic methods such as the color of colonies on CHROMagar Candida medium (CHROMagar, India, Cat no: 212961), germ-tube formation in serum and production of chlamydoconidia in corn meal agar (Merck, Germany) with 1% Tween 80.
7
Isolation and Identification of M. canis Dermatophytes
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The samples collected from skin and hairs were cultured on Sabouraud's dextrose agar (Merck Co.) medium containing chloramphenicol (0.05 g/L) and cycloheximide (0.5 g/L) (SCC) under sterile conditions. The plates were kept in a warm incubator at a temperature of 28°C for 14 days. The inoculated plates were checked every 3 days for any growth of dermatophytes. Ten fungal isolates of M. canis were identified and subcultured for further experiments. M. canis isolates were identified based on their cultural morphology and microscopic features.
8
Fungal Growth Cultivation Protocol
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The samples obtained were cultured in two series: sabouraud's dextrose agar (Merck) containing chloramphenicol (50 mg/L) + cycloheximide (500 mg/L) (SCC) and sabouraud glucose agar without cycloheximide (SC). The incubation temperature was 25–35°C for four weeks, and the cultures were monitored daily for observation of any colony‐forming growth. The mucoid colony represented the yeast fungi, and the mold colony revealed the filamentous fungi.
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