Depending on the weight of single ice cubes (ranging between 10 and 20 g approximately for the different shapes), about 25-50 cubes corresponding to 500 g were left thawing in a 1 l sterile Dhuram bottle at ambient temperature. Two 100 ml aliquots from each sample collected were separately analysed by membrane filtration in order to investigate unicellular and filamentous fungi. Both eukaryotic microbial groups were inoculated on malt agar (Oxoid, Milan, Italy) supplemented with 0Á1 g l À1 chloramphenicol (Sigma-Aldrich, Milan, Italy) to avoid bacterial growth. For the determination of yeasts the plates were incubated at 28°C for 48 h, while for moulds at 25°C for 7 d. When the number of colonies from a given sample exceeded the number of squares (n = 186) present on the membrane grid, or several colonies showed a clear confluent growth, aliquots of 1 ml from this sample were directly inoculated onto malt agar (Oxoid). Two 1-ml aliquots were collected after vigorous manual agitation of thawed ice.
Malt agar
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Malt agar is a culture medium used for the cultivation and enumeration of yeasts and molds. It provides nutrients and a solid substrate for the growth of these microorganisms.
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Lab products found in correlation
4 protocols using malt agar
1
Fungi Isolation from Thawed Ice Cubes
2
Mycological Diagnosis and Aspergillus Detection
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All clinical samples were analysed after conventional microbiological diagnostic tests had been performed. Routine mycological diagnostic tests consisted of culturing on malt agar (Oxoid, Basingstoke, UK) for seven days at 30 °C. In the case of fungal growth, susceptibility testing was performed by ETEST® for VOR (bioMérieux, Marcy-l′Étoile, France) and itraconazole (Liofilchem, Roseto degli Abruzzi, Italy). Minimal inhibitory concentrations were determined in accordance with EUCAST guidelines version 10.0 [20 ]. Furthermore, GM index was determined via Platelia Aspergillus EIA (BioRad, Marnes-la-Coquette, France) from BAL and serum samples, following the manufacturer’s instructions. Results from BAL samples with a GM index ≥1 and from serum samples with a GM index >0.5 were interpreted as positive.
3
Microbial Diversity in Fermented Juice
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Microorganisms responsible for the spoilage of the juice, mostly yeasts and molds, were detected during fermentation. In addition, the microbial counts of lactic acid bacteria (LAB) were also screened in triplicate before and after 24 h and 48 h of fermentation. Therefore, a representative amount of 10 mL from each juice sample was blended with 90 mL of sterilized 1/4 Ringer’s solution (Sigma-Aldrich, St. Louis, MO, USA) and subjected to serial dilutions.
The following tests were performed: (i) lactobacilli [Gram (+), catalase (−)] on acidified MRS agar (Oxoid Ltd., Hampshire, UK) at 37 °C for 48 h anaerobically (Anaerobic jar, Anerocult C, Merck, Rahway, NJ, USA); (ii) yeasts and molds on malt agar (Oxoid Ltd., Hampshire, UK) (pH was adjusted to 4.5 using a sterile solution of 10% lactic acid) at 30 °C for 48 h. All incubations were further extended up to 120 h; however, no extra colonies were observed. Gram staining and catalase tests were performed for LAB confirmation. Results are presented as a log of mean colony-forming units (CFU) per mL of each juice.
The following tests were performed: (i) lactobacilli [Gram (+), catalase (−)] on acidified MRS agar (Oxoid Ltd., Hampshire, UK) at 37 °C for 48 h anaerobically (Anaerobic jar, Anerocult C, Merck, Rahway, NJ, USA); (ii) yeasts and molds on malt agar (Oxoid Ltd., Hampshire, UK) (pH was adjusted to 4.5 using a sterile solution of 10% lactic acid) at 30 °C for 48 h. All incubations were further extended up to 120 h; however, no extra colonies were observed. Gram staining and catalase tests were performed for LAB confirmation. Results are presented as a log of mean colony-forming units (CFU) per mL of each juice.
4
Rasamsonia Detection in Cystic Fibrosis
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In total, 214 samples from 138 CF patients (one to four samples per patient) attending the
University Hospital Essen and the Clinic of the Ruhr, West German Lung Centre, Essen, Germany, in
2012 were tested for the presence of DNA from Rasamsonia species as mentioned
above. For culturing, 100 μL of the sputasol-pretreated respiratory samples were
inoculated on malt agar (Oxoid) and incubated aerobically at 36°C for 2 days and then
at 22°C for an additional 8 days.
In addition, 20 sputum samples from 15 CF patients followed up in Angers University Hospital,
Angers, France, were also analysed. Mycological examination of these samples performed in parallel
to the PCR assay consisted of the inoculation of 10-μL aliquots of the digested samples on
CHROMagar Candida (Becton-Dickinson, Franklin, NJ, USA), yeast extract-peptone-dextrose agar (YPDA)
supplemented with chloramphenicol and gentamicin (Becton-Dickinson), Dichloran-rose
bengale-chloramphenicol agar (DRBC), YPDA supplemented with chloramphenicol and cycloheximide, and
erythritol agar. Incubation was carried out at 37°C for 2 weeks, except for the last
two culture media, which were incubated at 25°C. Mould isolates were identified by cultural
and microscopic morphological characteristics.
University Hospital Essen and the Clinic of the Ruhr, West German Lung Centre, Essen, Germany, in
2012 were tested for the presence of DNA from Rasamsonia species as mentioned
above. For culturing, 100 μL of the sputasol-pretreated respiratory samples were
inoculated on malt agar (Oxoid) and incubated aerobically at 36°C for 2 days and then
at 22°C for an additional 8 days.
In addition, 20 sputum samples from 15 CF patients followed up in Angers University Hospital,
Angers, France, were also analysed. Mycological examination of these samples performed in parallel
to the PCR assay consisted of the inoculation of 10-μL aliquots of the digested samples on
CHROMagar Candida (Becton-Dickinson, Franklin, NJ, USA), yeast extract-peptone-dextrose agar (YPDA)
supplemented with chloramphenicol and gentamicin (Becton-Dickinson), Dichloran-rose
bengale-chloramphenicol agar (DRBC), YPDA supplemented with chloramphenicol and cycloheximide, and
erythritol agar. Incubation was carried out at 37°C for 2 weeks, except for the last
two culture media, which were incubated at 25°C. Mould isolates were identified by cultural
and microscopic morphological characteristics.
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