Folin–Ciocalteu’s reagent, 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonate) diammonium salt (ABTS), 6-hydroxy-2,5,7,8-tetramethyl-2-carboxylic acid (Trolox), 2, 2-diphenyl-1-picrylhydrazyl (DPPH), ferric chloride (FeCl3), sodium carbonate (Na2CO3), gallic acid (GA), and Ethanol were purchased from Sigma-Aldrich (Milan, Italy). Mueller–Hinton broth (MHB), Rose Bengal Chloramphenicol Agar (RBCA), Malt Extract Agar (MEA), Tryptic Soy Agar (TSA), Sabouraud Dextrose Agar (SDA), RPMI (Roswell Park Memorial Institute) 1640 medium, and purity-grade organic solvents (Ethanol, and Dimethyl Sulfoxide) were purchased from Sigma (Sigma-Aldrich, Milan, Italy).
Malt extract agar
Manufactured by Merck Group
Sourced in Germany, United States, United Kingdom, Italy
Malt extract agar is a microbiological growth medium used for the cultivation and isolation of various microorganisms, particularly yeasts and molds. It provides essential nutrients and a solidified substrate for the growth and development of these microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Potato dextrose agar (pda), by Merck Group (4 mentions)
Sabouraud dextrose agar, by Merck Group (3 mentions)
Ethanol, by Merck Group (3 mentions)
Malt extract, by Merck Group (3 mentions)
Mas 100 eco air sampler, by Merck Group (3 mentions)
Plate count agar, by Merck Group (3 mentions)
Tryptic soy agar, by Merck Group (2 mentions)
Rose bengal chloramphenicol agar, by Merck Group (2 mentions)
Avicel, by Merck Group (2 mentions)
Mueller hinton broth (mhb), by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Sodium chloride (nacl), by Merck Group (2 mentions)
2 6 dimethoxyphenol, by Merck Group (2 mentions)
Tryptic soy broth (tsb), by Merck Group (2 mentions)
Plate count agar, by BD (2 mentions)
Peptone, by Carl Roth (2 mentions)
Microcrystalline cellulose, by Thermo Fisher Scientific (2 mentions)
Formic acid, by Merck Group (2 mentions)
Methanol, by Merck Group (2 mentions)
Glycerol, by Merck Group (2 mentions)
56 protocols using malt extract agar
1
Antioxidant and Antimicrobial Assays
2
Isolation and Cultivation of Termitomyces goniospermum
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The fruiting bodies of the strain T. goniospermum PeruMyc2084 were collected in Pian Grande (Castelluccio di Norcia, PG, Italy) in June 2018. The Vaucher specimen (PeruMyc 2084) was identified on the basis of macromorphological and micromorphological features [28 ] and was deposited in the herbarium at the University of Perugia (Department of Chemistry, Biology and Biotechnology). In order to isolate mycelium in a pure culture, small pieces of context (about 10 mm3) were aseptically drawn from the fresh fruiting bodies and inoculated into Rose Bengal Chloramphenicol agar [30 ].
Incubation was performed in the dark at 25 °C for 14 days. Mycelium discs (5 mm in diameter) were then inoculated in each Petri dish [placed in the center of the Malt Extract Agar (Sigma-Aldrich, Milan, Italy) medium] under aseptic conditions. Free mycelium was obtained by culturing the isolated strain in 250 mL Erlenmeyer flasks containing 50 mL of 2% Malt Extract Broth (Sigma-Aldrich, Milan, Italy) supplemented with 1% glucose, pH 5.5. Four agar plugs (9 mm in diameter) were drawn from seven-day old cultures of T. goniospermum on Malt Extract Agar and inoculated into each flask. Incubation was performed statically at 25 °C for 10 days.
Incubation was performed in the dark at 25 °C for 14 days. Mycelium discs (5 mm in diameter) were then inoculated in each Petri dish [placed in the center of the Malt Extract Agar (Sigma-Aldrich, Milan, Italy) medium] under aseptic conditions. Free mycelium was obtained by culturing the isolated strain in 250 mL Erlenmeyer flasks containing 50 mL of 2% Malt Extract Broth (Sigma-Aldrich, Milan, Italy) supplemented with 1% glucose, pH 5.5. Four agar plugs (9 mm in diameter) were drawn from seven-day old cultures of T. goniospermum on Malt Extract Agar and inoculated into each flask. Incubation was performed statically at 25 °C for 10 days.
3
Cultivation of Trichophyton rubrum Strain
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T. rubrum strain CBS 119892 (WT) was kindly donated by the CBS-KNAW Fungal Collection (Westerdijk Fungal Biodiversity Institute). T. rubrum culture was carried out in Malt Extract Agar (MEA, 2 % malt extract (w/v), 2% glucose (w/v), 0.1% peptone (w/v) – Sigma Aldrich) at 28 °C. Conidia were recovered by scraping the mycelium from 15-day MEA plates flooded with sterilized 0.9% NaCl, followed by vortexing and filtration through glass wool. After centrifugation, the microconidia concentration was estimated by counting on the Neubauer chamber. Other media used in this study were: Sabouraud Dextrose Agar (SDA - 2% dextrose (w/v), 1% peptone (w/v), pH 5.7), Minimal Medium (MM) pH 5.0 (Cove, 1966 (link)), Potato Dextrose Agar (PDA - 0.4% potato extract (w/v) (Sigma Aldrich), 2% dextrose (w/v), pH 5.7), and Keratin Medium (KM - 2.5 g/L keratin powder from hooves and horns - MP Biomedicals, pH 5.5). The solid medium contained 2% agar (w/v).
4
Isolation and Identification of Apple Bacteria
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The sweet apples were collected from Edremit in Türkiye. Tryptic soy agar and malt extract agar were purchased from Sigma-Aldrich, Germany. HCCA Matrix was obtained from Bruker, United States. Crystal violet stain was purchased from Fluka, US.
5
Laccase-Catalyzed Oxidation of Vanilloids
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Potassium iodide (KI), 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), 3′,5-dimethoxy-4′-hydroxyacetophenone (acetosyringone, ACS), 4-hydroxy-3-methoxybenzaldehyde (vanillin), 5-iodovanillin, 2-hydroxy-3-methoxybenzaldehyde (o-vanillin), 4-hydroxy-3-ethoxybenzaldehyde (ethyl vanillin), 4′-hydroxy-3′-methoxyacetophenone (acetovanillone), methyl 4-hydroxy-3-methoxybenzoate (methyl vanillate), 4-hydroxy-3-methoxybenzoic acid (vanillic acid), 4-hydroxy-3-methoxybenzyl alcohol (vanillyl alcohol), α-naphthol, pyrogallol, malt extract agar, buffer components and solvents were purchased from Sigma-Aldrich (Buchs, Switzerland) in standard reagent grade. Freeze-dried preparations of Trametes versicolor laccase were purchased from Sigma-Aldrich and stored at −20°C before use. Activity of the commercial laccase preparations was verified with the substrate ABTS at pH 4 and room temperature and always ranged from 20 to 30 U mg−1.
6
Molecular Cloning Techniques Protocol
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Potato dextrose agar (PDA), yeast extract, sodium chloride, ammonium acetate and tryptone were from Merck chemicals (USA). Mycological peptone, malt extract agar (MEA), agar, water agar, ethanol, sucrose, Whatman No. 1 filter paper and sodium hypochlorite were from Sigma-Aldrich (USA). Not1 enzyme, Taq DNA Polymerase and detection kits were from Thermo™ Scientific (USA). Ampicillin, Isoprophylthio-β-D-galactoside (IPTG), chloroform, agarose gel, 5-bromo-chloro-3-indolyl-β-D-galactopyranoside (X-gal), isoamyl alcohol, glass beads and ethylene-diamine-tetra acetic acid (EDTA) were from Sigma-Aldrich (USA). Pure and ultra-pure water was from Molecular and Cell Biology laboratory, UCT (Millipore LTD, Bedford, MA, USA). Ligations were performed with the pGEM® T Easy vector and T4 DNA Ligase kit (Promega Corporation, USA).
7
Laccase Production from Orange Waste
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Orange waste used for laccase production was donated by a local restaurant and subsequently dried and milled. Cellic CTec2 enzymatic cocktail and commercial laccase (Novozymes NS-22127) were kindly donated by Novozymes® (Bagsvaerd, Denmark). The chromatography columns Hiprep Q FF and Superdex 75 10/300 GL were acquired from GE Healthcare Life Science (Chicago, IL, USA). Aminex HPX-87P column and Precision Plus Protein TM Standards were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Malt Extract Agar, Vanillin, 3,5-dinitrosalicylic acid (DNS), sodium carbonate, and the substrates for enzymatic activities, 3-ethylbenzothiazoline-6-sulphonic acid (ABTS), 2,6-dimethoxyphenol (DMP), xylan beechwood, carboxymethylcellulose (CMC), locust bean, debranched arabinan, β-glucan, p-nitrophenyl-α-L-arabinofuranoside, p-nitrophenyl-β-d -galactopyranoside, p-nitrophenyl-β-d -glycopyranoside, p-nitrophenyl-β-D-xylanopyranoside, and p-nitrophenyl-β-d -cellobioside, were purchased from Sigma-Aldrich (St. Louis, MO, USA). All reagents used for the assays were of analytical grade.
8
Recombinant Trichoderma reesei Cellulase Production
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Escherichia coli DH5α was used for plasmid construction and amplification. The uridine-auxotrophic strain T. reesei TU-6 (ATCC MYA-256) was used as a parental strain for recombinant strain construction throughout this work. All T. reesei strains were maintained on malt extract agar (Sigma-Aldrich, Madison, USA) supplemented with 10 mM uridine when necessary. For cellulase transcription and production analysis, T. reesei strains were pre-cultured in 1-l Erlenmeyer flasks on a rotary shaker (200 rpm) at 30 °C in 250 ml Mandels–Andreotti medium with glycerol (1%, v/v) as the carbon source for 36 h and further grown for another 12 h in the same fresh medium. Mycelia were harvested by filtration and washed twice with fresh Mandels–Andreotti medium with no carbon source. Equal amounts of mycelia were then transferred to a fresh medium containing 1% (w/v) Avicel (Sigma-Aldrich, Madison, USA), and incubation was continued for the indicated times.
9
Genetic Engineering of Trichoderma reesei
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Escherichia coli DH5α was routinely used for plasmid construction and amplification and was cultured in lysogeny broth with a rotary shaker (200 rpm) at 37 °C. A uridine–auxotrophic derivative strain T. reesei QM9414-Δpyr4 [54 (link)] was used as the parent strain for construction of Trrab7 knock-down and xyr1 or eg1 overexpression strains. All T. reesei strains were maintained on malt extract agar (Sigma Aldrich, USA).
For the gene transcription and cellulase production analysis, T. reesei strains were pre-cultured in 250 mL Mandels–Andreotti (MA) medium with 1% (v/v) glycerol as the carbon source for 36 h with a rotary shaker (200 rpm) at 30 °C as previously described [55 (link)]. Mycelia were harvested by filtration using the G1 funnel and washed twice with MA medium without any carbon source. Equal amounts of mycelia were then transferred to fresh MA medium without peptone containing 1% (w/v) Avicel (Sigma-Aldrich, USA) or other carbon sources as indicated, and the culture was continued for the indicated periods. The medium was supplemented with 10 mM uridine, 250 μM arginine, 120 μg/mL hygromycin B, and 20 nM copper ions when necessary.
For the gene transcription and cellulase production analysis, T. reesei strains were pre-cultured in 250 mL Mandels–Andreotti (MA) medium with 1% (v/v) glycerol as the carbon source for 36 h with a rotary shaker (200 rpm) at 30 °C as previously described [55 (link)]. Mycelia were harvested by filtration using the G1 funnel and washed twice with MA medium without any carbon source. Equal amounts of mycelia were then transferred to fresh MA medium without peptone containing 1% (w/v) Avicel (Sigma-Aldrich, USA) or other carbon sources as indicated, and the culture was continued for the indicated periods. The medium was supplemented with 10 mM uridine, 250 μM arginine, 120 μg/mL hygromycin B, and 20 nM copper ions when necessary.
10
Isolation and Characterization of Taxol-Producing Penicillium chrysogenum R16
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Thirty fungal isolates were isolated from the rhizosphere region of many leguminous plants cultivated in Sharkia Governorate (100 km north Cairo), Egypt; they were assayed for taxol production as reported previously [43 ]. The isolate number 16, which was isolated from the rhizosphere region of the plant soybean (Glycin max), was shown to produce the suspected taxol substance. This isolate grew on different media, viz., malt extract agar, yeast sucrose agar, Czapek–Dox agar and potato dextrose agar (all from Sigma) and its cultural characteristics were recorded as reported previously [18 ,44 (link),45 (link)]. Spore characteristics and morphology of hyphae were studied by microscopic examination of fungal hyphae stained with cotton blue in lactophenol. This isolate was characterized as belonging to Penicillium chrysogenum and designated as P. chrysogenum R16 (P. chrysogenum). Slope cultures of this R16 strain were prepared onto potato dextrose agar and stored in a refrigerator at 4 °C throughout the experiments. For inocula preparation, slope growth in test tubes was flooded with sterile peptone water with 0.1% Tween 20 and was gently scratched with a sterile needle loop; the inoculum was adjusted by aid of a hemocytometer at about 105 spores per milliliter [43 ,46 (link)].
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