Basidiomycota isolates of the class Agaricomycetes, order Polyporales, species Fomitopsis pinicola, Phlebia radiata, Trichaptum abietinum, and Junghuhnia luteoalba, and two species of the order Hymenochaetales from the genera Phellinus and Porodaedalea (Table 1 ), were obtained from the University of Helsinki Fungal Biotechnology Culture Collection (HAMBI/FBCC). The fungal strains were originally isolated from basidiocarps growing on decaying wood in boreal forest sites in Finland and identified morphologically. Their identity to species level was confirmed by phylogenetic analysis of the ribosomal DNA ITS region (ITS1+5.8S+ITS2 as the amplified region) (Fig A in S1 File ). One isolate of each species was selected for the co-culture experiments according to even and uniform hyphal growth on 2% malt extract agar (MEA) (2% w/V malt extract, Biokar, France and 2% w/V agar, Yliopiston Apteekki), pH 5.5±0.05, at 25°C, in the dark. The fungal isolates were pre-cultivated on MEA in 9 cm diameter petri dishes at 25°C, in the dark for one week before initiation of the co-cultivations.
Malt extract
Manufactured by Solabia
Sourced in France
Malt extract is a product derived from the malting process of cereal grains, primarily barley. It serves as a key ingredient in various food and beverage applications, providing fermentable sugars and other beneficial compounds.
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Lab products found in correlation
Yeast extract, by Solabia (2 mentions)
Tween 20, by Merck Group (1 mentions)
Potassium dihydrogen phosphate, by Merck Group (1 mentions)
Yeast extract, by Lab M (1 mentions)
Magnesium sulphate, by Merck Group (1 mentions)
Streptomycin sulphate, by Merck Group (1 mentions)
Peptone, by Solabia (1 mentions)
Sucrose, by Merck Group (1 mentions)
Nutrient broth, by Merck Group (1 mentions)
Peptone water, by Thermo Fisher Scientific (1 mentions)
Sodium lauryl sulfate, by Merck Group (1 mentions)
Glucose, by Merck Group (1 mentions)
8 protocols using malt extract
1
Cultivation of Basidiomycota Decomposers
2
Isolating and Cultivating Basidiomycete Fungi
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Isolates of Basidiomycota class Agaricomycetes order Polyporales species Fomitopsis pinicola and Phlebia radiata, and Trichaptum abietinum from the order Hymenochaetales were previously confirmed by internal transcribed spacer (ITS)-sequencing (Mali et al. 2017 ) and are deposited at the University of Helsinki HAMBI mBRC FBCC fungal culture sub-collection (https://www.helsinki.fi/en/infrastructures/biodiversity-collections/infrastructures/microbial-domain-biological-resource-centre-hambi ) (Table 1 ). The fungal isolates were maintained on malt extract agar (MEA) (2% w V−1 malt extract, Biokar Diagnostics, France; 2% w V−1 agar, Sigma; pH 5.5) at 25°C, in the dark for one week before initiating the wood cultivations.
3
Dichomitus squalens Cultivation for Enzyme Production
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Dichomitus squalens FBCC312 (A-670) was obtained from the Fungal Biotechnology Culture Collection (FBCC, email:fbcc@helsinki.fi ), Department of Food and Environmental Sciences, University of Helsinki, Finland. Fungus was maintained on 2% malt agar plates [2% (w/v) malt extract (Biokar, France), 2% (w/v) agar agar (Biokar, France)]. For inoculum, the fungus was cultivated in 75 ml liquid 2% (w/v) malt extract medium in 250 ml Erlenmeyer flasks for 8 days at 28°C, and homogenised in Waring blender. The homogenate (3–4 ml) was used to inoculate 100 ml of 1% Avicel medium, pH 6.0, which contained 2.5 g/l meat peptone (Lab M Limited, UK), 1 g/l yeast extract (Lab M Limited, UK), 1 g/l potassium dihydrogen phosphate (Sigma-Aldrich, Japan), 0.5 g/l magnesium sulphate (Merck, Germany) and 1% (w/v) Avicel® PH-101 cellulose (Fluka, Ireland) with or without 0.25% (w/v) Tween20 (Sigma Aldrich, Germany). The agitated (120 rpm) cultivations were performed in 250 ml baffled flasks and incubated for 29 days at 28°C. The cultivations were sampled three times a week for extracellular enzyme activity measurements. For enzyme purification, the culture liquids were collected after 6 to 10 days of cultivation and frozen at -20°C prior to purification.
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Dichomitus squalens FBCC312 (A-670) was obtained from the Fungal Biotechnology Culture Collection (FBCC, email:
4
Isolation and Identification of Bark Beetle Fungi
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Each bark beetle and phoretic mite was individually crushed and placed onto the surface of malt extract Agar (MEA; 2% malt extract from Biokar Diagnostics, Beauvais, France and 2% agar from Fisher Scientific, Mexico) in Petri dishes containing 0.05 g/l of streptomycin sulphate (Sigma-Aldrich, China). The plates were then incubated at 25°C for 2–4 weeks and observed daily for fungal growth. When fungal growth was observed, spore masses and/or fungal mycelia were transferred to fresh 2% MEA plates (without antibiotics) and sub-cultured until pure cultures were obtained. Purified fungal isolates were then examined under dissection microscope and grouped based on their colony characteristics. Depending on the size of the morphological group, 1–4 isolates from each group were chosen for identification based on DNA sequence comparisons. Representative isolates of ophiostomatoid fungi were deposited in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa.
5
Fungal Isolate Maintenance and Enzyme Production
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Fungal isolates (Table 1 ) were maintained on 2 % (w/v) malt-extract (Biokar Diagnostics, France) agar (2 % w/v agar-agar, Biokar Diagnostics, France) (MEA) plates at room temperature. For extraction of DNA, fungal isolates were cultivated on 2 % MEA plates for 14 days at 28 °C. For the determination of hyphal growth rates, one mycelium agar plug (7 mm in diameter) was inoculated in the center of each 2 % MEA plate and cultivated for 14 days at 28 °C - except in the case of the fungal isolates FBCC297, FBCC464, FBCC1283, FBCC422, FBCC423, FBCC359 and FBCC421, which were cultivated at 22 °C. For enzyme activity production, Phlebia spp. strains were cultivated as semi-solid liquid cultures in three parallel flasks containing 100 ml of low-nitrogen asparagine-succinate medium, pH 4.5 [31 (link), 35 (link)], without glucose but supplemented with 1 g (dry weight) of milled Norway spruce (Picea abies) wood as the sole carbon source. The semi-solid cultures were inoculated with four mycelial agar plugs (7 mm in diameter) from 7–14 days grown MEA plates, and incubated for 21 days at 28 °C in the dark as stationary cultures.
6
Patulin Biodegradation by Selected Yeasts
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The patulin biodegradation ability of the previously selected yeasts was tested adapting the protocol of Reddy et al. (2011) [47 (link)]. 20 mL of peptone malt extract broth (PM) (1% sucrose (Sigma, Buchs, Switherland), 0.5% yeast extract, 0.5% peptone, and 0.2% malt extract (Biokar diagnostics, Allonne, France) were poured into 100 mL flasks containing patulin standard from 100 µg/mL in acetonitrile (Libios, Vindry-sur-Turdine, France) in order to achieve a final concentration of 1000 µg/L of patulin. A volume of 100 µL of 108 CFU/mL of each yeast strain were inoculated in different flasks and incubated at 25 °C for 120 h at 200 rpm shaking conditions in the light. Controls consisted of noninnoculated broth supplemented with patulin. Cells were collected by centrifugation at 5000× g for 10 min at 4 °C at different incubation times, 24, 48, 72 h and 120 h. Cell free supernatant (CFS) was used for measuring patulin reduction. The patulin measured in the cell pellet was used to confirm if an adsorption in the cell wall or an absorption into the cell occurs. Four parallel repetitions were performed in triplicate.
7
Microbial Culture Preparation Protocol
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Isolates of E. coli (ATCC 25922), Salmonella spp. and Penicillium spp. were obtained from Microbiology Chair (University of La Plata, Argentina). Candida spp. was obtained from yeast collection of Microbiology Laboratory of CIATI (Center for Research and Technical
Assistance to Agri-food Industry).
Candida spp. was growth in broth malt containing malt extract (1%, Biokar, France), yeast extract (2%, Biokar, France) and glucose (1%, Merck, Germany). E coli and Salmonella spp were grown in a nutrient broth (Merck, Germany). All culture were incubated at 37ºC for 12 hours until reaching concentrations of 10 8 CFU ml -1 determined by optical density (OD).
Then, dilutions 1:10 were prepared from these inocula with sterile 0.1 % of peptone water (Oxoid) to obtain concentrations of 10 7 CFU ml -1 .
The inoculum of Penicillium spp. was prepared by growing the fungi on agar potato dextrose agar slants (Merck, Germany) for 7 days at 30ºC. After incubation, 10 ml of 0.01% (w/v) sodium lauryl sulfate (Merck, Germany) in 1% (w/w) sodium chloride solution were added to the tubes and spores were loosened by gently scraping with a spatula, and serial dilutions were made [34] . The cells were counted in a haemocytometer and diluted to a concentration of 10 5 spores ml -1 . Besides, a dilution 1:10 was also prepared with sterile 0.1% of peptone water (Oxoid).
Assistance to Agri-food Industry).
Candida spp. was growth in broth malt containing malt extract (1%, Biokar, France), yeast extract (2%, Biokar, France) and glucose (1%, Merck, Germany). E coli and Salmonella spp were grown in a nutrient broth (Merck, Germany). All culture were incubated at 37ºC for 12 hours until reaching concentrations of 10 8 CFU ml -1 determined by optical density (OD).
Then, dilutions 1:10 were prepared from these inocula with sterile 0.1 % of peptone water (Oxoid) to obtain concentrations of 10 7 CFU ml -1 .
The inoculum of Penicillium spp. was prepared by growing the fungi on agar potato dextrose agar slants (Merck, Germany) for 7 days at 30ºC. After incubation, 10 ml of 0.01% (w/v) sodium lauryl sulfate (Merck, Germany) in 1% (w/w) sodium chloride solution were added to the tubes and spores were loosened by gently scraping with a spatula, and serial dilutions were made [34] . The cells were counted in a haemocytometer and diluted to a concentration of 10 5 spores ml -1 . Besides, a dilution 1:10 was also prepared with sterile 0.1% of peptone water (Oxoid).
8
Microorganism Isolation and Identification
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Supernatants were stained with crystal violet and observed under the microscope. Isolation of the main microorganism distinguished (lactic acid bacteria (LAB), yeasts and molds) was performed in MRS (Difco, Detroit, USA), YGC (Merck, D-64271 Darmstadt, Germany) and Malt extract (Biokar, Beauvais, France) agar plates, respectively. All incubations were carried in aerobic conditions: 48 h at 30 °C for LAB and yeasts, and 5 days at 30 °C for fungus. Single colonies isolated in MRS and YGC were Gram stained. For mold screening, spores from the surface of each morphologically different colony obtained were scrapped off, extended and stained with lacto phenol cotton blue (Tiwari, Hoondal, & Tewari, 2009) .
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