The cellulolytic activity was determined as the ability of the fungal strains to degrade carboxymethylcellulose (CMC). The culture was carried out in CMC agar containing carboxymethylcellulose (CMC) 10 g/L (Sigma-Aldrich, St. Louis, MO, USA), NaNO3 6.5 g/L (POCH, Gliwice, Poland), K2HPO4 6.5 g/L (POCH, Gliwice, Poland), yeast extract 0.3 g/L (Difco, Sparks, USA), KCl 6.5 g/L (POCH, Gliwice, Poland), MgSO4·7H2O 3.0 g/L (Chempur, Piekary Śląskie, Poland), and agar 15 g/L (Biomaxima, Lublin, Poland). After the growth period, 1% Congo Red (Park Scientific, Northampton, UK) was added to the plates and incubated for 30 min at 20 °C. The excess dye was then poured off and the plates were flooded again with 1 M NaCl (POCH, Gliwice, Poland) to wash off the excess dye for 30 min at 20 °C. After this time, the excess solution was poured off and the emerging Echo zones were observed [69 (link)].
Agar
Manufactured by Biomaxima
Sourced in Poland
Agar is a solidifying agent derived from red algae. It is commonly used as a culture medium for the growth and isolation of microorganisms in microbiological laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Mgso4 7h2o, by Chempur (4 mentions)
Glucose, by Chempur (2 mentions)
Kh2po4, by Chempur (2 mentions)
Bacto peptone, by Merck Group (2 mentions)
Yeast extract, by Merck Group (1 mentions)
Agar, by Merck Group (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Pipes buffer, by Merck Group (1 mentions)
K2hpo4, by Chempur (1 mentions)
Mls 3781l, by Panasonic (1 mentions)
6 protocols using agar
1
Determination of Cellulolytic Activity
2
Yeast and Bacterial Strain Maintenance
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All strains used in this study are listed in Tab.S1. Y. lipolytica and E. coli strains were maintained as described in [25 (link),26 ]. E. coli JM109 strain, used for sub-cloning, and its derivatives were grown at 37 °C with 250 rpm shaking in LB medium ((g L − 1): yeast extract (BTL, Lodz, Poland), 5; bactopeptone (BTL), 10; NaCl (POCh, Gliwice, Poland), 5), supplemented with kanamycin (Sigma Aldrich, Merck KGaA, St. Louis, USA, 40 (μg mL−1)) and agar (Biomaxima, Lublin, Poland; 15 (g L − 1)), when required. Y. lipolytica Po1f strain (ura‐ leu‐, ATCC MYA2613) was used as a parental strain for co-transformations. Y. lipolytica Po1h strain (ura-, leu+) transformed with a URA3 marker cassette was used as a negative control strain for assessment of background fluorescence. Yeast strains were routinely maintained in a minimal yeast nitrogen base medium (YNB; (g L − 1): YNB (Sigma-Aldrich, Merck KGaA), 1.7; (NH4)2SO4 (PoCh), 5; glucose (PoCh), 20), or in a rich yeast extract-peptone-dextrose medium (YPD; (g L − 1): yeast extract, 10; bactopeptone, 20; glucose, 20), solidified with agar (15 (g L − 1)) when required, at 30 °C and with 250 rpm shaking, for liquid cultures.
3
Bacterial Culture Protocols
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The bacterial strains used in this study were Escherichia coli MC1061 and Staphylococcus aureus ATCC 29213 from the collection of the Institute of Microbiology, Department of Biology, UW. LB medium—Lysogeny Brith (BioMaxima, Lublin, Poland) was used for bacterial culture. Solid medium was obtained after solidifying LB with 1.5% agar (BioMaxima, Lublin, Poland).
4
Ferric Chelate Compounds Production Protocol
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The medium used to determine the ability to produce FeCCs was prepared according to the method proposed by Schwyn and Neilands [72 (link)]. The CAS agar was prepared in 860 mL of 0.1 M PIPES buffer (Merck, Darmstadt, Germany). The medium contained glucose 4.0 g/L (Chempur, Piekary Śląskie, Poland), KH2PO4 3 g/L (Chempur, Piekary Śląskie, Poland), NaCl 0.5 g/L (POCH, Gliwice, Poland), NH4Cl 1 g/L (POCH, Gliwice, Poland), MgSO4·7H2O 0.2 g/L (Chempur, Piekary Śląskie, Poland), and agar 15.0 g/L (Biomaxima, Lublin, Poland). Solutions of 10% acidic casein hydrolysate (POCH, Gliwice, Poland) (30 mL), 0.01 M CaCl2 (POCH, Gliwice, Poland) (10 mL), and a dark blue solution of CAS-complex (100 mL) prepared by mixing 60.5 mg chromazurol S (CAS) (Fulka, Göteborg, Sweden) (50 mL), 1 mM FeCl3·6H2O (POCH, Gliwice, Poland) in 10 mM HCl (POCH, Gliwice, Poland) (10 mL), and 72.9 mg of detergent—hexadecyltrimethylammonium bromide (HDTMA) (Sigma-Aldrich, Hamburg, Germany) (40 mL) were sterilised separately and added after autoclaving (MLS3781L, Sakata, Oizumi–Machi, Ora–Gun, Gunma Panasonic, Japan).
5
Amylolytic Activity Determination of Fungal Strains
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The amylolytic activity was determined as the ability of the fungal strains to degrade starch. The culture was conducted on Starch medium (AM agar) containing soluble starch 10 g/L (POCH, Gliwice, Poland), KH2PO4 0.5 g/L (Chempur, Piekary Śląskie, Poland), K2HPO4 0.5 g/L (Chempur, Piekary Śląskie, Poland), MgSO4·7H2O 0.2 g/L (Chempur, Piekary Śląskie, Poland), (NH4)2SO4 0.2 g/L (POCH, Gliwice, Poland), and agar 15 g/L (Biomaxima, Lublin, Poland). After the growth period, Lugol’s iodine (POCH, Gliwice, Poland) was added to the plates and incubated for 30 min at 20 °C. After this time, the excess solution was poured off and the emerging Echo zones were observed [71 (link)].
6
Phosphate Solubilization Assay Protocol
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The phosphate-solubilising activity was determined as the ability of the fungal strains to degrade insoluble phosphate forms. The culture was conducted on PS medium (PS agar) containing glucose 10 g/L (Chempur, Piekary Śląskie, Poland), asparagine 1 g/L (POCH, Gliwice, Poland), casein hydrolysate 0.2 g/L (POCH, Gliwice, Poland), MgSO4·7H2O 0.4 g/L (Chempur, Piekary Śląskie, Poland), K2SO4 0.2 g/L (POCH, Gliwice, Poland), and agar 15 g/L (Biomaxima, Lublin, Poland) in 800 mL of H2O. Solutions of Na3PO4·12H2O 10 g/100 mL (POCH, Gliwice, Poland) and CaCl2 22 g/100 mL (POCH, Gliwice, Poland) were sterilised separately and added after autoclaving (MLS3781L, Panasonic, Japan) [70 ].
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