2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) was purchased from Sigma‐Aldrich, Germany. Folin–Ciocalteau reagent was purchased from Merck Specialities Private Limited, India. Gallic acid was purchased from LOBA Chemie, India. Methanol was purchased from Fisher scientific, India. PCA agar, PDA agar, MRS agar, and VRBA agar media were purchased from Hi‐Media Laboratories, India. The spectrophotometer used was of Model GENESYSTM 10S Vis spectrophotometer from Thermo Scientific TM, Germany.
Pda agar
Manufactured by HiMedia
Sourced in India
PDA agar is a culture medium used for the isolation and enumeration of yeasts and molds. It provides a suitable environment for the growth of these microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Folin ciocalteau reagent, by Merck Group (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (1 mentions)
Gallic acid, by Loba Chemie (1 mentions)
Pca agar, by HiMedia (1 mentions)
Mrs agar, by HiMedia (1 mentions)
Chloramphenicol, by Merck Group (1 mentions)
4 protocols using pda agar
1
DPPH Antioxidant Assay Protocol
2
Identification of Crown Rot Pathogens
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The plants assessed for the level of stembase infestation and with symptoms characteristic for Fusarium, Gaeumannomyces, and Oculimacula were selected for further isolation and identification of causal agents of crown rot. Segments of 5 × 5 mm were removed from the basal part of the first internodes of the selected wheat tillers and were used to assess fungal pathogen species by culturing on a general media of 20% potato dextrose agar (PDA, potato 200 g, dextrose 20 g, agar 15 g) amended with lactic acid PDA. Prior to plating, stem segments were disinfected in sodium hypochlorite at 1% for 2 min and rinsed three times in sterile distilled water for 2 min every time. Five disinfected segments were placed on Water agar media amended with streptomycin sulfate as well as on PDA Agar (HiMedia, Einhausen, Germany) in order to identify predominant fungal species as causal agents of crown rot. Plates were incubated at 24 ± 1°C. Resulting fungal colonies were purified by single-spore isolation prior to final identification and DNA extraction. The identification based on morphological characteristics was performed using the keys of Barnett and Hunter (1972) and Leslie and Summerell (2006) .
3
Isolation and Characterization of Panus Mushroom
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The specimen was collected in the municipality of Santa Fe de Antioquia, located in the department of Antioquia, Colombia (6.56° N 75.83° W, 571 masl), with an average temperature of 28 °C. Sporomes of Panus were collected using an opportunistic approach of convenience sampling [28 ]. Macromorphological characters were described, including the fresh color, according to the Methuen Handbook of Colour [29 ]. Specimens were dried in a food dehydrator and placed in plastic bags for transport. All collections were deposited in the University of Antioquia herbarium (HUA). Isolation and culture were performed using small fragments of the pileus. The obtained pure cultures were preserved in potato dextrose agar medium (PDA agar, Himedia, Mumbai, India) at 4 °C until the beginning of the experiments. The strain was deposited in the Microorganism Collection of the School of Microbiology CM-EM-UdeA (CM-UDEA-H9, voucher basidiomata 2574a AMV).
4
Monitoring Cocoa Beans Fermentation Dynamics
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During the fermentation process, at intervals of 24 h, aliquots of cocoa beans were collected until the end of this stage, totalizing 168 h of natural fermentation (7 days). During the first 48 hours of fermentation, analyses were performed at four hours intervals because the predominant yeast activity in this period of time is elucidated in the literature [3, (link)6] .
Twenty grams of cocoa beans samples were aseptically macerated and homogenized in 180 mL 0.1% peptone water (Kasvi) for 2 min obtaining the dilution of 10 -1 . In sequence, decimal serial dilutions were taken until 10 -8 . Subsequently, 1 mL of each serial dilution was inoculated into sterile petri dishes via pour-plate technique and homogenised with PDA agar (Himedia) [8] (link) supplemented with chloramphenicol (Sigma-Aldrich Chemical Co., 100 mg/L). The inoculated plates were incubated at 30 °C for 48 h. The result of the plate count was expressed in log CFU g -1 of cocoa beans.
Ten colonies randomly chosen out of twenty to fifty colonies were collected after incubation (30 °C for 96 h) for each petri dish for all times of fermentation. The colonies were picked via the depletion technique twice in sterile petri dishes containing PDA agar and incubated for 72 h at 30 °C.
Twenty grams of cocoa beans samples were aseptically macerated and homogenized in 180 mL 0.1% peptone water (Kasvi) for 2 min obtaining the dilution of 10 -1 . In sequence, decimal serial dilutions were taken until 10 -8 . Subsequently, 1 mL of each serial dilution was inoculated into sterile petri dishes via pour-plate technique and homogenised with PDA agar (Himedia) [8] (link) supplemented with chloramphenicol (Sigma-Aldrich Chemical Co., 100 mg/L). The inoculated plates were incubated at 30 °C for 48 h. The result of the plate count was expressed in log CFU g -1 of cocoa beans.
Ten colonies randomly chosen out of twenty to fifty colonies were collected after incubation (30 °C for 96 h) for each petri dish for all times of fermentation. The colonies were picked via the depletion technique twice in sterile petri dishes containing PDA agar and incubated for 72 h at 30 °C.
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