The antimicrobial activity of E. beddomei essential oil was screened by the disc diffusion method according to Lu et al.40 (link) with some modifications. All bacterial pathogens were cultured in Mueller Hinton agar while C. albicans was cultured in potato dextrose agar (PDA). All bacterial and fungal pathogens were then sub-cultured in nutrient broth and potato dextrose broth (PDB), respectively. The bacterial pathogens were incubated at 37 °C for 24 h while the fungus pathogen was incubated at 28 °C for 48 h. The active bacterial pathogens were prepared in a nutrient broth until it matched the 0.5 McFarlane standard (1 × 108 colony-forming unit/mL). The bacterial pathogens were spread out on the dried surface of the nutrient agar plates. The serialized 6 mm diameter paper discs (Whatman, USA) were impregnated with 30 µL of E. beddomei essential oil solution (10 µg/mL) preparing in 10% DMSO. The paper discs were then placed on the nutrient agar plate. These plates were incubated at 37 °C for 24 h. The fungus plates were incubated at 28 °C for 48 h. The diameter of the zone inhibition was determined. Chloramphenicol (10 µg/mL) and 10% DMSO were used as the positive and negative controls, respectively. Each experiment was performed in replicates.
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9 protocols using paper disc
1
Antimicrobial Screening of E. beddomei Essential Oil
2
Bacterial Cellulose Purification and Peptide Immobilization
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To produce BC, K. xylinus ATCC 53524 was used. The strain was cultivated in stationary conditions for 7 days at 28 °C in 24-well using a Hestrin-Schramm (H-S) medium. Obtained BC carriers were rinsed with water. Next, to remove bacterial cells and media components, the BC carriers were purified in 0.1 M NaOH (POCH, Poland) for 90 min at 80 °C. After purification, BC disks were immersed in distilled water and incubated with shaking. The efficiency of BC purification was performed using Scanning Electron Microscope ZEISS EVO MA as we described it elsewhere [61 (link)]. The pH value was measured every 3 h. The washing procedure was continued until there was no change in pH. To calculate water content of BC, the carrier was dried at 37 °C. Every day the weight was assessed using electronic balance PA114CM/1 (Ohaus, Germany) until no further drop of weight was observed. The wet BC carriers were transferred to 24-well plate and immersed with 800 µL of 2 mg/mL of peptide. The plate was left for 24 h/4 °C. The control setting of this experiment was BC carrier absorbed with octenidine dihydrochloride (antiseptic substance of confirmed antimicrobial activity) and 0.9% NaCl, introduced to the BC carrier. Additionally, as carrier’s control setting, the peptides were introduced to paper discs (Whatmann, USA) of 6 mm diameter (used for conventional antibiotic therapy).
3
Antimicrobial Activity Screening of Oils
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The antimicrobial activity was performed using the disc diffusion method on Muller-Hinton (MH) and Sabouraud (SB) agar, following the CLSI recommendations [20 ]. Thus, the saline suspensions of microorganisms (NaCl 0.9%) were first adjusted to match the 0.5 McFarland standard (equivalent to 1 × 108 cfu/mL), and then inoculated onto plate surfaces with a sterile cotton swab. Whatman paper discs (about 5 mm in diameter) sterilized by autoclaving (121 °C for 15 min) were soaked with the test oil at different concentrations (30,40 and 50 μL/mL) and placed on the MH and SB agar surface. After 24 h of incubation at 37 °C and 48 h at 25 °C for bacteria and yeasts respectively, the diameter of inhibition growth zones was measured using a ruler. Each trial was repeated three times.
4
Antimicrobial Evaluation of Propolis
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The antimicrobial activity of propolis samples was evaluated in triplicate according to the procedure described by Kirby-Bauer [61 (link)], with slight modifications. The antimicrobial screening was performed by using Mueller–Hinton agar (MHA). The agar plate surface is inoculated by physiological inoculum (108 cfu·mL−1). The bacterial suspension was prepared according to the method explained previously [62 (link)]. Then, the paper discs (Whatman, 6 mm) were placed on the surface of the pre-inoculated agar and impregnated with 10 μL propolis samples (stock solution: 100 mg/mL). The inoculated plates were incubated at 37 °C for 24 h. The diameter of the inhibition zone was measured in mm.
5
Dextran Sulfate Stress on Gut
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Experimental and control flies were collected within 48 hr of eclosion at 18°C and moved to 29°C for 10 d on standard diet. Flies were then transferred to 5% sucrose (vehicle) or 5% sucrose−3% DSS (Fisher Scientific) on paper discs (Whatman) for 4 d. Media were changed daily. Guts were then dissected and analyzed using immunofluorescence and confocal imaging.
6
Chick Embryo Chorioallantoic Membrane Assay
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The CAM assay was performed according to Storgard et al.29 . Briefly, since the allantois of the chick embryo appears at about 3.5 days of incubation, eggs from local breeders were artificially incubated for 10 days. (37.5 °C, relative humidity: 55–65%). On the 10th incubation day, a false air chamber was created directly over the CAM, permitting its detachment from the shell membrane. To decrease the risk of infection, the whole eggs were cleaned and sterilised with 70% ethanol. The procedure was continued through a square incision of the eggshell over the CAM. The prepared window in a square form (2 cm2), was covered by a flexible film and then transported to the incubator. One hour after window opening, a paper disc (6 mm in diameter) (Whatman) was coated with target agents diluted in a total volume of 10 μL of PBS (0.1 and 1 μg anti-chemerin antibody, and 0.1 and 1 μg anti-CMKLR1 antibody) and placed in the central area of the corresponding window. The CAM surface was visually evaluated after 72 h of additional incubation using a stereomicroscope. We decided to use the surface of capillary blood vessels as the index of anti-angiogenic activity. Consequently, the prepared images were analysed using the Image J software to determine the percentage of the surface of the paper disc occupied by blood vessels.
7
Probiotic Antimicrobial Potency Evaluation
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The antimicrobial activity testing of probiotic-encapsulated cultures lyophilized powder against E. coli TISTR 073, S. aureus TISTR 029, and Ent. aerogenes TISTR 1540 was assayed using the swab paper disc method, according to the method of [28 (link)].
One milligram of their lyophilized powder was grown in the modified MRS broth supplemented with 0.2 % (w/v) dextrose (pH 6.0 ± 0.2), was inoculated with 1% (v/v) of an overnight culture, and incubated in anaerobic condition so as to rule out any inhibition due to hydrogen peroxide production at 37 °C for 18 h. The supernatants were harvested by centrifugation, sterilized by passing it through a 0.2 µm filter, and tested for antimicrobial activity against the strains of E. coli, S. aureus, and Ent. aerogenes using a paper disc diffusion method. Pathogen suspensions of approximately 0.2 mL (105–106 CFU/mL) after growing at 37 °C for 24 h in nutrient broth (Himedia™, Maharashtra, India) were spread with a sterile swap onto the surface of a nutrient agar. Then, 40 µL of each supernatant without the cells was dropped into the paper disc (7 mm diameter, Whatman), placed on the inoculated agar surfaces, with streptomycin discs (15 µg) used as a positive control. After the incubation, the plates measured the inhibition zone around a disc using calipers and expressed in millimeters (mm).
One milligram of their lyophilized powder was grown in the modified MRS broth supplemented with 0.2 % (w/v) dextrose (pH 6.0 ± 0.2), was inoculated with 1% (v/v) of an overnight culture, and incubated in anaerobic condition so as to rule out any inhibition due to hydrogen peroxide production at 37 °C for 18 h. The supernatants were harvested by centrifugation, sterilized by passing it through a 0.2 µm filter, and tested for antimicrobial activity against the strains of E. coli, S. aureus, and Ent. aerogenes using a paper disc diffusion method. Pathogen suspensions of approximately 0.2 mL (105–106 CFU/mL) after growing at 37 °C for 24 h in nutrient broth (Himedia™, Maharashtra, India) were spread with a sterile swap onto the surface of a nutrient agar. Then, 40 µL of each supernatant without the cells was dropped into the paper disc (7 mm diameter, Whatman), placed on the inoculated agar surfaces, with streptomycin discs (15 µg) used as a positive control. After the incubation, the plates measured the inhibition zone around a disc using calipers and expressed in millimeters (mm).
8
Antimicrobial Potential of Strain TW-1 Against E. coli and P. aeruginosa
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Antimicrobial activities of strain TW-1T against E. coli KACC 10,185 and P. aeruginosa KEMB 121–234 were examined by disc-diffusion and spotting method, respectively. Screening were done against these Gram-negative pathogens by spotting the colonies of strain TW-1T on R2A agar plates and incubated at 28 °C for 48 h. Crude product of culture extract was prepared by the culture supernatant of strain TW-1T to evaluate disc-diffusion test. Strain TW-1T was cultured in 300 mL of R2A broth at 28 °C (180 rpm for 5 days) into a 500 mL Erlenmeyer flask. Culture supernatant of strain TW-1T was extracted by equal volume of ethyl acetate (2 ×) with pH 2.0 and 10, respectively47 . Organic layer collected from extraction was completely evaporated by Rotary evaporator, (Eyela) and remained residue of crude product was dissolved in 500 µL of methanol. Then, 15 µL of crude product was diffused to a paper-disc (6 mm, Whatman) and antimicrobial activities against P. aeruginosa and E. coli KEMB were checked by measuring the inhibition zones. Trimethoprim/sulfamethaxazole (15 µg) and only methanol (15 µL) were used for positive and negative controls, respectively.
9
Entomopathogenic Fungal Conidia Toxicity Against Termites
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The toxicity of entomopathogenic fungal conidia against soil termites (Coptotermes sp.) was carried out using spray and bait method based on modified JIS K 1571, 2010. The both methods were carried out using 50 worker termites and 5 soldier termites. A total of 1 ml of entomopathogenic fungal spore suspension with a concentration of 10 7 was evenly sprayed on the surface of the termite's body in a separate container for each fungal strain and left for 1 minute. Then, the sprayed termites were placed into a 5 mm petri dish that has been lined with thick hard plaster of paris to maintain moisture. Meanwhile, the bait method was performed using paper disc (Whatman 8 mm in diameter) impregnated with 50 µl of entomopathogenic fungal conidia suspension and air dried for 1 minute. After that, the paper disc was put into a 5 mm Ø petri dish which had been coated with plaster of paris and contained 50 worker termites and 5 soldier termites. Each treatment was replicated 3 times and incubated at 25 °C. Number of dead termites was observed every 2 days for 14 days in each treatment. The percentage of termite mortality was calculated by the formula: -----------------------------x 100 55
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