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11 protocols using Paper discs

Antibacterial effects of L. plantarum strains were tested by the disc diffusion method. Four Gram-positive bacteria (SA, LM, SM, SS) and three Gram-negative bacteria (EC, PA, CS) were used as target bacteria. Overnight cultures of target bacteria were suspended in 0.9% saline with an optical density (OD) of 0.8 at 600 nm wavelength (OD600). Each bacterial suspension was spread on mixed agar medium of BHI and MRS at a 1:1 ratio in weight. After spreading bacterial suspensions, 8 mm paper discs (Advantec, Japan) were placed on agar plates, and 35 μL of each overnight lactobacillus broth culture was inoculated on the paper discs. After 24–48 h of incubation at 37°C, diameters of inhibition zones were measured. The diameter of the paper disc was subtracted from the total diameter of each measured inhibition zone.
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S. aureus cultured in nutrient broth medium was diluted onto agar plates to achieve an optical density at 600 nm (OD600) of 0.1 (corresponding to 1 × 108 CFU/mL). Petri dishes (87 × 15 mm) were sterilized and filled with agar medium for the cultivation of S. aureus. The agar medium composition was prepared according to the detailed composition and concentrations referenced in Section 4.6. Subsequently, 100 μL of the diluted bacterial culture was evenly spread onto each plate. Sterilized paper discs (8 mm, Advantec) were then placed uniformly on the agar surface. Peptide solutions, prepared at different concentrations, were applied onto the paper discs in 20 μL volumes. The plates were then incubated at 37 °C for 24 h, and the antimicrobial activity was measured by assessing the size of the inhibition zone (mm) around the paper disc.
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Intracellular ROS generation of R. quercus-mongolicae and R. solani was observed using the ROS-sensitive dye 2′,7′-dichlorofluorescin diacetate (≥97, Sigma-Aldrich, MO, USA). Nutrient agar (NA) (Difco, IL, USA) plates were prepared in Petri dishes (diameter 90 mm). Agar-mycelial plugs (diameter 5 mm) of R. querqus-mongalicae or R. solani were placed in the center of the dishes for incubation at 25 °C (one day for R. solani and four days for R. querqus-mongalicae). On agar-free lids, paper discs (diameter 8 mm, Advantec, Tokyo, Japan) with 10 mg carvacrol and thymol were placed. Controls received only 25% DMSO and 1% Tween 80. After treatment, treated and control NA plates were incubated for one day at 25 °C. After incubation, plugs from treated and control NA plate were stained with 3 μM 2′,7′-dichlorofluorescein diacetate for 1 h at 37 °C in the dark. Stained plugs were washed with phosphate-buffered saline (PBS, Sigma-Aldrich, MO, USA) diluted 10 times with distilled water, and observed with a confocal laser scanning microscope (CLSM) (excitation: 488 nm, emission: 490-560 nm, SP8X, Leica Microsystems, Germany)..
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Bacillus subtilis ATCC6633, Staphylococcus aureus 209PJC-1, Micrococcus luteus ATCC9341, and Escherichia coli NIHJ-2 were inoculated in 10 ml Mueller Hinton (MH) medium (BD Biosciences) and precultured for 1 day (180 rpm, 30 °C). Preculture (0.6 ml) was added to 6 ml of soft agar (1.6% nutrient broth (BD Biosciences), 0.5% agar). The solution was applied as an overlay to an agar plate (140 mm × 100 mm. Eiken chemical Co., LTD) containing 15 ml of MH agar. Twenty microliters of samples dissolved in DMSO (extracts from 0.2 ml culture) were transferred to paper discs (8 mm, ADVANTEC) and placed on an agar plate. After incubation at 30 °C for 1 day, the zones of growth inhibition were measured. The experiment was performed twice for the biological replicate. Gentamicin (5 µg) was used as positive control and DMSO (20 µL) was used as negative control.
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Susceptibility to antibiotics was examined using the disc-diffusion method with application of modified agar diffusion method described previously28 (link),29 (link). LAB inoculated in MRS agar were adjusted to approximately 108 CFU/mL and paper discs (Advantec, Tokyo, Japan) were dispensed. Each disc was treated with 10 μL of specific antibiotic. The concentrations of antibiotics tested are listed in Supplementary Table 1. The inhibition zone diameters were measured and evaluated in terms of sensitive, intermediate sensitive, and resistant according to the interpretative standard table (Supplementary Table 1). The 2013 Clinical and Laboratory Standards Institute criteria30 were used for interpretation.
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The paper disc assay (10 ) and the spot-on-the-lawn assay (11 ) were used to detect antifungal activities. Plates were prepared by adding mold (106 spores per 20 mL of PDA) up to a concentration of 1.5% (w/v). The spore solution was prepared as previously reported (12 (link)). For the paper disc assay, paper discs (diameter 8 mm; Advantec, Tokyo, Japan) on PDA plates were spotted with 100 μL of sample. The plates were incubated at 30°C for 48 h and examined for inhibition zones. For the spoton-the-lawn assay, 10~25 μL of sample was spotted onto the sensitive mold plates. Antifungal activity was expressed as the clear zone size (mm). The above experiment was performed in triplicate.
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Paper disc assay [13 ] was used for detection of antifungal activities. Plates were prepared by adding Penicillium brevicompactum strain FI02 culture medium (106 spores per 20 mL of PDA) up to a concentration of 1.5 % (w/v). For the paper disc assay, paper discs (diameter 8 mm; Advantec, Tokyo, Japan) on PDA plates were spotted with 100 μL of cinnamon extracts using different solvents. The plates were incubated at 30 °C for 48 h and examined for inhibition zones. Antifungal activity was expressed as clear zone size (mm). The above described experiment was performed in triplicate.
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The full-grown cultures of CNS stains or E. coli DH5α harboring the AhlS-expressing plasmid were inoculated in fresh LB medium (1% inoculum) containing 100 μM isopropyl-β-d-thiogalactopyranoside (IPTG). C6-HSL or C10-HSL was added at a final concentration of 20 μM (for CNS strains) or 10 μM (for E. coli). After incubation at 37 °C, cells were removed by centrifugation to obtain the supernatant. The residual AHLs in the supernatant were detected using C. violaceum CV026 and VIR07. An overnight culture of CV026 or VIR07 was mixed with 25 mL LB agar medium and poured into Petri dishes. AHL samples (20 μL) were placed in paper discs (8 mm in diameter, Advantec, Tokyo, Japan), which were subsequently placed onto LB agar plates containing CV026 or VIR07. Assay plates were incubated overnight at 30 °C and the residual amounts of AHL were calculated using the relationship equations based on the size of the purple-colored zones and the known quantities of AHLs [26 (link)].
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Lamiaceae plant essential oils or their constituents were applied to a paper disc (8 mm, Advantec, Tokyo, Japan) that was placed in the bottom lid of a glass cylinder (diameter, 5 cm; height, 10 cm) with a wire sieve fitted 3.5 cm above the bottom. The lid of the glass cylinder was then sealed with para-film (Pechiney Plastic Packaging Company, Chicago, IL, USA). Ten rice weevil adults (10–20 days old) were placed on the sieve, and this prevented direct contact of the rice weevils with the test oils or compounds. Glass cylinders were kept at 25 ± 1 °C and 60% RH. Adult rice weevils were considered dead if their appendages did not move when prodded with a paintbrush. Cumulative mortality was determined 48 h after treatment. All treatments were replicated five times.
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The following materials, chemicals, solvents, and drugs were used: Paper Disc (Advantec, India), Disk Diffusion Zone (Inhibitor) measuring ruler (Himedia, Chennai, India), Ethanol, Methanol (Fisher Scientific, Chennai, India), 96% Ethanol (Fisher Scientific, Gangnam, Seoul, South Korea), Mueller–Hinton Agar (Himedia, Chennai, India), Nutrient Broth (Himedia, Chennai, India), Mannitol salt agar (Himedia, Chennai, India), Triptic soy agar Phosphate buffer Saline (Fisher Scientific, Chennai, India), Antibiotics (ampicillin, vancomycin, penicillin, gentamicin, tetracycline, kanamycin, clindamycin, erythromycin, and novobiocin (Himedia, Chennai, India).
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