All chemicals were of reagent grade purity and were used without further purification. Silver sulfate (Ag2SO4), silver perchlorate (AgClO4), silver nitrate (AgNO3), 2-propanol ((CH3)2CHOH), polyvinyl alcohol (PVA, 87–90% hydrolyzed, MW 30,000–70,000 g/mole), potassium chloride (KCl, ≥99%) and sodium hydroxide (NaOH, ≥99.0%) were obtained from Sigma Aldrich (Saint Quentin Fallavier, France). Luria-Bertani (LB) broth powder and agar powder for the preparation of bacterial culture media were purchased from Becton, Dickinson and Co., France. S. aureus ATCC 27,217 bacteria strain was obtained from the American type culture collection (ATCC). The water used for the preparation of the solutions was purified using a Millipore MilliQ system at 18.2 MΩ·cm at 25 °C.
Agar powder
Manufactured by BD
Sourced in France, United States
Agar powder is a natural gelling agent derived from red algae. It is commonly used in microbiology and biotechnology laboratories as a solidifying agent for culture media. Agar powder forms a firm, transparent gel when dissolved in water and cooled, providing a suitable growth environment for a variety of microorganisms.
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Lab products found in correlation
Potassium chloride (kcl), by Merck Group (2 mentions)
Milli q system, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Driselase, by Merck Group (1 mentions)
Pda medium, by BD (1 mentions)
Peg4000, by Merck Group (1 mentions)
Miracloth, by Merck Group (1 mentions)
Tryptone, by BD (1 mentions)
Yeast extract, by BD (1 mentions)
Coomassie brilliant blue g 250, by Merck Group (1 mentions)
Congo red, by Merck Group (1 mentions)
Luria bertani medium, by BD (1 mentions)
Agno3, by Merck Group (1 mentions)
Bromocresol purple, by Merck Group (1 mentions)
Lactophenol blue, by Merck Group (1 mentions)
L cysteine hydrochloride anhydrous, by Merck Group (1 mentions)
Mrs broth, by BD (1 mentions)
Bacto tsb, by BD (1 mentions)
Epichlorohydrin, by Merck Group (1 mentions)
Hydrogen peroxide, by Daejung Chemicals (1 mentions)
8 protocols using agar powder
1
Silver-Based Antimicrobial Preparation
2
Fungal Strain P. chlamydosporia Fermentation
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The deuterium reagents were used from Cambridge Isotope Laboratories, Inc. (USA). The agar powder and PDA medium were used from Becton, Dickinson Co., Ltd. (USA). Yeast extracts and peptones were used from Oxoid, Inc. (UK). Driselase and PEG4000 were used from Sigma-Aldrich Trading Co., Ltd. (China). Miracloth was used from Merck-Calbiochem (Germany). The silica gel for column chromatography was used from Qingdao Haiyang Chemical Co., Ltd. (China). The monosaccharides and silanization reagents (L-cysteine methyl ester hydrochloride and N-trimethylsilylimidazole) were used from Shanghai Macklin Biochemical Technology Co., Ltd. (China). All molecular kits were purchased from Vazyme Biotech Co., Ltd. (China). All organic reagents were purchased from Thermo Fisher Scientific, Inc. (USA). The fungal strain P. chlamydosporia PC-170 was used for the study, and its solid (rice medium) fermentation method was the same as we described before (Qin et al., 2019 (link)).
3
Predicting Salmonella Biofilm Formation
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Salmonella isolates were assessed for differential colonial morphology, which can be used to predict bacterial biofilm formation traits (16 (link)). The base medium, derived from Luria-Bertani (LB) broth without salt, includes yeast extract (5 g/L; Becton, Dickinson) and tryptone (10 g/L; Becton, Dickinson). The final agar medium was made from the base medium supplemented with Congo red (40 mg/L; Sigma), Coomassie brilliant blue G-250 (20 mg/L; Sigma), and agar powder (2 g/L; Becton, Dickinson). Bacterial colonial morphology was examined after 48 h incubation at 35°C.
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4
Antibacterial Activity Evaluation
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AgNO3 was purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium hydroxide (NaOH) stock solution (1 M) was obtained from Daejung Chemicals (Daejung, Korea). Desiccated cell-free beef extract, nutrient broth (NB), Luria-Bertani (LB) medium and agar powder were purchased from Becton Dickinson (Pont-de-Claix, France). The Korean Culture Center of Microorganisms (KCCM, Seoul, Korea) supplied the MDR bacterial strains of S. aureus (KCCM 11335) and E. coli (KCCM 11234).
5
Quantification of Viable LAB and Fungal Species in Fermented Soybeans
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Viable cell numbers of LAB in the fermented soybean samples were determined using MRS agar plates containing 55 g/l MRS broth (Becton, Dickinson and Company) with 0.05% (w/v) L-cysteine-hydrochloride anhydrous (Sigma-Aldrich Co.), 20 g/l agar powder (Becton, Dickinson and Company), and 1.2 g/l bromocresol purple (Sigma-Aldrich Co.). The sample was diluted to 1/10 6 -1/10 9 . The diluted sample (100 µl) was inoculated onto MRS agar plates and the plates were incubated at 30°C for 2 days under anaerobic conditions. Isolation of fungal species from fermented soybean samples was determined using DRBC (Dichloran Rose Bengal Chloramphenicol) (Sigma-Aldrich Co.) agar plates with 0.01% (w/v) Chloramphenicol Selective Supplement (Sigma-Aldrich Co.). The sample was diluted to 1/10 7 . The diluted sample (100 µl) was inoculated onto DRBC agar plates and the plates were incubated at 30°C for 5 days under aerobic conditions. Then, the plates including the A. oryzae strain were morphologically selected and counted by staining with lacto phenol blue (Sigma-Aldrich Co.). The mean values and the standard deviation were calculated by duplicate independent trials.
6
Culturing vAh and E. coli Strains
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Tryptic Soy Broth (TSB) (Bacto TSB, BD) and Luria Broth (LB) prepared according to manufacturer's directions was routinely used as the culture medium for growth of vAh and Escherichia coli, respectively, with the addition of 1.5% agar powder (Alfa Aesar) for solid culture. When necessary for selection, media were supplemented with chloramphenicol (Cam) (25 μg/ml), tetracycline (Tet) (10 μg/ml), colistin (Col) (10 μg/ml), kanamycin (Kan) (30 μg/ml), or combinations of those antibiotics. Super Optimal Broth (SOB) (Difco SOB, BD) was used for culture of electrocompetent cells and Super Optimal Broth with Catabolite Repression (SOC) was used as a recovery medium following electroporation. vAh strains were cultured at 30°C and E. coli strains were cultured at 37°C.
Bacterial strain vAh ML09-119 was removed from cryogenic storage and inoculated into 25 ml TSB media and grown overnight at 30°C with shaking. An aliquot of overnight culture was transferred to 70 ml of TSB and grown at 30°C on an orbital shaker at 180 rpm to mid-log phase, ~16 h.
Bacterial strain vAh ML09-119 was removed from cryogenic storage and inoculated into 25 ml TSB media and grown overnight at 30°C with shaking. An aliquot of overnight culture was transferred to 70 ml of TSB and grown at 30°C on an orbital shaker at 180 rpm to mid-log phase, ~16 h.
7
Synthesis of Mucin-Based Biosensors
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Mucin (from porcine stomach, Type III), 11-mercapto-1-undecanol, epichlorohydrin, diethylene glycol dimethyl ether, potassium chloride, potassium hexacyanoferrate (II) trihydrate, potassium ferricyanide (III), sodium phosphate, and d -mannose were purchased from Sigma-Aldrich (St. Louis, MO, USA). Absolute ethyl alcohol, sodium hydroxide, acetone, sulfuric acid, sodium acetate, acetic acid, and hydrogen peroxide were from DaeJung Chemicals & Metals Co., Ltd. (Gyeonggi-do, Korea). Carboxyfluorescein diacetate (CFDA) was obtained from Dojindo Molecular Technologies, Inc. (Rockville, MD, USA), and potassium phosphate from Pure Chemicals Co., Ltd. (Kyoto, Japan). Lysogeny broth (LB) broth and agar powder were purchased from BD (BD Difco, Lawrence, KS, USA).
8
Characterizing Staphylococcal Swarming Dynamics
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A colony spreading assay was performed as previously described by Kaito et al and Tsompanidou et al, who showed a hyper-spreading phenotype of srtA KO strain [26] (link), [27] (link). In brief, Tryptic Soy Broth (TSB) (BD, BBL, Etten-Leur, the Netherlands) was supplemented with 0.24% Agar powder (BD, Difco, Etten-Leur, the Netherlands) to prepare TSA soft agar. Fifteen ml TSA soft agar was poured into plates with 8 cm diameter and dried for 20 min. In parallel, SH1000 WT and isogenic srtA S. aureus KO strains OD600 nm 0.05 were cultured during 24 hrs in TSB medium upon addition of either i) medium, ii) K(FITC)LPETG-amide or iii) K(FITC)EGTLP-amide (final volume of 50 µl), the final substrate concentration was 2 mM, 1 mM and 0.5 mM. After 24 hrs incubation 2 µl of individual cultures were gently spotted on the TSA soft agar plates and dried for 15 min. The plates were incubated during 20 hrs at 37°C. After incubation the spreading zones were examined and pictures were taken.
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