P. aeruginosa (ATCC 9027) and S. aureus (ATCC 6538) were grown overnight in Nutrient Broth (VWR, Avantor, Radnor, PA) at 37°C. OD was measured at 600 nm and adjusted to 1 × 107 CFU ml‐1 for S. aureus and 1 × 108 CFU ml‐1 for P. aeruginosa. To each 24‐well plates with or without a contact lens were transferred 500 µl of solutions composed of 100 µl aliquot of bacteria and Ozodrop® or Ozodrop® gel at 20% dissolved in Nutrient Broth, and as a control, a 100 µl aliquot of bacteria dissolved in Nutrient Broth. Biofilms formed by bacteria that did not undergo any treatment were used as controls. Daily disposable lenses composed of 31% nelfilcon A and 69% water (Dailies AquaComfort PLUS; Alcon Laboratories, Fort Worth, TX, USA) were used in the experiments and briefly rinsed in sterile phosphate‐buffered saline (PBS; VWR‐ Avantor, Radnor, PA, USA) before every assay. Contact lenses or plates were incubated at 37°C for 30 or 50 h. After treatment, culture fluid was discarded and plate wells or contact lens were gently washed with PBS to remove planktonic bacteria. The biofilm deposition was assessed by evaluating the amount of biomass formed on different supports through a crystal violet assay and bacterial viability was measured by MTT assay.
Nutrient broth
Manufactured by Avantor
Sourced in Belgium
Nutrient Broth is a general-purpose microbiology culture medium used for the growth of a wide variety of microorganisms. It provides essential nutrients and growth factors required for the cultivation of bacteria, fungi, and other microbes.
Automatically generated - may contain errors
3 protocols using nutrient broth
1
Antimicrobial Efficacy of Ozodrop on Biofilms
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P. aeruginosa (ATCC 9027) and S. aureus (ATCC 6538) were grown overnight in Nutrient Broth (VWR, Avantor, Radnor, PA) at 37°C. OD was measured at 600 nm and adjusted to 1 × 107 CFU ml‐1 for S. aureus and 1 × 108 CFU ml‐1 for P. aeruginosa. To each 24‐well plates with or without a contact lens were transferred 500 µl of solutions composed of 100 µl aliquot of bacteria and Ozodrop® or Ozodrop® gel at 20% dissolved in Nutrient Broth, and as a control, a 100 µl aliquot of bacteria dissolved in Nutrient Broth. Biofilms formed by bacteria that did not undergo any treatment were used as controls. Daily disposable lenses composed of 31% nelfilcon A and 69% water (Dailies AquaComfort PLUS; Alcon Laboratories, Fort Worth, TX, USA) were used in the experiments and briefly rinsed in sterile phosphate‐buffered saline (PBS; VWR‐ Avantor, Radnor, PA, USA) before every assay. Contact lenses or plates were incubated at 37°C for 30 or 50 h. After treatment, culture fluid was discarded and plate wells or contact lens were gently washed with PBS to remove planktonic bacteria. The biofilm deposition was assessed by evaluating the amount of biomass formed on different supports through a crystal violet assay and bacterial viability was measured by MTT assay.
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P. aeruginosa (ATCC 9027) and S. aureus (ATCC 6538) were grown overnight in Nutrient Broth (VWR, Avantor, Radnor, PA) at 37°C. OD was measured at 600 nm and adjusted to 1 × 107 CFU ml‐1 for S. aureus and 1 × 108 CFU ml‐1 for P. aeruginosa. To each 24‐well plates with or without a contact lens were transferred 500 µl of solutions composed of 100 µl aliquot of bacteria and Ozodrop® or Ozodrop® gel at 20% dissolved in Nutrient Broth, and as a control, a 100 µl aliquot of bacteria dissolved in Nutrient Broth. Biofilms formed by bacteria that did not undergo any treatment were used as controls. Daily disposable lenses composed of 31% nelfilcon A and 69% water (Dailies AquaComfort PLUS; Alcon Laboratories, Fort Worth, TX, USA) were used in the experiments and briefly rinsed in sterile phosphate‐buffered saline (PBS; VWR‐ Avantor, Radnor, PA, USA) before every assay. Contact lenses or plates were incubated at 37°C for 30 or 50 h. After treatment, culture fluid was discarded and plate wells or contact lens were gently washed with PBS to remove planktonic bacteria. The biofilm deposition was assessed by evaluating the amount of biomass formed on different supports through a crystal violet assay and bacterial viability was measured by MTT assay.
2
Pseudomonas and Staphylococcus Lung Infection
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Pseudomonas aeruginosa (PA01) or Staphylococcus aureus (QO51) was grown in nutrient broth (VWR, Leuven, Belgium) overnight before being refreshed for 3 hr and diluted to OD600 = 0.3 (P. aeruginosa) or OD600 = 5 (S. aureus) in sterile PBS. βENaC-Tg mice (3 weeks old) received a subcutaneous injection of I.6 CatS inhibitor (200 mg/kg) or vehicle control (4% DMSO in peanut oil, Sigma-Aldrich). After 20 min, mice received an intranasal delivery of 20 μl of either P. aeruginosa (OD 0.3), S. aureus (OD 5) or PBS control under isoflurane anaesthetic. Mice were sacrificed 6 hr later, and bronchoalveolar lavage (BAL) fluid collected and processed as described below. Lungs and spleens were collected and homogenised in sterile PBS. Samples were plated on cetrimide (Sigma-Aldrich) (P. aeruginosa) or mannitol (VWR, Leuven, Belgium) (S. aureus) agar for quantification of CFU.
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Pseudomonas aeruginosa (PA01) or Staphylococcus aureus (QO51) was grown in nutrient broth (VWR, Leuven, Belgium) overnight before being refreshed for 3 hr and diluted to OD600 = 0.3 (P. aeruginosa) or OD600 = 5 (S. aureus) in sterile PBS. βENaC-Tg mice (3 weeks old) received a subcutaneous injection of I.6 CatS inhibitor (200 mg/kg) or vehicle control (4% DMSO in peanut oil, Sigma-Aldrich). After 20 min, mice received an intranasal delivery of 20 μl of either P. aeruginosa (OD 0.3), S. aureus (OD 5) or PBS control under isoflurane anaesthetic. Mice were sacrificed 6 hr later, and bronchoalveolar lavage (BAL) fluid collected and processed as described below. Lungs and spleens were collected and homogenised in sterile PBS. Samples were plated on cetrimide (Sigma-Aldrich) (P. aeruginosa) or mannitol (VWR, Leuven, Belgium) (S. aureus) agar for quantification of CFU.
3
Dermatophytes and Bacterial Strains Protocol
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Pathogenic filamentous dermatophytes (collection of Pharmacology and Hygiene Section, Department of Biomolecular Sciences, University of Urbino) (T. mentagrophytes F6, T. rubrum F2, T. violaceum F11, E. floccosum F12) and the reference strain C. albicans ATCC 10231 were included. The dermatophytes were maintained on potato dextrose agar (PDA) (VWR) at 35 °C for 7 days, while C. albicans at 37 °C for 24 h.
In addition, five human clinical isolates of S. aureus (S. aureus HCS026, S. aureus 2/5, S. aureus 28/10, S. aureus 18/9, S. aureus HCS002 methicillin-resistant MRSA), as well as the clinical isolate P. aeruginosa C86, belonging to the strain collection of the above-mentioned Pharmacology and Hygiene Section, were used. The reference strains, S. aureus ATCC 43387, S. aureus ATCC 43300 (MRSA) and P. aeruginosa ATCC 27583 were also added. All the S. aureus strains were cultured in tryptone soy agar (TSA) (VWR, Milan, Italy) at 37 °C for 24 h, while Pseudomonas strains were grown in Cetrimide agar (VWR) at the same culture conditions. All the strains were stored at −80 °C in Nutrient broth (VWR) supplemented with 20% glycerol.
In addition, five human clinical isolates of S. aureus (S. aureus HCS026, S. aureus 2/5, S. aureus 28/10, S. aureus 18/9, S. aureus HCS002 methicillin-resistant MRSA), as well as the clinical isolate P. aeruginosa C86, belonging to the strain collection of the above-mentioned Pharmacology and Hygiene Section, were used. The reference strains, S. aureus ATCC 43387, S. aureus ATCC 43300 (MRSA) and P. aeruginosa ATCC 27583 were also added. All the S. aureus strains were cultured in tryptone soy agar (TSA) (VWR, Milan, Italy) at 37 °C for 24 h, while Pseudomonas strains were grown in Cetrimide agar (VWR) at the same culture conditions. All the strains were stored at −80 °C in Nutrient broth (VWR) supplemented with 20% glycerol.
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