Proteolytic activity was examined according to the method described by Kessler et al. (2014), Cheng et al. (2022), and Filloux et al. (2014) [18 (link),19 (link),20 (link)] but modified by using skim milk (Skim Milk Powder, Sigma-Aldrich, St. Louis, MO, USA) as a substrate. An overnight Luria Bertani (LB, Becton Dickinson, Holdrege, NE, USA) broth culture was spotted onto a 10% v/v skim milk agar plate and incubated at 37 °C for 24 h. The diameter of the transparent zone under and around the colony was measured and diameters > 5 mm were interpreted as positive for protease activity. The growth of the colony with a clear zone was described as a negative result.
Luria bertani lb
Manufactured by BD
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Luria-Bertani (LB) is a widely used nutrient-rich culture medium for the growth of bacteria, particularly Escherichia coli (E. coli). It provides the necessary nutrients and growth factors required for the cultivation of various bacterial species in a laboratory setting.
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Lab products found in correlation
Ampicillin, by Merck Group (2 mentions)
Tryptic soy broth tsb, by BD (2 mentions)
Tryptic soy broth (tsb), by BD (2 mentions)
Forma anaerobic chamber, by Thermo Fisher Scientific (2 mentions)
Skim milk powder, by Merck Group (1 mentions)
Lb broth, by BD (1 mentions)
Microbank, by Pro-Lab Diagnostics (1 mentions)
Tryptic soy agar tsa, by BD (1 mentions)
Polyethylene glycol peg, by Merck Group (1 mentions)
Maltodextrin, by Merck Group (1 mentions)
Sucrose, by Merck Group (1 mentions)
Chloramphenicol, by Merck Group (1 mentions)
Ciprofloxacin, by Merck Group (1 mentions)
Tobramycin, by Merck Group (1 mentions)
Erythromycin, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Kanamycin, by Nacalai Tesque (1 mentions)
Luria bertani agar, by BD (1 mentions)
37 protocols using luria bertani lb
1
Protease Activity Assay Using Skim Milk
2
Isolation and Identification of Vibrio parahaemolyticus
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After the incubation described above, bacterial cultures from enrichment APW with 3% (wt/vol) NaCl were subcultured on TCBS agar at 37°C for 18 to 24 h. Subsequently, one to three suspected green colonies of V. parahaemolyticus from direct TCBS plates and TCBS plates enriched by APW with 3% (wt/vol) NaCl were randomly selected. In total, three to five colonies were collected for each sample. The colonies obtained were biochemically characterized according to a published method (69 (link)). The identified isolates were then confirmed for species-specific ldh of V. parahaemolyticus by PCR (70 (link)). Pure cultures were preserved using 20% (vol/vol) glycerol in Luria-Bertani (LB, Becton Dickinson, Franklin Lakes, NJ) broth containing 3% (wt/vol) NaCl and stored at −80°C for further analysis.
3
Salmonella enterica Typhimurium Growth Protocol
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In this study, Salmonella enterica serovar Typhimurium (ATCC 14028) (ST) was used. ST was grown in Luria-Bertani (LB) (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) agar at 37 °C under aerobic conditions (Thermo Fisher Scientific Inc., Marietta, OH, USA). All experiments described were performed in LB broth (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) and were incubated under aerobic conditions for 24 h in 37 °C with continuous aeration at 150 rpm through the use of a shaking incubator.
4
Bacterial Strain Preparation Protocol
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Salmonella Typhimurium NBRC 12,529 and S. enteritidis NBRC3313 used in this study were obtained from the Biological Resource Center, National Institute of Technology and Evaluation (NBRC), Chiba, Japan. Enterohemorrhagic Escherichia coli O157:H7 No. 196 (stx1+/stx2+) was kindly provided by the Fukuoka City Institute of Health and Environment, Fukuoka, Japan. Bacterial strains were stored in a Microbank™ (Pro-Lab Diagnostics, Richmond Hill, ON, Canada) at −80 °C. Each strain kept in Microbank™ was streaked onto Tryptic Soy Agar (TSA; Becton, Dickinson and Company, Sparks, MD, USA) and incubated at 37 °C for 24 h. A single colony was inoculated into 5 mL of Luria Bertani (LB, Becton, Dickinson and Company, Sparks, MD, USA) broth and incubated overnight at 37 °C. Prior to use, the bacterial cultures were serially diluted to obtain the desired concentrations.
5
Antibiotic Resistance Study of Acinetobacter baumannii
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A clinical isolate of A. baumannii (strain ATCC 17978; “Ab17978”; originally isolated from an infant) was used in the present study. The strain was resistant to at least three different categories of antibiotics, including beta lactams. Luria–Bertani (LB, Becton Dickinson and Company, Sparks, MD, USA) agar and broth was used for bacterial culture. Ampicillin, tobramycin, chloramphenicol, erythromycin, ciprofloxacin, maltodextrin, sucrose, 3350 Da polyethylene glycol (PEG), and 400 Da PEG were procured from Sigma-Aldrich (St. Louis, MO, USA). tobramycin (Tob), chloramphenicol (Chl), erythromycin (Ery) and ciprofloxacin (Cip) were selected for sequential treatment because they represent four different classes of antibiotics with different mass (565, 323, 733, and 331 Da, respectively) and Log P characteristics (−6.2, 1.1, 2.7, and −1.1 units, respectively). Log P is the partial coefficient between n-octanol and water, and larger negative values represent more hydrophilic compounds. Log P and mass values used in this study were obtained from PubChem Compound https://www.ncbi.nlm.nih.gov/pccompound/ .
6
UPEC Strain UTI89 Preparation
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Uropathogenic Escherichia coli (UPEC) strain UTI89, a clinical cystitis isolate (Chen et al., 2009 ), was grown in Luria‐Bertani (LB; Becton Dickinson [BD]) broth statically overnight at 37°C. Cultures were centrifuged for 10 min at 7,500g at 4°C before being resuspended in sterile phosphate‐buffered saline (PBS) to a density of ~4 × 108 colony‐forming units (CFU)/ml.
7
Growth Conditions for Bacterial Strains
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Bacterial strains and plasmids used in this study are listed in Supplementary Table S1 . In general, bacterial strains were grown at 37°C with aeration and shaking at 250 rpm in Luria-Bertani (LB) (Becton Dickinson and Company, Franklin Lakes, NJ, United States) supplemented with appropriate antibiotics (ampicillin (Ap) at 200 μg/mL, kanamycin (Km) at 50 μg/mL, chloramphenicol (Cm) at 30 μg/mL, and tetracycline (Tc) at 5 μg/mL) when necessary. For microarray analyses, bacterial cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, United States) under gastrointestinal tract (GIT)-mimicking condition (Donnenberg and Kaper, 1992 (link)).
Porcine alveolar macrophage cell line 3D4/31 was maintained in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin–streptomycin, and non-essential amino acid (Thermo Fisher Scientific, Waltham, MA, United States) on a 100 mm × 20 mm culture dish (Corning, NY, United States) at 37°C under 5% CO2 atmosphere.
Porcine alveolar macrophage cell line 3D4/31 was maintained in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin–streptomycin, and non-essential amino acid (Thermo Fisher Scientific, Waltham, MA, United States) on a 100 mm × 20 mm culture dish (Corning, NY, United States) at 37°C under 5% CO2 atmosphere.
8
Cytotoxicity Assay for Campylobacter Strains
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C. upsaliensis strains, ATCC 43954, 40-1, 48-1, 99-1 and 102-1, examined previously [16 (link)] were used in
this study. Recombinant Escherichia coli strain BL21 (DE3) with cdt-II(Chcdt-II) genes of C. hyointestinalis in TH-pET vector (rChCDT-II) [17 (link)] and C. jejuni strain K328 with 667 and 51 bp deleted regions in the cdt gene cluster [15 (link)], were used as positive and negative controls, respectively, for cytotoxicity assay. Campylobacter strains were grown on
blood agar [blood base agar no. 2 (Oxoid Ltd., Basingstoke, U. K.)] supplemented with 5% (v/v) defibrinated horse blood (Nippon Bio-Supp. Center, Tokyo, Japan)
under anaerobic condition (10% CO2, 10% H2 and 80% N2) at 37°C for 48 hr. E. coli strains, JM109 and BL21
(DE3), were used as cloning and expression vectors, respectively, and they were grown aerobically in Luria-Bertani (LB; Becton, Dickinson and Co., Frankline
Lakes, NJ, U.S.A.) agar at 37°C overnight. E. coli strains carrying a recombinant plasmid were grown in LB broth containing 30
µg/ml kanamycin (Nacalai Tesque Inc., Kyoto, Japan) at 37°C with vigorous shaking.
this study. Recombinant Escherichia coli strain BL21 (DE3) with cdt-II(Chcdt-II) genes of C. hyointestinalis in TH-pET vector (rChCDT-II) [17 (link)] and C. jejuni strain K328 with 667 and 51 bp deleted regions in the cdt gene cluster [15 (link)], were used as positive and negative controls, respectively, for cytotoxicity assay. Campylobacter strains were grown on
blood agar [blood base agar no. 2 (Oxoid Ltd., Basingstoke, U. K.)] supplemented with 5% (v/v) defibrinated horse blood (Nippon Bio-Supp. Center, Tokyo, Japan)
under anaerobic condition (10% CO2, 10% H2 and 80% N2) at 37°C for 48 hr. E. coli strains, JM109 and BL21
(DE3), were used as cloning and expression vectors, respectively, and they were grown aerobically in Luria-Bertani (LB; Becton, Dickinson and Co., Frankline
Lakes, NJ, U.S.A.) agar at 37°C overnight. E. coli strains carrying a recombinant plasmid were grown in LB broth containing 30
µg/ml kanamycin (Nacalai Tesque Inc., Kyoto, Japan) at 37°C with vigorous shaking.
9
Culturing and Fermentation of Bacterial Strains
10
E. coli Growth and Culturing Protocol
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All bacterial strains used in this study appear in Table 1 . E. coli strains were cultured at 37 °C in Luria-Bertani (LB) (Becton & Dickinson, Sparks, MD, USA) broth or on Luria-Bertani Agar (Becton & Dickinson, Sparks, MD, USA). Wild-type (WT) strain refers to BW25113.
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