Salmonella hypermutators were screened as previously described (LeClerc et al., 1996 (link)). Briefly, single colonies of each Salmonella isolate grown on Luria–Bertani (LB) agar (BD, Cockeysville, MD, United States) were collected and cultured in 5 ml of LB broth (BD) at 37°C, with shaking at 100 rpm for 18–24 h. Then, 100 μl of 103-, 104-, 105-, and 106-fold diluted broth culture was spread in triplicate on LB agar (BD) plates containing 100 μg/ml rifampicin (Sigma, St. Louis, MO, United States) and 20 μg/ml nalidixic acid (Sigma), respectively. Meanwhile, 100 μl of 106-, 107-, and 108-fold diluted broth culture was spread in triplicate on antibiotic-free LB agar (BD) plates. After incubation (37°C, 18–24 h), the isolates yielding more than 50 separated colonies on each antibiotic-containing plate were selected as hypermutators. The mutation frequency was estimated by dividing the number of colony-forming units (CFUs) on antibiotic-containing plates by the number of CFU on antibiotic-free plates. All putative hypermutators were retested at least twice (LeClerc et al., 1996 (link); Yang et al., 2009 (link)). Salmonella SL226 was used as a positive control for hypermutator screening (Yang et al., 2009 (link)).
Lb agar
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LB agar is a solid growth medium used for the cultivation of bacteria. It is composed of tryptone, yeast extract, and sodium chloride, providing nutrients necessary for bacterial growth. LB agar solidifies at room temperature, allowing for the isolation and manipulation of bacterial colonies.
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Lab products found in correlation
Lb broth, by BD (9 mentions)
Luria bertani broth, by BD (9 mentions)
Rifampicin, by Merck Group (4 mentions)
Ampicillin, by Merck Group (4 mentions)
Streptomycin, by Merck Group (3 mentions)
Cefotaxime, by Merck Group (3 mentions)
Sodium chloride (nacl), by Merck Group (3 mentions)
Tetracycline, by Merck Group (2 mentions)
Hepes, by Merck Group (2 mentions)
Yeast extract, by BD (2 mentions)
Tryptic soy broth (tsb), by Merck Group (2 mentions)
Atcc baa 1605, by American Type Culture Collection (2 mentions)
Colistin sulfate, by Fujifilm (2 mentions)
Nalidixic acid, by Merck Group (1 mentions)
Macconkey agar, by BD (1 mentions)
Tryptic soy broth (tsb), by BD (1 mentions)
Sodium arsenite, by Merck Group (1 mentions)
Sodium arsenate, by Merck Group (1 mentions)
Vitek 2, by bioMérieux (1 mentions)
Rifampin, by BD (1 mentions)
60 protocols using lb agar
1
Screening Salmonella Hypermutators
2
Isolation and Characterization of Microbacterium invictum from Sludge
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Strain NIBRBAC000506063 T was isolated from a sludge sample collected in Yeosu-si (127°39'19.9"E, 34°48'10 "N), Jeollanam-do, Korea. The sludge sample was enrichment cultured for two days at 30°C in LB. Then, serial dilutions of the sample were spread on an LB agar (BD, Franklin Lakes, NJ, USA) plate and incubated at 30°C for 5 days under aerobic conditions, and selected colonies were transferred onto LB agar. Strain NIBRBAC000506063 T was puri ed by streaking more than thrice on LB agar. The bacterial culture was stored at -80°C in 20% (v/v) glycerol. Based on sequence similarity and phylogenetic analysis, the type strain Microbacterium invictum (Vaz-Moreira et al. 2009) was selected as the reference strain, and its characteristics were compared with those of strain NIBRBAC000506063 T .
3
Confirmation of mcr Gene Location
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To confirm mcr genes location biologically, conjugation was performed with an azide-resistant E. coli J53 strain. Selection of transconjuguants was done on MacConkey agar (Beckton Dickinson, Le Pont de Claix, France) supplemented with 120 mg/L sodium azide and 2 mg/L colistin. In case of unsuccessful conjugation, transformation of pure plasmid by electroporation method with Top10 electrocompetents E. coli (Thermo Fisher Scientific, Waltham, MA, United States) was performed and transformants were selected on LB agar (Beckton Dickinson, France) with 2 mg/L colistin. Plasmid curing was performed at high temperature on K. pneumoniae LH94 strain (Letchumanan et al., 2015 (link)). Then, the presence of mcr genes and plasmid types were controlled by standard PCR (Carattoli et al., 2005 (link); García-Fernández et al., 2009 (link); Rebelo et al., 2018 (link); Wang et al., 2018 (link)).
4
Bile Salts Tolerance Screening
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The strains and plasmids used in this study are listed in Table 1 . Frozen stock cultures were stored in Luria Bertani (LB) broth with 15% glycerol at −70°C. Working stock cultures were cultured on LB agar (Becton Dickinson, Sparks, MD, United States), incubated at 37°C for 20–24 h, and maintained at 4°C for up to 1 month. Overnight cultures were grown from a single colony in 5 ml of tryptic soy broth (TSB; Becton Dickinson) at 37°C for 20–24 h. For bile salts tolerance screening, TSB with 10% bile salts was inoculated with inocula ranging from103 to 105 CFU/ml. For survival and regrowth experiments, TSB with 10% bile salts was inoculated with approximately 106 CFU/ml and numbers of CFU/ml determined periodically during static incubation at 37°C.
5
Arsenic Tolerance Levels Determination
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The Tolerance levels to arsenic (TLA) for the isolates was determined by the agar dilution technique, on Luria Bertani (LB) agar (Becton Dickinson and Company, Maryland, MO, USA) plates supplemented with different concentrations of sodium arsenite (0.5–100 mM) (Sigma-Aldrich, San Luis, MO, USA) or sodium arsenate (0.5–1000 mM) (Sigma-Aldrich, San Luis, USA). Each plate was inoculated with cell suspensions from fresh pre-cultures at a final density of approximately 107 colony forming units (CFU) mL−1 and incubated for 24 h at 25 °C. To be used as control, LB agar plates were seeded with bacteria but without the metalloid. The criterion to consider bacterial strains as resistant or sensitive to As(III) or (As(V) was the one described by Rokbani et al. [35 (link)]. The bacterial strain capable to tolerate the highest arsenite and arsenate concentrations was selected to perform all the following experiments.
6
EHEC and Bacteroides growth kinetics
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For the growth kinetic experiments with EHEC NIPH-11060424 and B. thetaiotaomicron, a CFU ratio of 1:100 and 1:1 between the two species was used. In order to quantify viable bacterial cells from pure cultures and co-cultures, bacteria were enumerated on the basis of CFU ml-1 on LB-agar and Bacteroides Bile Esculin Agar (BBE-agar, Becton, Dickinson and company, Maryland, USA) at serial time points. The LB-agar was incubated aerobically to ensure growth of EHEC and BBE-agar was incubated anaerobically to ensure growth of B. thetaiotaomicron. Furthermore, growth kinetics was determined for EHEC grown in spent medium from B. thetaiotaomicron and B. fragilis as described above. For these latter experiments growth kinetics were determined by measuring optical density (OD) at 600 nm. All experiments were performed independently three times.
7
Isolate and Cultivate Rifampin-Resistant Shigella
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Shigella isolates were isolated by stool culture on Hektoen enteric agar (Remel, Lenexa, KS) and species determined by Vitek 2 (bioMérieux, Durham, NC). Rifampin-resistant mutants of clinical strains, used for functional studies, were created by plating a 10-µl loopful of cells from LB agar (Becton, Dickinson and Company, Sparks, MD) onto LB agar with Rifampin overnight at 37°C. The following antibiotics were used for selections: ampicillin (MilliporeSigma, Saint Louis, MO), 200 µg/ml; Rifampin (G-Biosciences, Saint Louis, MO), 100 µg/ml; and tetracycline (MilliporeSigma, Saint Louis, MO), 15 µg/ml. Bacterial strains used in this study are listed in Table S3 in the supplemental material.
8
Conjugation of CTX-M-65 Plasmids
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The transfer of blaCTX-M-65 plasmids was studied in a conjugation experiment following a previously established protocol [25 ] carried out on LB agar (Becton, Dickinson and Company) and in LB broth overnight at both 37°C and room temperature. The blaCTX-M-65-positive isolates, 98.1 (this study), and FC-471 [12 (link)] were used as donor strains. The nalidixic acid-resistant strain 711-nal [26 (link)] was used as a recipient. Transconjugants were selected on LB agar containing 50 mg/L nalidixic acid and 4mg/L ceftriaxone. Isolate PA3, which contains a transferable tetracycline resistance plasmid, was used as a positive control. PA3 transconjugants were selected on LB agar containing 50 mg/L nalidixic and 16 mg/L tetracycline.
9
Salmonella Strains and Culture Conditions
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Salmonella strains used in this study included Salmonella enterica sv Typhimurium strain 14028s, isolated from 4-week-old chickens in 1960 (Jarvik et al., 2010 (link)); Salmonella Enteritidis PT4 strain P125109, a clinical strain associated with a poultry outbreak in the United Kingdom (Toro et al., 2016 (link)); and Salmonella Newport strain C4.2, isolated from a tomato field in Virginia, United States (de Moraes et al., 2018 (link)). Single gene deletion (SGD) mutants of S. Typhimurium strain 14028s harbor a kanamycin (SGD-KanR) or chloramphenicol (SGD-CmR) resistance gene, oriented in the antisense and sense direction, respectively, in regard to the deleted gene (Porwollik et al., 2014 (link)). The strains were preserved at −80°C in Luria-Bertani broth (LB; Becton, Dickinson & Co. Sparks, MD, United States) containing 20% glycerol (Fisher Scientific, Fair Lawn, NJ, United States). Bacterial cultures were grown in LB or on LB agar containing 1.2% agar (Becton, Dickinson & Co.) at 37°C for 24 h unless indicated otherwise. When necessary, growth media were supplemented with 60 μg/ml kanamycin (LB + Kan; Fisher Scientific) or 20 μg/ml chloramphenicol (LB + Cm, Acros Organics, NJ, United States).
10
Isolation and Characterization of fRuSau02 Phage
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The bacterial strains used in this work are described in Table S1 . The collection of human isolates used in this study was provided by The Hospital District of Helsinki and Uusimaa Laboratories (HUSLAB), Finland. All staphylococcal and phage incubations were done at 37 °C using Luria Broth (LB) [23 ] medium. Soft agar medium included additionally 0.35 or 0.4% (w/v) agar (Becton Dickinson, Franklin Lakes, NJ, USA), and LB agar plates were solidified with 1.5% (w/v) of agar. fRuSau02 was isolated using a clinical S. aureus strain 13KP (Table S1 ) as a host, and the same strain was then used as a standard host strain for the phage. The phage lysates were produced from semiconfluent soft-agar plates as described elsewhere [23 ].
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