The Mtb H37Rv, MtbΔsufR, and sufR-Comp were grown in Middlebrook 7H9 broth (Becton, Dickinson and Company (BD), USA) medium supplemented with 0.2% glycerol, 0.5% BSA, 0.2% dextrose, and 0.085% NaCl (ADS) with 0.05% Tween 80 as described previously [23 ]. For culturing on solid medium, Mtb strains were cultured on 7H10/7H11 agar medium (Becton, Dickinson and Company (BD), USA) supplemented with 1x OADC (Becton, Dickinson and Company (BD), USA) and 0.2% glycerol. E. coli cultures were grown in LB medium (HIMEDIA, India). Whenever required, antibiotics were added to the culture medium (for E. coli, 100 μg/mL kanamycin (Amresco, USA) and 150 μg/mL hygromycin (Sigma-Aldrich, India); for Mtb strains, 50 μg/mL hygromycin).
Lb medium
Manufactured by HiMedia
Sourced in India
LB medium is a widely used growth medium for cultivating bacteria. It provides the necessary nutrients and support for the optimal growth and proliferation of various bacterial species.
Automatically generated - may contain errors
Lab products found in correlation
Middlebrook 7h9 broth, by BD (2 mentions)
Hygromycin, by Merck Group (1 mentions)
Kanamycin, by Avantor (1 mentions)
Kanamycin kan, by Avantor (1 mentions)
Lb broth, by HiMedia (1 mentions)
Qiaprep spin miniprep kit, by Qiagen (1 mentions)
Luria bertani agar, by HiMedia (1 mentions)
Rotary shaker, by Biosan (1 mentions)
8 protocols using lb medium
1
Mycobacterium tuberculosis Culturing Protocol
2
Antibacterial Activity of Biogenic Silver Nanoparticles
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The antibacterial properties of the as-synthesized biogenic Ag NPs were examined by carrying out a set of investigations to evaluate the growth of both Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacterial strains. The strain of both kinds of microorganisms were cultured in 5 mL LB medium (Hi Media) aseptically under laminar air-flow, followed by its sub-culturing into 50 mL LB of four sets of Ag NPs having concentrations of 2 mg L−1, 5 mg L−1, 8 mg L−1, and 10 mg L−1. At the same time, a control experiment having no Ag NPs was likewise done. The optical densities of the pathogens were recorded and plotted as a function of time. The bacterial expansion was observed (after interims of 2 h) by recording the OD600 nm in the UV-Vis spectrophotometer for the control and samples. The last OD600 nm was recorded after 24 h of growth.
3
Genomic Characterization of Klebsiella pneumoniae Isolates
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A collection comprising 32 clinical K. pneumoniae isolates from the Clinical Hospital №123 (Odintsovo, Russia) and 51 strains from the State Collection of Pathogenic Microorganisms and Cell Cultures, SCPM-Obolensk (State Research Center for Applied Microbiology and Biotechnology, Obolensk, Russia) were included in this study (Supplementary Table 3 ). All bacteria were grown in the Nutrient Medium No. 1 (SRCAMB, Obolensk, Russia), or in the lysogeny broth (LB) medium (Himedia, India) at 37°C. Bacterial identification was performed by MALDI-TOF mass spectrometry as described previously (Kornienko et al., 2016 (link)). The antibiotic susceptibility was tested using the disc diffusion method according to Clinical and Laboratory Standards Institute guidelines 28th edition (Clinical and Laboratory Standards Institute, 2018 ). Multilocus sequence typing (MLST) of K. pneumoniae strains was performed by determining the nucleotide sequences of seven housekeeping genes as described previously (Diancourt et al., 2005 (link)). The capsular type was determined by wzi gene sequencing (Brisse et al., 2013 (link)).
4
Cultivation of Mycobacterium tuberculosis Strains
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The Mycobacterium tuberculosis H37Rv, and derived strains (control and SufT-KD) were grown in Middlebrook 7H9 broth (Becton, Dickinson and Company, USA) medium supplemented with 0.2% glycerol, 0.5% BSA, 0.2% dextrose, and 0.085% NaCl (ADS) with 0.05% Tween-80 as described previously [65 (link)]. ATc was added in secondary culture between OD600 nm 0.1–0.2 at concentration of 200 ng/mL [36 (link)]. For culturing on solid medium, Mtb strains were cultured on 7H11 agar medium (Becton, Dickinson and Company, USA) supplemented with 1x OADC (Becton, Dickinson and Company, USA) and 0.2% glycerol. Whenever required, antibiotics were added to the culture medium, at concentration of 25 μg/mL kanamycin (KAN) (Amresco, USA) and 50 μg/mL hygromycin (HYG) (Sigma-Aldrich, USA). E. coli DH5α and BL21 strains were grown in LB medium (HIMEDIA, India) with antibiotic concentration of 50 μg/mL KAN and 100 μg/mL HYG.
5
Bacterial Metabolism and Pollutant Degradation
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LB medium, minimal medium (MM), and glucose were purchased from Himedia (Mumbai, India). Cetyltrimethylammonium bromide (CTAB), methylene blue, biphenyl, PCB-77 (3,4,3′,4′-tetrachlorobiphenyl), acetone, ethyl acetate, hexane, methanol (Empure, GC grade) was purchased from Sigma Aldrich (St. Louis, MO, USA).
6
Plasmid Transfer via Transformation and Conjugation
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Total Plasmid content was extracted by QIAprep Spin Miniprep Kit (Qiagen, Germany) and isolated plasmids were subjected to transformation by heat shock method using Escherichia coli DH5α as recipient. Transformants were selected on to the Luria Bertani agar (Hi-Media, Mumbai,India) containing 2 μg/ml of kanamycin. Conjugation assay was performed where the isolates harbouring 16S rRNA methyltransferase genes, acted as donor and azide resistant E.coli J53 was used as recipient. Mating was performed where both the donor and recipient cells were cultured in LB broth (Hi-Media) till it attained optical density at 600 nm (OD600) of 0.8–0.9. Cells were mixed at a ratio of 1:5 donor-to-recipient and transconjugant was selected on LB medium (Hi Media,Mumbai,India) containing 2 μg/ml of kanamycin and 100 μg/ml of sodium azide. Transformants and transconjugants were further screened for the presence of 16S methyltransferase genes, ESBLs and carbapenemase genes as obtained in parent isolates.
7
Bacterial Strains and Plasmids for Klebsiella Research
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The bacterial strains and plasmids used in this study are listed in Table 1 . E. coli DH5α, K. pneumoniae SM6, K. pneumoniae SM11 K. pneumoniae SM6Δ and K. pneumoniae SM11Δ were grown aerobically on LB medium (Himedia Laboratories, India) at 30°C. K. pneumoniae SM6 and K. pneumoniae SM11 were deposited at Microbial Culture Collection (MCC), Pune with accession numbers MCC2730 and MCC2716, respectively. For plate cultures, 1.5% agar was added to LB medium before autoclaving. Temperature sensitive mutants and temperature sensitive plasmid-carrying strains were grown at 28°C. Antibiotics ampicillin, erythromycin and kanamycin were added to the medium at a concentration of 50 μg ml-1, as and when required. Culture stocks were preserved in 50% (v/v) glycerol and stored at -20°C. Antibiotics, salts, reagents and carbon sources were acquired from Merck-Millipore, Germany.
8
Antimicrobial Potential of NM-CH-I15-I
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The antibacterial activity of the bacterium NM-CH-I15-I was tested against five Gram-positive (Arthrobacter sp. AK-5, Bacillus subtilis PY79, Brevibacterium flavum CCM 251, Lysinibacillus sp. AK-11, Streptomyces coelicolor M145), and five Gram-negative (Bacteroidetes sp. AK-13, Beta proteobacterium AK-23, E. coli, Ralstonia picketii MR-CH-I2, Stenotrophomonas chelatiphaga AK-32) bacterial strains (IMB SAS collection). Bacterium NM-CH-I15-I was grown in LB medium (HiMedia, India) in Erlenmeyer flask placed in a rotary shaker (200 rpm, Biosan, Lithuania) overnight at 30°C, the culture was 10-fold diluted in 10 mmol/l Tris-HCl (pH 7.0), and 5 ml aliquots were spotted in triplicate onto the surface of a LB agar plate. The plates were incubated overnight at 30°C, and then overlaid with 5 ml of LB soft agar (0.7% agar) seeded with 0.1 ml of an overnight culture of one of ten indicator strains. Plates were incubated for 12 h at 30°C and then checked for clear zones around the previously uncultured isolates.
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