The DH5α [F-, Φ 80 lacZΔM15, recA1, gyrA96, thi-1, hsdR17, (rk-, mk-), supE44, re1A1deoR, Δ(lacZYA-argF), U169 (Z. Burton, Michigan State, University East Lansing)] strain of Escherichia coli was stored as glycerol stock at −70°C, and for research purposes the culture was kept at 37°C for 24 h in LB broth (Biomaxima, Poland). In this work, plasmid pDS1 (2,833 bp, [AmpR; ori-pBR322; ori-ColE1] (NCBI—490681; courtesy of H. Heumann) was also applied.
Lb broth
Manufactured by Biomaxima
Sourced in Poland
LB broth is a commonly used nutrient-rich growth medium for culturing bacteria in a laboratory setting. It provides the essential nutrients and conditions required for the growth and proliferation of a wide range of bacterial strains.
Automatically generated - may contain errors
4 protocols using lb broth
1
Culturing DH5α E. coli with pDS1 Plasmid
2
Bacterial Culture Protocol in LB Broth
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Bacterial cells were cultivated in LB broth (Biomaxima, Lublin, Poland) at 37 °C with shaking at 120 rpm. Overnight cultures were diluted in fresh medium to an optical density (OD) of 0.05 and transferred to a 24-well plate (0.5 mL per well).
3
Microbiological Identification of Mastitic Milk
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Samples of mastitic milk were collected from farms from July 2021 to March 2022. The collected samples were submitted to standard microbiological procedures for initial multiplication and preliminary species identification: strains were isolated and then cultured on solid and liquid media. Specialized microbiological media (Biomaxima, Poland) were used to culture the strains: nutrient broth for general microbial culture, Mannitol Salt Lab-Agar, Edwards Lab-Agar with the addition of bovine blood, Chromogenic Uri-Color Lab-Agar, MacConkey Lab-Agar, Salmonella-Shigella Lab-Agar, Chromogenic Candida Lab Agar, Rose Bengal Lab-Agar, TBX Lab-Agar, Chromogenic Coliform Lab-Agar, LB Broth, Rogosa LS Lab-Agar, Reinforced Clostridial Medium, Legionella CYE Lab-Agar Base, Listeria acc., Oxford Lab-Agar Base, Enterococcus Confirmatory Lab-Agar, Eijkman Lactose Medium, Prototheca Isolation Medium according to Pore (1973),31 (link) and Sabouraud dextrose agar (all from Argenta, Poland). Subsequently, the selected colony samples were subjected to identification on a MALDI-TOF MS (Bruker, Poznań, Poland).
4
Bacterial and Fungal Culture Preparation
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Three reference isolates,
including the bacteria S. aureus Xen
30 and P. aeruginosa Xen 5 (Caliper
Life Sciences, Hopkinton, MA, USA), and the fungus C. albicans 1408 (Polish Collection of Microorganisms,
Polish Academy of Science, Wroclaw, Poland) were used. The analyzed
strains were cultured and maintained on the recommended selective
or selective-differential media, that is, Chapman agar (Biomaxima,
Poland) for S. aureus Xen 30, Cetrimide
agar (Thermo Scientific Oxoid, USA) for P. aeruginosa Xen 5, and Sabouraud Dextrose agar with chloramphenicol (Biomaxima,
Poland) for C. albicans 1408. Bacterial
cells of S. aureus Xen 30 and P. aeruginosa Xen 5 were cultured in an LB broth
(Biomaxima, Poland), whereas C. albicans 1408 was grown in a RPMI medium supplemented with MOPS andd -(+)-glucose (all from Sigma-Aldrich, USA) to mid-log phase at 37
°C in aerobic conditions to a final cell density of 108 colony-forming unit (cfu)/mL. At the end of incubation, an inoculum
of microorganisms was centrifuged at 2000 rpm for 10 min. The supernatant
was discarded and the sedimented bacteria or fungi were used for further
investigation.
including the bacteria S. aureus Xen
30 and P. aeruginosa Xen 5 (Caliper
Life Sciences, Hopkinton, MA, USA), and the fungus C. albicans 1408 (Polish Collection of Microorganisms,
Polish Academy of Science, Wroclaw, Poland) were used. The analyzed
strains were cultured and maintained on the recommended selective
or selective-differential media, that is, Chapman agar (Biomaxima,
Poland) for S. aureus Xen 30, Cetrimide
agar (Thermo Scientific Oxoid, USA) for P. aeruginosa Xen 5, and Sabouraud Dextrose agar with chloramphenicol (Biomaxima,
Poland) for C. albicans 1408. Bacterial
cells of S. aureus Xen 30 and P. aeruginosa Xen 5 were cultured in an LB broth
(Biomaxima, Poland), whereas C. albicans 1408 was grown in a RPMI medium supplemented with MOPS and
°C in aerobic conditions to a final cell density of 108 colony-forming unit (cfu)/mL. At the end of incubation, an inoculum
of microorganisms was centrifuged at 2000 rpm for 10 min. The supernatant
was discarded and the sedimented bacteria or fungi were used for further
investigation.
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