F. nucleatum subsp. nucleatum ATCC 23726, F. nucleatum subsp. nucleatum ATCC 25586, and F. nucleatum subsp. animalis 7_1 (Fna) were cultured on solid agar plates made with Columbia broth (Gibco) substituted with hemin (5 μg/ml) and menadione (0.5 μg/ml) (CBHK) under anaerobic conditions (90% N2, 5% H2, 5% CO2) at 37°C. Liquid cultures started from single colonies were grown in CBHK medium under the same conditions. For infections, overnight cultures were back-diluted 1:1000 and grown to mid-exponential phase [~0.5 OD600 (optical density at 600 nm)] before subsequent experiments unless otherwise noted.
Columbia broth
Manufactured by Thermo Fisher Scientific
Sourced in United States
Columbia broth is a general-purpose microbial growth medium. It supports the growth of a wide range of microorganisms, including bacteria and fungi. The broth provides the necessary nutrients and growth factors for microbial cultivation in a laboratory setting.
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7 protocols using columbia broth
1
Culturing Fusobacterium nucleatum Strains
2
Growth and Preparation of Anaerobic Bacteria
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Fusobacterium nucleatum subsp. nucleatum ATCC 23726 was grown under anaerobic conditions19 (link). Frozen cultures of bacteria were streaked on solid agar plates made with Columbia broth (Gibco) substituted with hemin (5 μg/mL) and menadione (0.5 μg/mL) (CBHK) and grown in an anaerobic chamber (90% N2, 10% H2, and 10% CO2) at 37 °C (Whitley A35 anaerobic workstation). Single colonies were then retrieved and inoculated in liquid cultures of CBHK media and grown for ~16 h anaerobically to reach a mid-exponential growth phase. The optical density at 600 nm was measured to be 0.5 (Implen OD600 DiluPhotometer) and the bacteria were resuspended in PBS and used for the experiments.
Escherichia coli TOP 10 was grown in Luria Broth (LB) in a shaker incubated at 37 °C overnight. Bacterial culture was diluted to an optical density (600 nm) of 0.5 before being used for experiments.
Escherichia coli TOP 10 was grown in Luria Broth (LB) in a shaker incubated at 37 °C overnight. Bacterial culture was diluted to an optical density (600 nm) of 0.5 before being used for experiments.
3
Fusobacterium nucleatum Cultivation and HCT116 Infection
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Culturing Fusobacterium nucleatum Fusobacterium nucleatum subsp. nucleatum ATCC 23726, Fusobacterium nucleatum subsp. nucleatum ATCC 25586, and Fusobacterium nucleatum subsp. animalis 7_1 ( Fna ) were cultured on solid agar plates made with Columbia Broth (Gibco) substituted with hemin (5 μg/mL) and menadione (0.5 μg/mL) (CBHK) under anaerobic conditions (90% N 2 , 5% H 2 , 5% CO 2 ) at 37 ˚C. Liquid cultures started from single colonies were grown in CBHK media under the same conditions. For infections, overnight cultures were back diluted 1:1000 and grown to mid-exponential phase (~0.5 OD 600 ) prior to subsequent experiments unless otherwise noted.
Culturing HCT116 CRC cells HCT116 (ATCC CCL-247) cells were purchased from ATCC and grown on tissue culture treated plates and flasks in McCoy's 5A media supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin. Cells were grown to no more than passage 15 at 37 ˚C with 5% CO 2 . For maintenance between infections, cells were passaged by gentle trypsinization and reseeding. For infection experiments, cells were grown to >90% confluency in 6 or 24 well plates. Unless otherwise indicated, all experiments were conducted with HCT116 media supplemented with 10% FBS.
Culturing HCT116 CRC cells HCT116 (ATCC CCL-247) cells were purchased from ATCC and grown on tissue culture treated plates and flasks in McCoy's 5A media supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin. Cells were grown to no more than passage 15 at 37 ˚C with 5% CO 2 . For maintenance between infections, cells were passaged by gentle trypsinization and reseeding. For infection experiments, cells were grown to >90% confluency in 6 or 24 well plates. Unless otherwise indicated, all experiments were conducted with HCT116 media supplemented with 10% FBS.
4
Streptococcus pneumoniae Growth Protocol
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The experimental work was performed with the R6D wild-type strain of Streptococcus pneumoniae (Hun663.tr4), as this was used in our previous studies of BriC (11 (link)). Colonies were grown from frozen stocks by streaking on TSA-II agar plates supplemented with 5% sheep blood (BD BBL, NJ, USA). Unless otherwise stated, streaked colonies were picked and inoculated in fresh Columbia broth (Remel Microbiology Products, Thermo Fisher Scientific, USA) whose pH was adjusted to 6.6 by the addition of 1 M HCl and thereafter incubated at 37°C and 5% CO2 without shaking. Antibiotics were not added to the growth medium under any assay conditions.
5
Anaerobic and Aerobic Growth of Oral Microbes
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XH001 monoculture and XH001/TM7x coculture were isolated from the oral cavity as described in the previous published study [13 (link)]. Strains were cultured in brain heart infusion (BHI, Thermo Fisher Scientific, NH) at 37°C under different oxygen conditions as specified in the main text: anaerobic (0% O2, 10% CO2, 5% H2, balanced with N2), microaerophilic (2% O2, 5% CO2, balanced with N2), and atmospheric conditions (∼21% O2, 0.04% CO2, 0.9% Ar, 78% N2). To acquire growth kinetics and phase contrast images, three independent cell cultures were grown under the specified oxygen condition for two passages (1 ml culture inoculated into 10 ml BHI and incubated 24 h each) before being reinoculated into 20 ml fresh BHI. The optical density at 600 nm (OD600) was measured using a spectrophotometer (Thermo Fisher Scientific, MA).
Fusobacterium nucleatum strains were cultured in Columbia broth (Thermo Fisher Scientific, MA) at 37°C under anaerobic condition (0% O2, 10% CO2, 5% H2, balanced with N2) for two passages.
Streptococcus mutans UA159 and MRSA were cultured inside BHI broth at aerobic condition (∼21% O2, 0.04% CO2, 0.9% Ar, 78% N2) for two passages.
Fusobacterium nucleatum strains were cultured in Columbia broth (Thermo Fisher Scientific, MA) at 37°C under anaerobic condition (0% O2, 10% CO2, 5% H2, balanced with N2) for two passages.
Streptococcus mutans UA159 and MRSA were cultured inside BHI broth at aerobic condition (∼21% O2, 0.04% CO2, 0.9% Ar, 78% N2) for two passages.
6
Growth of S. pneumoniae in vitro
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Frozen bacterial stocks were streaked onto Trypticase soy agar plates containing 5% sheep blood (BD BBL). All S. pneumoniae strains were grown in Columbia broth (Thermo Scientific) at 37°C with 5% CO2 without shaking. Medium was supplemented with antibiotics at 1 µg/ml for tetracycline and 100 µg/ml for spectinomycin.
7
Reverse Genetics Protocols for Influenza A H1N1pdm09 and Pneumococcus Strains
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MDCK (Madin-Darby canine kidney, obtained from ATCC) were grown at 37°C in 5% CO2 in MEM medium (Sigma) containing 5% Fetal Bovine Serum (FBS, HyClone), penicillin/streptomycin, and L-glutamine. Reverse genetics plasmids of A/California/07/2009 were a generous gift from Dr Jesse Bloom (Fred Hutch Cancer Research Center, Seattle) and were rescued as previously described (Lakdawala et al. 2011 (link)). The viral titers were determined by tissue culture infectious dose 50 (TCID50) using the endpoint titration method on MDCK cells for H1N1pdm09. The bacterial strains used in this study were graciously provided by Dr Hasan Yesilkaya (wild type Spn serotype 2 strain D39; Andrew et al. 2018 (link)) and Dr Jason Rosch (wild type Spn serotype 19F strain BHN97; Rowe et al. 2019 (link)). Bacteria were grown from frozen stocks by streaking on TSA-II agar plates supplemented with 5% sheep blood (BD). Cultures were generated in fresh Columbia broth (Thermo Fisher) and incubated at 37°C and 5% CO2 without shaking. Pneumococci in nasal washes and tissues were determined by plating serial dilutions onto blood agar plates incubated at 37°C overnight.
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