Bacteroides thetaiotaomicron VPI-5482, Bacteroides thetaiotaomicron VPI-5482 CPS385 (link), and Bacteroides fragilis NCTC 9343 were cultured as previously described84 (link). Briefly, these strains were grown in brain-heart infusion (BHI) broth (Becton Dickinson, Franklin Lakes, NJ) supplemented with 1 g/L cysteine, 5% w/v NaHCO3, and 5 mg/L hemin (BHIS), Varel-Bryant minimal media86 (link) supplemented with 28 mM glucose, or Bacteroides phage recovery medium (BPRM)87 (link) at 37° C in a Coy Type A vinyl anaerobic chamber in an atmosphere of 5% hydrogen, 20% carbon dioxide, and balanced nitrogen (Coy Lab Products, Grass Lake, MI). For growth on solid-media we used BHI agar supplemented with 10% defibrinated calf blood (Colorado Serum Company, Denver, CO). Escherichia coli S17-1 was grown in Lennox L broth (Fisher Scientific, Hampton, NH) with aeration at 37° C. Tetracycline was used at 2 μg/mL, gentamicin was used at 200 μg/mL, and ampicillin was used at 100 μg/mL.
Lennox l broth
Manufactured by Thermo Fisher Scientific
Sourced in Germany
Lennox L broth is a complex microbiological medium used for the cultivation of a variety of bacterial species. It provides the necessary nutrients and growth factors to support the growth of these organisms in a laboratory setting.
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Lab products found in correlation
Bhi broth, by BD (2 mentions)
Blood agar base 2, by Thermo Fisher Scientific (1 mentions)
Protino ni nta agarose, by Macherey-Nagel (1 mentions)
Isopropyl thiogalactose iptg, by Merck Group (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Air tight jars, by Thermo Fisher Scientific (1 mentions)
Anaerocult c gas generating bags, by Merck Group (1 mentions)
Pcr dna purification kit, by Qiagen (1 mentions)
Dmem 25 mm hepes, by Thermo Fisher Scientific (1 mentions)
Cisplatin, by Merck Group (1 mentions)
9 protocols using lennox l broth
1
Culturing Bacteroides and E. coli Strains
2
Culturing Bacteroides and E. coli
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Bacteroides thetaiotaomicron VPI-5482, Bacteroides thetaiotaomicron VPI-5482 CPS3, and Bacteroides fragilis NCTC 9343 were cultured as previously described 84 (link),85 (link). Briefly, these strains were grown in brain-heart infusion (BHI) broth (Becton Dickinson, Franklin Lakes, NJ) supplemented with 1 g/L cysteine, 5% w/v NaHCO3, and 5 mg/L hemin (BHIS), Varel-Bryant minimal media 86 (link) supplemented with 28 mM glucose, or Bacteroides phage recovery medium (BPRM) 87 (link) at 37° C in a Coy Type A vinyl anaerobic chamber in an atmosphere of 5% hydrogen, 20% carbon dioxide, and balanced nitrogen (Coy Lab Products, Grass Lake, MI). For growth on solid-media we used BHI agar supplemented with 10% defibrinated calf blood (Colorado Serum Company, Denver, CO). Escherichia coli S17-1 was grown in Lennox L broth (Fisher Scientific, Hampton, NH) with aeration at 37° C. Tetracycline was used at 2 μg/mL, gentamicin was used at 200 μg/mL, and ampicillin was used at 100 μg/mL.
3
Quantifying Salmonella Shedding in Poultry
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On D21, treatments began for each group of birds (n = 19 to 22 per group after removing non-shedders in each experiment). On D28, 35, and 42, approximately 0.5 g of freshly voided feces (from each bird) was briefly vortexed in 10 mL of Lennox L broth (Invitrogen, Carlsbad, CA). An aliquot of this mixture (100 μL) was incubated overnight at 37°C on XLD agar. The following d, white colonies with black centers were enumerated and CFU/g of feces was calculated based on a dilution factor equal to 100.
4
Salmonella Enumeration from Avian Intestines
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On D49, all remaining birds were euthanized and a 5 cm section (approximately 3 g) of distal intestine (between the cloaca and ceca) was aseptically removed from each bird and cut longitudinally. Each section was placed in 10 mL Lennox L broth (Invitrogen, Carlsbad, CA) and briefly vortexed to dislodge the Salmonella. An aliquot (100 μL) of this mixture was then dispersed onto XLD agar plates that were incubated overnight at 37°C. The following d, white colonies with black centers were enumerated and CFU/g of intestine was calculated based on a dilution factor equal to 100.
5
Culturing H. pylori and E. coli
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H. pylori strains were cultured on blood agar plates (blood agar base II; Oxoid, Wesel, Germany) containing 10% horse blood and supplemented with antibiotics (vancomycin [10 mg/liter], polymyxin B [3.2 mg/liter], amphotericin B [4 mg/liter], and trimethoprim [5 mg/liter]) as previously described (67 (link)). H. pylori liquid cultures were performed in brain heart infusion broth (BHI; BD Difco, Heidelberg, Germany) with yeast extract (2.5 g/liter), 10% heat-inactivated horse serum, and the same combination of antibiotics as on the blood agar plates (23 (link)). Escherichia coli strains were grown in LB medium (Lennox L broth; Invitrogen Life Technologies, Darmstadt, Germany) supplemented with ampicillin (200 μg/ml) and/or kanamycin (20 μg/ml) as described previously (41 (link)).
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6
Oral Salmonella Typhimurium Inoculation in Chicks
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All birds were confirmed to be Salmonella-free by fecal culture upon arrival. Specifically, one to 5 g of freshly voided feces from each chick was diluted in 10 mL of Lennox L broth (Invitrogen, Carlsbad, CA). After settling for one to 2 h at room temperature, an aliquot (100 μL) of this mixture was streaked onto and then incubated overnight at 37°C on Xylose Lysine Deoxycholate (XLD ) agar (Fisher Scientific, Pittsburgh, PA) that is selective for Salmonella, which appear as white colonies with black centers (Anderson et al., 2015 ). All pre-infection fecal samples were free of Salmonella.
On D2, 9, and 16, birds were orally inoculated with Salmonella Typhimurium strain LNWI (Wu et al., 2002 (link); Anderson et al., 2015 ). The dose increased from 2 × 108 CFU/bird on D2 (Anderson et al., 2015 ) to 4 × 108 CFU/bird on D9 to 8 × 108 CFU/bird on D16, and this procedure was done to maximize the likelihood of large intestinal carriage. The Salmonella inoculum was prepared and dosed as previously reported (Xiong et al., 2011 (link); Xiong et al., 2012 (link); Xiong et al., 2013 (link); Anderson et al., 2015 ). The inoculum was slowly introduced into the mouth of each bird using a pipette tip. Previous studies revealed that Salmonella is viable after incubation with either XPC (at the concentration equivalent to the dose used in this study) or the CON treatment (Anderson et al., 2015 ).
On D2, 9, and 16, birds were orally inoculated with Salmonella Typhimurium strain LNWI (Wu et al., 2002 (link); Anderson et al., 2015 ). The dose increased from 2 × 108 CFU/bird on D2 (Anderson et al., 2015 ) to 4 × 108 CFU/bird on D9 to 8 × 108 CFU/bird on D16, and this procedure was done to maximize the likelihood of large intestinal carriage. The Salmonella inoculum was prepared and dosed as previously reported (Xiong et al., 2011 (link); Xiong et al., 2012 (link); Xiong et al., 2013 (link); Anderson et al., 2015 ). The inoculum was slowly introduced into the mouth of each bird using a pipette tip. Previous studies revealed that Salmonella is viable after incubation with either XPC (at the concentration equivalent to the dose used in this study) or the CON treatment (Anderson et al., 2015 ).
7
Recombinant Fibronectin Fragment Production
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FN fragment FNIII7-10, RGD-deleted FN fragment FNIII7-10ΔRGD and synergy site mutated FN fragment FNIII7-10-mSYN were expressed from plasmid pET15b-FNIII7-10 and pET15b-FNIII7-10ΔRGD44 (link) (generated by R. Fässler) in E.coli BL21 (DE3) pLysS44 (link). Briefly, cells were grown in Lennox L broth (Invitrogen, Carlsbad, USA) supplemented with 100 μg ml–1 ampicillin (Sigma-Aldrich, Buchs, Switzerland) at 37 °C. Expression was induced with 1 mM isopropyl thiogalactose (IPTG, Sigma) at optical density (OD)600=0.6. Cells were collected after 4 h, re-suspended in buffer (20 mM Tris–HCl, 150 mM NaCl, pH 8.0), and broken by sonication. Cell debris was removed by ultracentrifugation at 40,000g for 45 min. The soluble protein fraction was bound to nickel-nitrilotriacetic acid resin (Protino Ni-NTA Agarose, MACHEREY-NAGEL, Düren, Germany) for 1 h at 4 °C. The resin was then loaded onto a column and washed with buffer (20 mM Tris–HCl, 150 mM NaCl, 10 mM imidazole, pH 8.0). FN fragments were eluted with elution buffer (20 mM Tris–HCl, 150 mM NaCl, 500 mM imidazole, pH 8.0). Peak fractions were pooled and dialyzed against washing buffer (20 mM Tris–HCl, 150 mM NaCl, pH 8.0). The protein concentration was adjusted to 1.0 mg ml–1 with dialyzing buffer and aliquots were stored at –20 °C.
8
Culturing H. pylori and E. coli
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H. pylori and Escherichia coli strains used in this study are listed in Supplementary Table 1 . Wild-type and mutant H. pylori strains were cultured from frozen stocks on blood-agar plates7 (link). For liquid cultures, bacteria were routinely grown in brain heart infusion broth (BHI, BD Difco, Heidelberg, Germany) with yeast extract (2.5 g l-1), 10% heat-inactivated horse serum and a mix of antibiotics (vancomycin (10 mg l-1), polymyxin B (3.2 mg l-1), amphotericin B (4 mg l-1), and trimethoprim (5 mg l-1)). Cultivation was performed at 175 r.p.m. and 37 °C in microaerobic atmosphere using air-tight jars (Oxoid, Wesel, Germany) and Anaerocult C gas generating bags (Merck, Darmstadt, Germany).
For DNA cloning experiment, E. coli strains DH5α, DH5α λpir and MC1061 were cultured in LB or on LB agar plates (Lennox L Broth, Invitrogen–Life Technologies, Darmstadt, Germany) supplemented with ampicillin (200 μg ml-1), kanamycin (20 μg ml-1) and/or chloramphenicol (20 μg ml-1) as required.
For DNA cloning experiment, E. coli strains DH5α, DH5α λpir and MC1061 were cultured in LB or on LB agar plates (Lennox L Broth, Invitrogen–Life Technologies, Darmstadt, Germany) supplemented with ampicillin (200 μg ml-1), kanamycin (20 μg ml-1) and/or chloramphenicol (20 μg ml-1) as required.
9
E. coli DNA Crosslinking Assay
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The E. coli strain Nissle 1917 used in this study was obtained from Dr. Ulrich Dobrindt (University of Münster). The clbA and clbP isogenic mutants were described previously (ref Ollier et Nat Comm). Before infection, the bacteria were grown overnight at 37°C with 240 RPM agitation in 5 mL of Lennox L broth (LB, Invitrogen) then diluted 1/20 in pre-warmed DMEM 25 mM Hepes (Invitrogen) and incubated at 37°C with 240 RPM agitation to reach exponential phase (OD600=0.4 to 0.5).
In vitro DNA crosslinking assay 3x10 6 bacteria or numbers given in the text were inoculated in 100 l of DMEM 25 mM Hepes, incubated at 37°C for 3.5 hours, then EDTA (1 mM) and 400 ng of linearized (BamHI) pUC19
DNA were added and further incubated 40 minutes. As controls, DNA was left uninfected or was treated with 100 or 200 M cisplatin (Sigma). Following a centrifugation to pellet the bacteria, the DNA was purified using Qiagen PCR DNA purification kit before analysis by denaturing gel electrophoresis.
In vitro DNA crosslinking assay 3x10 6 bacteria or numbers given in the text were inoculated in 100 l of DMEM 25 mM Hepes, incubated at 37°C for 3.5 hours, then EDTA (1 mM) and 400 ng of linearized (BamHI) pUC19
DNA were added and further incubated 40 minutes. As controls, DNA was left uninfected or was treated with 100 or 200 M cisplatin (Sigma). Following a centrifugation to pellet the bacteria, the DNA was purified using Qiagen PCR DNA purification kit before analysis by denaturing gel electrophoresis.
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