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13 protocols using BHI) broth

Enrichment and isolation of Listeria from these environmental samples were performed using a modified version of the USDA-FSIS MLG 8.10 method (36 ). Three grams of fresh soil or feces were added to 9 ml of buffered peptone water (Acumedia, Lansing, MI, USA) in a filtered stomacher bag and were vigorously shaken for 30 s. As a pre-enrichment step, the stomached homogenates remained in the filtered stomacher bag and were incubated overnight at 35°C. This pre-enrichment step was followed by two enrichments in University of Vermont Modified Listeria Enrichment Broth (UVM; Remel, Lenexa, KS, USA) and Fraser Broth (Oxoid CM0895, Basingstoke, UK), both requiring overnight incubation for 24 h at 30°C. One loopful of the Fraser’s enrichment culture was streaked on Listeria selective agar (LSA, Oxoid CM0856, Basingstoke, UK) for the isolation of Listeria colonies. These plates were incubated overnight at 30°C, and on each plate three Listeria-like colonies per positive samples were picked and kept for further identification tests. Stock cultures were prepared by growing Listeria strains in tripticase soy broth (TSB; Acumedia, Lansing, MI, USA) at 37°C. After washing in sterile water, the cell pellet was suspended in a brain heart infusion (BHI) broth (Acumedia, Lansing, MI, USA) with 25% of glycerol, aliquoted (300 µl in microtubes) and frozen at −80°C until further utilization.
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Aggregatibacter actinomycetemcomitans ATCC 700685 (strain HK1651, serotype b), ATCC 33384 (strain NCTC 9710, serotype c), ATCC 43717 (strain SUNYab 75, serotype a), ATCC 43719 (strain SUNYab 67, serotype c) were obtained from the American Type Culture Collection (Manassas). All A. actinomycetemcomitans strains used in this study were chloramphenicol and kanamycin sensitive. Bacteria were cultured in brain heart infusion (BHI) broth (Acumedia) and incubated at 37°C in an atmosphere supplemented with 5% CO2. E. coli 1354 (Barrett et al., 2008 (link)), a diaminopimelic acid (DAP) auxotroph, was cultured in LB broth (Acumedia) supplemented with 100 μg/mL DAP (Sigma) and incubated with aeration at 37°C.
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S. mitis (ATCC 49456), S. oralis (ATCC 35037),
and F. nucleatum (ATCC 25586) strains were purchased from the
American Type Culture Collection. S. mitis and S.
oralis
were cultured in brain heart infusion (BHI) broth (Acumedia)
in a humidified incubator with 5% CO2 at 37°C. F.
nucleatum
was cultured in BHI broth supplemented with 0.5% yeast
extract, 5 µg/mL hemin (Sigma-Aldrich), and 1 µg/mL vitamin K, and incubated at
37°C in an anaerobic chamber (Don Whitley Scientific).
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Cultures of S. mutans strain UA159 were grown overnight in brain heart infusion (BHI) broth (Acumedia, Lansing, MI) at 37 °C in 95% air/5% CO2. B. subtilis wild-type (WT) strain NCIB3610 was routinely maintained in Lysogeny broth (LB, Neogen, Lansing, MI). To generate starter cultures, one colony of B. subtilis from a fresh LB agar plate was grown as a suspension in LB via incubation at 37 °C/150 rpm for 5 h. All experiments were conducted using bacterial cells in the late exponential phase.
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Streptococcus mitis ATCC 49456, Streptococcus oralis ATCC 35037, and Streptococcus mutans UA159 were obtained from the American Type Culture Collection (Manassas, VA, USA). Bacteria were cultured in brain heart infusion (BHI) broth (Acumedia, Lansing, MI, USA), and incubated at 37 °C with 5% CO2 supplementation.
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S. mutans UA159 was cultivated in brain–heart infusion (BHI) broth (Acumedia, Lansing, MI, USA) [30 (link)]. The day before the experiment, 100 µL of a frozen (−80 °C) stock was inoculated in 10 mL BHI followed by incubation at 37 °C in a humidified incubator in the presence of 5% CO2 for 20 h. The chain formation and coccoid-to-ellipsoid morphology of the S. mutans culture were verified under light microscopy (Axio Lab.A1, Carl Zeiss GmbH, Jena, Germany) using the ×100 lens, and the optical density at 600 nm determined by Ultraspec 10 spectrophotometer (Amersham Biosciences, Amersham, UK), usually being in the range of 1.2–1.3. The purity of the culture was tested by seeding the bacteria on BHI-agar plates where they formed distinctive minute colonies that are strongly attached to the agar.
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Fungal isolates (n = 23) were grown on Sabouraud agar slants at room temperature for 7 days. Then, the total growth from each slant was transferred to a 500 mL Erlenmeyer flask containing 50 ml of Brain Heart Infusion (BHI) broth (Acumedia, USA), supplemented with dextrose (18 g/L), pH 8.0, and incubated at 37°C for 10 days, under gentle shaking. This procedure induced yeast formation. The codes and information for all isolates are summarized in Table 1.
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Teeth were then exposed to a cariogenic challenge in brain-heart infusion (BHI) broth (Acumedia, Neogen Corporation, Lansing, MI, USA), supplemented with 1% glucose (Labsynth Materiais para Laboratório Ltda., Diadema, SP, Brazil), 1% sucrose (Labcenter Materiais para Laboratórios, Campinas, SP, Brazil), 0.5% yeast extract (Oxoid Ltd., Basingstoke, HPH, UK), and S. mutans type strain ATCC 25175 (Fundação André Tosello, Campinas, SP, Brazil), standardized to 0.5 McFarland turbidity. Samples were incubated in anaerobic jars (Probac do Brasil, São Paulo, SP, Brazil) at 37 °C and subsequently stored in a bacteriological incubator (Sterilifer Indústria e Comércio Ltda., Diadema, SP, Brazil) for 15 days. During this period, BHI broth was replaced every 24 h (adapted from Carvalho et al. [21 (link)] and Lima et al. [22 (link)]).
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One EGCG tablet (Source Naturals, Scotts Valley, CA, USA) containing 700 mg EGCG, was dissolved in 10 ml of DDW by a 1 h shaking at 4 °C. The stock EGCG solution (70 mg/ml) was sterile filtered through a filtration device with a pore size of 0.22 μm (Merck Millipore, Darmstadt, Germany), and diluted 1:1 in brain heart infusion (BHI) broth (Acumedia, Neogen, Lansing, MI, USA) concentrated x2 to get a final BHI x1 concentration. Then serial dilution was done in BHI to achieve final concentrations of 0.13–17.6 mg/ml EGCG. Since the effective concentrations were in the range of 0.55–4.4 mg/ml, only data obtained with these concentrations are presented. The working solutions were used fresh. For planktonic growth, the bacteria were incubated in BHI, while for biofilm formation BHI was supplemented with sucrose to a 2% final concentration (BHIS) [33 (link)]. The diluted EGCG solutions in BHI/BHIS were filtrated through a filtration device with a pore size of 0.22 μm before use to remove any precipitates. Control bacteria received the same incubation conditions without EGCG (see below).
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F. nucleatum ATCC 23726 was obtained from the American Type Culture Collection, USA. F. nucleatum was cultured in brain heart infusion (BHI) broth (Acumedia, USA) supplemented with 0.5% yeast extract (Acumedia), 5 μg/mL hemin and 1 μg/mL vitamin K (Sigma-Aldrich, USA), and incubated at 37°C in an anaerobic chamber supplemented with 80% N2, 10% H2, and 10% CO2 (Don Whitley Scientific, USA).
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