To ensure exact exposure times, and by this exact determination of disinfection efficacy, 3 neutralization media were tested and validated depending on the disinfectant ingredient and its concentration (Table 2 ): (1) 1% tryptic soy broth (Oxoid, Germany), (2) 0.5% (w/v) sodium sulfite in 1% tryptic soy broth, or (3) 3% tryptic soy broth, 9% (v/v) Tween 80, 0.9% (w/v) lecithin, and 3.0% (w/v) histidine (Carl Roth GmbH, Germany).4 (link) Validation of neutralization media was performed in 3 independent experiments prior to carrier assays according to DIN EN 14347:2005 using the dilution-neutralization method.20
Tryptic soy broth (tsb)
Manufactured by Carl Roth
Sourced in Germany
Tryptic soy broth is a general-purpose microbiological culture medium used for the cultivation of a wide range of microorganisms, including bacteria and fungi. It provides the necessary nutrients and growth factors for the propagation of various microbial species.
Automatically generated - may contain errors
Lab products found in correlation
Proteinase k, by Thermo Fisher Scientific (2 mentions)
Tryptic soy broth (tsb), by Thermo Fisher Scientific (1 mentions)
Lecithin, by Carl Roth (1 mentions)
Histidine, by Carl Roth (1 mentions)
Deionized water, by Merck Group (1 mentions)
Tryptic soy agar, by Thermo Fisher Scientific (1 mentions)
Sodium chloride (nacl), by Carl Roth (1 mentions)
Sorval rc 6, by Thermo Fisher Scientific (1 mentions)
Gold chloride trihydrate haucl4, by Merck Group (1 mentions)
N2 gas, by Linde (1 mentions)
Mucin, by Merck Group (1 mentions)
Biospectrometer, by Eppendorf (1 mentions)
Anhydrous glycerol, by Merck Group (1 mentions)
Lysogeny broth lb, by Carl Roth (1 mentions)
Maldi tof ms, by Bruker (1 mentions)
Lysozyme, by Thermo Fisher Scientific (1 mentions)
Cefotaxime, by Avantor (1 mentions)
Chromid mrsa agar plates, by bioMérieux (1 mentions)
Chromid esbl, by bioMérieux (1 mentions)
10 protocols using tryptic soy broth (tsb)
1
Validated Neutralization of Disinfectants
2
Bactericidal Peptide Testing Protocol
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For bactericidal testing, logarithmically growing staphylococci were resuspended in PBS (Sigma) containing 0.1% TSB (Carl Roth) and colony-forming unit (CFU) was adjusted to 3 × 106 CFU/mL. Different concentrations of single peptides and their combinations were diluted in PBS containing 0.1% TSB and incubated with bacteria in triplicates for 3 h at 37 °C and 150 rpm orbital shaking. Subsequently, serial dilutions (10–1–10–4) of the bacterial suspensions were prepared in PBS and 20 µL of each dilution was spotted in duplicates onto TSB plates and incubated at 37 °C overnight. The next day, the number of CFU was analyzed and the percentage of viable bacteria was determined by normalizing to the untreated control (100%). Results are illustrated in a S. aureus killing curve. In each experiment, negative control replicates (PBS + 0.1% TSB) as well as sterility control replicates were included.
3
Genetic Manipulation of Streptomyces mirabilis
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S. mirabilis P16B-1 (2 (link)) is an environmental isolate from a former uranium mining site with high concentrations of soluble metal ions in the soil. A derivative lacking endogenous plasmids, S. mirabilis ΔpI, was obtained in this study by applying heat shock. The plasmid loss was monitored by Southern blotting as well as PCR using primers for plasmid-specific genes. The genetic model organism S. lividans TK24 (str−, SLP2 SLP3−) (65 ) was used as recipient in conjugation and transformation experiments. The streptomycetes were grown in mannitol soy medium (65 ), casein starch medium (CSA; 10 g/L starch, 1 g/L casein hydrolysate, and 0.5 g/L K2HPO4), minimal medium AM (66 (link)), tryptic soy broth (Carl Roth, Karlsruhe, Germany), or glucose yeast medium (GYM; DMSZ, Germany). Metal salts were added if indicated, and oxidative stress was exerted by adding H2O2 of up to 40 ppm depending on growth at lower concentrations.
Escherichia coli DH5α, ET12567 (pUZ8002; dam−, dcm−, hsdS−, cmr) (67 (link)), BW25113 (68 (link)) and TransforMax EC100D pir-116 (Epicentre Biotechnologies, Madison, WI, USA) with plasmids pSET152 (14 (link)), pIJ790 and pIJ773 (18 (link)), pTrc99A (69 (link)), and pKOSI (17 (link)) were used for cloning. E. coli was grown in complex media standard I, super optimal broth (SOB), or LB (all by Merck, Darmstadt, Germany).
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4
Preparation of Bacterial Cell Stocks for Metal Interactions
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Gold chloride trihydrate (HAuCl4, Sigma-Aldrich, Bornem, Belgium) was dissolved in deionized water (Merck Millipore, Burlington, MA, USA) to prepare a stock solution (5 g L−1). The experimental solutions were prepared from the stock by dissolving in 0.9% w/v NaCl (Carl Roth, Karlsruhe, Germany).
Frozen cells (20% glycerol at −80 °C) of S. oneidensis MR-1 (LMG 19005) and C. metallidurans CH34 (SCK-CEN, Mol, Belgium) were inoculated under aerobic conditions on tryptic-soy agar (Oxoid, England) at 28 °C. Individual colonies were inoculated in tryptic-soy broth (Carl Roth, Germany) aerobically at 28 °C agitated in a rotatory shaker (KS 400i, IKA, Staufen, Germany) set at 120 rpm. Cells were harvested at the stationary phase (after 72-h of incubation at an optical density, OD610, of ≈1) by centrifugation (10 min at 10,000 rcf, Sorval RC6+, Thermo Scientific, Waltham, MA, USA), washed twice with sterile 0.9% w/v NaCl, and finally concentrated to a value of OD610 ≈ 7 [32 (link)]. Concentrated cell stocks were made anoxic by degassing for 21 cycles of vacuum and filling with N2 gas (Linde, Munich, Germany). A control was set with non-viable cells for experimental validation by autoclaving the viable cell stocks for 21 min at 121 °C.
Frozen cells (20% glycerol at −80 °C) of S. oneidensis MR-1 (LMG 19005) and C. metallidurans CH34 (SCK-CEN, Mol, Belgium) were inoculated under aerobic conditions on tryptic-soy agar (Oxoid, England) at 28 °C. Individual colonies were inoculated in tryptic-soy broth (Carl Roth, Germany) aerobically at 28 °C agitated in a rotatory shaker (KS 400i, IKA, Staufen, Germany) set at 120 rpm. Cells were harvested at the stationary phase (after 72-h of incubation at an optical density, OD610, of ≈1) by centrifugation (10 min at 10,000 rcf, Sorval RC6+, Thermo Scientific, Waltham, MA, USA), washed twice with sterile 0.9% w/v NaCl, and finally concentrated to a value of OD610 ≈ 7 [32 (link)]. Concentrated cell stocks were made anoxic by degassing for 21 cycles of vacuum and filling with N2 gas (Linde, Munich, Germany). A control was set with non-viable cells for experimental validation by autoclaving the viable cell stocks for 21 min at 121 °C.
5
Mucin Degradation Ability of LAB
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The mucin degradation ability of the LAB was assessed using a previously reported method with slight modification (Daliri et al., 2022 (link)) Briefly, the LAB strains were grown in MRS broth supplemented with both 0.5% (w/v) glucose and 0.5% (w/v) mucin. After inoculation, the cultures were incubated at 37°C for 48 h under aerobic conditions. The bacterial growth was estimated every 6 h by measuring absorbance at 600 nm. E. coli ATCC 25922 was used as positive control and grown in Tryptic soy broth (Carl Roth, Karlsruhe, Germany) containing 0.5% (w/v) glucose supplemented with or without 0.5% (w/v) mucin (Sigma- Aldrich, Poznań, Poland) and cultured at 37°C for 48 h under aerobic conditions. The optical densities were measured using a spectrometer (Eppendorf Bio spectrometer®, Hamburg, Germany). The initial optical density value of the media was deducted from the final value for each test sample.
6
MDR Bacteria Screening in Fresh Meat
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As part of a small screening study, packaged fresh meat samples from various grocery stores were tested for different MDR bacteria. Briefly, the meat samples were opened under sterile conditions, two pieces of approximately 5 mm³ were cut out with a sterile scalpel (Braun, Melsungen, Germany) and transferred to 5 mL of tryptic soy broth (Carl Roth, Karlsruhe, Germany). The bacteria were then enriched under shaking conditions (130 rpm) at 37 °C overnight. Then, 100 µL of the bacterial suspension was plated onto different chromogenic selection plates and incubated overnight at 37 °C. The strain 27-ESBL-EC (internal designation PBIO3502) was cultivated on a CHROM-ESBL selection plate (Mast Diagnostica GmbH, Reinfeld, Germany) and the species E. coli was confirmed using MALDI-TOF MS (Bruker, Bremen, Germany). It was derived from a fresh bratwurst purchased from a German grocery store. All strains were stored at −80 °C in lysogeny broth (LB; Carl Roth, Karlsruhe, Germany) supplemented with 20% (v/v) glycerol (anhydrous; Merck, Darmstadt, Germany). Prior to use, one single colony of fresh overnight cultures on LB agar plates was inoculated in 5 mL of LB and grown under shaking conditions (130 rpm) at 37 °C overnight.
7
Genomic DNA Extraction from Strain Msb3
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Strain Msb3 was grown from cryo-stock in liquid culture overnight at RT in TSB (Carl Roth GmbH & Co., KG). 4 mL (OD600 = 0.8) were transferred into a 15 mL tube and centrifuged at 2400 × g for 8 min to sediment biomass. Cells were washed in TE and subsequently lysed using lysozyme (Thermo Fisher Scientific, Waltham, MA, United States). Lytic activity was stopped using Proteinase K (Thermo Fisher Scientific). This approach was chosen to prevent excessive shearing of DNA molecules in order to produce long sequencing reads. Extraction of total chromosomal and plasmid DNA was performed following a standard phenol/chloroform/isoamyl alcohol (PCI) extraction protocol as described below.
8
Isolation and Identification of Antibiotic-Resistant Bacteria
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In the laboratory, 100 mL of water samples were first pre-filtered using a sterilized gauze (PZN: 04046708, FESMED Verbandmittel GmbH, Frankenberg/Sa., Germany) to remove the fine particles. The pre-filtered water was free from any macro-particles, sediment and most fine particles. Thereafter, each sample was filtered using the EZ-Fit filtration system with 0.45 µm pore size filter membranes (merckmillipore, Darmstadt, Germany). After filtration, filter membranes were transferred to 10 mL TSB (Carl Roth GmbH, Karlsruhe, Germany) containing 2 µg/mL cefotaxime (VWR International, Darmstadt, Germany) followed by overnight incubation at 37 °C and shaking at 200 rpm. Depending on the turbidity level, dilution of the overnight cultures followed (up to 10,000 fold dilution). For each sample, 100 µL of the dilutions were plated on chromogenic media CHROMID CARB/OXA, CHROMID ESBL, CHROMID Colistin, and CHROMID MRSA agar plates (bioMérieux, Nürtingen, Germany), and incubated overnight at 37 °C. Putative antibiotic-resistant colonies of E. coli (red-purple colonies) and KEC (Klebsiella spp., Enterobacter spp., Citrobacter spp. (blue colonies)) were subcultivated until pure cultures were achieved. Single pure colonies were picked for further verification and characterization.
9
Bacterial Inoculation of Plant Seedlings
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Strains Msb3 and PsJN were grown from cryo-stock in liquid culture overnight at 28 °C in tryptic soy broth (TSB) (Carl Roth), shaking at 250 rpm. Bacteria were harvested at an OD600 of 0.6 and washed three times with 10 mM MgCl2. Bacterial suspensions of an OD600 of 0.1, 0.01 or 0.001 in MgCl2 were used for plant inoculations, which corresponds to approximately 3 × 106, 3 × 105 and 3 × 104 CFUs ml–1, respectively. Seedlings were dipped into the suspension for 10 seconds. They did not retain significant amounts of liquid (>100 µl) when they were removed. Dilutions with OD600 of 0.1 were used for the timeseries experiment, those with an OD600 of 0.01 were used for growth promotion assays and those with an OD600 of 0.001 for monitoring early infection dynamics.
10
Enterococci Growth Conditions
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The different bacterial strains used are summarized in Table 1 . Enterococci were grown at 37°C without agitation in tryptic soy broth (TSB; Carl Roth).
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