Nutrient broth

Manufactured by Carl Roth

Sourced in Germany

Nutrient broth is a general-purpose medium for the cultivation and growth of a wide range of microorganisms. It provides essential nutrients and growth factors required for the development of bacterial, fungal, and other microbial cultures.

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Nutrient Broth medium Nutrient Broth (NB,

9 protocols using nutrient broth

Pseudomonas aeruginosa Inhibition by Nanogels

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The nanogels (4 mg) were swollen over night at 37 °C under shaking conditions in 1 mL Nutrient broth (NB, Carl Roth).
Nutrient broth was inoculated with Pseudomonas aeruginosa and grown under shaking conditions (110 rpm) at 37 °C overnight. The overnight culture was diluted to an OD600 nm of 0.2 with NB and subsequently diluted with the nanogel suspension in a ratio of 1:2 (nanogel 2 mg/mL, PA OD 0.1). 500 µL of this mixture were plated in a 24-well plate (n = 4) (TPP, Techno Plastic Products AG, Trasadingen, Switzerland)) and cultivated under static condition overnight at 37 °C. Then the fluorescence of pyoverdin was detected using UV light (Dark Hood DH-50, BIOSTEP, FELIX 2000, Burkhardtsdorf, Germany).
All samples and the controls (PA in NB) showing fluorescence were diluted to 10⁻⁴–10⁻⁶ with PBS (Biochrom AG, Berlin, Germany). Samples without fluorescence were diluted to 10⁻². Subsequently, 100 µL of each sample was plated on cetrimid agar plates (Carl Roth) and stored overnight (37 °C).

Tang J.S., Rosencrantz S., Tepper L., Chea S., Klöpzig S., Krüger-Genge A., Storsberg J, & Rosencrantz R.R. (2019). Functional Glyco-Nanogels for Multivalent Interaction with Lectins. Molecules, 24(10), 1865.

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Bacterial Strains and Genetic Constructs

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Bacterial strains and plasmids generated in this study are listed in Table 1. S. plymuthica WS3236 was obtained from the Weihenstephaner collection of microorganisms (Chair of Microbial Ecology, Technical University of Munich Freising, Germany). S. plymuthica WS3236 was cultivated in nutrient broth (Carl Roth, Germany) at 28 °C (liquid cultures) or 30 °C (agar plates). Liquid cultures were incubated while shaking at 200 rpm. Under routine culture conditions unless otherwise specified, E. coli strains were grown in lysogeny broth (LB) medium (Carl Roth, Germany) supplemented with 50 µg/mL kanamycin.Table 1

Bacterial strains and plasmids used in this study

	Description	Reference or source
Strains
Escherichia coli DH5α	Host strain for cloning	NEB
Escherichia coli BL21	Heterologous expression strain	NEB
Serratia plymuthica WS3236	Native producer of sodorifen	ZIEL Institute Culture Collection
Plasmids
pET28b-ptetO-gfpv2(6029 bp)	Tetracycline inducible expression plasmid, ColE1, Kan^R, gfp reporter gene downstream of promotor	This study
pET28b-ptetO::sod_gfpv2(11,124 bp)	pET28b-ptetO_gfpv2 with sodABCD cloned as single fragment between Ptet_O and gfp	This study

Duell E.R., D’Agostino P.M., Shapiro N., Woyke T., Fuchs T.M, & Gulder T.A. (2019). Direct pathway cloning of the sodorifen biosynthetic gene cluster and recombinant generation of its product in E. coli. Microbial Cell Factories, 18, 32.

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Antimicrobial Activity of Lignin

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Inoculums of Staphylococcus aureus ssp. Aureus (DSM No 799), L. monocytogenes (DSM No 19094), and E. coli (DSM No 1576) were prepared by transferring a frozen culture to 10 mL of nutrient broth (Roth, Karlsruhe, Germany). Afterwards the broth was incubated at 37 °C for 24 h. In the beginning of each trial, the inoculum was diluted in physiological saline solution with tryptone (Oxoid, Hampshire, UK) to a final concentration of 10⁷ cfu/mL. 1 mL of the suspension was spread with a sterile spatula over the surface of a plate count agar plate (Roth, Karlsruhe, Germany). Three filter papers were impregnated with the antimicrobial agent, 0.1 g of lignin was dissolved in 1 mL DMSO and dried over a circular filter paper (0.5 cm of diameter) and three blank filter papers as references were applied on the inoculated agar plate. The agar plates were incubated for 24 h at 37 °C. A clear zone without microorganism growth (zone of inhibition) is related to the level of antimicrobial activity of the agent.

Alzagameem A., Klein S.E., Bergs M., Do X.T., Korte I., Dohlen S., Hüwe C., Kreyenschmidt J., Kamm B., Larkins M, & Schulze M. (2019). Antimicrobial Activity of Lignin and Lignin-Derived Cellulose and Chitosan Composites against Selected Pathogenic and Spoilage Microorganisms. Polymers, 11(4), 670.

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Bacterial Culture and Preparation Protocol

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Example 1

Characterized strains of Escherichia coli (ATCC® 8739™) and Staphylococcus aureus (ATCC® 6538™) were obtained from Leibniz Institute DSMZ, of Braunschweig, Germany. 20 ml of nutrient broth (item number x929.1, from Carl Roth GmbH+Co KG, of Karlsruhe, Germany) was inoculated with a single colony of E. coli. 20 ml of Caso broth (item number x938.1, from Carl Roth) was inoculated with a single colony of S. aureus. Both colonies were allowed to grow overnight at 37° C. while subject to agitation.

1 ml of each overnight culture was diluted in 20 ml of the respective broth, and allowed to grow at 37° C. for 3 hours or until OD₆₀₀(i.e., optical density measured at a wavelength of 600 nm, a measurement correlated to concentration of the bacteria) of 1 was obtained. Afterwards, the cells were harvested by centrifuging to form pellets, which were washed one time with phosphate buffered saline (PBS), and then resuspended in PBS to achieve desired concentrations.

US10139407B2. Methods for detecting bacteria using polymer materials (2018-11-27). Universiteit Maastricht [NL], Academisch Ziekenhuis Maastricht [NL]. Inventors: Bart Robert Nicolaas Van Grinsven [NL], Thomas Jan Cleij [NL].

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Cultivation of E. coli and B. subtilis

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Escherichia (E.) coli NEB 5α was obtained from New England Biolabs (Ipswich, MA, USA) and Bacillus (B.) subtilis ATCC 6051 from the Leibniz Institute DSZM-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). Both bacterial strains were cultured in nutrient broth (8 g/L, Carl Roth, Karlsruhe, Germany) at 37 °C for 24 h under shaking in a temperature-controlled incubator shaker (Innova42R New Brunswick, New York, NY, USA) as described before [12 (link)].

Heymich M.L., Srirangan S, & Pischetsrieder M. (2021). Stability and Activity of the Antimicrobial Peptide Leg1 in Solution and on Meat and Its Optimized Generation from Chickpea Storage Protein. Foods, 10(6), 1192.

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Bacterial Culture Preparation and Standardization

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Listeria innocua (DSM 20649), Escherichia coli (DSM 1116), and Pectobacterium carotovorum spp. carotovorum (DSM 30168) were stored as glass bead cultures at −80°C for long-term maintenance. One glass bead was given to 5 ml nutrient broth (Carl Roth GmbH & Co KG, Germany) and incubated for 24 h without shaking at 37°C (L. innocua, E. coli) or 30°C (P. carotovorum) to re-activate the bacteria. Afterwards, 100 ml nutrient broth was inoculated with bacteria suspension at a calculated optical density at 620 nm (OD₆₂₀) of 0.07 ml⁻¹. Bacteria were harvested in the early to mid-stationary growth phase (L. innocua and E. coli: 18 h at 37°C or P. carotovorum: 18 h at 30°C) by centrifugation at 3220 × g for 15 min at 4°C. The pelleted material was suspended in 50 mM phosphate buffered saline (PBS) to a cell concentration of approximately 9–10 log CFU ml⁻¹. PBS was prepared of 137 mM NaCl, 2.7 mM KCl, 40.6 mM Na₂HPO₄, and 7.1 mM KH₂PO₄. The pH was adjusted to 7.0 with HCl and finally filtered with a 0.2 μm membrane filter. All reagents were provided by Carl Roth GmbH & Co KG, Germany.

Fröhling A, & Schlüter O. (2015). Flow cytometric evaluation of physico-chemical impact on Gram-positive and Gram-negative bacteria. Frontiers in Microbiology, 6, 939.

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Biofilm Gelatinase Activity Assessment

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The gelatinase activity caused by extracellular enzyme produced by biofilm cells or cells dispersed from the biofilm was assessed according to Marra et al. (2007) (link). In brief, the suspension of cells (1 ml) was taken from the vicinity of the biofilm into microtubes. The biofilm-covered coupons were rinsed with saline and sonicated for 15 min in 1 ml of saline to remove biofilm cells from the surface of coupons. After that, both types of samples were injected thrice into different places into nutrient broth (Carl Roth, Germany) mixed with gelatin (Across Organics, USA) in a volume of 3×5 µl in tubes. The inoculated gelatin tubes were incubated for 72 h at 37 °C. After incubation, the tubes were put to 4 °C for 30 min. If the gelatin in tube remained liquified even after cooling down, the cells in the sample produced gelatinase and therefore the sample is gelatinase positive. If the gelatin in tube remained solid after cooling down, the cells in sample did not produce gelatinase and therefore are gelatinase negative. As negative control the experiment also contained gelatin tubes with injected saline instead of sample, which remained solid when cooled down after incubation. The experiments were carried out in triplicates and the results were qualitative

Kašparová P., Vaňková E., Paldrychová M., Svobodová A., Hadravová R., Jarošová Kolouchová I., Masák J, & Scholtz V. (2022). Non-thermal plasma causes Pseudomonas aeruginosa biofilm release to planktonic form and inhibits production of Las-B elastase, protease and pyocyanin. Frontiers in Cellular and Infection Microbiology, 12, 993029.

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Bacterial Culture and Enumeration Protocol

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Example 8

US10890584B2. Devices for detecting analytes using thermal waves and related methods (2021-01-12). Academisch Ziekenhuis Maastricht [NL], Universiteit Maastricht [NL]. Inventors: Bart Robert Nicolaas Van Grinsven [NL], Thomas Jan Cleij [NL].

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Antimicrobial Polymer Formulation Development

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Metronidazole, ciprofloxacin, chitosan (low molecular weight [MW]), PCL (MW, 80 kDa), and PEO (MW, 8 MDa) were from Sigma-Aldrich (China, UK or USA). Glacial acetic acid (100%), formic acid (98%-100%), potassium dihydrogen phosphate, sodium hydroxide, methanol, and acetonitrile were from Merck (Finland or USA). Standard culture media, nutrient broth, and agar-agar were from Roth (Germany), and brain-heart infusion broth was from Biolife (Italy). All of the chemicals were used as received, without further purification or modification. Drug-release experiments were performed using 50 mM phosphate buffer, pH 7.4 as the dissolution medium, and the samples were filtered using 0.2-µm filters (Minisart RC 15; Sartorius StedimBiotech GmbH, Germany).

Zupančič Š., Casula L., Rijavec T., Lapanje A., Luštrik M., Fadda A.M., Kocbek P, & Kristl J. (2019). Sustained release of antimicrobials from double-layer nanofiber mats for local treatment of periodontal disease, evaluated using a new micro flow-through apparatus. Journal of controlled release : official journal of the Controlled Release Society, 316.

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