Isopropyl-β-D-thiogalactopyranosid (IPTG) came from Applichem GmbH (Darmstadt, Germany), anhydrotetracycline (ATc) from Toku-e (Gentaur GmbH, Aachen, Germany), Luria-Bertani broth (LB) and LB agar plates were purchased from Roth (Karlsruhe, Germany). Components for the M9-minimal medium (di-sodium-hydrogenphosphate (33.9 g/L), potassium-dihydrogenphosphate (15 g/L), sodium chloride (2.5 g/L), ammonium-chloride (5 g/L)) came from Merck (Darmstadt, Germany). This medium was either supplemented with glucose (0.4%), thiamin (10 µg/mL, Roth) and nicotic acid (0.0025%, Roth) or tryptone (0.2%, BD Chemicals, Greenwood Village, CO, USA). 2,2′-bipyridine, 5 g, was bought from Roth. Human urine was donated by healthy individuals. The urine was filtered (0.2 µm pore size) and used on the day of donation.
Lb agar
Manufactured by Carl Roth
Sourced in Germany
LB agar is a nutrient-rich solid growth medium used for culturing bacteria. It provides the necessary nutrients and solidifying agent for bacterial growth and colony formation.
Automatically generated - may contain errors
Lab products found in correlation
Lb medium, by Carl Roth (5 mentions)
Ammonium chloride, by Merck Group (2 mentions)
Potassium dihydrogen phosphate, by Merck Group (2 mentions)
Luria bertani medium lb broth, by Carl Roth (2 mentions)
Columbia blood agar, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Luria bertani broth, by Carl Roth (1 mentions)
Disodium hydrogen phosphate, by Merck Group (1 mentions)
Lb broth, by Carl Roth (1 mentions)
Luria bertani medium, by BD (1 mentions)
Direct q8 system, by Merck Group (1 mentions)
Poly ethylene glycol diacrylate, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Thermo Fisher Scientific (1 mentions)
Gentamycin, by AppliChem (1 mentions)
Ampicillin, by AppliChem (1 mentions)
Disodium hydrogen phosphate, by Carl Roth (1 mentions)
Magnesium sulfate, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Calcium chloride, by Merck Group (1 mentions)
Potassium chloride (kcl), by Merck Group (1 mentions)
17 protocols using lb agar
1
Preparation of Minimal Medium for E. coli
2
E. coli Strain XL10 Gold Amplification
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E. coli strain XL10 Gold (StrataGene, La Jolla, USA/Agilent, Santa Clara, USA) (genomic information: endA1 glnV44 recA1 thi-1 gyrA96 relA1 lac Hte Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 tetR F’[proAB lacIqZΔM15 Tn10(TetR Amy CmR)]) was used for amplifications of DNA plasmids. The strain was grown in LB medium (Carl Roth, Karlsruhe, Germany) or on LB-agar (Carl Roth) with or without the respective antibiotics the strain carries resistances for (10 μg/ml tetracycline and 30 μg/ml chloramphenicol) or obtained resistances for (by transformation with plasmids) (usually either 100 μg/ml ampiciline or 25 μg/ml kanamycine). The strain was usually either grown at room temperature (RT) or at 37°C.
3
Bacterial Strains and Culture Conditions
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The following species were used in this study: S. aureus (strains 6850 (clinical sepsis isolate), USA300 (prototypic community-acquired S. aureus), LS1 (laboratory strain from infective arthritis in mice), Newman (clinical isolate from osteomyelitis), SH1000 (laboratory strain), Cowan I (human septic arthritis), and ST398 (prototypic livestock-acquired S. aureus)) and E. coli (strains 536 (prototypic uropathogenic E. coli [UPEC]), IHE3034 (newborn meningitis-associated E. coli [MNEC]), and MG1655 (commensal E. coli)) (Table 1 ).
Prior to the experiments, the bacteria were plated on lysogeny broth (LB) agar (Roth, Karlsruhe, Germany) plates and incubated overnight at 37 °C. Single colonies were taken to inoculate overnight cultures in LB medium (Roth) and bacteria were grown at 37 °C with shaking at 180 rpm.
Prior to the experiments, the bacteria were plated on lysogeny broth (LB) agar (Roth, Karlsruhe, Germany) plates and incubated overnight at 37 °C. Single colonies were taken to inoculate overnight cultures in LB medium (Roth) and bacteria were grown at 37 °C with shaking at 180 rpm.
4
Induction of Bronchus-Associated Lymphoid Tissue in Mice
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E. coli, strain DH5-α, was cultivated in Luria-Bertani (LB) medium (BD). Bacteria were plated on LB-agar (Carl Roth) and incubated overnight at 37 °C. A single colony culture was prepared in 10 ml of LB broth (Carl Roth) and incubated as above, on a shaker. The bacterial suspension was diluted 1:200 in 100 ml LB broth and incubated as above. Bacteria were harvested at an optical density of 600 (OD600), pelleted, washed and re-suspended in PBS. Colony forming units (CFU) were determined by plating different dilutions and counting using a Thoma cell counting chamber (Brand GmbH); thereafter, they were heat-inactivated (30 min at 50 °C and an additional 15 min at 85 °C) in a water bath. Heat-inactivation was confirmed by plating bacteria on LB-agar and Columbia-blood-agar (Oxoid) and incubating for 72 h. For induction of BALT, anesthetized mice were administered a single 30 μl suspension of 3 × 108 CFU, as determined by titration, intranasally.
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E. coli, strain DH5-α, was cultivated in Luria-Bertani (LB) medium (BD). Bacteria were plated on LB-agar (Carl Roth) and incubated overnight at 37 °C. A single colony culture was prepared in 10 ml of LB broth (Carl Roth) and incubated as above, on a shaker. The bacterial suspension was diluted 1:200 in 100 ml LB broth and incubated as above. Bacteria were harvested at an optical density of 600 (OD600), pelleted, washed and re-suspended in PBS. Colony forming units (CFU) were determined by plating different dilutions and counting using a Thoma cell counting chamber (Brand GmbH); thereafter, they were heat-inactivated (30 min at 50 °C and an additional 15 min at 85 °C) in a water bath. Heat-inactivation was confirmed by plating bacteria on LB-agar and Columbia-blood-agar (Oxoid) and incubating for 72 h. For induction of BALT, anesthetized mice were administered a single 30 μl suspension of 3 × 108 CFU, as determined by titration, intranasally.
5
E. coli Cloning and Protein Expression
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Escherichia coli DH5α was used as a host for recombinant plasmids, E. coli Rosetta 2 (DE3) [pLysSRARE2] (Merck KGaA, Darmstadt, Germany) was used for recombinant protein expression. The bacteria were incubated at 37 °C or 26 °C on either LB agar (Roth, Karlsruhe, Germany) or at 120–180 rpm in liquid LB medium (Roth, Karlsruhe, Germany) containing the appropriate antibiotics (100 μg/ml ampicillin or 100 μg/ml ampicillin and 34 μg/ml chloramphenicol) for plasmid stability. Stocks for long term storage at − 80 °C were prepared using 25% (v/v) glycerol and 0.5x LB medium.
6
Purification and Use of PEG Diacrylate
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All chemicals were purchased from Sigma-Aldrich
and used without further purification unless otherwise noted. Poly(ethylene
glycol) diacrylate (Mn = 250 g/mol) was purchased from Sigma-Aldrich and purified using
aluminum oxide 90 active neutral (0.063–0.200 mm, Merck KGaA).
Luria–Bertani medium (LB broth) and LB agar (Luria/Miller)
were purchased from Carl Roth. Throughout the whole study, Milli-Q
water was drawn from a Millipore Direct Q8 system (Millipore Advantage
A10 system, Schwalbach, with Millimark Express 40 filter, Merck, Germany)
with a resistivity 18.2 MΩ cm.
and used without further purification unless otherwise noted. Poly(ethylene
glycol) diacrylate (Mn = 250 g/mol) was purchased from Sigma-Aldrich and purified using
aluminum oxide 90 active neutral (0.063–0.200 mm, Merck KGaA).
Luria–Bertani medium (LB broth) and LB agar (Luria/Miller)
were purchased from Carl Roth. Throughout the whole study, Milli-Q
water was drawn from a Millipore Direct Q8 system (Millipore Advantage
A10 system, Schwalbach, with Millimark Express 40 filter, Merck, Germany)
with a resistivity 18.2 MΩ cm.
7
Chemo-enzymatic Hydrogel Functionalization
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Alginic acid sodium salt, glycidyl methacrylate,
triethylamine, tetrabutylammonium bromide, calcium chloride
hexahydrate, sodium chloride, 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone
(Irgacure 2959), hexamethyldisiloxane, Trizma base, Fluoresceinamine
isomer I, N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide sodium salt (Sulfo-NHS),
2-(N-morpholino)ethanesulfonic acid (MES) hydrate,
and N-(3-oxododecanoyl)homoserine lactone were
purchased from Sigma-Aldrich and used without further purification.
Hydrochloric acid was purchased from Fisher Chemical. N-Dodecanoyl-l -homoserine lactone-3-hydrazone fluorescein
was purchased from Biomol. Luria-Bertani medium (LB broth) and LB
agar were purchased from Carl Roth (Germany). Throughout the whole
study, Milli-Q water drawn from a Millipore Direct Q8 system with
Millimark Express 40 filter (Merck, Germany) was used.
triethylamine, tetrabutylammonium bromide, calcium chloride
hexahydrate, sodium chloride, 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone
(Irgacure 2959), hexamethyldisiloxane, Trizma base, Fluoresceinamine
isomer I, N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide sodium salt (Sulfo-NHS),
2-(N-morpholino)ethanesulfonic acid (MES) hydrate,
and N-(3-oxododecanoyl)homoserine lactone were
purchased from Sigma-Aldrich and used without further purification.
Hydrochloric acid was purchased from Fisher Chemical. N-Dodecanoyl-
was purchased from Biomol. Luria-Bertani medium (LB broth) and LB
agar were purchased from Carl Roth (Germany). Throughout the whole
study, Milli-Q water drawn from a Millipore Direct Q8 system with
Millimark Express 40 filter (Merck, Germany) was used.
8
Vesiculation Profiles of VRE Isolates
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Six different VRE isolates defined by MLST (ST80, ST117, ST192, ST203, ST721, and ST1489) were used for MV preparation. The isolates originated from a surveillance sample collection of the University Hospital Münster, Germany, and all of them were derived from routine anal swab samples. Based on previous investigations by Correa-Martinez et al. addressing the spread of VRE in the hospital environment41 (link), representative isolates were selected for the respective STs. E. faecalis ATCC 29212 and E. faecium ATCC 6057 served as vancomycin-susceptible reference strains; E. faecium ATCC 6057 was also used as a potential vesiductant.
For general cultivation purposes, all bacteria were grown on Columbia blood agar (Thermo Scientific, Waltham, USA) and incubated at 37 °C for approximately 24 h.
They were further cultured either in LB (Carl Roth, Karlsruhe, Germany) or on LB agar (Carl Roth) to investigate vesiculation under stressful growth conditions22 (link).
For general cultivation purposes, all bacteria were grown on Columbia blood agar (Thermo Scientific, Waltham, USA) and incubated at 37 °C for approximately 24 h.
They were further cultured either in LB (Carl Roth, Karlsruhe, Germany) or on LB agar (Carl Roth) to investigate vesiculation under stressful growth conditions22 (link).
9
Preparation of Bacterial Culture Media
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Luria-Bertani broth (LB) and LB agar were purchased from Roth (Karlsruhe, Germany). The 5×M9-minimal salt contained di-sodium hydrogen phosphate (33.9 g/L; Roth, Karlsruhe, Germany), potassium-dihydrogen phosphate (15 g/L; Merck, Darmstadt, Germany), sodium chloride (2.5 g/L; Merck, Darmstadt, Germany) and ammonium-chloride (5 g/L; Merck, Darmstadt, Germany). This M9-salt was diluted 1:5 and supplemented with magnesium sulfate (1 mM, Merck, Darmstadt, Germany), calcium chloride (0.1 mM; Merck, Darmstadt, Germany), glucose (0.4%, Merck, Darmstadt, Germany), thiamin-hydrochloride (10 µg/mL, Roth, Karlsruhe, Germany) and nicotinic acid (0.0025%, Roth, Karlsruhe, Germany). We bought McCoy and RPMI-1640 from Merck (Darmstadt, Germany) and FBS from Thermo Fisher Scientific (Waltham, MA, USA). Human urine was donated by healthy individuals. The urine was filtered (0.2 µm pore size) and used at the day of donation. Potassium chloride came from Merck (Darmstadt, Germany), potassium sulfate from Roth (Karlsruhe, Germany). Phorbol 12-myristate 13-acetat was bought from PromoCell (Heidelberg, Germany). We purchased E. coli LPS K235 from Merck (Darmstadt, Germany), kanamycin, ampicillin and gentamycin from AppliChem (Darmstadt, Germany) and chloramphenicol from Merck (Darmstadt, Germany).
10
Germ-Free Fly Maintenance Protocol
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