Bacterial strains were cultivated in Luria-Bertani (LB) and M9 minimal medium purchased from Becton, Dickinson & Co, Kehl, Germany. Ten milliliters of LB broth supplemented with 200 µg/mL ampicillin was inoculated with a single colony from a freshly streaked plate of Top10 containing BBa_T9002 and incubated for 18 h at 37 °C, shaking at 100 rpm. Each culture was then diluted 1:1000 into 20 mL M9 minimal medium supplemented with 0.2% casamino acids and 1 mM thiamine hydrochloride plus 200 µg/mL ampicillin (AppliChem GmbH, Darmstadt, Germany). The culture was maintained under the same conditions until the optical density measured at 600 nm (OD600) reached 0.15 (~5 h). Then 500 µL overnight culture and 500 µL 30% sterile glycerol were mixed together in cryotubes and stored at −80 °C. Before the biosensor assay was conducted, 40 µL bacteria from the glycerol stock vials was cultivated in 20 mL M9 medium plus 200 µg/mL ampicillin until the OD600 reached 0.04~0.07 (~4 h).
M9 minimal medium
Manufactured by BD
Sourced in Germany, United States
M9 minimal medium is a laboratory culture medium used for the growth of bacteria, particularly Escherichia coli. It provides the essential nutrients and minerals required for bacterial growth in a defined and standardized formulation.
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Lab products found in correlation
8 protocols using m9 minimal medium
1
Bacterial Cultivation and Glycerol Stock Preparation
2
Bacterial Culture and AHL Signaling
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Luria-Bertani bacteria culture broth, and 3-oxo-N-hexanoyl homoserine lactone (3OC6HSL, or generically denoted as AHL) and all other analytical grade chemicals were purchased from Sigma–Aldrich Chemie GmbH (Hamburg, Germany). M9 minimal medium was purchased from Becton, Dickinson and Company (Germany) and ampicillin (AppliChem GmbH, Germany).
3
Bacterial Culture for Biosensor Assay
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Bacterial strains were cultivated using on Luria-Bertani (LB) and M9 minimal medium (Becton, Dickinson and company, Germany). We inoculated 10 mL of LB broth supplemented with 10µL (200 µg/mL) ampicillin (AppliChem GmbH, Germany) with a single colony from a freshly streaked plate of E. coli Top10 containing BBa_T9002 and incubated the culture for 18 h at 37ºC, shaking at 100 r.p.m. Each culture was then diluted 1:1000 into 20 mL M9 minimal medium supplemented with 0.2% casamino acids and 1 mM thiamine hydrochloride plus 20µL (200 µg/mL) ampicillin. The culture was maintained under the same conditions until the OD600 reached 0.15 (~5 h). Then 500 µ L of the overnight culture were mixed with 500µ l 30% sterile glycerol together in the white plastic vials and stored at -80 C for future use. Before the biosensor assay, the bacteria cultivation was prepared by cultivating 40µL of thawed bacteria from the white plastic vial kept at -80 C unto 20 mL M9 medium plus 20µ l of ampicillin (200 g/mL). This is incubated at 37 C, 100 r.p.m until the OD600 reached 0.04~ 0.07 (~4 h).
4
Anaerobic Growth Assays of Evolved Strains
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In vitro growth assays were performed in M9 minimal medium (BD) supplemented with 20 mM xylose or glucose (Sigma). Wild-type and xylR evolved/transposon strains were first cultured at 37°C for 24 h in an anaerobic box (AnaeroPack system, Mitsubishi Gas Chemical Company) with disposable single-use anaerobic sachets (GasPak EZ Anaerobe Container System, BD). Then, strains were washed and resuspended at 105 CFU/ml in M9 with 20 mM sugar and incubated anaerobically in 96-well plates in 100 μl final volumes for 24 h. After 24 h, the OD at 600 nm was measured with a plate reader (Biotek).
5
S. Typhimurium Growth in Acidic Media
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Overnight S. Typhimurium cultures grown in Luria broth were washed in 1× PBS and normalized to an OD600 of 0.05 in M9 minimal medium (BD Biosciences, USA) buffered to pH 5.5 in the presence or absence of 0.5 mM S-nitroso-N-acetylcysteine (SNAC). SNAC was prepared as previously described (40 (link)). Briefly, the S-nitrosothiol precursor, N-acetyl-l -cysteine (Sigma Aldrich, St. Louis, MO), was dissolved in 1 N HCl and then mixed with sodium-nitrite (water) in equimolar concentrations. The SNAC solution was prepared fresh daily and used immediately. The cultures were grown aerobically at 37°C, and optical density measurements were taken every hour.
6
Reactivation of Lyophilized Probiotic
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Reactivation of a lyophilized probiotic, which was done for L. reuteri only, was performed by resuspending the powder in either MRS, M9 minimal medium (Difco, BD Biosciences, New Jersey, United States) with 2% w/v D-glucose (MM + gluc), or filter-sterilized saliva, followed by an incubation period of 2 or 24 h at 37°C before addition to discs. All reactivated probiotics were readjusted to 1 × 108 cells/mL and added to saliva-covered discs for 30 min and compared to the fresh and non-reactivated lyophilized probiotic.
7
Bacterial Strain Cultivation and Glycerol Stocks
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Bacterial strains were cultivated using on Luria-Bertani (LB) and M9 minimal medium purchased from BD GmbH (Heidelberg, Germany). We inoculated 10 mL of LB broth supplemented with 200 µg/mL ampicillin with a single colony from a freshly streaked plate of Top10 containing BBa_T9002 and incubated the culture for 18 h at 37 °C, shaking at 100 rpm. Glycerol stocks were prepared as described in our previous studies [38 (link)]. Briefly, a 500 µL aliquots of overnight bacterial culture were mixed with 500 µL 30% sterile glycerol in 1.5 mL plastic vials and stored at −80 °C. Prior to each experiment, an aliquot of a glycerol stock from the single culture was diluted 1:1000 into 20 mL M9 minimal medium supplemented with 0.2% casamino acids, 1 mM thiamine hydrochloride and 200 µg/mL ampicillin (AppliChem GmbH, city, Germany). The culture was maintained under the same conditions until the OD600 reached ~0.15 (~5 h).
8
Biofilm Formation Quantification Assay
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Qualitative biofilm assay was performed following the procedure described previously [27 ]. Briefly, overnight bacterial cultures were diluted to 1:100 in M9 minimal medium (BD Technologies) containing 10 μg/ml niacin or LB medium followed by inoculation into U-bottom 96-well plates (Denville) in triplicates. The plates were incubated at room temperature for 24 h at 37 °C. The plates were washed three times with distilled water and biofilms were stained with 0.1 % crystal violet for 15 min. After three washes with distilled water, the presences or absence of biofilms was evaluated.
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