Tryptone broth (TB) and agar, LB broth (LB) and agar (LBA) and suspension medium (SM) have been described (4 (link),28 (link)). TB top agar contained 0.75% Bacto-Agar (Becton-Dickinson) and TB bottom agar contained 1.0% Bacto-Agar. Tryptone agar plates (BBL YE) for the detection of clear and turbid plaques contained 0.2% yeast extract (Becton-Dickinson).
Bacto agar
Manufactured by BD
Sourced in United States, Germany, France, Japan, Switzerland, Netherlands, United Kingdom
Bacto agar is a solidifying agent used in microbiology and cell culture applications. It is derived from red seaweed and provides a solid growth medium for the cultivation of microorganisms and cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Bacto peptone, by BD (28 mentions)
Bacto yeast extract, by BD (26 mentions)
Yeast extract, by BD (24 mentions)
Bacto tryptone, by BD (12 mentions)
Kanamycin, by Merck Group (9 mentions)
Ampicillin, by Merck Group (8 mentions)
Glucose, by Merck Group (7 mentions)
Sodium chloride (nacl), by Merck Group (7 mentions)
Tryptone, by BD (7 mentions)
Yeast nitrogen base, by BD (6 mentions)
Peptone, by BD (5 mentions)
Glucose 6 phosphate (g6p), by Merck Group (5 mentions)
Tryptic soy broth (tsb), by BD (5 mentions)
Crystal violet, by Merck Group (5 mentions)
Acetylated trypsin, by Merck Group (4 mentions)
2 nitrofluorene, by Merck Group (4 mentions)
2 aminoanthracene, by Merck Group (4 mentions)
9 aminoacridine, by Merck Group (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Kh2po4, by Merck Group (4 mentions)
380 protocols using bacto agar
1
Tryptone and LB Media Preparation
2
Soft Agar Colony Assay
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24 h post-transfection, 5000 S2-007 cells or 7000 Panc-1 cells were mixed with DMEM containing 0.33 % Bacto Agar (Becton, Dickinson & Company, Sparks, NV, USA) and reseeded in 12-well cell culture plates coated with a bottom layer of 0.5 % Bacto Agar in DMEM. Viable colonies were counted 7–10 days after reseeding under a microscope with 10× magnification.
3
Bacterial Motility Assays in Agar
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Motility was assayed by the Plate-Based method as previously described (Filloux and Ramos, 2014 (link)). Swimming motility was assessed on 0.3% agar plates (3 g/l Bacto agar [Becton Dickinson] and 8 g/l Nutrient Broth [Becton Dickinson]). Overnight cultures grown at 37°C and 200 rpm in LB were point inoculated on the plates by depositing 1 μl of culture directly into the agar in the center of the plate. Plates were incubated face up at 37°C, and the swim diameter (in centimeters) was measured after at 16 h.
Swarming motility was assessed on 0.6% agar plates (6 g/l Bacto agar [Becton Dickinson], 5 g Bacto-peptone [Becton Dickinson], 3 g/l Yeast Extract [Sigma], and 5 g/l D. glucose). Overnight cultures grown at 37°C and 200 rpm in LB were point inoculated on the plates by depositing 1 μl of culture directly into the agar in the center of the plate. Plates were incubated face up at 37°C, and the swarming motility was examined after 16 h.
Twitching motility was assessed on 1.5% agar LB plates. Overnight cultures (37°C, 200 rpm; LB) were used to inoculate twitch plates by depositing 1 μl of culture directly into agar in the bottom of the plate. Plates were incubated face down at 37°C for 16 h, and the twitching motility visualized by fixing the culture with Water : Glacial acetic Acid : Methanol at a ratio of 4 : 1 : 5 (v/v) and stained with 0.1% crystal violet.
Swarming motility was assessed on 0.6% agar plates (6 g/l Bacto agar [Becton Dickinson], 5 g Bacto-peptone [Becton Dickinson], 3 g/l Yeast Extract [Sigma], and 5 g/l D. glucose). Overnight cultures grown at 37°C and 200 rpm in LB were point inoculated on the plates by depositing 1 μl of culture directly into the agar in the center of the plate. Plates were incubated face up at 37°C, and the swarming motility was examined after 16 h.
Twitching motility was assessed on 1.5% agar LB plates. Overnight cultures (37°C, 200 rpm; LB) were used to inoculate twitch plates by depositing 1 μl of culture directly into agar in the bottom of the plate. Plates were incubated face down at 37°C for 16 h, and the twitching motility visualized by fixing the culture with Water : Glacial acetic Acid : Methanol at a ratio of 4 : 1 : 5 (v/v) and stained with 0.1% crystal violet.
4
Cultivation of Extremophilic Bacteria
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D. radiodurans strains were grown at 30 °C in TGY broth composed of 0.5% tryptone (Difco Laboratories, Detroit, MI, USA), 0.3% yeast extract (Difco Laboratories, Detroit, MI, USA), and 0.1% glucose (Sigma-Aldrich, St. Louis, MO, USA) or on TGY plates with 1.5% Bacto-agar (Difco Laboratories, Detroit, MI, USA). E. coli strains, DH5α (Thermo Fisher Scientific, Waltham, MA, USA) and BL21(DE3) (Novagen, Darmstadt, Germany), were cultivated in Luria-Bertani (LB) broth (Difco Laboratories, Detroit, MI, USA) (1% tryptone, 0.5% yeast extract, 1% NaCl) or on LB medium with 1.5% Bacto-agar at 37 °C. Antibiotics were added to the medium if necessary: ampicillin (Sigma-Aldrich, St. Louis, MO, USA), 100 μg/mL (E. coli); kanamycin (Sigma-Aldrich, St. Louis, MO, USA), 50 μg/mL (E. coli) and 8 μg/mL (D. radiodurans); and chloramphenicol (Sigma-Aldrich, St. Louis, MO, USA), 3 μg/mL (D. radiodurans).
5
Rat-1 Cell Transformation Assay
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After retroviral transduction 5 x 103 transduced Rat-1 cells were suspended in ‘top-agar’, DMEM supplemented with 10% FCS and 0.25% bacto-agar (DIFCO Laboratories, Detroit, USA), and stacked in six-well plates filled with DMEM supplemented with 10% FCS and 0.5% bacto-agar (2 ml per well). After 15 days incubation at 37°C and 5% CO2 colonies were counted. The focus-formation assays were performed in 24-well plates. 4 x 104 transduced Rat-1 cells/well were plated. Unstained foci were photographed at day 15 using an AxioCam HRc system (Zeiss, Goettingen, Germany) with 10x magnification.
6
Cultivation of Streptococcus ferus and E. coli
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Bacterial strains and plasmids used in this study are listed in Table S1. S. ferus strains were derived from DSM20646 (37 (link)) were grown on Todd-Hewitt plates (TH, BD Biosciences) with 1.4% Bacto-Agar (BD Biosciences) and 0.2% yeast extract (VWR), in Todd-Hewitt broth with 0.2% yeast (THY), or in a chemically-defined medium (CDM) containing 1% glucose at 37°C with 5% CO2. The components and recipe for CDM used were as described previously (38 (link)). E. coli strains were grown on Luria-Bertani (BD Biosciences) plates with 1.4% Bacto-Agar at 37°C or in Luria-Bertani (LB) broth with shaking at 175 rpm. When required spectinomycin (50–100 μg/mL), erythromycin (0.5 μg/mL for S. ferus, 500 μg/mL for E. coli), or kanamycin (50–100 μg/mL) were added to S. ferus or E. coli culture media.
7
Bioluminescent Microbial Culture Media
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Two different media (trypticase yeast extract seawater agar (TYESA) and AB medium) were used in this study. A suitable agar medium (TYESA medium) necessary to support the growth and production of bioluminescent light was prepared by using 30 g of tryptone, 20 g of Bacto agar and 5 g yeast (BD Biosciences; San Jose, CA, USA), per liter of deionized water. To selectively support the growth of the bioluminescent microorganism, artificial seawater was prepared by adding 30 g/L of analytical grade sodium chloride (EMD Chemicals; Gibbstown, NJ, USA). One liter of AB medium was prepared using (3 mL glycerol, 5 g casitone, 3 g yeast extract (BD Biosciences; San Jose, CA, USA), 1.179 g potassium phosphate monobasic (Sigma-Aldrich; St. Louis, MO, USA), 5.395 g sodium phosphate dibasic dihydrate (Honeywell Fluka; Muskegon, MI, USA, 30 g sodium chloride (EMD Chemicals; Gibbstown, NJ, USA), and 18 g Bacto agar (BD Biosciences; San Jose, CA, USA).
8
Oatmeal Agar and M63 Overlay Media
9
Oatmeal Agar and M63 Overlay Media
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1x oatmeal agar: 72.5 g/L Difco oatmeal agar (BD). 1/2x oatmeal agar: 31.25 g/L Difco oatmeal agar (BD) and 6.25 g/L Bacto agar (BD), with ~100 μL/L 10M NaOH to adjust to pH 7. Media for overlays consisted of M63 salts (2 g/L (NH4)2SO4, 13.6 g/L KH2PO4, 0.5 mg/L FeSO47H2O) supplemented with 0.4% glucose, 0.02% casamino acids, 1 mM MgSO4, 1.5 mM thiamine, 7.5g/L bacto-agar (BD), adjusting pH to 7.0 with 1M NaOH. 1000x Thiamine was prepared at 1.5 M in H2O and filter sterilized.
10
Anchorage-Independent Cancer Cell Proliferation
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Anchorage-independent proliferation was quantified by soft agar assay. Cells were suspended in 1 mL of 0.36 % (w/v) bacto-agar (BD Biosciences) overlaid by a 0.6 % (w/v) bacto-agar support in 6-well plates in triplicates at densities of 5 × 103 cells per well for A375, A375.S2, A375Scr, A375 shFN2, A375.S2 Scr, A375.S2 shFN2 lines, respectively. Plates were incubated at 37 °C in normoxic conditions. Pathway inhibitors were used at the following concentrations: 3.0 μM cisplatin (DNA-crosslinking agent), 3.0 μM VX-11e (ERK inhibitor), 3.0 μM vemurafenib (BRAFV600E inhibitor) (Chemietek, Indianapolis, IN), and 50 nM paclitaxel (microtubule stabilizing agent) (Life Technologies). Pathway inhibitors were administered at the beginning of the experiment or initiated 14 days later. At the end of the experiment, wells were stained with 0.5 μg/mL of nitrotetrazolium blue chloride (Sigma-Aldrich). Colonies with > 20 μm in diameter were counted using the Bioreader Software and a BioSys BioCount 4000 Pro machine. Clonogenic survival for each line and condition was plotted as frequency of colony size normalized to a gaussian distribution. Experiments were performed in triplicate.
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