Batch cultivation was performed in Luria-Bertini broth (LB) (HiMedia Laboratories, Mumbai, India) containing, in g/L, tryptone 10, yeast extract 5, and NaCl 10 or in Super optimal broth (SOB) medium containing tryptone (HiMedia Laboratories, Mumbai, India) 20, yeast extract (Sigma Aldrich, St. Louis, MO) 5, NaCl 0.5, and KCl 0.186, with an initial culture optical density (OD600) of 0.05. The initial production medium was supplemented with 2.76 g/L glycerol, 10 mM MgCl2/MgSO4, and an appropriate antibiotic [23] (link).
Tryptone
Manufactured by HiMedia
Sourced in India, United States
Tryptone is a bacterial growth medium commonly used in microbiology laboratories. It is a nutrient-rich powder composed primarily of pancreatic digest of casein, which provides a source of amino acids, peptides, and other essential nutrients to support the growth of a wide range of microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Yeast extract, by HiMedia (11 mentions)
Sodium chloride (nacl), by Merck Group (6 mentions)
Sodium chloride (nacl), by HiMedia (3 mentions)
Bacto agar, by HiMedia (3 mentions)
Cacl2, by Merck Group (3 mentions)
Kh2po4, by Merck Group (3 mentions)
Glucose, by Merck Group (2 mentions)
Peptone, by HiMedia (2 mentions)
Glucose, by HiMedia (2 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (2 mentions)
Mgso4.7h2o, by Merck Group (2 mentions)
Feso4 7h2o, by Merck Group (2 mentions)
Na2hpo4, by Merck Group (2 mentions)
Yeast extract, by Merck Group (2 mentions)
Agar, by Thermo Fisher Scientific (1 mentions)
Potato extract, by Merck Group (1 mentions)
Ferric citrate, by Merck Group (1 mentions)
Sabouraud dextrose agar, by HiMedia (1 mentions)
Bacteriological agar, by HiMedia (1 mentions)
Tryptic soy agar, by HiMedia (1 mentions)
18 protocols using tryptone
1
Batch cultivation of microbes in LB or SOB
2
Isolation and Culturing of Fungal Isolates
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To retrieve a local isolate, a loopful of spores was scraped from moldy bread and cultured on potato dextrose agar (PDA). The PDA contained potato extract (4.0 g) (fresh, unpeeled potatoes), glucose (20 g) (Merck KGaA, Darmstadt, Germany), and agar (20 g) (Oxoid, Basingstoke, UK) in distilled water (1000 mL) [11 (link)]. The agar was dissolved by boiling the mixture, followed by sterilization before pouring it onto plates. Sabouraud dextrose agar (SDA) (HiMedia, Mumbai, India) was also used for the parallel culturing of the isolate. Both media were incubated for five to seven days at 25 ± 2 °C [12 (link)]. Then, a cork borer was used to inoculate a fungal disk on Aspergillus differentiation agar. This agar was prepared by adding 10 g of yeast extract (HiMedia, Thane, India), 15 g of tryptone (HiMedia), 0.5 g of ferric citrate (Merck KGaA), and 15 g of agar (Oxoid) to distilled water (1000 mL). The media were boiled and autoclaved, and the inoculated plates were incubated for five to seven days at 25 ± 2 °C [13 (link)].
3
Antimicrobial Activity Evaluation
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Culture media (Tryptic Soy Broth, Tryptic Soy Agar, Peptone, Yeast Extract, Tryptone, Glucose, and Bacteriological Agar) were purchased from HiMedia (Mumbai, India); ABTS from Sigma Aldrich Co. (St. Louis, MO, USA); and ciprofloxacin disks from Bioanalyse® (Ankara, Turkey).
4
Culturing Candida albicans and E. coli
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The strains used in this study were C. albicans-ATCC 10231 and E. coli-ATCC 25992 purchased from the Balai Besar Laboratorium Kesehatan. Overnight C. albicans cultures were grown in yeast extract–peptone–dextrose (YPD) medium (1% yeast extract [BD Biosciences, USA], 2% peptone [Oxoid Ltd, UK], and 2% dextrose [Conda Pronadisa, Spain]) at 30°C. Overnight E. coli cultures were grown in Luria–Bertani (LB) medium (1% tryptone [Himedia, India], 0.5% yeast extract, and 1% NaCl [Merck, Germany]) at 37°C.
5
Procurement of Reagents and Cell Lines
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Silver nitrate and copper sulfate (analytical grade) were obtained from Sigma Aldrich Bangalore, India. All bacteriological media components such as crystal violet, tryptone, yeast extract, glucose, and sodium chloride, and media such as nutrient both were procured from Hi-Media Laboratories Pvt. Ltd., Mumbai, India. The HeLa & HEK293 cell lines utilized in the experiment was acquired from American Type Culture Collection (ATTC, LG Promochem, Barce-lona, Spain).
6
Heterologous expression of Lip11 lipase
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Restriction enzymes were purchased from New England Biolabs (NEB), USA. Taq polymerase and T4 DNA ligase were purchased from Bangalore Genei, India. Gel extraction kit and plasmid isolation kit were purchased from Qiagen, India. Recombinant yeast strain P. pastoris X-33 harbouring Lip11 gene from Yarrowia lipolytica was taken from the laboratory culture collection. This strain has been submitted to Microbial Type Culture Collection (MTCC) with MTCC number 9517. Zeocine was from Invitrogen. The triacylglycerides, p-np esters used in the experiments were procured from Sigma Aldrich. Luria bertani, tryptone, yeast extract, yeast nitrogen base and methanol were purchased from Hi-Media. Sodium chloride was taken from Sisco Research Laboratories Pvt. Ltd. India (SRL). Glycosylation kit was procured from G Bioscience (USA).
7
Synthesis of Salmonella Aptamer Probe
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Chemicals used for the synthesis were of
analytical grade, purchased from different chemical companies, and
used without further purification. Sodium molybdate (Na2MoO4·2H2O with 99% purity) was purchased
from Fisher Scientific. Selenium (Se with 99.99% purity), hydrazine
hydrate (N2H4·H2O), potassium
ferricyanide (K3[Fe(CN)6]), potassium ferrocyanide
(K4[Fe(CN)6]), potassium chloride (KCl), and
glutaraldehyde were purchased from Sigma-Aldrich, India. For the preparation
of solid LB medium: solid agar, sodium chloride (NaCl), tryptone and
yeast extract of Hi-Media were used. All of the other chemicals were
of analytical reagent grade. Aliquots of bacterial strains were acquired
from Microbial Type Culture Collection and Gene Bank CSIR-IMTECH,
Chandigarh (3220-S. paratyphi). E. coli was grown at the Amity Institute of Nanotechnology
under sterilized conditions in the lab. The amino-group-modified aptamer
nucleotide sequence of Salmonella was synthesized by GCC Biotech (GCC
Biotech India Pvt Ltd, India). The 41-mer aptamer sequence was obtained
from the work of Xiaoyuan et al. (2014): 5′-NH2-TAT
GGC GGC GTC ACC CGA CGG GGA CTT GAC ATT ATG ACA-G-3′. The aptamer
probe solution of varying concentrations was prepared in Tris–ethylenediaminetetraacetic
acid (EDTA) buffer solution (10 mM Tris, pH 8.0, 1 mM EDTA).
analytical grade, purchased from different chemical companies, and
used without further purification. Sodium molybdate (Na2MoO4·2H2O with 99% purity) was purchased
from Fisher Scientific. Selenium (Se with 99.99% purity), hydrazine
hydrate (N2H4·H2O), potassium
ferricyanide (K3[Fe(CN)6]), potassium ferrocyanide
(K4[Fe(CN)6]), potassium chloride (KCl), and
glutaraldehyde were purchased from Sigma-Aldrich, India. For the preparation
of solid LB medium: solid agar, sodium chloride (NaCl), tryptone and
yeast extract of Hi-Media were used. All of the other chemicals were
of analytical reagent grade. Aliquots of bacterial strains were acquired
from Microbial Type Culture Collection and Gene Bank CSIR-IMTECH,
Chandigarh (3220-S. paratyphi). E. coli was grown at the Amity Institute of Nanotechnology
under sterilized conditions in the lab. The amino-group-modified aptamer
nucleotide sequence of Salmonella was synthesized by GCC Biotech (GCC
Biotech India Pvt Ltd, India). The 41-mer aptamer sequence was obtained
from the work of Xiaoyuan et al. (2014): 5′-NH2-TAT
GGC GGC GTC ACC CGA CGG GGA CTT GAC ATT ATG ACA-G-3′. The aptamer
probe solution of varying concentrations was prepared in Tris–ethylenediaminetetraacetic
acid (EDTA) buffer solution (10 mM Tris, pH 8.0, 1 mM EDTA).
8
Bacterial Strain Cultivation and Genetic Manipulation
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The bacterial strains used in this study are listed in Table 1 . All cultures were grown in Luria-Bertani (LB) medium consisting of 10 g of tryptone (Himedia, Mumbai, India), 10 g of NaCl (Merck, Mumbai, India) and 5 g of yeast extract (Himedia) per liter at 37°C with constant shaking at 180 rpm. Single colony cultures grown for 8 h-10 h served as pre-inoculum cultures for all experiments. Antibiotics were used at 30 μg/ml chloramphenicol, 50 μg/ml of streptomycin (Sigma, United States), 100 μg/ml ampicillin (Himedia). Single gene knockouts were generated using one step chromosomal gene inactivation as described using plasmids and primers in Tables 1 and 2 (Datsenko and Wanner, 2000 (link)). The antibiotic resistance cassette was then removed using pCP20 before being used.
9
Xylan and Arabinoxylan Extraction Protocol
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Kanamycin, ampicillin, and IPTG were purchased
from Sigma-Aldrich
Co. LLC., USA. The analytical-grade reagents and chemicals, namely,
sodium chloride, sodium acetate, glucose, yeast, extract, peptone,
tryptone, di-potassium phosphate, monopotassium phosphate, glycerol,
sodium carbonate, sodium bicarbonate, sodium potassium tartrate, sodium
sulfate, copper sulfate, ammonium molybdate, trifluoroacetic acid
(TFA), Coomassie Brilliant Blue G-250, and sodium arsenate were purchased
from Himedia Pvt. Ltd., India. The xylan substrate was purchased from
Carbosynth, UK, and rye arabinoxylan from Megazyme, Ireland.
from Sigma-Aldrich
Co. LLC., USA. The analytical-grade reagents and chemicals, namely,
sodium chloride, sodium acetate, glucose, yeast, extract, peptone,
tryptone, di-potassium phosphate, monopotassium phosphate, glycerol,
sodium carbonate, sodium bicarbonate, sodium potassium tartrate, sodium
sulfate, copper sulfate, ammonium molybdate, trifluoroacetic acid
(TFA), Coomassie Brilliant Blue G-250, and sodium arsenate were purchased
from Himedia Pvt. Ltd., India. The xylan substrate was purchased from
Carbosynth, UK, and rye arabinoxylan from Megazyme, Ireland.
10
Isolation and Characterization of PHB-producing E. coli Strain
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The E. coli strains and plasmids used in this study are listed in Table 1 . The PHB-producing C. necator strain A-04 (Chanprateep et al., 2008 (link)) was used to isolate the phaCABA–04 gene operon. All bacterial strains were grown at 37°C in Luria-Bertani (LB) medium supplemented with 100 μg/L ampicillin. The LB medium contained (per liter) 10 g of tryptone (Himedia, Mumbai, India), 5 g of yeast extract (Himedia, Mumbai, India) and 10 g of NaCl (Merck KGaA, Darmstadt, Germany). Stock cultures were maintained at –80°C in a 15% glycerol solution. The experiments were performed in a biosafety level 1 laboratory and by researchers and investigators who had undergone biosafety training.
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