A municipal waste-contaminated soil sample was used for the enrichment of culture in a liquid mineral salt medium (MSM [27 (link)]), supplemented with DnOP (0.5 g l−1) as the sole source of carbon and energy, and incubated at 28 °C on a rotary shaker (180 r.p.m.). Through several rounds of sub-culturing in liquid medium and subsequent plating on Luria-Bertani agar medium containing 1.8 % agar-agar (Hi-Media), a DnOP-degrading pure culture was isolated. For characterization of the isolate, designated as strain GONU, a combination of universal primers f27 and r1492 [28 (link)] was used to amplify the 16S rRNA gene. The gene was sequenced following the manufacturer’s instructions for Taq DNA polymerase-initiated cycle sequencing reactions using fluorescently labelled di-deoxynucleotide terminators with an ABI PRISM 377 automated sequencer (Perkin–Elmer Applied Biosystems). The 16S rRNA sequence was subjected to a homology search in blast (http://www.ncbi.nlm.nih.gov/BLAST ) to evaluate the phylogenetic affiliation of strain GONU. Then, the 16S rRNA gene sequences of the top 50 closest type strains were collected from GenBank, and aligned (ClustalX2), and a phylogenetic tree was reconstructed using the neighbour-joining method [29 (link)]. The tree was visualized and labelled in Tree Explorer v2.12.
Luria bertani agar medium
Manufactured by HiMedia
Sourced in India
Luria Bertani (LB) agar medium is a commonly used nutrient-rich growth medium for culturing bacteria. It provides the necessary nutrients and support for the growth and maintenance of a wide range of bacterial species. The medium is composed of peptone, yeast extract, and sodium chloride, which collectively create an optimal environment for bacterial proliferation.
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6 protocols using luria bertani agar medium
1
Isolation and Characterization of DnOP-Degrading Bacteria
2
Soil Bacteria Isolation Protocol
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Soil samples were aseptically collected from effluent adjacent dump backyard area of the Emergency Unit, Burdwan Medical College and Hospital, Burdwan, West Bengal, India (23.2489° N, 87.8536° E). Qualitative elemental analysis of the soil sample was analyzed using X-ray fluorescence spectroscopy (Artax, Bruker). For isolation of bacterial species, 1.0 g of soil sample was diluted gradually up to 10−4 in sterile saline Milli-Q (0.1% NaCl, HiMedia) and 100 μL of each dilution was plated onto Luria Bertani Agar medium (HiMedia) and incubated at 30 °C for 24 h61 (link).
3
Potato-Soybean Powder Synthesis and Characterization
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Polyaniline (PANI), carbon nanotubes (CNTs), reduced graphene oxide powder (rGO), and proton exchange membrane (PEM: Nafion 117) were purchased from Sigma–Aldrich. Luria Bertani (LB) agar medium was purchased from Himedia Labs (India). Potato powder containing 15–20% carbohydrate was prepared by crushing the cleaned potato in a mixer grinder, then extracting the fine paste into a glass Petri dish. The paste was then calcined in a hot air oven overnight at 60 ± 5 °C, and the resulting fine powder was cooled to room temperature. Soybean powder containing 36–58% protein was prepared from raw dried soybeans purchased from the local market. Dried soybeans were properly crushed using a mixer grinder to the finest powder form. Finally, the Potato and Soybean powders were mixed at 1:1 to produce a homogeneous Potato-Soybean (plant) powder and then stored at 4 °C for further use. Deionized water (18.2 MΩ cm) from Millipore Co. was used throughout the experiment. The study of collection of plants/plant material, are in compliance with relevant institutional, national, and international guidelines and legislation. All reagents used were of analytical grade and used as received without further purification.
4
Reactivation and Growth of MDR A. baumannii
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Clinical isolates of MDR A. baumannii described by Castilho et al. [43 (link)] identified as AB 02, AB 53, and AB 72 cryopreserved at −80 °C were reactivated in Luria Bertani (LB) agar medium (HiMedia) and grown at 37 °C for 24 h. A colony isolated from each strain was inoculated into 5 mL of LB broth medium (HiMedia, Pennsylvania, USA) until growth corresponded to 0.5 of the MacFarland scale. Some growth conditions were modified for each experiment to evaluate the different stages of biofilm formation described below.
5
Isolation and Characterization of Arthrobacter Strain P3B162T from Pokkali Rice Rhizosphere
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Strain P3B162T was isolated from the rhizosphere soil of a hybrid pokkali rice variety VTL-6 collected from Alappuzha in Kerala, India. A standard serial dilution procedure was followed in isolating the bacterial strain P3B162T. Briefly, two grams of soil attaching to the root portion was collected carefully and mixed with 20ml of sterile 0.85% (w/v) NaCl solution. The resulting rhizosphere soil suspension was then vortexed vigorously for 10-30min and was allowed to stand still for 5min. A fivefold serial dilutions were done with the above rhizosphere soil suspension and 0.1ml aliquots of each dilution was then spread plated onto 1/1000 dilutions of Luria Bertani (LB) agar medium (Himedia, India), pH 5.5 (maintained with 0.1N HCl). These plates were then incubated for 1–2 weeks. Strain P3B162T was regularly subcultured on full strength LB agar medium at 28°C for 3–4 days. For storage purposes, the strain was kept at 4°C as active plates for 2–3 weeks as short-term storage and as 10% glycerol suspensions stored in deep freezer at −80°C as long-term storage. The following reference strains were included in this study; Arthrobacter globiformis LMG 3813T, A. pascens LMG 16255T, A. liuii JCM 19864T, A. humicola DSM 25587T, A. oryzae DSM 25586T and A. cupressi DSM 24664T.
6
Plasmid-Mediated CTX-M-15 Gene Transfer
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Using a plasmid extraction kit (GeNei, Bangalore), the bacterial plasmid was extracted. The puri ed plasmid DNA was dissolved and stored at 4 o C in TE buffer. Conjugation experiments were carried out by a broth mating method using azide resistant (AzR) E. coli J53 as recipient and bla CTX-M-15 positive E. coli as the donor (wang et al. 2008). Pure colonies of donor and recipient cells were inoculated separately and incubated overnight at 37 o C with shaking. These overnight cultures were diluted in a fresh medium at 1:100 and each was grown to the early exponential phase. The mating combination was prepared by adding 0.1 ml of donor cells to 0.9 ml of recipient cells. The mixture was gently whirled for a few minutes, incubated at 37 o C for 6 h (without shaking). This was followed by plating on Luria-Bertani (LB) agar medium (Himedia, India) containing cefotaxime (2 mg/l). Trans-conjugants carrying the same bla CTX-M- 15 gene as their donor were veri ed by PCR.
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