Inoculated the Lactococcus lactis ssp. lactis C2 culture onto the petri dish with bottom agar by using inoculation loop for streaking. The streaking was used to isolate the pure strain of Lactococcus lactis ssp. lactis C2 from the stock culture. The inoculation loop was sterilized in a fire for 5-7 seconds and then cooled to dip into the starting culture for microbes inoculation on the plate.
2.2.Yeast cell preconditioning and inoculum preparation 1 g dry weight of yeast was resuspended in 100 mL of deionized water in an Erlenmeyer flask of 250 mL volume, at 30–35°C, for 30 min with NaCl 6% w/v. Inoculum for experimental fermentations was prepared
In 10 g dried sediment sample added 7 ml 0.2 M NH4Cl solution. A mixture of 100 ml hexane: acetone (1:1) was used as a solvent to extract pesticides with overnight shaking for 12 h on reciprocal or wrist action shaker at 180 rpm. The extract was carefully decanted through activated florisil column (2-3 cm), giving twice wash with25 ml hexane: acetone (1:1) to the sediments. The elute was then washed with 200 ml water and then again aqueous layer was extracted with 50 ml hexane. Finally the hexane layer was washed with 100 ml water and then evaporated to dryness with a vacuum rotary evaporator.
The final volume was adjusted with the same solvent to get concentration of 100 µg/ml. the solution was further diluted to get the concentration of 10 µg/ml, filtered through 0.45 µm filter tips , and aliquots of 20 µl from this solution was injected into HPLC by using an
Experimental procedure (real life) Complete the ‘experimental setup’ for the guinea pig ilium experiment as shown in Fig 1. The agonist, acetylcholine is added to the organ bath at a concentration of 1 x 10⁻⁸ M where it will follow the 3 minute cycle. After 30 seconds the results are recorded. After results are recorded, washing of the organ bath and ilium is conducted (note. Called wash out).
Heat the Agarose gel in a 65 °C water-bath to melt the agarose. After it melted, maintain its temperature at 55 °C until it is ready for use. 2. Transfer the spheroids from the 96-well plate to a 15 mL centrifugal tube using a 1000 μL pipette 3. centrifuge the tube for 5 min at 1000 rpm to form a pellet. 4.
Quantification and trypsin digestion of polypeptides Protein concentration was estimated by Bradford assay, and 100µg of total protein from each sample was subjected to in-solution trypsin digestion to generate peptides. Initially, treating the sample with 5µl of 100mM dithiothreitol in 50 mM ammonium bicarbonate for 30 min at 60ºC and alkylation with 200mM iodoacetamide in 50 mM ammonium bicarbonate at room temperature for 30 minutes reduced the protein disulphide bonds. Proteins were then digested with 4µg of sequencing grade-modified trypsin (Sigma) in 50 mM ammonium bicarbonate by incubating overnight at 37ºC. The trypsin digestion reaction was stopped by 1µl of 100% formic acid. The digested peptide solutions were centrifuged at 14000
Materials and methods Isolation and identification of Bacillus thuringiensis Bacillus thuringiensis was isolated from raw milk (Taif, KSA) on nutrient agar at 37oC for overnight. The supernatant of the bacterial isolate was screened for synthesis of AgNPs. The bacterial isolate was morphologically and biochemically characterized according to Bergy’s Manual of Systemic Bacteriology. Also, this bacterial isolate was further identified by 16S rRNA sequencing. The culture was maintained on a nutrient agar plate/slants at 4°C and as glycerol stocks 40% at −70°C.