Bacillus Thingiensis Lab Report

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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[21]. 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.
Preparation of bacterial supernatant for synthesis of nanoparticles
A loopful of the culture was inoculated into 250 ml sterile nutrient broth and incubated at 37°C for 24 hours. The
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The other factors like temperature and pH were constant. The production of AgNPs was analyzed using UV–visible spectroscopy.
2.3.3. Effect of pH on synthesis of AgNPs
To study the effect of pH on AgNPs production, different pH values (pH 8.0, 9.0, 10.0 and11.0) of the mixed reaction was used. pH was adjusted by 0.01 M HCl and 0.01 M NaOH. The other factor like supernatant concentration and temperature were constant. The production of AgNPs was measured by UV–visible spectroscopy.
Effect of the time of preparation of AgNPs
Time is an important factor for any biosynthesis reaction, especially in biochemical reactions. The effect of time on the synthesis of AgNPs was studied by using various time (1 day, 7 days, 14 days, 21 days and 2 months) with the fixed concentration of AgNO3 (0.001 M). pH was adjusted by 0.01 M HCl and 0.01 M NaOH solution. The other factor like volume of supernatant, concentration of AgNO3 (0.001 M) and temperature were constant at optimum conditions. The effect of time was analyzed by UV–visible
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coli (Table 1) by using the well agar diffusion method[26]. The multidrug resistant pathogens were initially propagated at 37 °C in nutrient broth. The overnight grown cultures were then again sub-cultured into nutrient broth media for 2 h till 0.01 OD. Subsequently, 100 μL of each culture was then spread uniformly onto nutrient agar plates. Wells of 6 mm diameter were made on agar plates using an agar well borer. The AgNPs (4.5, 9 and 13.5 g/ml) was loaded into the wells. The plates were incubated at 37 °C for 24 hours. Zone of inhibition was calculated by measuring the diameter of bacterial

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