The culture was then streaked on Asbhy’s N-free agar plates and incubated at 37 ºC for 24h. Production of ammonia by the isolate was quantified by the method of Goswami et al. (2014). Bacterial culture grown in Asbhy’s N-free liquid medium was centrifuged at 6000 rpm for 10 min and 0.2 ml culture supernatant was mixed with 1ml Nessler’s reagent and total volume was made 8.5 ml by adding ammonia free distilled water. Development of brown to yellow colour is indicative of ammonia production.
Then 1 ml from these bacterial solutions were added to 1 ml of MHII containing different concentration of ciprofloxacin 0.5x, 1x, 5x, 10x, 20x, 30x, 50x,75x and 100x MIC to each well in 12-well plate. Colony counts (CFU/ml) were determined at different time points (T0, T3, T24) by using appropriate dilutions of each culture. Using spot-plating method (32) 10µl was spotted on LB agar plates. However after 24 hour exposure the bacterial cell from dilutions of ≤ 1x102 were washed twice with sterile PBS prior to plating in triplicate. These were then incubated overnight at 37°C.
The minimum inhibitory concentration of abietic acid was studied by broth micro dilution method using 96-well microtitre plates.9 Test compound was dissolved in DMSO (1%) with the addition of Tween-80(0.5%) and diluted in Muller Hinton Broth to get a concentration range of 100-1000μg/mL. The solution was then two fold diluted in Muller Hinton Broth (100 μL), inoculated with bacterial strains and then incubated at 37°C for 24 h. The bacterial growth was measured as turbidity with a Cyberlab microplate reader at 405 nm. The minimum inhibitory concentration was defined as the lowest concentration of test compound that inhibits the growth of the test bacteria. DMSO assayed as the negative control at a concentration of 1% did not inhibit any of the strains tested. All tests were assayed in triplicate in three independent experiments and median values were used for MICs calculation.
3.3.4 Preparation of CaCl2 solution The molecular weight of CaCl2 was 111 g/mole. Weighed 11.1g CaCl2 and dissolved into 100ml distilled water. The final CaCl2 concentration would be 1M. CaCl2 solution was used to make bottom agar and initiate the infection cycle. 3.4 Selection of most sensitive strain to bacteriophages to make new stock culture Starting culture was prepared by inoculating 1ml (1×109 CFU/ml) stock Lactococcus lactis ssp.
The fresh intestine was collected, which was cut through lumen. The microsphers equivalent to 100 mg of the drug was taken on mucosal side of the intestine, was placed b/n donor and receptor compartment; in such a way that mucosal side is facing towards donor compartment. One ml of phosphate buffer pH7.4 was added to donor compartment, the receptor compartment is filled with 25 ml of phosphate buffer pH7.4. The rate of drug permeation was studied by using collection of receptor fluid at regular time intervals, and analysed for drug
Indole production test The ability of bacterial isolates to produce indole in broth was tested following the method described by Seeley and Vandermark, (1981). Each fresh isolate was inoculated to sterile trypton broth in test tubes and incubated at 37±1o C for 24-48 hours. After incubation, to these tubes 0.5 ml of kovac’s reagent was added and mixed well. Development of red ring in alcohol layer within few minutes was considered as positive test for indole production. Composition of Trypton broth medium Composition of Kovac’s reagent Trypton 10g Sodium chloride 5g Calcium chloride 1g Distilled water 1000ml PH 7.2 P- Dimethyl amino benzaldehyde
A flame test and a halide ion test were performed. To start with the flame test, weigh 1 gram of Unknown substance in an analytical balance using a scoopula and mixed it in with 20 mL of water into a 150 mL. Repeat the same step with NaC2H3O2. Repeat the same step for the flame test and record down data. Lastly is the halide ion test.
0.5 mL of AuNPs solution was added to the above mixture. The UV-Vis spectra were recorded with a time interval of 1 min in a scanning range of 200-600nm at ambient temperature (25±20C). Antimicrobial activity The agar disc diffusion method was employed for the determination of antimicrobial activity of the papaya leaf extract stabilized gold nanoparticles. The 0.1 ml of 108cfu/ml of different pathogenic bacteria suspension was spread on different plates nourished with LB media. Filter paper discs (5 mm in diameter) were placed on the plates and synthesized AuNPs and papaya leaf extract solution were impregnated in different concentrations then onto the discs.
After incubation, 200 µl of 100% ethanol was added to the lysate. The sample was then washed and centrifuged following the manufacturer’s recommendations. Nucleic acid was eluted with 100 µl of elution buffer provided in the kit. Oligonucleotide Primer. Primers used were supplied from Metabion (Germany) are listed in table (1).
Characterization of bacteriocins 6.1. Effect of temperature The effect of temperature on bacteriocin stability was determined by exposing 5 ml aliquots of CFS at 40C, Room temperature, 370C, 630C and 1000C for 30 minutes. Agar well diffusion method was performed to determine the residual activity in the heat-treated CFS17. 6.2. Effect of pH To check the pH stability of the bacteriocins, 5 ml aliquots of CFS was taken in sterile tubes and were adjusted to pH values of 2, 4, 7 and 9 busing 1N HCl or 1 N NaOH.