MATERIALS AND METHODS Bacterial strains and culture conditions Two S. aureus strains were used in the present study; S. aureus 8325-4 (SigB-) and SH1000 representing a SigB+.strain. Overnight cultures were grown in Luria Broth (LB) at 37°Cwith shaking at 150 rpm. Exposure to antibiotics was carried out as detailed below. Antibiotics Ciprofloxacin were purchased from Sigma-Aldrich CO. 10 mg/ml stock solution of antibiotic were prepared freshly with 0.1N HCl and stored at -20°C. During the experiment we diluted with sterile water 1:10 and 1:100 depending on the different drug concentration.
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. After incubation at room temperature for 30 minutes, pH in each tube was readjusted to 6.5 and the antimicrobial activity was determined by agar well diffusion method17. 6.3. Effect of proteolytic enzyme 5 ml of CFS was taken in a test tube and was treated with trypsin-chymotrypsin enzyme (10AU/ml) at pH 7. The test tubes with and without enzyme (control) were incubated at 370C for 1 hour.
Then, the leaves were grinded to powder by using domestic blender and the coarse powder is stored in airtight container. Preparation of crude extracts. The coarse powder was extracted with distilled water and methanol. For aqueous extraction process, the coarse powder was extracted with distilled water at a ratio of water to leaves of 10:1 (ml:g) for 30 min at 85 °C. The extraction process was repeated one more time.
After completion of the solubility tests, the appropriate solvent, cyclohexane, was selected for large-scale recrystallization. The unknown was weighed for the large-scale recrystallization then the same process was repeated to test the solubility. The solute dissolved after adding of about 60 mL of solvent, it was set aside to cool to induce crystal reformation. The solution was then seeded and set alone once more. When the process was complete, the crystals
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. lactis C2 culture into 25ml M17 medium in a test tube. The mixture was incubated overnight for 16h in a 32℃ water bath to get fresh Lactococcus lactis ssp. lactis C2 culture. Inoculated the Lactococcus lactis ssp.
Durham’s tubes filled with the broth were placed in each of the test tubes in an inverted position. Then the tubes were sterilized. After sterilization, the test tubes were inoculated with fresh bacterial cultures and one test tube was kept as control. The inoculated tubes were incubated at 37±1o C for 24-48 h (Aneja, 2001). After incubation, tubes were observed for the colour change in medium due to the production of acid and gas production.
Then, the funnel was fixed on a cylinder and 100 ml water was added to soil in the funnel slowly. Watch glass dish was used to cover the upper surface of the funnel to avoid losing any water by evaporation. The start time of each sample was recorded to calculate the amount of collect water in the cylinder after one hour. The retained water in the soil was calculated simply (100 – the collected water in the cylinder) with considering the amount of water in the cotton or ply tissue. Water content was estimated in additional samples (100 gm) by weighting the soil samples (W1) and afterwards drying them at 105 °C (W2).
A few of parasites were flattened on a clean slide under the slight pressure of a cover glass & fixed in Alcoholic Bouin,s fluid for 12 hours. Stains like Gover,s Carmine, Mayer’s Para carmine & Haemalum were used for the preparation of whole mounts for identification & the study of reproductive organs. The smears were prepared from the living material by puncturing the ovary with two fine steel needles under the stereoscopic binocular microscope. The semi fluid contents were allowed to spread over the clean slide & then the slide is inverted over the fixative without losing much time. After fixation with sublimate acetic the material is washed for 20-24 hours in running water.
Antibacterial assay The screening was done by disc diffusion method. The extracts were tested against Escherichia coli. A loopful of the pure bacterial culture was suspended in nutrient broth and incubated for 24 hours. Nutrient agar media was sterilized and poured into plates. After solidification, 0.1ml of the inoculum was spread over the agar evenly using L rod.
4.1 Extraction and Fractionation To determine the antimicrobial activity of Moringa oleifera , the finely grinded leave part of 500g was extracted using solvent extraction resulting in181.506g . Two different organic solvents were used in decreasing order of polarity to extract the crude fraction. 80g of dried crude is dissolved back in methanol for further extraction. The initial weights of petri plates before occupied with the fractions of extract and after the fractions completely dried were measured repeatedly. The mean value is recorded and used for further calculation.