Then the blower was turned on for sufficient duration and UV lights were switched on for one hour. LAF microbial contamination test (Settling plate method) A total of three petri dishes were prepared aseptically inside a laminar air flow (LAF), and then the petri dish was filled by pouring sterile Tryptone Soy Agar (TSA) liquid media and allowed to solidify. One test media was placed in the LAF cabinet, one test media is placed beside the LAF cabinet while the other the test media was placed near the door. The three petri dishes lid were opened and allowed to stand for 15 minutes and closed again. Then, the test media is then incubated at 37 ° C, for 18-24 hours.
On the second day of incubation, the plate was removed from the incubator and placed over a hot plate heating Iodine solids. The smoke of the Iodine stained the plate to display the presence or absence of a halo around the bacteria 2.12 Lipid Hydrolysis This test was done by making a single line streak inoculation on a tributyrin agar plate and allowing incubation. After the incubation period, the plate was observed for the presence or absence of a halo around the bacteria. 2.13 Gelatin Liquefaction A gelatin deep was deep stabbed and incubated. After incubation the tubes were placed in 4ºC for 30 minutes.
The colonies from serial dilution eight yielded (1x10-9) 78 CFU. The E. coli and Serratia colonies after the mixture showed yellow and variations of red in the colonies. They were too numerous to count. The smell was repugnant and there was some condensation on the plate top. Serratia and E. coli plated at a concentration of 1x10-3 (Figure 6 and 7) from the original mixture yielded scattered colonies on the media.
This organism was cultured under solid-state fermentation for 72 h using wheat bran as the substrate. After 72 h, crude enzyme was extracted from the culture medium. The fibrinolytic enzyme was purified from the crude sample by various steps: ammonium sulfate precipitation, dialysis, ion exchange chromatography, and casein-agarose affinity chromatography. All purification steps were performed at 4°C until otherwise stated. The crude enzyme was precipitated at 70% saturation of ammonium sulfate, and the protein was collected by centrifugation (10,000×g for 10 min).
Straight afterwards, saline solution was added (NaCl 0.16 M, pH=7) to the 10ml of the yolk solution with a Pasteur pipette, avoiding the sample diffusion, forming two phases and filling the tube completely. The ultracentrifugation was carried out during 19.5 h at 4ºC and 45000 rpm. (244.500 x g) in a Kontron Centrikon T-2190 ultracentrifuge in a TFT 50.38 rotor,
Placed the four test tubes into foam microtube holder and placed in water bath to incubate at 37 degrees Celsius for 45 minutes. Ms. Lovrien added 5 L of 10x loading solution to tubes labeled 1-4. Placed samples in the refrigerator overnight. After returning to class the next day, obtained a gel tray that contained 0.8% agarose with TAE buffer. Removed that comb and tape.
H and S were diluted 20x and P1 and P2 were diluted 2x to make up a total volume of 1ml each. 50ul of each diluted sample was pipetted into 8 wells of the microplate and 50ul of each protein standard was pipetted into 2 wells. 50ul were incubated with 50ul of alkaline copper reagent. 50ul of alkaline copper reagent was added to every well containing water, buffer, sample or standard and was incubated for 30 minutes. 100ul of Folin reagent was added to the wells and incubated for another 20 minutes.
The petri dish was incubated for 24Hrs at 35oC. The petri dish was observed to see if there was any bacterial growth and once the whole dish was full of bacterial colonies, it was observed under an UV lamp to see if there was fluorescence or not. I decided not to do the IMViC tests because the first two tests concluded on what the unknown bacteria
2 ml, 3 ml, 4 ml, and 5 ml solution was withdrawn and same procedure was repeated as above. Solutions were diluted to get concentration of 10 μg/ml, 20 μg/ml, 30 μg/ml, 40 μg/ml and 50 μg/ml neomycin sulphate respectively and scanned under the UV-VIS spectra were recorded between 400-800 nm. Absorbance was measured at 566nm. Similar procedure was used for the measurement of the drug content.