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 incubation mixture contained 2.5 ml of 1.2% (w/v) fibrin, 2.5 ml of 100 mM Tris–HCl buffer, 10 mM CaCl2 (pH 7.8), and 20 µg of enzyme. The incubation was carried out at 37°C for 30 min, and the reaction was stopped by adding 5 ml of 110 mM trichloroacetic acid containing 220 mM sodium acetate and 330 mM acetic acid. This reaction mixture was centrifuged at 3,000×g for 5 min, and the absorbance of the trichloroacetic acid (50 mM) soluble product was determined at 275 nm. One unit of fibrinolytic enzyme activity was defined as the amount of enzyme required to liberate 1 µg of L-tyrosine per minute at 37°C. The total protein determination was performed as described by Lowry et al.
One that fluoresced green under UV light, and one that did not fluoresce green under UV light. The third colony was taken from the LB/kanamycin plate, its ability to fluoresce is unknown. For each culture 1 mL of cell suspension was taken and added to a separate micro centrifuge tube. The cell suspensions were then centrifuged at 10,000 RCF for one minute to pellet the bacterial cells. The supernatant was then removed without disturbing the pellet.
All microorganism cultures were prepared from their respective slants. Cultures were grown over 24 hour at 37 °C and optical density was adjusted to 0.1, which corresponds to 108 CFU (colony forming unit)/ ml at 600 nm. About 100 µL bacteria were added to 100 ml of a nutrient broth solution to give bacteria concentration about 108 CFU/ ml. 4.2 Well diffusion method This method was employed to essay antimicrobial activity of pure PS nanofiber and Ag nano particle doped PS composite nanofibers. The bacterial suspension (100 µL 108 CFU) was spread uniformly on the nutrient agar plate and 50 µL solution of each of PS and Ag/ PS prepared in DMSO was loaded in each well on nutrient agar plate The plate was then put in incubator for 24 hour at 37 °C to record inhibition zone .
The selected streptomyces culture was inoculated into the broth. The tubes were incubated 4°C, 10°C, 20°C, 28°C, 37°C, 45°C, and 60°C for 8-10 days. The growth of the inoculated cultures was recorded after incubation. Effect of NaCl (Tresner et al., 1968) Yeast extract-malt extract agar was supplemented with Nacl at different concentrations 4%,6%,8%,10%(W/V) and autoclaved.After autoclaving, the medium wsa poured into sterilized petriplates.A loop full of spore suspensipn of each selected streptomyces species was streaked on the solidified agar plates divided into five sectors and incubated for 8-10 days. After the incubation period, the sodium chloride tolerance of the selected streptomyces was determined.
The swab was inoculated onto blood agar, MacConkey’s agar and nutrient agar, mannitol salt agar and Sabouraud's dextrose agar and was incubated at 370C for 24 hrs for bacterial culture and at room temperature for fungal isolates. Next day the colonies were picked up and preliminary identification was done and the bacterial isolates were identified based on standard protocols. LPCB was done for the identification of fungal isolates after 48 to 72 hrs. ANTIBIOTIC SUSCEPTABILITY TEST Antibiotic sensitivity test of the bacterial isolates was determined by the Kirby-Bauer disc diffusion method2. The following were the antibiotics used for the study-amikacin (AK 30), nalidic acid (NA 30), erythromycin (E 15), vancomycin (VA 30), tetracycline (TE 30), cefoxitin (CX 30), rifampicin (RIF 5) ciproflaxicine (Cip 5), ceftazidime (CAZ 30), cefotaxime (CTX 30), cepifime (Cpm 30), cefoperazone (CPZ 75).
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 media used in this experiment was Trypticase nitrate broth. The reagents used (A and B) were sulfanilic acid and alpha-naphthylamine (respectively). Using aseptic technique, the bacterium (16A and 16B) were inoculated into labeled broth test tubes. The tubes were incubated for 48 hours at 37 degrees Celsius. When the incubation was complete 5 drops of reagent A and 5 drops of reagent B were added to each of the broths.
TPR71H. From this study we found that the optimized parameters for the production of CGTase by Bacillus sp. TPR71H were Soluble Starch 3%, Yeast Extract 0.5%, K2HPO4 0.1%, Inoculum Level 3.5%, Inoculum Age 24h, Incubation Period 36h, rpm 220, Incubation Temperature 32°C and the pH of 7.5. M. Manoj Saravana Guru (2012) et. al.. Cyclodextrin glucosyltransferase (CGTase) producing alkalophilic bacteria were isolated from the water samples collected from Marneri pond of Tirunelveli, Tamil Nadu by serial dilution and plating method.
Lactobacillus lactis was inoculated in various media containing different vegetal source instead of beef extracts and was compared to the MRS culture medium as standard. These were incubated at 37oC for 72 hours wherein the growth was recorded every 24 hours using an undiluted, 10-4, 10-5 and 10-7 CFU/ml. The results of cultivation showed that the alternative test media using seed powders demonstrated better growth of colonies than the Man Rogosa Sharpe (MRS) cultre medium. Orange juice was used as an enrichment means for the enumeration of Alicyclobacillus acidoterrestis. In this process the inoculum was suspended in a medium containing orange juice and inoculated to different media such as K Agar, Yeast Starch Glucose Agar, Alicyclobacillus Agar, and Bacillus acidoterrestis Thermophilic Agar using duplicates for each run (Anjos et al, 2014).