We inoculated them with our stain and incubated them on shaker for 24 hours. After 24 hours incubation, we took O.D at 600nm and plotted a graph. To check effect of different temperatures on rhizospheric nitrogen bacterial growth: We took 6 flasks (100ml), and added 20ml L-broth in each flask and autoclaved it. We inoculated them with our stain. We selected different temperatures 4°C, 15°C, 37°C.
Buffer: Sorensen’s buffer solution. The ph must be at 6.8 for the May-Grunwald. Fig.1.4 May-Grunwald-Giemsa Stain http://i.ytimg.com/vi/GHBqhSXT-6I/maxresdefault.jpg Method Step 1: Fix for at least 30 seconds in absolute methanol. Step 2: Remove methanol by tilting the slide or by simply removing from the fixing jar. Step 3: Apply staining solution I freshly diluted with an equal part of buffer for 5 minutes on a horizontally positioned slide or in a jar.
The mixtures was filtered with Whatman #1 filter paper and the filtrate was used to repeat the extraction for another two times. Then, the methanol solvent in mixtures was removed using the Rotary Evaporator (EYELA, N1100) at 140° hPa; 60°C; speed 5. The distilled water and excessive methanol were removed with Freeze Dryer (GENEVAC LTO, EZ 2.3 ELITE) for a week. The crude obtained were weighed and stored at -20°C until use. 1.2 Partitioning method
1- Extraction method No. 1: Fifty grams of powdered aerial parts of portulaca oleracea were hydrolyzed by using reflux for 9 hr. with 300 ml of 2N hydrochloric acid then the extract cool at room temperature ,filter and wash the residue with 2N of ammonia solution. The residue dried overnight at 60ºC ,the final step involve the extraction of the residue with 250 ml of chloroform by using soxhlet ,the final extract cool at room temperature ,then filter and evaporate to dryness by using rotatory evaporator at 40ºC to yield (2.264 gm),as show in scheme (2-1). 184.108.40.206-Extraction method No.2: Fifty grams of powdered aerial parts of portulaca oleracea extracted by soxhlet with 500ml 0f 70% ethanol for 8 hr.
A single colony from each culture was carefully picked up with sterilized loop. Each colony was emulisified in a drop of sterilized distilled water placed on a glass slide, then mixed thoroughly to make a thin smear. The smear was air-dried, fixed by a flame, and then stained with crystal violet solution for 1-2 min. Subsequently, flooded with iodine for 1 min. and rinsed with alcohol 95% followed with tap water.
Punch disks of filter paper using a paper punch 2. Add the various amounts of medication solutions as shown in table 1.2 below to each filter disk using a micropipette 3. Place filter disks in a sealed container and leave for 24 hours Suspension of bacteria 1. Unseal the petri dishes 2. Add 1000 microliter of 0.8% of saline solution to the centre of the first petri dish using a micropipette 3.
Ethancridine lactate added to the serum or plasma to give a last convergance of 5.2gm every liter. Pretty nearly 90% of the included sum was disposed of with the encouraged proteins, while the rest of which guarantees the complete precipitation of undesirable protein, uprooted by assimilation on charcoal. To sum things up, 1.2% ethancridine lactate arrangement was added to the serum or plasma and the mixture was mixed for 30-40 minutes at 200c and after that the mixture was kept undisturbed for 1-2 hours. After precisely tapping the supernatant the hasten was centrifuged at 5600 g for 30 minutes and the supernatant was pooled. To the supernatant charcoal at the rate of 6g/liter was added to ingest the broke up ethancridine lactate.
Fraction I was discarded due to the presence of high fatty substances, whereas fraction II was analysed for the free flavonoids in each of the samples. Fraction III of each of the test samples was dried and hydrolysed by refluxing with 7% H2SO4 (10 ml/gm residue) for 5 hours on water bath. The mixture was filtered and the filtrate extracted with ethyl acetate in a separating funnel. The ethyl acetate layer was washed with distilled water till neutrality and dried in vacuum. The residues were taken up in small volumes of ethanol separately and then subjected to various tests for
After 8 days incubation, 5ml sterile distilled water was added on to the agar using aseptic techniques. The top of the agar was scrapped with a sterile hockey stick glass rod so that the spores was suspended into the solution. The concentration of the spore was determined by using hemocytometer. A proper dilution was carried out to get a spore concentration of 2 x 106. Each isolates was inoculated into 50ml cellulase enzyme production broth medium with inoculum size of 2 x 106 spore concentration.
Yeast Growth in YPD Agar: To prepare YPD agar, mentioned in Table 1 nutrient ingredients in given concentration were weighed and added to 200 ml of distilled water. The mixture was autoclaved (SMS ASL80 MSV) for 1.5 hours at 121°C. On sterile plates, 25-30 ml of the mixture was poured and left to cool down. The yeast cells were then streaked on the agar plates and the cultures were grown in a stationary incubator (S1-600R, Lab Companion) for 72 hours at 30°C. Yeast Growth in YPD: To prepare YPD liquid medium, glucose, peptone and yeast extract were weighed and added to 200 ml of distilled water.