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Macak-macak 0 cm (roots into the soil) Filled with water 2 inches above the ground (touch ground) Filled

with water 5 cm above the soil surface (hanging) Stagnant water 7 cm above the soil surface (hanging) c. enter Azolla into each glass and observe the growth every week

3) Isolation of non-symbiotic N


a. enter 10 g soil into 90 ml physiological saline, then shake until homogeneous (dilution 10-1)

b. take 1 ml solution of 10-1, put in 9 ml of physiological saline, then shake until homogeneous c. Do the same (dilution up to 10-5 10-2). dilution.

d. Isolation of fastening N bacteria using media YEMA, in the following way: e. To anchor bacteria N: Take a 0.1 ml solution of 10-2 and pour it into a

medium YEMA and blend solution into the whole medium using sterile dryglassky. Do the same until dillution of 10-5. b. These isolates incubation at room temperature, with a petri dish upside down position, for 4 -12 days. c. Count the number of colonies growing using a hand colony counter.
4) Calculation of oxidizer ammonium microbial and nitrate in the soil: a. Considering the soil as much as 10 grams with wax paper to keep the bacterial contamination from the outer side, turn on the Bunsen while considering the soil b. entering the soil sample into a 250 ml bottle containing 90 ml of dispersing solution and shakeing for 30 minutes at 50 rpm on a mechanical shaker c. entering the soil sample into 1 ml of soil suspension and make up to 10-6 dilution d. Inoculate ammonium and nitrite oxidizer cultures to ammonium and nitrit oxidizer media (at reaction tube ) with 0.1 ml of each dilution series e. Close the tube and inoculate for 4-5 weeks f. Observed changes in the tubes of each series B. Biota that affect the availability of nitrogen functional 1) Propagation, observation of spores and mycorrhizal infection a) Propagation of mycorrhizal (pot culture) a. Prepare media for pot culture there are zeolite and soil (Alfisols and Vertisols) with a ratio of 3:1 in the polybag. b. watering the Media and made a hole with 2 cm width and in 3 cm depth. plant the VAM starter (50-

100 spores spores / polybag) into the hole. c. then plant maize in the hole, (each hole 2 seed). d. look after the culture ,water it and give solution of Johnson as directed (table 3.1) e. Perform stressing according to treatment (treatment 1 is stresing performed at 1-month-old plants, treatment 2: is stresing performed at 3-month-old plants) f. Harvest Treatment of pot culture Vertisols (T1) Alfisols (T2) Stresing 1 T1S1 T2S1 Stresing 2 T1S2 T2S2 b) Separation of spores from the soil a. Mix the soil from the pot culture (100 grams) with water (250 ml) in a glass cup, then stir well. b. Pour the liquid (decanted) through a coarse sieve 60 micron and 90 micron (60 micron filter is above 90 micron filter) to separate the coarse particles. Collect the fluid that passing through the first filter. c. sieve the first filter results for the last time with the finest sieve (250 microns). Move substrat on the filter to reaction tube. d. Add a solution of glucose (sugar) and water with 1:2 (50gr: 100ml) ratio and then centrifuged for 5 minutes. e. Pour the clear solution of sentriguge result to the 250 micron sieve, sieve and upside down the sieve then water it with destillated water until all substrate deposited in petridish. f. Observe the substrate in a petri dish under a binocular microscope and observe the mycorrhizal spores. c) root Painting a. wash the roots of corn plants from pot culture (especially the fine roots or root hairs). Then soak in a 50% of alcohol b. Take the root that are stored on alkohol and wash thoroughly, then cut to a size of 1 cm c. Heat water in a pan (waterbath) d. prepare KOH suspension (20 ml), then enter the root was cut into a KOH solution and heated in a pan with 900C temperature for 5-10 minutes depending on thickness of the root or until the roots wilted e. Wash the roots using distilled water f. Soak the roots in 1 N HCl until the root look white g. Wash the roots again using distilled water h. Place the roots in a petridish and add the blue paint tryplan 0.05 then let stand for 1 hour to paint blue tryplan into the cell or reheat % 5 minutes i. observed Mycorrhizal infection under a microscope ( 5 pieces of roots) 2) Isolation of bacterial phosphate solvent a. Preparing the soil from plant roots affluent, setting up 125 or 259 ml erlenmeyer that consist of 90 ml of sterile physiological solution and then mixing with 10 g of soil into the erlenmeyer until homogeneous b. Dilute the suspension until 10-4 dilution

c. Inoculate dilution in petridish d. observed the colonies that formed after 1-2 days