G/LITRE 5.0 g KOH g 0.5 g 0.05 g 0.01 g MgS047H20 NaCl CaCl2

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1 A P P E N D IX -V III COMPOSITION OF USED MEDIA AND CHEMICAL REAGENTS 1. NITROGEN FREE BROMOTHYMOL BLUE (NFB) MEDIUM Dobereiner et al (1976) Same media was also used to check the effect of temperature and ph. Semi solid medium Malic acid 5.0 g KOH K2HPO4 FeS04 7H20 MnS04 7H g 0.5 g 0.05 g 0.01 g MgS047H20 NaCl CaCl2 Na2M o04 D/W g 0.02 g 0.01 g g 1000 ml Bromothymol blue 0.5% alcoholic solution Agar Agar ph adjusted to 6.6 to 7.0 For solid NFB medium Semi solid NFB medium Agar Yeast extract 2.0 ml 1.75 g 2 % % - :111: -

2 2. BMS AGAR (POTATO INFUSION AGAR MEDIUM) Components gms/litre Potatoes 200 g Mallic acid 2.5 KOH 2.0 Cane sugar 2.5 Biotine 2.1 Cook washed potatoes and filter through cotton. Prepare potassium malate by dissolving 2.5g malate in 50ml water adding 2 drops of Bromothymol blue (0.5% ethanol) and 2 g of KOH. Adjust ph to green colour. Add malate, sugar, biotine to potato filtrate and dilute to 1000ml. Solid media is obtained by adding 2% agaragar. 3. R. C. MEDIUM * Azospirillum cultures were isolated (Red, Scarlet colony) on the Roderquez Caceras (1982) Medium with the following composition. Components Gm/1. k2h po4 0.5 MgS NaCl 0.1 Yeast Extract 0.5 FeCl36H D. malic acid 5.0 KOH 4.8 Agar 20.0 ph (adjusted by 0.1NKOH) 7.0 Distilled water 1000ml Medium was autoclaved at 121 C for 15 minutes per litre, 15ml of 1:400 sterile aqueous solution of congo red was added aseptieally just before plating or use.

3 4. M EDIA USED FOR ACIDIFICATION OF PEPTONE BASED SUGAR MEDIUM Peptone M gs047h20 (NH4)2S04 FeCl3 6H20 M ns Bromothymolblue dissolved in KOH The medium made upto the Volume of 950ml adjusted to ph and sterlized by autoclaving at 15 lbs. After it has cooled, 50ml of 20% (w/v) solution of glucose is added aseptically. The development of yellow colour during incubation for 96 hours at 37 C indicates acidification. Slight growth in the form of a sediment can be detected by agitating the tubes. 5. OKON S MEDIUM : (Used for testing organic acid utilization) K 2H P g KH2P g M gs047h g NaCl g CaCl g FeCl g Na2M o04 2 H g Distilled water ml Adjust ph to 6.8 with NaOH and sterilized by autoclaving. Organic acids are added separately (0.5%).

4 6. KJELDHAL NESSELERIZATION METHOD : REAGENTS AND PROCEDURE The following reagents are used i) Acid digestion mixture : dissolve selenium oxide (Se02, 1 g) in distilled water (50ml) in a 100ml flask and add MAR grade concentrated sulphuric acid slowly to 100ml. ii) Nessler s Solution : Iodine (11.3g) is dissolved in water (10ml) containing 15g potassium iodide. To mercury (15g) in a glass bottle is added most of the iodine solution, the bottle is stoppered and the mixture shaken with cooling in a water bath until the supernatant liquid loses its yellow colour. The supernatant is then decanted into a flask (100ml) and a drop is tested for the presence of free iodine with 1% (w/v) starch solution. If no colour is obtained more of the iodine solution is added until a drop of the mixture gives a faint blue colour with starch. The solution is then diluted to 100ml and poured into 10% (w/v) sodium hydroxide solution (485ml). The turbid solution is filtered or the precipitate allowed to settle before use. Note that the alkaline solution should be stored in a hard glass bottle closed with a rubber stopper. iii) Standard nitrogen solution : dry Analar ammonium chloride (153mg) is dissolved in 100ml of distilled water in a Volumetric flask. 25ml of this solution and 10ml N sulphuric acid are diluted to 1000ml to provide a solution of which 1ml is equivalent to 10 fig nitrogen. Procedure Samples (0.5 ml) of a solution and blanks of distilled water are measured into Pyrex test tubes ( 6 in. X 5/8 in.) and acid catlyst solution (0.2 ml) added to each. The tubes are heated gently until water evaporates off and then more strongly so that the acid boils. After 4 h the tubes are removed from the heater and allowed to cool, to each is added distilled water (7 ml) and Neeler s reagent (3 ml) and after standing for exactly 5 min at room temperature the extinction values of the solutions are measured at 465 nm in a spectrophotometer set to give 100 % - : 114:-

5 transmission with the diluted blank digests. It is advisable to measure the extinction values of the diluted blank digests against distilled water to check that the values are reproducible. Duplicate Volumes of the standard ammonium chloride solution equivalent to 5, 10 and 20 jug of nitrogen are diluted to 7 ml with water, treated with Nesler s reagent (3 ml) and the extinction values of the series measured after 5 min against blanks of distilled water treated in a similar manner. The Beer-Lambert law is obeyed over the range 0-20 pg nitrogen in the sample. Calculation : Standard graph of absorption Vs ng of nitrogen fixed was drawn and test readings were referred to it.