PRODUCTION Of NUTRIENT SOURCES FOR RHIZOBIUM

containing 7.6 g of phenol and 8.3 g of sodium metabisulfite. The two solution were mixed and kept in
brown bottles.
7. Nitrite test reagents
Reagent I Dissolve 8 g of sulfanilic acid in either 1 liter of 5 N acetic acid
(1 part glacial acetic acid to 2.5 parts of water) or 1 liter of
dilute sulfuric acid (1 part concentrated acid to 20 parts of
water).
Reagent II Dissolved 5 g of α-naphthylamine in 1 liter of either 5 N acetic
acid or of diluted sulfuric acid (1:20), or dissolve 6 ml of
dimethyl-α-napthylamine in 1 liter of 5 N acetic acid.
� 8. Lowry’s Procedure for quantitation of proteins
�Reagents
�A 2% sodium carbonate in 0.1 N NaOH (sodium carbonate 21.2 g/L; 10 ml/L
NaOH 10 N)
�B 0.5% CuSO 4.5H 2O in 1% sodium citrate (sodium citrate in stead of tartrate,
modified by Leggeti-Bailey, 1967)
�C 1 N Folin phenol reagent (Folin phenol comes in 2 N and should be diluted
immediately before use)
�D 1 ml Reagent B+ 50 ml Reagent A (or similar ratio) (make up immediately
before use, good for one day) Standard, Bovine serum albumin at
concentration of 1 mg/ml sample volume total of 100 µl.
�Procedure
1)� Add 1 ml Reagent D to each of the standards and unknown tubes. Vortex immediately.
Incubate precisely 10 min at room temperature.
2)� Add 0.1 ml Reagent C (dilute 1:1 with deionized water). Vortex immediately. Incubate
30 min . (sample incubated longer than 60 min should be discarded.).
4)� Read absorbance at 750 nm.
5)� Plot standard curve and calculate the unknown.
�Standard : BSA standard at concentration of the 1000 µg/ml, 700 µg/ml, 500 µg/ml,