Define Procedure for Determination of Blood Glucose by Nelson Somogyi Method?
1. Label a series of Folins tubes with blank (B), standard (S1-S5) and sample (SA 1-2) and place them in a rack.
2. Preparation of Standard Glucose Solution: Weigh 50 mg of glucose and dissolve it in distilled water and make up the volume to 500 ml. This is your standard glucose solution.
3. Colour Development for standard solution: Take six different known concentrations of standard glucose solution (0.2, 0.4, 0.6, 0.8, and 1.0) in the first six Folins tubes to prepare a standard glucose curve. Add 1 ml of alkaline copper reagent. Shake well. Heat in boiling water bath, cool and add 1 ml of arsenomolybdate reagent.
4. Preparation of blood sample
1) Collect 3 ml blood and add a few crystals of sodium flouride.
2) Take 4.0 ml of ZnSO4 in a centrifugal tube.
3) Transfer 1 ml of the blood into the tube, and then add 4.0 ml of Barium hydroxide. Mix.
4) Centrifuge at 2500 rpm for 10 min. Check if the superhatant is clear.
5) Transfer the supernatant into another test tube.
6) Take 1.5 ml of this into a Follins tube.
7) Add 0.5 ml of distilled water & 2 ml of alkaline copper reagent.
8) Mix & put the tube in a boiling water bath for 20 min.
9) Cool tube immediately by cooling under tap water & then putting it in the cold water.
10) Add 2 ml of arsenomolybdate reagent molybdate reaput
11) Make up the volume to the 25 ml with distilled water.
12) Read the tubes at 540 nm. Calculate the amount of glucose from the standard curve.
13) Take the tubes in duplicate if you have enough blood samples.
5. For preparation of blank (B): 2ml of distilled water and 2 ml of alkaline copper reagent will be added to the tube B.
6. Determination of glucose content: Estimate the content of glucose in the blood sample from the experimental values and by using the standard curve.