13.3 Specimen Requirements
Plasma is the preferred sample for glucose estimation. Separate plasma from cells
rapidly to avoid glucose loss. It is stable for 8 h at 25C and 72 h at 4C. Preserve
plasma with sodiumfluoride. Whole blood may also be used, but whole blood levels
are 90% of total plasma glucose.
13.4 Reagents........................................
1.Phosphate buffer: Prepare 0.1 M potassium phosphate buffer of pH 7.0.
2.Enzyme reagent: Dissolve 18 mg of 4-aminoantipyrine and 36 mg solid phenol
in 100 ml of 0.1 M phosphate buffer. Add GOD (1500 U) and POD (100 U). Store
in brown bottle at 4C.
3.Stock glucose solution (100 mg/dl): Dissolve 100 mg glucose in 100 ml distilled
water.
4.Working glucose standard: Prepare glucose standards of concentration 25, 50,
75, and 100 mg/dl.
15.5 Procedure
- Take a set of test tubes in duplicates and label as B, S 1 ,S 2 ,S 3 ,S 4 ,S 5 , and T.
- Add 1.8 ml phosphate buffer and 1 ml enzyme reagent to all the test tubes. In
blank add 1.9 ml phosphate buffer and 1 ml enzyme reagent. - Add 0.1 ml of standard of concentration 10, 25, 50, 75, and 100 mg/dl to the tubes
labelled as S1,S2,S3,S 4 , and S 5 , respectively. Add 0.1 ml of serum sample in
tube T. - Mixed the content of the tubes thoroughly and place them at room temperature for
15 min.
Reagents Blank S 1 S 2 S 3 S 4 S 5 T
Phosphate buffer (ml) 1.9 1.8 1.8 1.8 1.8 1.8 1.8
Enzyme reagent 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Glucose standard (ml) – 0.1 0.1 0.1 0.1 0.1 –
Glucose concentration (mg/dl) – 10 25 50 75 100 –
Glucose amount (mg) 0.01 0.25 0.5 0.75 1.0
Serum/plasma (ml) ––––––0.1
Mix well and keep at room temperature for 15 min and take OD at 520 nm58 13 Estimation of Blood Glucose Levels by Glucose Oxidase Method