Blood Glucose Test_ Colorimetric Method
Blood Glucose Test_ Colorimetric Method


Glucose is the primary and major source of energy in the human body. It is derived from the breakdown of carbohydrates in the diet and in the body stored glycogen. Insulin , secretion  by islet cells in the pancreas of the human body , facilitates glucose entry  into the cells. A deficiency of insulin or a decrease in its effectiveness increases blood glucose . Mainly elevated serum glucose concentration is found in diabetes mellitus . Therefore , there are various method to determine blood glucose level to maintain the glucose level in  blood within the accepted range.

• Principle


The Glucose Oxidase enzymatic method is used commonly for the determination of blood glucose level. Glucose in the serum is oxidized by the enzyme glucose oxidase to gluconic acid with the liberation of hydrogen peroxide. The hydrogen peroxide is converted to water and molecular oxygen by the enzyme peroxidase. In the presence of an oxygen accepter or 4 – aminophenazone together with phenol , a pink colour (Quinoneimine ) is formed which is measured . The intensity of the pink colour formed is proportional to the glucose concentration in serum.

Blood Glucose Test_Principle

• Specimen

Serum must be separated from the red cells promptly to prevent glycolysis. The addition of Sodium fluoride / Potassium oxalate as an inhibitor of glycolysis and anticoagulant to the blood sample.

• Requirments

  • Glucose reagent
  • Demineralized water
  • Glucose standard
  • Test tubes
  • Graduated pipettes
  • Micropipettes
  • Spectrophotometer

• Procedure


Test tubes are labeled as Blank , Standard , and Test. Glucose reagent and Glucose standard

are warmed to room temperature before use. Then 3 ml glucose reagent is pipetted into all three tubes.30 µl  Demineralized water is pipetted into Blank , 30 µl Glucose standard is pipetted into Standard tube and 30 µl sample is pipetted into Test.

Blood Glucose measurement Test

All three tubes are mixed well and incubated at 37 0C for 10 minutes or at room temperature for 20 minutes. All tubes are shaken two or three times during the period to ensure adequate reaction. Absorbance is read at 510 nm against reagent blank within 15 minutes using spectrophotometer. The test is performed in triplicate to generate accurate results.The results are calculated using standard.

• Calculation




Glucose concentration = Absorbance of the Assay / Absorbance of the Standard X Concentration of the Standard

• Reference range

Glucose level in serum ( fasting )  –  < 100 mg/dL

• Limitation

  • Stability of reagents is protected if contaminations are prevented during use and reagents are stored tightly closed and protected from light after use.
  • As this is an enzymatic reaction , the factors that control enzymatic reactions should be kept under control  pH ,temperature, the period of incubation )
  • High serum bilirubin level may interfere with the glucose value of the assay.
  • Pipetting errors may interfere with the colour development of the reaction

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