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Estimation of blood glucose by Glucose oxidase method
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Materials Required:

 
  •  Collection of blood sample:

 

 

About 2ml of patient’s blood should be collected by venipuncture into a tube containing a mixture of ethylenediaminetetraacetic acid and sodium fluoride in the ratio of 1:2 (W/W). Five mg of the mixture is adequate for 2ml of blood. The tube should be thoroughly shaken for complete mixing.

 
  •  Preparation of anticoagulant mixture:

 

 

100mg of EDTA and 20mg of sodium fluoride should be mixed and ground into a fine powder using a blender. This should preferably do in a fume hood. The mixture should be stored in a clean container.

 
 

Reagents:

 

 

  1. 2N Sodium hydroxide (NaOH) - 8g of NaOH is dissolve and finally make up the volume to 100ml with distilled water.

  2. Sodium Sulphate- Zinc sulphate reagent – Dilute 55ml of the zinc sulphate solution (10g/100ml ZnSO4.7H2O) to 1 litre with the sodium sulphate solution (93mmol/liter).   

  3. Phosphate buffer 0.05M  pH-7.2

  4. Glucose oxidase reagent:  Prepare this reagent fresh by dissolving 25mg of glucose oxidase and 1% ortho - toluidine in the sodium phosphate buffer. Add a small quantity of peroxidase (2mg) and makeup to 250ml with the buffer. This solution is active for about 4 weeks if stored in a brown colored bottle at 4°C.

 

 

Procedure:

 
 
  • Preparation of Test: Pipette 0.1ml of blood into 1.8ml of sodium sulphate-zinc sulphate reagent in a centrifuge tube. Add 0.1ml of 2N Sodium hydroxide, centrifuge at 3000rpm for 5 minutes and take 0.5ml of supernatant in duplicate.

 

  •  Preparation of Blank: Take 0.5ml of distilled water.

 

  • Preparation of Standard: Prepare standard concentration of glucose (200mg/dl), use 0.5ml of a range of glucose solutions (50mg/dl, 100mg/dl, 150mg/dl and 200mg/dl) suitably diluted from standard.

 

 

                 I.    50mg/dl - 125µl glucose standard + 375µl distilled water

 

                 II.  100mg/dl - 250µl glucose standard + 250µl distilled water

 

                 III. 150mg/dl - 375µl glucose standard + 125µl distilled water

 

                 IV.  200mg/dl - 500µl glucose standard 

 

  • Add 5ml of the glucose oxidase reagent incubate for 1h at 37°C and read the extinction at 540nm against the reagent blank.

 

 

  • If the absorbance reading of the sample is too high, dilute the supernatant which was obtained earlier, 2x with distilled water and repeat the subsequent step.

 

 

 

 

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