Biochemical Techniques and Instrumentation

Procedure

1. Weigh 20 g of fish, homogenize and mix well with 50 ml of distilled water
2. Add 50 ml of 5% TCA solution and mix well
3. Centrifuge at 2500 rpm for 10 min and filter the supernatant
4. Pipette out 0.5 and 1.0 ml of supernatant into different test tubes
5. Similarly, pipette out 0.2, 0.4, 0.6, 0.8 and 1.0 ml of glucose working standard solution into different test tubes and adjust the volume to 1 ml using distilled water.
6. Pipette 1.0 ml of distilled water into a separate tube and this serves as ‘blank’ .
7. Add 10 ml of anthrone reagent to each tube and place the test tubes in a boiling water bath for 10-15 min.
8. Cool the tubes at room temperature for 30 min.
9. Determine the optical density at 620 nm in a Spectrophotometer
10. Draw a standard graph by plotting concentration of the standard on the X-axis versus absorbance on the Y-axis
11. From the graph, calculate the amount of carbohydrate present in the sample