Physiology of Finfish and Shellfish (2+1)

Procedure

Proedure

Deproteinize the known amount of tissue (I g) by grinding in 10% TeA. Transfer the extract in a centrifuge tube and make up the volume to 10 m1 with 10% TeA. Centrifuge the precipitate at3000 rpm and transfer 1 ml of the protein-free filtrate to a clean centrifuge tube graduated at 10 ml.

In a second similar tube and 5ml of standard lactic acid solution containing 0.01 mg of lactic acid per ml.In the third tube, place a little distilled water, which serves as a blank. To each tube add 1 ml of 20% copper sulphate solution and dilute to the 10 ml mark with distilled water. Add about 1 g of powdered calcium hydroxide to each tube,stopper and shake vigourously to mix the calcium hydroxide thoroughly. Allow to stand for 30 minutes, repeat the shaking at least once in between.Centrifuge the precipitate and transfer the supernatant from each tube to clean and dry test tubes. To each tube add 0.05ml of copper sulphate solution, followed by 6 ml of concentrated sulphuric acid. The sulphuric acid should be added drop by drop at first, mixing the contents of tubes well during the addition. After the acid has been added to all the tubes, place them upright in boiling water for 5 minutes, then transfer the tubes to cold water and cool to 20^cC or below. When the contents of the tubes are sufficiently cool, add 0.1 ml of the p-hydroxy diphenyl reagent. Disperse the reagent throughout the solution as quickly and uniformly by lateral shaking. Place the tubes in a beaker of water at 30°C and allow to stand for 30 minutes Finally place the tubes in vigorously boiling water fur exactly 90 seconds remove and cool in cold water to room temperature. Determine the optical density at 560 hm using water for setting the photometer at zero density.