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1. Remove bung & tubing from bell jar and stand in small bottle – DO NOT splash liquid onto base as it will contain traces of activity.

2. Fill bell jar with 1 L of tap water, add 2–3 crystals of potassium permanganate and swirl jar until dispersed.

3. Place bell jar back into tray and replace bung & tubing.

4. Switch on peristaltic pump making sure that the brass injection ports in the tubing are on low pressure side of pump – leave running to ensure thorough mixing.

5. Prepare 10 plastic scintillation vials by adding 10 cm3 of scintillation fluid to each.

6. Label 1 bottle as background, 3 as fluid activity (A), 3 as injection activity (B) and 3 as dilute activity (C).

7. Use a 5 cm3 syringe to withdraw ~5 cm3 of liquid from the furthest port on the tube from the pump and transfer it into an 11th plastic scintillation vial.

8. Use an automatic pipette and disposable tip to transfer 100 μL of this fluid into each of the 3 vials labelled A.

9. Add 500 μl of tritiated water (3HHO) to remaining 4.7 cm3 of liquid in you 11th vial.

10. Place fresh tip on the automatic pipette and transfer 100 μL of the labelled fluid put 100 μL into the 3 vials labelled B.

11. Use a 5 cm3 syringe fitted with a hypodermic needle to take up 4 cm3 of the tritiated fluid from the 11th vial and slowly inject this through the port nearest to the pump – recap the vial to avoid spillage.

12. DO NOT remove the syringe from the port – wait 1 minute then rinse the syringe by refilling it and then re-injecting this fluid back through the port.

13. Place the syringe in the yellow sharps bin provided.

14. Wait 30 minutes to ensure that the isotope is thoroughly mixed through the circulation apparatus.

15. Meanwhile use the liquid scintillation counter to measure the activity in the background vial, the 3 x A vials (fluid activity) and the 3 x B vials (injection activity) using protocol 1.

16. After 30 minutes use a 1 cm3 syringe to withdraw ~1 cm3 of fluid from the port furthest from the pump and transfer to the small glass beaker.

17. Accurately measure 100 uL of this diluted activity to the 3 vials labelled C (diluted activity).

18. Measure the activity on the scintillation counter as before.

19. Calculate the volume of the fluid in the circulation system (V2) from:-

A1V1 = A2V2

A1 = activity before injection (B)
A2 = activity after sampling (C)
V1 = volume injected into system