Electron Microscopy Centre

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1 Electron Microscopy Centre Title: STANDARD PROCEDURES TO USE THE CRITICAL POINT DRYER Equipment: BIO-RAD E3000 Critical Point Dryer Revision: 1.0 Effective Date: 01/10/2006 Author: X. Yang 1

2 Warning! Before you attempt to operate this equipment for the first time, please make sure you are aware of the precautions that you must take to ensure your own safety. Warning! This apparatus is a PRESSURE VESSEL and operates at pressures in the range of 800 to 2000 psi (54 to 136 bar) WHICH IS POTENTIALLY DANGEROUS IF HANDLED CARELESSLY. Warning! Operation of the instrument involves Carbon Dioxide (CO 2 ), which is deemed to be lethal if sufficient amount of gas has been inhaled. Please refer to MSDS information of CO 2 previous to the operation. 2

3 Standard Procedures to Use the Critical Point Dryer Understand the CPD Valves (Figure 1): 1. Inlet Valve 2. Vent Valve 3. Drain Valve Checkouts before a Drying run: 1. Check the cold/hot water line is connected to the apparatus with no visible leaks. 2. Check the CO 2 high pressure cylinder is connected through a gas line to the inlet valve of the CPD. 3. Make sure the door of the lab is wide opened and the exhaust fans are turned on. 4. Make sure the tissues required to be dried have been washed, fixed, and dehydrated in either ethanol or acetone to 100% according to proper drying protocol previous to CPD. 5. Make sure the CO 2 inlet valve on the CPD apparatus is closed. Procedures for critical point drying of tissues: 1. Ensure the apparatus is correctly installed and run cold water to cool the chamber to 20 C or lower. 2. Place tissue in metal baskets, or plastic containers, taking care to prevent the tissue samples from drying. The tissue should be in the solvent all the time. 3. Fill the transfer boat with enough of the dehydrating agent to almost cover the samples and then place the baskets/containers in the boat. Hints: in order to prevent the tissue from air drying during the transferring, you could also fill the liquid transfer boat with dehydrating agent, put the metal baskets of plastic container in the boat then transfer the tissue to the baskets. The dehydrating agent (100% alcohol or acetone) are highly evaporative so that you may have to keep filling the boat with dehydrating agent during the transferring if you have more than one tissue required to be dried in a run. 4. Open the back door of the CPD apparatus, load the transfer boat into the chamber and then screw the door back on. Make sure the door is tight. 3

4 5. Turn on the CO 2 cylinder valve and open the CO 2 inlet valve on the CPD apparatus. At the same time, slightly open the vent valve to avoid back-pressure and allow a quick fill. Note: Filling with liquid CO 2 gas should be rapid. The picture on the right demonstrates how to proper handle the three valves. Gloves should be worn all the time. Please refer to a separate manual for detailed high pressure gas cylinder operation information. Note: The valves seal by contact between a ground steel cone and a brass knife edge; this metal to metal contact should not be over tightened as this will impair the efficiency of the valve. 6. Leave the inlet valve fully open with the vent valve slightly open to maintain liquid level, and then open the drain valve at the bottom to remove most of the dehydrating fluid. Allow the chamber to flush for about 30 sec, and then close both the vent and drain valves. Leave the inlet valve open to maintain liquid CO 2 level. 7. Flush the apparatus as outlined above every half-hour for 30 sec or so, to allow specimens to infiltrate with CO 2 and remove dehydrating fluid. Large specimens will require about hr. total time, while small specimens will be done in about 1.5 hr. Remember to leave the room door wide open to dissipate the CO 2 gas that is being vented. 8. After flushing the bulk of the substitution liquid, close the inlet valve and lower the liquid level to just below the top of the boat by venting off excess gas. Note: It is very important to have to liquid level just above the top of the boat because it provides the best view of the liquid level and the disappearance of the liquid/gas boundary line would be a good indicator of CO 2 liquid to gas transformation. Note: For the safety issue, do not observe the liquid level by direct watching the viewing window, watch the reflection of the chamber in the mirror provided. 9. Run hot water through the water jacket and monitor the temperature and pressure, with all valves closed. 4

5 10. When the temperature reaches about 36 to 38 C, and the pressure rises to1200 lbs/sq. in. or slightly above, the liquid/gas boundary line will disappear and the specimens are above the critical point. 11. Once the critical point has been reached, vent the gas off slowly (over a 5 min. period) to avoid condensation. The temperature should remain around 40 C during this time. Note: Condensation can be caused by rapid venting or by the presence of acetone or amyl acetate. 12. Shut off the water, open the door, remove the boat and sniff it to check for residual dehydrating solvent. 13. After taking out the specimens, replace the boat and containers in the drawer, seal the CPD apparatus again, and turn off the CO 2 cylinder. 14. Record the usage of Critical Point Dryer on the log book. Reference: 1. Instruction Manual for E3000 Series Critical Point Drying Apparatus. BIO-RAD. 5