General Cautions

• Liquid Nitrogen is a cryogenic liquid and can cause severe frostbite. Always be cautious when working with it.
• Liquid nitrogen can quickly displace oxygen in a room. Always work in a well-ventilated area with an oxygen sensor.
• If you are unsure of any procedures, consult with NCCAT staff.

SOP

1. This SOP is for working with small amounts of liquid nitrogen: filling microscope dewars, cryo-loading, sample storage. It does not cover plunge freezing.
2. For delicate work such as grid loading, cryo gloves can be cumbersome. They do not need to be worn but be careful not to freeze your fingers.
3. Eye protection such as goggles or glasses should be worn.
4. Excess liquid nitrogen should be poured back into a storage dewar. Please do not dump it on the floor.
5. For the Tecnai F20, when doing a cryo cycle, remove the dewar and place on the floor. If there is a large amount of nitrogen, empty much of it into 10L dewar. Small amounts of liquid nitrogen can be allowed to evaporate naturally from the dewar. Leave it right-side up, uncovered, on the floor beside the table.
6. For the T12 Biotwin, when doing a cryo cycle, remove the cryo dewar and place it uncovered on the table.
7. When loading cryo samples, place the Styrofoam bin underneath the goniometer in order to prevent spillage onto the microscope controls.
8. When finished with the loading station, the small amount of lN2 can be emptied into a Styrofoam bin. This can be left to warm up or emptied into a 10L dewar.
9. At the end of the day, if you are the last user, empty the 4L dewars into the 10L dewars, and leave the 4L dewars to warm up so they are dry for the next user.
10. The general rule is that 4L dewars contain “clean, dry” nitrogen (samples) and the 10L dewars contain “wet” nitrogen (cryo dewars for microscopes).

General Cautions

• Liquid Nitrogen is a cryogenic liquid and can cause severe frostbite. Always be cautious when working with it.
• Liquid nitrogen can quickly displace oxygen in a room. Always work in a well-ventilated area with an oxygen sensor.
• If you are unsure of any procedures, consult with NCCAT staff.

SOP

1. Before dispensing nitrogen, wear appropriate personal protection such as cryo gloves, face mask or goggles. Wear appropriate laboratory foot wear such as closed toe shoes.
2. Dispensing should occur near an O₂ If alarm goes off during dispensing, close the valve and leave the area until O₂ level recovers. Do not continue dispensing while an alarm is going off.
3. Insert the tube from the 240L tank into the 4L or 10L dewar. If it does not fit inside the dewar, use an appropriate funnel to prevent spillage. Do not fill containers smaller than 4L from a 240L tank.
4. Open liquid valve to start dispensing liquid. Be careful that flow is not so quick that much spills over or flies back. This is particularly important for the 4L dewar.
5. Fill until dewar is full then close liquid valve. Be sure it is completely closed, but do not over tighten. Ask NCCAT staff for assistance if unsure.
6. If a loud whistling sound is heard during filling, this means that 240L tank is empty. Stop filling, wait for tank to cool, then move hose to a fresh tank. Mark empty tank as “empty” by removing the “in use” part of the tag. with a sign. Mark full tank as “in use” by removing the “full” part of the tag.
7. Let an NYSBC staff member know whenever you empty/change a tank so they can put the empty tanks in the appropriate place.
8. Only NYSBC staff members are permitted to move 160L/240L tanks.

General Cautions

• Liquid Nitrogen is a cryogenic liquid and can cause severe frostbite. Always be cautious when working with it.
• Liquid nitrogen can quickly displace oxygen in a room. Always work in a well-ventilated area with an oxygen sensor.
• If you are unsure of any procedures, consult with NCCAT staff.

SOP

1. Wear eye protection such as goggles or glasses throughout the experiment. Cryo gloves are not required since they are too bulky for delicate work. Be careful not to freeze your fingers.
2. Do not adjust the pressure on the regulator. Use the bleed valve to control gas flow.
3. Cool a chamber (thermos or bath of CP3/vitrobot) to liquid nitrogen temperature.
4. It is often easier to pre-cool the metal tip coming from the ethane tank to near liquid nitrogen temperature before dispensing. However, be careful since ethane remaining in tube can freeze solid, causing a blockage.
5. Open the main tank valve on the ethane tank. The bleed valve should remain closed until use.
6. Insert tip from ethane tube into filling container.
7. Slowly open bleed valve on ethane tank. Be careful not to spatter ethane.
8. As ethane liquifies, you will hear a loud noise. Filling too fast you will hear more gas hiss.
9. Fill about 5ml ethane (CP3)
10. When done filling, close bleed valve then close the ethane tank.
11. At the end of experiment, transfer remaining liquid ethane to fume hood.

General Cautions

• Uranium salts are highly toxic and emit beta radiation. Always wear eye protection, gloves and a lab coat when handling.
• Limit wet-work to designated negative-stain bench to limit possible spread of material.
• Use only designated pipetmen for handling liquid solutions.
• If you are unsure of any procedure, consult with NCCAT staff.

SOP

1. Only NYSBC staff are permitted to weigh uranyl salts
2. Any weighing of dry powder should be done in fume hood. You should wear a face mask to prevent inhalation of any fine powder.
3. Always wear goggles when handling liquids, particularly when filtering solutions.
4. Before beginning experiment, be sure all required materials (pipettes, gloves, Whatman paper, paper towels, grids, grid box and forceps) are on hand. Be sure waste container is not overflowing.
5. Discard filter paper used to blot stain in regular lab waste (biohazard bags)
6. Discard tips in tip waste for eventual disposal with sharps.
7. Discard excess uranyl acetate/formate into designated liquid waste container. Do not mix uranyl salts with other hazardous salts (e.g. lead, osmium, etc).
8. Use pH paper to measure pH. Dispose in lab waste.
9. When experiment is completed, discard all waste. Do not leave a messy bench for the next user.

Setup

1. Tank exchange should only be performed by NYSBC staff.
2. Tank exchange on the Krios is a 2 person operation and should generally be performed with other staff present, except in the case of emergencies.
3. Check that there is at least 1h remaining on the Krios before the next fill. Ensure that no sample exchange will occur during the tank exchange period. Allow about 15 minutes for the exchange. If a fill just occurred, allow time for the hoses to warm up before exchanging tank.
4. Close the gas valve and the liquid valve on the tank. If using a scale, note the final tank weight. Do not over-tighten valves.
5. Disconnect the gas and liquid lines from the tank.
6. Remove the tank from the room and place in the nitrogen dispensing area.
7. Bring a new tank into the room. Note that tanks weigh more than 600 lb and moving the tank into the room is a 2-person operation. If a scale is present, ensure that the tare is 0 and roll the tank carefully onto the scale.
8. Connect the gas and liquid lines. Open valves. Do not over-open: Leave valves one full turn away from fully open.
9. Purge the gas line for 30s.
10. Note the tank weight, tank tare, and tank pressure on the notepad. If pressure is <15 PSI, use the pressurizer to bring up to 20 PSI.
11. A second staff member should check that all operations were completed correctly. Ideally, a fill should occur before leaving to be absolutely sure everything was set up.