How to autoclave items for the lab

Autoclaving is used to sterilize items for lab and medical use. An autoclave is a large pressurized oven. It's safe when used properly, but precautions are necessary to avoid burns and explosions.

Autoclave safety

1. Don't screw lids on tightly! Lids should be loose enough that you would not want to flip your bottles upside down because the contents would leak and make a mess. 
• Stuff can explode due to the autoclave pressure changes, so for everyone's safety and to prevent autoclave equipment damage, keep lids loose. 
• This applies to empty bottles as well.
• Keep lids on about 10-30% tightness. Tight enough that lids won't fall off during transport to/from the autoclave, but loose enough that if the bottle fell over then liquids would leak. You need air to easily move in/out of the bottles during strong pressure changes.
  
  
2. Place all contents in an autoclave bin. Never place items directly on the autoclave grates or in random plastic bins. All containers (bins and bottles) must be autoclave-proof.
   
   
3. Use orange autoclave gloves (or similar heat-resistant gloves such as oven mitts) for loading and unloading the autoclave.
• Use these orange gloves for hot protection:
  
  
  
  
  
• Not these cryogloves... they are for cold protection:
  
  
  
  
  
4. Never force the autoclave doors open or closed. Especially if the autoclave is making clicking sounds! It might be in use and pressurized. Many autoclaves have a foot pedal for automatically opening and closing the door. Get training to prevent equipment damage.
   
   
5. Use a rolling cart to transport autoclaved items. It may be tempting to just use the orange gloves to transport the autoclave bin back to lab, but autoclaved items are HOT(!) and you don't want to risk dropping items and scalding yourself or someone else. 
   
   
6. Let items cool down to room temperature naturally. Leave stuff on the rolling cart or an empty bench to cool down. If needed, bottles with liquid contents can be cooled faster by adding water to the bin or running water outside the bottle. Once bottles are warm but comfortable to touch, you can tighten the lids and store them.

How to autoclave items

These instructions are meant as a reminder, not as a substitute for in-person training.

1. Run separate cycles for dry goods and liquids. They run on separate autoclave programs.
2. Prepare items in an autoclave bin. 
• Dry goods example: fill metal containers with glass pipettes, or fill refill pipette boxes with tips. Don't pile too many dry items on top of each other, otherwise the drying step of the autoclave cycle will be inefficient and your items will come out steamy and wet.
• Liquid items example: fill autoclave-proof lab bottles with water or media only up to the fill line (the ring around the bottle neck) and no further... anything else will boil over!
3. Add a small amount of "autoclave tape" to each item. 
• Autoclave tape is beige-color tape with invisible writing that changes color during heating. 
• Don't tape all around. Just a small piece is enough.
• The goal is to mark which items have/haven't been autoclaved (by tape color change) and which items have/haven't been opened after autoclaving (by placing tape where it needs to be disturbed upon first use). Typically we tape the sides of lids.
4. Place items on a rolling cart and bring orange gloves. Go to the autoclave room, e.g. SSB 126.
5. Check the autoclave status... is it running? If so, come back later. If not, continue.
6. Open the autoclave with the foot pedal. DON'T force the door open.
7. If there is stuff inside, move it out carefully. 
• Use your orange heat protection gloves and the rolling cart to move the HOT(!) items to the nearest empty rest area. 
• Keep items sufficiently far away from other equipment, chemicals, spray bottles, or anything else that is heat sensitive or can melt.
8. Place your items inside, avoiding touching the metal walls. Autoclaves are ovens.
9. Quick safety check:
• All bottles and containers are autoclave-proof? Ask if you're not sure.
• All bottle and container lids are loose? Autoclaves will pressurize and explode tightly closed bottles.
10. Close the autoclave with the foot pedal. DON'T force the door closed.
11. Click "Cycle Select" and pick an autoclave program.
• Dry goods use a "gravity cycle". For small and thin-walled items (glass Pasteur pipets, plastic pipette tips, small tubes), the shorter sterilization cycle is fine. 
• Liquid items use a "liquid cycle". For bottles 500 ml and less, the shorter sterilization cycle is fine (15-20 min at 121C). For larger bottles, use the longer sterilization cycle (30 min at 121C). Check the autoclave program line that says "sterilization time". 
12. Click "Run Cycle" and listen to the autoclave starting sounds. 
• It sounds like a steam engine with lots of clicks and clangs. That's normal.
13. When the "Time Remaining" countdown begins, go away and come back later. 
• If it's 5pm, you can come back the next morning.
14. Pick up your HOT(!) items with orange gloves and transport with a rolling cart. 
• DON'T CARRY hot items to different rooms. You risk spilling them and burning yourself or others. It is dangerous and against safety regulations. 
• Even if you are strong, people can bump into you. Always use the cart!
15. Close the autoclave door when done. Keep autoclaves closed to prevent equipment malfunction.
16. Leave your items somewhere safe to cool down. The rolling cart itself, a lab bench, an empty chemical hood are common spots. Keep hot items away from chemicals, equipment, pens, spray bottles, and anything else that could be damaged by heat.
17. Once items are comfortable to touch, store away. 
• They can be warm, but should not be hot. 
• You can tighten lids now for long-term storage. 

Random notes

• Culture media can be autoclaved but not all components will survive. 
• Salts, buffers, and simple nutrients in media can survive autoclaving. Bacteria culture media is commonly autoclaved to sterilize.
• Don't autoclave growth factors or delicate proteins. Beware autoclaving mammalian culture media which has more delicate components.
• Sugar solutions can caramelize. It's better to filter-sterilize sugar solutions, instead of autoclaving.
• Standard autoclaving does NOT remove RNases. Glassware needs 4+ hours of dry autoclaving to be free of RNases, and even then some particularly stable RNases can survive (e.g. RNaseA from miniprep kits).