Now all you need to do is prepare the bleach and make your sterile water. Both are very easy to do, the bleach can be made right before you start your tissue culture. The sterile water needs made ahead of time so it can cool to room temperature. Do not put in the fridge to help it cool, let it cool naturally. I prepare everything the day before I plan to use it to save time.

Lets start with the bleach. You need to make 10% bleach. Make it by mixing a ¼ cup household bleach with 2¼ cups water, add 3 drops liquid dish soap. Use a good name brand bleach, unscented if you can find it. I make mine in a quart jar then pour it into a pint jar before adding the dish soap. Fill the pint jar ¾ full then add the 3 drops liquid dish soap.

Now all you need is the sterile water. The sterile water is used to rinse the tissue free of the bleach. You need to get all of the bleach rinsed off of the tissue or it will kill new growth. You can rinse a couple cuttings in a pint jar but it wont take long to contaminate the water. I like using several babyfood jars for rinsing, use each one once. This helps prevent bleach buildup in the water and reduces the chances for airborne contaminates. Either way you want to use the 1 minute rule again, read on.

To sterilize water in the pint jar - Fill ¾ full with bottled or RO sodium free water, cap with the autoclave lid. Put it on slightly snug then back it off 1/4 turn or so to ensure pressure release. And when it cools it will shrink, if its not on loose it will be very hard to get off. Microwave on high for about 5 minutes, or whatever it takes to gently boil it for a full minute. You may have to turn the microwave on and off to cycle it to prevent boil over. I put it on a plate so I can remove it when it's done without having to touch it. Let it cool a minute in the microwave then transfer it to the sterile area. Stand and watch it, do not leave the room! Always watch or you can come back to huge mess.

To sterilize water in the babyfood jars - Fill each one, minimum of 6, 1/2 - 3/4 full with bottled or RO sodium free water. Cap with an autoclave cap. Place on a dinner plate and cover with the pyrex plate as you did with the media. It is important to put the same amount of water in each one so they all boil at the same time. Microwave at 50% power the same way you made the media. The goal is to bring them to a boil and keep them slightly boiling for a minute.

I usually make 6 or 8 babyfood jars and a pint jar of sterile water. You may need to make more depending on how many cuttings you are doing. You should do at least 2 - 8 minute rinses in the babyfood jars or 1 - 20 minute rinse in the pint jar. Gently swirl them around periodicly.

I get my alcohol set up and ready to go before hand too. You need the 90% rubbing alcohol for three things. 1) To spray your hands/arms and work area down. 2) To dip cuttings in to remove the waxy covering. 3) To sterilize your tools. You can pour the rubbing alcohol into a clean spray bottle or just screw the lid of a spray bottle onto the alcohol bottle. I generally use the same container to sterilize my tools and to dip the cuttings into. I use the tall babyfood jars, just fill with alcohol and cap with an autoclave cap.

You should have:
The sterile area set up and everything needed gathered up.
The media made and cooling.
10% bleach
Sterile water made and cooling.
Sprayable alcohol for you and the work area
Alcohol to dip tools and cuttings in.

So it should look something like this.

Now all you have to do is wait for everything to cool and your ready to collect your cuttings and get started on tissue culture. Click on "Let's Do Some TC" above to continue.