Consistent inputs are important for consistent outputs. In an experimental or culinary setting it’s important to continue to work with the same species or lineage of a particular microorganism. The best way to achieve this is to store samples of it long-term and then regrow it from a small culture into the quantities you need. Storing it for this purpose is called banking.
Because we wish to store these samples for a longer term (several weeks to a year) we must be extra cautious about sterilization. This storage term is also time in which contaminating bacteria can grow. Therefore a requirement for banking is a pressure cooker to properly sterilize our prepared samples. In particular, one that can reach 15 Psi, which excludes most electric pressure cookers like Instant Pots.
Materials
- Isolated culture you wish to bank
- Potato-Dextrose Agar (PDA) if banking yeast
- Glass test tubes (16mm+ diameter preferred)
- Inoculation loop
- Baking tray or other easily slant-able flat surface
- Pressure cooker
- Small funnel is helpful (optional)
- Mineral oil (optional)
Preparation
First, select the number of test tubes you wish to bank. One test tube per sample is needed, but of course it may make sense to have multiple test tubes per specimen you wish to bank. This would allow you to extra a sample without needing to replace your banked slant immediately.
Each test tube will need to be filled approximately half-full by volume of PDA. Calculate how much PDA will be required and prepare the potato and dextrose solution without agar.
When this solution is beginning to cool add the required amount of agar to it (2% by volume) and stir to dissolve it. We do not yet wish for it to be solidified as we’ll still need to pour it into our test tubes and sterilize it.
Now, with or without a funnel, fill each test tube to about half full with the warm PDA. Cap the test tubes to seal them, but only loose screwed. Ideally you should have a test tube rack to hold your tubes vertical, and this rack can fit inside of your pressure cooker. It may also be prudent to take advantage of this sterilization session to sterilize distilled water, mineral oil, other glassware, etc.
Fill the pressure cooker with near-boiling water, up to the level of the liquid within the test tubes. Seal the pressure cooker and bring it to 15 Psi for at least 15 minutes. Then, release the pressure and let the test tubes cool until they can be handled by hand, but just barely. We must reposition the agar filled flasks before it solidifies.
Slanting
Slanting is fairly self-descriptive: we allow agar in test tubes to solidify while slanted at an angle, such that the agar forms a bit of a triangle inside of test tube. The reason why we do this is twofold. First, we get much more surface area out of test tube in a small form factor! Secondly, we can easily inoculate or draw samples from it with an inoculation loop.
The simplest way to slant your test tubes is to take flat surface, like a cookie sheet or baking tray, and elevate one side of it. The tray should be angled quite low, perhaps 10-15°. Ideally, we want a minimal plug of agar media at the bottom of the tube with the horizon of the agar surface stretching to the threads of the lid of the test tube.
Once this surface is set simply lay your test tubes on it with the cap of the test tube directed into the elevated end of the surface. Make sure the lids are tightened all the way down. Leave them here until the agar has solidified.
Condensation
It is natural that as the hot agar solidifies condensation will form inside of the tube. This is problematic because it can prohibit proper streaking of microbiologia inside of the tube. The way to handle this is to wait until your agar has solidified, but is still warm, and to flip the tubes upside down onto their lids, standing completely vertically, perhaps in a test tube rack. This will allow the condensed water to collect at the lid which can be easily removed (when working near a flame!) when it’s time to inoculate them.
Streaking
Now it is time to streak out yeast culture onto the slants. As per usual you will need a flame, inoculation loop, a cooled and prepared slant, and of course some yeast to streak!
Light your flame, working as close to it as possible. Flame your loop and grab a loopful of your solution onto it. Grab your test tube to be streaking and remove the lid, keeping it near the flame. Flame the lip of your test tube to kill any microbes that might be on it. Insert your loop all the way to the bottom of the test tube, then touch your loop to the agar and pull it all the way up to the mouth of the test tube. Seal the test tube, and you’re done! This should give a strip of yeast along the length of the agar in the tube. Repeat as necessary. You can also use a zig-zag pattern as your drag the loop.
Let your streak test tubes grow at room temperature (ideally 22-28° C/ 72-82° F) for 24-48 hours to give your colonies some time to grow. After this period we should have yeast ready for long-term storage.
Freezing
Now that our growth is complete, we want to store the yeast for a long period of time. There are a few ways to do this, from simplest but shortest storage lifetime, to mildly less simple, but longest storage lifetime.
Don’t forget to label and date the tubes!
Simplest
Just wrap some vinyl (electrical) tape around where the lid and test tube meet and put it in your freezer. They will be stable for about 3 months without worry of losing your yeast. Parafilm is frequently used instead of electrical tape, but the tape in this case in just fine.
But we can store things for much longer! Re-culturing yeast stored like this is recommended every 6 months at the longest.
Distilled, sterile water
If you sterilized some distilled water in your pressure cooker you can fill the open-air half of your streaked test tube with it. This prolongs the storage of yeast by giving them an anaerobic environment to hibernate in.
Simply work near a flame, open the vessel containing your water and your test tube. Flame the lips of both of them and fill your streaked test tube with water. Close the lids. Tape the lid of the tube shut.
Note that you should always prefer to use mineral oil over water, given the option. Water can potentially dislodge your yeast from the substrate.
Congratulations, your cultures can now last for up to a year!
Sterilized mineral oil
Do the same as above, but with food-grade sterilized mineral oil. Congratulations, your cultures can now last from three up to thirty years! But, to be safe, you should still re-culture them every 2 years.