Inoculation loops are ideal for reaching into slants or precision selection of in agar plates and makes it simple to leave the agar behind. They are tool you should have and are inexpensive for generic. I prefer single strand nichrome wire instead of loops.
As far as how long cultures on agar will last is not easily answered. There are so many variables and to name a few, how aggressive the culture is, nutrient content, and depth of agar.
At room temp the mycelium metabolism is very active. It will continue to consume the nutrient agar and it will be depleted long before the culture dies.
If i were to store at room temp i would consider the nutrient and O2 free distilled water method then inoculate plates from it on a need to have basis.
If you must store on plates then i would pour as deep as possible. This would give a little more food but you will be surprised just how quickly a aggressive culture approaches the south glass. The depth in this case is more for moisture.
Another thing i would do is to go a little heavy on nutrient content @ 5%-8%. This will slow down the growth and consumption as well.
Also i would add some peptone or a cheap substitute such as soybean powder. This will spike the nitrogen content slowly growth down even more. The amounts will have to be determined by you and your culture. You will have to ease it in to higher nitrogen contents. I seen cultures take more then two weeks to colonize a 65 mm plate which otherwise would have grown in out 7 days.
The nitrogen will provide you a thicker mycelia mat. Nitrogen does encourage more biomass and this probably the most critical imo. Once a plate is colonized you will gradually start seeing the mycelia mat thinning out. You wont really notice until you see your agar surface through the mycelium.
Finally i would store in ziploc bags or another air tight container. All though i do this in addition to parafilm to stop the fridge from drying out my plates, a arid room can also dry out the plates.
Keep in mind your at room temp, the metabolism is very active so you will need to open the bag or container at least once a week depending on the size of the container/bags and quantity of cultures in there, you may need to do it more frequent.
I would recommend making slants also of master cultures also. You will have much higher maintenance then you would using cold storage but you should easily be able to pull off 4-6 months before having to transfer with a very slight slant in the tube. Also i would simmer some hobby sticks in a dextrose solution so that you have a chance to pull life from a slant that appears to had completely died off. Remember all it takes is a single cell to start a new colony or two compatible if so happened to pull a single monokaryotic cell, but if you can pull that off with scaple, heck if you can pull that in anyway, come to my house and help me breed some strains!
I dont see why you couldnt pull off at least a month after complete colonization of the plate.
It will be difficult maintaining a extensive library so work with only a few strains and print them in addition to slanting them. When your ready to work with another strain or few strains, stop expanding the current one on plates. This will leave you only transferring slants once every 3-6 months, maybe longer and your plates of just a few strains once every 45 days or so. Who knows you might get 90 days on plates after complete colonization.
Last thing to discuss is expanding. Slants are designed to give a relatively large enough area to develop a nice colony on the surface while minimizing cell division. By design they provide much more food and moisture respectively.
Distilled water storage. I would use a bat and cat jar also known as jelly jars. Put 2 ships on the lid and some shards of glass or something comparable to rip apart the mycelium.
Fill the jar with 2-3 ounces of water and pc. This will create a vaccum inside the jar assuming it is sealed properly.
Harvest mycelium without getting any agar, or as minimal amounts as possible as this only works with 0 nutes and 0 O2 present or trace amounts of each. If there is O2 then the culture will search for food and if there is nutes the culture will need O2 for cellular respiration.
So once you harvest your mycelium, place it in another jelly jar with glass shards. I use shards from a broken crown royal bottle. You only need 1 ship and a ge filter for this jar or you can go with 2 ships and use a airport syringe for this step.
Shake the hell out of the jar with the mycelium in it like it owes you a bunch of money.
Draw the myc water into a 10 cc syringe. Make sure you can see the mycelium in the syringe and hold it upright so that the mycelium settles in the bottom of the syringe.
When ready stick the syringe into the jar under vaccum and it will pull the contents of the syringe into the jar while any O2 that enters will be what was dissolved in the water which is ok.
Or you can buy glass vacationers and they will do the same thing. If they come with a chemical such as K3 EDTA a anticoagulant agent, you will need to wash and rinse it out.
I have stored cultures in slants before realizing this chemical was sprayed on the inside of the vacutainer. The mycelium was able to tolerate it but im not sure if it would be a food source or not.
I bought some tubes on ebay for super cheap to realize that they were polyethylene and the purple lid meant they had K2 EDTA. So i cant wash and steralize. I will be testing these to see if i can use these for distilled water storage. I figured i will fill several and then check viability a year later. If it works then the plastic sterile tubes would be the way to go.
If you use the jar then you can shake it up even more to dilute it. When your ready to grow it out again grab a few cc'ss and hit some plates up.
This method was designed for spores and or live cultures of NON spore producing fungi but a study that covered a span of 4 years showed that 95% of the 150+ or so cultures of various species, a dozen cultures from a dozen different species from the Basidiomycetes division were viable at 4 years.
Happy agar work!
Edited by Microbe, 01 June 2017 - 01:00 PM.
