Ultimate Guide to Spotting Contamination in Liquid Culture: Causes, Prevention, and Remedies
Now, that I have some time with the Kids being off school, I thought I would provide a detailed guide on spotting contamination within liquid culture. I always tell people that it is quite difficult to ever 100% know if your liquid culture has gone bad or if the mycelium took hold. The foolproof 100% way is to always bring the liquid culture mycelium and grow it on Sterile Agar Agar. By growing the liquid culture our on Agar Agar we’re able to visually and quickly see if it truly is mycelium, within 72 hours there should be visible growth on the Agar to give us our results of clean liquid culture or not. You can view our other guide on spotting contamination, there are visuals there for reference. Let’s begin on identifying or visual cues liquid culture can provide, there are two ways:
The mycelium grows quickly in a matter of 12 hours and/or looks odd. This is 100% contamination and mold (Picture 2)
This odd looking shape will look less like a fluff ball or circular jellyfish. (Picture 1)
The liquid culture looks cloudy. This is bacteria. (Picture 3)
In my experience, most all liquid cultures will contain some amount of bacteria unless you have gone with a clean room and flow hood, and in fact most all substrates do contain a tiny bit of bacteria that is introduced during the inoculation with spores or liquid culture. This is very easy to prove on agar.
The question isn't the absolute purity of the substrate, it's who gets the upper hand and conquers the liquid culture or any other substrate. Basically, who will win? That depends on how dirty the inoculate was with bacteria or if mold spores were introduced and how many.
A good liquid culture will often be a tiny bit cloudy and then clear up. This is because the bacteria starts to grow a tiny bit but the mycelium is so far ahead that it scours the liquid culture of bacteria and particulates and then dominates it (mycelium has been fighting bacteria like this for millions of years). You MUST allow the liquid culture to reach the stage where the liquid between the mycelium fragments looks clear. If it never reaches that stage, then it cannot be used safely. Here is a typical progression of a liquid culture:
The above picture will have multiple “Jellyfish” looking blobs as we use a magnetic stir bar, if you leave your mycelium undisturbed it will tend to form one Jellyfish/giant blob. They should look organized/structured, the small “tendrils” are straight.
Proper mycelium growth with Jelly Fish Circular growths.
Bacterial Contamination, the Liquid Culture is cloudy and after few days nothing grows.
If your liquid culture gets taken over by bacteria you’ll notice that the Mason Jar will be slightly pressurized. If you have a filter patch, you’ll be able to smell that it’s gone bad. Bacterial growth is one of the easier contaminations to identify as nothing visible will be growing.
With mold contaminated liquid culture, it is a little more difficult to identify with the naked eye as you’ll often think its just mycelium. What we will need to look for are the individual growths.
Was growth within 12 hours?
Do they look erratic? Almost like small cotton balls? With tendrils that are entangled?
Does it smell stale? - If there is a filter patch.
Did it develop any black or green color?
Mold Contamination, the Liquid Culture has sporadic growths that aren’t circular in nature.
Generally speaking, mycelium loses to mold, so you don't want to get that in there. Mycelium fares much better against a tiny bit of bacteria. A tiny bit of bacteria may make the water go cloudy at first but then is soon killed off and eaten by the developing mycelium and the water goes clear again.
A lot of bacteria, though, and the solution remains cloudy and the mycelium either doesn't develop at all or goes stagnant and fails to clear out the solution. Bacteria can be introduced very easily by infected spores or prints, so if you aren't cleaning your spores up through agar, you need to make relatively clean prints. That means must making them in a glove box or flow hood. If you don't, you may have a beautiful dark print, but once you make spore solution out of it, it will infect anything you inject it into with bacteria.
And I will repeat this again: you must wait until the mycelium has won over a liquid culture and had a chance to completely scour it of all bacteria. You'll know this has happened after the mycelium is dense in the solution and between it, the liquid is clear.
One last note before you head on your way, when inoculating with spores, make sure you shake your syringe thoroughly to disperse the spores evenly into the solution. You will only need a few spores to create a healthy liquid culture, I always tell people to distribute their spore syringe into a few jars to provide themselves some insurance if case one or two goes bad.