My new liquid culture went cloudy, help me spot mistakes in my process

[Talvara]

So I’m still just starting up, I’ve had a liquid culture of Lionsmane that has been doing well for the last 22 days (it hasn’t gone cloudy, stir it daily no complaints) it was made with a syringe I got from a Vendor. the two other strains I didn’t have any luck with, as both went cloudy and my attempts to isolate the mycelium on Agar failed.

Now 3 days ago I extracted 3 syringes of liquid culture from the lionsmane Culture, put some drops of each syringe on Agar plates, inoculated 3 jars of soaked/cooked/pressure cooked grains and injected the leftovers back into a freshly made liquid culture broth.

Looking at the newly made liquid culture broth it has gone cloudy, and looking at the petridishes I am fairly certain I am looking at bacterial contamination of the fluid.

here are the jars I’ve been using, i don’t have a self-healing injection port on there and am not using needles, instead i tàke out the filter inserted in the leurlock, screw the syringe on and inject, i do this in front of a small flow hood

the syringes don’t leave the oven bag before they are to be used in front of the flow hood,

The syringes themselves I first boil, suck up and expel boiling water 5~7 times. Then put them in an oven bag and include them in the pressure-cook alongside oven bagged Petri dishes and fresh made LC broth.

Extraction happens via the silicone tube that sticks out from the top (with its own filter) this tube goes down into the LC, When I do this I pinch off the tube as the filled syringe is decoupled from the leurlock at the end of the silicone tube

I’m not sure where Im going wrong

heres the agar plates, which I think indicate bacteria contamination. A small possibility is that this is just how LC broth stains the agar?

Finally, My pressure cooker only goes up to 1.2Bar, I pressure cook my liquid culture broth, syringed and petridishes for 40~45 minutes.

[Talvara]

I’ve been told elsewhere that it’s possible that I’ve overcooked my liquid culture broth causing caramelization, something that is known to make the broth cloudy after a couple of days to a week.

I’ve also been told that the Agar pictures I posted aren’t necessarily an indication of bacterial contamination, but could just be the Liquid Culture broth staining the Agar, I’ve marked one of these islands with a marker, and I am observing if it grows over time. So far it doesn’t look like the island has expanded (but that’s only been two days observing)

I’ve also been told that Lionsmane has a habit of looking like it’s not doing anything and then suddenly prove you wrong.

So I’m hopeful that things are salvageable, for the future I’ll avoid pressure-cooking my liquid culture broth, instead try a steam bath without elevated atmospheric pressure.

[KyleGabriel]

You most definitely want to pressure cook your broth. Pasteurization by simply heating to boiling temperature is insufficient for broth cultures. There are many endospore-forming bacteria that can withstand boiling temperatures for extended periods (e.g. 10+ hours). You need to reach sterilization conditions with a pressure cooker to ensure all microbes have been killed. Otherwise, even one contaminating cell can ruin your broth culture.

There are some components that may darken in a broth when sterilized with high temperature, such as when using cheap brewing malt extract, but this is not an issue. I’ve also never seen sterile broth become more turbid over time without some sort of microbial catalyst. You can always create a control sample of broth that you don’t inoculate a culture to and verify if it does or does not become turbid over time. If it does, then it suggests it is a natural process of whatever your broth is. If it doesn’t, it indicates the turbidity is caused by a microbe you inoculated to (or allowed to contaminate) your broth. If the control broth did increase in turbidity, you can also streak out a sample onto an agar plate and look for growth after incubation, to test if the turbidity is from a contamination of your control broth or is indeed an abiotic process. You can also stain a sample and view under a microscope to look for cell morphologies.

To sum it up, culturing in broth is much more difficult than solid media. You should be integrating testing in your workflow to determine if contamination is occurring. As a rule, I never trust cultures I receive from others to be pure isolates, unless I can absolutely trust that specific person. You should streak them yourself to verify, then use what grows from your streak plate as the starting culture for a broth. There’s simply too much uncertainty inoculating straight from another liquid culture that you received. Adding another step to ensure what you’re inoculating to your broth is pure is great piece of mind and eliminates that as the source of any future contamination when you’re trying to track down where it’s being introduced.

[Talvara]

This sounds like solid advice, making a control broth is definitely something I’ll try next time around, I am a little limited in the amount of prepared hardware I have lying around, I think I’ll have to acquire and make a few more liquid culture jar (lids).

I’ve started using Agar now aswell which I was avoiding for some time, I find Agar fairly intimidating and am always second guessing myself.

I have been able to isolate king oyster mycelium from vendor spores succesfully so I’m slowly getting more comfortable.

This is one of the plates I was able to make from the LC syringe I got from the vendor.

I took some wedges from an area that looked most clean.

Anyway, whats tripping me up is that the LC syringes I used for this latest batch of Liquid culture were from my own jar of LC which is doing great, nice and clear broth nicely colonized. so this wasn’t a case where I got some potential impurities from a vendor.

Somewhere between extracting the LC into the syringe and expelling it into the newly made broth I must have made a mistake. But I’m having trouble spotting where that could have happened. my initial thought was that I hadn’t sterilized my Syringes thoroughly enough. (I do reuse syringes after boiling and pressure cooking them, Generating plastic waste is something that I loathe, the parafilm is already making me feel guilty)

[KyleGabriel]

@Talvara

This is one of the plates I was able to make from the LC syringe I got from the vendor.

The mucoid culture on the plate looks like a bacterial or yeast contaminant. If you streak the sample with a loop, you can get individual colonies to form. This is ideal for isolation because you can select only those colonies that grow filamentously to subculture on a new plate to attempt to fully isolate the culture. Continue this subculturing until you’re relatively sure there’s only one organism growing.

@Talvara

I took some wedges from an area that looked most clean.

This looks good. You may want to subculture another sample near the margin, to a new plate, to be extra sure you’ve isolated it.

@Talvara

so this wasn’t a case where I got some potential impurities from a vendor

Liquid cultures can have very dilute contaminants in them. For a 50 ml syringe, for example, if there are only 5 viable contaminating cells, that would contaminate at most 5 of 25 liquid broths, if inoculating with 2 ml aliquots each. If inoculating from agar, after subculturing from your stock liquid broth, you can be assured there’s no contamination because you can physically see what’s growing on the plate you’re sampling from (which is impossible for liquid cultures).

@Talvara

Generating plastic waste is something that I loathe

Why not invest in glass Petri dishes that you can reuse?

@Talvara

I do reuse syringes

I wouldn’t recommend doing this, but if you have a sterilization method that works, then I see no reason not to.

[Talvara]

You’ve certainly given me some things to consider adding to my toolbox, thank you.

I already use glass Petri dishes, Borosilicate 100mm ‘Anumbra’ from Neolab. They’re a lab-ware vendor on Amazon that caters to the Netherlands. it’s just the Parafilm I used to wrap the Petri dishes that I feel guilty about.

It sounds like I’ll have to get used to the idea that inoculating liquid culture broth from Agar is the way to go forward, no problem. I just have to invest in some more glassware ;), my 10 dishes, 1 medium flask and 6 modified lid jars are proving to be a bottleneck when trying to work with 2 strains for me.