How To Know When Liquid Culture Is Done?

A couple weeks ago I made some liquid culture,
using 1000ml water and 8g honey as media ratio,
in jars with airport lids.

I inoculated the liquid media using slices of agar of oyster mycelium.

Everyday I put the jar on a magnetic stirrer until it whirlpools (for about 10-30 seconds),
and there is a metal bolt in the jar which helps it stir.

After about 2 weeks, here is what I am seeing:

I have a couple questions:

  1. what should the liquid culture look like when it is done / ready for use to inoculate other media? do these jars looks good?
  2. is this one whirlpool a day enough stirring?

I also tried inoculating some petri dishes with some of the liquid culture
– I maybe put too much LC into the dishes, but it appears to me that there is a layer of mycelium growing over the entire dish.

Hi notplants,

your LC looks great!
If your Petris are all white and there is no contam they are clean and good to go. I recommend about 5mL per jar of grains (I do 100g dry weight of cereal), even less LC might be enough.

1 Like

thanks @phylanx – since that post, I’ve tried taking the LC to grain with no luck yet (no mold no nothing) – but going to keep trying. I’m thinking perhaps I cooked the LC I had too long causing sugar to caremelize and maybe some mycelium grew at first but then it ran out of food and died out (totally speculative theory)

placing this here for any future readers https://ezmushroom.com/grow/liquid-culture-for-mushroom-cultivation-how-and-why/, as it seems like a nice guide

To me the most probable explanation is that you didn’t catch enough mycelial fragments. Try to shake the LC very well before taking anything with the syringe and then you have to see mycelium in the syringe. Everytime before inoculating a jar, shake the syringe as well - not too much so you don’t spritz LC around, but still with force :wink:

2 Likes

I can happily report that my LC tek (using water with 4% honey and silicon self-healing injector ports) is working,
these two rye grain jars were inoculated with LC and are now well colonized.

I think in my earlier experiment I was accidentally not putting enough honey (just a multiplication error). I got a tip from Ale that when using Malt extract in the LC medium, the LC changes color when it runs out of nutrients, which seems like a useful property. I’m curious to try it.

2 Likes

Hmm interesting i actually had the same question as you notplants.

I inoculated a 4% honey water solution with 1cc of various species about a week or go (plus minus)

Heres some photos of a Burmese strain, reishi and Turkey tail ( im not sure how long it takes to fully colonize about 100ml of New lc from 1ml LC inoculation tbh (I did NOT use agar wedges))

2 out of the 4 cultures had a bit more honey than the rest, hence the more golden amber colour.

I heard somewhere that i should colonize them over a month and then store them long term in the refrigerator. So thats my current plan…

Anyways im just curious as to how they looking in terms of health and knowing when the colonization is complete?

Note: i may be mistaken but some of the mycelium seems to be yellow/brown, could just be the honey though, idk…havent tested the culture out yet

Newbie here (lots of questions) :joy:

1 Like

@merlyn curious once you’ve tested it out how it goes.

I have a few LC jars going now too. My first batch (from agar) looks nice and cloudy after 2 weeks, and I tried taking some from the first batch to make a second batch of LC, and also a few jars of grain… will see how that goes.

The note about putting in the fridge after a month is helpful, I think I’ll try that timeline too with my initial batch of LC, and then hopefully keep the second generation around outside of the fridge for inoculating grain

also what types of lids are you using?

15 days old today…Turkey Tail (Trametes versicolor LC (inoculated with 2cc). This was by the far the most aggressive species to take to my honey broth. The jar contains about 100 - 150ml of LC

6 days old today…Reishi, Ganoderma lucidum. (Inoc w/1cc LC). This pretty much seems done already, the liquid seems quite clear and there seems to be a decent cloud of mycelium present.



I including some pictures of my lids that i use. Most of them are different but the premise is the same. A space for airflow (i mainly use polyfill and i like to add 1 layer of micropore tape over The fill, but that’s completely optional) and an injection port (i was buying self healing injection ports but now im just using RTV heat resistant silicone)

My handiwork with the RTV silicone is still a bit messy, and needs some tidying up but by far the more cost effective option that works.

I’m busy moving houses this week, but plan to do some more grain jars soon. I will keep you posted with the developments. For now I am using these LC’s as backup so I will probably end up storing them in the fridge until i need them.

1 Like

Here are some images of my latest round of LC… was enjoying looking at the sun and the courtyard through the lense of the golden liquid.

These are three jars of 4% agave broth, inoculated with an oyster agar wedge, after 3 weeks.
Interestingly two of them (A and B) have this more flaky appearance. And in one of them © the mycelium appears to have a finer appearance. I wonder what causes that.


[A]


[B]


[C]

And here’s an image of a second generation LC jar after one week, inoculated from a few ML from C.


[D]

Here are two agar plates I squirted LC on to test, after a week. I have to say I’m not really confident enough in my agar work for it to be a convincing test to me. If the agar looks contaminated I’m as likely to think it means I did a bad job with the agar as that the LC is contaminated. Also wonder if there’s a way I could be putting the LC into the agar so its a bit less, and not like full of liquid with random mycelium flecks everywhere.


[E]


[F]

@Merlyn are those marbles in the jars? do you just kind of roll them around to shake them?

I’ve been using magnetic stirrers because the space I work out of (top lab) already had some, so I’ve been using those, but I’m curious also to manage without in the future.

1 Like

Those are marbles indeed. I just give them a vigorous stirring each day, making sure i dont spill too much liquid through the polyfill on top.

Ill be getting some magnetic stir bars in the future but the marbles work fine for now. I find adding 2 marbles even, breaks up the myc faster.

2 Likes

How many drops are u adding to your agar? 1 drop should be suffice.

Also just beginning here and new to agar but so far had some moderate success. Been taking “the less is more” approach…

Your LC is looking amazing though, nice dense clouds forming there. Seems like a job well done to me :slight_smile:

1 Like

The marbles are a nice touch. Another option is small shards / pieces of glass (a nice way to reuse a broken drinking glass). Their cutting action helps to stimulate vigorous (re)growth in the mycelium.

2 Likes

using the syringe, somehow I felt like I need to get a bunch of liquid to make sure I get a fleck of mycelium. with a single drop, can you see the mycelium in there? or does pretty much all the liquid have some mycelium in it? also wondering if my syringe should be bigger. these are the syringes I was using: Romed Einmalspritzen + Einwegkanüle steril 5 ml 21 G x 1,5 bei praxy., 7,10 €
on the packaging: 5ml 21G x 1.5 0.8 x 40mm
not sure if this is a German metric or what exactly this means. I guess 0.8mm is the diameter of the needle

aw I’m now reading this as 21G probably means 21 gauge

@notplants managed to test some of my freshly made liquid culture out - in some grains, a few days ago.

@Merlyn this image is of grain after just a few days after being inoculated with liquid culture? looks great. and which species is this?

I’m still suspicious that something is going on with my liquid tek… I don’t trust it… this is one of my grain jars 12 days after innoculation with liquid culture (oyster)

The jar is Ganoderma lucidum @notplants inoculated on the 24th of February so that picture was taken when it was 7days old roughly.

I have not much experience with oyster mycelium but i assume the grey-ness in appearance implies immaturity over contamination.

However, 12 days does seem long to only be that far in the process, so im also a little suspicious for you based on that reason.

I guess we will just have to wait and see if the jar fully colonizes. Keep observing what pops up (or doesnt pop up). Holding thumbs and ill keep u posted with any updates on my side.

1 Like