Liquid To Liquid Contamination

[notplants]

When I’ve done transfers from Liquid to Agar, and Liquid to Grain, all appears to be clear. But when I’ve made transfers from Liquid to Liquid, so far I’m seeing the mycelium get all brown… doesn’t look good.

I’ve been using a fresh syringe, and transferring in a flow hood. The only thing I can think of right now, is to try to spray the top of the LC jar with alcohol before piercing it, which I don’t think I’ve been doing. Anyone else have any tips on what I might be doing wrong in my LC to LC transfers?

Here are some images of the clean tests:

[A] (I believe) mycelium everywhere from when I tested LC to agar with too much LC

[B] small bit of mycelium from the smaller test I did of just a drop of LC onto agar

[C] a close look will reveal white fuzzy mycelium growing in this landscape of grain

Trigger warning: the following images contain images of contamination

[D] after liquid to liquid transfer, mycelium strands have brownish tint / particles in it

[E] same as above

[F] large brown clump

[glyph]

Ah that’s a rough one, sorry @notplants . The first two LC photos gave me hope that maybe they weren’t contaminated but that brown clump in the third photo does not promising. Is your LC usually crystal clear or do you sometimes have a dusting of precipitate at the bottom?

When I’ve done transfers from Liquid to Agar, and Liquid to Grain, all appears to be clear

At least you’re getting good results with all the difficult steps! I have the highest contam rates when going from agar → LC.

I’ve been using a fresh syringe, and transferring in a flow hood. The only thing I can think of right now, is to try to spray the top of the LC jar with alcohol before piercing it, which I don’t think I’ve been doing.

Definitely spray the lid with alcohol and especially the injection site. Besides working with clean gear, this is the most important step. All of my LC → LC transfers are done in the open air and I basically never have issues (provided the first culture is clean, of course).

Since you’re not opening either of the vessels, I’d avoid using the flow hood for this step. Fresh needle, sterile syringe, alcohol’d injection site and you’re good to go.

[notplants]

Ok this is good to hear! I’ll try with alcohol spraying the port and hopefully that resolves the issue

I’ll have to take a closer look at some fresh LC to see about precipitate

[notplants]

Wanted to follow up on this,
after discussions with @Ale and Tuk who were seeing similar little bits in their LC,
we realized most of the ‘brown stuff’ I was seeing really was just precipitate from the honey, and not contamination. Not sure about that one brown ball (looks particularly suspect), but I think I dumped out a lot of other liquid culture which was probably not actually contaminated.

Here’s a colonized jar of grain which was inoculated from a second generation liquid culture (liquid to liquid):

I’ve inoculated a few other agar plates and grain jars from liquid that had brown bits in it and so far still just seeing white mycelium everywhere