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OfflineBlue Helix
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Liquid Culture - How to know if it's contaminated
    #8628727 - 07/13/08 09:53 AM (15 years, 8 months ago)

I've gotten a few questions as of late about how one knows if a liquid culture (LC) is contaminated.  There are two ways:

1) The mycelium grows in a matter of 12 hours and/or looks odd.  This is mold.

2) The liquid culture looks cloudy.  This is bacteria.

In my experience, most all liquid cultures DO contain some amount of bateria 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:


After a few days, either they MUSHROOM mycelium WINS:



OR the BACTERIA WINS:



OR the MOLD WINS (this looks also bacterial infected):



Generally 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.  For example this LC has some bacterial issues but it turned out clear as could be once the mycelium took over:




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.

PS - One last thing, don't make a spore solution so dense that it looks purple or something like that.  Bacteria love broken or damaged spores, and if there are enough of them in there, they'll chow down.  Keep the solution light to nearly clear.

Edited by Blue Helix (07/13/08 10:08 AM)

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Offlinerungi
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Re: Liquid Culture - How to know if it's contaminated [Re: Blue Helix]
    #9451150 - 12/17/08 05:10 PM (15 years, 3 months ago)

That mold looks like mushroom mycelium to me.  Is the rate of growth what you use as a criteria for identifying the mold as a contamination,or is it something visual.

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InvisibleJ3illy
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Re: Liquid Culture - How to know if it's contaminated [Re: rungi]
    #9451158 - 12/17/08 05:13 PM (15 years, 3 months ago)

Yea the mold and mushroom myc looks really similar.  I guess probably the best way to check is to just inoculate a single jar w/ the LC, unless there's other markers to look for.

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OfflineDamion5050
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Re: Liquid Culture - How to know if it's contaminated [Re: J3illy] * 1
    #9451177 - 12/17/08 05:16 PM (15 years, 3 months ago)

Best way IMO is to knock up a pint test jar cause it will colonize really fast.. Then you can just do G2G with the grain jar as well..

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Offlinerungi
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Re: Liquid Culture - How to know if it's contaminated [Re: Damion5050]
    #9491063 - 12/24/08 09:16 PM (15 years, 3 months ago)

Top picture:  How did you inoculate that liquid broth? Did you use chopped agar wedges?

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OfflineRECORD.V
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Re: Liquid Culture - How to know if it's contaminated [Re: Blue Helix]
    #13738787 - 01/05/11 11:58 PM (13 years, 2 months ago)

A few days ago ? Can you tell me exactly how many ago?

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InvisibleBarakanaten
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Re: Liquid Culture - How to know if it's contaminated [Re: RECORD.V]
    #13739248 - 01/06/11 02:55 AM (13 years, 2 months ago)

870 days ago...haha :oldthread:

no worries, I think I did that once too:lol:


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Offlinemasspan
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Re: Liquid Culture - How to know if it's contaminated [Re: Barakanaten]
    #13739700 - 01/06/11 07:18 AM (13 years, 2 months ago)

(...damn this lc is taking for fng ever, hope its strong)


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OfflineArush
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Re: Liquid Culture - How to know if it's contaminated [Re: rungi]
    #22309138 - 09/29/15 03:47 PM (8 years, 5 months ago)

What if nothing really happens?

My jars have sat for 2 weeks with no signs of any type of life.
No cloudiness or floaters. It only has honey sediment chunks floating around that looks partially white.

I did open one in a freshly sterile environment and took a whiff. It smelt like honey to a T.

I knocked up two half pint jars today to test them.


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Edited by Arush (09/29/15 03:48 PM)

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OfflineBlue Helix
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Re: Liquid Culture - How to know if it's contaminated [Re: Arush]
    #25414635 - 08/26/18 01:19 PM (5 years, 6 months ago)

Old reply but I figured I'd chime in here anyway since I'm such a big proponent of liquid cultures.  What if nothing happens?  It probably means you used a spore syringe for inoculation rather than spore print (or a dirty liquid culture not stored in a vacutainer).  If you do that, there is a much higher risk that bacteria will dominate the mix before the mycelium can release enough antibiotic to overtake it.  And when that happens you may or may not see cloudiness or mycelium strings (mycelium that grew then became bacterially contaminated). To prove it, one can put a drop on a petri dish and see what happens.

I am not a fan of spore syringes to inoculate liquid cultures.  They are fine for inoculating PF-style jars, but those tend to favor mycelium over bacteria whereas a liquid culture or agar surface favors both equally.  Even trough reputable vendors, I have found that spore syringes are often too bacterially contaminated to inoculate liquid cultures.

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Re: Liquid Culture - How to know if it's contaminated [Re: Blue Helix]
    #25461422 - 09/15/18 05:10 AM (5 years, 6 months ago)

I'm a total noob but I have tons of laboratory and clean room experience in the biotech sector. I absolutely love liquid culture so far as it is a great way to experiment and find a good process / recipe.

I have noticed that if you spin your culture a little bit too fast and for a little bit too long it'll also get cloudy I don't know if it's emulsifying the sugars with oxygen or if it's when mycelium first start to germinate and do not get a chance to Clump up thus making the solution cloudy? I have one right now that started out cloudy and his now cleared up as the mycelium slowly forms. It was a simple Karo syrup solution. I don't know what everyone else does but I do not pressure cook my LC. I water bath it for a long time. It is liquid it is coming from deionized water and supposedly sterile Karo syrup.

I think pressure cooking it is a bit over the top seeing as how I have canned food for 25 years and I know what non acidic food can be water bath in what acidic food as well. So basically if a food product can be water bath with no contamination, surely a jar of water and sugar can!


I have been doing a new Spore print method at least for me and simply taking a mason jar or a beaker and trimming a mushroom cap placing it on the bottom face down. And when it's time simply poking the cab removing it and adding the liquid culture. Oh I completely sterilize the jar prior to doing this and cover it with a sterilized watch glass. So you get all the Spore print without touching it.

I'm sure everyone else does this already but I came up with it the other day so I figured just in case LOL.


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InvisiblebodhisattaMDiscordReddit
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Re: Liquid Culture - How to know if it's contaminated [Re: Blue Helix] * 1
    #25462028 - 09/15/18 05:10 AM (5 years, 6 months ago)

This thread has been closed.

Reason:
Sugars don't emulsify with Oxygen

Water bath canned food has viable contaminants but they just don't germinate in the acidic conditions. There's absolutely nothing contaminant free about water bath canned food, they're there but we're relying on them not being able to grow in the anaerobic and acidic conditions which are not present in a LC.

Also this is a ten year old thread

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