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OfflineBlue Helix
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Liquid Culture - How to know if it's contaminated
    #8628727 - 07/13/08 11:53 AM (6 years, 3 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 12:08 PM)


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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 07:10 PM (5 years, 10 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 07:13 PM (5 years, 10 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]
    #9451177 - 12/17/08 07:16 PM (5 years, 10 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 11:16 PM (5 years, 9 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 - 01/06/11 01:58 AM (3 years, 9 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 04:55 AM (3 years, 9 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 09:18 AM (3 years, 9 months ago)

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


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Offlinegreencavefloat
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Re: Liquid Culture - How to know if it's contaminated [Re: Blue Helix]
    #15889346 - 03/02/12 09:43 AM (2 years, 7 months ago)

Quote:

Blue Helix said:

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. 

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:






Actually i change my mind this post is terrible. Really no way to tell if LC is contam just by looking at it. Just cos it clear dont mean it safe. Got to make sure your prinbt or agar are clean first and then, and only then, go to LC


Edited by greencavefloat (05/27/12 12:29 AM)


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