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Offlineinvitro

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Re: The Visual Signs Of A Contaminated Liquid Culture. [Re: SpitballJedi]
    #21770424 - 06/06/15 01:03 PM (8 years, 11 months ago)

Mikebearpig says trich can form dark specks. 

I also have another clue.  I have one culture which I think is good and one culture which is almost certainly some kind of contamination.  It's growth has been extremely rapid, maybe 20 times faster than normal.  Both cultures were transferred from old cultures (20ccs worth).  Both cultures had a few pieces stick to the side of the glass.  The one (I hope good) culture shows growth rings, about 5 or 6 of them, like a tree.  I think it's adding a ring every day, i just noticed this phenomenon today.  The other culture has no growth rings (on the colonies sticking to the glass).  The leading edges of the contam culture are rhizo and the slower culture tomentose.  I don't see any dark specks in the fast culture however.  I suspect the culture I'm infected with is penicillium but I don't know yet, I'm not that good at iding it.



I don't have a pic of the contamed culture, the lighting wasn't good and I moved the culture which knocked off all the side-colonies.

I'm about to whip up the fast culture and see if it will form an island and show it's true nature.

Edited by invitro (06/06/15 01:27 PM)

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Offlinecamplo
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Re: The Visual Signs Of A Contaminated Liquid Culture. [Re: invitro]
    #22934793 - 02/22/16 06:27 PM (8 years, 2 months ago)

So much good info here.
Thanks to Bodhisatta for playing devils advocate and Eats for his long time wisdom. Thanks to Hamloaf for making a current thread on the topic!
So, appropriately titled, visual signs of LC contamination;
My experience has been that if a LC is left undisturbed, bacteria will show itself by growing in colonize on the sides of the glass or floating growths show on the surface or in the media. Turbidity is easiest to detect unless the solution were cloudy from the get go. I've got a malt/coffee lc going that I have no idea whats going on, can't see shit.. I think mold would be less discernible, if left to grow a colony on the surface it would sporulate (provided enough GE) but it may take a long time to do that. I've never had mold in LC yet, so I dunno.

I do think a percentage of contam has to do with the grains, rather the LC. I think if you are going to use LC or any liquid based inoculate you should take the effort to know what moisture percentage your grains are at and adjust them to receive the inoculate if needed. I think its good advice to aim for a dryer post inoculate moisture content as well. Thats just some of my personal philosophy.

I am not certain how to feel about not being able to spot contams 100%. It has been said and I agree that it seems that with bacteria growth being so prolific that it would be impossible for it to not show itself in an LC. It seems... I've seen yeast in an LC. It grew on the grains that I PC'd inside the LC, little white specs. That LC was cloudy as well, most likely bacteria as well. That was my lesson on how grains, itself, provide another layer of insulation to the sterilizing heat and steam of the pressure cooker.

I like the idea of being able to know. Even if on agar I've heard tell that sometimes a contaminate did not express itself until a 3rd transfer. That scared me because I use agar but I don't do anything fancy with it other than germinate spores and for cloning.

There is also the concept that "contaminates" are always growing with our mycelium. Complete sterility is not exactly possible, to a certain extent, and that really we create "windows" which is more appropriate called, shelf-life.


So whats the answer?  How to tell visually if a LC is contamed: A motherfuckin microscope. Right? Squirt a little LC on the slide and view it. I think I'm on the right track at least. How expensive of a microscope would one need for such a task? If it was less than a couple hundred dollars, I could see myself getting one real soon.

thanks in advance


Bottom line is sterile technique really but you have to expect that even if everything is done perfect, if the contam isn't detectable you can't know its there. Even on agar it seems. Even more reason to use a microscope.


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Edited by camplo (02/22/16 06:32 PM)

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InvisiblePastywhyteMDiscord
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Re: The Visual Signs Of A Contaminated Liquid Culture. [Re: camplo]
    #22934810 - 02/22/16 06:30 PM (8 years, 2 months ago)

Being careful with LC is always good practice :thumbup:

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InvisiblebodhisattaMDiscordReddit
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Re: The Visual Signs Of A Contaminated Liquid Culture. [Re: Pastywhyte]
    #22934846 - 02/22/16 06:42 PM (8 years, 2 months ago)

Coors had a huge contamination problem from pediococcus once. it wouldn't grow on the media they used. so they thought everything was clean. Microscopically you couldn't really see it it's very small and if your cell counts are low then good luck finding it.

eventually they spoiled after months on the shelf since the organism grew so slow in the finished product. Before Miller owned Coors, Miller plated it on their beer media and the pediococcus grew and they knew what Coors problem was before Coors did

there was a soda and juice factory in town that released a slimy product to people a few years ago it was infected with leuconostoc and didn't make the product slime up right away.

lots of clear liquids with even more nutrition than a LC can look clean for a while then go bad. or harbor a bacteria that won't grow in the conditions it's in right now but might when you use it on spawn, or even during the spawn run for that matter.

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Offlinecamplo
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Re: The Visual Signs Of A Contaminated Liquid Culture. [Re: bodhisatta]
    #22934868 - 02/22/16 06:47 PM (8 years, 2 months ago)

I just googled compound microscope. Unless I'm mistaken, at those prices, why wouldn't you use a microscope!? I thought I was going to hurt my pocket book, looks like I'll be spending well under 100 dollars, especially if scoping LC is my only endeavor.


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Re: The Visual Signs Of A Contaminated Liquid Culture. [Re: camplo]
    #22934893 - 02/22/16 06:56 PM (8 years, 2 months ago)


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Offlinecamplo
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Re: The Visual Signs Of A Contaminated Liquid Culture. [Re: bodhisatta]
    #22934912 - 02/22/16 06:58 PM (8 years, 2 months ago)

Quote:

bodhisatta said:
The cheapest microscope I recommend is about 180
http://www.amscope.com/compound-microscopes/40x-1000x-led-lab-siedentopf-binocular-compound-microscope-with-3-d-double-layer-mechanical-stage.html





Could you elaborate on why thats the cheapest one you'd recommend? I know you know your stuff but for those of us un-enlightened on the topic, why? There are many scopes with seemingly enough magnifying power under 100 dollars.


Heres that same microscope for 150...thats right up my alley!
http://www.ebay.com/itm/like/381347257727?ul_noapp=true&chn=ps&lpid=82

Edited by camplo (02/22/16 07:05 PM)

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Re: The Visual Signs Of A Contaminated Liquid Culture. [Re: camplo]
    #22934983 - 02/22/16 07:15 PM (8 years, 2 months ago)

same thing, if you want to buy it on ebay go for it. the 25x eyepieces for these scopes are garbage and 2500x doesn't really work

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Offlinecamplo
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Re: The Visual Signs Of A Contaminated Liquid Culture. [Re: bodhisatta]
    #22935049 - 02/22/16 07:34 PM (8 years, 2 months ago)

If I don't want to use the 100x oil objective how much magnification would I need to see bacteria/contam in LC?
OK you've already answered that question here( https://www.shroomery.org/forums/showflat.php/Number/21952416#21952416 )....still not sure why this couldn't be done with a cheaper microscope.


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Re: The Visual Signs Of A Contaminated Liquid Culture. [Re: camplo]
    #22935060 - 02/22/16 07:38 PM (8 years, 2 months ago)

400x with stains. or 400x phase contrast(500+ dollar microscopes)

you could buy plate count agar and test your LC with that too.

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Offlinecamplo
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Re: The Visual Signs Of A Contaminated Liquid Culture. [Re: bodhisatta]
    #22935069 - 02/22/16 07:41 PM (8 years, 2 months ago)

So 400x phase contrast is the only way to see basteria without stain or oil. Thanks for your help.


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Re: The Visual Signs Of A Contaminated Liquid Culture. [Re: bodhisatta]
    #22935076 - 02/22/16 07:42 PM (8 years, 2 months ago)


400x brightfield


400x phase contrast

cheek cell. you can see the bacteria on the cheek cell in the phase one and the big rod shaped bacteria at 4:30 or bottom right.
on the phase one it's way easier to see the intracellular components too

you might be able to see some bacteria pretty well with 400x and no stains but not the other half of them

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Offlinecamplo
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Re: The Visual Signs Of A Contaminated Liquid Culture. [Re: bodhisatta]
    #22935343 - 02/22/16 09:00 PM (8 years, 2 months ago)

Well that idea died fast lol! Seems that microscopy isn't even a reliable way to search for contam in LC (This is just what I've read on the forums, if anyone disagrees speak up)? You'd be better to grow out a sample on agar sounds like. I think maybe I'm getting sucked into the "what ifs" and maybe I should just focus on good sterile technique.

My OCD is going out of control, what if I contam my grain/LC/LI/Agar upon checking to see if its contamed!>!>!:freakout:
Then you'd have to check it again, but what if I contamed it while I was checking it to see if I contamed it while checking it for contams!>!>!:shitmypants:
Then I'd have to check it to to see if I contamed it while checking to see if I contamed it while checking for contams:ewaahh:
I think I see a pattern...

So thus, I feel a focus on good sterile technique is King. From the experience of the past it can be said that you will achieve low contamination rates with good sterile technique but you will expect a small amount of contaminated LC. Checking your LC with agar might be suggested but is pointless if your sterile technique is no good for you'll just contam it later or during usage or while checking it even. This is true for all forms of inoculate. although some of the other forms allow a visual check right before usage. Even still with grain, a mold could be present and you wouldn't know it! You could have bacteria in your grains and not know it until you spawn to bulk and maybe it won't colonize or maybe it will fruit at 85% its potential and you never notice....maybe got shot, and if got ran over
:matrosjkacup:


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Edited by camplo (02/22/16 09:06 PM)

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Re: The Visual Signs Of A Contaminated Liquid Culture. [Re: camplo]
    #22935352 - 02/22/16 09:04 PM (8 years, 2 months ago)

Nothing can really be 100% guaranteed, procedure will always be a vector. But we can still giver a pretty good shot. When I test an LC I usually do 3 plates, 3 cakes, and a few quarts of grain. If those all fail its pretty small potatoes but once they prove clean I then try to use all the LC as soon as possible. LC sitting around is a bad idea IMO.

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Offlinecamplo
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Re: The Visual Signs Of A Contaminated Liquid Culture. [Re: Pastywhyte]
    #22935455 - 02/22/16 09:31 PM (8 years, 2 months ago)

Quote:

Pastywhyte said:
Nothing can really be 100% guaranteed, procedure will always be a vector. But we can still giver a pretty good shot. When I test an LC I usually do 3 plates, 3 cakes, and a few quarts of grain. If those all fail its pretty small potatoes but once they prove clean I then try to use all the LC as soon as possible. LC sitting around is a bad idea IMO.




Agreed. I don't like opening a LC more than once to be honest. Not just LC but anything containing sterile material. I'm amazed that I can keep going back into my master of agar successfully.

you can't be 100% sure your LC is contamed until you use it...
you can't be 100% sure your GRAIN is contamed until you use it...


Both of those statements are 100% correct lol Just like contams can hide in LC, they can hide in Grain as well, even if its more likely to happen with LC than Grain, it can happen.
I've read that to completely sterilize grain you'd have to PC for like 12 hours at 15psi. RR said that actually. I thought it was pretty interesting. Its like life really really really doesn't want to die or something.


Pasty you are way more cautious than I am my friend. Which is why I was looking to see if there was some "safer" way when there really isn't, outside of having impeccable sterile tech and a microscope to boot. Someone told me they have had mycelium on agar that finally showed a contam after 3 transfers. Even agar isn't safe? We are all doomed.!:mindblown:


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Re: The Visual Signs Of A Contaminated Liquid Culture. [Re: camplo]
    #22935488 - 02/22/16 09:40 PM (8 years, 2 months ago)

The worst I ever got burnt with dirty LC was 4 bags of grain. I was pissed to because the first test plate was clean but a second younger plate showed bacteria a few days later. The bloom happened after the firsr test. Funny thing was the grain still colonized fine except for a quarter size patch at the very bottoms of the bags. I also cased and top fruited the bags and got something like 5 flushes, they never did mold.

But I can see just how easy it would be to blow a huge grow and not even know it until too late. I treat LC with care and because of that I have yet to have a major fail with it.

I have also had several go bacterial before I even inoculated it. Temps got high for a few days while I was camping and had the AC off. Came home to some stank ass LC. Since then I take great care to colonize then in temps no higher than mid 70s and never let them sit for too long. I also sterilize them for longer than most, even malt or SITR LC which should be safe. I take no chances.

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Offlinecamplo
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Re: The Visual Signs Of A Contaminated Liquid Culture. [Re: Pastywhyte]
    #22936826 - 02/23/16 10:21 AM (8 years, 2 months ago)

I agree with increasing the the efficacy of the sterilization whether it be by adding time or additional temp/pressure. From my studying thermal death times decrease significantly with added heat.
(B.  sporothermodurans  D140 = 3.4–7.9s & z=13.1–14.2°C )
So at 125.8°C (258.44°F)  D125.8=79s for UHT (moist heat) conditions

(B. stearothermophilus D140=0.9s Z=9.1°C)

Bacillus subtilis - supposed common grain bacteria.
Moist Heat TDT
D121°C = 1min.  Z=58.3 (I got the Z from a different source than the TDT)
Dry Heat
D121°C = 121min

I think I'll by a microscope regardless of the information I read saying that you could miss seeing a contam with LC. Once again, nothing is 100% but it will help plus its pretty interesting to me so, I think I am going to buy the monocular version of the one Bod suggested, especially with it being half the price of the binocular one. The fact that you still pulled the grow off, with a slightly contamed LC versus some large spawn bags gives me hope, sounds like a tiny space of forgivable error. I am also inoculating a large amount of grain....you already know. lol. I think keeping tight regiments on moisture is also key to using LC. I think a dryer grain is less prone to contam as well.


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Re: The Visual Signs Of A Contaminated Liquid Culture. [Re: spacechildo] * 1
    #25179264 - 05/02/18 04:40 AM (6 years, 17 days ago)

Some contaminated lc. Honey lc,

I still look at them thinking...they look so good(the myc). But definately has the hazy look to the uncolonized area. In the third pic you can see the fog rising above the myc.

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Re: The Visual Signs Of A Contaminated Liquid Culture. [Re: BigSurMoon]
    #25180111 - 05/02/18 02:39 PM (6 years, 17 days ago)

Here goes a LC with several visual signs of contamination happening such as, but probably not limited to, discoloration, cloudiness, and a different looking type(s) of mycelial culture suspended in the media broth. 
 

The way that this LC has separate bits of mycelial culture suspended all through out the media is also a visual sign of a contaminated liquid culture.

Thanks for sharing, BigSurMoon.


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