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Offlinetrippleblack
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Using microscopes to indentify contaminated liquid culture
    #27001547 - 10/24/20 04:37 PM (3 years, 3 months ago)

Not sure if this is advanced; i don't think many will take this route, so assume the advance folks can only answer this question. 


I test liquid culture on agar before going bulk; this take several days to know if the culture is healthy enough to knock up grain bags.  Can i use a compound microscope to instantly check my liquid culture for contamination?

my main concern is bacteria. 

If this is a good route to take:

whats a good technique for analyzing this lc?  ie. a couple drops on a regular slide used to look for bacterial rods; or maybe a couple drops inside a petri slide...

thanks


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Re: Using microscopes to indentify contaminated liquid culture [Re: trippleblack] * 1
    #27002318 - 10/25/20 08:02 AM (3 years, 3 months ago)

Kind of. If your LC has a contamination of 1cfu per mL it's absolutely contaminated but you probably wouldn't catch it with a microscope. If your LC is at 100cfu/mL then your going to have an easier time. If its 10,000 units per mL it will be obvious.

Not all bacteria are rods either.

What would work best is if you took 1-5 mL of your LC and put it in a petri dish. Then pour agar into the plate with the LC. (Correct Pour temperature wont kill anything) then incubate and look for colonies. Of course the mycelium will grow and make it hard to see. So you would use cycloheximide to kill eukaryotic organisms and allows prokaryotic organisms to grow(bacteria) so then if you see any growth you know you have bacterial contamination


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Invisibleteknix
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Re: Using microscopes to indentify contaminated liquid culture [Re: trippleblack]
    #27002940 - 10/25/20 03:00 PM (3 years, 3 months ago)

You could use Crystal Violet stain to stain the cell walls to make it more visible.


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Offlinetrippleblack
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Re: Using microscopes to indentify contaminated liquid culture [Re: teknix]
    #27009458 - 10/28/20 11:12 PM (3 years, 2 months ago)

Thanks.  This remove all the guess work.  cycloheximide is a little pricey, but a gram will last a year.  I can't locate a better route to take than the two suggestions above. 

Violet Stain to do a visual inspection -because why not, then i will follow up with a cycloheximide nutrient agar dish.  Just tossed about 20+ spawn bags with bacteria from lc; i didnt see bacteria contam quick enough when i did a lc test on regular agar, earliest indication lc was iffy was a burnt smell; but i already knocked the bags up before the sniff.


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InvisiblebodhisattaMDiscordReddit
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Re: Using microscopes to indentify contaminated liquid culture [Re: trippleblack] * 1
    #27009632 - 10/29/20 04:31 AM (3 years, 2 months ago)

Honestly no one tests their LC. By the time you do all that you've entirely wasted the advantages of LC anyway. Just do g2g its faster, more reliable, and one less step for contamination


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Offlinetrippleblack
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Re: Using microscopes to indentify contaminated liquid culture [Re: bodhisatta]
    #27009815 - 10/29/20 08:18 AM (3 years, 2 months ago)

I see.

I used to do g2g, but running over 100+ bags g2g became inefficient.  For lc i'm going to use 2000 ml media bottles; currently i use 1000 ml jars.

I also deal with liquid culture on a semi commercial scale and I want to run quality assurance before sending them out.

In my situation would you still suggest no agar tests?


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InvisiblebodhisattaMDiscordReddit
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Re: Using microscopes to indentify contaminated liquid culture [Re: trippleblack]
    #27010060 - 10/29/20 10:34 AM (3 years, 2 months ago)

If you're going commercial scale I wouldn't use LC at all. You've got profits riding on your successfulness.



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Offlinetrippleblack
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Re: Using microscopes to indentify contaminated liquid culture [Re: bodhisatta]
    #27010128 - 10/29/20 11:04 AM (3 years, 2 months ago)

Ok.  commercially it the the sale of the liquid culture itself.

I can understand why commercial scale mushroom growers wouldn't use lc.  I'm rethinking my use of lc for bulk grows now.


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Re: Using microscopes to indentify contaminated liquid culture [Re: trippleblack] * 1
    #27010142 - 10/29/20 11:08 AM (3 years, 2 months ago)

Big huge farms use LC. And also have labs that run PCR to ensure clean cultures

But of course you can do whatever. I just personally think you're playing with fire.

There's a reason breweries and wineries almost all buy yeast rather than do in house propagation. They leave the culturing work to lab techs not plant employees


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Offlinetrippleblack
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Re: Using microscopes to indentify contaminated liquid culture [Re: bodhisatta]
    #27010161 - 10/29/20 11:16 AM (3 years, 2 months ago)

Ahhhhhh...... i just got a chub...

pcr...

thanks for that.  I was getting a pcr in near future for non lc stuff, I was pondering just yesterday if i can use a pcr to amplify and id bacterial contamination.

I am playing with fire.. thats why i'm trying to take the most professional approach as a amatuer.


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Re: Using microscopes to indentify contaminated liquid culture [Re: trippleblack] * 1
    #27010184 - 10/29/20 11:34 AM (3 years, 2 months ago)

Pcr is a great tool but tools suck if used improperly. You generally assume you'll run into specific contamination and have primers for those. But if you entirely miss the contamination that you have because of your protocols with your pcr machine then it's as good as a paperweight


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Re: Using microscopes to indentify contaminated liquid culture [Re: trippleblack]
    #27018546 - 11/03/20 12:26 AM (3 years, 2 months ago)

You could also try plating out some liquid on bacterial screening media, or a sterility test medium that is targeted towards bacterial vs. fungi. Or add in a broad-spectrum anti-fungal to emphasize bacterial growth.


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Re: Using microscopes to indentify contaminated liquid culture [Re: Bloo] * 1
    #27018874 - 11/03/20 08:17 AM (3 years, 2 months ago)

Like what the second post said?


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OfflineBloo
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Re: Using microscopes to indentify contaminated liquid culture [Re: bodhisatta] * 1
    #27022478 - 11/05/20 12:18 AM (3 years, 2 months ago)

Sort of, yes, but in industry we don't do a warm pour, we just plate it onto finished agar. At least that's how it's done with injectables for the European Medicines Agency, I don't know about FDA regulations in America, and for insect cell culture, and mammalian cell culture. There's probably more in the FDA's Bacteriological Analytical Manual (BAM).


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Re: Using microscopes to indentify contaminated liquid culture [Re: Bloo] * 1
    #27022606 - 11/05/20 04:55 AM (3 years, 2 months ago)

Depends what's being looked for. Spread plates work well for some things pour plates work well for other things like anaerobic organisms. Id be more interested in beer, wine, dairy qc methods than medical ones to adapt to mycology anyway


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Offlinetrippleblack
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Re: Using microscopes to indentify contaminated liquid culture [Re: bodhisatta]
    #27030699 - 11/09/20 07:46 PM (3 years, 2 months ago)

researching this tons..

It seems as if i can do a ok job with viewing bacteria through a phase contrast compound microscope, i' took route using gram staining techniques; if culture is loaded with bacteria i should be able to detect, but i'm sure their is a lower threshold. 

brew companies use cycloheximide agar plates, i'll be playing with this too, but it is a bit a dangerous.. i want a pcr for christmas, seems so efficient.


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OfflineColonna45
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Re: Using microscopes to indentify contaminated liquid culture [Re: trippleblack]
    #28219888 - 03/07/23 11:14 PM (10 months, 15 days ago)

Hey I'm sorry to be replying to you on an older thread but I had the same thoughts you did. LC to Agar is a little time consuming to make sure it's correct and I have a microscope so I could just take a look to see if I'm in good shape. Do you have any pictures of what LC under the scope should look like? If I posted some would you be able to help me understand what I'm looking for? I'm colorblind so I'm gonna have to teach my girlfriend too lol but my microscope can take pictures at least


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Re: Using microscopes to indentify contaminated liquid culture [Re: Colonna45]
    #28232188 - 03/16/23 02:57 PM (10 months, 7 days ago)

Quote:

Colonna45 said:
Hey I'm sorry to be replying to you on an older thread but I had the same thoughts you did. LC to Agar is a little time consuming to make sure it's correct and I have a microscope so I could just take a look to see if I'm in good shape. Do you have any pictures of what LC under the scope should look like? If I posted some would you be able to help me understand what I'm looking for? I'm colorblind so I'm gonna have to teach my girlfriend too lol but my microscope can take pictures at least




if you dont have a pcr then plating is the only option from what is posted above.  you totally pulled a dead thread up though but i kinda enjoyed the read.


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