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Shroomway
It's me!
Registered: 07/05/17
Posts: 438
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Why often no contamination on spore prints?
#24482413 - 07/14/17 09:58 AM (6 years, 8 months ago) |
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Hi all,
The subject line sais it all; why is it that there is quite often no visible contamination from a sporeprint when you scrape some of it on a agar plate. Or even if a syringe is made from it which is used on agar (which is an extra contamination possibility in itself).
To me this is quite hard to understand because if you expose an agar petri dish to open air for a few seconds, it is almost for sure contaminated.... Since spore prints are almost impossible to make in a completely sterile way (no matter which technique you employ) it is not hard to understand my question I hope/suppose.
Does anyone has an answer to this?
Thanks in advance! Shroomway
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woodrow
journeyman
Registered: 03/17/03
Posts: 142
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Re: Why often no contamination on spore prints? [Re: Shroomway]
#24484363 - 07/15/17 02:41 AM (6 years, 8 months ago) |
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Agar plates should not contam if you expose them to the air for a few seconds unless you are working in a drafty barn. Where did you get that idea?
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Shroomway
It's me!
Registered: 07/05/17
Posts: 438
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Re: Why often no contamination on spore prints? [Re: woodrow]
#24484437 - 07/15/17 04:34 AM (6 years, 8 months ago) |
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Hi, ...I found out by working with agar without a glove box/SAB.... At home, in (relatively) clean air.
If you treat an agar dish like a spore print (the most common and sufficient methods that are out there) it contaminates. Yet if you scrape some spores off a print and inoculate an agar plate with it, OFTEN there will be no contaminants growing on the agar
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TheMadHatter420
Trusted Farmer
Registered: 10/12/16
Posts: 12,941
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Re: Why often no contamination on spore prints? [Re: Shroomway]
#24491289 - 07/18/17 09:19 AM (6 years, 8 months ago) |
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You are all fucked up. You can NOT do agar work in the open air. That air is not even relatively clean. There are millions, if not billions of mold spores floating around any and all houses. Its there, you just can not see it. Those prints can and usually do carry contamination, it is more often bacteria as opposed to mold, unless print was taken from a moldy tub. What grows on the agar after scrapping spores id based off how clean that print was, such as from bacterial/moldy tub or from a clean healthy tub.
-------------------- JOIN THE POW WOW
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woodrow
journeyman
Registered: 03/17/03
Posts: 142
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Re: Why often no contamination on spore prints? [Re: TheMadHatter420]
#24493194 - 07/19/17 12:34 AM (6 years, 8 months ago) |
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Agar work can be done in open air with only an occasional contam in most environments. But, how do you you "treat an agar dish like a spore print"? Your explanation is not clear.
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Shroomway
It's me!
Registered: 07/05/17
Posts: 438
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Re: Why often no contamination on spore prints? [Re: woodrow]
#24493248 - 07/19/17 01:36 AM (6 years, 8 months ago) |
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Quote:
woodrow said: ...how do you you "treat an agar dish like a spore print"? Your explanation is not clear...
What I mean is: Take a mushroom cap and print it (usually it involves putting a bowl of some sort over it and letting it sit for a longer period of time).
Do the same with an agar petri dish (imagine having one the size of the mushroom cap).
Now there is a big chance the petri will be contaminated. So will the spore print probably, but that is not visible. Yet, if you use the spore print to inoculate (something) there is a pretty good chance you are not going to be troubled by contamination.
This is what I am curious about... I prefer working in a glove box myself...
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Bunya
Registered: 06/04/16
Posts: 601
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Re: Why often no contamination on spore prints? [Re: Shroomway] 1
#24493264 - 07/19/17 01:53 AM (6 years, 8 months ago) |
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The agar dish contains moist nutrients that encourage and support the growth of bacteria and fungi. The spore print has no nutrition or moisture if done corectly so the bacteria and fungi doesnt grow. If the print has been exposed to bacteria or bad spores there is a chance of them growing when you transfer to agar. Because you are taking a small sample from the print to agar, your odds of picking up undesirables is reduced. (Asuming done in a still air box).
-------------------- Bunya cones kill.
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Shroomway
It's me!
Registered: 07/05/17
Posts: 438
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Re: Why often no contamination on spore prints? [Re: Bunya]
#24493520 - 07/19/17 06:00 AM (6 years, 8 months ago) |
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Quote:
Bunya said: The agar dish contains moist nutrients that encourage and support the growth of bacteria and fungi. The spore print has no nutrition or moisture if done corectly so the bacteria and fungi doesnt grow. If the print has been exposed to bacteria or bad spores there is a chance of them growing when you transfer to agar. Because you are taking a small sample from the print to agar, your odds of picking up undesirables is reduced. (Asuming done in a still air box).
Yeah, you must be right. Because there is no moisture there is no grow and no spreading! I could have come up with that myself. Good one... thanks
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Solipsis
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Re: Why often no contamination on spore prints? [Re: Shroomway]
#24560206 - 08/17/17 11:18 AM (6 years, 7 months ago) |
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Usually it is a matter of which organisms get the headstart on something and dominate. Also true for example with meat: both bacteria and flies will want to eat meat, but usually one of them will get going on it first and 'win out'.
If you start brewing cider or beer it's really not in entirely sterile conditions, but you add a lot of yeast (preferably as an active starter) and the yeast will multiply and dominate. Contaminations become a problem mostly when somehow the number of active yeast drop and there are still nutrients there.
As said, in a spore print or syringe there are no nutrients. Not only that, but any contaminant unless hefty is incredibly outnumbered and will often be eaten for breakfast. The agar plate is not the same thing: there is a lot of nutrients there and not yet an established dominant species.
Another way of saying something like Bunya said, but IMO it's a little more sophisticated: the food available relative to the stage of colonization and the numbers of contenders.
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blackout
Registered: 07/16/00
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Re: Why often no contamination on spore prints? [Re: Shroomway]
#24560609 - 08/17/17 02:31 PM (6 years, 7 months ago) |
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Quote:
Shroomway said: Take a mushroom cap and print it (usually it involves putting a bowl of some sort over it and letting it sit for a longer period of time).
Do the same with an agar petri dish (imagine having one the size of the mushroom cap).
You put a bowl over the shroom, and the shroom itself acts like an umbrella too. Then you take the cap off the foil and close it, so it is only in open air for a few seconds. The petri exposed like this will likely contaminate in those few seconds.
The mistake is saying it's the same size as the petri, it is the same size but you do not use the entire print on the petri you are working with. Instead you take a tiny spec of the print in a clean environment and transfer to agar. A tiny % of the overall area of the print.
So a better comparison would be leaving your petri in open air for several seconds. Then immediately bringing that contaminated petri into an SAB, and immediately transferring a tiny spec of that contaminated plate to a clean plate. Now you have hugely reduced the likelihood of transferring contams.
In a few days time your orginal contaminated petri might have loads of visible contams, but these were microscopic to start with, and have just grow a lot bigger, so it can appear as though a huge % of the area had contams on it, but this is only since they have grown outwards and developed just like your germinating mushroom spores would have.
This is why I say the fair comparison is to immediately take a tiny spec from the contaminated plate, there is only a very small % of surface area where contams landed. When contams land on your print they have no real nutrition like a agar plate would, so they are not growing and increasing outwards.
EDIT- only see Bunya's post now, which was the pretty much the same...
Edited by blackout (08/17/17 02:41 PM)
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AlexSmitt
Smitty Mushroomjagermanjenson
Registered: 09/10/16
Posts: 953
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Re: Why often no contamination on spore prints? [Re: blackout]
#24588422 - 08/29/17 04:36 PM (6 years, 6 months ago) |
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I can't say this is the reason why you don't see contams on your prints when you put them to agar but I can say doing your own grow with proper sterile technique and keeping your growing space clean cuts contam risk, then the veil is added protection till it begins to tear. So with filters such as polyfil or filter disks being yet another contam barrier along side the brief hours the gills are exposed AND taking spore prints in a sab I can't imagine too many airborne bacteria/mold spores make it into a tub and then move upward onto the gills under the cap. Lets say that does happen, you then only use a small portion of that print, a portion that may not have dropped the contams that more than likely attached themselves to the gills anyway and wont drop. You don't need many spores to get started so that minute surface area you scrap/swab from is likely to not have something bad hanging around. once on agar there's still the chance its lying dormant and just piggy backing, and who knows if the bacteria occupying that space is even a threat, you may just get a little metabolites and have a less than optimal flush but still have a successful grow. Myc isn't as weak as everyone makes it seem, it can overcome some contams. All these reasons help reduce the risk on contams but ultimately it'll always be a gamble.
-------------------- it's only logical
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Shroomway
It's me!
Registered: 07/05/17
Posts: 438
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Re: Why often no contamination on spore prints? [Re: blackout]
#24590206 - 08/30/17 07:47 AM (6 years, 6 months ago) |
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Quote:
blackout said:
In a few days time your original contaminated petri might have loads of visible contams, but these were microscopic to start with, and have just grow a lot bigger, so it can appear as though a huge % of the area had contams on it, but this is only since they have grown outwards and developed just like your germinating mushroom spores would have.
This is why I say the fair comparison is to immediately take a tiny spec from the contaminated plate, there is only a very small % of surface area where contams landed. When contams land on your print they have no real nutrition like a agar plate would, so they are not growing and increasing outwards.
True... in essence Bunya indeed already answered my question. The absence of water and nutrients in a print (and of course the fact that only a tiny bit of spores are scraped off [as locally as you can]) makes the difference. To me, at least those things seem key...
@AlexSmitt: I do totally agree that you can prbably get away with a certain amount of contams. Depending on a lot of things (not unimportantly which contam). I do not agree though that there will not be a lot of contams underneath the veil when it is broken for a few hours or a night. I guess the question would be: how do you quantify: "a lot" or "not much". Enough contaminants though to make it a wise decision to transfer to agar before innoculating anything (except for cakes). So yeah, what is a lot? Interesting thread, good answers....
Thanks a lot! :)
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kingkc
enthusiast
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Re: Why often no contamination on spore prints? [Re: Shroomway]
#24590691 - 08/30/17 12:16 PM (6 years, 6 months ago) |
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My spore prints are always without any thing other than spores.
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Shroomway
It's me!
Registered: 07/05/17
Posts: 438
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Re: Why often no contamination on spore prints? [Re: kingkc]
#24590754 - 08/30/17 12:54 PM (6 years, 6 months ago) |
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Quote:
kingkc said: My spore prints are always without any thing other than spores.
Like AlexSmitt sais you are more likely getting away with the contams. There will probably be some there. Most pro's here check their agar inoculated LC's on agar before use. If your spore prints are so clean it means you could consistantly innoclulate LC's with them. That does not seem very likely to me. But maybe you are right. That just means you are very very good at what you are doing.
Edited by Shroomway (08/30/17 01:07 PM)
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bodhisatta
Smurf real estate agent
Registered: 04/30/13
Posts: 61,891
Loc: Milky way
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Re: Why often no contamination on spore prints? [Re: kingkc]
#24590775 - 08/30/17 01:08 PM (6 years, 6 months ago) |
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Quote:
kingkc said: My spore prints are always without any thing other than spores.
You're not Unless you're growing fruits in-vitro and also printing in a clean room.
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kingkc
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Re: Why often no contamination on spore prints? [Re: bodhisatta]
#24609764 - 09/06/17 07:27 PM (6 years, 6 months ago) |
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I am pretty good at making spore prints. And , most folks are pretty good at making spore prints too. While some like to use syringes and , they are popular , there is that one vector -spore syringes have to have a spore print first - which makes for twice handled for a syringe and once handled for a print.
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Solipsis
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Re: Why often no contamination on spore prints? [Re: kingkc] 1
#24610705 - 09/07/17 02:50 AM (6 years, 6 months ago) |
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One of the few ways to find out would be to dilute your spore syringe after shaking it and putting a lot of droplets on agar.
If you have excellent sterile procedure i think you could still get contams because the dilution should lighten the competition.
Normally there are likely to be such a masive amount of spores that they outcompete most contams. I think not getting any virulent contams from prints doesn't guarantee at all that technically your prints are perfectly clean.
That is just an assumption you are making.
The main problem seems to me how to test this, and undo the competition. I suggested doing a lot of plates to maximize the chance of finding contams after dilution of the suspension.
Edited by Solipsis (09/07/17 02:53 AM)
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kingkc
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Re: Why often no contamination on spore prints? [Re: kingkc]
#24711891 - 10/15/17 01:26 PM (6 years, 5 months ago) |
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I simply place all things on a relatively clean surface area , place the caps on individual pieces of cut up polymer covered paper , cover said area with anything reasonable and allow the spores to drop for about 24 hours or so and then fold closed and tape up. It may be an old fashioned way of doing things but I am an old fashioned kind of guy.
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