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OfflinePutACapInHisAss
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Agar Plates...Am I Lucky Or...? How is Your Plate Contam Rate?
    #23432383 - 07/11/16 03:19 PM (7 years, 6 months ago)

I've read some of the major posts with plenty of contamination pics. So far, I haven't seen anything like them on my plates after a few transfers. Heck, I didn't even see anything on my first plates from 4 different syringes and 2 sponsors.

Am I just really lucky so far? Or is it just likely that my myc has consistently overgrown contams that I have not seen?

I think my prevention procedures are fairly good but I cannot see how they could be anywhere near perfect. I engage quite a few of the recommended precautions but surely there must be something alive in my SAB.

Do any of you have a high number of plates on which you do not see any contaminants? What is your visible contam rate?


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Re: Agar Plates...Am I Lucky Or...? How is Your Plate Contam Rate? [Re: PutACapInHisAss]
    #23433334 - 07/11/16 09:25 PM (7 years, 6 months ago)

Could be luck. Vendor syringes should be pretty clean.
Out of my last 12 plates only one contaminated and those were made from a print > agar using a print from EYA


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Re: Agar Plates...Am I Lucky Or...? How is Your Plate Contam Rate? [Re: GreenRabbit]
    #23433477 - 07/11/16 10:06 PM (7 years, 6 months ago)

Well from spores is always a crap shoot. Ditto for cloning sometimes. But the best way to evaluate your technique is for the clean transfers when isolating or expanding to more plates. There if you screw up it appears as a satellite colony.

I am probably about 98% success rate for avoiding satellites. It would be better but I got hammered one night a few years ago and screwed up a few plates :rofldrunk:

Once you get your movements down you might see a spot pop up every 100 plates or so.


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Re: Agar Plates...Am I Lucky Or...? How is Your Plate Contam Rate? [Re: Pastywhyte]
    #23433533 - 07/11/16 10:20 PM (7 years, 6 months ago)

cotams happen no matter what but ya success rate mine pretty good
it best to take more xfers than u need to pad your bed :thumbup:
expericance is key the more you do the better you get :headbang3:


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Re: Agar Plates...Am I Lucky Or...? How is Your Plate Contam Rate? [Re: tripdawg420]
    #23433584 - 07/11/16 10:33 PM (7 years, 6 months ago)

Quote:

tripdawg420 said:
expericance is key the more you do the better you get :headbang3:




This. In fact a big part is developing the muscle memory. I took a few months off this year with zero agar work and let me tell you, when I went back to do some transfers I felt like a noob again. Practice is totally key.


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OfflinePutACapInHisAss
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Re: Agar Plates...Am I Lucky Or...? How is Your Plate Contam Rate? [Re: Pastywhyte]
    #23435054 - 07/12/16 02:08 PM (7 years, 6 months ago)

Quote:

Pastywhyte said:
There if you screw up it appears as a satellite colony.





You mean satellites of mycelium? I've had a couple of those. One with a little bit of agar attached grew some beautiful myc on the side wall inside my Pasty plate :smile:

As for the physical logistics of transfers, my biggest problem so far has been getting the myc off of my scalpel and onto the new plate. It often sticks fiercely! A couple of times I have resorted to using tweezers which isn't ideal--bringing in a second tool.

I have been considering trying a whole different approach. As some of you may have read, acidified bleach supposedly will kill just about any pathogen. I've been thinking, why not just have a shallow tray of this solution in my SAB and let the tools rest in it between uses. Then there might be less of a problem using the additional tool. This may not be a perfect solution but neither is a SAB. The real question is: would it be good enough?

With this method I could use the additional tool and also eliminate taking my hands in and out of the SAB for flame sterilization of the tools. This would save tons of time, reduce the likeliness of getting contams on my gloves outside the SAB, and push less air in and out of the box with far less movement involved.


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Re: Agar Plates...Am I Lucky Or...? How is Your Plate Contam Rate? [Re: PutACapInHisAss]
    #23435211 - 07/12/16 03:14 PM (7 years, 6 months ago)

Chemicals have no place in this work. Try to stab through the wedge into the reciting plate and drag the scalpel out in the same motion. Use a fresh(er) blade as well if that doesn't work.


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OfflinePutACapInHisAss
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Re: Agar Plates...Am I Lucky Or...? How is Your Plate Contam Rate? [Re: Pastywhyte]
    #23435570 - 07/12/16 05:32 PM (7 years, 6 months ago)

Quote:

Pastywhyte said:
Chemicals have no place in this work. Try to stab through the wedge into the reciting plate and drag the scalpel out in the same motion. Use a fresh(er) blade as well if that doesn't work.




I am sort of "anti-chemical" myself but you might also consider the broader view. Technically speaking, everything we can easily observe that is not radiation is a chemical. Hence the name "Periodic Table of Chemical Elements."

The real question is this: which chemicals are significantly harmful to humans and other life forms which will be exposed to them?

Acetic acid, the active ingredient in vinegar which is used to acidify bleach, is relatively innocuous and is generally considered to have serious health benefits when ingested in moderate amounts.

As for the bleach itself, that is highly debatable, but 100's of millions of people world wide use it as a common household cleaner and even swim in it, submersing their entire bodies, and occasionally swallowing small amounts unintentionally.

I'm not talking about using sodium cyanide or something else clearly known to be highly toxic to humans and other higher life forms!

And keep in mind, fire and fuels are dangerous to humans and other life forms too. Especially if your chemicals of choice for SAB sanitation work are alcohol based :wink:


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Re: Agar Plates...Am I Lucky Or...? How is Your Plate Contam Rate? [Re: PutACapInHisAss]
    #23435781 - 07/12/16 06:52 PM (7 years, 6 months ago)

You can argue semantics all day. Does not make it better procedure. Tools are flame sterilized and you shouldn't be messing around with chemicals during your transfers when you should be focusing on technique. Guess that is just my opinion, but I stand by it.

I have done 1000's of transfers. Never did I feel that dipping my tools in anything was a necessary or beneficial procedure. Bottom line is that flame sterilizes and chemicals mearly sanitize.


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Re: Agar Plates...Am I Lucky Or...? How is Your Plate Contam Rate? [Re: Pastywhyte]
    #23436456 - 07/12/16 11:00 PM (7 years, 6 months ago)

I have not seen mold/bacteria on a plate since my first couple rounds ever making plates....I did have a spiral galaxy of satellite colonies on a transfer plate last week....but nothing ever turned green or black


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OfflinePutACapInHisAss
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Re: Agar Plates...Am I Lucky Or...? How is Your Plate Contam Rate? [Re: Pastywhyte]
    #23436590 - 07/12/16 11:55 PM (7 years, 6 months ago)

Quote:

Pastywhyte said:
You can argue semantics all day.

Bottom line is that flame sterilizes and chemicals mearly sanitize.




I highly respect your opinion and am grateful for your tek but it is not just semantics.

Some chemicals are dangerous, some are semi-safe/dangerous, and others are not only safe, but literally form the very foundation of life itself. All living organisms are biochemical machines.

Also, if the article on acidified bleach is correct, and the science was properly performed, 100% of pathogens were destroyed. That is not just sanitation. 100% fits the definition of sterilization.

Beyond that, even if it only turned out to only be "extremely good sanitation," considering the fact that many people report success performing procedures in open air, "extremely good sanitation" is likely good enough for a very high rate of success.

Flame sterilizing tools once at the beginning of a sequence of procedures is simple and quick. Doing it 14 or more times in a session is quite tedious and time-consuming.

Quote:

Mycologist217 said:
I have not seen mold/bacteria on a plate since my first couple rounds ever making plates.





I haven't made very many plates yet but so far I have not seen anything green, black, or visually suspect as a contaminant myself...knock on wood!

Do you use a SAB, LFH, or other?


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Re: Agar Plates...Am I Lucky Or...? How is Your Plate Contam Rate? [Re: PutACapInHisAss]
    #23436611 - 07/13/16 12:05 AM (7 years, 6 months ago)

I would like to see that article. But even if it did kill 100% I would still be hesitant to use it. Flame kills all and the heat of the blade keeps it clean until in a safe place to use it. We can then quickly cool it when needed. But I don't like the idea of highly toxic materials coming into direct contact with my cultures. How do you ensure there is no residue on the blade after you dip it?

You might find it works for you. Fill your boots. But I can't envision a scenario where I am going to feel it's easier and as guaranteed as flame. Maybe I'm an old dog. But I still have a pretty decent success rate.


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OfflinePutACapInHisAss
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Re: Agar Plates...Am I Lucky Or...? How is Your Plate Contam Rate? [Re: Pastywhyte]
    #23436675 - 07/13/16 12:37 AM (7 years, 6 months ago)

Quote:

Pastywhyte said:
I would like to see that article.

I don't like the idea of highly toxic materials coming into direct contact with my cultures. How do you ensure there is no residue on the blade after you dip it?




Honestly, I wouldn't worry about any residue. Again, these are not the most toxic substances on Earth. One of them is commonly used as a health supplement. The other is quite regularly ingested by humans who swim in chlorinated pools and breathed in by people who use it as a household disinfectant. Again, this is not DDT or sodium cyanide!

Do people worry about alcohol residue?

As for the article, that is simple:

http://www.eurekalert.org/pub_releases/2006-02/asfm-vik021306.php

Here are the most relevant excerpts:

"During the same time period the acidified solution killed all of the spores on all of the surfaces.

'Diluted bleach at an alkaline pH is a relatively poor disinfectant, but acidified diluted bleach will virtually kill anything in 10 to 20 minutes,' says Miner.

Miner recommends first diluting one cup of household bleach in one gallon of water and then adding one cup of white vinegar."


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Re: Agar Plates...Am I Lucky Or...? How is Your Plate Contam Rate? [Re: PutACapInHisAss]
    #23436948 - 07/13/16 04:42 AM (7 years, 6 months ago)

Quote:

but acidified diluted bleach will virtually kill anything in 10 to 20 minutes





Quote:

But I can't envision a scenario where I am going to feel it's easier and as guaranteed as flame. Maybe I'm an old dog. But I still have a pretty decent success rate.




:whathesaid:

10-20mminutes???  no way, I got shit to do man!!  like swiping left :lmafo:.  I think flaming is easier dude.


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Re: Agar Plates...Am I Lucky Or...? How is Your Plate Contam Rate? [Re: blindingleaf]
    #23437072 - 07/13/16 06:21 AM (7 years, 6 months ago)

So wait they're only focusing on pathogens? What about the spores that we have worry about that can EASILY survive well any sanitizer. Also the resource says virtually every thing. Not everything (which flame guarantees). And the 10-20 minutes like leaf pointed out... sorry but I started out trying to do alcohol as a replacement for flame. It crashed and burned.


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Re: Agar Plates...Am I Lucky Or...? How is Your Plate Contam Rate? [Re: Mad Season]
    #23437229 - 07/13/16 07:49 AM (7 years, 6 months ago)

so... 10-20 mins vs 10-20(actually it's more like 5) secs of flame.... i know what im gonna keep doing.


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Re: Agar Plates...Am I Lucky Or...? How is Your Plate Contam Rate? [Re: mupetmower]
    #23437244 - 07/13/16 07:58 AM (7 years, 6 months ago)

I've never found flaming my tools to be tedious.....as pasty said early in this thread a lot of it comes down to muscle me pry...my left hand automatically comes out of my SAB and over to my alcohol lamp when I complete an action inside the SAB....it all feels natural after a while....unscrewing lids, holding jars, moving things around with my right hand, and manipulating tools (syringes, scalpel, swabs) in my left hand......I mean I can buy denatured alcohol clean burning fuel for so cheap that it wouldn't even be economical to use anything else to sterilize my stuff


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Re: Agar Plates...Am I Lucky Or...? How is Your Plate Contam Rate? [Re: Mycologist217]
    #23437291 - 07/13/16 08:21 AM (7 years, 6 months ago)

Vendor syringes ARE usually pretty clean and if youre using just a drop, contams are unlikely. I sometimes will get little contams on the side or something if i am using those regular, hard to work with petri dishes, and i might touch it by accident when closing it. I actually really like the "pastyplates" for that reason (Pastywhyte, feeding your ego lol!), they are alot harder to just accidentally contaminate. Petri dishes are so hard not to IMO. They dont snap shut, some liquid will pour out, etc...

When i made my first good agar culture, i actually had pre-made agar from someone else who had just PCed it. And while i didnt make the formula, i knocked 4 of those up and they were all good.

I mostly started using agar for specifically PE. This is because MS wouldnt colonize rye for like 10 days (for first signs) but on agar it seems to growing at a more normal rate. Using an isolate seems to "stabilize" the PE a bit. I think the next one will be.... Im thinking agar with PF Classic.


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Re: Agar Plates...Am I Lucky Or...? How is Your Plate Contam Rate? [Re: mupetmower]
    #23437296 - 07/13/16 08:23 AM (7 years, 6 months ago)

As the others stated the term virtually is the problem. A chemical that kills 99.999% actually leaves a big door for contamination open. Let's say you have a billion CFU's on your hands (probably a low estimate). You then use a chemical to sanitize them at 99.999% effectiveness. You now have a million CFU's that survived. That relatively good but still a disaster from our stance.

If the chemicals won't hurt the fungi, they won't do the job. We have more to worry about than just spores and endospores. Mycelium fragments from molds are everywhere and if the chemical won't kill those, that it is ineffective. If it can kill them then it will harm your culture as well. People have been dipping biopsy tissues in all sorts of things for ages in an attempt to clean them before putting to media. The result is always damage to the intended culture.

This topic comes up at least twice a year. So far I still remain unconvinced. If you still want to try then go for it. But understand why it's considered bad practice.


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Re: Agar Plates...Am I Lucky Or...? How is Your Plate Contam Rate? [Re: Pastywhyte]
    #23437304 - 07/13/16 08:26 AM (7 years, 6 months ago)

Quote:

I actually really like the "pastyplates" for that reason (Pastywhyte, feeding your ego lol )




:lolsy:

I actually never called them that, I always called em "mini rounds". But I guess the name is catchy cause it really took off.


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OfflinePutACapInHisAss
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Re: Agar Plates...Am I Lucky Or...? How is Your Plate Contam Rate? [Re: Pastywhyte]
    #23438090 - 07/13/16 03:15 PM (7 years, 6 months ago)

Quote:

blindingleaf said:
10-20mminutes??? no way, I got shit to do man!!  like swiping left :lmafo:.  I think flaming is easier dude.




My idea isn't to wait 10-20 minutes like in the case of surface sterilization mentioned in this article.

My idea is to start with flame sterilized tools and then keep them in the SAB using the solution in a convenient way between each action. The tools must be set down at times for opening containers, wiping down containers, etc. The tools can be set in a shallow container of this solution or possibly wrapped in a towel or cloth that is well soaked with it.

I can understand your reluctance. All I can tell you is that I have had significant success in the past without flame sterilization of syringes between procedures. Long ago I didn't realize that it was something meant to be repeated between each jar. I just did it once at the beginning. I still had a 90%+ success rate on jar colonization. Heck, as already mentioned, some people claim significant success in open air! It is not that outlandish of an idea, especially in a SAB or using a LFH.

Quote:

Mad Season said:
So wait they're only focusing on pathogens? What about the spores that we have worry about that can EASILY survive well any sanitizer. Also the resource says virtually every thing. Not everything (which flame guarantees).






First, the word pathogen is my word, it is not actually in the article. Second, in their trial they claim “the acidified solution killed ALL of the spores on all of the surfaces.” “All” is not “virtually all,” even though it is said in a later quote. Perhaps the use of the phrase “virtually all" is because in that sentence they say 10-20 mins, rather than the 20 mins where all were killed. I am talking about using it on a surface that should be extremely clean in the first place. Finally, “spores” means spores “that we have worry about.”

Quote:

Mycologist217 said:
I mean I can buy denatured alcohol clean burning fuel for so cheap that it wouldn't even be economical to use anything else to sterilize my stuff






Maybe it is because of the economy where you live. In the US bleach and vinegar are extremely cheap by the gallon—about $2 for a gallon of vinegar and $3 for a gallon of bleach. A quart of denatured alcohol is $8-10.

Quote:

Pastywhyte said:
If the chemicals won't hurt the fungi, they won't do the job. We have more to worry about than just spores and endospores. Mycelium fragments from molds are everywhere and if the chemical won't kill those, that it is ineffective. If it can kill them then it will harm your culture as well. People have been dipping biopsy tissues in all sorts of things for ages in an attempt to clean them before putting to media. The result is always damage to the intended culture.

This topic comes up at least twice a year. So far I still remain unconvinced. If you still want to try then go for it. But understand why it's considered bad practice.






This is a very good point, I hear you—fragments of mold (etc) would still be a problem, no doubt!



However, it is a small thing to wipe off the end of a scalpel with a sanitary towel after each procedure. I do this already and it is very likely to remove most or all such fragments.

Still, I really think it is a question of what is good enough. I am not trying to save people’s lives using tissue cultures. Also, I along with many others have had significant success with far less than optimal procedures. If it works consistently with a high rate of success then there is no argument.



Plus, while you may have an aversion to such relatively innocuous chemicals, I have an aversion to fire and moving my hands in and out of my SAB. I think the air and my body (which is also outside of the SAB) are as likely vectors for contamination as my tools.

And fire with alcohol in the SAB is a certain danger. Although, with such a possibly effective non-flammable sanitizer, I can think of no reason to continue using isopropyl in the SAB, or for wiping anything down, especially when it is also much more expensive.

I may feel differently when I am using a LFH in the future. If the air is clean and my gloves become less likely a vector because they are kept behind the containers in the airflow, what does it matter if they are contaminated while flaming outside of the clean space.


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Re: Agar Plates...Am I Lucky Or...? How is Your Plate Contam Rate? [Re: PutACapInHisAss]
    #23438141 - 07/13/16 03:43 PM (7 years, 6 months ago)

Quote:

PutACapInHisAss said:
Quote:

Mad Season said:
So wait they're only focusing on pathogens? What about the spores that we have worry about that can EASILY survive well any sanitizer. Also the resource says virtually every thing. Not everything (which flame guarantees).






First, the word pathogen is my word, it is not actually in the article. Second, in their trial they claim “the acidified solution killed ALL of the spores on all of the surfaces.” “All” is not “virtually all,” even though it is said in a later quote. Perhaps the use of the phrase “virtually all" is because in that sentence they say 10-20 mins, rather than the 20 mins where all were killed. I am talking about using it on a surface that should be extremely clean in the first place. Finally, “spores” means spores “that we have worry about.”



No it doesn't, it means dried bacterial spores, and it doesn't even list what types of them. I'm talking broad scale of everything. Saying it killed every bacterial spore they put on a surface doesn't mean much to me. Tell me what it killed and how long it took. Furthermore it takes 20 minutes to kill everything. So basically when we wipe it every minute, it'll just add the spores that are on it to the blade. There's a reason why they call it a sanitizer (disinfectant) over a sterilizer. Flame instantly kills everything.

Quote:

Miner and his colleagues compared the ability of alkaline (pH 11) and acidified (pH 6) bleach dilutions to disinfect surfaces contaminated with dried bacterial spores, considered the most resistant to disinfectants of all microbes. The alkaline dilution was practically ineffective, killing all of the spores on only 2.5 percent of the surfaces after 20 minutes. During the same time period the acidified solution killed all of the spores on all of the surfaces.

"Diluted bleach at an alkaline pH is a relatively poor disinfectant, but acidified diluted bleach will virtually kill anything in 10 to 20 minutes," says Miner. "In the event of an emergency involving Bacillus anthracis spores contaminating such environmental surfaces as counter tops, desk and table tops, and floors, for example, virtually every household has a sporicidal sterilant available in the form of diluted, acidified bleach."



My point being that there's much more to this than worrying about bacteria spores, which is what they focused on. And after 20 minutes they'll still be around, you should have gone through how many plates by then?


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Edited by Mad Season (07/13/16 04:10 PM)


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Re: Agar Plates...Am I Lucky Or...? How is Your Plate Contam Rate? [Re: PutACapInHisAss]
    #23438159 - 07/13/16 03:51 PM (7 years, 6 months ago)

You can use soap and water to wet the SAB :shrug:

End of the day it's your grow. I wish you well.


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Re: Agar Plates...Am I Lucky Or...? How is Your Plate Contam Rate? [Re: Pastywhyte]
    #23438212 - 07/13/16 04:12 PM (7 years, 6 months ago)

peroxyacetic acid + 26% h2o2 sanitizer basically will sterilize stainless steel surfaces if they're surgically clean before hand IE no soil at all, and the stainless is passivated and ready to go. but it's still a sanitizer.

diluting bleach in water acidifies it. 1 part bleach to 9 parts water is a very effective sanitizer too nearly a sterilizer on already surgically clean hard surfaces

doesn't really matter though with a SAB or even a FH to use a bunch of sanitizer or one that's particularly better than another. 70% iso works fine for wiping your jars down.

the biggest problem you'll have is eliminating yourself as a contaminant vector and you can't sterilize yourself even with gloves and masks or showering before hand. so to worry about the chemicals you're using totally misses the point, if you're having problems fix yourself not your chemical regimen. because the sanitizers are not saving your ass.


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OfflineGinge
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Re: Agar Plates...Am I Lucky Or...? How is Your Plate Contam Rate? [Re: bodhisatta]
    #25381087 - 08/11/18 07:38 AM (5 years, 5 months ago)

Okay, first of all.  Your counter arguments are respectful and thought out, from your perspective.  However, you asked for the perspective of other growers.  While ingenious new things are being discovered every day, millions of supposedly ingenious new things are failing.  I love experimentation, and I love taking what I have learned about mycology and applying it to the variety of situations I encounter.  BUUUUT, I have had a lower success rate with my own ideas or combinations of shroomery teks that I thought worked together, than I have had just following teks exactly.  Flaming your loop, needle or blade is THE transfer tool tek, if you come up with something more effective, CHEERS, but it has cost me a lot of time, money and fruits to experiment.  I also really think there is a lot of value in what Bod says about fixing the contamination vectors using known techniques instead of recreating something to compensate for those vectors.  You would be in a much better realm to experiment if you had really good technique to add to your new ideas.  I applaud your early success with plates, I had 1 contam of 32 plates on my first MS to agar run, 8 did not germinate however. I will be working with syringes tonight on agar, and I am hoping this is the last time I work with a vendor syringe! (unless I pc them for my LC's :hehehe:)


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InvisiblebodhisattaMDiscordReddit
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Re: Agar Plates...Am I Lucky Or...? How is Your Plate Contam Rate? [Re: Ginge]
    #25381098 - 08/11/18 07:46 AM (5 years, 5 months ago)

Check thread ages. You're asking questions to people who haven't been around in years and probably ain't coming back. The other thread you bumped was 7+ years old for an easy agar question.


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