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agar and air born bacteria


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#1 invisibilitysyndrome

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Posted 25 October 2021 - 11:43 AM

Ok yall...
So ive been having some serious issues with bacteria.

Have a sterilizer to do my sterile work.

Few questions for those that know

First off can bacteria travel airborn ?
This is my biggest question really..

Also for anyone that uses a steralizer are you aware of bacteria build up amd can the steralizer become a vector for contamination if it isnt properly cleaned. Its only used to steralize grain coir and water...

Ive been working with some prints to agar and im getting alot of bacteria. Some ru s are up to 80% contam. Mold has been less of a problem maybe 10 or 15 % at times .

Any one out there ha e any ideas about these things.


Even when i do my grain for spawn runs im getting bacteria. Its hard to say if the bacteria is coming from the agar plates im using. Even if they look good or if the grain is the problem or if its both.

Ive sortof gotten to the point im feeling that its the steralizer. Both of them I have 2 or if possibly bacteria is airborn and getting in that way..

Any ideas ill accept that can help me figure this out.
Thank all..

.
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#2 HrVanker

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Posted 25 October 2021 - 11:56 AM

Yes, bacteria can travel via air. Yeast is quite common, but many bacteria can travel by air.

What do you mean by sterilizer? Is it an autoclave or a PC? Or some other device? Honestly: if it's blowing air, and it's not a laminar flow hood, I wouldn't trust it. I'll take my ISO and a still air box any day.

Have you let an agar jar sit for a week or so unopened to be sure that it's really sterile?
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#3 Myc

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Posted 25 October 2021 - 12:59 PM

Archive a control plate when you pour next time.

If the control stays clean - you know your problem comes in after the agar is poured.

 

I would be interested to see pics of the bacteria - since I strongly suspect yeast is your contaminant. If you have a hand-loupe or microscope, yeast are very visible at 400x magnification (40x objective and 10x eyepieces when using a microscope).

 

You say you are working with prints. I treat all prints as if they are "dirty". Start on agar and transfer healthy tissue away from the contaminants to fresh, clean plates. Eventually you will get a clean cultivar with which to do further work.


Edited by Myc, 25 October 2021 - 01:02 PM.

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#4 invisibilitysyndrome

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Posted 27 October 2021 - 12:02 PM

Right on yall
so first off i was calling my autoclave a steralizer
I am working in a slill air environment. A small plastic room big enough for me and a table.
No flow hood. Ive had plenty of success with this set up for last 8 years.

Ive left agar alone to see if its good prior to pour and any transfers. It has been fine
What I'm noticing is that ive been pouring plates a bit hot and have way too much condensation that never
Gets reabsorbed back to agar so my feeling is that this water sitting in the plates is the vector
For the bacteria.

Yes ive started from prints but im in yo 2nd 3rd transfers now from what looked to be good healty clean
Plates and then a majority will go septic and one or 2 out of 10 will grow nice.

I believe its the condensatiin ill work on pics if thatll get some good info and possibly a microscope or somthing

Ive had plenty of success in the past with agar and all other aspects for that matter doing things this same way its just that this infection issue is new
And the only thing currently i can figure is the water.
I appreciate the response sofar. If I have clarified things a bit more id be interested in any more feed back.
Thanks yall

#5 Myc

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Posted 27 October 2021 - 03:04 PM

The condensate on the plates should be sterile - and should not be presenting a contamination vector.

 

I'm guessing there is something else - post sterilization - that you're missing.


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#6 simplegood78

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Posted 27 October 2021 - 03:04 PM

From "Radical Mycology" by Peter McCoy, Activated Charcoal can be added to agar at the rate of 10 grams per liter as a bacterial suppressant and spore germination promoter.

 

It will also impart a nice black color to your agar, which makes it easier to see the white wisps of mycellium.

 

Another option is after the agar has cooled below 140F/60C add food grade (3%) hydrogen peroxide at 6-10 ml per liter of agar.  If you add it before, the hydrogen peroxide will not survive your sterilization.


Edited by simplegood78, 27 October 2021 - 03:09 PM.

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#7 invisibilitysyndrome

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Posted 16 November 2021 - 10:47 AM

Ok so despite the fact that the water condensate should be sterile i believe thatas time passes it is giving the contaminant a place to enter. i do use para film to seal them but.....

I did one more agar pour after my last post and poured them cooler and have not an issue with them.
Also ill have to agree with the idea up top that its been yeast that has been the contaminate based on looks I still havent used the microscope.

The new plates are all looking good with no condensation and the jar that I poured from is contaminant free as well not a single spot and that jar was opened and closed 25 times to pour all the plates.

I spose this concludes this post because my personal conclusion is that while condensation should be harmless it should be avoided as much as possible. I really appreciate the good answers from yall.
I have also noticed that while ive had plenty of success in the past when I stop for a while and come back its super important to do exactly as ive done in th past because somthing as simple as condensation can throw everything off kilter and while believing something is harmless is not the same as proving that it is actually harmless and in my case I've proved that here in my lab it can and most likely will be harmfull.

Science is truly a wonderful thing. Thanks again y'all

#8 HrVanker

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Posted 16 November 2021 - 11:53 AM

If you are pouring agar w/o a flowhood, or very very clean technique, contamination comes easily.

For this reason (and laziness) I do no-pour jars. I have had jars dry out over 8mo, w/ no contam in sight. I do get some condensation, but I've only had 2 out of about 120 show signs of contamination. Plus the condensation can be easily removed in my SAB.

#9 invisibilitysyndrome

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Posted 16 November 2021 - 12:12 PM

So here is a pic of a plate as can be seen the contam is all around the edges and its like this on all the plates. On the edges where the condensation eventually comes in contact with the agar.

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  • 20211116_121107.jpg

Edited by invisibilitysyndrome, 16 November 2021 - 12:16 PM.

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#10 invisibilitysyndrome

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Posted 16 November 2021 - 12:19 PM

If you are pouring agar w/o a flowhood, or very very clean technique, contamination comes easily.
For this reason (and laziness) I do no-pour jars. I have had jars dry out over 8mo, w/ no contam in sight. I do get some condensation, but I've only had 2 out of about 120 show signs of contamination. Plus the condensation can be easily removed in my SAB.

Ive only begun to have issues after i started pouring plates when the agar was too hot.
Not prior or very little and never like this and like i said earlier my new set didnt contaminate when plates were poured cooler.

Edited by invisibilitysyndrome, 16 November 2021 - 12:20 PM.


#11 bezevo

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Posted 16 November 2021 - 12:20 PM

you could  make a still air box to use in you little mini clean room also ..

 

good luck solving your issue

 

Bez


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#12 Salty117

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Posted 16 November 2021 - 12:28 PM

Do you sterilize the plates before pouring or are they fresh out of packaging, never before used? And have you tried putting some of your spores to the new plates yet?


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#13 HrVanker

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Posted 16 November 2021 - 12:43 PM

Yeah, it may have been too early for me to be posting. But I've had my coffee now so here I go again:

You are right, it does look like the contam is wicking in through the condensation. If lower temps don't have condensation but contam because of the temps, and higher temps contam because of moisture wicking, it seems like another solution or variable would be the key to success.

Perhaps dew-point is to blame, since that dictates how moisture forms from vapor, at particular temperatures in a given area/location.

At some point, running a pc, supplies, time, troubleshooting etc. will add up to a lot more time/money than just going with something that works. My feeling after seeing this thread is: perhaps plates are better used in a cleaner lab environment, than a clean home environment.

Personally, I love my 4oz jelly jars. They don't look as cool as plates, but they get the job done, and they work well in a non-lab environment. I've only had 2 jars contam, and it was mold that showed after transfers.

#14 invisibilitysyndrome

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Posted 16 November 2021 - 02:04 PM

So basically what im saying now is that pouring plates at a cooler temp avoiding the condensation has resolved the issue.

When i see green mold i know ive not been clean or my procedure was lacking this contam is different and most likely yeast as stated above by anither member and entering somehow through the condensation.

Also to be clear and not to be rude but im not concerned about my set up or cleanliness as these things have not been an issue.
My original questiin was referring to airborn bacteria and turns out in this case it seems to be air born yeast.

Why green mold dosent come thru the condensation I do not know but seems to only be the one particular organism that is effecting my plates.

Here is a pic of my small clean/still air room i do my work in. Ive used this for years and didnt have an issue untill pouring hot plates leading to my question about airborn bacteria..

Sorry about pic being sideways but in reality it is straight up and down sealed from cealing to the floor and when cleaned out of dust etc and ive showered and precations taken ince inside the air is still and by moving slow and methodically ive achieved very good success rates with out a flow hood so just saying. I dont have a microscope to actually analyze the organism to see if it is actually yeast but it does look much like it. Regardless by pouring cooler plates it seamd to have solved that issue.

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#15 invisibilitysyndrome

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Posted 16 November 2021 - 02:18 PM

https://mycotopia.ne...ibilitysyndrome

So here is a link to a thread I did 5 years ago if anyone is interested in reading.
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#16 Myc

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Posted 17 November 2021 - 10:28 AM

I didn't think to include microscopy photos of yeast.

The cells in this photo are part of a ginger bug for making craft beverages. They are loaded onto a hemocytometer for counting purposes.

The smaller - very tiny cells are the size of beer yeast. The larger cells........I'm still exploring to find out what variant is present.

 

20200223_114132.jpg

 

It is not inconceivable that yeast have been telegraphing through the condensate. They thrive in solution. The condensate likely has enough sugar content to support growth.


Edited by Myc, 17 November 2021 - 10:33 AM.

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#17 Arathu

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Posted 17 November 2021 - 07:30 PM

And air movement is about impossible to stop too....the movement of micro organisms and many other "things" in the air is assured.....

 

Life is incredibly tenacious........ and delicate....if I don't use Parafilm on my plates here I will have very few successful adventures.....

 

I work in front of my hood and then stash things in large Ziplock's so I can see what's happening without letting airflow in, even with my plates filmed....I follow aseptic protocol in the air stream of the hood for any opening of the bags, close inspection, transfers, and etc.

 

Condensation can be a pain in the rear if not controlled, pouring agar at a cooler temperature helps especially if the plates are warmed a bit before pouring, they can be dried and cooled in a good HEPA stream too... I like a little warm and very consistent temp for incubating my plates.....change of temperatures is bad news in my experiences...

 

I need a new lab facility to do work in.....I've been thinking about it lately for working on my edibles library......I think I need a POD dropped here...insta-lab.

 

A


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#18 Myc

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Posted 18 November 2021 - 10:55 AM

Plus One for Parafilm !!!

I've always used it and rarely ever had problems. Still using the same box of film that I purchased 10 years ago. Pretty cheap insurance if you ask me.


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