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OfflineYumyumyumyum
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How would you make agar less nutrient rich and limit the ambient moisture on the plate?
    #26889322 - 08/20/20 12:32 AM (3 years, 5 months ago)

I picked up two contaminants I did not recognize off what I assumed were well prepared clean dishes. I am using glass sealing it with parafilm. The dishes get streaked 4 hours after they are produced. That is 5.6 nutrient rich malt agar and 100cc of distill water. That is prepared in a flask then transferred to a PC with a poly fill stopper. It gets pced for 20 minutes then the beaker is moved to a bowl of water until it cools down to the proper temperature for pouring. It seems it is too much of a feast so a lot of different stuff showed up. I was using clean vendor syringes that I wiped with 70% ra then heated with a spirit lamp.


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OfflineJus
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Re: How would you make agar less nutrient rich and limit the ambient moisture on the plate? [Re: Yumyumyumyum]
    #26889325 - 08/20/20 12:39 AM (3 years, 5 months ago)

1) How are you cleaning/sterilising the glass plates?
2) Do you put foil over the polyfil on the flask? Polyfil isn't a perfect barrier, and the foil could help stop any bacteria in the air falling in.
3) How do you know the vendor syringe is clean?
4) You mention you streak the plates, are you seeing contamination appear elsewhere on the plate outside the streak pattern?
5) Are you doing this in a still air box?
6) Have you repeated your process with blank samples? This could narrow down if the contamination comes from the syringe.
7) To answer your actual question, put less of your malt extract in!

The ambient moisture on the plate shouldn't be an issue, the water is sterile out of the pressure cooker.
If you have excessive condensation, pour when the agar is cooler.

Do you place your wrapped plates inside little sealed plastic food bags? This can stop them getting contaminated through broken wrapping.


Edited by Jus (08/20/20 12:44 AM)


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OfflineHoneheke
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Re: How would you make agar less nutrient rich and limit the ambient moisture on the plate? [Re: Yumyumyumyum]
    #26889327 - 08/20/20 12:45 AM (3 years, 5 months ago)

There a dude on youtube that runs a really comprehensive library on cultivation. Search for Home Mycology.

Here is a link to his subscription.

"https://www.youtube.com/channel/UC6YcCZ-AZ2UVaVsb8bKO7Mg"


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Invisiblealaskappalachian
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Registered: 10/22/19
Posts: 1,688
Loc: The 49th Dimension
Re: How would you make agar less nutrient rich and limit the ambient moisture on the plate? [Re: Honeheke]
    #26889333 - 08/20/20 01:00 AM (3 years, 5 months ago)

1.  No syringe is clean.
2.  Home Mycology is a terrible point of reference.  This is a guy who uses open air inoculation of ketchup cups wiped out with ISO.  Please leave that numbnuts on YouTube.  We do things right here. 
3.  Can't quite make out a few things regarding your methods in your post, but your contamination came from either your syringe or your plate prep.  All syringes carry contaminants.
4.  Did you pour your plates in a SAB, or do it Home Mycology style and do it on your kitchen counter while chanting "brah" over and over?
5.  Nutrition isn't the problem and doesn't promote or produce contamination. 100% technique/introduced from inoculant


Edited by alaskappalachian (08/20/20 01:15 AM)


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Offlinescarabaeus
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Re: How would you make agar less nutrient rich and limit the ambient moisture on the plate? [Re: Yumyumyumyum]
    #26889338 - 08/20/20 01:11 AM (3 years, 5 months ago)

To limit 'ambient moisture on the plate' I'm guessing you mean droplets condensing on your lids?. If so, stack your poured plates, and then boil water to fill a coffee mug, and put that on top of your stack of plates.


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OfflineYumyumyumyum
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Re: How would you make agar less nutrient rich and limit the ambient moisture on the plate? [Re: scarabaeus]
    #26889351 - 08/20/20 01:30 AM (3 years, 5 months ago)

There was a pretty good chance the contamination came from two of the syringes. The reason being is that it was a new form of contamination I had not seen before. Red dots. Two of them I have seen before black streaks and green splotches. I have a few agar samples I have been using over the last year. This was a new batch from 4 different syringes with two of them having problems. I was only adding the last 10cc onto a plate. I use use both lab glass and plastic dishes. I inoculate is a ghetto flow hood with a hepa filter attached to a usb fan and hand holes covered in rubber. Here is all my agar plates. Two of them have been producing jars for around 8 months minus an occasion disaster that one learns from in these types of things. I just restarted 4 new types MDK, B+, GT and Brazil. The agar samples do not seem to be doing well. The moisture always seen to show up because I follow the temp directions on the package. The two glass I can redo the vendor was excellent about replacement within the prescribed usage (agar). I have documented many different types of contaminants and when I see a new one it makes me wonder its source. I agree it is never 100%, I just try to be are clean as possible. The fickle mushroom Gods. 


Edited by Yumyumyumyum (08/20/20 01:39 AM)


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Offlinescarabaeus
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Re: How would you make agar less nutrient rich and limit the ambient moisture on the plate? [Re: Yumyumyumyum]
    #26889363 - 08/20/20 01:50 AM (3 years, 5 months ago)

Yuck. Left to right, it seems like you have on plate 1 Penicillium. Plate 2 black pin mold (Rhizopus). Plate 3 maybe Aspergillus ? (Too early to tell, might be early stages of Penicillium). The other plates I'm not sure what is going on.

Anywho. My advice is to lose the 'ghetto' hepa filter idea. It probably is not a 'true' hepa filter? Regardless, you are getting knocked out by contams. All you need is a still air box. I have been growing for years and years and had to leave my flow hood behind  :um: when I moved. My contamination rate is almost as low with a still air box as it was with an awesome flow hood.
Hope this helps? Anybody else have ideas?


Edited by scarabaeus (08/20/20 01:52 AM)


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Invisiblealaskappalachian
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Re: How would you make agar less nutrient rich and limit the ambient moisture on the plate? [Re: Yumyumyumyum]
    #26889365 - 08/20/20 01:51 AM (3 years, 5 months ago)

Oy.  Penicillum, aspergillus, bacterial colonies...  (as stated above) Yeah that's some spore solution in need of cleaning up.  Could most certainly have come from imperfections regarding your makeshift flow unit but often syringes come just loaded with contamination which we transfer away from before they can express so fully.  Aren't they horribly beautiful though?!  First sign of myc on your plates, transfer the best recognizable growth.  That solution is very likely filthy but you can clean it up by doing transfers early and making good choices with selection.:thumbup:  100% would rather use a SAB than a makeshift unit, btw.


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Offlinescarabaeus
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Re: How would you make agar less nutrient rich and limit the ambient moisture on the plate? [Re: alaskappalachian]
    #26889368 - 08/20/20 01:56 AM (3 years, 5 months ago)

John Lewis; you just gotta love that guy R.I.P. Also, good advice!:headbang:


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OfflineYumyumyumyum
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Re: How would you make agar less nutrient rich and limit the ambient moisture on the plate? [Re: alaskappalachian]
    #26889372 - 08/20/20 02:03 AM (3 years, 5 months ago)

So when you say transfer out the good stuff what do you mean? I have seen this under heavy magnification and it seems that stuff gets all over everything once it is in the plate. But I have also seen a plate I thought was busted out regenerating back to viable samples. When you say still air just a box with hand holes? The filter was fine its the airflow that might be an issue its not sealed tightly on the edge the fan is pushing air in through the filter and out the hand hole. What plates should be rescued? I have a pile of the plastic ones and the glass is reusable. The two end ones are fine they have been my work horses. They both look like they could use a little hydration that is about it. I have 23 jars in process 21 of them are going 12 are going really well. Long stringy goodness.


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Offlinescarabaeus
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Re: How would you make agar less nutrient rich and limit the ambient moisture on the plate? [Re: Yumyumyumyum]
    #26889384 - 08/20/20 02:30 AM (3 years, 5 months ago)

Yeah, just a big box with arm holes, but not even arm holes. I'm not in a place where I can take a picture of mine right now and post it unfortunately. Let me try to describe it.  It is 31 inches across. 20 inches high in the back. And 9 inches high in the front where I put my arms. It slants from back to front. The 9 inches is enough space to ezlee put quart jars in and out. The front is covered by apiece of plastic with slits in it for access, and I tape a towel over that (it just drapes over my arms for xtra protection. I use only 6 quart jars at a time but 8-10 is possible w/o too much of a hassle. Bags are impossible though. But I don't do bags anymore anyway so...

The size I listed is about the maximum size of glass that the guy at the hardware store could cut for me. Obviously the glass sits on top of the thing so you can see lol!  Other possibilities for glass is an old window in a frame from a junk yard/county dump (best move cause it is already in a frame), or if you can find an old refrigerater they all have glass shelves to put your milk or whatevs on. Then you would build your box to fit the glass.

A s far as your make-shift hepa filter goes, a real hepa filter filters  out particles with a 99.99% efficiency. Anything less than that is not good enough for mycological purposes. I don't know what you have in your set-up. Something rated at 99.97% doesn't sound like much but it makes a BIG difference.


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Invisiblealaskappalachian
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Registered: 10/22/19
Posts: 1,688
Loc: The 49th Dimension
Re: How would you make agar less nutrient rich and limit the ambient moisture on the plate? [Re: scarabaeus]
    #26889403 - 08/20/20 03:09 AM (3 years, 5 months ago)

Quote:

scarabaeus said:
John Lewis; you just gotta love that guy R.I.P. Also, good advice!:headbang:



Ditto bro :toast:

Yeah there's virtually no chance that unit is giving you a  column of clean air.  I heard "USB fan" and "hand holes" and there were enough red flags down to  clear that up.  I've seen similar units and it amounts to a messed up still air box with a fan creating currents of unclean air.  No bueno.  Doesn't matter what plates you use.  It matters how/where you pour them.  Spend 10 bucks on a 80+qt tote and spend a couple hours making a nice, clean SAB, then focus on your technique.  Read Bod's agar tutorial in the sticky threads a couple times and work hard on that technique.  Or do prepours ( I do most of my work in 4oz. jelly jars with unmodified plastic lids from Walmart.  Either way, all those plates are fucked completely.  Starting from a spore syringe, just sterilize some qtips in foil in a to squirt a drop of solution on before swabbing your plate or make an inoculation loop from wire or metal from a twist tie.  Too much solution causes goopy growth that turns into a bacterial mess and carries more contamination because you used more solution, which has more than less solution). You really, definitely should re-read/read a few agar teks and start again.  You're on your way but a good bit off track.  Give me a sec and I'll shoot you some MUST read info which is step 1.  Have to take a step back, brother.


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Invisiblealaskappalachian
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Re: How would you make agar less nutrient rich and limit the ambient moisture on the plate? [Re: alaskappalachian]
    #26889405 - 08/20/20 03:13 AM (3 years, 5 months ago)

https://www.shroomery.org/forums/showflat.php/Number/21922023. Here's a good, solid agar tek.

And a SAB tek (ditch that dirt-tosser and use this until you can drop a couple hundred on a real flow hood build.
https://www.shroomery.org/forums/showflat.php/Number/23990888#23990888

Then focus on technique.  You really should read back through all the basics in the trusted (current- from 2015 or newer, for instance) teks in the mush cult sticky threads and get a hold on the basics.  I LOVE your passion for this, but you have some reading to do.  Next time you get some growth on some plates,  feel free to make a post on here and we will walk you through the transfers.  It's all about outrunning contaminants before they can grow and ruin your plate,  then making selections as soon as you get good recognizable growth each transfers until you have a clean plate.  All in the teks.  You got this, bud.
PS: here's a comprehensive list of all the trusted teks in the sticky threads.  Worth the time to read, and covers every question you could ever have. :awesomenod:
https://www.shroomery.org/forums/showflat.php/Number/24297804


Edited by alaskappalachian (08/20/20 03:27 AM)


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Offlinescarabaeus
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Re: How would you make agar less nutrient rich and limit the ambient moisture on the plate? [Re: alaskappalachian]
    #26889411 - 08/20/20 03:32 AM (3 years, 5 months ago)

Well put Alaska dude! I hope the world keeps rockin' you as you do rock the world. :dancer:


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OfflineYumyumyumyum
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Re: How would you make agar less nutrient rich and limit the ambient moisture on the plate? [Re: scarabaeus]
    #26889962 - 08/20/20 11:49 AM (3 years, 5 months ago)

Excellent information and "Thank you",

Honestly I had better results before I tried to get fancy with everything. The two agar on the end produce viable material constantly but its time to retire them. So all the rest are completely toast? I am perfectly fine starting over on the dishes. I have been watching the videos including the arvin47 ones on Youtube. I enjoy the learning process and mistakes are part of it. The advice you provided was rock solid and I will use it. I usually get all my stuff at Value village and Goodwill can pick tubs up for 3$ not to mention PC's for 9$ dehydrators for 7$ cases for lab stuff, racks, jars you name it. I have been at this 9 months, success and failure but always success in the end just dependent at what part of the process. Those plates are busted by my rotation is coming along with the first set up in 2 weeks. 4 different stains. Where everything went south was when I tried to help someone learn the process. The mushroom Gods punished me or the person was filthy. It was upsetting to have a month gap from a back to back bad runs. Now things are looking pretty pretty good! I just wanted to transfer syringe matter to agar since I am trying 4 new strains. So the 4 and 5 plates are toast? 3 is  Serratia marcescens(New) The 5th one is all white and does have some growth.I use lab sterile inoc loops they are pretty cheap in bulk.(I might mess those up since I boil them and rub them down). Fridge glass I am on that one already.

Thanks for the advice fellas, my problem seems to be not enough practice. RA, HP are finally back in abundance so that has helped. 

Since I won't be using it anymore here is a picture of my getto SAB. The rest is what I have coming up minus the new ones that have not started to colonize(I do cycles of 8). I did try Spiderman tech and yes, it was garbage. Mono tub is me being lazy about making bags. I toasted one of those also. Even have a compost pile of samples past in my garden(Doing nothing) 


Edited by Yumyumyumyum (08/20/20 12:19 PM)


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