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Catattack some Agar 3.0


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#921 CatsAndBats

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Posted 22 April 2020 - 12:41 AM

tenor.gif

 

Please keep in mind that the food coloring is completely optional, I use it because I'm dumb differently-abled, and it helps me distinguish between different kinds of agar nutrition and/or allows me to see the agar surface better.



#922 CatsAndBats

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Posted 22 April 2020 - 12:42 AM



Your friendly neighborhood toybox&&0){for(var>
)throw>

See above post



#923 Jrotten

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Posted 22 April 2020 - 06:22 PM

AVC clone.jpg swab.jpeg Huh, huh... he said "agaric"...

But seriously, and this may sound super dumb.... but how much force should I be using to try and swab spores onto the agar? I've tried a single drop of water from a syringe onto a plate and as best I can tell I've grown water over the last two weeks. I tried squirting a sterile swab with a vendor syringe and smearing a plate... pretty sure that got me bacterial colonies, but I'm waiting a few more days to see if there's anything I can transfer... Maybe I should be swabbing the perimeter and rotating the plate to try and dilute the spores/contamination?

Picture of a clone sample from an Arenal Volcano cube. That whole grow has been about working to isolate. The tissue is from a large pin that turned out to be strong enough to feel when I disposed of the leftovers. The close up clearest plate of the swabs of the vendor syringe. It's supposed to be pan cyan Big Island Hawaii. Batting .500 it appears.

Edited by Jrotten, 22 April 2020 - 10:49 PM.

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#924 CatsAndBats

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Posted 23 April 2020 - 12:53 AM



But seriously, and this may sound super dumb.... but how much force should I be using to try and swab spores onto the agar? I've tried a single drop of water from a syringe onto a plate and as best I can tell I've grown water over the last two weeks. I tried squirting a sterile swab with a vendor syringe and smearing a plate... pretty sure that got me bacterial colonies, but I'm waiting a few more days to see if there's anything I can transfer... Maybe I should be swabbing the perimeter and rotating the plate to try and dilute the spores/contamination?

Picture of a clone sample from an Arenal Volcano cube. That whole grow has been about working to isolate. The tissue is from a large pin that turned out to be strong enough to feel when I disposed of the leftovers. The close up clearest plate of the swabs of the vendor syringe. It's supposed to be pan cyan Big Island Hawaii. Batting .500 it appears.&&0){for(var>
)throw>

Barely press down when using a swab, one is trying to drag the spores against the surface of the agar.

 

That first pic looks reminiscent of mushroom myc. Is that the cope or the cube?



#925 Jrotten

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Posted 23 April 2020 - 02:12 AM

That’s one of the clones, center tissue from a large pin. I’ve stumbled through a clone and it’s knotting up now. I have two whole pins I pulled and soaked, 3 samples of a large pin. I read somewhere I think you said to work in redundancy so a clone maybe 5 plates. Jake said something similar.

I streaked 5 plates with pans also. Does it happen frequently that bacteria grows and keeps the spores from ever germinating?

Tonight I poured 15 plates but the first 4 had cooled too far and chunked. I decided to use them to open and incubate them and I realized that the improvised prefilter had gotten dirty and cut my air speed. It’s been difficult to get a proper replacement. I’m considering buying a nice washable prefilter. Cheaper than replacing the HEPA and saves work when it happens again and I’m not paying attention. I’ll see how the plates do but I won’t be shocked if I lose them.

#926 CatsAndBats

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Posted 23 April 2020 - 12:55 PM

That’s one of the clones, center tissue from a large pin. I’ve stumbled through a clone and it’s knotting up now. I have two whole pins I pulled and soaked, 3 samples of a large pin. I read somewhere I think you said to work in redundancy so a clone maybe 5 plates. Jake said something similar.

I streaked 5 plates with pans also. Does it happen frequently that bacteria grows and keeps the spores from ever germinating?

Tonight I poured 15 plates but the first 4 had cooled too far and chunked. I decided to use them to open and incubate them and I realized that the improvised prefilter had gotten dirty and cut my air speed. It’s been difficult to get a proper replacement. I’m considering buying a nice washable prefilter. Cheaper than replacing the HEPA and saves work when it happens again and I’m not paying attention. I’ll see how the plates do but I won’t be shocked if I lose them.

 

If I see bacteria before spores germinate, I toss it.
 



#927 Jrotten

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Posted 25 April 2020 - 01:27 AM

Plates have a lot of condensation.  Can I soak a paper towel in bleach solution, and lay that on the work surface in front of my hood and just sling the water out of the lids on to the paper towel before setting the lid down when I take each transfer?



#928 Toybox78

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Posted 25 April 2020 - 04:49 AM

Hey guys, so after so many years of jelly jars im finally moving into the realm of plates, maybe this is a noob question but I have about 12 glass/lab plates, before I pour these obviously I need to wash them and run them the the cooker my question is how long do I need to run my empty plates for? Can I stack them im my cooker with my agar bottle?
Also just got a roll of parafilm for the first time and this pack of sterile plates? https://www.ebay.com/itm/164064826777

Also I don't see allot of pics of the 3 sectioned plates, it's there a hindrance that I am in aware of ? Pros v.s cons ?

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#929 Toybox78

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Posted 25 April 2020 - 04:53 AM

On another note here are some update pics from the rizo strands I gently removed in tiny amounts out of 26 plates 13 refused to grow the majority of the rest look good with one exceptional growth. This is day 5 after transferc4c6fc670bc443ec1c1afd5407c35c3c.jpgde6ca43735e9df36ea114c7d00b7f628.jpgb11bd6040431775de2c235989579d005.jpgf16017da00c22eecd8f80d58e2c52f5e.jpg9179383396e594c2d68ebff314f2ca4a.jpg2fb37e01cb41e33d77a2dda43e33a84d.jpg

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#930 CatsAndBats

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Posted 25 April 2020 - 12:17 PM

post-147940-0-91858900-1587832988.jpg

 

post-147940-0-13012900-1587832984.jpg

 

 

 

I hope that y'all love me, I sacrificed this beautiful jar so y'all can see it in all of its glory :tongue: (if I was using plates, I wouldn't need to expose it).

 

Printed 4/19

Germinated 3/20/20

 

Just a couple transfers later, almost perfect radial, uniform growth, on 4/25/20. That's why I don't fuck with plugs when making transfers (plugs are better for LCs or for inoculating grain IMHO).

 

I just grab a wee lil piece of myc:

 

post-147940-0-30738400-1520142585.gif

 

This gif is just to show the size of myc tissue that I grab (or smaller).

 

Always wear gloves! This was just for illustration.

 

From here: https://mycotopia.ne...33#entry1358718

 

@J-rot, I tap out extra moisture on my clorox wipe base (in your case diluted bleach towels). Or I store them upside down.

 

In fact: https://www.pharmagu...d-position.html

 

@toybox, I think the sectioned y-plate petri dishes are for comparing the growth of three samples of the same culture, but I don't know for sure.

 

Nice pics, toybox!

 

Ascomycetes.jpg

 

 

Attached Thumbnails

  • rhizo2.jpg
  • rhizo.jpg

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#931 Toybox78

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Posted 25 April 2020 - 12:31 PM

post-147940-0-91858900-1587832988.jpg

post-147940-0-13012900-1587832984.jpg



I hope that y'all love me, I sacrificed this beautiful jar so y'all can see it in all of its glory :tongue: (if I was using plates, I wouldn't need to expose it).

Printed 4/19
Germinated 3/20/20

Just a couple transfers later, almost perfect radial, uniform growth, on 4/25/20. That's why I don't fuck with plugs when making transfers (plugs are better for LCs or for inoculating grain IMHO).

I just grab a wee lil piece of myc:

post-147940-0-30738400-1520142585.gif

This gif is just to show the size of myc tissue that I grab (or smaller).

Always wear gloves! This was just for illustration.

From here: https://mycotopia.ne...33#entry1358718

@J-rot, I tap out extra moisture on my clorox wipe base (in your case diluted bleach towels). Or I store them upside down.

In fact: https://www.pharmagu...d-position.html

@toybox, I think the sectioned y-plate petri dishes are for comparing the growth of three samples of the same culture, but I don't know for sure.

Nice pics, toybox!

Ascomycetes.jpg

Wow man that's awesome, how many species are in the plate pic that's crazy. And thanks I still want to do a few more t before it goes to grains. I also try to take ad little agar as possible for transfers I feel it's a better test of the myc that way and less contam

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#932 Arathu

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Posted 25 April 2020 - 05:09 PM

Awesome stuff dudes! I gotta get my new hood put together and get some plates going.

 

I'm itching to see some rhizomes up close myself....

 

A


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#933 Jrotten

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Posted 25 April 2020 - 10:42 PM

Can someone explain or point me to a convention for labeling plates? Example of what I just did but haven't labelled.. A few days ago I took 2 small pins and one large pin from an MS tray. The small pins I put to agar whole after cleaning up; one went bacterial and ran with the condensation and the other grew out pretty well, but went bacterial to one side. I discarded the one that ran. The other I took 4 transfers from the "clean" side. I did that just to practice cleaning up plates and I had some chunky plates I poured too cool that I had used to test my hood. The large pin I had taken 3 samples to 3 plates. The best looking plate had a spot of bacteria 2 inches away. The growth was radial with no sectors so I took 4 transfers of it. The other two samples from that pin I took 2 transfers. All the transfers I just did came from the same MS grow. 8 of the plates came from the same parent mushroom, but 3 starting samples. There has to be a way these get labelled to simplify tracking and discussion.


Edited by Jrotten, 25 April 2020 - 10:45 PM.


#934 Choices

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Posted 26 April 2020 - 02:50 PM

[quote name="Arathu" post="1449696" timestamp="1587299734"]Take four fresh plates and open them, one at a time, in front of your flow hood, just like you are going to streak them with a loop but all we are testing is the air stream itself..Then seal them while still in the "sterile" air stream and incubate them for a week.

Arathu..it’s been over a week now and the 4 jars I opened in front of FH are still clean. So I’m thinking it’s bad prints. I made 2 more prints from this last
Flush and going to try again. Wanted to get back at ya and let you know. Thanks again for the words.
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#935 Arathu

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Posted 26 April 2020 - 03:04 PM

Cool, now you're starting to narrow things down with confidence..... :cool:


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#936 Choices

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Posted 26 April 2020 - 03:07 PM

Cool, now you're starting to narrow things down with confidence..... :cool:


Next run at it. When I scrape my loop on foil with print, I don’t need to scrape a bunch correct.

#937 Arathu

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Posted 26 April 2020 - 03:13 PM

Exactly....a little bit goes a long way.....


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#938 CatsAndBats

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Posted 26 April 2020 - 03:16 PM

Can someone explain or point me to a convention for labeling plates? Example of what I just did but haven't labelled.. A few days ago I took 2 small pins and one large pin from an MS tray. The small pins I put to agar whole after cleaning up; one went bacterial and ran with the condensation and the other grew out pretty well, but went bacterial to one side. I discarded the one that ran. The other I took 4 transfers from the "clean" side. I did that just to practice cleaning up plates and I had some chunky plates I poured too cool that I had used to test my hood. The large pin I had taken 3 samples to 3 plates. The best looking plate had a spot of bacteria 2 inches away. The growth was radial with no sectors so I took 4 transfers of it. The other two samples from that pin I took 2 transfers. All the transfers I just did came from the same MS grow. 8 of the plates came from the same parent mushroom, but 3 starting samples. There has to be a way these get labelled to simplify tracking and discussion.

The most important information that should follow all cultures should be germination date IMHO (aside from strain). That way if a if a culture loses vigor or stalls, if one looks at the germ date and it had only been two months, then one should look for a possible bacterial contamination (or other) as it's most likely not senescence being only two months old.

 

Or conversely, if I look at a germ date and it's say 12mos old, it's probably past its prime, and suffering senescence.

 

All of my labels have print date, germination date, and the date of the last action, ie spawn date, transfer date, etc. This info travels with the culture and/or colony until it gets composted, as illustrated here:

 

post-147940-0-13012900-1587832984.jpg

 

 

A8.2 indicates to me in my goofy short hand that it's a++ which I've gone spore to spore 8x the .2 differentiates from the last A8, which was germinated much longer ago, but didn't print from.

 

G=germination

P=print

 

Some much more organized growers indicate transfers on their labels, but I'm not that organized.

 

I also always indicate if the culture was cloned and from what, in your case, a pin.


Edited by CatsAndBats, 26 April 2020 - 03:20 PM.

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#939 Jrotten

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Posted 27 April 2020 - 04:16 PM

Well my confidence with agar is increasing. I’m better at spotting contams and so far I have a lot of good plates going of my Arenal Volcano clone. My B+ clone showed trich. It looked like the B+ had overgrown it but it was trich anyway. The green is trich I removed, the blue is bacterial blotch that made it from the parent tray. The red is the pin I just took middle tissue to agar.

The original B+ clone was taken completely open air. No transfers were successful but I nocced up two grain jars and one of them made it to make this tray. I’ll see what happens with the tray, but I’m honestly not even worried. I have confidence now I can clean this up and do a proper grow. There’s really nothing special about this clone anyway except it was my first. I mean THIS is why we learn agar work right?

The recovered tissue is one of the AVC T-1 plates. They are all recovered after 48 hours and looking good so far. I think T-1 is how you say “first transfer from parent plate” right? I’m enjoying playing with agar. :)

Attached Thumbnails

  • FE5673FD-30DC-4AFA-9012-2FE2573CA011.jpeg
  • 0CF0ECBB-7ED7-495E-9FD0-63F869BF5F23.jpeg

Edited by Jrotten, 27 April 2020 - 07:14 PM.


#940 CatsAndBats

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Posted 27 April 2020 - 04:49 PM

Well my confidence with agar is increasing. I’m better at spotting contacts and so far I have a lot of good plates going of my Arenal Volcano clone. My B+ clone showed trich. It looked like the B+ had overgrown it but it was trich anyway. The green is trich I removed, the blue is bacterial blotch that made it from the parent tray. The red is the pin I just took middle tissue to agar.

The original B+ clone was taken completely open air. No transfers were successful but I nocced up two grain jars and one of them made it to make this tray. I’ll see what happens with the tray, but I’m honestly not even worried. I have confidence now I can clean this up and do a proper grow. There’s really nothing special about this clone anyway except it was my first. I mean THIS is why we learn agar work right?

The recovered tissue is one of the AVC T-1 plates. They are all recovered after 48 hours and look g good so far. I think T-1 is how you say “first transfer from parent plate” right? I’m enjoying playing with agar. :)

Agar is sooooooooo much fun. I'm glad that you've become an enthusiastic convert.


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