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Getting my feet wet again: a grow log


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#1 Elenchus

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Posted 08 July 2019 - 04:01 PM

Hello all,

 

I am pleased to present my first ever grow log here. Would love to get some good feedback and hopefully show some success down the line.

 

I've been on a five year break from this hobby, and it feels good to be back at it. I was lucky enough to be gifted with 3 PE clones on agar, as well as two syringes: one of PESA and another of Malabar.

 

I decided to make some BRF jars, which I PCed at 15 psi for 30mins, to test out the spores. On the first run, I made 4 jars and innoced three of them with approx 2ml each of solution. I set the fourth aside as a control. On day 4-5, signs of growth were noted along with what looked like bacterial contam. The control jar showed no signs of contam. 

 

A side note: I innoced these jars in open air, flame sterilizing in-between jars and wiping with 70% iso in-between holes. 

A further note: I have a sourdough culture at home, which I thought might be causing some problems with airborne contaminants.

 

Currently, around day 19 after inoculation, they appear to be consuming whatever bacterial contam is in the jars at the innoc points. Photos attached.

 

On another trial-run, I decided to prepare a 4% dextrose solution and PC at 5 psi for 20mins. This LC was innoced with a wedge of PE clone taken from agar in a SAB while in a cleaned room. My clean work is not as good as it once was, but the LC looks clean as far as I can tell. Pic attached.

 

Finally, I innoced 5 more jars with PESA using approx 1.5ml each. These were innoced in a SAB in the same conditions as the agar transfer, just to be safe. However, after 4-5 days, the same yellowing around the innoc points occurred next to myc, which also germinated around the same time. Pic attached.

 

 

 

Now, my main question is whether you all think it was my methods or the syringes? I do not know where they came from so I have my reasons to doubt them. But I also have reasons to doubt my own procedure.

 

...which is why some ideas/views/thoughts would be appreciated.

 

Peace n Love

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#2 roc

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Posted 08 July 2019 - 08:13 PM

Hard one to call.

 

2 ml may have added to much moisture which will foster bacterial growth.

 

Not knowing the history or preparation of syringes throws another twist in determining if it was syringe related.

 

I'm leaning towards to much moisture if the bacteria is overtaken and no other contamination shows it's ugly head.


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#3 Elenchus

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Posted 08 July 2019 - 08:30 PM

Hard one to call.

 

2 ml may have added to much moisture which will foster bacterial growth.

 

Not knowing the history or preparation of syringes throws another twist in determining if it was syringe related.

 

I'm leaning towards to much moisture if the bacteria is overtaken and no other contamination shows it's ugly head.

 

Appreciate the input, roc. I was aiming for faster colonization on these cakes, but maybe I should have just stuck to 1ml.

 

The third photo is the PESA, which were innoced with only 1-1.5 each and still showed the same yellowing on the grains. I also put a small extra layer of dry verm at the bottoms of these jars to collect moisture because I noticed the rice was a bit too compact/stuck together at the bottom of the first set of jars. 

 

The second photo is the Malabar, which is beginning to overtake the bacteria, I presume.


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#4 Moonless

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Posted 09 July 2019 - 09:51 AM

I did the same 2 ml mistake before, all but one were bacteria contaminated too much, one of them overtook the bacteria (this jar had but a small patch of goop gop). Have you considered making a Still Air Box? Perhaps next time you are gathering supplies it would be beneficial to make one real quick. I made mine out of a storage container and used a large can of beans to drill a hole for my arms. Will you be doing another round of jars soon? I think better start soon in case some jars don't make it. IMO it seems like they might be able to over take it, however there might be a small patch that wont colonize. If a jar is contaminated, throwing it into a hole outside is a good bet.

 

EDIT:

Looks like PESA is doing well, Malabar might be able to do it. Please update us when you can, I would love to see the progress!


Edited by Moonless, 09 July 2019 - 09:52 AM.


#5 Elenchus

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Posted 09 July 2019 - 04:53 PM

I did the same 2 ml mistake before, all but one were bacteria contaminated too much, one of them overtook the bacteria (this jar had but a small patch of goop gop). Have you considered making a Still Air Box? Perhaps next time you are gathering supplies it would be beneficial to make one real quick. I made mine out of a storage container and used a large can of beans to drill a hole for my arms. Will you be doing another round of jars soon? I think better start soon in case some jars don't make it. IMO it seems like they might be able to over take it, however there might be a small patch that wont colonize. If a jar is contaminated, throwing it into a hole outside is a good bet.

 

EDIT:

Looks like PESA is doing well, Malabar might be able to do it. Please update us when you can, I would love to see the progress!

Moonless- I do have a still air box. 

 

The main reason I think it could be the syringes is because I inoculated the PESA in the SAB while in a bleach-sanitized room after several mistings of 10%bleach solution to the air (perhaps too much, I hate inhaling that stuff). I also did a transfer from an agar jar (PE) to an LC jar in the same conditions (in the SAB). The LC looks clean. But I wont know for sure until I test it out on a few jars of grain (either rye or millet).

 

I figure that the bacteria needs to come from somewhere, right? After taking such precautions (inoculating in the SAB/cleaned room), I am thinking that I just have bad syringes.

 

But I could be wrong. You are all right that I should not have deviated from the prescribed 1ml.

Must have lost some patience over the years.

 

 

Here's the overall plan for PE:

Agar clone >> LC >> Grain >> Bulk Sub >>Clone to agar >>attempt further isolation/store>>repeat

 

And for the PESA/Mal cakes (if they survive):

 

MS >> BRF >> Clone/Print >>LC >> Grain >> Bulk

From Prints attempt isolation of sub-strains >> test for fruiting abilities

 

 

Updates to come


Edited by Elenchus, 09 July 2019 - 04:54 PM.


#6 Elenchus

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Posted 09 July 2019 - 05:00 PM

Here's some more pics for y'all:

 

(not recent because I'm out of town)

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#7 jkdeth

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Posted 09 July 2019 - 06:19 PM

I wouldn't worry too much about the amount of inoculant. I don't think that's the issue.

The cakes look like they were too wet to start with. Can you detail your cake prep?

Could very well be the syringe was dirty, because they're all dirty, some are just a lot cleaner than others.
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#8 Elenchus

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Posted 09 July 2019 - 07:24 PM

Jkdeth- here's the prep, briefly

 

On the first round I made all the BRF mix for 4 jars at once using the correct ratio (1:1:2). This, I think, was a mistake. Another problem was that the verm I bought was med-large in size, which meant it held less water. I noticed that there was some excess water at the bottom of the bowl I did the mixing in, so I added extra verm to soak it up. Jars were loaded and wiped clean, etc. 

 

On the second round I mixed each jar separately (1/4 cup rice, 1/4 cup water, 1/2 cup verm). I also separated whatever small-grade verm I could out of the bag from the bottoms and used that to make the substrate part of the jars. This made it so that no excess water was left over after mixing. Noticing some moisture problems on the first round of jars, I decided to add a small layer of larger grade verm at the bottoms to collect moisture and prevent an overly compact substrate (I'm not sure if this was a good idea or what, never done it before). I loaded the jars in the same way as above.

 

I can't see anything in particular wrong with the second prep method.


Edited by Elenchus, 09 July 2019 - 07:26 PM.


#9 Elenchus

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Posted 14 July 2019 - 10:28 PM

A quick update:

 

the Malabar strain has made some invirto pins/primordia at about 80% colonization (with bacteria):

 

What should I do? 

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Edited by Elenchus, 14 July 2019 - 10:33 PM.


#10 Elenchus

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Posted 14 July 2019 - 10:30 PM

Here's some more pics

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Edited by Elenchus, 14 July 2019 - 10:36 PM.

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#11 Elenchus

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Posted 08 August 2019 - 04:46 PM

Life has been rather hectic, so this project had to rest on the back burner for awhile. Now I'm back at it.

Time for a full-sized update:

 

 

Two Malabar cakes reached 90% colonization and were crumbled in a res-effect casing and placed outdoors.

20190808_170115.jpg

 

Another reached about 75%. The un-colonized grain was removed and the rest was cumbled and placed under the upper layer of soil in an outdoor potted plant. There's already a small pin poking through:

20190808_170126.jpg

 

Only 2 PESA cakes reached around 95% colonization.

20190805_115315.jpg

 

These cakes were birthed and the un-colonized bits were removed with a flame sterilized blade. They are now dunking in 1:100 bleach/water. The plan is to try to fruit them in a small chamber with perlite, double-end casings, etc. Not sure if it's a great idea, but the reasoning is that maybe the spore prints would be cleaner if taken from fruits in this way.

 

Finally the PE lc was tested on 4 jars of millet (my preferred grain). Each jar was inoculated with 2ml of live tissue solution. One jar just started showing growth:

20190808_175012.jpg 20190808_175008.jpg

 

Can anyone tell if the grain is at optimum moisture content? I no longer have an eye for water content. 

 

Still not sure what went wrong with these cakes, but as long as I can get a print and some live tissue from at least one specimen I'd consider it a success. The goal at this moment is to get a stock of prints and isolates/clones. 



#12 Elenchus

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Posted 08 August 2019 - 05:08 PM

Agar work:

 

I finally sourced from agar powder and malt but when I dissolved the malt in water it looked way too dark to work with. So, I made some PDA instead.

20190801_220530.jpg 20190801_220503.jpg

 

I made way too much so I ended up filling the jelly jars half way with agar. The rest was sterilized in a pint jar (about 300ml). Can this be melted, repoured into more jelly jars and then re-sterilized or does it not last beyond more than one PC run??

 

I was gifted 3 jelly jars of PE on MEA by a FAOF. I was told that two of them were 2nd transfers and one was a clone.

 

From these I transferred 1 single wedge each onto three new PDA jelly jars. 

20190808_185007 (2).jpg 20190805_115247.jpg 20190805_115103.jpg

I believe the first pic is transfer from clone. Seems to be uniform linear growth. It appears to be showing contams as well. I will transfer away asap.

 

I was also given one GT on MEA and was told it was 1 transfer away from MS. From this I transferred onto two new PDA jars.

20190808_180944.jpg 20190808_185047.jpg

Possible contams were noted as well as some rizomorphic sectors, which was transferred onto three new jars. One shown here:

20190808_185123.jpg

 

My goal with these is to transfer until mostly uniform growth is noted, label ISO A, B, C, etc, then test in bulk substrates and select for best phenotype, aggressive growth, etc.

 



#13 Elenchus

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Posted 08 August 2019 - 05:36 PM

I was also given some new toys:

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Can anyone link me with teks on how to use them? Is the RTV going to work the same as GE silicone II caulk? 

Any help much appreciated

 

I tried using the rubber stopper/filter set up one one of my LC jars

20190808_192541 (2).jpg

 

But I did not use silicone caulk to seal the hole. I made them just a bit smaller than the size of the pieces and them just stuffed them in. Seems to be leaking though so it was not a good idea. The double rubber stopper setup will probably be caulked in before use.



#14 Elenchus

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Posted 11 August 2019 - 03:35 PM

Here's some progress on the PESA cakes:

20190811_172622.jpg 20190811_172628.jpg

 

They appear to be recovering nicely from the bleach dunk. The areas where infected grain was removed are fluffing back up with mycelium and there are a couple pins. 

 

Not sure if anyone is following this thread, but I will keep updating for now.






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