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OfflineSMmack
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Update 2: "Still not going great" * 1
    #28649086 - 02/06/24 02:06 PM (11 months, 4 days ago)

My main takeaway previously was that there were no trends to how things failed, I'm really good at failing grows and all previous samples went bad in different ways, but all things considered my techniques aren't actually that bad. This month I think I've proved that my spore syringes have probably been spat in because they are terrible when compared to my own poorly taken spore prints, compare the other samples to F.

Failed grows from last month:
1) GT T-3 11/01 Trich.  2) APE-Revert T-6 11/01 Yeast & Trich.  3) GT T-3 ?tomentose 16/01 Good old funcicola again & some trich starting to pop up.


The GT I sent to bulk as a trial. One has cobweb and stalled, the other smells wack, it went really wet with bacillus, makes your eyes water, but a total of 2 tiny GT pins before stalling.

When I see a pin in 1KG of bacteria spawn.




This month:
I needed to get more organised so I started a new naming system. Just give each new spore transfer a letter and track back the strain later on. I'm also using LC as a crutch to help identify bacteria contamination, and possibly as a inoculation method, should anything actually grow at any point.

A: GT T-2 culture. 1) Agar looked OK I think  2)Agar-LC came out clear and not baccy, getting excited. 3+4) LC-agar test, second pic seems a bit iff with two different growths in the centre.  5) LC-grain 24/01 Trich end of grow.


B: APE-R T7 (yep it's the only good one I had) 1+2) Agar-Agar 24/01 Looks good. 3) Agar-LC 24/01 lots of sediment on the bottom and no real growth around agar wedge ?Bacc.  4)Same jar after stirring Definitely ?Bacc.  5)Good agar-grain though???
Don't worry, I imagine it'll get worse in a week.


C: GT T2 1+2+3) Agar-Agar 24/01 Looks good. 4) Agar-LC 24/01 There may be some myc floating round in that, but not on the wedge yet. 5)Agar-Grain 24/01 Slower growth, less obvious than B, but looks good so far.


D: GT T0 Spore syringe on inoculation loop, Looks like the spores are crap and the bacteria isn't. Two spots of Maybe growth, but not worth using.


E: APE-R T0 24/01 syringe on inoculation loop, I'll transfer (1) 6 o'clock to two new plates, Probably right on the leading edge but if I see any signs of other growth I'll fall back in slightly.


F: GT T0 spore print directly to agar 24/01. (1) I'm seeing a couple different styles of growth but I couldn't tell you which look good and which look trichy as I haven't developed that eye yet, (2) a little bit of uniform growth, (3) nothing. Where would you take from?


G: APE-R T0 spores 16/01 Might have stalled so I don't want to transfer from the leading edge, I'll take a transfer from just inside the egde.



I also have a small fruit from an old puck, I could let grow out and try and clone. My clones have always ended up notoriously poorly though.


Questions:
- Do I shake B & C now to accelerate results potentially getting a contam takeover or let them grow out to see when and if contam forms?
- Where do I start taking transfers from on F?

Edited by SMmack (02/06/24 02:18 PM)

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Invisiblemeta_mmxxii
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Re: Update 2: "Still not going great" [Re: SMmack]
    #28649105 - 02/06/24 02:31 PM (11 months, 4 days ago)

You appear to me to be having issues with grain sterility, either they are not getting completely sterile in the PC process, or it is getting contaminated from stuff getting in.
Are you using filter lids? If so the source of your contamination is coming from your filter as you are leaving the foil on your jars.
That will promote contamination on your filter and then seep into your grains causing the contamination issue you are having.


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OfflineSMmack
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Re: Update 2: "Still not going great" [Re: meta_mmxxii]
    #28649783 - 02/07/24 12:39 AM (11 months, 3 days ago)

I usually cover the top side with a filter patch and bottom side with micropore to keep the filter clean, some are still micropore both sides. Yes I leave the foil on as that was recommended to me.

I run a few control jars with every inoculation. At 1.30hrs 15psi I got trich in a control jar after 6 weeks, which is a bit random when contam usually shows up in the first week.

This new batch runs for 2hrs @15psi.

How does leaving the foil on cause contamination?

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InvisibleBaba Yaga
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Re: Update 2: "Still not going great" [Re: SMmack]
    #28649804 - 02/07/24 01:47 AM (11 months, 3 days ago)

I'd say condensation between foil and lid and the filter never really getting dry could be a vector. Not sure if this is what really happens but is what springs to mind. The point of having a filter is to keep bad stuff out while letting oxygen in so why use foil. Lotkid does recommend to use at least 4 layer of micropore tape when used as filter on grain jars. I use 3 layers on my no pour dishes.


What does your SAB look like? How big are your arm holes? Feels to me like stuff is getting in during inoculation.

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OfflineSMmack
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Re: Update 2: "Still not going great" [Re: Baba Yaga]
    #28649853 - 02/07/24 03:50 AM (11 months, 3 days ago)

Quote:

Baba Yaga said:
What does your SAB look like? How big are yor arm holes? Feels to me like stuff is getting in during inoculation.





I'll take the foil off I get the wet filter patch theory but I'd seen more advice regarding keeping it on than taking it off. Jar A is definitely from contaminated LC, as my slants got trich as well the other god knows how many jars I can't say.

SAB gets clinell wiped, 10% bleach, dry, 70% iso, put all the stuff in, spray iso mist, leave to settle for 20-40minutes before use.


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OfflineBrokenHeart
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Re: Update 2: "Still not going great" [Re: SMmack]
    #28649887 - 02/07/24 05:23 AM (11 months, 3 days ago)

I love the effort, now let's see about the effects.

In many ways your studies are further than my own.  There are some differences in our processes.

  I PC by venting the steam after lock engagement for ten minutes, then PCing at 15 for 2 hours for 1qt grain jars.  Then after cooling to a reasonable warmth I shock the jars and removed the foil.

  In addition to stopping all fans, furnace,  computers even, I let my prepared SAB sit for 2 hours and during, move like my hands are under water slow methodical.

  Last time I cut my wait time in half, hurried through my SAB work and increased my failure rate by 30%.

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InvisibleBaba Yaga
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Re: Update 2: "Still not going great" [Re: BrokenHeart]
    #28649964 - 02/07/24 07:41 AM (11 months, 3 days ago)

I see a few possible vectors that could cause your contamination problems.

First your arm hols on your SAB are looking too small. They should be somewhere around 7" or otherwise too much air turbulence is created when putting your arm(s) in and out unless you are doing it very slowly. Not sure about the lid on your SAB as well, looks like it has those little ridges for ventilation if I'm not mistaken. Every time you catch the side of the arm hole when removing your arm then air will get sucked in through the gap between lid and tub rim. That fit should be tighter.

I see that you have only one layer of micropore tape on the ventilation holes of your no pour plates. I would double this up and I would cut the tape off the roll instead of tearing it off as I think this causes the membrane to stretch and widens pore size of the tape.

You are keeping your plates upside down in the SAB and it looks like you are having quite a bit of condensation in those plates. Do you ever get water trapped between the lid and the rim of these condiment cups? I am using more or less the same type of cups and when ever pooling water from the agar surface gets trapped there I do not continue to use that plate. It's because this water will have leached nutrients from the agar and is an ideal breeding ground for for contamination. This is why I only ever turn plates upside down which I use as donors for transfers, any other plates will stay right side up.

Have a look at the spots I have circled on those photos of yours. Is this just light reflections or is this where aforementioned water got trapped between lid and rim? I see colors there which seem to resemble what is contaminating the plates.


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OfflineSMmack
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Re: Update 2: "Still not going great" [Re: BrokenHeart]
    #28651246 - 02/08/24 03:38 AM (11 months, 2 days ago)

Quote:

BrokenHeart said:
I love the effort, now let's see about the effects.

In many ways your studies are further than my own.  There are some differences in our processes.

  I PC by venting the steam after lock engagement for ten minutes, then PCing at 15 for 2 hours for 1qt grain jars.  Then after cooling to a reasonable warmth I shock the jars and removed the foil.

  In addition to stopping all fans, furnace,  computers even, I let my prepared SAB sit for 2 hours and during, move like my hands are under water slow methodical.

  Last time I cut my wait time in half, hurried through my SAB work and increased my failure rate by 30%.




This is a better description of how I do mine, just not removing the foil. I’ve left it for hours but didn’t notice a difference in contam rates.

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OfflineSMmack
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Re: Update 2: "Still not going great" [Re: Baba Yaga]
    #28651310 - 02/08/24 06:13 AM (11 months, 2 days ago)

A lot of stuff to think about here, thanks so much.

Quote:

Baba Yaga said:
I see a few possible vectors that could cause your contamination problems.

First your arm hols on your SAB are looking too small. They should be somewhere around 7" or otherwise too much air turbulence is created when putting your arm(s) in and out unless you are doing it very slowly. Not sure about the lid on your SAB as well, looks like it has those little ridges for ventilation if I'm not mistaken. Every time you catch the side of the arm hole when removing your arm then air will get sucked in through the gap between lid and tub rim. That fit should be tighter.




The arm holes are 6.5” diameter, couldn’t find a coffee pot with 7” exactly, I’ll see if there are any in work running empty. The lid doesn’t fit great, better than previous SAB’s but my solution was to just not hit the lid and stop if I did, I’m getting a lot less random spots of contam in my agar since using this one, but am not happy about the edges of the SAB as you said. Any ideas on what I can use to pad, or seal the edges?

Quote:

I see that you have only one layer of micropore tape on the ventilation holes of your no pour plates. I would double this up and I would cut the tape off the roll instead of tearing it off.



Jars are 5x tape or filters, I want to get plastic lids because the metal ones keep rusting after even one use. I’ll double up on the agar tape, but my untouched control plates are all clean.

Quote:

You are keeping your plates upside down in the SAB and it looks like you are having quite a bit of condensation in those plates. Do you ever get water trapped between the lid and the rim of these condiment cups?




Yes. I used to use Petri dishes for no-pour but noticed there was a vacuum broken every time I opened the plate for the first time. I figured this was pulling contam into the plate and so recently moved to condiment cups with a hole to prevent vacuum creation. Due to the height of the cup I’ve noticed loads of condensation on the agar. I used to store plates upside down in the lab so I just carried on doing it because I didn’t like having water available to spread any contaminants across the entire plate. But I guess that doesn’t work if your not in a sterilised air filtered room.

Quote:

This is why I only ever turn plates upside down which I use as donors for transfers, any other plates will stay right side up.



Why turn these upside down, Because they’ve dried out by the time they’re ready to use?

Quote:

Have a look at the spots I have circled on those photos of yours. Is this just light reflections or is this where aforementioned water got trapped between lid and rim? I see colors there which seem to resemble what is contaminating the plates.




Good catch. I had a look a couple days post photos and those patches are now definitely growing green where the water is trapped. This could be where a lot of my issues are coming from.

It is possible this was happening to my Petri dishes aswell, however as so shallow in comparison the growth was just wicking straight into the agar rather than pooling in the join. Does just keeping them upright and letting the condensation evaporate though the tape prevent this problem?

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InvisibleBaba Yaga
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Re: Update 2: "Still not going great" [Re: SMmack]
    #28651641 - 02/08/24 12:38 PM (11 months, 2 days ago)

Ah OK, 6.5 inches is big enough, just looked small. For the lid you could try the foam tape for sealing drafty windows, they sell it in all sort of sizes.


Quote:

Why turn these upside down, Because they’ve dried out by the time they’re ready to use?




Nah, I crack the lid and turn the dish upside down just before I get to work. I can then pick the cup up with one hand to take the transfer and put it back down on it's lid so it doesn't stay uncovered between transfers. The cup doesn't slide around when I cut the wedge. When the cup was still "wet" when turning it I will clean it out and reuse for the next agar batch, if the pooling condensation had already dried up I might keep it around in case I need to take more transfers.


Quote:

Good catch. I had a look a couple days post photos and those patches are now definitely growing green where the water is trapped. This could be where a lot of my issues are coming from.

It is possible this was happening to my Petri dishes aswell, however as so shallow in comparison the growth was just wicking straight into the agar rather than pooling in the join. Does just keeping them upright and letting the condensation evaporate though the tape prevent this problem?





IMO yes, steam that condensates directly in the gap between lid and cup shouldn't have any nutrients in it since its evaporated water, but if it's condensation that pooled on the agar first then it will have nutrients in it and has a high potential to cause contamination. Letting the pooling water dry up does prevent this, never had problems turning dishes upside down and keep using them once the pooling water dried up.

I had random contam problems as well from time to time where single dishes or whole batches would grow a lot of mold. After some trouble shooting I got to the conclusion that nutrients got trapped in that gap. IMO this can happen as discussed above, or when agar boils over in your PC (e.g. knocking the weight or force cooling under cold water) or if not washing dishes which had been used upside down while they were still "wet".


FWIW, I just work with the condensation in the cup, the agar surface is never totally level and the puddle tends to stay on one side or stcks to the cup wall and doesn't bother me. When making the the plates I take the cups out about 20 min after the pressure lock on my 8 liter PC dropped so they are still quite warm but not super hot anymore. I take them out, give them a quick wipe with a clean sponge which I use for nothing else than that job and stack them. I still get droplets on the walls that way but much less on the lids. I'd definitely would put 2 layers of MP tape over the vent hole when taking them out warm though.


Edited by Baba Yaga (02/08/24 12:55 PM)

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OfflineSMmack
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Re: Update 2: "Still not going great" [Re: Baba Yaga]
    #28669116 - 02/21/24 03:45 AM (10 months, 20 days ago)

Quote:

Baba Yaga said:
FWIW, I just work with the condensation in the cup, the agar surface is never totally level and the puddle tends to stay on one side or stcks to the cup wall and doesn't bother me. When making the the plates I take the cups out about 20 min after the pressure lock on my 8 liter PC dropped so they are still quite warm but not super hot anymore. I take them out, give them a quick wipe with a clean sponge which I use for nothing else than that job and stack them. I still get droplets on the walls that way but much less on the lids. I'd definitely would put 2 layers of MP tape over the vent hole when taking them out warm though.



The SAB was 5" actually, I increased it to 6".

I used a remaining 20 plates which have been sat in the cupboard ready to go but this time kept them right side up. All of them ended up like this. 


Then I ran a new batch with 2 lots of mp tape.
Day 2, Samples A C D E & F. There are white trichy dots forming again on the agar transfers, 10 plates, I expect this will spread to match above.


However I also did a fruit clone from an old BRF puck using tweezers on the same batch and the same session in the SAB, 5 plates, no white trichy dots?

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InvisibleBaba Yaga
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Re: Update 2: "Still not going great" [Re: SMmack]
    #28670052 - 02/21/24 04:58 PM (10 months, 20 days ago)

OK the second batch looks way better. I think this a mixture of problems and hopefully not turning the cups over will eliminate the full plate contamination. The rest seems to be a matter of improving sterile technique.

How are you opening the cups?

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OfflineSMmack
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Re: Update 2: "Still not going great" [Re: Baba Yaga]
    #28670758 - 02/22/24 02:22 AM (10 months, 19 days ago)

Quote:

Baba Yaga said:
OK the second batch looks way better. I think this a mixture of problems and hopefully not turning the cups over will eliminate the full plate contamination. The rest seems to be a matter of improving sterile technique.

How are you opening the cups?




In terms of sterile technique, compared to two years ago I'm loads better than when I was just flaming a needle and stabbing bags of ready rice. But I haven't been able to grow anything since. I slow my movements compared to using laminar flow, crack open both lids, take donor lid off, transfer to new plate by pivoting the lid open, then return the lid of the donor. The only thing I can think of to make a big change is to take it into the flow hood in work, but that's not really an option.
Day 3: more trich spots now, I think it won't be long until it looks just like the previous batch.


It does look like aerated trich rather than growing trich, whether its been washed around the surface or something. It is possible its coming through the gap in the lid, Cling film? It's also possible it's coming through the air vent still, 1 layer of tape before PC and 2 layers after?

The contam is definitely being introduced during transfers, However like I have over the past year, I get the feeling the contam is coming from the spores. When you consider that:
  • There is trich in 30/30 agar transfers. But no trich visible on the masters.
  • There is now trich in 2/5 clones, but much less (so far) than agar transfers.
  • I put agar in grain, and tilted the jar to cover the wedge rather than shaking. The trich shows up around the wedge rather than on the surface where you would expect it to if it was introduced via the air.

    9/9 grain jars got contam like this.


IDK it almost seems like my donor agar is the source. Maybe I should just go back to glass petri dishes that don't have air holes at all, but keep them right side up. I might run a new batch with petri's but do one transfer of just clean agar to agar.

Edited by SMmack (02/22/24 02:29 AM)

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InvisibleBaba Yaga
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Re: Update 2: "Still not going great" [Re: SMmack]
    #28671268 - 02/22/24 11:47 AM (10 months, 19 days ago)

Switching to a different type of plate would definitely be a good move if you want to get to the bottom of this.

FWIW this is how I open my cups.



Pry open a short section and then twist the lid which should "pop off".

Edited by Baba Yaga (02/22/24 03:56 PM)

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OfflineSMmack
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Re: Update 2: "Still not going great" [Re: Baba Yaga] * 1
    #28672881 - 02/23/24 02:05 PM (10 months, 18 days ago)

Quote:

Baba Yaga said:

Pry open a short section and then twist the lid which should "pop off".




I open mine to a max similar angle to yours except I just break the seal around the edge before pulling off. Before I did the same thing but upside down. Whether it drew trich into the lid in the same way but just not into the agar, which is why I got trich starting in the lid, just caused slower "invisible" trich. Obvs that would mean my SAB is filled with trich, bacillus, and mould. Which I don't really get where that's coming from.

I'll do a full proper A-Z tek-nique review post in the coming week from agar and grain prep, to transfers with controls when I next get in the SAB. Will link you in.

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