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Offlineinsutama
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Pictures of my agar possible contams advice please
    #25378698 - 08/10/18 01:46 AM (5 years, 7 months ago)

So about 5 days ago i inoculated some dog food agar with a spore syringe in a SAB i had major condensation issues in the mini rounds. I took a few cuts of the leading edge of the mycelium from one container. The other container im sure is all contaminated can you guys please clarify if both these agar dishes are contaminated or if it just looks this way because there was way too much moisture in them. It wouldn't make sense that the were both 100% contaminated because i used the same syringe on 5 jars of grain and there all doing good with no contams. thanks.




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Re: Pictures of my agar possible contams advice please [Re: insutama]
    #25378766 - 08/10/18 02:56 AM (5 years, 7 months ago)

Grain is good at hiding bad things. like trying to play where's Waldo with a pop-up book from hell.

I would continue working with the top plate and toss the other.  do you have others plates left over that aren't contaminated from the same batch of agar?

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Re: Pictures of my agar possible contams advice please [Re: liloldme]
    #25378798 - 08/10/18 03:49 AM (5 years, 7 months ago)

:whathesaid: you wont know until your first flush if they had contams you can't see or recognize


Are these your plates from spore inoculation?
Next time, use a loop. If you are using spore solution, place a teeny drop on the plate and swipe it out into a zig zag pattern with a sterilized loop. Same going from print to plate.

This gives you a better idea of where germination should be occurring, in that zig zag pattern. Transfer as soon as you see healthy growth.

On your top plate, you have blobs of bacteria all over the agar, and a very fluffy culture. It could be fine butvit also could have spread right over bacteria, making it very hard to seperate


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Re: Pictures of my agar possible contams advice please [Re: mushpunx] * 1
    #25378868 - 08/10/18 05:04 AM (5 years, 7 months ago)

It's hard for me to tell if it's potato flakes or blobs of bacteria, I'm wondering if his extra plates are displaying these blobs is where I was going with the next question. I like pouring my plates a week ahead of time and let them sit at room temperature to be safe. That's more than enough time to let anything bad colonize so you know the plates are good to go, or not..

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Re: Pictures of my agar possible contams advice please [Re: liloldme]
    #25378885 - 08/10/18 05:16 AM (5 years, 7 months ago)

Yea that's not a bad idea!

I think they are blobs


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Re: Pictures of my agar possible contams advice please [Re: mushpunx]
    #25378894 - 08/10/18 05:23 AM (5 years, 7 months ago)

I'm leaning towards blobs as well because they're not on the bottom plate.

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Offlineinsutama
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Re: Pictures of my agar possible contams advice please [Re: liloldme]
    #25379082 - 08/10/18 07:52 AM (5 years, 7 months ago)

I did toss both the plates but i took 2 transfers from the first plate the second one i threw out and didnt take any transfers.

All the other containers i made with this same batch of no pour i kept in the fridge and there all contam free so if these are contaminated it would of had to happen when i inoculated them.

What are blobs ?

Edited by insutama (08/10/18 07:54 AM)

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Re: Pictures of my agar possible contams advice please [Re: insutama]
    #25379275 - 08/10/18 09:59 AM (5 years, 7 months ago)

Blobs of bacteria. The little yellow dots on your place that look like boogers. Sometimes its a thin layer of scum that's hard to see,other times it's blobs.
They'll be on the surface. You should be able to tell the difference between bacteria and sediment from agar mix because sediment will be there after you pour/after the no pours are set.

Mold colonies (if they're green/sporulating I'd suggest just tossing that plate.
  Before the mold spodulates , it will look like a white, to off white spots.

This is why its important to not let your culture get too big that grows into the mold/bacteria.
Its also why its important to swipe your spores with a loop


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Offlineinsutama
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Re: Pictures of my agar possible contams advice please [Re: mushpunx]
    #25379365 - 08/10/18 10:43 AM (5 years, 7 months ago)

What about the second picture whats up with that is that bacteria mold or mycelium ?

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Re: Pictures of my agar possible contams advice please [Re: insutama]
    #25380782 - 08/11/18 12:44 AM (5 years, 7 months ago)

Not much there to get excited about.. You mentioned you have grain jars that are almost finished colonizing? You could do a grain to agar transfer to clean things up with a few rounds of plates.

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OfflineManifoldPrime
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Re: Pictures of my agar possible contams advice please [Re: mushpunx]
    #25380800 - 08/11/18 01:18 AM (5 years, 7 months ago)

Quote:

mushpunx said:
Are these your plates from spore inoculation?
Next time, use a loop. If you are using spore solution, place a teeny drop on the plate and swipe it out into a zig zag pattern with a sterilized loop. Same going from print to plate.

This gives you a better idea of where germination should be occurring, in that zig zag pattern. Transfer as soon as you see healthy growth.





Second this OP. Or (I haven't done this myself) you could make a divot in the center of the plate with the back of a scalpel/loop and  put a dot of spore solution in the middle of the plate. So the solution stays in the middle, your inoculation point is obvious , and anything that grows outside of it you can disregard/avoid.

Then, do 2-3 transfers and post pics for us to verify.


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Offlineinsutama
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Re: Pictures of my agar possible contams advice please [Re: ManifoldPrime]
    #25382889 - 08/12/18 05:07 AM (5 years, 7 months ago)

thanks for the feedback guys. Ill post pictures of the agar with the slices i took from the first picture once there is more growth and if there no good ill throw them out and try a piece of grain from my grain jars try that out.
I also have some older spore prints i took about 5 years ago i could try using them.

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Offlineinsutama
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Re: Pictures of my agar possible contams advice please [Re: insutama]
    #25390444 - 08/15/18 03:26 PM (5 years, 7 months ago)

So this is my update on the slices i took i now made one more transfer but i think for some reason they keep getting contaminated i dont get why im using a SAB and clean procedures. any advice on what the issue could be ?




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OfflineDorfnob
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Re: Pictures of my agar possible contams advice please [Re: insutama]
    #25390672 - 08/15/18 05:23 PM (5 years, 7 months ago)

I think you have to take samples a few times before you get a clean plate. keep at it. :thumbup:

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Offlineinsutama
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Re: Pictures of my agar possible contams advice please [Re: Dorfnob]
    #25390689 - 08/15/18 05:28 PM (5 years, 7 months ago)

this will be my third now hopfully they turn out better

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OfflineDorfnob
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Re: Pictures of my agar possible contams advice please [Re: insutama]
    #25390767 - 08/15/18 06:02 PM (5 years, 7 months ago)

practice makes perfect but you already know that. I've taken the hobby up again and I'm right where your at. don't sweat it too much it's supposed to be fun.:peace:

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Re: Pictures of my agar possible contams advice please [Re: Dorfnob]
    #25390812 - 08/15/18 06:20 PM (5 years, 7 months ago)

You can see the bacteria spots are growing all over the plates in your last few pics. In fact some are growing on the walls of the dish. This tells me that it is not your inoculant but is in your sterile procedure or possibly contaminating while cooling down.

You may be moving your arms too fast in the box, maybe you have some airflow in the room with the box, something like that. Wash your arms up to the elbows before starting work.


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Re: Pictures of my agar possible contams advice please [Re: insutama]
    #25390922 - 08/15/18 07:11 PM (5 years, 7 months ago)

Quote:

insutama said:
So this is my update on the slices i took i now made one more transfer but i think for some reason they keep getting contaminated i dont get why im using a SAB and clean procedures. any advice on what the issue could be ?









These look pretty un savable. Bacteria absolutely everywhere, from the way the myc looks its infected for sure.
I'm guessing it grew up and over bacteria on the plate.

Start a new culture from germination. Make sure to swipe spores in a zig zag pattern and transfer clean growth as soon as you see it.
On the next plates, try to transfer when its about the size of a dime to a nickel.
Subsequent cultures that will be clean you can grow out more, to about 2/3rds of the plate before using or placing in cold storage.

Read thru Bodhisatta's pages on agar work.


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Offlineinsutama
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Re: Pictures of my agar possible contams advice please [Re: mushpunx]
    #25390973 - 08/15/18 07:33 PM (5 years, 7 months ago)

thanks guys ill have to make some new jars and try again

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InvisibleCaps McGee
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Re: Pictures of my agar possible contams advice please [Re: liloldme]
    #25391158 - 08/15/18 08:50 PM (5 years, 7 months ago)

Almost nothing (except a few cold loving molds)will grow out refrigerated... so leaving them there will not eliminate contamination as a possibility...
Quote:

liloldme said:
I like pouring my plates a week ahead of time and let them sit at room temperature to be safe.



I pour ahead of time as well, they sit in my SAB, which doubles as storage for equipment and unused/ready to use Petri dishes with the lid, when it's not flipped over, straight onto the table top

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Offlineinsutama
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Re: Pictures of my agar possible contams advice please [Re: Caps McGee]
    #25395368 - 08/17/18 10:17 PM (5 years, 7 months ago)

How do you check your growth in the mini rounds is it safe to take lid off and look then put it back on. I find i cant even tell there contaminated until i take lid off to make my cuttings and i dont really want to take lids off them just to look at them because this could cause contams on it own. Im starting to think agar is alot more difficult that it sounds at least for me i have done dozens of grain jars and monos with multi spore and had 80 - 90% success however so far i have had 100% failure with agar lol

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Re: Pictures of my agar possible contams advice please [Re: insutama]
    #25395374 - 08/17/18 10:22 PM (5 years, 7 months ago)

I try to trust my technique and not fiddle with my plates... but you can open pasty plates turned down at an angle (in a SAB) to check them out... if it's completely grown out, it's as resistant as a fully colonized substrate... I prefer pouring in petris... couldn't keep condensation down in the pasty's

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Re: Pictures of my agar possible contams advice please [Re: Caps McGee]
    #25395606 - 08/18/18 03:25 AM (5 years, 7 months ago)

Quote:

Caps McGee said:
I try to trust my technique and not fiddle with my plates... but you can open pasty plates turned down at an angle (in a SAB) to check them out... if it's completely grown out, it's as resistant as a fully colonized substrate... I prefer pouring in petris... couldn't keep condensation down in the pasty's




It maybe be resistant at that point but if you are going to be using it to inoculate anything you need to treat it the same as you would a blank plate.


If you are going to be working with agar and grains, you gotta learn to open lids. Gotta learn how to work in the SAB proper

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Re: Pictures of my agar possible contams advice please [Re: mushpunx]
    #25395684 - 08/18/18 05:14 AM (5 years, 7 months ago)

I don't feel the need to peek lol... consequently, I use petris so not a huge deal viewing when the hunger strikes

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Re: Pictures of my agar possible contams advice please [Re: insutama]
    #25395940 - 08/18/18 09:28 AM (5 years, 7 months ago)

Quote:

insutama said:
How do you check your growth in the mini rounds is it safe to take lid off and look then put it back on. I find i cant even tell there contaminated until i take lid off to make my cuttings and i dont really want to take lids off them just to look at them because this could cause contams on it own. Im starting to think agar is alot more difficult that it sounds at least for me i have done dozens of grain jars and monos with multi spore and had 80 - 90% success however so far i have had 100% failure with agar lol




This is where no-pour style plates like these are kinda shit and traditional petri dishes are better: Actually being able to see what is happening in your plates. If you wanna full peak you gotta break out the SAB (or flowhood, but if you have a flowhood and are doing no-pour plates you are a mystery to me) and even then your vision is somewhat obscured by the SAB transparency.


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Re: Pictures of my agar possible contams advice please [Re: sandman420]
    #25395950 - 08/18/18 09:32 AM (5 years, 7 months ago)

I peer through the arm holes with my chin tucked while breathing through my nose...

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Offlineinsutama
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Re: Pictures of my agar possible contams advice please [Re: Caps McGee]
    #25396554 - 08/18/18 02:15 PM (5 years, 7 months ago)

Good advice thanks ya for now im putting the agar work on hold i got 4 jars of spawn that will be ready any day so im gonna spawn them to my mono tub then maybe try playing with a fresh batch of agar and new inoculation

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Re: Pictures of my agar possible contams advice please [Re: insutama]
    #25396664 - 08/18/18 03:31 PM (5 years, 7 months ago)

:rockon: :rockon: :rockon:

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