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OfflineRo0k
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First Agar plates/test pictures. And then a question (Update)
    #22332654 - 10/04/15 01:09 PM (8 years, 3 months ago)

****Update 1****


Last night I mixed up my first batch of agar in my SAB using RR's Agar video and tek.  350mL of water for 8g of agar worked great.  Put plenty of water in my PC and used one of my SHIP lids with some micropore over the filter hole and it worked like a charm.  No boil-over, it was easy to handle and pour.  Where I deviated was I used bleach in my SAB instead of soapy water for this one, figuring that since I was working with a substance essentially designed to feed bacteria, the more dead any floaties are the better. 

So I poured the plates (stacked them on a small plate so they were off the bottom of my SAB) covered them back up with the sleeve and left them till morning.

So far, so good.  I made 10 plates and they turned out great with no air bubbles and nothing growing (yet).  Fairly clear too:



I put 8 plates aside for spares and to use for more isolation/strains.  So what I did was wrap them with masking tape and put them in individual baggies in a covered ziploc container.  They're all wrapped and upside-down in the containers to prevent contam from beaded water and are sitting happily in my fridge.





I used 2 plates and put 8-10 drops of spores from a syringe I'd made two days prior from a print from 2013... I figure 48 hours to re-hydrate is enough.  It's a good test of both whether the syringe is contaminated and/or whether my agar technique is good.






The two test plates are now sitting in my incubator on top of a spare lid to get them off the bottom of the box.  And before you ask why I'm using an incubator, here's the temperature this morning in my place:




Now the Questions.

Both of my inoculated plates are sitting in the incubator wrapped in micropore tape and in their own individual baggy, but they're starting to condense due to the temperature differential and the water in the spore syringe I assume. 

How do I handle this? 
Do I crack the bag?
Do I take them out and hope the tape is enough to prevent contam? 
Do I leave it as-is trusting that the condensation will settle?
Do I flip the plates upside-down to prevent dripping (I'd thought of this, but won't the spores fall off the agar?)

I'm accepting all answers/critiques on my technique.


Edited by Ro0k (10/07/15 05:01 PM)


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InvisiblePastywhyteMDiscord
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Re: First Agar plates/test pictures. And then a question [Re: Ro0k]
    #22332769 - 10/04/15 01:35 PM (8 years, 3 months ago)

Why did you wrap them in tape. That ain't going to help condensation none. Why are you keeping them in the fridge. Do that when you have something growing on them that you want to stall.


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OfflineRo0k
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Re: First Agar plates/test pictures. And then a question [Re: Pastywhyte]
    #22332845 - 10/04/15 01:55 PM (8 years, 3 months ago)

Everything I did i did to limit contams as much as possible. So the wrap is to prevent mold/bacteria from finding its way insode the dishes. Fridge is to limit growth of any potential contams and isolate them as much as possible because I won't be using these plates again for at least another week. I'd hate to go to use the plates to isolate a strain and find out that they've all been colonized by bacteria.


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Re: First Agar plates/test pictures. And then a question [Re: Ro0k]
    #22332940 - 10/04/15 02:12 PM (8 years, 3 months ago)

Your thinking is wrong. First if there are live microbes in the unused plates you want them to germ and grow. That way you cansee them and not use those plates. Otherwise it ends up in your grow and it fails. You don't want tape on the edges of your plates either, its a sticky hard too work with mess. Use parafilm or saran wrap. They breathe enough for condensation to dissapate and prevent an anerobic environment.


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OfflineRo0k
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Re: First Agar plates/test pictures. And then a question [Re: Pastywhyte]
    #22332977 - 10/04/15 02:18 PM (8 years, 3 months ago)

I see. So no individual bagging and leave em at room temperature. Can I still leave them in the containers unbagged?


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Re: First Agar plates/test pictures. And then a question [Re: Ro0k]
    #22332981 - 10/04/15 02:19 PM (8 years, 3 months ago)

You can bag em and wrap the edges in saran or parafilm. But no tape and leave at room temp until you have a culture on them that you want to save.


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Re: First Agar plates/test pictures. And then a question [Re: Pastywhyte]
    #22333627 - 10/04/15 05:10 PM (8 years, 3 months ago)

8 to 10 drops per plate? Correct me if I'm wrong, but wouldn't you like to know how your plates are going to perform under normal conditions? You would know within 4 days with 1 drop if your syringe was clean and you might still have a culture worth working with after. With 8 to 10 drops that plate is going to be a mess.


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OfflineRo0k
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Re: First Agar plates/test pictures. And then a question [Re: Kalistis]
    #22335856 - 10/05/15 07:19 AM (8 years, 3 months ago)

Quote:

Caelistis said:
8 to 10 drops per plate? Correct me if I'm wrong, but wouldn't you like to know how your plates are going to perform under normal conditions? You would know within 4 days with 1 drop if your syringe was clean and you might still have a culture worth working with after. With 8 to 10 drops that plate is going to be a mess.





Oh. Well we'll see in a few days. Worse comes to worse I chalk it up to a lesson learned. So like... 2-3?


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Re: First Agar plates/test pictures. And then a question [Re: Ro0k]
    #22335875 - 10/05/15 07:27 AM (8 years, 3 months ago)

Yea man 8 drops is way too much. When it germinates it will make it super hard to distinguish clean myc from anything else

With  innoculations from spore solution, drop it onto a sterile cotton swab or flamed loop and do a little swipe on the plate. Otherwise the liquid will roll around causing growth all over the place vs where you made the swipe.. which makes it much harder to find contams , the culture might grow right over bacteria or mold colonies

Lose the bleach too. If its dirty wash it out before you work but just use soapy water spritZ, you just need the air to be still. The only reason we spray soapy water is to help catch and trap any airborn contams

Even if the syringe doesn't show contams on agar you'll still want to make a transfer or two and use your agar for innoculations over the syringe.. unless you are doing BRF cakes


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Edited by mushpunx (10/05/15 07:39 AM)


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Offlinemushpunx
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Re: First Agar plates/test pictures. And then a question [Re: mushpunx]
    #22335909 - 10/05/15 07:45 AM (8 years, 3 months ago)



Oh, since you don't have parafilm yet probably, here's what I do when Im out. Cut a 1.5 inch section off a roll of cling wrap and tightly wrap the edge of the plate.

This is a really sloppy wrap job in the pic tho :facepalm: but its all I have for example.

Fresh poured plates can be put in stacks of 3-4 un wrapped in brand new zip lock bags (they're sterile) . Then wrapped after theyre inoculated.

I usually don't do that though unless I poured a lot of plates. If its just a stack I just give a quick wrap with cling wrap, and use parafilm only after theyre i nnoculated. Saves money :shrug:


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OfflineRo0k
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Re: First Agar plates/test pictures. And then a question [Re: mushpunx]
    #22336106 - 10/05/15 08:54 AM (8 years, 3 months ago)

Parafilm is expensive as eff and hard to find here. Gonna try the saran wrap method first. But on the next set of dishes, don't want to chamce contams while I've got them wrapped and stored atm. I'll just deal with the mess and do it better next time.


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OfflineRo0k
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Re: First Agar plates/test pictures. And then a question [Re: mushpunx]
    #22337190 - 10/05/15 02:48 PM (8 years, 3 months ago)

Quote:

mushpunx said:
Yea man 8 drops is way too much. When it germinates it will make it super hard to distinguish clean myc from anything else

With  innoculations from spore solution, drop it onto a sterile cotton swab or flamed loop and do a little swipe on the plate. Otherwise the liquid will roll around causing growth all over the place vs where you made the swipe.. which makes it much harder to find contams , the culture might grow right over bacteria or mold colonies

Lose the bleach too. If its dirty wash it out before you work but just use soapy water spritZ, you just need the air to be still. The only reason we spray soapy water is to help catch and trap any airborn contams

Even if the syringe doesn't show contams on agar you'll still want to make a transfer or two and use your agar for innoculations over the syringe.. unless you are doing BRF cakes





Wouldn't the spores get stuck to the swab instead of the plate tho?


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OfflineNDStepp84
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Re: First Agar plates/test pictures. And then a question [Re: Ro0k]
    #22337234 - 10/05/15 02:57 PM (8 years, 3 months ago)

Some, but I think you may be underestimating how many spores are in a drop of spore solution, check this out, http://www.shroomery.org/forums/showflat.php/Number/21365919/fpart/1/vc/1


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Re: First Agar plates/test pictures. And then a question [Re: Ro0k]
    #22338108 - 10/05/15 06:55 PM (8 years, 3 months ago)



quote]Ro0k said:
Parafilm is expensive as eff and hard to find here. Gonna try the saran wrap method first. But on the next set of dishes, don't want to chamce contams while I've got them wrapped and stored atm. I'll just deal with the mess and do it better next time.





If you poured them clean then they won't contam if you don't open them in open air. Keeping them  wrapped helps them stay closed.

If they do grow contams while theyre being stored it was a bad pour. See this photo? Its a stack of plates I wrapped and slid back into its sleeve on the side of my flow hood. Its one of 5 stacks of 20 I poured last month, not one was bad and they've kept just fine.

Pouring isn't so hard. If you haven't, watch the agar section of letsgrowmushrooms to get a better idea of proper sterile tek

:thumbup:


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OfflineRo0k
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Re: First Agar plates/test pictures. And then a question [Re: mushpunx]
    #22339959 - 10/06/15 07:24 AM (8 years, 3 months ago)

Pouring is no problem... At least so far 36 hoirs in and no signs of contam in the poured plates. I did 2 more dishes last night by dropping enoigh drops onto the sterile q-tip to wet it then swiping the plate. I'll see if that generates better results because i've pretty much written off the first 2.

My one inpleasant discovery is that my SAB isn't tall enough for a stack of 20 plates. I have to buy sleeves of 10 insread.


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Re: First Agar plates/test pictures. And then a question [Re: Ro0k]
    #22340068 - 10/06/15 08:10 AM (8 years, 3 months ago)

Quote:

Ro0k said:
Pouring is no problem... At least so far 36 hoirs in and no signs of contam in the poured plates. I did 2 more dishes last night by dropping enoigh drops onto the sterile q-tip to wet it then swiping the plate. I'll see if that generates better results because i've pretty much written off the first 2.

My one inpleasant discovery is that my SAB isn't tall enough for a stack of 20 plates. I have to buy sleeves of 10 insread.





Building a bigger still air box will make life so much easier.

But most people seperate the sleeve of 20 into two or 3 stacks, its too tricky to lift that whole stack up to pour!!

Oh, and even if you don't pour the whole sleeve of 20 then just wrap the unused plates and store in fresh zip lock until you're gunna use them


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Invisibleblindingleaf
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Re: First Agar plates/test pictures. And then a question [Re: mushpunx]
    #22340263 - 10/06/15 09:14 AM (8 years, 3 months ago)

:whathesaid:



always pour a full sleeve.  u will be thankful later when u need dishes.  store in plastic gallon zip locks.  two stacks of ten each.  they keep for a while at room temp.  longer in the fridge



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Re: First Agar plates/test pictures. And then a question [Re: Ro0k]
    #22340478 - 10/06/15 10:27 AM (8 years, 3 months ago)

Quote:

Ro0k said:
Quote:

Caelistis said:
8 to 10 drops per plate? Correct me if I'm wrong, but wouldn't you like to know how your plates are going to perform under normal conditions? You would know within 4 days with 1 drop if your syringe was clean and you might still have a culture worth working with after. With 8 to 10 drops that plate is going to be a mess.





Oh. Well we'll see in a few days. Worse comes to worse I chalk it up to a lesson learned. So like... 2-3?




Good morning! You can take a look at my pics in my gallery, but all of my plates started from MS.... less than a drop. Like others have mentioned, there are loads of spores in a single drop, and more in the case of agar doesn't mean better, just more work. I accidentally squirted 3 drops on one plate and that plate was a complete loss. There was so much overgrowth that I couldn't determine a healthy spot to isolate.

I have also experimented with technique using the syringe with needle directly to agar and syringe to inoculation loop then to agar. Honestly, all worked. I did get some contaminant my last few plates which I think had to due to my technique and how I put my lid back on the plate.

I decided that just putting a drop on the plate is fine. I feel like solution to loop to agar increases the potential for contamination, especially given I am juggling the steps and bound to fuck something up. But that is just me.


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Re: First Agar plates/test pictures. And then a question [Re: Kalistis]
    #22340498 - 10/06/15 10:33 AM (8 years, 3 months ago)

And honestly, I am brand new to this and I go through plates like water. My advice is to mix and pour a lot! I mixed enough for 20 plates my first go and only got like 12 out of it because I was struggling to see what I was doing in my SAB. I have a lot of agar pouring experience in a laboratory setting, but this is a completely different ball park. I really wish I had a flow hood. The second two batches I doubled my batches- which helped a lot.


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Re: First Agar plates/test pictures. And then a question [Re: Kalistis]
    #22346432 - 10/07/15 04:52 PM (8 years, 3 months ago)

Just some updates on the progress of my agar plates. 

First some good news: after 4 days there appears to be ZERO growth on any of my poured plates, both those inside the fridge and those left in room temperature.  I'm extremely happy about this because it means that, at least so far, my sterile procedures when pouring are good.  So far. 

Well, I inoculated a few plates on 10/04 (pictured in the first post with the micropore tape) and did 2 more on 10/05 using a different technique (no parafilm yet till this weekend).  If you'll recall, the first 2 plates I poured were a giant fustercluck where I put 6-8 drops on each and let the water slosh around and it was terrible.  I have since learned my lesson and modified my procedure.  The next two plates I tested were different strains, and instead of dripping the water right onto the plate, I dripped 5-6 drops onto a sterile swab till it had a nic bead of liquid, then swabbed the plates in a predictable zig-zag pattern.  It worked much better - mostly.  I still may have used too much liquid.  I also wrapped those new ones in cling wrap instead of the micropore tape and I'm seeing much faster results.  I'm unsure of whether this is related to a strain (the best performing one is a strain from a large country in South America) or I managed to get some magical combination of agar mix, spore potency and air flow.  Regardless, I'm happy with this and I think it's going to be a good performer.

To reduce the chance of getting myself in trouble, we'll call the plates below (starting at 9:00) 1, 2 (12:00) and 3 (6:00).  The ones wrapped in micropore tape are the first set.



Plates 1 and 2 were the first batch I did and since it was such a nightmare, they're performing poorly.... a little growth after 3 days, but nothing to write home about.  It could be I didn't leave the syringes to rehydrate long enough, but likely my atrocious technique.  I still think I can get a culture or two off these to isolate, so I'll wait and see.  I don't believe it to be bacteria, but time will tell.  The spotting is most definitely because I dropped the water and let it slosh around, and not necessarily bacteria.  I'll know in a week, and they're wrapped well enough to isolate if I need to. 



This last one has really taken off in the last 48 hours.  Huge, aggressive growth.  If the below is in fact spore germination and I didn't bungle the smear, I expect to be able to get a bunch of really good cultures off it. 

I'm actually a little paranoid about this one since it's doing so much better and developing so much faster than all the rest.  Wouldn't mind some feedback on this, but it's definitely following my streaking pattern.



I'm excited, a bit uneasy,  but I'm learning a ton.  Agar is definitely the way to start this hobby.  Wish I'd known that 2 months ago when I started.


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Re: First Agar plates/test pictures. And then a question [Re: Ro0k]
    #22346776 - 10/07/15 06:21 PM (8 years, 3 months ago)

That last plate looks like hella bacteria colonies.


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Re: First Agar plates/test pictures. And then a question [Re: Pastywhyte]
    #22347240 - 10/07/15 07:51 PM (8 years, 3 months ago)

Quote:

Pastywhyte said:
That last plate looks like hella bacteria colonies.




Yea I second this it looks like no germination, but bacteria colonies completely following your streaks

  You used a swab? Was it sterile, like opened from  sterile packaging or wrapped in foil and run thru PC cycle?


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Edited by mushpunx (10/07/15 07:52 PM)


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OfflineRo0k
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Re: First Agar plates/test pictures. And then a question [Re: mushpunx]
    #22347384 - 10/07/15 08:13 PM (8 years, 3 months ago)

Yeah. Opemed from a pack of sterile swabs. I was afraid of this. Hrmmm.... Into isolation it goes, then.


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Re: First Agar plates/test pictures. And then a question [Re: Ro0k]
    #22347394 - 10/07/15 08:16 PM (8 years, 3 months ago)

I doubt anything is going to germinate in that.


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Re: First Agar plates/test pictures. And then a question [Re: Ro0k]
    #22347396 - 10/07/15 08:17 PM (8 years, 3 months ago)

Quote:

Ro0k said:
Yeah. Opemed from a pack of sterile swabs. I was afraid of this. Hrmmm.... Into isolation it goes, then.





Isolation? No its not gunna spread to the other plates. But toss it, you dont even have germination and already have that much bacteria. I think its un redeeemable man

Did you make the syringe yourself?

Ah Pasty beat me


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Edited by mushpunx (10/07/15 08:17 PM)


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Re: First Agar plates/test pictures. And then a question [Re: mushpunx]
    #22347641 - 10/07/15 08:56 PM (8 years, 3 months ago)

Quote:

mushpunx said:
Quote:

Ro0k said:
Yeah. Opemed from a pack of sterile swabs. I was afraid of this. Hrmmm.... Into isolation it goes, then.





Isolation? No its not gunna spread to the other plates. But toss it, you dont even have germination and already have that much bacteria. I think its un redeeemable man

Did you make the syringe yourself?

Ah Pasty beat me




Nope.  Actually the first two wrapped in micropore are from a syringe I made.  So that seems to have worked okay.  The lower one is from a syringe I bought.  I'm going to try again tomorrow on another plate and remove the swab from the equation.  See if it's the syringe.

What do you think of if I sterilize a knife (I use a propane torch) then score the centre of the plate about half an inch (or cut a shallow x) and put a drop in there?  Figure it'll work?


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Re: First Agar plates/test pictures. And then a question [Re: Ro0k]
    #22348973 - 10/08/15 07:38 AM (8 years, 3 months ago)

That'd be fine too I guess , swabbing it on just keeps the liquid from ro. Dring around and uses way less solution lowering your chance of contams

You can also use an inoculation loop, you can make one out of wire and an e xacto knife handle even.
Flame sterilize it, cool it on the agar, flame your needle, cool it by squirting some liquid out, drip onto loop, swipe on agar.

Im just saying you don't want the liquid all over the place and the myc growing over any bacteria


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