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Invisiblewoofwoof
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Re: Diving into agar pouring your own? [Re: woofwoof]
    #26498718 - 02/22/20 04:19 PM (3 years, 11 months ago)



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InvisibleInfiniteDreams
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Re: Diving into agar pouring your own? [Re: woofwoof]
    #26498753 - 02/22/20 04:56 PM (3 years, 11 months ago)

Another vote for pouring your own.  I made plenty of mistakes on my first attempt, and still had 95% success.  Every time after that was easy since I knew what to expect.  Bod's guide and c10h12n2o's guide are gold


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OfflineIgloomis
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Re: Diving into agar pouring your own? [Re: InfiniteDreams]
    #26499503 - 02/23/20 07:31 AM (3 years, 10 months ago)

How many plates do people typically start with (I'm inoculating from a syringe)? I should probably expect 1-4 transfers if I'm just looking for clean MS, not going for monoculture, right?

I have ordered everything I need to pour my own but I already have 15 pre-poured plates. I was planning to inoculate 2 or 3 of them, and I want to be sure I have enough plates. Do I even need to start with 2 or 3, or is one plate an adequate starting point?

I have always worked with MS syringes but this time, once I reap the harvest, it's Clonesville for me.


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Invisiblec10h12n2o
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Re: Diving into agar pouring your own? [Re: Igloomis]
    #26499899 - 02/23/20 12:44 PM (3 years, 10 months ago)

I always buy a case at a time, cheaper that way

Depends how many cultures you run at a time. Agar work has a tendency to multiply. For example I started a project recently with about 12 plates, and on t2 it's already become more like 80.. but you might not be as OCD as me


--------------------

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche


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OfflineIgloomis
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Re: Diving into agar pouring your own? [Re: c10h12n2o]
    #26500049 - 02/23/20 02:47 PM (3 years, 10 months ago)

Quote:

c10h12n2o said:
...but you might not be as OCD as me



Definitely not :wink:

For this run I'm trying out Bod's unmodified tub Tek and I just want to make sure I have clean mycelium to inoculate 6 quarts. I've been using spores straight from the syringe up until now and, despite being ultra-careful in trying to create sterile conditions, I lost half of my previous 8 (modified) tubs to contamination.

So, dipping my toe into agar before attempting a swan dive.


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Invisiblec10h12n2o
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Re: Diving into agar pouring your own? [Re: Igloomis]
    #26500051 - 02/23/20 02:51 PM (3 years, 10 months ago)

If it was me, I would use the streaking method in my guide, use a drop or two of solution per plate, for each variety you are working with. Then transfer myc a few times to make sure its clean, then do either agar to grain master jar (then grain to grain to make more jars), or make a liquid inoculant and use that to make grain masters (NOT a liquid culture)


--------------------

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche


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OfflineIgloomis
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Re: Diving into agar pouring your own? [Re: c10h12n2o]
    #26502229 - 02/24/20 08:00 PM (3 years, 10 months ago)

Quote:

c10h12n2o said:
If it was me, I would use the streaking method in my guide, use a drop or two of solution per plate, for each variety you are working with. Then transfer myc a few times to make sure its clean, then do either agar to grain master jar (then grain to grain to make more jars), or make a liquid inoculant and use that to make grain masters (NOT a liquid culture)



C10, I have a question about your streaking technique. I have seen pictures of a lot of plates that have a blob in the middle with the sectors fanning out from there. I can see how streaking covers more of the surface but doesn't it make it more difficult to tell what's going on with mycelium development?


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Invisiblec10h12n2o
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Re: Diving into agar pouring your own? [Re: Igloomis]
    #26502471 - 02/25/20 12:23 AM (3 years, 10 months ago)

The purpose of the streaking technique I'm talking about is basically to do a serial dilution on the agar surface. This means that in zone 1, there will be lots of spores (and possibly contams), while each subsequent zone will have less spores and less potential contams, and they will be spread farther apart

Basically you would transfer myc to a new plate as soon as you can. This is a good idea in general when working with spores, since they are typically filthy.

Keep in mind that MS cultures have countless strains intermingled within the colony. On a serial streak, there will be more in zone one colonies, far less in zone 5.

Typically I transfer healthy colonies as soon as I can, often 2 or 3 to a plate. So I might take 5 transfers from a streak plate.

It's not about surface area, it's about trying to get clean colonies , and optionally colonies with less intermingled strains.

You dont really want to let the myc develop too much before transferring it from a spore plate. You'd be watching the development on a subsequent plate, after the transfers.  The goal is to get clean , well organized mycelium,  so typically you should do at least 2 or 3 transfers after the spore plate before actually using the myc to inoculate, since this reduces the possibility of contams from filthy spores, and let's you select aggressive/rhizo/healthy sectors (whatever you are looking for).

Lemme see if I have something on hand and I will post examples


--------------------

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche


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Invisiblec10h12n2o
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Re: Diving into agar pouring your own? [Re: c10h12n2o]
    #26502488 - 02/25/20 12:58 AM (3 years, 10 months ago)

This is what it is supposed to accomplish:


The goal isnt to cover the plate with myc, but rather to isolate clean colonies with less genetic diversity and contams than you would get from simply dropping spores on the plate would result in

You then transfer these colonies and grow them out on other plates, and continue transferring good looking sectors until you have clean, well organized myc

I dont have any great examples unfortunately since my streak plates are all way past the point where i transferred colonies, but maybe you can tell from these pics :


This was the streak plate. I only had germination in zones 1-3 , and I transferred 6 of those those  colonies almost as soon as I noticed them. If you look close you can tell when I did the transfers, about 10 days ago


This is one of the plates I transferred the initial colonies to (transfer #1), and you can see where i took transfers from these about 6 days ago


Here are 2 plates from transfer #2 . Notice how much better organized and rhizomorphic (and faster) these are than the previous plates. They are also much cleaner since there are no spores, so less vectors for contam

These last plates are probably safe to inoculate with, but I will probably do a few more transfers

Does this make sense? Are you getting an idea of the purpose of this technique? It gives us many genetically different colonies to choose from and test/grow out


--------------------

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche


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OfflineCatsLoveHouseMusic
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Re: Diving into agar pouring your own? [Re: c10h12n2o]
    #26502508 - 02/25/20 01:46 AM (3 years, 10 months ago)

It’s kind of like, rather than trying to find the nice families in a big city, you’re spreading it out and finding them in small villages. Definitely learned something today. Good question, good answer. I’m glad I bought a loop as well today, I actually got it because of your tek and you talking about it. I’ll be starting my agar when my items arrive the 26-27. Thanks for the good convo guys. Catch you tomorrow.


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Invisiblec10h12n2o
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Re: Diving into agar pouring your own? [Re: CatsLoveHouseMusic]
    #26502548 - 02/25/20 03:07 AM (3 years, 10 months ago)

That's a great analogy for it. Cities also harbor more disease (contams)

In a big drop of solution, there can be thousands of strains comingling to form the visible mycelium. By spreading and diluting that drop into different zones, the concentration of spores and contams is lower in each new zone.

Often what people think is tomentose myc is actually many different kinds of myc growing through each other.

Serial streaking/dilution helps by creating myc with fewer intermingled strains per colony, and also fewer contams. So it can be really useful whether you #1 just want clean myc (should be the goal for newbies), #2 want to grow out several cultures from the same sporeprint/syringe on agar to find aggressive/rhizo growth, or #3 want to work towards isolates (or at least well organized cultures)

If you could germinate only 2 compatible spores, you would have an isolated strain. But since there are countless spores per drop of solution, unless you do a serial dilution you will have colonies made up of countless intermingled strains

Even with serial dilution streaking you will still most likely have multispore colonies even in the later zones, because spores clump together. But each colony will be genetically distinct from other colonies, and comprised of far fewer strains per colony than they would be if the spores were not spread out. This is great for transferring out lots of different genetically distinct colonies to fresh plates, then picking the best looking ones to move forward with


--------------------

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche


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Invisiblec10h12n2o
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Re: Diving into agar pouring your own? [Re: c10h12n2o]
    #26502550 - 02/25/20 03:13 AM (3 years, 10 months ago)

Also the to illustrate the reason for transferring colonies from the streak plate asap:

Notice in the pic of my (overgrown) streak plate, you can see where in transferred colonies, but now that it has grown out you cannot distinguish those colonies from the countless other colonies that have grown over and through the ones I chose to transfer. This is why you transfer them early before they become intermingled and indistinguishable


--------------------

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche


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OfflineIgloomis
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Re: Diving into agar pouring your own? [Re: c10h12n2o]
    #26502683 - 02/25/20 07:17 AM (3 years, 10 months ago)

Quote:

c10h12n2o said:
Does this make sense? Are you getting an idea of the purpose of this technique? It gives us many genetically different colonies to choose from and test/grow out



Yes, it makes perfect sense. Thank you so much!


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Re: Diving into agar pouring your own? [Re: c10h12n2o]
    #26502691 - 02/25/20 07:23 AM (3 years, 10 months ago)

Quote:

c10h12n2o said:
Also the to illustrate the reason for transferring colonies from the streak plate asap:

Notice in the pic of my (overgrown) streak plate, you can see where in transferred colonies, but now that it has grown out you cannot distinguish those colonies from the countless other colonies that have grown over and through the ones I chose to transfer. This is why you transfer them early before they become intermingled and indistinguishable



So do you want to grab a small clean colony from the first plate when it's about as developed as the first picture, or do you let it develop a little more, somewhere between the first and later picture?


Edited by Igloomis (02/25/20 08:03 AM)


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Re: Diving into agar pouring your own? [Re: Igloomis]
    #26502868 - 02/25/20 10:06 AM (3 years, 10 months ago)

Just 3 more things:

1- Thanks, CatsLoveHouseMusic for starting this thread.

2- C10, what temperature do you recommend for growing cultures?

3- C10, I am going to do my first inoculation this weekend. Will you please stay tuned to this channel so I can post pics and get your feedback?

Thanks again! Oh, and did I mention how much I love your flow hood?


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Invisiblec10h12n2o
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Re: Diving into agar pouring your own? [Re: Igloomis]
    #26503021 - 02/25/20 11:48 AM (3 years, 10 months ago)

Quote:

Igloomis said:
Quote:

c10h12n2o said:
Also the to illustrate the reason for transferring colonies from the streak plate asap:

Notice in the pic of my (overgrown) streak plate, you can see where in transferred colonies, but now that it has grown out you cannot distinguish those colonies from the countless other colonies that have grown over and through the ones I chose to transfer. This is why you transfer them early before they become intermingled and indistinguishable



So do you want to grab a small clean colony from the first plate when it's about as developed as the first picture, or do you let it develop a little more, somewhere between the first and later picture?




The first pic in the thread is actually of bacteria, but the same principle applies.

The streak plate I posted is WAY past the point you'd take transfers. Typically I transfer colonies when they are smaller than a pinhead, basically as soon as possible

You could wait longer and probably not have issues, but the colonies will quickly overlap and intermingle, losing their genetic distinctiveness

Again this is bacteria not myc, but this is about the point I would transfer colonies, with the colonies I would transfer circled in red (very tiny)


Quote:

Igloomis said:
Just 3 more things:

2- C10, what temperature do you recommend for growing cultures?

3- C10, I am going to do my first inoculation this weekend. Will you please stay tuned to this channel so I can post pics and get your feedback?

Thanks again! Oh, and did I mention how much I love your flow hood?




2. You are fine just storing them in room temperature.  Sometimes I keep them closer to 80 is I'm I'm a hurry, and technically you could go as high as 85 but contams benefit from that more than the myc does

3. Sure no problem, pm me if I miss it and I'll come post in the thread again

Thanks, I love it too :smile:


--------------------

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche


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OfflineIgloomis
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Re: Diving into agar pouring your own? [Re: c10h12n2o]
    #26507925 - 02/28/20 10:20 AM (3 years, 10 months ago)

Just did my first attempt at inoculating a dish.

I'm using pre-poured MEA plates from a mycology specialist supplier to simplify my first try. I kept the dishes in the refrigerator until the spores arrived. The refrigeration (I assume) had caused condensation to form on the lidsof the plates. One of them had minimal condensation, so I inoculated that one and left the others in my SAB to warm up.

How concerned should I be about condensation?

Also, I had a bit of trouble gauging the rotations of the plate between loop flamings. I think next time I'll put a mark at 12 o'clock for reference.

ETA: As I was inoculating I noticed my syringe said Golden Teacher. Damn, I was sure I ordered B Plus. I checked the order and sure enough, they sent me the wrong spores. I have grown GTs before; this was supposed to be my graduation to something new.


Edited by Igloomis (02/28/20 10:38 AM)


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Re: Diving into agar pouring your own? [Re: Igloomis]
    #26507937 - 02/28/20 10:28 AM (3 years, 10 months ago)

Yeah I use a sharpie and right on my plates around the putter edge on the face and back of plates. I make all kinds of notes and sector labels. Then when I wrap the plate in my little roll of plastic cling wrap the sharpie ink is protected from when I wipe it down with ISO later.



Edited by Sockadin (02/28/20 10:29 AM)


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OfflineCatsLoveHouseMusic
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Re: Diving into agar pouring your own? [Re: Sockadin]
    #26508066 - 02/28/20 11:54 AM (3 years, 10 months ago)

So you don’t streak plates? May I ask why? Curious


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Re: Diving into agar pouring your own? [Re: CatsLoveHouseMusic]
    #26509988 - 02/29/20 04:27 PM (3 years, 10 months ago)

How long until I should expect to see growth, given a 72-ish F incubation temperature?


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