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randy420rhoads


Registered: 02/24/07
Posts: 535
Last seen: 11 years, 5 months
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Oysters not pinning
#14051169 - 03/01/11 09:07 PM (12 years, 10 months ago) |
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After a mishap with my oyster culture syring I was only able to knock up 4 jars successfully using the Pf tek on BRF( just a couple so I can clone to agar then grow in bulk). According to RR's videos you must leave them in the jars for 1 week after full colonization before birthing (or they will just sit in the FC for a week anyway) wich I did. after a little over a week in the jars after 100% I birthed, dunked for 24 hours then rolled in dry verm. I placed the cakes in idividual plastic milk jugs with 1-2 inches of perlite.
They've been in the FC for a week now with no pinning, or even mycelial knotting. They've almost covered the dry verm case in mycellium. The temps are between 60-72 with light misting and fanning 5-10 times a day. What's going on
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Humility
Working on it



Registered: 10/07/08
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Did they smell fresh, like mushrooms when you smelled them?
You placed the cakes in individual plastic milk jugs? Oysters are very sensitive to CO2 buildup; they need lots of fresh air all the time. That doesn't sound like a healthy environment for them.
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randy420rhoads


Registered: 02/24/07
Posts: 535
Last seen: 11 years, 5 months
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Re: Oysters not pinning [Re: Humility]
#14051402 - 03/01/11 09:47 PM (12 years, 10 months ago) |
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Yes. I grew cubensis years back, these a different yet similiar smell. A much more pleasant one distinctly healthy mushroomy without the tang of cubensis.
I know they are sensitive to CO2 buildup I open and fan at least 5 times a day mostly an upwards of 10 times a day if not more. Is that not a sufficient rate? They still smell very healthy and are colonized the rolled casing, just not pinning.
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BlueLightRain
WhoaUnbrokenChain



Registered: 01/14/11
Posts: 354
Last seen: 9 years, 26 days
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Hmmmm. Pinning development is truly strain specific. For instance I have a strain of Pl. pulmonarius that pinned a couple days after I exposed it to air. I have a strain of Pl. ostreatus that doesn't fruit for a week after being exposed to fresh air. You could be close.
Also, I never misted my Pl. pulm the first time it grew (and never misted the fruit either!) and I only misted the Pl. ostreatus once to twice a day. You might be drowning it. Let it breath more and give it a bit of a break from the water. Too much water can be restricting.
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randy420rhoads


Registered: 02/24/07
Posts: 535
Last seen: 11 years, 5 months
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It's sprinkling out i'm leaving it outside (under cover) with the lid off. Maybe It just needs air.
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randy420rhoads


Registered: 02/24/07
Posts: 535
Last seen: 11 years, 5 months
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Well just checked the one outside. Some weird shit going on. Looks like 2 different contams dont understand how they could have done this overnight on a fully colonized cake.
One spot looks gray, the other looks like caramalized MYA. Both spots are roughly dime sized on part of it.
FInally one of my indoor cakes has 7 tine primordia forming on it wich i'm thankfull foor but why so little i thought oysters had huge numbers of pins?
Also noticed they formed on the base in between the jar lid and cake (I set the cakes on upside down jar lids w/ rings. My best guess is they fruited between there because it was more humid. Does this soudns correct?
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RogerRabbit
Bans for Pleasure



Registered: 03/26/03
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Oysters won't pin in a milk jug. The CO2 level is too high. If they're non-contaminated and a viable culture, you'll see pins with them outside in the rain. RR
-------------------- Download Let's Grow Mushrooms semper in excretia sumus solim profundum variat "I've never had a failed experiment. I've only discovered 10,000 methods which do not work." Thomas Edison
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randy420rhoads


Registered: 02/24/07
Posts: 535
Last seen: 11 years, 5 months
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Ok i'll take that info for the next batch for sure, but they did start pinning. Did you read the most recent post above yours?
And I don't understand how the Co2 level can be too high if i fan say every hour?
Edited by randy420rhoads (03/02/11 11:55 AM)
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BlueLightRain
WhoaUnbrokenChain



Registered: 01/14/11
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Last seen: 9 years, 26 days
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Pins will develop according to how much sustenance is available to the oysters. If its half a pint of substrate, you'll probably get a small pin flush. If it's 20 pounds of substrate, well whoa momma hold on!
Oysters love fresh air...I've seen them malform in shotgun terrariums.
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randy420rhoads


Registered: 02/24/07
Posts: 535
Last seen: 11 years, 5 months
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AS long as I can get a good tissue sample/ prints from at least one.
How do you print clone? Just like cubensis? Cut down the stipe under sterile conditions, removed some mycellium and place on agar?
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BlueLightRain
WhoaUnbrokenChain



Registered: 01/14/11
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Yeah if you want to start your own strains from scratch, take some prints. If you want to clone exactly what has grown, remove the innards of the mushroom and transfer to agar. This comes with a hole host of difficulties. Your best bet is to make some prints. This way you can watch the mycelium on the plates and transfer the strains that grow most vigorously.
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randy420rhoads


Registered: 02/24/07
Posts: 535
Last seen: 11 years, 5 months
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I have some agar plates i filled up last night so i'll probablly do both.
RR never got back to me about this question.... HE made it seem like you want or it's at least ok to get contams on agar, then seperate the healthy mycellium to another agar plate. Is that true?
The lids on my plate also seem pretty loose like they'd let airflow in and compromise sterility, is it normal for them to be so loose?
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BlueLightRain
WhoaUnbrokenChain



Registered: 01/14/11
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Yeah I've found its difficult to avoid some kind of contamination when cutting out a mushroom's innards. Expect bacteria of some sort then keep transferring the fastest healthiest looking mycelium away until you get a pure culture.
I'm pretty sure all brands have loose lids, but they make a tight seal where the rim contacts the lid. Pick up some 2" Flat Twine at Ace hardware and use that to wrap your plates. I believe it's wood-based cellophane. It breathes and works the same as parafilm and its cheaper. It stretches well and clings to plastic phenomenally, as well as sticking to itself.
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randy420rhoads


Registered: 02/24/07
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Last seen: 11 years, 5 months
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Appreciate all the advice!
So after I transfer my first clone expect contams and just keep transfering. Got it. Is it a bad idea to try and use some as spawn (a wedge for a rye bag in this case) before transfering and getting a completely clean plate? Or use as spawn before the plate is 100% colonized?
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BlueLightRain
WhoaUnbrokenChain



Registered: 01/14/11
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Quote:
randy420rhoads said: Is it a bad idea to try and use some as spawn (a wedge for a rye bag in this case) before transfering and getting a completely clean plate? Or use as spawn before the plate is 100% colonized?
Respectfully, yes. Since you're likely to have contaminants on your agar plate then you're even more likely to get contaminants on your rye grain, given that it has more surface area that it can touch. Take the time to isolate the mycelium and make it pure and you'll have an endless bounty of fungus from which to draw. If you try and jump the gun you'll have to start all over. I recommend next time you get spores from a syringe, make some drops across a bunch of plates. This way you can isolate many different strains and select those that are most vigorous.
You don't have to wait until your plate is 100% colonized to use it. In fact I recommend once it is almost completely colonized to then create more plates. Each plate can then either create more plates (we are talking exponential factors here!) or be used to create grain spawn. Think of the possibilities!
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randy420rhoads


Registered: 02/24/07
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Last seen: 11 years, 5 months
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Wow so streak some spores on an agar plate with an inoculation loop rather than making syringes growing then cloning?
If I do a steak of spores wont there be dozens in not hundreds on strains on such a small surface area it would be hard to tell wich is healthier?
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BlueLightRain
WhoaUnbrokenChain



Registered: 01/14/11
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Yeah if you make a nice solid culture bank to draw from you can do this an endless number of times. Let me do some simple math for you. Once I discovered this I was a completely changed man. You can take one of those syringes and inoculate a few jars -OR- you can drop some spores on several plates. Once you isolate a strain, you can allow it to colonize your plate, lets label this p1. Once this mycelium (p1) has colonized the plate you can then comfortably make 20 plates from little wedges you transfer to other plates (call them p2). If you then allow each of these wedges to grow out and colonize a plate, you have enough to mycelium to inoculate a bunch of jars. But you probably only need one plate to inoculate around 10 jars. You can then take the other 19 plates and make 20 more plates PER plate. So you then have 20 times 20 = 400 plates of mycelium (p3). Do this again and you now have 8000 plates (p4). In case you are worried that you will use 8000 plates in the next couple years, consider expanding each plate another 20 times. Now you have 160,000 plates (p5) of mycelium (a culture bank) from which you can inoculate your jars. Each jar can also be expanded. You don't have to wait for all 160,000 jars to grow before you start a project though! After p2 gets going you can create a cycle where you are always expanding your culture bank while simultaneously inoculating jars. With a vigorous strain, you can go from 1 plate to 160,000 plates in a month! Over 3 million plates in 5 weeks! Marvelous!
This is mind blowing and I'm always fascinated by this! This is why its better to use your spore syringes you purchased and your prints you've acquired to make agar plates first instead of using them to inoculate jars directly.
To your second question: Yes you are right. As soon as little white spots of mycelium crop up on your plate (be sure its mycelium and not a contaminant), transfer each spot to other plates. Then look for the denser, sometimes ripply shoots and cut those out and transfer. Keep doing this until each individual strain appears to have the same observable and unique features. This can take a couple of transfers to obtain a p1.
This is from dropping a spore solution onto agar. The solution ran around the edges of the plate whenever I tilted it so you see it growing pretty much everywhere on the plate. Notice the tufts that are growing out more quickly then everything else? Use those!

I simply used the sections that were showing the fastest growth.

Then I transferred all of those sections to individual plates to watch each one grow and track its rate of growth. From there I selected the ones that were most vigorous and expanded those. (I coudn't find cheap plastic petri plates at the time so I used gerber baby food jars, sterilized them then dropped the sterile agar inside...worked fine, but just gave poor visibility and little surface area. Plastic petri plates are superior)
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randy420rhoads


Registered: 02/24/07
Posts: 535
Last seen: 11 years, 5 months
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Appreciate you taking the time to write all the good info down for me.
All of them except the one I had outside started fruiting and look quite healthy.
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BlueLightRain
WhoaUnbrokenChain



Registered: 01/14/11
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Yay! Do you have pictures?
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randy420rhoads


Registered: 02/24/07
Posts: 535
Last seen: 11 years, 5 months
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Seeing as you took the time to help me out so much I can definately take the time to snap a few.
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BlueLightRain
WhoaUnbrokenChain



Registered: 01/14/11
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Nice Tapatio! Nice pins too, of course. Looks like you have some aborts at the bottom of the cake, and a couple of happy clusters fruiting from the middle region. I'm not sure how much nutrition those clusters have but looks like they can mature a bit more. Enjoy!
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randy420rhoads


Registered: 02/24/07
Posts: 535
Last seen: 11 years, 5 months
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I sure hope so the biggest isn't even the size of my pinky! They should have plenty i'd assume being the first flush from a dunked cake. How can you tell they're aborts I think they're still growing.
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BlueLightRain
WhoaUnbrokenChain



Registered: 01/14/11
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Yeah I hope they are still growing. They just have that appearance most aborts get from my experience. Please do tell if they develop more! I recommend taking some spore prints from your mature oyster mushrooms so you can start again.
If those cakes were inoculated with spore syringes then each cake may have anywhere from 1 to 1000 strains, each competing for a section of nutrients. The one that is fruiting probably has the largest territory of the cake. The advantage of isolating strains is that you give one strain an entire block of nutrients from which to grow, hence larger and more abundant fruits.
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randy420rhoads


Registered: 02/24/07
Posts: 535
Last seen: 11 years, 5 months
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Wow did not know that. Greedy bastards don't share lol....
How long should I let it go before printing, How do you tell when it's no mature since there is no veil?
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BlueLightRain
WhoaUnbrokenChain



Registered: 01/14/11
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Hmmm...To be certain I would say wait until the cap starts to make wrinkles or folds in the margin. If you've waited too long you'll probably still get some prints and if you pick it too soon you'll probably still get prints. You don't need a heavy spore load to have success making new strains but more spores from which to draw always helps. Oysters eject tons of spores for just about their entire fruiting cycle.
If you place your oysters over a dark colored table or paper you'll see it becoming covered with a cream, gray, or lilac film. Those are spores! I came home to my first batch absolutely coating my entire kitchen counter. It turned from white tile to gray tile.
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randy420rhoads


Registered: 02/24/07
Posts: 535
Last seen: 11 years, 5 months
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I always liked knocking up jars with spore solution but I guess that would be stupid to continue to do. Just have to practice with the agar i'm not sure if mine is still good or if I need to order more...whole last (and my very first) batch of plates was trash.
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randy420rhoads


Registered: 02/24/07
Posts: 535
Last seen: 11 years, 5 months
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I'm wondering if my agar wasn't sterile , or even if it was if it is now. I'm going to use it in jars instead of plates. I didn't have any sort of tape sealing the last batch of plates do you think that's why they all had contams? The loose lids?
I'm about the try the agar in 1/2 pints now would it be better to use the regular metal can lids or the plastic ones? I know the plastic are better for gas exchange but do I want that with agar too?
Edited by randy420rhoads (03/06/11 05:13 PM)
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BlueLightRain
WhoaUnbrokenChain



Registered: 01/14/11
Posts: 354
Last seen: 9 years, 26 days
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There are a lot of questions to be answered, and in time you'll know what to do. I'll try and help along the way. First off, you have a bunch of time between when the oyster is ready to harvest, when your prints are made, and when it's time to make some cultures. Once the prints are made you can save and utilize them within a year, and reports have been made that prints have been utilized for several years after they were made. Once the prints are made there is no reason to rush the process. Consider the tortoise versus the hare.
My point is that because you have all of this time, now is a great opportunity to do some research. I'd like to see some pictures of your agar plates that were completely messed up. If you don't have any available can you describe them? Were they covered in streaks of pastel colored ooze? Or did they have symmetrical, circular growths? Description is key here because it will tell you what you are battling.
I've found when it comes to pouring and transferring agar a zen state of mind is necessary. When I'm in a hurry and I want it done as quickly as possible most often I cause contaminated plates. If I'm focused and careful my success rate is reassuring.
There is an easy fix to the fact that you don't have tape for your agar plates. When I first started I didn't have a lot of money to spend on the hobby so I discovered Flat Twine (the 2 inch wide variety), sold at Ace hardware, and probably several other stores, worked perfectly well to wrap plates. It stretches, allows the fungus to breathe, and sticks to itself. If you use plastic petri plates it works even more because it adheres to the plastic very well. Best of all, its cheap ($6-$7USD) for something like 170 feet versus parafilm which does the same thing but can be 3 to 5 times as expensive based on price and shipping and handling.
What kind of laboratory setup do you have? What does your glove box look like? Is it made out of plastic, cardboard, or some other material? How do you clean it, and how do you clean your tools? If you want to expand you ability to make mushrooms I highly recommend taking the time to sit down and do some research. What kind of recipe are you using for your agar? How do you sterilize it?
If you don't want to buy a sleeve of plastic petri plates, consider buying baby food jars like I did. At about 55 cents a piece, they did the job, they are fit for a pressure cooker, and they seal. They have disadvantages though: the inability to look directly down on your media, and the lack of surface area compared to a petri plate.
Yes you can use canning jars as your agar surface. You could simply pour a minute amount of agar into a few jars. Once you sterilize them allow them to cool on their side, then make careful transfers. This is a big use of space though, hence why petri plates are standard and most advantageous.
It's easy to get caught up in the rush of making more mushrooms but by making a plan and following through with care and quality you have the ability to make a whole lot more. Your most important step is making prints on aluminum foil within a container, which is as close to sterile as you'll get for prints, without an elaborate setup.
Take some prints and eat and enjoy the rest of the fruits of your labor!
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randy420rhoads


Registered: 02/24/07
Posts: 535
Last seen: 11 years, 5 months
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The contam on my dishes was circual symetrical growth of clear slime. I can upload pics if you'd like but they are all trash. I started over and poured agar into 1/2 pints then resterilized jar and agar so shouldn't have any problems there. I think i'll just use jars from now on because I have a mini fridge with about 100 in it and they just take up that space empty anyway. Plus I never use more than a few seeing as I hope to get into rye gran in filter patch spawn bags. Jars just seem safer to me and the space is not an issue.

My gloves box is a clear rubbermaid container with 2 holes on one side in wich I put plastic PVC pipe for armholes and duct taped the heavy duty rubber cleaning gloves. On the other side I have another hole cover in duct tape I can open to pass a flame sterilized needle back and forth through should I happen to be using the pf tek. But for most things( Like agar) it's completely sealed off from and outside air.
The jars I poured yesterday apear to be free from contams. Since rye is so cheap and I have a bunch of bags... After I harvest and take prints could I crumble a spent pf cake into a bag as spawn just to kill time and mess around while i'm taking the tortoise approach.
I definately want to do it the slow way isolate a good strain to grow much better but in the mean time i'm getting hungry for whatever I can eat...
Edited by randy420rhoads (03/07/11 01:24 PM)
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BlueLightRain
WhoaUnbrokenChain



Registered: 01/14/11
Posts: 354
Last seen: 9 years, 26 days
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Quote:
randy420rhoads said: The jars I poured yesterday apear to be free from contams. Since rye is so cheap and I have a bunch of bags... After I harvest and take prints could I crumble a spent pf cake into a bag as spawn just to kill time and mess around while i'm taking the tortoise approach.
Yes and no. If the oysters were grown 100% in vitro without any exposure to air, then yes if you transfer them in a sterile glove box. Generally no though because you've already exposed your pf cake to the air, which means millions of mold spores have descended up on it, which when mixed around on your grain will germinate and contaminate the substrate.
Yeah the little slime spots on your agar appear to be bacteria.
If the ceiling of your glove box is tall enough, I have suggestion for you. Drill a hole about 1/2 inch to 1 inch in diameter in the ceiling and stuff it really good with some polyfil that you've soaked in hydrogen peroxide. You only need to soak it once to eliminate any dwelling contaminant spores that may descend upon your work area. Drill this hole directly above the area where your alcohol lamp will be so that when the lamp is burning it allows gas to escape through the polyfill. Caution, though, if the ceiling is too low then you will melt the polyfil, potentially make a mess, or even worse a fire.
The point of doing this is to allow you to sterilize your scalpel inside the glove box, which means you won't have to move it in and out of your glove box (which is what I picked up from your description of your transfer sessions - if I understood you correctly?) If the ceiling is too low, I recommend hunting for a larger plastic container or even using one that is wide and turning it on its side, as long as the side gives you enough work space surface area. The idea is to keep your work tools, instruments, and media all within the sterile environment.
Also - on your agar and jars...it's a great way to do it if you have the space, since you'll be able to sterilize the agar within the jar and allow it to cool on its side (or upright if they are shallow) without ever exposing it to air (until you make transfers). This might be overkill but I sterilize my batches of agar for 45 minutes on 15psi. Never had contaminant issues. I've heard other people doing it for 20 minutes. I haven't tested that, but I've found 45 minutes works, and it's really not an extensive period of time.
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