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Invisiblefreespeech
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Premixed agar preparation tek - preparing agar with jars and petri dishes * 2
    #13450919 - 11/07/10 11:10 PM (10 years, 11 months ago)

RogerRabbit's various teks have always proven to be well-explained and effective for me. That's why I wanted to do his agar tek when I got into agar. I have a pretty basic pressure cooker that holds four quart jars, standing up, with not much headroom. I think it's 8 or 10 quarts. Trying to follow RR's tek, I looked high and low for a suitable bottle with a neck, but I was unable to find one. So I started preparing agar in quart jars and haven't looked back. This method is otherwise similar to RogerRabbit's tek, which is demonstrated in detail on his DVD (go buy it now if you don't have it yet.)

Here's how I prepare agar using quart jars and pour them in a glovebox.

Supplies
  • Clean mason jars with bands and lids. I use one wide mouth quart jar per 25 grams of agar. You can probably use just about any type of jar you have, but don't fill any jar more than half or 2/3 full. It takes two quart jars for me to fill a sleeve of 25 petri dishes.
  • Filter discs or your choice of pressure-cookable jar filtration. I've never tried polyfill but I think it would work fine.
  • Aluminum foil.
  • Pressure cooker. You'll also need a means to keep the jars off the bottom of the pressure cooker. I use a little metal rack. You can use spare mason jar bands if you don't have anything else.
  • Your choice of premixed agar powder. I use 50 grams of MEA (malt extract agar) to prepare a sleeve of 25 petri dishes.
  • Water. My agar's recipe calls for 25g of premixed MEA and 500ml of water per batch, which fills one quart jar to just over half full - perfect. I use distilled water when I've thought ahead enough to buy some, but filtered tap water works for me in a pinch.
  • Sterile petri dishes.
  • Glove box for pouring agar.
  • Gloves, alcohol and whatever other supplies your sterile technique requires.


Instructions
1. Using your metal cutting tool of choice, cut a large tab out of the side of each of your jar lids. We'll be pouring the agar out through this hole. These specially-prepared lids can be washed and reused for future agar preparations.

You can see what my lids look like in Step 4, below. I've found that the hole in this design is a bit too large. I'd recommend going a little smaller.


2. Measure out agar and water, according to the recipe of your agar, and pour them into your jar(s). Don't fill any jar more than about 2/3 full of the agar/water mixture. The recipe for my agar calls for 25g agar and 500ml water, and I make one batch of this recipe per quart jar.

3. Mix well. I use a spoon to stir the mixture up, and I make sure to scrape the tip of the spoon around the edge of the bottom of the jar where the agar powder might be hiding.


4. Put the lids that we prepared in Step 1 on the jars, rubber side down.


5. Put a filter disc on top of the jar lid.


6. Screw the band onto the jar, securing the filter disc and lid in place.


7. Add a foil cap to the top of each jar.


8. Put the jars in your pressure cooker. The jars should not touch the bottom of the pressure cooker.

9. Fill the pressure cooker with enough cold water that the water level comes up to just under halfway up the mason jars. In this photo, the water looks lower than it actually is.


10. Sterilize according to the directions of your premixed agar. Mine calls for 35 minutes at 15psi.

11. When sterilization is complete, turn off the stove and walk away. Let the pressure drop naturally and slowly. Prepare your glovebox by cleaning and sanitizing it, if necessary.

12. Once pressure has dropped and some time has passed, bring the pressure cooker to your work area, and transfer the hot jars into the clean glovebox.


13. Clean and sanitize your work area and self. Take a shower, brush your teeth, use mouthwash. I usually go right from the shower into my work area, having thrown on a clean t-shirt and fresh shorts.

13. Let the jars sit in the glovebox until they are cool enough to handle for an extended period. Do not let them cool to near room temperature, because the agar will start to solidify. Pouring chunky agar is not good. When the jars are ready to be poured, get as sterile as you can. Put on your gloves and sterilize them and your forearms as best you can. I use a spray bottle filled with 70% isopropyl alcohol, and I liberally spray this all over my work area and self. I spray alcohol onto my gloved hands and wipe my hands together whenever I've removed my hands from the glovebox.

14. Open your sleeve of petri dishes and put them into the glovebox. I arrange one sleeve of 25 petris into about 3 stacks.


15. Remove the foil from a jar. Unscrew the band from the jar and remove the filter disc. Then screw the band back onto the jar. Once you've poured all the agar from this first jar, you'll remove the foil and filter disc from the second jar, and so on until you're done pouring.

16. Pour your agar. Use your smart hand to grab the jar by the bottom. Use your dumb hand to lift a stack of empty petris in a way that exposes the inside of the bottom-most petri dish. Holding the jar by the bottom, pour agar into the exposed dish. Set the stack of dishes back down, closing the now filled petri dish. Move your hand one dish up the stack and pour the next dish. Repeat until you've poured as much as you have or need.

I've found it to be rather important to pour with the correct grip as shown in this photo.


17. I finish up by removing everything from the glovebox except for the poured dishes, and then covering the holes of my glovebox with a sheet of tyvek that I've sprayed with Lysol. I tape the tyvek in place.


That's it! Let the dishes cool in the glovebox and then do your thing with them. You can keep extra dishes in the fridge in a ziploc bag for a little while.


Edited by freespeech (11/08/10 02:21 AM)


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OfflinegitranduhShutr
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Re: Premixed agar preparation tek - preparing agar with jars and petri dishes [Re: freespeech]
    #13616489 - 12/11/10 10:44 AM (10 years, 10 months ago)

Hi i am interested in your agar tek, specifically your contamination rate from doing it in the glove box, in your pics it looks like your gb is not airtight with just big hole for your arms to fit in, basically i want to know how effective this has been from protecting against contams. I Have A similar glove box but i have gloves caulked in the holes so there is no air flow in box when working, I want to start cloning using agar and was just unsure if contams would be a huge problem, also my grow is contained in my bedroom closet so i dont have much room(I have a flow hood that just needs to be put together but i have no room for it here. so sorry if this is rambling any info or advice appreciated thanks


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Invisiblefreespeech
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Re: Premixed agar preparation tek - preparing agar with jars and petri dishes [Re: gitranduhShutr]
    #13617349 - 12/11/10 03:30 PM (10 years, 10 months ago)

Quote:

gitranduhShutr said:
Hi i am interested in your agar tek, specifically your contamination rate from doing it in the glove box, in your pics it looks like your gb is not airtight with just big hole for your arms to fit in, basically i want to know how effective this has been from protecting against contams. I Have A similar glove box but i have gloves caulked in the holes so there is no air flow in box when working, I want to start cloning using agar and was just unsure if contams would be a huge problem, also my grow is contained in my bedroom closet so i dont have much room(I have a flow hood that just needs to be put together but i have no room for it here. so sorry if this is rambling any info or advice appreciated thanks




The goal of the glovebox is to provide relatively still air. It doesn't have to be airtight or have any complex filtration system. I get very few contams from the pouring of agar using my setup. I have used this same glovebox for all my mycology work and it seems to be reasonably effective, although space can get tight in there. RR says you don't even need to extensively sanitize the glovebox before you work, because the stillness of the air is what prevents mold spores from being agitated into your project. I, however, still clean my glovebox before using it, and hose it down with alcohol or Lysol. It definitely takes practice to work in a glovebox; being able to work quickly and efficiently is key.

If you have a different setup that works well for you, then go with that, but I think that having actual gloves caulked into the glovebox is not necessary. I would worry that an airtight glovebox would actually cause more airflow within the box as you work. I know that others have done this before, but I've never done it so I can't personally comment.

To make the holes in my glovebox, I use an empty metal coffee can. I turn a burner to high on my electric stove, and set the metal coffee can bottom side down on the red hot burner. Once the coffee can is very hot, I can use it to punch perfectly round holes in the plastic of my glovebox.

Good luck! Contamination during the pouring phase has never been much of an issue for me -- I get all my contams when I go to work with the agar and I introduce contams from scraping spores off of a sporeprint or something. Which is, of course, the point of agar: being able to isolate away from contaminants.


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OfflinegitranduhShutr
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Re: Premixed agar preparation tek - preparing agar with jars and petri dishes [Re: freespeech]
    #13666655 - 12/21/10 06:17 PM (10 years, 9 months ago)

ok so after pouring agar into petri's in your gb, how many trays on average contam at this stage(before adding spores/myc/tissue)? thats awesome if its hardly ever, anyway thanks friend this tek has been helpful


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Invisiblefreespeech
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Re: Premixed agar preparation tek - preparing agar with jars and petri dishes [Re: gitranduhShutr]
    #13667647 - 12/21/10 09:51 PM (10 years, 9 months ago)

Zero.

In any case, I use the petris within a day or two, or else put them in the fridge, so any contamination is likely to still be microscopic. I've also kept poured but unused petris on a shelf at room temperature for weeks with no noticeable contamination, indicating that the process of pouring and storage is fairly sterile.


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Offlinesgfcchamber
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Re: Premixed agar preparation tek - preparing agar with jars and petri dishes [Re: freespeech]
    #15566230 - 12/24/11 11:52 AM (9 years, 9 months ago)

Oh man, this is genius!!  Thanks for commenting on my other thread pointing out that you had a link to this one (I would have never clicked "Link" in your sig, haha).

Anyway, this solution to my bottle of agar being too large for both my PC & my GB is an excellent one.  Significantly better than going out and buying both a new PC & GB.

I'll be pouring more plates this afternoon and will without a doubt be doing this.

Five mushies for you my friend!!


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Offlinethefun69
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Registered: 04/21/12
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Re: Premixed agar preparation tek - preparing agar with jars and petri dishes [Re: freespeech]
    #18546912 - 07/12/13 03:05 AM (8 years, 3 months ago)

Excellent tek.


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OfflineCrispyth
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Registered: 06/22/13
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Re: Premixed agar preparation tek - preparing agar with jars and petri dishes [Re: thefun69]
    #18547036 - 07/12/13 04:13 AM (8 years, 3 months ago)

I am just now trying to learn to work with agar and this is very helpful. Thanks alot.


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