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Agar for the masses - Banzai Style!

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#1 BuckarooBanzai


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Posted 28 July 2007 - 02:58 AM

Through a totally odd series of coincidences, a FOAF science teacher needed 120 PDA agar plates cheap (she needed ‘em free, actually) for a bread mold experiment in her 7th grade science class. While making ‘em up, I thought, “Wow…this is the kind of thing I should document for Mycotopia…”

In writing this up, I realize that this is probably going to be my last full on Banzai Institute Tek. This took forever to get down and it still has huge holes. My focus just isn’t what it once was. Please pardon the spelling/grammatical errors that follow. Please pardon this not being up to the standards of the rest of the Banzai Institute.

Thank you “old fogies” for teaching me this stuff and giving me a forum to regurgitate your teachings as “new” information with my little Teks.

Amazing, how far this place has come in such a short time.

Thank you for being, Mycotopiates. You have touched my life and more others than you shall ever know. You gave me superpowers – you gave me the ability to make the magic. You are each and every one Gods to me.

Thank you.

#2 mycobri


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Posted 28 July 2007 - 03:01 AM

nice to see ya doing your thing BB :)

#3 BuckarooBanzai


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Posted 28 July 2007 - 03:03 AM

Okay, first things first…why play with agar? Because agar is geeky science fun!

Agar might just be that “next level” of cultivation fun you are looking for. Agar is also utterly indispensable for cloning…

Seriously, agar in Petri dishes provides a very unique environment for growing cultures. When you combine agar and Petri dishes, you can experiment with a lot of cultures in a very small space. When you work with 20 plates, a 50% failure/contamination rate still means 10 usable plates!!!! And 5 of those plates can go in the fridge for use later, while five spawn a new generation of fruit!

Since an agar plate is a two dimensional surface, growing on agar makes it very easy to spot contaminants…and remove them! You can cut healthy growth out of a “dirty” plate and put just the strong/healthy stuff on a clean plate.

Usually, the nasties will come back on the second plate, but weaker and slower. And psilo mycelia is SOOOO fast when incubated at 84-85F, it outgrows most contaminants.

Ya cut out the good from the bad and “transfer to a new plate” one more time…usually no more nasties after 4 transfers…unless your sterile procedure sucks…

This is just ONE WAY to “clean up a culture.” But if ya do it – if ya master “cleaning up cultures” - ya can clone any mushroom (even hard/dried fungi from a REALLY good bag can be brought back to life, cleaned up from nasties and then FRUITED AGAIN…using agar…voice of experience…).

Agar also makes cloning MUCH easier.

Finally, an agar plate (or a slant) provides a great storage media for those particularly choice cultures you don’t want to loose.

Grow out 5 clean plates. Use 3 to start grain jars. Leave the other two sealed and toss ‘em in the fridge – after they are fully Parafilm sealed. You may just have discovered a new and very valuable sub-strain. Why chance loosing it? Keep a plate in the fridge. A new sub-strain’s value might not be obvious for a month or more…

An important note: agar is just a gelling media: agar is not food. Pure agar will not grow mycelium. You must add some food (sugar, etc.) for agar to be a growing media.

Another important note – there are LOTS of other ways to clean up and store cultures than just using plates and slants. But the focus of this is agar plates, so I’ll stick to that.

The only real problem with agar is the expense of purchasing pre-poured plates contrasted with the difficulty (or, perceived difficulty) of pouring them yourself. $20 for 20 pre-poured plates (or an %80 contamination rate on plates you pour yourself) keeps many people from playing with agar.

Enter the Banzai agar Tek.

Follow this process and you can have 90% clean plates using just a glove box. A true HEPA station helps, but IS NOT REQUIRED.

#4 BuckarooBanzai


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Posted 28 July 2007 - 03:09 AM

Sterile Petri dishes (plastic)
Sterile 60mL syringe and 16 gauge needle
Sterile airport syringe
2 Quart (or one ½ gallon) canning jars
Jar lid with 2 silicone injection ports (to fit above jar/jars)
Metal mesh strainer
Agar/Nutrients (recipes at end)
1 Liter water (spring is good, tap works)
Glove Box, gloves, good basic sterile technique
Silicone (sterilizable) heat pads – at least two
Mini “blowtorch” lighter

For your first foray into agar, I would strongly suggest using a pre-mixed agar/nutrient formula. The Potato Dextrose Agar (PDA) or Light Malt Extract Agar (MEA) available from are both excellent choices.

I have a slight preference for PDA, but MEA works damned well. Play around. Let experience and feelings be yer guide.

There are a million ways to do this right and about 23 to do it wrong…if you follow instructions and don’t deviate from them until you understand the WHYs of the instructions. Don't make changes until you understand WHY you want to change a variable.

Once you get the WHYs, well then you develop (and hopefully write up) your own Teks…

If you haven’t already discovered airport syringes and silicone injection ports, go read up on them now:

Seriously, if you don't already know that thread like mantra, re-read it and book mark it. It is revolutionary. It is the core function that makes this "easy agar Tek" workable.

Airport breather syringes and silicone injection ports are easy to make and will make a HUGE difference in your success rates.

I cannot emphasize this enough.

Silicone injection ports are CHEAP/EASY to make. Silicone injection ports, combined with a simple pressure cooker and canning jars, put so much “professional level” mycology in the hands of the home experimenter that it boggles the mind.

I still think the combination of airport syringe and silicone injection ports are the greatest single development I have seen, during my brief time on this scene, in home mycology.

Read up on ‘em and make a couple…you’ll never go back…

And thank Hippie3. He brought these to the scene.

Edited by Sidestreet, 14 February 2017 - 07:00 AM.

#5 BuckarooBanzai


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Posted 28 July 2007 - 03:12 AM

Step One: Make a double silicone injection port for your quart (or half gallon) jar

Did ya read the link above?

You want a lid that fits your jar that has two injection ports in it. One port will be for you to suck OUT agar. One port will be for the airport syringe to let back IN filtered air. Obviously, ya gotta drill holes in the lid before putting on the silicone…but you already read the link above, RIGHT?

Your lid should look like this:


That particular lid had a Hillbilly syringe refill kit under it at first, so he deserves props on manufacture. The point is, that lid has been used MANY times and is still totally valid. Thick silicone blobs are the way to go.

You want BIG silicone ports.

Your jar will eventually be filled with very hot agar and it will roll around a good bit as you suck agar out of it. Thin ports will leak and/or tear loose from the lid. Use a THICK blob of silicone on the top (and on the bottom) of the lid.

Edited by Sidestreet, 02 October 2016 - 03:12 PM.

#6 BuckarooBanzai


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Posted 28 July 2007 - 03:22 AM

Step two: Make up 1 Liter of agar


Some agar recipes are at the end of this, but a pre-mixed PDA/MEA works best for your first few attempts. A little agar goes a long way. Don’t overestimate your measurements thinking “more is better.”


As a for instance, if you were preparing 1 liter of PDA, I would need to add 39 grams of powdered mix to 1 liter of water. Just follow the recipe.

Don’t mess with the recipe, at first. Experience will teach you to make better agar. Recipes/mixes will also teach you to make better agar.

Recipes/mixes are MUCH FASTER in terms of learning to make “better agar” instead of “crap agar.”

HUGE HINT: go past 10% sugar (dextrose, malt, wheat, whatever) and you WILL make “crap agar” that grows only yeasts.

Stick to the recipe! There is a reason I’m repeating that for the umpteenth time…

So, to make 1 liter of PDA, add 1L of water to a pan and heat it to a rolling boil.

SLOWLY – about 3-5 grams at a time - add the powdered agar mix.

Let the powder “float” on the water surface for a minute or so, until it starts to “melt” and sink into the water. Be aware that you will have to increase the stove temperature to keep it boiling as you add agar.

A “proper” agar mixture boils at a MUCH higher temperature than plain tap water. Be careful turning up the heat – if your agar mix froths over, you will likely start a fire on your stove top.

If you are stupid/lazy/likely to ignore the pan of agar and let it froth over, have a fire extinguisher (or a disposable house).

Stir CONSTANTLY after the agar starts to hydrate/sink into the pan.

DON’T stir until it starts to melt/sink.

Let the dry agar mix hydrate for a moment or so before stirring. Add the additional agar mix SLOWLY. It takes about 10 minutes to add 39 grams of agar to 1 liter of water and not get a ton of clumps.

Clumps are bad for this process and must be avoided at all costs. If ya get clumps, don’t worry, you will filter them out later.

BE AWARE: boiling agar is a LOT hotter than boiling water (note how you have to keep turning the stove up to keep the agar liquid boiling). Spill some hot agar on your hand and you will regret it very deeply. You will also probably drop whatever you are holding and make a HUGE mess…this is the voice of experience talking…be respectful of boiling agar. ‘Nuff said.

You probably will get clumps the first few times you mix up agar. Clumps are the enemy of this process, however. Clumps will clog the syringe later and totally ruin everything. Clumps must be avoided – or filtered out.

You filter out the clumps by pouring the hot agar through one of these:
Posted Image

That is a very cheesy metal filter that can be acquired in virtually any grocery store for less than $3. A filter like this is CRITICAL for this Tek unless you already know how to mix up a batch of agar with no lumps.

Note the tabs at the top and the handle at the bottom. Those will hold the strainer on top of your quart (or half gallon) jar while you pour the red hot agar out of the pan.

Don’t try to strain agar lumps out with a coffee filter – it just won’t work.

Rinse the clumps out of your metal strainer with very hot tap water immediately after you use it or your strainer will be ruined. Once agar clumps dry out and harden…well, good luck removing them without tearing the screen.

Seriously – rinse that strainer immediately. It will be HOURS before the pan of hot agar hardens. That shit on your strainer will turn to rock in a few moments.

Now look at the agar in the jar you just poured. Swirl it around gently (DON’T shake it and mix in air). Any chunks remaining?

If ya have visible chunks, run it through the strainer again.

Chunks big enough to see will prevent this Tek from working.

NEVER fill an agar jar more than 2/3 full before pressure cooking/sterilizing. It will boil over and make a HIDEOUS mess inside your pressure cooker. Seriously – no more than 2/3 full. ½ full is safer…again, this is the voice of experience…

All agar jars should be 2/3 full (or less) before sterilizing.
If you are using quart jars, you should split the 1 liter of liquid agar between two of them. About 500mL in a quart jar is the maximum amount you should use. Don’t take the chance of boil over.

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#7 BuckarooBanzai


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Posted 28 July 2007 - 03:26 AM

Step Three: Sterilize the agar

Put your ported lids on the agar jars and then screw the bands down loosely. NOT TIGHT. Pressure must be allowed to escape during the sterilization run or the jars could explode.

Do NOT put the rings on tightly.

Agar can be fully sterilized by 15-30 minutes at 15psi. Cooking agar is one of those times you will really appreciate the difference between a pressure cooker and an All American sterilizer. All Americans really are the shit…

After the sterilization run, let the cooker cool until you can either remove the rocker weight or open the stop cock without blowing pressure.

Don’t let it cool more than two hours, just let it get cool enough to open without blowing pressure. One hour is my FOAF’s usual wait time from “heat off” until “agar removal.”

NEVER quick cool an agar run.

Never let a ton of pressure blow off quickly. The agar will boil over and make a HUGE mess.

As SOON as you open the cooker/sterilizer, tighten the canning jar’s metal band(s).

#8 BuckarooBanzai


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Posted 28 July 2007 - 03:40 AM

Step Four: Learn to work with Parafilm

First off, Parafilm is the SHIT for sealing stuff. It is indispensable with Petri dishes and slants. Plastic wrap and/or masking tape can work but they are a POOOOOOOOOR substitute.

Parafilm sticks to itself – that is why it makes a good wrap for the plates. Parafilm also makes a stack of plates stick together! Pull them apart gently and you won’t have an issue. Jerk them apart and one or the other might tear its seal loose. Pull plates apart gently!

Don’t worry about buying a whole roll of Parafilm for your first time out (whole rolls are expensive, but they last FOREVER). Folks on eBay will sell you small amounts (at inflated prices, but cheaper than whole rolls) that are perfectly usable. HINT: on eBay, you are buying “stuff that will be used to mask a plastic model’s canopy/wing while painting.” If you look for mycology Parafilm on eBay…well, just go ahead and lube up yer butt. You are about to get bent over.

As a total side note, if any of you do paint miniature or R/C airplane models, Parafilm is fucking AMAZING as a masking material for making lines/shapes. Parafilm will NEVER (and seriously, I mean NEVER – even on foam) lift the undercoat color when you remove it. Parafilm is really neat shit.

Regardless, a strip of Parafilm (cut from a roll) looks like this (this is enough Parafilm for 10 plates):

Parafilm is two layers: the paper with shit printed on it and the “filmy” stuff underneath.

You want to cut it up to look like the strips on the right:

The strips on the right are what you will use for sealing the Petri dishes.

You MUST learn to prepare/stretch the Parafilm before trying to use it.

Parafilm does NOT come ready to use. It sounds hard to “prepare” it, but it is really quite easy.

Basically, you slowly (about 3 seconds) stretch the Parafilm to almost three times it’s original size while grasping both ends firmly. As it stretches, you can see it change visual qualities. You can see the “thick” spots disappear and stretch out. This is what you are shooting for: After all the “thick spots” disappear, you have a ready to use piece of Parafilm.

If you stretch past all the “thick” spots stretching out and disappearing, the film will tear. Torn film should be wadded up and disposed of.

It should look something like this (pardon the shots – best I could do)…

Removing the paper cover and expose the Parafilm:

Get a GOOD grip on both ends and start pulling slowly and equally in opposite directions:

As it stretches, note how parts of it change appearance. To the right is an area that hasn’t stretched enough. To the left is an area that has stretched enough. Compare this pic to the pic below and you will see the “thick area” to the right disappear:


Note how uniform it looks – almost ready to use; hold the current stretch for about 3 seconds to “set” the stretch:


Totally uniform in thickness and held that way for 3 seconds – this strip is ready:


Edited by Sidestreet, 24 September 2016 - 10:18 AM.

#9 BuckarooBanzai


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Posted 28 July 2007 - 03:46 AM

Step Five: Squirt (don’t pour) the plates

This one is tricky, because the jar of hot agar you will be dealing with is VERY hot.

Have some sterile heat pads in your glove box to set the jar on (hint: silicone heat pads are great because they can be sterilized in the same cooker run as the agar).

Have another heat pad to handle the hot jar. Have at least two sterile heat pads or you’ll burn your fingertips and/or melt your glove box.

Insert the airport syringe through one silicone port and wait a second or two for the pressure to equalize. You should hear a slight “inrush” of air.

Insert your 60mL syringe needle through the other port and suck it (slowly) full of agar. Don’t rush – even red hot agar is thick. The syringe WILL fill with agar but you MUST be patient!

It looks a little like this (note the silicone hot pads under the agar jar):

The airport equalizing air pressure is on the top, the 60mL syringe (half full of agar) is on the bottom.

Both syringes are inserted through the silicone ports, insuring the agar remains sterile.

Once the syringe is full, tear open your Petri dish bag (inside the glove box) and make a stack of them. One 60mL syringe will make at least 6 plates. Make stacks accordingly. Wire tie up the bag of unused dishes.

Do NOT leave unused dishes exposed to dirty air or you might as well throw them away.

Working as quickly as possible, lift the Petri lid just enough to allow the syringe to enter and “squirt” in about 10mL of agar.

After you get good at this, 8mL will be enough, but start with 10. Again, don’t remove the lid, just lift it a tiny bit…just enough to get the syringe tip in WITHOUT touching the syringe tip against the Petri dish.

It looks like this:

Once filled with agar, it is no longer a Petri dish. It is now properly referred to as a “plate” or an “agar plate.”

HINT: The less agar you can use and still cover the bottom of the dish, the less problems you will have later with condensation (a LEVEL glove box is critical to using only a little agar). Less than a millimeter thickness is enough – as long as it covers the plate bottom completely, you have enough. More is better than less if your glove box sits on an angle. You want to fully cover the bottom of the plate – no exposed plastic. 6mL plates are my FOAF’s standard at this point with a perfectly level HEPA work station…agar mix goes a LONG way using only 6mL per plate…

Don’t let the syringe tip touch anything except the silicone port (when refilling).

After any syringe contact, flame sterilize the syringe with the mini blowtorch until it GLOWS before continuing. Waste a little agar squirting it through the flaming hot tip to cool it quickly. Better to waste a little agar cooling the syringe than to have it jam/clog…voice of experience…

Swirl the plate gently after filling to make sure the agar covers the bottom (very important if your glove box doesn’t sit on a level surface). Don’t uncover the plate. Just move it in a gentle circle to spread the agar around. If your box is on an angle and ya need more to fully cover the bottom as the plate dries, add more agar – and pay attention to how much “extra” you need to add so you get the next plate right on the first “squirt.”.

Place the next clean plate on top of the just filled plate, lift the top slightly, and fill it up in the same fashion. When the syringe empties, go back to the beginning of this step and start again.

Flame sterilize if the syringe tip touches ANYTHING (always sterilize after hitting the silicone port for a refill).
Repeat until you are either exhausted, out of agar, or out of Petri dishes.

Let the plates cool (in the glove box) for 1 hour before sealing them.

Cool the plates in a stack – only the top one will get bad condensation inside. This can be avoided by having a (sterilized) coffee cup of hot liquid to set on top of the top plate.

You can also use other “throw away” plates from previous experiments on top to prevent condensation. Or you can ignore the condensation. Condensation makes no real difference other than visual inspection of the plate. Way condensed plates still grow just fine.

If you don’t do something to avoid condensation, the top plate will have a TON of water vapor on it when you are finished. Personally, my FOAF just tosses the top plates into the “use as top plate next time” bin and moves on.

When plates are this cheap and easy to make, why fuck with the heavily “condensated” ones? Make two “uselessly condensed” plates to put on top of all stacks from that point on. All the plates under them will be crystal clear…

Edited by Sidestreet, 02 October 2016 - 03:13 PM.

#10 BuckarooBanzai


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Posted 28 July 2007 - 03:52 AM

Step Six: Seal the plates and store them

This is where the Parafilm comes into play.

You already spent some time learning to stretch and wrap with it, right???

Parafilm is really critical to sealing and keeping your plates clean (uncontaminated) for the next few months, stored in the fridge, until you are ready to use them. If you are gonna go this far, don’t skimp on sealing with masking tape/plastic wrap.

Pulling plates out of the crisper 2 months later, when you need them, and finding them brown/grey/green with mold just sucks.

Start out by making sure the agar is hard. If is still flowing at all on the plate, wait another 15 minutes. The agar MUST be completely solidified before the plate is Parafilm sealed.

Stretch a single strip of Parafilm. Wrap it around the side of the plate and grab it, on the top and bottom of the plate, with your fingertips:

While holding your fingertips down (mashing the film onto the dish top/bottom) stretch the Parafilm and wrap about 1/3 of the dish. Keep the Parafilm parallel to the dish at all times. It is critical that the Parafilm overlap both the top and bottom of the dish all the way around:

Keep stretching (gently stretching) and wrapping. The Parafilm should be slightly taunt the whole time, so it makes a good seal with the plate. Don’t stretch it too taunt or it will tear – just keep it tight:

At the end, just mash the edges of the Parafilm to itself. Now roll the plate in your hand to mash the sides of the Parafilm down all the way around.

HINT: Practicing with your Parafilm is incredibly important. DON’T have your first experience with it inside your glove box…voice of experience…

An “average” strip of Parafilm will wrap an “average” Petri dish two full revolutions plus some spare. If you don’t get two full layers of Parafilm around the plate, use additional pieces. You can’t put on too many layers – but you can put on too few. One layer is NOT enough for effective sterile storage.

One layer of Parafilm is designed to breathe. Two layers are designed to be a seal. Keep this in mind when using Parafilm.
Parafilm can be used in this exact same fashion to seal slants or flasks or jars (or just about anything else with a weirdly shaped opening). Just use two layers to seal or one layer to “breathe.”


Edited by Sidestreet, 02 October 2016 - 03:13 PM.

#11 BuckarooBanzai


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Posted 28 July 2007 - 03:53 AM

Step 7: Seal up the plates and put them in cold storage for later use

Put the hardened and Parafilm sealed plates into nice thick freezer baggies and toss them in the fridge. They should be good for at least 3 months. 9 or longer once you get your sterile technique totally down.

When taking plates out of storage, examine each one closely under bright light for contamination before wasting a culture sample.

So there ya go. Best I could come up with for “cheap and easy agar plates for the masses.”

Use this knowledge for the betterment of mankind and share it with whomever you want/can.

Claim it as your own, if you want.

Just get it out there.

#12 BuckarooBanzai


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Posted 28 July 2007 - 03:55 AM

Agar recipes:

NONE OF THESE I CAN PERSONALLY GUARANTEE. This is cut and paste stuff that SEEMS valid, but I can’t testify to the validity of any of these recipes. Personally, my FOAF prefers pre-mixed PDA from

Amaranth* Soy Agar

20 grams amaranth flour
20 grams soy flour
9.5 grams agar
500 mL distilled water


EntheoGenesis No. 442

10 grams amaranth flour
10 grams brown rice flour
10 grams potato flour
10 grams soy flour
2 grams malted barley
9.5 grams agar
500 mL distilled water


Oatmeal Neopeptone Agar

40 grams oatmeal or oat flour
2 grams neopeptone (optional)
9.5 grams agar
500 mL distilled water


Modified Sabouraud's Medium

25 grams barley flour
5 grams dextrose
2 grams neopeptone (optional)
1 gram yeast extract
9.5 grams agar
500 mL distilled water


Cornmeal Dextrose Agar

25 grams yellow cornmeal
2.5 grams dextrose
9.5 grams agar
500 mL distilled water


Barley Malt Extract Agar

40 grams barley flour
2 grams malt extract
1 - 2 grams yeast extract (optional)
9.5 grams agar
500 mL distilled water


Dr. Pollock's Modified Agar*

10 grams dried dog food
10 grams amaranth flour
2 grams dextrose or malt extract
9.5 grams agar
500 mL distilled water

#13 BuckarooBanzai


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Posted 28 July 2007 - 04:21 AM

Thanks again, all.

Banzai out...
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#14 Guest_vinz_*

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Posted 28 July 2007 - 05:51 AM

nice one BB!:bow:
love the agar thru syringe idea :)

#15 TVCasualty


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Posted 28 July 2007 - 07:42 AM

Thanks again, all.

Banzai out...

Oh no, thank you! :bow:

You've mentioned how you arrived here a noob a few years ago and we can all see how you quickly learned vast amounts of information about mycology, and have always been very gracious about pointing out the fact that what you've done was made possible by those who came before (something some people seem to forget). Anyway, just thought I'd mention that by the time I got here, you had already become a source of the knowledge that only a few years before you had been seeking.

Sorry to hear it may be the last installment to your Institute (which was the inspiration for my slowly-developing "TV Guide" -only one entry so far, but check back in 6 months!), but I know how life keeps on changing... You did manage to close the circle- coming here with a mind like an empty jar, filling up your jar from those further along, and finally filling the jars of others before you (may) depart. That is ideal, and I hope to do it that way too, though I think my jar is cracked!

You have certainly left your mark- Mycotopia wouldn't be the same without you! :love:

Oh, and great agar TEK, by the way! (see, I'm back on-topic :D)

#16 troutlips


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Posted 28 July 2007 - 07:56 AM

Great post BB, thank you!
I'm sure that you just saved me and a lot of other people a lot of time and aggrevation with the benefit of your experience.
Well done sir! :bow:

#17 reverend trips

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Posted 28 July 2007 - 08:04 AM

Another :bow:

Using a syringe to pour out the agar nice and thin is a great idea!

Thanks for another great tek Buck:bow:

#18 safTman


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Posted 28 July 2007 - 09:02 AM

Well done BB :eusa_clap
great write up!

#19 Hippie3



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Posted 28 July 2007 - 09:12 AM


using your method
in combination with those new ball jars
[ see ]
also fitted with airported lids
would be an interesting synthesis
allowing total sterility in open air.
dbzeroone mentions the possibility in that thread link i gave.

Edited by Sidestreet, 02 October 2016 - 03:15 PM.
fixed link, removed old bbs code

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