How to Prepare & Pour Agar Dishes for Mushrooms
Fun fact, the word “agar” comes from agar-agar, the Malay name for red algae.
The History of Agar in Microbiology
Agar (pronounced ‘ay-gar’ by some, ‘ah-grr’ by others) is derived from seaweed, it was first put to use in microbiology in 1982 and quickly replaced gelatin as the media thickener of choice. Its higher melting temperature allowed incubation at warmer temperatures without liquefying the media.
When enriched with sugar and starch, agar becomes the perfect vehicle to transport mycelium. Everything from tissue samples (clones) from wild or store bought fruitbodies to spores, colonized grains, colonized bark, and even pins (immature fruit bodies) can be used to inoculate a petri dish which has been prepared for use with mushrooms.
Many growers fear the microscopic world and avoid agar initially (having heard countless horror stories of contamination resulting in homes being burnt down and rebuilt as a result …I am teasing) however nearly everyone eventually graduates to agar when they feel comfortable. For me, it took me 6 months before I felt confident enough to work with agar and I purchased some pre-poured plates online. I took them to the field and successfully cloned a beautiful Laetiporus cincinnatus (white pored chicken of the woods) in the field in the open air. The culture shriveled and died as I had no idea what to do next, but it was successful and inspired me to continue onward. Fast forward 3 years and I’ve started a culture business using a still air box, fast forward 6 years from there and here I am typing to you now.
The microscopic world is frightening at first, but once you understand why and how things work (within reason, we’re amateurs here) you can learn to avoid contamination through proper sterile procedure and practice. You will not likely have major successes until you’ve made a few mistakes, and that is okay! Don’t worry about contaminating your grows, just know that if and when you do everything as you should, you can use petri dishes to help you grow in nearly any habitable conditions.
Types of Agar for Mushroom Growing
Like everything now days, it seems there is no shortage of folks trying out new things and coming up with their own recipes for using agar to grow mushrooms. There are a few staple recipes I’d like to mention, and then my staple recipe I would like to share. Some recipes you may or may not have heard of:
- MEA (Malt Extract Agar) – Great for Agaricus & other coprophiles (dung lovers). Avoid for lignophiles (wood loving mushrooms) as the extra nutrition encourages contamination & sectoring across many species.
- PDYA (Potato Dextrose Yeast Agar) – A standard recipe for many growers, this works well though yeast is not necessarily required
- PDA (Potato Dextrose Agar) – Same as above, sans yeast!
- DFA (Dog Food Agar) – Using dry kibble in place of starch, I’ve found this to really bring out color in species like Pycnoporus cinnabarinus (cinnabar red polypore) and Laetiporus sulphureus (chicken of the woods!) in culture suspecting the broad spectrum of nutrients found in dog food to enable other biological processes within the mycelium that they’re not able to do with MEA, PDA or other less nutritional medias.
- PHA (Potato Honey Agar) – A personal favorite, as I have honey on hand all year round!
Note that dextrose (aka candy makers sugar) is common in one form or another in each recipe, and differs chemically from table sugar (sucrose) which is not recommended for culture media. The formulation for each varies with respect to the scale of the pour at hand, but in general I am not one to keep large amounts of candy making sugar on hand, though I do love honey so for my recipe we’ll use honey, but for each step I’ve used weights (1ml honey ~ 1g) and you can replace with a sugar of your choice. If you’re not sure which agar to buy, check out this page of my website for recommendations.
Potato Honey Agar
My personal favorite staple recipe for agar (aside from dog food agar on rare occasions) – other than working very well, it’s fun to say! “What type of agar do you use?” — “Me? Oh just Potato, Honey!”
Petri dishes generally come in sleeves of 10,20 & 25, here is the recipe for each:
Preparing & Working With Agar
Now that you’re equipped with a basic understanding of what agar is, and some idea of what you might want to use for your own recipe, you’re probably wondering what to do next! In short, the next steps are sterilizing the agar, pouring & finally sealing your dishes. It is absolutely worth mentioning that this must be done in sterile conditions, any attempts to try otherwise will provide colorful rainbows of contaminants and a lesson in ‘I told you so!’ that is fun, but can be a waste of time if not prepared for it, so, I told you so!
You’ll need a vessel to sterilize in, a pressure cooker (preferably 15PSI), petri dishes and some time.
A standard quart sized mason jar holds 800ML of liquid comfortably, in general people are preparing batches of 20 plates & using 500ML of water. Out of the box a mason jar is well suited for a trip through the pressure cooker after mixing the dry ingredients per your recipe requirements. Once mixed, it’s as simple as sterilize and pour! It’s really not that simple, and does take some practice to get right, so don’t be discouraged if you’re struggling at first, like riding a bike it will come to you in time!
When choosing a vessel to pour from in the average household, I prefer the standard regular mouth jars for pouring (when choosing jars) compared to the wide mouth jars more commonly found in growing due to the ease of extracting grains or PF style cakes from the wider opening. Presently I find myself gravitating towards 1L flasks for agar work compared to the jar as there can sometimes be issues with dripping agar on the sides. This can be minimized if you’re careful in your pours and learn to tip your jars back upright quickly after each pour to disallow opportunity for drips in the first place!
The concern with drips in the first place is potentially contaminating the whole pour if a drip or stream of agar pours down the jar and you inadvertently cross contaminate the entire pour from there on out simply due to not being careful about what you’ve touched/are touching/have touched when working in sterile conditions. Cross contamination can and does happen and when working in sterile conditions it’s important to work deliberately, with intent. Say that again with me, “When working in sterile conditions it’s important to work deliberately, with intent!”
Sterile Working Conditions?
Now, what are sterile conditions exactly? It could be anything from a still air or glove box, a laminar flow hood or even a Bunsen burner updraft cone. All methods work, some have pros & cons, but many growers begin in a still air or glove box & they function very well on a small scale. When working with improvised sterile setups, 70% Isopropyl Alcohol is often the best choice to sanitize working areas without the harsh effects of bleach & has the lowest risk of accidentally setting yourself on fire. Additionally, the increased water content of 70% ISO allows the alcohol to sanitize more effectively by sitting longer before evaporation.
It’s important to sanitize all things entering into whatever sterile work area you have setup for pouring petri dishes. It’s often a learning process in and of itself to become aware of the many ways you can unintentionally cross contaminate items, but when in doubt, ISO out! I find a spray bottle helps to apply liberal amounts of ISO as necessary in a fine mist.
Pouring Petri Dishes
At a later date, this section will be replaced with a video. I don’t believe text alone can satisfy the need for a comprehensive guide to pouring, and the best way to do that is to show you. But – so as to not let you down completely, I’ll walk you though the high level overview but first, an important note on the pressure cooker. It’s crucial to allow the pressure cooker to cool naturally, without giving into the urge to manually vent pressure to expedite experiments. It generally takes around 30-45 minutes for my pressure cooker to cool enough to reach atmospheric pressure to avoid the agar boiling out your vessel upon opening. This can lead to a big mess and a potential burn hazard. In that time you are waiting, you can be preparing your sterile work area, supplies and getting yourself prepared to pour your dishes.
- Get your pack of petris out and into the sterile work area.
- Don your gloves, clean yourself up with ISO, hands arms elbows, all that, do your hands last and use extra ISO.
- After removing agar from pressure cooker, allow to cool to tolerable temperatures for touching (thick gloves help), sanitize your jar or flask (on the outside) and place into sterile work area, open sanitized packing for petri dishes, separate stack into stacks of 10.
- You can pour hot agar, but will have issues with condensation, some growers flip their plates for storage to manage condensate. You may also have issues melting plates (white discoloration on lids) by pouring too hot, allowing to cool until touchable is best.
- Loosen your jar lid (remove foil for flasks) & break the seal (it’s pressurized, go slowly and deliberately) and be ready to move.
- Set the jar lid to the side & with your non-dominant hand, grab the bottom most dish of a stack of 10 and lift the top of that dish and the 9 above it up high enough to pour.
- With your dominant hand, gently tip the jar over to pour enough agar into the dish to cover the surface of the dish, place the stack of 9 & the top of the working dish down, leaving the top of that dish and bringing the top of the next dish up with your stack as you go along working from the bottom up.
- In summary, you’re pouring from the bottom dish upwards and leaving stacks of 10 to cool.
This is the quickest way to pour dishes that I have found, others may have other processes but I find this works really well for bulk runs of petri dishes.
Now – you’ll need to let them cool in your sterile work area until they’ve solidified, and this means when you return after doing so, you need to sanitize yourself thoroughly. Don’t let things sit too long in a laminar flow hood (dishes dry out) or still air box (contaminants are going to build up in the air again!) before attending to them, let them cool and get them sealed!
Sealing & Storing Petri Dishes
Parafilm is a waxy film which is used commonly in etymology, plant grafting, modeling & other areas – but in our case, it is used to seal our dishes tight. Parafilm is cut into 1″x4″ strips, stretched & wrapped 2-3x around the outside of each dish allowing gas exchange (GE) while keeping contaminants out. This results in happier, healthier cultures & less issues with contaminants. The casual home grower may not have Parafilm on hand, but at $25+- for a 250′ roll, it took me about a half a year of running a culture business to go through one of them. I expect most home growers will be set on a roll! For those who don’t want to use or are otherwise averse to Parafilm, resealable plastic sandwich bags can be used with equal success & some growers even use suran or plastic wrap (though I’ve not tested it I have heard & read good things) to seal their petri plates. Store in the fridge for best results, in a sealed bag. It’s best to use them ASAP, don’t let them sit longer than a month & good luck with your experiments! If you’ve made it this far, but don’t feel like pouring your own is a good choice for you maybe a pre-poured petri dish solution is best for you? Ha! Doubt it. If I’m any good at teaching you’re excited and ready to go! In that case, check out my recommended petri dishes, agar and other recommended supplies for working with agar for mushrooms. Happy growing, good luck with your experiments, and thank you for reading! I can only do better if you let me know! Drop me a comment below and I’ll be happy to help with your needs as best I can!