This blog will cover a simple recipe -- detailing how to make agar plates or culture slants, using our agar powder and nutrient agar mix together.
For many, making your own agar plates may be a difficult task without the proper equipment. Please read through this blog and decide if pouring your own plates is right for you - or if ordering ready-made plates is more suitable.
We have pre-poured >> sterile agar plates and >> sterile culture slants ready to go.
What is agar?
Agar is a gelatinous substance derived from seaweed. It is used as a solidifying
agent and is essentially a plant-derived version of gelatin. The more agar the firmer the final mix will be.
Agar alone has little to no nutritional value so for growing mushrooms, moulds or bacteria we must add in a nutrient base.
Why use agar plates?
Agar plates and slants are excellent for maintaining and storing pure mushroom cultures, germinating your own mushroom spores or cloning a wild mushroom. You can visibly see if you have a clean culture which can then be used to inoculate liquid culture or >> sterile grain spawn.
Agar plates can also be used to check if liquid culture or grain spawn is contaminated - or not - by adding a few drops, or singular grains, onto a plate and watching to see how the mycelium grows. If you have green, yellow or other colours other than white as pictured to the right then it may indicate you have an issue with that batch of innoculum.
Mushroom cultures can be stored for many years on agar in a vial called a >> culture slant.
Step-by-step: how to make, mix and pour your own plates or slants:
This guide will be to make 1.5L of agar. Adjust recipe as necessary for smaller or larger volumes, see per 100ml rates below.
You will need:
>> Agar powder.
Pot and spoon.
Pressure cooker that goes to 15psi.
>> Laminar flow hood or Still air box.
Clean work space.
>> Sterile petri dishes or >> Culture slant vials.
1-2L plastic or glass bottle that can handle 121c.
Tin Foil.
Scales.
Heat source / stove top.
The Steps:
Prepping the mix:
When mixing the dry powders into the water be sure to add the powders to cold water. If you add the agar powder to hot water it will instantly clump - and take a very long time, and a lot of stirring, to fully dissolve later.
I personally prefer a slightly more solid agar that I can easily pick up with a scalpel when cut. If you want a softer, more jelly-like agar, use the lower ratio of agar powder per 100ml below. The nutrient ratio does not change with more or less agar.
Measure out 1.5L COLD water
Mix in 50g agar powder (3.33g per 100ml- if you want a softer agar you can reduce agar powder to 2g/100ml).
Mix in 60g Agar nutrient mix (4g per 100ml)
Begin heating the water - constantly stirring as you do so. It is important to constantly stir the mix as the agar easily sticks and burns on the bottom of the pot.
Bring the mix to a light simmer and continue sitting for about 10-15 minutes - patience is key here as the agar takes a fair while to fully dissolve.
After plenty of stirring, you will visibly see and feel the mix thicken as the agar fully dissolves.
If you wish you can add colours to your agar at this point with a few drops of food colouring.
Adding agar mix to your pouring vessel for pressure cooking:
Now we have our agar and nutrients fully dissolved into a light amber solution. It is time to prepare the pressure cooker and pour the agar mix into a suitable vessel for pressure cooking and pouring.
There are a few considerations when choosing what vessel you want to use - consider your work space and how much room you have. Remembering once the agar has been sterilised you are going to pour it into a vertical stack of >> petri dishes - one by one -and it must be kept in a sterile environment the entire time.
Wine bottles can work well as they have a nice long neck making it easy to hold and pour from a distance, but may be difficult to work with in a small still air box. The small hole at the top also helps reduce the chance of contamination. Be sure to warm the glass before pouring hot liquid in to avoid cracking the bottle.
1L plastic food grade jugs with a handle also work well - especially for pouring in to tight culture vials where accuracy is critical. These are my vessel of choice. Be sure to check your jug can handle the temperature in the pressure cooker (121c). These jugs do have a large open top so I make a simple tinfoil lid pictured below.
I cut the tinfoil at the front making a horn over the pouring point: this makes it easy to peel the foil back after pressure cooking - without physically touching anywhere that will be in contact with the liquid agar. This greatly reduces chances of contamination.
Once the agar is at pouring temperature I peel the foil back as pictured above. You do not need to peel the foil far back at all - as you will be pouring slowly and accurately. The foil will tear easily.
If you are working with a laminar flow hood be sure to keep your hands behind any openings to the agar -- so the airflow does not potentially blow contaminants from your hands into the vessel.
If using a glass bottle tear off a good bit of tinfoil and wrap this around the lid and stem of the bottle: this will stop any water entering the bottle during the pressure cooking and also helps keep the glass underneath clean while cooling. If you are not using a laminar flow hood I would wrap half the bottle so you have plenty of foil to remove later.
Fill your vessel up to a maximum of 3/4 full - the last 1/4 allows for any expansion if the agar solution begins to boil while in the PC. We will do our best to avoid boiling the solution during the sterilisation stage in the next steps.
Pressure cooking the agar mix:
The agar mix must be sterilised. In my opinion there is no other way to reliably sterilise agar other than using a pressure cooker that can reach 15psi. If you do not have a pressure cooker I would advise buying ready made >> agar plates instead.
Put your vessel in the pressure cooker, be sure it has enough water for a 25minute run. 8-10cm of water is plenty - or 1/4 of the way up the vessel. If using glass again be sure cold glass is not going into hot water.
Secure the pressure cooker lid as per manufacturer's instructions and turn on the heat.
Keep an eye on the PC, once the cooker reaches a full 15psi you can start a timer for 25min (it is critical that the timer is NOT started until 15psi is reached).
At this point you want to carefully adjust the temperature so the steam rocker is just slowly venting - keeping the cooker at a steady 15psi. We want to avoid aggressive venting of steam as this can result in the agar boiling inside. Agar does not boil like water it more foams and expands in volume quite quickly.
A nice steady rock of the steam rocker is what we are after.
Cooling the PC correctly to ensure a sterile result
After 25mins of cooking turn off the heat and allow the PC to begin cooling naturally. The agar inside is now fully sterilised. Do not remove the steam pressure valve.
As the pressure cooker cools the air inside it begins to contract - once the pressure goes back down to 0 it is going to begin sucking dirty air back inside which can contaminate your agar. Atmospheric air is a soup of floating spores, pollen etc, which we don't want in our now sterile agar.
Dirty air being sucked back into the PC is the number 1 reason I see people fail spectacularly with their agar journey and is a reasonably simple problem to mitigate.
Laminar flow hood method:
Once the pressure gets down below 5PSI we want to move our PC into a space with as clean air as possible, the ideal is in front of a running >> laminar flow hood.
If you have a laminar flow hood you can open the PC once it has reached 0 PSI and cool the agar in the clean air stream.
Still air box method:
If you don't have a laminar flow hood then we need to make a small simple filter that can go over the steam vent once the pressure gauge reaches 0PSI. A piece of surgical tubing or clear-water pipe onto a syringe filter is a cost-effective solution.
If you are working with a still air box I would recommend leaving the agar in the PC with the makeshift filter attachment until the cooker is down to about 60deg.
At this point you can open the PC and move the agar into the still air box - do this quickly as we want to reduce exposure to unclean air as much as possible.
Pouring the agar:
Agar contaminates very easily so do not touch inside the bottle or anywhere the liquid agar will contact: nothing should touch the liquid agar from now on that is not fully sterile. Only handle the outside of the vessel well away from the pouring points.
Now the agar is sterilised and cooling, it is time to prepare our plates or culture slants for pouring. There is a limited time window to work within here so it is important to have everything prepared so once you start pouring you can do so uninterrupted.
It is important to not pour agar too hot. Often beginners will pour the agar when it is still 80-99c -- this is dangerous as agar is thick, dense and sticky, and will cause very serious burns if you have a spillage.
Pouring agar when it is very hot will result in a lot of steam coming off the hot agar, this will make plates or slants with a lot of condensate/water in the finished product. A little condensate is no problem, but we don't want a swimming pool in our agar plates or slants.
To avoid large volumes of water in our plates or slants simply allow the agar to naturally cool in the vessel. To check the temperature use an infrared no touch temperature reader or do it by hand feel. DO NOT stick a temperature probe into the liquid, this will almost always contaminate the agar solution.
The ideal temperature to pour agar is in the 45-60deg range, so you should be able to hold your hand on the outside of the agar vessel without pain.
Start pouring once the agar reaches the top-end of this range, you will have about a 10-15minute window before the agar starts getting too cool and begins to clump as it sets. Work efficiently but do not rush and make mistakes. It's more important to take your time and keep things clean.
If your agar begins solidifying and you have not finished you can run it back through the PC -- you will need to do a full 25min run again. As long as you have done your pre prep and everything is ready to go pouring time should not be an issue.
Each petri dish only needs about 15-30ml of liquid, just enough to cover the bottom with the dish 1/4-1/3 full.
When pouring into the empty plates start at the bottom picking the entire stack up and pouring the bottom plate, sit them back on top and pick up the entire stack again pouring plate two, repeat this process until all plates have been poured.
Setting agar and finishing up:
Now you have your agar plates poured allow them to sit and cool in your clean space for 30mins or so -- the agar will then be set and firm.
If you want to reduce the amount of condensation you can leave plates or slants in front of the flow hood for a few hours. This will help evaporate some of the moisture.
If poured at the correct temperature you won't get much more condensate than pictured below, which the agar will reabsorb overtime when sealed.
You can now inoculate the plates or slants with your desired culture.
If you wish to store the plates for later use, you can easily do so by wrapping each plate with >> agar wrapping film -- going round each plate at least 2 full times. This seals in moisture, locks out dirty air and keeps everything clean until you wish to use the plate.
With any agar work you do I advise always wrapping at least 1 plate, labelling it control with the date and just keep it aside. This plate will act as a control which you can keep an eye on, use it to check and be sure your pouring work has come out contaminant free.
Contamination will usually show up by day 3-5 (with plates stored at 18-24deg).
This way if you inoculate plates right away and they contaminate but your control plate is clean you are able to more accurately isolate if it was your transfers that were the issue, or your agar sterilisation and pouring procedure that was the issue.
Being able to isolate points of contamination will save a lot of time and frustration while learning to work with agar.
