Agar – Preparation

Green & Blue PDA

In our previous guide we covered what agar is, and gave an overview of some of the different types and what they’re used for.

This article will aim to explain how to prepare your own agar for use in petri dishes or making your own master culture slants.  

You can prepare agar!

There are just a few rules to follow, and some things you need to know.

Let’s break it down…

Table of Contents

Preparing Agar

First thing’s first, decide on your recipe!  Below is a recipe for MEA, a staple used by mycologists the world over.  Alternatively, you could use an Agar Pre-Mix.  We sell sachets of pre-measured agar mixture in a range of formulae.  

Let’s have a look how to prepare your agar mixture.

Malt Extract (Yeast) Agar

  • 10g Agar
  • 10g Malt Extract
  • (Optional) 2g Nutritional Yeast
  • 500ml Water
This recipe makes 500ml/20 plates. 

Agar Pre-Mixes

Mix!

  • In a suitably sized jug, prepare 500ml boiling water.  
  • Keep stirring the water, and sprinkle in your dry ingredients slowly.  This will help to avoid the dreaded clumping which will serve to leave bits and clumps in your finished plates.  
  • Keep stirring!  Taking the time here to make sure it’s 100% dissolved will pay off later!
  • Once dissolved, add your mixture to your pouring vessel of choice.

The ideal pouring vessel is a Pyrex Media Bottle (pictured below).  Available in 500ml (20 plates) or 1L (40 plates), these are autoclave safe, and feature a pouring-ring to help avoid drips.

Specialist glassware such as this though can be expensive (£10+ per bottle!) so when just getting started, there are some other options worth exploring.

A 500ml beer bottle (clear preferably) makes a fine stand-in.  You will need to cap the bottle with aluminium foil before sterilising.  The narrow opening of beer bottles is useful for agar pouring as you reduce the opening into which contaminants could fall.  These bottles are however quite prone to dripping – practice is the only way to avoid it!

Pyrex Media Bottle

Pyrex Media Bottle

Agar Beer Bottle

Beer Bottle

Sterilise!

Once your bottle of agar is prepared, it needs to be sterilised.  This is absolutely crucial to your success.  Sterilisation is the process of killing all the bacteria and mould/fungi spores in the mixture by heating to 121˚c for 15 minutes.  

We use a pressure cooker or autoclave to bring the pressure up to 15psi, and the temperature to 121˚c, for 15 minutes.  This ensures there are no contaminants in the agar.

Once your agar has cooled sufficiently, you can pour your plates!  50-65Ëšc is the magic range.   Agar begins to solidify at 40-50Ëšc, and once this happens you’ll start seeing lumps in your plates – not the end of the world, but ugly!

As a rule of thumb, once your pouring bottle is cool enough to handle without burning yourself, you can begin pouring.  When you’re first starting out, it’s best to err on the hot side to avoid solidifying before you’re finished.  Pouring too hot though will result in more condensation, which reduces plate visibility and may increase chances of contamination spreading. 

Now we pour!

If you’re not prepared, this is the tricky bit.  It comes down to having your work area prepared and clean.

Clean is the crucial word here!  You need to work in as sterile of an environment as is possible, to avoid introducing airborne contaminants to your freshly-poured plates.  For this reason, professional mycologists carry out their agar work in front of a Laminar Flow Hood – a device which blows a stream of sterile air in which to work without fear of contaminants. 

The affordable alternative available to the amateur is a Still Air Box (or SAB).  Effectively this is a box with arm holes, where you can work isolated from the air-currents in the room which could carry contaminants into your agar.

Still Air Box with hinged arm-hole covers

Laminar Flow Cabinet

Pouring plates successfully relies on having your work area prepared, and working methodically and deliberately.

  • Clean your work area with a bleach solution, or 70% Isopropyl Alcohol.
  • Clean the outer sleeve of your petri dishes, and only open them inside your sterile work area once you’re ready to pour.
  • Stack the plates – you may find it easiest to work with two stacks of 10 rather than one tall tower of 20.
  • Clean the outside of the pouring vessel with 70% alcohol.

It’s very important that from this point, aseptic technique is employed.  Nothing ‘dirty’ (that’s you, sorry!) should ever pass above something ‘clean’ (agar bottle, petri dishes) as contaminants may fall into your nice clean plates.

When pouring, we work from the bottom plate up by lifting the lid of the bottom plate, with the other empty plates on top.  Pour enough agar to cover the bottom of the plate to a thickness of 3-6mm (~12-15ml in a standard 90mm petri dish).

Work deliberately and quickly without being hasty.  Replace the lid as soon as you’ve finished pouring, and move up to the next plate until completed.

Close up your SAB if that’s what you’re using. 

Take a breather!

Now it’s just a case of waiting for them to solidify and dry.  The plates will be solid in 40-60 minutes.  Drying time may take several hours depending on how hot your agar was when you poured, relative humidity in the area they’re drying in etc.  

Seal them up!

Once dry, it’s a good idea to seal your plates if not using immediately. 

The best thing for the job by far is Parafilm – a stretchy paraffin-based laboratory film.  Wrapping plates is a bit of a skill in itself, but the only way is to practice!  

Some use micropore tape, but it is cumbersome to apply compared to parafilm and is better suited to other uses.  It’s sticky, and you may find yourself fumbling with a length long enough to wrap a plate.  It’s also less convenient to un-wrap when you’re ready to use.

Cling-film is another diy suggestion.  It may be cheaper than lab film but for the negligible difference in cost, I’d rather not risk the plates I just laboured over!

What You'll Need

As with anything there’s more than one way to get started.  But broadly speaking, you’ll need:

  • Agar Agar powder – food-grade is most common and perfectly fine for mycology work, though you can pay extra for lab-grade which is finer and results in a marginally clearer plate.
  • Sugar – Dextrose (also sold as brewing sugar at homebrew shops), Malt Extract (light or extra-light is preferable for clarity), Honey etc. 
  • Optionally: yeast, potato flakes (or broth), soy peptone, antibiotics, activated charcoal, or any number of recipe-dependant extras.
  • Or – Pre-mix Agar, which contains pre-measured ingredients – just add to water!
  • Pouring vessel – Media bottles, beer bottles etc.
  • Sterile petri dishes, test tubes (for slants), or even autoclavable PP5 takeaway pots.
  • Somewhere clean to work – SAB, flowhood etc.
  • 70% Isopropyl alcohol to sanitise with.
 

If you’re keen to jump straight in, you could always cut out some variables and go with Pre-Poured Plates.  This gives you opportunity to work with plates of known quality whilst you practice your transfers and sterile technique.

Summary

Hopefully this guide has given you a good overview of what goes into producing your own agar plates.  All it takes is a little preparation, a steady hand, and somewhere clean to work.

 
Maybe you’d rather leave the hard work to us, and focus on the fun part? 

Is there anything you’d like to see us cover in future guides?

Feeling a bit stuck on something?

Leave a comment below, or drop us a line! Â