How To Prepare Agar

Learn To prepare agar plates for mushroom cultivation

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What Is Agar?

Agar is produced from a red algae, known as agar-agar in Malay. This jelly-like substance has  been used for numerous applications as an ingredient in desserts, laxative, appetite suppressant, stabiliser in jams, preserves, ice-cream and as a clarifying agent in brewing.

It’s vegetal origins make it a popular substitute for animal gelatin amongst those following a vegan diet.

It’s qualities are believed to have originally been observed in 1658 by a Japanese inkeeper, Mino Tarōzaemon who had noticed how his seaweed soup had gelled upon being left overnight. Despite this it wasn’t until late in the 19th century that it was used as a solidified medium for microbiology. This use was first noted by German microbiologist Walther Hesse after the suggestion made by his wife, Fannie Hesse in 1882.

 

Why Use Agar To Cultivate Mushrooms?

Agar is used in the cultivation of mushrooms in order to store cultures for long-term use and create mono-cultures free from contamination. 

It’s strongly recommended to germinate spores on agar before use. Spores are often accompanied by pathogens like yeast and mould. By cultivating mycelium on agar plates we can visually identify contaminants with ease allowing for the cultivator to make a clean transfer to a new agar plate.

Agar plates are also used for making fungal clones. A clean fragment of the mushroom fruiting body can be placed on agar to grow rather than germinating spores. This gives the cultivator and advantage in isolating any potential contaminants if present. 

Do You Need A Flow Hood?

Using a laminar flow hood is greatly beneficial in the production of agar plates. Sterilised media is highly susceptible to contamination. In the absence of a flow hood all sterile transfers should be undertaken in an enclosed space with minimal airflow such as a bathroom.

Alternatively, a Bunsen burner may be used to create an upward draft to work within. All precautions need to be taken to reduce the likelihood of contamination. The use of gloves and a face mask is strongly recommended.

What You'll Need

In this tutorial we will provide the quantities to prepare 40 agar plates.

  • 20 grams agar
  • 20 grams light malt extract
  • 1 L water
  • Media bottle or jar with hole in lid

Step 1. Weigh Dry Ingredients

Using scales weigh light malt extract and agar powder. 

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Step 2. Dissolve Dry Ingredients

Add agar and light malt extract to boiled water. Stir continuously until dissolved. 

Step 3. Sterilise

Pour dissolved agar and light malt extract mixture into bottle for sterilising. Place lids on bottles and seal with a layer of aluminium foil.

Sterilise agar mixture for 20 minutes at 15PSI. Allow to completely depressurise or wait 1 hour minimum before opening pressure cooker.

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Step 4. Cool Agar In Water Bath

Place bottle of hot agar mixture in a warm water bath. Use a thermometer to gauge temperature. The agar mixture will measure the same temperature as the water bath. 

Cool agar to 55 degrees Celsius before pouring plates to reduce condensation.

Step 5. Pour Plates

Prepare work space to pour plates using aseptic technique.  Mentally rehearse before removing the lid off the agar mixture to ensure that the work is done quickly and effectively. 

Stack petri dishes 5 plates high. Remove lid off agar mixture. Start by pouring the bottom plate on the stack by lifting its lid with the additional 4 empty plates. Repeat this action working up each stack without disturbing the already poured plates. Pour approximately 20 ml of agar into each plate. 

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Step 6. Seal & Label

Wait for the agar to cool below 40 degrees. Seal with plastic wrap or parafilm. Label and store for later use.

Plates should be left at room temperature for 3 to 4 days to verify the absence of contaminants before refrigeration. Unsealed or poorly sealed plates may dehydrate and should be discarded.

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