Making Liquid Culture, it’s a 2-step process. First, make the jar with nutrient medium and then sterilise it. Finally, it is inoculated with mycelium.

Steps

Step 1

Preparing sterilised Jar (Using microwave technique)

Get a desired size jar (Max 1 litre) depending on how much Liquid culture you need to make. The best Jar size for home use is around 500 ml.
The jar would need a lid with an injection port and air filter. You can use the stick-on ones. Alternatively, use a rubber and plastic air filter that is stuck by applying high temp-resistant RTV silicon. Here is a video on how you can achieve this:

If you make Liquid culture using the microwave sterilisation technique, it is essential that you use plastic lids only. They are also better in the long term as they do not rust and contaminate your LC.

Tip: Do not start a liquid culture from a spore syringe. Instead, Begin from a colonised agar plate that has no visible contamination. It can be achieved by transferring it from plate to plate multiple times.
Only fill 60 to 70% of the jar with water, as some space is required on top during sterilisation. Moreover, it will support the liquid culture to absorb oxygen from the air during colonisation.

Liquid Culture Preparation Instructions

1. Boil water in a kettle or a pan.
2. In the meantime, thoroughly clean the inside of the jar, lid and marble/ magnetic stirrer bar with soap and hot water. Ensure all soap residue is removed.
3. Place the marble/ stirrer bar in the jar first and then add the boiling water and the Dry LC mix powder. You may be required to adjust the powder’s ratio according to the water you will use.
4. Using a clean spoon, stir until the powder is completely dissolved. Place the lid on top again and tighten it completely and then loosen it a bit (Very slightly). Please do not use a metal spoon, as it may crack the jar.
5. Place the jar in a microwave and heat for 2 minutes or until it starts to boil(do not let the water flow out of the jar). Allow 1 minute to rest and repeat 3 more times. Subsequent boiling will only require few seconds.
6. Leave the jar in the microwave and let it cool down until it’s good to handle with bare hands. Ensure that the Stickon (if you are using one) Silicon injection port and air filter are placed firmly on the jar by pressing it down with clean hands. On top of that, tilt the jar to ensure no leakage occurs. If there is leakage, tighten the lid firmly.
   Tip: It’s best to prepare sterilised jars 2 weeks before inoculation. Thus, if it turns milky, it can be disposed of. Use only the jars that have remained clear and did not turn milky. It’s valuable to take a photo after sterilisation and compare the jar with the photos after 2 weeks. Ensure labelling the jar so that you know when they were sterilised.

Step 2 Inoculation

1. Ensure your workspace has minimal airflow for the procedure. Sanitise the area to remove any potential contaminants from entering the cultures. Use a spray bottle with Isopropyl alcohol 70% and a clean cloth for this step.
2. A small room with a table is suggested as a workspace. Ensure your windows and doors are closed and that the Air conditioner is off. It includes sanitising the inside and outside of the still air box if you have one.
3. Spray the outside of the jar and the lid with isopropyl alcohol.
4. Clean the scalpel and blade with isopropyl alcohol. Once the alcohol has evaporated from the scalpel, sterilise it using a flame until the scalpel blade is red hot.
5. Loosen the lid but do not open it completely. It needs to be loose enough to open it using one hand later.
6. Cut a small agar wedge from a desired sector on your plate and open the jar lid with your other hand. Only open it slightly(not completely) and place the agar wedge inside.
7. Close the lid entirely and leave it for 2 weeks to colonise.
8. You have successfully made your LC, if the water is clear, and you see a jelly-like white substance growing (Mycelium). On the other hand, if the water turns milky, the jar is contaminated.
9. Most of the time, the mycelium will not grow in contaminated jars. Occasionally, however, mycelium and contamination grow side by side.
10. If you notice any colour other than white, such as black or green, growing in the jar, discard it.

Tip: It is recommended to test the jar on an agar plate before you colonise your grain bag. This additional preventive measure will prevent contamination of grain spawn and substrate bags. Do not use MEAG plate, as it won’t show bacterial contamination if any is present, use MEA plate. If you do not have an agar plate, you can inoculate some grain bags and observe if they contaminate.

Don’t have time to make your own pre-sterilized jar? We’ve got you covered! We sell pre-sterilized jars for pickup only—check them out if you’re interested!

Jae

Owner of Rootlab