Liquid Culture Procedure

Ingredients:

  • Gram scale
  • Pint-size (473 ml) mason jar with lid (prepared with rubber plug and vent hole installed)
  • Warm water
  • Light corn syrup (clear, not dark)
  • Small rock or piece of broken glass
  • Micropore tape
  • Pressure cooker
  • Source of spores or mycelium
  • Luer lock syringe (I use 30-60cc)
  • 4” luer lock blunt tip dispensing needle
  • Luer lock syringe tip cap
  • Permanent marker
  • 70% isopropyl alcohol (rubbing alcohol)
  • Optional:
    • Magnetic stir plate with stir bars
    • Flow hood or still air box

Discussion:

This procedure is used to expand a spore solution or mycelium culture into a volume many times its original size. The source of spores or mycelium should be free of contamination, and ideally tested for quality on a plate of agar before use in the liquid culture. Regardless of agar testing, the liquid culture should be closely monitored for abnormal colors or growths; most mushroom mycelium is colored white and grows in a cloudy clump when undisturbed. Common contaminants are often colored brown, green or black, and grow on the surface of the liquid or in clumps that don’t break up easily when disturbed. Liquid cultures that are suspected of being contaminated with competing organisms should be discarded. 

This procedure is written to be used with a single US quart-size jar, but can easily be used with smaller jars. Taller jars may be used, but you may experience difficulty in withdrawing the culture into syringes. If using this procedure for more than one jar, maintain the ratio of 9 ml of nutrients to 300 ml of water. 

The optional equipment will make this task easier, but add expense. A magnetic stir plate is a great investment if you plan on performing this procedure often, and is also helpful when preparing agar. A still air box is handy to have around for this and many other mushroom cultivation techniques, and is inexpensive to build. Flow hoods are quite expensive and require dedicated space; many mushroom cultivators have successfully done this work for years without the use of a flow hood.

Directions:

 

Step

 

Principle

1.

Add 300g of warm water to the mason jar.

 

Warm water is used to help the sugars in the corn syrup melt; this makes it easier to mix. 

2.

Add 12g of light corn syrup to the water and stir to form a solution.

 

This is equivalent to 4% corn syrup:water ratio. 

 

The corn syrup provides the nutrients needed for the mycelium to grow in the liquid culture. 

3.

Place the piece of rock or broken glass in the jar.

 

 

 

If using the optional magnetic stir bar, place this in the jar instead of the rock/glass.

 

Mycelium tends to grow in clumps in the jar. A piece of material in the jar is helpful to break up the mycelium later to allow for more ease in sucking the solution into a syringe. 

 

The magnetic stir bar is used in conjunction with a magnetic stir plate. This tool makes the process easier, but isn’t a recommended investment for the weekend mushroom cultivator. 

4.

Install the prepared lid and ring on the jar.

 

 

5.

Place a piece of micropore tape over the vent hole on the lid. 

 

The mycelium will need some oxygen while colonizing the solution. The mycelium will also produce a small amount of gasses that will need to have a vent path. Micropore tape over the vent hole allows for this exchange of air/gas, but prevents bugs, dust or other contaminants from entering the jar. Micropore tape also holds up well in the pressure cooker. 

6.

Pour about 2cm of water into the bottom of the pressure cooker. 

 

Water in the bottom of the pressure cooker is necessary for proper functioning of the pressure cooker. 

7.

Place the mason jar in the pressure cooker and install the pressure cooker lid.

 

 

8.

Pressure cook at 15psi for 15 minutes.

 

15psi results in a temperature of approx 121C (250F) in the pressure cooker. 15 minutes at this temperature is sufficient to kill competing organisms in the solution.

9.

 

 

Allow the pressure cooker to naturally cool to room temperature before removing the jar.

 

Do not reduce pressure by ventilating or force cooling the pressure cooker as this could warp the vessel. 

10.

Add the source of spores or mycelium. If injecting spores or mycelium from a syringe, spray the injection port with 70% isopropyl alcohol before injecting. If using an agar culture, this should be done in a still-air box or under a flow hood with proper sanitization techniques.

 

This inoculates the solution with what will become the new liquid culture. 

11.

Place the jar in a location that is about 21-27C (70-81F). 

 

This temperature range is ideal for mycelium growth. 

12.

Check for signs of growth after about 7 days. Mix by swirling the jar (be careful not to wet the vent hole).

 

Mixing helps the mycelium access all the available nutrients in the solution and provides some fresh oxygen to the solution.

13.

After about 21 days, you should see signs of growth slowing. The jar may be refrigerated at this point, or dispensed into syringes.

 

 

14.

To dispense into syringes:

 

 

14.a

   Mix the liquid cultures by swirling the jar.

 

 

14.b

   Spray the lid's vent port (covered with micropore tape) with isopropyl alcohol.

 

This disinfects the vent port to minimize the chances of contamination during the following step. 

14.c

   Press the 4” dispensing needle through the tape and into the solution. 

 

 

14.d

   Connect a syringe to the dispensing needle and fill the needle to full capacity. Attempt to suck up some visible mycelium into the syringe.

 

 

14.e

   Disconnect the syringe from the needle, invert the syringe and push out any air from the syringe. Install cap on syringe. 

 

 

14.f

   Repeat step 14a-14.e until the jar is empty. 

 

 

15.

The jar or syringes may be stored in the refrigerator for up to 6 months.

 

The mycelium begins to lose viability after about 6 months.