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Key Points for Preparing Liquid Culture in Shake Flasks

(1) Culture Medium Formula:

Potato 200g, glucose 20g, peptone 5g, potassium dihydrogen phosphate 2.0g, magnesium sulfate 1.0g, 

vitamin B₁ 1 tablet, pH 6.8～7.0, water 1000ml.

(2) Preparing the Culture Medium:

Boil the potatoes over low heat for 30 minutes (the standard is that they are cooked but not mushy). 

Then filter the mixture through 6 layers of gauze. Pour the filtrate into a pot, add the various ingredients

weighed according to the formula, continue heating and stirring until completely dissolved, then stop 

heating and add water to make up the volume. Dispense the culture medium into Erlenmeyer flasks,

filling each 500ml flask with 150ml of culture medium. Add 10 glass beads to each flask, then seal the 

flask opening with a silicone breathable stopper.

(3) Sterilization and Cooling: 

Place the Erlenmeyer flasks in an autoclave, cover them with newspaper, and after venting, start timing 

when the temperature reaches 121℃ and maintain this temperature for 30 minutes. When the pressure

is about to drop to zero, start releasing the pressure, open the lid, and cool the flasks to below 30℃. 

Remove the flasks and place them in a clean bench to cool.

(4) Shake Flask Inoculation: 

Select one freshly cultured slant culture tube. Under aseptic conditions, quickly inoculate each flask with

a 2-3cm² piece of mother culture. Each mother culture can inoculate 5 shake flasks.

(5) Incubation: 

The inoculated flasks can be placed on a shaker for incubation, or placed at a constant temperature of 25℃

for 48 hours to ensure no contamination and that aerial mycelia have extended into the culture medium 

before placing them on a shaker for further incubation. In a rotary culture medium, the shaking frequency is

180 r/min, the culture temperature is 25℃, and the culture time is generally 5-6 days. When the mycelial balls

evenly cover the transparent orange-yellow nutrient solution, the culture is stopped.