GB/T 38568-2020Determination of growth phenotype for industrialmicrobial strain—Microdroplet turbidity (English PDF)

3.1 Reference strain

The identified stock strain, whose fermentation performance is clear, for fermentation production.

7.1 Strain activation

7.1.1 Prepare solid plates and liquid culture media corresponding to industrial microorganisms according to the methods that are given in GB/T 14926.43. Use a sterile inoculating loop to dip the reference strain and the strain preservation bacteria solution of the to-be-evaluated strain respectively; draw a line on the solid plate; select the corresponding temperature and other conditions to perform cultivation according to the production process parameters, until a single colony grows; respectively pick 3 reference strains and 3 single colonies of to-be-evaluated strains; inoculate them into a test tube that contains 5 mL of liquid medium; select the corresponding temperature and speed according to the production process parameters; cultivate to the logarithmic phase.

7.1.2 Pick a monoclone of the microorganism strain from the activated plate petri dish; place it in a container of 5 mL of liquid medium; select the corresponding temperature and speed according to the production process parameters; cultivate to the stable period. After removing the supernatant, add the PBS solution and repeat the washing 3 times.

7.2 Selection of microbial liquid concentration

Use the basal medium to dilute to obtain a cell suspension concentration of about 6 x 10^5 CFU/mL.

7.3 Preparation of agarose solution

Use the analytical balance to weigh 0.02 g of ultra-low melting point agarose; place it in a sterile EP tube; add 1 mL of sterilized basal medium to it; heat to dissolve; prepare it into a 2% (mass concentration) solution. Use a 0.22 µm sterile filter to filter while it is hot; prepare it when necessary.

7.4 Single cell droplet generation

7.4.1 Respectively take 500 µL of the microbial liquid and the agarose solution in equal volumes and mix them.

7.4.2 Take a chip; respectively add 200 µL of fluorocarbon oil to the oil phase inlet of the chip and 100 µL of low melting point agarose to the water phase inlet; connect the syringe to the chip outlet.

7.4.3 Place the chip on the hot plate (temperature stability of ±1°C, range at 30 °C ~ 37 °C); fix the plunger of the syringe at the 2 mL mark; let the liquid enter the chip through the negative pressure in the syringe; start to generate droplets. It takes about 2 minutes to generate droplets. […]