Standard number: GB/T 38568-2020
Standard name: Determination of growth phenotype for industrial microbial strain
—Microdroplet turbidity English name: Determination of growth phenotype for industrial microbial strain—Microdroplet turbidity
Standard format: PDF
Release time: 2020-03-31
Implementation time: 2020-03-31
Standard size: 1.88M
Standard introduction: 1 Scope
This standard specifies the method for determining the growth phenotype of industrial microbial strains by microdroplet turbidity.
This standard is applicable to the determination of growth phenotypes of bacteria, fungi and microalgae strains for industrial fermentation.
2 Normative references
The following documents are essential for the application of this document. For all dated references, only the dated version applies to this document. For any undated referenced document, the latest version (including all amendments) shall apply to this document.
GB/T6682 Specifications and test methods for water used in analytical laboratories
GB/T14926.43 Staining methods, culture media and reagents for bacteriological detection of experimental animals
This standard specifies the method for determining the growth phenotype of industrial microbial strains by microdroplet turbidity method. This standard is applicable to the determination of growth phenotypes of bacteria, fungi and microalgae strains used in industrial fermentation.

ICS07.080
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National Standard of the People's Republic of China
GB/T38568—2020
Determination of growth phenotypefor industrialmicrobial strain-Microdropletturbidity
Published on 2020-03-31
State Administration for Market Regulation
National Administration of Standardization
Implementation on 2020-03-31
This standard was drafted in accordance with the rules given in GB/T1.1-2009. This standard was proposed and managed by the China National Institute of Standardization. The drafting units of this standard are: Qingdao Institute of Bioenergy and Process Technology, Chinese Academy of Sciences, and China National Institute of Standardization. The main drafters of this standard are: Ma Bo, Ge Anle, Wang Xixian, Ji Yuetong, Xu Jian, and Ma Aijin. KAa-JoakAn
GB/T38568-—2020bzxz.net
1 Scope
Determination of growth phenotype of industrial microbial strains
Microdroplet turbidity method
This standard specifies the method for determining the growth phenotype of industrial microbial strains by microdroplet turbidity method. This standard is applicable to the determination of growth phenotype of bacteria, fungi and microalgae strains for industrial fermentation. 2 Normative references
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GB/T38568-—2020
The following documents are indispensable for the application of this document. For all dated references, only the dated version applies to this document. For any undated referenced document, the latest version (including all amendments) shall apply to this document GB/T6682 Specifications and test methods for water used in analytical laboratories GB/T14926.43 Terms and definitions for staining methods, culture media and reagents for bacteriological detection of experimental animals
The following terms and definitions apply to this document. 3.1
reference strain
Reference strain
An identified preserved strain with clear fermentation performance used for fermentation production. Principle
Single-cell encapsulated droplets are generated through microfluidic chips and cultured. The growth rate is calculated based on the area of ​​the bacterial body in the droplets. Reagents or materials
Unless otherwise specified, all reagents used are analytically pure. 5.1 Water
Conform to the secondary water specified in GB/T6682.
5.2 Basic culture medium
Prepare according to the method given in GB/T14926.43. Or select commercial pre-made culture medium and add water to prepare it strictly according to the product instructions.
3PBS buffer·pH7.4
Weigh 0.27g potassium dihydrogen phosphate (KH,PO), 1.42g disodium hydrogen phosphate (NaHPO), 8g sodium chloride (NaCI), and 0.2g potassium chloride (KCI). Add the above ingredients to about 800ml of deionized water. Stir thoroughly to dissolve, then add concentrated hydrochloric acid to adjust the pH to 7.4, and finally adjust to 1La
GB/T38568—2020
4Ultra-low melting point agarose
Gel point is 8℃~17℃
5Fluorocarbon oil.
Contains surfactant.
Instruments
Thermostat: ±1℃.
Centrifuge: 500g4000g.
Inverted microscope: with imaging module.
PTFE catheter: inner diameter 0.5mm.
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6.5PDMS microfluidic chip: height 100μm, connected to at least 50 culture chamber structures at the back, each chamber size is 1.5mmx1mm: the linear structures on both sides of the oil phase are the same, each side is 29.2mm long and 40m wide; the linear structure of the water phase is 4.54mm long and 60μm wide.
6Spectrophotometer.
6.7Hot plate.
7 Determination steps
7.1 Strain activation
7.1.1 Prepare the corresponding solid plates and liquid culture media for industrial microorganisms according to the method given in GB/T14926.43, use a sterile inoculation loop to dip the reference strain and the seed preservation solution of the strain to be evaluated, streak on the solid plate, select the corresponding temperature and pH value according to the production process parameters, and culture until single colonies grow: pick 3 reference strains and 3 single colonies of the strain to be evaluated, respectively, and inoculate them into test tubes containing 5mL of liquid culture medium. Select the corresponding temperature and speed according to the production process parameters, and culture to the logarithmic phase. 7.1.2 Pick a single clone of the microbial strain from the activated plate culture blood and place it in a container of 5mL of liquid culture medium, select the corresponding temperature and speed according to the production process parameters, culture to the stable period, remove the supernatant and add PBS solution, and repeat the washing 3 times. 7.2
Selection of concentration of microbial culture liquid
Dilution with basic culture medium to obtain a cell suspension concentration of about 6×10°CFU/mlL7.3
3 Preparation of agarose solution
Use analytical balance to weigh 0.02g ultra-low melting point agarose and place it in a sterile EP tube, and add 1ml of basal culture medium of ignited bacteria, heat and dissolve, and prepare a 2% (mass concentration) solution. Filter with a 0.22um sterile filter head and prepare it before use. 7.4 Single cell droplet generation
7.4.1 Take 500μl of microbial culture liquid and agarose solution in equal volumes and mix them evenly. 7.4.2 Take a chip, add 200l of fluorocarbon oil to the oil phase inlet of the chip and 100l of low melting point agarose solution to the water phase inlet, and connect the syringe to the chip outlet. 7.4.3 Place the chip on a hot plate (temperature stability ±1°C, range 30°C~37°C), fix the piston of the syringe at 2mL.2
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GB/T38568-—2020
degree), and use the negative pressure in the syringe to make the liquid enter the chip, and droplets begin to occur. It takes about 2 minutes for droplets to occur. 7.4.4 Take another chip and repeat the above steps, one for the strain to be identified and the other for the reference strain. This step was repeated three times. 7.5
Single cell droplet culture
After the droplet generation was completed, remove the pipette tip and polytetrafluoroethylene tube on the chip. Place the two chips in a 4℃ refrigerator to solidify for 20 minutes and then place them in a culture III containing water in a 37℃ constant temperature box for 6 hours. 6 Chip imaging
Place the chip under an inverted microscope and image it under a 10× objective lens. Obtain at least 20 pictures, no less than 2000 droplets and save them in bmp format.
Data processing
Use software to process the pictures, count the number of droplets in each picture and the diameter of each droplet, and calculate the plane area of ​​the plaque in the picture.
Result analysis
Result calculation
The relative difference in plaque area is calculated according to formula (1): d
Wherein:
Relative difference in plaque area:
The area of ​​bacteria in a single droplet in the image; R
The diameter of a single droplet;
The number of gel droplets.
Result analysis
......(1)
The similarity between the candidate strain and the reference strain is evaluated based on the relative difference in plaque area. The smaller the absolute value of the d value, the closer it is to the turbidity of the gel droplet represented by the standard signal. A negative d value indicates that the growth rate is lower than the reference strain, and a positive d value indicates that the growth rate is higher than the reference strain. The absolute difference between the three independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean.
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