GB/T 38483-2020.Determination of antibacterial activity for microbial secondary metabolites-Inhibition zone method.
1 Scope
GB/T 38483 specifies the method for determining the antibacterial activity of secondary metabolites of microbial antibiotics by the inhibition zone method.
GB/T 38483 is applicable to the determination of antibacterial activity of secondary metabolites of microbial antibiotics.
2 Normative references
The following documents are indispensable for the application of this document. For any dated referenced document, only the dated version applies to this document. For any undated referenced document, its latest version (including all amendments) applies to this document.
GB/T 6682 Specifications and test methods for water for analytical laboratories
GB 19489 Laboratory biosafety requirements
3 Terms and definitions
The following terms and definitions apply to this document.
3.1
Median inhibitory concentration
IC50
The concentration at which the inhibition rate of bacteria reaches 50%.
3.2
Antibacterial activity
The ability to inhibit bacterial growth and reproduction.
3.3
Inhibition zone
The zone of sterile reproduction at the boundary between the surface of the solid culture medium and the sample.
4 Principle
The activity is determined by calculating the IC50 by using the size of the inhibition zone formed by the diffusion of the secondary metabolite to be tested in the agar plate to inhibit the growth of the surrounding bacteria.
5 Laboratory biosafety requirements
Should comply with the provisions of GB 19489.
6 Reagents or materials
Unless otherwise specified, all reagents used are of analytical grade.
6.1 Water. GB/T 6682 Grade 1 water.
6.2 LB liquid medium. Weigh 10.0 g of tryptone, 5.0 g of yeast extract and 10.0 g of sodium chloride, then add the above ingredients to 1 000 mL of distilled water, boil to dissolve, adjust the pH to 7.0 ± 0.2, dispense into conical flasks, and sterilize at 121 °C for 20 min.
6.3 LB solid medium. Weigh 10.0 g of tryptone, 5.0 g of yeast extract, 10.0 g of sodium chloride, and 20.0 g of agar, then add the above ingredients to 1 000 mL of distilled water, boil to dissolve, adjust the pH to 7.0 ± 0.2, dispense into 250 mL conical flasks, and sterilize at 121 °C for 20 min.
This standard specifies the method for determining the antibacterial activity of secondary metabolites of microbial antibiotics by the inhibition zone method. This standard is applicable to the determination of antibacterial activity of secondary metabolites of antibiotics of microbial origin.

ICS07.080
National Standard of the People's Republic of China
GB/T38483—2020
Determination of antibacterial activity for microbial secondarymetabolites-Inhibition zone method2020-11-19Issued
State Administration for Market Regulation
National Administration of Standardization
Issued
Implementation on 2020-11-19
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. Drafting units of this standard: China University of Metrology, China National Institute of Standardization Main drafters of this standard: Shentu Xuping, Ye Zihong, Ma Aijin, Yu Xiaoping, Xu Yipeng, Cui Haifeng, Zhang Yafen Sa
GB/T38483—2020
1 Scope
Determination of antibacterial activity of secondary metabolites of antibiotics of microbial origin
Inhibition zone method
GB/T38483—2020
This standard specifies the method for determining the antibacterial activity of secondary metabolites of antibiotics of microbial origin by the inhibition zone method. This standard applies to the determination of antibacterial activity of secondary metabolites of antibiotics of microbial origin. 2 Normative references
The following documents are indispensable for the application of this document. For all dated references, only the dated version applies to this document. For all undated references, the latest version (including all amendments) applies to this document. GB/T6682 Specifications and test methods for water used in analytical laboratories GB19489 Laboratory biosafety requirements
3 Terms and definitions
The following terms and definitions apply to this document. 3.1
Median inhibitory concentration
ICso
The concentration at which the inhibition rate of bacteria reaches 50%3.2
Antibacterial activity
antibacterial activity
The ability to inhibit bacterial growth and reproduction.
Inhibition zone
inhibition zone
The zone of sterile reproduction at the boundary between the surface of the solid culture medium and the sample. 4 Principle
The activity is determined by calculating the ICso based on the size of the inhibition zone formed by the diffusion of the secondary metabolite to be tested in the agar plate to inhibit the growth of the surrounding bacteria.
Laboratory biosafety requirements
Should comply with the provisions of GB19489.
Reagents or materials
Unless otherwise specified, all reagents used are analytically pure. 1
GB/T38483—2020
6.1 Water. GB/T6682 Grade 1 water.
6.2LB liquid culture medium. Weigh 10.0g of trypsin, 5.0g of yeast extract, and 10.0g of sodium chloride, then add the above ingredients to 1000mL of distilled water, boil to dissolve, adjust the pH to 7.0±0.2, dispense into conical flasks, and sterilize at 121℃ for 20min. 6.3LB solid culture medium. Weigh 10.0g of trypsin, 5.0g of yeast extract, 10.0g of sodium chloride, and 20.0g of agar, then add the above ingredients to 1000mL of distilled water, boil to dissolve, adjust the pH to 7.0±0.2, dispense into 250mL conical flasks, and sterilize at 121℃ for 20min.
6.4 Standard strains of indicator bacteria:
Gram-positive bacteria: Staphylococcus aureus ATCc6538. Gram-negative bacteria: Escherichia coli ATCC11229. Other standard strains can also be used as test bacteria, and the specific selection can be made according to the actual situation. 7 Instruments and equipment
Constant temperature incubator: temperature deviation within ±1°C. Sterile culture dish: with ceramic tile cover, diameter 90mm. 7.3 Sterile Oxford cup: stainless steel, standard specifications: inner diameter 6mm, outer diameter 8mm, height 10mm. 7.4 Inhibition zone measuring instrument: accuracy 0.1mm. Spectrophotometer: detection wavelength 600nm.
Sterile conical flask: capacity 100mL, 250mL. Sterile pipette: 1mL (with 0.01mL scale), 10mL (with 0.1mL scale) or micropipette. 8
Operation steps
8.1 Strains activation
Inoculate the strain into a test tube containing 2mL of LB liquid medium and culture at 37℃±1℃ for 12h~18h. Use an inoculation loop to pick up the bacterial suspension and inoculate it onto the LB solid plate by streaking method, and culture it in a constant temperature incubator at 37℃±1℃ for 18h24h. Then pick a single colony from the plate and inoculate it into the slant of the LB solid medium test tube, and culture it in a constant temperature incubator at 37℃±1℃ for 18h~24h. Then store the test tube slant in a refrigerator at 1℃~4℃ as a preserved bacteria. The storage period shall not exceed one month, and the culture shall be subcultured once a month, and the number of subcultures shall not exceed 10 generations. 8.2 Preparation of bacterial suspension
8.2.1 Take the preserved bacteria with an inoculation loop and inoculate them onto the LB solid plate by streaking method, and culture at 37℃±1℃ for 24h. Note: The LB solid plate should be stored at 1℃~4℃ and used within 1 week. 8.2.2 Take 20mL of LB liquid culture medium and add it to a sterile conical flask with a capacity of 100mL. Take a single colony on the plate in 8.2.1 with an inoculation loop and inoculate it into the LB liquid culture medium, and culture it at 37℃±1℃ for 12h18h. 8.2.3 Use LB liquid culture medium to adjust the bacterial concentration after culture to 1X10°CFU/mL～5×10\CFU/mL and use it as the test bacterial suspension. Use a spectrophotometer to determine the ODoo of the test bacterial suspension to be 0.5～0.65 or (other) appropriate methods to determine the concentration of the bacterial suspension. Note: The test bacterial suspension should be stored at 1℃～4℃ and used within 4h. 8.3 Preparation of test samples
Polar samples are directly dissolved in water (non-polar samples are fully dissolved by adding a certain concentration of surfactant), and a certain concentration of mother solution is prepared. It is filtered through a sterile 0.45μm filter membrane, and then diluted step by step with sterile water (or the corresponding surfactant) at a concentration of 2 or 5 times to prepare at least 5 different concentrations of test solutions; when diluting step by step, it should be ensured that there are both concentration points with an inhibition rate greater than 50% and concentration points with an inhibition rate less than 50%, and the solution is prepared and used immediately. 2
8.4 Preparation of test plates
GB/T38483—2020
Dilute the bacterial suspension in 8.2.3 with LB solid culture medium cooled to about 55℃ at a ratio of 1:10, mix thoroughly, take 15mL into a sterile culture dish, gently spread the plate to make the bacterial solution evenly spread, and make a test plate after it solidifies for use. 5 Determination of antibacterial activity
Gently place the sterile Oxford cup on the test plate prepared in 8.4, and then use a sterile pipette to add 200μL of the test sample solution of different concentrations prepared in 8.3 to the Oxford cup, and add 200μL of the corresponding solvent to the blank control group. Repeat each treatment 5 times. Place the plate upright in a constant temperature incubator at 37℃±1℃ for 12h~18h, take it out, and measure the diameter of the inhibition zone formed by the test sample and the blank control group with an inhibition zone measuring instrument.
Result calculation
The inhibition rate is calculated according to formula (1):
Where:
I—inhibition rate;
D,-D2Www.bzxZ.net
×100%
.(1)
D,—the diameter of the inhibition zone formed in the test metabolite plate, in millimeters (mm); D——the diameter of the inhibition zone formed in the blank control plate, in millimeters (mm). The average of five parallel samples is the final result, and the calculation result is retained to two decimal places. According to the logarithmic value of the concentration of the drug and the probability value of the corresponding inhibition rate, a regression curve Y = aX + b is obtained, where Y is the probability value of the inhibition rate, X is the logarithmic value of the concentration of the drug, a is the slope of the regression curve, b is a constant, and the R2 value of the regression curve is given to calculate the IC5o values ??of the test agents for Escherichia coli and Staphylococcus aureus. 10
Repeatability
The absolute difference between two independent determination results obtained under repeated conditions should not exceed 15% of the arithmetic mean.
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