GB/T 38480-2020.Determination of antifungal activity for microbial secondary metabolites-Mycelial growth rate method.
1 Scope
GB/T 38480 specifies the method for determining the antifungal activity of secondary metabolites of microbial antibiotics by mycelial growth rate method.
GB/T 38480 is applicable to the determination of antifungal 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
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 fungal growth reaches 50%.
3.2
Antifungal activity
The ability to inhibit the growth and reproduction of fungi.
4 Principle
Mix the secondary metabolites to be tested with the culture medium. The growth rate of the colonies on the culture medium is used to measure the ability of the secondary metabolites to inhibit filamentous fungi. The activity is determined by calculating IC50.
5 Reagents or materials
Unless otherwise specified, all reagents used are of analytical grade.
5.1 Water. GB/T 6682 Grade 1 water.
5.2 Potato dextrose agar (PDA). Weigh 3.0 g potato powder, 20.0 g glucose and 20.0 g agar, then add the above ingredients to 1 000 mL distilled water, boil and dissolve, pH natural, dispense into 250 mL conical flasks, sterilize at 121℃ for 20 min, and set aside. Commercial culture medium available on the market can also be used.
5.3 Standard strain of indicator bacteria: Rhizoctonia solani Kuhn ATCC76145.
Depending on the application field of microbial antibiotic secondary metabolites, other strains can also be used as test bacteria.
This standard specifies the method for determining the antifungal activity of microbial antibiotic secondary metabolites using the mycelial growth rate method. This standard is applicable to the determination of antifungal activity of microbial antibiotic secondary metabolites.

ICS07.080
National Standard of the People's Republic of China
GB/T38480—2020
Mycelial growth rate method for secondary metabolites of antibiotics from microorganisms
Determination of antifungal activity for microbial secondary metabolites-Mycelial growth rate method
Issued on 2020-11-19
State Administration for Market Regulation
National Administration of Standardizationwww.bzxz.net
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 GB/T38480—2020
Main drafters of this standard: Shentu Xuping, Ye Zihong, Ma Aijin, Yu Xiaoping, Cui Haifeng, Zhang Yafen, Xu Yipeng1
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1Scope
Determination of antifungal activity of secondary metabolites of antibiotics of microbial origin
Mycelial growth rate method
GB/T38480—2020
This standard specifies the method for determining the antifungal activity of secondary metabolites of antibiotics of microbial origin by mycelial growth rate method. This standard applies to the determination of antifungal activity of secondary metabolites of antibiotics of microbial origin 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 for analytical laboratories Terms and definitions
The following terms and definitions apply to this document. 3.1
medianinhibitory concentrationMedian inhibition concentration
ICsa
The concentration at which the inhibition rate of fungal growth reaches 50%3.2
Eantifungal activity
Antifungal activity
The ability to inhibit the growth and reproduction of fungi.
Principle
Mix the secondary metabolites to be tested with the culture medium, and measure the ability of the secondary metabolites to inhibit filamentous fungi by the growth rate of the colonies on the culture medium, and determine the activity by calculating IC Reagents or materials
Unless otherwise specified, all reagents used are analytical grade 5.1 water. GB/T6682 grade water.
5.2 Potato dextrose agar (PDA). Weigh 3.0g potato powder, 20.0g glucose, and 20.0g agar, then add the above ingredients into 1000mL distilled water, boil and dissolve. The pH is natural, and the mixture is divided into 250mL conical flasks, sterilized at 121℃ for 20min, and set aside. Commercial culture medium available on the market can also be used. 5.3 Standard strain of indicator bacteria: Rhizoctonia solani Kuhn ATCC76145 According to the different application fields of secondary metabolites of microbial antibiotics, other strains can also be used as test bacteria 1
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GB/T38480—2020
6 Instruments and equipment
Constant temperature incubator: temperature deviation within ±1℃6.1
2 Clean bench.
3 Sterile culture dish: diameter 90mm.
6.4 Measuring instrument: vernier caliper, accuracy o.1mm. 6.5 Sterile punch: inner diameter 5mm±0.1mm5 Sterile pipette: 1mL (with 0.01mL scale), 10mL (with 0.1mL scale) or micropipette. 6.6
7 Operation steps
7.1 Indicator bacteria culture
Pour 15mL of PDA solid culture medium that has been melted and sterilized at 121℃ and cooled to about 55℃ into a sterile culture dish, wait for it to solidify and make a PDA plate. Pick a small amount of mycelium from the frozen strain of Rhizoctonia solani with a sterile inoculation needle and inoculate it in the middle of the PDA plate. Cultivate it in a constant temperature incubator at 28℃±1℃ until the mycelium covers the entire plate and set aside. The above operations are all carried out in a clean bench. 7.2 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 prepared into a mother solution of a certain concentration, filtered through a sterile 0.45um filter membrane, and then diluted with sterile water (or the corresponding surfactant) by 2 or 5 times the concentration to prepare at least 5 test solutions of different concentrations. 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%. Prepare and use them immediately. 7.3 Preparation of test plates
Dilute the test samples prepared in 7.2 with PDA culture medium cooled to 55℃±1℃ at a ratio of 1:9, mix thoroughly, and use a sterile pipette to take 10mL each and transfer it to sterile culture blood, gently shake the culture blood to make it evenly spread, and make a test plate after it solidifies. The plate prepared by mixing the corresponding solvent and PDA is used as a blank control plate for standby use. 7.4 Determination of antibacterial activity
Use a sterile puncher (inner diameter 5mm ± 0.1mm) to punch holes at the same radius edge of the activated indicator bacteria plate, and use sterile tweezers to take the bacterial cake and place it in the middle of the test plate prepared in 7.3. Repeat each treatment 5 times. Place the plate in a constant temperature incubator at 28℃±1℃ for 24h~48h, take it out, and measure the colony diameter using the ten-degree cross method. The average value of the two measurements is the colony diameter. 8 Calculation of results
The inhibition rate is calculated according to formula (1):
Where:
1—inhibition rate;
(D,-5)—(D2—5)
X100%
D, -5
Dl—the diameter of the colony formed in the blank control plate, in millimeters (mm); Dz—the diameter of the colony formed in the secondary metabolite plate for testing, in millimeters (mm). The average of five parallel samples is the final result, and the calculation result is retained to two decimal places 2
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（1)
GB/T38480—2020
According to the logarithmic value of the concentration of the agent 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 agent, a is the slope of the regression curve, b is a constant, and the R2 value of the regression curve is given. The IC5 value of the test agent against Rhizoctonia solani is calculated.
Repeatability
The absolute difference between two independent determination results obtained under repeated conditions shall not exceed 15% of the arithmetic mean. rrKaeerkAca-
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