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A minimum bactericidal concentration determination method based on trace natural compounds

A natural compound and bactericidal concentration technology, applied in the direction of microorganism-based methods, microorganism measurement/inspection, biochemical equipment and methods, etc., can solve the problems of large amount of medicine, difficulty in meeting the needs of experimental determination, and inapplicability. To achieve the effect of increasing reliability

Active Publication Date: 2021-10-15
HENAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

"However, this experimental method is not suitable for the determination of MBC from trace amounts of natural products, because in the study of antibacterial and bactericidal natural products, the above method requires a relatively large amount of medicine.
As we all know, the samples of natural products obtained by extraction and separation are often difficult to meet the needs of experimental determination due to the small amount of samples.

Method used

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  • A minimum bactericidal concentration determination method based on trace natural compounds
  • A minimum bactericidal concentration determination method based on trace natural compounds
  • A minimum bactericidal concentration determination method based on trace natural compounds

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Experimental program
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preparation example Construction

[0031] 1. Preparation of liquid to be tested

[0032] Take 70% methanol solution to dissolve the natural compound drug to be tested, and use the multiple dilution method to prepare the 70% methanol mixture of the drug to be tested into different solubility of the drug solution to be tested, and place it in the refrigerator for subsequent use;

[0033] The natural compound drug to be tested is obtained by in vitro high-throughput screening method, has a clear antibacterial spectrum and the minimum inhibitory concentration MIC value is less than 256ug / mL; the concentration of the drug solution to be tested is 625ug / mL, 256ug / mL, 128ug / mL, 78ug / mL, 64ug / mL, 32ug / mL, 16ug / mL, 8ug / mL, and 4ug / mL.

[0034] 2. Preparation of standard bacterial solution

[0035] Inoculate the selected strain into a test tube containing liquid medium, put it into a shaker for shaking, and then prepare the strain to obtain the OD 600 The bacterial liquid with the best absorbance value at that time is...

Embodiment 1

[0052] A method for determining the minimum bactericidal concentration based on trace natural compounds, comprising the following steps:

[0053] Step 1: Preparation of the drug solution to be tested

[0054] 1. Liquid preparation: 70% methanol, compound scorpion extract; 2. Dissolve compound 2 extracted from scorpion with 70% methanol; 3. Prepare the drug extracted from scorpion by multiple dilution method to a concentration of 625ug / mL for No. ① respectively; ②No. 256ug / mL ③No. 128ug / mL; ④No. 78ug / mL; ⑤No. 64ug / mL; ⑥No. 32ug / mL;

[0055] Step 2: Preparation of standard bacterial solution

[0056] 1. Inoculate the selected Staphylococcus aureus and Pseudomonas aeruginosa into test tubes containing liquid medium, and put them on a shaker for 4 hours; 2. Prepare the bacterial solution in 1 to correspond to different strains OD value, ready to use. Finally determine the OD of different strains corresponding to the optimal bacterial concentration 600 The absorbance was measur...

Embodiment 2

[0071] A method for determining the minimum bactericidal concentration based on trace natural compounds, comprising the following steps:

[0072] Step 1: Preparation of the drug solution to be tested

[0073] 1. Liquid preparation: 70% methanol, compound 5 extracted from Earthworm; 2. Dissolve compound 5 extracted from Earthworm in 70% methanol solution first; 3. Extract compound 5 from Earthworm in multiples The dilution method is prepared to the concentration of ①625ug / mL; ②256ug / mL; ③128ug / mL; ④78ug / mL; ⑤64ug / mL; ⑥32ug / mL; Finally, put it in the refrigerator and set aside;

[0074] Step 2: Preparation of standard bacterial solution

[0075] 1. Inoculate the selected strain of Pseudomonas aeruginosa into a test tube with liquid medium, put it into a shaker and shake for 5 hours; 2. Prepare the bacterial liquid in 1 to the original OD value corresponding to the strain, and now Ready to use.

[0076] Finally determine the OD of different strains corresponding to the optima...

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Abstract

The invention relates to a method for measuring the minimum bactericidal concentration based on trace amounts of natural compounds, comprising the following steps: 1. taking 70% methanol solution to dissolve the medicine of the natural compound to be tested, and preparing the medicine liquid to be tested with different solubility by a multiple dilution method; 2. , inoculate the selected bacterial species into a test tube containing a liquid medium, put it into a shaker for shaking, and then prepare to obtain the bacterial species at OD. 600 3. Determination of minimum inhibitory concentration (MIC); 4. Determination of minimum bactericidal concentration (MBC) Bacteria on the blank solid medium, placed in a constant temperature incubator for cultivation, and observed the culture results. After 24 hours, the minimum concentration of the drug solution without bacterial growth is the bactericidal concentration of the drug solution to be tested. The method of the invention can conveniently, quickly and accurately obtain the MBC value of a trace amount of antibacterial compounds.

Description

technical field [0001] The invention relates to the technical field of antibacterial compounds, in particular to a method for determining the minimum bactericidal concentration based on trace amounts of natural compounds. Background technique [0002] The minimum bactericidal concentration refers to the lowest drug concentration that can kill bacteria in the culture medium (that is, kill 99.9% of the tested microorganisms), usually expressed by MBC (Minimum bactericidal concentration) value. The determination method of MBC in microbiology is generally based on the minimum inhibitory concentration MIC value of the substance, and the minimum concentration of antibacterial drugs that can reduce the growth of viable bacteria by more than 99% is determined by the test tube dilution method. The specific method is: "dissolve the drug, and use sterilized water to make a 1000 μg / mL drug stock solution. On a 96-well cell culture plate, add 100 μL of 3% NaCl nutrient broth to each row ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/18C12R1/49C12R1/445C12R1/385C12R1/125C12R1/19C12R1/01
CPCC12Q1/18G01N2333/21G01N2333/245G01N2333/265G01N2333/31G01N2333/32G01N2333/36
Inventor 尹卫平刘华清吕碧玉白洁朱培文胡永鑫何纪虎朱丽妍苏会浩秦天
Owner HENAN UNIV OF SCI & TECH
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