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Micropore plate biological detection method for rapidly detecting bacteriostatics activity

A technology of antibacterial substances and biological detection, which is applied in the field of bioengineering, can solve the problems of time-consuming and long-term errors, and is not suitable for rapid detection of large-scale samples, and achieve the effects of reducing equipment costs, saving reagents, and overcoming cumbersome steps

Inactive Publication Date: 2009-04-22
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method needs to detect each sample separately, which takes a long time, and there are errors caused by different sample addition and detection times, and it is not suitable for rapid detection of large batches of samples.

Method used

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  • Micropore plate biological detection method for rapidly detecting bacteriostatics activity
  • Micropore plate biological detection method for rapidly detecting bacteriostatics activity
  • Micropore plate biological detection method for rapidly detecting bacteriostatics activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Rapid detection of polymyxin E, an antibacterial substance in Paenibacillus polymyxa 1.541 fermentation broth.

[0031] The detection method is:

[0032] Produce bacterium-polymyxa Paenibacillus (Paenibacilluspolymyxa) 1.541 slant (slant medium (g / L): glucose 1.0, (NH 4 ) 2 SO 4 1.0, sodium citrate 1.0, KH 2 PO 4 1.0, MgSO 4 0.125, agar 15) and inoculate a ring into 30mL seed medium (seed medium (g / L): glucose 25, beef extract 5, yeast extract 5, NaCl2.5, KH 2 PO 4 0.8,K 2 HPO 4 0.8, MgSO 4 0.125) in a 250mL Erlenmeyer flask, 200r / min, 30 ℃ shaker cultivation 24h, then inoculate the seed culture solution to 30mL fermentation medium (fermentation medium (g / L): soluble starch 50, Glucose 15, (NH 4 ) 2 SO 4 10. MgSO 4 0.25, KH 2 PO 4 0.8, NaCl 1.0, CaCO 3 10) in a 250mL Erlenmeyer flask, fermented at 30°C and 300r / min. Take respectively 10ml of the fermented liquid of culture 24h and 32h, adjust its pH to about 2.0 with 1M sulfuric acid, ce...

Embodiment 2

[0041] Example 2: Rapid detection of the bacteriostatic substance-bacitracin in the fermentation broth of Bacillus licheniformis 1.521.

[0042] Use an inoculation loop to inoculate a loop from the slant of bacitracin-producing bacteria Bacillus licheniformis 1.521 (slant medium (g / L): peptone 1, beef extract 1, NaCl 05, agar 2.2) to 30 mL of seed medium (seed Medium (g / L): soybean meal 2, starch 1, CaCO 3 0.5) in a 250mL Erlenmeyer flask, 240r / min, 37 ℃ shaker cultivation 16h, then inoculate the seed culture solution to 30mL fermentation medium (fermentation medium (g / L): soybean meal 10 , Starch 4, (NH 4 ) 2 SO 4 0.1, CaCO 3 0.6, MgSO 4 ·7H 2 O 0.002, MnSO 4 ·H 2 (0.001) in 250mL Erlenmeyer flask, 37 DEG C, carry out fermentation under the condition of 240r / min, take respectively 5mL of the fermented liquid of cultivating 32h, 40h and 48h, centrifuge 10min at 3000r / min, then use bacterial filter to filter and sterilize. The resulting supernatant diluted 20 times w...

Embodiment 3

[0048] Example 3: Rapid detection of natamycin, an antibacterial substance in the fermentation broth of Streptomyces gilvosporeus (Streptomyces gilvosporeus) ATCC 13326.

[0049] The detection method is:

[0050] Using an inoculation loop from the natamycin-producing bacteria—Streptomyces chrysosporium ATCC 13326 slant (slant medium (g / L): glucose 10, peptone 5, yeast powder 3, malt extract powder 3, agar powder 15, pH7 .0) Inoculate a ring from the top into a 250mL Erlenmeyer flask containing 30mL seed medium (seed medium (g / L): glucose 20, peptone 6, yeast powder 6, NaCl10, pH7.2), at 29°C, 200r / min shake culture 32h, make thalline be in rapid growth phase, then inoculate seed liquid with 10% inoculum amount in 50mL fermentation medium (fermentation medium g / L: peptone 19.5, yeast powder 4.5, glucose ( 50% glucose (sterilized separately) 40, pH 7.5) in a 500mL Erlenmeyer flask, fermented at 29°C and 200r / min, respectively took 1ml of the fermentation broth cultured for 40h,...

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Abstract

The invention relates to a micro plate biological detection method for rapid detecting bacteriostatic activity of antibacterial substance; in the method, a sample to be tested which has higher antibacterial properties on sensitive indicator bacteria is used; the micro plate with good standardization and parallelization is taken as a detection plate with high flux; after sensitive bacteria indicator solution and the sample to be tested are fully blended and are cultured for a proper time, the bacteriostatic activity of the sample is rapid detected by an ELIASA determining the change of the turbidity of the culture liquid in each hole of the micro plate. The detection method has simple operation, rapid analyzing speed, large primary treatment capacity, high reliability of the result, and overcomes the shortcoming that the existing biological detection method has complex steps and is often interfered by human factors. The detection method has similar analytical accuracy with high performance liquid chromatography but can better reflect the bacteriostatic activity of the sample in a visualized way, can be popularized to high flux screening work of producing bacteria of antibacterial substance.

Description

technical field [0001] The invention belongs to the breeding of bacterial strains producing antibacterial substances in the field of bioengineering, in particular to a microporous plate detection method for rapidly detecting the antibacterial activity of antibacterial substances. Background technique [0002] Antibacterial substances can be isolated from natural strains from the environment, including prokaryotes, eukaryotes, or genetically engineered bacteria produced by fermentation or directly extracted from natural products. At present, high performance liquid chromatography and biological detection methods are usually used to detect It detects. [0003] High-performance liquid chromatography is often used to accurately quantify the concentration of samples, but cannot directly obtain the minimum inhibitory concentration (MIC) that reflects the antibacterial activity of the sample. In addition, this method has relatively high requirements for equipment and consumables, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/17C12Q1/18
Inventor 骆健美王敏刘峰刘丹李建姝
Owner TIANJIN UNIV OF SCI & TECH
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