Growth effect and acute and chronic toxicity detection method of luminous bacteria

A technology for luminescent bacteria and toxicity detection. It is used in chemiluminescence/bioluminescence, color/spectral property measurement, and analysis by chemical reaction of materials. It can solve the problems of low test efficiency, single evaluation method, and limited number of samples. , to achieve the effect of saving manpower, simple process, comprehensive and accurate detection effect

Inactive Publication Date: 2019-11-12
NANJING COLLEGE OF INFORMATION TECH
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Problems solved by technology

[0004] However, the current detection methods for the toxicity of luminescent bacteria usually only have the acute (5-30 min) toxicological endpoint of luminescent toxicity detection, which can only evaluate the acute effect of the substance to be tested on the luminescence of luminescent bacteria, thus ignoring the delay caused by the substance on luminescent bacteria Toxic effects, or effects other than luminescence, such as growth
In addition, the existing bacterial toxicity detection method is to use a luminometer to test the luminous intensity, the evaluation method is single, and the number of samples tested each time is limited, and the test efficiency is low

Method used

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  • Growth effect and acute and chronic toxicity detection method of luminous bacteria
  • Growth effect and acute and chronic toxicity detection method of luminous bacteria
  • Growth effect and acute and chronic toxicity detection method of luminous bacteria

Examples

Experimental program
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Effect test

Embodiment 1

[0053] A method for detecting the growth effect and acute and chronic toxicity of luminescent bacteria, comprising the following steps:

[0054] Step 1. Collect the soil to be tested: select 4 different lakes, use a grab sampler to collect 0-10cm sediments on the surface, pass through a 1.8mm mesh sieve to remove impurities, air-dry and crush until the particle size is less than 2mm for later use, and number each sediment 1 , Precipitation 2, Precipitation 3 and Precipitation 4.

[0055] Step 2. Preparation of aqueous phase experimental samples: Mix 3 g of sediment with 30 mL of 3% (m / v) NaCl solution, shake in a shaker at 20°C for 12 hours, and filter with a 0.45 μm filter membrane to obtain the mother liquor of the aqueous phase extract , with 3% NaCl solution as the diluent, the prepared aqueous phase extract mother liquor was diluted according to the 1:2 serial gradient dilution method to obtain 100%, 50%, 25% and 12.5% ​​aqueous phase experimental samples.

[0056] Step ...

Embodiment 2

[0073] A method for detecting the growth effect and acute and chronic toxicity of luminescent bacteria, comprising the following steps:

[0074] Step 1. Prepare the organic phase experimental sample: use the soil to be tested in Step 1 of Example 1, mix 1 g of sediment with 30 mL of acetone and hexane mixed at a volume ratio of 1:1, and heat the mixture in a microwave digestion furnace at 115 ° C. After treatment for 20 minutes, the extract was passed through a 0.45 μm filter membrane, the filtrate was evaporated to dryness with a nitrogen blower, and the residue was dissolved in 4 mL of dimethyl sulfoxide (DMSO) to obtain an organic phase extract; before the experiment, the organic phase extract was dissolved in 3% NaCl solution to a final concentration of 1%, this solution is the mother liquor of the organic phase extraction solution; the 3% NaCl solution is used as the diluent, and the mother liquor of the organic phase extraction solution is diluted according to the 1:2 ser...

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Abstract

The invention discloses a growth effect and acute and chronic toxicity detection method of luminous bacteria. The method comprises the steps of: preparing an experiment sample, a negative control sample and a positive control sample; preparing a luminous bacteria solution; adding the luminous bacteria solution into a 96-pore cell culture board, then placing the 96-pore cell culture board on a microplate reader, and measuring an initial optical density value and an initial bioluminescence value 30 minutes later; adding the experiment sample, the negative control sample and the positive controlsample into the luminous bacteria solution, measuring the optical density value and the bioluminescence value every 30 minutes within the first 1 hour, and then measuring the optical density value and the bioluminescence value every 1h, and performing the test for not less than 24h; obtaining an optical density mutation time and a bioluminescence mutation time; and calculating an acute luminescence inhibition rate, a chronic luminescence inhibition rate and a growth inhibition rate. The growth effect and acute and chronic toxicity detection method disclosed by the invention can be used for simultaneously evaluating the growth effect, and acute and chronic toxic effects of a test substance on the luminous bacteria, the accuracy is high, high throughput of the toxicity test is achieved, andthe test efficiency is high.

Description

technical field [0001] The invention belongs to the technical field of environmental detection, and in particular relates to a method for detecting the growth effect and acute and chronic toxicity of luminescent bacteria. Background technique [0002] Luminescent bacteria are chemoautotrophic microorganisms that can spontaneously emit blue-green light during normal metabolism. The luminescence mainly depends on the bioluminescence enzyme system composed of NAD(P)H: FMN oxidoreductase and luciferase. The luminescence intensity It is closely related to intracellular metabolism. When the activity of luminescent bacteria is higher, the ATP content of cell metabolism is higher, and the luminescence is stronger. Conversely, if the cell activity of luminescent bacteria is inhibited by certain factors, the ATP content will respond and decrease accordingly, which will cause the luminescence of luminescent bacteria to become weaker or even stop luminescence. The traditional luminesce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/76G01N21/31G01N1/34G01N1/38
CPCG01N1/34G01N1/38G01N21/31G01N21/763
Inventor 袁楠楠鲍安平
Owner NANJING COLLEGE OF INFORMATION TECH
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