阅读说明：本技术 一种基于多种微生物的急性毒性试验方法及其试剂盒 (Acute toxicity test method based on multiple microorganisms and kit thereof ) 是由 唐小标 于 2018-07-13 设计创作，主要内容包括：本发明公开了一种基于多种微生物的急性毒性试验方法,包括：从国家环境微生物专业菌种库中获取30-40个不同种属的菌株为材料,备用；然后根据每一颗菌株与有毒化合物反应的剂量效应关系、系统发育多样性以及对2,3,5-三苯基四氮唑的响应稳定性的原则筛选了10-15株细菌和1株酵母菌放入器皿中进行培养作为受试菌；然后利用脱氢酶活性法检测待测样品对上述细菌和酵母菌中培养获得集训的脱氢酶活性变化最终来评价待测样品的毒性大小。本发明选取最合适的菌种进行培养成受试菌,然后与多种微生物反应,最终快速的测试出微生物的毒性,相对灵敏度更高,效果更好,速度更快。本发明还公开了一种基于多种微生物的急性毒性试验的试剂盒。(The invention discloses an acute toxicity test method based on multiple microorganisms, which comprises the following steps: acquiring 30-40 strains of different species from a national environmental microorganism professional strain library as materials for later use; then 10-15 strains of bacteria and 1 strain of yeast are screened and put into a vessel to be cultured as test bacteria according to the dose-effect relationship of the reaction of each strain and the toxic compound, the phylogenetic diversity and the principle of response stability to 2, 3, 5-triphenyltetrazolium; and then detecting the activity change of the dehydrogenase cultured in the bacteria and the yeast by using a dehydrogenase activity method to obtain a trained dehydrogenase activity, and finally evaluating the toxicity of the sample to be detected. The invention selects the most suitable strains to culture into the tested strains, then reacts with a plurality of microorganisms, finally tests the toxicity of the microorganisms rapidly, and has higher relative sensitivity, better effect and higher speed. The invention also discloses a kit for acute toxicity test based on various microorganisms.)

1. An acute toxicity test method based on multiple microorganisms is characterized by comprising the following steps:

step 1, acquiring 30-40 bacterial strains of different species from a national environmental microorganism professional bacterial strain library as materials for later use;

step 2, screening 10-15 strains of bacteria and 1 strain of yeast to be placed into a vessel to be cultured as test bacteria according to the dose-effect relationship of the reaction of each strain and the toxic compound, the phylogenetic diversity and the principle of response stability to 2, 3, 5-triphenyltetrazolium;

and 3, detecting the activity change of the dehydrogenase cultured in the bacteria and the yeast by using a dehydrogenase activity method to obtain a trained dehydrogenase activity to finally evaluate the toxicity of the sample to be detected.

2. The acute toxicity test method based on multiple microorganisms according to claim 1, wherein the vessels for culturing the test bacteria in step 2 are 96-well plates which are distributed up and down to form a double layer, and the upper and lower layers of the 96-well plates are hinged at one end.

3. The acute toxicity test method based on multiple microorganisms according to claim 1 or 2, wherein in step 1, 35 strains of different species are obtained as materials from national environmental microorganism specialty strain banks; and (5) standby.

4. The method for acute toxicity test based on multiple microorganisms according to claim 1 or 2, wherein in step 2. Then screening 12 strains of bacteria and 1 strain of yeast to be put into a vessel to be cultured as test bacteria according to the dose-effect relationship of the reaction of each strain and the toxic compound, the phylogenetic diversity and the principle of response stability to the 2, 3, 5-triphenyltetrazolium; the selection mode of the 12 strains of bacteria and 1 strain of yeast is to select the dose-effect relationship difference of the reaction of each strain and the toxic compound to be arranged in sequence, the phylogenetic excellent condition to be arranged in sequence, the difference value of the response stability of the 2, 3, 5-triphenyl tetrazole to be arranged from large to small, and finally select the first 12 bacteria and 1 strain of yeast.

5. The utility model provides a kit based on acute toxicity test of multiple microorganism, includes the bacterial strain of examination, the culture solution of cultivateing the bacterial strain of examination, the household utensils and the 2, 3, 5-triphenyl tetrazole of cultivateing the bacterial strain of examination, its characterized in that, still include casing (1), display (2) and controller (3), be equipped with a plurality of detecting channel (4) and contrast passageway (5) in casing (1), all be equipped with one observation window (6) in each detecting channel (4) and contrast passageway (5), be equipped with photoelectric detection device (7) and detector (8) in each detecting channel (4), photoelectric detection device (7), detector (8) and display (2) all are connected with controller (3).

6. The acute toxicity test method and kit based on multiple microorganisms according to claim 5, wherein the vessel for culturing the test strains is a 96-well plate with two layers formed by distributing the upper and lower layers, and the upper and lower layers of the 96-well plate are hinged at one end.

Technical Field

The invention relates to the technical field of tests on acute toxicity and kits thereof, in particular to an acute toxicity test method based on multiple microorganisms and a kit thereof.

Background

Microorganisms include bacteria, viruses, fungi, and some small protists, microscopic algae, etc., which are small and closely related to humans. The food can be widely applied to various fields such as food, medicine, industry and agriculture, environmental protection and the like. In textbooks in the mainland area of china, microorganisms are classified into the following 8 major groups: bacteria, viruses, fungi, actinomycetes, rickettsia, mycoplasma, chlamydia, spirochete. Microbial assays have unparalleled advantages over other biological assay methods in toxicity testing. The development time of the microorganism is short, and the test time can be greatly shortened; the operation capacity is small, the tested organism quantity is large, and the contamination effect of the whole population can be reflected, but not the individual effect; the microorganism adaptability is strong, and the method can be used for measuring various environmental samples with different sensitivity requirements; no ethical dispute exists. The current microbiological detection methods for water quality detection mainly include the following methods: bioluminescence, growth inhibition, respirometry, enzymatic activity, and nitrification rate. However, the current method can only detect a single microorganism, which results in low detection efficiency and long detection time.

Disclosure of Invention

The present invention is directed to solving the above-mentioned problems of the prior art and providing a method and a kit for acute toxicity test based on various microorganisms.

In order to achieve the above purpose, the present invention provides a method for acute toxicity test based on multiple microorganisms, which comprises the following steps:

step 1, acquiring 30-40 bacterial strains of different species from a national environmental microorganism professional bacterial strain library as materials for later use;

step 2, screening 10-15 strains of bacteria and 1 strain of yeast to be placed into a vessel to be cultured as test bacteria according to the dose-effect relationship of the reaction of each strain and the toxic compound, the phylogenetic diversity and the principle of response stability to 2, 3, 5-triphenyltetrazolium;

and 3, detecting the activity change of the dehydrogenase cultured in the bacteria and the yeast by using a dehydrogenase activity method to obtain a trained dehydrogenase activity to finally evaluate the toxicity of the sample to be detected.

Further, the vessels used for culturing the test bacteria in the step 2 are 96-well plates which are distributed up and down to form a double layer, and the upper layer and the lower layer of the 96-well plate are both in a form that one end is hinged.

Further, in step 1, 35 strains of different species are obtained from a national environmental microorganism professional strain library as materials; and (5) standby.

Further, in step 2. Then screening 12 strains of bacteria and 1 strain of yeast to be put into a vessel to be cultured as test bacteria according to the dose-effect relationship of the reaction of each strain and the toxic compound, the phylogenetic diversity and the principle of response stability to the 2, 3, 5-triphenyltetrazolium; the selection mode of the 12 strains of bacteria and 1 strain of yeast is to select the dose-effect relationship difference of the reaction of each strain and the toxic compound to be arranged in sequence, the phylogenetic excellent condition to be arranged in sequence, the difference value of the response stability of the 2, 3, 5-triphenyl tetrazole to be arranged from large to small, and finally select the first 12 bacteria and 1 strain of yeast.

The invention also discloses a kit for acute toxicity tests based on multiple microorganisms, which comprises a test strain, a culture solution for culturing the test strain, a vessel for culturing the test strain and 2, 3, 5-triphenyltetrazolium, and is characterized by further comprising a shell, a display and a controller, wherein a plurality of detection channels and comparison channels are arranged in the shell, an observation window is arranged in each detection channel and each comparison channel, a photoelectric detection device and a detector are arranged in each detection channel, and the photoelectric detection device, the detector and the display are connected with the controller.

Further, the utensil for culturing the tested strains is a 96-pore plate which is distributed up and down to form a double layer, and the upper layer and the lower layer of the 96-pore plate are both in a form that one end is hinged.

According to the acute toxicity test method based on multiple microorganisms and the kit thereof, the most appropriate strains are selected for culturing into tested bacteria, then the tested bacteria react with the multiple microorganisms, finally, the toxicity of the microorganisms is rapidly tested, and the method has the advantages of higher relative sensitivity, better effect and higher speed.

Description of the drawings:

FIG. 1 is a schematic diagram showing the structure of a kit for acute toxicity test based on various microorganisms in example 1.

In the reference symbols: 1. a housing; 2. a display; 3. a controller; 4. a detection channel; 5. a control channel; 6. an observation window; 7. a photoelectric detection device; 8. and a detector.

Detailed Description

The invention will be further described with reference to the following examples.