A trace counting method for detecting exogenous microorganisms in soil
A technology for the number and counting method of microorganisms, which is applied in the field of tracer counting for detecting the number of exogenous microorganisms in soil, can solve the problem of inability to count and quantify exogenous microorganisms, and achieves the effects of convenient and rapid operation, good stability and high sensitivity
Active Publication Date: 2019-03-05
NANKAI UNIV
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However, these techniques are aimed at staining and counting all bacteria in water, soil, and sediment, and cannot count and quantify a certain exogenous microorganism added.
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[0027] A trace counting method for detecting the number of exogenous microorganisms in soil, using Bacillus subtilis as the starting strain, and obtaining the mutagenic strain B38 with high tolerance and adsorption capacity to heavy metal cadmium through ultraviolet mutagenesis for 5 minutes, the steps are as follows:
[0028] 1. Preparation of reagents
[0029] 1) Preparation of physiological saline: Dissolve sodium chloride in distilled water, the dosage ratio of sodium chloride to distilled water is 0.85g:100mL, and the prepared 0.85wt% normal saline is autoclaved at 121°C for 30 minutes before use;
[0030] 2) Preparation of 4’,6’-diamidino-2-phenylindole (DAPI) stock solution: DAPI powder was prepared into a 1.0 mg / mL stock solution with double distilled water, and stored at -20°C for later use;
[0031] 3) Preparation of 4',6'-diamidino-2-phenylindole (DAPI) working solution: DAPI stock solution was diluted to 0.1 μg / mL with phosphate buffer solution of pH=7.4 to prepare...
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The invention relates to a tracing and counting method for detecting quantity of exogenous microorganisms in soil. The tracing and counting method comprises the following steps: 1) preparing dye stock solution and working solution; 2) activating and culturing bacteria, centrifuging to obtain target bacteria, fixing cells, adding dye working solution, and dyeing in the dark place, so that dyed bacteria are prepared; 3) adding the dyed bacteria into soil to be cultured, collecting a soil sample after culture, carrying out oscillating extraction to obtain the target bacteria, diluting, then filtering with a black nuclear pore filter membrane, then placing on a glass slide, and sealing the glass slide; and 4) observing and counting under ultraviolet light by adopting a fluorescent microscope, and converting the quantity obtained by counting into the quantity of microorganism cells contained in per kg of dry soil. The tracing and counting method provided by the invention has the advantages that a fluorescent dyeing counting technology is combined with a fluorescent labelling technology, and growth and propagation conditions of the exogenous microorganisms added into the soil are traced and quantified; and the used fluorescent dye is convenient and fast to operate, extremely high in sensitivity, good in stability and extremely low in usage amount, so that a practical and reliable soil exogenous microorganism tracing and counting method is provided.
Description
technical field [0001] The invention belongs to the field of environmental microbiology, in particular to a trace counting method for detecting the quantity of soil exogenous microorganisms. Background technique [0002] Soil is the foundation of life, and agriculture is the material basis for human survival. However, at present, the problem of soil pollution in my country and the world has become quite serious. Bioaugmentation technology has become an effective method for in-situ repair of polluted soil. means. Bioaugmentation technology is to apply exogenous microorganisms that have a certain ability to treat target pollutants to the target soil, and use their metabolic reactions to repair them. Whether exogenous microorganisms can grow and reproduce normally is the key to the success or failure of microbial enhancement technology. However, at present, whether the added exogenous microorganisms can survive for a long time and maintain their reproductive ability in the tre...
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IPC IPC(8): C12Q1/06C12R1/125
CPCC12Q1/06
Inventor 王婷孙红文王恺毛洪钧
Owner NANKAI UNIV