Rapid detection method of Thraustochytrium based on flow cytometry detection technology
A thraustochytrium and flow cytometry technology, which is applied in measuring devices, particle and sedimentation analysis, individual particle analysis, etc., can solve the problems of low efficiency and poor detection accuracy of Thraustochytrium, so as to improve efficiency and reduce errors , time-saving effect
Active Publication Date: 2019-04-19
TIANJIN UNIV +1
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[0006] In order to solve the problems existing in the prior art, the present invention provides a rapid assay method for Thraustochytrium based on flow cytometry technology, which overcomes the problems of poor accuracy and low efficiency in the prior art.
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[0027] The present invention will be described in detail below in conjunction with the accompanying drawings.
[0028] 1. Rapid determination of Thraustochytrium in cultured samples
[0029] The invention provides key technical support for the thraustochytrium high-density fermentation test and the real-time monitoring of the thraustochytrium quantity in the production process.
[0030] The following are the best implementation steps for using the present invention to quickly determine the number of Thraustochytrium in high-density fermentation (artificial culture environment).
[0031] (1) Sampling and pretreatment
[0032] Take an appropriate volume of shaken bacteria solution, dilute it 1000 times with 0.22μm filtered TE buffer, take 1ml and add it to a sterile flow cytometry sample tube, add 10μl of 0.22μm filtered acridine yellow solution (0.02mg / ml), vortexed briefly, and then stained at room temperature for 30 min in the dark. At the same time, the 0.22 μm filtered ...
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The invention discloses a quick thraustochytrids determination method based on a flow cytometry technology. The method comprises the following steps of 1 sampling and sample pretreating, wherein a sample is diluted or immobilized with degerming liquid, the sample to be tested is dyed with an acridine yellow solution in a light shading mode, and meanwhile fluorescent microsphere suspension liquid is prepared to serve as an internal reference; 2 flow cytometry conducting, wherein the sample is tested with a flow cytometer which is provided with a 488-nm laser; 3 internal reference fluorescent microsphere counting, wherein the fluorescent microsphere suspension liquid is diluted with pure water, subjected to vortex oscillation, mixed to be uniform and then filtered to a black polycarbonate film to make a slide, and absolute counting is conducted on internal reference fluorescent microspheres through a fluorescent microscope; 4 thraustochytrids abundance calculating, wherein flow data which mainly comprises ring doors and counting of thraustochytrids is analyzed through FlowJo software or CellQuest software. According to the method, the high automation and process degree is achieved, errors brought by manual operation can be reduced, and therefore the higher precision and stability are achieved.
Description
technical field [0001] The invention belongs to the technical field of development, utilization and industrialization of marine biological resources and the technical field of marine environmental element monitoring, and particularly relates to a rapid quantitative detection technology for a class of important marine microorganisms (thraustochytrium) in natural or artificial environments. Background technique [0002] Thraustochytrids are unicellular heterotrophic fungal protists. Its main cell feature is that it has an ectoplasmic reticulum, and the cell wall contains proteins and sulfated polysaccharides, and the cell size is about 2-20 μm. This is an important class of marine microorganisms, which are widely distributed in marine and estuary habitats, and have a significant regulatory effect on marine ecology. In addition, Thraustochytrium is also an important source of active substances such as polyunsaturated fatty acids, polysaccharides, carotenoids, and various extra...
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IPC IPC(8): G01N15/14
CPCG01N15/14G01N2015/149
Inventor 汪光义谢宁栋段瑛博马文猛陈子熙
Owner TIANJIN UNIV