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Proto green algae micro quantitation method

A prochlorococcus, image technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, fluorescence/phosphorescence, etc., can solve the problems of unobservable fluorescence, high cost of Prochlorococcus, etc., and achieve easy operation. , the effect of the simple method

Inactive Publication Date: 2007-09-12
XIAMEN UNIV
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The purpose of the present invention is to overcome the rapid quenching of Prochlorococcus under common EM settings, the fluorescence is blocked by Synechococcus and cannot be observed under the initial field of view of cyanobacteria, and the limitations of Prochlorococcus counting using flow cytometry Insufficient such as the expensive expenses needed, provide a new method of Prochlorococcus counting, this method makes Prochlorococcus can be carried out in the ordinary laboratory with EM, easy to operate

Method used

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Examples

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Embodiment Construction

[0013] 1. Pre-filter the sampled sample with a 20 μm sieve to remove large organic organisms and inorganic particles, and then fix it with paraformaldehyde (PFA) with a final concentration of 2% for 15 minutes, and then use a final concentration of 5 μg / ml of DAPI was stained for 30 minutes, after which the cells were filtered onto a black polycarbonate (PC) membrane (Whatman) with a pore size of 0.2 μm, and 1 / 4 of the membrane sample was cut out, and the cells were prepared with the cell facing up.

[0014] 2. Use EM to observe the preparation, and the fluorescent excitation block used is as follows. DAPI-stained cells were used as controls, and the fluorescent excitation block used was: 330-390nm excitation, 440-490nm emission, 400-430nm spectroscopic (Zeiss Filter set02) (Kolber et al., 2001; Zhang and Jiao, 2004) ( DAPI-settings). The fluorescent excitation blocks used by cyanobacteria (mainly including Synechococcus and Prochlorococcus) cells are: BP 546±12nm excitation...

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Abstract

The micro- quantitative method of Prochlorococcus, which includes the steps: the sample is filtered in advance, fixed, double-chain DNA fluorescence dye, filtered again to make the sheet. Observe by the floor fluorescence microscope with red-sensitive electronic coupling package camera and mercury light. Observe and take picture for the blue bacterium and dyestuffs image in the same visual field by 2 groups of different fluorescence exciting block. Obtain the image on the time sequence of the blue bacterium and dyestuffs image after the microscope focusing. Distinguish and analyze the cells in the picture to get a new binary digital image. Calculate to obtain the abundance degree of Prochlorococcus.

Description

technical field [0001] The invention relates to a quantitative method for marine phytoplankton, in particular to a quantitative method for marine phytoplankton Prochlorococcus by adopting a falling fluorescence microscope method based on time series observation, automatic imaging and digital analysis. Background technique [0002] Prochlorococcus is the newly discovered prokaryotic autotrophic photosynthetic organism with the highest abundance in the ocean so far, which has great ecological, evolutionary and resource environmental significance. However, the cells of Prochlorococcus are extremely small, the fluorescence is very weak, and under the normal setting of epifluorescence microscope (EM) (for example, the excitation light is 450-480nm), the quenching is very fast, and it is almost impossible to observe, let alone accurately Quantification (Sieracki et al., 1995; Partensky et al., 1999; Schwalbach and Fuhrman, 2005). In addition, Synechococcus and Prochlorococcus are...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/04C12Q1/68G01N21/64
Inventor 焦念志张瑶陈瑶
Owner XIAMEN UNIV