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Fluorescence microscope viewing and counting method for bacteria in viable but non-culturable state

A fluorescence microscope and counting method technology, applied in biochemical equipment and methods, preparation of test samples, enzymology/microbiology devices, etc. The problem of narrow scope, etc., achieves the effect of good fluorescence observation effect, shortened observation period, and convenient material acquisition.

Inactive Publication Date: 2009-01-14
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(2) The scope of application is narrow, only applicable to Gram-negative bacteria, but not applicable to Gram-positive bacteria
(3) Can not truly reflect the number of live bacteria
After acridine orange staining, it is difficult to effectively determine whether it is a live or dead bacteria that shows orange-yellow or orange-red fluorescence
(4) There are many types of materials used, and it is inconvenient to obtain materials
For example, Iraqi black is a black dye used in the printing and dyeing industry, but it is rarely sold in major biochemical and biological companies in China

Method used

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  • Fluorescence microscope viewing and counting method for bacteria in viable but non-culturable state
  • Fluorescence microscope viewing and counting method for bacteria in viable but non-culturable state
  • Fluorescence microscope viewing and counting method for bacteria in viable but non-culturable state

Examples

Experimental program
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Effect test

Embodiment 1

[0021] 1. First, pre-treat the VBNC bacterial observation sample, and the concentration of the bacterial solution before VBNC bacterial induction is 10 6 cells / mL, take the number of cultivable bacteria as 1×10 6 Put 1mL of VBNC bacteria solution per mL into a sterile centrifuge tube, centrifuge at 8000r / min for 2min, discard the supernatant; then wash the bacteria with sterile normal saline and precipitate twice, each time at 8000r / min, centrifuge After 2 minutes, the supernatant was discarded; when washing the bacteria, the bacterial culture solution should be removed to prevent the culture solution from producing self-luminescence and interfering with the observation results; finally, the washed precipitate should be placed at room temperature for immediate observation;

[0022] 2. Next, check and treat the hydrophobicity of the microporous membrane. First, completely immerse the microporous membrane with a diameter of 25mm and a diameter of 0.2um in sterile saline for 2 mi...

Embodiment 2

[0030] 1. First, pre-treat the VBNC bacterial observation sample, and the concentration of the bacterial solution before VBNC bacterial induction is 10 5 cells / mL, take the number of cultivable bacteria as 1×10 5 Put 1mL of VBNC bacteria solution per mL into a sterile centrifuge tube, centrifuge at 8000r / min for 2min, discard the supernatant; then wash the bacteria with sterile normal saline and precipitate twice, each time at 8000r / min, centrifuge After 2 minutes, discard the supernatant; store the cells at 4°C and use them within 2 days;

[0031] 2. Next, check and treat the hydrophobicity of the microporous membrane. First, completely immerse the microporous membrane with a diameter of 25mm and a diameter of 0.2um in sterile saline for 2 minutes. Facing upward, carefully observe the surface of the microporous filter membrane. If there is no water mark on the surface of the microporous filter membrane, it means that the filter membrane is hydrophobic and should be replaced;...

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Abstract

The invention relates to an observing and counting method of germs in a viable but non-culture state with a fluorescent microscope, which is characterized in that the method comprises the following concrete steps: (1) an observing sample of the VBNC germs is preprocessed; (2) the hydrophobic nature of a microporous filtering film is examined and processed; (3) the microporous filtering film is dyed; (4) the sample of the VBNC germs is fluorescently dyed; (5) a fluorescent sample of the VBNC germs is prepared; and (6) the counting of the VBNC germs is implemented with the fluorescent microscope. The observing and counting method of the germs in the viable but non-culture state with the fluorescent microscope simplifies operation processes, overcomes the disadvantages of narrow application range, difficult material preparation and inaccurate observing result in the prior art, solves the technical difficulty in the research field of the VBNC germs, and raises the practical operation level of practitioners.

Description

Technical field: [0001] The invention relates to a fluorescent microscope observation and counting method for live bacteria in a non-culturable state, belonging to the field of biotechnology. Background technique: [0002] Fluorescence microscope observation and counting are the most basic technical means used in the study of bacterial viable non-culturable state (Viable but Non-culturable, VBNC). At present, the existing technology for VBNC bacterial fluorescence observation and counting at home and abroad is mainly derived from an article entitled "Using Nuclear Pore Filters for Bacterial Fluorescence Counting" published in "Applied and Environmental Microbiology" in 1977 by Hobbie et al. " article. One of the highlights of this technology is the use of polycarbonate nuclear pore filter membrane instead of cellulose microporous filter membrane for the determination of total bacterial count. At the same time, acridine orange and nalidixic acid were used to stain the bacte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N15/14G01N21/76G01N1/30C12M1/34
Inventor 李影钱爱东段锐王伟利孙晓媛候凤
Owner JILIN AGRICULTURAL UNIV
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