CN106350570B

CN106350570B - Real-time observation method for single cell of spore-forming bacteria

Info

Publication number: CN106350570B
Application number: CN201610956670.XA
Authority: CN
Inventors: 朱春节; 孙国萍; 许玫英; 郭俊
Original assignee: Guangdong Institute of Microbiology
Current assignee: Guangdong Detection Center of Microbiology of Guangdong Institute of Microbiology
Priority date: 2016-10-27
Filing date: 2016-10-27
Publication date: 2020-04-10
Anticipated expiration: 2036-10-27
Also published as: CN106350570A

Abstract

The invention discloses a real-time observation method for single cells of spore-forming bacteria. Inoculating spore-producing bacteria into a culture medium to be cultured until spores are formed, then killing all vegetative cells to obtain the spores, then inoculating the spores into a semisolid culture medium, then transferring the spores into a culture dish special for a laser confocal microscope together, and observing single cells of the spore-producing bacteria in real time by using the laser confocal microscope. The invention establishes a method for real-time on-line observation of the growth cycle of a single cell of a spore-forming bacterium by collecting pure spores, optimizing spore germination conditions, optimizing inoculum size and controlling agar concentration of a culture medium. The real-time observation method of the invention does not need repeated sampling, ensures the accuracy of the observation data and has simple operation. Can realize the recording of the growth cycle of a single cell of the spore-forming bacteria and provide a new way for the on-line research of the physiological characteristics of synchronously growing cells.

Description

Real-time observation method for single cell of spore-forming bacteria

The technical field is as follows:

the invention belongs to the technical field of microorganisms, and particularly relates to a real-time observation method for single cells of spore-forming bacteria.

Background art:

spore bacteria widely exist in the environment such as atmosphere, water body, soil, animal and plant body, etc. Spore bacteria are easy to culture and have multiple functions, so that the spore bacteria are one of the most common model organisms in scientific research. The development of bacteriology is promoted by the research of biological attributes, spore formation and germination and genetic differentiation mode systems. Meanwhile, the characteristics of spore formation, antibiotic production, flocculant production, protease and amylase production and the like make the bacillus subtilis have special importance in the fields of biological pesticides, medicine development, pollutant degradation, food processing and the like. Developing a suitable research method is beneficial to strengthening the basic research of the spore bacteria and further accelerating the application of the spore bacteria.

Within a bacterial population, individuals may be at different stages of growth, leading to inconsistent growth, division, and even metabolic activity. In order to study biochemical and cytological changes of a certain cell or a certain growth period of a spore bacterium, it is necessary to establish a synchronized culture technique to keep the asynchronous cells in the population in the same growth state as much as possible.

Spores can germinate and reenter the cell growth cycle under appropriate conditions, and the method has the inherent excellent condition for realizing synchronous culture. However, there is currently no effective method for obtaining a suitable amount of pure spores, inducing spore germination, and enabling real-time online observation of changes in growth or phenotypic characteristics of individual cells.

Laser confocal microscopy (LSM) uses laser as a light source, employs a conjugate focusing principle and apparatus, and utilizes a computer to perform digital image processing, analysis and output on an observed object. Compared with a common microscope, the microscope has the advantages that the tomography and imaging can be carried out on the sample, and the signal interference of a non-focusing plane is avoided. In addition, due to the continuous photographing function, the LSM can realize real-time online observation of cell properties, and avoid the influence of a fussy sampling process on cells. Although currently, LSM is widely used in the fields of cell and molecular biology, tumor drug screening, and brain and neuroscience, it is rarely used in bacterial research.

The invention content is as follows:

the invention aims to provide a real-time observation method for single cells of spore-forming bacteria, which can observe physiological characteristic changes in the growth cycle of the single cells from spores.

A real-time observation method for single cells of spore-forming bacteria is characterized by comprising the following steps:

inoculating spore-producing bacteria into a culture medium to be cultured until spores are formed, killing all vegetative cells to obtain the spores, inoculating the spores into a semi-solid culture medium, transferring the spores into a culture dish special for a laser confocal microscope, and observing single cells of the spore-producing bacteria in real time by using the laser confocal microscope.

Preferably, the real-time observation method of the single cell of the spore-forming bacteria comprises the following specific steps: inoculating spore-producing bacteria on an LB flat plate, culturing at 28-30 ℃ for 7-10 days, then placing the LB flat plate at 80-105 ℃ and drying until no live vegetative cells are seen in microscopic examination, taking a culture medium containing lawn from a dried flat plate and adding the culture medium into an LB liquid culture medium, scattering the culture medium containing the lawn, thermally shocking at 30 ℃ for 30min, immediately carrying out ice bath for 15min after thermal shock, then inoculating the culture medium into a semisolid culture medium according to the inoculation amount of 1-2.5% of the volume fraction, transferring the semisolid culture medium into a special culture dish of a laser confocal microscope, and observing single cells of the spore-producing bacteria in real time by the laser confocal microscope at 30 ℃.

Preferably, the semi-solid medium is an LB medium containing 0.5 mass percent of low-melting-point agarose.

Selecting a semi-solid culture medium, and culturing in a culture dish which is not completely anaerobic so as to realize the real-time online observation of the growth and the propagation of the single cells under the conditions of not influencing the growth of the bacteria and reducing the motility of the bacteria.

Preferably, the spore-forming bacteria are Bacillus subtilis or Bacillus proteus (Lysinibacillus varians).

The invention establishes a method for on-line observation of the growth cycle of a single cell of a spore-forming bacterium by collecting pure spores, optimizing spore germination conditions, optimizing inoculum size and controlling agar concentration of a culture medium. The real-time observation method of the invention does not need repeated sampling, ensures the accuracy of the observation data and has simple operation. Can realize the recording of the growth cycle of a single cell of the spore-forming bacteria and provide a new way for the on-line research of the physiological characteristics of synchronously growing cells.

Description of the drawings:

FIG. 1 is a confocal microscope photograph showing the complete cell cycle of a single cell of Lysinibacillus varians, (a) to (f) are photographs taken of the succession of spores germinating at the initial stage of cultivation to spores again growing again, and the scale in the figure is 5 μm;

FIG. 2 is a confocal microscope photograph showing the complete cell cycle of a single cell of Bacillus subtilis, and (a) to (f) are photographs taken continuously from germination of spores at the initial stage of culture to reproduction of spores again, with a scale of 5 μm.

The specific implementation mode is as follows:

the following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1: bacillus proteus (Lysinibacillus varians) GY32 cell cycle observations

Coating the GY32 of Bacillus proteus (Lysinibacillus varians) on LB plate, culturing at 30 ℃ for 7d, then baking the plate at 105 ℃ for 60min, and microscopic examining the yield of spores. No motile bacteria are seen in the bacterial colony subjected to microscopic examination at the high temperature of 105 ℃, and only incomplete bacteria and spores with bacillus outlines are seen. Indicating that the high temperature treatment can kill all vegetative cells. Taking the culture medium containing the lawn from the dried plate, adding the culture medium containing the lawn into 1mL of LB liquid culture medium, scattering the lawn, thermally shocking at 30 ℃ for 30min, and immediately carrying out ice bath for 15 min. And (3) adding 20 mu L of the bacterium solution subjected to ice bath into 2mL of LB semisolid culture medium containing 0.5% mass fraction of low-melting-point agarose, uniformly mixing, finally adding 200 mu L of the bacterium solution to a special culture dish of a laser confocal microscope, and carrying out real-time online observation by using the laser confocal microscope at the temperature of 30 ℃. Microscopic examination shows that the real-time observation method can record the unique cell morphological change cycle of the spore formed again finally after the single spore of the Bacillus proteus GY32 germinates from the spore to the filament cell and further the rod cell is split (figure 1). The method can realize the observation of the process from the germination of single spores to the re-sporulation, thereby recording the complete cell cycle of single cells.

Example 2: bacillus subtilis 168 cell cycle observation

Coating Bacillus subtilis 168 on LB plate, culturing at 28 deg.C for 10 days, baking at 80 deg.C for 60min, and examining spore yield. No motile bacteria are seen in the bacterial colony microscopic examination baked at 80 ℃, and only incomplete bacteria and spores with bacillus outlines are seen. Indicating that the high temperature treatment can kill all vegetative cells. Taking the culture medium containing the lawn from the dried plate, adding the culture medium containing the lawn into 1mL of LB liquid culture medium, scattering the lawn, thermally shocking at 30 ℃ for 30min, and immediately carrying out ice bath for 15 min. Adding 50 mu L of the bacterium solution subjected to ice bath into 2mL of LB semisolid culture medium containing 0.5 mass percent of low-melting-point agarose, uniformly mixing, finally adding 200 mu L of the bacterium solution into a special culture dish of a laser confocal microscope, and carrying out real-time online observation by using the laser confocal microscope at the temperature of 30 ℃. Microscopic examination shows that the real-time observation method can record the typical characteristics that most common bacillus germinates from the spores to rod-shaped cells and propagates in large quantities in real time and forms the spores again (figure 2). The method can realize the observation of the process from the germination of single spores to the re-sporulation, thereby recording the complete cell cycle of single cells.

Claims

1. A real-time observation method for single cells of spore-forming bacteria is characterized by comprising the following steps: inoculating spore-producing bacteria into a culture medium to be cultured until spores are formed, killing all vegetative cells to obtain the spores, thermally stimulating at 30 ℃ for 30min, immediately carrying out ice bath for 15min after thermal stimulation, inoculating the spores into a semi-solid culture medium, transferring the spores into a special culture dish of a laser confocal microscope, and observing single cells of the spore-producing bacteria in real time by using the laser confocal microscope; the semi-solid culture medium is an LB culture medium containing 0.5 mass percent of low-melting-point agarose.

2. The real-time observation method of claim 1, wherein the real-time observation method of the single cell of the spore-forming bacteria comprises the following specific steps: inoculating spore-producing bacteria on an LB flat plate, culturing at 28-30 ℃ for 7-10 days, then placing the LB flat plate at 80-105 ℃ and drying until no live vegetative cells are seen in microscopic examination, taking a culture medium containing lawn from a dried flat plate and adding the culture medium into an LB liquid culture medium, scattering the culture medium containing the lawn, thermally shocking at 30 ℃ for 30min, immediately carrying out ice bath for 15min after thermal shock, then inoculating the culture medium into a semisolid culture medium according to the inoculation amount of 1-2.5% of the volume fraction, transferring the semisolid culture medium into a special culture dish of a laser confocal microscope, and observing single cells of the spore-producing bacteria in real time by the laser confocal microscope at 30 ℃.

3. The real-time observation method according to claim 1 or 2, wherein the spore-forming bacteria are bacillus subtilis or bacillus proteus (Lysinibacillus varians).