JP5935766B2 - Microbial sample observation method - Google Patents

Description

  The present invention relates to a method for observing a microorganism sample.

Microbial observation is performed using a scanning electron microscope (hereinafter referred to as SEM) in order to clarify the surface morphology of microorganisms, the mode of division, the manner of growth, the manner of attachment to the surface of an object, and the type of assembly. Microbial observation using such a scanning electron microscope (hereinafter referred to as SEM) is an indispensable technique for biological research and development. Usually, in SEM observation, a microorganism sample is placed in a vacuum. Therefore, when observing a biological sample such as a microorganism, the microorganism sample is fixed, dehydrated, etc. in advance so that the structure of the microorganism sample does not change even in the vacuum. It needs to be processed. There are many standard microbial sample processes for SEM observation, such as fixation, dehydration, replacement, lyophilization, staining, metal coating, etc., depending on the characteristics of the microbial sample, and all processes are completed. It takes a minimum of 2-3 days to do this. As a method for fixing, dehydrating, replacing, and lyophilizing a microorganism sample, a method of sequentially performing the following 1 to 6 steps is known (Non-Patent Documents 1 to 3).
1) Pre-fixation step: The microorganism sample is immersed in a glutaraldehyde solution (2.5 mass% to 5 mass%) for several hours to 24 hours.
2) Washing step: The microorganism sample is washed several times with a phosphate buffer.
3) Post-fixation step: The microorganism sample is immersed in an osmium tetroxide solution (1-2% by mass) for several hours.
4) Washing step: The microorganism sample is washed several times with a phosphate buffer.
5) Dehydration step: The microorganism sample is immersed in 50, 70, 85, and 95% by mass of ethanol for 15 minutes each, and further immersed in absolute ethanol for 15 minutes three times.
6) Substitution process, drying process: After immersing the microorganism sample in isoamyl acetate for 15 minutes, critical point drying is performed.

  In the above pre-fixing step, when glutaraldehyde is used as a fixing solution, it is known that the best observation results can be obtained by changing the pH to alkaline during the fixing operation while maintaining high dissolved oxygen. Yes. However, since live microorganisms that have just been collected breathe, the dissolved oxygen and pH of the fixative may be lowered during fixation, and the microorganism sample may not be fixed well. Therefore, in general, it is necessary to take measures such as increasing the amount of the fixing solution in which the microorganism sample is immersed, replacing the sample with a fresh fixing solution in the middle of fixing, or aeration (Non-Patent Document 4).

  In addition, when observing plant bacteria, plaque, and environmental microorganism aggregates (biofilms), the structures and positional relationships of living organisms other than microorganisms and aggregates of microorganisms with different structures and components are observed. Since it is necessary to fix at a time while maintaining the above, skilled techniques and more complicated processes are required (Non-Patent Documents 1 to 3).

  Thus, the preparation of a sample for SEM observation of microorganisms is complicated and takes time. Observing skill is necessary for observation. In addition, since a large amount of a protein denaturant, such as a fixative or a volatile solvent, is used, sufficient attention must be paid to the safety of workers and consideration of the environment of waste.

Scanning electron microscope for medical biology, supervision: Shichirou Miyazawa Medical Publishing Center Electron Microscope Sample Preparation, Kansai Electron Microscope Application Technology Study Group KINPODO Scanning electron microscope, Japan Electron Microscope Society Kanto branch Abstracts of Medical Biological Electron Microscopy, 22nd Annual Conference and General Meeting

  This invention is made | formed in view of this situation, Comprising: It aims at providing the method of observing a microorganism sample easily, without performing a complicated pretreatment.

  The inventors of the present invention have considered that the necessity of complicated sample processing when observing a microbial sample by ordinary SEM observation is due to the fact that the microbial sample must be placed in a vacuum. Based on the above, paying attention to an environment-controlled scanning electron microscope (hereinafter referred to as ESEM) that enables observation of a microbial sample in an atmosphere, and pre-treating a microbial sample if the ESEM is used under specific conditions I thought that it would be possible to observe without any change. Specifically, if a microbial sample can be observed under saturated water vapor pressure, it is considered that the microbial sample can be observed as it is without pretreatment without drying. However, when observing a microorganism sample, it is necessary to observe a structure of the order of μm or less. In order to observe with the spatial resolution for observing such a level structure using ESEM, there are the following problems. In the ESEM, since an electron beam passes through the atmosphere, a low-energy electron beam scatters electrons due to collision with the atmospheric gas, and a good image cannot be obtained. Therefore, in order to suppress scattering of electrons due to the atmosphere, ESEM generally uses a high acceleration voltage of 15 kV or higher. If a high energy electron beam generated at a high acceleration voltage is used, scattering of electrons in the atmosphere can be suppressed. On the other hand, the diffusion region of the electron beam in the microorganism sample becomes large, and sufficient spatial resolution cannot be obtained. Since microorganisms are mainly composed of light elements, the diffusion region is large when a high-energy beam is used for microbial sample observation, and the spatial resolution is significantly degraded.

  Therefore, intensive studies were conducted to solve the above problems. As a result, the Schottky electron gun has a high brightness and a beam having a small probe diameter, and the acceleration is reduced to suppress the diffusion of electrons in the microorganism sample, and the working distance is shortened. Then, it was found that the microstructure of the microbial sample can be observed by ESEM under saturated vapor pressure without drying the microbial sample by suppressing the scattering of electrons due to the atmosphere.

The present invention has been made based on such findings, and the gist thereof is as follows.
[1] Using an environmentally controlled scanning electron microscope with a Schottky electron gun, under the conditions of an acceleration voltage of 2 kV to 5 kV and a working distance of 5 mm to 8 mm under saturated water vapor pressure in a temperature range of 0 ° C to 5 ° C. A method for observing a microorganism sample, comprising observing the microorganism sample.

  According to the present invention, it is possible to observe a microbial sample without complicated pretreatment.

It is the figure which observed the microorganism sample.

In the present invention, ESEM is used to observe a microorganism sample under saturated vapor pressure. And when observing a microorganism sample using ESEM, ESEM shall have a Schottky type electron gun. Furthermore, the observation conditions are a saturated water vapor pressure, a temperature range of 0 ° C. to 5 ° C., an acceleration voltage of 2 kV to 5 kV, and a working distance of 5 mm to 8 mm. Details will be described below.
In the present invention, a Schottky electron gun is used. By using a high-luminance Schottky electron gun and a beam with a small probe diameter, a small-diameter beam can be obtained while maintaining a sufficient electron beam luminance, and the structure is finer than the μm order or μm order. Can be observed.

  By observing under saturated water vapor pressure, microorganisms can be prevented from drying out.

  As described above, when observing a microbial sample with an ESEM, there are two problems: scattering of electrons due to the atmosphere and a large diffusion area of the beam in the microbial sample, making it impossible to obtain sufficient spatial resolution. There is. In response to this problem, in the present invention, first, the acceleration voltage is set to 5 kV or less to suppress the diffusion of the electron beam in the microorganism sample. Lowering the acceleration voltage may cause scattering of the electron beam by the atmosphere, so the lower limit is 2 kV.

  When the acceleration voltage is lowered, scattering of the electron beam by the atmosphere becomes a problem. Therefore, it was considered to suppress scattering of the electron beam by the atmosphere by shortening the path of the electron beam in the atmosphere by bringing the microorganism sample close to the objective lens.

  Here, in the present invention, the observation temperature is also important because it is premised on observation under saturated water vapor pressure. Therefore, the acceleration voltage was set to 3 kV, and the relationship between the temperature, the working distance, and the electron beam scattering suppression, that is, the image was investigated. The obtained results are shown in Table 1. In Table 1, “◯ Δ ×”, which is the evaluation result of the image, is the same as the evaluation criteria of the examples described later.

From Table 1, it can be seen that a good image can be obtained when the working distance is 5 mm or more and 8 mm or less in the temperature range of 0 ° C to 5 ° C. When the working distance is too short, the detector and the sample come into contact with each other and an image cannot be obtained. If the working distance is too long, a good image cannot be obtained due to scattering of the electron beam. Further, when the temperature is high, the saturated water vapor pressure rises, so that a good image cannot be acquired.
Based on the above, the working distance is 5 mm or more and 8 mm or less.

  Moreover, it is set as the temperature range of 0 degreeC-5 degreeC. If it is less than 0 degreeC, water will freeze. On the other hand, if it exceeds 5 ° C., the saturated water vapor pressure becomes high and the electron beam is scattered by the atmosphere, so that a good image cannot be obtained.

  By applying the above conditions, the microorganism sample can be observed without any special pretreatment.

  The specific procedure is as follows. First, the microorganism sample is cooled to 0 ° C. to 5 ° C. with a cooling holder, and the microorganism sample is introduced into the sample chamber of the ESEM. Next, the atmosphere in the sample chamber of the ESEM is replaced with water vapor, and the sample chamber is filled with water vapor having a saturated water vapor pressure. An ESEM having a Schottky electron gun is used. Further, the acceleration voltage is set to 2 kV or more and 5 kV or less in order to suppress diffusion of the electron beam in the microorganism sample. Next, the microorganism sample is observed close to the objective lens.

Examples of the present invention will be described.
ESEM used was Quanta 250 FEG manufactured by FEI. Bacteria (Bacillus subtilis) was placed in a cooled sample holder, and the bacteria (microorganism sample) were observed under the conditions shown in Tables 2 and 3. The atmosphere was water vapor and was observed under saturated water vapor pressure at each temperature. At pressures above the saturated water vapor pressure, the bacteria (microorganism sample) will condense, while at pressures below the saturated water vapor pressure, the bacteria (microorganism sample) will dry out and keep the bacteria (microorganism sample) in good condition for observation. I couldn't.
FIG. 1 (a) shows the results observed under the conditions of Table 2 Example No. 25 (example of the present invention). Bacteria (microorganism sample) can be observed in good condition, and as a result, the morphology of the bacteria (microorganism sample) can be identified.
FIG. 1B shows an example (reference example) in which bacteria (microorganism sample) are placed in a normal SEM vacuum. In FIG. 1 (b), it can be seen that the bacteria do not remain in their original form.
FIG. 1 (c) shows the results of observation under the conditions of Table 3 Example No. 74 (Comparative Example). ESEM observation itself is difficult, and it can be seen that bacteria (microorganism sample) are not visualized at all.
FIG. 1 (d) shows the results observed under the conditions of Table 2 Example No. 40 (Comparative Example). It turns out that the form identification of bacteria (microorganism sample) is difficult.
The obtained observation results are shown in Tables 2 and 3 together with the conditions. In Tables 2 and 3, the evaluation of the images is ○ when the image as shown in FIG. 1A is obtained, × when the observation is difficult as shown in FIG. A case where it was difficult to identify the form of the bacteria as in 1 (d) was marked with Δ.

  From Tables 2 and 3, it can be seen that under the observation conditions of the examples of the present invention, the bacteria (microorganism sample) can be observed in a good state without pretreatment.

Claims (1)

  Using an environmentally controlled scanning electron microscope with a Schottky electron gun, a microorganism sample under saturated water vapor pressure, at a temperature range of 0 ° C to 5 ° C, under an acceleration voltage of 2 kV to 5 kV and a working distance of 5 mm to 8 mm A method for observing a microorganism sample, characterized in that