JP2008286591A - Treatment method of sem microbial sample - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a pretreatment method and a device thereof for performing SEM observation of a microbial sample. <P>SOLUTION: This treatment method of the microbial sample is for preparing a sample for a scanning electron microscope for performing successively each process, namely, (1) a collection-installation process, (2) an immobilization process or a cleaning-immobilization process, (3) a cleaning process, (4) a dehydration process, (5) a replacement process, and (6) a drying process; and after collecting and installing the microbial sample on a liquid-permeable film, at least (2) the immobilization process or the cleaning-immobilization process, (3) the cleaning process and (4) the dehydration process are performed, while sucking the sample from a lower part of the film. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for treating a microbial sample for the preparation of a scanning electron microscope (SEM) specimen and an apparatus for carrying out this method.

  Microorganism observation using a scanning electron microscope (SEM) reveals the surface structure of microorganisms, changes in surface structure after treatment with drugs, etc., division mode, adhesion mode to host and object surfaces, growth mode, and formation 3 It is effective for various purposes, such as clarifying the positional relationship between a dimensional structure and adjacent microorganisms.

  When observing using an actual SEM, it is necessary to place the sample in a vacuum due to the characteristics of the microscope. Therefore, when observing a biological sample such as a microorganism, it is necessary to prepare the sample by performing a process such as fixing and dehydrating the sample in advance so that the structure and surface state of the sample do not change even in a vacuum. The sample preparation technique and technique are major factors that influence the quality of the SEM image. Until now, in order to obtain SEM images of microorganisms and samples containing microorganisms, a complicated and long pre-treatment step as described below has been required.

  There are a number of processes for sample preparation of microorganism samples for SEM observation, such as sample collection, fixation, dehydration, replacement, lyophilization, staining, metal coating, etc., and all processes are completed. It takes a minimum of 2-3 days to do this.

  Microbial sampling: 1) Filter paper support method: A method in which the bacterial solution is filtered using a suction filter and collected on the filter paper (Non-patent Document 1). 2) Aluminum foil support method: Bacteria on the aluminum foil 3) Agar block support method: a method of cutting out bacteria grown on an agar medium together with agar (Non-patent document 1), 4) Collodion membrane support method: on the membrane Method of dropping and adsorbing bacterial solution (Non-patent document 1), 5) Method of attaching to hard surface: Method of dripping and adsorbing bacterial solution after soaking glass, metal, etc. with polylysine etc. (Non-patent document 1) Etc.

In general, a method of sequentially performing the following steps 1 to 6 is known for fixing, dehydrating, replacing, and lyophilizing a collected microorganism sample (Non-Patent Documents 1 to 3).
1) Pre-fixing step: The sample is immersed in a glutaraldehyde solution (2.5% to 5%) for several hours to 24 hours.
2) Washing step: The sample is washed several times with a phosphate buffer.
3) Post-fixing step: The sample is immersed in an osmium tetroxide solution (1-2%) for several hours.
4) Washing step: The sample is washed several times with a phosphate buffer.
5) Dehydration step: The sample is immersed in 50, 70, 85, and 95% ethanol for 15 minutes each and further immersed in absolute ethanol for 15 minutes three times.
6) Substitution process, drying process: After immersing the sample in isoamyl acetate for 15 minutes, critical point drying is performed.

Further, when mold spores are samples, the number of pretreatment steps is further increased as in the following steps 1 to 8, because they are less structurally fixed than bacteria. (Non-Patent Document 1)
1) Spore collection step and washing step: The spores on the agar medium are suspended in a phosphate buffer or the like, and centrifugation and washing are repeated several times to obtain a spore suspension.
2) Pre-fixation affirmation: A fixative solution (glutaraldehyde solution (2.5% to 5%)) is added to the spores collected by centrifugation and prefixed for 2 to 4 hours.
3) Washing step: Centrifugation is performed after the fixing step, and the supernatant is suspended by replacing with a phosphate buffer to perform washing (this operation is repeated several times).
4) Post-fixing step: An osmium tetroxide solution (1-2%) is added to the spores collected by centrifugation, and fixed after 2 hours.
5) Washing step: Centrifugation after the post-fixing step, the supernatant is replaced with a phosphate buffer, suspended, and washed (this operation is repeated several times).
6) Installation step: The post-fixed spore suspension is dropped using a suction filter, and the post-fixed spore is adsorbed on a filter paper or the like, and installed on the filter paper.
7) Dehydration step: The sample is immersed in 50, 70, 85, and 95% ethanol for 15 minutes each and further for 15 minutes in absolute ethanol for 3 times.
8) Substitution process, drying process: After immersing the sample in isoamyl acetate for 15 minutes, critical point drying is performed.

  In general, when glutaraldehyde used as a fixing solution is used, the best result can be obtained by changing the pH to alkaline during the fixing operation while maintaining high dissolved oxygen. However, since living microorganisms that have just been collected respire, the dissolved oxygen and pH of the fixing solution may be lowered during fixation, and the sample may not be fixed well. Therefore, generally, it is necessary to take measures such as increasing the amount of the fixing solution in which the 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, dental plaque, and environmental microorganism aggregates (biofilms), the structure and positional relationship of the organisms other than microorganisms and the aggregates of microorganisms with different structures and constituents are determined. Since it is necessary to fix at once while maintaining, a skillful technique and a more complicated process are required (Non-Patent Documents 1 to 3).

As described above, the method for preparing a sample for SEM observation of microorganisms is complicated, time consuming, and requires a skillful technique, so that it has low versatility. 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 method, 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

  The present invention relates to a sample processing method for preparing an SEM observation specimen of a microorganism sample.

  As a result of studying a sample pretreatment method for SEM specimen preparation, the present inventors have 1) a sampling / installation step, 2) a fixing step or a washing / fixing step, 3) a washing step, 4) a dehydration step, 5 In the processing step of sequentially performing the replacement step and 6) the drying step, after the sample is collected and placed on the liquid permeable membrane, at least 2) the fixing step or the washing / fixing step, 3) the washing step, and 4) the dehydration step By carrying out suction from the lower part of the membrane, it is possible to easily fix the sample without moving the sample without destroying the structure of the sample, shortening the time required for the entire process, improving the ease of operation, etc. I found.

That is, this invention provides the invention which concerns on the following (1)-(6).
(1) For preparation of a scanning electron microscope specimen that sequentially performs 1) collection / installation process, 2) fixing process or cleaning / fixing process, 3) cleaning process, 4) dehydration process, 5) replacement process, and 6) drying process. After the microorganism sample is collected and placed on the liquid permeable membrane, at least 2) a fixing step or a washing / fixing step, 3) a washing step, and 4) a dehydration step are performed on the membrane. A method for treating a microorganism sample, which is carried out while aspirating from the lower part.
(2) The processing method according to (1), wherein the processing solution used in each step is dropped from the upper part of the sample.
(3) 1) The processing method according to the above (1) or (2), wherein the sampling / installation step is performed while sucking from the lower part of the membrane.
(4) 4) The treatment method according to any one of the above (1) to (3), wherein in the dehydration step, 25 to 100% by volume of a dewatered solution in several stages of concentration is sequentially dropped stepwise from a low concentration.
(5) Further, 5) The processing method according to any one of (1) to (4), wherein the substitution step is performed while suctioning from the lower part of the film.
(6) 6) The processing method according to any one of (1) to (5), wherein the drying step is freeze-drying.
(7) The processing method according to any one of (1) to (6), wherein the steps 1) to 5) are performed using a suction filtration device.
(8) A microorganism sample for preparing a scanning electron microscope specimen for carrying out the processing method according to any one of (1) to (7) above, including suction filtration means, processing droplet dropping means, and control means thereof. Processing equipment.

According to the treatment method of the present invention, it is not necessary to move the microorganism sample, and since the treatment solution required for each step is infiltrated and replaced in sequence, the microorganism itself, the aggregate of microorganisms, and the microorganisms are removed. The sample can be easily fixed without destroying the structure of the attached biological sample or the like, and the surface state thereof is also good.
In addition, according to the present invention, it is possible to shorten the time required for all processes, improve the ease of operation, and reduce the amount of various processing solutions to be used. The processing method of the present invention is efficient, safe, and environmental protection. Excellent from a viewpoint.

  The method for treating a microbial sample of the present invention is used in preparation of a specimen of a scanning electron microscope. 1) Collection / installation step, 2) Fixing step or washing / fixing step, 3) Cleaning step, 4) Dehydration step, 5) Replacement step And 6) In the sample pretreatment in which the drying step is sequentially performed, after the sample is collected and placed on the liquid permeable membrane, the steps 2) to 6) are sequentially performed without moving the sample as it is, and at least 2) The fixing step or the washing / fixing step, 3) the washing step, and 4) the dehydration step are performed while sucking from the lower part of the membrane, and the sample is infiltrated / replaced with the processing solution of each step. Further, the step 1) or 5) or all the steps may be performed while sucking from the lower part of the film.

In the treatment method of the present invention, 1) the sampling / installation step is performed by providing a suction device (for example, a suction filtration device) including a suction container, a suction funnel, a suction pump, and a tube connected to the suction container with a membrane capable of transmitting liquid. This is a process of setting and collecting and setting a microorganism sample to be observed on this. In the main collection / installation step, the sample may be dropped and collected on the liquid permeable membrane while sucking from the lower part of the membrane according to the state of the microorganism sample. Specifically, the microorganism suspension solution prepared in advance is dropped on the liquid permeable membrane while sucking, and the liquid permeability is maintained while maintaining the positional relationship of the microorganisms (biofilm). What is necessary is just to employ | adopt methods, such as mounting on a filter.
2) The fixing step or the washing / fixing step is a step of fixing the sample. Depending on the type of microbial sample, the sample may be washed in advance with a washing solution so that the sample can be easily fixed and then fixed. By performing this step while sucking, it becomes possible to perform the fixing operation while keeping the dissolved oxygen of the fixing solution high, and it is also possible to gradually change the pH of the fixing solution dripped simultaneously to the alkali side. Therefore, it is possible to easily obtain a good sample for observation by avoiding a decrease in dissolved oxygen amount and a pH decrease during fixation.
3) The cleaning step is a step of cleaning the fixed sample using a cleaning solution.
4) The dehydration step is a step of dehydrating the fixed sample that has been washed using the dewatered solution.
5) The replacement step is a step of replacing the dewatered solution in the fixed sample with a replacement solution.
6) The drying step is a step of performing freeze-drying after freezing the substituted fixed sample or the membrane on which the sample is prepared, and drying the sample.

  In addition, it is preferable not to dry a sample until it reaches 6) a drying process after collection and installation of the sample.

The treatment solution (fixing solution, washing solution, dewatering solution, replacement solution) used in each step is preferably added dropwise to the sample collected and set on the liquid permeable membrane.
By continuously dropping the treatment solution manually while aspirating the collected microorganisms, the treatment solution can be quickly penetrated and replaced without moving the sample. In addition, even when an aggregate is formed, uniform processing can be performed in a short time while maintaining the positional relationship between adjacent microorganisms.

Examples of the microorganism sample used in the present invention include microorganisms or samples containing microorganisms, and specifically, microorganisms such as bacteria, yeasts, filamentous fungi, and aggregates (biofilms) composed of them, or those A living body to which is attached.
Examples of living organisms to which microorganisms are attached include human tissues, human cultured cells, animal tissues, animal cells, plant tissues, plant cells, and insects.
Further, when these are collected and set on a membrane, microorganisms separated and collected from the natural world may be used as they are or by culturing them by a known culture method.
The microorganism may belong to any species or genus of bacteria, yeast, and filamentous fungi. For example, in the case of bacteria, Escherichia coli , Staphylococcus aureus , Pseudomonas aeruginosa ), Bacillus subtilis (Bacillus subtilis), Streptococcus (Streptococus), lactic acid and the like, Saccharomyces (Saccharomyces) genus in yeast, Candida (Candida) genus Rhodotorula (Rhodotorula) genus, and the like, if the filamentous fungus Alternaria (Alternaria) genus Aspergillus (Aspergillus) sp., Cladosporium (Cladosporium) genus Fusarium (Fusarium) genus Paecilomyces (Paecilomyces) genus Penicillium (Penicillium) genus, former (Phoma) genus, Yurochiumu (Eurotium) genus, Chaetomium (Chaetomium) genus Mucor (Mucor) genus and Byssochlamys (Byssochlamys) genus, and the like.

The liquid permeable membrane used for sample collection / installation corresponds to a filter that can hold, permeate or permeate the processing solution used in each processing step.
The material of the film is, for example, polypropylene (PP), polystyrene (PS), polyvinyl chloride (PVC), methyl methacrylate (MMA), acrylonitrile-butadiene-styrene copolymer (ABS), styrene acrylonitrile (SAN), Examples include polycarbonate (PC), poly (ethylene terephthalate) (PET), polytetrafluoroethylene (PTFE), vinylidene fluoride (PVDF), polyester (PES), glass fiber, cellulose, nylon, and the state thereof is, for example, Any of a fibrous shape, a net shape, a film shape, a paper shape, a non-woven fabric shape and the like may be used.
The membrane needs to have pores that pass through the treatment solution and do not allow the target microbial sample to pass through. The diameter of such a hole is 0.1 μm depending on the size of the target sample. It is 10-10 micrometers, Preferably it is 0.2-5 micrometers.
The film thickness is preferably 50 μm to 1 mm, more preferably 100 to 500 μm, from the viewpoint of ease of processing and handling during freeze-drying and SEM observation.
As the membrane, for example, commercially available products such as an isopore membrane (manufactured by Millipore), an omnipore membrane (manufactured by Millipore) and a cyclopore membrane (manufactured by Whatman) can be used.

As the treatment solution (fixing solution, washing solution, deaqueous solution, replacement solution) used in the present invention, it is preferable to use a solution filtered with a sterilization filter. The pore size of the sterilizing filter is 0.1 μm to 10 μm, preferably 0.2 to 5 μm.
The temperature when using the treatment solution is 1 to 25 ° C, preferably 4 to 10 ° C.

As the fixing solution, for example, a glutaraldehyde aqueous solution, a paraformaldehyde aqueous solution, or the like can be used. The concentration of the glutaraldehyde aqueous solution is 1 to 25% by mass, preferably 1 to 5% by mass, and the pH of the solution is 6 to 11, preferably 7 to 9. The concentration of the paraformaldehyde aqueous solution is 0.1 to 10% by mass, preferably 0.5 to 4% by mass.
In addition, these aqueous solutions may be appropriately mixed and used, and more commonly used buffer solutions may be mixed and used. Examples of the mixed solution include 2.5% by mass glutaraldehyde, 2.0% by mass formaldehyde, and a Karnovsky fixing solution having a 0.1M cacodylic acid composition.
Further, after fixing the microorganism sample with the fixing solution, it may be further fixed with osmic acid.

As the washing solution, for example, a phosphate buffer solution, a sodium cacodylate buffer solution, a HEPES buffer solution, or the like can be used.
The concentration of the phosphate buffer is 10 mM to 1 M, preferably 0.05 M to 0.5 M, and the pH of the solution is 6 to 11, preferably 7 to 9. The concentration of the sodium cacodylate buffer is 10 mM to 1 M, preferably 0.05 M to 0.5 M, and the pH of the solution is 6 to 11, preferably 7 to 9. The concentration of the HEPES buffer is 10 mM to 1 M, preferably 0.05 M to 0.5 M, and the pH of the solution is 6 to 11, preferably 7 to 9.

As the dewatering solution, for example, ethanol, acetone or the like can be used.
The concentration of ethanol is 25 to 100% by volume, preferably 50 to 100% by volume, and the concentration of acetone is 25 to 100% by volume, preferably 50 to 100% by volume. Examples of the concentration group include 70, 80, 90, 95, 100% by volume, and the like, and a method of sequentially dropping these in a stepwise manner from a low concentration is preferable.

  As the replacement solution, for example, t-butyl alcohol, isoamyl acetate, or the like can be used.

  The amount of treatment solution used in each step (fixing solution, cleaning solution, dewatered solution, replacement solution), treatment time, and suction amount vary depending on the size of the sample and the constituent microorganism species, but are performed under conditions that do not damage or deform the sample. . For example, the amount of the fixing solution used is 0.01 to 10 mL, preferably 0.1 to 5 mL, the treatment time is 1 to 120 minutes, preferably 30 to 60 minutes, and the pH of the fixing solution to be dropped is alkalinized. It can also be changed sequentially. The suction speed at this time is 0.0001 mL / min to 10 mL / min, preferably 0.01 mL / min to 1 mL / min, and the sample is immersed in the fixing solution for a longer time by slowing the suction. Is also possible.

  Moreover, the usage-amount of a washing | cleaning solution is 0.01-100 mL, Preferably it is 0.1-10 mL, and processing time is 1 minute-120 minutes, Preferably it is 5-15 minutes. The suction speed at this time is 0.001 mL / min to 10 mL / min, preferably 0.01 mL / min to 1 mL / min.

  Moreover, the usage-amount for each density | concentration of a dewatering solution is 0.01-50 mL, Preferably it is 0.1-5 mL, Processing time is 1 minute-120 minutes, Preferably it is 5-15 minutes. The suction speed at this time is 0.001 mL / min to 10 mL / min, preferably 0.01 mL / min to 1 mL / min.

  Moreover, the usage-amount of a substitution solution is 0.01-50 mL, Preferably it is 0.1-5 mL, and processing time is 1 minute-120 minutes, Preferably it is 5-15 minutes. The suction speed at this time is 0.001 mL / min to 10 mL / min, preferably 0.01 mL / min to 1 mL / min.

  Drying is performed after a dehydration process or a substitution process. Usually, freeze-drying is performed after freezing the sample or the membrane on which the sample is prepared. For each sample or membrane on which the sample is prepared, a known critical point drying device or t-butyl alcohol freeze-drying device is used. Substitution and drying may be performed.

After the drying process, the sample is appropriately cut and cleaved according to the observation purpose, placed on the observation sample stage, and subjected to a conductive treatment process that performs metal coating, carbon coating, etc. with an ion sputtering apparatus or vacuum deposition apparatus, etc. Obtain an electron microscope specimen.
The prepared specimen is observed by SEM.

  The processing method of the present invention requires at least suction means such as a suction filtration device, but further, a combination of processing solution dropping means such as a processing solution dropping device and a control means for controlling them by a series of operations. Can also be automated. The control means is programmed with the method of the present invention, such as the order of the steps, the dropping amount of the processing solution required for each step, the dropping speed, and the suction speed.

The suction filtration apparatus includes at least a suction container, a suction funnel, a tube connected to the suction container, and a suction pump, and continuously contacts the processing liquid with the sample on the membrane while sucking. A commercial product such as a suction filtration system (NALGENE) may be used as the apparatus.
Here, the suction container is of any material and shape as long as it can withstand the suction pressure by the suction device, can store the sucked processing solution, and has a hole that can be connected to the suction tube on the side surface. For example, it is in the shape of a cup made of a material such as glass, plastic, metal, or earthenware.
The suction funnel may be made of any material as long as it can withstand the suction pressure by the suction device and can be installed with the liquid permeable membrane of the present invention. For example, the suction funnel is made of a material such as glass, plastic, metal, and ceramics. .
The suction funnel is composed of a pedestal-shaped upper part on which a liquid-permeable membrane is installed and a lower part that can be connected to the upper part of the suction container to seal the suction container. The pedestal has a surface on which the membrane is installed. The surface has a hole through which the treatment solution dropped from the top can pass.
The connecting tube material may be of any material and shape as long as it can withstand the suction pressure and satisfies the above-described suction speed. For example, the connection tube material has a tubular shape such as silicon, rubber, or vinyl chloride.
The suction pump may be anything as long as it satisfies the above-described suction speed and time. For example, an electric pump, a manual pump, an aspirator, an electric pipetter, or the like can be used as the suction pump.

Below, the specific operation procedure using the processing method of the present invention is shown.
The suction filtration apparatus 1 includes a suction container 2, a suction funnel 3 provided with a surface 4 for installing a membrane, a connection tube 5 and a suction pump (not shown) (see FIG. 1).
1) Install a filter on the surface 4 of the suction funnel 3. Next, the sample is placed on the filter by dripping the microorganism suspension solution prepared in advance on the filter while sucking or placing a microorganism aggregate (biofilm) on the filter.
Subsequent operations are performed while suctioning through the connection tube 5 with a suction pump, taking care not to dry the sample on the filter.
2) The cleaning liquid is dropped to clean the sample (can be omitted depending on the sample characteristics).
3) Fix the sample by dropping the fixative.
4) Wash the sample by dropping the cleaning solution.
5) Dehydrated water 50, 70, 80, 90, 95, 100% by volume are added dropwise sequentially to perform dehydration.
6) The replacement solution is dropped to replace the solution in the sample.
In addition, the process of 2-5) carries out the pre-process of the process of washing | cleaning-replacement by dripping various solutions on a sample, controlling using a process solution dripping apparatus (a computer controllable pump etc.). All can be done automatically. The apparatus may be anything as long as it can accurately flow a small amount of liquid. For example, a thin micropump (Takasago Electric Co., Ltd.), a micro diaphragm pump 5013 (Namiki Seisakusaku Co., Ltd.) and the like are commercially available. You may use goods.

  Thereafter, the sample is taken out from the suction filtration device together with the filter, and after freezing the sample in a freezer or the like, the sample is dried with a freeze dryer to obtain a pretreated sample of the SEM specimen. Next, the sample is cut and cut according to the purpose of observation, placed on the observation sample stage, metal-coated with an ion sputtering apparatus or the like, a sample is obtained, and SEM observation is performed on the sample.

Hereinafter, the present invention will be described in more detail with reference to examples.
[Example 1] Microscopic observation of bacteria by scanning electron microscope (SEM)
Using SEM, Staphylococcus aureus IFO13276 was observed in the following steps.
1) Sample installation After culturing S. aureus on SCD agar medium at 30 ° C for 24 hours, the emerged colonies were suspended in sterile physiological saline using platinum ears so that the bacterial count would be 10 ⁶ cfu / mL. A bacterial suspension was prepared.
Next, a membrane filter (Millipore, ISOPORE, Cellulose, pore size 0.2 μm) is installed in the suction filtration system (NALGENE, pore size 0.45 μm), and 0.1 mL of the bacterial solution adjusted while aspirating is dropped on the membrane filter. did.
SCD (Soybean Casein Digest) Medium: Casein Peptone 15g / L, Soybean Peptone 5g / L, NaCl 5g / L, Ager 15g / L

2) Washing Immediately drop 1.0 ml of HEPES Buffer (Irvine Scientific, 4 ° C., 10 mM, pH 7.0) for 5 minutes while sucking at a suction speed of 0.2 m / min before the sample surface dries. Went.

3) Fixation Immediately with 2.0 mL of 2.5% glutaraldehyde / HEPES solution (adjusted with 4 ° C, HEPES Buffer (10 mM, pH 7.0)) while aspirating at a suction speed of 0.066 mL / min before the sample surface dries. It was dripped continuously and fixed for 30 minutes.

4) Cleaning Before the sample surface dries, 5.0 mL of HEPES Buffer (Irvine Scientific, 4 ° C, 10 mM, pH 7.0) is immediately added dropwise and cleaned for 15 minutes while aspirating at a suction speed of 0.33 mL / min. Went.

5) Dehydration Before the surface of the sample dries, 5.0 ml of 50% ethanol is added dropwise continuously for 15 minutes while sucking at a suction speed of 0.33 ml / min, and then 70%, 80%, 90%, Dehydration was performed by adding dropwise 95% and 100% ethanol (dehydrated in advance with a dehydrating agent (Molekularsieb MERCK)).

6) Replacement Before the surface of the sample was dried, 2.0 mL of t-butyl alcohol (30 ° C.) was immediately dropped continuously for 10 minutes while performing suction at a suction speed of 0.2 mL / min.

7) Freezing and drying Remove the sample together with the filter, freeze it in the freezer for 15 minutes, set the frozen sample in the freeze dryer ES-2030 (Hitachi) set at the sample chamber temperature -10 ° C, and dry the sample. (Approximately 20-30 minutes).

8) Observation The dried filter was attached to an aluminum SEM sample table with carbon double-sided tape, and platinum and panadium coating treatment was performed with a discharge current of 20 mA and a discharge time of 80 seconds using Ion Sputter E-1030 (Hitachi). Observation was performed with a scanning electron microscope S-4300SE / N, Hitachi.

As described above, S. aureus could be easily observed in a short time. The process of the present invention and the conventional process are shown in Table 1. The observed SEM image is shown in FIG.
As shown in Table 1, according to the method of the present invention, the pretreatment process was simple and the operation time was shortened. The amount of processing used was also reduced.
It was possible to observe the surface structure and morphology as shown in FIG.

[Example 2] Observation of microbial aggregate (biofilm) by scanning electron microscope (SEM)
Observation of the aggregate (biofilm) of bacteria, yeast, and filamentous fungi formed on the floor using SEM was performed in the following steps.

1) Sample installation HEPES Buffer (Irvine Scientific, 10 mM, pH 7.0) was added dropwise at 1.0 mL / cm ² to a film-like colored stain (biofilm) generated on a vinyl chloride floor in a residence for 30 minutes. After leaving it to stand, a film-like colored stain having a size of about 0.5 mm × 0.5 mm was peeled off from the floor using a silicon spatula for cell peeling. The peeled colored stain was gently placed on a membrane filter (Millipore, ISOPORE, cellulose, pore size 0.2 μm), and the membrane filter was placed on a suction filtration system (NALGENE, pore size 0.45 μm).

2) Washing Immediately before the surface of the sample was dried, 1.0 ml of HEPES Buffer (Irvine Scientific, 4 ° C., 10 mM, pH 7.0) was continuously added dropwise for washing for 5 minutes.

3) Fixation Immediately drop 2.0 ml glutaraldehyde (4 ° C, 2.5%: adjusted with Irvine Scientific [10 mM, pH 7.0]) immediately while sucking before the sample surface dries. Fixing was performed for 30 minutes.

4) Cleaning Immediately before the surface of the sample was dried, 5.0 mL of HEPES Buffer (Irvine Scientific, 4 ° C., 10 mM, pH 7.0) was immediately added dropwise for washing for 15 minutes.

5) Dehydration Immediately after the sample surface is aspirated, 5.0 mL of 50% ethanol is added dropwise continuously for 15 minutes, and then 70%, 80%, 90%, 95%, 100% (dehydrated). Dehydration was performed by adding dropwise the ethanol (previously dehydrated with Molekularsieb MERCK).

6) Replacement Immediately before the surface of the sample was dried, 2.0 mL of t-butyl alcohol (30 ° C.) was continuously dropped for 10 minutes while performing suction.

7) Freezing and drying Remove the sample together with the filter, freeze it in the freezer for 15 minutes, set the frozen sample in the freeze dryer ES-2030 (Hitachi) set at the sample chamber temperature -10 ° C, and dry the sample. (Approximately 20-30 minutes).

8) Observation The dried filter was attached to an aluminum SEM sample table with carbon double-sided tape, and platinum and panadium coating treatment was performed with a discharge current of 20 mA and a discharge time of 80 seconds using Ion Sputter E-1030 (Hitachi). Observation was performed with a scanning electron microscope S-4300SE / N, Hitachi.

  The SEM image observed as described above is shown in FIG. The film-like colored stain on the floor is a biofilm structure in which various microorganisms are laminated. It is observed that the lower layer is composed mainly of bacteria, the middle layer is yeast, and the upper layer is composed of mold. It was done. Thus, according to the present invention, it was possible to observe while maintaining various shapes and structures of microorganisms.

FIG. 1 shows a schematic diagram of a pretreatment device for carrying out the method of the invention. FIG. 2 shows a scanning electron micrograph of S. aureus. FIG. 3 shows a scanning electron micrograph of a section of a biofilm formed on the floor.

Claims (8)

  1) Collection / installation process, 2) Fixing process or washing / fixing process, 3) Cleaning process, 4) Dehydration process, 5) Replacement process and 6) Drying process. After collecting and setting a microorganism sample on a liquid permeable membrane, at least 2) a fixing step or a washing / fixing step, 3) a washing step, and 4) a dehydration step are sucked from the lower part of the membrane. A method for treating a microorganism sample, which is performed while   The processing method according to claim 1, wherein a processing solution used in each step is dropped onto an upper portion of the sample.   1) The processing method according to claim 1 or 2, wherein the sampling and setting step is performed while sucking from the lower part of the membrane.   4) The processing method according to any one of claims 1 to 3, wherein, in the dehydration step, a dewatering solution having a concentration of several steps of 25 to 100% by volume is dropped in a stepwise manner from a low concentration.   5) The processing method according to any one of claims 1 to 4, wherein the replacement step is performed while sucking from the lower part of the film.   6) The treatment method according to any one of claims 1 to 5, wherein the drying step is freeze-drying.   The processing method according to any one of claims 1 to 6, wherein the steps 1) to 5) are performed using a suction filtration device.   A processing apparatus for a microorganism sample for preparing a scanning electron microscope specimen for carrying out the processing method according to any one of claims 1 to 7, comprising suction means, processing droplet dropping means, and control means thereof.