JPH1014595A - Observation of microbial sample - Google Patents

Abstract

PROBLEM TO BE SOLVED: To facilitate the observation of a microorganism such as the one derived from urine, blood or feces, the one used in the food industry, e.g. Sake (Japanese fermented mildly alcoholic beverage made from rice wine) brewing or brewing or chemical industry by dispersing a microbial sample in an aqueous solvent and observing the microbial sample under a scanning type probe microscope. SOLUTION: An aqueous solvent comprising an aqueous solution, etc., containing polyL-lysine or the polyL-lysine and paraformaldehyde is dropped onto a glass substrate such as a cover glass for a microscope having 15mm diameter and air-dried and a suspension of a microorganism is then dropped thereonto and air-dried to prepare a microbial sample and the dispersion state of the microbial sample is observed under a scanning type probe microscope. Thereby, the infectivity of the microorganism is attenuated and a microorganism derived from various test materials such as urine, blood or feces, a yeast, etc., utilized in the field of food industry such as Sake brewing or brewing or the chemical industry in the microbial sample can readily be observed in a state of a high density and high dispersion.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a scanning probe microscope for analyzing microorganisms derived from various test materials such as urine, blood, and feces, and yeasts used in the fields of food and chemical industries such as sake brewing and brewing. Related to how to observe.

[0002]

2. Description of the Related Art Conventionally, a disk inspection method has been known as a representative method for inspecting microorganisms and the like separated from various inspection materials. In this method, a filter paper disk having absorbed a certain amount of an antibiotic is placed on an agar medium coated with a test bacterium and cultured. If the test bacterium is resistant to the antibiotic, the test bacterium grows over the entire surface of the agar medium.
On the other hand, when the test bacterium is sensitive to the antibiotic, an area called a "blocking circle" where the test bacterium does not grow is formed concentrically with the filter paper disk. Since the diameter of this inhibition circle is considered to be correlated with the minimum growth inhibitory concentration of the test bacterium, the diameter of this inhibition circle is measured, and whether the test bacterium is a susceptible or resistant bacterium is determined by its size or presence or absence. Has been determined. Further, as a method for measuring yeast and the like, measurement of yeast biomass, test measurement of biochemical properties of yeast such as enzyme activity, and the like are known.

However, all of the above methods involve the cultivation of microorganisms, so that there is a problem that the test requires a long time.
Therefore, the applicant of the present application has proposed that the yeast and the like be replaced with an atomic force microscope (AF).
M), etc. (Japanese Patent Application No. 7-276401). This method
Thawed yeast or the like that has been frozen and stored is placed on a glass plate as it is, dried to obtain a sample, and observed with a scanning probe microscope.

[0004]

In general, the scanning probe microscope has a very narrow observation field of view, and the observation position is a place where the probe accidentally descends. Can not. When observing microorganisms using such a scanning probe microscope, it is desirable that there be many microorganisms to be observed everywhere. That is, it is desirable to prepare a high-density and highly dispersed microorganism sample.

Accordingly, an object of the present invention is to provide a method for preparing a microorganism sample of high density and high dispersion and observing the sample with a scanning probe microscope.

[0006]

In order to solve the above-mentioned problems, the present invention is characterized in that a microorganism sample is dispersed in an aqueous solvent and the microorganism sample is observed with a scanning probe microscope. Here, as the aqueous solvent, an aqueous solution containing an amino acid having a basic R group, for example, poly-L-lysine can be used, but is not limited thereto. Further, poly-L-lysine or the like may be used alone or may be used by adding a solvent for enhancing the activity. Examples of the solvent that enhances the activity include, but are not limited to, paraformaldehyde, glutaraldehyde, and formalin. The concentration of the poly-L-lysine solution to be added is 0.001% to 0.1%.
%, Preferably 0.01%. If the content is 0.001% or less, there is no effect of dispersing microorganisms, and if the content is 0.1% or more, the surface of the dispersed sample is not smooth. Further, the solvent for enhancing the activity such as paraformaldehyde is preferably about 100 times the concentration of the poly-L-lysine solution.

As a method of dispersing a microorganism sample in an aqueous solvent, a method of coating a substrate with a solution containing the aqueous solvent and then dropping and dispersing the microorganism sample or a method of adding an aqueous solvent to the microorganism sample in advance is used. good. As the substrate, a transparent plate such as glass or polyacrylamide can be used.

The scanning probe microscope used in the present invention includes an atomic force microscope (AFM), a scanning tunnel microscope (STM), a laser force microscope (LFM), a magnetic force microscope (MFM), and a scanning ion conductance. Examples include, but are not limited to, a microscope (SICM). Here, the AFM detects the interatomic force acting between the sample and the probe and scans the surface of the sample so that the force becomes constant. Similarly, the STM determines the tunnel current flowing between the sample and the probe. , LFM indicates the resonance frequency of the probe,
MFM detects a magnetic force between the sample and the probe, and SICM detects an ion current between the sample and the probe, and scans the surface of the sample so that these are constant. Commercially available devices can be used for these devices.

The microorganism samples to be observed in the present invention include microorganisms derived from various test materials such as urine, blood and feces, yeasts used in the fields of food and chemical industries such as sake brewing and brewing, and the like. Including everything.

[0010]

【Example】

<Example 1: When using an aqueous solution of poly-L-lysine>
20 μl of 0.01% poly-L-lysine (Sigma) aqueous solution
1 was dropped on a glass substrate (cover glass of a microscope) having a diameter of 15 mm and air-dried (left at room temperature for 1 hour). To this, 10 μl of a suspension of yeast (ATCC287) cultured on an agar medium was dropped and air-dried (left at room temperature for 1 hour).
Microbial samples were prepared. FIG. 1A shows the result of observing the dispersion state of this sample with an optical microscope. For comparison, FIG. 1 (b) shows the result of observing only the cells with an optical microscope without dropping the aqueous solution of poly-L-lysine onto the cover glass.

As is clear from FIGS. 1 (a) and 1 (b), when the aqueous solution of poly-L-lysine is not coated, the yeast which looks black is agglomerated at the center, whereas when coated, the yeast is uniformly dispersed. You can see that it is doing. The same results were obtained when the solution containing poly-L-lysine was not added dropwise to the cover glass in advance, and poly-L-lysine was added to the solution containing yeast so that the final concentration was 0.01%.

Then, the microorganism sample dispersed in the aqueous solution of poly-L-lysine was subjected to an atomic force microscope (SPM-9 manufactured by Shimadzu Corporation).
Observation at 500) gave good results because the sample was dispersed.

<Example 2: Using an aqueous solution containing poly-L-lysine and paraformaldehyde> The same yeast-containing solution as in Example 1 was used so that the final concentration was 0.01%.
0.01% poly-L-lysine (Sigma) aqueous solution, 1%
Of paraformaldehyde (manufactured by Nacalai Tesque) was added. 10 μl of this solution was sampled, and the diameter was 15 mm.
Was dropped on a glass substrate (cover glass of a microscope) and air-dried (left at room temperature for 1 hour). FIG. 2A shows the result of observing this sample with an optical microscope. For comparison,
FIG. 2B shows the result of observing only the cells using an optical microscope without dropping an aqueous solution containing poly-L-lysine and paraformaldehyde onto the cover glass.

As apparent from FIGS. 2 (a) and 2 (b), when poly-L-lysine and paraformaldehyde were not added to the yeast-containing solution, the yeast which appeared white was aggregated, whereas It can be seen that are dispersed uniformly.

Thereafter, when the microorganism sample dispersed in an aqueous solution containing poly-L-lysine and paraformaldehyde was observed with an atomic force microscope (SPM-9500, manufactured by Shimadzu Corporation), good results were obtained because the sample was dispersed. Was done.

[0016]

According to the present invention, a microorganism sample can be prepared at a high density.
It can be observed in a highly dispersed state. In particular, when poly-L-lysine and paraformaldehyde are used in combination, the dispersing effect is large, and the effect of weakening (sterilizing) the infectivity of microorganisms also occurs.

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 (a) A diagram in which a solution containing poly-L-lysine was previously dropped onto a cover glass to observe yeast. (B) A diagram in which yeast was observed without using poly-L-lysine.

FIG. 2 (a) A diagram of yeast observed by adding poly-L-lysine and paraformaldehyde to a solution containing yeast. (B) A diagram of yeast observed without using poly-L-lysine and paraformaldehyde.

Claims (2)

[Claims] 1. A method for observing a microorganism sample, comprising dispersing the microorganism sample in an aqueous solvent and observing the microorganism sample with a scanning probe microscope. 2. The method for observing a microorganism sample according to claim 1, wherein the aqueous solvent is poly-L-lysine or an aqueous solution containing poly-L-lysine and paraformaldehyde.