Microbial image analysis method

Abstract

[Problem] To provide a microorganism image analysis method capable of capturing information on the fine morphology of individual microorganisms with the resolution of an electron microscope and evaluating the ratio of microorganisms. [Solution] A microorganism image analysis method according to one aspect of the present invention comprises a first step of acquiring an image of a specimen obtained by staining a sample containing microorganisms using an electron microscope, a second step of acquiring a luminance profile relating to the brightness of the image, a third step of setting a first standard luminance range in the profile that satisfies a first condition relating to luminance as an area in which a first group of microorganisms exists, and setting a second standard luminance range in the profile that satisfies a second condition relating to luminance as an area in which a second group of microorganisms exists, and a fourth step of calculating the ratio of microorganisms existing within each of the first and second standard luminance ranges. [Selected Figure] Figure 1

Description

[0001] METHOD OF ANALYSIS OF MICROBIAL ANALYSIS

[0002] CONTEXT OF THE INVENTION

[0003] 1. Field of the invention

[0004] The present invention relates to a method for analyzing images of microbes.

[0005] 2. Description of the state of the art

[0006] With concerns about the global spread of antimicrobial-resistant bacteria, the identification of a microbe causing an infectious disease and an infectious disease test such as an antimicrobial susceptibility test and the like are becoming increasingly important. Here, as a method for identifying a microbe and measuring susceptibility to an antimicrobial, a drug susceptibility test method is known in which a microbe in a liquid is incubated with a dye and then collected by filtration through a filter, and a viability test is used to enumerate a microbe stained with a substance that stains both a living cell and a dead cell in a field of view of an optical microscope and a microbe stained with a substance that stains only a dead cell (refer to JP-A-2001-509008).

[0007] SUMMARY OF THE INVENTION

[0008] According to JP-A-2001-509008, the problem is that only a method using an optical microscope is described, and the finer morphology of a microbe is not observed.

[0009] Therefore, an object of the present invention is to provide a method for analyzing images of microbes that can evaluate a proportion of a microbe by obtaining information on the fine morphology of an individual microbe with the resolution of an electron microscope.

[0010] A method for analyzing images of microbes according to one embodiment of the present invention comprises: a first step for obtaining an image of a specimen in which a sample having microbes is colored, by means of an electron microscope; a second step for obtaining a brightness profile relating to the brightness of the image; a third step for defining a first standard brightness range that satisfies a first condition relating to the brightness of the profile as a region where a first group of microbes exists, and defining a second standard brightness range that satisfies a second condition relating to the brightness of the profile as a region where a second group of microbes exists; and a fourth step for calculating a proportion of the microbe that exists in each of the first and second standard brightness ranges.

[0011] According to the present invention, it is possible to provide a method for analyzing images of microbes which makes it possible to evaluate a proportion of a microbe by obtaining information on the fine morphology of an individual microbe with the resolution of an electron microscope.

[0012] BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 is a diagram illustrating an image of microbes having different image brightness and image brightness range; FIG. 2 is a diagram illustrating an example of the image brightness range; FIG. 3 is a diagram illustrating an example of microbe image classification; FIG. 4 is a diagram illustrating a viability test result of a microbe after storage treatment; FIG. 5 is a diagram illustrating a viability test result of a microbe exposed to oxygen; FIG. 6 is a diagram illustrating electronic staining; and FIG. 7 is a diagram illustrating an image analysis result of a microbe treated with an antibacterial agent. DESCRIPTION OF EMBODIMENTS

[0014] The background of the present invention will be described before describing an embodiment.

[0015] As a first aspect of the prior art, one of the important basic techniques is microscopic observation, which uses an image of an individual microbe and a group of microbes. For example, in a clinical examination, a difference in cell wall structure of bacteria is stained with a dye and observed with a light microscope, so that Gram-positive and Gram-negative bacteria are separated and classified into bacilli and cocci on the basis of the appearance of the bacteria, and information associated with bacterial species such as staphylococcus, streptococcus and their equivalents can be obtained from an approximate form of aggregates of isolated and cultured bacteria.

[0016] In addition to identification by this staining, it is hoped to gain information about the fine morphology of the individual microbe. However, the resolution of a light microscope typically used in clinical examination is about several hundred nanometers, so even if it is possible to obtain the approximate shape of the microbe and its staining information, it is not easy to identify a difference in morphology between individual microbes.

[0017] An electron microscope is used here. Since the wavelength of an electron beam is much shorter than that of visible light, a high-resolution image can be obtained, and even fine morphology that cannot be captured with a light microscope can be observed. Examples of fine morphology include a structure specific to the microbe's species, a structure observed during cell division, abnormal morphology caused by the influence of a drug, and their equivalents.

[0018] Since a state-of-the-art electron microscope requires large-sized mechanisms such as a mechanism that applies a high voltage to generate a stable electron beam, a mechanism to keep the interior of the microscope in a vacuum state to prevent scattering of the emitted electron beam, and their equivalents, it is difficult to use the state-of-the-art electron microscope in a daily microbial test as in clinical practice or its equivalents. However, in recent years, a benchtop electron microscope, which was obtained by improving the state-of-the-art electron microscope, has been developed, and it is also planned to be applied to a microbial sample.

[0019] One of the problems with an observation method using the electron microscope is that the staining method is limited. For the optical microscope, dyes corresponding to various purposes such as cell biology, histology, pathology and their equivalents are developed, improved, and various types of staining methods for staining a component and function of a cell and tissue with different colors are established.

[0020] For example, the electron microscope uses a method to visualize a structure of the entire microbe such as a cell membrane, a nucleus and their equivalents through the contrast of black and white images by treating a sample with a dye containing a heavy metal such as uranium, lead, platinum, osmium and their equivalents, or a method to visualize the location of a specific molecule with a marker made of fine gold particles. However, there are few reports of dyes coloring the specific component of the microbe and its function and presenting a contrast with an electron beam.

[0021] As described above, although it is desirable to use a method that combines both light microscope staining and high-resolution electron microscope observation, there is no practical method for everyday microbial testing.

[0022] An example of a second aspect of the prior art is a test for the viability of a microbe, which is one of the important test items in a microbial test. For example, when developing a treatment method for a drug, heat, gas, and the like to remove the microbe, or when selecting a therapeutic agent for infectious diseases and the like, the viability of a microbe in a sample treated with these treatments is measured, thereby determining a treatment type, treatment method, treatment intensity, treatment concentration, and the like.

[0023] There are various viability testing methods, and an ordinary method is a culture method such as colony forming unit and its equivalents. In colony forming evaluation, a certain amount of a microbial suspension of known concentration is thinly spread on an agar medium and cultured for about one day, the number of bacterial colonies developing in a certain period of time is taken as the number of bacteria surviving at the beginning of the culture, a colony forming unit (CFU / ml) is calculated from the number of colonies relative to a liquid amount of inoculated bacteria, and the calculated colony forming unit is taken as the viability at the beginning of the culture.

[0024] The culture method is the most widely used method for testing the viability of a microbe. However, the problems are that it takes one or more days to perform the culture to form the colony, informed judgment is required to visually determine colony formation, and the culture method cannot be applied to a microbe whose culture conditions such as the type of medium and its equivalents are unknown.

[0025] In order to solve the problems of the culture method described above, a method using the optical microscope and its equivalents that does not require culture has been developed. Examples of the method include a method for detecting a function such as the enzymatic activity of a living cell and its equivalents with a chromogenic substrate, a method for performing staining and detection with a difference in permeability to cell membrane substances of a living cell and a dead cell, a method for detecting a morphological change, and their equivalents. The methods described above are excellent in terms of speed, because there is no need to wait for the culture time until the bacteria grow and form a visible colony.

[0026] Here, in conjunction with the first and second aspects described above, information on the fine morphology of a microbe with the resolution of the electron microscope, such as a structure specific to the species of the microbe, a structure observed during cell division, an abnormal morphology caused by the influence of a drug and the like are harvested, and the usefulness of a technique for performing a viability test on the same specimen is also considered. For example, a type and a state are harvested from morphological information of a biofilm-forming microbe, treatment is performed on the biofilm with an antimicrobial, a bactericide and the like that affect the viability of the microbe, and the viability of a treated specimen and the viability of an untreated specimen are compared with each other, thereby making it possible to evaluate the effect of the antimicrobial and the bactericide in situ (in situ observation).However, the viability testing method using electron microscope is not put into practice in microbial tests such as clinical and environmental tests.

[0027] The reason why the viability test using the electron microscope is difficult is that in the case of an ordinary electron microscope, there is an observation pretreatment to preserve the morphology of a microbe to be observed under vacuum. Since a protein crosslinking agent such as glutaraldehyde and its equivalents is used in advance to chemically fix the microbe and then dry the microbe, at the time of fixing the microbe, the microbe loses a viability function. Therefore, it is not possible to perform a viability test as described in the optical detection example that visualizes a viability function of a truly living microbe such as a difference and its equivalents in enzyme activity and biological membrane permeability by staining.

[0028] Therefore, as one of the methods to observe fine morphology and determine life and death with the same specimen, there is a method to determine the morphology associated with the life and death of the microbe, for example, a method to determine membrane damage, collapse of an intracellular microstructure and their equivalents by observing an ultrathin slice using a transmission electron microscope. However, this method requires mastered technique and laborious work. In order to calculate viability by observing a statistically significant number of microbes, a huge amount of work is required, so it is difficult to apply this method to a daily microbial test.

[0029] An example of another method is correlative light and electron microscopy, in which a color image obtained by the light microscope and a morphological image obtained by the electron microscope are combined with each other. In this method, although it is possible in principle to process the same sample and observe the same sample under an electron microscope after determining whether the sample is alive or dead under an optical microscope, operations such as sample preparation, image superposition, and their equivalents are not simple, and therefore difficult to apply to everyday microbial testing.

[0030] In the following embodiments, as a technique for collecting the information on the fine morphology of the microbe with the resolution of the electron microscope and performing the viability test with the same sample, an aqueous solution of phosphotungstic acid (hereinafter referred to as PTA) is used as a dye which strongly adheres to a microbe undergoing morphological damage associated with the life and death of the microbe, and an image exhibiting compositional contrast is acquired with the backscattered electrons of a scanning electron microscope (hereinafter referred to as SEM), thereby providing a method for evaluating a proportion of the microbe corresponding to viability.

[0031] Here, the proportion is acquired such that, when a microbe in a suspension and the like is stained with an aqueous PTA solution, a difference in staining intensity between the microbe not undergoing the morphological damage associated with the life and death of the microbe and the microbe undergoing the morphological damage associated with the life and death of the microbe is detected as a difference in image brightness, and each microbe is quantified. This proportion is defined as viability according to the present invention. As described above, since observation preprocessing such as fixation and the like is performed, the proportion of living microbes at the time of SEM image acquisition is not indicated, but the embodiment shows that a proportion equivalent to viability by a prior art method is obtained.

[0032] A method of using PTA as a dye in the present invention will be described. Generally, in electronic staining at the time of observing a biological sample under an electron microscope, dyes such as uranium acetate aqueous solution, lead salt aqueous solution, platinum blue and the like are often used. These dyes are excellent dyes that enable intracellular structures such as a cell membrane, a nucleus and the like to be visualized by contrasting an image using a difference in affinity with a cell-forming component, but are not used for a staining method for identifying the life and death of the microbe. Uranium is a radioactive substance and is limited by its availability and handling.Uranium, lead, and platinum are not suitable dyes for routine microbial testing due to their toxicity, stability in solution, cost of reagents, and their equivalents.

[0033] Therefore, regarding the selection of the dye, since PTA is a reagent that is relatively cheap, easy to store, and easy to use as described above, the inventor considers that PTA could be applied to the electronic staining of a microbe in a daily microbial test. PTA is widely used for the preparation of microscopic specimens. For example, PTA is used as a staining mordant for a light microscopy specimen in a pathological examination, and used as a negative stain for observing a microbe under an electron microscope. It is known that PTA is also used for positive staining at the time of observing a cell under an electron microscope, and PTA can separately stain basic proteins, glycoproteins, and polysaccharides by adjusting the concentration and pH of a solution and binding it to a carbohydrate portion of the glycoprotein.It has been reported that when PTA is used for SEM observation of bacteria, Gram-positive bacteria and Gram-negative bacteria have different staining intensities.

[0034] Here, the inventor discovered that after bacteria with known sensitivity are treated with an antibacterial agent exhibiting bactericidal action that damages the cell membrane and cell wall, a PTA-stained specimen is prepared, a backscattered electron image of the SEM is obtained, individual bacteria having high image brightness are identified in the specimen, and a proportion of individuals having high image brightness increases as the antibacterial treatment time elapses.

[0035] It is known that since a characteristic of a backscattered electron image is that the compositional contrast of a substance can be obtained in addition to the surface shape contrast of a specimen, a greater amount of PTA is bound to bacteria with a cell membrane and cell wall damaged by the bactericidal action of the antibacterial agent than to living bacteria. This result suggests that morphological damage associated with the life and death of bacteria can be identified with PTA staining.

[0036] Therefore, the inventor carefully studied a sample preparation method comprising a PTA staining step and a condition for observing backscattered electrons using the SEM. As an example, a specimen is prepared by placing a microbe treated with a bactericide and its equivalents and a control microbe not treated with the bactericide on the equipment. The specimen is fixed with 2.5% glutaraldehyde for 5 minutes, the specimen is stained with an aqueous PTA solution at a concentration of 10% by weight for about 2 minutes, and a backscattered electron image is acquired at an accelerating voltage of 5 kV to 10 kV using the SEM.

[0037] As a result, the microbe contained in the specimen treated with the bactericide is mainly classified into two groups, a microbe having a high image brightness and a microbe having a low image brightness. The image brightness of the latter microbe having the low image brightness is identical to the image brightness of the control microbe specimen not treated with the bactericide. In other words, it has been found that in a group of microbes treated with a bactericide, it is possible to identify, by staining, bacteria undergoing the morphological damage caused by the bactericidal action and bacteria not undergoing the morphological damage, and to determine a proportion.

[0038] Based on the findings described above, the inventor provides a method for knowing the type of a microbe and a morphological characteristic thereof with an electron microscope, and for easily identifying a microbe undergoing morphological damage associated with life and death and a microbe not undergoing the morphological damage by the image brightness arising from the intensity of electron staining using PTA.

[0039] Hereinafter, detailed embodiments will be described. A procedure, drug concentration, treatment time, image analysis procedure and their equivalents described above are examples, and do not limit the scope of the present invention.

[0040] [First embodiment]

[0041] A first embodiment shows an example of classifying a microbe image based on a difference in image brightness of a stained microbe in an electron microscope image of the microbe. FIG. 1 illustrates an example in which a microbe 1 (102) having weak staining and weak image brightness and a microbe 2 (103) having strong staining and strong image brightness are mixed by means of a schematic diagram of electron microscope images (101) of a specimen in which the microbe is placed on the equipment and stained. In this example, the microbe image is classified into two groups including microbe 1 and microbe 2 based on the difference in image brightness.

[0042] An example of an electron microscope image (104) in which microbe 1 (102) exists and an example of an electron microscope image (105) in which microbe 2 (103) exists are backscattered electron images obtained such that a Pseudomonas aeruginosa strain is placed on a track-etched polycarbonate membrane, the Pseudomonas aeruginosa strain is fixed with 2.5% glutaraldehyde and stained with an aqueous PTA solution at a concentration of 10% by weight, a specimen in which Pseudomonas aeruginosa is placed on the membrane is inserted into an SEM sample chamber, the accelerating voltage of the SEM is set to 5 kV, and the brightness contrast at the time of image generation is set to be constant.Since the SEM image generation conditions are constant, the difference in brightness between microbe 1 (102) in image (104) and microbe 2 (103) in image (105) arises from a difference in PTA staining intensity.

[0043] As an example, the etched track membrane made of a plastic material is used as equipment, and the equipment is not limited thereto. Here, from the perspective of subsequent image analysis, it is desirable for a user to select equipment made of a material having an image brightness different from that of the colored microbes 1 and 2 to be analyzed in the backscattered electron image.

[0044] A brightness profile (106) associated with the brightness of the image (104) and a brightness profile (107) associated with the brightness of the image (105) are difference histograms obtained such that the number of pixels for each brightness is obtained for each image in which microbe 1 or microbe 2 is placed on the membrane, and the number of pixels for each brightness in a membrane image in which the microbe is not placed is subtracted therefrom.

[0045] For example, an image region of microbe 1 is characterized by a standard brightness range 1 (108) having a peak 1 of the brightness profile (106) relating to the brightness, an image region of microbe 2 is characterized by a standard brightness range 2 (102) having a peak 2 of the brightness profile (107) relating to the brightness, such that the images of microbe 1 and microbe 2 are classified.

[0046] As described above, when the brightness contrast of the SEM is set to be constant at the time of acquisition of the image (104), the image (105), an image in a different region of the same specimen, and images of a plurality of specimens prepared with the same material and method, the standard brightness range 1

[0047] (108) which characterizes the image region of microbe 1 and the standard brightness range 2

[0048] (109) which characterizes the image region of microbe 2 can be set to the same value, when an image analysis is performed.

[0049] FIG. 2 illustrates examples (201 and 202) of setting the standard brightness range. 201 and 202 are the same histograms and are brightness profiles associated with the electron microscope image brightness. As illustrated in the schematic diagram (101) of FIG. 1, since 201 and 202 arise from the image in which microbes having different image brightness are mixed, peak 1 and peak 2 exist in examples 201 and 202.

[0050] Another peak located to the left of peak 1 corresponds to a region of the electron microscope image of the equipment in which the microbe is placed, and this range is thus excluded to determine the standard brightness range that characterizes the image of microbes.

[0051] As shown in histogram 1 (201), standard brightness range 1 including peak 1 arising from an image of microbe 1 is determined, and a standard brightness range including peak 2 arising from an image of microbe 2 is determined within a range that does not overlap with standard brightness range 1, such that microbe 1 characterized by standard brightness range 1 and microbe 2 characterized by standard brightness range 2 are classified.

[0052] Alternatively, as illustrated in histogram 2 (202), standard brightness range 1 including both peak 1 arising from the image of microbe 1 and peak 2 arising from the image of microbe 2 is determined, and standard brightness range 2 including peak 2 without including peak 1 is defined, thereby classifying all microbes contained in the image, microbe 2 characterized by standard brightness range 2, and microbe 1 obtained by subtracting microbe 2 from all microbes.

[0053] FIG. 3 illustrates an example in which the image of microbes is classified based on a difference in brightness of the electron microscope image by means of image analysis software, and an image mask is generated for each classification. As illustrated in the schematic diagram (101) of FIG. 1, in an electron microscope image (301) of a microbial specimen, microbe 1 (102) having weak coloration and weak image brightness and microbe 2 (103) having strong coloration and strong image brightness are mixed.

[0054] As illustrated in histogram 2 (202) of FIG. 2, the mask (302) is a masked image generated such that the brightness range of the equipment is excluded, the image is binarized by setting a threshold value from the standard brightness range 1 including both peak 1 arising from the image of microbe 1 and peak 2 arising from the image of microbe 2, and particles substantially smaller than microbes 1 and 2 are then removed using a particle analysis algorithm. As illustrated in histogram 2 (202) of FIG. 2, the mask (303) is a masked image generated such that the image is binarized by setting a threshold value from the standard brightness range 2 including peak 2 without including peak 1, and particles substantially smaller than the microbe are then removed using a particle analysis algorithm.By means of the image analysis software, the individual microbe classified within mask 1 and mask 2 can be identified, and the number of microbes, an image area of ​​microbes and the number of pixels can be obtained to calculate a proportion.

[0055] As described above, the microbial specimen is stained to generate the backscattered electron image, the brightness profile relating to the image brightness of microbe 1 (104) having weak staining and weak image brightness and microbe 2 (105) having strong staining and strong image brightness is acquired, and the image of microbes is classified by the image brightness range, such that the proportion of microbe 1 and microbe 2 in the specimen can be easily calculated.

[0056] In the first embodiment, although the difference in the staining intensity of the microbe is assigned to two types, and two types of standard brightness range in the brightness profile relating to the image brightness are determined, the types of standard brightness range may amount to more than two.

[0057] [Second embodiment]

[0058] <Test de viabilité d’un microbe>

[0059] A second embodiment describes an example in which the proportion of a microbe obtained by the method of the present invention is compared with the viability of the microbe obtained by the colony forming unit, which is a state-of-the-art method of a viability test, and a flow cytometry method which is a culture-free method.

[0060] As an example, bacteria (Akkermansia muciniphila) are grown under anaerobic conditions on agar medium for 48 hours and isolated. Then, the isolated bacteria are suspended at a bacterial concentration of approximately 10 10 CFU / ml in storage medium containing an antioxidant, and are frozen and lyophilized at -80°C for 24 hours.

[0061] After being frozen and freeze-dried, the bacteria were stained with 10% PTA for 5 minutes at 37°C, the bacterial suspension was spread on a glass slide by centrifugation and dried at room temperature, and SEM observation was performed at an accelerating voltage of 10 kV. Images of 500 bacterial cells were obtained, classified into two types according to the staining intensity, and the proportion was obtained.

[0062] In the colony-forming unit, once frozen and lyophilized bacteria are diluted 10-step under anaerobic conditions using anaerobic PBS, inoculated onto a Columbia blood agar plate, and cultured at 37 °C for 48 to 72 hours. Bacterial viability is calculated as the ratio of the number of bacteria colonized by the culture to the number of bacteria at the beginning of the culture.

[0063] FIG. 4 illustrates a result obtained by measuring the viability of bacteria after freezing and freeze-drying with the method of the present invention (scanning electron microscopy), the flow cytometry method, or the colony-forming unit. As a result of the comparison, there is no statistically significant difference in the viability obtained by the three types of method, whether after freezing or after freeze-drying.

[0064] As another example, bacteria (Akkermansia muciniphila) are suspended in Mueller Hinton medium (MHB) or a medium containing an antioxidant, and then left at room temperature for one hour under anaerobic or aerobic conditions.

[0065] FIG. 5 illustrates results of a proportion of bacteria obtained by the method of the present invention and a viability obtained by the colony-forming unit, in the same manner as that of the example described above. As a result of the comparison, there is no statistically significant difference in the viability obtained by the two methods under either condition.

[0066] As illustrated in these examples, a correlation is observed between the viability obtained by the state-of-the-art viability test method for the microbe and the proportion of the microbe obtained by the method of the present invention. In the method of the present invention, by observing the morphology of the individual microbe with the resolution of the electron microscope, it is also possible to analyze the presence or absence of the antioxidant in the medium and the abnormal morphology caused by exposure to oxygen. In other words, it can be seen that, according to the embodiment, the type and morphological characteristic of the microbe can be observed under the electron microscope and the viability can be evaluated.

[0067] [Third embodiment]

[0068] A third embodiment describes an example of quantifying the effect of performing a treatment having a bactericidal action on a microbe by analyzing an electron microscope image of the microbe stained with PTA.

[0069] As an example, in FIG. 6, a strain of Pseudomonas aeruginosa is used as a material, bacteria in a control group not treated with the antibacterial agent and bacteria in a test group treated with the antibacterial agent colistin are cultured at 37°C for 30 minutes, the bacteria are collected on a track-etched membrane and fixed with 2.5% glutaraldehyde, a specimen is prepared by performing electron staining for 2 minutes using an aqueous solution of PTA having a weight concentration of 10% as a dye and an aqueous solution of platinum blue for comparison, and a backscattered electron image is obtained by SEM observation at an accelerating voltage of 5kV.

[0070] In the specimen stained with the PTA aqueous solution, a bacteria image with low image brightness is observed in the control group, and a bacteria image with high image brightness is observed in the test group. On the other hand, in the specimen stained with the platinum blue aqueous solution, the image brightness of the bacteria in both the control group and the test group is strong, and no significant difference is observed between the control group and the test group.

[0071] As another stain not shown in the figure, in a specimen stained with PTA aqueous solution whose pH is adjusted to a neutral value, the brightness of the bacteria image in the test group is as low as in the control group. On the other hand, in sodium tungstate aqueous solution or ammonium molybdate aqueous solution, no positive staining image is observed in both the control group and the test group. Therefore, it can be seen that no significant difference is observed between the control group and the test group in these three types of staining.

[0072] As described above, by selecting the PTA aqueous solution having a weight concentration of 10% as the dye, the bacteria in the control group not treated with the antibacterial agent and the bacteria in the test group treated with the antibacterial agent and thus affected can be stained based on the difference in image brightness.

[0073] In FIG. 6, the influence of colistin on Pseudomonas aeruginosa is detected by PTA staining, but the purpose of the embodiment is not limited to this combination. As another example, when Escherichia coli or Pseudomonas aeruginosa is treated with the β-lactam antibiotic imipenem having bactericidal action, bacteria having high image brightness are enhanced by PTA staining.

[0074] As described above, when an electron stain that increases the staining intensity of a microbe susceptible to bactericidal action is selected, it is possible to classify images of bacteria undergoing damage by bactericidal action and bacteria not undergoing damage based on a difference in image brightness under an electron microscope. In particular, strong PTA staining for colistin-treated bacteria is considered to be associated with life and death caused by damage to the membrane and cell wall. In other words, by classifying and quantifying bacteria stained with PTA, the bactericidal effect of the antibacterial agent colistin can be analyzed and evaluated.

[0075] The embodiment is not limited to this example, and when a user takes advantage of the fact that the intensity of the electronic coloring varies depending on the state of the microbe at the time of performing the treatment that affects the microbe, a control group image and a test group image are obtained, a microbe image brightness range that characterizes a control group microbe image and a microbe image brightness range that characterizes a test group microbe image having a brightness different from that of the control specimen are determined, and the control group and test group microbe images are classified and the proportion is obtained, such that the effect of the treatment affecting the microbe can be analyzed and evaluated.

[0076] For example, as an action of the antibacterial agent, many antibacterial agents that affect the cell wall of bacteria, prevent the growth of bacteria, or exhibit bactericidal action are developed. Since a human cell does not have a cell wall, a compound that selectively acts on the cell wall of bacteria should have low side effects on humans. The method of this embodiment can be used to evaluate the effect of such compounds, or to screen a new compound based on a mechanism of action.

[0077] In order to treat an infectious disease and control an infection in a hospital, it is important to quickly determine whether the pathogenic bacteria are resistant or not, and a drug susceptibility test is performed for each bacterium isolated from a patient. In a state-of-the-art method, resistance is determined from a minimum inhibitory concentration (MIC) value that inhibits the growth of bacteria, and it takes about one day of cultivation to obtain the MIC. On the other hand, in this embodiment, it is possible to analyze the presence or absence of damage caused by the influence of the antibacterial agent, and to quickly determine the susceptibility of the pathogenic bacteria.

[0078] As another example, this embodiment can also be used to evaluate a bacteriophage that infects bacteria and causes bacteriolytic action. A phage destroys the membrane and peptidoglycan to kill bacteria. Phage therapy is being studied as one of the treatments for multidrug-resistant bacteria, and requires a method for rapidly selecting a combination of phages specific for pathogenic bacteria from a phage library in the environment of artificially modified phages. In this embodiment, the bacteriolytic property of the phage is evaluated, thus enabling it to be used for phage screening.

[0079] As another example, when the microbe is exposed to heat or oxygen, including not only bacteria, but also fungi and archaea, and the deterioration of viability caused by damage to the microbe is assessed, the assessment can be used to select storage conditions for the microbe or a sterilization method.

[0080] As a specific example of analysis of treatment affecting the microbe, FIG. 7 shows the result of treatment of Pseudomonas aeruginosa with the antibacterial agent colistin and the analysis of the time variation of the influence of colistin on the image brightness of bacteria.

[0081] A strain of Pseudomonas aeruginosa with known colistin sensitivity from the MIC value measured by a state-of-the-art method and a strain of Pseudomonas aeruginosa with known colistin sensitivity are used. A bacterial solution suspended in a medium at an initial concentration of 10 6 / ml is treated with colistin at 2 mg / l, which is the concentration that distinguishes between susceptible and resistant bacteria by the state-of-the-art method, is treated and cultured at 37°C, and a certain amount of the bacterial solution is sampled over time.

[0082] A track-etched polycarbonate membrane with a hole diameter of 0.2 μm is used as the specimen equipment for SEM observation of bacteria, and the bacteria are uniformly collected in a certain area on the membrane. Conductivity is imparted to the specimen by pre-spraying platinum-palladium on the membrane surface.

[0083] After washing a medium component on the bacteria collection surface with physiological saline, the bacteria are treated and fixed for 5 minutes with a 2.5% glutaraldehyde fixation solution having a protein crosslinking effect to prevent morphological change of the bacteria. After washing the excess fixation solution with water, the bacteria are treated with a PTA aqueous solution having a weight concentration of 10% for 2 minutes and stained. After washing the excess dye with water, the bacteria are dried on the track-etched membrane to form a specimen for SEM observation.

[0084] The SEM observation conditions are set at an accelerating voltage of 5 kV, backscattered electron detection, and a magnification of 7000 times. The reason why the accelerating voltage is set at 5 kV is that a hole image of the membrane tracked by an electron beam transmitted through a bacterial cell does not overlap with an image of Pseudomonas aeruginosa. The reason for setting the magnification to 7000 times is to observe the morphology of Pseudomonas aeruginosa, and the embodiment is not limited to this.

[0085] As an observation step, before observing the bacterial specimen, a brightness standard sample is used and the brightness contrast of the SEM is set to be constant so that the brightness contrast of the SEM is made constant in the image acquisition of all specimens. Therefore, the image analysis to be performed later becomes easier.

[0086] The backscattered electron image series (701) in FIG. 7 is an example of an image acquired by SEM observation using a colistin-sensitive strain of Pseudomonas aeruginosa. The backscattered electron image (702) is an example of an image obtained by SEM observation of microbe 1 observed in a specimen at 0 minutes of culture. The backscattered electron image (702) shows Pseudomonas aeruginosa unaffected by colistin, and is the same as that of an untreated control specimen. The backscattered electron image (703) is an example of an image obtained by SEM observation of microbe 2 observed after 8 minutes of culture, and shows Pseudomonas aeruginosa whose membrane and cell wall are damaged by the influence of colistin and stained with PTA.

[0087] The brightness profile relative to the brightness of the image (702) of microbe 1 is acquired to determine the image brightness range 1 (108) illustrated in FIG. 1. The brightness profile relative to the brightness of the image (703) of microbe 2 is acquired to determine the image brightness range 2 (107) illustrated in FIG. 1.

[0088] Then, SEM observation is performed on each specimen from which the backscattered electron image series (701) was obtained and one specimen in the control group prepared at the same time, and twenty-five images per specimen and images of 300 to 500 bacterial cells are acquired. The number of images taken and the number of bacterial cells to be imaged are not limited to the numbers indicated above as long as the reproducibility of the quantitative analysis can be ensured.

[0089] An image region of microbes is extracted from all images taken for each specimen, and is classified into microbe 1 characterized by image brightness range 1 (108) and microbe 2 characterized by image brightness range 2 (107), thus determining whether an individual microbe is an individual unaffected by colistin or an individual whose cell membrane and cell wall are damaged by the influence of colistin. The classified individuals are counted, and a proportion of microbe 1 is calculated by counting the total number of microbes 1 and 2 as the total number of microbes. Since microbe 1 is unaffected by the influence of colistin and is considered to be alive, a change in the proportion of microbe 1 corresponds to a change in viability.

[0090] Graph (704) shows the change over time in the proportion of microbe 1 contained in bacteria sampled from the control group, and graph (705) shows the change over time in the proportion of microbe 1 contained in bacteria sampled from the colistin-treated test group. In the control group, the proportion of microbe 1 barely changes, but in the test group, the proportion of microbe 1 is reduced to 80% after 8 minutes, 60% after 30 minutes, and 0% after 60 minutes. This result indicates that the strain of Pseudomonas aeruginosa to be analyzed is sensitive to colistin.

[0091] Furthermore, when the colistin-resistant strain of Pseudomonas aeruginosa is treated with colistin in the same way as the sensitive bacteria, the proportion of microbe 1 in the control group corresponding to graph (704) and the proportion of microbe 1 in the test group corresponding to graph (705) are barely reduced, and are about 95%, even after 60 minutes. This result indicates that colistin has no influence on the strain of Pseudomonas aeruginosa to be analyzed.

[0092] As described above, it is possible to show a difference between sensitive and resistant bacteria by quantitatively analyzing and comparing the influence of colistin treatment on Pseudomonas aeruginosa, and the result of determining sensitivity and resistance is consistent with the determination based on MIC value which is the state-of-the-art method.

[0093] While it takes about one day of cultivation to obtain the MIC with the state-of-the-art method, depending on the embodiment, it is possible to quickly determine the sensitivity and resistance of pathogenic bacteria by analyzing the presence or absence of morphological damage caused by the influence of the antibacterial agent.

[0094] Here, features of the specimen in the embodiment will be described. Other viability tests using a microscopic observation technique are described, and a reagent for determining life and death dedicated to the optical microscope is based on the enzyme activity of the living cell and the selective permeability of the cell membrane, and the liveness at the time of observation is a prerequisite for staining. Furthermore, since a target to be stained with PTA is morphologically damaged in the process of killing bacteria, the target can be stained and observed even after being fixed with glutaraldehyde and the like and stored, thereby improving the practicality of the viability test.Since desktop SEM can be used in recent years, desktop SEM can be applied to daily microbial testing such as infectious disease testing for microbe, environmental monitoring, and food safety testing.

Claims

23 Demands 1. A method for analyzing images of microbes, comprising: a first step for obtaining an image of a specimen in which a sample containing microbes is stained, using an electron microscope; a second step for obtaining a brightness profile relative to a range of brightness distribution of the image; a third step for defining a first standard brightness range that satisfies a first condition relating to the brightness of the profile as a region where a first group of microbes exists, and defining a second standard brightness range that satisfies a second condition relating to the brightness of the profile as a region where a second group of microbes exists;and a fourth step used to identify individual microbes that exist in each of the first and second standard brightness ranges and / or to calculate a proportion of the microbe that exists in each of the first and second standard brightness ranges.

2. A method for analyzing microbe images according to claim 1, wherein the first condition includes a first peak in the brightness profile and does not include a second peak that is different from the first peak, the second condition does not include the first peak and includes the second peak, and the fourth step is used to identify the life or death of an individual microbe and / or individual microbes that are morphologically damaged in each of the first and second standard brightness ranges and / or to calculate the viability of microbes and / or a proportion of a microbe that is morphologically damaged on the basis of the number of microbes and / or an image area of ​​microbes, and the number of image pixels in each of the first and second standard brightness ranges.

3. Microbe image analysis method according to claim 1, wherein the specimen is a spread specimen which is prepared from a microbial suspension in order to prevent microbes and solid matter from overlapping.

4. Microbe image analysis method according to claim 1, wherein the first step is used to perform a chemical fixation operation on microbes using at least one compound among glutaraldehyde, formalin and an alcohol in order to stop the morphological modification and preserve the shape of the microbes.

5. Microbe image analysis method according to claim 1, wherein the first step is used to stain the sample using a phosphoric acid solution.

6. Microbe image analysis method according to claim 5, wherein the phosphotungstic acid solution is an acidic solution which is adjusted from pH 0.0 to pH 7.

0.

7. Microbe image analysis method according to claim 5, wherein the phosphotungstic acid solution is an acidic solution which is adjusted from pH 0.0 to pH 3.

0.

8. Method for analyzing microbe images according to claim 5, in in which the concentration of the phosphotungstic acid solution is from 0.1% to 20%.

9. Microbe image analysis method according to claim 5, wherein the concentration of the phosphotungstic acid solution is from 2% to 10%.

10. Kit for implementing the microbe image analysis method according to claim 5, comprising: a solution containing chemical fixatives which contains at least one compound from glutaraldehyde, formalin, and an alcohol; a dye which contains at least phosphotungstic acid; and a washing solvent which contains at least one element from physiological saline, water, and a buffer solution.

11. A method for analyzing microbe images, comprising: a first step for obtaining a first image of a control specimen in which a sample containing microbes is stained without performing any treatment that affects the microbes, using an electron microscope; a second step for obtaining a second image of a test specimen in which a sample containing microbes is stained without performing any treatment that affects the microbes, using the electron microscope; a third step for obtaining a brightness profile relative to the brightness of the first image, and a first peak of the brightness of the profile; and a fourth step for obtaining a brightness profile relative to the brightness of the second image, and a second peak of the brightness of the profile.

12. Method for analyzing images of microbes according to claim 11, in 26 in which the third step is used to obtain a first standard brightness range including the first peak based on a microbial species, a type of treatment that affects microbes and a type of dye, and the fourth step is used to obtain a second standard brightness range including the second peak based on a microbial species, a type of treatment that affects microbes and a type of dye.

13. Microbe image analysis method according to claim 12, further comprising: a fifth step for obtaining the number of microbes, the microbe image area and / or the number of pixels and calculating a proportion of the microbe that exists in each of the first and second standard brightness ranges, and quantifying an effect of treatments that affect microbes by comparing the control specimen and the test specimen.

14. Microbe image analysis method according to claim 11, wherein the treatment affecting the microbes consists of giving the microbes at least one treatment among an antimicrobial, heat, oxygen, detergent, and bacteriophage.

15. Microbe image analysis method according to claim 12, wherein the dye is phosphotungstic acid.

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