Classifications

• • G—PHYSICS
  • G06—COMPUTING OR CALCULATING; COUNTING
  • G06V—IMAGE OR VIDEO RECOGNITION OR UNDERSTANDING
  • G06V20/00—Scenes; Scene-specific elements
  • G06V20/60—Type of objects
  • G06V20/69—Microscopic objects, e.g. biological cells or cellular parts
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/18—Testing for antimicrobial activity of a material
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
• • G—PHYSICS
  • G06—COMPUTING OR CALCULATING; COUNTING
  • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
  • G06T7/00—Image analysis
  • G06T7/0002—Inspection of images, e.g. flaw detection
  • G06T7/0012—Biomedical image inspection
• • G—PHYSICS
  • G06—COMPUTING OR CALCULATING; COUNTING
  • G06V—IMAGE OR VIDEO RECOGNITION OR UNDERSTANDING
  • G06V10/00—Arrangements for image or video recognition or understanding
  • G06V10/70—Arrangements for image or video recognition or understanding using pattern recognition or machine learning
  • G06V10/74—Image or video pattern matching; Proximity measures in feature spaces
  • G06V10/75—Organisation of the matching processes, e.g. simultaneous or sequential comparisons of image or video features; Coarse-fine approaches, e.g. multi-scale approaches; using context analysis; Selection of dictionaries
  • G06V10/758—Involving statistics of pixels or of feature values, e.g. histogram matching
• • G—PHYSICS
  • G06—COMPUTING OR CALCULATING; COUNTING
  • G06V—IMAGE OR VIDEO RECOGNITION OR UNDERSTANDING
  • G06V20/00—Scenes; Scene-specific elements
  • G06V20/60—Type of objects
  • G06V20/69—Microscopic objects, e.g. biological cells or cellular parts
  • G06V20/693—Acquisition
• • G—PHYSICS
  • G06—COMPUTING OR CALCULATING; COUNTING
  • G06V—IMAGE OR VIDEO RECOGNITION OR UNDERSTANDING
  • G06V20/00—Scenes; Scene-specific elements
  • G06V20/60—Type of objects
  • G06V20/69—Microscopic objects, e.g. biological cells or cellular parts
  • G06V20/695—Preprocessing, e.g. image segmentation
• • G—PHYSICS
  • G06—COMPUTING OR CALCULATING; COUNTING
  • G06V—IMAGE OR VIDEO RECOGNITION OR UNDERSTANDING
  • G06V20/00—Scenes; Scene-specific elements
  • G06V20/60—Type of objects
  • G06V20/69—Microscopic objects, e.g. biological cells or cellular parts
  • G06V20/698—Matching; Classification
• • G—PHYSICS
  • G06—COMPUTING OR CALCULATING; COUNTING
  • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
  • G06T2207/00—Indexing scheme for image analysis or image enhancement
  • G06T2207/10—Image acquisition modality
  • G06T2207/10056—Microscopic image
• • G—PHYSICS
  • G06—COMPUTING OR CALCULATING; COUNTING
  • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
  • G06T2207/00—Indexing scheme for image analysis or image enhancement
  • G06T2207/30—Subject of image; Context of image processing
  • G06T2207/30004—Biomedical image processing
  • G06T2207/30024—Cell structures in vitro; Tissue sections in vitro
• • G—PHYSICS
  • G06—COMPUTING OR CALCULATING; COUNTING
  • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
  • G06T2207/00—Indexing scheme for image analysis or image enhancement
  • G06T2207/30—Subject of image; Context of image processing
  • G06T2207/30242—Counting objects in image

Definitions

• the present invention relates to a method for analyzing images of microbes.
• a drug susceptibility test method 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 a light microscope and a microbe stained with a substance that stains only a dead cell (se refer to JP-A-2001-509008).
• 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.
• an object of the present invention is to provide a microbe image analysis method which 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.
• a method for analyzing images of microbes comprises: 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 relating to the brightness of the image; a third step for defining a first standard luminosity range which meets 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 luminosity range which meets a second condition relating to the profile brightness 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.
• 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 a electronic microscope.
• 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 classification of images of microbes
• FIG. 4 is a diagram illustrating a viability test result of a microbe after a 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 coloring
• FIG. 7 is a diagram illustrating an image analysis result of a microbe treated with an antibacterial agent.
• one of the important basic techniques is microscopic observation, which uses an image of an individual microbe and a group of microbes.
• microscopic observation uses an image of an individual microbe and a group of microbes.
• 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 based on the appearance of 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.
• an electron microscope is used. As 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 an optical microscope can be observed. Examples of fine morphology include species-specific structure of the microbe, structure observed during cell division, abnormal morphology caused by the influence of a drug, and their equivalents.
• a state-of-the-art electron microscope requires large-scale mechanisms such as a mechanism that applies a high voltage to generate a beam stable electron beam, a mechanism for keeping the inside of the microscope in a vacuum state to prevent scattering of the emitted electron beam, and their equivalents that make it difficult to use the electron microscope's state of art in a daily microbial test as in clinical practice or its equivalents.
• a benchtop electron microscope which has been achieved by improving the state-of-the-art electron microscope, has been developed, and it is also planned to apply it to a microbial sample.
• staining method is limited.
• stains corresponding to various purposes such as cell biology, histology, pathology and their equivalents are developed, improved, and various kinds of staining methods for staining component and function of a cell and a tissue with different colors are drawn.
• the electron microscope uses a method of visualizing a structure of the whole microbe such as a cell membrane, 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 of visualizing the location of a specific molecule with a marker consisting of fine gold particles.
• a dye containing a heavy metal such as uranium, lead, platinum, osmium and their equivalents
• An example of a second aspect of the prior art is a test for viability of a microbe, which is one of the important test items in a microbial test.
• a test for viability of a microbe which is one of the important test items in a microbial test.
• the viability of a microbe in a sample treated with these treatments is measured, thereby determining treatment type, treatment method, treatment intensity, treatment concentration, and their equivalents.
• an ordinary method is a culture method such as a colony forming unit and its equivalents.
• a culture method such as a colony forming unit and its equivalents.
• a certain amount of a microbial suspension of known concentration is thinly spread on an agar medium and cultured for about a day, the number of colonies of bacteria growing in a certain period of time is considered as the number of bacteria surviving at the start of cultivation, a colony forming unit (CFU/ml) is calculated from the number of colonies against a liquid amount of seeded bacteria, and the calculated colony forming unit is taken as the viability at the start of the culture.
• CFU/ml colony forming unit
• the culture method is the most widely used method to test the viability of a microbe.
• 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 medium type and its equivalents are unknown.
• a method using the optical microscope and its equivalents which does not require culture has been developed.
• the method include a method for detecting a function such as 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 substances of cell membranes of a living cell and a dead cell, a method for detecting a morphological change, and the like.
• the methods described above are excellent in terms of speed, because it is not necessary to wait for the culture time until the bacteria grow and form a visible colony.
• pieces of information about the fine morphology of a microbe with the resolution of the electron microscope such as a species-specific structure of the microbe, an observed structure during cell division, abnormal morphology caused by drug influence and their equivalents are harvested, and the usefulness of a technique to perform viability test on the same specimen is also considered.
• a type and condition is harvested from morphological information of a biofilm-forming microbe, treatment is performed on the biofilm with an antimicrobial, bactericide, and their equivalents 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, thus making it possible to evaluate the effect of the antimicrobial and the bactericide in situ (in situ observation).
• the viability test method using the electron microscope is not practiced in microbial tests such as clinical and environmental tests.
• the reason that 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.
• a protein cross-linking 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 assay as described in the optical detection example that visualizes a viability function of a truly living microbe such as a difference and its equivalents at the l enzymatic activity and biological membrane permeability by staining.
• An example of another method is correlative optical and electron microscopy in which a colored image obtained by the optical microscope and a morphological image obtained by the electron microscope are associated with each other.
• this method although it is possible in principle to process the same sample and observe the same sample under the electron microscope after determining whether the sample is alive or dead under the light microscope, operations such as sample preparation , image overlay and their equivalents are not straightforward, and therefore difficult to apply to everyday microbial testing.
• an aqueous solution of phosphotungstic acid (hereinafter PTA ) is used as a stain that strongly adheres to a microbe undergoing morphological damage associated with the life and death of the microbe, and an image showing compositional contrast is acquired with the backscattered electrons of a scanning electron microscope (referred to below). after SEM), thus providing a method for assessing a proportion of the microbe corresponding to viability.
• PTA phosphotungstic acid
• the proportion is acquired such that when a microbe in a suspension and its equivalents are stained with an aqueous solution of PTA, a difference of 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 brightness of image, and each microbe is quantified.
• This proportion is defined as the viability according to the present invention.
• the proportion of living microbes at the time of SEM image acquisition is not shown, but the embodiment shows that a proportion equivalent to the viability by a method of the state of the art is obtained.
• a method of using PTA as a colorant in the present invention will be described.
• dyes such as an aqueous solution of uranium acetate, an aqueous solution of lead salt, platinum blue and their equivalents are often used.
• These stains are excellent stains for visualizing intracellular structures such as a cell membrane, nucleus, and their equivalents by image contrast using a difference in affinity with a cell-forming component, but are not not used for a staining method to identify 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 daily microbial testing due to their toxicity, stability in solution, cost of reagents, and their equivalents.
• PTA is a reagent which is relatively cheap, easy to store and easy to use as described above, the inventor considers that PTA could be applied to staining electronics of a microbe in a daily microbial test.
• PTA is widely used for the preparation of microscopic samples. For example, PTA is used as a staining mordant for light microscopy specimen in pathological examination, and used as a negative stain for microbe observation at the electronic microscope.
• PTA is also used for positive staining at the time of observing a cell by 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 part 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.
• the inventor found that after bacteria with known susceptibility are treated with an antibacterial agent exhibiting bactericidal action that damages cell membrane and cell wall, a PTA-stained specimen is prepared, an image of backscattered electrons from the SEM is obtained, individual bacteria with high image brightness are identified in the specimen, and a proportion of individuals with high image brightness increases as the antibacterial treatment time elapses.
• 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 solution of PTA at a concentration of 10% by weight for approximately 2 minutes, and a backscattered electron image is acquired at an accelerating voltage of 5 kV to 10 kV using SEM.
• the microbe contained in the specimen treated with the bactericide is mainly classified into two groups, a microbe having high image brightness and a microbe having 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.
• the inventor provides a method for knowing the type of a microbe and a morphological characteristic thereof with an electron microscope, and easily identifying a microbe undergoing associated morphological damage. to life and death and a microbe not undergoing morphological damage by image brightness arising from the intensity of electron staining using PTA.
• 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 low coloration and low image brightness and a microbe 2 (103) having strong coloration and high image brightness are mixed by means of a schematic diagram electron microscope images (101) of a specimen in which the microbe is placed on the equipment and stained.
• the image of microbes is classified into two groups including microbe 1 and microbe 2 based on the difference in image brightness.
• 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 in such a way that a strain of Pseudomonas aeruginosa is placed on a polycarbonate membrane with etched tracks, the strain of Pseudomonas aeruginosa is fixed with 2.5% glutaraldehyde and stained with an aqueous solution of PTA at a concentration of 10 % by weight, a specimen in which Pseudomonas aeruginosa is placed on the membrane is inserted into a SEM sample chamber, the accelerating voltage of SEM is set to 5 kV, and the brightness contrast at the time of generation of images is set to be constant. As the conditions for generating SEM images are constant, the difference in brightness between microbe 1 (102) in image (104) and microbe 2 (103) in image (105) arises from a difference PTA staining intensity.
• the etched track membrane made of a plastic material is used as the equipment, and the equipment is not limited thereto.
• a brightness profile (106) associated with image brightness (104) and a brightness profile (107) associated with image brightness (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.
• an image region of microbe 1 is characterized by a range of standard luminosity 1 (108) having a peak 1 of the luminosity profile (106) relative to the luminosity
• an image region of the microbe 2 is characterized by a standard luminosity range 2 (102) comprising a peak 2 of the luminosity profile (107) relative to brightness, such that the images of microbe 1 and microbe 2 are classified.
• the standard luminosity range 1 when the SEM brightness contrast is set to be constant at the time of acquisition of the image (104), image (105), image in a different region of the same specimen, and of images of a plurality of specimens prepared with the same material and the same method, the standard luminosity range 1
• FIG. 2 illustrates examples (201 and 202) of adjusting the standard luminosity range.
• 201 and 202 are the same histograms and are brightness profiles associated with the brightness of the electron microscope image. As shown in the schematic diagram (101) of FIG. 1, since 201 and 202 arise from the image in which microbes with different image brightness are mixed, peak 1 and peak 2 exist in examples 201 and 202.
• peak 1 Another peak located on 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 range of standard luminosity which characterizes the image of microbes.
• the standard brightness range 1 including peak 1 from an image of microbe 1 is determined, and a standard brightness range including peak 2 from an image of microbe 2 is determined within a range that does not overlap with standard luminosity range 1, such that microbe 1 characterized by standard luminosity range 1 and microbe 2 characterized by standard luminosity range 2 are classified .
• the range of brightness standard 1 including both peak 1 resulting from the image of microbe 1 and peak 2 resulting from the image of microbe 2 is determined, and the standard brightness range 2 including peak 2 without including peak 1 is defined , which thus makes it possible to classify all the microbes contained in the image, the microbe 2 characterized by the standard luminosity range 2, and the microbe 1 obtained by subtracting the microbe 2 from all the microbes.
• FIG. 3 illustrates an example in which the image of microbes is classified based on a difference in brightness of the electron microscope image using image analysis software, and an image mask is generated for each classification.
• FIG. 1 in an electron microscope image (301) of a microbial specimen, microbe 1 (102) having low staining and low image brightness and microbe 2 (103) having high staining and high image brightness picture are mixed.
• the mask (302) is an image with mask generated in such a way that the brightness range of the equipment is excluded, the image is binarized by defining a threshold value from the standard brightness range 1 comprising at the times peak 1 from the image of microbe 1 and peak 2 from the image of microbe 2, and particles significantly smaller than microbes 1 and 2 are then removed using a particles.
• 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.
• the image analysis software the individual microbe classified inside the mask 1 and the 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.
• the microbial specimen is stained to generate the backscattered electron image, the brightness profile relative to the image brightness of the microbe 1 (104) having low coloration and low image brightness and microbe 2 (105) having strong coloration and high image brightness is acquired, and the microbe image is classified by the range of brightness d image, so that the proportion of microbe 1 and microbe 2 in the specimen can be easily calculated.
• the types of standard luminosity range can amount to more than two.
• 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.
• art of a viability assay, and a flow cytometry method which is a culture-free method.
• bacteria As an example, bacteria (Akkermansia muciniphila) are cultured under anaerobic conditions on an agar medium for 48 hours and isolated. Then, the isolated bacteria are suspended at a bacterial concentration of approximately 10 10 CFU/ml in a storage medium containing an antioxidant, and are frozen and freeze-dried at ⁇ 80° C. for 24 hours.
• the bacteria After being frozen and lyophilized, the bacteria are stained with 10% PTA for 5 minutes at 37°C, the bacterial suspension is spread on a glass slide by centrifugation and dried at room temperature, and SEM observation is performed. at an acceleration voltage of 10 kV. Images of 500 bacterial cells are obtained, classified into two types based on staining intensity, and the proportion is obtained.
• frozen and freeze-dried bacteria are diluted in 10 steps under anaerobic conditions using anaerobic PBS, inoculated onto a Columbia blood agar plate, and cultured at 37°C for 48 to 72 hours.
• the viability of the bacteria is calculated as the ratio between the number of bacteria colonized by the culture and the number of bacteria at the start of the culture.
• FIG. 4 illustrates a result obtained by measuring the viability of bacteria after freezing and lyophilization 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, either after freezing or after lyophilization.
• bacteria As another example, bacteria (Akkermansia muciniphila) are suspended in Mueller Hinton (MHB) medium or medium containing an antioxidant, and then left at room temperature for one hour under anaerobic or aerobic conditions.
• MLB Mueller Hinton
• 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 in either condition.
• a third embodiment describes an example of quantification of 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.
• a strain of Pseudomonas aeruginosa is used as the 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 electronic staining for 2 minutes using an aqueous solution of PTA having a concentration by weight of 10% as a dye and an aqueous solution of platinum blue for comparison
• a backscattered electron image is obtained by SEM observation at an accelerating voltage of 5 kV.
• 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 therefore affected can be colored based on the difference in image brightness.
• the influence of colistin on Pseudomonas aeruginosa is detected by PTA staining, but the purpose of the embodiment is not limited to this combination.
• Escherichia coli or Pseudomonas aeruginosa is treated with the ⁇ -lactam antibiotic imipenem with bactericidal action, bacteria with high image brightness are enhanced by PTA staining.
• 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 which affects the microbe, a control group image and a test group image are obtained, a microbe image brightness range which characterizes a control group microbe image and a microbe image brightness range which characterizes a microbes of the test group having a brightness different from that of the control specimen are determined, and the images of microbes of the control group and the test group are classified and the proportion is obtained, so that the effect of the treatment affecting the microbe can be analyzed and evaluated.
• the antibacterial agent many agents antibacterials which affect the cell wall of bacteria, inhibit the growth of bacteria or exhibit bactericidal action are being developed. Since a human cell does not have a cell wall, a compound that acts selectively on the cell wall of bacteria should have low side effects in humans.
• the method of this embodiment can be used to assess the effect of such compounds, or to screen a new compound based on a mechanism of action.
• a drug susceptibility test is performed for each bacteria isolated from a patient.
• resistance is determined from a minimum inhibitory concentration (MIC) value that inhibits the growth of bacteria, and it takes about a day of culture to obtain the MIC.
• MIC minimum inhibitory concentration
• 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 investigated as one of the treatments for multidrug resistant bacteria, and requires a method to rapidly select a combination of phages specific to pathogenic bacteria from a phage bank in the environment of artificially modified phages.
• the bacteriolytic property of the phage is assessed, thus allowing it to be used for phage screening.
• evaluation can be used to select conditions of storage of the microbe or a method of sterilization.
• FIG. 7 shows the result of the treatment of Pseudomonas aeruginosa with the antibacterial agent colistin and the analysis of the time variation of the influence of colistin by the image brightness of bacteria.
• a strain of Pseudomonas aeruginosa whose sensitivity to colistin is known from the MIC value measured by a method of the state of the art and a strain of Pseudomonas aeruginosa whose sensitivity to colistin is known 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 sensitive bacteria and resistant bacteria by the state method. of the art, is treated and cultured at 37°C, and a certain amount of the bacterial solution is sampled over time.
• a track-etched polycarbonate membrane having a hole diameter of 0.2 ⁇ m is used as the specimen equipment for SEM observation of bacteria, and the bacteria are uniformly collected in a certain area on the membrane.
• a conductivity is conferred on the specimen by prior vaporization of platinum-palladium on the surface of the membrane.
• the bacteria After washing a component of the medium on the bacteria collection surface with physiological saline solution, the bacteria are treated and fixed for 5 minutes with a 2.5% glutaraldehyde fixing solution having a protein cross-linking effect in order to to prevent the morphological modification of the bacteria. After washing the excess fixing solution with water, the bacteria are treated with an aqueous solution of PTA having a concentration by weight of 10% for 2 minutes and stained. After washing off the excess dye with water, the bacteria are dried on the etched track membrane to form a specimen for SEM observation.
• the SEM observation conditions are established at an acceleration voltage of 5 kV, the detection of backscattered electrons, and a magnification of 7000 times.
• the reason for which the accelerating voltage is set to 5 kV is that a hole image of the membrane with tracks etched 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 thereto.
• the SEM brightness contrast is set to be constant such that the SEM brightness contrast is made constant in the acquisition images of all specimens. Therefore, image analysis to be performed later becomes easier.
• the series of backscattered electron images (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 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.
• the brightness profile relative to the brightness of the image (702) of microbe 1 is acquired to determine the range of image brightness 1 (108) shown in FIG. 1.
• the brightness profile relative to the brightness of image (703) of microbe 2 is acquired to determine the range of image brightness 2 (107) shown in FIG. 1.
• a microbe image region 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), thereby determining whether an individual microbe is an individual unaffected by colistin or an individual whose 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 not affected 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.
• the graph (704) shows the variation over time of the proportion of microbe 1 contained in the bacteria sampled from the control group
• the graph (705) shows the variation over time of the proportion of microbe 1 contained in the bacteria sampled from the colistin-treated test group.
• the proportion of microbe 1 hardly 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.

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