FI94645B - Method and apparatus for studying the activity of microbial growth inhibiting agents - Google Patents

Description

94645

Method and apparatus for studying the activity of antimicrobial agents

The invention relates to a method for examining the activity of antimicrobial agents set out in the preamble of appended claim 1 and to an apparatus for carrying out the method according to the preamble of appended claim 5.

10 Antimicrobials, such as antibiotics, play an important role in the treatment of infectious diseases. Antibiotics are defined as compounds produced by microbes that have an inhibitory effect on certain (harmful) microbes without adverse effects in 15 host organisms. In other words, antibiotics are selectively toxic.

The susceptibility of bacteria to different antibiotics varies from species to species. The effect of antibiotics on bacteria and, conversely, the sensitivity of bacteria to antibiotics is described by the term antibacterial spectrum (to which bacterium the antibiotic is effective) and antibiotic spectrum (to which antibiotics the bacterium in question is sensitive).

25 Choosing the right antibiotic has a big impact: on the outcome of treatment in infectious diseases. However, the selection of the most effective antibiotics for a particular bacterium cannot be made in vivo or on the basis of the above-mentioned indicators alone, but must be tested under laboratory conditions with a susceptibility analysis. Naturally, the final "correct" antibiotic * treatment will be determined on a clinical basis once the best options have been identified in an in vitro assay.

35 Susceptibility testing is not a one-off, ie susceptibility testing is performed on the same bacterium time and time again. This is due to e.g. diversity of bacterial strains and new antibiotics.

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The susceptibility analysis aims to grow the bacterium under '• standard conditions' (temperature, growth time, concentrations, etc.) in the presence of the antibiotic. However, the result of the susceptibility test is indicative only, as laboratory conditions are different from those in the infection center.

Sensitivity assays mainly use two types of methods: dilution and diffusion methods. The dilution method allows the bacteria to grow at varying antibiotic concentrations to determine the Minimum Inhibitory Con-15 concentration (MIC).

The diffusion method is more used than the dilution method. In this context, the Paper Disk 20 Method (PDM) is also discussed. In PDM, the bacterium is grown in an agar plate containing medium. Prior to the actual growth, i.e. incubation, paper discs containing various antibiotics are added to the plate. The bacterial growth is then allowed to develop under standard conditions in a heat cabinet (temperature 35 ° C to 37 ° C).

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From the paper disc, the antibiotic diffuses into the bacterial culture, to which a bacterial-free circular zone, a barrier ring, is formed proportional to the action of the antibiotic. The diameter of this barrier ring indicates the susceptibility of the bacterium in question. antibiotic. However, the size of the diameter does not absolutely indicate the efficacy of the antibiotic, but the final choice also depends on the application, the bacterium (e.g. growth rate) 35 and the antibiotic (e.g. diffusion rate, molecular weight, charge, side effects). Locking rings are provided

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94645 limit values for each bacterium. Both the limit values of different antibiotics for the same bacterium and the limit values of the same antibiotic for different bacteria naturally vary.

5 The agar dish used as a growth medium is about 14 cm in diameter. The bottom of the dish is coated with a medium into which the cultured bacterial strain is applied. Paper discs (5 mm in diameter) containing the antibiotic are placed on top of the bacterial layer. The discs are placed 10 mechanically in a specific location. The number of discs varies depending on the bacterium being tested and the group of antibiotics used (currently a maximum of 14).

15 Until now, stop rings have been measured completely manually, for example with a caliper or ruler. The measurement results are stored simultaneously in the laboratory information system. When done in this way, routine work takes a lot of time and the measurement phase binds two 20 employees. In addition, there is variation in measurement results between different meters. Also the same meter. may interpret the zones differently as different measurements. Barrier rings do not always have sharp edges, but sliding borders often appear. Especially in 25 such cases, scattering occurs between different meters.

The object of the invention is to eliminate the above-mentioned drawbacks and to present a method by which the measurement of the above-mentioned areas from the growing media can be accelerated and the results made uniform. The object of the invention is also to provide a device with which the above-mentioned objects are achieved. To achieve this object, the method according to the invention is mainly characterized by what is set forth in the characterizing part of appended claim 1, and the device according to the invention is characterized by what is set forth in the characterizing part of appended claim 5.

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The invention uses an automatic measurement system based on pattern recognition. The agar plate on the light table is imaged with a video camera, after which the 5 video signals are digitized on an image memory card in a microcomputer. The digitized image is then analyzed so that based on the distribution of the grayscale values of the pixels (pixels 1. pixels), the grayscale values corresponding to the corresponding dots 10 are located within the region where the antibiotic has affected the microbial growth. The system can also be used to "search" for antibiotic discs, after which any circles formed around them can be identified. The program designed to implement the invention uses a maximum of 20 to 25 seconds and also provides a variety of options for printing, retrieving and saving images, as well as editing the results obtained.

The invention will now be described in more detail with reference to the accompanying figures, in which Figure 1 shows a typical object to be examined by the invention, Figure 2 schematically shows the principle of determining the spectral range of the method used in the invention, Figure 3 schematically shows the principle of determining the radius candidate of the method.

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5 94645 Figure 4 shows an apparatus for carrying out the method, and 5 Figure 5 shows an example of the information displayed on the apparatus during the implementation of the method.

The detection of the blocking ring is in three stages. First, all the antibiotic discs are identified and then the blocking rings formed around them. Finally, the blocking ring number, clarity percentage, and blocking ring diameter are printed. The identification of antibiotic discs is quite simple, but since the darkness of the inhibitory ring varies with culture and medium, the identification of the inhibitory-15 ring, on the other hand, is considerably more complex. In order to achieve the best dynamics, a pattern recognition based on spectral analysis has been concluded.

20 The principle of the method, which is implemented with a device, which will be described in more detail later, is described below.

The medium is initially imaged with a CCD camera. In digital-25 operation, the image taken with a CCD camera is divided into so-called

; the pixels. A pixel is a pixel with a constant grayscale inside. The grayscale value is expressed as an integer. The smaller the pixels in which the image is constructed (resolution) and the larger the grayscale-30 scale, the better the image quality. The number of pixels and the proportions of the image are expressed by an image matrix, which in this application is 768 x 512. The grayscale of each individual pixel in the image matrix is expressed by the number 0 ... 255 (0 = black, 255 = white).

Computer-based pattern recognition is based on a threshold value, i.e. the so-called threshold setting. The shape of the object to be identified is only recognized if 35 6 94645 the grayscale value of the surrounding background is smaller and the grayscale value of the object (character distinguishable from the background) is greater than the threshold value, or vice versa. For example, if the background grayscale varies between 5 50 ... 100 and the subject's grayscale between 150 ... 250, the threshold value should be between 101 ... 149 for the subject's outline to be correctly identified. If the threshold is between 51 ... 100, part of the background is counted as belonging to the target and if threshold 10 is set between 150 ... 249, part of the target is counted as belonging to the background. If, on the other hand, the threshold is greater than 250 or less than 50, no object is detected at all.

15 Determining the correct threshold value is therefore key in character recognition. Only after recognizing the contours of the part can the properties of the part (degree of circularity, area, minimum and maximum radii, etc.) be analyzed. Thus, in normal character recognition 20, the grayscale of the objects must first be known and then •• objects with a certain criterion are extracted from these objects.

Because the grayscale values of the bacterial growth and the bacteria-free region of the agar plates vary widely, no constant threshold of ·· 'can be determined.

On the other hand, the edges of the agar plates and the inhomogeneity of the bacterial growth as well as the cutting of the blocking rings cause the blocking rings to often deviate from the circular shape. As a result, a separate pattern recognition mechanism has been developed for these.

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Although antibiotic discs are "dipped" onto an agar plate with a special machine, the discs are not always located in exactly the right place. Antibiotic disc shifts occur especially during the set-up phase and during the transfer and handling of agar plates.

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Figure 1 shows a typical circular medium A formed by an agar plate. Antibiotic discs 1-13 are marked in the figure. 14. The disc 14 is placed in the area between the discs 3, 4 and 10.

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The analyzer searches the disc for an area of approximately 16 cm2 around the permitted location (center of interest). An area of interest is a square area that is processed and whose pixels are initially analyzed. The measuring cup must therefore always be placed in the same position and in approximately the same position in relation to the camera. The easiest way to position the dish and camera correctly is with the live image displayed on the monitor of the device and the grid placed on it, and this can be done once at the beginning of 15, after which the shots can be performed on standard-sized plates without separate focus. The support base and the cup facing the camera can then have suitable markings for placing the cups in the correct positions so that the discs 20 come in approximately the same places. Usually the discs in the cup stand out clearly from their surroundings. However, due to the large variation in background grayscale, no standard value is used here, and the antibiotic disk is searched for at five different threshold-25 values (140, 120, 100, 80, and 60). Since the shape and size of the • antibiotic disc do not vary (0.5 cm in diameter) and its grayscale value varies only slightly (a small variation is due to automatic exposure), the antibiotic disc can be identified by the following 30 pixel data: - Grayscale value is less than 140 - The ratio of the maximum length to the maximum width is between 1.1 ... 0.9 - The area is between 0.8 ... 0.4 cm2 This examination is performed 14 times, ie the surroundings of each possible permitted antibiotic disc are searched for in the vicinity of the above criteria. These 35 8 94645 criteria reduced any debris and text that would otherwise have been interpreted as an antibiotic disc based on, for example, its grayscale value alone.

5 Once all the antibiotic discs have been found, analyze the grayscale value distribution of the whole image in the 1st spectrum according to the principle shown in Figure 2. The spectrum provides indicative information on the darkness of the bacterial growth. Find the peak of the spectrum, as well as the darkest and lightest point where 10 spectra intersect the 0.2 * maximum value limit. The area between these is the area of interest in the spectrum, i.e. the threshold value of each blocking ring is searched for from this area.

After determining the spectral range of interest in Figure 2, to analyze the environment of each antibiotic disc found, a threshold value is sought that is between the bacterial growth and the grayscale values of the bacterial-free region. In this case, the blocking ring is interpreted 20 correctly. However, the difference in determining the range of interest is that due to the inhomogeneity of bacterial growth and illumination, as well as the shadow created by the edge of the agar plate, no single correct threshold can be set, but a separate threshold must be defined for each blocking ring.

Two larger (normal-distributed) peaks can be observed in Figure 2, one corresponding to the value distribution of the bacterial-free region and the other to the grayscale-30 value of the bacterial growth. The threshold value should therefore be set between the two. The main problem with this method is that it is not possible to see directly from the spectrum which peak corresponds to the bacterial-free and which to the bacterial region, because the bacterial-free region may be darker or lighter-35 pi than the background (unchanged bacterial growth). Sometimes, on the other hand, several different minima arise between the two peaks depending on the spectral resolution used and how close the e.m. two spikes

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9 94645 each other are. These two problems can be eliminated by the procedure shown below with reference to Fig. 3, which can be called the STAM method (spectral-based threshold value determination). The purpose of procedure 5 is thus to determine a threshold value for each blocking ring. The determination of the threshold value of an individual blocking ring proceeds as follows: 1) Determining the grayscale value of the blocking ring 10 Set the area of interest so that its center point is the same as the center point of the antibiotic disc and its area is 1 cm2. In this case, the spectrum shows a peak of the antibiotic disc and the bacteria-free region, of which only the latter falls in the range of interest of the spectrum (cf. Fig. 3, point 1).

2) Monitoring the evolution of the barrier ring peak The area of interest is grown until its area is 25 cm2, continuously monitoring the grayscale peak of the bacteria-free region in the spectrum (cf. Figure 3, point 2).

This is done in order to know as surely as possible which peak of the spectrum corresponds to the peak of the blocking ring. The size of the area of interest is not affected by the size of the barrier ring. In the future, 25 areas of interest will not be changed.

3) Define three threshold candidates for the inhibitory ring Like the grayscale of the barrier ring, the grayscale of the bacterial growth appears in the spectrum as a relatively high peak 30 (Figure 3, item 2). In this case, the treshold value corresponds to the minimum in the spectrum between these two peaks. However, there are several minima in the spectrum and there is no certainty about the peak formed by the bacterial region. In order to obtain more reliable results, a method has been developed in which three threshold candidates are determined instead of one threshold value.

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The procedure is as follows: - If the bacterial growth is darker than the blocking gas, consider the interval of interest in the spectrum from the spectral peak of the blocking ring to the minimum grayscale value of the interval of interest in spectrum 5. If the bacterial growth is lighter than the blocking ring, consider the interval of the spectral point of interest from the spectral peak of the blocking ring to the maximum grayscale value of the spectral point of interest. 10 At the beginning of the analysis, the user is asked if the barrier ring is darker or lighter than the bacterial growth.

- Group the values of the pixels of the considered region of the spectrum into five equal division intervals 15 (Figure 3, point 3). For example, if the bacterial growth is darker than the blocking ring, the peak of the blocking ring is defined at 150 and the minimum spectral spacing is 100, then the spectral values are divided into classes 101-110, 111-120, 121-130, 131-20 140 and 141-150 ( resolution is ten).

- Search for the minimum (height of adjacent spikes greater than the spike in question) from the spike of the blocking ring. If three minima are not found in the first round, the resolution of the spectrum 25 is improved (division interval is reduced) until three minima are found or the division interval is one (original spectrum).

The grayscale value of each minimum found is a thres-30 hold candidate. If three threshold candidates are not found, default values are used for the missing threshold candidates.

Em. the method has ended with e.g. for the following reasons: 35 - The location of the spectral peaks formed by the blocking ring and the bacterium varies (the grayscale value is not constant).

Il 11 94645 - The size of the peaks is proportional to the size of the bacterial growth and the barrier ring, so it is not possible to deduce from the height or size of the peaks where the spike is from.

5 - There are many interference peaks in the spectrum.

- Spectrum peaks are not sharp. If the gray shades of the bacteria-free and barrier ring are close to each other, they will mix with each other.

10 4) Define three radius candidates for the blocking ring

Measure the distance from the center of the antibiotic disc to the nearest point where the profile of the image (i.e., the grayscale value of the pixels as a function of distance) intersects the threshold candidate value (Figure 3, item 15 4). This distance is called the radius candidate, and thus there are three of them on each blocking ring (one for each threshold candidate). This measurement is performed by a pixel data processing technique known per se.

20 5) Select the correct radius From the radius candidates, the one with the highest clarity percentage is selected.

25 An exception is blocking rings that touch the edge of the ‘agar dish. In this case, all pixels that are more than 6 cm from the center of the bowl must be pruned.

30 At the end of the analysis, the blocking ring is still determined. the clarity of the limit, expressed as a percentage. The percentage clarity of the blocking ring is determined by the formula: 35 grayscale fulkopix. ^ _ Grayscale (inner pix.t * 100

The outer pixels are taken at a distance of 5 pixels from the beam candidate from outside the beam candidate, and the inner pixels are taken at a distance of 5 pixels from inside the beam candidate. If the clarity is too low 5 (less than 10%) the program determines whether the k.o. blocking ring zero or oversized. If the grayscale value of the spectral peak of the barrier ring is less than the maximum grayscale value of the threshold candidates of the other barrier rings, the barrier ring is set to zero. Otherwise it is defined as 48 10 mm (= oversized). If the barrier ring is darker than the bacterial growth, the grayscale value is compared in a similar manner to the minimum grayscale value of the threshold candidates of the other barrier rings. One hundred percent clarity is obtained by a circle with a bacteria-free region (block-15 ring) of pure white (grayscale value 255) and a surrounding bacterial growth of pure black (grayscale value 0), or vice versa.

At the end of the analysis, a window will appear on the monitor 20 of the PC belonging to the device, from which the diameter of each antibiotic disc in millimeters and the percentage of clarity can be read. The program also draws the blocking rings and antibiotic disc number it has defined on the so-called agar plate. overlay graphics. Through the result window 25, the results can be easily edited and finally accepted, in which case the results are saved in the file block.xls or the result is discarded. An example of printing is shown in Figure 5.

30 The following is one example of the measuring device and ancillary programs in question. The parts listed are not the only ones in question and can be replaced by compatible parts from other manufacturers that are compatible.

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94645

The measuring device shown in Figure 4 consists of the following modules: - Computer (PC) MikroMikko 5 CX486 (reference C) 5 - Image digitization card DT3851 - CCD camera Panasonic WV-BL600 (reference K) - Color monitor Panasonic BT-H1450Y, not necessary (reference M ) - Planilux light table (reference L).

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The illuminating surface of the light table L is covered with a plate P with an opening for placing the dish in the correct position.

MikroMikko 5 is an ICL 80486-based microcomputer 15 with a 210MB hard disk, a 1.44MB disk drive (3.5 ”) and a VGA graphics card (ATI). In addition to the CPU, the configuration includes a keyboard, VE17C monitor (17 ") and mouse. The operating environment is Windows 3.1.

20 Image digitization card In the DT2867, the image is digitized with a 768 x 512 matrix with an resolution of 8 bits (256 different shades of gray). The card has an M / V input (15-pin male right-angle D-shell connector) for digitizing images and an RGB output (15-pin female high-density right-25 angle D-shell connector) for digitized and real-time image for presentation. The image digitization card is connected to the PC's AT bus in the same way as other option cards.

The WV-BL600 is a light-sensitive 30 CCD camera designed as a surveillance camera. The camera uses a fixed-focal auto iris lens (12mm 1: 1.4 WV-LA12B2) and AGC for better dynamics. The CCD camera has a BNC connector for a video signal. The camera is mounted above the light table with a special stand.

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The agar plates are illuminated from below, allowing the antibiotic discs and barrier rings to stand out from the bacterial growth. Even light is required from the light table, whereas normal ambient lighting does not interfere with the measurement.

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A Panasonic BT-H1450Y color monitor is connected to the RGB output of the micro digitizer card with BNC connectors. The monitor displays either the dish captured by the camera in real time (the signal circulates through the image memory card) or an image stored in 10 image memories. However, the program also allows a digitized still image to be displayed in a Windows window, eliminating the need for an external monitor.

15 The system requires the following software to work: - Microsoft Windows 3.1 (user interface)

Microsoft DOS 5.0 (operating system) esto.exe (executable program) 20 - dt2867.dll (digital card driver)

The program has a Windows-based interface to it and thus needs both the Microsoft DOS operating system and the Windows interface.

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Esto.exe is an actual driver with a size of about 100 kbytes. The exe program consists of five C language programs (app.c, apputil.c, glinit.c, imagutil.c, and tooldemo.c). If 30 changes are made to the program, C-language programs must be compiled with blocking.Ink, which contains the correct options for linking • (n.bat is a dos-based program).

The Dt2867.dll file is required as the driver for the digitization card 35, but if other digitization cards are used (the method has also been tried with the Data Translation QuickCapture card), k.o. card 15 94645 dll file. The device also works without a digitization card, with some exceptions. The program automatically retrieves the correct dll file from the dlls subdirectory of the working directory.

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In addition to automatic measurement, the program includes several other commands related to file, measurement, windowing, and analysis that do not in themselves constitute an invention.

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When the camera, light table, PC monitor and computer, and any external monitor and printer are switched on, the machine automatically starts the ERA application (ERA = estoren-15 gas analyzer). If the application is not set to start automatically and the machine is in Windows mode, the blocking ring analyzer will start like any other Windows application. When the application starts, select whether to use an external monitor. Selection 20 can be performed independently of the system. The system can thus be used as a single monitor, regardless of whether a digitization card, camera or external monitor is connected.

25 The File menu contains commands used to control ·· file and print. Open Image ...

The command can be used to retrieve images stored in files and display them in the active buffer. Similarly, the Save Image ... command saves an image of 30 active buffers to a file. Images can be in TIFF, PCX or IRIS format. The Print Image command allows you to take a paper print from the active buffer.

35 The Window menu contains some of the most common commands for editing and arranging Windows windows. These commands are only available when using a single-monitor system. Windows can also be edited - 94645 as in other Windows applications (stretch, scroll, move, shrink, etc.). The New Window command opens a new window and copies the image from the active window. In this case, the second window can be used for 5 ns. as an overlay window, i.e. measurements are made on one window while the other remains clean. The Overlap and Side by Side commands are used to rearrange windows. The Close All command closes all windows and the Enlarge Image command maximizes the active 10 windows. Open When the New Buffer option is on, a new window opens for each new image.

The Tools menu contains all the most important commands in this application. These commands 15 can be used to analyze and measure stop rings and to calibrate the ERA (stop ring analyzer) to operation.

The Take Picture command takes still pictures with the camera. The Analyze command launches an analysis program that automatically measures the 20 agar dishes in the still image as mentioned in the previous paragraph.

The Result command returns the result of the most recent measurement. The measurement result automatically appears with the Analyze and Measure commands. The Clear command clears the overlay graphic from the active buffer. The Measure 25 command can be used to "manually" measure the diameter of a circle. The measurement is made with the mouse by pressing the left mouse button at the center of the desired circle and dragging the rubber band larger or smaller than the circle with the mouse. Measurement result 30 immediately appears as a Result or Information window, unlike the Measure All command, where the measurement - * result is obtained by pressing the right mouse button.

In a single-monitor system, the measurement takes place directly in the window. In this case, Measure All works as an option that is not on by default. The Grid option places a grid over the image to set the camera to the correct position. The grid can be turned off

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17 94645 by re-entering the Grid command. When the Live View option is on, the external monitor will display the so-called the live image. This makes it easier to adjust the camera. The live image changes to a still image with the Analyze and Take Image-5 commands.

The application is terminated, like other Windows applications, either from the control menu screen or with the Quit ERA command (file menu).

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Example of basic use of a blocking ring analyzer 1) Turn on the monitor, camera, and light table. The computer is then started, which automatically starts the blocking ring analyzer (defined in autoexec.bat and in the Startup group window).

2) Place the first agar plate on the light table and take a picture. If the image is correct, go to step 20 in step 4.

3) If the camera has shifted or there is no resolution, align the camera by placing a live image and a grid and focusing the image from the camera.

4) Analyze the retaining rings. The program asks (messagewin-25 Dow) if the bacterial growth is lighter than the blocking ring, which is answered yes / no / cancel (cancel = abort analysis).

5) At the end of the analysis (after approx. 20 seconds), the results are displayed in a table, in which case the results can be edited either with the button keys or with the mouse, after which the results are accepted.

6) Accept the results and place the next dish on the light table and analyze it, etc.

7) Close the blocking ring analyzer and Windows and 35 close the PC, monitor, camera and light table.

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The invention can be applied to the study of changes in microbial growths caused by all microbial growth inhibitors. The agent may also be added to the substrate in other ways than as antibiotic discs. The condition is that the added substance causes a noticeable change in the gray tones of the substrate.

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Claims (8)

19,94645 Claims; A method for studying the activity of antimicrobial agents, which investigates the change in the medium caused by the substance added to the microbial medium (A), characterized in that - the medium is imaged and the resulting image is divided into pixels whose grayscale values are stored, 10. the distribution of the stored grayscale values (spectrum), which indicates the number of pixels by grayscale value, is formed by determining the range of grayscale values in the field , and determining a grayscale value corresponding to this minimum as a threshold value, - the location of the pixels implementing the threshold value determines the characteristic of the change area, such as the diameter of the antibiotic-induced barrier ring. 25 A method according to claim 1, characterized in that the pixels corresponding to the substrate (1-14) containing the added growth inhibitory substance are searched for in the pixels of the growth medium (A) by searching for those pixels which implement a predetermined threshold value of grayscale values. · A method according to claim 2, characterized in that the area to be analyzed around the substrate (1-14) containing the added growth inhibitor is gradually enlarged to obtain successive grayscale distributions, by means of which the peaks and minima of the distribution are monitored. 20 94645 Method according to one of the preceding claims 1 to 3, characterized in that more than one minimum of grayscale values is searched for in the distribution, preferably refining the resolution gradually until the desired number of minimum points between the examined spectra is observed, after which the clarity of each limit is calculated. the corresponding code is selected. 10 Apparatus for testing the activity of antimicrobial agents on a microbial medium (A), characterized in that it comprises a support for placing the microbial medium, 15. a camera (K) facing the support for imaging the medium (A), - means for dividing the image into pixels ) and means for determining and storing the grayscale values of the pixels; to determine a threshold value, wherein the means are arranged to search for a minimum of 30 spectra representing the number of pixels as a function of grayscale values, located within the range of pixels and between the distribution peaks formed by the pixels of the normal microbial growth, and registering a grayscale value corresponding to this minimum as a threshold value, 35. means for calculating a quantity describing the potency of the growth inhibitor using the pixels corresponding to the threshold value, and • · · means for storing said quantity. 21 94645 Device according to claim 5, characterized in that it comprises a display device for displaying the results. 5 Device according to Claim 6, characterized in that the display device is arranged to display graphically the image captured by the camera (K) and the results of the analysis of the pixels in the same image (Fig. 5). 10 Device according to one of Claims 5 to 7, characterized in that the base for accommodating the growing medium is a light table (L) comprising a stand on which a camera 15 (K) facing the light table is attached. • · «• 94645 22