JPH06508021A - Improvements in measuring biomass and differentiation in microbial organisms or related matters - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] ``Measurement of existence and transformation in living organisms, as or in conjunction with them. The present invention relates to a method for measuring cells of microbial organisms, particularly cell volume or biomass, and For example, fermentation broth allows for more detailed monitoring of differentiation at the cellular site of microbial organisms. Bacteria, yeast, or fungi in the fermentation process can be monitored during the fermentation process. It's about how you can do it.

In certain industries, such as pharmaceutical manufacturing, e.g. In particular, biochemical manufacturing processes, such as bacteria, yeast, or fungi, are used in the production of The desired microorganisms are grown in broth, and the desired There is an increasing use of a manufacturing process called "fermentation" to produce products. industrial In the process, it is necessary to optimize the fermentation process, so it is necessary to conditions, especially changes in cell volume or biomass in the broth, as well as the process of cell differentiation. It is necessary to measure.

At present, there are very few methods for measuring biomass that are satisfactory. The easiest way is to This method measures the optical density of ions. This method uses optical density and biological presence. It is necessary to dilute the broth to obtain an accurate correlation between the amounts, but this method The method uses broth to collect well-dispersed individual cells, e.g. bacteria, in a clear medium. It can only be applied to processes that include a. So this method is limited in its applicability. The morphology of living organisms is more complex, such as mycelium. When they do, the organisms are often entangled. In such a situation The living body forms a pellet with a size of 1 mm to several millimeters. There is. Under these conditions, optical density cannot be an accurate measure of biomass. Therefore, the biomass can be determined by centrifuging the sample and measuring the amount of solid matter. Measured. Another method is to filter the broth, dry the solids, and weigh them. There is.

However, fermentation broths often contain insoluble solids in addition to cellular organisms. (when using cheap materials to increase production efficiency) Various methods cannot distinguish between the amount of solid matter and the amount of biomass present. Furthermore, Emit items such as the amount of sample in the cytoplasm, vacuolar sites, lytic sites, or growth sites. Since no technology exists that allows for characterization during fermentation, this method of measurement is If it is possible to add this to the basic biological abundance measurement method, it will be possible to understand the fermentation process in more detail. It is useful because it can be monitored and managed.

In the present invention, the step of illuminating the sample to obtain an image of the sample, identifying individual regions within the image area by comparison with a degree threshold; The process of measuring the areas of identified parts within the area of the image A method is provided for measuring cell volume in a biological organism. The present invention an optical system consisting of a light source, a microscope and a camera to obtain an image of the sample; a comparator that predetermines the brightness of the region and compares it to a threshold; consisting of an adding means for adding up the total area of the various parts with respect to a pre-measured threshold value. We also provide equipment for measuring the cell volume of living cells in samples. It is something to do.

Measurements can be made to detect microbial microorganisms such as vacuoles or growth sites by setting appropriate thresholds. It is performed on a certain part (e.g. the tip of a fibrous organism) or In some cases, it is performed on the entire body, excluding the parts that may be affected. growth area They can also be selected by dyeing.

For example, using roundness parameters to better monitor biological growth. It is also possible to include a step of measuring the morphology of cells or parts (e.g. vacuoles) .

Biological biomass is calculated by multiplying the measured value by the density.

The method and apparatus of the present invention are particularly effective against various fibrous organisms, such as filamentous fungi and prokaryotes. Useful. The method is also applicable to industrial fermentations where pharmaceutical media, chalk, molasses, etc. are present. It is also useful when measuring non-fibrous organisms in broth containing insoluble solids. It is for use.

Predetermined thresholds are designed to include the entire cellular material or exclude large vacuoles or lytic sites. or - the second time covers the entire area of cellular material and the second time covers large vacuoles and A single measurement is performed to measure the area excluding the lytic site.

In this way, in the present invention, the measurement of cell volume is based on the area of cell material within the sample. and, if necessary, use the results to measure cell volume. The biomass is determined for samples containing living organisms in the form of mycelium. It can be measured in various ways.

Direct measurement of area improves the accuracy of final cell volume/biomass measurements. go up

It is possible to characterize the development of an organism using the total volume of the cytoplasmic and lytic sites. This allows for more detailed monitoring of the progress of the fermentation process.

Quantification of hyphal sites allows calculation of rates of change. The proportion of such areas and their changes The rate is related to the rate of product formation and other metabolic parameters such as maintenance counts. There is.

The present invention provides a solution to target cells excluding non-dissolved solids that may be present in the sample. Satisfies preset roundness parameters such that measurements are limited to certain areas. The method may also include the step of excluding from the measurement the portion of the image that is present.

Therefore, the present invention provides a solution for non-fine materials where the material is of low quality and confounds conventional dry solids measurement methods. suitable for successfully carrying out fermentation processes involving cell material.

The image can be digitized into an array of pixels, and the area can be calculated by integrating the array. You can also assign an array to a grayscale that is selected and sets the threshold to the desired value. Ru.

The present invention deals with areas darker than a preset level, or such dark areas and their surrounding areas. It is also possible to exclude predefined additional sites from the measurement. By such means Therefore, it is possible to exclude the area where insoluble solid particles cover and overlap the cell body. Ru. However, the intersection between organisms can also be darkened, e.g. by creating a threshold in size. This can be removed from the image by smaller than the body).

Once you have calculated the visible area of a cellular organism, you can use various methods to calculate, for example, the area of the organism. Assuming that it has a geometric form whose volume can be calculated by measuring Therefore, the volume can be reduced. For example, for mycelium-like organisms, hyphae are cylindrical. It is assumed that the volume is given by: Volume = π x Diameter of hyphae x Area x O, 25 Therefore, if the diameter of hyphae is measured, the volume is calculated using the average diameter and area measurements. considered to be statistically significant The average diameter can be calculated by measuring the number of hyphae samples, for example 100 samples. . By multiplying the volume by the density, it can be converted to the amount of living organisms present. For various living organisms Since the density value is known for the cytoplasmic region and the degenerated region, the drying of such region Weight can be calculated.

This method can also be applied to determine the volume and morphology of vacuoles in cells in a sample. . In this way, the total volume of the vacuole is calculated, and the morphology of the vacuole (spherical or cylindrical) is also calculated. The roundness can be determined using the roundness parameter.

The present invention can also include a step of measuring the volume of the cell growth site in the sample. . Staining and combinations that can selectively stain either growth areas or non-growth areas This is possible by increasing the brightness threshold for processing images by .

Both the measurement of the total volume of the vacuole and the measurement of the area of the growing site compared to the area of the non-growing site. By carrying out this method, a more accurate measurement of the progress of fermentation is possible.

In the example of the present invention described below, the diameter of hyphae is measured manually. However, it is also possible to use automated equipment for gag measurements.

It is used to identify living organisms from the background and to separate various parts of living organisms. The setting of the threshold for the intensity of the light used, the sensitivity of the optical system (camera and microscope used), Depends on the form. However, industrial production, which is the primary target of the present invention, In this case, the threshold value is determined at the time of setting and is not changed (periodic inspection and correction). (except for drift due to equipment and materials that can be monitored). Therefore, the present invention Automatic monitoring of living organisms and the volume of cytoplasmic and lytic material Automatically monitor the progress of fermentation (batch, batch, or continuous) how to monitor and optimize the process, for example through feedback control. This is what we provide.

The invention will now be explained in more detail using non-limiting examples and with reference to the following drawings. Ru: Figure 1 explains the identification of hyphal parts in penicillin fermentation; FIG. 2 explains the steps in one embodiment of the present invention; Figures 3A-C illustrate each step used in the present invention; Figure 4 shows the correlation between dry cell weights obtained by conventional filtration methods and measurements used in the present invention. Figure 5 shows the relationship between the dry cell weight obtained by the conventional filtration method and the present invention. It is a graph comparing the fermentation time course with measurement using; Figure 6 shows the difference between dry cell weight obtained by conventional filtration method and measurement using the present invention. This is a graph comparing two fermentation time courses; Figure 7 shows the volume of all parts and cytoplasmic parts, and the development of hyphal density in the case of the fermentation shown in Figure 6. It is a graph showing the fermentation time course; Figure 8 shows fermentation of dry cell weight measured by conventional filtration method and measured using the present invention. This is a graph comparing the passage of time; Figure 9 shows the volume of the cytoplasmic region and degenerated region in the batch fermentation, and the hyphal density. It is a graph showing the fermentation time course; Figure 10 shows the dry weight of lactose/drug culture fermentation measured by conventional filtration method and this product. It is a graph comparing the fermentation time course with the measured value using bright light; Figure 11 shows cell volume and hyphal density over time in lactose/drug medium fermentation. FIG. 12 is a block diagram of a second embodiment of the present invention. .

Examples of the present invention are described below, along with descriptions of individual examples describing comparisons with conventional methods. It is stated.

To monitor the progress of fermentation using filamentous fungi, monitor each stage of cell degeneration. It is effective to Traditional models of cell development suggest that two types of cells are produced during fermentation. It is thought that there are cells, one of which is a "healthy" cell completely filled with cytoplasm. ``cells'' and the other are degenerated cells that have lost their cytoplasm.However, in the present invention, the intermediate stage A different way of thinking is adopted that takes into account the cells of the floor. That way of thinking is both comprehensive contains “healthy” cells and cells that are partially degenerated but without large vacuoles. The other is the degenerated region where all cells have no cytoplasm and large vacuoles. Ru. The total of both sites is the total hyphal site, and a comparison of each site is shown in Figure 1.

To measure cell volume and calculate biomass using the present invention, fermentation broth sample for analysis. One way to prepare samples is to kill the organism and then This method involves treating the sample with a preservative to prevent growth. Then dilute the sample (the dilution rate depends on the nature of the broth, which is often semi-solid). Bibble a small amount, e.g. 4 microliters, onto a glass slide and cover. Cover with bar glass to prevent drying. Imaging with the microscope by illuminating the field under the coverslip Images are formed using a video camera that is connected to and controlled by an analysis device.

The amount of sample used, the use of preservatives, the slide preparation method, the magnification of the microscope, etc. depend on the characteristics of the sample. It can be changed by

Divide the field of view into pixels and determine what level of grayscale each pixel is at . The threshold is first set so that the background can be distinguished from the presence of cells, and the second set cells containing large amounts of cytoplasm, degenerated cells, and vacuolar sites. It will be done. In this way, two relevant thresholds are set. The number of pixels in each part is the entire field of view. are aggregated against. Then move the slide to image the new field of view and Repeat this operation until the entire area under the bar glass is treated. This overrepresentation is the second Shown in the flowchart in Figure.

For media that do not contain non-dissolved solids, the processing phase is as shown in Figure 3A. If the image exceeds the threshold, for example, using the roundness parameter, all particles can be removed. be removed. However, most fermentation broths contain undissolved solids that are not included in the measurement range. It is possible to include it and remove it. There are several ways to do this, but The degree parameter (inversely proportional to the area divided by the perimeter raised to the sixth power multiplied by 4π) One of them is the method used. Parameters are based on the organism used and the expected rigidity. Selected according to body type, easy to identify rounded, non-dissolved solids This method is particularly suitable for fibrous organisms. However, the size of the solid This also helps in identification, so it is possible to identify Dissolved solids (using the fact that they become darker than the level of formation) and those located in the periphery It is possible to identify. The process steps are shown in Figures 38 and 0. statue first creates a threshold to select dark areas including solid objects. The part where two hyphae overlap However, by taking advantage of the fact that such a part is smaller than the solid, for example, this Remove such overlapping parts from the solid image. The position of the solid on the image then includes the entire area of the particle. (see description below).

Hyphal sections can be further removed from the image using size and roundness parameters. Ru. If what remains is a solid image, subtract it from the basic threshold of the mycelial image and measure it. The mycelium itself remains.

Once the areas of all the cell parts and the cytoplasm part are obtained, the living body has a regular geometric shape. If it is treated as an object, its volume can be estimated. In the case of hyphae, it is a cylinder. Measure the diameter of the hyphae observed in the image. Appropriate to the value thus obtained Multiplying the density values gives the biomass. Whole cell site and cytoplasmic site The difference between the two is thought to indicate the amount of degraded material and the amount of product produced.

The above method is suitable for filamentous fungi, even if the concentration of non-dissolved solids is It is possible to estimate the biomass even in cases where the amount reaches 30 grams per torr. It has been proven that 0.03 to 38 grams per liter of Bacterial concentration was successfully measured.

Examples of fermentations and analyzes using the method of the invention compared to conventional methods are shown below.

Pencil [um chrysoghanu + s p-1 in defined medium or It was grown in a batch culture method using a technical medium of cutose/drug medium. Limited culture In the case of soil, a turbine agitator rotating at 800 times per minute (tip speed 3.35 minutes) is used. 1011 culture tank (Chemap^, G, 5 Mannedorf, Switzerland) so that the initial value is 101 spores, Q-1. were inoculated with spore inoculum. Fermentation was carried out twice under different conditions: A was fermented at 26°C; Se B was fermented at 30°C.

Turbine agitator with 950 revolutions per minute (tip speed 3.35 ■ Air with three 3-1 5Q culture tank with a flow rate of 0.05vvw+ (LllFermentation Lt d, UK) in a lactose/drug medium and grown in a non-limited seed medium. A 10% (V/V) 54-hour spread inoculum was inoculated. Lactose/drug culture The industrial medium contains 30gQ- of insoluble solids when measured by the filtration dry weight method described below. 'Contained.

Air flow rate with a stirrer of 1300 revolutions per minute (tip speed 4, 6 ms-') Mou and Cooney (Bioteeh, Bi The preparation buffer was prepared using the medium described by Pen1eillus + chrysogenum was grown using the latch method. Group The preparation speed of lucose is 7.08 throughout fermentation! hr-', but gluco After 26 hours of fermentation time, the concentration of the base is 485 g Q-' to 638 g Q. −’ was increased. A vegetative inoculum of Ig cell weight Q-' was used. Culture after sterilization The solids content of the soil was approximately 2.5 gQ-'. Time to estimate biomass Samples were taken from the fermenter at intervals. To remove errors associated with slide creation, From shake flask culture of Penicillium Chrysogenum Two samples were used. This sampling was done at 30.5 hours and 90 hours after spore inoculation. went. Fill the flask with 10% (v/v) defined medium and place on an orbital shaker at 26°C. (G25 type incubator shaker, New Brunsviek 5 scientific .

Watford, UK; 200 times per minute, amplitude 5 cm).

For comparison with the conventional method of filtering and measuring dry weight, 5 d of broth was dried/weighed in advance. llatman's CF high grade glass fine fiber filter Whatman In Ltd., Maidstone, UK). sample is distilled It was washed with 10 ml of water and dried at 105°C until a constant weight was obtained. Measurement results are attached This is shown in the drawing.

To estimate biomass from fermentations using the present invention, equal volumes of fixative (5 0% V/V ethanol 200 mQ with 40% formaldehyde 13IIQ and ice Mix with 5 mQ of acetic acid. Fixed samples are usually prepared using a fixative. Then dilute the solution further by 10 times, or 20 times if solids are present. limited Two sets of samples are made from the culture fermentation. This is pre-applied to the lactose/drug medium. The final concentration of undissolved solids in combination with the seeded sample is lactose/drug fermentation medium. Dilute the sample directly to the same concentration as the 20-fold dilution. Ru. During this procedure, the defined medium is diluted 20 times.

Pipette 4Ji from each diluted sample onto a microscope slide, 324+wi” Cover with a cover glass. Seal the coverslip to prevent the sample from drying out during the procedure do. To eliminate errors during slide preparation, samples from flasks incubated for 90 hours with shaking were used. Ten slides were made from the samples and both shake flask samples (305 and 90 hours) were prepared. ) was diluted 10 times and one sample was measured from each.

To measure the hyphae, we use general-purpose image analysis software called MagiseanMDs. yqtem (Joyce Loabal Ltd, , Gatehead.

Polyvarli microscope and field illuminator (Leica Cambridge) in UK) Ltd., UK) was used. 5-Microscope of automatic stage with increments I attached it to a mirror and controlled the x, y, and z axes and illuminance. of hyphae for each sample. Three things were measured: the total area, the area of the cytoplasmic part of the hyphae, and the average diameter of the hyphae.

To measure the area of hyphae, the magnification of the microscope is set to 100x and all the hyphae present are The program was designed to cover the entire area of the coverslip so that hyphae could be measured. I missed it. A video camera attached to the microscope transmits the video signal of the field of view to an image analysis system. where both space and tone are digitized and sent to the pixel array. Ru. Each pixel has a grayscale record representing the tone at that pixel location in the original image. Bell is assigned. Since the image is digitized, it can be compared with the darkness value, In other words, all pixels at a predetermined Lareta gray scale level are targeted. It is processed. The threshold values of this system have a scale of 0 to 63 (64 levels).

The thresholds used for all sites were 0 to 54. Masking of binary images is All problematic objects can be processed further and the background removed. is set to When measuring the total area, all mycelial material must be in the cytoplasmic area. The threshold level (0 to 49 in this system) for the measurement of the area of the large liquid To be included in the binary image, cells and lytic sites are removed from the binary image. Threshold the captured images. Undesirable objects such as hyphae as well as medium particles If the binary image is thresholded to contain The artifacts are removed using the roundness parameter. The roundness of this system is Particles with a value greater than 0.35 are excluded. However, lactose/drug medium industrial Subsequent treatments of the media are intended to exclude media particles that are on the mycelial aggregates.

There are two types of particles that should be excluded: 1. Particles trapped in the hyphal network 2. Particles that exist freely outside the mesh.

Once the image has been captured, select the threshold range from 0 to 26 to Set the threshold to detect dark areas. The intersections of mycelial networks are also often dark. Since it falls into this range of grayscale, we need to exclude it from the binary image. is necessary. The intersection exceeding the threshold is smaller than 20 pixels and is a dark area caused by the solid medium. Therefore, all objects with an area smaller than 25 pixels are excluded.

In order to set a threshold for only the dark areas caused by medium particles, the area covered by such areas must be undissolved. It is necessary to expand to encompass the entire particle. The technique of expansion is a particle It is used to determine the area covered. This technique is used to draw objects in a binary image. It means adding elements.

Two levels of pixels are added to the area where the threshold value has been set. In the first stage, the sum with value 1 is Replace all pixels with crosses (3x3 pixels), then replace all existing pixels with a value of 1. Replace pixels with 3x3 boxes. This technique detects pixels that exceed the threshold within the field of view. Expand to cover all visible media particles. but Small pieces of hyphae may still be present in the binary image, so it can be detected by measurement. Determine which of the following two ranges the size belongs to: 1. Area is 290 to 80,000 pixels, circularity is 0.2 to 0.8 2. Area is 200 to 40,000 pixels, circularity is 0.75 to 1.0° The above values are used to remove hyphal sections that exceed the threshold from the image. non-soluble A binary image containing solid particles is stored in an image memory. Brightness, size, roundness The threshold value can be changed depending on the system. Darkness where particles are completely different from hyphae If it has the same value as the hyphae, it can be excluded using only the threshold value, but if it has the same threshold value as the hyphae, If so, there is a problem. If this situation occurs due to lactose/medicinal medium fermentation, Another technique than expansion is to expand the dark threshold range to encompass all particles. is required.

The captured image is thresholded for the whole or cytoplasmic region and has a roundness of 0.35. Elimination parameters are set to remove all larger objects. mycelium mesh The stored binary image containing particles from is subtracted from this binary image to determine the grain size of the media. child is removed.

The area of objects and hyphae remaining in the processed image is calculated based on the total area of the object and hyphae present in the two-dimensional image. Apply the scale factor during addition to calculate the area in square microns by adding the elements. It is measured by using Once the field of view has been measured, the stage of the microscope is The camera automatically moves to the next field of view and repeats this until the entire area of the cover glass is covered. data is accumulated.

To estimate the cell volume, assume that the hyphae are cylindrical and calculate from the following formula: Volume = π x Diameter of hyphae x Area x 0.25 In this example, the magnification of the microscope is set to 400x. Touch two opposing points on the image of the hyphal wall of the hyphae with a light pen, which were arbitrarily selected. The average diameter of the hyphae of each sample was manually determined by. The distance between two points is an image Calculated by the analyzer and stored in a data file. Repeat this operation for each sample. Repeat at least 100 times using new points on the same hyphae and different hyphae.

The volumes calculated for the whole hyphae, the cytoplasmic part, and the degenerated part are the differences between them. is calculated. The results are calculated per liter of fermentation broth, taking into account the dilution factor of the sample. Convert the cell volume to P.

Once the volume of each part is determined, it can be converted to an estimated value of dry weight. Mycelia density value (dry weight) for enieillium chrysogenum The amount g/cell volume d) is given in the literature, but for the cytoplasmic part it is 0.35 g. /cd, and 0.18g/cj for degenerated sites. Therefore, the volume is as follows It is converted to the biomass: dry weight (g/+1) = (volume of cytoplasmic part ( ci/Q) xo, a 5 (g/C1)) + (volume of degenerated site (c11/1) o, 18 (g/cd)) The attached drawings show the results compared with measurements using conventional methods.

Statistical analysis was performed using analysis of variance with a 95% confidence level for dilution and slide preparation. No particular difference was observed in the distribution of mycelial material between the two slide samples obtained by repeating the procedure. It became clear that something was wrong. For 90 hour samples, pipette onto the slide. The % error for dilution is 8.3% at the 95% confidence level and 7.3% for dilution. It was. In the case of young cells (30.5 hours) the dilution error was 4%. aggregate The percentage of material present in the form of 96% for the 90 h sample, 30,5 h It was 76% for the sample.

The following is a comparison of the dry cell weight estimated by the method of the present invention and the value measured by the conventional filtration method. The results of several fermentation experiments described below are pooled and shown in FIG. The correlation is It shows that the data fit a straight line very well, and the correlation coefficient in this case is (95% The confidence level was 0.99. This fact corresponds to the dry cell weight estimated by the present invention. The results show that the amounts and dry weights measured using conventional methods are in good agreement.

Figures 5 and 6 show the conventional method (0) and fermentation time for fermentation A and 8 in defined medium. It is a comparison of the amount of living organisms measured by the method (x) of the present invention. In Figure 4, the cell Growth occurs for up to 45 hours when the offspring germinate and the hyphae begin to lyse. No. In the second fermentation example (Figure 5), the situation is different and the growth continues until later. symbol mouth shows the results of estimation using the method of the present invention when solids are present in the sample. (see below). Existence of organisms estimated from image analysis in both fermentations The amounts are in good agreement with the values obtained using conventional methods. In fermentation A (Figure 4), lysis occurs. Due to this, the dry weight starts to decrease after 62 hours, but the method of the present invention It can be said that it fits well with the phenomenon of

The time course of the volume of total hyphae and cytoplasmic parts measured by the method of the present invention is shown for fermentation B. This is shown in FIG. Volume of total hyphae (・) and cytoplasm (■) at 30 hours after spore inoculation. The difference is the same up to that point, but the appearance is different after that because vacuoles are observed.

The time course of the average hyphal density (Δ) is also shown, and this value is initially 0. 35g/w", but after the volumetric images started to differ, it remained constant throughout the rest of the fermentation period. The measured value of mycelial diameter in fermentation B was 4.3. 5.2-. Such differences depending on the region cannot be detected using conventional methods. stomach.

Figure 5 shows the dry weight obtained when lactose/drug medium is added to the fermentation B sample. An image of the amount is also shown. Measurements of biological status obtained using the present invention are Unaltered by the presence of soluble solids.

Figure 8 shows the time course of the amount of biomass present in the batch fermentation method. According to the present invention The estimated dry weight (X) is in good agreement with the value measured by the conventional method (0). Service In mixed batch fermentation, small amounts of undissolved solids are initially present in the medium; No such influence was observed on the dry weight image. Therefore, the first sample was taken at 16.25 hours after inoculation. Considering that it is something in between, solid matter has been destroyed, and even if it exists, However, it is considered to be a very small amount. Total cell volume (・) and hyphal density (Δ) in this fermentation is shown in Figure 9.

Total cell volume steadily increases over time. However, the cytoplasmic site The volume (■) increases rapidly until 64 hours, but the rate of increase in volume decreases after that. are doing. The average diameter of the hyphae of the samples ranged from 6.8 to 5.2p.

The time course of lactose/drug culture fermentation was measured by the conventional method (O) and the method of the present invention (×). The determined results are shown in Figure 10. Up to 65 hours, solids are mycelia when measured by filtration. After that, the dry weight increases up to 97 hours, but after that the dry weight increases. The amount of precipitation has decreased slightly. The biomass estimated by image analysis method is up to 90 hours after inoculation. It increases steadily and then decreases rapidly. Figure 11 shows the origin of lactose/drug medium. Total hyphal volume of fermentation (,・), cytoplasmic part (■), degenerated part (b), hyphal density (△) It shows the passage of time. Total hyphae increased steadily until 90 hours, at which point 1 It becomes a roughly constant value of 15 cd. The volume of the cytoplasmic region also increases up to 97 hours, but After that, it decreases rapidly. Average of Samples Obtained from Lactose/Drug Media Fermentation Hyphal diameter ranges from 4 to 6p. Conventional methods involve cytoplasmic sites and degenerated sites. No information is obtained, only the total biomass in the absence of solids. It is.

In this way, lactose/drug medium fermentation can be determined by measuring mycelial volume. The growth of mycelia was monitored (Figure 11). Since the inoculum is mycelium, vacuole production is It has already begun, and the cytoplasmic volume and total hyphal volume have already taken on different values. The statue is emitted Throughout the fermentation period, the volume of the branching site decreases markedly, but the total cell volume remains small. As long as the cell wall is stable, the value remains almost constant. This phenomenon also appears in mycelial density. When cells die and lysis begins, the hyphal density decreases. Concentration of non-dissolved solids Measurements using mycelial techniques in lactose/drug culture fermentations with large The advantage of the measurement method used is that the volume of the mycelium can be directly determined without measuring solid particles. This shows that it is possible to

The main error in this method occurs during slide preparation through repeated dilutions. This is the process of placing the 41i sample on the slide (to avoid dilution errors, place a pipette on the slide). The difference in morphology and dispersion between the two samples is hidden behind the error of loading the sample with a sample. Therefore, the error will be larger for the 90-hour sample than for the 30.5-hour sample). Book When using the method of the invention, the error in estimating the current status of organisms due to sample preparation is ±8. 4%, compared to the error of the conventional method with a 95% confidence limit of ±12 to 2%. The error is larger when the dry weight is small.

In the above example, the sample has a biomass in the range of 0.68 to 38 g/Q before dilution. This technique is used to estimate biomass. Minimum cell concentration that can be used If dilution is performed taking into account that o, os4gz/l, the resolution will simply be diluted. It will only improve. This method works with up to 30 g/II of non- Since it is not affected by the presence of soluble solids (Figures 6 and 10), whole cells, cells Being able to measure the quality and volume of degenerated hyphal cells is extremely effective for fermentation research. Ru.

Next, a second embodiment of the present invention will be described. In this example, we will examine the characteristics of biological development. Methods are provided that add extra steps that allow for more complete knowledge. Special This method is useful for cells that are completely filled with cytoplasmic material, cells with vacuoles, and degenerated cells. Cells and empty cells can be completely distinguished. We have already mentioned filamentous microorganisms. As mentioned above, different parts of the hyphae are distinguished, i.e., different biological stages and stages of life. The body has a complex structure, and growth may be limited to the tip of the hyphae, for example. If it were, growth would occur over a wider area and the age distribution of cells would be smaller than that of non-filament microorganisms. It can be difficult to tell the difference. This makes it difficult to construct a mathematical model of the identification process. Therefore, controlling the fermentation process has become extremely difficult.

In this example of the invention, samples were prepared and diluted in a manner similar to that described above. The sample is mounted on a slide and covered with a colored or burr glass. The sample is then The image is formed using a microscopic camera and an image analyzer, and in this example, the magnification is 200. Double it. The image analysis process is shown in Figure 12 and consists of six stages. Yes: (1) Settings, (II) Gray image processing, (m) Image detection, (mV) II Original image processing, (V) image measurement, (VI) data analysis.

(1) Two-legged lice The program requires the user to set two types of parameters = ( Limited involvement in a) hardware parameters and (b) vacuole and hyphal measurements Image processing parameters. The former includes the microscope lamp illuminance and autofocus parameters. – includes correction factors for the objective used and the scan pattern of the slide. . Image processing parameters such as the detection level of hyphae and vacuoles (see below) and morphology factors are set to limit the filtration operation described below. these The parameters are fermentation specific. Default values are stored in the sample file. It can be stored.

A correction for uneven slide illumination (“shading correction”) is established during setup and can be manually The editor is selected.

This step is required only once for each measurement setup.

Steps (n) to (V) described below are performed after all selected slides and fields of view have been analyzed. It is repeated until the end.

(n) Gray image processing A digitized gray image (512x512 pixels) are captured. Each pixel is divided into groups from O (indicating black) to 255 (indicating white). It has a ray scale value. The image has a medium gray level, especially around the edges of the object. "Description" is performed to improve the quality so that the gray level of the outline is sharp except for It will be done.

(m)L rises Problematic objects, such as mycelium in the depicted gray image, have their gray level be “detected” by the

Two detection levels (selected during configuration) are used.

At one level, all hyphae except vacuolar sites and empty cells are picked up. now At one level, vacuoles, empty cells, and some background areas are detected.

The result of such processing is a binary image that masks the problematic gray image part. . These two images are named “Image 1” and “Image 2” respectively. Sequential image processing is performed on these two-dimensional images.

(rV) binary [1ffi dan 亙1 Image 1 is a pixel defect in the binary image due to non-uniform intensity of the original gray image. Reinforced to repair defects that mainly result in losses. Located between tightly intertwined hyphae Small background areas are stitched together by this operation and filter HY PI (roundness >2.0) is applied to eliminate such undesirable effects.

This criterion is used because the background that has an adverse effect has a smaller roundness than the defect. It can be repaired.

The enhanced binary image captures numerous small non-hyphae objects such as debris, media particles, dust, etc. detection due to non-uniform illumination of the slide. Contains the area of the background. Even minute air bubbles become defects. in contact with mycelium Such untouched materials have a preset roundness factor (1,0≦perfect roundness). degree≦3.0) and erased with filter HYP2. Almost like a circle than a hyphae. This method is effective for any object.

Since connected areas in the image are treated as a whole, this filter It is not valid for non-hyphal objects that are in contact with (in the image). are in contact For non-hyphal objects, repeat the “release” operation to peel off pixels that protrude from the image. This allows identification and removal. Relatively thin fibrous mycelia look like this However, wide non-hyphal objects remain. These remaining non-hyphal objects When deleted from the original image, hyphae without vacuoles are obtained in "Image 3". Obsi 1 If the manual editing function, editor EDD HYP, has been selected in advance, this step It can be used in This editor breaks in binary images due to poor detection. It can be used to correct defects such as hyphae that appear in cells. However, such defects is considered rare, and manual editing slows down the process.

Image 2 (of the vacuole and empty cell) also shows the background and many undesirable objects. Contains. The former has a gray level similar to that of the vacuole, thus forming image 2. detected at times. Most of the background and many unwanted objects are can be removed by presetting the roundness factor (filter V in Figure 12). AC: Roundness>2.0; 3゜l≦Area(-”)<78.5).Select in advance. For example, the optional vacuole editor EDVAC can be used at this stage to detect Vacuoles missed due to poor image quality can be captured in image 2.

Small background artifacts similar in shape and size to certain objects, especially vacuoles The structure cannot be deleted from image 2 using filter VAC as is. image 3<m of threads) to image 4 (of vacuoles containing background artifacts) and vacuoles are protected, resulting in complete wear and tear of remaining artifacts outside the hyphae. (filter HYPV). The beautiful image obtained, Image 5 is an image of hyphae + vacuole. From the difference between images 3 and 5, an “image” of only vacuoles 6″ is obtained.

(V) Image measurement The first thing to measure for each object in the image is the perimeter and area. Original data is converted to volume using an appropriate model of hyphae and vacuoles. Hyphae containing vacuoles are cylindrical If we consider that the pixels are counted and the area and perimeter are calculated, we can calculate the slanted edge when measuring the perimeter. By correcting , we can derive a two-dimensional projection of the diameter. volume of hard hyphae can be calculated in this way.

The shape of the vacuole is often close to a sphere, but if it is elongated, it can also be close to a short cylinder. Yes (especially empty cells). The volume of the vacuole and empty cell is given by: (a) When close to a sphere (circularity < 1.3) Vv = (πd,”)/6 (b) If it is elongated or cylindrical (1,3≦roundness<2.0) V v” (W d vA v) / 4 where d is the equivalent diameter of the vacuole close to the sphere ( [4A/π]””), dv is the diameter (width) of the cylindrical vacuole ([P-(P''-16A)'''']/4), A is the projected area of the vacuole.

Other derived values include roundness and equivalent diameter (of the vacuole). Volume fraction of each part is calculated.

(VT) 7' Nimdanhan Statistical analysis of the data following measurement of the entire field yields mean values, standard deviations, and parameters. A histogram of parameters is obtained.

The preset values of various parameters used in this example are Pen1eill Optimal identification of hyphae, vacuoles, and empty cells in iu@chrysogenui test fermentation This is the setting for fermentation, and the settings need to be changed for other fermentations. Ru.

This embodiment of the invention allows the distribution of vacuoles to be monitored during fermentation and the results Monitor the effect of various fruit parameters (e.g. glucose supply) on fermentation and can be controlled. Characterizing fungal differences to this extent Conventionally, it was impossible to Get results in minutes.

Next, a third embodiment of the present invention will be described. In this example, the above method was applied to mycelia. It has been expanded to measure growth (mainly apical) and non-growth sites. p, CIB Preliminary test fermentation of ysoBnuw+ Manipulating the addition of nutrients resulted in mycelial differentiation. (i.e. the ratio of vacuoles to empty cells and growing to non-growing areas) The inventor has revealed that this can be done. Therefore, these parts should be modeled. Nitering allows for more detailed control of fermentation.

In this embodiment of the invention, samples obtained from fermentation are diluted and stained. for example , using a neutral red solution will make the growth areas darker (and become non-growth areas). Can be identified. The images generally represent the first and second embodiments of the invention. processed in the same way, but with an additional threshold to single out the growth sites of the cells. There is. For example, various staining agents such as fluorescent staining agents, fluorescent staining is Although there is a problem with making the light brighter, it can be used. Examples of test fermentation and aspects of the present invention The sample preparation method for this example is described below.

U plate Hole”l Industrial strain of Penicillium ehrysogenu (Ss+ith KIineBeaeham Pharmaceutical ple, UK) in a 5Q fermenter using complex medium (Mouand Cooney, 1993). Made it grow.

The fermentor was inoculated with 32 hour old vegetative medium. Stirring, aeration, minimum dissolved oxygen level (adjusted with a gas blender) are 800 rpm, lvv, and 40% air saturation. It was peaceful.

5% (W/V) phenylacetic acid supplemented with 5% (W/V) ammonium sulfate. Subsequently, it was fed to the fermenter. After 24 hours, glucose supply was stopped for 6 hours and then We met again. This pulsed flow was maintained throughout the entire production process of the fermentation.

Sample made from W4 The samples collected from the fermentation were diluted with a 20% (W/V) sucrose solution to reach the final concentration of biomass. It was diluted from 1 to 1.5 g/Q. Adjust the pH of the diluted sample by adding dilute sulfuric acid. Adjusted to 3.0. Neutral Red Solution (BDHLidl, UK) approx. 1- was added to the diluted sample 1-. After 5 minutes, pipet one drop of each sample onto the slide. and covered with a cover glass.

Images can be captured and processed in a similar manner to the second embodiment.

For the above samples, sample collection and diameter measurement must be done manually, but non-dissolved Measuring mycelial volume and estimating biomass even in industrial fermentations containing high concentrations of sexual solids can be performed completely automatically. This technique can be controlled according to its results. can be applied online to the fermentation conditions to be determined, e.g. by taking a sample and Complete equipment including means for processing and means for controlling fermentation conditions according to measurement results. It can be applied to automatic hunt rolls.

Figure 1 “Axis-healthy” cells Partially degenerated degenerated cells Cells Figure 3B Figure 3C Figure 4 Copy and translation of amendment 1) Written submission (Kokuhogumi Article 84-8) September 21, 1993 Day 1

Claims (18)

[Claims] 1. The process of illuminating the sample so that an image of the sample can be obtained, at a preset brightness level. The process of identifying various parts within the field of view by comparing them with a threshold value of the The process of measuring fibrous microorganisms in a sample consists of the following steps: A method of measuring cell volume in living organisms. 2. 2. The method of claim 1, wherein the threshold level is set to include only vacuoles. 3. 2. The method of claim 1, wherein the threshold level is limited to the step of measuring the growth site. 4. A claim comprising the step of staining the sample to distinguish growth sites from non-growth sites The method described in 3. 5. Claim 1, wherein the predetermined level is set to encompass whole cellular material. How to put it on. 6. Predetermined levels are set to exclude large vacuoles and lytic sites. The method according to claim 1. 7. 7. A method according to any preceding claim, including the step of removing solid particles from the image. 8. Exclude parts of the image that satisfy the preset roundness parameters from the measurement. It also includes a step of limiting the measurement to the target cell site by The method according to any one of claims 1 to 7. 9. 9. The method according to claim 1, comprising the step of digitizing the image into an array of pixels. Method. 10. Pixels are assigned to a gray scale and thresholds are set on this scale. The method according to claim 9. 11. Areas darker than the preset level are detected and excluded from the measurement area. The method according to any one of claims 1 to 10. 12. The dark area in question and a preset additional area around it are removed from the measurement area. 12. The method of claim 11, wherein the method is removed. 13. The measured area is multiplied by the width parameter to derive a measurement representing the volume of the cell. 13. The method according to claim 1, further comprising: 14. A light source for illuminating the sample, a microscope and a camera for imaging the sample. A comparator to compare the brightness of the image area with a predetermined threshold level. total area of the area identified with reference to the rater and a predetermined threshold level. measuring the cell volume of filamentous microorganisms in a sample consisting of an addition means for adding equipment for. 15. Multiple thresholds can be added so that the addition means can add areas corresponding to different thresholds. 15. The apparatus according to claim 14, further comprising setting means for setting. 16. The cell volume is measured according to the method according to any one of claims 1 to 13. The biomass in the sample consists of determining the biomass by multiplying the product by the density parameter. How to measure the existing amount. 17. A step comprising the step of measuring cell volume according to any one of claims 1 to 13. How to monitor the progress of the fermentation process for optimization. 18. Device according to claim 14 or 15 for measuring cell volume in fermentation broth. Use of.