JPH0430798A - Counting of viable microorganism and device therefor - Google Patents

Abstract

PURPOSE:To quickly and correctly count number of viable microorganism in a ready operation by impregnating a filter attached of viable microorganism in a specimen with a bacteriolytic reagent and a reagent emitting light with ATP, and converting luminescence from ATP derived from the viable microorganism into image signal with a two-dimensional photon-detecting means. CONSTITUTION:A fixed amount of specimen liquid or gas is previously filtered and the resultant filter 2 is laid on a sample stage 3 on a base stage 1. Then, a mixed solution of a reagent dissolving cell membrane, an enzyme emitting light in the presence of adenosine triphosphate(ATP), and a substrate is introduced from a reagent- injecting path 4, thus the filter 2 is impregnated with the solution. A light-emitting reaction by the ATP derived from the viable microorganism and the enzyme-substrate at an attaching position of the viable microorganism is performed, then a distributing image of photon on the face of the filter attaching the viable microorganism is converted to an electrical image signal using a two-dimensional photon-detecting means, composed of a camera lens 7, a close-up photography ring, an image intensifier 10 and a camera 12. Thus, the signal subjected to an image integration and number of picture elements having high brightness or groups of the elements is counted to detect the number of attached viral microorganism.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a viable bacteria counting method and apparatus for counting the number of microorganisms and somatic cells in a liquid or gas.

[Conventional technology]

Here, country of origin is treated as a general term for somatic cells, bacteria, microorganisms, etc., but measuring the number of viable bacteria is used for quality control and in various fields such as food, brewing, medicine, clinical practice, and semiconductors. This is extremely important in terms of environmental management, etc.

Conventionally, the following methods have been used to measure, for example, the number of microorganisms or the activity of microorganisms.

(1) Colony counting method A method in which the number of microorganisms is determined by visually counting colonies grown on an agar medium.

+2+ i! I-degree counting method A method of optically measuring the light scattering phenomenon caused by corpses floating in a culture solution and determining the number of microorganisms from the turbidity.

(3) AT'P (adenosine triphosphate) measurement method A method of measuring the number of microorganisms 'IIFI from the amount of luminescence of ATP using bioluminescence such as luciferin-luciferase.

(4) Impedance measurement method A method of measuring microbial activity by measuring changes in electrical impedance in a fermentation medium due to the metabolism of microorganisms.

[Problem to be solved by the invention]

However, the methods (1) to (4) above have the following problems.

(1) Colony counting method The operation is complicated and it takes a long time to grow (24 to 48
time) and the measurement accuracy is poor.

(2) Turbidity counting method Although the sample solution must have low spectroscopic absorption, in practice, a medium containing solids or a medium with a high degree of coloration is often used, and accurate Difficult to measure.

(31A TP measurement method: Fluorescent substances and ATP coexisting in the culture medium may cause background light, and the amount of ATP contained in each microorganism or cell varies depending on the type and condition, making it difficult to accurately determine the number of bacteria.) It is difficult to

(4) Impedance measurement method Impedance measurement cannot be performed under conditions with aeration and agitation, and there may be salts in the sample solution that are conductive. It also cannot be measured when If is present.

The present invention has been made in view of the above-mentioned points, and its purpose is to provide a method and an apparatus for measuring the number of viable bacteria quickly, accurately, and with high sensitivity using simple operations. be.

[Means to solve the problem]

above&! In order to solve this problem, the method of the present invention involves filtering a predetermined amount of sample liquid or gas and applying live bacteria to the filter in the presence of a reagent for dissolving cell membranes and adenosine triphosphate PII (ATP). A mixed solution of an enzyme and a substrate that emits light is impregnated onto the surface of the filter where live bacteria are attached, and a luminescence reaction is caused by ATP derived from the live bacteria and the flllK-substrate. Convert it into an electrical video signal using a dimensional photon detection means, and count high brightness pixels or a set of pixels in the image obtained by integrating this video signal for a predetermined time, or set the above video signal in advance. A plurality of photon integral images obtained by repeating image integration at predetermined time intervals are each binarized using the same method, and the logical This counting method involves performing a product operation, displaying the results of these operations on one screen by superimposing the screens or performing a logical sum operation, and then counting high-intensity pixels or a set of pixels in the final image obtained. In addition to the filter, the apparatus of the present invention includes, in addition to the filter, a reagent injection means for impregnating the filter with a mixed solution, an imaging optical means for imaging at least a part of the viable bacteria adhesion surface of the filter, and Two-dimensional photon detection means that amplifies the distribution image of scattered photons to a two-dimensional high sensitivity, and imaging means that converts the two-dimensional distribution image of photons output from the two-dimensional photon detection means into an electrical video signal. , an image integration circuit that integrates the video signal output from the imaging means for a predetermined time; an image processing circuit that performs arithmetic processing on the integrated image data obtained from this circuit to extract and count high-luminance pixels or a set thereof; It includes an output means for displaying the extracted image and the counting results.

[Effect]

In the present invention, a filter obtained by filtering a certain amount of sample liquid or gas in advance and adhering live bacteria is impregnated with a reagent that dissolves cell membranes and a mixed solution of an enzyme and a substrate that emit light in the presence of ATP. causing a luminescent reaction between ATP and the enzyme-substrate to occur at the attachment position of the living bacteria,
The generated photons are converted into electrical video signals using a one-dimensional photon detection means, and the presence of viable bacteria is detected as a two-dimensional distribution of i! fraction.

That is, by accumulating photons on a pixel-by-pixel basis, the S/N ratio against background light caused by fluorescent substances in reagents and random noise from a high-sensitivity camera is improved.

Eventually, a group of high-intensity pixels with a large number of detected photons centered around the attached position of viable bacteria will appear for the number of attached viable bacteria, and by counting them, it is possible to determine the number of bacteria. . In addition, in the present invention, in order to remove shot noise that occurs two-dimensionally and randomly, and to extract only live bacteria from photon images that are continuously generated from the same location due to a luminescent reaction, Repeat image integration at intervals to obtain multiple photon integrated images, disulfide each of these photon integrated images using the same method, perform a sequential AND operation between two temporally consecutive binary screens, and It is also possible to display the results of the calculations on one screen by superimposing them or by performing a logical sum operation, and then count high-intensity pixels or a set of pixels in the finally obtained image.

〔Example〕

Hereinafter, the present invention will be explained based on examples.

In the first step of the method of the present invention, for example, a certain amount of sample liquid or gas containing microorganisms is filtered through a filter that does not allow microorganisms to pass through. , ATP, which is a high-energy substance inside cells, is extracted, and then a luciferin-luciferase mixture solution extracted from fireflies is gently added or impregnated onto the filter to cause bioluminescence as shown in the following formula. be.

Adenylluciferin +0! -Oxydylsiferin 10A
MP + CIJz + light However, PP is birophosphoric acid, A
MP is adenosine-phosphate At this time, since the above luminescent reaction occurs mainly in the vicinity of adherent cells with a high ATPi degree of 1, luminescent points will be dispersed on the filter at positions that coincide with adherent cells.

The second step of the method of the present invention is to two-dimensionally observe the above-mentioned light emission process using a high-sensitivity camera.

Since the amount of light emitted per cell is 8ii+ weak, the camera here needs to be extremely sensitive. In the -g product example, a combination of an ultra-high sensitivity image intensifier called a dimensional photon counter and a COD video camera was used.

This image intensifier has Multi-Champful Play (MCP) cut out within two pages, and is
It has a detection sensitivity of 108 photons/f1259-, and is capable of amplifying Hell's abrasive images by a few photons to the extent that they can be visualized sufficiently even with the sensitivity of a normal CCD camera.

Therefore, the above-mentioned light-emitting point can be observed as a series of photons at a certain point.

The third step of the method of the present invention involves converting successive photons emanating from the above-mentioned cell attachment sites into sufficiently small i! The process is to integrate temporally and create an image at the falcon's position. This makes it possible to improve the S/N ratio with respect to electrical random noise (shot noise) and background light due to fluorescent substances contained in the reagent. The integration time may be adjusted to the above-mentioned luminescence reaction time, and approximately 1 to 2 minutes is sufficient.

Finally, the obtained integral image may be binarized and the luminescent points due to ATP extracted from the cells may be counted through image processing.

Next, the apparatus to which the method of the present invention is applied and its operation will be described. FIG. 1 is a schematic diagram showing, in partial cross-section, the configuration of a measuring section of an apparatus to which the method of the present invention is applied. In Fig. 1, the measurement unit is installed on a base l, and two reagent injection paths 4 are connected to the inside of a sample stage 3 on which a filter 2, which has previously filtered a certain amount of sample liquid or gas, is mounted. but.

Only one reagent injection path 4 is shown in FIG.

One of these reagent injection paths 4 is for the ATP extraction reagent, and the other is for the oxygen-substrate mixed solution. A drainage channel 5 for cleaning the upper part of the sample stage 3 runs through the center of the sample stage 3.

Above the filter 2, the hood 6, camera lens 7, and close-up ring 8 are placed in this order when viewed from the filter 2, and covered with a dark box 9 to keep it completely dark during measurement. Ultra-high sensitivity image intensifier 10, relay lens 11 and C, which together with camera lens 7 and close-up ring 8 constitute a two-dimensional photon detection system
A movable stage 13 is connected to the CD camera 12 and attached to the ultra-high sensitivity image intensifier 10, and the movable stage 13 is defined by a support 14. camera lens 7
A close-up ring 8 forms an image of the surface of the filter 2 onto the photocathode 15 of the ultra-high sensitivity image intensifier 10 at a predetermined magnification. The photons reaching the photocathode 15 are photoelectrically converted and electronically amplified inside the ultra-high-sensitivity image intensifier 10, and reproduced on the fluorescent surface 16 as a bright spot with constant brightness. The image on the fluorescent surface 16, that is, the photon distribution image on the filter 2, is transmitted by the relay lens 11 to the damage element 17 of the CCD camera 12, and can be output as a video signal.

FIG. 2 is a system block diagram showing an outline of the electrical system of the viable bacteria counting device of the present invention. As shown in FIG. 2, this device includes the measurement section shown in FIG. Ultra-high sensitivity image intensifier-10 sensations! It is comprised of a high-voltage power supply section 20 for the IN section, overall control of these, image processing such as binarization, extraction of light-emitting points, and counting, and a II/III output section for displaying and recording the results. Video signal 18A output from measuring section 18fJ-p
is converted into a video signal by the video camera controller 22 and transmitted to the 'N image integration field 23. Here, the 1 degree signal of the video signal is A/D converted, stored in the video frame memory 24, and lii! Add each i side and rewrite the memory contents. When the integration for the preset time is completed, the microcomputer 25 performs image processing such as smoothing two-image correction and binarization on the integrated image data of the video frame memo IJ24, and displays the integrated image data on the CRT.
26. Furthermore, the microwave computer 2
5 counts the light emitting points of the image-processed binary image based on the criteria described later, and causes the printer 27 to output the counted light-emitting points. Also,
By operating the keyboard 28, the final binary image and the counting results can be recorded on a storage medium by the external storage device 29.

Next, a determination method for counting light emitting points from the finally obtained binary image will be described. The effective function number of the image stored in the memory and output is 480 (vertical) x 512 (horizontal). FIG. 3 is a schematic diagram showing an enlarged part of a binary image, and each square surrounded by a square corresponds to a pixel. Of these, the one shaded part is! ! The element ii is a high brightness part. The outfit of the present invention!
In this way, the number of pixels occupied by a light emitting point per cell varies by several pixels. This is the 1st fA
This reflects the resolution layer of the imaging optical system of the measurement unit shown in 2 and the degree of spread of ATP seeped out from the cells.

Therefore, when using the device of the present invention, a set of adjacent high-intensity pixels is determined to be one light-emitting point.

As described above, as a result of measuring general bacteria using the viable bacteria counting device of the present invention, a good correlation with the colony counting method was obtained in the range of 10' to 10', especially for samples with low bacterial counts.
Can be measured with high accuracy.

By the way, noise in the two-dimensional photon detection means used as a detector in this method may stochastically remain as an image even when the detector is cooled, and may cause measurement errors. The photon image captured by the CCD camera includes (11 photons emitted from living bacteria121iiiiii image)3 photons emitted from the charging surface as noise (3)3 photons emitted from the fluorescence emitted from the solution
There are bottles, and only (1) of this a! ! ! It is necessary to measure the raw number accurately and with high sensitivity by reducing the influence of shot noise.

To this end, in the third step of the method of the present invention already described, continuous photons emitted from the microorganism adhesion position are image-integrated repeatedly at predetermined time intervals in sufficiently small pixel units to obtain a plurality of photon integral images. Binarize each of these photon integral images using the same method, perform a logical product operation between two temporally consecutive binary images, and combine the results of these calculations by superimposing them or combining them by a logical sum operation. The number of attached microorganisms can be determined by displaying the image on one screen and counting the high-intensity pixels or aggregations thereof in the final image obtained.

FIG. 4 is a schematic diagram showing the process of extracting only attached microorganisms from a binary screen. The large circles in FIG. 4 each represent a binary screen, with OI to 0. are the respective times o”
``'tl+ t, ~F+ 5~L3+ j3~t4 and t4~[, is a photon integrated binary image obtained by binarizing the image integrated in the darkness, A, ~A# are 0.~OS sequentially zi
This is a sequential AND image obtained by performing an AND operation on each side. Further, S is a logical sum image in which A1-A4 are logically summed on one screen. The C mark and the mark shown in each screen are a photon image and a synotonoid image due to a luminescent reaction, respectively, and a symbol a=i is attached to some of them. Since shot noise occurs two-dimensionally randomly and sporadically, if the integration interval is shortened, the probability that a bright spot will appear at the same position on two screens is extremely low.

On the other hand, photon images generated continuously from the same place for a certain period of time (reaction time) due to a luminescence reaction and corresponding to the attachment position of microorganisms or cells can be generated over two or more screens by keeping the integration time at least equal to or less than the luminescence reaction time. For example, shot noise images a, d on screen 01 and shot noise images f, j on screen 0□ are at different positions, and photon images c, g, h
will be at the same location. Therefore, if these two images are logically multiplied, all other than point image b (or g) and point image C (or h) at the same location will be removed.

As described above, the viable bacteria counting device, counting method, and counting method of the present invention can be used to accurately count viable bacteria in a short period of just a few minutes with a simple operation, and can be used in foods, brewing, etc.
It will greatly contribute to quality control and environmental management in fields such as pharmaceuticals and semiconductors.

[Effects of the Invention] According to the present invention, as described in the Examples, the presence of a reagent for dissolving cell membranes and ATP in a filter to which live bacteria are attached after filtering a certain amount of sample liquid or gas. A mixed solution of an enzyme and a substrate that emits light is impregnated in the bottom, and a luminescent reaction between ATP derived from the living bacteria and the enzyme substrate occurs at the attachment position of the living bacteria, and the photons generated are detected two-dimensionally to detect the presence of the living bacteria. Since the presence of bacteria is observed as a two-dimensional photon distribution, it is now possible to count the number of viable bacteria without being affected by variations in the amount of ATP per cell due to differences in cell type and shape S (activity).

ATP luminescence derived from living bacteria occurs continuously for a certain period of time and at the same location, while background light due to fluorescent light in the reagent and high temperature. The noise of the camera is extremely random, and by integrating the number of photons for each pixel over a given period of time,
ATP using conventional photomultiplier for photon counting
The SN ratio can be improved compared to the measurement method. Alternatively, continuous photons emitted from the viable bacteria adhesion position are repeatedly image-integrated at predetermined time intervals in sufficiently small pixel units to obtain multiple photon-integrated images, and each of these photon-integrated images is binarized using the same method. , performs a sequential AND operation between two temporally consecutive binary screens, overlaps the west side of these operation results, or displays it on a second screen by a logical OR operation, and the final image obtained is By counting high-intensity pixels or aggregations thereof to determine the number of attached microorganisms, the S/N ratio can be significantly improved.

As described above, in the present invention, by performing highly sensitive detection at the photon level, it is possible to detect several native countries without multiplying them.

In other words, in order to measure the number of viable bacteria at the level of a few cells, conventionally, the colony counting method required at least 24 hours of culture time, but according to the present invention, the number of viable bacteria at the level of a few cells can be measured without forming colonies. Since the number of bacteria can be measured within a few minutes, it will greatly contribute to quality control and environmental control in various fields such as food production, pharmaceuticals, and semiconductors.

[Brief explanation of the drawing]

Fig. 1 is a schematic diagram showing a partial cross-section of the measuring section of the country of origin counting device of the present invention, Fig. 2 is a system block diagram showing the electrical system of the viable bacteria counting device of the present invention, and Fig. 3 is a schematic diagram showing the measuring section of the country of origin counting device of the present invention. FIG. 4 is a schematic diagram showing a partially enlarged binary image in the method of the present invention, and FIG. 4 is a schematic diagram showing the process of extracting only attached microorganisms from the binary screen in the method of the present invention. 1: Base, 2: Filter, 3: Sample stage, 4: Reagent injection channel, 5: Drain channel, 6: Hood, 7: Camera lens, 8: Close-up ring, 9: Dark box, 10. Super high road image in Tensifier-111: Relay lens, 12:C
CD camera, 13: Movable stage, 14: Support, 15:
Photocathode, 16: Fluorescence! , 17: side image element, U, measurement section,
18A: video signal, frame: image integration section, 20: high voltage power supply section, 21. 22: Video camera controller, 23; Image integration circuit, 24. Video frame memory, 25: Microcomputer, 26: CR
T, 27: Printer 2nd: Keyboard, 29: External storage device. Deputy, ′, Iwasu Yamaguchi

Claims (1)

[Scope of Claims] 1) A reagent for dissolving cell membranes and an enzyme that emits light in the presence of adenosine triphosphate (ATP) and A mixed solution of the substrate is impregnated to cause a luminescent reaction between ATP derived from the viable bacteria and the enzyme-substrate to occur at the position where the viable bacteria adhere, and a two-dimensional photon detection means is used to detect the photon distribution image on the surface of the filter to which the viable bacteria is attached. The method is characterized in that the video signal is integrated into an electrical video signal for a predetermined period of time, and high-luminance pixels or aggregations thereof are counted to determine the number of attached viable bacteria. Bacteria counting method. 2) A plurality of photon integral images obtained by repeatedly integrating the video signal according to claim 1 at a predetermined time interval are binarized using the same method, and two or more temporally consecutive images are obtained. Performs a logical product operation while sequentially moving one screen at a time between binary screens, and displays the results of these calculations on one screen by superimposing the screens or performing a logical sum operation,
A viable bacteria counting method characterized by counting pixels or groups of pixels with high brightness in the last obtained image to determine the number of adherent viable bacteria. 3) a. A filter that has previously filtered a certain amount of sample liquid or gas and has viable bacteria attached to it; b. The presence of a reagent and adenosine triphosphate (ATP) that holds this filter in place and dissolves cell membranes. a reagent injection means for impregnating the filter with a mixed solution of an enzyme and a substrate that emit light at the bottom; c. an imaging optical means for forming an image of at least a part of the viable bacteria adhesion surface of the filter; d. by the imaging optical means; A two-dimensional photon detection means that two-dimensionally amplifies the obtained photon distribution image with high sensitivity, and an imaging means that converts the two-dimensional photon distribution image output from the two-dimensional photon detection means into an electrical video signal. , e. An image integration circuit that integrates the video signal output from this imaging means for a predetermined time; f. Processes the integral image data obtained from this circuit to extract and count high-luminance pixels or a set thereof. A viable bacteria counting device comprising: an image processing circuit; g; output means for displaying an extracted image and counting results;