WO2005098022A1

WO2005098022A1 - Bacteria counting method and bacteria counter

Info

Publication number: WO2005098022A1
Application number: PCT/JP2005/005375
Authority: WO
Inventors: Tomonari Kogure; Kazuhiro Yamada; Hiraku Sasaki; Minoru Sakata; Akihiko Tokida
Original assignee: Bussan Nanotech Research Institute, Inc.
Priority date: 2004-04-06
Filing date: 2005-03-24
Publication date: 2005-10-20
Also published as: JPWO2005098022A1

Abstract

A method for counting bacteria comprising the steps of extracting ATP in bacteria from bacteria in solvent flow, touching the ATP in bacteria thus extracted to liquid emitting light under the presence of ATP in bacteria, measuring an optical signal generated by the contact by at least one light detecting means provided at the touching part of the light emitting liquid and the ATP in bacteria or on the downstream side thereof, and then counting bacteria based on the intensity of the measured optical signal, characterized in that a threshold level is set and bacteria is counted by assuming the number of times when the intensity of measured optical signal exceeds the threshold level as the number of bacteria. A method for counting bacteria and a bacteria counter capable of counting bacteria quickly and conveniently with high accuracy can thereby be provided.

Description

Specification

Bacteria counting method and bacteria counting device

Technical field

The present invention relates to a bacteria counting method and a bacteria counting device.

Background art

[0002] Conventionally, as a method for counting bacteria, a culture method in which bacteria are grown and the number of colonies (communities) in a petri dish reaching several hundreds is visually observed or counted by a colony counter is known.

[0003] In addition, bacteria suspended in a culture solution are passed through tubules, and individual bacteria are irradiated with a laser beam to convert bacterial properties (such as size and DNA content) into optical parameters for identification. Cytometry (FCM) t methods are also known.

[0004] Further, there is disclosed a method for measuring the number of bacteria by preliminarily associating the luminescence intensity due to the reaction between ATP in the bacterium and luciferin luciferase with the number of bacteria, and estimating the luminescence intensity as well as the number of bacteria ( Patent Document 1 or Patent Document 2).

[0005] In addition, different fluorescent dyes are added to both the live bacteria (live bacteria) and the dead bacteria (dead bacteria) and the DNA of the dead bacteria, and fluorescent staining is performed! A method of measuring the number of bacteria (here, the number of viable bacteria) by optically measuring the number of fluorescent dyes is disclosed (see Patent Document 3).

Patent Document 1: Japanese Patent Application Laid-Open No. 06-237793

Patent Document 2: JP-A-2000-157295

Patent Document 3: JP 2001-286296 A

Disclosure of the invention

Problems to be solved by the invention

[0006] However, the conventional culture method has a problem in that it takes several days to grow the bacteria, and a rapid bacterial count cannot be obtained. Therefore, the conventional culturing method is an inconvenient counting method in the case where it is quickly determined whether or not the product can be shipped based on the test result, particularly in a food test. [0007] FCM also has the disadvantage that the equipment is expensive and parallel measurement cannot be performed! Therefore, the counting throughput is low, and it is only used in limited areas in medical and biological research laboratories. Was.

[0008] On the other hand, the bacterial counting method using ATP in bacteria is quick and simple, and solves the above-mentioned problems. However, this method has the following reasons: the counting accuracy is low. There was a problem.

(1) Since the amount of ATP possessed varies depending on the type of bacteria, if the sample contains two or more types of bacteria, the accuracy of the counting result will deteriorate in some cases.

(2) The amount of ATP possessed by the same strain varies greatly depending on its nutritional status. Therefore, even if only one kind of bacteria is contained in the specimen, an error of up to about 1000% occurs.

[0009] Further, in the above-described method of counting bacteria by fluorescent staining of DNA, there is no practical means for efficiently removing only background DNA, so that cells derived from living organisms other than bacteria exist. However, there is a disadvantage that the counting accuracy is lowered.

[0010] Therefore, it is an object of the present invention to provide a bacteria counting method and a bacteria counting device that are quick, simple, and capable of increasing the counting accuracy.

Means for solving the problem

[0011] To achieve the above object, the bacterial counting method of the present invention extracts bacterial ATP from bacteria in a flow of a solvent and emits light in the presence of the extracted bacterial ATP and the bacterial ATP. A light signal generated upon contact with the luminescent liquid was measured by at least one or more light detection means provided at a contact portion between the luminescent liquid and the ATP in the bacterium or downstream thereof. A bacteria counting method for counting bacteria based on the intensity of the optical signal, wherein a threshold is set, and when the intensity of the measured optical signal exceeds the threshold, the number is determined as the number of bacteria and the bacteria are counted. It is characterized by counting.

[0012] According to the bacterial counting method of the present invention, the threshold is set when the intensity of the optical signal generated due to the contact between the ATP in the bacteria and the luminescent liquid is measured by the light detection means. Detection of noise due to diffusion and irregular reflection of light can be eliminated, and highly accurate counting measurement can be performed. Further, according to the bacteria counting method of the present invention, bacteria are counted based on the measured light signal, so that the number of bacteria can be measured quickly and easily. In addition, bacterial Light intensity varies depending on the type and its nutritional status. Even in such a case, the bacteria counting method can count the bacteria by setting the threshold appropriately.

[0013] Preferably, the threshold value is set based on the intensity of the optical signal measured without extracting ATP in bacteria. If the optical signal is measured without extracting ATP in the bacterium, the intensity of the background optical signal can be ascertained, and a more accurate counting measurement can be performed.

[0014] Further, it is preferable to set a threshold value according to the type of bacteria. As described above, the luminescence intensity generally differs depending on the type of bacteria. Therefore, when it is desired to count and measure bacteria including particularly weak luminescence intensity, the bacteria can be counted and measured by lowering the threshold.

[0015] Further, it is preferable to set a plurality of the threshold values according to the type of the bacteria. In this case, even if there are many types of bacteria, by setting the threshold value to an appropriate value, counting can be performed while identifying many bacteria.

[0016] Further, before extracting the ATP in the bacterium from the bacterium, it is preferable to decompose the bacterial floc with a decomposing solution. Degrading bacterial flocs before extracting ATP in bacteria can prevent multiple bacteria from being counted as one. Therefore, the number of bacteria and the number of luminescence equal to or higher than the threshold can be accurately associated with each other, and the counting accuracy can be further improved.

[0017] Further, before extracting the ATP in the bacterium from the bacterium, it is preferable to widen an interval between the bacterium with a diluent. When light from multiple bacteria is detected by the photodetector at the same time, accurate counting may be difficult if the light enters the detection range at the same time. By spreading it, such troubles can be prevented. Therefore, the counting accuracy can be further improved.

[0018] Further, it is preferable that before extracting the ATP in the bacterium from the bacterium, free ATP is removed with an ATP remover, and after the removal, the ATP remover is inactivated with an inert solution. No. According to this method, free ATP that may be contained in the sample solution can be removed before extracting ATP in the bacterium, so that only the desired ATP in the bacterium can be counted. That is, free ATP is not counted. Therefore, to improve the counting accuracy Can do. An ATP remover may destroy bacterial ATP if it has been in contact with bacterial ATP for a long period of time.However, after bacterial ATP is extracted from bacteria, ATP is removed using an inactivation solution. Since the agent is inactivated, it is possible to accurately count the original bacterial ATP in the bacteria to be counted.

[0019] Furthermore, it is preferable that after extracting the bacterial ATP, the ATP is amplified with an amplification solution. A reaction system for amplifying ATP is disclosed in, for example, Japanese Patent Application Laid-Open No. 2001-299390 (Title of Invention: "Method of Amplifying ATP in a Chain and Method of Examining Trace ATP Using the Method"). ing. If the ATP in the bacterium is amplified after the extraction of the ATP in the bacterium in this way, it becomes possible to use low-cost and low-sensitivity photodetection means, such as a photodiode and a phototransistor. The light detecting means can be formed on a substrate made of silicon or gallium arsenide with a high degree of integration by using a semiconductor process. That is, the detection cost is reduced and the degree of integration of the light detection means is increased.

[0020] The present invention also provides a contact portion for contacting an extract for extracting ATP in bacteria, a luminescent solution that emits light in the presence of ATP in bacteria, and a sample solution containing the bacteria. A light detection means for measuring the intensity of the optical signal generated in the step (c), wherein the intensity of the measured optical signal exceeds a threshold set according to the intensity of the measured optical signal. It is characterized by having a control device that counts the bacteria with the number as the number of bacteria.

According to this bacteria counting device, the extraction liquid, the luminescent liquid and the sample liquid are brought into contact at the contact portion, and the intensity of the optical signal generated at the contact portion is measured by the light intensity measuring device, that is, the light detecting means. You. Then, when the intensity of the measured optical signal exceeds a threshold value set according to the measured intensity of the optical signal, the number of bacteria is counted as the number of bacteria. In this way, since the threshold value is set when measuring the intensity of the optical signal generated due to the contact between the ATP in bacteria and the luminescent solution, it is possible to exclude the detection of noise due to light diffusion and diffuse reflection. This makes it possible to perform highly accurate counting measurement. Further, according to the bacteria counting apparatus of the present invention, bacteria are counted based on the measured optical signal, so that the number of bacteria can be measured quickly and easily. Furthermore, the luminescence intensity of bacteria varies depending on the type and its nutritional status. Even in such cases, this bacteria counting device counts bacteria by setting the threshold appropriately. It is possible to do.

[0022] Further, the bacteria counting device may be configured to emit light by a reaction between the first well containing the specimen liquid, the second well containing the extract, the third well containing the luminescent liquid, ATP in bacteria, and the luminescent liquid. Where the light emitting portion, the first channel connecting the first well and the light emitting portion, the second channel connecting the first channel and the second well, and the first channel and the third well are formed. Preferably, a microchannel chip having a third channel to be connected is provided, the channel width of the first channel is 1 mm or less, and the first channel is the contact portion.

[0023] According to the bacteria counting device having such a structure, the sorting operation as in the related art is not required, and there is an effect that convenience is increased. That is, in the microchannel chip, since the first well containing the sample liquid and the light emitting section are connected by the first channel, the sample liquid can be sent to the light emitting section in a few seconds, and the second Since the channel and the third channel are connected to the first channel, when the sample liquid is sent to the first developer, the extract contained in the second well and the luminescent liquid contained in the third well Can be sequentially sent to the first channel. For this reason, by mixing each of the extract and the luminescent liquid in the first channel, the reaction between the sample liquid and the extract and the luminescent liquid can be performed efficiently.

[0024] Further, since the channel width of the first to third channels is 1 mm or less, the diffusion and mixing of the sample solution, the extract solution, and the luminescent solution in the channels are performed in an extremely short time, so that the reaction is instantaneous. Can be done. As a result, bacteria can be counted and measured in seconds, which previously required at least 1-2 hours.

[0025] The microchannel chip further includes a fourth well for containing a decomposing solution for decomposing bacterial flocs, and a fourth channel connecting the first channel and the fourth well, wherein the fourth channel is provided. It is preferable to be connected to the first channel upstream of the second channel.

By providing the fourth channel for sending the decomposition solution into the microchannel chip as described above, it is possible to introduce the decomposition solution into the first channel from the fourth channel. In other words, by decomposing bacterial flocs before extracting intracellular ATP, multiple bacteria can be combined into one. Can be prevented from being counted as bacteria. That is, erroneous counting of bacterial ATP can be prevented. Therefore, the number of bacteria and luminescence can be accurately matched, and the counting accuracy can be further improved.

[0027] Further, the microchannel chip further includes a fifth well for containing a diluent for widening the interval between bacteria, and a fifth channel connecting the first channel and the fifth well, and the fifth channel Is preferably connected to the first channel upstream of the second channel.

[0028] Providing the fifth channel for feeding the diluent into the microchannel chip as described above allows the diluent to be introduced into the first channel with the fifth channel force. In other words, when light from multiple bacteria is detected by the light detection means at the same time, counting may be difficult if the light enters the detection range at the same time.However, by increasing the distance between the bacteria before extracting ATP in the bacteria, Such troubles can be prevented. Therefore, the counting accuracy can be further improved.

[0029] Further, the microchannel chip contains a sixth layer containing a removing solution containing an ATP removing agent for removing free ATP and a deactivating solution containing a deactivating agent for inactivating the ATP removing agent. A seventh channel connecting the first channel and the sixth well, and a seventh channel connecting the first channel and the seventh well, wherein the sixth channel and the seventh channel are the third channel. Preferably, it is connected to the first channel upstream of the two channels.

[0030] By thus providing the sixth channel for sending the removing liquid into the microchannel chip and the seventh channel for sending the inactivating liquid, the sixth channel force removing liquid can be supplied to the seventh channel. Plain inert liquid can be introduced into the first channel. That is, since free ATP that may be contained in the sample solution can be removed before extracting ATP in bacteria, only desired ATP in bacteria can be counted. That is, free ATP is not counted. Therefore, the counting accuracy can be further improved. An ATP remover may destroy ATP in bacteria if it has been in contact with ATP in bacteria for a long time.However, after ATP in bacteria has been extracted from bacteria, ATP is removed with an inactive solution. Since the agent is inactivated, it is possible to accurately count the original bacterial ATP in the bacteria to be counted.

[0031] The microchannel chip contains an amplification solution containing an amplification agent that amplifies ATP. It is preferable that the device further include an eighth channel and an eighth channel connecting the first channel and the eighth channel, wherein the eighth channel is connected to the first channel downstream of the second channel.

[0032] By thus providing the eighth channel for sending the amplification solution into the microchannel chip, the eighth channel force can also introduce the amplification solution into the first channel. That is, if the ATP in the bacterium is amplified after the extraction of the ATP in the bacterium, the use of low-cost and low-sensitivity photodetection means, such as a photodiode or a phototransistor, becomes possible. The light detecting means can be formed on a substrate such as silicon or gallium arsenide with a high degree of integration by using a semiconductor process. As a result, the detection cost is reduced and the degree of integration of the light detection means is increased.

[0033] Also, a second channel containing the microchannel chip force extract, a third level containing the luminescent liquid, a fourth level containing the decomposed liquid, a fifth level containing the diluent, and the removing liquid. A 6th well containing the inactive solution, an 8th well containing the amplification solution, and at least one of the 2nd through 8th wells in that well. It is preferable that the corresponding extract, the luminescent solution, the decomposition solution, the diluting solution, the removing solution, the inert solution or the amplification solution is accommodated.

[0034] By storing the liquid corresponding to the well in the second to eighth wells in advance, the labor for introducing the liquid at the time of counting the bacteria can be omitted, and the counting and measurement can be easily performed. In addition, it is possible to prevent a problem that the liquid to be introduced is mistaken or another bacterium invades from the outside. The invention's effect

According to the bacterial counting method and the bacterial counting device of the present invention, counting accuracy can be increased quickly and simply.

Brief Description of Drawings

FIG. 1 is a perspective view showing a microchannel chip which is a main part of one embodiment of a bacteria counting device of the present invention.

FIG. 2 is a partial cross-sectional view along a thickness direction of a main body constituting the microchannel chip of FIG. 1.

FIG. 3 is a partial plan view showing a part of the microchannel chip of FIG. 1. FIG. 4 is a block diagram showing a configuration of a light detection device and a control device.

FIG. 5 is a cross-sectional view showing first and second modes of a first channel, a second channel, and a third channel.

FIG. 6 is a graph showing the relationship between the emission peak intensity and the threshold value when two types of bacteria are present.

FIG. 7 is a schematic view showing another embodiment of the bacteria counting device of the present invention.

Explanation of symbols

[0037] 1 ······················································································································································································································· s neither- '' and-',,,,,,,,,,,,,,,,, and, respectively, and 、, 7… 7th 、, 8… 8th ゥ, 9 ··· Reaction field (light-emitting part), 9a… Photodetector (light-detecting means), 10 ··· Microchannel chip, 10a… Main body , 10b ... cover, 11 to 1st channel (contact part), 12 ... 2nd channel, 13 ... 3rd channel, 14 ... 4th channel, 15 ... 5th channel, 16 ... 6th channel, 17th channel, 7th channel, 18th channel, 8th channel, 21a, 2 lb channel, 23a, 23b, 24a—24c substrate, 35 hollow part, 36 air vent hole , 40 ... Control unit, 41 "'AZD converter, 42 ... Comparison unit, 43"' RAM, 44-ROM, 45 ... I / O interface, d ... Channel width, II, 12 ... Threshold, P1, Ρ2 ····· Emission peak. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, preferred embodiments of a bacteria counting device according to the present invention will be described in detail with reference to the drawings.

FIG. 1 is a perspective view showing a microchannel chip which is a main part of the first embodiment of the bacteria counting device of the present invention. As shown in FIG. 1, the bacteria counting device of the present embodiment includes a microchannel chip 10, and the microchannel chip 10 has a rectangular flat main body 10a and a cover 10b that covers the surface of the main body 10a. And On one surface of the main body 10a, a first well 1 and a reaction field (light emitting part) 9 are formed at both ends, and the first well 1 accommodates a sample liquid containing bacteria. ing. The first well 1 and the reaction field 9 are connected by a first channel (contact part) 11 formed on one surface of the main body 10a. The liquid is sent from the first well 1 to the reaction field 9 via the first channel 11.

Further, a second well 2 and a third well 3 are formed on the main body 10 a along the first channel 11. The second well 2 is designed to contain a bacterial fluid extract for extracting ATP in bacteria, and the third well 3 contains a luminescent solution that emits light in the presence of ATP in bacteria.

Further, the second well 2 is connected to the extraction liquid introducing portion 31a of the first channel 11 by the second channel 12, and the third well 3 is connected to the reaction field 9 by the third channel. Both the second channel 12 and the third channel 13 are formed on one surface of the main body 10a. Then, the reaction field 9 is disposed downstream of the extract introduction part 31a. Therefore, when the sample liquid is sent to the reaction field 9 through the first channel 11, the extract is sent from the second well 2 to the second channel 12, and the light is emitted from the third well 3 to the third channel 13. The liquid is sent. The sent extract solution and luminescent solution are mixed with the detection solution in the first channel 11 to perform a desired reaction. Note that, in the bacteria detection device of the present embodiment, the reaction field 9 also serves as the luminescent liquid introduction part 31b.

FIG. 2 is a partial cross-sectional view along the thickness direction of the main body 10a. In FIG. 2, the cross-sectional shape of the first channel 11 is rectangular, and the channel width d of the first channel 11 is 1 mm or less. The “channel width” refers to a length in a direction perpendicular to the thickness direction of the main body and in a direction perpendicular to the extending direction of the channel. When the cross-sectional shape of the first channel 11 is rectangular, it refers to the horizontal length, and when it is trapezoidal or triangular, it refers to the maximum length in the thickness direction of the main body 10a. In the case of a circular or semicircular shape, the length is the diameter, and in the case of an elliptical shape, the length is the longest diameter in the thickness direction of the main body 10a. Although not shown, the cross-sectional shapes of the second channel 12 and the third channel 13 are the same as those of the first channel 11, and the channel width is the same as that of the first channel 11.

The first well 1, the second well 2 and the third well 3 are holes formed along the vertical direction with a surface force so as to be able to store the sample liquid, the extract liquid and the luminescent liquid.

Further, a cavity 35 is formed in the main body 10 a on the side opposite to the first well 1 with respect to the reaction field 9. The cavity 35 has an air vent 36 extending to the edge of the main body 10a.

Further, the above-described cover 10b has the second well 2, the third well 3, the first channel 11, the second channel 12, and the second well 1, except that the first well 1 is opened for introducing the sample liquid. The third channel 13 is provided so as to be sealed. The cover 10b is preferably transparent. Good. In this case, it is convenient to visually check whether or not the liquid in each of the wells 1, 2, and 3 has been introduced into the first channel 11.

In the microchannel chip 10, as shown in FIG. 3, a light detector 9 a is arranged adjacent to the reaction field 9. Specifically, the light detection section 9a is provided on the side opposite to the cover 10b with respect to the main body section 10a. The control device 40 is electrically connected to the light detection unit 9a. Here, the control device 40 will be described with reference to FIG.

FIG. 4 is a block diagram showing a configuration of the light detection unit 9a and the control device 40. As shown in FIG. 4, the control device 40 includes an AZD converter 41, a comparison / determination unit 42, a RAM 43, a ROM 44, and an input / output interface 45. The A / D converter 41 converts an analog signal of the light intensity detected by the light detection unit 9a into a digital signal, and the RAM 43 stores threshold data input via the input / output interface 45. It is. The ROM 44 stores a program for counting the number of bacteria as the number of bacteria when the strength of the digital signal obtained by the AZD converter 41 exceeds the threshold data stored in the RAM 43. The comparison / determination unit 42 compares the threshold data stored in the RAM 43 with the light intensity obtained by the AZD converter 41. If the light intensity exceeds the threshold data stored in the RAM 43, the number of bacteria is counted. , And stored in the RAM 43, or output to an output unit such as a monitor via the input / output interface 45. Further, the input / output interface 45 is for outputting data on the number of bacteria obtained by the comparison / determination unit 42 and for storing the input threshold data in the RAM 43 via the comparison / determination unit 42.

[0048] In the bacterium counting device configured as described above, the second well 2 contains the extract, the third well 3 contains the luminescent liquid, and the main body 10a is covered with the cover 10b. When the sample liquid is introduced from the outside and the cavity portion 35 is pressed on the cover 10b, air escapes from the space of the cavity portion 35 through the air vent hole 36. If the pressure is removed after closing the air vent hole 36 by some method (for example, by using a rubber stopper), the space in the cavity 35 becomes negative pressure. Then, the sample solution of the first well 1, the extract of the second well 2, and the luminescent solution of the third well 3 are introduced into the first channel 1, and the sample solution, the extract, and the luminescent solution flow together in the flow. Contacted. At that time, the contact between the sample solution and the extract solution The ATP in the bacteria is extracted therefrom, and the luminescent solution emits light due to the contact between the ATP in the bacteria and the luminescent solution, and an optical signal due to the emission is detected by the light detection unit 9a.

At this time, one threshold is set according to the intensity of the detected optical signal. Here, a value larger than the noise level and smaller than the maximum detected optical signal is set as the threshold. Hereinafter, this threshold is referred to as a set threshold. The threshold value is input by storing it in the RAM 43 via the input / output interface 45.

After setting the threshold value in this manner, the analog signal of the optical signal detected by the light detection unit 9a is converted into a digital signal optical signal by the AZD converter 41, and the comparison determination unit 42 converts the analog signal into an optical signal. , And is compared with the set threshold value stored in the RAM 43. If the comparison / determination unit 42 determines that the light intensity exceeds the set threshold, the number of bacteria is counted as one. The counted bacterial count data is output to an output unit such as a monitor via the force stored in the RAM 43 and the input / output interface 45. When the light intensity is determined to be equal to or less than the set threshold by the comparison determination unit 42, the number of bacteria is not counted. Then, the above operation is repeated to count bacteria.

[0051] When the bacteria are counted in this manner, the threshold is set to a level higher than the noise level when measuring the intensity of the optical signal generated due to the contact between the ATP in the bacteria and the luminescent solution. Therefore, noise detection due to light diffusion and diffuse reflection can be eliminated, and highly accurate counting and measurement can be performed. Also, the intensity of the optical signal due to the luminescence of the bacterium may vary depending on the nutritional status, even if the bacteria are of the same species. Since the measurement is performed and the threshold is set according to the measured intensity, it is possible to accurately count the bacteria even if the bacteria are of the same species. Furthermore, bacteria generally have different optical signal intensities when they are of different types. However, according to this bacteria counting method, even in such a case, by setting the threshold to an appropriate value, only the specific bacteria can be detected. Counting can also be performed selectively.

[0052] In addition, when the bacteria are counted using the bacteria counting device having the above-described structure, the sorting operation as in the related art is not required, and there is an effect that convenience is increased. That is, since the first well 1 containing the sample liquid and the reaction field 9 are connected via the first channel 11 in the microchannel chip 10, the sample liquid is sent to the reaction field 9 in a few seconds. In Since the second channel 12 and the third channel 13 are connected to the first channel 11 when the test solution is sent from the first well 1 to the reaction field 9, the extraction contained in the second well 2 is performed. The liquid and the luminescent liquid contained in the third well 3 can be sequentially sent to the first channel 11. Then, the extraction liquid and the luminescence liquid are mixed in the first channel 11, whereby the reaction between the sample liquid and the extraction liquid and the luminescence liquid can be performed efficiently.

Further, since the channel width d of the first to third channels 11 to 13 is 1 mm or less, the diffusion and mixing of the sample solution, the extract solution, and the luminescent solution on the channel are performed in a very short time. By doing so, the reaction can be performed instantaneously. As a result, the conventional method requires at least one and a half hours, and can detect bacteria in a few seconds, and thus can count bacteria in an extremely short time. More preferably, the channel width d is 0.1-500 / zm.

[0054] In the microchannel chip 10, the main body 10a is hermetically closed by the cover 10b, so that invasion of bacteria from the outside is sufficiently suppressed. Therefore, it is extremely convenient because it does not require inspection in a clean environment or strict disinfection as in the past.

As the photodetector 9a, a known detector can be used, and for example, a CCD, a photodiode, a phototransistor, a photomultiplier, an image intensifier, or the like can be used. The light detection unit 9a detects light generated by the reaction of ATP in bacteria with the luminescent solution in the reaction field 9. Therefore, the installation position of the light detection unit 9a is not particularly limited, but is preferably installed near the reaction field 9. Note that the light detection unit 9a can be installed in the main body 10a of the microchannel chip 10.

[0056] Examples of the bacteria contained in the sample solution include, but are not particularly limited to, bacteria that may cause food poisoning (eg, Vibrio parahaemolyticus, Salmonella, Staphylococcus aureus, Bacillus cereus, or Bacillus pylobacter).

[0057] The component contained in the extract may be any component that can extract ATP in bacteria, and may be lysozyme, a mixture of ethanol and ammonia, methanol, ethanol, a surfactant (benzetonium chloride, benzalco-chloride). Pharm, Triton X-100, etc.), trichloroacetic acid, perchloric acid, phage, and antibiotics.

[0058] Further, the component contained in the luminescent solution may be any component that emits light in the presence of ATP, and examples thereof include luciferase and luciferin. [0059] The sample liquid, extract liquid and luminescent liquid may be a suspension or emulsion as long as they are liquid.

FIGS. 5A and 5B are cross-sectional views showing the first, second, and third modes of the first channel 11, the second channel 12, and the third channel 13. FIG.

[0061] The channel 21a of the second embodiment shown in Fig. 5 (a) is a flow channel formed by laminating two base materials. That is, a groove is provided in a lower portion of one base material 23a and an upper portion of the other base material 23b, and the channels 21a are formed by bonding. The channel 21b of the third embodiment shown in FIG. 5 (b) is formed by laminating three base materials. That is, a channel 21b is formed by providing a through groove in the base material 24b as an intermediate layer, and laminating the base materials 24a and 24c on the upper and lower portions so as to sandwich the base material 24b. The channel 21a and the channel 21b correspond to the first channel 11, the second channel 12, and the third channel 13, respectively.

[0062] These substrates 23a, 23b, 24a-24c can be arbitrarily determined, and may be glass such as quartz glass or Pyrex (registered trademark) glass, polymers such as PDMS, polycarbonate or polyimide, iron, stainless steel, and aluminum. Metal such as nickel, copper or the like, or silicon or the like can be used. When laminating, the respective substrates may be of the same type or different types.

[0063] The channel width of the first channel 11, the second channel 12, and the third channel 13 is 1 mm or less.

[0064] Since the channel width is within the above range, when the sample liquid, the extract liquid, and the luminescent liquid are mixed in this channel, the contact surface area per unit volume increases, so that the reaction can be promoted. . That is, since the diffusion control can be minimized, a quick reaction can be realized. On the other hand, if the channel width exceeds 1 mm, the diffusion control cannot be sufficiently reduced, so that the object of the present invention cannot be achieved. A more preferred channel width is 0.1-500πι.

For example, in the microchannel chip 10 according to the first embodiment, the force of storing the extraction liquid in the second well 2 and the luminescent liquid in the third well 3 can be reversed. Use it. That is, when the sample liquid of the first well 1 is sent to the reaction field 9 through the first channel 11, the luminescent liquid is sent from the second well 2 through the second channel 12, and further the third well 3 is sent. Similarly, when the extract is sent through the third channel 13, the sample liquid in the first well 1 is sent to the reaction field 9 through the first channel 11. A mixed liquid of the extract and the luminescent liquid may be sent through the flow passage 12.

[0067] Since the extract is a solution for extracting ATP in bacteria, it does not react even when mixed with a luminescent solution containing no bacteria, while the luminescent solution emits light in the presence of ATP. This is because the reaction solution does not contain ATP and does not react even when mixed with the extract. In addition, when the sample liquid and the luminescent liquid are mixed and then mixed with the extract, the ATP in bacteria can be extracted from the sample liquid and emit light at the same time. Further, when the extract solution and the luminescent solution are mixed and then mixed with the sample solution, the number of wells and the number of channels can be reduced, and the bacteria counting device can be simplified.

[0068] The present invention is not limited to the above embodiment! For example, in the above embodiment, the threshold value is set independently of the type of bacteria, but it is preferable that the threshold value be set according to the type of bacteria. As described above, the luminescence intensity generally varies depending on the type of bacteria. Therefore, when it is desired to count and measure bacteria including particularly weak luminescence intensity, the bacteria can be counted and measured by lowering the threshold.

[0069] Furthermore, it is preferable to set a plurality of the thresholds according to the type of the bacteria. In this case, even if there are many types of bacteria, by setting the threshold value to an appropriate value, counting can be performed while identifying many bacteria. For example, when there are two kinds of bacteria, as shown in FIG. 6, two kinds of luminescence peaks PI and P2 are observed according to the two kinds of bacteria. In this case, one threshold II is set to a threshold lower than the emission peak P1, and the other threshold 12 is set to a value smaller than the emission peak P2 and larger than the emission peak P1. When the bacteria are counted in this manner, the luminescence peaks exceeding II can be counted, and the bacteria corresponding to the luminescence peaks P1 and P2 can be counted.By counting the luminescence peaks exceeding 12, only the luminescence peak P2 can be counted. It becomes possible to count the number of bacteria. Therefore, by taking the difference between the two counts, the number of bacteria corresponding to the emission peak P1 is calculated. As a result, bacteria corresponding to each of the luminescence peaks PI and P2 can be counted, and two types of bacteria can be distinguished. In the above embodiment, the force using the microchannel chip 10 having only the first level 1, the second level 2, and the third level 3 As shown in FIG. 7, the micro channel chip 10 In addition, a fourth well 4 containing a decomposing solution that decomposes bacterial flocs, a fifth well 5 containing a diluent that increases the distance between bacteria, and a removing solution containing an ATP remover that removes free ATP are stored. A sixth layer 6 containing a deactivating solution containing an inactivating agent for inactivating the ATP removing agent, a seventh layer 7 containing an inactivating solution containing an inactivating agent for inactivating the ATP removing agent, and a second container containing an amplifying solution containing an amplifying agent for amplifying ATP And a fourth channel 14 for connecting the first channel 11 to the fourth well 4 and a fifth channel for connecting the first channel 11 to the fifth well 5 corresponding thereto. 15, 6th channel connecting 1st channel 11 to 6th channel 6, 16th channel And a seventh channel 17, and the first channel 11 and the eighth channel 18 that connects the eighth Ueru 8 connects Le 11 and a seventh Ueru 7.

In this case, the decomposing solution, the diluting solution, the removing solution, the inactivating solution, and the amplifying solution can be instantaneously introduced into the first channel 11. That is, the bacterial floc can be decomposed by introducing the decomposing solution, and the distance between the bacteria can be increased by introducing the diluting solution. Further, unnecessary free ATP can be removed by the removing solution, and an excess removing solution can be inactivated by the inactivating solution. By introducing these liquids, extremely accurate counting and measurement can be performed. Furthermore, if ATP in bacteria is extracted from the bacteria with an extract solution, and luminescence is caused by the luminescent solution, and then the amplification solution is introduced, the bacterial count can be measured even with low-sensitivity light detection means. Further, if such a device is used, the detection cost is reduced and the degree of integration of the light detection means is increased.

[0072] These decomposing liquids, diluting liquids, removing liquids, inactivating liquids, and amplifying liquids can be introduced appropriately in accordance with the desired accuracy in counting bacteria, and can be omitted as necessary. It is also possible.

[0073] The components contained in the decomposition solution are not particularly limited !, but Tris-Hcl, HEPES,

PBS, MES, Tricine and the like.

[0074] Examples of the components contained in the removal solution include adenosine phosphate deaminase, avirase, alkaline phosphatase, acid phosphatase, hexokinase, and adenosine triphosphatase. [0075] Examples of the components contained in the inactive solution include coformycin, EDTA, dithiothreitol, ammonium sulfate, HEPES, MES, and Tricine.

[0076] The components contained in the amplification solution are not particularly limited !, but are adenylate kinase, polyphosphate kinase, polyphosphate, adenosine monophosphate, pyruvate orthophosphate dikinase, phosphoenolpyruvate, and pyrroline. Acids and magnesium ions are exemplified.

Further, in the above embodiment, an example of bacterial counting using the microchannel chip 10 is shown, but the present invention is not limited to the one using the microchannel chip 10. In short, what is necessary is just to use a substance having a contact portion that allows the sample liquid, the extract liquid and the luminescent liquid to come into contact with each other. Test tubes, flasks and the like having such a contact portion are exemplified.

Further, in another embodiment of the present invention, the 2nd to 8th wells correspond to the extraction liquid, the luminescent liquid, the decomposing liquid, the diluting liquid, the removing liquid, the inactivating liquid, and the amplification liquid corresponding to the corresponding liquid tank. Can be stored in advance. If these solutions are stored in advance, the bacteria can be counted simply by introducing the sample solution, and the measurement can be easily performed with high accuracy regardless of the environment.

Industrial applicability

The bacterial counting method and the bacterial counting device of the present invention can be performed quickly and easily, and can increase the counting accuracy. Therefore, particularly when it is desired to quickly determine whether or not a product can be shipped based on the inspection result, such as in a food inspection. Etc. can be suitably used.

Claims

The scope of the claims

[1] Bacterial force in the flow of solvent Extracts ATP in bacteria, contacts the extracted bacterial ATP with a luminescent solution that emits light in the presence of the ATP in the bacteria, and generates light generated by the contact. A bacterium count that measures a signal with at least one or more light detecting means disposed at a contact portion between the luminescent liquid and the ATP in the bacterium or a downstream side thereof and counts the bacterium based on the measured intensity of the optical signal. The method,

A bacterium counting method, comprising setting a threshold, and counting the bacterium with the number as the number of bacteria when the intensity of the measured optical signal exceeds the threshold.

2. The bacteria counting method according to claim 1, wherein the threshold is set based on the intensity of an optical signal measured in a state where the ATP in the bacteria is not extracted.

[3] The bacteria counting method according to claim 1 or 2, wherein the threshold value is set according to the type of bacteria.

4. The method according to claim 1, wherein a plurality of the thresholds are set according to the type of the bacteria.

V. The method for counting bacteria according to any one of the preceding claims.

[5] The method for counting bacteria according to any one of [14] to [14], wherein the bacterial force is also used to decompose bacterial flocs with a decomposition solution before extracting the ATP in the bacteria.

[6] The method for counting bacteria according to any one of claims 15 to 15, wherein, before extracting the ATP in the bacteria, the bacterial force also increases the distance between the bacteria using a diluent.

[7] The method for removing free ATP with an ATP removing agent before extracting the ATP in the bacterium and extracting the ATP in the bacterium, and inactivating the ATP removing agent with an inactive solution after the removal. The method for counting bacteria according to any one of claims 16 to 16.

[8] The bacterial counting method according to any one of [17] to [17], wherein the ATP is amplified by an amplification solution after extracting the ATP in the bacterium.

[9] Bacterial power: an extract for extracting intracellular ATP, a light emitting solution that emits light in the presence of the intracellular ATP, and a contact portion for contacting a sample liquid containing the bacteria;

Light detection means for measuring the intensity of an optical signal due to light generated at the contact portion or downstream thereof, and a bacteria counting device comprising:

The threshold value set according to the measured optical signal intensity is increased when the measured optical signal intensity increases. A bacteria counting device comprising: a control device that counts bacteria when the number of bacteria turns, the number of bacteria being counted.

[10] A first column containing the sample liquid,

A second well containing the extract,

A third column containing the luminescent liquid,

A light-emitting portion that emits light by the reaction between the bacterial ATP and the luminescent solution,

A first channel connecting the first well and the light emitting unit,

A second channel connecting the first channel and the second well; and

Third channel connecting the first channel and the third well

A microchannel chip having

The channel width of the first channel is lmm or less, and

10. The bacteria counting device according to claim 9, wherein the first channel is the contact portion.

[11] The microchannel chip,

A fourth well containing a digestion solution that degrades the bacterial flocs;

A fourth channel connecting the first channel and the fourth well;

Further comprising

11. The bacteria counting device according to claim 10, wherein the fourth channel is connected to the first channel on an upstream side of the second channel.

[12] The microchannel chip comprises:

A fifth well containing a diluent to increase the spacing between the bacteria;

A fifth channel connecting the first channel and the fifth well;

Further comprising

12. The bacteria counting device according to claim 10, wherein the fifth channel is connected to the first channel on an upstream side of the second channel.

[13] The microchannel chip comprises:

A sixth layer containing a remover containing an ATP remover for removing free ATP, and a seventh column containing a deactivator containing an inactivator for inactivating the ATP remover. Enore,

A sixth channel connecting the first channel and the sixth well,

A seventh channel connecting the first channel and the seventh well,

Further comprising

13. The cell counting device according to claim 10, wherein the sixth channel and the seventh channel are connected to the first channel on an upstream side of the second channel. .

[14] The microchannel chip comprises:

An eighth well containing an amplification solution containing an amplification agent for amplifying ATP,

An eighth channel connecting the first channel and the eighth well,

Further comprising

14. The bacteria counting device according to claim 10, wherein the eighth channel is connected to the first channel on a downstream side of the second channel.

[15] The microchannel chip comprises:

A fourth well containing a decomposing solution for decomposing the bacterial flocs, a fifth well containing a diluent for increasing the distance between the bacteria, and a sixth well containing a removing solution containing an ATP remover for removing free ATP. A bottle selected from the group consisting of a seventh column containing an inactivating solution containing an inactivating agent for inactivating the ATP removing agent and an eighth column containing an amplifying solution containing an amplifying agent for ATP. And at least one

A channel that connects the well and the first channel;

In the well, a liquid corresponding to the well among the liquids selected from the group consisting of the extract, the luminescent solution, the decomposition solution, the diluting solution, the removing solution, the inert solution, and the amplifying solution. 11. The bacteria counting device according to claim 10, wherein the bacteria are stored.