JP2588113B2 - Viable cell count measuring device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a viable cell count measuring apparatus. More specifically, the present invention relates to a viable cell count measuring apparatus comprising growing a specific microorganism in a specimen using a liquid selective medium and capturing fluorescence emitted from the microorganism.

[0002]

2. Description of the Related Art Conventionally, such microorganism species are identified based on physiological and biochemical properties. On the other hand, the number of viable bacteria is determined by ^measuring a sample at 10 ⁻¹ , 10 ⁻² ,. , 10 ^-8 times, inoculate a certain amount of this diluted solution on an agar plate medium, and after culturing for a certain period of time (24 to 48 hours), adjust the dilution ratio to the number of colonies appearing on this agar plate. It was required to be multiplied.

[0003] However, such a completely manual microbial testing method requires a testing period of 2 to 5 days, considerable skill, and may cause measurement differences between technicians or inspectors. Are known. In addition, the cost of testing is high due to the use of large volumes of media and dishes and the high labor costs of skilled technicians.

[0004] Microbial tests such as identification of microbial species and measurement of the number of viable bacteria are indispensable in departments such as clinical tests, food tests and pharmaceutical tests, and there is a high need for speeding up, labor saving and automation.

[0005] For this reason, various kinds of automated machines are being developed in the clinical examination department. For example, on a plate agar rotating at 50 rpm, a plater that smears the sample solution from the center to the outside, the smeared agar medium is cultured, and the colonies formed on the plate medium are irradiated with He-Ne laser light. Using a viable cell counting device consisting of a colony counter for counting and a data processor, or a cartridge containing various culture media for property inspection, the identification of microorganism species and the measurement of bacterial amount by turbidimetry are fully automated. A viable cell count measuring device that can be used or a similar device using a microplate instead of this cartridge has been trial manufactured.

However, most of these instruments are used only for microbial tests such as urinary tract infection, and cannot be measured when samples are often turbid as in food tests. In some cases, sufficient accuracy may not be obtained. In addition, since each method requires a culture period of 24 to 48 hours, rapid measurement is impossible, and it cannot meet the needs for urgent inspection.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide a viable cell count measuring apparatus which can be used in a wide range of fields, is highly accurate and low-cost, and requires only a few hours of inspection time.

[0008]

Means for Solving the Problems As a result of extensive experiments and research conducted by the present inventors for many years, microorganisms in a liquid selective medium are irradiated with excitation light, and the intensity of fluorescence emitted from the microorganisms is measured. Then, the microorganisms in the liquid selective medium are cultured, the grown microorganisms are irradiated with excitation light, the intensity of the fluorescence emitted from the microorganisms is measured, and the difference in the fluorescence intensity before and after the culture is determined to determine the microorganism species. It has been found that identification and the viable cell count of the microorganism can be performed with high accuracy and in a short time. The present invention has been completed based on such findings.

[0009]

The living cells of the microorganism are coenzyme NADH (reduced form of nicotinamide adenine dinucleotide) and NADP
H (reduced form of nicotinamide adenine dinucleotide phosphate) universally. Irradiation of these coenzymes with excitation light produces fluorescence.

Since the intensity of the fluorescence depends on the species of each microorganism, the number of viable bacteria, and the amount of coenzyme contained in each viable cell, a specimen whose microbial species and the number of viable bacteria before culture are known before culture is used. And the fluorescence intensity after culture, and the difference (Δ
Find I). The amount of coenzyme contained per living cell before culturing is proportional to the value obtained by subtracting the background fluorescence intensity derived from the liquid selection medium from the fluorescence intensity before culturing, and dividing the result by the known number of living bacteria. Because of the relationship, this value can be regarded as the amount of coenzyme per viable cell number before culturing.

Therefore, based on the known sample, the fluorescence intensity difference ΔI
When creating the database of s, for example, a two-dimensional storage table is used. In this table, the number of viable cells is taken in the vertical column, and the amount of coenzyme per one viable cell number before cultivation obtained by the above method is taken in the horizontal column. The number of known viable bacteria is divided into, for example, 10 ¹ , 10 ² , 10 ³ ,..., 10 ⁿ , and the amount of coenzyme is normalized by the maximum value.
1,0.2,0.3, ..., 0.9,1.0. Then, for example, a known microbial species having a viable cell count of 10 ¹ and a coenzyme amount of 0 is diluted 1-fold in n stages (for example, 11 stages), and the fluorescence intensity before and after culturing is measured for each dilution stage, Fluorescent intensity differences ΔIs ₁ , ΔIs ₂ ,..., ΔIs ₁₁ are obtained. This data collection is carried out for each viable cell count and each coenzyme amount to form a database. The dilution step varies depending on the microplate used. For example, for a 96-well microplate, a maximum of 11 dilution steps are possible.

With respect to a specimen of the same microorganism species whose number of bacteria is unknown, the fluorescence intensity before and after the culture is measured in the same dilution step, and the fluorescence intensity differences ΔIu ₁ , ΔIu ₂ ,.
Seek ΔIu _11.

In order to determine the viable cell count of microorganisms in an unknown sample, the “sag (distance)” between the standard data of the known sample and the actually measured data of the unknown sample is defined, and it is 0 or 0. The number of microorganisms corresponding to the standard data closest to is estimated as the number of viable microorganisms in the unknown sample.

For example, the characteristics (ΔIu ₁ , ΔIu ₂ ,
...., one unknown samples U with a? Iu _n), characterized _{(ΔIs 1, ΔIs 2, ·····} , when compared to the known standard sample S is .DELTA.Is _n), the "Toda Rip (distance) "D" is obtained by the following relational expression.

If a data matrix as described above is created for each microbial species and stored in a database, the number of viable bacteria can be accurately and quickly estimated by searching and referring to the database from the measured values of unknown samples. Therefore,
The reliability of the measuring device of the present invention increases as the amount of data in the database increases.

The growth process of a microorganism can be generally divided into an induction period, a logarithmic period, a arrest period and a death period. At each stage, the amount of coenzyme per cell of the microorganism changes. The database is created using a specimen whose microbial species, viable cell count and coenzyme amount before culture are known. Therefore, the species of the microorganism in the unknown sample is identified, the number of viable cells thereof is estimated, and the amount of the coenzyme is also estimated secondarily, so that the growth stage of the microorganism in the sample can be predicted.

According to the present invention, the identification of the microorganism species in the unknown sample and the measurement of the viable cell count thereof are performed by using only the specific microorganism species for growth and growth.
This is made possible by using a selective medium that can be grown. The selection medium is a medium in which a specific microorganism species can be more advantageously grown than other microorganism species by adding a specific substrate, an antibiotic or the like.

For example, in an unknown sample, Bifidobacterium
In the case where a bacterium belonging to the genus (Bifidobacterium) (so-called bifidobacterium) is present, and if so, the number of viable bacteria, a culture medium having the following composition is used as a selective medium. Lab-lemco powder (Oxoid)
2.4 g / l, proteose peptone no. 3 (Difco
10.0 g / l, tryptic case (BBL) 5 g
/ L, yeast extract (Difco) 5.0 g / l, liver exudate (Mitsuoka, 1969) 150 ml / l, raffinose 4
g / l, saline solution A (Mitsuoka et al., 1965) 10 ml /
1, 5 ml / l of salt solution B (Mitsuoka et al., 1965), 5 ml / l of antifoaming agent (manufactured by Dow Corning, 10%), 1 g / l of Tween 80, 1 g / l of L-cysteine HCl.H ₂ O.
5 g / l, sodium propionate 15 g / l, colimycin ⁽¹⁰⁶ units, 1%) 12ml / l. When preparing this selective medium, components other than colimycin are mixed and mixed.
Sterilize at 5 ° C for 20 minutes. Colimycin is added aseptically immediately before use. Since only bacteria belonging to the genus Bifidobacterium can grow in this selective medium, if the unknown specimen emits fluorescence, the microorganism species in the unknown specimen is identified as a bacterium belonging to the genus Bacterium.

As described above, an unknown sample is inoculated into a selective medium capable of growing only a specific microorganism species, cultured, and the fluorescence intensity is measured. If there is a difference in the fluorescence intensity before and after the culture, the microorganism species is identified. This is because the microorganisms are present in the unknown sample and proliferated, so that the microorganism species in the unknown sample can be easily identified.

In practice, it is difficult to predict what kind of microbial species is present in the unknown sample, so that the unknown sample is inoculated on all selective media, cultured, and grown on which selective medium. It is confirmed based on the difference in fluorescence intensity before and after the culture, and the microorganism species is identified.

According to the study of the present inventors, only Escherichia coli can be specifically grown by using a selective medium having the following composition. Meat extract 3.0 g Peptone 10.0 g Casein 5.0 g Lactose 15.0 g Sucrose 10.0 g Sodium deoxycholate 1.0 g Sodium thiosulfate 2.5 g Sodium citrate 1.0 g Ammonium citrate 1.0 g Purified water 1000 ml

In the case of Salmonella, they grow specifically in a selective medium having the following composition. Meat extract 3.0 g Proteose peptone 12.0 g Lactose 12.0 g Sucrose 12.0 g Salicin 2.0 g Bile salts 15.0 g Sodium chloride 5.0 g Sodium thiosulfate 6.8 g Ammonium citrate 0.8 g Sodium deoxycholate 2.0g purified water 1000ml

The selection medium used for S. aureus has the following composition: Yeast extract 2.5 g Peptone 10.0 g Keratin 30.0 g Lactose 2.0 g Mannit 10.0 g Sodium chloride 75.0 g Dipotassium phosphate 5.0 g Lithium chloride 5.0 g Phenol ethyl alcohol 25 ml Purified water 1000 ml

The selection medium for Campylobacter has the following composition: Proteose peptone 15.0 g Yeast extract 5.0 g Liver extract 2.5 g Sodium chloride 5.0 g Vancomycin 10 mg Polymyxin B 2500 units Trimtobrim 5 mg Cephalosin 15 mg Amphotericin B 2 mg Purified water 1000 ml

For yeast, a selective medium having the following composition is used. Bacto potato dextrose bouillon (Difco) 39.0 g 10% tartaric acid 14.0 ml Purified water 1000 ml

The selection medium is preferably a liquid medium. In the case of a liquid medium, it is possible to automatically dispense a fixed amount using a micropipette or the like.

[0027]

An embodiment of the present invention will be described below in more detail with reference to the drawings.

FIG. 1 is a block diagram showing an example of the viable cell count measuring apparatus of the present invention. This apparatus basically includes a microplate dilution / measurement unit 10 and a data processing unit 20. FIG.
In the microplate dilution / measurement section 10, the microplate is sent from the loader cassette 30 to the automatic dilution / stirring apparatus 40 as indicated by the solid arrow in the figure, where the inoculation, dilution, and stirring of the sample (sample) are performed. Simultaneously, they are transferred to the unloader cassette 32. The unloader cassette 32 also serves as a loader cassette that supplies a microplate to the adjacent automatic fluorescence measurement device 50. The microplate having undergone the fluorescence measurement is transferred to another unloader cassette 34. The fluorescence intensity measurement by the automatic fluorescence measurement device 50 is performed on the microplate before culture, then transferred to the incubator 36 and cultured for a predetermined time, and after the culture, the fluorescence intensity measurement is performed again. The incubator does not need to be particularly incorporated in the apparatus itself, and a usual thermostat or the like provided in an ordinary laboratory can be used. In FIG. 1, the thick arrow indicates the movement of the microplate after the culture. Conventional means such as a conveyor is used for transferring the microplate. The transfer of the cassette between the unloader / loader cassette 32 and the unloader cassette 34 and the incubator 36 is performed as desired.
A robot or the like can be used.

The fluorescence intensity measurement signal before culture (indicated by a dashed line in the figure) and the fluorescence intensity measurement signal after culture (indicated by a dashed line in the figure) from the automatic fluorescence measuring device 50 are transmitted to the data processor 2.
0 to the processor 60. The external storage device 7 is obtained from the measurement signal and information on the type of the selection medium used.
By searching and referring to the database stored in the database 0, the identification of the microorganism species existing in the specimen and the number of viable cells of the microorganism species are estimated. The processor 60 includes an external storage device 70 and a console 8 having a CRT and a keyboard.
Connected to 0. All operations can be performed by inputting commands from the console 80. Dashed arrows in the figure indicate the flow of control signals and data. That is, FIG.
As can be understood from the control line indicated by the control dotted line,
The processor 60 converts the values measured by the automatic fluorescence measurement device 50
Search and refer to the database of the external storage device 70 based on
Data processing and automatic dilution / stirring equipment
The microplate 90 is loaded on
Generating and selecting different liquid selective media
Control to dilute in multiple stages, this diluted medium
The microplate 90 with
Controls the loading and controls the automatic fluorescence measurement device 50
Plate with diluted medium loaded
Control for measuring the fluorescence intensity for each of the 90 media
Each of the cultures obtained at this time by the automatic fluorescence measurement device 50
The fluorescence intensity of the ground is obtained as the first fluorescence intensity,
Unloads the microplate 90 from the setting device 50
Control and measure the first fluorescence intensity
Control the loading of the plate into the automatic fluorescence measurement device 50
And after the culture loaded on the automatic fluorescence measurement device 50
The fluorescence intensity was measured for each medium of the microplate 90.
Control, and the automatic fluorescence measurement device 50
The obtained fluorescence intensity of each medium is obtained as a second fluorescence intensity.
And according to the difference between the second fluorescence intensity and the first fluorescence intensity.
This is a process for calculating the number of viable bacteria that have proliferated.

As described above, the fluorescence intensity was measured before culture,
By subtracting the value from the measured fluorescence intensity after culture, the background is removed and a signal corresponding to the increase in the number of viable cells of the specific microorganism species grown in the selection medium is obtained. On the other hand, if there is no difference between the fluorescence intensities before and after the culture, it means that the microorganism species corresponding to the selective medium is not present in the unknown sample.

FIG. 2 is a perspective view of the microplate 90 used in the apparatus of the present invention. The microplate 90 is made of, for example, a polymer material such as polycarbonate or a transparent material such as glass. The microplate 90 may be made of opaque plastics, ceramics, ceramics, or the like.

On the front surface of the microplate 90, 96
The holes (8 rows × 12 columns) are provided. The shape of the hole is not particularly limited. Any shape such as a sliver, a V, a column or a prism can be used. Of course, microplates other than 96 wells can be used. One row on the front side of the microplate is a space into which the sample liquid is injected, and a predetermined amount of the liquid selective medium is injected into the remaining row for dilution in advance.

FIG. 3 is a plan view of an example of the viable cell count measuring apparatus of the present invention, and FIG. 4 is a view taken in the direction of arrow A in FIG. A loader cassette 30 (the left side in the figure) and an unloader / loader cassette 32 are provided at both ends of the transport system 41 of the automatic dilution / stirring device 40. The drive transmission system of the transport system 41 can be constituted by conventional means such as a conveyor, a chain, and a belt. Each cassette can store, for example, ten microplates 90 and has a miniature warehouse type structure. The unloader cassette 34 in FIG. 1 has the same structure. The microplate can be put in the incubator 36 together with the cassette. The cassette can be moved up and down by using conventional means such as a ball screw mechanism or an elevator. In FIG.
48 is a base, and 49 is a gantry.

At the tip of a drive system 43 that can move forward and backward along the guide rail 42, a micro-dispensing device 44, for example, a micro-syringe or the like, whose number corresponds to the number of sample holes in the microplate 90, is attached. . This micro amount dispenser 4
A sterilized disposable tip 45 is attached to the tip of 4. The chip 45 is supplied from a chip supply unit 46. The microplate 90 can be fed not only horizontally but also vertically as shown in FIG. In the case of horizontal feeding, a maximum of 8 samples can be diluted to 10 ⁻¹¹ times, and in the case of vertical feeding, a maximum of 12 samples can be diluted to 10 ⁻⁷ times. Micro-dispensing means 44 by eccentric motor 47
By vibrating the tip 45 at the tip of the above, the liquid in the hole is stirred to make the concentration uniform. Stirring means other than the eccentric motor can naturally be used. For example, the purpose of stirring can be achieved by repeating the operation of sucking and discharging the liquid in the hole with a syringe several times.

The syringe stroke of the sample liquid dispenser is set so as to have a predetermined dilution ratio in accordance with the amount of the selective medium injected into each hole, and the dispenser is sterilized while repeatedly moving back and forth. Loading of chips, dilution and stirring at the setting stage,
Until the last chip is discarded automatically. For the dilution method, for example, 45 μl of the selection medium is injected into each hole of the selection medium row, and 5 μl of the sample is collected from the hole of the sample row with a pipettor, poured into the first selection medium hole, and stirred. . 5 μl is collected from the obtained 10 ^-1 dilution, poured into the next selective medium well, and stirred. This gives a ^10-2 fold dilution. 5 μl is collected from the 10 ⁻² -fold diluted solution, poured into the next selective medium well, and stirred. Thus, a ^10-3 dilution is obtained. By repeating this operation, it is possible to prepare the dilutions of up to 10 ^-7 or 10 ^{-1 1} ×.
As described above, the test with a large number of dilution ratios for one sample can be detected in a wide range of 10 ¹ to 10 ¹¹ / ml of viable cells of the bacteria to be measured in the unknown sample. And to estimate an approximate value of the viable cell count. For example, when no fluorescence intensity difference is detected for a diluted sample of 10 ⁻⁹ times or more, the number of viable bacteria in the unknown sample is estimated to be about 10 ⁸ to 10 ⁹ .

FIG. 5 is a schematic diagram showing the principle of fluorescence detection by the optical system in the automatic fluorescence measurement device 50. The principle of fluorescence detection itself is known, and basically an excitation filter,
The observation system has an absorption filter, and a dichroic mirror is used instead of the half mirror.

As shown in FIG. 5, from the illumination light including light of various wavelengths emitted from the light source 51, only light in the wavelength range necessary for generating fluorescence is extracted by the excitation filter 52. Is transmitted. The transmitted light is reflected downward by 90 ° by the dichroic mirror 53, and then reflected by the objective lens 54.
a in the direction opposite to the normal direction, and reaches the specimen in the hole of the microplate 90 as excitation light. Part of the fluorescence emitted in an arbitrary direction by the excitation light irradiation enters the objective lens 54a. The dichroic mirror 53 transmits the fluorescence having a longer wavelength than the excitation light without reflecting it. Therefore, the objective lens 5
The fluorescence entering 4a passes through the dichroic mirror 53 and passes through the absorption filter 55a. The absorption filter 55a also cuts off a slight stray light of the excitation light, and allows only the fluorescence to reach the photomultiplier tube 56a.

Since the microplate 90 is made of a transparent light-transmitting material, the fluorescent light emitted in any direction by the irradiation of the excitation light travels in both directions above and below the microplate 90. Therefore, the objective lens 54b, the absorption filter 55b, and the photomultiplier tube 56b are also provided below the microplate 90. Since the fluorescence generated from the microorganism is extremely weak, the detection accuracy is improved by capturing and measuring the fluorescence on both the front and back sides of the microplate. However, the use of light transmissive microplates is not a requirement of the present invention. Therefore, it is also possible to use a light-impermeable microplate and capture and measure fluorescence only from above.

As the light source 51, for example, an ultra-high pressure mercury lamp is used. A bright line spectrum having a wavelength in the range of 365 nm to 546 nm is mainly used as the excitation light. In the present invention, an excitation light having a peak wavelength of 366 nm in the range of about 340 nm to about 390 nm is used. In addition, a xenon lamp, a halogen lamp, or the like can be used as the light source.
The wavelength of 400 nm or more of the illumination light emitted from the light source is cut by the excitation filter 52 to obtain excitation light having a peak wavelength of about 366 nm within a range of about 340 nm to about 390 nm.

The dichroic mirror 53 is an interference filter having such a characteristic that when arranged at an angle of 45 degrees with respect to the optical axis, light having a wavelength shorter than a certain wavelength is reflected and light having a longer wavelength is transmitted. . In the present invention, the excitation wavelength range is set to the short wavelength side and the fluorescence wavelength range is set to the long wavelength side.

The absorption filters 55a and 55b are provided to prevent the excitation light reflected and transmitted without being absorbed by the microorganism from being incident on the photomultiplier tube 56, and to transmit only a specific wavelength among the fluorescent light. Has been established.
In the present invention, about 430 nm to 490 nm, preferably about 440 nm
nm to about 480 nm, more preferably 450 nm to 470 nm,
Most preferably, it captures fluorescence having a wavelength in the range of 455 nm to 465 nm.

The measurement signals from the photomultiplier tubes 56a and 56b are transmitted to the processor 60. Processor A
The measurement signal is converted into a digital value by a / D converter (not shown), and the fluorescence intensity is calculated by an arithmetic circuit (not shown). Based on the calculation result, a database stored in an external storage device is searched and referred to while calculating the “sag (distance)” D, and standard data in which the D is closest to 0 is found. This gives the viable cell count. The result can be displayed on the CRT screen of the console or output to a printer (not shown) if desired.

The device of the present invention can be used for both aerobic bacteria and anaerobic bacteria. When the apparatus of the present invention is used for anaerobic bacteria, the measuring apparatus may need to be operated in an inert gas atmosphere such as carbon dioxide gas or nitrogen gas.

[0044]

As described above, according to the apparatus of the present invention, the microorganisms in the liquid selective medium are irradiated with excitation light, the intensity of fluorescence emitted from the coenzyme of the microorganisms is measured, and the microorganisms are grown. The microorganism is irradiated with excitation light, the intensity of the fluorescence emitted from the coenzyme possessed by the microorganism is measured, the difference in the fluorescence intensity before and after the culture is determined, and this value is created in advance for the known number of viable bacteria and the fluorescence intensity. By applying the data to the database, the microorganism species in the unknown sample can be identified, and the viable cell count of the microorganism can be quickly measured.

As described above, the present invention is the first device to measure the number of viable microorganisms from the fluorescence emitted from the coenzyme of the microorganism without using any fluorescent label. In fact, many methods and devices for measuring microbial load by using fluorescent labels are known. For example, JP-A-61-1
No. 86854, No. 61-21084, No. 60-165
Nos. 86 and 57-132899. The use of fluorescent labels has the advantage of increasing the detection sensitivity, but on the other hand, measurement errors associated with the use of fluorescent labels also occur. For example, some microorganisms do not bind to the added fluorescent label, which leads to uncertainty in use. Further, not only microorganisms but also fluorescent labels that bind to the medium components have been a major cause of measurement errors. For this reason, an extra burden such as performing the work of removing the culture medium before the measurement is imposed. Furthermore, the stoichiometric amount of the fluorescent label relative to the number of microorganisms present cannot be determined before the measurement, so that the fluorescent label must necessarily be added in excess. Then, fluorescence is also generated from the fluorescent label remaining without binding to the microorganism, which is also a major cause constituting a measurement error. In addition, some fluorescent labels are unstable, and may be decomposed under the influence of a substance such as oxygen in the air or a temperature before or during the measurement, resulting in erroneous measurement. Therefore, in the apparatus of the present invention, there is no problem of measurement error associated with the use of such a conventional fluorescent label.

According to the viable cell count measuring apparatus of the present invention,
Accurate dilution of the sample liquid containing live bacteria, identification of microorganism species, and measurement of the number of viable bacteria can be performed completely automatically without manual intervention.

In the apparatus of the present invention, since a microplate is used, a large amount of samples can be processed simultaneously with a small amount of medium, so that the test cost per sample is reduced to about 1/20 as compared with the conventional agar plate method. You.

Since the liquid selective medium is used and transported in a cassette, there is little risk of bacterial contamination by airborne bacteria. For this reason, the apparatus of the present invention does not require a clean room or a clean bench, and is simple and convenient.
A microorganism species can be identified quickly, accurately and inexpensively, and the viable cell count of the microorganism species can be measured.

The apparatus according to the present invention is not limited to the field of clinical examination, but also relates to food-related, cosmetic-related, distribution-related, and health center-related. It can be used for identification of microorganism species and measurement of the viable cell count of the microorganism species in a wide range of fields such as pharmaceuticals.

[Brief description of the drawings]

FIG. 1 is a block diagram of an example of a viable cell count measuring device of the present invention.

FIG. 2 is a perspective view of a microplate used in the present invention.

FIG. 3 is a plan view of an example of the viable cell count measuring device of the present invention.

FIG. 4 is a view taken in the direction of arrow A in FIG. 3;

FIG. 5 is a schematic diagram showing the principle of fluorescence detection by an optical system in the automatic fluorescence measurement device.

[Explanation of symbols]

Reference Signs List 10: microplate dilution / measurement unit, 20: data processing unit, 30: loader cassette, 32: unloader / loader cassette, 34: unloader cassette, 36: incubator, 40: automatic dilution device, 41: transport system, 4
2: Guide rail, 43: Drive system, 44: Dispenser, 45
... disposable chip, 46 ... chip supply part, 47 ... eccentric motor, 48 ... base, 49 ... mount, 50 ... automatic fluorescence measurement device, 51 ... light source, 52 ... excitation filter, 53 ... dichroic mirror, 54a and 54b ... objective lens , 5
5a and 55b ... absorption filters, 56a and 56b
... Photomultiplier tube, 60 ... Processor, 70 ... External storage device, 80 ... Console, 90 ... Microplate

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical indication location C12Q 1/10 7823-4B C12Q 1/10 1/14 7823-4B 1/14 (72) Inventor Shinpei Suzuki 2-6-2 Otemachi, Chiyoda-ku, Tokyo Hitachi Electronics Engineering Co., Ltd. (72) Inventor Koichi Takachi 2-6-1, Otemachi, Chiyoda-ku, Tokyo Hitachi Electronics Engineering Co., Ltd. (72) Inventor Isao Endo 2-1 Hirosawa, Wako-shi, Saitama Pref., RIKEN (72) Inventor Teruyuki Nagato 2-1 Hirosawa, Wako-shi, Saitama Pref. RIKEN (72) Inventor Yoshimi Benno 2-1 Hirosawa, Wako-shi, Saitama Pref. RIKEN

Claims (4)

(57) [Claims] 1. The method according to claim 1 , wherein each of the specimens containing the microorganism to be measured is collected.
A microplate for receiving the sample.
In the hole of the black plate, use the following five types of liquid selective media.
Diluted in multiple steps for each liquid selective medium and agitated.
Automatic dilution / stirring device, and the diluted sample has a peak at the start and end of culture.
The sample is irradiated with excitation light having a wavelength of 366 nm,
The intensity of fluorescence emitted from living organisms at wavelengths of 430 to 490 nm
An automatic fluorescence measuring device for measuring the degree of microbial activity, an incubator for culturing the microorganisms in the diluted sample , and a known microorganism type having a known viable cell count before culture.
Multiple stages using the respective liquid selective medium
Before and after incubation for each dilution step
The fluorescence of each microbial species determined by measuring the intensity
Database on light intensity difference and viable cell count was stored
An external storage device, and an external storage device based on the measurement value of the automatic fluorescence measurement device.
Search and refer to the database of
At the same time, the micro-play
Load the door or to generate the five types of liquid election 択培land
Control to dilute these media into the plurality of stages
The microplate with this diluted medium
And control the loading of the
The diluted fluorescence loaded into the automatic fluorescence measurement device.
For each of the media on the microplate with
Control to measure the fluorescence intensity using the automatic fluorescence measurement device.
The fluorescence intensity of each of the culture media obtained at this time by the
The fluorescence intensity obtained from the automatic fluorescence measurement device
Controlling the unloading of the microplate, the first
The above-mentioned microplate after culture in which the fluorescence intensity of
Control the loading to the automatic fluorescence measurement device,
The microphone after culture loaded on the fluorescence measurement device
The fluorescence intensity is measured for each of the above media in the plate.
Control and obtain at this time by the automatic fluorescence measurement device.
Obtaining the fluorescence intensity of each of the culture media as a second fluorescence intensity,
And a difference between the second fluorescence intensity and the first fluorescence intensity
Processor that calculates the number of viable bacteria that have proliferated
Consists of a, the a liquid selective medium, Rab - Remco, proteose peptone No. 3, trypticase, yeast extract, liver leachate, raffinose, saline A, saline B, defoamers, Tween 80, L-cysteine HCl · _H 2 O, bi hydrate genus consisting of sodium propionate and colimycin Liquid selective medium for bacteria, Meat extract, peptone, casein, lactose, saccharose, sodium deoxycholate, sodium thiosulfate, sodium citrate, ammonium citrate, and purified water for Escherichia coli, meat extract, proteose peptone , Lactose, sucrose, salicin, bile salts, sodium chloride, sodium thiosulfate,
Bacterial liquid selection medium for Salmonella consisting of ammonium citrate, sodium deoxycholate and purified water, yeast extract, peptone, keratin, lactose, mannitol,
A liquid selection medium for Staphylococcus aureus, comprising sodium chloride, dipotassium phosphate, lithium chloride, phenolethyl alcohol and purified water, proteose peptone,
A liquid selective medium for Campylobacter comprising yeast extract, liver extract, sodium chloride, vancomycin, polymyxin B, trimtobrim, cephalosin, amphotericin B and purified water, and a yeast comprising bacto-potato dextrose broth, 10% tartaric acid and purified water A viable cell count measuring device characterized by using a liquid selective medium for bacteria. 2. A liquid selective medium for Bacteria belonging to the genus Bacterium, Rab-Remco 2.4 g / l, Proteose Peptone No. 310.0 g / l, trypticase 5 g / l, yeast extract 5.0 g / l, liver exudate 150 ml / l, raffinose 4 g / l, saline solution A 10 ml / l, saline solution B 5 ml / l, defoamer 5 ml / l , Tween 80 1
g / l, L-cysteine HCl · H ₂ O 0.5 g / l,
Sodium propionate 15 g / l and colimycin ⁽¹⁰⁶ units, 1%) a 12 ml / l, E. coli liquid selection medium, meat extract 3.0 g / l, peptone 10.0 g / l, casein 5.0 g / l , Lactose 1
5.0 g / l, sucrose 10.0 g / l, sodium deoxycholate 1.0 g / l, sodium thiosulfate 2.5 g
/ L, sodium citrate 1.0 g / l, ammonium citrate 1.0 g / l and purified water 1000 ml. The liquid selective medium for Salmonella is 3.0 g / meat extract.
11. Proteose peptone 12.0 g / l, lactose
0 g / l, white sugar 12.0 g / l, salicin 2.0 g /
l, bile salts 15.0 g / l, sodium chloride 5.0 g
/ G, sodium thiosulfate 6.8 g / l, ammonium citrate 0.8 g / l, sodium deoxycholate 2.0 g / l and purified water 1000 ml. The liquid selective medium for Staphylococcus aureus is yeast extract 2 .5
g / l, peptone 10.0 g / l, keratin 30.0 g
/ L, lactose 2.0g / l, mannitol 10.0g / l,
75.0 g / l sodium chloride, dipotassium phosphate5.
0 g / l, lithium chloride 5.0 g / l, phenol ethyl alcohol 25 ml / l and purified water 1000 ml. The liquid selective medium for Campylobacter is 15.0 g / l proteose peptone, 5.0 g / l yeast extract. , Liver extract 2.5 g / l, sodium chloride 5.0 g / l, vancomycin 10 mg / l, polymyxin B2500
Unit: Trimtoblim 5mg / l, Cephalosin 15m
g / l, amphotericin B 2 mg / l and purified water 1
2. The viable cell count measuring apparatus according to claim 1, wherein the liquid selective medium for yeast comprises 39.0 g / l of bacto potato dextrose broth, 14 ml / l of 10% tartaric acid and 1000 ml of purified water. 3. An automatic fluorescence measuring device, wherein the peak wavelength is 36
A light source and an excitation filter for obtaining 6 nm excitation light; and reflecting the excitation light toward the diluted sample, the diluted sample.
Longer wavelength than the excitation light emitted from the microorganisms in the
Dichroic mirror transmitting fluorescence and wavelength 43
2. The viable cell count measuring apparatus according to claim 1, comprising : an absorption filter for cutting light having a wavelength other than 0 to 490 nm; and a photomultiplier tube for capturing light transmitted through the absorption filter . 4. A light-transmitting material having a transparent microplate.
And above and below the microplate.
Below and below the wavelength 4 emitted by the microorganism 30nm ~
The viable cell count according to claim 1, wherein the intensity of fluorescence at 490 nm is measured.
Setting device.