JPH0630627B2 - Viable count method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a viable cell count measuring method. More specifically, the present invention relates to a method for measuring the viable cell count, which comprises using a liquid selective medium to grow only a specific microbial species in a sample and capturing the fluorescence emitted from the microorganism.

[Prior Art] Conventionally, identification of such microbial species has been carried out based on physiological and biochemical properties, while the viable cell count is 10 ^-1 , 10 ^-2 , ..., 10 ^-8 times dilution, inoculate a fixed amount of this diluted solution on agar plate medium, and after culturing for a fixed time (24 to 48 hours), multiply the number of colonies appearing on this agar plate by the dilution rate. It was wanted.

However, it is also known that such a completely manual microbial test method requires a test period of 2 to 5 days and considerable skill, and that a measurement difference by an engineer or an inspector occurs. There is. Furthermore, the cost of testing is high due to the use of large amounts of medium and petri dishes and the high labor costs of skilled technicians.

Microbial tests such as identification of microbial species and measurement of viable cell count are indispensable in clinical inspection, food inspection, drug inspection, etc.
There is a high need for speed, labor saving, and automation.

For this reason, various types of automated machines are being developed in the clinical laboratory department. For example, a plate that smears the sample solution from the center to the outside is cultivated on the agar plate medium rotating at 50 rpm, and the agar plate that has been smeared is cultured, and the colonies formed on the plate medium are irradiated with He-Ne laser light. Completely automatic identification of microbial species and measurement of bacterial quantity by nephelometry using a viable cell count measuring device consisting of three devices, a colony counter for counting and a data processor, or a cartridge containing various culture media for property inspection. A viable cell count measuring device that can be used, or a similar device that uses a microplate instead of this cartridge, has been prototyped.

However, most of these devices are only used for microbiological tests such as urinary tract infections, and it is impossible to measure when samples are often turbid such as food tests, and , In some cases, sufficient accuracy cannot be obtained. Furthermore, since any of the methods requires a culture period of 24 to 48 hours, rapid measurement is not possible, and it is not possible to meet the needs that require an urgent test.

[Problems to be Solved by the Invention] An object of the present invention is to provide a viable cell count measuring method which can be used in a wide range of fields, has high accuracy and low cost, and requires a test time of about several hours.

[Means for Solving the Problems] As a result of the extensive experiments and researches conducted by the present inventors over the years, the microorganisms in the 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 was discovered that the number of viable cells of the microorganism can be measured with high accuracy and in a short time. The present invention has been completed based on these findings.

[Action] Living cells of microorganisms universally have the coenzymes NADH (reduced form of nicotinamide adenine dinucleotide) and NADPH (reduced form of nicotinamide adenine dinucleotide phosphate). When these coenzymes are irradiated with excitation light, fluorescence is generated.

Since the intensity of fluorescence depends on each microbial species, the number of viable bacteria, and the amount of coenzyme contained in one viable cell, the microbial species and the number of viable bacteria before culturing are known in advance for samples before and after culturing. Fluorescence intensity is measured, and the difference (ΔI) is determined. The amount of coenzyme contained per living cell before culturing is
Since the background fluorescence intensity derived from the liquid selective medium is subtracted from the fluorescence intensity before culturing, and the value is proportional to the value obtained by dividing the background fluorescence intensity by the above-mentioned known viable cell count, this value per viable cell count before culturing Can be regarded as the amount of coenzyme.

Therefore, when creating a database of the fluorescence intensity difference ΔIs based on a known sample, for example, a two-dimensional storage table is used. In this table, the number of viable cells is shown in the vertical column, and the amount of coenzyme per number of viable cells before culturing obtained by the above method is shown in the horizontal column. The known viable cell count is, for example, 10
^{It is} divided into ¹ , 10 ² , 10 ³ , ..., 10 ⁿ , and the amount of coenzyme is standardized by its maximum value, for example, 0, 0.1, 0.2, 0.3, ..., 0.
Divide into 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 equally diluted in n steps (for example, 11 steps), and the fluorescence intensity before and after the culture is measured for each dilution step. Fluorescence intensity difference ΔIs ₁ , ΔIs ₂ ,
…, ΔIs ₁₁ is calculated. This data is collected for each viable cell count and each coenzyme amount, and is used as a database. The dilution step will vary depending on the microplate used. For example, with a 96-well microplate, a maximum of 11 dilution steps are possible. Fluorescence intensities before and after culture are measured at the same dilution stage for specimens of the same microbial species and unknown number of bacteria, and the fluorescence intensity differences ΔIu ₁ , ΔIu ₂ , ...
…, ΔIu ₁₁ is calculated.

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

For example, the characteristics (ΔIu ₁ , ΔIu ₂ , ..., ΔI
One unknown specimen U having u _n ) has a characteristic (ΔIs ₁ ,
When compared with a known standard sample S that is ΔIs ₂ , ..., ΔIs _n ), its “sag (distance) D” is obtained by the following relational expression.

If the data matrix as described above is created for each microbial species and made into a database, the viable cell count can be accurately and quickly estimated by grinding and referring to the database from the measured values of unknown samples. Therefore, the more the amount of data in the database is, the more reliable the measurement method of the present invention is.

Generally, the growth process of a microorganism can be divided into an induction period, a logarithmic period, a quiescent period and a death period, but the amount of coenzyme per cell possessed by the microorganism also changes at each stage. The database is prepared by using a sample in which the microbial species, the viable cell count, and the amount of coenzyme before culturing are known. Therefore, the microbial species in the unknown sample is identified, and the viable cell count is estimated,
The amount of the coenzyme is secondarily estimated, and it becomes possible to predict the growth stage of the microorganism in the sample.

Identification of a microbial species in an unknown sample and measurement of the viable cell count thereof according to the present invention are possible by using a selective medium capable of growing and growing only a specific microbial species.
The selective medium is a medium in which a specific microbial species is allowed to grow more advantageously than other microbial species by adding a specific substrate, antibiotics or the like.

For example, in an unknown sample, Bihidobacteria (Bifidobact
If there is a bacterium belonging to the genus erium (so-called Behizus bacterium), and if there is any number of viable bacteria, a selective medium having the following composition is used.
Lab-lemco powder (manufactured by Oxoid)
2.4 g / 1, Proteose Peptone NO.3 (manufactured by Difco) 10.0 g / 1, Tripty Case (manufactured by BBL) 5 g
/ 1, yeast extract (manufactured by Difco) 5.0 g / 1, liver exudate (Mitsuoka, 1969) 150 ml / 1, raffinose 4
g / 1, salt solution A (Mitsuoka et al., 1965) 10 ml / 1
Salt solution B (Mitsuoka et al., 1965) 5 ml / 1, antifoam agent Dow Corning, 10%) 5 ml / 1, Tween 80 1
g / 1, L-Cysteine HCl · H ₂ O 0.5g / 1
Sodium propionate 15 g / 1 and colimycin (10 ⁶ units, 1%) 12 ml / 1. When preparing this selective medium, mix components other than colimycin at 115 ° C.
Sterilize for 20 minutes. Colimycin is added aseptically just before use. Since only the bacteria of the genus Bihidobacteria can grow in this selective medium, if the unknown sample fluoresces, the microbial species in the unknown sample is identified as a bacterium of the genus Bihidobacteria.

As mentioned above, an unknown sample is inoculated into a selective medium in which only specific microbial species can grow, cultured, and the fluorescence intensity is measured.
If there is a difference in fluorescence intensity before and after the culture, it is because the corresponding microbial species is present in the unknown specimen and proliferated, and the microbial species in the unknown specimen can be easily identified.

In practice, it is difficult to predict what kind of microbial species is present in an unknown sample, so inoculate the unknown sample into all selective media, culture, and in which selective medium was grown,
Confirmation based on the difference in fluorescence intensity before and after culturing will identify the microbial species.

According to the study by 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 White sugar 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, it grows specifically in a selective medium having the following composition.

Meat extract 3.0 g Proteose peptone 12.0 g Lactose 12.0 g White sugar 12.0 g Salicin 2.0 g Bile salt 15.0 g Sodium chloride 5.0 g Sodium thiosulfate 6.8 g Ammonium citrate 0.8 g Sodium deoxycholate 2.0 g Purified water 1000 ml 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 Mannitol 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 Campylobacter The selective medium for 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 Trimtobrym 5 mg Cephalosin 15 mg Amphotericin B 2 mg Purified Water 1000 ml For yeast, a selective medium with the following composition Is used.

Bacto potato dextrose broth (manufactured by Difco) 39.0 g 10% Tartaric acid 14.0 ml Purified water 1000 ml The selective medium is preferably a liquid medium. If it is a liquid medium, it is possible to automatically dispense a fixed amount with a micropipette or the like.

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

FIG. 1 is a block diagram of an example of an apparatus used for carrying out the viable cell count measuring method of the present invention.

This device is basically a microplate dilution / measurement unit 10
And a data processing unit 20.

In the microplate dilution / measurement unit 10 of FIG. 1,
The microplate is sent from the loader cassette 30 to the automatic diluting / stirring device 40 as indicated by the solid arrow in the figure, where the inoculation, dilution and stirring of the sample (sample) are simultaneously performed and transferred to the unloader cassette 32. It This unloader cassette 32 is attached to the adjacent automatic fluorescence measuring device 5
It also serves as a loader cassette that supplies the microplate to the 0. The microplate after the fluorescence measurement is transferred to another unloader cassette 34. Automatic fluorescence measurement device 5
The fluorescence intensity measurement by 0 is performed on the microplate before culturing, then transferred to the incubator 36 and cultured for a predetermined time, and after the culturing, the fluorescence intensity measurement is performed again. The incubator does not have to be incorporated in the apparatus itself, and a commonly used incubator or the like provided in an ordinary laboratory can be used. In FIG. 1, the bold arrows indicate the movement of the microplate after culturing. A conventional means such as a conveyor is used to transfer the microplate. Unloader /
Loader / cassette 32 and unloader cassette 34
If desired, a robot or the like can be used to transfer the cassette between the incubator 36 and the incubator 36.

The pre-culture fluorescence intensity measurement signal (indicated by a dashed-dotted line arrow in the figure) and the post-culture fluorescence intensity measurement signal (indicated by a bold-dotted line arrow in the figure) from the automatic fluorescence measurement device 50 are transmitted to the processor 60 of the data processing unit 20. The database stored in the external storage device 70 is searched and referenced from the measurement signal and the information about the type of the selected culture medium to identify the microbial species present in the sample and to determine the viable cell count of the microbial species. presume. The processor 60 includes an external storage device 70 and a C
It is connected to a console 80 having an RT and a keyboard. All operations can be performed by inputting commands from the console 80. The dashed arrows in the figure show the flow of control signals and data.

By performing fluorescence intensity measurement before culturing and subtracting that value from the fluorescence intensity measured value after culturing, the background is removed and a signal corresponding to the increase in the viable cell count of the specific microbial species grown in the selective medium. Is obtained. On the other hand, if there is no difference in the fluorescence intensity before and after the culture, it means that the microbial 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 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, pottery, or the like.

The front surface of the micro plastic 90 is provided with 96 holes (8 rows × 12 columns). The shape of the hole is not particularly limited. Any shape such as a rib shape, a V shape, a columnar shape or a prismatic shape can be used. Needless to say, 9
Microplates other than 6 wells can also be used. The first row on the front side of the microplate is a space for injecting the test liquid, and a fixed amount of liquid selective medium is preliminarily injected in the remaining row for dilution.

FIG. 3 is a plan view of an example of an apparatus used to carry out the viable cell count measuring method of the present invention, and FIG. 4 is a view on arrow A in FIG.

A loader cassette 30 (on the left side in the drawing) and an unloader / loader cassette 32 are arranged at both ends of a transport system 41 of the automatic diluting / stirring device 40. The drive transmission system of the transport system 41 can be constituted by a conventional means such as a conveyor, a chain, a belt or the like. Each cassette can store, for example, 10 microplates 90, and has a miniature warehouse type structure. The unloader cassette 34 of FIG.
Also have the same structure. The microplate together with the cassette can be placed in the incubator 36. The cassette can be moved up and down by using conventional means such as a ball screw mechanism or an elevator. In FIG. 3, reference numeral 48 is a base and 49 is a mount.

An extremely small amount dispenser 44 such as a microsyringe having a number corresponding to the number of sample holes of the microplate 90 is attached to the tip of the drive system 43 that can move back and forth along the guide rail 42. A sterilized disposable tip 45 is attached to the tip of the microvolume dispenser 44. Chip 4
5 is supplied from the chip supply unit 46. The microplate 90 can be vertically fed as well as horizontally fed as shown in FIG. In the case of horizontal feed, a maximum of 8 specimens is 10
It is possible to dilute up to ^-11 times, and it is possible to dilute up to 12 specimens up to 10 ^-7 times in the case of vertical feeding. The eccentric motor 47 vibrates the tip 45 of the tip of the ultra-small amount dispensing means 44 to stir the liquid in the hole to make the concentration uniform. Naturally, stirring means other than the eccentric motor can 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 according to the amount of selective medium injected into each hole so that the predetermined dilution ratio is achieved, and the sterilized chip is loaded while the dispenser repeatedly moves back and forth. , Dilution and stirring at the setting stage and the final chip disposal are automatically performed. The dilution method is, for example, injecting 45 μ of selective medium into each hole of the selective medium row, collecting 5 μ of the sample from the hole of the sample row with a dispenser, pouring this into the first selective medium hole, and stirring. . Obtained 10 ^-1
Collect 5 μl from the double dilution, pour it into the next selective medium hole, and stir. Then, a 10 ^-2 times diluted solution is obtained. 5 μm of this 10 ⁻² -fold diluted solution is sampled, poured into the next selective medium hole, and stirred. Thus, a 10 ⁻³ fold dilution is obtained. By repeating this operation, 10 ^-7 or 10
^{-Up to 11} times dilution can be prepared. In this way, testing with a large number of dilution ratios for one sample can be performed for a wide range of viable cell count levels of 10 ¹ to 10 ¹¹ / ml in the unknown sample to be measured. And to predict the approximate value of the viable cell count. For example, if no difference in fluorescence intensity is detected in a diluted sample of 10 ^-9 times or more, the number of viable bacteria in the unknown sample is about 1
It is estimated to be about 0 ⁸ to 10 ⁹ .

FIG. 5 is a schematic diagram showing the principle of fluorescence detection by the optical system in the automatic fluorescence measuring device 50.

The principle itself of fluorescence detection is known, and basically, an illumination system has an excitation filter and an observation system has an absorption filter, and a dichroic mirror is used instead of a half mirror.

As shown in FIG. 5, illumination light including light of various wavelengths emitted from the light source 51 is extracted and transmitted by the excitation filter 52 only in the wavelength range necessary for generating fluorescence. To do. This transmitted light is dichroic mirror 53.
After being reflected by 90 ° downward by, the light passes through the objective lens 54a in the direction opposite to the normal direction and serves as excitation light in the microplate 9
Reach the specimen in the 0 hole. A part of the fluorescence emitted in any direction by the excitation light irradiation enters the objective lens 54a. The dichroic mirror 53 transmits the fluorescence having a wavelength longer than that of the excitation light without reflecting the fluorescence. Therefore, the fluorescence entering the objective lens 54a passes through the dichroic mirror 53 and passes through the absorption filter 55a. The absorption filter 55a cuts off a little stray light of the excitation light and allows only fluorescence to reach the photomultiplier tube 56a.

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

As the light source 51, for example, an ultrahigh pressure mercury lamp is used.
Mainly, an emission line spectrum having a wavelength in the range of 365 nm to 546 nm is used as the excitation light.
Excitation light with a peak wavelength of 366 nm in the range of 40 nm to about 390 nm is used. Besides, 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 366 nm within the range of about 340 nm to about 390 nm.

When the dichroic mirror 53 is arranged at an angle of 45 degrees with respect to the optical axis, it reflects light having a wavelength shorter than a certain wavelength,
It is an interference filter having a characteristic of transmitting long wavelength light. In the present invention, the excitation wavelength range is set on the short wavelength side and the fluorescence wavelength range is set on the long wavelength side.

The absorption filters 55a and 55b are provided in order to prevent the excitation light reflected and transmitted without being absorbed by the microorganisms from entering the photomultiplier tube 56, and for transmitting only a specific wavelength of fluorescence. There is. In the present invention, it is about 430 nm to 490 nm, preferably about 440 nm to about 480 nm.
nm, more preferably 450 nm to 470 nm, most preferably fluorescence having a wavelength in the range of 455 nm to 467 nm is captured.

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

The method of the present invention can be used for both aerobic and anaerobic bacteria. When carrying out the method of the present invention with respect to anaerobic bacteria, it may be necessary to operate the measuring device under an atmosphere of an inert gas such as carbon dioxide gas or nitrogen gas.

[Effects of the Invention] As described above, according to the method of the present invention, a microorganism in a liquid selective medium is irradiated with excitation light, and the intensity of fluorescence emitted from a coenzyme contained in the microorganism is measured and proliferated. Irradiating the microorganisms with excitation light, measuring the intensity of fluorescence emitted from the coenzyme possessed by the microorganisms, obtaining the difference in fluorescence intensity before and after the culture, this value is prepared in advance for known viable cell count and fluorescence intensity. By applying it to the database, it is possible to fix the microbial species in the unknown sample and to rapidly measure the viable cell count of the microorganism.

Thus, the present invention is the first method to measure the viable cell count of a microorganism from the fluorescence emitted from the coenzyme of the microorganism without using any fluorescent label. In fact, there are many known methods for measuring microbial load by using fluorescent labels. For example, JP-A-61-186854 and 61-
No. 21084, No. 61-16586 and No. 57-1
It is disclosed in Japanese Patent No. 32899. The use of a fluorescent label has the advantage of increasing the detection sensitivity, but on the other hand, it causes a measurement error associated with the use of the detection label. For example, some microorganisms do not bind to the added detection label, which adds uncertainty to their use. In addition to the microorganisms, there are also fluorescent labels that bind to the medium components, which was a major cause of measurement error. For this reason, an extra burden is imposed such as removing the medium before the measurement. Furthermore, the stoichiometric amount of fluorescent label relative to the number of microorganisms present cannot be determined prior to the measurement, so the fluorescent label must necessarily be added in excess. Then, fluorescence is also generated from the fluorescent label remaining without being bound to the microorganism, which is also a major cause of 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 the temperature before or during the measurement, which may cause an erroneous measurement. Therefore, the method of the present invention does not cause any measurement error problem associated with the use of such conventional fluorescent labels.

Further, according to the viable cell count measuring method of the present invention, accurate dilution of a sample liquid containing viable cells and identification of microbial species and measurement of viable cell number can be performed completely automatically without human intervention. it can.

Since a microplate is used in the present invention, a large amount of a sample can be simultaneously treated with a small amount of medium, so that the inspection cost per sample can be reduced to about 1/20 as compared with the conventional agar plate method.

Since liquid selective medium is used and it is transported in a cassette, there is little risk of bacterial contamination by airborne bacteria. Therefore, in the present invention, a clean room or a clean bench is not particularly required, and the microbial species can be identified easily, quickly, accurately and inexpensively, and the viable cell count of the microbial species can be measured.

The method of the present invention is not limited to the field of clinical examination, but is applicable to food, cosmetics, distribution, and public health centers. It can be used for identification of microbial species and measurement of viable cell count of the microbial species in a wide range of fields such as pharmaceuticals.

[Brief description of drawings]

FIG. 1 is a block diagram of an example of an apparatus used to carry out the method for measuring the viable cell count of the present invention, FIG. 2 is a perspective view of a microplate used in the present invention, and FIG. FIG. 4 is a plan view of an example of an apparatus used to carry out the viable cell count measuring method, FIG. 4 is a view taken in the direction of arrow A in FIG. 3, and FIG. 5 shows the principle of fluorescence detection by an optical system in the automatic fluorescence measuring apparatus. It is a schematic diagram showing. 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 ... Conveying system, 4
2 ... Guide rail, 43 ... Drive system, 44 ... Dispenser, 45
Disposable chip, 46 ... Chip supply unit, 47 ... Eccentric motor, 48 ... Base, 49 ... Frame, 50 ... Automatic fluorescence measuring device, 51 ... Light source, 52 ... Excitation filter, 53 ... Dichroic mirror, 54a and 54b ... Objective lens , 5
5a and 55b ... Absorption filter, 56a and 56b
... Photomultiplier tube, 60 ... Processor, 70 ... External storage device, 80 ... Console, 90 ... Microplate

Front Page Continuation (72) Inventor Shinpei 2-6-2 Otemachi, Chiyoda-ku, Tokyo Niitsu Ritsuden Engineering Co., Ltd. (72) Inventor Koichi Takachi 2-6-2 Otemachi, Chiyoda-ku, Tokyo Japan Inside Ritsuden Engineering Co., Ltd. (72) Inventor Isao Isawa No. 1 Hirosawa, Wako City, Saitama Prefecture RIKEN (72) Inventor Teruyuki Nagamune No. 2 Hirosawa Hirosawa, Wako City, Saitama Prefecture (72) Inventor Hajime Asama, 2-1, Hirosawa, Wako-shi, Saitama, RIKEN (72) Inventor, Yoshimi Higano, 2-1-1, Hirosawa, Wako, Saitama (56) Reference JP-A-61-186854 (JP) , A) JP 61-21084 (JP, A) JP 60-16586 (JP, A)

Claims (2)

[Claims] 1. A love-lemco, proteose peptone No.
3, tryptic case, yeast extract, liver exudate, raffinose, saline solution A, saline solution B, antifoaming agent, Tween 8
Liquid selection medium for Bhidobacteria consisting of 0, L-cysteine HCl · H ₂ O, sodium propionate and colimycin, meat extract, peptone, casein, lactose, sucrose, sodium deoxycholate, sodium thiosulfate, citric acid Liquid selective medium for E. coli consisting of sodium acidate, ammonium citrate and purified water, meat extract, proteose peptone, lactose, sucrose, salicin, bile salt, sodium chloride, sodium thiosulfate,
Bacteria liquid selective medium for Salmonella consisting of ammonium citrate, sodium deoxycholate and purified water, yeast extract, peptone, keratin, lactose, mannitol,
Liquid selective medium for Staphylococcus aureus consisting of sodium chloride, dipotassium phosphate, lithium chloride, phenol ethyl alcohol and purified water, proteose peptone, yeast extract, liver extract, sodium chloride, vancomycin, polymyxin B, trimtobrim, cephalosin , A liquid selective medium for Campylobacter consisting of amphotericin B and purified water, and a liquid selective medium for yeast consisting of bactopotato dextrose broth, 10% tartaric acid and purified water, inoculated with a microorganism and uniformly mixed, The microorganism in the liquid medium is irradiated with excitation light having a peak wavelength of 366 nm, and the microorganism itself measures the intensity of fluorescence emitted from the coenzyme in its living cells at a wavelength of 430 to 490 nm, and then,
The microorganisms in the liquid selective medium are cultured, the grown microorganisms are irradiated with excitation light having a peak wavelength of 366 nm, and the microorganisms themselves emit a wavelength 4 from a coenzyme contained in their living cells.
The fluorescence intensity of 30 to 490 nm is measured, the difference between the fluorescence intensities before and after the culture is determined, and from the obtained value, it is confirmed that the microorganism has grown in a specific selective medium of the liquid selective medium. A method comprising identifying a microbial species corresponding to a specific selection medium, and at the same time obtaining a corresponding viable cell number by searching and referring to a database relating to the viable cell number and fluorescence intensity prepared in advance for the same microbial species. Bacteria count method. 2. A liquid selective medium for a bacterium of the genus Bihidobacteria is Lab-Lemco 2.4 g /, proteose peptone.
No.3 10.0g /, tryptic case 5g /, yeast extract 5.0g /, liver exudate 150ml /, raffinose 4g /, salt solution A 10ml /, salt solution B 5ml
/, Antifoaming agent 5 ml /, Tween 80 1 g /, L-
Cysteine HCl · H ₂ O 0.5 g /, sodium propionate 15 g / and colimycin (10 ⁶ units, 1%) 12 ml / consisted of. Escherichia coli liquid selection medium was meat extract 3.0 g /, peptone 10.0 g /, Casein 5.0g /, lactose 1
5.0 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 /, and purified water 1000 ml. The bacterium liquid selection medium for Salmonella is meat extract 3.0 g /
, Proteose peptone 12.0 g /, lactose 12.
0 g /, sucrose 12.0 g /, salicin 2.0 g /
, Bile salt 15.0g /, sodium chloride 5.0g
/, Sodium thiosulfate 6.8 g /, ammonium citrate 0.8 g /, sodium deoxycholate 2.0 g / and purified water 1000 ml. The liquid selective medium for Staphylococcus aureus is yeast extract 2.5.
g /, peptone 10.0g /, keratin 30.0g
/, Lactose 2.0 g /, mannitol 10.0 g /,
Sodium chloride 75.0 g /, dipotassium phosphate 5.
0 g /, lithium chloride 5.0 g /, phenol ethyl alcohol 25 ml /, and purified water 1000 ml. The liquid selective medium for Campylobacter was 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 / and purified water 1000
The liquid selective medium for yeast comprises 39.0 g of Bacto potato dextrose broth / 14 ml of 10% tartaric acid / 1000 ml of purified water, and the viable cell count according to claim 1. Measuring method.