JP3208892B2 - Method for measuring viable cell count - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to food BACKGROUND OF THE, pharmaceutical, cosmetics, water to be used in fields such as electronic industry, raw materials, intermediates, or adenosine triphosphate the number of bacteria of viable bacteria present in such products ( The present invention relates to a method for measuring quickly, simply and with high sensitivity by detecting ATP).

[0002]

2. Description of the Related Art As is well known in the fields of foods, pharmaceuticals, cosmetics and the like, it is extremely important to control viable bacteria in water, raw materials, intermediates or products. Also, the quality of water used in the electronics industry is important, and it is necessary to keep track of the number of viable bacteria in the water. Therefore, in those industrial fields, it is essential to measure the viable cell count. To measure the number of these viable cells, a so-called plate method has conventionally been used, in which viable cells in a specimen are cultured on an agar plate medium to form colonies and detected. In addition, a long time of 24 to 72 hours is required for the determination, and a simpler method has been desired.

[0003] measure the amount of and various proposals to answer this demand is made, for example, adenosine triphosphate cells which had been filtered current bacteria filter (ATP) in a luminometer,
From this, the biomillescence method for calculating the number of cells (Japanese Patent Application Laid-Open No. 2-163098) or, in the case of a sample liquid with a small number of viable cells, filtration and concentration with a filter and measurement with a luminometer (Japanese Patent Application Laid-Open No. 2-57197) and the like. However, these methods require the presence of viable bacteria (for example, 10 ³ or 10 ⁴ cells / ml or more) exceeding the detection limit of the luminometer in the measurement container. There is a problem that the sample must be filtered.

Accordingly, the present inventors have solved the above problems .
In order to develop a filter consisting of a number of small hydrophilic filtration membrane surrounded by a number of hydrophobic partition walls, and extract it performs addition of reagents emitting liquid such as a spray method, and a high sensitivity the sample And invented a method of applying the method to a bioluminescence image analysis system (Japanese Patent Application No. 3-40615).
No. and PCT / JP92 / 00145).

[0005] However, in this method, in the step of extracting ATP from the captured and collected viable cells, the conventional extractant used is a surfactant whose main component is a surfactant. The decomposing enzyme was also extracted, and the extracted ATP was decomposed, and the extracted components tended to diffuse to the vicinity due to high affinity with the filtration membrane. Therefore, there is a disadvantage that extraction while maintaining the high concentration becomes difficult. Saritote, than with a strong extractant, such as trichloroacetic acetic acid and perchloric acid becomes strongly acidic still these extractants on film, there is a problem that inhibits the enzymatic reactions performed by subsequently, extracting Later it was necessary to add enough buffer to make it neutral. Therefore, the use amount of these extractants increases, dilution, diffusion, and outflow occur, and the ATP detection sensitivity decreases, and the outline of the luminescent point near the capture point of live bacteria is somewhat blurred. Was left.

[0006]

The present invention relates to the above-mentioned A
In order to solve the problem of the ATP extraction step in the viable cell count method by TP detection, an ATP extractant which can efficiently extract ATP from viable bacteria and is excellent in clarifying the outline of a luminescent spot during light emission. An object of the present invention is to provide an excellent method for measuring viable bacteria by developing and improving the ATP extraction method using the same, and combining this with a bioluminescence image analysis system.

[0007]

Means for Solving the Problems In order to solve the above problems, as a result of diligent studies on various extractants used for separating various components from cells in biochemistry, a volatile compound having a boiling point of 120 ° C. or less was obtained. It has been found that the extractant is excellent for extracting ATP from viable bacteria, and that good results can be obtained when the extractant is volatilized under heating.
The present invention has been reached.

[0008] That is, the present invention relates to a multi-walled partition wall.
Consists of a number of small hydrophilic filtration membrane compartments that are substantially enclosed
The sample solution is filtered through a hydrophilic membrane filter, and the viable bacteria contained in the sample solution are captured on the filtration membrane, and then the adenosine triphosphate in the viable bacteria is placed on the viable bacteria. While spraying a volatile extractant having a boiling point of 120 ° C. or lower for extraction, or thereafter, after evaporating the liquid at room temperature or by heating, the luciferin-luciferase luminescent reagent solution is sprayed onto the living bacteria. It is intended to provide a method for measuring the number of viable bacteria in a specimen, which comprises emitting light and measuring the amount of emitted light or the number of luminescent spots using a luminescence measuring device.

Hereinafter, the present invention will be described in detail. The ATP extractant used in the present invention includes alcohols, ethers, esters, methane, ethane or methylene, halogen derivatives of ethylene, acetonitrile, triethylamine and the like having a boiling point of 120 ° C. or lower. Of these, methanol and ethanol are particularly preferred.

In addition, hydrochloric acid or an organic acid having a boiling point of 120 ° C. or less or ammonium hydroxide (NH
_The addition of 0.1 to 5% of an alkaline substance such as 4OH) or an organic amine having a boiling point of 110 ° C. or less further improves the extraction effect. However, while volatile hydrochloric acid and organic acids enhance the extraction effect, the activity of the luminescent enzyme is reduced if a small amount remains, so that it is necessary to perform evaporation more completely. Must be neutralized by spraying. Therefore, among these additives, ammonium hydroxide which has high volatility and has no emission inhibition is particularly preferable.

[0011] These ATP extractants are extracted from room temperature to 80
C., preferably 40-70.degree. C. to evaporate. It is convenient to spray the extractant into fine particles using an ultrasonic sprayer (for example, manufactured by Matsushita Electric Works) or the like, and it is effective to add just the required amount.

[0012] The membrane filter used in the present invention comprises:
Hydrophilic plastic-based materials such as hydrophilic polytetrafluoroethylene, hydrophilic polyvinylidene fluoride, hydrophilic polysulfone, hydrophilic polycarbonate, hydrophilic polyamide, hydrophilic polyethylene, hydrophilic polypropylene, and acetyl cellulose and nitrocellulose. It is a precise hydrophilic filtration membrane in the form of a film or sheet made of a material having uniform microporosity and having a pore size of 0.1 to 1 μm.

Japanese Patent Application No. 3-40615 (PCT / JP92 / 0014) discloses these membrane filters.
It is preferable to use one having a large number of hydrophilic sections surrounded by hydrophobic fine section walls such as lattice or circle described in 5). FIG. 1 shows a plan view of the membrane filter. Other than the above, a membrane filter suitable for a sample solution having a viable cell count on the filtration membrane of 10 ² or more is described in Japanese Patent Application No. 2-253093.
0.1 to 1 μm pore size at the bottom of φ plastic cylinder,
Preferably, a filter cup equipped with a 0.2 to 0.45 μm filter membrane is also used.

Next, a method for capturing live bacteria and a method for measuring the same according to the present invention will be described. First, a specimen is filtered using a filter provided with a membrane filter in a cup-shaped container or the like in which the specimen is placed, and viable bacteria are captured on the filter. Next, the above-mentioned ATP extractant is sprayed from the upper surface of the membrane filter into fine particles over a spraying time of about 5 seconds to 5 minutes using, for example, an ultrasonic sprayer manufactured by Matsushita Electric Works.
Extract P. The temperature is from room temperature to 80 ° C., preferably 40 to
70 ° C. is applied. Note that the spraying time is appropriately selected depending on the types of viable bacteria and the extractant.

The heating is carried out at the time of spraying or after spraying. A preferred method is to spray the extractant in the form of fine particles at around room temperature, and then remove the remaining extractant from room temperature to 80 ° C, 40 to 70 ° C.
To quickly evaporate. Subsequently, a luciferin-luciferase-based luminescent reagent is further added from above, and when the viable cell count is high (10 ² or more / membrane) such as when a luminometer is used, the luciferin is added to the filter cup with a pipette, Alternatively, it is added using a usual atomizer to emit light. When the number of viable bacteria is small (10 ² or less /
The film is required to be sprayed with an ultrasonic sprayer or the like, and therefore, is required to be sprayed with a finer amount.

In order to carry out the present invention more effectively, a luciferin-luciferase-based luminescent reagent is sprayed or added with a pipette (for example, when the number of viable bacteria is large, 1
(In the case of 0 ² or more / in the case of film), a method of increasing the concentration of the luminescent reagent when emitting light is adopted. This is because, when the concentration of the luminescent reagent is increased, the speed of the luminescent reaction is increased, and as a result, the amount of emitted light is increased. This method has a particularly low viable count,
That is, it is more effective when applied to a bioluminescence image analysis system at 10 ² or less / film, particularly 50 or less / film.

That is, the concentration of the luminescent reagent is set to 2 to 10 of the standard concentration.
It is preferable that the concentration be increased by a factor of 3, especially 3 to 6 times.
No. By doing so, the light emission speed increases and the light emission
The bell goes up and the detection speed goes up. Here is the standard concentration
Conventional luminescent reagents (for example, Kikkoman Corporation)
Luciferin, a commercially available luciferin-luciferase
LU). Specifically, one example
For example, Kikkoman's luminescent reagent (trade name: Lucifer)
LU ) is usually used by diluting 70 mg of the lyophilized product thereof by adding 5 ml of water thereto. When this is used at a high concentration of about 3 to 6 times the ordinary concentration as described above , The luminescence time after spraying the luminescence reagent can be shortened, and the luminescence amount can be increased almost linearly in proportion to the concentration.

The light emitted from the filter is measured by a luminometer, for example, a luminescence reader BLR-201 (improved type) manufactured by Aloka, or a bioluminescence image analysis system [eg, ARGUS- manufactured by Hamamatsu Photonic Co., Ltd.] 50 / CL (with a tapered fiber)], the bright spot is imaged, and the number of viable bacteria can be measured.

The latter improved bioluminescence image analysis system can process weak luminescence with high sensitivity as compared with a conventional measurement device, and can process and analyze data quickly. An extremely excellent method for measuring viable bacteria can be realized in synergy with the effect of the addition.

The outline of the system is as shown in FIG. 3, in which (A) is an overall view and (B) is a partially enlarged view thereof. In this system, a sample holder 9 for supporting a filter (specimen) 5 after being treated with the above-mentioned extraction reagent and luminescent reagent, a total reflection plate 6, a light-shielding housing 8, and a luminescence device 2 arranged as close as possible to the filter. It is composed of a taper fiber 7 for detecting a dimension, an ultra-high-sensitivity television camera 10 comprising an optical amplifier and an image pickup tube, a camera controller 11, an image processor 12, a data analysis device 13, and a television monitor 14, particularly Hamamatsu Photonics ( ARGUS-50 / CL (trade name) manufactured by K.K. and having a taper fiber input type or a similar measurement function is preferable. A cooled solid-state image sensor (CCD) is used as the ultra-high-sensitivity TV camera, and is approximately -30.
By cooling to −120 ° C., a television camera that can suppress noise from the camera itself and accumulate weak light emission can be used.

For example, a cooling C manufactured by Hamamatsu Photonics Co., Ltd.
There is a CD digital imaging system. Alternatively, the operation can be performed by inverting the tapered fiber 7 and the ultra-high-sensitivity television camera 10 of the imaging section, and disposing a filter holder on which a sample is set, and operating the same.

It is desirable to set the specimen 5 and the tapered fiber 7 as close as possible. Thereby, the measuring sensitivity is significantly improved. In addition, in order to automate the measurement, a sprayer for the extraction reagent and the luminescence reagent, a transport device for the specimen, and the like can be combined and set as needed.

The measurement was carried out by placing the specimen holder 9 holding the luminescent treated specimen (the filter holding live bacteria) in close contact with the tapered fiber surface, and using an ultra-sensitive television camera, camera controller and image processor. Dimensionally photons for 30-180 seconds, for example 12
It accumulates for 0 seconds, captures the light emission from the cells, processes the image with a data analysis device, eliminates the fine light emission noise, and displays only the strong light emission derived from live bacteria on a television monitor. Since the light emission other than the cells is eliminated as noise by this processing, the measured number of bright spots substantially matches the number of viable bacteria.

[0024]

According to the present invention, a suitable ATP extractant is used in which live bacteria captured on a good membrane filter are sprayed at the same position and are easily volatilized.
At room temperature to 80 ° C., preferably 40 to 80 ° C.
After heating to 70 ° C. for rapid volatilization, the number of viable bacteria is measured by directly spraying a luminescent reagent from above and emitting light.

[0025] Thus, the extraction agent as in the case of using an extraction agent to the base resin a surfactant aqueous solution used conventionally
No components remain, and in the present invention, a suitable extractant and its volatilization do not substantially inhibit the extractant from remaining on the membrane filter.
Since the activity of the TPase is inhibited, there is no decay of light emission, and there is no diffusion of ATP, so that the bright spot near the location where live bacteria are present becomes sharp. As a result, the detection sensitivity is greatly improved, and the measurement time and labor are greatly reduced.

When a membrane filter (filtration membrane) composed of a number of hydrophilic small sections surrounded by a number of hydrophobic section walls is used as a filter, all the viable bacteria are well dispersed in each section. Is captured. Similarly, the extract (ATP extract) and the luminescent reagent solution (luciferin-luciferase system) spray-added from above remain in the compartment, and are not dispersed or diluted outside the compartment. Has been made. Therefore, the luminescent cell component is also maintained at a high concentration in the vicinity of the location where live bacteria exist, so that the above-mentioned effects are further improved and extremely sensitive measurement is possible.

Further, when the sample (membrane filter) which emits light in this way is applied to a suitable bioluminescence image analysis system, the bright spot can be measured with high sensitivity and selectively. This makes it possible to easily and easily eliminate luminescence noise from light, and to quickly and easily measure even when the number of viable bacteria is small (for example, when there are several viable bacteria / film).

[0028]

The present invention will be described below with reference to examples.

Example 1 Saccharomyces cerviche
es cerevisiae, IFO 0209) was cultured overnight in a glucose-peptone medium (manufactured by Eiken Chemical Co., Ltd.), and then diluted with a phosphate buffer (pH 7.5) to obtain a test solution (about 10 ² viable bacteria). Per ml) was placed on the bottom of a polystyrene cylinder with a biopore membrane (0.
Millicell-CM which is a culture cup equipped with 4 μm)
After filtering and collecting bacteria using a trade name (manufactured by Nippon Millipore Co., Ltd.), 90% ethyl alcohol was sprayed on the bacteria on the membrane of Millicell-CM in the form of fine particles for 30 seconds using an ultrasonic sprayer manufactured by Matsushita Electric Works. ATP is extracted from

Next, after the solvent was volatilized at 50 to 60 ° C., a luminous reagent (Lucifer-LU, manufactured by Kikkoman Corp.) was sprayed for 20 seconds to emit light, and a luminometer (luminescence reader BLR) was used. −
201 improved type, manufactured by Aloka Co., Ltd.), the number of viable bacteria was calculated by measuring the amount of emitted light for 1 minute, and the results are shown in Table 1. For comparison, the results of measuring the viable cell count by the standard agar plate method using potato dextrose agar (manufactured by Nissui Pharmaceutical Co., Ltd.) are also shown.

[0031]

[Table 1]

Example 2 Using the Millicell-CM used in Example 1, Escherichia coli (Es
cherichia coli, IFO13898) m
-TGE medium (DIFCO Laboratories)
Overnight culture in a phosphate buffer (pH 7.0).
After filtering and collecting 5 ml of the test solution (about 10 ³ viable cells / ml) diluted in 5), extraction and luminescence treatment were carried out in the same manner as in Example 1 except that the solvent was evaporated at room temperature for 3 minutes. The number of viable bacteria was calculated and the results are shown in Table 2 together with the results of the standard agar plate method.

[0033]

[Table 2]

Example 3 Saccharomyces cerviche was prepared in the same manner as in Example 1 using Millicell-GVWP (manufactured by Nippon Millipore Limited) equipped with a hydrophilic PVDF membrane.
es cerevisiae, IFO 0209), methanol was sprayed on the membrane obtained by filtration and collection of the overnight culture diluent for 30 seconds in the same manner as in Example 1, and then the solvent was volatilized at 50 to 60 ° C. The luminescence was measured in the same manner as in Example 1, the luminescence was measured using a luminometer, and the number of viable bacteria was calculated.
It was shown to.

[0035]

[Table 3]

Example 4 Saccharomyc
es cerevisiae, IFO 0209) using a glucose / peptone medium was diluted day and night with a phosphate buffer (pH 7.5) to obtain about 20 viable cells / ml and used as a sample liquid. 1 ml thereof was filtered and washed using a hydrophilic Durapore filtration membrane with a special lattice (25 mmφ, manufactured by Nippon Millipore) to collect viable bacteria on the filtration membrane, and the membrane was removed from the filter and dried.

Next, 90% ethyl alcohol was sprayed thereon in the form of fine particles for 30 seconds in the same manner as in Example 1 to obtain AT.
After P is extracted and the solvent is further volatilized at 50 to 60 ° C., a luminescent reagent (Lucifer-LU, manufactured by Kikkoman) is sprayed into fine particles for 10 seconds to emit light. This was applied to an image analyzer (Argus 50 / CL, manufactured by Hamamatsu Photonics), and after accumulating photons for 2 minutes, image processing was performed and only bright spots above the threshold were imaged on a TV monitor, and the number of viable bacteria was measured. did. The results are shown in Table 4 as a comparative experiment. The same sample solution was cultured at 30 ° C. for 48 hours by a standard agar plate method using a potato dextrose agar medium, and the number of viable cells was measured.

[0038]

[Table 4]

Example 5 Staphylococcus aureus
occus aureus IFO 3060) was cultured overnight in a normal bouillon medium (manufactured by Eiken Chemical Co., Ltd.), and diluted with a phosphate buffer (pH 7.5) to about 30 viable cells / ml to prepare a sample solution. Using 1 ml of the same filtration membrane as in Example 4, collecting bacteria, extracting ATP, and performing luminescence treatment in the same manner, imaging only bright spots derived from live bacteria with an image analyzer, and measuring the number of viable bacteria. The results are shown in Table 5. Table 5 also shows the results obtained by culturing the same sample solution by a standard agar plate method using a normal bouillon agar medium (manufactured by Eiken Chemical Co., Ltd.) at 37 ° C. for 48 hours and measuring the number of viable bacteria.

[0040]

[Table 5]

Example 6 Saccharomyces
es cerevisiae, IFO 0209) was cultured and diluted in the same manner as in Example 4 to obtain about 30 viable cells /
A sample solution of ml was prepared. 1 ml thereof was filtered and collected using the same filtration membrane as in Example 4, and then methanol containing 3% of triethylamine was sprayed into fine particles for 30 seconds to form ATP.
Was extracted. After standing at room temperature for 3 minutes, the luminescent reagent was sprayed for 10 seconds to emit light in the same manner as in Example 4, and the solvent was removed from 50 to 6 minutes.
After volatilization at 0 ° C., the sample was supplied to an image analyzer to accumulate photons for 2 minutes, and only bright spots derived from live bacteria having a threshold or more were imaged, and the number of viable bacteria was measured. Further, the same sample solution was cultured at 30 ° C. for 48 hours by a standard agar plate method using a peptone dextrose agar medium, and the number of viable cells was measured.

[0042]

[Table 6]

Example 7 Staphylococcus aureus
occus aureusIFO 3060) similarly cultured as in Example 5 was used as specimens was prepared so as viable cells 10 ³ cells / ml performs dilution. After filtering and collecting 5 ml of the ATP, the ATP was extracted in the same manner as in Example 1 using 90% ethyl alcohol containing 1% of ammonium hydroxide (aqueous ammonia), and a luminescent reagent (Lumi-
PM) was sprayed for 20 seconds and emitted light to measure the amount of emitted light. On the other hand, the same sample solution is filtered by the same amount, and another ATP extraction reagent (N
Extraction was carried out using RB Lumac Co., Ltd., and a luminescent reagent (Lumit-PM Lumac Co.) was added to emit light, and the amount of luminescence was compared and measured. Table 7 shows the results.

[0044]

[Table 7]

Example 8 A hydrophilic filter membrane with a special lattice (47 mmφ, Nippon Millipore Limited) was used in the same manner as in Example 4 by adding 0.5 μl of an aqueous solution containing 4 × 10 ^-13 , 4 × 10 ^-14 , and 2 × 10 ^-14 g ATP. Made)
And dried. Next, a reagent having a concentration four times that of a conventional luminescent reagent (manufactured by Kikkoman Corporation) is sprayed on the fine particles for 10 seconds to emit light. Similarly, photon accumulation was performed for 2 minutes using an image analyzer. Table 8 shows the results together with the results measured using a common luminescent reagent for comparison.
It was shown to.

[0046]

[Table 8]

Example 9 Saccharomyces cerviche (Sac)
charomycescerevisiae, IFO
0209) (about 100 cells / ml). 0.2 ml of this was added to a hydrophilic filtration membrane with a special lattice (47 mmφ, manufactured by Nippon Millipore Limited), and filtered and dried. Then, the extraction reagent was sprayed for 30 seconds in the same manner as in Example 1 to perform ATP.
Is extracted. Subsequently, a luminescent reagent having a usual concentration of 4 times is sprayed thereon in the form of fine particles for 10 seconds to emit light. Similarly, after accumulating photons for 2 minutes using an image analyzer, image processing was performed and only bright spots were imaged to measure the number of viable bacteria. The results are shown in Table 9 together with the results of measurement using a common luminescent reagent for comparison.

[0048]

[Table 9]

As can be seen from the tables in the above Examples, the standard agar plate method, which takes a long time, and the simple method of the present invention show approximate measured values, indicating that the present invention can be used for measuring the viable cell count. .

[0050]

The present invention provides a method for measuring the viable cell count by the ATP bioluminescence method, which comprises using a specific extractant and a volatilization method which can be volatilized for the extraction of ATP of viable bacteria captured and collected on a membrane filter. It has the effect of enabling high-efficiency extraction and preventing the enzyme (luciferase) activity from being inhibited in the subsequent luminescence step and the adverse effect of the ATPase. For this reason, the viable cell count can be more quickly measured with higher sensitivity than the conventional method.

Further, the present invention uses an improved bioluminescence image which uses a membrane filter composed of small compartments having raised compartment walls, uses a spray method as a sample addition method, and can detect weak luminescence with high sensitivity and quickly. By using the analysis system, the number of viable cells can be measured much faster than the conventional method, and the measurement time can be greatly reduced. Has the effect of being able to carry out quickly.

[Brief description of the drawings]

FIG. 1 is a plan view of a membrane filter used in the method of the present invention and a partially cutaway enlarged view thereof.

FIG. 2 is a partial cross-sectional view of a membrane filter.

FIG. 3 is a schematic diagram of a bioluminescence image analysis system used in the method of the present invention and a partially enlarged view thereof.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 Filter 2 Section 3 Section wall 4 Frame 5 Filter (sample) after processing with an extraction reagent and luminescent reagent 6 Total reflection plate 7 Tapered fiber 8 Light shielding housing 9 Specimen holder 10 Ultra-high sensitivity television camera 11 Camera controller 12 Image processor 13 Data analyzer 14 TV monitor

Continuation of the front page (72) Inventor Jung Jong-won 2-36-30 Megalon Maruyamadai, Konan-ku, Yokohama-shi, Kanagawa 602 (56) References JP-A-59-113899 (JP, A) JP-A-4- 30798 (JP, A) JP 46-30783 (JP, B1) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12Q 1/00-1/66 C12M 1/26-1/34

Claims (10)

(57) [Claims] 1. A method according to claim 1, wherein the plurality of hydrophobic compartment walls are substantially enclosed.
The sample solution is filtered through a hydrophilic membrane filter composed of a number of small hydrophilic filtration membrane sections, and the viable bacteria contained in the sample solution are captured on the filtration membrane.
While spraying a volatile extractant having a boiling point of 120 ° C. or less for extracting adenosine triphosphate in the viable cells onto the viable cells , or after spraying the liquid at room temperature or heating, luciferin -Add luciferase luminescence reagent solution to live bacteria
A method for measuring the number of viable bacteria in a specimen, wherein the number of viable cells is measured by using a luminometer to measure the amount of luminescence or the number of luminescent spots. 2. A hydrophilic membrane filter according to claim 1 Symbol placement measurement method viable count of a filter cup bottom hydrophilic filtration membrane of the cylinder of plastic is attached. 3. The method according to claim 1, wherein the hydrophilic membrane filter is made of hydrophilic polytetrafluoroethylene, polyvinylidene fluoride, polycarbonate, polyamide, polysulfone,
3. The method according to claim 1, wherein said method is made of a material selected from polyethylene, polypropylene, their polymer alloys, acetylcellulose, and nitrocellulose. 4. A viable count of the measuring method according to any one of claims 1-3 volatile extraction agents of adenosine triphosphate is methanol or ethanol. 5. A viable count according to any one of adenosine triphosphate claim volatile extractant phosphoric acid is obtained by adding ammonium hydroxide 0.1-5% by weight in methanol or ethanol 1-4 Measurement method. 6. viable count measurement method according to any one of claims 1 to 5 used a luminescent reagent was concentrated to 3-6 times the usual concentration. 7. A method of measuring the viable count according to any one of claims 1 to 6, heating of the liquid volatilization is performed at 40 to 70 ° C.. 8. A luminescence measuring device the measuring method of the number of viable bacteria according to any one of claims 1 to 7, which is a bioluminescent image analysis system. 9. The television camera head of the bioluminescence image analysis system includes a tapered fiber, an optical amplifier, and an image pickup tube.
9. The method according to claim 8 , wherein the method comprises a tapered fiber and a cooled CCD television camera. 10. The method of claim 8 bioluminescence image analysis system to erase the emission noise which is not derived from viable cells, measuring the number of viable bacteria by performing an image processing for displaying only the bright spots shows the above light emission threshold Or the method for measuring the viable cell count according to 9 .