WO2002092202A1

WO2002092202A1 - Method of treating filter membrane, filter membrane, and method of rapidly counting number of microorganism with the filter membrane

Info

Publication number: WO2002092202A1
Application number: PCT/JP2002/004733
Authority: WO
Inventors: Tatsuya Sakakibara; Toshinori Igarashi; Seiji Murakami; Yoshio Monji
Original assignee: Kikkoman Corporation; Nihon Millipore K.K.
Priority date: 2001-05-16
Filing date: 2002-05-16
Publication date: 2002-11-21

Abstract

A method of treating a filter membrane or water-absorbing zone, characterized by contacting the membrane or zone with at least one member selected from the group consisting of an organic solvent, sterilized water containing an organic solvent, sterilized water, enzyme solution containing an adenosine phosphatase, acid, alkali, surfactant, and bleaching agent to remove the adenosine phosphate adherent to the filter membrane or water-absorbing zone.

Description

Description Filtration membrane treatment method, filtration membrane and use of the filtration membrane

Method for rapid measurement of bacterial count

The present invention relates to a method for treating a filtration membrane or a water absorption zone. Further, the present invention relates to a method for measuring microorganisms in a sample solution quickly, simply, and with high sensitivity. More specifically, the present invention relates to a method for measuring microorganisms captured on a filtration membrane by using an ATP conversion reaction reagent and a bioluminescent reagent. Background art

In the fields of food, pharmaceuticals, cosmetics, etc., the control of viable bacteria in the water, raw materials, intermediates and products used is very important. Also, quality control of water used in the electronics industry is important, and it is necessary to keep track of the number of bacteria in it. Therefore, measurement of viable cell count is essential in these industrial fields.

For this reason, various proposals have been made regarding a rapid detection method of bacteria. For example, if the number of viable bacteria in the sample solution is sufficiently large, a small amount should be collected in a small test tube.If the number of viable bacteria is slightly small, the sample solution should be filtered through a filtration membrane to capture microorganisms. After the filter membrane is immersed in a very small amount of sterile water or the like and a part of the liquid in which the microorganisms are suspended is collected in a small test tube.

A method of measuring by the biomillescence method is known (Japanese Unexamined Patent Publication No. 2-57197, Japanese Unexamined Patent Publication No. 2-163098, etc., Misao Haruta et al. P. 58, Science Forum (1985) Japan.

However, these methods, when the viable count is less sample solution (e.g. 10 ³ to 1 0 ⁴ / ml or less) in the detected amount of ATP for the following measurement limit luminometer is extremely difficult.

Therefore, the present applicants first filtered a sample solution containing microorganisms through a filtration membrane, captured the microorganisms in the sample solution on the filtration membrane, extracted biological components in the microorganisms,

Add the ATP conversion reagent and bioluminescent reagent, and measure the luminescence generated on the filtration membrane. We have filed an application for "Method for Measuring Microorganisms" (Japanese Patent Application Laid-Open No. H11-69994) regarding the method for measuring organisms. The filtration membrane used in this method, for example, a membrane filter comprising a number of small hydrophilic filtration membrane sections surrounded by a number of hydrophobic compartment walls described in Patent No. 3133328, and JP-A-6-237793. When the liquid extraction reagent and the liquid luminescent reagent are sprayed in a mist, the hydrophobic filter membrane described in the item (1) adheres to the point of attachment of live bacteria in ultrafine particles due to surface tension. It is excellent in that the extracted luminescent component is not diffused but is held in the form of ultrafine particles to emit light.

However, even in the absence of bacteria, noise luminescent spots or background luminescence may occur. Therefore, if such noise luminescent spots or packed ground emission can be suppressed, it is expected that emission generated on the filtration membrane will be detected with higher sensitivity. Disclosure of the invention

An object of the present invention is to provide a rapid, simple, and highly sensitive method for measuring microorganisms captured on a filtration membrane. In particular, an object of the present invention is to prevent contamination of adenosine phosphates such as AMP, ATP or RNA, which is a cause of noise luminescent spots and background luminescence, appearing on these membranes in the above-described measurement method using a bioluminescence reaction. By eliminating them, the appearance of noise luminescent spots and background luminescence is eliminated, and more accurate and rapid bacterial counts can be measured.

Thus, the present inventors have conducted intensive studies on a rapid, simple, and highly sensitive measurement method of microorganisms captured on filtration membranes, and as a result, the bright spot 原因 the background of background light emission appearing on these filtration membranes Found that the filtration membrane was contaminated with AMP and ATP. Furthermore, if this contamination is washed or removed by means such as electrophoresis or chemical or enzymatic modification or decomposition, noise luminescent spots and background luminescence will not appear, resulting in a more accurate and rapid bacterial count. They found that measurement was possible, and completed the present invention.

That is, the present invention is selected from the group consisting of an organic solvent, sterilized water containing an organic solvent, sterilized water, an enzyme solution containing an adenate esterase, an acid, an alkali, a surfactant, and a bleaching agent. At least one filter membrane or water absorption zone. A method for treating a filtration membrane or a water absorption zone, comprising removing adenosine phosphate attached to the filtration membrane or the water absorption zone by touching the filter membrane or the water absorption zone. Examples of the organic solvent include ethanol. Bleaching agents include hypochlorous acid, hypobromite and hypoiodite, and salts thereof, such as sodium hypochlorite, potassium hypochlorite, calcium hypochlorite, barium hypochlorite, Methyl hypochlorite, Copper hypochlorite, Silver hypochlorite, Sodium hypobromite, Potassium hypobromite, Calcium hypobromite, Barium hypobromite, Methyl hypobromite, Next Copper hypobromite, silver hypobromite, sodium hypoiodite, potassium hypoiodite, calcium hypoiodite, barium hypoiodite, methyl hypoiodite, copper hypoiodite, and And silver iodate. Tween80 is mentioned as a surfactant. Also

Adenosine phosphates may be removed by combining a bleaching agent and a surfactant and bringing them into contact with a filtration membrane or a water absorption zone. In this case, the bleaching agent may be hypochlorous acid, hypobromous acid or hypoiodic acid, or a salt thereof (eg, sodium hypochlorite, potassium hypochlorite, calcium hypochlorite, hypochlorous acid) Barium, methyl hypochlorite, copper hypochlorite, silver hypochlorite, sodium hypobromite, potassium hypobromite, calcium hypobromite, barium hypobromite, hypobromite Methyl, copper hypobromite, silver hypobromite, sodium hypoiodite, potassium hypoiodite, calcium hypoiodite, barium hypoiodite, methyl hypoiodite, hypoiodite Copper or silver hypoiodite) or a combination thereof is preferably combined using Tween80 as a surfactant. At this time, 0.0

It is preferred to combine the bleach at a concentration of 00005 to 5% with Tween 80 at a concentration of 0.0001 to 5%. It is particularly preferable to use sodium hypochlorite as a bleaching agent and Tween80 as a surfactant. In this case, the effective chlorine concentration is 0.00000 to 5

More preferably, sodium hypochlorite at a concentration of% and Tween 80 at a concentration of 0.0001 to 5% are combined. It is particularly preferable to combine sodium hypochlorite having an effective chlorine concentration of 0.0005 to 0.5% with Tween80 having a 0.0001 to 5% concentration. Further, adenosine phosphates may be removed by bringing a bleaching agent into contact with a filtration membrane or a water absorption zone. In this case, it is preferable to use the above-mentioned type of bleaching agent, and it is more preferable to use a bleaching agent having a concentration of 0.0005 to 0.5%. No. Further, the above treatment of the present invention is preferably performed at a temperature of 10 to 150 ° C. During this treatment, it is also preferable to apply autoclaving.

Furthermore, the present invention is a filtration membrane or a water absorption zone treated by the above treatment method. Furthermore, the present invention is a method for measuring the number of bacteria in a sample, comprising the following steps.

(1) The first step of capturing the cells in the sample on the filtration membrane by adding the sample to the filtration membrane according to claim 3.

(2) Second Step of Detecting Adenosine Phosphate Esters Contained in Bacteria Captured on Filtration Membrane Hereinafter, the present invention will be described in detail.

This application is intended to cover Japanese Patent Application No. 2001-146474, which is a priority document.

First, the filtration membrane to be washed in the present invention is not particularly limited as long as it can capture microorganisms, and includes, for example, a commercially available hydrophilic or hydrophobic filtration membrane. '' Examples of hydrophilic filtration membranes include hydrophilic polytetrafluoroethylene, hydrophilic polyvinylidene difluoride, hydrophilic polysulfone, hydrophilic polycarbonate, hydrophilic polyamide, hydrophilic polyethylene, hydrophilic polypropylene, etc. A film-like or sheet-like material made of a hydrophilic plastic material or a material such as acetyl cellulose or nitrocellulose is used. Examples of the hydrophobic filtration membrane include, for example, PVDF (polyvinylidene difluoride), PTFE (polytetrafluoroethylene), PE (polyethylene), and the like, or a relatively large hydrophilic hydrophobic membrane such as PC (Polycarbonate), PP (polypropylene), PA (polyamide), PS (polysulfone) and the like can be used. Also, RMD Special Membrane Fill Yuichi (PVDF film with hydrophobic lattice) and polycarbonate film manufactured by Nippon Millipore are preferred. Next, the method for cleaning a filtration membrane of the present invention will be described. First, examples of the solution for washing the filtration membrane include organic solvents such as methanol, ethanol, propanol, butanol, isopropanol, acetonitrile, acetone, and dimethyl sulfoxide, or sterilized water containing these organic solvents. Alternatively, only sterilized water may be used. Also optional It is possible to use sterile water in which the above-mentioned buffer or salt is dissolved, or to use a solution in which these are mixed with an organic solvent. The concentration and type of these organic solvents are not particularly limited, but preferably 5 to 95% ethanol, particularly preferably 10 to 90% ethanol.

Further, a surfactant may be further added to these cleaning liquids before use. Examples of the surfactant include any one selected from Tween, SDS, SPAN, Triton, benzalkonium chloride, benzethonium chloride, and the like, and the concentration used is not particularly limited. Preferably, Tween80 is at least 0.0001% and at most 50%, more preferably at least 0.001% and at most 30%.

Further, an acid or an alkali may be added to these cleaning liquids before use. Any acid or alkali can be used. For example, among acids, hydrochloric acid and acetic acid are particularly preferred, and the concentration to be used is 0.1% or more, preferably 1% or more. As the alkali, ammonia, sodium hydroxide, potassium hydroxide and the like are preferable, and the concentration to be used is 0.1% or more, preferably 1% to 30%. When cleaning the filtration membrane, it is desirable to use a pre-filter or a mesh spacer to prevent contamination by dust and the like. It is also desirable that the pre-filter and spacer be washed in advance with an organic solvent or sterile water containing these organic solvents.

Next, as a method for drying the filtration membrane after the above-mentioned washing, air drying, dry heat and the like can be mentioned. When drying the filtration membrane, it is preferable to put it in a petri dish to dry so that dust and the like do not adhere.The drying temperature is not particularly limited, but preferably 40 ° C or more, particularly preferably 45 ° C or more. Appropriate.

Further, a method of drying by blowing a clean wind through a HEPA filter or the like is also preferable. The temperature at this time is also not particularly limited, but is preferably 30 ° C or more, and particularly preferably 40 ° C or more.

In addition, the adenosine phosphates of the filtration membrane can be treated by enzymatically or chemically modifying or decomposing them into substances not related to the reaction system. The reagent to be used is not particularly limited, but is preferably an acid or an alkali, in addition to an enzyme such as adenosine phosphate deaminase which widely degrades adenosine phosphates. Among acids, hydrochloric acid or acetic acid is particularly preferred, and the concentration used is 0.1% or more, preferably

1% or more and 30% or less is appropriate. For alkali, ammonia, sodium hydroxide

, Potassium hydroxide and the like are preferable, and the concentration to be used is 0.1% or more, preferably 1% or more and 30% or less. The temperature during the reaction is not particularly limited, but is 40 ° C or more, especially

60 ° C. or higher is preferred. It is also particularly preferable to carry out autoclave treatment at a temperature of 100 ° C or higher (for example, 121 ° C).

Further, as a chemical method, adenosine phosphate esters in the filtration membrane may be decomposed using a bleaching agent or the like. Bleaching agents include oxidizing bleaches and reducing bleaches. Oxidative bleaching agents include peroxides and chlorine compounds. Examples of the peroxides include hydrogen peroxide, sodium peroxide, sodium perborate, potassium permanganate, and the like. Examples of the chlorine compound include salad powder, sodium hypochlorite, sodium chlorite and the like. Examples of the reducing bleaching agent include iodide, sodium bisulfite, hydrosulfite, and sodium dithionite. Also, potassium bisulfite, sodium hyposulfite, potassium pyrosulfite, nitrogen dioxide, benzoyl peroxide, ammonium persulfate, chlorine dioxide and the like can be used. Examples of the bleaching agent include hypochlorous acid, hypobromite, hypoiodic acid, and salts thereof, such as sodium hypochlorite, potassium hypochlorite, calcium hypochlorite, and hypochlorous acid. Barium, methyl hypochlorite, copper hypochlorite, silver hypochlorite, sodium hypobromite, potassium hypobromite, calcium hypobromite, barium hypobromite, hypobromite Methyl, copper hypobromite, silver hypobromite, sodium hypoiodite, potassium hypoiodite, calcium hypoiodite, barium hypoiodite, methyl hypoiodite, copper hypoiodite or It is preferable to use silver hypoiodite, and it is more preferable to use sodium hypochlorite. The concentration, temperature, time, pH, etc., when these bleaching agents are applied are not particularly limited. The bleaching agent may be used alone to degrade adenosine phosphates in the filtration membrane. In this case, it is preferable to use a bleaching agent having a concentration of 0.0005 to 0.5%. Further, an appropriate surfactant may be used in combination. In that case, the bleaching agent may be hypochlorous acid, hypobromite, hypoiodic acid, or a salt thereof, such as sodium hypochlorite, potassium hypochlorite, calcium hypochlorite, hypochlorous acid. Barium chlorate, Methyl hypochlorite, copper hypochlorite, silver hypochlorite, sodium hypobromite, potassium hypobromite, calcium hypobromite, barium hypobromite, methyl hypobromite, next Copper hypobromite, silver hypobromite, sodium hypoiodite, potassium hypoiodite, calcium hypoiodite, barium hypoiodite, methyl hypoiodite, copper hypoiodite or Although it is preferable to use silver iodate, it is more preferable to use sodium hypochlorite, and it is more preferable to use a sodium hypochlorite having an effective chlorine concentration of 0.00000 to 5%. It is particularly preferred to use sodium hypochlorite at a concentration of 0.0005-0.5%. Further, it is preferable to use sodium hypochlorite and Tween80 in combination, and it is preferable to use sodium hypochlorite having an effective chlorine concentration of 0.00000 to 5% and Tween80 at a concentration of 0.0001 to 5%. . In particular, it is preferable to use sodium hypochlorite having an effective chlorine concentration of 0.0005 to 0.5% in combination with Tween80 having a 0.0001 to 5% concentration. When a bleaching agent and a surfactant are used in combination, it is preferable to use a bleaching agent having a concentration of 0.0005 to 0.5% and a surfactant having a concentration of 0.0001 to 5%. The temperature during the reaction is arbitrary, but is preferably iOt or more, preferably 30: or more, particularly preferably 40 ° C. or more and 150 or less. In this treatment, autoclaving is also preferable. When an enzyme is used, for example, adenosine phosphate deaminase solution, or adenosine phosphate deaminase and another enzyme (eg, avirase, alkaline phosphatase, acid phosphatase, hexokinase or adenosine triphosphatase, or a combination thereof) A mixed solution or the like can be used. The final concentration of adenosine phosphate deaminase is at least 0.001 l / ml, preferably at least 0.01 U / ml and at most 10 U / ml.

The pH during the reaction is preferably from pH 4.5 to pH 8.5, particularly preferably from pH 5 to pH 8. The temperature during the reaction is preferably from 25 to 60 ° C, more preferably from 30 to 55 ° C.

In addition, the liquid-contacting parts such as the water absorption zone of the sprayer used for measurement, that is, the parts that come into contact with the luminescent reagent and the extraction reagent are contaminated with adenosine phosphates. May cause ground emission. Therefore, it is necessary to remove adenosine phosphates by an enzyme treatment or the like in advance. As a water absorption zone Examples thereof include those used for Matsushita Electric Works ultrasonic sprayer (EW622) and those used for automatic sprayer manufactured by Nippon Millipore. Various methods are available for treating the water-absorbing zone, such as a washing method, a method of chemically or enzymatically modifying or decomposing the same as in the above-described treatment method of the filtration membrane, but enzymatic treatment is preferred. The enzyme to be used is, for example, adenosine phosphate deaminase solution or adenosine phosphate deaminase and other enzymes (eg, apyrase, alkaline phosphatase, acid phosphatase, hexokinase, adenosine triphosphatase). A mixed solution or the like can be used. The final concentration of adenosine phosphate kinase is 0.001 U / ml or more, preferably 0.01 U / ml or more and 10 U / ml or less. The pH during the reaction is preferably from pH 4.5 to pH 8.5, and particularly preferably from pH 5 to pH 8. The temperature during the reaction is preferably 25 ° C or higher and 60 ° C or lower, but is more preferably 30 ° C or higher and 55 ° C or lower.

Next, as a method for drying the water absorption zone after the above treatment, air drying, dry heat, or the like can be used. When drying the water absorption zone, it is preferable to put it in a petri dish and dry it so that dust etc. do not adhere.The drying temperature is not particularly limited, but preferably 40 ° C or more, particularly preferably 45 ° C or more. Appropriate.

Further, a method of drying by blowing a clean wind through a HEPA filter or the like is also preferable. The temperature at this time is also not particularly limited, but is preferably 30 ° C or more, and particularly preferably 40 ° C or more. Next, a method for quickly measuring the number of bacteria using the above-mentioned filtration membrane will be described. In the present invention, first, microorganisms in a sample solution are captured on a filtration membrane.

The microorganism according to the present invention means yeasts, molds, mushrooms, bacteria, actinomycetes, unicellular algae, viruses, protozoa, animals or non-differentiated cells and tissue cultures.

The sample liquid is not particularly limited, but examples include the following.

Beverages: peel, wine, juice, etc.

Food: meat, fish, vegetables, etc.

Industrial products: Pharmaceuticals, cosmetics, semi-finished products, or raw materials for these

Seawater, river water, industrial water, pharmaceutical water, sewage, soil

Body fluid: urine, feces, blood, sputum, pus, etc.

Cultures of the above microorganisms, etc.

Further, a solution in which the above sample solution is suspended in an appropriate solvent (for example, distilled water, physiological saline, phosphate buffer solution, Tris buffer solution, sodium acetate buffer solution, etc.) may be used as the sample solution. When the sample solution contains a solid content, the sample solution is suspended in an appropriate solvent (eg, distilled water, physiological saline, phosphate buffer, Tris buffer, acetate buffer, etc.) or a mixer. A solution that has been homogenized by, for example, and used as a solution can be used. The means for capturing the microorganisms on the filtration membrane is not particularly limited, but, for example, is by adding a sample solution which seems to contain microorganisms to the filtration membrane. In the present invention

“Adding the sample solution to the filtration membrane” means applying the sample solution to the filtration membrane, specifically, filtering the sample solution using the filtration membrane, and spotting the sample solution on the filtration membrane.

(Dropping), applying or spraying the sample liquid on the filter membrane, and immersing the filter membrane in the sample liquid. When a sample solution is added to the filtration membrane, any of these methods may be used, or some of these methods may be used in combination.

As a method for filtering a sample solution, for example, a filter membrane after washing by the above method is attached to a cup-shaped filtration container (for example, Milliflex filter unit manufactured by Nippon Millipore Co., Ltd., Sterifil), and the sample solution is suction-filtered A method of capturing microorganisms on a filtration membrane is employed. After the filtration is completed, remove the filtration membrane from the filter, and if necessary, wash and air-dry the filtration membrane. When the microorganism is an animal cell, protoplast, or the like, it is preferable to use an isotonic solution such as a physiological saline solution for washing the filtration membrane in order to prevent cell rupture. After filtering the sample, the filter membrane may be placed on an agar medium containing adenosine phosphate deminase, cultured for an appropriate time, dried, and sprayed with an extraction reagent. As a result, the amount of luminescence from bacteria can be increased.

The filtration membrane capturing the microorganisms is brought into contact with a reagent containing ATP-degrading enzyme (hereinafter referred to as “ATP scavenger”), and ATP other than ATP contained in the microbial cells (hereinafter referred to as “background AT

It is desirable to disassemble PJ). Treating the filtration membrane with an ATP scavenger reduces background luminescence in the bioluminescence reaction and increases the sensitivity of microbial measurement. Is improved.

Examples of the ATP scavenger include adenosine phosphate deaminase solution, adenosine phosphate deaminase and other enzymes (eg, apyrase, alkaline phosphatase, acid phosphatase, hexokinase or adenosine triphosphase, or a combination thereof). And the like can be used. The final concentration of adenosine phosphate kinase in the ATP scavenger is 0.001 U / ml or more, and preferably 0.01 to 10 U / ml.

When the filter membrane is brought into contact with the ATP scavenger, the ATP scavenger may be dropped or sprayed on the filter membrane or the filter membrane may be immersed in the ATP scavenger.

After erasing the backdarand ATP, it is desirable to remove or deactivate the ATP scavenger on the filtration membrane. The removal of the ATP scavenger is carried out by washing the filter membrane with ATP-depleted ultrapure water or a buffer solution. Further, the ATP scavenger can be deactivated by acting an inhibitor against the scavenger. .

When the ATP scavenger is an adenosine phosphate deaminase solution, for example, coformycin can be used as an inhibitor of the scavenger. Coformycin is known as an inhibitor of adenosine phosphate deaminase (THE JOURNAL OF ANTIBI0TI CS, SE. A Vol. 20 No. 4 227-231).

When an inhibitor of the ATP scavenger is allowed to act, the inhibitor may be dropped or sprayed onto the filtration membrane. More preferably, the inhibitor may be added to the ATP extractant described below and added or sprayed onto the filtration membrane. Next, the biological components in the microorganisms captured on the filtration membrane are extracted. The method for extracting the biological component is not particularly limited. For example, a known ATP extractant may be dropped or sprayed on the filtration membrane. ATP extractants include, for example, a mixture of ethanol and ammonia

, Methanol, ethanol, surfactants (benzetonium chloride, benzalcodium chloride, Triton X100, etc.), trichloroacetic acid, perchloric acid, etc. can be used. In particular, a mixed solution of ethanol and ammonia is suitable as an ATP extractant because it is easy to volatilize after the ATP extraction operation and inhibits the activity of the ATPase.

The ATP extractant is sprayed using, for example, an ultrasonic sprayer (Matsushita Electric Works) for 5 seconds to 5 seconds. It may be done for about a minute. After ATP extraction, if necessary, the filtration membrane is dried to remove the ATP extractant.

The biological component in the microorganism according to the present invention means a substance contained in the microorganism and involved in the ATP conversion reaction or the bioluminescence reaction of the present invention. Such substances include, for example, adenosine phosphate (ATP, AMP, ADP, cyclic AMP, RNA, etc.), PPDK, phosphoenolpyruvate, pyrophosphate, luciferin, luciferase, pyruvate kinase, acetate acetate Creatine kinase, or cyclic 3 ', 5'-nucleotide phosphodiesterase. In addition, substances involved in the production of the above-mentioned biological components are also included in the biological component of the present invention. Next, the ATP conversion reagent and the bioluminescent reagent are dropped or sprayed on the filtration membrane from which the biological components in the microorganisms have been extracted. In order to prevent the extracted biological component from flowing out, it is preferable to spray the reagent using an ultrasonic sprayer manufactured by Matsushita Electric Works.

The ATP conversion reaction reagent referred to in the present invention is a reagent containing a substance involved in the ATP conversion reaction, for example, pyruvate orthophosphate dikinase (hereinafter, “PPDK”). The ATP conversion reaction referred to in the present invention refers to any reaction system in which ATP is generated during the reaction, and includes, for example, a reaction system in which AMP is directly generated from AMP.

One of the substances that contribute to the ATP conversion reaction is, for example, an enzyme that catalyzes the ATP conversion reaction. Enzymes that catalyze this ATP conversion reaction include, for example, pyruvate orthophosphate dikinase (PPDK) and phosphoenol pyruvate synthetase.

In the present invention, for example, the following ATP conversion reaction can be used by using a substance involved in the ATP conversion reaction.

① Generate ATP from AMP using a combination of adenylate kinase and pyruvate kinase.

② Polyphosphate: ATP is generated by combining AMP phosphotransferase and adenylate kinase (Applied and Environmental Microbiology, May

2000, 2045-2051).

③ ADP-dependent (AMP-Forniing) Generating AMP from ADP using darcokinase ATP is generated from AMP using pyruvate orthophosphate dikinase (Journal of Biochemis try 128, 1079-1085 2000).

A Generating ATP from AMP using a combination of polyphosphate kinase and adenylate kinase (EC 2.7.4.3) (Patent 2001- 211900)

A ATP is produced from AMP using a combination of AMP-PolyP phosphotransferase (AMP-Polyphosphate kinase) and Polyphosphate kinase (Polyphosphate kinase).

Specifically, the ATP conversion reaction reagent in the present invention is, for example, a reagent containing the following substances (a) to (2).

(Ii) PPM

(Mouth) Phosphoenolpyruvate

(8) Pyrophosphate

(Ii) Magnesium ions or other metal ions

PPDK is a known enzyme that catalyzes the reaction of producing ATP, pyruvate and phosphoric acid by acting on AMP, phosphoenorubyruvic acid and pyrophosphate in the presence of magnesium ion, and also catalyzes the reverse reaction.

ATP is generated by the reaction between the above reagent and AMP in the reaction system. The AMP in the reaction system as referred to herein is that contained as a biological component in the microorganism and that generated with the consumption of ATP in the bioluminescence reaction.

Further, in the present invention, other substances may be added to the ATP conversion reaction reagent containing the substances (a) to (2). For example, when cyclic 3 ', 5'-nucleotide phosphodiesterase is added, ATP is generated not only from AMP but also from cyclic AMP.

In addition, when pyruvate kinase is added to the ATP conversion reaction reagent containing the substances (a) to (2), ATP is generated not only from AMP but also from ADP.

In addition, a reagent which is a combination of the above-mentioned substances (a) to (2) and another substance and which contains a substance which participates in the reaction that generates ATP during the reaction is an ATP conversion reaction reagent of the present invention. include. The physicochemical properties and production method of PPDK, which is one of the substances involved in the ATP conversion reaction, are known, and their availability is relatively easy. Examples of plant-derived PPDK include enzymes derived from corn leaves [Biochemis try 12, 2862-2867 (1973)] and sugarcane leaves [The Biochemical Journal 114, 117-125 (1969)].

Microbial sources include, for example, Propionibacterium shermanii (Biochemistry 10, 721-729 (1971)), Acetobacter xylinum (Ace tobacter xyl inum) [Journal of Bacteriology (1970)], Bacteroides symbiosus [Me thods in Enzymolog y 42, 199-212 (1975)] and the genus Microbispora [eg Microbispora thermorosea IF0 14047] and the like.

As the PPDK of the present invention, for example, a natural PPDK purified from the above organism can be used. In addition, a mutant PPDK in which one or more (eg, one or several) amino acids in the amino acid sequence of the natural PPDK are introduced, deleted, or substituted with a mutation within a range not to lose the enzyme activity. Can also be used.

Natural PPDK can also be obtained by genetic engineering techniques. That is, the native PPDK gene may be introduced into an appropriate vector-one host system, and PPDK may be collected from the obtained culture of the recombinant microorganism. In addition, the PPDK gene can be obtained from maize (J. Biol. Chem. 263, 11080-11083 (1988)), Flaveria trinervia (FEBS Let t. 273, 116-121 (1990)), etc. It has already been cloned from living things.

When a mutant PPDK is used, the PPDK can be obtained from a mutant of the above organism.

. In addition, mutant PPM can be obtained by genetic engineering techniques. That is, it is added to one or more (eg, one or several) bases in the base sequence of the natural PPDK gene.

A mutant PPDK gene into which mutations such as deletion, substitution, etc. have been introduced is prepared and introduced into an appropriate vector-host system, and a mutant PPDK gene is collected from a culture of the obtained recombinant microorganism. I just need. Methods for introducing a mutation into a gene include, for example, a natural PPDK gene and a mutagenic agent (hydroxylamine, nitrite, sulfurous acid, 5-promouracil, etc.)

) Can be used. In addition, UV irradiation, cassette mutation,

A site-specific mutagenesis method using a PCR method can be widely used. Furthermore, It is also possible to construct a mutant PPDK gene having a mutation at a desired site by annealing the chemically synthesized DNA.

The bioluminescent reagent in the present invention is not particularly limited as long as it is a reagent containing a substance involved in a bioluminescent reaction. The bioluminescent reagent of the present invention is, for example, a reagent containing a substance involved in a luciferin-luciferase luminescence reaction, and specifically, for example, a reagent containing the following substances (a) to (c) It is.

(B) Magnesium ions or other metal ions

(Mouth) Luciferin

(8) Rireshifera Ize

A luminescence reaction occurs when the above reagent reacts with ATP in the reaction system. At that time, ATP is consumed and AMP is generated. The ATP in the reaction system as referred to herein includes those contained as biological components in microorganisms and those produced by the ATP conversion reaction.

In the present invention, as the luciferin and luciferase, those derived from, for example, insects (Genji firefly, Heike firefly, North American firefly, Hikarimemushimushi, Tsubotaru) can be used.

Luciferase is added to or deleted from one or more amino acids in the amino acid sequence of natural luciferase purified from the luminescent tissue of the above organism, natural luciferase prepared by genetic engineering techniques, or natural luciferase. Mutant luciferase into which a mutation such as loss or substitution has been introduced can be used.

When the biological component is RNA, AMP is generated from RNA by adding liponuclease (RNase) to the reaction system, and the AMP becomes ATP in the ATP conversion reaction.

In the conventional measurement method using a bioluminescent reagent, a biological component involved in the bioluminescence reaction is mainly ATP, and there is a problem that the amount of luminescence is attenuated with consumption of the ATP. On the other hand, in the present invention, by utilizing the ATP conversion reaction, ATP is newly generated from various adenosine phosphates (AMP, cyclic AMP, ADP, RNA, etc.) and consumed in the bioluminescence reaction. ATP is also played. As a result, the luminescence amount and the luminescence time are increased, so that microorganisms can be measured quickly, easily and with high sensitivity. The conditions of the ATP conversion reaction and the bioluminescence reaction (final concentration of each component contained in the reagent, reaction time, temperature, etc.), the order of dropping or spraying the above reagents are not particularly limited. What is necessary is just to set suitably. That is, the ATP conversion reaction reagent and the bioluminescent reagent may be used as a mixture, or may be sequentially added to the filtration membrane.

From the viewpoint of simplification of the operation, it is preferable to use a mixture of the ATP conversion reaction reagent and the bioluminescent reagent. In that case, for example, a mixed solution having the following composition can be used.

(B) PPDK; 0.001 l / ml or more, preferably 0.002 to 100 U / ml

(Mouth) Phosphoenolpyruvate; final concentration of 0. ImM or more, preferably 0.5 to 8. OmM (c) Pyrophosphoric acid; final concentration of 1. OM or more, preferably 50 to 5000 M Magnesium ion; final concentration 1. OmM or more, preferably 5.0 to 100 mM

(E) Luciferin; final concentration 5. O ^ M or more, preferably 50.0 to 10000 M

(F) Luciferase; final concentration 0.1 mg / ml or more, preferably 0.5 to 20 mg / nil (g) Ammonium sulfate; final concentration 0.5 ImM or more, preferably 0.5 to 100 mM

(H) dithiothreitol; final concentration 0.5 O lmM or more, preferably 0.05 to 10 mM (ii) EDTA; final concentration 0. ImM or more, preferably 0.5 to 10 mM

(N) HEPES or Tricine buffer (ρΗ7 · 0-8.5); final concentration 1 OmM or more, preferably 20-200 mM

By using the above mixed solution, ATP is regenerated from AMP generated by consumption of ATP in the bioluminescence reaction, so that the luminescence amount and the luminescence time are increased.

In the above-mentioned mixed solution, ammonium sulfate, dithiothreitol (DTT), EDTA, HEPES or Tricine is a substance that is preferably added to improve the stability or buffer capacity of the solution. . As such a substance, perserum albumin, sucrose and the like can be used.

In the present invention, when ATP is generated from cyclic AMP, which is a biological component in a microorganism, cyclic 3 ′, 5′-nucleotide phosphodiesterase (0.01 μl / ml or more, preferably 0.02 to 10 l / ml).

The ATP conversion reagent and bioluminescent reagent contain adenosine lysate, an ATPase. Acid deaminase may be added. If ATP is contaminated in the above reagent, ATP in the reagent will be eliminated by the addition of adenosine phosphate kinase. In that case, it is preferable to add coformycin to the ATP extractant. In this way, when the above reagent is added to the filtration membrane, decomposition of ATP derived from microorganisms can be prevented.

When adding adenosine phosphate deaminase to the above mixed solution of the ATP conversion reaction reagent and the bioluminescent reagent, add it so that the concentration is 0.01 l / ml or more, preferably 0.05 to 10 U / ml. do it.

Next, by measuring the luminescence generated on the filtration membrane, it becomes possible to determine the presence or absence of microorganisms in the sample solution or to measure the viable cell count. The amount of luminescence generated on the filtration membrane can be measured by a luminometer, for example, a luminescence reader BLR-201 (improved type) manufactured by Aroka. In addition, the number of viable bacteria is measured by imaging the bright spots on the filtration membrane using a bioluminescence image analysis system device, for example, ARGUS-50 / CL (with taper fiber: manufactured by Hamamatsu Photonics KK). It is also possible. BEST MODE FOR CARRYING OUT THE INVENTION

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

In the examples, a PPM luminescence reagent (a mixed reagent of an ATP conversion reaction reagent and a bioluminescence reagent) was used as the reagent of the present invention. In addition, HSLU (bioluminescent reagent not containing ATP conversion reagent) was used as a reagent of the conventional method.

1. Composition of reagent used

(1) ATP extractant

ATP release reagent containing coformycin (derived from St reptomyces genus) at 0.1 OlmM, manufactured by Nippon Millipore

(2) PPDK luminescence reagent

Luciferin 0.75 mM

Luciferase 3.6 mg / ml PPDK 0.47U / ml

EDTA 2Na lmM

DTT 0.2mM

Magnesium acetate lOmM

BSA 0.2%

Sucrose 0.4%

Potassium pyrophosphate 0.3mM

PEP potassium salt 1.lmM

Adenosine phosphate deaminase 0.2U / ml

Tricine 25mM (pH7.8) ■

(3) HSLU

Luciferin 0.5mM

Genzipo Luciferase 0.5mg / ml

Magnesium sulfate 10mM

EDTA 1. OmM

DTT 1. OmM

Sucrose 0.37%

HEPES 50mM (pH7.5)

2. Method for measuring enzyme activity

The method for measuring the activity of the enzyme used in the examples is shown below.

(DPPDK (from Microbispora thermorosea IF014047)

50 mM with 3 mM magnesium sulfate, 25 mM ammonium sulfate, 2 mM 2-mercaptoethanol, 2 mM pyrophosphate, 2 phosphoenolpyruvate and 0.1 lmM AMP

BIS-TRIS PROPANE buffer (pH 6.8) 180 1

After reaching 7 ° C, add 20 / xl of enzyme solution with appropriate activity and let react for 15 minutes.

Boil for 3 minutes in boiling water to stop the reaction. Appropriate dilution of this reaction solution

1 into a test tube, and add 50 Lucifer 250 (Kikkoman) solution to it. Add 1 drop and measure the amount of luminescence. Separately, prepare a graph in which the relationship between the ATP standard solution of a known concentration and the amount of emitted light is examined. Using this graph, calculate the moles of ATP generated per minute at 37 ° C, and use this value as the activity unit in the enzyme solution used. The amount of enzyme that produces 1 mol of ATP per minute in 37 is 1 unit (U)

(2) Adenosine phosphate deaminase (from Aspergillus genus)

Dissolve ATP to 80 in lOOmM sodium acetate buffer (containing lmM EDTA) (pH 5.0) to obtain a substrate solution. Add 100 l of the enzyme solution to 3 ml of the substrate solution, and react at 30 ° C for 30 minutes. Thereafter, the reaction is stopped by adding 60% perchloric acid 100, and the absorbance 0D at 265 nm is measured. The amount of enzyme whose 0D value changes by 2.4 per minute is defined as 1 unit (U).

(3) Avila Ize grade VI I I (from potato) (Sigma)

At 6.5 and 30 ° C, the amount of enzyme that releases 1.0 Omol of inorganic phosphoric acid per minute from ATP is defined as 1 unit (U).

(Example 1)

A filtration membrane RMD special membrane filter manufactured by Nippon Millipore Co., Ltd. (with 0.45 ^ m hydrophobic lattice, diameter 47mm) was immersed in 70% ethanol. This is set in a filter equipped with a polypropylene pre-filter (0.6 m), manufactured by Nippon Millipore Co., Ltd. and washed with 50 ml of 70% ethanol, and washed with 100 ml of 70% ethanol in advance. did. Thereafter, the membrane was washed twice with 100 ml of 20% ethanol, the membrane filter was removed from the filter, turned over, and set again in the filter. Further, the membrane was washed with 30 ml of ultrapure water, removed from the filter, placed in a sterile petri dish, and dried at 50 ° C for 15 hours. The PPM luminescent reagent was applied to the membrane before washing by an ultrasonic sprayer manufactured by Matsushita Electric Works (EW622). After setting in a RMDS device manufactured by Millipore Japan, luminescence was integrated for 5 minutes and measured at bit 0-4. The washed membrane was treated in the same manner and compared.

As a result, the film before cleaning emitted light over the entire surface, but the film after washing did not show any noise luminescent spots or back light emission. (Example 2)

A filtration membrane RMD special membrane filter manufactured by Nippon Millipore Co., Ltd. (0.45 m with hydrophobic grid, diameter 47 mm) was immersed in 70% ethanol. Then, it was placed in 200 ml of 10% acetic acid and placed in a 200 ml pressure bottle and capped. This was autoclaved at 121 ° C for 200 minutes. After the temperature dropped, the membrane was taken out and rinsed three times with ultrapure water to remove excess acetic acid.

The membrane before the treatment was sprayed with a PPM luminescent reagent using an ultrasonic atomizer (EW622) manufactured by Matsushita Electric Works, and set on a RMDS device manufactured by Millipore Japan. After that, the luminescence was integrated for 5 minutes and measured at bit 0-4. The treated film was similarly treated and compared.

As a result, the entire surface of the film before the treatment emitted light, but no noise luminescent spot and no background emission were observed in the film after the treatment.

(Example 3)

RMD special membrane filter 1 (manufactured by Nippon Millipore, 0.45 Mm with hydrophobic lattice, diameter 471M1) as a filtration membrane was immersed in 70% ethanol. Thereafter, the filter was set on a filter, and 1 Oml of a 10 mM HEPES buffer solution (pH 7.0) containing 1.ml/ml of adenosine phosphate deaminase was passed through to wash out excess 70% ethanol. Next, the plate was immersed in 20 ml of lOmM HEPES buffer (pH 7.0) containing 1.0 U / ml adenosine phosphate deaminase in a sterile petri dish and treated at 50 ° C for 6 hours. After the treatment, only the filter membrane was transferred into a new sterile petri dish and dried at 50 ° C overnight.

The PPDK luminescence reagent was sprayed on the membrane before the treatment with an ultrasonic sprayer (EW622) manufactured by Matsushita Electric Works, and set on a RMDS device manufactured by Millipore Japan. After that, the luminescence was integrated for 5 minutes and measured at bit 0-4. The treated film was similarly treated and compared.

As a result, the film before the treatment emitted light over the entire surface, and 20 noise luminescent spots were observed. However, the film after the treatment showed no noise luminescent spot and no background light emission.

(Example 4)

Nippon Millipore RMD Special Membrane Filter (0.45 m hydrophobic) 100 pieces with a lattice and 47 imn in diameter were immersed in 70% ethanol. Then, the mixture was put in a 200 ml mixed solution of 0.0005% sodium hypochlorite and 0.01% Tween80 with an effective chlorine concentration of 200 ml and sealed in a 200 ml pressure-resistant bottle. This was treated at 60 ° C for 5 hours. After the treatment, the filtration membrane was taken out and rinsed sufficiently with ultrapure water to remove excess sodium hypochlorite.

As a result, the film before the treatment emitted light from the entire surface, but the film after the treatment did not show any noise luminescent spot or background light emission. This method was excellent in that a large number of films could be processed at once.

(Example 5)

Mixing 100 filter membranes made by Nippon Millipore RMD special membrane filter (0.45 z in hydrophobic grid, diameter 47 mm) with effective chlorine concentration 0.0005% sodium hypochlorite and 0.01% Tween80 The solution was placed in 200 ml and placed in a 200 ml pressure bottle and capped. This was treated at 60 ° C for 6 hours. After the treatment, the filtration membrane was taken out and rinsed sufficiently with ultrapure water to remove excess sodium hypochlorite.

(Example 6)

100 filter membranes made by Nippon Millipore RMD special membrane filter (0.45 m with hydrophobic lattice, diameter 47 mm) Effective chlorine concentration 0.0005% Potassium hypochlorite, 0.011%

Tween80 mixture was placed in 200 ml and placed in a 200 ml pressure bottle and capped. This was treated at 60 ° C for 6 hours. After treatment, remove the filtration membrane, rinse thoroughly with ultrapure water and remove excess Potassium hypochlorite was removed.

(Example 7)

Nippon Millipore RMD Special Membrane Filter (0.45 m with hydrophobic lattice, diameter 47 mm) as filter membrane 100 pieces of effective chlorine concentration 0.05% sodium hypochlorite 200 ml and put in 200 ml pressure bottle I closed the lid. This was treated at room temperature for 6 hours. After the treatment, the filtration membrane was taken out and rinsed sufficiently with ultrapure water to remove excess sodium hypochlorite.

(Example 8)

Nihon Millipore RMD Special Membrane Filter (0.45 Mm with hydrophobic lattice, diameter 47 mm) is a filtration membrane. Effective chlorine concentration 0.005% sodium hypochlorite, 0.01% TweenSO mixed solution Put in 200ml, put in a 200ml pressure bottle and capped. This was treated at room temperature for 4 hours. After the treatment, the filtration membrane was taken out and rinsed sufficiently with ultrapure water to remove excess sodium hypochlorite.

The membrane before the treatment was sprayed with a PPM luminescent reagent using an ultrasonic atomizer (EW622) manufactured by Matsushita Electric Works, and set on a RMDS device manufactured by Millipore Japan. After that, the light emission is integrated for 5 minutes and bit 0-4 Measured. The treated film was similarly treated and compared.

(Example 9)

Nippon Millipore RMD Special Membrane Filter (0.45 / m with hydrophobic lattice, diameter 47mm), which is a filtration membrane, put 200 sheets of effective chlorine concentration 0.005% sodium hypochlorite in 200ml and put it in a 200ml pressure bottle I put it in and closed it. This was treated at room temperature for 15 hours. After the treatment, the filtration membrane was taken out and rinsed sufficiently with ultrapure water to remove excess sodium hypochlorite.

The PPDK luminescence reagent was sprayed on the membrane before the treatment with an ultrasonic sprayer (EW622) manufactured by Matsushita Electric Works, and set on a RMDS device manufactured by Millipore Japan. Thereafter, luminescence was integrated for 5 minutes and measured at Mt 0-4. The treated film was similarly treated and compared.

(Example 10)

The water absorption zone for an ultrasonic atomizer (EW622) manufactured by Matsushita Electric Works was immersed in sterilized ultrapure water containing 0.1 μg / ml of adenosine phosphate deaminase, and treated with 3rc for 1 hour. This was placed in a sterile petri dish and dried at 50 ° C for 15 hours. This was immersed in 2 ml of ultrapure water and stirred vigorously. The obtained solution 5a1 was added to PPDK luminescence reagent 1001, and the measurement was performed using a Lumitester C-100. The measured value was 520 RLU. On the other hand, when the untreated water absorption zone was measured in the same manner, the measured value was 146838 RLU. Background luminescence was sufficiently reduced by treating the water-absorbing zone with adenosine phosphate deaminase. This was used in the following experiments.

(Example 11) A filtration membrane RMD special membrane filter manufactured by Nippon Millipore Co. (0.45 / 1 m with hydrophobic grid, diameter 47 mni) was immersed in 5% Tween80. Set this in a filter equipped with a polypropylene pre-filter (0.6 / xm) and MF mesh filter AP32, manufactured by Nippon Millipore Co., Ltd., previously washed with 5% Tween80 and 100 ml, and made up to 100 ml with 5% Tween80. After washing twice, the membrane filter was removed from the filter, turned over, and set back in the filter. Further, it was washed with 30 ml of ultrapure water to remove excess Tween80, removed from the filter, placed in a sterile petri dish, and dried at 50 ° C for 15 hours.

The membrane before washing was sprayed with a PPDK luminescent reagent using an ultrasonic atomizer (EW622) manufactured by Matsushita Electric Works, and set on a RMDS device manufactured by Millipore Japan. After that, the luminescence was integrated for 5 minutes and measured at bit 0-4. The washed membrane was treated in the same manner and compared.

As a result, the film before the treatment emitted light on the entire surface, but the film after the treatment did not show any noise luminescent spots or background emission.

(Example 12)

A filtration membrane, a polycarbonate membrane (0.45 xm) manufactured by Nippon Millipore Co., Ltd., was immersed in 70% ethanol. This is set in a filter equipped with a polypropylene pre-filter (0.6 / ζπι), manufactured by Nippon Millipore Co., Ltd., and MF mesh spacer AP32, which has been washed with 100 ml of 70% ethanol. And washed. Thereafter, the membrane was washed twice with 100 ml of 20% ethanol, the membrane filter was removed from the filter, turned over, and set again in the filter. Further, it was washed with 30 ml of ultrapure water, removed from the filter, placed in a sterile dish, and dried with for 15 hours.

As a result, the film before cleaning emitted light over the entire surface, and 24 noise luminescent spots were observed. However, the film after cleaning showed no noise luminescent spot and no background light emission.

(Example 13)

Nippon Millipore RMD special membrane filter (0.45 m hydrophobic) A grid with a diameter of 47 mm) was immersed in 70% ethanol. This was set in a filter equipped with Nippon Millipore's polypropylene prefill Yuichi (0.6 m) and MF mesh spacer AP32 previously washed with 100% 70% ethanol and 50 ml of 70% ethanol. And washed. After that, the membrane was washed twice with 100 ml of 20% ethanol, the membrane filter was removed from the filter, turned over, and set again in the filter. Further, the membrane was washed with 30 ml of ultrapure water, removed from the filter, placed in a sterile petri dish, and dried at 50 ° C for 15 hours. HSLU was sprayed on the membrane before washing with an ultrasonic atomizer (EW622, manufactured by Matsushita Electric Works). Then, it was set on a RMDS device manufactured by Japan Millipore. After that, the luminescence was integrated for 5 minutes and measured at bit 0-4. The washed membrane was treated in the same manner and compared.

As a result, five noise bright spots were detected in the membrane before washing, but no noise bright spot and no background emission were observed in the filtered membrane after washing.

(Example 14)

The membrane filter prepared in Example 5 was set on a filter, and washed twice with 20 ml of ultrapure water. Then, add 10 ml of the washing solution (0.05% glucose, 0.05% fructose, 1 mM HEPES pH 7.0), and add the bacterial dilution (5% sucrose, 0.05% glucose, 0.05% fructose, 10% HEPES, 10 mM HEPES). Escherichia coli ATCC25922 appropriately diluted with pH 7.0) was added. Next, the plate was washed twice with 10 ml of a washing solution, and then washed with 10 ml of a washing solution containing 0.1 mg / ml adenosine phosphate deaminase. The membrane was removed from the filter, set in a holder, placed in a petri dish, opened slightly, and air-dried. After drying, the ATP extractant was sprayed with a Matsushita Electric Works ultrasonic sprayer (EW622) for 5 seconds and air-dried. Thereafter, a PPDK luminescence reagent was sprayed for 5 seconds with an ultrasonic atomizer (EW622) manufactured by Matsushita Electric Works, and set on an RMDS device to integrate the luminescence amount for 5 minutes. The number of appearing bright spots was measured with bitO-4. On the other hand, the number of bacteria (Colony Forming Unit) (CFU) measured by pour culture on a standard agar medium was measured and shown in Table 1.

As a result, the number of bright spots that appeared and the CFU measured by the pour culture method were in good agreement even with a small number of bacteria. Table 1.Results of bacterial count detection when using the membrane after washing Example

The present invention (piece) pour culture method (GFU)

0 0

twenty two

5 5

8

10 10

15 16

21 20

26 28

36 35

51 53

66 67

76 75

(Comparative example)

Set the unwashed membrane in the filter, and then add 10 ml of the washing solution (0.05% glucose, 0.05% fructose, ImM HEPES pH 7.0), and add the bacterial dilution (5% sucrose, 0.05% Escherichia coli ATCC25922, appropriately diluted with glucose, 0.05% fruc 1 ^ 1 +, lOmM HEPES pH 7.0) was added. Next, the plate was washed twice with 10 ml of a washing solution and then with 10 ml of a washing solution containing 0.1 lU / ml adenosine phosphate deaminase. The membrane was removed from the filter, set in a holder, placed in a petri dish, opened slightly, and air-dried. After drying, the ATP extractant was sprayed with a Matsushita Electric Works ultrasonic sprayer (EW622) for 5 seconds and air-dried. Then, the PPDK luminescent reagent was sprayed for 5 seconds with an ultrasonic atomizer (EW622) manufactured by Matsushita Electric Works, set on an RMDS device, and the luminescence was integrated for 5 minutes. The number of bright spots was measured using MtO-4. On the other hand, the number of bacteria (CFU) measured by pour culture on a standard agar medium was measured and shown in Table 2.

As a result, the number of bright spots appeared also included noise bright spots, and did not match the CFU measured by the pour culture method. Table 2 Results of detection of bacterial count when using unwashed filtration membrane Comparative example

Comparative example (piece) Pour culture method (CFU)

13 0

19 2

27 5

24 8

36 10

30 16

28 20

43 28

49 35

82 53

80 67

86 75

All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety. Industrial applicability

According to the present invention, a method for washing a filtration membrane, and a method for measuring the number of bacteria using the washed filtration membrane are provided.

In the conventional method, it was difficult to accurately measure the number of bacteria due to background luminescence derived from ATP, AMP, etc. attached to the membrane and noise spots, but in the present invention, a membrane substantially free of ATP and AMP was used. By using, measurement can be performed without noise luminescent spots. As a result, it is possible to measure the number of microorganisms quickly, simply, and with high sensitivity and accuracy.

Claims

Scope of request Selected from the group consisting of organic solvents, sterilized water containing organic solvents, sterilized water, enzyme solutions containing adenosine phosphate esterase, acids, alkalis, surfactants and bleaching agents A method for treating a filtration membrane or a water absorption zone, comprising contacting at least one of the filtration membrane and the water absorption zone with a filtration membrane or a water absorption zone to remove adenosine phosphates attached to the filtration membrane or the water absorption zone. 2. The method according to claim 1, wherein the organic solvent is ethanol. Bleaching agents are hypochlorous acid, hypobromite, hypoiodite, sodium hypochlorite, potassium hypochlorite, calcium hypochlorite, barium hypochlorite, hypochlorous acid Methyl, copper hypochlorite, silver hypochlorite, sodium hypobromite, potassium hypobromite, calcium hypobromite, barium hypobromite, methyl hypobromite, copper hypobromite, Consists of silver hypobromite, sodium hypoiodite, potassium hypoiodite, calcium hypoiodite, barium hypoiodite, methyl hypoiodite, copper hypoiodite and silver hypoiodite 2. The method according to claim 1, wherein the method is at least one selected from the group. The method according to claim 1, wherein the surfactant is Tween80. The method according to claim 1, wherein the bleaching agent and the surfactant are brought into contact with a filtration membrane or a water absorption zone. Bleaching agents are hypochlorous acid, hypobromite, hypoiodite, sodium hypochlorite, potassium hypochlorite, calcium hypochlorite, barium hypochlorite, methyl hypochlorite, Copper hypochlorite, Silver hypochlorite, Sodium hypobromite, Potassium hypobromite, Calcium hypobromite, Barium hypobromite, Methyl hypobromite, Copper hypobromite, Hypochlorite From the group consisting of silver bromate, sodium hypoiodite, potassium hypoiodite, calcium hypoiodite, barium hypoiodite, methyl hypoiodite, copper hypoiodite and silver hypoiodite The method according to claim 5, wherein at least one selected is selected, and the surfactant is Tween80. 7. The method of claim 6, wherein the bleach is at a concentration of 0.0005-0.5% and the surfactant is at a concentration of 0.0001-5%. A method for treating a filtration membrane or a water absorption zone, comprising contacting a bleach with a filtration membrane or a water absorption zone to remove adenosine phosphates adhering to the filtration membrane or the water absorption zone. Bleaching agents are hypochlorous acid, hypobromite, hypoiodite, sodium hypochlorite, potassium hypochlorite, calcium hypochlorite, barium hypochlorite, methyl hypochlorite, Copper hypochlorite, Silver hypochlorite, Sodium hypobromite, Potassium hypobromite, Calcium hypobromite, Barium hypobromite, Methyl hypobromite, Copper hypobromite, Hypoxia From the group consisting of silver bromate, sodium hypoiodite, potassium hypoiodite, calcium hypoiodite, barium hypoiodite, methyl hypoiodite, copper hypoiodite and silver hypoiodite 9. The method of claim 8, wherein the method is at least one selected. 9. The method of claim 8, wherein the bleach is at a concentration of 0.0005-0.5%. The method according to any one of claims 1 to 10, wherein the processing temperature is 10 to 150 ° C. 2. A filtration membrane treated by the method according to any one of claims 1 to 11. 3. A water absorption zone treated by the method according to any one of claims 1 to 11. 4. A method for measuring the number of bacteria in a sample, comprising the following steps (1) and (2).

(1) The first step of adding the sample to the filtration membrane according to claim 3 to capture the cells in the sample on the filtration membrane.

(2) The second step of detecting adenosine phosphates contained in the cells captured on the filtration membrane