JP6404072B2 - Acanthamoeba disinfection evaluation method - Google Patents

Description

The present invention relates to a disinfection evaluation method for Acanthamoeba. Specifically, the present invention relates to a disinfection evaluation method for Acanthamoeba using the MTT method.

Acanthamoeba (Acanthamoeba) is a general term for microorganisms of the genus Acanthamoeba, which is a kind of protists that exist widely in nature, and inhabits soil, water, etc., and may also inhabit tap water. Acanthamoeba is widely known to be a pathogen of infectious diseases, and causes, for example, keratitis and granulomatous encephalitis.

If you suffer from Acanthamoeba keratitis, you will not only feel intense pain in your eyes, but if it becomes severe, you may open a hole in the cornea and cause blindness. Acanthamoeba keratitis is particularly frequent in contact lens users, and one of the factors is inappropriate use of contact lenses and care methods.

When using contact lenses, cleaning with contact lens care products or the like is required except for single-use contact lenses. Among contact lenses, soft contact lenses made of a water-containing material, in particular, tend to be a hotbed for Acanthamoeba as well as fungi and bacteria. Therefore, soft contact lenses are required to be disinfected with contact lens care products.

Under such circumstances, in the care product for soft contact lenses, the disinfection effect is very important, and the disinfection effect is confirmed by various disinfection evaluation methods. However, although guidelines for the disinfection evaluation method for fungi and bacteria have already been established, there is no fixed method for the disinfection evaluation method for Acanthamoeba.

One of the methods for evaluating disinfection of Acanthamoeba is the Spearman-Karber method. This method serially dilutes Acanthamoeba sterilized with a predetermined disinfectant solution and then cultivates, and the disinfection ability is determined from the number of surviving Acanthamoeba (positive number) and the number of dead Acanthamoeba (negative number) in each diluted solution. It is a method to evaluate. However, in this method, a large difference is likely to occur in the determination of the positive number and the negative number, and the accuracy of the evaluation result is low. Therefore, a method for accurately grasping the number of surviving acanthamoeba is desired.

For example, the MTT method is known as a method for measuring the number of living cells. In the MTT method, 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyltetrazolium bromide (hereinafter referred to as MTT) is reduced by mitochondrial dehydrogenase in living cells, and 3,5 -This method utilizes the production of diphenyl-1- (4,5-dimethyl-2-thiazolyl) formazan (hereinafter referred to as MTT formazan). In the MTT method, MTT is added to a test sample, and the number of viable cells in the test sample is determined from the amount of MTT formazan obtained.

In Non-Patent Document 1, as a method for measuring the number of HeLa cells using the MTT method, an MTT solution is added and reacted in a culture solution of HeLa cells, and a formazan solution (hydrochloric acid / isopropyl alcohol solution) is added to the recovered HeLa cells. Alternatively, DMSO) is added, and the amount of MTT formazan produced by the reaction is measured as the absorbance with a microplate reader to describe the method of measuring the number of cells. A similar method is also described in Patent Document 1.

In Patent Document 2, by combining WST-1, which is the same water-soluble tetrazolium compound as MTT, and 1-MPMS, which is an electron carrier, it is possible to easily measure the growth state and activity of microorganisms compared to the case of using MTT. Is described.

On the other hand, Non-Patent Document 2 describes a culture method using a PYG medium mainly composed of proteose peptone, which is a kind of peptones, as a culture method for Acanthamoeba. Non-Patent Document 3 describes various peptones.

JP-A-5-244993 JP 2001-204492 A

“Dojindo Laboratories Cell Proliferation Measurement Cell Staining Protocol”, [online], Dojindo Laboratories, Inc. [searched on September 18, 2014], Internet <URL: http://www.dojindo.co.jp /technical/pdf/catalog-project.pdf>, pp. 12-18 Investigative Ophthalmology and Visual Science, March 2011, Vol.52, No.3, pp.1793-1799 “BD Bionutrients Technical Manual Advanced Bioprocessing 3rd Edition”, [online], Japan Becton Dickinson, [searched September 18, 2014], Internet <URL: http: // https: //www.bdj.co. jp / hkdqj2000001lwn0-att / 67-001-00_BD-Bionutrients-technical-manual.pdf>

The methods described in Non-Patent Document 1 and Patent Document 1 are based on the colorimetric determination of MTT. In these methods, dimethyl sulfoxide, isopropanol containing dimethyl sulfoxide, dimethylformamide, or the like is used as a solvent for obtaining an MTT formazan solution. However, organic solvents such as dimethyl sulfoxide and dimethylformamide are highly soluble in MTT formazan, but they invade the body and parts of the analyzer and are highly fat-soluble and highly permeable to the skin. There are problems with its use, such as being exposed and being flammable, limiting its use. Therefore, it is conceivable to use lower alcohols such as methanol and isopropanol instead of the solvent having these problems. However, lower alcohols have low solubility in MTT formazan, and there is a problem that MTT formazan cannot be extracted efficiently.

By the way, the method for measuring the number of living cells using the MTT method is a method for obtaining the number of living cells from the amount of MTT formazan generated by reducing MTT by dehydrogenation in mitochondria in living cells as described above. And culturing the living cells. As described in Non-Patent Document 2, for cultivation of Acanthamoeba, a PYG medium mainly containing proteose peptone, which is a peptone-containing substance, and further containing a yeast extract and glucose is generally used. However, PYG medium is highly reactive with MTT, and MTT is reduced by medium components in addition to reduction by mitochondrial dehydrogenation in living cells. Therefore, using PYG medium, the number of viable cells of Acanthamoeba is reduced. There is a problem that it cannot be measured accurately.

Patent Document 2 describes that the amount of MTT formazan in E. coli produced by culturing Escherichia coli in Mueller Hinton medium and then subjecting to MTT is measured for absorbance. However, it is described that when MTT is used as a tetrazolium compound, even if an electron carrier coexists, the formazan produced is lower in detection sensitivity than WST-1 secreted extracellularly. Furthermore, in Patent Document 2, when Escherichia coli is cultured in the Mulan Hinton medium, there is a problem that Acanthamoeba cannot be cultured in the Mulan Hinton medium. Non-Patent Document 3 does not describe peptones suitable for the growth of Acanthamoeba.

As described above, the current MTT method as it is cannot be said to be suitable for a method for evaluating disinfection against Acanthamoeba. Therefore, the present invention accurately performs the culturing of Acanthamoeba and the production reaction of formazan, and by using a solvent that is safe for the analyzer and the human body, provides a safe, accurate, and highly sensitive disinfecting effect on Acanthamoeba. It is an object of the present invention to provide a method for evaluation.

As a result of intensive studies on the above problems, the present inventors have found that a medium containing a hydrolyzate of milk protein or an extract of animal tissue other than the stomach as a main component in place of the PYG medium has a high growth rate of Acanthamoeba. And it discovered that the reduction effect with respect to MTT which coexists was low.

Next, the present inventors have a high possibility that organic solvents such as hexane, acetone, tetrahydrofuran, dimethylformamide, and dimethyl sulfoxide, which are generally used as extraction solvents for MTT formazan, will invade the measuring apparatus main body and parts. Since the analysis apparatus and analysis method that can be used for quantitative measurement of the extract solution are limited, the use of lower alcohols with less of these problems was examined. In particular, even when a lower alcohol was used as an extraction solvent, a process that can dissolve MTT formazan was studied. As a result, MTT formazan-containing acanthamoeba obtained by reacting cultured Acanthamoeba with MTT was analyzed. MTT formazan was successfully extracted by collecting the solid phase and then passing the lower alcohol through physical stress such as centrifugal force and pressure.

The present invention is based on the above-mentioned findings and successful examples, and uses a medium mainly composed of components that are proliferative to Acanthamoeba and less reducible to MTT, and is used as an extraction solvent for MTT formazan. This was completed as a method for evaluating the disinfection effect against Acanthamoeba, which increased the extraction efficiency of MTT formazan while using alcohol.

Therefore, according to this invention, the evaluation method of the disinfection effect with respect to an acanthamoeba including the following process (1)-(4) is provided.
(1) The step of culturing the Acanthamoeba subjected to the disinfection treatment using a medium containing a hydrolyzate of milk protein or an extract of animal tissue other than the stomach as a main component in the presence of MTT (2) Step of collecting Acanthamoeba on solid phase (3) Step of extracting MTT formazan from Acanthamoeba collected on said solid phase by applying physical stress to said solid phase and applying MTT formazan soluble solvent (4) The process of evaluating the disinfection effect with respect to Acanthamoeba of the said disinfection process by the amount of the extracted MTT formazan

Preferably, the animal tissue extract other than the stomach is an extract of at least one animal tissue selected from the group consisting of heart and brain.

Preferably, the milk protein hydrolyzate is casein or whey hydrolyzate.

Preferably, the physical stress is centrifugal force or pressure.

According to another aspect of the present invention, there is provided a method for measuring the number of living cells of Acanthamoeba, comprising the following steps (A) to (D).
(A) A step of culturing Acanthamoeba using a medium containing a hydrolyzate of milk protein or an extract of animal tissue other than stomach as a main component in the presence of MTT (B) The cultured Acanthamoeba is solid-phased (C) Step of extracting MTT formazan from Acanthamoeba collected on the solid phase by applying physical stress to the solid phase and applying an MTT formazan soluble solvent to the solid phase (D) Determining the number of living cells of Acanthamoeba by the amount of MTT formazan prepared

According to the method of the present invention, the disinfection effect on acanthamoeba can be evaluated safely, accurately, and with high sensitivity, so that contact lens care reduces the rate of occurrence of an eye disease caused by acanthamoeba of a contact lens wearer. Further development of products and anti-acanthamoeba agents can be expected.

FIG. 1 is a diagram showing a calibration curve for calculating the concentration of a living Acanthamoeba. FIG. 2 is a diagram showing the amount of Acanthamoeba decrease in the control and sample.

Details of the present invention will be described below.
The method of the present invention relates to a method for evaluating the disinfection effect on Acanthamoeba, and includes at least the following steps (1) to (4).
(1) The step of culturing the Acanthamoeba subjected to the disinfection treatment using a medium containing a hydrolyzate of milk protein or an extract of animal tissue other than the stomach as a main component in the presence of MTT (2) Step of collecting Acanthamoeba on solid phase (3) Step of extracting MTT formazan from Acanthamoeba collected on said solid phase by applying physical stress to said solid phase and applying MTT formazan soluble solvent (4) The process of evaluating the disinfection effect with respect to Acanthamoeba of the said disinfection process by the amount of the extracted MTT formazan

The method of the present invention is a method suitable for measuring the number of living cells of Acanthamoeba based on the MTT method widely used as a method for measuring the number of living cells. The method of the present invention is characterized by the medium used for culturing Acanthamoeba and the extraction step of the produced MTT formazan.

In step (1) in the method of the present invention, Acanthamoeba subjected to disinfection is cultured using a medium containing a hydrolyzate of milk protein or an extract of animal tissue other than the stomach as a main component in the presence of MTT. It is a process to do.

The disinfection treatment provided to the Acanthamoeba is not particularly limited. For example, to kill the Acanthamoeba such as contacting the Acanthamoeba with a disinfectant, heating the Acanthamoeba, or physically crushing the Acanthamoeba. However, the disinfection treatment is preferably performed by bringing Acanthamoeba into contact with a disinfectant.

The medium used in the step (1) contains a hydrolyzate of milk protein, an extract of animal tissues other than the stomach, or both as main components. The hydrolyzate of milk protein is not particularly limited as long as it is obtained by hydrolyzing protein derived from milk of mammals such as cows and pigs by using an acid or an enzyme, for example, casein hydrolyzate, Examples include whey hydrolyzate. Examples of extracts of animal tissues other than the stomach include brain, heart and both extracts. Among these, a hydrolyzate of milk protein and an extract of animal tissue other than the stomach preferably have a low carbohydrate content.

Specific examples of hydrolysates of milk proteins and extracts of animal tissues other than the stomach include casamino acids, casein digests, casitoles, lactalbumin hydrolysates, triptycase, tryptone, brain heart infusion broth, etc. These can be used alone or in combination of two or more. Among these, when considering the reactivity with MTT and the growth of Acanthamoeba, it is preferably casamino acid, casein digest, trypticase, tryptone, brain heart infusion broth, more preferably casamino acid, casein digest. , Tryptone and brain heart infusion broth, more preferably casamino acid, tryptone and brain heart infusion broth having a relatively low carbohydrate content.

The method for obtaining the hydrolyzate of milk protein and the extract of animal tissue other than the stomach is not particularly limited. For example, a milk protein hydrolyzate can be prepared by treating milk such as milk itself, casein, or whey under conditions known to those skilled in the art using an acid such as hydrochloric acid or an enzyme such as pepsin, and if necessary, drying treatment such as lyophilization. It can obtain by providing. Extracts of animal tissues other than the stomach can be obtained by steaming organs other than the stomach such as the brain and heart with purified water, and if necessary, subjecting them to a drying treatment such as lyophilization. Commercially available milk protein hydrolysates and extracts of animal tissues other than the stomach may be used. Examples of these commercially available products include those described in Non-Patent Document 3.

The amount of the milk protein hydrolyzate and the extract of animal tissue other than the stomach with respect to the total amount of the medium at the time of preparation as the liquid medium is not particularly limited as long as it is an amount capable of growing Acanthamoeba. 0.1 to 90 W / V%, preferably 0.25 to 75 W / V%, more preferably 0.5 to 50 W / V%, and still more preferably 1.0 to 10 W / V%. . When the blending amount of the hydrolyzate of milk protein and the extract of animal tissue other than the stomach is less than 0.1 W / V%, the growth ability cannot be imparted to Acanthamoeba, and the blending amount is 90 W / V. If the percentage exceeds 100%, the growth of Acanthamoeba may be suppressed due to the influence of osmotic pressure or the like.

In addition to the hydrolyzate of milk protein and the extract of animal tissues other than the stomach, the medium used in the step (1) considers the influence on the growth ability of Acanthamoeba, for example, pH 5.0 to 8.0, A buffering agent that preferably has a pH of 5.5 to 7.5, more preferably 6.0 to 7.0 may be added.

The buffer is not particularly limited as long as it does not inhibit the growth ability of Acanthamoeba, and examples thereof include a phosphate buffer, a borate buffer, a citrate buffer, and a trishydroxyaminomethane buffer. Of these, a phosphate buffer is preferably used in consideration of the growth of Acanthamoeba.

If the amount of buffering agent is too small, the desired buffering effect cannot be obtained. On the other hand, if the amount of buffering agent is too large, there is concern that precipitation in the medium and growth of Acanthamoeba may be inhibited. Therefore, the blending amount of the buffer with respect to the total amount of the medium is, for example, 0.005 to 20 W / V%, preferably 0.01 to 10 W / V%.

In the medium used in the step (1), in addition to the above components, various additives such as extracts, sugars, inorganic substances, salts and hydrates thereof can be blended to the extent that the object of the present invention is not impaired. .

Specific examples of additives include yeast extract as extract; glucose, sucrose, glycogen, glucan, fructan, etc. as saccharide; ammonium iron sulfate (II) / hexahydrate, magnesium sulfate / seven water as inorganic substances Examples include, but are not limited to, Japanese hydrate, calcium chloride, calcium chloride dihydrate, and the like.

For example, the milk protein hydrolyzate and the extract of animal tissue other than stomach and other components in the medium used in the step (1) are mixed with, for example, the milk protein hydrolyzate and extract of animal tissue other than the stomach. Is 20 to 50 parts by mass, and the total amount of other components is 1 to 5 parts by mass. The amount of the hydrolyzate of milk protein and the extract of animal tissue other than the stomach is preferably 70 to 99 W / W%, preferably 80 to 98 W / W%, based on the total amount of the medium components. Is more preferable, and it is still more preferable that it is 90-96W / W%.

Specific examples of the medium used in the step (1) include 10-30 parts by mass of milk protein hydrolyzate or extract of animal tissue other than stomach, 0.1-5 parts by mass of yeast extract, 0.01-0. 1 part by mass, magnesium salt 0.0001 to 0.01 part by mass, calcium salt 0.00001 to 0.0001 part by mass, iron salt 0.00001 to 0.0001 part by mass, phosphate 0.0001 to 0.001 It is a culture medium which consists of a mass part and 0.0001-0.01 mass part of citrate. Such a medium is useful as a medium for measuring the number of viable cells of Acanthamoeba because it has the ability to grow Acanthamoeba and has a low reactivity with MTT. Further, a kit containing such a medium and a tetrazolium compound such as MTT, WST-1, triphenyltetrazolium, tetrazolium violet, nitroblue tetrazolium, tetranitroblue tetrazolium, tetrazolium blue chloride, p-iodonitrotetrazolium, It is useful as a kit for measuring the number of living cells of Acanthamoeba.

The medium used in step (1) can be prepared according to a generally known medium preparation method, and is not particularly limited. For example, it is prepared by adding a hydrolyzate of milk protein and an extract of animal tissue other than stomach and other components to purified water simultaneously or by adding each component before and after confirming the solubility of each component. Can do. The medium used in step (1) is a powdered medium obtained by mixing a hydrolyzate of milk protein and an extract of animal tissue other than the stomach and other components, a hydrolyzate of milk protein and an animal other than the stomach. Liquid medium obtained by dissolving tissue extract and other ingredients in water, milk protein hydrolyzate and extract of animal tissues other than stomach and other ingredients were further coagulated by adding agar etc. Includes solid media and the like.

In step (1), Acanthamoeba is cultured by adding MTT to the medium. The MTT added to the medium may be either MTT itself or an MTT solution in which MTT is dissolved. The solvent used for the preparation of the MTT solution is not particularly limited as long as it does not affect the growth ability of Acanthamoeba and the production of MTT formazan. Examples thereof include Ringer's solution, other physiological saline, and purified water. .

The conditions for culturing Acanthamoeba are not particularly limited as long as they are suitable for the growth of Acanthamoeba and the production of MTT formazan. For example, the conditions are 20-30 ° C., preferably about 25 ° C., several hours to several tens of hours. The condition is preferably 1 to 24 hours.

In step (2) in the method of the present invention, the Acanthamoeba cultured in step (1) is collected on a solid phase.

The solid phase used in the step (2) is not particularly limited as long as it can collect the Acanthamoeba, and examples thereof include a filter medium having a sufficiently small pore size with respect to the Acanthamoeba size. The pore size of the filter medium is preferably 1 μm or less, more preferably 0.5 μm or less, because the Acanthamoeba size is 10 μm or more.

As a specific example of the step (2), filtration is performed by applying an Acanthamoeba-containing solution from one side of a filter medium that is a solid phase and applying a physical stress such as centrifugation or pressure to extract a liquid component from the other side of the filter medium. The step of collecting the Acanthamoeba on the solid phase by performing is included, but is not limited thereto.

In step (3) in the method of the present invention, the Acanthamoeba collected in Step (2) is subjected to physical stress and passed through the MTT formazan-soluble solvent through the solid phase collected in Acanthamoeba. Extract MTT formazan from amoeba.

As the MTT formazan-soluble solvent for extracting MTT formazan in the step (3), it is preferable to use a lower alcohol that is relatively safe for the analyzer and the human body. However, lower alcohols have relatively low solubility in MTT formazan. Therefore, when extracting MTT formazan using a lower alcohol, even if a solvent having a relatively low solubility in MTT formazan is used by applying physical stress, dimethyl sulfoxide or the like having a relatively high solubility in MTT formazan can be used. The extraction efficiency of MTT formazan is comparable to that obtained when a mixture of a solvent or lower alcohol isopropanol and hydrochloric acid or sodium hydroxide is used. Therefore, by using lower alcohol and physical stress, the Acanthamoeba collected in the solid phase can be crushed and MTT formazan accumulated inside and outside the Acanthamoeba can be extracted into the lower alcohol.

As a specific example of extraction of MTT formazan in step (3), lower alcohol is applied from one side of the filter medium collecting Acanthamoeba, and physical stress is applied from one side to the other side, Although it is possible to obtain a MTT formazan-containing material as an extract from the other side of the filter medium by performing filtration through which the lower alcohol is passed through the filter medium, the present invention is not limited thereto. Examples of the physical stress applied at this time include pressure and centrifugal force, and centrifugal force is preferably used.

If the applied physical stress is too weak, MTT formazan cannot be extracted, and if the physical stress is too strong, the solid phase such as filter media may be destroyed. The centrifugal speed is preferably 100 to 10,000 rpm, more preferably 500 to 5,000 rpm, and still more preferably 1,000 to 3,000 rpm. When the physical stress is a pressure, the pressure is a stress equivalent to the centrifugal force.

When the extraction solvent is an organic solvent, time for applying physical stress is important. When the time is too short, physical stress sufficient to extract MTT formazan cannot be obtained, and when the time is too long, extraction is performed. The solvent may evaporate, which is not preferable anyway. Therefore, the time for applying physical stress is preferably 30 seconds to 10 minutes, more preferably 1 minute to 7 minutes and 30 seconds, and even more preferably 1 minute 30 seconds to 5 minutes.

The physical stress can be applied in accordance with a generally known physical stress load operation, and is not particularly limited. For example, an operation using a device such as a centrifuge, a pump, a syringe, or an aspirator is used. Can be carried out alone or in combination of two or more of these operations.

In step (4) in the method of the present invention, the disinfection effect on Acanthamoeba produced by the disinfection treatment is evaluated based on the amount of MTT formazan extracted in step (3). When the amount of MTT formazan extracted is small, it can be evaluated that the disinfection effect on Acanthamoeba by disinfection is large, and on the other hand, when the amount of MTT formazan extracted is large, it is evaluated that disinfection effect on Acanthamoeba by disinfection is small it can. At this time, in the step (1), if a system using an Acanthamoeba that has not been subjected to disinfection is used as a comparative control in addition to a system using an Acanthamoeba that has been subjected to disinfection, an accurate disinfection effect can be determined. become able to. In addition, the disinfection effect can be selected by comparing with the existing disinfection treatment, for example, by comparing the disinfection effect of the existing disinfectant with a high disinfection effect against Acanthamoeba. It becomes possible. Thus, as another aspect of the present invention, there is provided a screening method for an anti-acanthamoeba agent using the method for evaluating the disinfection effect on an acanthamoeba of the present invention.

In the method of the present invention, Acanthamoeba is extracted to a solid phase before extracting MTT formazan, and an excess culture solution (reaction solution) is removed. Therefore, the tetrazolium compound to be used is preferably MTT. If the method of the invention is modified, other tetrazolium compounds instead of MTT, such as WST-1, triphenyltetrazolium, tetrazolium violet, nitroblue tetrazolium, tetranitroblue tetrazolium, tetrazolium blue chloride, p-iodonitrotetrazolium Or a method using a tetrazolium compound.

Another aspect of the present invention is a method for measuring the number of viable cells of Acanthamoeba, comprising the following steps (A) to (D).
(A) A step of culturing Acanthamoeba using a medium containing a hydrolyzate of milk protein or an extract of animal tissue other than stomach as a main component in the presence of MTT (B) The cultured Acanthamoeba is solid-phased (C) Step of extracting MTT formazan from Acanthamoeba collected on the solid phase by applying physical stress to the solid phase and applying an MTT formazan soluble solvent to the solid phase (D) Determining the number of living cells of Acanthamoeba by the amount of MTT formazan prepared

Steps (A) to (D) of the measurement method of the present invention can be performed with reference to the respective items of steps (1) to (4) of the evaluation method of the present invention.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited or limited by these examples, and the present invention takes various modes as long as the problems of the present invention can be solved. be able to.

Evaluation on the MTT formazan extraction method was carried out under the conditions shown in Table 1, and the results are shown in Table 2. Moreover, the performance of the culture medium prepared by the formulation shown in Table 3 was evaluated, and the results are shown in Table 4. Furthermore, disinfection evaluation was implemented on the MTT formazan detection conditions shown in Table 5, and the result is shown to FIGS. Hereinafter, these details will be described.

[1. MTT formazan extraction method]
(1) Preparation of MTT formazan dispersion 0.001 g of MTT formazan (Tokyo Chemical Industry) was weighed and transferred to a PP container. To this, 10 mL of Ringer solution 1/4 concentration (hereinafter referred to as 1/4 Ringer solution) was added, and MTT formazan was sufficiently dispersed by irradiating ultrasonic waves to prepare an MTT formazan dispersion.

(2) Recovery of MTT formazan by centrifugal force 0.3 mL of the MTT formazan dispersion was fractionated into a microtube (Cosmo Spin Filter H, Nacalai Tesque) with a hydrophilic PTFE filter having a pore diameter of 0.45 μm attached in advance. The microtube was centrifuged at 1,500 rpm for 5 minutes to apply a stress due to centrifugal force, and MTT formazan was collected on a filter. The microtube containing the 1/4 Ringer's solution was removed and a filter collecting MTT formazan was attached to a new microtube, and 0.3 mL of extraction solvent (methanol, propanol or dimethyl sulfoxide) was added from above the filter. By centrifuging the microtube at 1,500 rpm for 5 minutes, MTT formazan collected in the filter was dissolved and recovered in the extraction solvent. As shown in Table 1 to be described later, among the MTT formazan recovery systems carried out in this way, the recovery systems using methanol and propanol as extraction solvents were set as Examples 1 and 2, respectively, and the recovery systems using dimethyl sulfoxide were compared. Example 3 was used.

(3) Recovery of MTT formazan by pressure 0.3 mL of MTT formazan dispersion was dispensed into a PP syringe (Cosmonis filter S, Nacalai Tesque) equipped with a hydrophilic PTFE filter having a pore diameter of 0.45 μm. Next, pressure was applied by pushing the syringe so that the extrusion speed was 30 seconds / drop, MTT formazan in the MTT formazan dispersion was collected on a filter, and the 1/4 Ringer solution was removed. Add 0.3 mL of the extraction solvent (propanol) into the syringe and apply pressure by pushing the syringe so that the extrusion speed is 30 seconds / drop, so that the MTT formazan collected in the filter is put into the extraction solvent. Dissolved and collected. As shown in Table 1 described later, the MTT formazan recovery system carried out in this way was designated as Example 3.

(4) Recovery of MTT formazan by no stress 0.3 mL of MTT formazan dispersion was fractionated into a microtube (Cosmo Spin Filter H, Nacalai Tesque) to which a hydrophilic PTFE filter having a pore diameter of 0.45 μm was previously attached. The microtube was centrifuged at 1,500 rpm for 5 minutes to apply a stress due to centrifugal force, and MTT formazan was collected on a filter. After removing the microtube containing the 1/4 Ringer's solution and attaching a filter collecting MTT formazan to a new microtube, add 0.3 mL of extraction solvent (methanol or propanol) from the filter and let stand for 5 minutes. I put it. After standing, the extraction solvent was recovered from the upper part of the filter without applying stress. As shown in Table 1 to be described later, of the MTT formazan recovery system carried out in this way, the recovery systems using methanol and propanol as extraction solvents were designated as Comparative Examples 1 and 2, respectively.

(5) After extracting the evaluation results, the recoverability of MTT formazan was evaluated for the following three items.

1) Residual MTT Formazan When the filter collected with the remaining MTT formazan was visually confirmed, it was determined to be “conforming” when the blue-violet MTT formazan did not remain, and “non-conforming” when it remained.

2) When visually confirming the remaining syringe and microtube of the extraction solvent, it is determined as “compatible” if the extraction solvent does not remain, and “not compatible” if the extraction solvent remains without passing through the filter. "

3) Compare the chemical resistance of the extraction solvent to polyether ether ketone (PEEK), which is highly resistant to polyether ether ketone. If there is no, it was determined as “non-conforming”.

The results of the above three items collected and evaluated for Examples 1 to 3 and Comparative Examples 1 to 3 are shown in Table 2 below. As shown in Table 2, Examples 1 to 3 are all suitable for the above three items, and show that there is no problem in the influence on the MTT formazan extraction method and the analysis apparatus and its components.

[2. Culture medium]
(6) Preparation of medium According to the formulation shown in Table 3, 1,000 mL of purified water was weighed into the mixture obtained by mixing each component for the media of Examples 4 to 6 and Comparative Examples 4 to 6 of the present invention. It prepared by stirring so that each component might become uniform at room temperature. The obtained medium was sterilized, and each sterilized medium was prepared. In addition, “Brain Heart Infusion Broth” in Table 3 was obtained by decomposing animal tissues derived from the brain and heart, and “Proteau Speptone” represents animal tissues derived from the stomach of cattle or pigs. It was obtained by decomposition. Commercial products (manufactured by Becton Dickinson) were used for tryptone, casamino acid, brain heart infusion broth, proteose peptone, malt extract and phyton.

(7) Evaluation result 4) Reactivity with MTT After dispensing 0.2 mL of each sterilized medium of Examples 4-6 and Comparative Examples 4-6 to a 48-well culture plate, 0.02 mL of MTT solution was further added, It left still for 24 hours in a 25 degreeC incubator. The MTT solution was prepared by adding 25 mg of MTT to 5 ml of 1/4 Ringer's solution with reference to the description on page 12 of Non-Patent Document 2.

After standing, all the contents in each well are transferred to a spin filter (Cosmo Spin Filter H, Nacalai Tesque), centrifuged at 1,500 rpm for 5 minutes, and then filtered using methanol as an extraction solvent. The MTT formazan collected above was extracted by centrifugal filtration under the same conditions. The extract was detected by HPLC under the conditions shown in Table 5, and the MTT formazan area value was determined.

By comparing the obtained MTT formazan area value with the MTT formazan area value obtained for Comparative Example 4 which is a PYG medium, the MTT reactivity of each medium was determined by the following equation as the MTT formazan relative ratio.

When the MTT reactivity was less than 20%, the medium was judged to be a medium with low reactivity to MTT.

5) Acanthamoeba proliferating ability Acanthamoeba pre-cultured was collected. Acanthamoeba was recovered by adding 1/4 Ringer's solution to the collected Acanthamoeba, washing, and then removing the supernatant by centrifugation. Acanthamoeba suspension was prepared from the collected Acanthamoeba using the media of Examples 4 to 6 and Comparative Examples 4 to 6 so that the concentration of Acanthamoeba was 500 cells / mL. Dispense 5 mL of the Acanthamoeba suspension into a T-25 culture flask and incubate in a 25 ° C. incubator for 3 days. Collect the Acanthamoeba in the flask and count the number of Acanthamoeba using a hemocytometer. did. The number of Acanthamoeba cultivated using each medium was compared with the number of Acanthamoeba of Comparative Example 4 which is a PYG medium.

When the Acanthamoeba proliferative ability was in the range of 80 to 120%, the medium was judged to be a medium capable of achieving the Acanthamoeba proliferative ability equivalent to PYG.

The evaluation results of the above-mentioned two items carried out for Examples 4 to 6 and Comparative Examples 4 to 6 are shown in Table 4 described later. As Table 4 shows, Examples 4 to 6 can be evaluated as media that have low reactivity with respect to MTT and can achieve the growth ability of Acanthamoeba equivalent to PYG in any of the above two items. .

[3. Disinfection evaluation]
The pre-cultured Acanthamoeba was collected and suspended in a 1/4 Ringer solution to 1.0 × 10 ⁷ cells / mL, which was used as an inoculum stock solution.

0.1 mL of the inoculum stock solution was inoculated into 10 mL of 1/4 Ringer's solution and prepared so that the initial Acanthamoeba concentration was 1 × 10 ⁵ cells / mL, and this was used as a standard sample. A comparative control was prepared using Dulbecco's phosphate buffer instead of 1/4 Ringer's solution. A sample prepared using Dulbecco's phosphate buffer containing 5.0 μg / mL of polyhexanide hydrochloride instead of the 1/4 Ringer solution was used.

After standing at 25 ° C. for 4 hours, 1 mL each of the standard sample, the comparative control and the sample was put into a 15 mL polypropylene centrifuge tube containing 9 mL of an inert agent-containing solution (¼ Ringer solution containing 10% (V / V) polysorbate 80). By adding, a 10-fold diluted solution in which polyhexanide hydrochloride was inactivated by neutralization was prepared.

The whole amount of the 10-fold diluted solution was centrifuged at 5,000 rpm for 10 minutes to precipitate the acanthamoeba. After removing the supernatant, 10 mL of the medium of Example 4 described in Table 3 was added, and the Acanthamoeba was resuspended to prepare a 10-fold diluted solution (1).

After separating 1 mL of the 10-fold diluted solution (1) into a new centrifuge tube, 9 mL of a new medium of Example 4 was added to prepare a 10-fold diluted solution (2). The same operation was further repeated twice to prepare a dilution series for the 10-fold diluted solution (3) and the 10-fold diluted solution (4).

0.2 mL of each of the prepared 10-fold diluted solutions (1) to (4) was seeded on a 48-well culture plate and cultured at 25 ° C. for 1 week. After the culture, the medium in the well was replaced with 0.2 mL of a new medium of Example 4, and 0.5% (W / V) MTT-dissolved 1/4 Ringer solution was added to 0.02 mL / well. . The plate was then allowed to react overnight at 25 ° C.

The reaction product containing the MTT formazan-containing Acanthamoeba was collected on the filter by centrifuging at 1,500 rpm for 5 minutes using a spin filter (Cosmo Spin Filter H, Nacalai Tesque). After exchanging the microtube, 0.4 mL of methanol was added to the filter, and the mixture was centrifuged at 1,500 rpm for 5 minutes, and MTT formazan in Acanthamoeba was eluted in methanol.

The absorbance of the obtained methanol eluate was measured by HPLC under the conditions of Table 5 described later, and the logarithmic value (MTT formazan area value) of the absorbance value obtained using the standard sample and the Acanthamoeba concentration (cells / (see FIG. 1). From the obtained calibration curve, the viable Acanthamoeba concentration of the comparative control and the sample was calculated, and the viable Acanthamoeba concentration was subtracted from the initial Acanthamoeba concentration to obtain the respective Acanthamoeba decrease amount. The results are shown in FIG. As shown in FIG. 2, it can be seen that the acanthamoeba decreased in the sample containing the disinfectant compared to the control.

From the above results, it was shown that according to the method of the present invention, an Acanthamoeba evaluation method applying the MTT method can be obtained.

The method of the present invention can promote further development of contact lens care products and anti-acanthamoeba agents that reduce the incidence of eye diseases caused by acanthamoeba in contact lens wearers, and diseases associated with acanthamoeba Can contribute to the health and well-being of those affected or at risk of being affected.

Claims (4)

The evaluation method of the disinfection effect with respect to an acanthamoeba including the following process (1)-(4).
(1) Acanthamoeba subjected to disinfection treatment using a medium containing as a main component an extract of milk protein hydrolyzate or at least one animal tissue selected from the group consisting of heart and brain in the presence of MTT However, the medium is not a PYG medium containing 2% bactocasitone (2) A step of collecting the cultured Acanthamoeba on a solid phase (3) A physical stress is applied to the solid phase A step of extracting MTT formazan from the Acanthamoeba collected on the solid phase by applying an MTT formazan soluble solvent (4) The disinfection effect on the Acanthamoeba of the disinfection treatment is evaluated by the amount of the extracted MTT formazan Process The evaluation method according to claim 1, wherein the milk protein hydrolyzate is casein or whey hydrolyzate. The physical stress, a centrifugal force or pressure, the evaluation method according to any one of claims 1-2. A method for measuring the number of viable cells of Acanthamoeba, comprising the following steps (A) to (D).
(A) A step of culturing Acanthamoeba using a medium containing, as a main component, a hydrolyzate of milk protein or an extract of at least one animal tissue selected from the group consisting of heart and brain in the presence of MTT , However, the medium is not a PYG medium containing 2% bactocasitone (B) a step of collecting the cultured Acanthamoeba on a solid phase (C) physical stress is applied to the solid phase to add an MTT formazan soluble solvent. Applying MTT formazan from the Acanthamoeba collected on the solid phase by applying (D) determining the number of viable cells of Acanthamoeba according to the amount of MTT formazan extracted