JP3325876B2 - Evaluation method for antibacterial property of surface to be evaluated - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for evaluating antibacterial properties of a surface to be evaluated. INDUSTRIAL APPLICABILITY The present invention is suitable for use in a method for evaluating an antibacterial function of a ceramic product, a glass product or the like having an antibacterial function.

[0002]

2. Description of the Related Art In recent years, ceramic products and the like having an antibacterial function have been put on the market from a sanitary point of view, and it is required to establish a method for evaluating the antibacterial function of these products.

Heretofore, a film adhesion method has been widely adopted as such an antibacterial evaluation method. In this method, the evaluation is performed in the following procedure.

[0004] First, a test piece having a surface to be evaluated is placed in a sterile petri dish, a certain amount of the inoculum is inoculated on the surface to be evaluated, and a cover film is placed on the surface of the test piece, and a cover is placed thereon. To save for a certain period of time.

[0005] Thereafter, a bacterial solution adhering to the test piece and the coated film is sufficiently washed out into a petri dish using a fixed amount of SCDLP medium. Then, a fixed amount is collected from the washed out measurement solution, and agar plate culture (at a temperature of 35 ± 1 ° C., 40 to 40 ° C.) using an SA medium (standard agar medium) under a certain condition.
Culture for 48 hours).

[0006] The number of viable bacteria is determined by determining the number of colonies after culture, and the antibacterial function is evaluated.

[0007]

However, the above-mentioned conventional method for evaluating antibacterial properties has a problem that the measured values vary considerably. This tendency is particularly large when the surface to be evaluated is a water-repellent surface. The inventor of the present invention has made intensive studies on the dispersion of measured values in the antibacterial evaluation method, and as a result, has found that the dispersion is caused by the following.

[0008] That is, in the above-mentioned method, a certain amount of the bacterial solution for inoculation is inoculated on the surface to be evaluated, and a coating film is applied to spread the bacterial solution over a certain area.

However, a hydrophobic film such as polyethylene (PE) has been used as a conventional covering film. When the surface to be evaluated is a hydrophilic surface, a certain amount of the inoculated bacterial solution for inoculation is easily repelled on the coating film side, but spreads to some extent over the entire surface of the coating film through the hydrophilic surface to be evaluated. This spread is not always stable. When the surface to be evaluated is a water-repellent surface, the inoculation bacterial solution is easily repelled on both the coating film side and the surface to be evaluated, and is difficult to spread over the entire surface of the coating film. For this reason, the contact area of the bacterial solution for inoculation with the surface to be evaluated is different depending on how the covering film is covered or pressed, and the number of viable bacteria is also widely varied.

The present invention has been made in view of the above-mentioned conventional circumstances, and has a method for evaluating antibacterial properties of a surface to be evaluated.
An object of the present invention is to provide a method capable of accurately evaluating an antibacterial function with little variation in measured values.

[0011]

According to the method of the present invention for evaluating the antibacterial activity of a surface to be evaluated, a predetermined amount of a bacterial solution for inoculation is inoculated on the surface to be evaluated.
After covering with a covering film and covering it, a first step of storing under a certain condition, and after the first step, bacteria adhering to the surface to be evaluated and the covering film are removed using a certain medium. Wash out into the measurement solution and measure the number of viable bacteria in the measurement solution.
And the step of evaluating the antibacterial property of the surface to be evaluated, wherein the coating film has hydrophilicity.

In the method of the present invention, first, as a first step, a predetermined amount of a bacterial solution for inoculation is inoculated on a surface to be evaluated, a covering film is put on the surface, a lid is placed on the surface, and the surface is stored under certain conditions. . Next, as a second step, bacteria adhering to the surface to be evaluated and the coating film are washed out in the measurement solution using a constant medium, and the number of viable bacteria in the measurement solution is measured. In the first step, since the coating film has hydrophilicity, the coating film is easily wetted by the inoculation bacterial solution, and the inoculation bacterial solution spreads over the entire coating film. For this reason, the area where the bacterial solution for inoculation comes into contact with the surface to be evaluated easily matches the area of the coating film regardless of the difference in the method of covering or pressing the coating film. As a result, the variation in the number of viable bacteria is reduced, and the obtained measured value accurately reflects the antibacterial function of the surface to be evaluated.

Therefore, according to the method for evaluating antibacterial property of a surface to be evaluated according to the present invention, it is possible to accurately evaluate the antibacterial function with little variation in measured values.

As the coating film according to the present invention, in addition to a film having hydrophilicity itself, a material which does not have hydrophilicity itself and which has been subjected to hydrophilic treatment can be used. It is also possible to employ a material which has been subjected to a hydrophilic treatment on a material which itself has hydrophilicity. If the coating film itself has antibacterial properties, it is not suitable for use.

Examples of the coating film having hydrophilicity per se include, for example, polycarbonate (PC) having been subjected to hydrophilic treatment.
Films and cellulose mixed ester films can be selected.

On the other hand, even if the material itself does not have hydrophilicity, as a coating film subjected to hydrophilic treatment, P
A hydrophobic film made of E or the like may be contacted with an aqueous solution of polyvinylpyrrolidone (PVP), washed with water, and then dried. PVP in the wash water can be reused. Even in the latter case, the coated film is easily wetted by the inoculation bacterial solution, and the above-described effect can be obtained. For this reason, even if the material of the covering film is hydrophobic, it can be used, and the range of choice of the covering film can be expanded, so that the evaluation cost can be reduced.

The contact between the hydrophobic film and the aqueous PVP solution can be carried out by an immersion method, a spray method, a coating method, a mist method or the like. The higher the concentration of the PVP aqueous solution, the more reliable the hydrophilic treatment is possible. Specifically, the concentration of the PVP aqueous solution is
It is preferably at least 1.0 × 10 ⁻⁴ mol / L, more preferably at least 1.0 × 10 ⁻³ mol / L, even more preferably at least 1.0 × 10 ⁻² mol / L. The contact is suitably performed for 5 minutes or more.

[0018] A porous covering film may be employed. In this case, the surface of the coating film becomes rough due to the pores that are open at the surface of each of the porous pores, thereby reducing the water contact angle, thereby realizing hydrophilicity. For this reason, it is also preferable to employ a coating film whose surface is roughened. In addition, since the porous covering film having continuous pores is rich in air permeability, it is possible to prevent a decrease in the amount of dissolved oxygen in the inoculum bacterial solution between the covering film and the surface to be evaluated, and to prevent aerobic bacteria. It is also possible to perform evaluation using.

When the coating film is porous, it is preferable that the coating film is subjected to a water-absorbing treatment before the coating film is coated on the inoculum. In this case, it is possible to reduce errors caused by water absorption from the inoculating bacterial solution, as compared with a case where the water absorption treatment is not performed in advance. It is preferable that the liquid used for the water absorption treatment is a physiological saline solution, since it does not affect the growth of bacteria. More preferably, the liquid used for the water absorption treatment is a medium for preparing a bacterial liquid for inoculation. In this case, not only does not affect the growth of bacteria, but the nutrient concentration in the inoculum is not changed at all, and higher accuracy can be maintained.

Examples of the water absorption treatment include natural water absorption in which the coated film is completely immersed in the liquid, and forced water absorption in which the liquid is transmitted through the coated film by increasing or decreasing the pressure. In the water absorption treatment, it is preferable that no excess liquid is left on at least the surface of the coating film that comes into contact with the inoculating bacterial liquid. For this reason, when natural water absorption, forced water absorption, or the like is employed, it is preferable to wipe off the liquid remaining on at least the surface of the coating film that comes into contact with the inoculum bacterial solution with sterile filter paper or the like. It is also possible to employ a method in which a coating film is placed on the inoculation bacterial solution inoculated on the surface to be evaluated, and the solution is replenished from above the coating film. In this case, the replenishing amount of the solution is obtained by calculating the amount of water absorption from the opening density of the coating film.

Further, in the first step, a second coating film for suppressing evaporation of the inoculating bacterial solution may be put on the upper surface of the coating film. In this case, even if the opening density of the covering film is high and the inoculation bacterial solution may easily evaporate in a large amount from the opening of the covering film, the evaporation of the inoculating bacterial solution is suppressed by the second covering film. Therefore, high accuracy can be ensured. As the second film, a film having a low aperture density and having no water absorption is employed. In addition, before and after the coating film is brought into contact with the bacterial solution for inoculation, the coating film can be covered with the second coating film.

The porous covering film has an opening diameter of 1.0
μm or less, more preferably 0.4 μm or less.
μm or less. This is because both Escherichia coli and Staphylococcus aureus easily penetrate into the opening having a diameter exceeding 1.0 μm, and at the time of washing, these bacteria remain in the coating film and cause fluctuations in the measured value.

According to the test results of the inventors, the hydrophilicity of the coated film is preferably such that the water contact angle is 30 ° or less. If this is the case, the coating film is easily wettable by the inoculation bacterial solution,
This is because the bacterial solution for inoculation can be easily spread over the entire coating film.

The present invention is particularly effective when the surface to be evaluated is a water-repellent surface. If the surface to be evaluated is a water-repellent surface, the inoculation bacterial solution is easily repelled to the surface to be evaluated, but is easily wetted by the hydrophilic coating film, and thus spreads over the entire coating film. For this reason, the area where the bacterial solution for inoculation is in contact with the surface to be evaluated easily matches the area of the coating film.

According to the test results of the inventors, the sum of the water contact angle of the surface to be evaluated and the water contact angle of the coating film is 15%.
It is preferably 0 ° or less, preferably 130 ° or less, more preferably 110 ° or less. In this case, the inoculation bacterial solution is easily wettable on the surface to be evaluated and / or the coating film, and as a result, the inoculation bacterial solution can be spread over the entire coating film.

[0026]

Embodiments of the present invention will be described below.

Preparation of test pieces:

(1) Silver powder (99% silver purity) as an antibacterial agent
Above, an average particle size of 10 μm) was prepared, and this antibacterial agent 0.5
% Is applied to the sanitary ware before firing. And
The fired sanitary ware was cut into squares of 50 ± 2 mm square (within a thickness of 10 mm), and these were used as test pieces A to F.

(2) The sanitary ware after firing is subjected to the following water repellent treatment. That is, a non-woven fabric is impregnated with a commercially available water-repellent treatment solution containing a perfluoroalkyl group-containing organosilicon compound, and the surface of the sanitary ware after firing is rubbed about 10 times with the non-woven fabric, whereby the surface is subjected to a water-repellent treatment. Apply liquid. The water-repellent treatment liquid applied to the surface is dried for about 10 minutes. Thereafter, the water-repellent treatment liquid remaining on the surface is removed with ethanol to complete the water-repellent treatment. Similarly, the sanitary ware after the water repellent treatment is cut, and the test pieces G to
L.

(3) The surface of each of the test pieces A to J is lightly lightened with a local gauze or absorbent cotton impregnated with ethanol.
After wiping three times, dry (pre-treatment). Three of these were prepared, and the following antibacterial properties were evaluated.

Culture of test bacteria:

(1) The test bacteria were used in NA medium (normal agar medium)
And cultured at a temperature of 35 to 37 ° C. for 16 to 24 hours (pre-preculture).

(2) The bacterium pre-cultured in (1) above is subjected to NA
One platinum loop was transplanted to the medium, and the temperature was 35 to 37 ° C and 16 to 2
The cells were cultured for 0 hours (pre-culture).

Preparation of bacterial solution for inoculation:

The NB medium (normal broth medium) was diluted 500-fold with a phosphate buffer to prepare a "1 / 500NB medium" whose pH was adjusted to 7.0 ± 0.2. To give a bacterial solution for inoculation.

Test operation:

(1) Test pieces A to A which have not been subjected to a water-repellent treatment
F (3 each) and the test pieces GL to L (3 each)
) Is placed in a sterile petri dish, and the surface to be evaluated is inoculated with 0.4 ml (including 1.0 to 5.0 × 10 ⁵ bacteria) of the inoculum bacterial solution, and a 40 mm × 40 mm coated film is placed thereon. , And stored under conditions of a temperature of 35 ± 1 ° C. and a relative humidity of 90% or more.

The following were used as the covering films.

Test Specimens A and G: Stomacker film commercially available for microbial testing (“Stromacker 400”)
Plastic bag for mold (reinforced PE (organo: 180mm × 300
mm × 0.09 mm)) ”is aseptically cut into a size of 40 ± 2 mm square (water contact angle of 100 mm).
Every time). )

Test pieces B and H: improved stomacher film (the above stomacher film was converted to PV having a molecular weight of 25,000).
It is completely immersed in a P aqueous solution (5.0 × 10 ⁻³ mol / L) for 5 minutes, washed with water and dried to impart hydrophilicity (water contact angle 30 °). )

Test pieces C and I: membrane filter 1
(Hydrophilic membrane filter (PC, 0.2μ aperture)
m))

Test pieces D and J: membrane filter 2
(Hydrophilic membrane filter (made of PC, opening 1.0μ
m))

Test pieces E and K: membrane filter 3
(Hydrophilic membrane filter (made of PC, opening 1.0μ
m), in which 10 mL of the 1/500 NB medium was subjected to suction filtration, and the portion in contact with the inoculum solution was wiped off with sterile filter paper immediately before use. )

Test pieces F and L: membrane filter 4
(After the membrane filter 3 is placed on the inoculation bacterial solution inoculated on the surface to be evaluated, the above-mentioned stomacher film of the same size is placed thereon as the second coating film.)

It should be noted that the water contact angles of the membrane filters 1 to 4 could not be measured because of their water absorption.

Measurement of viable cell count:

(1) Three sterile petri dishes are prepared, and the same amount of the inoculum as that inoculated to each of the test pieces A to L is added, and a coating film is placed thereon. After this, SCD
Immediately using LP medium (10 ml), bacteria adhering to the respective coated films are thoroughly washed out into a Petri dish.
The number of viable cells in 1 ml of the washed liquid was measured by an agar plate culture method (cultured at a temperature of 35 ± 1 ° C. for 48 hours) using an SA medium (standard agar medium). Immediately after, the average value of the control section) was determined, and a value obtained by multiplying the average value by 10 was designated as X.

The dilution at the time of measuring the number of viable cells was performed using sterile phosphate buffered saline.

(2) The average value of the three viable cell counts ("control group") measured in the same manner as in the sterile petri dish described in the preceding section was determined for the control group sterile dishes (3 pieces) 24 hours after storage. , And a value obtained by multiplying it by 10 was set as Y.

(3) With respect to the test pieces A to L (three each) 24 hours after storage, the bacteria adhering to the test pieces A to L and the coated film, respectively, using an SCDLP medium (10 ml) were placed in a sterile petri dish. Wash out enough. Agar plate culture method using SA medium (cultured at a temperature of 35 ± 1 ° C for 48 hours)
As a result, the number of viable bacteria in 1 ml of the washed out measurement liquid was measured, and the average value of the three viable cell counts (“test group”) was obtained.

<Display of test results>

The “increase / decrease value difference” was calculated by the following equation. The second digit after the decimal point was truncated.

Log (Y / X) -log (Z / X) = 1
og (Y / Z)

<Test Success Conditions>

When all of the following four test conditions are satisfied, the test is considered valid.

(1) “Control plot immediately after inoculation” and “Control plot”
For each of the three viable bacterial counts, calculate according to the following formula,
The calculated value is 0.2 or less.

(Highest log value−lowest log value) / (log average value) ≦ 0.2

(2) The extinction rate of Y (average value of “control group”) to X (average value of “control group immediately after connection”) is 90.
% Or less.

{(X-Y) / X} × 100 ≦ 90

(3) For each of the three viable cell counts in the “control group immediately after inoculation”, their average value was 1.0 to 5.0 × 10 ⁵
/ Sheet.

(4) The viable cell count of each of the three cells in the “test plot” is 1.0 × 10 ³ / sheet or more.

<Test strain>

(1) Staphylococcus aureus IFO12
732 (ATCC 6538P)

(2) Escherichia coli IFO3972
(ATCC8739)

<Preparation for Test>

Apparatus, equipment and materials:

(1) Pipette (milk pipette, female pipette capable of dispensing at least 1 ml and 10 ml, or automatic pipettor)

(2) Thermostat (a model that can be operated with an accuracy within ± 1 ° C)

(3) Desiccator

(4) Sterilized Petri dish (80 mm to 10 mm in inner diameter)
0mm, height 15mm ~ 25mm)

(5) Underlay film (coating film is 5
Cut to size of 0mm square or more)

Medium and the like:

(1) Ordinary broth medium (NB medium)

Meat extract: 5.0 g Peptone: 10.0 g Sodium chloride: 5.0 g Purified water: 1,000 ml pH: 7.0-7.2

(2) Normal agar medium (NA medium) NB medium (1) supplemented with 1.5% agar

(3) Standard agar medium (SA medium) Yeast extract: 2.5 g Tryptone: 5.0 g Glucose: 1.0 g Agar: 15.0 g Purified water: 1,000 ml pH: 7.1 ± 0.1

(4) SCDLP medium Peptone made of casein: 17.0 g Peptone made of soybean: 3.0 g Sodium chloride: 5.0 g Potassium hydrogen phosphate: 2.5 g Glucose: 2.5 g Lecithin: 1.0 g Polysorbate 80: 7.0 g Purified water: 1,000 ml pH: 6.8-7.2

(5) Ethanol (purity 99% or more)

(6) Phosphate buffered saline

34 g of KH ₂ PO ₄ is dissolved in 500 ml of purified water, adjusted to pH 7.2 with 1N NaOH, and made up to 1000 ml with purified water. 1.25 ml of this solution is diluted 800 times with physiological saline (0.85% NaCl) to 1000 ml.

<Evaluation>

In the film adhesion method of the test pieces A to L,
Table 1 shows the proportion of the spread area of the inoculation bacterial solution when the covering film was put on and covered with the inoculation bacterial solution inoculated on the surface of each of the test pieces A to L.

[0083]

[Table 1]

As can be seen from Table 1, when the modified stomacher film and the hydrophilic coated films of the membrane filters 1 to 4 were used, 100% regardless of whether or not the test piece had been subjected to the water repellent treatment. (Specimens BF, HL). On the other hand, when a coated film having hydrophobicity of the stomacher film was used, 100% when the test piece was not subjected to the water-repellent treatment.
% Of the test piece (test piece A), but when the water-repellent treatment is applied, the spread is only 80 to 95% (test piece G). It was easy.

Further, from the results in Table 1, it can be seen that when the hydrophilic coated film was used, the coated film was more easily wetted by the inoculated bacterial solution than when the hydrophobic coated film was used. The solution spreads over the entire coating film, and it can be seen that the area where the bacterial solution for inoculation contacts the surface to be evaluated matches the area of the coating film.

Next, the results of the antibacterial evaluation of the test pieces A to L by the film adhesion method are shown in Table 2. Here, it was assumed that the antibacterial effect was obtained when the average increase / decrease value difference was 2.0 or more.

[0087]

[Table 2]

As can be seen from Table 2, when the modified Stomacher film and the hydrophilic coated films of the membrane filters 1 to 4 were used for both Escherichia coli and Staphylococcus aureus, the antimicrobial performance was obtained regardless of whether or not the test piece had water repellency. No difference was observed (test pieces BF, HL). On the other hand, when the hydrophobic coating film of the stomacher film was used, and when the test piece was not subjected to the water-repellent treatment, the value of the antibacterial evaluation when the hydrophilic coating film was used was obtained ( The test piece A) did not show any antibacterial effect in the test piece subjected to the water repellent treatment (test piece G).

Also, from the results in Table 2, when the hydrophilic coating film is used, the value of the obtained number of viable bacteria is small and the value accurately reflects the antibacterial performance of the surface to be evaluated. Understand.

Therefore, the method for evaluating antibacterial property of a surface to be evaluated according to the present embodiment makes it possible to accurately evaluate the antibacterial effect with little variation in measured values. In particular, this method
It can be seen that this is particularly effective when the surface to be evaluated is a water-repellent surface.

Further, from Tables 1 and 2, when the hydrophilicity of the coated film is 30 ° or less, the coated film is easily wetted with the inoculating bacterial solution and the inoculating bacterial solution can be easily coated. It can be seen that it can be spread all over. Further, from the test piece G, it is understood that the sum of the water contact angle of the surface to be evaluated and the water contact angle of the coating film is desirably 150 ° or less.

Further, it is understood from the test pieces E and K that the membrane filter having water absorbency can be subjected to a water absorption treatment in advance.

Further, it is understood from the test pieces F and L that it is also effective to apply a water-absorbing treatment to a water-absorbing membrane filter in advance and to stack a non-water-absorbing stroma film.

────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Haruyuki Mizuno 5-1-1 Koiehonmachi, Tokoname-shi, Aichi Inax Inc. (56) References JP-A-10-276798 (JP, A) (58) Survey Field (Int.Cl. ⁷ , DB name) C12Q 1/00-1/24 G01N 33/15

Claims (8)

(57) [Claims] 1. A first step of inoculating a predetermined amount of a bacterial solution for inoculation on a surface to be evaluated, covering with a cover film and covering the same, and then storing under a certain condition; A step of washing out the bacteria adhering to the surface to be evaluated and the coating film using a culture medium into a measurement solution, and measuring the number of viable bacteria in the solution to be evaluated. The method for evaluating antibacterial properties of a surface to be evaluated, wherein the coating film has hydrophilicity. 2. The method according to claim 1, wherein the coating film is subjected to a hydrophilic treatment. 3. The method according to claim 1, wherein the coating film is porous. 4. The method for evaluating antibacterial property of a surface to be evaluated according to claim 3, wherein the coated film is subjected to a water absorption treatment in advance. 5. The method for evaluating antibacterial properties of a surface to be evaluated according to claim 3 or 4, wherein in the first step, a second coating film for suppressing evaporation of the inoculum is coated on the upper surface of the coating film. . 6. The hydrophilicity of the coating film is such that the water contact angle is 30 °.
The method for evaluating antibacterial activity of a surface to be evaluated according to claim 1, 2, or 3, wherein: 7. The method for evaluating antibacterial property of a surface to be evaluated according to claim 1, wherein the surface to be evaluated is a water-repellent surface. 8. The method according to claim 6, wherein the sum of the water contact angle of the surface to be evaluated and the water contact angle of the coating film is 150 ° or less.