JPH0924089A - Evaluation method for antibacterial function of antibacterial textile product and evaluation kit therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method as well as an evaluation kit capable of easily evaluating the antibacterial function of a textile product so far difficult in terms of the evaluation thereof, such as water-repellent fabric containing an antibacterial agent not easily eluted, and fabric with post-treated surface. SOLUTION: In this evaluation method for the antibacterial function of an antibacterial textile product. and the evaluation kit including a sterilized weight to be placed on evaluated fabric, an agar culture medium, sterilized reference cloth and bacteria liquid are sprayed on antibacterial fabric and then, the bacteria liquid thereon is dried. Thereafter, the antibacterial fabric is placed on the agar culture medium and cultured. In addition, the colony number of bacteria formed on the medium is measured.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for evaluating antibacterial properties of antibacterial textiles and an evaluation kit used for the evaluation.

[0002]

2. Description of the Related Art The main antibacterial test methods for textile products that have been carried out in the past are as follows: 1) The fungus resistance test method of JIS Z 2911, which tests using 4 kinds of fungi.

2) AATCC Test Method 90 (1981 Halo-Tes
t) AATCC Broth agar medium (pH 6.8) is autoclaved and cooled to 45 ° C. For 150 ml of this medium, the test bacterial solution (Staphylo-coccus aureus 209ATCC 6
538, Klebsiella Pneumoniae ATCC 4352). Dispense 15 ml each into a petri dish with a diameter of 9 cm,
Solidify. Next, gently attach the circular sterilization test cloths (processed cloth and unprocessed cloth) each having a diameter of 28.6 mm with sterile tweezers so that they are sufficiently brought into contact with the agar plates inoculated with the test bacteria in advance. Incubate the agar plates at 37 ° C for 18-24 hours. The evaluation method is to measure the diameter of the fungus inhibition zone around the test cloth from the back side of the agar plate and calculate by the following formula. W = (T−D) / 2 W is the diameter of the stop zone: mm T is the diameter of the test cloth and the stop zone: mm D is the diameter of the test cloth: mm

3) AATCC Test Method 100 Unprocessed fibers cut to a diameter of 4.8 cm and processed fibers (the number of test cloths is the amount that exactly absorbs 1 ml of bacterial solution) were put into an 8-ounce wide-mouth bottle with a screw cap, Autoclave sterilized by autoclaving, and then a test bacterial solution (Staphylococcus aureus 209 ATCC 6538,
Inoculate 1 ml of Klebsiella Pneumoniae ATCC 4352).
Next, immediately after inoculation and after culturing at 37 ° C for 18 to 24 hours, add 100 ml of neutralizing solution (solution for washing out test bacteria) to the unprocessed cloth and processed cloth, and shake vigorously for 1 minute. To wash out the bacteria. The viable cell count of the treated cloth and the untreated cloth (control) is measured, and the reduction rate of the viable cell count due to the chemical agent of the processed cloth is calculated by the following formula. In addition, the viable cell count was measured by using this washout solution to make a continuous dilution solution, and then using Trypton Glucose extract (TG
E.) Perform by plate dilution method (incubate at 37 ° C for 48 hours) on agar medium. Bacterial reduction rate (%) = B (or C or (B + C) / 2)
-A / B (or C or (B + C) / 2) x 100 A was inoculated on the processed cloth, and the number of bacteria was obtained after culturing for 18 to 24 hours, and B was obtained immediately after inoculated on the processed cloth. The number of bacteria, C is the number of bacteria obtained from the one immediately after inoculating the untreated cloth, and if B and C are not the same, the larger number, B and C
If is not so different, use (B + C) / 2.

4) Bacterial Growth Inhibition Test Method Unprocessed cloth (control cloth) cut into squares with a diameter of about 20 mm or about 18 mm and processed cloth of about 0.2 g (6 samples of unprocessed cloth for each test cloth) , 4 pieces of processed cloth are used in a vial bottle with a screw of about 30 ml each and autoclaved at 121 ° C. for 15 minutes under high pressure steam sterilization. Preliminarily cultivated in normal agar medium for 24 to 48 hours, then shaken or statically cultivated at 35 to 37 ° C. to obtain 1 × 10 ⁸ cells / ml (test bacteria are S. aureus, B. Subtilis, K. Pneumonia
e, E. coli) was diluted with buffered saline, and finally diluted with broth medium to 5 × 10 ⁵ to 3 × 10 ⁶ cells / ml, and 0.2 ml was dropped on each test cloth. Inoculate evenly.
Next, 20 ml of sterilized buffered saline was added to the unprocessed cloth and the processed cloth immediately after inoculation and after culturing at 35 to 37 ° C. for 18 hours, and shaken vigorously 25 to 30 times with a shaking width of about 30 cm. To wash out the bacteria. This dispersed bacterial solution is AATCC Test Method 100
Perform the same operation as above and serially dilute to make a 10-fold dilution series. Unprocessed cloth was collected immediately after inoculation, 100, 1000 times diluted (A, 3 samples), collected after 18 hours of culture, 10,000,
100,000-1 million times diluted (B, 3 samples) Processed cloth collected after 18 hours of culture, 10, 100, 1000 times diluted (C, 4 samples)
The viable cell count of each is measured. The judgment is the average value of B ÷ the average value of A> 10, the lowest value of B> the highest value of A, the highest value of C <A
If it is the lowest value of, it is evaluated that there is a growth inhibitory effect of the test bacteria used.

5) Shake Frask method Nutrient broth was used, and the test bacterial solution was precultured by shaking at 37 ° C. for 18 hours (the test bacterial solution was the same S. aureus as AATCC 90).
K. Pneumoniae) is adjusted to 1.5 to 3.0 × 10 ⁵ cells / ml, and this is inoculated with 5 ml of 70 ml of a culture solution diluted 800 times with 0.5 M phosphate buffer of sterilized pH 7.2. Next, autoclave high pressure steam sterilization (121
1.5 ± 0.1 g of sample (15 minutes at ℃) (sample size is not specified, but make it fine) and shake for 1 hour at 37 ℃ to forcibly contact the sample with the test bacteria. Incubate. The number of viable bacteria is measured by using this culture medium to make a serial dilution, and performing plate dilution on Trypton Glucose Extract agar medium. The evaluation method was to measure the viable cell count before shaking (before adding the sample) and after 1 hour of shaking culture by the plate dilution method, and the bacterial reduction rate relative to the viable cell count before shaking was calculated by the following formula: calculate. Bacterial reduction rate (%) = 100 x (BA) / BA is the number of bacteria per 1 ml in the Erlenmeyer flask after shaking B is the number of bacteria per 1 ml in the Erlenmeyer flask before shaking Effect If the average value of the bacterial reduction rate of 2 samples (blank (cultured at the same time without adding a sample)) is X and the average value of the bacterial reduction rate of 2 samples is Y, then X is ,
It is determined that the growth of bacteria was suppressed. However, -10 <X
If <10, this test is considered valid. There are five types of methods.

These methods have the following drawbacks. That is, 1). In the AATCC Test Method 90 of 2), a) antibacterial agent permeates into the agar plate, and the spreading agent does not diffuse rapidly into the agar plate, the plate is left at 5 ° C for 16 to 18 hours and then 37 ° C. In 6
It can be evaluated by a method of culturing for ~ 8 hours, but it cannot be compared under the same conditions as fibers not left at low temperature. And the like. 2). The method of Nakajima et al., Which is an improved version of 2) AATCC Test Method 90 (using two kinds of bacteria, observing the growth condition of the agar plate after removing the test cloth on the agar that had been cultured) The method of determining antibacterial activity) has the drawbacks that the growth state of bacteria is unclear, the measured values vary, the reproducibility is poor, and the antibacterial activity is difficult to measure. 3). With the AATCC Test Method 100 method of 3), a) it is not possible to test a large number of samples at one time, and the laboratory time is long. b) Antibacterial properties cannot be evaluated with antibacterial fibers that are water repellent and do not elute antibacterial agents. c) The type of neutralizer for washing out the test bacteria is not specified. And the like. Four). In the bacterial growth inhibition method of 4), antibacterial properties cannot be evaluated with antibacterial fibers that are water-repellent and do not elute antibacterial agents. This is because even if the bacterial growth is suppressed by bacteriostatics, if broth medium rich in nutrients is used in the inoculum, the antibacterial fiber that does not elute the antibacterial agent is 18 times more inoculated after the inoculum. It has the drawback of increasing in number. Five). With the Shake Frask method in 5), a) it is not possible to test a large number of samples at once, and the laboratory time is long. b) Since the culture solution is a 800-fold diluted phosphate buffer, it lacks the nutrients necessary for the growth of microorganisms. c) Culture time is 1
Due to the short time, the test bacteria can hardly grow. d) Since the test fiber is shaken in a large amount of liquid, it is different from the actual wearing condition. And the like. 6). In all of the evaluation methods 1) to 5) above, the evaluation bacteria are limited to wet bacteria, and the evaluation method for dry bacteria has not yet been established.

[0008]

SUMMARY OF THE INVENTION The inventors of the present invention
In view of the above problems, the present invention has been achieved by earnestly studying with the goal of solving the following problems a) to e). a) The antibacterial activity of fibers that are water-repellent and do not elute antibacterial agents can be accurately evaluated. b) Fibers whose surface is post-processed can also be evaluated easily. c) A large number of samples can be processed at one time. d) The inspection method is simple. e) Establish an evaluation method for antibacterial textile products using dry bacteria.

[0009]

[Means for Solving the Problems] That is, according to the present invention, a bacterial solution is sprayed onto an antibacterial fiber, and after drying the bacterial solution on the antibacterial fiber,
A method for evaluating the antibacterial property of an antibacterial fiber product, which comprises culturing the antibacterial fiber provided with the bacterium on an agar medium and measuring the number of colonies of the bacterium formed on the agar medium. The evaluation method is excellent in that the antibacterial property can be easily evaluated even with fibers in which the antibacterial agent is not eluted.

To explain the evaluation method of the present invention in more detail, the following operating procedure can be illustrated. That is, 1). A sterilizing agent is sprayed inside the sealed box used for spraying the bacterial solution to sterilize the inside of the box. 2). After a certain period of time, place a plurality of petri dishes, each of which has the same size as the inner diameter of the petri dish and contains one pre-sterilized fiber product to be evaluated, (with the lid open) inside the box of 1). 3) Spray the bacterial solution inside the box of 2). 4) Aseptically cover the Petri dish inside the box in 3). 5) Cultivate the petri dish of 4) in an incubator at a certain temperature for a certain period of time to dry the bacteria on the evaluated fiber product. 6) Place the test fiber product of 5) on the agar medium (face with the bacteria on the agar side). 3). Place a pre-sterilized weight for adhering the textile on the agar surface onto the textile to be tested in 6). 4). Cover the agar medium and incubate in an incubator at a constant temperature for a fixed time. 5). Count the number of bacterial colonies formed on the agar medium.

The bacterium used in the present invention is not particularly limited, but a bacterium that forms colonies of Gram-positive bacteria, Gram-negative bacteria, fungi and the like is preferable. The agar medium may be AATCC agar medium, normal agar medium, Sabouraud agar medium, etc., but is not necessarily limited thereto. Further, it is preferable to adjust the number of bacteria to be sprayed so as to be in the range of 200 to 1000 cells / plate. In the case of cloth, the size of the fiber product is preferably equal to the inner diameter of the petri dish used. If the fiber product is in the form of threads, stick it on the entire surface of a cloth or paper of the same size as the inner diameter of the petri dish, or stick it with a thread of the same quality, and then bundle it with the same size as the petri dish. It is preferable to use. The weight used to attach the fiber product to the agar surface is preferably a glass plate with the same size as the inner diameter of the petri dish used and a thickness of about 2 mm, but the fiber product is attached to the agar surface. The weight used for the purpose is not limited to this. Subsequent culture in an incubator is preferably carried out at 37 ° C. for about 18 to 24 hours for bacteria and at 27 ° C. for about 72 hours for fungi, but is not necessarily limited thereto. The number of bacterial colonies formed on the agar medium may be measured by a manual method using a handy colony counter, but a method using an image analyzer is preferable. A kit configured to carry out the present invention is required to include an agar medium, sterilized standard fibers (antibacterial processed fiber, unprocessed fiber), and a weight for bringing the agar surface into close contact with the fiber to be evaluated. However, other items necessary for measurement may be included.

[0012]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Example 1 An example of evaluation of the antibacterial effect of methicillin-resistant Staphylococcus by a fiber product treated with antibacterial and water repellent is shown below. 1). 20 ml of AATCC broth agar was dispensed into a petri dish with a diameter of 9 cm and coagulated. 2). Test bacterium after 24 hours culture (S. aureus methicillin resistance)
The solution is diluted with buffered saline to adjust the number of bacteria to a constant value (1.00
× 10 ⁵ cells / ml). 3) 100 ml of the bacterial solution prepared in 2) was transferred to a previously sterilized sprayer. 4). Box for spraying bacterial solution (586 mm x 550 mm x 1,100
(mm) was sprayed with an antiseptic solution to sterilize the inside. 5). 24 hours later, put in a petri dish inside the box of 4),
Pre-sterilized antibacterial fiber product (antibacterial fiber product) 1
Two pieces were placed, the lid of the dish was opened, and then the lid of the box was closed. 6) The bacterial solution prepared in 3) was sprayed on the antibacterial fiber product to be evaluated 4) through the small hole formed on the top of the box. 7). After 2 hours, the dish that had been subjected to the operation of 6) was capped, taken out, and cultured in an incubator at 37 ° C. for 2 hours. 8). The antibacterial textile product dried in 7) was gently placed using sterile tweezers with the side with the bacteria facing toward the AATCC broth agar side. 9). On the agar medium subjected to the operation of 8), a circular sterilized glass plate having the same size as the inner diameter of the dish was gently placed with sterile tweezers. 10). The agar medium which had been subjected to the operation of 9) was capped and cultured in an incubator at 37 ° C for 18 hours. 11). The number of bacterial colonies formed on the agar medium was counted. 12). The operations 1) to 11) were performed using a fiber product that was not subjected to antibacterial treatment, and the number of colonies of the bacteria formed on the agar medium was counted.

The results are shown in Tables 1 and 2. Table 1
As shown in Table 1, the number of colonies of the unprocessed fiber was smaller than the number of colonies of the control, but the result was that the number of bacteria on the antibacterial processed fiber product shown in Table 1 was significantly higher. That is, in the process of drying the sprayed bacterial liquid, dead bacteria were also recognized, but a significant difference was found in the amount of dead bacteria between the antibacterial processed fiber product and the unprocessed fiber product, so this difference It is obvious that this is due to the difference between the presence and absence of processing, and it was proved that the method for evaluating the antibacterial property of the antibacterial fiber according to the present invention was accurate. The number of colonies was measured using an image analyzer (Image Analyzer V2 manufactured by Toyobo Co., Ltd.).
) Was used for the automatic binarization method.

Example 2 An example of evaluation of the antibacterial effect against Escherichia coli by the antibacterial processed fiber product is shown below. The same procedure as in Example 1 was carried out except that the bacterium used was changed to Escherichia coli. The results are shown in Tables 3 and 4.
As shown in Table 3, the number of colonies of the unprocessed fiber was smaller than the number of colonies of the control, but the number of colonies of the unprocessed fiber was significantly higher than the number of bacteria on the antibacterial processed fiber product. The number of colonies of unprocessed fibers was generally lower than when methicillin-resistant staphylococci was selected as the evaluation bacterium, but other trends were the same as when methicillin-resistant staphylococci was selected as the evaluation bacterium. That is, it was proved that the method for evaluating the antibacterial property of the antibacterial fiber according to the present invention was accurate.
In addition, the number of colonies was measured by an automatic binarization method using an image analyzer (manufactured by Toyobo Co., Ltd., Image Analyzer V2).

(Example 3) Evaluation of antibacterial effect against fungus by antibacterial processed fiber product The fungus (Candida albicans) was used as a substitute, the medium was changed to Sabouraud agar medium, the number of sprayed bacteria was changed, and the culture time was 72 hours. Was carried out in the same manner as in Example 1 except that the culture temperature was changed to 27 ° C. The results are shown in Tables 5 and 6. As shown in Table 5, the number of colonies of the unprocessed fiber was almost equal to the number of colonies of the control, and although it was smaller than the number of colonies of the control, it was higher than the number of bacteria on the antibacterial processed fiber product. It was That is, although some were killed in the process of drying the sprayed bacterial solution, a difference was found in the amount of killed bacteria between the antibacterial processed fiber product and the unprocessed fiber product. It is obvious that this is due to the difference between the presence and absence of, and it was proved that the method for evaluating the antibacterial property of the antibacterial fiber according to the present invention is accurate. In addition, the number of colonies was measured by an automatic binarization method using an image analyzer (manufactured by Toyobo Co., Ltd., Image Analyzer V2).

(Example 4) In order to investigate the degree of variation when an antibacterial test was conducted by the method according to the present invention, a retest was conducted in the same manner as in Example 1. The results are shown in Tables 7 and 8. The results shown in Tables 7 and 8 showed the same tendency as the results shown in Tables 1 and 2. Therefore, it was proved that the method for evaluating the antibacterial property of the antibacterial fiber according to the present invention was accurate. In addition, the number of colonies was measured by an automatic binarization method using an image analyzer (manufactured by Toyobo Co., Ltd., Image Analyzer V2).

(Example 5) In order to investigate the degree of variation when an antibacterial test was conducted by the method according to the present invention, a retest was conducted in the same manner as in Example 2. The results are shown in Table 9 and Table 10. The results shown in Tables 9 and 10 showed the same tendency as the results shown in Tables 3 and 4. Therefore, it was proved that the method for evaluating the antibacterial property of the antibacterial fiber according to the present invention was accurate. In addition, the number of colonies was measured by an automatic binarization method using an image analyzer (manufactured by Toyobo Co., Ltd., Image Analyzer V2).

(Comparative Example 1) Investigation of accuracy of Shake Frask method 1) 1.5 × 10 ⁵ cells / ml of a test bacterial solution (K. Pneumoniae) pre-cultured by shaking for 18 hours at 37 ° C. using Nutrient broth
5 ml was inoculated into 70 ml of culture solution diluted 800 times with sterilized 0.5 M phosphate buffer of pH 7.2. 2) Next, add 1.5 ± 0.1 g of a sample autoclaved by autoclave (15 minutes at 121 ℃) to this solution (the size of the sample is not specified, but it was made fine) and tested. The cells were shaken at 37 ° C. for 1 hour in order to bring them into contact with each other forcibly. 3) The viable cell count was measured by making a serial dilution using this culture medium and performing plate dilution on Trypton Glucose Extract agar medium. 4) The viable cell count before shaking (before adding the sample) and after 1 hour of shaking culture was measured by the plate dilution method, and the bacterial reduction rate relative to the viable cell count before shaking was calculated by the following formula. . Bacteria reduction rate (%) = 100 x (BA) / BA is the number of bacteria per ml in the Erlenmeyer flask after shaking B is the number of bacteria per 1 ml in the Erlenmeyer flask before shaking Effect If the average value of the bacterial reduction rate of two samples (blank (cultured at the same time without adding the sample) with shaking) is X and the average value of bacterial reduction rate of the two samples is Y, then X is determined as follows. ,
It was determined that the growth of bacteria was suppressed. However, -10 <
This test was considered valid when X <10. In addition, the number of colonies was measured by an automatic binarization method using an image analyzer (manufactured by Toyobo Co., Ltd., Image Analyzer V2).

The results are shown in Table 11. As shown in Table 11, the sterilization rate of the water repellent gown manufactured by Company H (without antibacterial treatment) was 99.8%, and the Shake Frask method is not always accurate (the effect of water repellent chemicals is affected). Received). In addition, S. aureus as an evaluation bacterium,
When the water repellent gown manufactured by Company H was evaluated for antibacterial properties by the method according to the present invention, the results shown in Table 12 were obtained.
The accuracy of the method according to the invention has been demonstrated.

[0020]

[Table 1]

[0021]

[Table 2]

[0022]

[Table 3]

[0023]

[Table 4]

[0024]

[Table 5]

[0025]

[Table 6]

[0026]

[Table 7]

[0027]

[Table 8]

[0028]

[Table 9]

[0029]

[Table 10]

[0030]

[Table 11]

[0031]

[Table 12]

(Embodiment 6) An example of a kit configured to carry out the present invention is shown below. a) Agar medium (AATCC broth agar medium, Sabouraud agar medium) b) Sterilized standard fiber (antibacterial processed fiber, unprocessed fiber) c) Glass plate for adhering the fiber to be evaluated to the agar surface (sterilized product) d) Conrage Stick e) Tweezers f) Fungus (S. aureus, Escherchia coli) cultivated on the agar slope
, Candida albicans)

[0033]

INDUSTRIAL APPLICABILITY According to the present invention, a) the antibacterial activity of a fiber which is water-repellent and in which an antibacterial agent is not eluted can be accurately evaluated. b) The antibacterial activity of the fibers post-treated on the surface can be easily evaluated. c) A large number of samples can be processed at one time. d) The inspection method is simple. There is an advantage.

Claims (3)

[Claims] 1. A bacterial solution is sprayed onto an antibacterial fiber, the bacterial solution on the antibacterial fiber is dried, and then the antibacterial fiber to which the fungus has been added is placed on an agar medium and cultivated. A method for evaluating the antibacterial property of an antibacterial textile product, wherein the number of formed colonies of the bacterium is measured. 2. The method for evaluating the antibacterial property of an antibacterial fiber product according to claim 1, wherein an image analysis device is used to measure the number of colonies. 3. An evaluation kit comprising an agar medium, a sterilized standard fiber, and a weight, which is configured to evaluate the antibacterial property of the antibacterial fiber product according to claim 1.