HG/T 3663-2000 Test method for antibacterial performance of rubber shoes (agar plate method) HG/T3663-2000 standard download decompression password: www.bzxz.net

Request number: 7262-2000
HG/T3663-2000
This standard is specially formulated to meet the requirements of the determination of the antibacterial performance of rubber shoes. The formulation of this standard refers to the standard AATCC90-1977 (R1981) of the American Association of Textile Chemists and Dyers. When rubber shoes are processed with antibacterial and deodorizing treatment, antibacterial agents can be applied to sponge rubber, insole fabric glue and upper materials. When determining the antibacterial performance of the product, samples can be taken from the parts that have been treated with antibacterial and deodorizing treatment. The test bacteria are Staphylococcus aureus, Escherichia coli, and Bacillus subtilis. These three bacteria are common bacteria with strong resistance to the outside world. They are the main microorganisms that cause foot odor and are also the bacteria used in the disinfection effect detection method of disinfectants. Therefore, the above three bacteria were selected in the test in the standard.
After testing, it was found that the diameter of the sample had no obvious effect on the test results, so the sample size was set to (16.0±0.2) mm. This standard was proposed by the Technical Supervision Department of the former Ministry of Chemical Industry of the People's Republic of China. This standard is under the jurisdiction of the Rubber Shoes Technical Committee of the National Rubber and Rubber Products Standardization Committee. The drafting unit of this standard: Shanghai Rubber Shoes Research Institute. The main drafters of this standard: Lv Zhiqiang, Xu Dejia 9
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Chemical Industry Standards of the People's Republic of China
Testing Method for Antibactarial Activity of Rubber Shoes (Agar Plate Method)
This standard specifies the test method for antibacterial activity of rubber shoes and their components with antibacterial and deodorizing treatment. This standard is applicable to the determination of antibacterial activity of rubber shoes and their components. 2 Referenced Standards
HG/T3663—2000
The provisions contained in the following standards constitute the provisions of this standard through reference in this standard. When this standard is published, the versions shown are valid. All standards will be revised, and parties using this standard should explore the possibility of using the latest versions of the following standards. GB4789.28-1994 Food Hygiene Microbiological Inspection, Staining Methods, Culture Media and Reagents GB15981-1995 Evaluation Methods and Standards for Disinfection and Sterilization Effects 3 Principles
Using the diffusion effect of antibacterial materials (substances) in rubber shoes and their components in agar culture medium, that is, when the concentration is maintained during the diffusion process, the growth of bacteria can be inhibited, thereby producing a transparent inhibition zone. According to the size of the inhibition zone, the level of antibacterial performance of rubber shoes can be measured.
4 Definitions
This standard adopts the following definitions:
4.1 Antibacterial and anti-stinking processing Antibacterial and anti-stinking processing refers to the processing of adding antibacterial materials (substances) to rubber shoes and their components to inhibit the growth of bacteria in the shoe cavity and on the feet during the wearing process.
4.2 Antibacterial clearzone Antibacterial materials (substances) hinder bacterial growth and thus form a transparent area around the sample in the culture dish. 4.3 Antibacterial activity Antibacterial activity The property of inhibiting bacterial growth and reproduction, or killing bacteria. 4.4 Antibacterial width of antibacterial clearzone Antibacterial materials (substances) hinder bacterial growth and thus form a transparent area around the sample in the culture dish 5 Test bacteria
5.1 Staphylococcus aureus (ATCC6538). 5.2 Escherichia coli (8099 or ATCC25922). State Administration of Petroleum and Chemical Industry 2000-05-23 Approved standard Dig by the website a.basoo.co Customer standard Industry information free download 2000-12-01 Implementation
HG/T3663—1999
5.3 Bacillus subtilis var. niger spores (ATCC9372). 6 Instruments and equipment
6.1 Constant temperature incubator.
6.2 Pressure steam sterilizer.
6.3 Refrigerator.
6.4 Inoculation loop (diameter of tip ring is about 4mm).
6.5 Pipette (1.0mL).
6.6 Flat plate (9 cm).
6.7 Glass test tube (g12mm×100mm).
6.8 Pipette.
6.9 Erlenmeyer flask. bzxz.net
6.10 Alcohol lamp.
6.11 Platen.
6.12 Vernier caliper (division: 0.02mm). 6.13 Circular cutter for preparing sample, inner diameter of cutter: (16.0±0.2)mm. 7 Culture medium, nutrient broth and physiological saline
7.1 Nutrient agar culture medium (semi-solid culture medium) 7.1.1 Ingredients
Beef extract
Sodium chloride
Distilled water
7.1.2 Preparation
15g (0.5g if preparing semi-solid culture medium) 1000mL
Except agar, dissolve other ingredients in distilled water, adjust pH to 7.2-7.4 with 4.0mol/L sodium hydroxide solution, add agar, heat to dissolve, divide into flasks, and sterilize by high pressure at a temperature not lower than 120℃ for 30min. 7.2 Plate preparation
Take out about 15mL of the nutrient agar culture medium in 7.1.2 and pour it into sterilized plate. After the nutrient agar solidifies, turn the plate over and incubate it in a (36±1)℃ constant temperature box for (24±2)h. If the sterilized nutrient agar is not used immediately, it should be stored at 4-10°C for no more than 10 days. Before use, open the plate and place it upside down in a (36±1)°C incubator for 15-30 minutes. 7.3 Preparation of slant culture medium
Add about 2 mL of nutrient agar in 7.1.2 into a sterile test tube, then tilt the test tube to allow the nutrient agar to cool and solidify. If the prepared slant culture medium is not used immediately, it should be stored at 4-10°C for no more than 2 weeks. 7.4 Nutrient broth
7.4.1 Ingredients
Protein
Beef extract
Sodium chloride
Distilled water
7.4.2 Preparation
1000mL
Mix the above ingredients and adjust the pH value to 7.2~7.4 (adjust with 4.0mol/L sodium hydroxide solution) after dissolution. Then, inject 2mL of each of 12
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into the test tube, plug with cotton, and sterilize at a temperature not lower than 120℃ for 30min. If the sterilized nutrient broth is not used immediately, it should be stored at 4~10℃, and the storage time should not exceed 2 weeks. 7.5 Physiological saline
Add 8.5g sodium chloride to 1000mL distilled water to make physiological saline. Dispense into bottles, 100 mL per bottle, and sterilize by autoclave at a temperature not lower than 120℃ for 30 minutes. Cool to room temperature for later use. 8 Preservation of bacteria
8.1 Preservation on agar slant
Inoculate the bacteria on a nutrient agar slant, place it in a (36±1)℃ constant temperature incubator for 18-24 hours, and store it in a (4±1)℃ refrigerator. It can generally be stored for about 1 month.
8.2 Preservation by semi-solid puncture
Put the bacteria in a semi-solid culture medium by puncture, and after constant temperature incubation at (36±1)℃ for 18-24 hours, take it out and add an appropriate amount of sterilized liquid paraffin to cover the culture medium, isolate it from the air, and place it in a (4±1)℃ refrigerator for 3-6 months. If long-term storage is required, subculture once every 36 months.
8.3 Isolation and culture
Working bacteria strains should be isolated from single colonies on agar plates every 3 to 6 months. Select typical colonies and inoculate them on ordinary agar slants. They can replace the original working bacteria strains.
9 Samples
9.1 Take samples from the parts where the antibacterial and deodorant agents are applied. 9.1.1 Upper sampling
Upper sampling Take one sample each from the front and back uppers of the shoes. 9.1.2 Insole sampling
Take one sample each from the forefoot and heel of the insole. 9.2 Use a (16 ± 0.2) mm round cutter to cut the sample. 10 Test steps
10.1 Preparation before the test
10.1.1 Test tubes, conical flasks, pipettes, transfer pipettes and culture dishes should be washed and dried in advance. Plug the test tube, conical flask and pipette with cotton plugs, and wrap the pipette and pipette tightly with paper. Sterilize all the above utensils at a temperature not lower than 120℃ for 30 minutes, and then dry them for later use.
10.1.2 Measure the actual diameter of the sample with a vernier caliper and record it. 10.2 Preparation of bacterial suspension
10.2.1 Transfer an inoculation loop from the bacteria stored in 8.1 to the slant culture medium and incubate at (36±1)℃ for 18~24h. 10.2.2 Transfer an inoculation loop from the slant culture bacteria in 10.2.1 to the nutrient broth tube in 7.4.2 and incubate at (36±1)℃ for 18~24h.
10.3 Test
10.3.1 Use a pipette to aspirate 0.1mL of 10.2.2 Staphylococcus aureus, Escherichia coli and 0.2mL of Bacillus subtilis var. niger spores (the biofilm of Bacillus subtilis var. niger spores must be broken and mixed in advance) and dilute each into 100mL of sterile saline.
10.3.2 Use a pipette to suck out the diluted bacterial solution in 10.3.1 and spread it evenly on the sterile culture medium in 7.2, and dry the excess bacterial solution. 10.3.3 Use sterilized tweezers to pick up the sample in 9.2 and gently place it close to the center of the culture medium in 10.3.2. Be careful not to damage the surface of the culture medium.
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10.3.4 Place the culture medium in 10.3.3 in a constant temperature culture of (36±1)℃ for 18 to 24 hours. 11. Test results
11.1 Observe from the bottom of the culture medium and measure the diameter of the sample's inhibition zone with a vernier caliper. 11.2 Calculation of test results
The width of the inhibition zone is calculated according to formula (1):
W-(TD)/2
Wherein: w-
width of the inhibition zone, mm;
Tthe shortest diameter of the inhibition zone, mm;
Dthe diameter of the sample, mm.
11.3 Representation of test results
The number of tests for each sample shall not be less than 2, and the test results shall be expressed as the arithmetic mean (the value shall be retained to 1 decimal place). 12 Test report
The test report shall include the following contents:
a) Sample name;
b) Test number;
e) Bacteria species name;
d) Test results;
e) Test date;
f) Tester and reviewer.
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