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GB/T 15981-2021 English PDF (GB 15981-1995)

GB/T 15981-2021_English: PDF (GB/T15981-2021)
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GB/T 15981-2021English230 Add to Cart 0--9 seconds. Auto-delivery Evaluating method for the efficacy of sterilization for disinfection equipment Valid GB/T 15981-2021
GB 15981-1995English155 Add to Cart 0--9 seconds. Auto-delivery Evaluating method and standard for the efficacy of disinfection and sterilization Obsolete GB 15981-1995


BASIC DATA
Standard ID GB/T 15981-2021 (GB/T15981-2021)
Description (Translated English) Evaluating method for the efficacy of sterilization for disinfection equipment
Sector / Industry National Standard (Recommended)
Classification of Chinese Standard C59
Classification of International Standard 11.080
Word Count Estimation 11,119
Date of Issue 2021-12-31
Date of Implementation 2022-07-01
Older Standard (superseded by this standard) GB 15981-1995
Drafting Organization Environmental and Health-Related Product Safety Institute, Chinese Center for Disease Control and Prevention
Administrative Organization National Health Commission
Regulation (derived from) National Standard Announcement No. 17 of 2021
Proposing organization National Health Commission of the People's Republic of China
Issuing agency(ies) State Administration for Market Regulation, National Standardization Administration

BASIC DATA
Standard ID GB 15981-1995 (GB15981-1995)
Description (Translated English) Evaluating method and standard for the efficacy of disinfection and sterilization
Sector / Industry National Standard
Classification of Chinese Standard C50
Classification of International Standard 11.080
Word Count Estimation 14,163
Date of Issue 1996-01-23
Date of Implementation 1996-07-01
Drafting Organization Chinese Preventive Medical Sciences, Institute of Epidemiology and Microbiology
Administrative Organization Ministry of Health infectious disease prevention and control management office
Proposing organization Ministry of Health of the People Republic of China
Issuing agency(ies) State Bureau of Technical Supervision, Ministry of Health of the People Republic of China
Summary This Chinese standard specifies the technical standards Autoclave Sterilization testing and evaluation methods, This method is suitable for steam sterilization equipment sterilization effect evaluation.


GB/T 15981-2021 NATIONAL STANDARD OF THE PEOPLE’S REPUBLIC OF CHINA ICS 11.080 CCS C 59 Replacing GB 15981-1995 Evaluating method for the efficacy of sterilization for disinfection equipment ISSUED ON. DECEMBER 31, 2021 IMPLEMENTED ON. JULY 01, 2022 Issued by. State Administration for Market Regulation; Standardization Administration of the People's Republic of China. Table of Contents Foreword... 3  1 Scope... 5  2 Normative references... 5  3 Terms and definitions... 5  4 Identification test for efficacy of sterilization for disinfection equipment... 6  Annex A (informative) Reagents and medium recipes... 18  Evaluating method for the efficacy of sterilization for disinfection equipment 1 Scope This document specifies the test equipment, test procedures, evaluation regulations and precautions for the identification test of the efficacy of sterilization for pressure steam sterilizer, dry heat sterilizer (cabinet), ethylene oxide sterilizer, low temperature steam formaldehyde sterilizer, hydrogen peroxide gas plasma sterilizer. This document is applicable to the evaluation of the efficacy of sterilization for pressure steam sterilizers, dry heat sterilizers (cabinet), ethylene oxide sterilizers, low temperature steam formaldehyde sterilizers, and hydrogen peroxide gas plasma sterilizers. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. GBZ 2.1, Occupational exposure limits for hazardous agents in the workplace - Part 1.Chemical hazardous agents WS/T 649, Hygienic requirement for medical purpose-low temperature steam and formaldehyde sterilizers WS/T 683, Microbiological requirements for disinfection test 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 D value Under the set exposure conditions, the time required to kill 90% of the total number of specific test microorganisms. 3.2 carrier Support for test microorganisms. 3.3 fully loaded When using the disinfection equipment, the maximum allowable load placed in the manner specified in the manufacturer's instructions. 3.4 process challenge device; PCD A specially designed device to simulate the sterilization of articles, resistant to a specific sterilization process, and used to evaluate the effectiveness of the sterilization process. 4 Identification test for efficacy of sterilization for disinfection equipment 4.1 Identification test for efficacy of sterilization for pressure steam sterilizer 4.1.1 Test equipment 4.1.1.1 Test bacterial tablet. Bacillus stearothermophilus spore (ATCC 7953 or SSI K31 strain) bacterial tablet (cloth or filter paper). The amount of recovered bacteria is ≥1×105 CFU/tablet. Under the condition of 121℃±0.5℃, D value is ≥1.5min. Self- contained biological indicators that meet the above requirements may also be used. 4.1.1.2 Standard biological test bag. 23cm×23cm×15cm ordinary cotton bag (30±6 warp threads per 10mm, 27±5 weft threads per 10mm). The mass is 1.5kg±0.045kg. 4.1.1.3 Ventilation storage box. The volume is 22cm×13cm×6cm. 4.1.1.4 Fully loaded items. The items specified in the instruction manual. 4.1.1.5 Reagents. Phosphate buffered saline (PBS, see A.1 of Annex A); tryptone soy agar medium (TSA, see A.3); bromocresol purple peptone medium (see A.4). 4.1.2 Test steps The test steps are as follows. a) Put the two bacterial tablets into sterilized small paper bags (can also use biological indicators directly). b) Place the paper bag containing the tablet or two self-contained biological indicators of Bacillus stearothermophilus spores (ATCC 7953 or SSI K31 strain) in the center of the standard test pack. Make standard biological test packs (disposable standard biological test packs that meet the requirements of 4.1.1.1 can also be used). c) For lower exhaust pressure steam sterilizers, place biological test packs in the 4.2.1 Test equipment 4.2.1.1 Test bacterial tablet. The spore suspension and spore tablets of Bacillus subtilis var. black for laboratory use are prepared according to the method of WS/T 683.The amount of bacteria recovered from the tablet is ≥1×106 CFU/ tablet. Under the condition of temperature of 160℃±2℃, D value is ≥2.5min. Self-contained biological indicators that meet the above requirements may also be used. The bacterial carrier is a stainless- steel sheet with a diameter of 12mm, or a glass slide with an area of 10mm×10mm. If necessary, add or switch to other carriers. 4.2.1.2 Reagents. Phosphate buffered saline (PBS, see A.1); tryptone soy broth (TSB, see A.2); tryptone soy agar (TSA, see A.3). 4.2.1.3 Fully loaded items. The items specified in the instruction manual. 4.2.2 Test steps The test steps are as follows. a) Take 2 bacterial tablets for each test as a group. Lay flat in sterile petri dishes. Do not overlap. Cover them. Put them in each layer of the sterilizer (cabinet). The inner, middle and outer 3 points of the diagonal line are placed diagonally across adjacent layers. Put items in the cabinet until it is fully loaded. b) Close the appliance (cabinet) door. Power it on. Run 1 cycle according to the design program time of the sterilizer (cabinet) for sterilization. After sterilization is completed, the cabinet door can be opened only when the temperature of the sterilizer naturally drops below 50°C. Remove the dish. Take out the bacterial tablets and inoculate into a test tube containing 5.0mL of TSB. Incubate for 7d in a constant temperature incubator at 36°C ± 1°C. Conduct daily observations from the third day. Those with wrinkled biofilm on the turbid surface indicate bacterial growth and are determined as positive. Those with clarification indicate sterile growth and are determined as negative. For the broth tube that is difficult to determine, take 0.1mL~0.2mL of the suspension and inoculate the TSA plate. Apply evenly with a sterilized L stick. Incubate in a constant temperature incubator at 36°C ± 1°C. Conduct smear staining after 48h. Observe the colony morphology under the microscope. Or further refer to the relevant standards to do other tests to determine whether the grower is the test bacteria. If there is contamination by non- test bacteria, the cause shall be found, and the test shall be repeated. c) For bacterial count control group, take 2 pieces of the same batch of test bacterial tablets at room temperature. After the test group is sterilized and inoculated, immediately transfer into test tubes containing 5.0mL of PBS, respectively. Vibrate and beat 80 times. According to the method shown in WS/T 683, count the viable bacterial culture. d) For positive control group, put 2 pieces of bacteria tablets in the same batch at room temperature. After the test group is sterilized and inoculated, immediately transfer into test tubes containing 5.0mL of TSB. Put in incubator for 7d. From the 3rd day onwards, observe the results simultaneously with the test group. e) For negative control group, inoculate 2 pieces of uncontaminated carrier into 5.0mL of TSB, respectively. Put in incubator for qualitative culture. Observe for bacterial growth. f) Repeat the test for 5 times. 4.2.3 Evaluation regulations 4.2.3.1 To determine that the efficacy of sterilization of the sterilizer is qualified, it must meet the requirements at the same time. In the 5 sterilization tests, the amount of recovered bacteria in the control group is ≥1×106 CFU/tablet in each test. There is bacterial growth in the positive control group. There is aseptic growth in the negative control group. All test bacterial tablets are aseptically grown. 4.2.3.2 When self-contained biological indicators are used for evaluation, the results are determined according to the instruction manual. 4.2.4 Precautions 4.2.4.1 Strictly abide by aseptic technique regulations. 4.2.4.2 The test shall be carried out under full load conditions. 4.3 Identification test for efficacy of sterilization for ethylene oxide sterilizer 4.3.1 Test equipment 4.3.1.1 Test bacteria tablets. Bacillus subtilis var. Niger (ATCC 9372) spores or biological indicators thereof. When the ethylene oxide concentration is 600mg/L±30mg/L, the temperature is 54℃±1℃ and the relative humidity is 60%±10%, the D value is ≥2.5min (use ethylene oxide mixed gas) or D value is ≥ 2.0min (use 100% ethylene oxide pure gas). The bacterial carrier is filter paper. The area is 10mm×10mm. Add or use other carriers if necessary. The amount of recovered bacteria shall be greater than or equal to 1×106 CFU/tablet. Self-contained biological indicators that meet the above requirements may also be used. 4.3.1.2 Polyethylene plastic bag. The size is 60mm×40mm. The material thickness is 0.15mm~0.25mm. 4.3.1.3 Reagents. Phosphate buffered saline (PBS, see A.1); tryptone soy broth (TSB, see A.2); tryptone soy agar (TSA, see A.3). 4.3.1.4 Fully loaded items. The items specified in the instruction manual. g) Place 2 pieces of uncontaminated carrier in the test tubes that contain 5.0mL of TSB, respectively. Put in incubator for qualitative culture. Observe if there is bacterial growth. Use as a negative control group. h) Repeat the test for 5 times. 4.3.3 Evaluation regulations 4.3.3.1 To determine the sterilization effect of the sterilizer is qualified, it must meet the following requirements. The amount of recovered bacteria in the control group shall be greater than or equal to 1×106 CFU/tablet. There is bacterial growth in the positive control group. There is aseptic growth in the negative control group. All the test groups are aseptically grown. 4.3.3.2 For TSB whose results are difficult to judge, take 0.1mL~0.2mL of the suspension to inoculate the TSA plate. Apply evenly with a sterile L stick. Incubate in a constant temperature incubator at 36°C ± 1°C. Conduct smear staining after 48h. Observe colony morphology under microscope. Or further refer to the relevant standards to do other tests to determine whether there is growth or whether the growth is the test bacteria. If it is the test bacteria, it will be judged as unqualified for sterilization. If it is non-test bacteria, the test shall be repeated. 4.3.3.3 When self-contained biological indicators are used for evaluation, the results are determined according to the instruction manual. 4.3.4 Precautions 4.3.4.1 Ethylene oxide is flammable, explosive and toxic. To ensure the safety of the test, the operators and test personnel shall be familiar with the performance of ethylene oxide and the operating procedures of the equipment. Strictly follow the safety rules. When using steel cylinders and gas tanks to store ethylene oxide, the valve shall be opened slowly. Do not let the liquid spray out suddenly. Fire and explosion-proof measures shall be taken at the operation site. No open flame operations and electric sparks are allowed. It is strictly forbidden to enter the site wearing shoes with metal soles, so as to prevent safety accidents caused by friction and sparks. 4.3.4.2 The working environment shall be well ventilated. The maximum allowable concentration of ethylene oxide in the air at the work site is 2mg/m3.If a person inhales too much ethylene oxide gas, it will cause poisoning symptoms. Severe cases can cause pulmonary edema. If symptoms of poisoning appear, leave the scene immediately and rest in a well-ventilated place. Mild cases breathe fresh air until symptoms clear up. Severe cases shall be sent to hospital for treatment in time. 4.4 Identification test for efficacy of sterilization for low temperature steam formaldehyde sterilizer 4.4.1 Test equipment 4.4.1.1 Test bacterial tablets. Stearothermophilus (ATCC 7953 or SSI K31) spores; microorganisms whose resistance meets the requirements of WS/T 649.The bacterial content is 1×106 CFU/tablet~5×106 CFU/tablet. Self-contained biological indicators that meet the above requirements may also be used. Bacteria carriers are. 1) metal sheet. stainless steel disc with a diameter of 12mm~15mm; 2) glass sheet. 10mm×10mm glass sheet; 3) plastic sheet. 10mm×10mm PTFE plastic sheet. 4.4.1.2 Sterilization process challenge device (PCD). It is composed of a 1.5m-long, 2mm inner diameter PTFE blind end tube and a receiving cavity for the bacteria tablets at the blind end. Put the bacterial carrier into the PCD. The PCS is double-layer packed. For the carriers that cannot be placed in the PCD, they are packaged with sterilized packaging materials and placed in the small load unit and the full load unit according to the method of WS/T 649. 4.4.1.3 Reagents. Phosphate buffered saline (PBS, see A.1); tryptone soy agar medium (TSA, see A.3); bromocresol purple peptone medium (see A.4). 4.4.1.4 Fully loaded items. The items specified in the instruction manual. 4.4.1.5 Neutralizer (There is no device that removes residual formaldehyde gas after sterilization. Use a certified neutralizer during the test). 4.4.2 Test steps The test steps are as follows. a) Bacillus stearothermophilus spore suspension and bacterial tablets are prepared according to the method in WS/T 683.Place the double-layer-packed PCD evenly in the sterilization chamber according to the requirements specified in the instruction manual. Record the distribution. b) Under the condition of small load, for the sterilizer with the volume of the sterilization chamber is less than 60L, at least 7 microbial carriers shall be placed. For the 60L~100L sterilizer, at least 11 microbial carriers are placed. For sterilizers larger than 100L, on this basis, add 1 microbial carrier for every 100L increase in volume. The selection of placement point shall first consider the position that it is the most difficult to sterilize. The rest are distributed evenly in the sterilizer chamber. c) Sterilize according to the formaldehyde concentration specified in the instruction manual, 0.5 times the effective action time (half of the sterilization treatment time in the sterilization cycle), and the temperature inside the device. d) After the sterilization procedure, take out the contamination carrier under aseptic conditions. Put the Bacillus stearothermophilus spore carrier into the bromocresol purple peptone culture solution containing the neutralizer (the self-contained biological indicator is operated according to the instruction manual). Culture in a constant temperature incubator at 56℃±2℃ for 7d as the test group. e) Put 2 spore carriers of Bacillus stearothermophilus for the same batch of test into test tubes containing 5.0mL of diluent. Vibrate and beat 200 times for each. According to the method shown in WS/T 683, count the viable bacteria culture. Calculate the amount of bacteria recovered from the test bacterial tablet. f) Put two spore carriers of Bacillus stearothermophilus for the same batch of test into the bromocresol purple peptone culture solution. Culture in a constant temperature incubator at 56℃±2℃ for 7d as a positive control group. g) Put 2 uncontaminated bacterial carriers in the same batch into the bromocresol purple peptone culture solution. Cultured in a constant temperature incubator at 56°C ± 2°C for 7d as a negative control group. h) Repeat the test 5 times. 4.4.3 Evaluation regulations 4.4.3.1 To determine that the sterilization effect of the sterilizer is qualified, it must meet the requirements at the same time. The amount of testing recovered bacteria of the bacterial count control group in each test is 1×106 CFU/tablet~5×106 CFU/tablet. There is bacterial growth in the positive control group. There is aseptic growth in the negative control group. There is aseptic growth in the test group. 4.4.3.2 When self-contained biological indicators are used for evaluation, the results are determined according to the instruction manual. 4.4.4 Precautions 4.4.4.1 Formaldehyde is toxic. The working environment shall be well ventilated. The maximum allowable concentration in the air of the workplace shall meet the requirements of GBZ 2.1. 4.4.4.2 To eliminate residual formaldehyde, natural ventilation can be used. Or use 25% ammonia water to heat and evaporate or spray for neutralization. 4.4.4.3 If the low temperature steam formaldehyde sterilizer has multiple sterilization procedures, each sterilization procedure shall be verified separately. 4.5 Identification test for efficacy of sterilization for hydrogen peroxide gas plasma sterilizer 4.5.1 Test equipment 4.5.1.1 Test contamination carrier. Bacillus stearothermophilus (ATCC 7953 or SSI K31) spores, with qualified resistance identification. The contamination carrier is a stainless-steel needle with a diameter of ≤0.4mm. The limit is that the lumen is not ......


GB 15981-1995 GB NATIONAL STANDARD OF THE PEOPLE’S REPUBLIC OF CHINA ICS 11.080 C 59 Evaluating method and standard for the efficacy of disinfection and sterilization ISSUED ON: JANUARY 23, 1996 IMPLEMENTED ON: JULY 01, 1996 Issued by: State Bureau of Technical Supervision; Ministry of Health of PRC. Table of Contents 1 Scope ... 3  2 Reagent ... 3  3 Indicator bacteria ... 3  4 Chemical indicator ... 4  5 Technical requirements ... 4  6 Testing method ... 4  7 Results judgment and evaluation ... 4  8 Scope ... 5  9 Indicator bacteria ... 5  10 Physical indicators ... 5  11 Testing method ... 6  12 Judgment criteria ... 6  13 Scope ... 7  14 Physical and chemical index ... 7  15 indicator microorganisms ... 7  16 Testing method ... 7  17 Evaluation criteria of disinfection effect ... 8  Appendix A Neutralization effect test of neutralizer (Supplement) ... 9  Appendix B Qualitative disinfection test of disinfectant (Supplement) ... 13  Appendix C Quantitative disinfection test of disinfectant (Supplement) ... 16  Appendix D Organic matter protection test (Supplement) ... 19  Appendix E Hepatitis B surface antigen destruction test (Supplement) ... 21  Additional information: ... 26  Evaluating method and standard for the efficacy of disinfection and sterilization Article 1 -- Evaluation method and standard for the efficacy of pressure steam sterilization 1 Scope This method specifies the technical standards for pressure steam sterilization and the testing methods for evaluating the sterilization effect. This method is suitable for evaluating the sterilization effect of pressure steam sterilization equipment. 2 Reagent The reagents used in this standard are analytically pure (AR), unless otherwise specified; the water is distilled water. 2.1 Peptone. 2.2 Glucose. 2.3 Bromocresol purple alcohol solution: Take 2.0 g of bromocresol purple. Dissolve it in 100 mL of 95% ethanol. 2.4 Preparation of bromocresol purple peptone water culture medium: Dissolve 10.0 g of peptone and 5.0 g of glucose in 1000 mL distilled water. Adjust the pH to 7.0 ~ 7.2. Then add 0.6 mL of 2% bromocresol purple alcohol solution. Shake it uniformly. According to 5 mL/tube, pack and seal it. Put in a pressure steam sterilizer, to sterilize it at 115 °C for 40 min, to prepare for use. 3 Indicator bacteria Bacillus stearothermophilus spores (ATCC 7953 or SSI K31) bacterial tablets, containing 5 x 105 ~ 5 x 106 cfu/tablet; the time D121 required to kill 90% of microorganisms at 121 °C is 1.3 ~ 1.9 min; the killing time (KT value) is ≤ 19 min; the survival time (ST value) is ≥ 3.9 min. bromocresol purple peptone water culture medium inoculated with each indicator bacteria tablet does not change color, it is judged as qualified for sterilization. When the bromocresol purple peptone water medium inoculated with one of the indicator bacteria tablet turns from purple to yellow, it is judged as unqualified for sterilization. 7.2 When the color of the chemical indicator becomes the same as the standard color for sterilization, or when it is melted, it is used as the reference standard for sterilization. Article 2 -- Evaluation methods and standards of efficacy of ultraviolet surface disinfection 8 Scope This method specifies the wavelength and intensity of ultraviolet rays used for surface disinfection, as well as physical indicators and biological testing methods for evaluating the disinfection effect. This method is suitable for the evaluation of the disinfection effect on the surface of objects directly irradiated by ultraviolet rays. 9 Indicator bacteria 9.1 Escherichia coli (8099 or ATCC 25922). 9.2 Bacillus subtilis black variant spores (ATCC 9372). 10 Physical indicators 10.1 When the voltage is 220 V, the ordinary 30 W straight tube ultraviolet lamp has, at the use conditions of room temperature of 20 ~ 25 °C, an ultraviolet radiation intensity of 253.7 nm (1 m vertical) shall be ≥ 70 µW/cm2. 10.2 When the voltage is 220 V, the high-intensity ultraviolet lamp has, at the use conditions of the room temperature of 20 ~ 25 °C, an ultraviolet radiation intensity of 253.7 nm (1 m vertical) shall be ≥ 200 µW/cm2. 10.3 The radiation dose is calculated according to formula (1): Dose (μW • s/cm2) = Intensity (μW/cm2) x time (s)………………………… (1) 12.2 When it reaches the physical testing standard, it is used as the reference standard for disinfection. Article 3 -- Evaluation methods and criteria of disinfection effect of liquid disinfectants 13 Scope This method specifies the biological testing methods and evaluation criteria for the disinfection effect of disinfectants. This method is suitable for evaluating the disinfection effect of disinfectants on various objects. 14 Physical and chemical index Put the disinfectant in a water bath at 20 ± 2 °C. Determine the shortest time (min) required to kill the indicator microorganisms to achieve disinfection or sterilization at the use concentration. 15 indicator microorganisms 15.1 Bacteria 15.1.1 Bacterial propagule: Staphylococcus aureus (ATCC 6538), Escherichia coli (8099 or ATCC 25922). 15.1.2 Bacterial spores: Bacillus subtilis black variant spores (ATCC 9732). 15.2 Fungus: Candida albicans (ATCC 10231). 15.3 Hepatitis B surface antigen: Purified antigen (1.0 mg/mL). 16 Testing method 16.1 Neutralization test (see Appendix A). 16.2 Qualitative disinfection test of disinfectant (see Appendix B). 16.3 Quantitative disinfection test of disinfectant (see Appendix C). 16.4 Sterilization energy test of disinfectant (see Appendix D). Appendix A Neutralization effect test of neutralizer (Supplement) A1 Summary of contents In order to accurately evaluate the killing effect of disinfectants on microorganisms, it is required to select an appropriate neutralizer in the disinfection test. The selected neutralizer can not only stop the disinfectant's microbicidal killing effect in time, but also the neutralizer itself and its reaction product with the disinfectant (hereinafter referred to as neutralized product) still need no inhibition or killing effect on microorganisms, meanwhile there is no adverse effect on the culture medium. A2 Medium and reagents A2.1 Nutrient agar medium Ingredient: Peptone 10.00 g Beef extract 3.00 g Sodium chloride 5.00 g Agar 15.00 g Distilled water 1000.00 mL Preparation method: In addition to agar, the other ingredients are dissolved in distilled water. Adjust the pH to 7.2 ~ 7.4. Add the agar and heat to dissolve it. Filter it pack contain it. After being subject to pressurized steam action at 121 °C for 30 minutes, sterilize it to prepare for use. A2.2 0.03 mol/L phosphate buffer (pH 7.2 ~ 7.6, hereinafter referred to as PBS). Ingredients: Disodium hydrogen phosphate: 2.84 g Potassium dihydrogen phosphate: 1.36 g Distilled water: 1000.00 mL Preparation method: Dissolve disodium hydrogen phosphate and potassium dihydrogen phosphate in distilled water at a pH of 7.2 ~ 7.4; pack it separately; sterilize it in pressure steam at 121 °C for 30 minutes to prepare for use. Appendix B Qualitative disinfection test of disinfectant (Supplement) B1 Summary of contents The qualitative disinfection test is a test method to determine whether there is bacterial growth in the sample after being subject to the action from the disinfection factor. It is used for the identification of the sterilization effect of the disinfection factor and the preliminary evaluation of the sterilization effect of the disinfectant. B2 Medium and reagents B2.1 Ordinary broth medium B2.1.1 Ingredients: Peptone: 10.00 g Sodium chloride: 5.00 g Meat extract: 1000.00 mL B2.1.2 Preparation method: Add peptone and sodium chloride into the meat extract. Dissolve at low temperature. Adjust the pH to weakly alkaline. Boil it. Filter it clean. Adjust the pH so that it will be 7.2 ~ 7.4 after sterilization. Sterilize it in pressurized steam to prepare for use. B2.2 Reagent B2.2.1 Diluent: 0.03 mol/L PBS (pH 7.2 ~ 7.4) containing 1% peptone. B2.2.2 Sterilized distilled water. B2.2.3 Neutralizer: Select it according to Appendix A of this standard. B3 Equipment B3.1 Sterilized graduated pipette (1.0, 5.0, 10.0 mL). B3.2 Sterilized test tube. B3.3 Sterilized conical flask. B3.4 Alcohol lamp. B3.5 Constant temperature water bath tank. Appendix C Quantitative disinfection test of disinfectant (Supplement) C1 Summary of contents Quantitative disinfection test is a test method to determine the number of microorganisms remaining in a sample after being affected by disinfection factors; the result is expressed in terms of killing rate. It is used to evaluate the killing effect of disinfectants. C2 Medium and reagents C2.1 Ordinary nutrient agar medium: It is prepared according to A2.1 of this standard. C2.2 Reagent C2.2.1 Diluent: 0.03 mol/L PBS (pH 7.2 ~ 7.4) containing 1% peptone. C2.2.2 Sterilized distilled water. C2.2.3 Neutralizer: Choose according to Appendix A of this standard. C2.2.4 0.03 mol/L PBS (pH 7.2 ~ 7.4). C2.2.5 Eluent: PBS containing neutralizer, 1% peptone, 0.1% Tween 80. C3 Equipment C3.1 Sterilized graduated pipette (1.0, 5.0, 10.0 mL). C3.2 Sterilized test tube. C3.3 Sterilized conical flask. C3.4 Sterilized plates (9 cm in diameter). C3.5 Constant temperature water bath tank. C3.6 Constant temperature incubator. C3.7 Alcohol lamp. C3.8 Colony counter. Appendix D Organic matter protection test (Supplement) D1 Summary of contents The organic matter protection test is to determine the disinfectant's killing effect on microorganisms under the condition of organic matter protection, expressed as the killing rate. The result is compared with the disinfectant's quantitative disinfection test, to evaluate the influence of organic matter on the disinfectant's bactericidal ability. D2 Medium and reagents D2.1 Ordinary nutrient agar medium, which is prepared according to A2.1 of this standard. D2.2 Reagents: D2.2.1 Diluent: Same as C2.2.1. D2.2.2 Sterilized distilled water. D2.2.3 Neutralizer: Select it according to Appendix A of this standard. D2.2.4 0.03 mol/L phosphate buffer (pH 7.2 ~ 7.4) (abbreviated as PBS). D2.2.5 Eluent: Physiological saline which contains 1% peptone and 0.1% Tween 80. D2.2.6 Add calf serum to the bacterial suspension, to make its final concentration at 10%. D3 Equipment Same as C3 of this standard. D4 Test method D4.1 Before the test, count the viable bacteria in the bacterial solution. Use diluent to dilute it. Add calf serum, to make the final serum content of 10% and the bacterial count of 5 x 105 ~ 5 x 106 cfu/mL, which is used as a test bacteria suspension. D4.2 The following steps are the same as C4.1.2 ~ C4.1.8 of this standard. Appendix E Hepatitis B surface antigen destruction test (Supplement) E1 Summary of contents The hepatitis B surface antigen (HBsAg) destruction test is a test method to evaluate the ability of disinfection factors to inactivate hepatitis B virus (HBV) using the antigen activity of HBsAg as an indirect marker. It is suitable for evaluating the disinfection effect of chemical disinfectants and ultraviolet rays on HBV. E2 Reagent E2.1 Calf serum (56 °C, 30 min inactivation). E2.2 0.01 mol/L phosphate buffered saline (PBS, pH 7.2 ~ 7.4). E2.3 Purified HBsAg (1.0 mg/mL). E2.4 Reagents for solid-phase radioimmunoassay, requiring specificity 100%, precision CV ≤ 15%, sensitivity ≤ 1.0 ng/mL, linearity r > 0.95. E2.5 Enzyme-linked immunosorbent assay reagents, requiring 100% specificity, precision CV ≤ 15%, sensitivity ≤ 3.2 ng/mL, linearity r > 0.95. E3 Equipment E3.1 Pipette: It is composed of test tube, pipette (4 kinds: 0.1, 1.0, 5.0, 10.0 mL), micro sample injector (100.0 μL). E3.2 Carrier (1.5 cm diameter stainless steel sheet). E3.3 r-immune counter. E3.4 Enzyme-linked immunoassay instrument. E3.5 Low temperature refrigerator (-30 °C ~ -70 °C). E4 Preparation of HBsAg suspension E4.1 Take 9.4 mL of 0.01 mol/L PBS (pH 7.2 ~ 7.4). Add 0.6 mL of calf serum, to prepare a suspension containing 6% calf serum. Then take 9.0 mL of the PBS and add 1.0 mL of purified HBsAg suspension which has a concentration d. 0.9 mL of PBS containing 10% calf serum + 0.1 mL of HBsAg suspension; e. 0.9 mL of neutralizer containing 10% calf serum + 0.1 mL of HBsAg suspension; f. 0.9 mL of neutralized product solution + 0.1mL of PBS. Only in groups c, d, e are the measured values of HBsAg activity similar (with a difference of less than 10%) and are significantly more than those of group b. The HBsAg activity of group a cannot be detected or the detected activity is significantly less than that of group b. The group f's negative control is normal, then it can prove that the selected neutralizer and its dosage are appropriate. E6.3 When conducting disinfection tests, adjust the amount of neutralizer appropriately according to the principle of neutralization of the equivalent of disinfectant and neutralizer. After the neutralization effect is measured according to the procedures of E6.2 again, the antigenic destruction test can be carried out. E7 Destruction test method E7.1 Suspension method The suspension method refers to a test method in which HBsAg interacts with a disinfectant in a suspension and the effect of antigenic destruction is observed. E7.1.1 The concentration of HBsAg suspension is 104 times the sensitivity of the testing reagent. If the sensitivity of the testing reagent is 1 ng/mL, the concentration of HBsAg shall be 10 μg/mL. E7.1.2 Take 2.7 mL of PBS containing 5% calf serum and add it to 0.3 mL of HBsAg suspension at a concentration of 100 μg/mL. Mix well. E7.1.3 Take 20 mL of calf serum and add it to 80 mL of sterilized neutralizer solution. Mix well. E7.1.4 Destruction test: Mix the disinfectant with 1.25 times the concentration of the predetermined disinfectant with the HBsAg suspension in a ratio of 4:1. The volume of the mixed solution is not less than 1.5 mL. Then place it in a water bath at 20 ± 2 °C for a specified time. Immediately take 0.3 mL of the mixed solution and mix it with an equal volume of 20% calf serum neutralizer. Let it action for 10 ~ 30 min. Take samples to determine the activity of residual HBsAg. Make two parallel determinations for each sample, 0.1 mL for each. Take the average value to determine the destruction effect. Observe 3 concentrations for each disinfectant; observe 4 action times for each concentration. The test is repeated 5 times. activity of residual HBsAg. Take 2 sets for each sample, 0.1 mL for each set. Take the average value to determine the antigenic destruction effect. Observe 3 concentrations for each disinfectant. Observe 4 action times for each concentration. The test is repeated 5 times. When observing the effect of ultraviolet rays destroying HBsAg, place the contaminated carrier directly under the action factor. After the action reaches the prescribed dosage, move the carrier into a test tube containing 1.0 mL of 10% calf serum PBS. Knock and shake 200 times. Take samples to test the activity of residual HBsAg. Take 2 sets for each sample, 0.1 mL for each set. Take the average value to determine the destruction effect. The test is repeated 5 times. E7.2.3 Positive control When observing the effect of disinfectant on destroying HBsAg, add 50 μL of test disinfectant to a test tube containing 1.0 mL of 10% calf serum neutralizer. Act for 10 ~ 30 minutes to prepare a neutralized product solution. Take 1.0 mL of the neutralized product solution and add it to a large test tube. Then move in the drip-stained HBsAg carrier. Knock and shake 200 times. Test 3 sets for each sample, 0.1 mL for each set. Take the average value as the positive control value. When observing the effect of ultraviolet rays destroying HBsAg, directly move the carrier contaminated with HBsAg into a test tube containing 1.0 mL of 10% calf serum PBS (pH 7.2 ~ 7.4). Knock and shake 200 times. Test 3 sets for each sample, 0.1 mL for each set. Take the average value as the positive control value. E7.2.4 Negative control When observing the effect of disinfectant destroying HBsAg, add 50 μL of disinfectant to a test tube containing 1 mL of 10% calf serum neutralizer. Let it act for 10 ~ 30 min. Take samples for testing. Test 3 sets for each sample, 0.1 mL for each set. Take the average value as the negative control value. When observing the effect of ultraviolet rays destroying HBsAg, the negative control is PBS containing 10% calf serum (pH 7.2 ~ 7.4). Test 3 sets for each sample, 0.1 mL for each set. Take the average value as the negative control value. It shall be noted that the negative control sample of the kit cannot be used for the negative control. E8 Judgement of destruction effect S/N < 2.1 is taken as the eligibility criterion for HBsAg antigenic destruction. ......

Similar standards: GB 15979-2024  GB 15982-2012  
Similar PDFs (Auto-delivered in 9 seconds): GB/T 15981-2021  GB 15982-2012  GB 15979-2024  GB 26373-2010