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Basic dataStandard ID: WS/T 683-2020 (WS/T683-2020)Description (Translated English): (Microbiological requirements for disinfection test) Sector / Industry: Health Industry Standard (Recommended) Classification of Chinese Standard: C50 Word Count Estimation: 13,149 Date of Issue: 2020-07-20 Date of Implementation: 2021-02-01 Regulation (derived from): State-health communication (2020) No. 14 Issuing agency(ies): National Health Commission WS/T 683-2020: (Microbiological requirements for disinfection test)---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order.(Microbiological requirements for disinfection test) ICS 11.080 C 50 WS People's Republic of China Health Industry Standard Microbiological requirements for disinfection test Issued by the National Health Commission of the People's Republic of China ForewordThis standard was drafted in accordance with the rules given in GB/T 1.1-2009. Drafting organizations of this standard. China Center for Disease Control and Prevention, Environmental and Health Related Product Safety Institute, Jiangsu Provincial Center for Disease Control and Prevention, Chinese People’s Liberation Army Center for Disease Control and Prevention, Beijing Municipal Center for Disease Control and Prevention, Shandong Province Center for Disease Control and Prevention, Heilongjiang Province Disease Prevention and Control Center, Guangzhou Customs Technology Center. The main drafters of this standard. Shen Jin, Duan Hongyang, Wang Lin, Zhang Liubo, Xu Yan, Wei Qiuhua, Wang Jiaqi, Tong Ying, Cui Shuyu, Li Tao, Li Yan, Lin Ling, Wu Xiaosong, Yu Li, Sun Huihui, Liao Ruyan, Yang Bin, Han Jie, Li Li, Zhu Tingting. Microbiological requirements for disinfection test1 ScopeThis standard specifies the requirements for the preparation of disinfection test microorganisms, culture media, passage and preservation, bacterial suspension and bacterial carrier. The microorganisms used in disinfection tests mentioned in this standard only include bacteria, fungi, mycobacteria and bacterial spores, and do not involve viruses and Alternatives to other microorganisms (eg enzymes, antigens, nucleic acids, etc.). This standard applies to the use and preservation of various microorganisms (except viruses and substitutes) for disinfection testing.2 Normative referencesThe following documents are indispensable for the application of this document. For dated reference documents, only the dated version applies to this document. For undated references, the latest version (including all amendments) applies to this document. GB 18281.5 Biological indicators for sterilization of medical and healthcare products Part 5.Biological indicators for low-temperature steam formaldehyde sterilization GB/T 33417 Biological indicator test method for hydrogen peroxide gas sterilization GB/T 33419 Test method for biological indicator of ethylene oxide sterilization GB/T 33420 Test method for pressure steam sterilization biological indicator Disinfection technical specifications (2002 edition) Ministry of Health (Health Law Jianfa [2002] No. 282)3 Test microorganisms3.1 Source The test microorganisms are derived from a qualified strain preservation institution and can be identified by conventional testing methods. 3.2 Bacteria 3.2.1 Staphylococcus aureus The disinfection test uses Staphylococcus aureus as a representative of pyogenic cocci in bacterial propagules. The commonly used strain is ATCC 6538; when the disinfectant disinfects the skin in a simulated field test, ATCC 27217 is used. 3.2.2 Escherichia coli In the disinfection test, Escherichia coli was used as the representative of the intestinal bacteria in the bacterial propagation body, and the commonly used bacteria in the test was 8099; Use 8099 or NCTC 10538 for hand disinfection simulation field test. 3.2.3 Pseudomonas aeruginosa The disinfection test uses Pseudomonas aeruginosa as the most commonly isolated bacterial multiplier in nosocomial infections. Table, the test strain is ATCC 15442. 3.2.4 Staphylococcus albicans The disinfection test uses Staphylococcus albus as a representative of bacteria in the air, and the test strain is 8032. 3.3 Fungi 3.3.1 Candida albicans The disinfection test uses Candida albicans as a representative of pathogenic fungi, and the commonly used strain is ATCC 10231. 3.3.2 Aspergillus niger In the disinfection test, Aspergillus niger was used as a representative of pathogenic fungi, and the commonly used species in the test was ATCC 16404. 3.4 Mycobacterium The disinfection test is based on Mycobacterium chelonae subsp.abscessus, Mycobacterium avium (Mycobacterium avium subsp.avium) or Mycobacterium terrae as human Mycobacterium tuberculosis The representative of the test strain is Mycobacterium chelonae abscess subspecies CMCC (B) 93326 or ATCC.19977, Mycobacterium avium ATCC 15769, Mycobacterium ATCC 15755. 3.5 Bacterial spores 3.5.1 Bacillus subtilis black var. spores In the disinfection test, Bacillus subtilis var. niger was used as the representative of bacterial spores. The commonly used strain is ATCC 9372; when it is used to indicate the effect of dry heat sterilization, it meets the requirements of the "Disinfection Technical Specification" (2002 edition); When indicating the sterilization effect of ethylene oxide, it meets the requirements of GB/T 33419. 3.5.2 Bacillus stearothermophilus spores Geobacillus stearothermophilus (Geobacillus stearothermophilus) is an indicator of sterilization effect, and the strain is ATCC 7953 or SSI K31; when used to indicate the effect of pressure steam sterilization, it meets the requirements of GB/T 33420; when used to indicate the effect of hydrogen peroxide gas sterilization, Meet the requirements of GB/T 33417; when used to indicate the effect of low-temperature steam formaldehyde sterilization, it meets the requirements of GB 18281.5. 3.5.3 Bacillus pumilus spores Bacillus pumilus is an indicator bacteria for sterilization or disinfection by ionizing radiation. The strain is E 601 or ATCC 27142. Meet the requirements of "Disinfection Technical Specification" (2002 edition). 3.6 Other According to the requirements of the disinfection test, other strains with equivalent resistance can be selected for the test.4 Medium4.1 Select the appropriate medium and reagents for the subculture and culture of bacterial species. See Appendix A for the medium and reagents required for the disinfection test. 4.2 The solid slant culture medium and flat plate prepared by the laboratory should be kept under refrigeration, and the storage period should not exceed 3 months, if there is water shortage, dryness, etc. In case, destroy after sterilization. The liquid culture medium prepared by the laboratory should be sealed and refrigerated, and the storage period should not exceed 3 months. Turbid, destroyed after sterilization. 4.3 Commercial plates, slopes and culture media are stored in accordance with the product instructions and used within the validity period.5 Passage and preservation5.1 Recovery and Passage 5.1.1 Open the freeze-dried strain tube in an aseptic manner, and add an appropriate amount of culture solution (select the culture solution according to the strain, such as nutrient broth for bacteria, Candida albicans select Sandcastle liquid medium, Mycobacterium select Sutong comprehensive liquid medium, Aspergillus niger select Malt extract nutrient broth Medium), blow and suck several times to melt and disperse the bacteria. Take a test tube containing 5.0 mL～10.0 mL of the corresponding culture solution, drop a little strain Incubate at an appropriate temperature for a specified time (generally 36 ℃ ± 1 ℃ for 18 h ~ 24 h, Aspergillus niger is 30 ℃ ± 1 ℃ Raise for 42 h to 48 h), this is the first generation culture. 5.1.2 Use an inoculating loop to take the first-generation culture, or take an appropriate amount of bacterial liquid/ceramic beads from the bacterial cryopreservation tube, and inoculate it on the corresponding medium plate (according to Strain selection medium, such as bacterial selection nutrient agar medium, Candida albicans selection sandcastle agar medium, mycobacterium selection modified Luo Medium or other commercial mycobacteria special compound agar medium, Aspergillus niger selects malt extract agar medium), at a suitable temperature Cultivate for a specified time (generally 36 ℃ ± 1 ℃ for 18 h ~ 24 h, mycobacterium 36 ℃ ± 1 ℃ for 72 h, Aspergillus niger Bacteria cultured at 30 ℃ ± 1 ℃ for 42 h～48 h), this is the second generation culture. 5.1.3 Pick typical colonies from the second-generation culture and inoculate them on the corresponding medium slope or plate (select the medium slope according to the strain, such as fine Bacteria selection nutrient agar slant, Candida albicans selection Sandcastle agar slant, mycobacterium selection modified Roche medium or other commercial branches Bacillus special compound agar slant, Aspergillus niger selection malt extract agar medium plate), cultivate at a suitable temperature for a specified time (one Generally, 36 ℃±1 ℃ culture for 18 h～24 h, Mycobacterium 36 ℃±1 ℃ culture for 72 h, Aspergillus niger 30 ℃±1 ℃ culture for 42 h～ 48 h), this is the third generation culture. 5.1.4 Take the third-generation culture, inoculate it on the corresponding medium slant, and cultivate it at a suitable temperature for the specified time. This is the fourth-generation culture. 5.1.5 Cultivate to the required number of generations according to the above method, and indicate the basic information such as the name of the bacteria, the number of the bacteria, the number of generations, and the date of passage on the test tube during passage. 5.2 Save 5.2.1 Preservation of bacterial strain slope 5.2.1.1 Bacteria used for passage should be stored in slant refrigeration. 5.2.1.2 Inoculate the strain on a suitable solid slant medium, cultivate it at a suitable temperature for the specified time, and transfer it after it has grown sufficiently Store in the refrigerator for future generations. This method is used for short-term preservation of experimental bacterial species, and check the contamination and mutation at any time. If abnormal conditions are found, it will be destroyed after sterilization. 5.2.1.3 The storage time is related to the type of bacteria, the general storage time is not more than 9 weeks, and Pseudomonas aeruginosa and mycobacteria are not more than 6 weeks. If during the storage period, the inclined surface is lack of water, dry and cracked, it shall be destroyed after sterilization. 5.2.2 Storage of cryopreservation tubes Bacteria can be frozen in glycerin, liquid paraffin, porcelain beads, etc. The appropriate freezing method is selected according to its characteristics. Refer to the appendix for common methods B. The cryopreservation tube indicates basic information such as the name of the strain, the number of the strain, and the date of cryopreservation. The cryopreservation tube of strains should not be stored at -80 ℃ for more than 18 months. After a period of time, the strain cryopreservation tube can be recovered and then frozen again. 5.2.3 Vacuum freeze-dry storage Using vacuum freezing technology, the bacteria are freeze-dried into bacterial powder and stored for a long time. The strain tube indicates the strain name, strain number, date, etc. This information is stored frozen at -20 ℃±2 ℃.6 Preparation of bacterial suspension6.1 Preparation of bacterial propagule suspension 6.1.1 Take the 3rd to 8th generation nutrient agar medium and cultivate the fresh slant culture for 18 h to 24 h, and use a 5.0 mL pipette to suck 3.0 mL～5.0 mL diluent (except physiological saline for acidic electrolyzed water, tryptone saline solution for all others) is added to the inclined tube, Repeatedly blow and suck to wash off the lawn. Use a 5.0 mL pipette to transfer the eluate to another sterile test tube, mix with an electric mixer for 20 s, or in hand Beat 80 times on the palm to make the bacteria evenly suspended. 6.1.2 The prepared bacterial suspension is cultured and counted with live bacteria, and the result is diluted with the dilution solution to the required concentration, and the suspension quantitatively kills the test At this time, the concentration of the bacterial suspension is 1×108 CFU/mL～5×108 CFU/mL. 6.1.3 The suspension of bacterial propagules should be kept refrigerated for later use and used on the same day. 6.1.4 When contamination is suspected, use methods such as colony morphology, Gram staining and biochemical tests for identification. 6.2 Preparation of spore suspension of Bacillus subtilis var. black 6.2.1 Open the strain tube in an aseptic manner, add an appropriate amount of nutrient broth medium, and blow several times to melt and disperse the strain. Take 5.0 mL～10.0 mL nutrient broth culture medium test tube, drop a little strain suspension, and incubate at 36 ℃±1 ℃ for 18 h～24 h. Take with inoculation loop The bacterial suspension of the first generation culture is streaked and inoculated on a nutrient agar medium plate, and incubated at 36 ℃ ± 1 ℃ for 18 h to 24 h. Pick up The typical colony in the second-generation culture is inoculated in nutrient broth medium and incubated at 36 ℃ ± 1 ℃ for 18 h to 24 h, which is the third-generation culture. 6.2.2 Use a 10.0 mL pipette to draw 5.0 mL to 10.0 mL of the 18 h to 24 h nutrient broth culture of the 3rd to 5th generation, and inoculate it in Romania. The surface of the nutrient agar medium in the flask (or cell culture flask), shake it to cover the surface of the medium, and suck out the excess broth culture. Roche flasks (or cell culture flasks) are placed in a constant temperature incubator at 36 ℃ ± 1 ℃ for 5 to 7 days. 6.2.3 Use an inoculating loop to take a little moss and apply it on the glass slide, and stain it with the modified spore staining method. Improved spore staining method. use an inoculation loop to take the lawn Spread on the glass slide, after natural drying, fix the bacteria on the glass slide by flame heating; put the smear into a petri dish, put two layers of filter paper on the slide, drip Add a sufficient amount of 5.0% malachite green water solution, cover the plate, and heat at 55 ℃ ± 1 ℃ for 30 min. Take it out, remove the filter paper, rinse with tap water Remove residual liquid; add 0.5% saffron aqueous solution, wash with water after 1 min, and check with microscope after drying. The spores are green and the bacteria are red. In the microscope (oil (Microscopy) Under microscopy, when the spore formation rate reaches more than 90%, the following treatments can be performed. Otherwise, continue to place it at room temperature for a certain period of time until After reaching the above spore formation rate, perform the following treatments. 6.2.4 Add 10.0 mL of sterile distilled water to the Roche flask (or cell culture flask), gently scrape the lawn with an L stick, aspirate it, and then add 5.0 Rinse the surface of the culture medium with mL of sterile distilled water and aspirate. Concentrate the bacterial suspension sucked out twice in a sterile Erlenmeyer flask containing glass beads and shake for 5 min. 6.2.5 Place the flask in a water bath at 45°C for 24 hours to allow the bacteria to self-dissolve and break the chain and disperse into individual spores. 6.2.6 Filter the spore suspension with sterile cotton or gauze to remove agar clots. 6.2.7 Place the spore suspension in a sterile centrifuge tube and centrifuge at 3000 r/min for 30 min. Discard the supernatant, add distilled water and suck to make buds Cells are resuspended. Centrifugation and resuspension washing, this step is repeated 3 times. 6.2.8 Put the washed spore suspension into a flask containing an appropriate amount of small glass beads, and bathe in a water bath at 80 ℃ for 10 min (or 30 min at 60 ℃). To kill the remaining bacterial propagules. After cooling to room temperature, shake well and distribute and store in refrigerator for later use. The validity period is 6 months. 6.2.9 When the spore suspension is used, the live bacteria should be cultured and counted first. 6.2.10 When contamination is suspected, use methods such as colony morphology, Gram staining and biochemical tests for identification. 6.3 Preparation of fungal suspension 6.3.1 Preparation of Candida albicans suspension 6.3.1.1 Take the 3rd to 6th generation sandcastle agar medium slant fresh culture (18h-24h), use a 5.0 mL pipette to suck 3.0 Add mL～5.0 mL of the diluent into the oblique interview tube, repeatedly blow and suck, and wash the lawn. Use a 5.0 mL pipette to transfer the eluate to another sterile test In the tube, mix with an electric mixer for 20 s, or beat 80 times on the palm of your hand to suspend Candida albicans evenly. 6.3.1.2 The prepared bacterial suspension is cultured and counted with live bacteria, and the result is diluted with the dilution solution to the required concentration, and the suspension is tested for quantitative killing During the test, the concentration of the bacterial suspension was 1×107 CFU/mL～5×107 CFU/mL. 6.3.1.3 Bacterial suspension is stored in refrigeration and used on the same day. 6.3.1.4 When contamination is suspected, identification shall be carried out by methods such as colony morphology, Gram staining and biochemical tests. 6.3.2 Preparation of Aspergillus niger spore suspension 6.3.2.1 Pick a typical colony from the second generation culture, inoculate it in the nutrient broth medium of malt extract, and place it at 30 ℃ ± 1 ℃ constant temperature culture Cultivate in the incubator for 42 h to 48 h, which is the third generation culture. 6.3.2.2 Use a 10.0 mL pipette to draw 5.0 mL～10.0 mL of the third generation culture, inoculate the Roche flask, and shake the bacteria liquid to cover the malt Extract the surface of the agar medium, aspirate the excess broth culture liquid, and place it in a constant temperature incubator at 30 ℃ ± 1 ℃ for 42 h～48 h. 6.3.2.3 Add 5.0 mL to 10.0 mL of 0.05% (V/V) Tween 80 saline solution to the Roche flask culture to scrape the Aspergillus niger Bacteria conidia in the solution, transfer the spore suspension into a triangular flask with glass beads, shake gently for 1 min, filter to remove the hyphae, show Under the microscope (400 times), observe whether there are still hyphae. If there is, centrifuge at 5000 r/min～6000 r/min for 20 min. Again at Observe under the microscope (400 times), repeat the above steps if necessary. 6.3.2.4 Aspergillus niger conidia suspension should be stored in cold storage for no more than 2 days. Before use, mix well and observe under a microscope (400 times). Check if there are spores sprouting, if so, do not use it. 6.3.2.5 The prepared bacterial suspension is cultured and counted with live bacteria, and the result is diluted with the dilution solution to the required concentration, and the suspension is tested for quantitative killing During the test, the concentration of the bacterial suspension was 1×107 CFU/mL～5×107 CFU/mL. 6.4 Preparation of mycobacterial suspension 6.4.1 Take the third-generation slant culture and connect it to the modified Roche medium or other commercial mycobacterial compound agar medium slant. Subsequent passage, the culture method is the same as that of the third generation. Take the fresh 72-hour culture from the 5th to the 6th generation, and use a 5.0 mL pipette to suck 3.0 mL～ Add 5.0 mL of diluent to the oblique interview tube, repeatedly blow and suck, and wash the lawn. Use a 5.0 mL pipette to transfer the eluate to a glass containing 6 g～7 g Mix the glass beads in a sterile conical-bottomed test tube with an electric mixer for at least 5 min. Inhale the bacterial liquid into another test tube to make a bacterial suspension, cool Store and save, use on the day. 6.4.2 The prepared bacterial suspension is cultured and counted with live bacteria, and the result is diluted with the dilution solution......Tips & Frequently Asked Questions:Question 1: How long will the true-PDF of WS/T 683-2020_English be delivered?Answer: Upon your order, we will start to translate WS/T 683-2020_English as soon as possible, and keep you informed of the progress. The lead time is typically 1 ~ 3 working days. 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