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Evaluting Method for Efficacy of Antibacterial and Bacteriostasis
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Basic data
| Standard ID | WS/T 650-2019 (WS/T650-2019) |
| Description (Translated English) | Evaluting Method for Efficacy of Antibacterial and Bacteriostasis |
| Sector / Industry | Health Industry Standard (Recommended) |
| Classification of Chinese Standard | C50 |
| Classification of International Standard | 11.080 |
| Word Count Estimation | 20,247 |
| Date of Issue | 2019 |
| Date of Implementation | 2019-07-01 |
| Issuing agency(ies) | National Health Commission |
WS/T 650-2019: Evaluting Method for Efficacy of Antibacterial and Bacteriostasis
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Evaluting Method for Efficacy of Antibacterial and Bacteriostasis
ICS 11.080
C 50
WS
People's Republic of China Health Industry Standard
Antibacterial and antibacterial effect evaluation method
Published on.2019 - 01 - 30
2019 -07 - 01 implementation
National Health and Wellness Committee of the People's Republic of China
Foreword
This standard is written in accordance with the provisions of GB/T 1.1-2009.
This standard is provided by Jiangsu Provincial Center for Disease Control and Prevention, China Center for Disease Control and Prevention, Environmental and Health-related Product Safety Institute, Shandong Province
The Center for Disease Control and Prevention and the Heilongjiang Provincial Center for Disease Control and Prevention are responsible for drafting.
The main drafters of this standard. Xu Yan, Tan Zhi, Zhang Liubo, Chen Yueying, Wu Xiaosong, Lin Ling, Sun Qihua, Cui Shuyu, Zhao Huawei, Wu
Ke, Zhang Wensheng, Wang Yurong, Wu Xiao, Shen Yi, Li Yan, Sun Wei, Wang Ling, Wang Wei, Wang Xiaolei, and Yan Hongliang.
Antibacterial and antibacterial effect evaluation method
1 Scope
This standard specifies the selection principles and uses of antibacterial and bacteriostatic evaluation methods.
This standard is applicable to the identification of antibacterial and bacteriostatic effects of products with antibacterial and/or bacteriostatic functions.
2 Normative references
The following documents are indispensable for the application of this document. For dated references, only the dated version applies to this article.
Pieces. For undated references, the latest edition (including all amendments) applies to this document.
GB 15979 Hygienic standard for single-use hygiene products
FZ/T 73023 antibacterial knitwear
Disinfection Technical Specifications (2002 Edition) Ministry of Health (Wei Fa Jian Fa [2002] No. 282)
3 Terms and definitions
The following terms and definitions apply to this document.
3.1
Antibacterial antibacterial
The use of chemical or physical methods to kill or hinder the growth and reproduction of bacteria can reduce the number and activity of the process.
3.2
Bacteriostatic bacteriostasis
The process of inhibiting or hindering the growth and reproduction of bacteria and its activity by chemical or physical means.
3.3
Sustained antibacterial action
The antibacterial product with antibacterial effect is applied to the surface of the object, and it still kills or hinders the growth and reproduction of bacteria for more than 7 days.
3.4
Neutralizer
In the microbicide test, the disinfectant used to eliminate the test microorganisms and disinfectants, and the disinfectant remaining on the surface of the microorganisms,
It removes the agent that inhibits and kills microorganisms.
4 Selection principles of evaluation methods
4.1 Difference between antibacterial test and antibacterial test
Antibacterial test. The antibacterial effect of the antibacterial product on bacteria and fungi is determined. The neutralizing agent is required to terminate the bactericidal action during the test.
Bacteriostatic test. The antibacterial effect of the antibacterial product on bacteria and fungi was determined, and the neutralizing agent was not needed to terminate the antibacterial action during the test.
4.2 Select the appropriate evaluation method based on product performance
4.2.1 Principles for selection of methods for evaluating bacteriostasis
Antibacterial products are selected for antibacterial effect evaluation methods. Liquid antibacterial products, selective suspension bacteriostatic test, paste or semi-solid gel,
Viscous antibacterial products select carrier soaking quantitative antibacterial test, wet wipes or other products containing their own antibacterial ingredients
Test, antibacterial ring test for soap solid antibacterial products, textile selective dipping bacteriostatic test containing soluble antibacterial ingredients, containing continuous
Antibacterial washing solution for bacteriostatic action was selected for retention and bacteriostatic test.
4.2.2 Selection principle of antibacterial effect evaluation method
Antibacterial products are selected for evaluation of antibacterial effects. Liquid antibacterial product selection suspension quantitative sterilization test, gelatinous or paste antibacterial product
Select carrier soak quantitative bactericidal test, wet wipes or other products containing antibacterial ingredients to select carrier sterilization test, containing soluble anti-drug
The textiles of the bacterial components were selected for the impregnation test.
Textiles, non-woven fabrics, fibers, etc. containing insoluble antibacterial ingredients, after being identified as containing no soluble antibacterial ingredients, are shaken by flasks
test.
Tiles, plastics, metals, paints, etc. containing insoluble antibacterial ingredients, using a film test; for application to the surface, after drying
Continue antibacterial tests for products that continue to be antibacterial.
5 Evaluation methods
5.1 Antibacterial effect
5.1.1 Suspension quantitative inhibition test
5.1.1.1 Scope of application
It is suitable for the determination of microbial bacteriostatic effect of liquid antibacterial preparations such as sanitary lotion and antibacterial spray.
5.1.1.2 Reagents, media and equipment
5.1.1.2.1 Test strain
Staphylococcus aureus (ATCC 6538), Escherichia coli (8099), Candida albicans (ATCC 10231) and according to bacteriostatic agents
Other strains used for specific purposes.
5.1.1.2.2 Reagents
Diluent. 0.03 mol/L phosphate buffered saline (PBS) (pH 7.2-7.4), medium. Staphylococcus aureus and large intestine rod
The culture was carried out using a nutrient agar medium, and the culture of Candida albicans was carried out using Sabouraud agar medium.
5.1.1.2.3 Equipment
Constant temperature water bath, timer, Class II biological safety cabinet, etc.
5.1.1.3 Test procedure
The fresh slant culture of the test bacteria for 24 hours was washed with PBS and diluted with PBS to about 5.0×10 5 CFU/mL to 4.5×10 6 CFU/mL bacterial suspension.
spare. Take a sterile test tube, first add 5.0 mL of sample (use the original solution or diluent according to the instructions for use), set 20 ° C ± 1 ° C water bath
After 5 minutes, add 0.1 mL of the test bacterial suspension, mix quickly and time immediately. The bacteria to be tested interact with the sample to the specification
At a fixed time, inoculate 1.0 mL of the test bacteria and the sample mixture to inoculate 2 plates, and pour the medium. When the amount of bacteria cannot be counted, do 10 with PBS.
Double dilution, select appropriate dilution to absorb 1.0 mL to inoculate 2 plates, and count the viable culture. Simultaneously replace the sample with PBS
Parallel test as a positive control. The number of colonies recovered from the positive control was between 1.0 x 104 CFU/mL and 9.0 x 104 CFU/mL. Take the same batch
PBS and medium were used as negative controls. All test samples and control samples were cultured at 36 °C ± 1 °C, and the bacterial propagules were cultured for 48 hours.
Results. Candida albicans culture for 72 hours to observe the final results. The test was repeated 3 times and the inhibition rate was calculated.
5.1.1.4 Calculation of inhibition rate
10
AA
X ...(1)
In the formula.
X-bacteriostatic rate, %;
The amount of bacteria recovered in the A0-positive control group, the unit is CFU/mL;
A1 - The amount of bacteria recovered in the test group, the unit is CFU/mL.
5.1.1.5 Result determination
The inhibition rate is ≥50%～90%, and the bacteriostasis effect is determined; the inhibition rate is ≥90%, and it has strong antibacterial effect.
5.1.2 Carrier immersion quantitative inhibition test
5.1.2.1 Scope of application
It is suitable for the determination of microbial bacteriostasis effect of paste or semi-solid gel and viscous antibacterial products.
5.1.2.2 Reagents, media and equipment
The carrier is a 10 mm × 10 mm skim white plain cloth, and the degreasing method is carried out according to the "Disinfection Technical Specification" (2002 edition).
Force steam sterilization standby, electronic balance (d = 0.01g), the same as 5.1.1.2.
5.1.2.3 Operation steps
The fresh slant culture of the test bacteria for 24 hours was washed with PBS and diluted with PBS to about 5.0×10 6 CFU/mL to 5.0×107 CFU/mL.
The suspension is ready for use. Dilute 10 μl of the bacterial suspension on a sterile carrier with a micropipette, dry at 36 °C ± 1 °C or air dry at room temperature for use.
The sample was weighed in a sterile dish at a rate of 5 g/tablet, placed in a water bath at 20 ° C ± 1 ° C for 5 min, and the carrier was inoculated with sterile forceps to complete the carrier.
Fully immersed in the sample and timed immediately. The carrier to be infected and the sample interact with the sample for the specified time, respectively, and the carrier is added to 5.0.
In a mLPBS tube, mix and shake, and wash the test bacteria, respectively, and take 1.0 mL of the sample solution, and measure the number of viable cells according to the viable culture counting method.
Each tube was inoculated with 2 plates. If the number of colonies growing on the plate is large, it can be diluted 10 times with PBS, and then cultured for live bacteria.
count. Take 10.0 g of the control sample containing the bacteriostatic component of the homogenous material of the test sample and soak two bacterial carriers for parallel test.
Sexual control. The amount of recovered bacteria in the positive control was 1.0 x 104 CFU/table to 9.0 x 104 CFU/tablet. The same batch of PBS and medium were used as negative controls.
All the test samples and the control samples were cultured at 36 °C ± 1 °C, and the bacterial propagules were cultured for 48 hours. The culture of Candida albicans was observed for 72 hours.
result. The test was repeated 3 times and the inhibition rate was calculated.
5.1.2.4 Calculation of inhibition rate
10
AA
X ...(2)
In the formula.
X-bacteriostatic rate, %;
The average number of colonies of A0-control sample, the unit is CFU/tablet;
A1 - The average number of colonies of the test sample, the unit is CFU/tablet.
5.1.2.5 Result determination
The inhibition rate is ≥50%～90%, and the bacteriostasis effect is determined; the inhibition rate is ≥90%, and it has strong antibacterial effect.
5.1.3 Carrier antibacterial test
5.1.3.1 Scope of application
It is suitable for micro-inhibition products such as wet wipes, non-woven masks, sanitary napkins, pads, diapers, etc. containing dissolution-inhibiting components.
Determination of the effect of bacteria.
5.1.3.2 Reagents, media and equipment
See 5.1.1.2.
5.1.3.3 Test procedure
The fresh slant culture of the test bacteria for 24 hours was washed with PBS, and diluted with PBS to about 105 CFU/mL to 106 CFU/mL to prepare a bacterial suspension.
The test sample and the control sample were separately cut into 20 mm × 30 mm pieces under sterile conditions using a sterile scissors. 0.1 mL was added during the test
Bacterial suspension. The control samples were sterilized by pressure steam before infection. Take a sterile plate and take 2 test pieces with sterile forceps. Do not overlap and set at 20 °C.
A water bath of ±1 ° C for 5 min, 0.1 mL of the test bacterial suspension was added dropwise to each of the tablets, and the time was immediately counted. The test bacteria are contacted with the sample to explain
At the specified time of the book, the samples were collected and added to 5.0 mL PBS tubes and mixed. Oscillate and elute, respectively absorb 1.0 mL of sample solution, press
The number of viable cells was determined by viable culture counting method, and two tubes were inoculated per tube solution. If the number of colonies growing on the plate is large, it can be 10 times series.
After dilution, the viable culture count was performed.
At the same time, two samples of the control sample containing the bacteriostatic component of the homogenous material of the test sample were used instead of the test piece to be tested as a positive control.
The amount of recovered bacteria in the positive control was 1.0×104 CFU/table to 9.0×104 CFU/tablet; the same batch of PBS and medium were used as negative control.
All test samples and control samples were cultured at 36 °C ± 1 °C, bacterial propagule culture for 48 h, and Candida albicans culture for 72 h.
result. The test was repeated 3 times and the inhibition rate was calculated.
5.1.3.4 Calculation of inhibition rate
See formula (2).
5.1.3.5 Result determination
The inhibition rate is ≥50%～90%, and the bacteriostasis effect is determined; the inhibition rate is ≥90%, and it has strong antibacterial effect.
5.1.4 inhibition zone test
5.1.4.1 Scope of application
It is suitable for the antibacterial effect of solid antibacterial products with dissolution-producing substances and can be made into 5 mm diameter tablets. It can also be used for identification.
Do not contain soluble bacteriostatic substances in the antibacterial samples.
5.1.4.2 Reagents, media and equipment
Vernier calipers, others see 5.1.1.2
5.1.4.3 Test procedure
The fresh slant culture of the test bacteria for 24 hours was washed with PBS and diluted to 5.0×10 5 CFU/mL to 5.0×10 6 CFU/mL for use.
Preparation of antibacterial tablets. Dissolved solid antibacterial products, directly made into discs with a diameter of 5 mm and a thickness of not more than 4 mm (blocks), 4 pieces each
As a group.
Preparation of Negative Control Samples. Samples of the same material containing no bacteriostatic components were prepared, and wafers (blocks) of the same size as the test group were prepared.
Use a sterile cotton swab to draw a concentration of 5.0 × 105 CFU/mL ~ 5.0 × 106 CFU/mL test bacterial suspension, in a suitable medium plate
Apply evenly 3 times. For each application, the plate should be rotated 60°, and finally the cotton swab is applied around the edge of the plate for one week. Cover the plate and set the room temperature
Dry for 5 min.
One test plate was placed in each test, and four test pieces and one negative control sample were placed on each plate for a total of five pieces. Sterile forceps
The sample piece is placed on the surface of the plate, and the negative control sample piece is attached to the center of the plate. The test piece is attached to the periphery, and after being placed, the sterile tweezers are used.
Gently press the sample to fit snugly against the surface of the plate. The center of each sample is more than 25 mm apart and 15 mm or more from the circumference of the plate. Cover up
The plate was placed at 36 ° C ± 1 ° C for 16 h to 18 h, and the test was repeated 3 times.
Use a vernier caliper to measure the diameter of the inhibition ring (including the patch) and record it. When measuring the inhibition zone, you should choose uniform and completely sterile growth.
The bacteria ring is carried out and its diameter should be measured by the outer edge of the inhibition zone.
5.1.4.4 Result determination
Negative control samples should be produced without inhibition zone. If the inhibition zone diameter of the test sample is >7 mm, it is judged to have antibacterial effect; the inhibition zone diameter is ≤7 mm.
It was judged to have no bacteriostatic effect.
Three replicates (12 samples in total) had bacteriostatic effects and were judged as qualified.
5.1.5 Impregnation inhibition test
5.1.5.1 Scope of application
It is suitable for the determination of the antibacterial effect of dissolution bacteriostatic fabrics (such as bacteriostatic towels, masks, underwear, etc.).
5.1.5.2 Reagents, media and equipment
5.1.5.2.1 Test strains see 5.1.1.2.1
5.1.5.2.2 Sample
Cut a circular sample with a diameter of 5 cm from the edge of the sample 10 cm or more and 1 m above the cloth end, and take 3 samples separately.
Packed in 3 conical flasks, cover the bottle for use (the number of samples required depends on the fiber type and fabric weave to absorb 1 mL of bacteria)
And there is no residual liquid in the conical flask. In the same method, the same material as the sample was cut but the bacteriostatic control fabric was not included, and 2 pieces were separately packed in 2
In a conical flask, cover the bottle mouth and sterilize at 121 ° C for 15 min.
5.1.5.2.3 Reagents and media
0.03 mol/L PBS (pH 7.2-7.4), broth medium, nutrient agar medium, Sabouraud medium.
5.1.5.2.4 Preparation of bacterial suspension
The preserved strains were inoculated into the nutrient agar plates by the inoculation loop, cultured at 36 ° C ± 1 ° C for 24 h, and the typical colonies were transplanted to contain
The broth medium is incubated in an Erlenmeyer flask at 36 ° C ± 1 ° C for 24 h, and the broth is serially diluted with the broth to make the bacterial suspension contain 1.0.
×105 CFU/mL to 5.0×10 5 CFU/mL.
5.1.5.3 Test procedure
Take 1 mL of the bacterial suspension and add 2 parts of the prepared conical flask sample and 1 control fabric to ensure uniform distribution, and in the conical flask.
Leave no excess liquid and seal the bottle to prevent evaporation from causing bacterial death. Cone in a sample containing the inoculum suspension and the control fabric
Add 100 mL of PBS to the vial, shake the vortex shaker for 1 min to wash the bacteria, and pipette 1.0 mL of sample solution or 10 times serial dilution solution.
Two plates were used as the number of bacteria on the "0" contact time sample and the control fabric.
Another flask containing the inoculum suspension sample was incubated at 36 ° C ± 1 ° C for 20 h ± 2 h, added to 100 mL PBS, and vortexed.
The bacteria were washed by shaking for 1 min, and 1.0 mL of the sample solution or 10 times of the serial dilution solution was used to inoculate 2 plates, respectively, as a test group.
In the negative control group, the sample was not inoculated with the bacterial suspension, 100 mL of PBS was added at the contact time of “0”, and the sample was shaken for 1 min on a vortex shaker.
Inoculate the plate.
For the positive control group, another conical flask containing the control fabric was inoculated, and after inoculating 1 mL of the bacterial suspension, it was cultured at 36 ° C ± 1 ° C for 20 h ± 2 h.
Add 100 mL of PBS, shake the vortex shaker for 1 min to wash the bacteria, and inhale 1.0 mL of sample solution or 10 times serial dilution to inoculate 2 flats.
Dish. The negative and positive control samples were incubated with the test group samples at 36 ° C ± 1 ° C for 48 h, and the number of colonies was counted, and the test was repeated 3 times.
5.1.5.4 Calculating the inhibition rate
2/)][(C
]2/)[(C
CBB
ACBB
Or or
Or or
...(3)
In the formula.
X-bacteriostatic rate, %;
A-number of bacteria on the test group, the unit is CFU/mL;
B-"0" contact time sample number of bacteria, the unit is CFU/mL;
C-"0" contact time control the number of bacteria on the fabric, the unit is CFU/mL;
If the difference between "B" and "C" is large, take a larger value; if "B" and "C" are not much different, take the average.
5.1.5.5 Result determination
The conditions for the establishment of the test required. the average recovery amount of the contact time of the “0” contact time was 1×103 CFU/mL～5×103 CFU/mL;
The sex control should be aseptically grown, and the number of positive control bacteria is significantly higher than that of 0 contact time.
The inhibition rate of each test was ≥50%, and the sample was judged to have a bacteriostatic effect.
5.1.6 retention bacteriostatic effect test
5.1.6.1 Scope of application
It is suitable for the identification of the antibacterial effect of antibacterial lotion antibacterial products with continuous antibacterial action.
5.1.6.2 Reagents, media and test equipment
Test strain. Staphylococcus aureus (ATCC 27217)
Test product. The test article consisted of 25 sets of sequentially numbered samples. 2 bottles per set, one for the test sample and the other for the anti- (repression)
A control sample of the microbial agent.
25 bottles containing no bacteriostatic agent (for the test adjustment phase).
Trypsin soy broth (TSB), trypsin soy agar medium (TSA), sheep blood, defibration treatment, 0.075
Mol/L phosphate buffer, 70% alcohol, metal cylinder (2.2 cm diameter, height 3 cm), disposable inoculating loop (4 mm diameter),
Small plastic bowl (2.2 cm in diameter, 2.5 mm in height), tape (Darapore, manufactured by 3M), nylon scraping stick, polyethylene glycol monoxin
Phenylphenyl ether (Traton X-100; Triton-X 100) 500 mL, topical antibiotic ointment, glass bending stick, skin disinfectant, etc.
5.1.6.3 Test procedure
5.1.6.3.1 Adjustment phase
From 7 days to 14 days before the start of the test, the subjects used daily soap, shampoo and body wash without anti-bacterial ingredients to wash their hands.
Take a bath, this phase lasts at least 7d, but no more than 14d.
5.1.6.3.2 Cleaning stage
During the cleaning phase for 3 days, the subjects washed one side of the forearm with a bacteriostatic lotion sample with continuous bacteriostatic action every day, and washed the other with the control sample.
One side of the forearm, the cleaning process is as follows.
First clean the left arm and moisten the inside of the forearm with running water at a temperature of 35 ° C to 37 ° C; 3 mL of the sample solution in the palm of the hand; from wrist to elbow
Rub up and down for 60s; rinse the forearm with flowing water for 15s, do not rub; use a paper towel to dry the forearm, do not rub; use no antibacterial ingredients
Repeat the above steps to clean the right arm; wash the forearm 3 times daily, at least 1 h each time, in the test procedure described above.
After the last cleaning, record the time and check the retention effect after 12 hours. After the 9th wash of the forearm, the subject did not
Can bathe, shower or wash the forearm until the end of the test.
5.1.6.3.3 Test phase
At 12h or 24h after the last wash, a test area will be drawn on each forearm of the subject, and the test area will be inoculated and packaged.
And recovering viable bacteria, the specific steps are as follows.
Staphylococcus aureus (ATCC 27217) was continuously transferred for 3 generations, and the 3rd generation culture was inoculated into tryptic soy broth (TSB).
The medium was cultured at 36 ° C ± 1 ° C for 20 h ± 2 h. Then appropriately dilute the bacterial suspension with TSB so that the concentration of the bacterial suspension is about 108 CFU/mL~
CFU/mL.
Inoculation. In the middle of each forearm of the subject (not at the wrist and elbow folds), use a glass with an ink diameter of 3.00 cm.
The glass cylinder is fastened to the skin and a test area is divided. 10 μL of the above bacterial suspension was taken using a pipette and inoculated in the forearm test area (number of colonies)
For 106 CFU/test area ~ 107 CFU/test area), use a disposable inoculation loop to coat the inoculum into a circle so that it should be adjacent to the edge of the test area.
There is a distance of 4 mm to 5 mm.
Packing. Immediately after inoculation, use a small plastic bowl to fasten the area above the dyeing area, then tape the small plastic bowl to the skin and record the packet.
time.
Recovering viable bacteria. The area inoculated on the forearm was sampled 2 h ± 5 min after inoculation. Place the metal tube in the middle of the test area.
Do not touch the edges that are covered with ink. Pipette 1 mL of 0.075 mol/L phosphate buffer containing 0.1% Triton-X 100 into a metal can.
Scrape the skin in the area with a nylon scraping rod for 60s, pipette the liquid into the tube, and add 1 mL with 0.1% Triton
X-100 0.075 mol/L phosphate buffer, the skin in this area is scraped for the second time for 30 s, the second scrubbing liquid, note
Into the test tube containing the first scrubbing liquid.
Disinfection treatment after the test in the experimental area. After sampling the experimental area after the antibacterial sample group is cleaned, 70% alcohol is needed to eliminate the experimental area.
poison. Then, the experimental area after the control group without the bacteriostatic component was sampled and disinfected in the same manner. After the experiment, disinfect with skin
The agent disinfects the two forearms, rinses with water after treatment, wipes dry, and then applies a small amount of antibiotic ointment.
Inoculation and culture. Each sample was inoculated, and the sample was serially diluted 10 times with 0.0375 mol/L phosphate buffer.
Appropriate dilution 0.1 mL was inoculated on the surface of 2 TSA plates containing 5% sheep blood, spread wit...
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