An antibacterial coating antibacterial performance detection method

0 With the improvement
people's living standard, health has gradually become the main aspect of improving the quality of life. The epidemic of infectious diseases such as SARS and avian influenza has sounded the alarm to the Chinese people, and controlling and eliminating the spread of germs has become a hot research topic in various fields. The development of antibacterial coatings is of great significance for promoting public health and improving the level of national health
china
. Jiangsu Chenguang Coatings Co., Ltd. actively participated in the Chinese General Institute of Building Materials Scientific Research is responsible for drafting and attributing to the National Technical Committee for The Standardization of Coatings and Pigments" industry standards, and combined with the actual experience of enterprises put forward a number of amendments. The industry standard was reviewed on 5 January 2007. Based on the latest industry standards of Antibacterial Coatings, this paper introduces and discusses the improved experimental operation method of antibacterial coating antibacterial performance testing in view of the actual situation of small and medium-sized coating enterprises in China.
1 Experimental Part
1.1 Test Raw Materials and Instruments
1.1.1 Materials
Experimental Strains: Staphylococcus aureus ATCC6538, E. coli ATCC25922, Jiangsu Province Health and Epidemic Prevention Station. Nano antibacterial inner wall latex paint, antibacterial anti-mold anti-algae anti-corrosion multi-functional nano-environmental protection coating, nano-silver antibacterial inner wall coating, nano-composite multi-functional interior wall coating, nano-antibacterial ultraviolet curing paint, nano-antibacterial water-based wood paint, are Jiangsu Morning Light Coatings Co., Ltd. products. PBS phosphate buffer (0.03 mol/L, pH=7.2 to 7.4),
sterile test with semen, nutritional agar culture, MH agar culture.
1.1.2 Instruments and equipment
scale straws, capillary straws, digital continuous adjustable pipelets (referred to as piped guns, 10 to 1 000 μL), sterilized flat dishes, sterilized test tubes, sterilized transfer Pipes, inoculation rings, alcohol lamps, microscopes (1 μL), polyethylene film (40±2) mm× (40±2) mm, thickness 0.05 to 0.10 mm. Centrifuges, electric mixers, thermostats (37±1) degrees C, refrigerated boxes (0 to 5 degrees C), ultra-clean workbench, bio-optical microscopes, pressure steam sterilizers, electrothermal drying tanks.
1.2 Experimental method
1.2.1 bacteria preservation and resuscing
inoculated the species on the slope of the nutritional agar culture, cultured at (37±1) degrees C, and then stored at (0 to 5) degrees C (no more than 1 month) as a sloped preservation bacteria. The beveled preservation bacteria were transferred to the plate nutrition agar culture base and cultured at (37±1) degrees C at 24 h. Fresh bacterial cultures transferred within 3-14 generations should be used in the test.
1.2.2 bacteria suspension preparation
with inoculation ring from the transfer medium to take a small amount (scratch 1 to 2 rings) of fresh bacteria, add phosphate buffer (PBS), and do 10 times increment Dilution, select the concentration of bacteria liquid for (5.0 to 10.0) ×105 cfu/mL dilution as the experimental bacteria solution, according to GB/T
4789.2 method of operation.
1.2.3 Paint model preparation
coating model preparation according to GB/T 1756, application and drying method according to the actual use of thickness and method. The test model is 50 mm×50 mm in size and can be used in the actual substrate of the optional coating (e.g. metal plate and plastic plate), which can be used when the substrate is water-resistant or moisture-absorbing. Painted models need to be disinfected before testing.
1.2.4 Test Step
Take 0.2 mL of bacterial droplets to a negative blank sample (Number O), a paint blank control sample without antibacterial components (Number A) and an antibacterial coating sample (Number B), each sample for at least 5 parallel experiments. The sterilized mulch film is clamped with sterilized tweezers and covered with samples O, A and B respectively, so that the bacteria liquid evenly touches the sample and is placed in the sterilized flat dish, cultured 24 h in a thermostat with a relative humidity greater than 90% and a temperature (36±1) degrees C. The cultured 24 h after the sample O, A, B and its covering film removed, respectively, into the sterilized 100 mL beoth, and added 50 mLPBS solution, repeatedly rinse each sample and cover film (preferably with tweezers clamped film flushing), fully shake, get dilution of 1 00 bacteria suspension, take 1 mL dilution of 100 bacteria suspension in the sterilization test tube, with PBS solution for 10 times gradient dilution, to obtain dilution of 10-1, 10-2, 10-3 bacterial suspension. Each dilution gradient of 100, 10-1, 10-2, 10-3 bacteria suspension 1 mL, inoculated in the nutritional agar medium (NA), cultured at (37±1) degrees C 24 to 48 h live bacteria count, according to GB /T 4789.2 method to determine the number of live bacteria.
1.3 Test Results Calculation and Error Analysis
1.3.1 Antibacterial Rate Calculation
Culture negative control sample O, blank control sample A and antibacterial coating sample B culture 24 h After the actual recovered live bacteria values are recorded as values O, A and B (cfu/tablet), the antibacterial rate of antibacterial coatings is calculated at the press:
antibacterial rate R(%) - 100× (A-B) A
1.3.2 Experimental results error analysis
Sample A's 5 parallel live bacteria values should be in line with the (highest to value - lowest pair value) / average live bacteria value to value not greater than ×0.3× In the live bacteria count due to technology and operation caused by the number of fallout error rate (between plates, dilution) should not exceed 10%, the error rate is calculated according to the following formula.
(1) between plates (parallel models at the same dilution) error rate calculation formula:
2 results and discussion
experimental operation according to standard antibacterial testing methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Two 10x gradient dilutions in steps, using the 1.2.4 step as an example, are done by grouping the test tubes on the test tube racks by the number required, with 4.5 mL of dilution added to each tube. Each group is labeled 10-1, 10-2, 10-3 from left to right. Mix the bacterial suspension sample with an electric mixer for another 20 s (or 80 times with force in the palm of your hand), then use an ear-washing ball and straw to absorb 0.5 mL into a 10-1 tube, and mix 10-1 with an electric mixer for 20 s (or with force in the palm of your hand) 80 times), mix well, and then draw 0.5 mL into the 10-2 tube, and so on, until the last tube (each sample has 3 sets of controls, each control 5 groups parallel, gradient dilution to 10-3, that is, 45 test tubes), and all operations need to be carried out in a strictly sterile state. The operation process is cumbersome and time-rifty, the workload is heavy, and the dependence on the operator's operating level and proficiency is very high, which directly affects the detection efficiency and the accuracy of the results.
for the above, when gradient dilution is carried out, replace it with a pipet gun, e.g. in the 1.2.4 step to precisely absorb 50 μL at a time, added to a 1.5 mL centrifuge tube filled with 450 μL dilution. Because the pipelet gun can absorb the same volume continuously with a single capacity, and the centrifuge tube is easier to oscillate uniformly because of its small size, it simplifies the operation, reduces the working intensity and reduces the system error. The straws and centrifuges of the pipe transfer gun are disposable supplies, which also reduce the workload of pre-test preparation and post-treatment. In addition, the use of prefabribribrio culture base flat dishes and then coated flat-plate live bacteria rapid count, can reduce the live bacteria count time to 10 to 12 h, thereby improving the sample detection rate. Taking the nano-antibacterial water-based wood lacquer produced by Jiangsu Chenguang Coatings Co., Ltd. as an example of E. coli ATCC25922, the improved method is compared with the test results of the original method in Table 1.
table 1 nanometer antibacterial water-based wood paint different antibacterial rate
Note: 1, including experimental preparation, equipment sterilization, sterile operation and equipment cleaning, including the entire operation process time.
2, refers to the difference between the average of the experimental test results and the detection results of the Guangdong Microbiological Analysis and Testing Center.
In Table 1, three groups of skilled personnel (i.e., trained by a professional department or with relevant professional basis and with more than 12 months of experimental experience) and non-skilled personnel (without any professional background or foundation, only 3 weeks of temporary training) were used to compare the same samples. Because of the gradient dilution of bacterial fluid by pipe shift gun and centrifuge tube, the experimental operation is simplified, the experimental time is shortened, and the error rate between plate and dilution is effectively reduced, so that the test results of unskilled personnel also meet the requirements of experimental error, thus reducing the dependence of the test results on the operation of personnel. Another advantage of using improved methods is that the contamination rate of germs in the experimental process is reduced. In Table 1, the error rate between dilution degrees of the improved method is significantly higher than that between plates, while the results of the traditional method are reversed. This is because the inter-plate error rate is more sensitive to human operation, and the pipe and centrifugal tube of the pipe shift gun are organic plastic products, compared to the inorganic glass straws and test tubes used in traditional methods, its adsorption to bacteria is strong, increasing the system error of the multiplile dilution process.
in order to further improve the operating efficiency and reduce the working intensity of gradient dilution, the experimental comparison of gradient dilution ratio of 1:10, 1:100 and 1:1,000 times was carried out with a liquid shift gun and microscope. As shown in Figure 1, through 6 different antibacterial coating samples to do 3 different dilution gradient ratio of antibacterial testing, it is found that with the increase of dilution gradient ratio, the average error of the experiment increased, especially according to the 1:1,000 times gradient ratio dilution detection error significantly increased, the dilution error rate between more than 10%. At this dilution multiplification rate, a trace sampler is required to absorb 1.0 μL bacteria liquid into the centrifuge tube containing 999 μL dilution, the absorbed bacteria liquid accumulates too low, the sampling uniformity is poor, resulting in the sampling of the relative error is magnified, resulting in an increase in system error. Therefore,
dilution gradient multiply of the test should not exceed 100.
key operating step that affects the accuracy of the test results is to add the bacteria to the sample in step 1.2.4. The number of live bacteria in the drip liquid should be between 1.0×105 and 1.0×106cfu, and the amount of bacteria added should be between 50 and 200 μL, less than 50 It is easy to cause the evaporation loss of water in the bacterial fluid in the culture process is too large, resulting in a negative blank control sample of the number of live bacteria is too low; The selection of model substrates also need to pay attention to avoid the impact on test results, with porous structure, water absorption and moisture absorption capacity or strong adsorption force of the substrate will make the sample of the bacteria fluid washed out not thoroughly, blank control sample of the number of live bacteria is low; For Jiangsu Chenguang Coatings Co., Ltd.'s nano antibacterial inner wall latex paint, antibacterial anti-mold anti-algae anti-corrosion multi-functional nano-environmental protection coating, nano-silver antibacterial antibacterial inner wall coating, nano-composite multi-functional interior wall coating, nano-antibacterial ultraviolet curing paint and nano-antibacterial water-based wood paint and other 6 kinds of antibacterial coatings were tested with traditional methods and improved methods, the results of which are compared with the authoritative test results of Guangdong Microbiological Analysis and Testing Center. The results of the improved method are closer to the results
guangdong microbiological analysis and testing center, which shows that the accuracy of the test method is reliable.
the evaporation loss of water in the culture process is too large, resulting in a negative blank control sample of the number of live bacteria is too low; The selection of model substrates also need to pay attention to avoid the impact on test results, with porous structure, water absorption and moisture absorption capacity or strong adsorption force of the substrate will make the sample of the bacteria fluid washed out not thoroughly, blank control sample of the number of live bacteria is low; For Jiangsu Chenguang Coatings Co., Ltd.'s nano-antibacterial inner wall latex paint, anti-bacterial anti-mold anti-algae anti-corrosion multi-functional nano-environmental protection coating, nano-silver antibacterial inner wall coating, nano-composite multi-functional interior wall coating, nano-antibacterial ultraviolet curing paint and nano-antibacterial water-based wood paint and other 6 kinds of antibacterial coatings were tested with traditional methods and improved methods, the results of which are compared with the authoritative test results of Guangdong Microbial Analysis and Testing Center. The results of the improved method are closer to
the Guangdong Microbiological Analysis and Testing Center, which shows that the accuracy of the test method is reliable.3 Conclusion
In accordance with the industry standards of Antibacterial Coatings, refer to the Disinfection Technical Specification (2002 edition), QB/T 2591-2003 Antibacterial Plastics - Antibacterial Performance Test Methods and Antibacterial Effects The antibacterial performance testing method in JC/T 897-2002 antibacterial ceramic products, combined with the actual and requirements of small and medium-sized coating enterprises, introduces the antibacterial performance testing method of antibacterial coatings as an example. By improving the operation method, under the premise of ensuring the accuracy of the test results, not only improves the detection efficiency, but also reduces the dependence of the test results on the level of operation and proficiency of the personnel. Using this antibacterial performance testing method, a full set of hardware equipment investment can be controlled within 100,000 yuan, equipped with testing personnel 1 to 2 people, after 2 to 3 weeks of training, can meet the enterprise's general product performance testing requirements, is very suitable for the vast number of small and medium-sized paint enterprises.