LY/T 1926-2010Test method for evaluating the antibacterial performance of wood(bamboo)-based panels and products (English PDF)

4.1 Principle

The antibacterial test method refers to the filming method used in the WS/T 650-2019 standard, that is, by inoculating bacteria on the sample to be tested the surface of the product is then covered with a plastic film, so that the bacteria evenly and fully contact the surface of the sample to be tested, and pass through under the specified conditions.

After culturing for a certain period of time to reach a certain number of bacteria, use the plate viable bacteria count method to calculate the number of bacteria in the tested sample.

A method to evaluate the antibacterial effect by calculating the antibacterial rate.

4.2 Test conditions

The bacteria testing laboratory shall meet the requirements of GB 19489.

4.3 Main instruments and equipment

-Constant temperature incubator (can keep the temperature at 37±2°C)-Refrigerator (can keep the temperature at 0-5°C)-Ultra-clean workbench (cleanliness level not less than 100)-Biological optical microscope (200~400×)-Pressure steam sterilizer (can maintain a temperature of 121°C and a pressure of 103kpa)-Electric heating drying oven (can keep the temperature at 160-180°C)-Inoculation loop (diameter not more than 4mm)

4.4 Cover film

Polyethylene film, size 40mm×40mm×0.05mm, soak in 75% ethanol solution for 1min, then use sterile water or Rinse the eluate, dry it naturally and set aside.

4.5 Medium

4.5.1 Liquid LB medium

Yeast extract 5.0g

Tryptone 10.0g

Sodium chloride 5.0g Preparation method. add the above ingredients into 1000mL distilled water at once, heat to dissolve, then use 0.1mol/L sodium hydroxide solution Adjust the pH to between 7.0 and 7.2, and place it in a pressure steam sterilizer for sterilization at 121°C for 30 minutes.

4.5.2 Solid LB medium

Add 15g agar to 1000mL liquid LB medium, heat to melt, adjust pH with 0.1mol/L sodium hydroxide solution the value is between 7.0 and 7.2, and then placed in a pressure steam sterilizer for sterilization at 121°C for 30 minutes.

4.6 Reagents

4.6.1 Disinfectant

75% ethanol disinfectant.

4.6.2 Eluent

Normal saline containing 0.85% sodium chloride. Adjust pH with 0.1mol/L sodium hydroxide solution or 0.1mol/L hydrochloric acid solution the value is between 7.0 and 7.2, and then placed in a pressure steam sterilizer for sterilization at 121°C for 30 minutes.

4.6.3 Culture medium

Liquid LB medium/physiological saline solution, the concentration of E. coli inoculum is 1/500 (volume ratio), golden the concentration of Staphylococcus chromosome inoculum is 1/100 (volume ratio). In order to facilitate the dispersion of bacteria, 0.2% of sterile surface activity can be added Agent (such as Tween 80). Adjust the pH value with 0.1mol/L sodium hydroxide solution or 0.1mol/L hydrochloric acid solution to make it in (25 ±1) The temperature is between 7.0-7.2 at °C. After sub-packaging, place it in a pressure steam sterilizer at 121°C for 30 minutes.

4.7 Detection of bacteria

The tested bacteria should meet the requirements of Table 1.

Note. According to user requirements, other strains can be added as strains for testing, but the strains used need to be managed by the national-level strain preservation Provided by the management center.

4.8 Preparation and processing of test pieces

4.8.1 Negative control sample

A sterile culture plate with a diameter of 90mm, number A.

4.8.2 Blank control sample

Artificial board or wood (bamboo) without anti-bacterial treatment made of the same material and the same processing technology as the tested sample the product is cut into a sample with a size of (50±1)mm×(50±1)mm, and the number is B.

4.8.3 Antibacterial treatment of wood-based panels or wood (bamboo) product samples

Select various man-made panels or wood (bamboo) products that have undergone antibacterial treatment, and cut into a size of (50±1) mm×(50±1) mm sample, numbered C.

4.8.4 Number of samples

The number of samples of each type of the above negative, blank control, and antibacterial treatment should be no less than 6.Surface should be carried out before testing for disinfection, wipe the surface of the sample with 75% ethanol solution, rinse with sterile water after 1 min, and dry it naturally for later use.

4.9 Testing procedure

4.9.1 Strain preservation

Strains were inoculated on the slant of nutrient medium, cultured at 37±2°C for 24h, and preserved at 0-5°C (not more than 1 Month), as a slant to preserve bacteria.

4.9.2 Strain activation

Transfer the slant preserved strains to the plate medium and incubate at 37±2°C for 24h, transfer once a day, no more than Two weeks. The fresh bacterial culture transferred within 24 hours should be used for the test.

4.9.3 Preparation of bacterial suspension

Use an inoculation loop to take a small amount (scrape 1-2 loops) of fresh bacteria from the 4.9.2 medium and add it to the inoculation solution. After culturing for 24 h, Make 10-fold increasing dilutions in sequence, and choose the dilution with the bacterial concentration of 5.0×105 CFU/mL~10.0×105 CFU/mL as the test bacteria liquid, operate according to the method specified in GB/T 4789.2-2016.

4.9.4 Sample test

Take 0.2 mL test bacterial solution 4.9.3 and drop it on the blank control sample (B) and antibacterial treatment artificial board or wood (bamboo) products.

On the sample (C), at the same time, take 0.2 mL of sterile water and drip it on the negative control sample (A). Use sterile tweezers to pick up the sterile cover the membrane is respectively covered on sample A, sample B and sample C. It must be spread flat so that the bacteria evenly contact the sample; put it in a sterilized dish, Incubate for 24 h under the conditions of ±2°C and relative humidity >90%. Make 3 parallels for each sample.

Take out the samples cultured for 24 hours, add 20 mL of eluent to each piece, and wash sample A, sample B, sample C and cover film repeatedly (It is best to use tweezers to pick up the film and rinse). After shaking well, take 0.5 mL of the washing solution into a sterilized petri dish, and pour into 15 mL~ in 20 mL of solid medium cooled to 45±2°C, shake well and solidify, incubate at 37°C±2°C for 24 to 48 hours, then count the activity for the number of bacteria, operate according to the method specified in GB/T 4789.2-2016.

The above test was repeated once.

4.10 Test result requirements and calculation

4.10.1 Test result requirements

Calculate the actual number of viable bacteria of samples A, B and C after 24 hours of culture according to the dilution factor.

Collect the values of viable bacteria and record them as Na, Nb, and Nc, respectively, to ensure that the test results meet the following requirements at the same time, otherwise the test is invalid.

(A) The negative control should grow aseptically (Na is 0);

(B) The three parallel viable bacteria values of the control sample shall meet the following requirements.

(Highest value-lowest value)/average number <= 0.3;

(C) The control sample should not have obvious antibacterial effect, and the actual recovered colony value Nb should not be less than 1.0×104CFU/piece.

4.10.2 Antibacterial rate calculation

Nb. The average number of recovered bacteria in the blank control sample, CFU/tablet;

Nc. The average number of bacteria recovered from antibacterial wood-based panels or wood (bamboo) products, CFU/piece.

5.2 Antibacterial performance classification standards

The antibacterial performance grading standard shall be carried out according to Table 2.