GB/T 24128-2018Plastics -- Assessment of the effectiveness of fungistatic compounds in plastics formulations (English PDF)

3.1 Plastic susceptible to fungal attack

Plastic that contains in its formulation one or more nutrients that support fungal growth.

3.2 Fungistat

Compound that prevents fungal growth on a material that is normally susceptible to fungal attack.

5.1 Instruments

Sterilize all glassware and other instruments that come into contact with media and/or reagents (except sterile instruments) by one of the following methods:

Method A: use an autoclave (see 5.1.2), sterilize at 121°C for at least 15min;

Method B: use a dry heat sterilizer (see 5.1.2), sterilize at 180°C for at least 30min, sterilize at 170°C for at least 1h, or sterilize at 160°C for at least 2h;

Method C: use a membrane filtration system with a pore size of 0.45um.

5.1.1 Incubator, the temperature can be maintained at 24 degrees C+/-1 degrees C, and the relative humidity can

be maintained at 85% or above.

5.1.2 Sterilization equipment

5.1.2.1 Moist heat sterilization, suitable autoclave.

5.1.2.2 For dry heat sterilization, the thermal oven shall maintain a temperature specified in

Method B.

5.1.2.3 Membrane filtration sterilization, a membrane filtration device with the pore size

specified in Method C.

5.1.3 Analytical balance, accurate to +/-0.1mg.

5.1.4 Laboratory centrifuge, with the speed of 2000r/min~5000r/min.

5.1.5 Counting plate (direct counting under microscope).

5.1.6 Microscope, the magnification can reach 100x.

5.1.7 pH meter, with an accuracy of +/-0.1pH unit, with temperature calibration function.

5.1.8 Vortex shaker, with the speed of 2000r/min~2500r/min.

5.1.9 Glass containers: test tubes, flasks or bottles of appropriate capacity.

5.1.10 Culture dish, with a diameter of 90mm ~ 100mm and a depth of at least 15mm.

5.1.11 Standard pipettes, with standard volume of 1.0mL and 15.0mL, calibrated automatic

pipettes.

5.1.12 Standard graduated cylinder, with a minimum capacity of 30mL.

5.1.13 Glass beads, with diameter of 3mm~5mm.

5.2 Medium and reagents

All reagents shall be analytically pure and/or suitable for use in microbiological assays.

5.2.1 Water

The used water shall be distilled or deionized water with a conductivity of less than 1uS/cm.

5.2.2 Malt extract agar (MEA)

Malt extract: 30.0g;

Soy peptone: 3.0g;

5.3.1.3 Paecilomyces varioti: CGMCC3.4253 or CBS628.66;

5.3.1.4 Penicillium funiculosum: CGMCC3.3875 or ATCC9644;

5.3.1.5 Trichoderma longibrachiatum: CGMCC3.4291 or ATCC13631.

Other test fungus (such as Aspergillus terreus, Aureobasidium pullulans) can be added according to product use and needs, and all used strains shall be listed in the test report.

NOTE: CGMCC is the China General Microbiological Culture Collection Center; ATCC is the American Type Culture Collection; and CBS is the Centraalbureauvoor Schimmeicultures.

5.3.2 Culture conditions

In 5.3.1.1 and 5.3.1.3 to 5.3.1.5, the strain shall be cultured on the slant of malt extract agar (5.2.2) in a test tube; and culture at 24 degrees C +/- 1 degrees C for 14 days ~ 21 days.

In 5.3.1.2, the strain shall be cultured on Chaetomium globosum agar (5.2.3) medium; and culture at 24 degrees C +/- 1 degrees C for 14 days ~ 21 days.

Cultures for storage shall be kept on agar slants, preferably lyophilized or cryopreserved.

6.1 Shape and size

Sterilize the slicer that meets the requirements; and cut out a circular piece with a diameter of 1cm ~ 4cm or a square piece with a side length of 1cm ~ 4cm from each test sample as required.

The thickness of the specimen shall not exceed 10mm.

6.2 Number of specimens

At least 3 specimens are prepared for each test sample for test evaluation.

7.1 Cleaning

Hold the test sample with tweezers; wash it repeatedly (with a brush if necessary) and store it in a clean container and dry it naturally. Use tweezers for all subsequent processing to avoid sample contamination.

7.2 Labeling and storage

Labels or markers may interact with the plastic during testing; so store samples separately in airtight containers (such as culture dishes) at room temperature; label the container rather than the sample.

8.1 Test temperature

Prepare and adjust the state of the sample according to the Class-2 atmospheric conditions [temperature 23 degrees C +/- 2 degrees C and relative humidity (50 +/- 10) %] in ISO 291:2008.

8.2 Pour the medium

After sterilization, pour 20 mL of nutrient salt agar (5.2.7) into each culture dish; allow it to solidify and dry until there is no visible moisture on its surface.

8.3 Placement of specimen

Place the test specimen discs separately, as flat as possible, in the middle of the solidified medium.

If the thickness of the specimen exceeds 5mm, punch holes in the agar with a hole punch of the same size as the specimen; and put the specimen into the agar hole. The punch should be sterilized, such as flame sterilization.

8.4 Inoculation of specimen

8.4.1 Preparation of spore suspension

Prepare a mixed spore suspension from a cultured spore culture as follows:

Add (see 5.3.2) 5 mL of wetting agent (5.2.4) to each culture tube; gently scrape the surface of the spore medium with a sterile inoculation loop to obtain a spore suspension; and gently shake the culture tube to disperse the spores; pour the spore suspension into a sterile conical flask containing 10 ~ 20 glass beads; and repeat the washing for three times according to the above process. Each spore suspension with glass beads is then shaken with a shaker (5.1.8) and filtered through a thin layer of sterile cotton or glass wool to eliminate residual hyphae.

Centrifuge the filtered spore suspension aseptically and discard the supernatant. Resuspend the sediment with 50 mL of nutrient salt solution (5.2.6); centrifuge again; and suspend the sediment with 100 mL of the same solution (5.2.6).

Measure the concentration of each spore suspension with a counting plate, and the number of spores shall be at least 5x106 pieces/mL.

Before use, mix together equal amounts of each spore suspension and mix with a shaker (5.1.8).

Single spore suspensions can be stored for up to 4 days at 4°C; up to 2 months at -18°C; or up to 12 months at -196°C.