GB/T 21510-2024 English PDF (GB/T21510-2024)

GB/T 21510-2024: Antimicrobial property testing and evaluation methods for nano-inorganic materials
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GB/T 21510-2024
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 11.080.99
CCS G 70
Replacing GB/T 21510-2008
Antimicrobial property testing and evaluation methods for
nano-inorganic materials
ISSUED ON: JULY 24, 2024
IMPLEMENTED ON: FEBRUARY 01, 2025
Issued by: State Administration for Market Regulation;
Standardization Administration of the People’s Republic of China.
Table of Contents
Foreword ... 3
1 Scope ... 5
2 Normative references ... 5
3 Terms and definitions ... 5
4 Test method ... 6
5 Antibacterial property evaluation ... 7
6 Test report ... 7
7 Safety operation requirements ... 8
Appendix A (Normative) Test method for antibacterial property of powdered nano-
inorganic materials - Oscillation method ... 9
Appendix B (Normative) Test method for antibacterial properties of porous and non-
porous materials containing nano-inorganic antibacterial components - Oscillation
method ... 14
Appendix C (Normative) Test method for antibacterial property of non-porous materials
containing nano-inorganic antibacterial components - Film method ... 16
Appendix D (Normative) Test method for antibacterial property of porous materials
containing nano-inorganic antibacterial components - Absorption method ... 19
Bibliography ... 22
Antimicrobial property testing and evaluation methods for
nano-inorganic materials
1 Scope
This document specifies the test methods, antimicrobial property evaluation, test report
and safety operation requirements for the antibacterial property of nano-inorganic
materials.
This document applies to nano-inorganic materials with antibacterial functions, as well
as products with nano-inorganic materials as antibacterial components (structural units),
such as fibers, fabrics, plastics, coatings and ceramics. The antibacterial property testing
and evaluation of other materials can also be carried out in accordance with this
document.
2 Normative references
The following documents are referred to in the text in such a way that some or all of
their content constitutes requirements of this document. For dated references, only the
version corresponding to that date is applicable to this document; for undated references,
the latest version (including all amendments) is applicable to this document.
GB 4789.2, National food safety standard -Microbiological examination of food:
Aerobic plate count
GB/T 30544 (all parts), Nanotechnologies - Vocabulary
3 Terms and definitions
For the purposes of this document, the following terms and definitions, as well as those
given in GB/T 30544 (all parts), apply.
3.1
antibacterial
The process of killing microorganisms such as bacteria and fungi and/or hindering their
growth, reproduction and activity by chemical or physical methods.
3.2
nano-inorganic materials
Inorganic materials whose three-dimensional spatial scale has at least one dimension at
the nanoscale (1 nm ~ 100 nm).
4 Test method
4.1 Procedure
4.1.1 The test of the antibacterial property of powdered nano-inorganic materials shall
be carried out according to the method specified in Appendix A.
4.1.2 The test of the surface antibacterial property of non-porous products containing
nano-inorganic antibacterial components shall be carried out according to the method
specified in Appendix B or Appendix C.
4.1.3 The test of the antibacterial property of porous products containing nano-
inorganic antibacterial components shall be carried out according to the method
specified in Appendix B or Appendix D.
4.2 Test data processing
Multiply the number of colonies on each plate by the dilution factor to get the actual
number of colonies recovered from the sample.
4.3 Calculation of test results
4.3.1 Calculation of colony counts
Multiply the colony counts on each plate by the dilution factor to get the actual number
of bacteria recovered from the sample.
4.3.2 Calculation of antibacterial rate
Calculate the antibacterial rate R according to Formula (1).
Where:
R – antibacterial rate;
A – the average number of bacteria recovered after the control sample has been in
contact with the test bacteria for a certain period of time, in colony forming units
per milliliter (cfu/mL);
B – the average number of bacteria recovered after the test sample has been in contact
with the test bacteria for a certain period of time, in colony forming units per
milliliter (cfu/mL).
microbial culture collection centers or national corresponding culture collection
management centers.
A.2.5 Control samples
Silicon dioxide powder, the powder size is required to be no more than 100 nm and the
purity to be 98% ~ 99%. It has no antibacterial effect and has no influence on the
determination of the test results.
A.3 Test procedure
A.3.1 Preparation of bacterial slants
A.3.1.1 Bacterial activation
Take a tube of dry culture; open it under sterile operation; add an appropriate amount
of nutrient broth with a capillary pipette; gently pipette several times to melt and
disperse the culture. Take a test tube containing 5.0 mL ~ 10.0 mL of nutrient broth;
drop a small amount of bacterial suspension into it; culture it at 37 ℃ ± 1 ℃ for 18 h ~
24 h.
A.3.1.2 Separation
Use an inoculating loop to take the bacterial suspension of the first-generation culture;
streak it onto a nutrient agar plate; culture it at 37 ℃ ± 1 ℃ for 18 h ~ 24 h.
A.3.1.3 Purification
Pick a typical colony from the second-generation culture mentioned above; inoculate it
on a nutrient agar slant; culture it at 37 ℃ ± 1 ℃ for 18 h ~ 24 h to obtain the third-
generation culture.
A.3.1.4 Preservation of bacterial species
Inoculate the species on a slant of nutrient agar medium; culture at 37 ℃ ± 1 ℃ for 24
h; then store at 0 ℃ ~ 5 ℃. Generally, transfer the species once no more than one month.
When contamination is suspected, identification shall be carried out using methods such
as colony morphology, Gram staining and biochemical tests.
A.3.2 Test steps
A.3.2.1 Preparation of bacterial suspension
Take 18 h ~ 24 h fresh culture of the nutrient agar medium slant of the third to eighth
generations of bacterial species; use a 5.0 mL pipette to draw 3.0 mL ~ 5.0 mL of 0.03
mol/L phosphate buffer into the slant test tube; repeatedly aspirate and blow to wash
off the bacterial lawn. Transfer the washed bacterial solution to another test tube; use
an oscillator to mix it; use 0.03 mol/L phosphate buffer to dilute it to an appropriate
concentration (about 105 cfu/mL). The bacterial vegetative suspension shall be stored
in a 4 °C refrigerator for use and shall not be stored for more than 4 h.
A.3.2.2 Preparation of control group sample solution
Weigh 0.50 g ± 0.05 g of the control sample powder and put it into a conical flask; add
95 mL of phosphate buffered saline containing 0.1% (mass fraction) Tween-80; mix
well; then, add 5.0 mL of the pre-made bacterial suspension.
A.3.2.3 Preparation of test sample solution
Weigh 0.50 g ± 0.05 g of the test sample powder and put it into a conical flask; add 95
mL of phosphate buffered saline containing 0.1% (mass fraction) Tween-80; mix well;
then, add 5.0 mL of the pre-made bacterial suspension.
A.3.2.4 Viable count of control sample at “0” contact time
Before shaking, dilute the control sample solution appropriately; aspirate 1.0 mL and
inoculate it into a sterile plate; inoculate 3 plates in parallel with each sample solution;
pour in nutrient agar medium dissolved at 45 ℃ ~ 55 ℃; turn the plate over after the
agar medium solidifies; place the plate in a constant temperature incubator at 37 ℃ ±
1 ℃ to count the colonies.
A.3.2.5 Oscillating contact culture
Fix the conical flasks containing the control sample and the test sample on the shaker
of a constant temperature shaking incubator. Under the condition of an action
temperature of 37 ℃ ± 1 ℃, shake at a speed of 150 r/min. The materials that need to
be diluted before use should be shaken for 1 h ~ 4 h, and the materials that do not need
to be diluted and used directly should be shaken for 4 h ~ 24 h.
A.3.2.6 Viable count after oscillation contact for a certain period of time
After appropriate dilution of the shaken control sample and test sample, take 1.0 mL of
each sample and inoculate it in a sterile plate. Inoculate 2 plates in parallel with each
sample. Pour the dissolved nutrient agar medium at 45 ℃ ~ 55 ℃. After the agar
medium solidifies, turn the plate over and place the plate in a constant temperature
incubator at 37 ℃ ± 1 ℃ to count the viable bacteria.
A.3.2.7 Negative control group
For the negative control group, respectively take dilution solution and culture medium
from the same batch of test samples and place in a constant temperature incubator at
37 ℃ ± 1 ℃ for culture. Observe for contamination.
A.3.2.8 Observations
Appendix B
(Normative)
Test method for antibacterial properties of porous and non-porous materials
containing nano-inorganic antibacterial components - Oscillation method
B.1 Applicability
This test method is applicable to the determination of the antibacterial property of
porous and non-porous materials (including dissolvable and non-dissolvable fibers,
fabrics, plastic powders, microporous filter materials, etc.) containing antibacterial
components of nano-inorganic materials.
B.2 Test equipment and materials
B.2.1 Test equipment, test apparatuses and standard species for testing
The requirements for test equipment, test apparatuses and standard species for testing
shall comply with the provisions of A.2.1 ~ A.2.4.
B.2.2 Control samples
The control sample is a sample cut from pure cotton plain white cloth (32 yarns). The
sample itself has no antibacterial effect and has no effect on the determination of the
test results. Degreasing treatment shall be carried out before the test: boil the pure cotton
plain white cloth in water containing detergent for 30 minutes; rinse with tap water 3
times; then boil with distilled water for 5 minutes; rinse, dry and iron; before cutting,
remove the warp and weft yarns according to the size of the prepared sample; then cut
according to the yarn drawing marks.
B.3 Test procedure
B.3.1 Preparation of bacterial slants
The preparation of bacterial slant shall comply with the requirements of A.3.1.
B.3.2 Test steps
B.3.2.1 Select the sterilization method according to the sample. Generally, use high-
pressure steam sterilization; place the sample in an appropriate container and place it in
a high-pressure sterilizer for sterilization (121°C, 103 kPa, 20 min). If the sample is not
suitable for sterilization by high-pressure steam, it can be sterilized by other methods
such as high-temperature moist heat, dry heat or ethylene oxide. However, the
sterilization method used shall not affect the antibacterial performance and test results,
and shall be stated in the report.
Appendix C
(Normative)
Test method for antibacterial property of non-porous materials containing nano-
inorganic antibacterial components - Film method
C.1 Applicability
This test method is applicable to the determination of the antibacterial property of non-
porous materials (such as plastics, ceramics, paint films, plates, metals and other hard
surface materials) containing antibacterial components of nano-inorganic materials.
C.2 Test equipment and materials
C.2.1 Test equipment
Type A2 secondary biological safety cabinet, constant temperature incubator (37 ℃ ±
2 ℃), pressure steam sterilizer (pressure 103 kPa, temperature 121 ℃), electric constant
temperature dry oven (0 ℃ ~ 250 ℃), refrigerator (2 ℃ ~ 8 ℃), microwave oven
(output power ≥ 700 W), ultraviolet lamp (30 W, 253.7 nm).
C.2.2 Test apparatuses
Erlenmeyer flask (capacity 500 mL, 250 mL), petri dish (diameter 90 mm), test tube
(18 mm × 180 mm), measuring cylinder (100 mL), pipette (10 mL, 5 mL, 1 mL), pipette
(accuracy 0.01 mL), alcohol lamp, test tube rack, 70% ethanol and polyethylene film,
etc.
C.2.3 Culture medium, reagents and standard bacterial species for testing
Culture media, reagents and standard bacterial species for testing shall comply with the
requirements of A.2.3 and A.2.4.
C.3 Test procedure
C.3.1 Preparation of bacterial slants
The preparation of bacterial slant shall comply with the requirements of A.3.1.
C.3.2 Test steps
C.3.2.1 Preparation of covering film
The covering film is made of polyethylene film with a size of (40±2) mm × (40±2) mm
and a thickness of 0.05 mm ~ 0.10 mm. If the test sample is small, the size of the
covering film can be reduced according to its surface area to prevent the bacterial
suspension from overflowing.
C.3.2.2 Control samples
Use sanitary high-density polyethylene (HDPE) to perform injection molding on the
control sample to a standard size of (50±2) mm × (50±2) mm and a thickness of no
more than 5 mm. It is required that it itself has no antibacterial effect and has no
influence on the determination of the test results.
C.3.2.3 Preparation of test group samples
Make the test samples into standard size of (50±2) mm × (50±2) mm. If the test samples
are smaller, they shall not be less than 20 mm × 20 mm.
C.3.2.4 Sample pretreatment
Take the control sample and the test sample; use 70% ethanol solution to wipe their
surfaces; rinse with sterile distilled water after 5 minutes; dry naturally. If the sample is
not suitable for treatment with disinfectants, it can be directly rinsed with sterile
distilled water or disinfected by other methods according to the characteristics of the
sample, but it shall not affect its antibacterial properties and interfere with the test
results.
C.3.2.5 Preparation of bacterial suspension
Take 18 h ~ 24 h fresh culture of the nutrient agar medium slant of the third to eighth
generations of bacterial species; use a 5.0 mL pipette to draw 3.0 mL ~ 5.0 mL of 0.03
mol/L phosphate buffer into the slant test tube; repeatedly aspirate and blow to wash
off the bacterial lawn. Transfer t...