QB/T 2738-2023 English PDF (QBT2738-2023)

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QB/T 2738-2023: Evaluating methods for antibacterial and bacteriostatic efficacy of daily chemical products
QB/T 2738-2023
QB
LIGHT INDUSTRY STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 71.100.40
CCS Y 40
Replacing QB/T 2738-2012
Evaluating methods for antibacterial and bacteriostatic
efficacy of daily chemical products
ISSUED ON: APRIL 21, 2023
IMPLEMENTED ON: NOVEMBER 01, 2023
Issued by: Ministry of Industry and Information Technology of PRC
Table of Contents
Foreword ... 3
1 Scope ... 5
2 Normative references ... 5
3 Terms and definitions ... 5
4 Basic requirements for laboratories and aseptic operations for testing the effects of
antibacterial and bacteriostatic daily chemical products ... 7
5 Sample collection ... 8
6 Principles for evaluating the antibacterial and bacteriostatic efficacy of daily chemical
products ... 8
7 Test methods for antibacterial and bacteriostatic efficacy of daily chemical products
... 9
8 Stability test method for antibacterial and bacteriostatic efficacy of daily chemical
products ... 38
Appendix A (Informative) Statistical test examples ... 39
References ... 42
Evaluating methods for antibacterial and bacteriostatic
efficacy of daily chemical products
1 Scope
This document describes the detection method of the antibacterial and bacteriostatic
efficacy of daily chemical products with special hygiene functions; stipulates the
evaluation criteria.
This document is applicable to the testing and evaluation of the antibacterial and
bacteriostatic properties of common detergents. The testing and evaluation of the
antibacterial and bacteriostatic properties of other daily chemical products are selected
according to the purpose.
2 Normative references
The contents of the following documents constitute the essential terms of this document
through normative references in the text. Among them, 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
QB/T 2153 Industrial oleic acid
QB/T 2850 General technical requirements for antibacterial and bacteriostatic
detergents
3 Terms and definitions
The following terms and definitions apply to this document.
3.1
Antibacterial
The process of killing bacteria or hindering bacterial growth and activity by
chemical or physical methods.
4 Basic requirements for laboratories and aseptic operations
for testing the effects of antibacterial and bacteriostatic daily
chemical products
4.1 Microbiological laboratories shall adopt a closed layout; the building shall be easy
to clean and disinfect. To avoid contamination, the test shall be carried out under
relatively positive pressure clean conditions. When pathogenic bacteria are used as
indicator bacteria due to special needs, they shall be carried out in a biological safety
cabinet (negative pressure).
4.2 Before the start of the test, the table and the indoor floor shall be cleaned by wet
methods; then the air in the laboratory shall be disinfected by ultraviolet rays or other
methods.
4.3 Experimenters shall wear work clothes, masks, hats; when conducting sterility tests,
they shall enter the laboratory after air showering. Then, wear sterile isolation clothes,
hats, masks correctly.
4.4 The sterile pipette shall be replaced every time a different sample is drawn; the
inoculation loop (needle) shall be burned and sterilized on the flame before it can be
used again.
4.5 Unless otherwise specified, only reagents and distilled water or deionized water or
water of equivalent purity, that are confirmed to be analytically pure, shall be used in
the analysis.
4.6 Reagents that require sterility, such as distilled water, phosphate buffer, culture
medium, bovine serum albumin, standard hard water, neutralizer, etc., shall be sterilized
or filtered.
4.7 Before using sterile equipment and reagents, check whether the container or
packaging is intact. If damaged, it shall not be used.
4.8 Sterile equipment and reagents in use shall not be exposed to the air for a long time.
4.9 When pipetting or inoculating, the mouth of the test tube and the agar plate shall be
close to the flame to prevent contamination.
4.10 All used contaminated equipment shall be immediately placed in a container
containing disinfectant, to prevent contamination of the surrounding environment and
clean items.
4.11 If the microbial culture is accidentally broken or other experimental
non-oxidizing fungicides);
c) 1.36 g potassium dihydrogen phosphate, 2.83 g disodium hydrogen phosphate,
3.0 g lecithin, 20.0 g Tween (80), 1000 mL distilled water (for non-oxidizing
fungicides);
d) 20.0 g Tween (80), 10.0 g sodium thiosulfate, 1000 mL PBS (for oxygen-type
fungicides).
7.2.2.5 Preparation of bacterial suspension
7.2.2.5.1 Preparation of bacterial propagule suspension
The preparation steps of bacterial propagule suspension are as follows.
a) Take the freeze-dried bacterial tube; open it under sterile operation; add an
appropriate amount of nutrient broth; gently blow and aspirate several times to
melt and disperse the bacterial strain. Take a test tube containing 5.0 mL ~ 10.0
mL of nutrient broth medium; add dropwise a small amount of bacterial
suspension; culture it at (36 ± 1) °C for 18 h ~ 24 h. Use an inoculation loop to
take the bacterial suspension of the first generation culture; streak it on the
nutrient agar medium plate; culture it at (36 ± 1) °C for 18 h ~ 24 h. Pick the
typical colony in the second generation culture above; inoculate it on the nutrient
agar slant; culture it at (36 ± 1) °C for 18 h ~ 24 h, which is the third generation
culture.
b) Take the fresh culture of the 3rd ~ 6th generation of the nutrient agar medium
slant (18 h ~ 24 h); use a 5.0 mL pipette to draw 3.0 mL ~ 5.0 mL of diluent into
the slant test tube; blow and suck repeatedly to wash off the bacterial moss. Then,
use a 5.0 mL pipette to transfer the washing liquid to another sterile test tube; mix
(oscillate) with an electric mixer for 20 seconds or tap on the palm 80 times, to
make the bacteria evenly suspended.
c) The preliminary bacterial suspension shall first be roughly measured by bacterial
concentration turbidimetric determination; then diluted with diluent to the
required concentration.
d) The bacterial propagule suspension shall be stored in a 4 °C refrigerator for future
use; it shall not be kept overnight for use on the same day.
e) When contamination is suspected, it shall be identified by colony morphology,
Gram staining, biochemical tests.
7.2.2.5.2 Preparation of Candida albicans suspension
The preparation steps of Candida albicans suspension are as follows:
a) Take a freeze-dried bacterial tube; open it with aseptic operation; use a capillary
pipette to add an appropriate amount of Sandcastle liquid culture medium into the
tube; gently blow and aspirate several times to melt and disperse the bacterial
strain. Take a test tube containing 5.0 mL ~ 10.0 mL of Sandcastle liquid culture
medium; add dropwise a small amount of bacterial suspension into it; culture it
at (36 ± 1) °C for 18 h ~ 24 h. Use an inoculation loop to take the bacterial
suspension of the first generation culture; streak it on the Sandcastle agar medium
plate; culture it at (36 ± 1) °C for 18 h ~ 24 h. Pick out the typical colonies in the
above second generation culture; inoculate them on the Sandcastle agar slant;
culture them at (36 ± 1) °C for 18 h ~ 24 h, which is the third generation culture.
b) Take the fresh culture of the 3rd ~ 6th generation of the strain on the sandcastle
agar medium slant for 18 h ~ 24 h; use a 5.0 mL pipette to draw 3.0 mL ~ 5.0 mL
of PBS diluent; add it to the slant test tube; blow and suck repeatedly to wash off
the bacterial moss. Then, use a 5.0 mL pipette to transfer the washing liquid to
another sterile test tube; mix it with an electric mixer for 20 seconds or tap it on
the palm of your hand 80 times, to make the Candida albicans evenly suspended.
c) The bacterial suspension shall be stored in a 4 °C refrigerator for future use. It
shall not be kept overnight on the same day.
d) When contamination is suspected, it shall be identified by colony morphology,
Gram staining, biochemical tests. Colony morphology can be directly observed
under a microscope. The bacterial morphology can be directly observed under a
high-power microscope after smearing, or it can be observed after staining with
ink shade method (mixing the bacteria with black ink on the glass slide and
pushing it into a film).
7.2.3 Neutralizer identification test
7.2.3.1 General
In order to accurately evaluate the killing effect of the sample on microorganisms, it is
required to select an appropriate neutralizer in the sterilization test. The selected
neutralizer can not only stop the inhibitory and killing effect of the antibacterial wash
on microorganisms in time, but also the reaction product of the neutralizer itself and the
sample (i.e., the neutralization product) has no inhibitory or killing effect on
microorganisms, has no adverse effect on the culture medium.
7.2.3.2 Test grouping
The test is divided into the following 4 groups:
a) Group 1: 5.0 mL neutralizer + bacterial solution → culture;
b) Group 2: (0.5 mL antibacterial agent + 4.5 mL neutralizer) + bacterial solution
V - Arithmetic mean of colonies between the 3 groups, in colony forming units per
milliliter (CFU/mL);
v - Arithmetic mean of colonies in each group, in colony forming units per milliliter
(CFU/mL).
7.2.3.4.2 Group 4 has sterile growth.
7.2.3.4.3 Qualified evaluation is obtained after 3 consecutive tests.
7.2.4 Sterilization test operation steps
7.2.4.1 Dilute the test bacterial suspension (7.2.2.5) with PBS solution. The required
concentration is as follows: take 0.1 mL and drop it into 5.0 mL of control sample
solution (PBS). The number of recovered bacteria is 1×104 CFU/mL ~ 9×104 CFU/mL.
7.2.4.2 Dilute the test sample with sterile standard hard water to the specified
concentration.
7.2.4.3 Pipette 5.0 mL of the test sample solution or its dilution into a sterile test tube;
keep it at 20 °C for 5 minutes (30 °C for soap products).
7.2.4.4 Pipette 0.1 mL of the test bacterial solution into the test tube containing 5.0 mL
of the sample; mix quickly; start the timer immediately.
7.2.4.5 After the set time, take 0.5 mL of the mixture of the test bacteria and the sample;
add it to 4.5 mL of the sterilized neutralizer; mix well.
7.2.4.6 After 10 minutes of neutralization, take 1 mL of the sample solution (or take the
diluted solution of 2 ~ 3 dilutions after appropriate dilution); place it in a sterile plate.
Inoculate two sterile plates with each sample solution or dilution. Pour 15 mL ~ 20 mL
of nutrient agar medium (bacteria) or Sabouraud agar medium (Candida albicans),
which was cooled to 40 °C ~ 45 °C; rotate the plate to make it fully uniform; turn the
plate over after the agar solidifies; culture at (36 ± 1) °C for (48 ± 2) h (bacteria) or (72
± 3) h (Candida albicans); then count the viable colonies.
7.2.4.7 Replace the test sample with PBS and follow the above steps as the control
sample.
7.2.4.8 Repeat the experiment 3 times; calculate the arithmetic mean.
7.2.5 Calculation formula
The sterilization rate is calculated according to formula (2); the result is expressed as a
percentage (%), with two decimal places.
7.4.2.7 Glass petri dish covered with filter paper.
7.4.2.8 Bacteria preservation tube.
7.4.2.9 Bacteria culture box.
7.4.2.10 Vortex oscillator.
7.4.2.11 Cotton cloth (32 yarns/cm×32 yarns/cm plain woven cotton cloth).
7.4.2.12 Pins, tweezers, sterile gloves, 3 mm ~ 5 mm glass beads, stopwatch, 2.5 cm ~
3.8 cm carrier cloth (see preparation of test cotton cloth).
7.4.3 Reagents
7.4.3.1 Culture medium (commercial qualified commercial culture medium): Nutrient
agar, tryptone soy agar (TSA), tryptone soy broth (TSB).
7.4.3.2 Bovine serum albumin solution (concentration 3%): Weigh 3.0 g of bovine
serum albumin; add 100 mL of distilled water; filter and sterilize with a microporous
filter membrane (pore size 0.45 μm) after dissolving; store in a refrigerator for later use.
7.4.3.3 Standard hard water (7.2.2.3).
7.4.3.4 Non-ionic wetting agent: Weigh 5.0 g of alkylphenol polyoxyethylene ether, 5.0
g of sodium carbonate; add 1000 mL of distilled water to dissolve it.
7.4.3.5 Washing liquid: Take 1.5 g of non-ionic wetting agent (7.4.3.4), 1.5 g of sodium
carbonate; add 3000 mL of distilled water to dissolve it.
7.4.3.6 Neutralizer (qualified by neutralizer identification test, same as 7.2.3).
7.4.3.7 Tween 80 (filter sterilization).
7.4.4 Test preparation
7.4.4.1 Preparation of test cotton cloth
Add about 300 g of test cotton cloth to 3 L washing liquid (7.4.3.5); heat and boil for 1
h. Take out the cotton cloth and wash it in boiling deionized water for 5 min. Then put
it in cold deionized water for 5 min, to remove the residual washing liquid; finally dry
the cotton cloth.
7.4.4.2 Preparation of test cotton cloth and rotating bracket
Take the treated cotton cloth; cut it into 5 cm wide and (15 ± 1) g strips; insert one end
of it into the horizontal outer edge of the test rotating bracket; then wrap it between the
three horizontal brackets with sufficient tension for 12 full circles; fix the other end of
the strip to the previous circle of strips with a stainless steel pin. Finally, sterilize it with
121 °C pressure steam for 15 min; prepare for use.
7.4.4.3 Preparation of bacterial suspension
Dilute the test bacterial suspension (7.2.2.5) with PBS solution, the required
concentration is 1×108 CFU/mL ~ 5×108 CFU/mL; then add an equal volume of bovine
serum albumin solution (3%).
7.4.4.4 Preparation of bacterial carrier
Each carrier cloth piece (7.4.2.11) is inoculated with 20 µL of bacterial suspension,
placed back in the culture dish, covered, dried in a (36 ± 1) °C...