QB/T 2738-2012 (QB/T 2738-2023 Newer Version) PDF English
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Evaluating methods for antibacterial and bacteriostatic efficacy of daily chemical products
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Test methods for evaluating daily chemical products in antibacterial and bacteriostatic efficacy
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QB/T 2738-2012: PDF in English (QBT 2738-2012) QB/T 2738-2012
QB
LIGHT INDUSTRY STANDARD OF
THE PEOPLE’S REPUBLIC OF CHINA
ICS 71.040.99
Classification No.: Y40
Filing No.: 36712-2012
Replacing QB/T 2738-2005
Test methods for evaluating daily chemical products
in antibacterial and bacteriostatic efficacy
ISSUED ON: MAY 24, 2012
IMPLEMENTED ON: NOVEMBER 01, 2012
Issued by: Ministry of Industry and Information Technology of PRC
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Normative references ... 4
3 Terms and definitions ... 4
4 Basic requirements for laboratory and aseptic operation to test the effect of
antibacterial and bacteriostatic daily chemical products ... 6
5 Collection of sample ... 7
6 Evaluation principles of antibacterial and bacteriostatic efficacy of daily
chemical products ... 7
7 Inspection methods for antibacterial and bacteriostatic efficacy of daily
chemical products ... 8
8 Methods for testing the stability of antibacterial and bacteriostatic efficacy of
daily chemical products ... 29
Appendix A (Informative) Method for determining the content of antibacterial
and bacteriostatic active ingredients ... 31
Test methods for evaluating daily chemical products
in antibacterial and bacteriostatic efficacy
1 Scope
This standard specifies the testing methods and evaluation criteria, for the
antibacterial and bacteriostatic efficacy of daily chemical products, which have
special sanitary functions.
This standard is applicable to the antibacterial and bacteriostatic performance
tests, of common detergents and human skin cleaning products. For other daily
chemical products, it may use it selectively, according to their purposes.
2 Normative references
The following documents are essential to the application of this document. For
the dated documents, only the versions with the dates indicated are applicable
to this document; for the undated documents, only the latest version (including
all the amendments) is applicable to this standard.
Disinfection technical specifications [2002]
GB 15979-2002 Hygienic standard for disposable sanitary products
GB 15981-1995 Evaluating method and standard for the efficacy of
disinfection and sterilization
QB/T 2739-2005 Preparations of standard volumetric solutions of general
test methods for washing products
3 Terms and definitions
The following terms and definitions apply to this document.
3.1
Antibacterial
The process of using chemical or physical methods, to kill bacteria or hinder
the growth and reproduction of bacteria and their activity.
3.2
inhibition and killing effects of microorganism.
3.9
Product of neutralization
Refers to the product, after the action of neutralizer and bactericide.
4 Basic requirements for laboratory and aseptic
operation to test the effect of antibacterial and
bacteriostatic daily chemical products
4.1 The microbiology laboratory shall adopt a closed layout. The building shall
be easy to clean and disinfect. To avoid contamination, it shall be carried out
under relatively positive pressure and clean conditions. When pathogenic
bacteria are used as indicator bacteria, due to special needs, it shall be carried
out in a biological safety cabinet (negative pressure).
4.2 Before the start of the test, the countertops and indoor floors shall be
cleaned by a wet method; then the air in the laboratory shall be disinfected, by
ultraviolet or other methods.
4.3 Experimental personnel shall wear work clothes, masks, caps; when
performing sterility inspection, they must enter the laboratory after being air-
drenched. Then, properly wear sterile gowns, caps, masks.
4.4 The sterile pipette shall be replaced, every time a different liquid is taken.
The inoculation loop (needle) shall be burnt and sterilized, on the flame, before
it can be used again.
4.5 Unless otherwise specified, only reagents which is confirmed to be
analytically pure and distilled water or deionized water or water of equivalent
purity, are 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.,
need to be sterilized or filtered for degerming.
4.7 For aseptic equipment and reagents, the container or package must be
checked for integrity before use; those with damage shall not be used.
4.8 The sterile equipment and reagents being used shall not be exposed to the
air, for a long time.
4.9 When pipetting liquid or inoculating, the test tube's port and agar plate shall
be close to the flame, to prevent contamination.
4.10 All used contaminated equipment shall be immediately put into a container,
which is filled with disinfectant, to prevent pollution to the surrounding
environment and cleaning materials.
4.11 In the event of accidental breakage of microbial cultures or leakage of other
test microorganisms, no matter whether it is pathogenic or not, the
contaminated and possibly affected areas shall be disinfected immediately.
4.12 After all tests are over, indoor air and environmental surfaces shall be
disinfected as usual.
5 Collection of sample
In order to make the samples have good representativeness, at least 12
samples of the smallest sales packaging shall be randomly selected, from the
three shipping packages of the same batch number, of which 4 samples are
retained, 4 samples are tested for bacteriostatic or sterilization performance, 4
samples are tested for stability. The smallest package sampled shall not be
broken; it shall not be opened before inspection.
6 Evaluation principles of antibacterial and
bacteriostatic efficacy of daily chemical products
6.1 Sterilization test method
The sterilization test method in this standard is used to assess the antibacterial
effect of the product.
6.2 Bacteriostatic test method
The bacteriostatic test method in the standard is used to assess the
bacteriostatic effect of the product.
6.3 Indicator microorganisms for inspection
According to the product implementation standard OR the application scope of
the manual, carry out the selection and testing of the test bacteria in Table 1
(provided by the national or provincial strain collection and management center).
It may also follow the use requirements of the product, to add other strains, as
the test bacteria.
phosphate, 10.0g of lecithin, 10.0g of glycine, 30.0g of Tween (80),
1000mL of distilled water.
3) 1.36g of potassium dihydrogen phosphate, 2.83g of disodium hydrogen
phosphate, 3.0g of lecithin, 20.0g of Tween (80), 1000mL of distilled
water.
4) 20.0g of Tween (80), 10.0g of sodium thiosulfate, 1000mL of PBS.
Note: 1) is used for chlorine-type fungicides; 2) and 3) are used for non-oxidizing
fungicides; 4) is used for oxygen-type fungicides.
f) Preparation of bacterial liquid
(1) Preparation of bacterial propagule suspension
1) Take the freeze-dried strain tube. Open it under aseptic operation. Use
a capillary pipette to add an appropriate amount of nutrient broth.
Gently blow and suck several times, to melt and disperse the strain.
Take a test tube, which contains 5.0mL ~ 10.0mL of nutrient broth
medium. Add a little strain suspension dropwise. Incubate it at 37°C, for
18h ~ 24h. Use an inoculating loop, to take the bacterial suspension of
the first generation of culture. Streak and inoculate it on a nutrient agar
medium plate. Incubate it at 37°C for 18h ~ 24h. Pick the typical
colonies from the aforementioned second-generation culture. Inoculate
it on a nutrient agar slant. Incubate at 37°C for 18h ~ 24h, which is the
third-generation culture.
2) Take the 3rd ~ 14th generation of the fresh culture (18h ~ 24h) on the
nutrient agar medium slant. Use a 5.0mL pipette, to take 3.0mL ~ 5.0mL
of the diluent, into the slant tube. Repeatedly blow and suck. Wash off
the lawn. Subsequently, use a 5.0mL pipette, to transfer the washing
solution to another sterile test tube. Use an electric mixer, to mix
(vibrate) it for 20s. OR shake it on the palm for 80 times, to make the
bacteria evenly suspended.
3) Preliminarily prepared bacterial suspension, first use the bacterial
concentration turbidimetric method, to roughly measure the bacterial
concentration, THEN use the diluent, to dilute it to the required
concentration.
4) The suspension of bacterial propagules shall be stored in a refrigerator
at 4°C for later use. It shall be used on the day when it is prepared; it
shall not be overnight.
5) When contamination is suspected, it shall be identified by colony
morphology, Gram staining, biochemical test, etc.
(2) Preparation of Candida albicans suspension
1) Take the freeze-dried strain tube. Open it through aseptic operation.
Use a capillary pipette, to add a proper amount of Sabouraud liquid
medium to the tube. Gently blow and suck several times, to melt and
disperse the strain. Take a test tube, which contains 5.0mL ~ 10.0mL of
Sabouraud liquid medium. Add a little bacterial suspension dropwise.
Incubate at 37°C for 18h ~ 24h. Use an inoculating loop, to take the
bacterial suspension of the first generation of culture. Streak to
inoculate it on a Sabouraud agar medium plate. Incubate at 37°C for
18h ~ 24h. Pick the typical colonies from the second-generation culture
mentioned above. Inoculate it on the Sabouraud agar slant. Incubate at
37°C for 18h ~ 24h, which is the third-generation culture. Seal it and
store it at 4°C. The shelf life shall be not more than 6 weeks.
2) During the test, take the third-generation slant culture for continuous
passage on the Sabouraud agar slant; the method is the same as the
third generation. Take the 5th or 6th generation of fresh culture (18h ~
24h) from the Sabouraud agar medium slant. Use a 5.0mL pipette to
suck 3.0mL ~ 5.0mL of PBS diluent. Add it into the slant test tube.
Repeatedly blow and suck it, to wash off the lawn. Subsequently, use a
5.0mL pipette, to transfer the washing solution into another sterile test
tube. Use an electric mixer, to mix it for 20s. OR shake it on the palm
for 80 times, to make the Candida albicans be suspended evenly.
3) The bacterial suspension shall be stored in a refrigerator at 4°C for later
use. It shall be used on the day when it is prepared; it shall not be
overnight.
4) When contamination is suspected, it shall be identified by colony
morphology, Gram staining, biochemical test, etc. The colony
morphology can be directly observed, by a microscope. The
morphology of the bacteria can be observed directly, by a high-power
microscope, after the smear; OR can be observed, after staining by the
ink shadow method (mix the bacteria and black ink uniformly on the
glass slide; push it into a thin film).
7.2.3 Identification test method of neutralizer
In order to accurately evaluate the killing effect of the tested sample on
microorganisms, it is required to select an appropriate neutralizer, in the
sterilization test. The selected neutralizer can stop the antimicrobial lotion's
inhibitory effect on microorganisms in time, meanwhile the reaction products
between the neutralizer itself and the tested sample (that is, the neutralization
product) have no inhibition or killing effect on the microorganism, nor adverse
effects on the culture medium.
neutralization product, BETWEEN the neutralizer AND the test sample, is non-
toxic to the indicator bacteria, it is judged as the neutralizer for the test sample.
7.2.4 Operation steps of sterilization test
a) Use the PBS solution to dilute the test bacteria suspension (7.2.2.f). The
required concentration is as follows: take 0.1mL; drop it in 5.0mL of control
sample solution (PBS); the number of recovered bacteria is 1x104 cfu/mL
~ 9x104 cfu/mL;
b) Use the sterile standard hard water, to dilute the test sample to the
specified concentration:
c) Take 5.0mL of the original test sample OR its diluted solution. Put it into a
sterile test tube. Keep it at 20°C for 5min (30°C for soap products);
d) Pipette 0.1mL of the test bacteria solution, into a test tube, which contains
5.0mL of sample. Mix it quickly. Make timekeeping immediately;
e) After acting for the set time, take 0.5mL of the mixture of the test bacteria
and the sample. Add it to 4.5mL of sterilized neutralizer. Mix it uniformly.
f) After 10min of neutralization, pipette 1mL of the sample solution (or after
appropriate dilution, take the diluent of 2 ~ 3 dilution gradients). Place it in
a sterilized culture dish. Inoculate two sterile plates, for each sample
solution or dilution. Pour 15mL of nutrient agar medium (bacteria) or
Sabouraud agar medium (Candida albicans), which was cooled to 40°C ~
45°C. Turn the plate, to make it fully uniform. Turn the plate over, after the
agar is solidified. After culturing at (35±2)°C for 48h (for bacteria) or 72h
(for Candida albicans), count the viable colonies;
g) Use the PBS to replace the test sample. Operate according to the above
steps, to make it as a control sample;
h) Repeat the test 3 times. Find the average value.
7.2.5 Calculation formula
The sterilization rate is calculated according to formula (1).
Sterilization rate (%) = [(I - II) / I] x 100 ………………………. (1)
Where:
I - The average number of colonies in the control sample;
II - The average number of colonies in the test sample.
chemical products (suspension quantitative method)
7.3.1 Equipment
Same as 7.2.1.
7.3.2 Reagents
Same as 7.2.2.
7.3.3 Operation steps of bacteriostatic test
a) Use the PBS solution to dilute the test bacteria suspension (7.2.2.f). The
required concentration is as follows: drop 0.1mL into 5.0mL of control
sample solution (PBS); the number of recovered bacteria is 1x104 ~ 9 x104
cfu/mL;
b) Use sterile standard hard water to dilute the test sample to the specified
concentration;
c) Take the stock solution of test sample OR 5.0mL of its diluted solution. Put
it into a sterile test tube. Keep it at a constant temperature of 20°C, for
5min (30°C for soap products);
d) Pipette 0.1mL of test bacteria liquid, into a test tube, which contains 5.0mL
of sample. Mix it quickly. Start timekeeping immediately;
e) After acting for the set time, take 0.5mL of the mixed solution of test
bacteria and sample. Add it to a 4.5mL sterilized PBS test tube. Mix it
thoroughly;
f) After standing for 10min, pipette 1mL of the sample solution (or after
appropriate dilution, take diluent of 2 ~ 3 dilution gradients). Place it in a
sterile plate. Inoculate two sterile plates for each sample solution or
dilution gradient. Pour 15mL of nutrient agar medium (bacteria) or
Sabouraud agar medium (Candida albicans), which was cooled to 40°C ~
45°C. Turn the plate, to make it fully uniform. Turn the plate over after the
agar is solidified. Culture it at (35±2) °C for 48h (bacteria) or 72h (Candida
albicans). Count the viable colonies;
g) Use PBS to replace the test sample. Operate according to the above steps
at the same time, as the control sample;
h) Repeat the test 3 times. Calculate the average value.
7.3.4 Calculation formula
The bacteriostatic rate is calculated according to formula (8).
Weigh 15.0g of tryptone, 5.0g of soy peptone, 5.0g of sodium chloride. Add
1000L of distilled water to dissolve it. Adjust the pH to 7.2 ± 0.2. Sterilize it
by pressure steam at 121°C, to prepare for later use;
d) Bovine serum albumin solution (3%):
Weigh 3.0g of bovine serum albumin. Add 100mL of distilled water to
dissolve it. Use a microporous membrane (pore size 0.45μm) to filter and
sterilize it. Store it in the refrigerator for later use.
e) Standard hard water (same as 7.2.2.d)
f) Non-ionic wetting agent
Weigh 5.0g of alkylphenol polyoxyethylene ether, 5.0g of sodium
carbonate. Add 1000mL of distilled water to dissolve it.
g) Washing liquid:
Pipette 1.5g of non-ionic wetting agent (7.4.3.f), 1.5g of sodium carbonate.
Add 3000mL of distilled water to dissolve it.
h) Neutralizer (passed the neutralizer identification test, same as 7.2.3)
i) Tween 80 (filter sterilization)
7.4.4 Test preparation
a) Preparation of test cotton cloth
Add about 300g of test cotton cloth to 3L washing liquid (7.4.3.g), Heat and
boil for 1h. Take out the cotton cloth. Wash it in boiling deionized water for
5min. Then put it in cold deionized water for 5min, to remove the remaining
washing liquid. Finally dry the cotton cloth.
b) Preparation of test cotton cloth and rotating support
Take the treated cotton cloth. Cut it into a cloth strip, which has a width of
5cm AND a weight of (15±1) g. Insert one end of it into the outer edge of
the horizontal direction of the test rotating support. Then wind 12
completed circles, with sufficient tension, between the 3 horizontal
supports. Use a stainless steel pin to fix the other end of the cloth strip, to
the previous cloth strip. Finally, sterilize it by pressure steam at 121°C for
15min, to prepare for use.
c) Preparation of bacterial suspension
Use PBS solution to dilute the test bacteria suspension (7.2.2.f), to a
concentration of 1x108 cfu/mL ~ 5x108 cfu/mL. Then add an equal volume
of bovine serum albumin solution (3%).
d) Preparation of bacteria carrier
Inoculate 20μL of bacterial suspension to each piece of carrier cloth
(7.4.2.1). Put back into the culture dish. Apply the lid. Dry it in an incubator
at (35 ± 2) °C for 20min.
e) Preparation of test samples:
20min before the start of the test, put a glass jar, which contains 265mL of
hard water, into the water bath, to keep constant temperature to the test
temperature (25±1) °C. Then add the tested sample, according to the
specified concentration (that is, the action concentration of the
antibacterial and bacteriostatic test). Mix and dissolve it. Keep the
temperature of the solution consistent with the test temperature (25±1) °C.
7.4.5 Operation steps of sterilization or bacteriostatic test
a) Put the 2 pieces of bacteria carrier [7.4.4d)] between the 6th and 7th layers
of the rotating support. Put the 3rd piece between the 7th and 8th layers of
the cloth;
b) Aseptically put the rotating unit (support, cloth strip and bacteria carrier)
into the glass jar, which contains the test sample. Cover it;
c) Fix the glass jar on the shaker. Let it roll and rotate, to wash it to the set
time (that is, the action time of the antibacterial and bacteriostatic test).
Then remove the glass jar;
d) Take out the rotating unit in an aseptic manner. Remove 3 pieces of
bacteria carrier. Put them into the test tube, which contains 30mL of
neutralizer (when testing the bacteriostatic effect of the bacteriostatic
product, use 30mL of PBS containing 0.5% Tween 80 instead of the
neutralizer; the other operation steps are the same as the test for testing
the antibacterial products). Mix it in a shaker for 10s. Then shake 200
times. Use PBS to make a 10-fold serial dilution. Choose the sample liquid
of appropriate dilution, to inoculate the TSB plate. Inoculate two plates for
each dilution gradient;
e) Use the PBS containing 0.5% Tween 80 to replace the test sample.
Operate according to the above steps, as the control sample group;
f) Bacterial count control group: Add 3 pieces of bacteria carrier to a PBS test
tube, which contains 30mL of 0.5% Tween 80. Shake for 10s in a shaker.
Then shake 200 times. Use PBS to make a 10-fold serial dilution. Take
......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.
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