GB/T 15981-2021_English: PDF (GB/T15981-2021)
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Evaluating method for the efficacy of sterilization for disinfection equipment
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Evaluating method and standard for the efficacy of disinfection and sterilization
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Standard ID | GB/T 15981-2021 (GB/T15981-2021) | Description (Translated English) | Evaluating method for the efficacy of sterilization for disinfection equipment | Sector / Industry | National Standard (Recommended) | Classification of Chinese Standard | C59 | Classification of International Standard | 11.080 | Word Count Estimation | 11,119 | Date of Issue | 2021-12-31 | Date of Implementation | 2022-07-01 | Older Standard (superseded by this standard) | GB 15981-1995 | Drafting Organization | Environmental and Health-Related Product Safety Institute, Chinese Center for Disease Control and Prevention | Administrative Organization | National Health Commission | Regulation (derived from) | National Standard Announcement No. 17 of 2021 | Proposing organization | National Health Commission of the People's Republic of China | Issuing agency(ies) | State Administration for Market Regulation, National Standardization Administration | Standard ID | GB 15981-1995 (GB15981-1995) | Description (Translated English) | Evaluating method and standard for the efficacy of disinfection and sterilization | Sector / Industry | National Standard | Classification of Chinese Standard | C50 | Classification of International Standard | 11.080 | Word Count Estimation | 14,163 | Date of Issue | 1996-01-23 | Date of Implementation | 1996-07-01 | Drafting Organization | Chinese Preventive Medical Sciences, Institute of Epidemiology and Microbiology | Administrative Organization | Ministry of Health infectious disease prevention and control management office | Proposing organization | Ministry of Health of the People Republic of China | Issuing agency(ies) | State Bureau of Technical Supervision, Ministry of Health of the People Republic of China | Summary | This Chinese standard specifies the technical standards Autoclave Sterilization testing and evaluation methods, This method is suitable for steam sterilization equipment sterilization effect evaluation. |
GB/T 15981-2021
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 11.080
CCS C 59
Replacing GB 15981-1995
Evaluating method for the efficacy of sterilization for
disinfection equipment
ISSUED ON. DECEMBER 31, 2021
IMPLEMENTED ON. JULY 01, 2022
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 Identification test for efficacy of sterilization for disinfection equipment... 6
Annex A (informative) Reagents and medium recipes... 18
Evaluating method for the efficacy of sterilization for
disinfection equipment
1 Scope
This document specifies the test equipment, test procedures, evaluation regulations and
precautions for the identification test of the efficacy of sterilization for pressure steam
sterilizer, dry heat sterilizer (cabinet), ethylene oxide sterilizer, low temperature steam
formaldehyde sterilizer, hydrogen peroxide gas plasma sterilizer.
This document is applicable to the evaluation of the efficacy of sterilization for pressure
steam sterilizers, dry heat sterilizers (cabinet), ethylene oxide sterilizers, low
temperature steam formaldehyde sterilizers, and hydrogen peroxide gas plasma
sterilizers.
2 Normative references
The following referenced documents are indispensable for the application of this
document. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
GBZ 2.1, Occupational exposure limits for hazardous agents in the workplace - Part
1.Chemical hazardous agents
WS/T 649, Hygienic requirement for medical purpose-low temperature steam and
formaldehyde sterilizers
WS/T 683, Microbiological requirements for disinfection test
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1 D value
Under the set exposure conditions, the time required to kill 90% of the total number of
specific test microorganisms.
3.2 carrier
Support for test microorganisms.
3.3 fully loaded
When using the disinfection equipment, the maximum allowable load placed in the
manner specified in the manufacturer's instructions.
3.4 process challenge device; PCD
A specially designed device to simulate the sterilization of articles, resistant to a specific
sterilization process, and used to evaluate the effectiveness of the sterilization process.
4 Identification test for efficacy of sterilization for disinfection
equipment
4.1 Identification test for efficacy of sterilization for pressure steam sterilizer
4.1.1 Test equipment
4.1.1.1 Test bacterial tablet. Bacillus stearothermophilus spore (ATCC 7953 or SSI K31
strain) bacterial tablet (cloth or filter paper). The amount of recovered bacteria is
≥1×105 CFU/tablet. Under the condition of 121℃±0.5℃, D value is ≥1.5min. Self-
contained biological indicators that meet the above requirements may also be used.
4.1.1.2 Standard biological test bag. 23cm×23cm×15cm ordinary cotton bag (30±6
warp threads per 10mm, 27±5 weft threads per 10mm). The mass is 1.5kg±0.045kg.
4.1.1.3 Ventilation storage box. The volume is 22cm×13cm×6cm.
4.1.1.4 Fully loaded items. The items specified in the instruction manual.
4.1.1.5 Reagents. Phosphate buffered saline (PBS, see A.1 of Annex A); tryptone soy
agar medium (TSA, see A.3); bromocresol purple peptone medium (see A.4).
4.1.2 Test steps
The test steps are as follows.
a) Put the two bacterial tablets into sterilized small paper bags (can also use
biological indicators directly).
b) Place the paper bag containing the tablet or two self-contained biological
indicators of Bacillus stearothermophilus spores (ATCC 7953 or SSI K31 strain)
in the center of the standard test pack. Make standard biological test packs
(disposable standard biological test packs that meet the requirements of 4.1.1.1 can
also be used).
c) For lower exhaust pressure steam sterilizers, place biological test packs in the
4.2.1 Test equipment
4.2.1.1 Test bacterial tablet. The spore suspension and spore tablets of Bacillus subtilis
var. black for laboratory use are prepared according to the method of WS/T 683.The
amount of bacteria recovered from the tablet is ≥1×106 CFU/ tablet. Under the condition
of temperature of 160℃±2℃, D value is ≥2.5min. Self-contained biological indicators
that meet the above requirements may also be used. The bacterial carrier is a stainless-
steel sheet with a diameter of 12mm, or a glass slide with an area of 10mm×10mm. If
necessary, add or switch to other carriers.
4.2.1.2 Reagents. Phosphate buffered saline (PBS, see A.1); tryptone soy broth (TSB,
see A.2); tryptone soy agar (TSA, see A.3).
4.2.1.3 Fully loaded items. The items specified in the instruction manual.
4.2.2 Test steps
The test steps are as follows.
a) Take 2 bacterial tablets for each test as a group. Lay flat in sterile petri dishes. Do
not overlap. Cover them. Put them in each layer of the sterilizer (cabinet). The
inner, middle and outer 3 points of the diagonal line are placed diagonally across
adjacent layers. Put items in the cabinet until it is fully loaded.
b) Close the appliance (cabinet) door. Power it on. Run 1 cycle according to the
design program time of the sterilizer (cabinet) for sterilization. After sterilization
is completed, the cabinet door can be opened only when the temperature of the
sterilizer naturally drops below 50°C. Remove the dish. Take out the bacterial
tablets and inoculate into a test tube containing 5.0mL of TSB. Incubate for 7d in
a constant temperature incubator at 36°C ± 1°C. Conduct daily observations from
the third day. Those with wrinkled biofilm on the turbid surface indicate bacterial
growth and are determined as positive. Those with clarification indicate sterile
growth and are determined as negative. For the broth tube that is difficult to
determine, take 0.1mL~0.2mL of the suspension and inoculate the TSA plate.
Apply evenly with a sterilized L stick. Incubate in a constant temperature incubator
at 36°C ± 1°C. Conduct smear staining after 48h. Observe the colony morphology
under the microscope. Or further refer to the relevant standards to do other tests to
determine whether the grower is the test bacteria. If there is contamination by non-
test bacteria, the cause shall be found, and the test shall be repeated.
c) For bacterial count control group, take 2 pieces of the same batch of test bacterial
tablets at room temperature. After the test group is sterilized and inoculated,
immediately transfer into test tubes containing 5.0mL of PBS, respectively. Vibrate
and beat 80 times. According to the method shown in WS/T 683, count the viable
bacterial culture.
d) For positive control group, put 2 pieces of bacteria tablets in the same batch at
room temperature. After the test group is sterilized and inoculated, immediately
transfer into test tubes containing 5.0mL of TSB. Put in incubator for 7d. From the
3rd day onwards, observe the results simultaneously with the test group.
e) For negative control group, inoculate 2 pieces of uncontaminated carrier into
5.0mL of TSB, respectively. Put in incubator for qualitative culture. Observe for
bacterial growth.
f) Repeat the test for 5 times.
4.2.3 Evaluation regulations
4.2.3.1 To determine that the efficacy of sterilization of the sterilizer is qualified, it must
meet the requirements at the same time. In the 5 sterilization tests, the amount of
recovered bacteria in the control group is ≥1×106 CFU/tablet in each test. There is
bacterial growth in the positive control group. There is aseptic growth in the negative
control group. All test bacterial tablets are aseptically grown.
4.2.3.2 When self-contained biological indicators are used for evaluation, the results
are determined according to the instruction manual.
4.2.4 Precautions
4.2.4.1 Strictly abide by aseptic technique regulations.
4.2.4.2 The test shall be carried out under full load conditions.
4.3 Identification test for efficacy of sterilization for ethylene oxide sterilizer
4.3.1 Test equipment
4.3.1.1 Test bacteria tablets. Bacillus subtilis var. Niger (ATCC 9372) spores or
biological indicators thereof. When the ethylene oxide concentration is
600mg/L±30mg/L, the temperature is 54℃±1℃ and the relative humidity is 60%±10%,
the D value is ≥2.5min (use ethylene oxide mixed gas) or D value is ≥ 2.0min (use 100%
ethylene oxide pure gas). The bacterial carrier is filter paper. The area is 10mm×10mm.
Add or use other carriers if necessary. The amount of recovered bacteria shall be greater
than or equal to 1×106 CFU/tablet. Self-contained biological indicators that meet the
above requirements may also be used.
4.3.1.2 Polyethylene plastic bag. The size is 60mm×40mm. The material thickness is
0.15mm~0.25mm.
4.3.1.3 Reagents. Phosphate buffered saline (PBS, see A.1); tryptone soy broth (TSB,
see A.2); tryptone soy agar (TSA, see A.3).
4.3.1.4 Fully loaded items. The items specified in the instruction manual.
g) Place 2 pieces of uncontaminated carrier in the test tubes that contain 5.0mL of
TSB, respectively. Put in incubator for qualitative culture. Observe if there is
bacterial growth. Use as a negative control group.
h) Repeat the test for 5 times.
4.3.3 Evaluation regulations
4.3.3.1 To determine the sterilization effect of the sterilizer is qualified, it must meet
the following requirements. The amount of recovered bacteria in the control group shall
be greater than or equal to 1×106 CFU/tablet. There is bacterial growth in the positive
control group. There is aseptic growth in the negative control group. All the test groups
are aseptically grown.
4.3.3.2 For TSB whose results are difficult to judge, take 0.1mL~0.2mL of the
suspension to inoculate the TSA plate. Apply evenly with a sterile L stick. Incubate in
a constant temperature incubator at 36°C ± 1°C. Conduct smear staining after 48h.
Observe colony morphology under microscope. Or further refer to the relevant
standards to do other tests to determine whether there is growth or whether the growth
is the test bacteria. If it is the test bacteria, it will be judged as unqualified for
sterilization. If it is non-test bacteria, the test shall be repeated.
4.3.3.3 When self-contained biological indicators are used for evaluation, the results
are determined according to the instruction manual.
4.3.4 Precautions
4.3.4.1 Ethylene oxide is flammable, explosive and toxic. To ensure the safety of the
test, the operators and test personnel shall be familiar with the performance of ethylene
oxide and the operating procedures of the equipment. Strictly follow the safety rules.
When using steel cylinders and gas tanks to store ethylene oxide, the valve shall be
opened slowly. Do not let the liquid spray out suddenly. Fire and explosion-proof
measures shall be taken at the operation site. No open flame operations and electric
sparks are allowed. It is strictly forbidden to enter the site wearing shoes with metal
soles, so as to prevent safety accidents caused by friction and sparks.
4.3.4.2 The working environment shall be well ventilated. The maximum allowable
concentration of ethylene oxide in the air at the work site is 2mg/m3.If a person inhales
too much ethylene oxide gas, it will cause poisoning symptoms. Severe cases can cause
pulmonary edema. If symptoms of poisoning appear, leave the scene immediately and
rest in a well-ventilated place. Mild cases breathe fresh air until symptoms clear up.
Severe cases shall be sent to hospital for treatment in time.
4.4 Identification test for efficacy of sterilization for low temperature steam
formaldehyde sterilizer
4.4.1 Test equipment
4.4.1.1 Test bacterial tablets. Stearothermophilus (ATCC 7953 or SSI K31) spores;
microorganisms whose resistance meets the requirements of WS/T 649.The bacterial
content is 1×106 CFU/tablet~5×106 CFU/tablet. Self-contained biological indicators
that meet the above requirements may also be used. Bacteria carriers are. 1) metal sheet.
stainless steel disc with a diameter of 12mm~15mm; 2) glass sheet. 10mm×10mm glass
sheet; 3) plastic sheet. 10mm×10mm PTFE plastic sheet.
4.4.1.2 Sterilization process challenge device (PCD). It is composed of a 1.5m-long,
2mm inner diameter PTFE blind end tube and a receiving cavity for the bacteria tablets
at the blind end. Put the bacterial carrier into the PCD. The PCS is double-layer packed.
For the carriers that cannot be placed in the PCD, they are packaged with sterilized
packaging materials and placed in the small load unit and the full load unit according
to the method of WS/T 649.
4.4.1.3 Reagents. Phosphate buffered saline (PBS, see A.1); tryptone soy agar medium
(TSA, see A.3); bromocresol purple peptone medium (see A.4).
4.4.1.4 Fully loaded items. The items specified in the instruction manual.
4.4.1.5 Neutralizer (There is no device that removes residual formaldehyde gas after
sterilization. Use a certified neutralizer during the test).
4.4.2 Test steps
The test steps are as follows.
a) Bacillus stearothermophilus spore suspension and bacterial tablets are prepared
according to the method in WS/T 683.Place the double-layer-packed PCD evenly
in the sterilization chamber according to the requirements specified in the
instruction manual. Record the distribution.
b) Under the condition of small load, for the sterilizer with the volume of the
sterilization chamber is less than 60L, at least 7 microbial carriers shall be placed.
For the 60L~100L sterilizer, at least 11 microbial carriers are placed. For sterilizers
larger than 100L, on this basis, add 1 microbial carrier for every 100L increase in
volume. The selection of placement point shall first consider the position that it is
the most difficult to sterilize. The rest are distributed evenly in the sterilizer
chamber.
c) Sterilize according to the formaldehyde concentration specified in the instruction
manual, 0.5 times the effective action time (half of the sterilization treatment time
in the sterilization cycle), and the temperature inside the device.
d) After the sterilization procedure, take out the contamination carrier under aseptic
conditions. Put the Bacillus stearothermophilus spore carrier into the bromocresol
purple peptone culture solution containing the neutralizer (the self-contained
biological indicator is operated according to the instruction manual). Culture in a
constant temperature incubator at 56℃±2℃ for 7d as the test group.
e) Put 2 spore carriers of Bacillus stearothermophilus for the same batch of test into
test tubes containing 5.0mL of diluent. Vibrate and beat 200 times for each.
According to the method shown in WS/T 683, count the viable bacteria culture.
Calculate the amount of bacteria recovered from the test bacterial tablet.
f) Put two spore carriers of Bacillus stearothermophilus for the same batch of test
into the bromocresol purple peptone culture solution. Culture in a constant
temperature incubator at 56℃±2℃ for 7d as a positive control group.
g) Put 2 uncontaminated bacterial carriers in the same batch into the bromocresol
purple peptone culture solution. Cultured in a constant temperature incubator at
56°C ± 2°C for 7d as a negative control group.
h) Repeat the test 5 times.
4.4.3 Evaluation regulations
4.4.3.1 To determine that the sterilization effect of the sterilizer is qualified, it must
meet the requirements at the same time. The amount of testing recovered bacteria of the
bacterial count control group in each test is 1×106 CFU/tablet~5×106 CFU/tablet. There
is bacterial growth in the positive control group. There is aseptic growth in the negative
control group. There is aseptic growth in the test group.
4.4.3.2 When self-contained biological indicators are used for evaluation, the results
are determined according to the instruction manual.
4.4.4 Precautions
4.4.4.1 Formaldehyde is toxic. The working environment shall be well ventilated. The
maximum allowable concentration in the air of the workplace shall meet the
requirements of GBZ 2.1.
4.4.4.2 To eliminate residual formaldehyde, natural ventilation can be used. Or use 25%
ammonia water to heat and evaporate or spray for neutralization.
4.4.4.3 If the low temperature steam formaldehyde sterilizer has multiple sterilization
procedures, each sterilization procedure shall be verified separately.
4.5 Identification test for efficacy of sterilization for hydrogen peroxide gas plasma
sterilizer
4.5.1 Test equipment
4.5.1.1 Test contamination carrier. Bacillus stearothermophilus (ATCC 7953 or SSI
K31) spores, with qualified resistance identification. The contamination carrier is a
stainless-steel needle with a diameter of ≤0.4mm. The limit is that the lumen is not
......
GB 15981-1995
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 11.080
C 59
Evaluating method and standard for the efficacy of
disinfection and sterilization
ISSUED ON: JANUARY 23, 1996
IMPLEMENTED ON: JULY 01, 1996
Issued by: State Bureau of Technical Supervision;
Ministry of Health of PRC.
Table of Contents
1 Scope ... 3
2 Reagent ... 3
3 Indicator bacteria ... 3
4 Chemical indicator ... 4
5 Technical requirements ... 4
6 Testing method ... 4
7 Results judgment and evaluation ... 4
8 Scope ... 5
9 Indicator bacteria ... 5
10 Physical indicators ... 5
11 Testing method ... 6
12 Judgment criteria ... 6
13 Scope ... 7
14 Physical and chemical index ... 7
15 indicator microorganisms ... 7
16 Testing method ... 7
17 Evaluation criteria of disinfection effect ... 8
Appendix A Neutralization effect test of neutralizer (Supplement) ... 9
Appendix B Qualitative disinfection test of disinfectant (Supplement) ... 13
Appendix C Quantitative disinfection test of disinfectant (Supplement) ... 16
Appendix D Organic matter protection test (Supplement) ... 19
Appendix E Hepatitis B surface antigen destruction test (Supplement) ... 21
Additional information: ... 26
Evaluating method and standard for the efficacy of
disinfection and sterilization
Article 1 -- Evaluation method and standard for the efficacy of
pressure steam sterilization
1 Scope
This method specifies the technical standards for pressure steam sterilization
and the testing methods for evaluating the sterilization effect.
This method is suitable for evaluating the sterilization effect of pressure steam
sterilization equipment.
2 Reagent
The reagents used in this standard are analytically pure (AR), unless otherwise
specified; the water is distilled water.
2.1 Peptone.
2.2 Glucose.
2.3 Bromocresol purple alcohol solution: Take 2.0 g of bromocresol purple.
Dissolve it in 100 mL of 95% ethanol.
2.4 Preparation of bromocresol purple peptone water culture medium: Dissolve
10.0 g of peptone and 5.0 g of glucose in 1000 mL distilled water. Adjust the pH
to 7.0 ~ 7.2. Then add 0.6 mL of 2% bromocresol purple alcohol solution. Shake
it uniformly. According to 5 mL/tube, pack and seal it. Put in a pressure steam
sterilizer, to sterilize it at 115 °C for 40 min, to prepare for use.
3 Indicator bacteria
Bacillus stearothermophilus spores (ATCC 7953 or SSI K31) bacterial tablets,
containing 5 x 105 ~ 5 x 106 cfu/tablet; the time D121 required to kill 90% of
microorganisms at 121 °C is 1.3 ~ 1.9 min; the killing time (KT value) is ≤ 19
min; the survival time (ST value) is ≥ 3.9 min.
bromocresol purple peptone water culture medium inoculated with each
indicator bacteria tablet does not change color, it is judged as qualified for
sterilization. When the bromocresol purple peptone water medium inoculated
with one of the indicator bacteria tablet turns from purple to yellow, it is judged
as unqualified for sterilization.
7.2 When the color of the chemical indicator becomes the same as the standard
color for sterilization, or when it is melted, it is used as the reference standard
for sterilization.
Article 2 -- Evaluation methods and standards of efficacy of
ultraviolet surface disinfection
8 Scope
This method specifies the wavelength and intensity of ultraviolet rays used for
surface disinfection, as well as physical indicators and biological testing
methods for evaluating the disinfection effect.
This method is suitable for the evaluation of the disinfection effect on the
surface of objects directly irradiated by ultraviolet rays.
9 Indicator bacteria
9.1 Escherichia coli (8099 or ATCC 25922).
9.2 Bacillus subtilis black variant spores (ATCC 9372).
10 Physical indicators
10.1 When the voltage is 220 V, the ordinary 30 W straight tube ultraviolet lamp
has, at the use conditions of room temperature of 20 ~ 25 °C, an ultraviolet
radiation intensity of 253.7 nm (1 m vertical) shall be ≥ 70 µW/cm2.
10.2 When the voltage is 220 V, the high-intensity ultraviolet lamp has, at the
use conditions of the room temperature of 20 ~ 25 °C, an ultraviolet radiation
intensity of 253.7 nm (1 m vertical) shall be ≥ 200 µW/cm2.
10.3 The radiation dose is calculated according to formula (1):
Dose (μW • s/cm2) = Intensity (μW/cm2) x time (s)………………………… (1)
12.2 When it reaches the physical testing standard, it is used as the reference
standard for disinfection.
Article 3 -- Evaluation methods and criteria of disinfection
effect of liquid disinfectants
13 Scope
This method specifies the biological testing methods and evaluation criteria for
the disinfection effect of disinfectants.
This method is suitable for evaluating the disinfection effect of disinfectants on
various objects.
14 Physical and chemical index
Put the disinfectant in a water bath at 20 ± 2 °C. Determine the shortest time
(min) required to kill the indicator microorganisms to achieve disinfection or
sterilization at the use concentration.
15 indicator microorganisms
15.1 Bacteria
15.1.1 Bacterial propagule: Staphylococcus aureus (ATCC 6538), Escherichia
coli (8099 or ATCC 25922).
15.1.2 Bacterial spores: Bacillus subtilis black variant spores (ATCC 9732).
15.2 Fungus: Candida albicans (ATCC 10231).
15.3 Hepatitis B surface antigen: Purified antigen (1.0 mg/mL).
16 Testing method
16.1 Neutralization test (see Appendix A).
16.2 Qualitative disinfection test of disinfectant (see Appendix B).
16.3 Quantitative disinfection test of disinfectant (see Appendix C).
16.4 Sterilization energy test of disinfectant (see Appendix D).
Appendix A
Neutralization effect test of neutralizer
(Supplement)
A1 Summary of contents
In order to accurately evaluate the killing effect of disinfectants on
microorganisms, it is required to select an appropriate neutralizer in the
disinfection test. The selected neutralizer can not only stop the disinfectant's
microbicidal killing effect in time, but also the neutralizer itself and its reaction
product with the disinfectant (hereinafter referred to as neutralized product) still
need no inhibition or killing effect on microorganisms, meanwhile there is no
adverse effect on the culture medium.
A2 Medium and reagents
A2.1 Nutrient agar medium
Ingredient: Peptone 10.00 g
Beef extract 3.00 g
Sodium chloride 5.00 g
Agar 15.00 g
Distilled water 1000.00 mL
Preparation method: In addition to agar, the other ingredients are dissolved in
distilled water. Adjust the pH to 7.2 ~ 7.4. Add the agar and heat to dissolve it.
Filter it pack contain it. After being subject to pressurized steam action at 121 °C
for 30 minutes, sterilize it to prepare for use.
A2.2 0.03 mol/L phosphate buffer (pH 7.2 ~ 7.6, hereinafter referred to as PBS).
Ingredients: Disodium hydrogen phosphate: 2.84 g
Potassium dihydrogen phosphate: 1.36 g
Distilled water: 1000.00 mL
Preparation method: Dissolve disodium hydrogen phosphate and potassium
dihydrogen phosphate in distilled water at a pH of 7.2 ~ 7.4; pack it separately;
sterilize it in pressure steam at 121 °C for 30 minutes to prepare for use.
Appendix B
Qualitative disinfection test of disinfectant
(Supplement)
B1 Summary of contents
The qualitative disinfection test is a test method to determine whether there is
bacterial growth in the sample after being subject to the action from the
disinfection factor. It is used for the identification of the sterilization effect of the
disinfection factor and the preliminary evaluation of the sterilization effect of the
disinfectant.
B2 Medium and reagents
B2.1 Ordinary broth medium
B2.1.1 Ingredients: Peptone: 10.00 g
Sodium chloride: 5.00 g
Meat extract: 1000.00 mL
B2.1.2 Preparation method: Add peptone and sodium chloride into the meat
extract. Dissolve at low temperature. Adjust the pH to weakly alkaline. Boil it.
Filter it clean. Adjust the pH so that it will be 7.2 ~ 7.4 after sterilization. Sterilize
it in pressurized steam to prepare for use.
B2.2 Reagent
B2.2.1 Diluent: 0.03 mol/L PBS (pH 7.2 ~ 7.4) containing 1% peptone.
B2.2.2 Sterilized distilled water.
B2.2.3 Neutralizer: Select it according to Appendix A of this standard.
B3 Equipment
B3.1 Sterilized graduated pipette (1.0, 5.0, 10.0 mL).
B3.2 Sterilized test tube.
B3.3 Sterilized conical flask.
B3.4 Alcohol lamp.
B3.5 Constant temperature water bath tank.
Appendix C
Quantitative disinfection test of disinfectant
(Supplement)
C1 Summary of contents
Quantitative disinfection test is a test method to determine the number of
microorganisms remaining in a sample after being affected by disinfection
factors; the result is expressed in terms of killing rate. It is used to evaluate the
killing effect of disinfectants.
C2 Medium and reagents
C2.1 Ordinary nutrient agar medium: It is prepared according to A2.1 of this
standard.
C2.2 Reagent
C2.2.1 Diluent: 0.03 mol/L PBS (pH 7.2 ~ 7.4) containing 1% peptone.
C2.2.2 Sterilized distilled water.
C2.2.3 Neutralizer: Choose according to Appendix A of this standard.
C2.2.4 0.03 mol/L PBS (pH 7.2 ~ 7.4).
C2.2.5 Eluent: PBS containing neutralizer, 1% peptone, 0.1% Tween 80.
C3 Equipment
C3.1 Sterilized graduated pipette (1.0, 5.0, 10.0 mL).
C3.2 Sterilized test tube.
C3.3 Sterilized conical flask.
C3.4 Sterilized plates (9 cm in diameter).
C3.5 Constant temperature water bath tank.
C3.6 Constant temperature incubator.
C3.7 Alcohol lamp.
C3.8 Colony counter.
Appendix D
Organic matter protection test
(Supplement)
D1 Summary of contents
The organic matter protection test is to determine the disinfectant's killing effect
on microorganisms under the condition of organic matter protection, expressed
as the killing rate. The result is compared with the disinfectant's quantitative
disinfection test, to evaluate the influence of organic matter on the disinfectant's
bactericidal ability.
D2 Medium and reagents
D2.1 Ordinary nutrient agar medium, which is prepared according to A2.1 of
this standard.
D2.2 Reagents:
D2.2.1 Diluent: Same as C2.2.1.
D2.2.2 Sterilized distilled water.
D2.2.3 Neutralizer: Select it according to Appendix A of this standard.
D2.2.4 0.03 mol/L phosphate buffer (pH 7.2 ~ 7.4) (abbreviated as PBS).
D2.2.5 Eluent: Physiological saline which contains 1% peptone and 0.1%
Tween 80.
D2.2.6 Add calf serum to the bacterial suspension, to make its final
concentration at 10%.
D3 Equipment
Same as C3 of this standard.
D4 Test method
D4.1 Before the test, count the viable bacteria in the bacterial solution. Use
diluent to dilute it. Add calf serum, to make the final serum content of 10% and
the bacterial count of 5 x 105 ~ 5 x 106 cfu/mL, which is used as a test bacteria
suspension.
D4.2 The following steps are the same as C4.1.2 ~ C4.1.8 of this standard.
Appendix E
Hepatitis B surface antigen destruction test
(Supplement)
E1 Summary of contents
The hepatitis B surface antigen (HBsAg) destruction test is a test method to
evaluate the ability of disinfection factors to inactivate hepatitis B virus (HBV)
using the antigen activity of HBsAg as an indirect marker.
It is suitable for evaluating the disinfection effect of chemical disinfectants and
ultraviolet rays on HBV.
E2 Reagent
E2.1 Calf serum (56 °C, 30 min inactivation).
E2.2 0.01 mol/L phosphate buffered saline (PBS, pH 7.2 ~ 7.4).
E2.3 Purified HBsAg (1.0 mg/mL).
E2.4 Reagents for solid-phase radioimmunoassay, requiring specificity 100%,
precision CV ≤ 15%, sensitivity ≤ 1.0 ng/mL, linearity r > 0.95.
E2.5 Enzyme-linked immunosorbent assay reagents, requiring 100% specificity,
precision CV ≤ 15%, sensitivity ≤ 3.2 ng/mL, linearity r > 0.95.
E3 Equipment
E3.1 Pipette: It is composed of test tube, pipette (4 kinds: 0.1, 1.0, 5.0, 10.0
mL), micro sample injector (100.0 μL).
E3.2 Carrier (1.5 cm diameter stainless steel sheet).
E3.3 r-immune counter.
E3.4 Enzyme-linked immunoassay instrument.
E3.5 Low temperature refrigerator (-30 °C ~ -70 °C).
E4 Preparation of HBsAg suspension
E4.1 Take 9.4 mL of 0.01 mol/L PBS (pH 7.2 ~ 7.4). Add 0.6 mL of calf serum,
to prepare a suspension containing 6% calf serum. Then take 9.0 mL of the
PBS and add 1.0 mL of purified HBsAg suspension which has a concentration
d. 0.9 mL of PBS containing 10% calf serum + 0.1 mL of HBsAg suspension;
e. 0.9 mL of neutralizer containing 10% calf serum + 0.1 mL of HBsAg
suspension;
f. 0.9 mL of neutralized product solution + 0.1mL of PBS.
Only in groups c, d, e are the measured values of HBsAg activity similar (with
a difference of less than 10%) and are significantly more than those of group b.
The HBsAg activity of group a cannot be detected or the detected activity is
significantly less than that of group b. The group f's negative control is normal,
then it can prove that the selected neutralizer and its dosage are appropriate.
E6.3 When conducting disinfection tests, adjust the amount of neutralizer
appropriately according to the principle of neutralization of the equivalent of
disinfectant and neutralizer. After the neutralization effect is measured
according to the procedures of E6.2 again, the antigenic destruction test can
be carried out.
E7 Destruction test method
E7.1 Suspension method
The suspension method refers to a test method in which HBsAg interacts with
a disinfectant in a suspension and the effect of antigenic destruction is observed.
E7.1.1 The concentration of HBsAg suspension is 104 times the sensitivity of
the testing reagent. If the sensitivity of the testing reagent is 1 ng/mL, the
concentration of HBsAg shall be 10 μg/mL.
E7.1.2 Take 2.7 mL of PBS containing 5% calf serum and add it to 0.3 mL of
HBsAg suspension at a concentration of 100 μg/mL. Mix well.
E7.1.3 Take 20 mL of calf serum and add it to 80 mL of sterilized neutralizer
solution. Mix well.
E7.1.4 Destruction test: Mix the disinfectant with 1.25 times the concentration
of the predetermined disinfectant with the HBsAg suspension in a ratio of 4:1.
The volume of the mixed solution is not less than 1.5 mL. Then place it in a
water bath at 20 ± 2 °C for a specified time. Immediately take 0.3 mL of the
mixed solution and mix it with an equal volume of 20% calf serum neutralizer.
Let it action for 10 ~ 30 min. Take samples to determine the activity of residual
HBsAg. Make two parallel determinations for each sample, 0.1 mL for each.
Take the average value to determine the destruction effect. Observe 3
concentrations for each disinfectant; observe 4 action times for each
concentration. The test is repeated 5 times.
activity of residual HBsAg. Take 2 sets for each sample, 0.1 mL for each set.
Take the average value to determine the antigenic destruction effect. Observe
3 concentrations for each disinfectant. Observe 4 action times for each
concentration. The test is repeated 5 times.
When observing the effect of ultraviolet rays destroying HBsAg, place the
contaminated carrier directly under the action factor. After the action reaches
the prescribed dosage, move the carrier into a test tube containing 1.0 mL of
10% calf serum PBS. Knock and shake 200 times. Take samples to test the
activity of residual HBsAg. Take 2 sets for each sample, 0.1 mL for each set.
Take the average value to determine the destruction effect. The test is repeated
5 times.
E7.2.3 Positive control
When observing the effect of disinfectant on destroying HBsAg, add 50 μL of
test disinfectant to a test tube containing 1.0 mL of 10% calf serum neutralizer.
Act for 10 ~ 30 minutes to prepare a neutralized product solution. Take 1.0 mL
of the neutralized product solution and add it to a large test tube. Then move in
the drip-stained HBsAg carrier. Knock and shake 200 times. Test 3 sets for each
sample, 0.1 mL for each set. Take the average value as the positive control
value.
When observing the effect of ultraviolet rays destroying HBsAg, directly move
the carrier contaminated with HBsAg into a test tube containing 1.0 mL of 10%
calf serum PBS (pH 7.2 ~ 7.4). Knock and shake 200 times. Test 3 sets for each
sample, 0.1 mL for each set. Take the average value as the positive control
value.
E7.2.4 Negative control
When observing the effect of disinfectant destroying HBsAg, add 50 μL of
disinfectant to a test tube containing 1 mL of 10% calf serum neutralizer. Let it
act for 10 ~ 30 min. Take samples for testing. Test 3 sets for each sample, 0.1
mL for each set. Take the average value as the negative control value.
When observing the effect of ultraviolet rays destroying HBsAg, the negative
control is PBS containing 10% calf serum (pH 7.2 ~ 7.4). Test 3 sets for each
sample, 0.1 mL for each set. Take the average value as the negative control
value.
It shall be noted that the negative control sample of the kit cannot be used for
the negative control.
E8 Judgement of destruction effect
S/N < 2.1 is taken as the eligibility criterion for HBsAg antigenic destruction.
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