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GB/T 15981-2021 PDF in English


GB/T 15981-2021 (GB/T15981-2021, GBT 15981-2021, GBT15981-2021)
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GB/T 15981-2021: PDF in English (GBT 15981-2021)

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
......
 
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.