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GB 15981-1995 English PDF (GB15981-1995)

GB 15981-1995 English PDF (GB15981-1995)

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GB 15981-1995: Evaluating method and standard for the efficacy of disinfection and sterilization

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.
GB 15981-1995
GB
NATIONAL STANDARD OF THE
PEOPLE 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 ??W/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 ??W/cm2.
10.3 The radiation dose is calculated according to formula (1):
Dose (??W ?€? s/cm2) = Intensity (??W/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 ??L).
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 ??g/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 ??g/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 ??L 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 ??L 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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