CN116590379A - Steam sterilization and chemical disinfection effect verification method for live-toxic wastewater treatment system - Google Patents
Steam sterilization and chemical disinfection effect verification method for live-toxic wastewater treatment system Download PDFInfo
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- 238000004659 sterilization and disinfection Methods 0.000 title claims abstract description 70
- 230000001954 sterilising effect Effects 0.000 title claims abstract description 58
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- 230000000694 effects Effects 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 28
- 238000004065 wastewater treatment Methods 0.000 title claims abstract description 24
- 238000012795 verification Methods 0.000 title claims abstract description 9
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- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 18
- KFSLWBXXFJQRDL-UHFFFAOYSA-N Peracetic acid Chemical compound CC(=O)OO KFSLWBXXFJQRDL-UHFFFAOYSA-N 0.000 claims description 18
- 239000000645 desinfectant Substances 0.000 claims description 18
- 239000001963 growth medium Substances 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 12
- 239000013642 negative control Substances 0.000 claims description 12
- 239000013641 positive control Substances 0.000 claims description 12
- 239000000523 sample Substances 0.000 claims description 12
- 239000002609 medium Substances 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 8
- 244000063299 Bacillus subtilis Species 0.000 claims description 7
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 7
- 238000001514 detection method Methods 0.000 claims description 7
- 230000003472 neutralizing effect Effects 0.000 claims description 7
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- 231100000331 toxic Toxicity 0.000 claims description 7
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- 241000193385 Geobacillus stearothermophilus Species 0.000 claims description 6
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- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 3
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- PODWXQQNRWNDGD-UHFFFAOYSA-L sodium thiosulfate pentahydrate Chemical compound O.O.O.O.O.[Na+].[Na+].[O-]S([S-])(=O)=O PODWXQQNRWNDGD-UHFFFAOYSA-L 0.000 claims description 3
- 239000003053 toxin Substances 0.000 claims description 3
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
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Abstract
The invention discloses a method for verifying steam sterilization and chemical sterilization effects of a live wastewater treatment system, which comprises the following steps: (1) preparation of instrument equipment; (2) preparing a test material; (3) a heat distribution device arrangement; (4) thermal profile data analysis; (5) observing steam sterilization effect; (6) The chemical dosing system is used as an emergency measure for emergency chemical disinfection verification. The invention has the advantages that: the temperature distribution condition of different positions during steam sterilization of the live-poison wastewater treatment system can be directly observed through heat distribution, and meanwhile, the steam sterilization and chemical sterilization effects are verified by using the biological indicator.
Description
Technical Field
The invention relates to the technical field of biological safety, in particular to a method for verifying steam sterilization and chemical disinfection effects of a live-toxic wastewater treatment system.
Background
The waste water discharged from the chemical shower, autoclave, animal raising and the like in the protection areas of the three-level and four-level biosafety laboratory should be sterilized. The live toxicity wastewater treatment system is used as one of the key protection devices of a high-grade biosafety laboratory, and the sterilization effect verification is carried out, so that the wastewater can meet the discharge requirement, and the biosafety of the laboratory can be ensured. The toxic waste water treating system adopts high temperature inactivating treatment, and has continuous and sequencing processes. The living toxicity wastewater treatment system of the national Kunming high-grade biological safety primate animal experiment center is a sequencing batch type, and mainly comprises a stainless steel tank body, a chemical dosing system, a control system, an operating system, matched water, electricity, gas and the like, wherein the system comprises 3 systems with the capacity of 1m 3 The container consists of 3 containers with the same functions, and the containers are stored one by one and are prepared one by one, and sterilization treatment is carried out in turn. The chemical dosing system is used as an emergency measure for emergency chemical disinfection, and peracetic acid, hydrogen peroxide and the like can completely kill bacillus subtilis black variant spore suspension, can be used for soaking and disinfecting high-grade biosafety laboratory wastewater, and ensures the safety of drainage in case of system failure.
However, in the prior art, steam sterilization and chemical sterilization of a live-toxic wastewater treatment system cannot directly ensure whether the sterilization is complete or not, and the verification mode is inaccurate.
Disclosure of Invention
The invention aims to provide a method for verifying steam sterilization and chemical disinfection effects of a live wastewater treatment system.
In order to solve the technical problems, the technical scheme of the invention is as follows:
1. a method for verifying steam sterilization and chemical sterilization effects of a live wastewater treatment system comprises the following steps:
(1) Preparing an instrument: the instrument and equipment comprises a live toxin wastewater treatment system, a temperature verification instrument, a constant temperature incubator and a biological safety cabinet;
(2) Test material preparation: the test materials comprise biological indicators, chemical disinfectants, culture mediums and neutralizers;
biological indicator classes include bacillus stearothermophilus biological indicators, self-contained biological indicators, bacillus subtilis black variant spore suspensions;
the chemical disinfectant is prepared from 2% hydrogen peroxide and 0.1% peracetic acid;
the culture medium adopts RM/100Red release culture medium containing soybean casein digestion culture medium and color indicator;
the neutralizer is prepared by sodium thiosulfate pentahydrate;
(3) Heat distribution device arrangement: binding the calibrated wired temperature probe and biological indicator on a detection fixture device, then placing the wired temperature probe and biological indicator into a tank body, wherein the liquid level in the tank body is 75%, 4 detection positions are arranged on the fixture, the No. 1 is positioned at an insertion port of the detection fixture device, the No. 2 position is positioned on the liquid level, the No. 3 and the No. 4 positions are positioned in the liquid level, the No. 4 position is close to the bottom of the tank body relative to the No. 3 position, and the No. 2, the No. 3 and the No. 4 positions are bound with the 4 temperature probes; by setting a steam sterilization program, observing the heat distribution in the tank body in the steam sterilization process;
(4) Analysis of heat distribution data: respectively averaging the data of the 4 temperature probes at the positions 1, 2, 3 and 4 as the temperature data of the positions, respectively selecting the temperature data within 10 minutes before and after the temperature reaches the set temperature action time, wherein the time is an abscissa, and the temperature is an ordinate to be plotted;
(5) And (3) observing steam sterilization effect: 1 self-contained biological indicators are bound at the position 1, the biological indicators which are not sterilized are used as positive controls, and the biological indicators which are sterilized by steam but do not crush the culture medium are used as negative controls; 2. binding 1 biological indicator at the positions 3 and 4 respectively, setting an unsterilized biological indicator as a positive control, and taking the unsterilized biological indicator as a blank control without spore suspension and the sterilized biological indicator as a negative control; respectively setting three sterilization procedures of 121 ℃, 20min,124 ℃, 20min and 137 ℃ and 6min for sterilization and observation;
(6) The chemical dosing system is used as an emergency measure for emergency chemical disinfection: adopting a spore suspension soaking test, adding a proper amount of chemical disinfectant into a tank, respectively preparing 2% hydrogen peroxide and 0.1% peracetic acid, uniformly mixing after the addition, and respectively taking 10ml of the mixture and placing the mixture into a sterilizing centrifuge tube for later use; referring to the evaluation method and standard of disinfection and sterilization effect, adding 2.5ml of disinfectant into 0.25ml of spore suspension, mixing, soaking and disinfecting, taking out 0.5ml of mixed solution at 2 time points of 10min and 30min, adding 4.5ml of neutralizer, neutralizing for 10min, taking out 0.5ml of mixed solution, adding RM/100Red release culture medium, and culturing at 37 ℃ for 7d; the test was repeated 3 times; 0.25ml of spore suspension is added into RM/100Red release culture medium as positive control; as a negative control, 0.5ml of medium of the neutralizing agent was added.
Preferably, the number of the steam sterilization effect observation tests in the step (5) is 3.
Preferably, the uniform mixing operation time is 5min after the chemical disinfectant in the step (6) is added.
Preferably, the biological indicator of step (5) is removed after the end of the procedure and the results are observed after incubation at 56℃for 48 h.
Preferably, the Bacillus stearothermophilus biological indicator is 1.0X10A 6 Spores/Unit～5.0×10 6 Spores/Unit。
Preferably, the self-contained biological indicator is 1.0X10 gauge 6 Spores/Unit～5.0×10 6 Spores/Unit。
Preferably, the Bacillus subtilis black variant spore suspension has a specification of 1.0X10 6 cfu/0.1ml～5.0×10 6 cfu/0.1ml。
The invention has the beneficial effects that:
the temperature distribution condition of different positions during steam sterilization of the live-poison wastewater treatment system can be directly observed through heat distribution, and meanwhile, the steam sterilization and chemical sterilization effects are verified by using the biological indicator.
Drawings
FIG. 1 is a diagram of a steam sterilization temperature probe and biological indicator arrangement;
FIG. 2 is a thermal profile of a different set-up procedure for steam sterilization;
FIG. 3 is a photograph of the effectiveness of steam sterilization of a biological indicator;
fig. 4 is a photograph of the chemical disinfection effect of a biological indicator.
Detailed Description
The following describes the embodiments of the present invention further with reference to the drawings. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
Examples
A method for verifying steam sterilization and chemical sterilization effects of a live wastewater treatment system comprises the following steps:
(1) Preparing an instrument: the instrument and equipment comprises a live toxin wastewater treatment system, a temperature verification instrument and a constant-temperature incubator;
live wastewater treatment systems, available from the company RICHSTAR, spain; KAYE valve temperature Validator, available from KAYE corporation, usa; a constant temperature incubator, available from Thermo Scientific, usa; biosafety cabinet, available from us Thermo Scientific company.
(2) Test material preparation: the test materials comprise biological indicators, chemical disinfectants, culture mediums and neutralizers;
biological indicators include Bacillus stearothermophilus (Bacillus stearothermophilus, ATCC 7953)Biological indicators (2.6X106 Spores/Unit) and +.>Self-contained biological indicators (1.9X106 Spores/Unit); bacillus subtilis black variant (Bacillus atrophaeus, ATCC 9372) spore suspension, 1.9X106 cfu/0.1ml. All available from the company Mesa Labs, USA.
The chemical disinfectant adopts peroxyacetic acid (binary package type) and 30% hydrogen peroxide, which are both domestic commercial products.
The medium was RM/100Red Release medium (containing soy casein digestion medium and color indicator) available from Mesa Labs, inc. of America.
The neutralizing agent is prepared from sodium thiosulfate pentahydrate, and is a domestic commercial product.
(3) Heat distribution device arrangement: because the live toxic wastewater treatment system has no fixed verification port, a detection fixture device is designed, and the calibrated wired temperature probe and biological indicator are bound on the device and then placed in a tank body, referring to fig. 1. The liquid level in the tank body is 75% of the volume, and the No. 2 position is on the liquid level; 3. the No. 4 position is in the liquid level, and the No. 4 position is close to the bottom of the tank body relative to the No. 3 position. 2. And 4 temperature probes are bound at the positions 3 and 4. 3 procedures (Table 1) were set up to observe the heat distribution in the tank during steam sterilization.
TABLE 1 steam sterilization procedure
| Sequence number | Temperature (. Degree. C.) | Program time (min) | Setting temperature (. Degree. C.) | Tank water level (%) |
| Ⅰ | 121 | 20 | 121 | 75 |
| Ⅱ | 121 | 20 | 124 | 75 |
| Ⅲ | 134 | 6 | 137 | 75 |
(4) Analysis of heat distribution data: and respectively averaging the data of the 4 temperature probes at the positions 2, 3 and 4 to obtain temperature data of the position, respectively selecting temperature data within 10 minutes before and after the temperature reaches the set temperature action time, and plotting the time as an abscissa and the temperature as an ordinate. When the sterilization temperature of the system is set to 121 ℃, the temperatures of the positions 3 and 4 can reach 121 ℃, and the temperature of the position 2 is only about 118 ℃ (fig. 2-A). The temperature in the liquid surface is about 3 ℃ different from the temperature on the liquid surface, and the temperature of the No. 2 position on the liquid surface can reach 121 ℃ (figure 2-B) and 134 ℃ (figure 2-C) only when the set temperature is set to 124 ℃ and 137 ℃.
(5) And (3) observing steam sterilization effect: position 1 binds 1A self-contained biological indicator, wherein the biological indicator which is not sterilized is used as a positive control, and the biological indicator which is sterilized by steam but does not crush the culture medium is used as a negative control; the positive control color changed from purple to yellow and turbidity indicated bacterial growth, and the negative control and test groups were considered sterile growth with the purple clear color unchanged. 2. The positions 3 and 4 are respectively bound with 1 +.>Biological indicators, non-sterilized biological indicators are set as positive controls, no spore suspension is contained, the non-sterilized biological indicators are used as blank controls, no spore suspension is contained, and the sterilized biological indicators are used as negative controls. The positive control color changed from purple to yellow and turbidity indicated bacterial growth, the blank control color was purple, the negative control and test group were yellow-purple and clear, and aseptic growth was considered. Sterilization procedures II and III were selected for sterilization and observation, and the experiment was repeated 3 times. The biological indicator was taken out after the end of the procedure, and the results were observed after culturing at 56℃for 48 hours according to the specifications of the biological indicator.
In the experiment, the biological indicators at 121 ℃ and 134 ℃ are sterilized for 20min and 6min, the biological indicators at 2, 3 and 4 are sterilized for 20min at 121 ℃ and 20min to be purple (figure 3-A), the biological indicators at 134 ℃ are sterilized for 6min to be yellow (figure 3-B), and the biological indicators at 4 positions are completely killed (table 2).
Table 2 2 different steam sterilization procedures sterilization effect
(6) The chemical dosing system is used as an emergency measure for emergency chemical disinfection: the chemical sterilization adopts a spore suspension soaking test, and a proper volume of sterilization stock solution is respectively added into a tank to prepare 2% hydrogen peroxide and 0.1% peracetic acid (table 3). Mixing for 5min after the addition, and placing 10ml into a sterilizing centrifuge tube for standby. Referring to the evaluation method of disinfection and sterilization effect and Standard, appendix B, adding 2.5ml of disinfectant into 0.25ml of spore suspension, mixing, soaking and sterilizing, taking out 0.5ml of mixed solution at 2 time points of 10min and 30min respectively, adding into 4.5ml of neutralizer, neutralizing for 10min, taking out 0.5ml of mixed solution, adding into RM/100Red release culture medium, and culturing at 37 ℃ for 7d. The test was repeated 3 times. 0.25ml of spore suspension is added into RM/100Red release culture medium as positive control; as a negative control, 0.5ml of medium of the neutralizing agent was added. The positive control medium changed from red to yellow and turbidity indicated bacterial growth; the negative control medium remained red transparent and was considered sterile grown; test group media remained red-orange in color and transparent, considered to be completely sterilized.
Table 3 3 disinfectant preparation table
| Type of disinfectant | Concentration of stock solution | Water level (%)/volume (L) in tank | Adding volume of stock solution (L) |
| 2% hydrogen peroxide | 30％ | 50/500 | 33.3 |
| 0.1% peracetic acid | 15％ | 60/600 | 4 |
In the experiment, the culture medium of the test group has red transparent color after the reaction of 0.1% peracetic acid and 2% hydrogen peroxide for 10min and 30min (figure 4), and 3 disinfectants can completely kill bacillus subtilis black variant spores after more than 10min (table 4).
Sterilizing effect of 4 3 disinfectants with different concentrations
The embodiments of the present invention have been described in detail above with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, and yet fall within the scope of the invention.
Claims (7)
1. A method for verifying steam sterilization and chemical sterilization effects of a live wastewater treatment system is characterized by comprising the following steps of: the method comprises the following steps:
(1) Preparing an instrument: the instrument and equipment comprises a live toxin wastewater treatment system, a temperature verification instrument, a constant temperature incubator and a biological safety cabinet;
(2) Test material preparation: the test materials comprise biological indicators, chemical disinfectants, culture mediums and neutralizers;
biological indicator classes include bacillus stearothermophilus biological indicators, self-contained biological indicators, bacillus subtilis black variant spore suspensions;
the chemical disinfectant is prepared from 2% hydrogen peroxide and 0.1% peracetic acid;
the culture medium adopts RM/100 RedRelease culture medium containing soybean casein digestion culture medium and color indicator;
the neutralizer is prepared by sodium thiosulfate pentahydrate;
(3) Heat distribution device arrangement: binding the calibrated wired temperature probe and biological indicator on a detection fixture device, then placing the wired temperature probe and biological indicator into a tank body, wherein the liquid level in the tank body is 75%, 4 detection positions are arranged on the fixture, the No. 1 is positioned at an insertion port of the detection fixture device, the No. 2 position is positioned on the liquid level, the No. 3 and the No. 4 positions are positioned in the liquid level, the No. 4 position is close to the bottom of the tank body relative to the No. 3 position, and the No. 2, the No. 3 and the No. 4 positions are bound with the 4 temperature probes; by setting a steam sterilization program, observing the heat distribution in the tank body in the steam sterilization process;
(4) Analysis of heat distribution data: respectively averaging the data of the 4 temperature probes at the positions 1, 2, 3 and 4 as the temperature data of the positions, respectively selecting the temperature data within 10 minutes before and after the temperature reaches the set temperature action time, wherein the time is an abscissa, and the temperature is an ordinate to be plotted;
(5) And (3) observing steam sterilization effect: 1 self-contained biological indicators are bound at the position 1, the biological indicators which are not sterilized are used as positive controls, and the biological indicators which are sterilized by steam but do not crush the culture medium are used as negative controls; 2. binding 1 biological indicator at the positions 3 and 4 respectively, setting an unsterilized biological indicator as a positive control, and taking the unsterilized biological indicator as a blank control without spore suspension and the sterilized biological indicator as a negative control; respectively setting three sterilization procedures of 121 ℃, 20min,124 ℃, 20min and 137 ℃ and 6min for sterilization and observation;
(6) The chemical dosing system is used as an emergency measure for emergency chemical disinfection: adopting a spore suspension soaking test, adding a proper amount of chemical disinfectant into a tank, respectively preparing 2% hydrogen peroxide, 5% mixed quaternary ammonium salt and 0.1% peracetic acid, uniformly mixing after the addition, and respectively taking 10ml of the mixture and placing the mixture into a sterilizing centrifuge tube for later use; referring to the evaluation method and standard of disinfection and sterilization effect, adding 2.5ml of disinfectant into 0.25ml of spore suspension, mixing, soaking and disinfecting, taking out 0.5ml of mixed solution at 2 time points of 10min and 30min, adding 4.5ml of neutralizer, neutralizing for 10min, taking out 0.5ml of mixed solution, adding RM/100 RedRelease culture medium, and culturing at 37 ℃ for 7d; the test was repeated 3 times; 0.25ml of spore suspension was added to RM/100 RedRelease medium as positive control; as a negative control, 0.5ml of medium of the neutralizing agent was added.
2. The method for verifying the steam sterilization and chemical disinfection effects of a toxic wastewater treatment system according to claim 1, wherein the method comprises the following steps: and (3) observing the steam sterilization effect in the step (5).
3. The method for verifying the steam sterilization and chemical disinfection effects of a toxic wastewater treatment system according to claim 1, wherein the method comprises the following steps: and (3) uniformly mixing the chemical disinfectant in the step (6) for 5min after the chemical disinfectant is added.
4. The method for verifying the steam sterilization and chemical disinfection effects of a toxic wastewater treatment system according to claim 1, wherein the method comprises the following steps: the biological indicator in the step (5) is taken out after the program is finished, and the result is observed after the biological indicator is cultured for 48 hours at 56 ℃.
5. The method for verifying the steam sterilization and chemical disinfection effects of a toxic wastewater treatment system according to claim 1, wherein the method comprises the following steps: the concentration of the bacillus stearothermophilus biological indicator is 1.0x10 6 Spores/Unit～5.0×10 6 Spores/Unit。
6. The method for verifying the steam sterilization and chemical disinfection effects of a toxic wastewater treatment system according to claim 1, wherein the method comprises the following steps: self-contained biological indicator concentration of 1.0X10 6 Spores/Unit～5.0×10 6 Spores/Unit。
7. The method for verifying the steam sterilization and chemical disinfection effects of a toxic wastewater treatment system according to claim 1, wherein the method comprises the following steps: the concentration of the black variant spore suspension of the bacillus subtilis is 1.0x10 6 cfu/0.1ml～5.0×10 6 cfu/0.1ml。
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