CN113862170B - Biological indicator for monitoring sterilization effect and preparation method thereof - Google Patents
Biological indicator for monitoring sterilization effect and preparation method thereof Download PDFInfo
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- CN113862170B CN113862170B CN202110336247.0A CN202110336247A CN113862170B CN 113862170 B CN113862170 B CN 113862170B CN 202110336247 A CN202110336247 A CN 202110336247A CN 113862170 B CN113862170 B CN 113862170B
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- 239000000090 biomarker Substances 0.000 title claims abstract description 43
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 230000001954 sterilising effect Effects 0.000 title claims description 52
- 238000004659 sterilization and disinfection Methods 0.000 title claims description 37
- 230000000694 effects Effects 0.000 title claims description 23
- 238000012544 monitoring process Methods 0.000 title claims description 8
- 239000001963 growth medium Substances 0.000 claims abstract description 57
- 230000004763 spore germination Effects 0.000 claims abstract description 17
- 238000004519 manufacturing process Methods 0.000 claims abstract description 8
- YUDPTGPSBJVHCN-JZYAIQKZSA-N 4-Methylumbelliferyl-alpha-D-glucopyranoside Chemical compound C1=CC=2C(C)=CC(=O)OC=2C=C1O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O YUDPTGPSBJVHCN-JZYAIQKZSA-N 0.000 claims description 28
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 claims description 26
- 239000002609 medium Substances 0.000 claims description 25
- 230000001580 bacterial effect Effects 0.000 claims description 24
- 239000000243 solution Substances 0.000 claims description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 241000233866 Fungi Species 0.000 claims description 16
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 16
- 229960003767 alanine Drugs 0.000 claims description 15
- 229960004295 valine Drugs 0.000 claims description 15
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 14
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 14
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 claims description 12
- QNAYBMKLOCPYGJ-UWTATZPHSA-N L-Alanine Natural products C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 claims description 12
- 239000012153 distilled water Substances 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 11
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 10
- 241000193385 Geobacillus stearothermophilus Species 0.000 claims description 10
- 239000004472 Lysine Substances 0.000 claims description 10
- 229940024606 amino acid Drugs 0.000 claims description 10
- 235000001014 amino acid Nutrition 0.000 claims description 10
- 150000001413 amino acids Chemical class 0.000 claims description 10
- 239000002994 raw material Substances 0.000 claims description 10
- 238000002386 leaching Methods 0.000 claims description 8
- 239000010413 mother solution Substances 0.000 claims description 8
- 238000005303 weighing Methods 0.000 claims description 8
- CABMTIJINOIHOD-UHFFFAOYSA-N 2-[4-methyl-5-oxo-4-(propan-2-yl)-4,5-dihydro-1H-imidazol-2-yl]quinoline-3-carboxylic acid Chemical compound N1C(=O)C(C(C)C)(C)N=C1C1=NC2=CC=CC=C2C=C1C(O)=O CABMTIJINOIHOD-UHFFFAOYSA-N 0.000 claims description 7
- WJJMNDUMQPNECX-UHFFFAOYSA-N Dipicolinic acid Natural products OC(=O)C1=CC=CC(C(O)=O)=N1 WJJMNDUMQPNECX-UHFFFAOYSA-N 0.000 claims description 7
- 229930091371 Fructose Natural products 0.000 claims description 7
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 7
- 239000005715 Fructose Substances 0.000 claims description 7
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 7
- 235000019766 L-Lysine Nutrition 0.000 claims description 7
- 229910021380 Manganese Chloride Inorganic materials 0.000 claims description 7
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 claims description 7
- 210000004556 brain Anatomy 0.000 claims description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 239000008103 glucose Substances 0.000 claims description 7
- 235000002867 manganese chloride Nutrition 0.000 claims description 7
- 239000011565 manganese chloride Substances 0.000 claims description 7
- 229940099607 manganese chloride Drugs 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- 235000011164 potassium chloride Nutrition 0.000 claims description 7
- 239000001103 potassium chloride Substances 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 7
- 239000012137 tryptone Substances 0.000 claims description 7
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 6
- 229920000053 polysorbate 80 Polymers 0.000 claims description 6
- 229960003646 lysine Drugs 0.000 claims description 5
- WPLOVIFNBMNBPD-ATHMIXSHSA-N subtilin Chemical compound CC1SCC(NC2=O)C(=O)NC(CC(N)=O)C(=O)NC(C(=O)NC(CCCCN)C(=O)NC(C(C)CC)C(=O)NC(=C)C(=O)NC(CCCCN)C(O)=O)CSC(C)C2NC(=O)C(CC(C)C)NC(=O)C1NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C1NC(=O)C(=C/C)/NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)CNC(=O)C(NC(=O)C(NC(=O)C2NC(=O)CNC(=O)C3CCCN3C(=O)C(NC(=O)C3NC(=O)C(CC(C)C)NC(=O)C(=C)NC(=O)C(CCC(O)=O)NC(=O)C(NC(=O)C(CCCCN)NC(=O)C(N)CC=4C5=CC=CC=C5NC=4)CSC3)C(C)SC2)C(C)C)C(C)SC1)CC1=CC=CC=C1 WPLOVIFNBMNBPD-ATHMIXSHSA-N 0.000 claims description 5
- 238000009631 Broth culture Methods 0.000 claims description 4
- 125000005997 bromomethyl group Chemical group 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 2
- 239000002068 microbial inoculum Substances 0.000 claims description 2
- 239000012452 mother liquor Substances 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 42
- 230000004044 response Effects 0.000 abstract description 39
- 239000002253 acid Substances 0.000 abstract description 4
- 210000004215 spore Anatomy 0.000 description 30
- 230000035784 germination Effects 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 238000004090 dissolution Methods 0.000 description 6
- 238000011068 loading method Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 3
- 235000004279 alanine Nutrition 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 235000018977 lysine Nutrition 0.000 description 3
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- 244000005700 microbiome Species 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 3
- 239000010452 phosphate Substances 0.000 description 3
- 239000004474 valine Substances 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000000306 component Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
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- 238000004383 yellowing Methods 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 1
- 125000003412 L-alanyl group Chemical group [H]N([H])[C@@](C([H])([H])[H])(C(=O)[*])[H] 0.000 description 1
- 125000001176 L-lysyl group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C([H])([H])C([H])([H])C([H])([H])C(N([H])[H])([H])[H] 0.000 description 1
- 125000003580 L-valyl group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(C([H])([H])[H])(C([H])([H])[H])[H] 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
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- 238000004458 analytical method Methods 0.000 description 1
- 210000004666 bacterial spore Anatomy 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
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- 235000013305 food Nutrition 0.000 description 1
- 239000012869 germination medium Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
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- 238000010186 staining Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/045—Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/22—Testing for sterility conditions
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The invention discloses a culture medium for spore germination and growth, a biological indicator prepared by the culture medium and a corresponding preparation method thereof. According to the invention, the spore germination acid production time is faster through the specific culture medium, the response value detected by the biological indicator reader is higher, the response time is shorter, and the spore germination detection result is faster and more accurate compared with the mature biological preparation in the current market, so that the biological indicator reader has popularization and application values.
Description
Technical Field
The invention particularly relates to a biological indicator for monitoring a sterilization effect and a preparation method thereof.
Background
Sterilization refers to a measure of using strong physical and chemical factors to make all microorganisms inside and outside any object lose their growth and reproduction ability forever. The common methods for sterilization include chemical reagent sterilization, radiation sterilization, dry heat sterilization, wet heat sterilization, filtration sterilization and the like, and the degree of thoroughly sterilization is limited by the resistance of microorganisms to sterilization modes, sterilization time and sterilization mode strength. Because sterilization is a necessary condition for obtaining pure culture, sterilization technology is used in various fields such as food industry, medicine field and agricultural planting, and the sterilization effect is directly related to quality safety of industry and production benefit.
The biological indicator is a special living microorganism product, and can be used for confirming the performance of sterilization equipment, verifying a sterilization program, monitoring the sterilization effect in the production process and the like. The existing biological indicators commonly used at present have long detection time, and the working efficiency of industries related to sterilization processes is reduced.
Disclosure of Invention
In order to solve the problems, the invention provides a culture medium for spore germination and growth, which is prepared from the following raw materials in percentage by weight:
0.5 to 1.5 percent of tryptone, 0.5 to 1.5 percent of brain heart leaching broth, 0.2 to 0.4 percent of fructose, 0.1 to 0.3 percent of glucose, 0.05 to 0.15 percent of potassium chloride, 0.05 to 0.15 percent of phosphate, 0.05 to 0.2 percent of tween 80, 0.3 to 0.7 percent of alanine, 0.05 to 0.15 percent of lysine, 0.05 to 0.15 percent of valine, 0.17 to 0.25 percent of 2, 6-pyridinedicarboxylic acid, 0.10 to 0.18 percent of CaCl 2, 0.0005 to 0.0015 percent of manganese chloride, 0.01 to 0.03 percent of 4-methylumbelliferyl-alpha-D-glucopyranoside (4-MUG), 0.001 to 0.003 percent of broma powder purple, and the balance of aqueous solution with pH of 7.4 to 7.8.
Further, the material is prepared from the following raw materials in percentage by weight:
1% of tryptone, 1% of brain heart leaching liquid broth culture medium, 0.3% of fructose, 0.2% of glucose, 0.1% of potassium chloride, 0.1% of phosphate, 0.1% of tween 80, 0.5% of alanine, 0.1% of lysine, 0.1% of valine, 0.21% of 2, 6-pyridine dicarboxylic acid, 0.14% of CaCl 2, 0.001% of manganese chloride, 0.02% of 4-methylumbelliferyl-alpha-D-glucopyranoside (4-MUG), 0.002% of broma powder purple and the balance of aqueous solution with the pH value of 7.4-7.8.
Still further, the phosphate is dipotassium hydrogen phosphate; the alanine is L-alanine; the lysine is L-lysine; the valine is L-valine.
The invention also provides a biological indicator comprising spores and the aforementioned medium; the volume colony count ratio of the culture medium to the spores is 0.7mL: 1X 10 5～1×106 CFU.
Further, the volume colony count ratio of the culture medium to the spores is 0.7mL: 1X 10 6 CFU.
Still further, the spore is a dormant body of bacillus stearothermophilus Bacillus stearothermophilus.
Still further, the spores are attached to a carrier; the carrier is a paper sheet with the specification of 0.5-1 multiplied by 1-3 cm.
Further, the paper sheet was a Testy paper having a specification of 0.5X2 cm.
The invention also provides a preparation method of the culture medium, which comprises the following steps:
(1) Weighing the raw materials according to the proportion;
(2) Dissolving 4-MUG in dimethyl sulfoxide to obtain 4-MUG solution; taking L-alanine, L-lysine and L-valine, adding 5% of water according to the formula amount, and dissolving to obtain an amino acid solution; dissolving the rest raw materials in 95% water, adjusting pH to 7.4-7.8, and sterilizing to obtain mother liquor;
(3) Mixing the amino acid solution obtained in the step 2) with the 4-MUG solution, filtering and sterilizing, and mixing with the mother solution of the culture medium obtained in the step 2) to obtain the culture medium.
Further, the water is distilled water.
The invention also provides a preparation method of the biological indicator, which comprises the following steps:
a. taking spores, and attaching the spores to a carrier to obtain fungus slices;
b. and c, respectively placing the bacterial slices in the step a and the culture medium in the same biological indicator container to obtain the microbial inoculum.
Further, the spores are attached to a carrier on one side; the carrier is a paper sheet with the specification of 0.5-1 multiplied by 1-3 cm, preferably, the specification of 0.5 multiplied by 2cm; the volume colony count ratio of the culture medium to the spores is 0.7mL: 1X 10 5～1×106 CFU, preferably 0.7mL: 1X 10 6 CFU; the side, to which the spores are attached, of the bacterial sheet faces the pipe wall of the biological indicator container and is fixed in the biological indicator container; the spores are dormant bodies of bacillus stearothermophilus Bacillus stearothermophilus.
The invention finally provides application of the biological indicator in preparing a reagent for monitoring biological sterilization effect.
Further, the agent is an agent that monitors chemical agent sterilization, radiation sterilization, dry heat sterilization, wet heat sterilization and/or filter sterilization, preferably wet heat sterilization.
Further, the moist heat sterilization is boiling water, steam and/or steam autoclaving, preferably steam autoclaving.
The brain-heart leaching liquid broth is a culture medium for culturing nutrition-demanding bacteria, and comprises the following formula:
The biological indicator for monitoring the biological sterilization effect provided by the invention has the advantages that the spore germination acid production time is faster through the specific culture medium, the response value detected by the biological indicator reader is higher, the response time is shorter, and the spore germination detection result is faster and more accurate compared with the existing market mature biological preparation as a whole, so that the biological indicator has popularization and application values.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1 self-made fungus sheet
FIG. 2 test Medium response curves
FIG. 3 results of germination of bacterial spores
Detailed Description
EXAMPLE 1 preparation of the Medium for spore germination growth of the present invention
The formula of the culture medium comprises: 10g of tryptone, 10g of brain heart leaching liquid broth culture medium, 3g of fructose, 2g of glucose, 1g of potassium chloride, 1g of dipotassium hydrogen phosphate, 1g of tween 80, 5g of L-alanine, 1g of L-lysine, 1g of L-valine, 2, 6-pyridinedicarboxylic acid, 2.1g of CaCl 2, 1.4g of manganese chloride, 10mg of 4-methylumbelliferyl-alpha-D-glucopyranoside (4-MUG), 200mg of broma powder purple, and adding distilled water to 1000mL, and adjusting the pH to 7.4-7.8.
Preparation method
1) Weighing 200mg of 4-MUG according to the proportion, and dissolving with 0.5mL of dimethyl sulfoxide to obtain 4-MUG solution; weighing L-alanine, L-lysine and L-valine according to a proportion, and adding 50mL of water for dissolution to obtain an amino acid solution;
2) Taking the rest raw materials, adding 950mL of distilled water for dissolution, and sterilizing at 121 ℃ for 20min to obtain a culture medium mother solution.
3) Mixing the amino acid solution with the 4-MUG solution at a clean place, filtering and sterilizing by a sterilizing filter membrane, and adding the mixture into a culture medium mother solution to obtain the culture medium for spore germination and growth.
EXAMPLE 2 preparation of the Medium for spore germination growth of the present invention
The formula of the culture medium comprises: 5g of tryptone, 5g of brain heart leaching liquid broth, 2g of fructose, 2g of glucose, 0.5g of potassium chloride, 0.5g of dipotassium hydrogen phosphate, 800.5g of tween, 3g of L-alanine, 0.5g of L-lysine, 1g of L-valine, 1.7g of 2, 6-pyridine dicarboxylic acid, 1.0g of CaCl 2, 100mg of manganese chloride, 5mg of 4-methylumbelliferyl-alpha-D-glucopyranoside (4-MUG), 10mg of bromomethyl powder, and adding distilled water to 1000mL, and adjusting the pH to 7.4-7.8.
Preparation method
1) Weighing 4-MUG according to the proportion, and dissolving with 0.5mL of dimethyl sulfoxide to obtain 4-MUG solution; weighing L-alanine, L-lysine and L-valine according to a proportion, and adding 50mL of water for dissolution to obtain an amino acid solution;
2) Taking the rest raw materials, adding 950mL of distilled water for dissolution, and sterilizing at 121 ℃ for 20min to obtain a culture medium mother solution.
3) Mixing the amino acid solution with the 4-MUG solution at a clean place, filtering and sterilizing by a sterilizing filter membrane, and adding the mixture into a culture medium mother solution to obtain the culture medium for spore germination and growth.
EXAMPLE 3 preparation of the Medium for spore germination growth of the present invention
The formula of the culture medium comprises: 15g of tryptone, 15g of brain heart leaching liquid broth, 4g of fructose, 3g of glucose, 1.5g of potassium chloride, 1g of dipotassium hydrogen phosphate, 2g of tween 80, 7g of L-alanine, 1.5g of L-lysine, 1.5g of L-valine, 2.5g of 2, 6-pyridine dicarboxylic acid, 1.8g of CaCl 2, 15mg of manganese chloride, 300mg of 4-methylumbelliferyl-alpha-D-glucopyranoside (4-MUG), 30mg of bromomethyl powder, and adding distilled water to 1000mL, and adjusting the pH to 7.4-7.8.
Preparation method
1) Weighing 4-MUG according to the proportion, and dissolving with 0.5mL of dimethyl sulfoxide to obtain 4-MUG solution; weighing L-alanine, L-lysine and L-valine according to a proportion, and adding 50mL of water for dissolution to obtain an amino acid solution;
2) Taking the rest raw materials, adding 950mL of distilled water for dissolution, and sterilizing at 121 ℃ for 20min to obtain a culture medium mother solution.
3) Mixing the amino acid solution with the 4-MUG solution at a clean place, filtering and sterilizing by a sterilizing filter membrane, and adding the mixture into a culture medium mother solution to obtain the culture medium for spore germination and growth.
EXAMPLE 4 preparation of biological indicators according to the invention
A. taking 1X 10 6 CFU bacillus stearothermophilus spores, and loading one side of the spores on 0.5X 2cm of Telekulare paper to obtain fungus flakes;
b. And c, respectively placing the bacterial sheet in the step a and 0.7mL of the culture medium prepared in the embodiment 1 in the same biological indicator container, wherein the side, on which spores are attached, of the bacterial sheet faces the pipe wall of the biological indicator container, and fixing the bacterial sheet in the biological indicator container.
EXAMPLE 5 preparation of biological indicators according to the invention
A. Taking 1X 10 5 CFU bacillus stearothermophilus spores, and loading one side of the spores on 0.5X 2cm of Telekulare paper to obtain fungus flakes;
b. And c, respectively placing the bacterial sheet in the step a and 0.7mL of the culture medium prepared in the embodiment 1 in the same biological indicator container, wherein the side, on which spores are attached, of the bacterial sheet faces the pipe wall of the biological indicator container, and fixing the bacterial sheet in the biological indicator container.
The advantageous effects of the present invention are described below by way of test examples.
Test example 1 evaluation of the Effect of the sterilizing biological indicator of the present invention
1. Method of
1. Detection of Medium response Effect contrast
And verifying with a commercially available standard bacterial sheet and a self-made bacterial sheet, and detecting fluorescent signal response of the bacterial sheet and a detection culture medium by the machine and exploring the response result of the detection culture medium to spores.
(1) Preparing a detection culture medium according to the set culture medium components.
(2) Group 1:1 standard indicator is sold on the market, when the standard indicator is used, a glass small pipe filled with a detection culture medium is broken, so that bacteria slices are contacted with the detection culture medium, and then the small pipe is inserted into a small hole of a detector for detection;
group 2: taking out the detection culture medium in the commercial standard indicator, replacing the detection culture medium with 0.7mL of self-made detection culture medium, shaking the indicator small tube, and performing on-machine detection;
Group 3: adding 0.7mL of self-made detection culture medium into a small tube filled with self-made fungus slices, and performing machine detection after shaking;
The preparation method of the fungus tablet comprises the following steps: activating and adding value to Bacillus stearothermophilus Bacillus stearothermophilus (named Bacillus stearothermophilus 6X 1320) purchased from China center for type culture collection, fermenting, collecting spores, taking spore liquid containing 1X 10 6 CFU to a marked carrier, loading on one side, loading in a 1.5mL EP tube, and drying for later use, wherein the carrier is 0.5X 2cm of Tibet paper; when the prepared bacterial sheet is used, the carrier loaded with spores is assembled into a small container tube with the first surface facing the tube wall of the biological indicator container.
(3) A biological sterilization indicator response curve is derived, comparing the commercially available standard indicator with the homemade test media indicator response curve.
2. Detection of germination efficacy of Medium on spores
① Exploration of acid production effect of thalli: and (3) when the temperature of the indicator detector is constant at 58 ℃, adding 0.7mL of germination culture medium and a little spore liquid into the small detection tube, shaking the small detection tube, inserting the small detection tube into a small hole of the indicator detector for culture, observing and observing the color change of the culture medium in the small tube every 20min, and recording the time length for starting yellowing and complete yellowing.
② Germination effect exploration: 0.7mL of germination medium is added into the detection small tube, a small amount of spore liquid is sucked into the small tube, and the mixture is uniformly mixed. The germination status (time of bacterial cell production) of spores in the medium was observed by staining every 15 min.
3. Comparison of the response effects of the bacterial tablets of the sterilization biological indicator
Experiments response conditions of self-made fungus sheets were investigated by adding the cultured spore liquid to the paper sheets.
(1) And (3) preparation of fungus flakes: 1X 10 6 CFU of 6X1320 spore solution was added to 0.5X 2cm of the marked support and single-sided loaded. The loaded paper sheet is placed in a 1.5mL EP tube and dried for use, and when in use, the paper sheet loaded with spores is assembled into the small tube of the container with the paper sheet facing the wall of the biological indicator container.
(2) Sterilization biological indicator response effect comparison: the group comparison was performed according to the commercially available standard indicator, homemade pellet + homemade detection medium, homemade pellet + tampon + commercially available standard medium, homemade pellet + tampon + homemade medium. The cotton plug is a part for fixing the bacterial sheet at the bottom of the detection small tube, and when in detection, the bacterial sheet is contacted with a detection culture medium and put into a reader for machine-on detection;
(3) And (3) deriving a response curve of the sterilization biological indicator, comparing the response curve of the standard indicator with the response curve of the experimental group, and comparing the response conditions of each group to confirm the response effect of the fungus sheet.
2. Results and analysis
1. Detection of Medium response Effect contrast
The component proportions of the detection culture medium are determined according to the results of the early test data, and are specifically as follows:
10g of tryptone, 10g of brain heart leaching liquid broth culture medium, 3g of fructose, 2g of glucose, 1g of potassium chloride, 1g of dipotassium hydrogen phosphate, 5g of L-alanine, 1g of L-lysine, 1g of L-valine, 1g of tween 80, 2.1g of 2, 6-pyridine dicarboxylic acid, 1.4 g of CaCl 2, 10mg of manganese chloride, 20mg of brommethyl powder purple and 0.2g of 4-MUG. Distilled water is filled to 1L, and the pH is adjusted to 7.4-7.8; the preparation method is the same as in example 1.
The results of comparing the response of the test medium of the present invention with the response of the test medium in a commercially available standard indicator are shown in Table 1 and FIG. 2.
TABLE 1 detection of Medium response status
As can be seen from table 1 and fig. 2: the detection medium has a good response effect, wherein the signal response of the self-made fungus sheet and the self-made detection medium is slightly better than that of the self-made detection medium and the commercial standard indicator fungus sheet. The signal response results of the commercial standard indicator and the homemade detection culture plus commercial indicator fungus sheet show that the homemade culture medium is superior to the commercial indicator detection medium.
2. Detection of germination efficacy of Medium on spores
A small amount of Bacillus stearothermophilus 6X1320 spore solution was added to a vial containing 0.7mL of detection medium and detected by a biological sterilization indicator reader, the results are shown in Table 2 and FIG. 3.
Table 2 detection of Medium parameters (min) for spore germination efficacy
From table 2 and fig. 3, it can be seen that the medium fluorescence response was detected: peak is reached for 3-6 min; after 30min, the bacterial cells begin to appear, and after 120min-140min, most bacterial cells are changed; the color starts to change after 60min and turns yellow completely after 160min-180min, so that the detection culture medium has higher response speed and better germination effect in fluorescent signals;
3. biological indicator fungus tablet response effect contrast
The results of the response effect of the biological indicator fungus flakes are shown in Table 3
TABLE 3 results of bacterial plaque response
As can be seen from Table 3, the results of the commercial standard indicator experiments were positive within 30min, but the overall response was lower with a small slope of the curve; the self-made fungus sheet has high response value and good response effect. It is worth mentioning that the signal response of the bacterial tablet prepared by the invention is still unchanged (peak is reached at 0min or 3 min) after the bacterial tablet is placed for 90 days at normal temperature.
Conclusion: ① The response of the tamponade group is higher than the non-tamponade group, so that the use of tampons to immobilize the bacterial sheet can increase the response of the biological sterilization indicator. ② The response effect of the self-made detection culture medium is better than that of the detection culture medium of a commercially available standard indicator, and the practical requirement can be met.
In conclusion, the culture medium for spore germination and growth provided by the invention has the advantages that the spore germination and acid production time is faster, the detection response value is higher, the response time is shorter on a biological indicator reader, and the spore germination detection result is faster and more accurate compared with the current market maturation biological indicator, so that the biological indicator has popularization and application values.
Claims (8)
1. A culture medium for spore germination and growth of bacillus stearothermophilus, which is characterized in that: the material is prepared from the following raw materials in percentage by weight:
1% of tryptone, 1% of brain heart leaching liquid broth culture medium, 0.3% of fructose, 0.2% of glucose, 0.1% of potassium chloride, 0.1% of dipotassium hydrogen phosphate, 0.1% of tween 80, 0.5% of L-alanine, 0.1% of L-lysine, 0.1% of L-valine, 0.21% of 2, 6-pyridine dicarboxylic acid, 0.14% of CaCl 2, 0.001% of manganese chloride, 0.02% of 4-methylumbelliferyl-alpha-D-glucopyranoside (4-MUG), 0.002% of bromomethyl powder and the balance of distilled water, and regulating the pH to 7.4-7.8.
2. A biological indicator comprising spores and the medium of claim 1; the volume colony count ratio of the culture medium to the spores is 0.7mL: 1X 10 5~1×106 CFU;
The spores are attached to a carrier; the carrier is made of special strong paper with the specification of 0.5-1 multiplied by 1-3 cm;
the spore is dormant of bacillus stearothermophilus.
3. The biological indicator of claim 2, wherein: the specification of the special strong paper is 0.5 multiplied by 2cm.
4. A method of preparing the medium of claim 1, wherein: it comprises the following steps:
(1) Weighing the raw materials according to the proportion of claim 1;
(2) Dissolving 4-MUG in dimethyl sulfoxide to obtain 4-MUG solution; taking L-alanine, L-lysine and L-valine, adding distilled water with the formula amount of 5% to dissolve the L-alanine, the L-lysine and the L-valine to obtain an amino acid solution; dissolving the rest raw materials in 95% distilled water, adjusting pH to 7.4-7.8, and sterilizing to obtain mother liquor;
(3) Mixing the amino acid solution obtained in the step 2) with the 4-MUG solution, filtering and sterilizing, and mixing with the mother solution of the culture medium obtained in the step 2) to obtain the culture medium.
5. A method of preparing a biological indicator according to claim 2 or 3, characterized in that: it comprises the following steps:
a. taking spores, and attaching the spores to a carrier to obtain fungus slices;
b. respectively placing the bacterial slices in the step a and the culture medium in the claim 1 in the same biological indicator container to obtain the microbial inoculum;
the spores are attached to the carrier on one side; the carrier is made of special strong paper with the specification of 0.5-1 multiplied by 1-3 cm; the volume colony count ratio of the culture medium to the spores is 0.7mL: 1X 10 5~1×106 CFU;
The side, to which the spores are attached, of the bacterial sheet faces the pipe wall of the biological indicator container and is fixed in the biological indicator container;
the spore is dormant of bacillus stearothermophilus.
6. The method of manufacturing according to claim 5, wherein: the specification of the special strong paper is 0.5 multiplied by 2cm; the volume colony count ratio of the culture medium to the spores is 0.7mL: 1X 10 6 CFU.
7. Use of the biological indicator of claim 2 in the preparation of a reagent for monitoring the effect of biological sterilization, characterized in that: the reagent is a reagent for monitoring damp heat sterilization.
8. Use according to claim 7, characterized in that: the reagent is a reagent that monitors steam autoclaving.
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