CN101974607B - Reagent and kit for detecting sensitivity of culture medium - Google Patents
Reagent and kit for detecting sensitivity of culture medium Download PDFInfo
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- CN101974607B CN101974607B CN2010105188201A CN201010518820A CN101974607B CN 101974607 B CN101974607 B CN 101974607B CN 2010105188201 A CN2010105188201 A CN 2010105188201A CN 201010518820 A CN201010518820 A CN 201010518820A CN 101974607 B CN101974607 B CN 101974607B
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- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 46
- 230000035945 sensitivity Effects 0.000 title claims abstract description 11
- 239000001963 growth medium Substances 0.000 title abstract description 3
- 239000007864 aqueous solution Substances 0.000 claims abstract description 32
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 28
- 210000002966 serum Anatomy 0.000 claims description 27
- 241000894006 Bacteria Species 0.000 claims description 24
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 18
- 238000004108 freeze drying Methods 0.000 claims description 15
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 14
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 14
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 14
- 229930195725 Mannitol Natural products 0.000 claims description 14
- 229920002472 Starch Polymers 0.000 claims description 14
- 229930006000 Sucrose Natural products 0.000 claims description 14
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 14
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 14
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 14
- 235000011187 glycerol Nutrition 0.000 claims description 14
- 235000013905 glycine and its sodium salt Nutrition 0.000 claims description 14
- 239000000594 mannitol Substances 0.000 claims description 14
- 235000010355 mannitol Nutrition 0.000 claims description 14
- 235000020183 skimmed milk Nutrition 0.000 claims description 14
- 239000008107 starch Substances 0.000 claims description 14
- 235000019698 starch Nutrition 0.000 claims description 14
- 239000005720 sucrose Substances 0.000 claims description 14
- 239000001888 Peptone Substances 0.000 claims description 12
- 108010080698 Peptones Proteins 0.000 claims description 12
- 235000019319 peptone Nutrition 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 229910019142 PO4 Inorganic materials 0.000 claims description 10
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 10
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 claims description 10
- 229940061607 dibasic sodium phosphate Drugs 0.000 claims description 10
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 10
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 10
- 239000010452 phosphate Substances 0.000 claims description 10
- 229910052700 potassium Inorganic materials 0.000 claims description 10
- 239000011591 potassium Substances 0.000 claims description 10
- 239000000463 material Substances 0.000 claims description 9
- 241000191967 Staphylococcus aureus Species 0.000 claims description 8
- GNBVPFITFYNRCN-UHFFFAOYSA-M sodium thioglycolate Chemical group [Na+].[O-]C(=O)CS GNBVPFITFYNRCN-UHFFFAOYSA-M 0.000 claims description 8
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 claims description 6
- 239000000428 dust Substances 0.000 claims description 6
- 230000003204 osmotic effect Effects 0.000 claims description 6
- 230000001681 protective effect Effects 0.000 claims description 6
- 239000003112 inhibitor Substances 0.000 claims description 5
- 241000222122 Candida albicans Species 0.000 claims description 4
- 241000193470 Clostridium sporogenes Species 0.000 claims description 4
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims description 4
- 229940095731 candida albicans Drugs 0.000 claims description 4
- 230000000968 intestinal effect Effects 0.000 claims description 4
- 235000019154 vitamin C Nutrition 0.000 claims description 4
- 239000011718 vitamin C Substances 0.000 claims description 4
- -1 Ox blood serum Chemical compound 0.000 claims description 3
- 229930003427 Vitamin E Natural products 0.000 claims description 3
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 claims description 3
- 238000012856 packing Methods 0.000 claims description 3
- 235000019165 vitamin E Nutrition 0.000 claims description 3
- 229940046009 vitamin E Drugs 0.000 claims description 3
- 239000011709 vitamin E Substances 0.000 claims description 3
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 7
- 238000010790 dilution Methods 0.000 abstract description 3
- 239000012895 dilution Substances 0.000 abstract description 3
- 241001052560 Thallis Species 0.000 abstract 2
- 238000009776 industrial production Methods 0.000 abstract 1
- 239000011814 protection agent Substances 0.000 abstract 1
- 239000003381 stabilizer Substances 0.000 abstract 1
- 239000000126 substance Substances 0.000 abstract 1
- 238000009777 vacuum freeze-drying Methods 0.000 abstract 1
- 239000000725 suspension Substances 0.000 description 26
- 244000005700 microbiome Species 0.000 description 17
- 238000000034 method Methods 0.000 description 15
- 238000004321 preservation Methods 0.000 description 10
- 230000000694 effects Effects 0.000 description 8
- 238000007789 sealing Methods 0.000 description 8
- 230000003019 stabilising effect Effects 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 238000007689 inspection Methods 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229930003268 Vitamin C Natural products 0.000 description 2
- 230000001332 colony forming effect Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 241000233866 Fungi Species 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 230000005059 dormancy Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000011169 microbiological contamination Methods 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000002417 nutraceutical Substances 0.000 description 1
- 235000021436 nutraceutical agent Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a reagent and a kit for detecting the sensitivity of a culture medium. The reagent comprises a component A and a component B which are independently packaged, wherein the component A is a freeze-dried substance prepared by vacuum freeze drying of first aqueous solution containing thalli and a thallus protection agent, and the component B is second aqueous solution containing a stabilizing agent capable of keeping the sleep state of the thalli. The reagent is suitable for industrial production; and when the reagent is used for detection, steps of gradual dilution, counting and the like are saved, the probability of causing infected microbe pollution is greatly reduced, the accurate number of the microbes can be ensured so as to meet the requirements specified by pharmacopeia, and the detection cost is also greatly reduced.
Description
Technical field
The present invention relates to substratum detection technique field, relate in particular to a kind of reagent and test kit that is used to detect substratum sensitivity.
Background technology
Microbiological culture media is that microorganism growth is bred needed nutraceutical matrix.In medicine and food test process, often to check the sensitivity and the suitability of substratum.To use sterility test method inspection medicine, medical apparatus, raw material, auxiliary material, and whether microbiological contamination of relative article like Chinese Pharmacopoeia regulation, check that used substratum should meet the sensitivity requirement that pharmacopeia is stipulated.Chinese Pharmacopoeia is also stipulated and will be checked that non-regulation sterilization preparation and raw material thereof, auxiliary material receive the degree of microbial contamination with the limit test of microbe method.Inspection item comprises bacterial count, fungi count, yeast count and control bacterium number.In addition, when carrying out the limit test of microbe of product, also to carry out the checking of bacterium, mould and yeast method of counting, whether be suitable for the The determination of this product to confirm the method that is adopted.No matter used substratum when carrying out limit test of microbe is finished product substratum, dehydrated medium, or by the substratum of substratum prescription preparation, all should before formal experiment, carry out the suitability inspection of substratum.
The Chinese Pharmacopoeia regulation: when carrying out limit test of microbe, test must not surpass 5 generations (the lyophilize bacterial classification that obtains from bacterial classification preservation center was the 0th generation) with the passage number of bacterial strain; When preparing, bacterium liquid should the bacterial classification that be cultured to logarithmic phase be diluted to the bacteria suspension that every 1ml contains 50～100cfu bacterium number (or spore count) with 0.9% aseptic sodium chloride solution; Bacteria suspension can use in 24 hours if be kept at 2～8 ℃ if at room temperature place and should in 2 hours, use; During the suitability inspection, get the bacterium liquid of 50～100cfu, inject aseptic plate, pour into corresponding substratum immediately, mixing solidifies and cultivates certain hour, counting under the rearmounted suitable temp.
Reviewer regular meeting runs into a lot of problems in real work.At first, the extent of dilution of bacteria suspension is difficult to grasp.The pharmacopeia regulation will dispose the bacteria suspension that 1ml contains 50～100cfu bacterium number (or spore count); If use the microscope direct-counting method, though quick and convenient, counting is not only difficult and what obtain is total count; Not only comprise mikrobe alive; Also comprised dead mikrobe, and the therefore normal and dull and stereotyped colony-forming unit of cultivating (colony forming unit, cfu) inconsistent; If use dull and stereotyped viable bacteria counting method, could count after needing to cultivate, culturing bacterium needs 12 hours at least, and yeast that incubation growth is slower or mould then need the longer time.Though bacteria suspension is preserved down at 2～8 ℃ and is allowed in 24 hours, to use; But be difficult to guarantee that bacterium counts up to full unanimity, and plate count to drop on the possibility of 50～100cfu fully less, often also need dilute adjustment concentration again; Complex operation, spended time is long.Next, but the shelf time of bacteria suspension is shorter.Even, can only use at most 24 hours 2～8 ℃ of preservations with a collection of diluent, if will after 24 hours, use, culture bacteria liquid dilutes again again, and counting not only bothers, and wastes a large amount of financial resources, material resources and manpower again.
Summary of the invention
The invention provides a kind of reagent that is used to detect substratum sensitivity, utilize this reagent can carry out rapid detection, solved the problem of traditional method length consuming time.
A kind of reagent that is used to detect substratum sensitivity; The A component and the B component that comprise independent packing; Wherein the A component is the lyophilized products of being processed through vacuum lyophilization by the mycetome and protectant first aqueous solution of thalline, and the B component is second aqueous solution that contains the stablizer that can let thalline keep dormant state.
Described thalline position streptococcus aureus, dust Xi Shi intestinal bacteria, Pseudomonas aeruginosa, subtilis, clostridium sporogenes, Candida albicans or black mold.
Described thalline protective material is mainly used in and prevents that thalline is dead in freeze-drying process, generally selects in skim-milk, trehalose, sucrose, N.F,USP MANNITOL, Ox blood serum, glycerine, glycocoll and the Zulkovsky starch one or more.The purpose that thalline protective material and thalline are processed the aqueous solution is for they are mixed, thus the protectant consumption of thalline can adjust according to actual needs, as long as can make its dissolving.
Described thalline protective material is respectively 0～10% skim-milk, 0～5% trehalose, 0～5% sucrose, 0～8% N.F,USP MANNITOL, 0～10% Ox blood serum, 0.5～5% glycerine, 0.1～0.5% glycocoll and 0～5% Zulkovsky starch by weight percent concentration in first aqueous solution and constitutes.Preferred, be respectively 5～8% skim-milk, 2～3% trehalose, 2～3% sucrose, 2～5% N.F,USP MANNITOL, 5～8% Ox blood serum, 1～3% glycerine, 0.2～0.3% glycocoll and 2～3% Zulkovsky starch by weight percent concentration in first aqueous solution.
Cell concentration is 4000～85000cfu/ml in described first aqueous solution; After first aqueous solution of selecting 60 μ l carries out vacuum lyophilization; The A components dissolved that is prepared into is in 5ml second aqueous solution time, and the gained cell concentration just meets the requirement that pharmacopeia detects.
Described lyophilized products is spherical, and water cut is no more than 3%.First aqueous solution of when the system ball, getting 60 μ l is injected in the spherical mould that internal diameter is 3mm, carries out vacuum lyophilization after the freeze forming demoulding at low temperatures.This spherical lyophilized products because the increase of lyophilized products surface-area is easy to drying, shortens freeze-drying time when freeze-drying.Because spherical lyophilized products has good rigidity, the convenient preservation is difficult for disperseing, and keeps good surface appearance, also helps the stable of lyophilized products microbe population during storage.Spherical lyophilized products is easy to dissolving in use, and prevented from caking keeps the homogeneity of dissolving back mikrobe.
Saidly comprise osmotic pressure poiser and inhibitor for stablizer; Using the purpose of inhibitor is in order to let thalline be in the environment of an anaerobic; Be in dormancy state and can be dead, the purpose of using the osmotic pressure poiser is in order to let thalline be in the suitable osmotic pressure environment.
Preferably, described osmotic pressure poiser is the mixture of peptone and inorganic salt, and described inhibitor is Thioglycolic acid sodium salt, vitamins C or vitamin E.
Described B component is to contain the aqueous solution that weight percent concentration is the Thioglycolic acid sodium salt of 0.1～0.9% sodium-chlor, 0.1～0.5% Sodium phosphate, dibasic, 0.1～0.5% potassium primary phosphate, 0.1～1.0% peptone and 1～5%.
The present invention also provides a kind of test kit that the arbitrary described reagent of claim 1～9 is used to detect substratum sensitivity that comprises, and this test kit comprises two containers that are respectively applied for packing A component and B component.
Reagent of the present invention can suitability for industrialized production, utilizes this reagent to detect, and has save steps such as gradient dilution and counting; The probability that causes living contaminants descends greatly; And can guarantee that microbe population is accurate, and reach the requirement of pharmacopeia regulation, detect cost in addition and reduce greatly.
Embodiment
Embodiment 1
Streptococcus aureus and 30 microlitres that 30 microlitres are contained 250cfu contain the aqueous solution that weight percent concentration is respectively the Zulkovsky starch of 10% skim-milk, 3% trehalose, 5% sucrose, 5% N.F,USP MANNITOL, 5% Ox blood serum, 5% glycerine, 0.1% glycocoll and 5%; Being added to internal diameter is in the spherical mould of 3mm, carries out vacuum lyophilization after the freeze forming demoulding at low temperatures.After treating the complete desorb of moisture, the sealing bottleneck is prepared into spherical lyophilized products A, and water cut is 1.0%.
The aqueous solution 5ml that will contain weight percent concentration and be 0.1% sodium-chlor, 0.1% Sodium phosphate, dibasic, 0.1% potassium primary phosphate, 0.1% peptone and 0.1% Thioglycolic acid sodium salt packs in the serum bottle; Seal the back and under the pressure of 0.1MPa, sterilized 20 minutes, be prepared into reagent B.
Detect: spherical lyophilized products A preserved 60 days down at 25 ℃, every 1 bottle of mensuration streptococcus aureus quantity wherein of getting at a distance from 10 days, or under 4 ℃, preserved 18 months, get 1 bottle of mensuration streptococcus aureus quantity wherein at quarterly intervals.The mensuration of streptococcus aureus quantity is used dull and stereotyped viable bacteria counting method.The result sees table 1.
5ml reagent B shakes up after adding to spherical lyophilized products A, suspension preserved down at 2 ℃, and the viable cell concentrations of whenever measuring in the suspension at a distance from 1 day, the mensuration of streptococcus aureus quantity is used dull and stereotyped viable bacteria counting method.The result sees table 2.
The preservation effect of the spherical lyophilized products A of table 1. (cfu/ bottle)
The stabilising effect (cfu/ml) of table 2. reagent B
Detected result shows that spherical lyophilized products A preserved 50 days down at 25 ℃, or preserves 12 months down at 4 ℃, and the mortality ratio of streptococcus aureus is less than 10%.After 5ml reagent B adds to spherical lyophilized products A, pulvis is dissolved rapidly, become the microorganism cells suspension of homogeneous after shaking.This suspension was preserved 8 days down at 2 ℃, and the microorganism cells number change is no more than 10%.
Embodiment 2
Dust Xi Shi intestinal bacteria and 30 microlitres that 30 microlitres are contained 2500cfu contain the aqueous solution that weight percent concentration is the Zulkovsky starch of 20% skim-milk, 10% trehalose, 10% sucrose, 15% N.F,USP MANNITOL, 20% Ox blood serum, 10% glycerine, 1% glycocoll and 10%; Add in the serum bottle (or ampere bottle), carry out vacuum lyophilization.After treating the complete desorb of moisture, the sealing bottleneck is prepared into pulvis A, and water cut is 2%.
The aqueous solution 5ml that will contain weight percent concentration and be the Thioglycolic acid sodium salt of 0.9% sodium-chlor, 0.5% Sodium phosphate, dibasic, 0.5% potassium primary phosphate, 1.0% peptone and 0.5% packs in the serum bottle; Seal the back and under the pressure of 0.1MPa, sterilized 30 minutes, be prepared into reagent B.
Method with embodiment 1 detects, and the result is shown in table 3 and table 4:
The preservation effect of table 3. pulvis A (cfu/ bottle)
The stabilising effect (cfu/ml) of table 4. reagent B
Detected result shows that pulvis A preserved 20 days down at 25 ℃, or preserves 12 months down at 2 ℃, and the colibacillary mortality ratio of dust Xi Shi is less than 10%.After 5ml reagent B adds to pulvis A, pulvis is dissolved rapidly, become the microorganism cells suspension of homogeneous after shaking.This suspension was preserved 10 days down at 2 ℃, and the microorganism cells number change is no more than 10%.
Embodiment 3
Dust Xi Shi intestinal bacteria and 30 microlitres that 30 microlitres are contained 400cfu contain concentration expressed in percentage by weight be respectively 10% skim-milk, 5% trehalose, 1% sucrose, 10% N.F,USP MANNITOL, 20% Ox blood serum, 1% the aqueous solution of glycerine, 1% glycocoll and 5% Zulkovsky starch; Add in the serum bottle (ampere bottle), carry out vacuum lyophilization.After treating the complete desorb of moisture, the sealing bottleneck is prepared into pulvis A, and water cut is 1.8%.
With weight percent concentration is not that the vitamin C water solution 5ml of 0.9% sodium-chlor, 0.5% Sodium phosphate, dibasic, 0.5% potassium primary phosphate, 1.0% peptone and 1% packs in the serum bottle; Seal the back and under the pressure of 0.1MPa, sterilized 30 minutes, be prepared into reagent B.
Method with embodiment 1 detects, and the result is shown in table 5 and table 6:
The preservation effect of table 5. pulvis A (cfu/ bottle)
The stabilising effect (cfu/ml) of table 6. reagent B
Detected result shows that pulvis A preserved 20 days down at 25 ℃, or preserves 12 months down at 2 ℃, and the colibacillary mortality ratio of dust Xi Shi is less than 10%.After 5ml reagent B adds to pulvis A, pulvis is dissolved rapidly, become the microorganism cells suspension of homogeneous after shaking.This suspension was preserved 8 days down at 4 ℃, and the microorganism cells number change is no more than 10%.
Embodiment 4
Pseudomonas aeruginosa and 30 microlitres that 30 microlitres are contained 5000cfu contain the aqueous solution that weight percent concentration is respectively the Zulkovsky starch of 10% skim-milk, 5% trehalose, 2.5% sucrose, 10% N.F,USP MANNITOL, 5% Ox blood serum, 0.5% glycerine, 0.5% glycocoll and 2.5%; Add in the serum bottle (ampere bottle), carry out vacuum lyophilization.After treating the complete evaporate to dryness of moisture, the sealing bottleneck is prepared into pulvis A, and water cut is 1.7%.
The aqueous solution 5ml that weight percent concentration is respectively the vitamin E of 0.5% sodium-chlor, 0.3% Sodium phosphate, dibasic, 0.3% potassium primary phosphate, 0.6% peptone and 0.5% packs in the serum bottle; Seal the back and under the pressure of 0.1MPa, sterilized 30 minutes, be prepared into reagent B.
Method with embodiment 1 detects, and the result is shown in table 7 and table 8:
The preservation effect of table 7. pulvis A (cfu/ bottle)
The stabilising effect (cfu/ml) of table 8. reagent B
Detected result shows that pulvis A preserved 20 days down at 25 ℃, or preserves 12 months down at 4 ℃, and the mortality ratio of Pseudomonas aeruginosa is less than 10%.After 5ml reagent B adds to pulvis A, pulvis is dissolved rapidly, become the microorganism cells suspension of homogeneous after shaking.This suspension was preserved 10 days down at 4 ℃, and the microorganism cells number change is no more than 10%.
Embodiment 5
Subtilis and the 30 microlitre weight percent concentration that 30 microlitres are contained 5000cfu are respectively the aqueous solution of the Zulkovsky starch of 10% skim-milk, 3% trehalose, 10% sucrose, 7.5% N.F,USP MANNITOL, 5% Ox blood serum, 5% glycerine, 0.1% glycocoll and 5%; Being added to internal diameter is in the spherical mould of 3mm, carries out vacuum lyophilization after the freeze forming demoulding at low temperatures.After treating the complete evaporate to dryness of moisture, the sealing bottleneck is prepared into spherical lyophilized products A, and water cut is 1.2%.
The aqueous solution 5ml that weight percent concentration is respectively the Thioglycolic acid sodium salt of 0.1% sodium-chlor, 0.5% Sodium phosphate, dibasic, 0.2% potassium primary phosphate, 0.9% peptone and 5% packs in the serum bottle; Seal the back and under the pressure of 0.1MPa, sterilized 30 minutes, be prepared into reagent B.
Detect: spherical lyophilized products A preserved 80 days down at 25 ℃, every 1 bottle of mensuration subtilis quantity wherein of getting at a distance from 10 days, or under 4 ℃, preserved 24 months, get 1 bottle of mensuration subtilis quantity wherein at quarterly intervals.The mensuration of subtilis quantity is used dull and stereotyped viable bacteria counting method.
5ml reagent B shakes up after adding to spherical lyophilized products A, suspension preserved down at 2 ℃, and the viable cell concentrations of whenever measuring in the suspension at a distance from 2 days, the mensuration of subtilis quantity is used dull and stereotyped viable bacteria counting method.
The preservation effect of the spherical lyophilized products A of table 9. (cfu/ bottle)
The stabilising effect (cfu/ml) of table 10. reagent B
Detected result shows that spherical lyophilized products A preserved 60 days down at 25 ℃, or preserves 24 months down at 4 ℃, and the mortality ratio of subtilis is less than 5%.After 5ml reagent B adds to pulvis A, pulvis is dissolved rapidly, become the microorganism cells suspension of homogeneous after shaking.This suspension was preserved 10 days down at 4 ℃, and the microorganism cells number change is no more than 10%.
Embodiment 6
Clostridium sporogenes and the 30 microlitre concentration expressed in percentage by weights that 30 microlitres are contained 5000cfu are respectively the aqueous solution of the Zulkovsky starch of 18% skim-milk, 3% trehalose, 6% sucrose, 7% N.F,USP MANNITOL, 5% Ox blood serum, 15% glycerine, 0.2% glycocoll and 5.5%; Add in the serum bottle (ampere bottle), carry out vacuum lyophilization.After treating the complete evaporate to dryness of moisture, the sealing bottleneck is prepared into pulvis A, water cut 1.6%.
The aqueous solution 5ml that weight percent concentration is respectively the peptone of 0.7% sodium-chlor, 0.1% Sodium phosphate, dibasic, 0.5% potassium primary phosphate and 0.4% packs in the serum bottle, seals the back and under the pressure of 0.1MPa, sterilizes 30 minutes, is prepared into reagent B.
Detection method is with embodiment 5, and detected result is shown in table 11 and table 12:
The preservation effect of table 11. pulvis A (cfu/ bottle)
The stabilising effect (cfu/ml) of table 12. reagent B
Detected result shows that pulvis A preserved 80 days down at 25 ℃, or preserves 24 months down at 4 ℃, and the mortality ratio of clostridium sporogenes is less than 5%.After 5ml reagent B adds to pulvis A, pulvis is dissolved rapidly, become the microorganism cells suspension of homogeneous after shaking.This suspension was preserved 10 days down at 4 ℃, and the microorganism cells number change is no more than 5%.
Embodiment 7
Candida albicans and the 30 microlitre concentration expressed in percentage by weights that 30 microlitres are contained 3000cfu are respectively the aqueous solution of the Zulkovsky starch of 10% skim-milk, 5% trehalose, 6% sucrose, 12% N.F,USP MANNITOL, 14% Ox blood serum, 7.5% glycerine, 0.3% glycocoll and 6%; Add in the serum bottle (ampere bottle), carry out vacuum lyophilization.After treating the complete evaporate to dryness of moisture, the sealing bottleneck is prepared into pulvis A, water cut 1.9%.
The vitamin C water solution 5ml that weight percent concentration is respectively 0.2% sodium-chlor, 0.4% Sodium phosphate, dibasic, 0.4% potassium primary phosphate, 0.2% peptone and 0.5% packs in the serum bottle; Seal the back and under the pressure of 0.1MPa, sterilized 30 minutes, be prepared into reagent B.
Detection method is with embodiment 1, and the result is shown in table 13 and table 14:
The preservation effect of table 13. pulvis A (cfu/ bottle)
The stabilising effect (cfu/ml) of table 14. reagent B
Detected result shows that pulvis A preserved 20 days down at 25 ℃, or preserves 12 months down at 4 ℃, and the mortality ratio of Candida albicans is less than 10%.After 5ml reagent B adds to pulvis A, pulvis is dissolved rapidly, become the microorganism cells suspension of homogeneous after shaking.This suspension was protected 10 days down at 4 ℃, and the microorganism cells number change is no more than 5%.
Embodiment 8
Black mold and the 30 microlitre concentration expressed in percentage by weights that 30 microlitres are contained 4200cfu are respectively the aqueous solution of the Zulkovsky starch of 13% skim-milk, 3% trehalose, 9% sucrose, 7% N.F,USP MANNITOL, 10% Ox blood serum, 5% glycerine, 0.5% glycocoll and 7%; Being added to internal diameter is in the spherical mould of 3mm, carries out vacuum lyophilization after the freeze forming demoulding at low temperatures.After treating the complete evaporate to dryness of moisture, the sealing bottleneck is prepared into spherical lyophilized products A,, water cut is 1.2%.
With weight percent concentration is that the aqueous solution 5ml of 0.7% sodium-chlor, 0.1% Sodium phosphate, dibasic, 0.3% potassium primary phosphate, 1.0% peptone, 3% Thioglycolic acid sodium salt packs in the serum bottle; Seal the back and under the pressure of 0.1MPa, sterilized 30 minutes, be prepared into reagent B.
Detection method is with embodiment 5, and the result is shown in table 15 and table 16:
The preservation effect of the spherical lyophilized products A of table 15. (cfu/ bottle)
The stabilising effect (cfu/ml) of table 16. reagent B
Detected result shows that spherical lyophilized products A preserved 60 days down at 25 ℃, or preserves 18 months down at 4 ℃, and the mortality ratio of black mold is less than 10%.After 5ml reagent B adds to spherical lyophilized products A, lyophilized products is dissolved rapidly, become the microorganism cells suspension of homogeneous after shaking.This suspension was preserved 10 days down at 4 ℃, and the microorganism cells number change is no more than 10%.
Claims (7)
1. reagent that is used to detect substratum sensitivity; It is characterized in that; The A component and the B component that comprise independent packing; Wherein the A component is the lyophilized products of being processed through vacuum lyophilization by the mycetome and protectant first aqueous solution of thalline, and the B component is second aqueous solution that contains the stablizer that can let thalline keep dormant state;
Wherein, described thalline is streptococcus aureus, dust Xi Shi intestinal bacteria, Pseudomonas aeruginosa, subtilis, clostridium sporogenes, Candida albicans or black mold;
Described thalline protective material is one or more in skim-milk, trehalose, sucrose, N.F,USP MANNITOL, Ox blood serum, glycerine, glycocoll and the Zulkovsky starch;
Described stablizer comprises osmotic pressure poiser and inhibitor; Described osmotic pressure poiser is the mixture of peptone and inorganic salt, and described inhibitor is Thioglycolic acid sodium salt, vitamins C or vitamin E.
2. reagent as claimed in claim 1; It is characterized in that described thalline protective material is respectively 0～10% skim-milk, 0～5% trehalose, 0～5% sucrose, 0～8% N.F,USP MANNITOL, 0～10% Ox blood serum, 0.5～5% glycerine, 0.1～0.5% glycocoll and 0～5% Zulkovsky starch by weight percent concentration in first aqueous solution and constitutes.
3. reagent as claimed in claim 2; It is characterized in that described thalline protective material is respectively 5～8% skim-milk, 2～3% trehalose, 2～3% sucrose, 2～5% N.F,USP MANNITOL, 5～8% Ox blood serum, 1～3% glycerine, 0.2～0.3% glycocoll and 2～3% Zulkovsky starch by weight percent concentration in first aqueous solution and constitutes.
4. reagent as claimed in claim 1 is characterized in that, the concentration of described thalline in first aqueous solution is 4000～85000cfu/ml.
5. reagent as claimed in claim 1 is characterized in that, described lyophilized products is spherical, and water cut is no more than 3%.
6. reagent as claimed in claim 1; It is characterized in that described B component is to contain the aqueous solution that weight percent concentration is respectively the Thioglycolic acid sodium salt of 0.1～0.9% sodium-chlor, 0.1～0.5% Sodium phosphate, dibasic, 0.1～0.5% potassium primary phosphate, 0.1～1.0% peptone and 1～5%.
7. one kind comprises the test kit that the arbitrary described reagent of claim 1～6 is used to detect substratum sensitivity.
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| CN106244459A (en) * | 2016-08-31 | 2016-12-21 | 沈阳化工研究院有限公司 | A kind of pseudomonas dry powder and preparation method thereof |
| CN106635802B (en) * | 2016-11-22 | 2019-10-18 | 浙江泰林生命科学有限公司 | The solid-state freeze-drying object and preparation method and application method of a kind of quantitative bacterial strain |
| CN109971673A (en) * | 2019-02-28 | 2019-07-05 | 浙江宝录检测技术有限公司 | Clostridium sporogenes employment and suitability test (E & ST) bacterial strain and preparation method thereof |
| CN109735469A (en) * | 2019-02-28 | 2019-05-10 | 中国检验检疫科学研究院 | Escherichia coli employment and suitability test (E & ST) bacterial strain and preparation method thereof |
| CN109971672A (en) * | 2019-02-28 | 2019-07-05 | 浙江宝录检测技术有限公司 | Pseudomonas aeruginosa employment and suitability test (E & ST) bacterial strain and preparation method thereof |
| CN109868238A (en) * | 2019-02-28 | 2019-06-11 | 浙江宝录检测技术有限公司 | Bacillus subtilis employment and suitability test (E & ST) bacterial strain and preparation method thereof |
| CN109797108A (en) * | 2019-02-28 | 2019-05-24 | 浙江宝录检测技术有限公司 | Aspergillus niger employment and suitability test (E & ST) bacterial strain and preparation method thereof |
| CN109735470A (en) * | 2019-02-28 | 2019-05-10 | 中国检验检疫科学研究院 | Staphylococcus aureus employment and suitability test (E & ST) bacterial strain and preparation method thereof |
| CN111321084A (en) * | 2020-04-10 | 2020-06-23 | 商城北纳创联生物科技有限公司 | Aspergillus niger quantitative bacterial tablet and preparation method thereof |
| CN114214198A (en) * | 2021-12-22 | 2022-03-22 | 中溶科技股份有限公司 | Strain preservation protective agent and preparation method and application thereof |
| CN115677039A (en) * | 2022-11-03 | 2023-02-03 | 重庆大学 | Storage method for maintaining performance and structural stability of aerobic granular sludge |
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