CN110093398A - A kind of spore piece elution method of inspection - Google Patents
A kind of spore piece elution method of inspection Download PDFInfo
- Publication number
- CN110093398A CN110093398A CN201910374647.3A CN201910374647A CN110093398A CN 110093398 A CN110093398 A CN 110093398A CN 201910374647 A CN201910374647 A CN 201910374647A CN 110093398 A CN110093398 A CN 110093398A
- Authority
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- China
- Prior art keywords
- bacteria suspension
- screw tube
- spore
- inspection
- phosphate buffer
- Prior art date
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Links
- 238000000034 method Methods 0.000 title claims abstract description 22
- 238000007689 inspection Methods 0.000 title claims abstract description 14
- 238000010828 elution Methods 0.000 title description 8
- 241000894006 Bacteria Species 0.000 claims abstract description 44
- 239000000725 suspension Substances 0.000 claims abstract description 35
- 239000008363 phosphate buffer Substances 0.000 claims abstract description 16
- 238000010790 dilution Methods 0.000 claims abstract description 14
- 239000012895 dilution Substances 0.000 claims abstract description 14
- 229920001817 Agar Polymers 0.000 claims abstract description 4
- 239000008272 agar Substances 0.000 claims abstract description 4
- 229910019142 PO4 Inorganic materials 0.000 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 6
- 239000010452 phosphate Substances 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 5
- 239000000872 buffer Substances 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims 2
- 238000011010 flushing procedure Methods 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 3
- 239000003480 eluent Substances 0.000 description 7
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 7
- 229920000053 polysorbate 80 Polymers 0.000 description 7
- 230000008859 change Effects 0.000 description 5
- 239000011324 bead Substances 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 210000003739 neck Anatomy 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- C12Q1/06—Quantitative determination
-
- 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
- C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Toxicology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of spore pieces to elute the method for inspection, include the following steps: that 1) 10ml phosphate buffer and several beades that sterilized are encased in screw tube, spore piece is put into screw tube, fully shake until the spore piece be dissolved completely in phosphate buffer, obtain 10ml bacteria suspension；2) in aseptic operating platform, it is placed in the screw tube equipped with 9ml phosphate buffer with liquid-transfering gun from 1ml is drawn in the 10ml bacteria suspension that step 1) dissolves, it sufficiently shakes up to obtain 10ml and once dilutes bacteria suspension, gradually dilute by this method, until completing the 6th dilution；3) 1ml bacteria suspension is drawn respectively from above-mentioned screw tube in culture dish, and is poured into after 30ml agar medium mixes respectively and be put into incubator culture；4) whether the clump count observed on culture dish after cultivating is consistent with theory.By the above-mentioned means, the present invention it is simple to operation, can be avoided bacteria suspension it is sticky and blistering, it is ensured that bacterium colony is evenly distributed, improve detection precision.
Description
Technical field
The present invention relates to bacterium piece detection technique fields, elute the method for inspection more particularly to a kind of spore piece.
Background technique
Bacterium piece (spore piece) method of inspection on the market is that using Tween 80 eluant, eluent, (Tween 80 concentration exists first at present
Between 0.01%-0.1%) in open elution container afford bacteria suspension, then using physiological saline to bacteria suspension into
Row gradually dilutes.Since Tween 80 has oiliness, using can make bacteria suspension sticky after such eluant, eluent and blister, bacterium colony is caused to divide
Cloth is uneven, influences to detect precision；The dissolution and degradation of spore piece also will affect the pH value hair of normal saline dilution liquid simultaneously
It is raw to change, to influence the growing environment of bacterium bacterium colony；Furthermore open elution container is used, sterile working is unfavorable for.
Summary of the invention
The invention mainly solves the technical problem of providing a kind of spore pieces to elute the method for inspection, is able to solve existing spore
Piece elutes drawbacks described above present in the method for inspection.
In order to solve the above technical problems, one technical scheme adopted by the invention is that: a kind of spore piece elution inspection is provided
Method includes the following steps:
1) 10ml phosphate buffer and several beades that sterilized are encased in screw tube, using the tweezers clamping that sterilized
Spore piece is put it into the screw tube, is then fullyd shake, until the spore piece is dissolved completely in phosphate-buffered
In liquid, 10ml bacteria suspension is obtained；
2) in aseptic operating platform, 1ml is drawn from the 10ml bacteria suspension that step 1) dissolves with liquid-transfering gun and is placed in a dress
It in the screw tube for having 9ml phosphate buffer, sufficiently shakes up to obtain 10ml and once dilutes bacteria suspension, and marked on tube wall
10-1, then draw with same method 1ml from the screw tube and once dilute bacteria suspension and be placed in another equipped with 9ml phosphate
In the screw tube of buffer, 10ml secondary dilution bacteria suspension is obtained after sufficiently shaking up, and 10 are marked on tube wall-2, with this side
Formula gradually dilutes, and until completing the 6th dilution, obtains six dilution bacteria suspensions of 10ml, and mark 10-6Until；
3) from above-mentioned 10-1~10-61ml bacteria suspension is drawn in screw tube respectively in six culture dishes, then to each culture
30ml agar medium is poured into ware respectively and is shaken up, then culture dish is placed into incubator, carries out training in 48 ~ 72 hours
It supports；
4) whether the clump count observed on culture dish after cultivating is consistent with theory.
In a preferred embodiment of the present invention, in step 1), the pH value of the phosphate buffer is 7.1 ~ 7.3.
In a preferred embodiment of the present invention, in step 1), the diameter of the bead that sterilized is 1 ~ 2mm.
In a preferred embodiment of the present invention, in step 3), the temperature in the incubator is controlled at 20 ~ 25 DEG C.
The beneficial effects of the present invention are: the present invention is using phosphate buffer and several bead substitution Tween 80s that sterilized
Eluant, eluent elutes spore piece, can be avoided that bacteria suspension is sticky and blistering, it is ensured that bacterium colony is evenly distributed, and it is accurate to improve detection
Degree；It is gradually diluted using phosphate buffer substitution physiological saline simultaneously, eliminates Tween 80 eluant, eluent and spore piece is eluted
Dissolve after bacteria suspension so that the PH of dilution is changed, guarantee that the growing environment of bacterium does not change；Furthermore spiral spiral shell is used
Test tube is revolved as container, can prevent liquid from splashing out during the shaking process, can also be convenient for sterile working, bacteria suspension is avoided to dilute
Pollution risk in journey.
Specific embodiment
The preferred embodiments of the present invention will be described in detail below so that advantages and features of the invention can be easier to by
It will be appreciated by those skilled in the art that so as to make a clearer definition of the protection scope of the present invention.
The embodiment of the present invention includes:
A kind of spore piece elution method of inspection, includes the following steps:
1) 10ml pH value is encased in screw tube for 7.1 ~ 7.3 phosphate buffer and several beades that sterilized, is adopted
With having sterilized, tweezers clamping spore piece is put it into the screw tube, is then fullyd shake, until the spore piece is complete
It is dissolved in phosphate buffer, obtains 10ml bacteria suspension；
2) in aseptic operating platform, 1ml is drawn from the 10ml bacteria suspension that step 1) dissolves with liquid-transfering gun and is placed in a dress
It in the screw tube for having 9ml phosphate buffer, sufficiently shakes up to obtain 10ml and once dilutes bacteria suspension, and marked on tube wall
10-1, then draw with same method 1ml from the screw tube and once dilute bacteria suspension and be placed in another equipped with 9ml phosphate
In the screw tube of buffer, 10ml secondary dilution bacteria suspension is obtained after sufficiently shaking up, and 10 are marked on tube wall-2, with this side
Formula gradually dilutes, and until completing the 6th dilution, obtains six dilution bacteria suspensions of 10ml, and mark 10-6Until；
3) from above-mentioned 10-1~10-61ml bacteria suspension is drawn in screw tube respectively in six culture dishes, then to each culture
30ml agar medium is poured into ware respectively and is shaken up, then culture dish is placed into incubator, carries out training in 48 ~ 72 hours
It supports；
4) whether the clump count observed on culture dish after cultivating is consistent with theory.
Wherein, the diameter of the bead that sterilized is 1 ~ 2mm.
Temperature in the incubator is controlled at 20 ~ 25 DEG C.
The configuration method for the phosphate buffer that pH value described in the present embodiment is 7.1 ~ 7.3 are as follows: take potassium dihydrogen phosphate
0.06g adds 0.1mol/L sodium hydroxide solution 3ml, is diluted with water to 10ml to obtain the final product.
A kind of spore piece elution method of inspection of the invention is simple to operation in summary, using phosphate buffer and several
The bead that sterilized substitutes Tween 80 eluant, eluent, can be avoided using Tween 80 (concentration 0.01%-0.1%) or other eluant, eluents
Caused bacteria suspension is sticky and blisters, and causes bacterium colony to be unevenly distributed, testing result is avoided error occur；Using PH7.1-7.3
Phosphate buffer replace physiological saline (0.9% sodium chloride), eliminate Tween 80 eluant, eluent to spore piece elution dissolve after
Bacteria suspension makes the PH of dilution change, and guarantees that the growing environment of bacterium does not change；Using screw tube, concussion process is prevented
Middle liquid splash is also convenient for sterile working, reduces and causes pollution risk of bacteria suspension during Sterile dilution using bulk container.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
The above description is only an embodiment of the present invention, is not intended to limit the scope of the invention, all to utilize this hair
Equivalent structure or equivalent flow shift made by bright description is applied directly or indirectly in other relevant technology necks
Domain is included within the scope of the present invention.
Claims (4)
1. a kind of spore piece elutes the method for inspection, which comprises the steps of:
1) 10ml phosphate buffer and several beades that sterilized are encased in screw tube, using the tweezers clamping that sterilized
Spore piece is put it into the screw tube, is then fullyd shake, until the spore piece is dissolved completely in phosphate-buffered
In liquid, 10ml bacteria suspension is obtained；
2) in aseptic operating platform, 1ml is drawn from the 10ml bacteria suspension that step 1) dissolves with liquid-transfering gun and is placed in a dress
It in the screw tube for having 9ml phosphate buffer, sufficiently shakes up to obtain 10ml and once dilutes bacteria suspension, and marked on tube wall
10-1, then draw with same method 1ml from the screw tube and once dilute bacteria suspension and be placed in another equipped with 9ml phosphate
In the screw tube of buffer, 10ml secondary dilution bacteria suspension is obtained after sufficiently shaking up, and 10 are marked on tube wall-2, with this side
Formula gradually dilutes, and until completing the 6th dilution, obtains six dilution bacteria suspensions of 10ml, and mark 10-6Until；
3) from above-mentioned 10-1~10-61ml bacteria suspension is drawn in screw tube respectively in six culture dishes, then to each culture dish
It is middle to pour into 30ml agar medium respectively and shake up, then culture dish is placed into incubator, carries out training in 48 ~ 72 hours
It supports；
4) whether the clump count observed on culture dish after cultivating is consistent with theory.
2. spore piece according to claim 1 elutes the method for inspection, which is characterized in that in step 1), the phosphate is slow
The pH value of fliud flushing is 7.1 ~ 7.3.
3. spore piece according to claim 1 elutes the method for inspection, which is characterized in that in step 1), the glass that sterilized
The diameter of glass pearl is 1 ~ 2mm.
4. spore piece according to claim 1 elutes the method for inspection, which is characterized in that in step 3), in the incubator
Temperature control at 20 ~ 25 DEG C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910374647.3A CN110093398A (en) | 2019-05-07 | 2019-05-07 | A kind of spore piece elution method of inspection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910374647.3A CN110093398A (en) | 2019-05-07 | 2019-05-07 | A kind of spore piece elution method of inspection |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110093398A true CN110093398A (en) | 2019-08-06 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910374647.3A Pending CN110093398A (en) | 2019-05-07 | 2019-05-07 | A kind of spore piece elution method of inspection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110093398A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101100645A (en) * | 2007-06-20 | 2008-01-09 | 南京工业大学 | A kind of fumaric acid producing bacteria and its mutagenesis screening method and application |
| CN101497916A (en) * | 2009-01-23 | 2009-08-05 | 东华大学 | Method for detecting mould fungus inhibition effect of leather material and product thereof |
| US7871764B1 (en) * | 2008-03-18 | 2011-01-18 | The United States Of America As Represented By The United States Department Of Energy | Universal nucleic acids sample preparation method for cells, spores and their mixture |
-
2019
- 2019-05-07 CN CN201910374647.3A patent/CN110093398A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101100645A (en) * | 2007-06-20 | 2008-01-09 | 南京工业大学 | A kind of fumaric acid producing bacteria and its mutagenesis screening method and application |
| US7871764B1 (en) * | 2008-03-18 | 2011-01-18 | The United States Of America As Represented By The United States Department Of Energy | Universal nucleic acids sample preparation method for cells, spores and their mixture |
| CN101497916A (en) * | 2009-01-23 | 2009-08-05 | 东华大学 | Method for detecting mould fungus inhibition effect of leather material and product thereof |
Non-Patent Citations (4)
| Title |
|---|
| W.ELLIOTT HORNER等: "Basidiospore allergen release:Elution from intact spores", 《J ALLERGY CLIN IMMUNOL》 * |
| 孟令利: "大豆分离蛋白中嗜热菌孢子的检验", 《漯河职业技术学院学报》 * |
| 王清平等: "动物性凉拌食品中微生物指标检测与分析", 《新疆畜牧业》 * |
| 黄遵锡等: "《植酸酶的研究》", 31 March 2000, 云南科学技术出版社 * |
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Application publication date: 20190806 |
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