CN101864386A - Method for enriching seawater bacteria by asepsis cellulose filter membrane - Google Patents
Method for enriching seawater bacteria by asepsis cellulose filter membrane Download PDFInfo
- Publication number
- CN101864386A CN101864386A CN 201010174899 CN201010174899A CN101864386A CN 101864386 A CN101864386 A CN 101864386A CN 201010174899 CN201010174899 CN 201010174899 CN 201010174899 A CN201010174899 A CN 201010174899A CN 101864386 A CN101864386 A CN 101864386A
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- China
- Prior art keywords
- filter membrane
- seawater
- cellulose filter
- bacteria
- asepsis
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Abstract
The invention relates to a method for enriching seawater bacteria by an asepsis cellulose filter membrane, comprising the following steps: (1) sterilizing a syringe and a syringe needle type filter; (2) putting the asepsis cellulose filter membrane in a sterilized filer; (3) filling seawater into the filter by the syringe, and enriching seawater bacteria; and (4) putting the cellulose filter membrane enriched with seawater bacteria on a culture medium, and cultivating the seawater bacteria. The culture method of the invention can overcome the defects that seawater contains few bacteria which are hard to cultivate; a majority of bacteria in seawater are separated and cultivated so as to provide possibility for comprehensively researching bacteria in seawater, and the invention has an important application significance.
Description
Technical field
The invention belongs to seawater bacteria separation and Culture field, particularly relate to a kind of method of utilizing the asepsis cellulose filter membrane enriching seawater bacteria.
Background technology
Abundant marine microorganism has been bred in vast marine site on the earth.The ocean area reaches 3.6 * 10
8Km
2, account for earth surface long-pending 71%, take up an area of 97% of ball Total Water, be water body geographical unit maximum on the earth.The vast oceans are cradles of life, according to statistics, in the ocean, find and biological nearly 300,000 kinds of name, account for 80% of earth species, wherein microbe species has more than 1,000,000 kinds, and the present marine microorganism of studying and identifying only accounts for 5% of all marine microorganism kinds.Unique ocean environment Microbial resources that are richly stored with, the species diversity of marine microorganism and environment heterogeneity of living in have determined its meta-bolites diversity, for the mankind provide of a great variety, molecular structure novelty, the complicated and special natural radioactivity product of physiologically active of chemical constitution.Investigation marine microorganism resource is the prerequisite and the basis of exploitation Microbial resources.It then is by substratum that marine microorganism is cultivated, and the bacterium in seawater and the oceanic sediment is isolated and cultured, and then study to reach the purpose of development and use marine microorganism resource.Because the diversity of marine microorganism, how the requirement of separation and Culture than higher, is set up a kind of separating marine method of microorganism all sidedly fully again, be the focus that everybody studies.
At present, the method of the most frequently used separation seawater bacteria is exactly direct coating method, promptly getting quantitative seawater directly is coated on the substratum with spreading rod, the shortcoming of this method is: because the bacteria containing amount in the seawater is very little, be difficult in this way bacteriums all in the seawater is is all isolated and cultured, do not reach the purpose of sufficient separation and Culture marine microorganism.
Summary of the invention
Technical problem to be solved by this invention provides a kind of method of utilizing the asepsis cellulose filter membrane enriching seawater bacteria, this method can overcome because of institute's bacteria containing amount in the seawater few, the shortcoming that is difficult to cultivate, cultivate again by first enrichment bacterium, most of bacterium in the seawater can be isolated and cultured, for the bacterium of studying in the seawater provides possibility comprehensively, has important practical application meaning.
A kind of method of utilizing the asepsis cellulose filter membrane enriching seawater bacteria of the present invention comprises:
(1) syringe, needle-based filter sterilised
Syringe, needle-based strainer is good with paper bag respectively, carry out autoclaving;
(2) in strainer, put into asepsis cellulose filter membrane
Filter membrane is packed into after pre-treatment in the above-mentioned needle-based strainer, and filter membrane and strainer contact surface are fully fitted, and do not stay the slit, when preventing to filter around leakage, a strainer is put into a filter membrane;
(3) filter enriching seawater bacteria
Earlier draw seawater with syringe, then by the needle-based strainer, enriching seawater bacteria on asepsis cellulose filter membrane;
(4) cultivate seawater bacteria
With enrichment the cellulose filter membrane of seawater bacteria place on the Zuo Beier substratum, the contact surface of cellulose filter membrane and substratum is fully fitted, and bacterium on the filter membrane can fully be absorbed nourish and grow, and carries out the cultivation of seawater bacteria.
The model of the syringe in the described step (1) is 10 milliliters a plastic injector, and the model of needle-based strainer is the interchangeable film needle-based of SwinnexPP strainer (diameter of can packing in the strainer is the filter membrane of 25mm).
Autoclaved condition in the described step (1) is 121 ℃ of temperature, time 30min.
Filter membrane pre-treatment in the described step (2) is meant the distilled water immersion of asepsis cellulose filter membrane with 70 ℃, makes it all moistening, anhydrates behind the 4h, spends the night with 70 ℃ distilled water immersion again, cleans once with 40 ℃ of distilled water immersions before the use again.The aperture of the asepsis cellulose filter membrane in the described step (2) is 0.45um.
Seawater in the described step (3) is taken from 120 ° to 123 ° of East Sea east longitudes, the marine site that north latitude is 28 ° to 31 °.
The prescription of Zuo Beier (ZoBell) substratum in the described step (5) is peptone 5g, agar 15g, and yeast extract paste 1g, FePO40.01g, artificial seawater 1000ml, the medium pH value is 7.6～7.8, cultivates based on 121 ℃ of autoclaving 20min.Described artificial seawater prescription is as follows: NaCl 23.476g, MgCl
26H
2O 10.6359g, Na
2SO
43.917g, CaCl
21.102g, KCl 0.664g, NaHCO
30.192g, KBr 0.096g, H
3BO
30.026g, SrCl
26H
2O 0.0403g, NaF 0.072g, NaNO
30.050g, NaH
2PO
40.005g, Na
2SiO
39H
2O 0.010g, MnCl
24H
2O 0.0003g, FeC
6H
5O
73H
2O 0.00054g, distilled water 1000ml.
Beneficial effect
Cultural method of the present invention can overcome because of bacteria containing amount in the seawater few, the shortcoming that is difficult to cultivate is cultivated by first enrichment bacterium again, and the most of bacterium in the seawater can be isolated and cultured, for the bacterium of studying in the seawater provides possibility comprehensively, has important practical application meaning.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Embodiment 1
Utilize the asepsis cellulose filter membrane enriching seawater bacteria, concrete steps are as follows:
(1) syringe, needle-based filter sterilised: syringe and needle-based strainer are earlier good with paper bag respectively, put into Autoclave again, in 121 ℃, and autoclaving 30 minutes;
(2) in the strainer of bacterium that went out, put into asepsis cellulose filter membrane
Asepsis cellulose filter membrane will clean earlier, and filter membrane is lain against in the cleaning vessel, with about 70 ℃ distilled water immersion, makes all moisteningly, anhydrates behind the 4h, spend the night with 70 ℃ distilled water immersion again, before the use more once with 40 ℃ of distilled water immersions cleanings;
With aseptic nipper the filter membrane that cleans is packed into and to go out in the strainer of bacterium, filter membrane and strainer contact surface will fully be fitted, and do not stay the slit, when preventing to filter around leakage, a strainer is put into a filter membrane;
(3) filter enriching seawater bacteria: draw seawater with syringe earlier, draw the 10ml seawater at every turn, then by needle-based strainer (be in the syringe seawater be injected in the strainer), enriching seawater bacteria on asepsis cellulose filter membrane through syringe needle;
(4) cultivate seawater bacteria: with aseptic nipper the cellulose filter membrane of having collected seawater bacteria is placed on the Zuo Beier substratum, the cultivation seawater bacteria, the contact surface of cellulose filter membrane and substratum is fully fitted, and bacterium on the filter membrane can fully be drawn nourish and grow.
Embodiment 2
Configuration Zuo Beier substratum, concrete steps are as follows:
(1) configuration artificial seawater: with NaCl 23.476g, MgCl
26H
2O 10.6359g, Na
2SO
43.917g, CaCl
21.102g, KCl 0.664g, NaHCO
30.192g, KBr 0.096g, H
3BO
30.026g, SrCl
26H
2O 0.0403g, NaF 0.072g, NaNO
30.050g, NaH
2PO
40.005g, Na
2SiO
39H
2O 0.010g, MnCl
24H
2O 0.0003g, FeC
6H
5O
73H
2O0.00054g, distilled water 1000ml, fully dissolving.
(2) configuration Zuo Beier substratum: with peptone 5g, agar 15g, yeast extract paste 1g, FePO40.01g adds respectively and is equipped with in the artificial seawater beaker of 1000ml;
(3) heating substratum: the substratum that will add the desired nutritional material is heated to boiling, and agar is fully dissolved;
(4) adjust pH: when the substratum after to be heated is cooled to 60 ℃ of left and right sides, with pH survey meter adjust pH to 7.6～7.8;
(5) medium sterilization:, put into Autoclave, in 121 ℃ of autoclaving 20min with the substratum branch 250ml triangular flask of packing into.
Claims (8)
1. method of utilizing the asepsis cellulose filter membrane enriching seawater bacteria comprises:
(1) syringe, needle-based filter sterilised
Syringe, needle-based strainer is good with paper bag respectively, carry out autoclaving;
(2) in strainer, put into asepsis cellulose filter membrane
Filter membrane is packed into after pre-treatment in the above-mentioned needle-based strainer, and filter membrane and strainer contact surface are fully fitted, and do not stay the slit, when preventing to filter around leakage, a strainer is put into a filter membrane;
(3) filter enriching seawater bacteria
Earlier draw seawater with syringe, then by the needle-based strainer, enriching seawater bacteria on asepsis cellulose filter membrane;
(4) cultivate seawater bacteria
With enrichment the cellulose filter membrane of seawater bacteria place on the Zuo Beier substratum, the contact surface of cellulose filter membrane and substratum is fully fitted, and carries out the cultivation of seawater bacteria.
2. a kind of method of utilizing the asepsis cellulose filter membrane enriching seawater bacteria according to claim 1, it is characterized in that: the model of the syringe in the described step (1) is 10 milliliters a plastic injector, and the model of needle-based strainer is the interchangeable film needle-based of a Swinnex PP strainer.
3. a kind of method of utilizing the asepsis cellulose filter membrane enriching seawater bacteria according to claim 1 is characterized in that: the autoclaved condition in the described step (1) is 121 ℃ of temperature, time 30min.
4. a kind of method of utilizing the asepsis cellulose filter membrane enriching seawater bacteria according to claim 1, it is characterized in that: the filter membrane pre-treatment in the described step (2) is meant the distilled water immersion of asepsis cellulose filter membrane with 70 ℃, make it all moistening, anhydrate behind the 4h, spend the night with 70 ℃ distilled water immersion again, clean once with 40 ℃ of distilled water immersions again before the use.
5. a kind of method of utilizing the asepsis cellulose filter membrane enriching seawater bacteria according to claim 1 is characterized in that: the aperture of the asepsis cellulose filter membrane in the described step (2) is 0.45um.
6. a kind of method of utilizing the asepsis cellulose filter membrane enriching seawater bacteria according to claim 1 is characterized in that: the seawater in the described step (3) is taken from 120 ° to 123 ° of East Sea east longitudes, the marine site that north latitude is 28 ° to 31 °.
7. a kind of method of utilizing the asepsis cellulose filter membrane enriching seawater bacteria according to claim 1, it is characterized in that: the prescription of the Zuo Beier substratum in the described step (5) is peptone 5g, agar 15g, yeast extract paste 1g, FePO40.01g, artificial seawater 1000ml, the medium pH value is 7.6～7.8, cultivates based on 121 ℃ of autoclaving 20min.
8. a kind of method of utilizing the asepsis cellulose filter membrane enriching seawater bacteria according to claim 7 is characterized in that: described artificial seawater prescription is as follows: NaCl 23.476g, MgCl
26H
2O 10.6359g, Na
2SO
43.917g, CaCl
21.102g, KCl 0.664g, NaHCO
30.192g, KBr 0.096g, H
3BO
30.026g, SrCl
26H
2O 0.0403g, NaF 0.072g, NaNO
30.050g, NaH
2PO
40.005g, Na
2SiO
39H
2O 0.010g, MnCl
24H
2O 0.0003g, FeC
6H
5O
73H
2O0.00054g, distilled water 1000ml.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010174899 CN101864386A (en) | 2010-05-14 | 2010-05-14 | Method for enriching seawater bacteria by asepsis cellulose filter membrane |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010174899 CN101864386A (en) | 2010-05-14 | 2010-05-14 | Method for enriching seawater bacteria by asepsis cellulose filter membrane |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101864386A true CN101864386A (en) | 2010-10-20 |
Family
ID=42956313
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010174899 Pending CN101864386A (en) | 2010-05-14 | 2010-05-14 | Method for enriching seawater bacteria by asepsis cellulose filter membrane |
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| Country | Link |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018061035A1 (en) | 2016-09-30 | 2018-04-05 | Council Of Scientific & Industrial Research | A device for collecting water sample to concentrate diversified bacteria from different water depth |
| CN117106678A (en) * | 2023-10-24 | 2023-11-24 | 中国海洋大学 | Enrichment culture of marine organism film bacteria and method for obtaining genome thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101538567A (en) * | 2008-03-20 | 2009-09-23 | 杭州优思达生物技术有限公司 | Method for quickly processing filter-type micro nucleic acid clinical samples |
-
2010
- 2010-05-14 CN CN 201010174899 patent/CN101864386A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101538567A (en) * | 2008-03-20 | 2009-09-23 | 杭州优思达生物技术有限公司 | Method for quickly processing filter-type micro nucleic acid clinical samples |
Non-Patent Citations (2)
| Title |
|---|
| 《海水化学》 19861231 大连水产学院 人工海水 农业出版社 , 1 * |
| 《环境工程微生物学》 20040630 李建政 微生物生长量的测定方法 化学工业出版社 , 1 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018061035A1 (en) | 2016-09-30 | 2018-04-05 | Council Of Scientific & Industrial Research | A device for collecting water sample to concentrate diversified bacteria from different water depth |
| US10690569B2 (en) | 2016-09-30 | 2020-06-23 | Council Of Scientific & Industrial Research | Device for collecting water sample to concentrate diversified bacteria from different water depth |
| CN117106678A (en) * | 2023-10-24 | 2023-11-24 | 中国海洋大学 | Enrichment culture of marine organism film bacteria and method for obtaining genome thereof |
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Open date: 20101020 |