CN100552021C - A kind of method for preserving methane-oxidizing bacteria - Google Patents
A kind of method for preserving methane-oxidizing bacteria Download PDFInfo
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- CN100552021C CN100552021C CNB2006100745306A CN200610074530A CN100552021C CN 100552021 C CN100552021 C CN 100552021C CN B2006100745306 A CNB2006100745306 A CN B2006100745306A CN 200610074530 A CN200610074530 A CN 200610074530A CN 100552021 C CN100552021 C CN 100552021C
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- Prior art keywords
- methane
- oxidizing bacteria
- paraffin oil
- preserving
- thalline
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Abstract
The invention discloses a kind of method for preserving methane-oxidizing bacteria.This method is to collect the methane-oxidizing bacteria thalline, and it with behind the paraffin oil mixing of sterilization, is preserved down at-4 ℃～-90 ℃.Because paraffin oil can not be mixed bacterium as utilization of carbon source by great majority, so this method can reduce the opportunities for contamination of assorted bacterium widely; In addition, only needing during use mixture with thalline and paraffin oil be added directly into to cultivate in the substratum gets final product.The present invention will play a significant role in the preservation of methane-oxidizing bacteria and suitability for industrialized production thereof.
Description
Technical field
The present invention relates to the method for preserving of microorganism, particularly relate to a kind of method for preserving methane-oxidizing bacteria that can effectively prevent living contaminants.
Background technology
Methane-oxidizing bacteria is the very special microorganism of occurring in nature one class, and it is the unique carbon source and energy with methane.Though some methane-oxidizing bacteria also can utilize methyl alcohol simultaneously, all methane-oxidizing bacterias all can not utilize many carbon compounds.Studies show that it is in the large-scale industrial production of Chemicals (Sweet natural gas or biogas etc.) of raw material that methane-oxidizing bacteria can be widely used in methane, also can be applied to the improvement of organic pollutant in the environment, even can be used for effectively preventing the methane gas blast in the colliery.At present, this bacterial classification does not have special store method, general just adds behind the glycerine in-70 ℃ of freezing preservations as other microorganism.Because glycerine is difficult to remove fully in follow-up operation, and the speed of growth of methane-oxidizing bacteria is much more slowly than other assorted bacterium, therefore, assorted bacterium just utilization glycerine wherein causes living contaminants as the carbon source raised growth.Another kind of method for preserving is that methane-oxidizing bacteria strain is stored on the plate culture medium that contains methane, inoculates on another flat board after cultivating, and does not so stop.Though this method can be avoided living contaminants and can make methane oxidation keep higher activity, wastes time and energy, and implements very inconvenient.
Therefore press for a kind of easyly, and can effectively prevent the method for preserving methane-oxidizing bacteria of living contaminants.
Summary of the invention
The purpose of this invention is to provide a kind of simple to operately, and can effectively prevent the method for preserving methane-oxidizing bacteria of living contaminants.
Method for preserving methane-oxidizing bacteria provided by the present invention is to collect the methane-oxidizing bacteria thalline, and it with behind the paraffin oil mixing of sterilization, is preserved down at-4 ℃～-90 ℃.
In above-mentioned method for preserving, the OD of described methane-oxidizing bacteria and paraffin oil mixture
600Value is 0.3-1.5.
The sterilising conditions of described paraffin oil can be at 121 ℃ and heated 15-25 minute down.
The container that can be used for preserving methane-oxidizing bacteria is diversified, any through sterilization and low temperature resistant container all can, be the plastic containers of 200 microlitres～500 milliliter as volume, be preferably the plastic centrifuge tube of 500 microlitres～1.5 milliliter.
Above-mentioned method for preserving is suitable for simultaneously to the methane-oxidizing bacteria through liquid culture and solid culture.
When need using, only need the thalline of preservation together poured into to cultivate in the substratum together with paraffin oil get final product, because paraffin oil except that the culture presevation that can be used for methane-oxidizing bacteria, also has the effect of this bacteria growing of promotion.
The invention provides a kind of method for preserving methane-oxidizing bacteria.This method is the method that replaces glycerine to carry out culture presevation with paraffin oil.Because paraffin oil can not be mixed bacterium as utilization of carbon source by great majority, so this method can reduce the opportunities for contamination of assorted bacterium widely; In addition, only need mixture with thalline and paraffin oil be added directly into to cultivate in the substratum during use and get final product, easily and fast.The present invention will play a significant role in the preservation of methane-oxidizing bacteria and suitability for industrialized production thereof.
Below in conjunction with specific embodiment the present invention is described in further detail.
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment, and all percentage concentrations are mass percent concentration, and the solvent in all substratum is deionized water.
NMS culture medium prescription: contain NaNO in every liter of substratum
30.85 gram, KH
2PO
40.53 gram, Na
2HPO
42.17 gram, K
2SO
40.17 gram, MgSO
47H
2O 0.037 gram, CaCl
22H
2O 0.007 gram, micro-stock solution 2mL, 1mmol/L H
2SO
40.5 milliliter, FeSO
47H
211.2 milligrams of O, CuSO
45H
22.5 milligrams of O (solid medium adds 1.5% agar again), pH 7.0.
The prescription of trace element stock solution is: contain ZnSO in every liter of solution
47H
2O 0.2042 gram, MnSO
44H
2O0.223g, H
3BO
30.062g, Na
2MoO
42H
2O 0.048g, CoCl
26H
2O 0.048g, KI 0.083g.
The liquid culture of embodiment 1, methane-oxidizing bacteria
One, the acquisition of the single bacterium colony of methane-oxidizing bacteria
Transfering loop picking methane-oxidizing bacteria with sterilization preserves liquid (Methylosinus trichosporium) OB3b (Sullivan JP et al.Methanotrophs, Methylosinus trichosporium OB3b, sMMO, and their application to bioremediation, Crit.Rev.Microbiol., 1998,4 (24): 335-373; Takeguchi M et al., Properties of the membranes containing theparticulate methane monooxygenase from Methylosinus trichosporium OB3b, Biometals, 1998,11:229-234), method with line is inoculated in NMS flat-plate solid media surface with it, in culture dish, add the methane gas of 5-10 milliliter every day by the strainer of 0.2 μ with syringe, after around cultivating under 30 ℃, there is tangible bacterium colony to produce on the flat board.
Two, the liquid culture of methane-oxidizing bacteria
1) get 50 milliliters culturing bottle, 10 milliliters of the NMS liquid nutrient mediums of packing into are 121 ℃ of sterilizations 20 minutes down;
2) the single bacterium colony of the methane-oxidizing bacteria that on flat board, grows of step 1 of picking, it is inoculated in the NMS liquid nutrient medium, add the methane gas of 5-20 milliliter with the strainer of syringe by 0.2 μ every day, under 30 ℃, 150rpm around the shaking culture, and the OD of substratum
600After value is about 0.1-1.0, promptly can be used for preserving.
The preservation in paraffin oil of embodiment 2, methane-oxidizing bacteria
The methane-oxidizing bacteria bacterium liquid that embodiment 1 cultivates is poured in 1.5 milliliters of centrifuge tubes of sterilization for 1.5 milliliters, behind centrifugal 10 minutes of the 8000rpm, supernatant discarded, in super clean bench or with the method for aseptic technique, the thalline of collecting is added in 500 microlitre paraffin oils of sterilization (121 ℃ heating 20 minutes) down, on the vortex vibrator, vibrate, thalline is uniformly dispersed in paraffin oil, puts into-20 ℃ of refrigerators then and preserve.
The use of the methane-oxidizing bacteria of embodiment 3, paraffin oil preservation
Get 50 milliliters of culturing bottles, pack into 10 milliliters of NMS liquid nutrient mediums, after sterilizing 20 minutes under 121 ℃, from refrigerator, get the methane-oxidizing bacteria that an effective paraffin oil was preserved 90 days, the bacterium liquid that will thaw in super clean bench all is poured in the NMS liquid nutrient medium, and add the methane gas of 5-20 milliliter with the strainer of syringe by 0.2 μ every day, and shaking culture is after three days under 30 ℃, 150rpm, substratum presents muddy shape, its OD
600Value is about 0.1-0.3, and methane-oxidizing bacteria is cultivated successfully, shows with the inventive method methane-oxidizing bacteria is carried out preservation, can make the higher biological activity of thalline long term maintenance.
Claims (3)
1, a kind of method for preserving methane-oxidizing bacteria is to collect the methane-oxidizing bacteria thalline, and it with behind the paraffin oil mixing of sterilization, is preserved down at-4 ℃～-90 ℃.
2, method for preserving according to claim 1 is characterized in that: the OD of described methane-oxidizing bacteria and paraffin oil mixture
600Value is 0.3-1.5.
3, method for preserving according to claim 1 and 2 is characterized in that: the sterilising conditions of described paraffin oil is for heating 15-25 minute down at 121 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100745306A CN100552021C (en) | 2006-04-27 | 2006-04-27 | A kind of method for preserving methane-oxidizing bacteria |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100745306A CN100552021C (en) | 2006-04-27 | 2006-04-27 | A kind of method for preserving methane-oxidizing bacteria |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1844368A CN1844368A (en) | 2006-10-11 |
| CN100552021C true CN100552021C (en) | 2009-10-21 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2006100745306A Expired - Fee Related CN100552021C (en) | 2006-04-27 | 2006-04-27 | A kind of method for preserving methane-oxidizing bacteria |
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| CN (1) | CN100552021C (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101670238B (en) * | 2009-09-07 | 2012-09-05 | 清华大学 | Method for removing methane in environment |
| CN107227345A (en) * | 2017-05-17 | 2017-10-03 | 董克海 | A kind of microbiological specimens DNA preserves liquid |
-
2006
- 2006-04-27 CN CNB2006100745306A patent/CN100552021C/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
|---|
| 微生物的保存方法. 王成怀.微生物学免疫学进展. 1979 |
| 微生物的保存方法. 王成怀.微生物学免疫学进展. 1979 * |
| 菌种的保存方法介绍. 吴茂红等.江西医学检验，第22卷第6期. 2004 |
| 菌种的保存方法介绍. 吴茂红等.江西医学检验，第22卷第6期. 2004 * |
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| CN1844368A (en) | 2006-10-11 |
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Granted publication date: 20091021 Termination date: 20120427 |