CN105462856B - A kind of simple and easy method of purification of bacterial pollution filamentous fungi - Google Patents
A kind of simple and easy method of purification of bacterial pollution filamentous fungi Download PDFInfo
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- CN105462856B CN105462856B CN201510988939.8A CN201510988939A CN105462856B CN 105462856 B CN105462856 B CN 105462856B CN 201510988939 A CN201510988939 A CN 201510988939A CN 105462856 B CN105462856 B CN 105462856B
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Abstract
The invention discloses a kind of simple and easy methods of purification of bacterial pollution filamentous fungi, include following operating procedure: (1) pouring into the culture medium after heating and melting in culture dish, with a thickness of 1-2mm；(2) it will access in gained plating medium, cultivate 2-3 days with the purpose fungi of bacterium mixed growth；(3) it is cultivated 5-7 days under the conditions of the plating medium with fungus colony being placed on 30-35 DEG C；(4) culture medium with fungi is cut into bulk, is forwarded to new plating medium and is cultivated；(5) picking step (4) culture gained colony edge mycelia, which is transferred in new plating medium, cultivates, and can be obtained the purpose fungi of purifying.The method of the present invention is not had to other appliance arrangements and assisted, can obtain purer fungi by simple and direct operation to the method for purifying and separating of germ contamination filamentous fungi without adding additional antibiotic.Purification effect is good, and purification process is easy to operate, consumptive material is few, at low cost.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of simple side for purifying the filamentous fungi being contaminated by bacterial
Method.
Background technique
Fungi is many kinds of in nature, can much cause human body, livestock and plant disease, can also cause the mould of food
Become.Some can be used for industrial production fermentation and extraction secretory product etc..Obtaining single targeted fungal pure culture is carry out one
The basis of serial scientific research and application.But since microbe species are more in environment, purpose fungi is easily contaminated, especially
It is the pollution of bacterium.Currently, the filamentous fungi that is contaminated by bacterial of purifying there are two main classes method, the first kind: mainly in culture medium
The method of middle addition antibiont inhibits the growth of bacterium；Second class: there is the ability for penetrating agar using hypha,hyphae, using one
Bacterium on fixed apparatus removal fungi.First kind method primary operational process: 1) cold by agar medium heating and melting
But to 50 DEG C or so；2) antibiotic is added under aseptic condition, pours into culture dish after shaking up and plating medium is made；3) purpose is accessed
Fungi；4) it is cultivated 2-4 days under the conditions of thermophilic；5) picking colony edge mycelia is transferred on new plating medium, cultivates 2-4
It；6) step 5) 1-2 times is repeated to differ.Second class method, as application number 200910235086.5 " it is a kind of purifying by bacterium dirt
The device and method of the filamentous fungi of dye ", primary operational process: 1) purify the system of fungi collecting layer culture medium (special culture medium)
Make and sterilizes；2) production and sterilizing of purification devices, device include sealed membrane, purifying cup, purification column etc.；It 3) will be by bacterium dirt
The fungi access purification devices of dye are simultaneously cultivated；4) fungi for purifying culture layer is transferred in culture medium, obtains purifying
Purpose fungi.
The above method exist for seriously polluted by bacterium or with bacterium combine closely growth fungi separating effect it is inadequate
It is good, or the relatively complicated problem that operates.Frequent certain physicochemical characters using also easy change purpose fungi of antibiotic,
So that fungi character after purification is different from original bacterial strain, subsequent research work is influenced.Purifier apparatus device is utilized simultaneously
Relatively complicated, the purifying of a small amount of bacterial strain is still dealt with, but for the population research of fungi, is implemented and had difficulties.And it is used for
The filamentous fungi of scientific research or industrial applications usually require to transfer in laboratory conditions culture expand it is numerous, but in the process
It is highly susceptible to the pollution of bacterium in environment.Therefore, it solves the problems, such as the fungi being contaminated by bacterial, is greatly improved scientific research
And the efficiency of the factorial production.
The information disclosed in the background technology section is intended only to increase the understanding to general background of the invention, without answering
When being considered as recognizing or imply that the information constitutes the prior art already known to those of ordinary skill in the art in any form.
Summary of the invention
The purpose of the present invention is to provide a kind of simple and easy methods of purification of bacterial pollution filamentous fungi, to overcome existing skill
Antibiotic must be additionally used in art, so that fungi physicochemical character obtained by influencing, purification effect is poor, purification process is cumbersome, consumptive material
Big disadvantage.
To achieve the above object, technical method provided by the invention is as follows:
A kind of simple and easy method of purification of bacterial pollution filamentous fungi, includes following operating procedure:
(1) it makes Thin cell layer base plate: the culture medium after heating and melting being poured into culture dish, is formed in culture dish
With a thickness of the plating medium of 1-2mm；
(2) it will be accessed in step (1) in gained plating medium with the purpose fungi of bacterium mixed growth, 25-28 DEG C of temperature
Degree lower culture 2-3 days；
(3) culture dish of the plating medium with fungus colony behind in step (2) culture 2-3 days is gone into hot environment
Culture 5-7 days；
(4) culture medium with fungi behind in step (3) culture 5-7 days is cut into bulk, is forwarded to new plate training
Feeding base is cultivated；
(5) picking step (4) culture gained colony edge mycelia, which is transferred in new plating medium, cultivates, and can be obtained pure
The purpose fungi of change.
Wherein, the culture medium is PDA culture medium, i.e. potato dextrose agar.
Wherein, the temperature of hot environment described in step (3) is 30-35 DEG C.
Wherein, culture dish described in step (3) is sealed without sealed membrane, i.e., need to only cover culture dish lid, not additionally
Lid edge is sealed using sealed membrane.
Wherein, step (4) operates on aseptic operating platform.
Wherein, step (4), in step (5) in condition of culture and step (2).
There is more apparent difference in the resistance of fungi and bacterium, fungi belongs to eucaryote, have preferable degeneration-resistant
Property cell wall；And bacterium belongs to prokaryotes, cell-free wall, resistance is poor compared with fungi.The method of the present invention is same by fungi and bacterium
When in high temperature without nutrition (culture medium has been dried) under the conditions of, bacterium is because resistance is poor and dead, but fungi still has life
Long vigor achievees the purpose that purify fungi by cultivating again.
Compared with prior art, the invention has the following advantages:
The method of the present invention does not have to addition volume by simple and direct operation to the method for purifying and separating of germ contamination filamentous fungi
Outer antibiotic does not have to other appliance arrangements and assists, can obtain purer fungi.Purification effect is good, purification process is easy to operate,
Consumptive material is few, at low cost.
Specific embodiment
It is described in detail With reference to embodiment, it is to be understood that protection scope of the present invention is not by specific
The limitation of embodiment.
Embodiment 1
A kind of simple and easy method of purification of bacterial pollution filamentous fungi, operating procedure are as follows:
(1) it makes Thin cell layer base plate: the PDA culture medium after heating and melting being poured into culture dish, in culture dish
Form the PDA plate culture medium with a thickness of 2mm；
(2) step will be accessed with the purpose fungi sugarcane top rot bacterium (Fusarium sacchari) of bacterium mixed growth
(1) it in gained PDA plate culture medium, is cultivated 3 days at a temperature of 25 DEG C；
(3) culture dish of the PDA plate culture medium with fungus colony behind in step (2) culture 3 days is placed on 32 DEG C
Cultivate 7 days in incubator, i.e., when PDA culture medium is dry, when the abacterial wet shape in surface, wherein culture dish is only with culture
Ware cap covers avoid the pollution of other microorganisms in environment, do not use sealed membrane and seal lid edge, make culture medium water
Dividing can be evaporated；
(4) PDA culture medium with fungi behind in step (3) culture 7 days is arrived with aseptic operation on aseptic operating platform
It is cut into fritter, new PDA plate culture medium is forwarded to and is cultivated, condition of culture is identical as in step (2), i.e. culture temperature
Degree is 25 DEG C, and incubation time is 3 days；
(5) picking step (4) culture gained colony edge mycelia, which is transferred in new PDA plate culture medium, cultivates, and can obtain
The sugarcane top rot bacterium (Fusarium sacchari) that must be purified, condition of culture is identical as in step (2), i.e. cultivation temperature
It is 25 DEG C, incubation time is 3 days.
Embodiment 2
A kind of simple and easy method of purification of bacterial pollution filamentous fungi, operating procedure are as follows:
(1) it makes Thin cell layer base plate: the PDA culture medium after heating and melting being poured into culture dish, in culture dish
Form the PDA plate culture medium with a thickness of 1mm；
(2) the purpose fungi red rot of sugar cane bacterium (Colletotrichum falcatum) with bacterium mixed growth is connect
In PDA plate culture medium obtained by entering in step (1), cultivated 2 days at a temperature of 28 DEG C；
(3) culture dish of the PDA plate culture medium with fungus colony behind in step (2) culture 2 days is placed on 33 DEG C
Cultivate 6 days in incubator, i.e., when PDA culture medium is dry, when the abacterial wet shape in surface, wherein culture dish is only with culture
Ware cap covers avoid the pollution of other microorganisms in environment, do not use sealed membrane and seal lid edge, make culture medium water
Dividing can be evaporated；
(4) by the PDA culture medium aseptic operation knife with fungi behind in step (3) culture 6 days on aseptic operating platform
It is cut into fritter, new PDA plate culture medium is forwarded to and is cultivated, condition of culture is identical as in step (2), i.e. culture temperature
Degree is 28 DEG C, and incubation time is 2 days；
(5) picking step (4) culture gained colony edge mycelia, which is transferred in new PDA plate culture medium, cultivates, and can obtain
The red rot of sugar cane bacterium (Colletotrichum falcatum) that must be purified, condition of culture is identical as in step (2), that is, trains
Supporting temperature is 28 DEG C, and incubation time is 2 days.
Embodiment 3
A kind of simple and easy method of purification of bacterial pollution filamentous fungi, operating procedure are as follows:
(1) it makes Thin cell layer base plate: the PDA culture medium after heating and melting being poured into culture dish, in culture dish
Form the PDA plate culture medium with a thickness of 1mm；
(2) it will access and walk with the purpose fungi fusarium graminearum (Fusarium graminearum) of bacterium mixed growth
Suddenly it is cultivated 3 days at a temperature of 26 DEG C in gained PDA plate culture medium in (1)；
(3) culture dish of the PDA plate culture medium with fungus colony behind in step (2) culture 3 days is placed on 35 DEG C
Cultivate 5 days in incubator, i.e., when PDA culture medium is dry, when the abacterial wet shape in surface, wherein culture dish is only with culture
Ware cap covers avoid the pollution of other microorganisms in environment, do not use sealed membrane and seal lid edge, make culture medium water
Dividing can be evaporated；
(4) by the PDA culture medium aseptic operation knife with fungi behind in step (3) culture 5 days on aseptic operating platform
It is cut into fritter, new PDA plate culture medium is forwarded to and is cultivated, condition of culture is identical as in step (2), i.e. culture temperature
Degree is 26 DEG C, and incubation time is 3 days；
(5) picking step (4) culture gained colony edge mycelia, which is transferred in new PDA plate culture medium, cultivates, and can obtain
The fusarium graminearum (Fusarium graminearum) that must be purified, condition of culture is identical as in step (2), i.e. culture temperature
Degree is 26 DEG C, and incubation time is 3 days.
Embodiment 4
A kind of simple and easy method of purification of bacterial pollution filamentous fungi, operating procedure are as follows:
(1) it makes Thin cell layer base plate: the PDA culture medium after heating and melting being poured into culture dish, in culture dish
Form the PDA plate culture medium with a thickness of 2mm；
(2) it will be accessed with the purpose fungi sigatoka bacterium (Exserohilum rostratum) of bacterium mixed growth
In step (1) in gained PDA culture medium plate, cultivated 2 days at a temperature of 27 DEG C；
(3) culture dish of the PDA plate culture medium with fungus colony behind in step (2) culture 2 days is placed on 30 DEG C
Cultivate 7 days in incubator, i.e., when PDA culture medium is dry, when the abacterial wet shape in surface, wherein culture dish is only with culture
Ware cap covers avoid the pollution of other microorganisms in environment, do not use sealed membrane and seal lid edge, make culture medium water
Divide evaporation；
(4) by the PDA culture medium aseptic operation knife with fungi behind in step (3) culture 7 days on aseptic operating platform
It is cut into fritter, new PDA plate culture medium is forwarded to and is cultivated, condition of culture is identical as in step (2), i.e. culture temperature
Degree is 27 DEG C, and incubation time is 2 days；
(5) picking step (4) culture gained colony edge mycelia, which is transferred in new PDA plate culture medium, cultivates, and can obtain
The sigatoka bacterium (Exserohilum rostratum) that must be purified, condition of culture is identical as in step (2), that is, cultivates
Temperature is 27 DEG C, and incubation time is 2 days.
The aforementioned description to specific exemplary embodiment of the invention is in order to illustrate and illustration purpose.These descriptions
It is not wishing to limit the invention to disclosed precise forms, and it will be apparent that according to the above instruction, can much be changed
And variation.The purpose of selecting and describing the exemplary embodiment is that explaining specific principle of the invention and its actually answering
With so that those skilled in the art can be realized and utilize a variety of different exemplary implementation schemes of the invention and
Various chooses and changes.The scope of the present invention is intended to be limited by claims and its equivalents.
Claims (4)
1. a kind of simple and easy method of purification of bacterial pollution filamentous fungi, which is characterized in that include following operating procedure:
(1) it makes Thin cell layer base plate: the culture medium after heating and melting being poured into culture dish, forms thickness in culture dish
For the plating medium of 1-2mm；
(2) it will be accessed in step (1) in gained plating medium, at a temperature of 25-28 DEG C with the purpose fungi of bacterium mixed growth
Culture 2-3 days；
(3) culture dish of the plating medium with fungus colony behind in step (2) culture 2-3 days is gone to temperature is 30-35
DEG C hot environment culture 5-7 days, the culture dish without sealed membrane seal；
(4) culture medium with fungi behind in step (3) culture 5-7 days is cut into bulk, is forwarded to new plating medium
It is cultivated；
(5) picking step (4) culture gained colony edge mycelia, which is transferred in new plating medium, cultivates, and obtains the purpose of purifying
Fungi.
2. the simple and easy method of purification of bacterial pollution filamentous fungi according to claim 1, it is characterised in that: the culture medium
It is all PDA culture medium.
3. the simple and easy method of purification of bacterial pollution filamentous fungi according to claim 1, it is characterised in that: step (4) is in nothing
It is operated on bacterium station.
4. the simple and easy method of purification of bacterial pollution filamentous fungi according to claim 1, it is characterised in that: step (4), step
(5) in condition of culture and step (2).
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| CN201510988939.8A CN105462856B (en) | 2015-12-25 | 2015-12-25 | A kind of simple and easy method of purification of bacterial pollution filamentous fungi |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102864221A (en) * | 2012-08-25 | 2013-01-09 | 福建农林大学 | Polymerase chain reaction (PCR) detection method for colletotrichum falcatum went |
| WO2013113033A1 (en) * | 2012-01-27 | 2013-08-01 | Tulane University | Postharvest production and enhancement of resveratrol and piceatannol in sugarcane |
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2015
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013113033A1 (en) * | 2012-01-27 | 2013-08-01 | Tulane University | Postharvest production and enhancement of resveratrol and piceatannol in sugarcane |
| CN102864221A (en) * | 2012-08-25 | 2013-01-09 | 福建农林大学 | Polymerase chain reaction (PCR) detection method for colletotrichum falcatum went |
Non-Patent Citations (3)
| Title |
|---|
| Gene Cloning, Purification, and Characterization of a Heat-Stable Phytase from the Fungus Aspergillus fumigatus;LUIS PASAMONTES等;《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》;19970531;第63卷(第5期);第1696-1700页 * |
| 斜面菌种的纯化;陈士瑜;《食用菌》;19841231(第4期);第10页 * |
| 福建坛紫菜赤腐病的病程及病原鉴定;刘一萌等;《福建农林大学学报(自然科学版)》;20130131;第42卷(第1期);第18-22页，尤其是第19页第4段 * |
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