CN105018346B - It is a kind of for freezing the hollow cap and cryopreservation methods of solid culture filamentous fungi - Google Patents
It is a kind of for freezing the hollow cap and cryopreservation methods of solid culture filamentous fungi Download PDFInfo
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- CN105018346B CN105018346B CN201510430876.4A CN201510430876A CN105018346B CN 105018346 B CN105018346 B CN 105018346B CN 201510430876 A CN201510430876 A CN 201510430876A CN 105018346 B CN105018346 B CN 105018346B
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Abstract
The present invention relates to a kind of for freezing the hollow cap and cryopreservation methods of solid culture filamentous fungi, belongs to cell preservation technique research field.The present invention is the improvement on the basis of cryopreservation methods of existing solid culture filamentous fungi.Improvement is: using diameter between 3mm~6mm, cover is covered with the hollow cap of quil and co-cultures with to preservation filamentous fungi in solid culture primary surface, covering is moved to completion strain in frozen stock solution to the hollow cap of preservation filamentous fungi and frozen by the cover that covering hollow cap is grown to filamentous fungi.The hollow cap that cover of the invention is covered with quil is made of resin material according to a conventional method.The beneficial effects of the present invention are: fungus block is effectively prevented to the carrying of solid medium and prepares the possible pollution of bacteria suspension, with easy to operate, it is low in cost, and can satisfy the advantages of specific culture requires, technical support is provided for the preservation of filamentous fungi.
Description
Technical field
The invention belongs to cell preservation technique research fields, and in particular to a kind of for freezing solid culture filamentous fungi
Hollow cap and cryopreservation methods.
Background technique
Filamentous fungi is important microbial resources, and the filamentous fungi of many monoids is scientific research or production bacterial strain,
Great influence is produced to human production life, there is biggish social economic value.Preservation to filamentous fungus strain resource
It not only protects bio-diversity, more human research and plays important function using these strains.Currently, the guarantor of microorganism
Mainly there is following four method: (1) nitrogen ultra low temperature preserving process in hiding, which is suitable for liquid culture and solid culture simultaneously
Preservation；(2) lyophil preservation method, though the method is suitable for the preservation of liquid and solid culture, two class cultures are both needed to
First it is prepared into the bacteria suspension for meeting preservation requirement；The operation of (3) -80 DEG C of sharp freezing preserving process, the method is protected with nitrogen ultra low temperature
Hiding method is similar, is only storage conditions (mainly temperature) difference；(4) preserving process is periodically transplanted, which is used for solid culture
Short term storage, need periodically to strain recovery and preservation again, it is time-consuming and laborious.Wherein, nitrogen ultra low temperature preserving process and -80 DEG C of low temperature
Freeze preserving process because operating the simple and preservation time longer preservation for being widely used in each quasi-microorganism.
Filamentous fungi for preservation mostly uses solid culture mode, is primarily due to solid culture and is easy to fungi growth characteristics
Observation, convenient for discovery pollution, to improve the quality of preservation.Currently, the preservation for carrying out solid culture filamentous fungi is to draw
It takes or picking fungus block, or is prepared into bacteria suspension and then is frozen.The exploitation of PRO-LABDIAGNOSTICS company
MICROBANK freezes systemWhat it is for solid culture filamentous fungi is also to need first to draw and take according to aforesaid operations when freezing
Fungus block or bacteria suspension addition freeze in solution, have then added hole pearl and have been mixed, cell has been attached to inside the pearl of hole
With external surface.There are certain defects and limitations in this kind of cryopreservation methods, be primarily due to: 1. drawing and takes or the bacterium of picking
Block be used for culture presevation when, fungus block carry culture medium be present in frozen stock solution, carry out strain recovery when can be by these culture mediums
It brings into new culture medium, can have an adverse effect to the Spawn incubation for having particular/special requirement；2. the filamentous fungi of solid culture exists
Pollution risk is larger when preparing bacteria suspension, easily leads to preservation failure.
At present about the cryopreservation methods of filamentous fungi it has been reported that but by literature search, do not find and the content of present invention phase
Similar open source literature report.
Summary of the invention
It is an object of the invention to improve the deficiency of the prior art, provide a kind of for freezing solid culture filamentous fungi
Hollow cap and using the easy to operate, low in cost of the hollow cap and can satisfy specific culture requirement solid culture it is Filamentous true
The cryopreservation methods of bacterium provide technical support for the preservation of filamentous fungi.
First aspect present invention is related to a kind of for freezing the hollow cap 3 of solid culture filamentous fungi, and bottom 34 is one
Plane and its internal there are hollow 33 be connected to the bottom 34.
In a preferred embodiment of the present invention, the hollow cap 3 is hemispherical hollow cap, round table-like hollow cap, cylinder
Shape hollow cap, coniform hollow cap or rectangular-shape hollow cap.
In a preferred embodiment of the present invention, quil 31 is furnished on the cover 32 of the hollow cap 3.The shape of the quil 31
Shape is unrestricted, vertical, bending or racemosus wooden fork shape.
In a preferred embodiment of the present invention, the length of the quil 31 is 1mm-4mm.
In a preferred embodiment of the present invention, the diameter of the hollow cap 3 is between 3mm~6mm.
In a preferred embodiment of the present invention, the hollow cap 3 is made of resin material.
Second aspect of the present invention is related to a kind of cryopreservation methods for solid culture filamentous fungi, and this method includes following steps
It is rapid:
A. solid medium tablets are prepared and are inoculated with to preservation filamentous fungi；
B. the sterilization treatment hollow cap according to a first aspect of the present invention；
C. the hollow cap bottom is placed in downwards to the solid culture primary surface around preservation filamentous fungi vaccination；
D. culture is described to preservation filamentous fungi, until filamentous fungi growth covers the cover or described hollow of the hollow cap
Quil on cover cover is obtained with the hollow cap to preservation filamentous fungi；
E. above-mentioned be transferred to the hollow cap 3 to preservation filamentous fungi is frozen in solution, it will be described after mixing
It is frozen to preservation filamentous fungus strain.
Wherein, the preparation of solid medium tablets involved in the cryopreservation methods, sterilizing, strain inoculation, Spawn incubation, strain
It freezes etc. and to be all made of the art routine operation, repeats no more herein.
In a preferred embodiment of the present invention, in above-mentioned steps c, the hollow cap and described to preservation filamentous fungi
The distance of vaccination is 0.3cm~1.0cm.
In a preferred embodiment of the present invention, that shifts in step e is described with the sky to preservation filamentous fungi
The quantity of heart cover is 4-6.
It is described hollow according to being directly taken out after routine operation recovery strain when the filamentous fungi frozen needs to carry out recovery
Cover is seeded to corresponding culture medium.
Compared with prior art, the beneficial effects of the present invention are:
1, the quil 31 being covered on the cover 32 for freezing the hollow cap 3 of solid culture filamentous fungi of the invention
It can either play the role of adhering to mycelia, and transfer mycelia can be played, the liquid that can be directly used for strain freezes, is not required to
Draw and take or picking fungus block and the operation such as prepare bacteria suspension, when effectively preventing freezing fungus block to the carrying of solid medium and
Prepare the possible pollution of bacteria suspension.
2, the hollow cap 3 for freezing solid culture filamentous fungi of the invention is made of resin material, Ke Yizhi
Row high-temperature sterilization is tapped into, and it can be used repeatedly, environmental protection is inexpensive.
3, cryopreservation methods of the invention have the advantages that easy to operate, low in cost and can satisfy specific culture requirement,
Technical support is provided for the preservation of filamentous fungi.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of the hollow cap for freezing solid culture filamentous fungi of the invention.
Fig. 2 is the schematic diagram that filamentous fungi co-cultures on solid medium with the hollow cap.
Wherein, each appended drawing reference has following meanings:
1- solid medium；2- waits for preservation filamentous fungi vaccination；3- hollow cap；4- plate；31- quil；32- cover；
33- is hollow；The bottom 34-.
Specific embodiment
The contents of the present invention are further illustrated below in conjunction with the drawings and specific embodiments, but should not be construed as to of the invention
Limitation.Without departing from the spirit and substance of the case in the present invention, it modifies or replaces to made by the method for the present invention, step or condition
It changes, all belongs to the scope of the present invention.
Embodiment 1: aspergillus flavus freezes
1. preparing Sabouraud glucose agar, appropriate above-mentioned training is poured into after autoclave sterilization in superclean bench
Base is supported into aseptic flat board, to its natural cooling, obtains Sabouraud glucose agar solid plate；
2. being inoculated with aspergillus flavus from cryopreservation tube or solid medium tablets to the Sabouraud glucose agar solid
On plate；
3. the hollow cap after sterilizing is placed in around aspergillus flavus vaccination with tweezers, obtain co-culturing plate
(as shown in Figure 2)；The wherein hollow cap diameter 4mm, the hollow cap are 1.0cm at a distance from the aspergillus flavus vaccination；
Wherein, used hollow cap 3 as shown in Figure 1, its be hemispherical hollow cap, be made of resin material, bottom 34 be one
Plane and its it is internal there are hollow 33 be connected to the bottom 34, be furnished with quil 31 on cover 32.
It is cultivated 3 days~5 days 4. the co-cultivation plate is placed in 26 DEG C of constant incubators, until mycelia growth covering is extremely
Quil on the hollow cap cover, obtains the hollow cap for being covered with mycelia；
5. drawing 1mL 15% (v/v) glycerol frozen stock solution to be added in cryopreservation tube, 4~6 institutes then are taken with aseptic nipper
It states and is covered with the hollow cap of mycelia and is put into frozen stock solution；
6. covering the pipe lid of the cryopreservation tube, mixes gently and the cryopreservation tube is put into program temperature reduction box afterwards for several times, into
Row is subsequent to freeze freezing for operation completion strain.
Embodiment 2: Paecilomyces lilacinus freezes
1. preparing potato dextrose agar, poured into superclean bench after autoclave sterilization appropriate above-mentioned
Culture medium, to its natural cooling, obtains potato dextrose agar solid plate into aseptic flat board；
2. being inoculated with Paecilomyces lilacinus from cryopreservation tube or solid medium tablets to the potato dextrose agar culture
On base solid plate；
3. the hollow cap after sterilizing is placed in around Paecilomyces lilacinus vaccination with tweezers, obtain co-culturing flat
Plate；The wherein hollow cap diameter 3mm, the hollow cap are 0.5cm at a distance from the Paecilomyces lilacinus vaccination；
It is cultivated 3 days~5 days 4. the co-cultivation plate is placed in 26 DEG C of constant incubators, until mycelia growth covering is extremely
Quil on the hollow cap cover is empty, obtains the hollow cap for being covered with mycelia；
5. 1mL 15% (v/v) glycerol frozen stock solution is added in cryopreservation tube, then taken described in 4~6 with aseptic nipper
The hollow cap for being covered with mycelia is put into frozen stock solution；
6. covering the pipe lid of the cryopreservation tube, mixes gently and the cryopreservation tube is put into program temperature reduction box afterwards for several times, into
Row is subsequent to freeze freezing for operation completion strain.
Wherein, aspergillus flavus involved in the embodiment of the present invention (Aspergillus flavus) and Paecilomyces lilacinus
(Paecilomyces lilacinus) is common filamentous fungi, can be commercially available from Spawn preservation organization both domestic and external.
Claims (2)
1. a kind of hollow cap (3) for freezing solid culture filamentous fungi, which is characterized in that its bottom (34) be a flat surface and
It is internal with hollow (33) that are connected to the bottom (34)；Quil (31) are furnished on its cover (32)；The quil (31)
Length be 1mm-4mm；The diameter of the hollow cap (3) is between 3mm~6mm；
The hollow cap (3) is hemispherical hollow cap, cylindric hollow cap or coniform hollow cap；It is made of resin material.
2. a kind of cryopreservation methods for solid culture filamentous fungi, which is characterized in that this method includes the following steps:
A. solid medium tablets are prepared and are inoculated with to preservation filamentous fungi；
B. sterilization treatment hollow cap according to claim 1；
C. the hollow cap bottom is placed in downwards to the solid culture primary surface around preservation filamentous fungi vaccination；The sky
Heart cover is with described to be 0.3cm~1.0cm at a distance from preservation filamentous fungi vaccination；
D. culture is described to preservation filamentous fungi, until filamentous fungi growth covers the cover or the hollow cap cover of the hollow cap
Quil on face is obtained with the hollow cap to preservation filamentous fungi；
E. the hollow cap (3) by above-mentioned with described to preservation filamentous fungi, which is transferred to, freezes in solution, after mixing will it is described to
Preservation filamentous fungus strain freezes；The quantity with the hollow cap to preservation filamentous fungi is 4-6.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510430876.4A CN105018346B (en) | 2015-07-21 | 2015-07-21 | It is a kind of for freezing the hollow cap and cryopreservation methods of solid culture filamentous fungi |
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| CN201510430876.4A CN105018346B (en) | 2015-07-21 | 2015-07-21 | It is a kind of for freezing the hollow cap and cryopreservation methods of solid culture filamentous fungi |
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| CN105018346A CN105018346A (en) | 2015-11-04 |
| CN105018346B true CN105018346B (en) | 2019-03-08 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101416059A (en) * | 2003-07-17 | 2009-04-22 | 格洛伯塞尔解决方案公司 | Automated cell culture system and process |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101416059A (en) * | 2003-07-17 | 2009-04-22 | 格洛伯塞尔解决方案公司 | Automated cell culture system and process |
Non-Patent Citations (1)
| Title |
|---|
| 载体吸附培养生产白僵菌分生孢子的样机;朱金国 等;《杀虫微生物》;19921031;173-178页 |
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