CN110024622A - A kind of preparation method by macro fungi fructification switching strain - Google Patents
A kind of preparation method by macro fungi fructification switching strain Download PDFInfo
- Publication number
- CN110024622A CN110024622A CN201910329207.6A CN201910329207A CN110024622A CN 110024622 A CN110024622 A CN 110024622A CN 201910329207 A CN201910329207 A CN 201910329207A CN 110024622 A CN110024622 A CN 110024622A
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- fructification
- macro fungi
- preparation
- strain
- culture medium
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/40—Cultivation of spawn
Abstract
The invention discloses a kind of preparation methods by macro fungi fructification switching strain, the tender macro fungi fructification refrigeration of the vigorous children of field grown is taken back first, fructification is uniformly sprayed on 75% alcohol after carefully being rinsed with distilled water to carry out disinfection, then it half-and-half breaks to cap and is split into two halves along stem, the stem and cap tissue that size is about 0.5cm × 0.5cm are cut with the sharp cutter of sterilizing, it is transferred on the solid potato culture medium PDA of addition streptomysin, finally vaccinated culture dish is placed in constant incubator, mycelium germination situation is observed after keeping dark condition to cultivate 7~10 days in 25 DEG C, white fluffy mycelia (contain culture medium) of the picking far from living contaminants point makees to expand culture into freshly prepd PDA culture medium, to acquisition single culture.The present invention carries out fructification switching strain using the PDA culture medium and tissue isolation of addition streptomysin, and then obtains purifying strain, easy to operate, time saving and energy saving.
Description
Technical field
The present invention relates to a kind of technology of preparing of strain more particularly to a kind of systems by macro fungi fructification switching strain
Preparation Method.
Background technique
Macro fungi refers to a kind of higher fungus that can form meat or colloid fructification, is species diversity in nature
Biota the most abundant.It rises in energy and the substance circulation of the ecosystem as the main decomposition person in the ecosystem
Conclusive effect, in addition to this, many macro fungis belong to edible and medicinal fungi, nutritive value with higher and medicinal valence
Value.With deepening continuously for modern biotechnology, gradually from the macro -examinations such as the morphological feature of macro fungi and institutional framework
It is raised to molecular biology field.Therefore, obtaining pure fungi strain by the switching of macro fungi fructification becomes key technique.
The organized partition method of method, spore separation method, mushroom wood partition method etc. of the switching of macro fungi fructification are obtained at present.
Wherein, tissue isolation is that acquisition pure culture is separated by the portion of tissue of fructification, this kind of method pure culture obtained
Stabilization characteristics of genetics, offspring are not susceptible to make a variation, and are the simple and effective methods of breeding excellent species.But most of macro fungi
The content of mycelia is relatively fewer in entity, and the pollution of bacterium is highly prone in switching process, this has seriously affected strain point
From success rate.
Summary of the invention
The object of the present invention is to provide a kind of preparation methods by macro fungi fructification switching strain, to macro fungi
Its hypha,hyphae of pure culture after entity sample is transferred, substantially increases the success rate of macro fungi fructification pure culture.
The purpose of the present invention is what is be achieved through the following technical solutions:
Preparation method by macro fungi fructification switching strain of the invention, comprising steps of
(1) macro fungi fructification sterilizes: the macro fungi fructification obtained in field refrigeration being taken back, is rushed with distilled water
Fructification is uniformly sprayed on 75% alcohol after washing to carry out disinfection；
(2) it is inoculated with: half-and-half breaking to cap and be split into two halves along stem, cut fritter stem and cap with the sharp cutter of sterilizing
Tissue is transferred on the solid potato culture medium PDA of addition streptomysin；
(3) it isolates and purifies: in 25 DEG C of constant temperature incubations, 7~10 days observation mycelium germination situations, picking white fluffy mycelia
Make to expand culture into freshly prepd PDA culture medium together with culture medium, until obtaining single culture.
As seen from the above technical solution provided by the invention, provided in an embodiment of the present invention by macro fungi fructification
The preparation method of switching strain, quick, economic, effective, separation strain success rate height, suitable for most of macro fungi, and
It is technically simple, it is easy to universal.
Detailed description of the invention
Fig. 1 is that the fructification of the preparation method provided in an embodiment of the present invention by macro fungi fructification switching strain is cut
Fungus block schematic diagram.
Fig. 2 is bacterium in the culture medium of the preparation method provided in an embodiment of the present invention by macro fungi fructification switching strain
Block places schematic diagram.
Specific embodiment
With reference to the attached drawing in the embodiment of the present invention, technical solution in the embodiment of the present invention carries out clear, complete
Ground description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on this
The embodiment of invention, every other implementation obtained by those of ordinary skill in the art without making creative efforts
Example, belongs to protection scope of the present invention.The content being not described in detail in the embodiment of the present invention belongs to this field professional technique
The prior art well known to personnel.
Preparation method by macro fungi fructification switching strain of the invention, preferable specific embodiment is:
Comprising steps of
(1) macro fungi fructification sterilizes: the macro fungi fructification obtained in field refrigeration being taken back, is rushed with distilled water
Fructification is uniformly sprayed on 75% alcohol after washing to carry out disinfection；
(2) it is inoculated with: half-and-half breaking to cap and be split into two halves along stem, cut fritter stem and cap with the sharp cutter of sterilizing
Tissue is transferred on the solid potato culture medium PDA of addition streptomysin；
(3) it isolates and purifies: in 25 DEG C of constant temperature incubations, 7~10 days observation mycelium germination situations, picking white fluffy mycelia
Make to expand culture into freshly prepd PDA culture medium together with culture medium, until obtaining single culture.
The macro fungi fructification need to keep fresh, and it is tender that when picking selects the involute children in vigorous growth, cap edge
Fructification is as the tissue separation material for examination.
The stem and cap tissue size is 0.5cm × 0.5cm.
The configuration method of the bell potato solid medium PDA of the addition streptomysin:
It weighs peeled potatoes 200g to be cut into small pieces, is put into pot after being heated to boiling and keeps with 1200mL distilled water
It is filtered after 15min；
Weigh glucose 20g, agar 15~20g, KH2PO43g, MgSO4·7H2O1.5g is heated in filtrate, and again
To being completely dissolved, benefit is filled with water to 1000mL；
It is transferred to cooling in rear superclean bench after the PDA culture medium sterilization treatment routinely configured, is mixed into when 50 DEG C
0.2g streptomysin sufficiently shakes up, and is poured into sterilized 100mm culture dish while hot.
The sterilising conditions are autoclave sterilization, and the PDA culture medium and culture dish by blade, routinely configured is put into
It sterilizes in high-pressure sterilizing pot, 121 DEG C of holding 30min.
The superclean bench need to carry out alcohol wipe, ultraviolet disinfection and ventilation process work in advance.
The inoculation, expand incubation and inverted plate in the superclean bench for closing ultraviolet light and ventilation process into
Row, and remain close to alcolhol burner flame vicinity.
The constant temperature incubation keeps dark no light in constant incubator, in incubation.
The white fluffy mycelia is the hypha,hyphae of target fructification, during the cultivation process vulnerable to mould, thin
Bacterium pollution selects the mycelia remote apart from points of contamination during picking mycelia, while the mycelia shifted should include partial medium.
The process that isolates and purifies is limited without number, is repeated, until obtaining pure culture.
Preparation method by macro fungi fructification switching strain of the invention is tender big by the vigorous children of field grown first
The refrigeration of type fungus sporophore is taken back, and is uniformly sprayed on fructification with 75% alcohol after carefully being rinsed with distilled water and carries out disinfection,
Then it half-and-half breaks to cap and is split into two halves along stem, cut the stem that size is about 0.5cm × 0.5cm with the sharp cutter of sterilizing
With cap tissue, it is transferred on the solid potato culture medium PDA of addition streptomysin, is finally set vaccinated culture dish
In constant incubator, mycelium germination situation is observed after keeping dark condition to cultivate 7~10 days in 25 DEG C, picking is far from miscellaneous bacteria dirt
The white fluffy mycelia (containing a small amount of culture medium) for contaminating point makees to expand culture into freshly prepd PDA culture medium, until obtaining single
Strain.
It is characteristic of the invention that the shortcomings that overcoming in traditional switching process vulnerable to outside contamination, increases substantially large-scale true
The success rate of mushroom entity switching strain.The improvement that streptomysin carries out PDA culture medium is added, reduces and isolates and purifies strain process
In germ contamination；Using the easy to operate, quick of tissue isolation and advantage that transmissibility shape is stable, realize to large size
The high efficiency of fungus sporophore switching strain.
Specific embodiment: bolete fructification strain transfer
The embodiment of the present invention is described in further detail with reference to the accompanying drawing, as shown in Figure 1 and Figure 2:
(1) fructification acquires: in August, 2018 carries out the collecting work of fructification in Yanqing County of Beijing mountain area, it should be noted that selects
More young tender, the bolete fructification of cap edge curls inward is several, gently takes, is put into portable together with a small amount of soil in root
Refrigerator is taken back.
(2) it is cut into small pieces preparation of culture medium: weighing peeled potatoes 200g, is boiled with appropriate distilled water, in whole process
Filtered through gauze is used after continuing 30min.Glucose 20g, agar 15~20g, KH are added in filtrate2PO43g, MgSO4·
7H2O1.5g, which is again heated to, is completely dissolved (benefit is filled with water to 1000mL).Autoclave sterilization pot is put it into sterilize in 121 DEG C
30min uses cooling in the superclean bench of alcohol wipe, ultraviolet disinfection and ventilation process in advance after being transferred quickly to, to
0.2g streptomysin is mixed at about 50 DEG C sufficiently to shake up, and is poured into sterilized 100mm culture dish while hot.Pay attention to adding strepto-
Element and inverted plate process are required to close to alcolhol burner flame vicinity.
(3) bolete is transferred: the bolete taken back gently being rinsed with distilled water, and removes other unrelated with fructification
Impurity is sprayed on bolete fructification surface with 75% alcohol and carries out disinfection.Tissue separation is carried out in superclean bench, along ox
Liver bacterium stem, which is half-and-half broken to cap, to be split into two halves, and the tissue for cutting 0.5cm × 0.5cm with the sharp cutter of sterilizing is several.By its
It is transferred to the center solid potato culture medium PDA of addition streptomysin respectively, a culture medium can place 3~4 tissue blocks, altogether
4~5 culture mediums of switching.
(4) isolation and purification culture: mycelium germination situation, table are observed in 25 DEG C dark culturing 7~10 days in constant incubator
It is now white fluffy mycelia occur around fructification block.Mycelia (containing a small amount of culture medium) on picking culture medium far from miscellaneous bacteria connects
Kind, again in 25 DEG C of dark culturings, repeats screening into new PDA culture medium to obtain most pure strain.
To sum up, should at least have following advantages by the preparation method of macro fungi fructification switching strain:
1. should be easy to operate by the preparation method of macro fungi fructification switching strain, it is easy to learn, operation is quick.
2. should by macro fungi fructification switching strain preparation method can according to different fungal species carry out in due course,
Suitable ground is adjusted flexibly.
3., should be by the preparation side of macro fungi fructification switching strain compared with traditional solid potato culture medium PDA
Method can effectively inhibit the pollution of bacterium, improve purifying success rate.
4. this method is by utilizing the easy to operate, fast of tissue isolation compared with other fructifications transfer strain technology
Victory and the stable advantage of transmissibility shape realize the high efficiency to macro fungi fructification switching strain.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Within the technical scope of the present disclosure, any changes or substitutions that can be easily thought of by anyone skilled in the art,
It should be covered by the protection scope of the present invention.Therefore, protection scope of the present invention should be with the protection model of claims
Subject to enclosing.
Claims (10)
1. a kind of preparation method by macro fungi fructification switching strain, which is characterized in that comprising steps of
(1) macro fungi fructification sterilizes: the macro fungi fructification obtained in field refrigeration being taken back, after distilled water flushing
Fructification is uniformly sprayed on 75% alcohol to carry out disinfection；
(2) it is inoculated with: half-and-half breaking to cap and be split into two halves along stem, cut fritter stem and cap group with the sharp cutter of sterilizing
It knits, is transferred on the solid potato culture medium PDA of addition streptomysin；
(3) it isolates and purifies: in 25 DEG C of constant temperature incubations, 7~10 days observation mycelium germination situations, picking white fluffy mycelia and training
Feeding base is made to expand culture together into freshly prepd PDA culture medium, until obtaining single culture.
2. the preparation method according to claim 1 by macro fungi fructification switching strain, which is characterized in that described
Macro fungi fructification need to keep fresh, and when picking selects the involute tender fructification of children in vigorous growth, cap edge and is used as examination
Tissue separation material.
3. the preparation method according to claim 1 by macro fungi fructification switching strain, which is characterized in that described
Stem and cap tissue size are 0.5cm × 0.5cm.
4. the preparation method according to claim 1 by macro fungi fructification switching strain, which is characterized in that described
Add the configuration method of the bell potato solid medium PDA of streptomysin:
It weighs peeled potatoes 200g to be cut into small pieces, be put into pot with 1200mL distilled water after keeping 15min after being heated to boiling
Filtering；
Weigh glucose 20g, agar 15~20g, KH2PO43g, MgSO4·7H2O1.5g is again heated to completely in filtrate
Dissolution, benefit are filled with water to 1000mL；
It is transferred to cooling in rear superclean bench after the PDA culture medium sterilization treatment routinely configured, is mixed into 0.2g chain when 50 DEG C
Mycin sufficiently shakes up, and is poured into sterilized 100mm culture dish while hot.
5. the preparation method according to claim 1 or 4 by macro fungi fructification switching strain, which is characterized in that institute
The sterilising conditions stated are autoclave sterilization, and the PDA culture medium and culture dish by blade, routinely configured is put into high-pressure sterilizing pot
Middle sterilizing, 121 DEG C of holding 30min.
6. the preparation method according to claim 1 by macro fungi fructification switching strain, which is characterized in that described
Superclean bench need to carry out alcohol wipe, ultraviolet disinfection and ventilation process work in advance.
7. the preparation method according to claim 1 or 4 by macro fungi fructification switching strain, the inoculation are expanded
Big incubation and inverted plate carry out in the superclean bench for closing ultraviolet light and ventilation process, and remain close to alcohol
Lamp flame vicinity.
8. the preparation method according to claim 1 by macro fungi fructification switching strain, which is characterized in that described
Constant temperature incubation keeps dark no light in constant incubator, in incubation.
9. the preparation method according to claim 1 by macro fungi fructification switching strain, which is characterized in that described
White fluffy mycelia is the hypha,hyphae of target fructification, during the cultivation process vulnerable to mould, germ contamination, in picking bacterium
The mycelia remote apart from points of contamination is selected during silk, while the mycelia shifted should include partial medium.
10. the preparation method according to claim 1 by macro fungi fructification switching strain, which is characterized in that described
Isolate and purify process without number limit, repeat, until obtain pure culture.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111670752A (en) * | 2020-06-29 | 2020-09-18 | 十堰市农业科学院(十堰市农业科学技术研究推广中心、丹江口库区十堰生态农业研究院) | Method for separating mushroom fruiting body tissues |
| CN111670751A (en) * | 2020-06-29 | 2020-09-18 | 新疆农业科学院植物保护研究所 | Culture medium suitable for culturing wild petiole nail ash wrapped strains |
| CN111727810A (en) * | 2020-06-29 | 2020-10-02 | 新疆农业科学院植物保护研究所 | Special culture medium and culture method for wild petiole nail ash wrapped strain |
| CN113455289A (en) * | 2021-08-05 | 2021-10-01 | 河南省农业科学院植物营养与资源环境研究所 | Method for rapidly detecting fruiting performance of edible mushroom tissue isolated strain |
| CN113455287A (en) * | 2021-07-07 | 2021-10-01 | 重庆市酉阳县琦睿峰食用菌有限责任公司 | Method for directly separating morchella tissue by PDA (personal digital Assistant) plate to obtain pure strain |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111670752A (en) * | 2020-06-29 | 2020-09-18 | 十堰市农业科学院(十堰市农业科学技术研究推广中心、丹江口库区十堰生态农业研究院) | Method for separating mushroom fruiting body tissues |
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| CN111727810A (en) * | 2020-06-29 | 2020-10-02 | 新疆农业科学院植物保护研究所 | Special culture medium and culture method for wild petiole nail ash wrapped strain |
| CN111727810B (en) * | 2020-06-29 | 2021-12-21 | 新疆农业科学院植物保护研究所 | Special culture medium and culture method for wild petiole nail ash wrapped strain |
| CN113455287A (en) * | 2021-07-07 | 2021-10-01 | 重庆市酉阳县琦睿峰食用菌有限责任公司 | Method for directly separating morchella tissue by PDA (personal digital Assistant) plate to obtain pure strain |
| CN113455289A (en) * | 2021-08-05 | 2021-10-01 | 河南省农业科学院植物营养与资源环境研究所 | Method for rapidly detecting fruiting performance of edible mushroom tissue isolated strain |
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Application publication date: 20190719 |