CN102668874A - Method for tissue isolation of wild edible fungus strain by culture medium inversion suspension method - Google Patents
Method for tissue isolation of wild edible fungus strain by culture medium inversion suspension method Download PDFInfo
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- CN102668874A CN102668874A CN2011104320931A CN201110432093A CN102668874A CN 102668874 A CN102668874 A CN 102668874A CN 2011104320931 A CN2011104320931 A CN 2011104320931A CN 201110432093 A CN201110432093 A CN 201110432093A CN 102668874 A CN102668874 A CN 102668874A
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Abstract
The invention relates to a method for tissue isolation of a wild edible fungus strain by a culture medium inversion suspension method. The method comprises the following steps of: wrapping a fruiting body by using an adhesive tape, cleaning the outer surface of the fruiting body by using 75 percent alcohol, longitudinally cutting the fruiting body, cutting and taking a tissue block at a thick position of the fruiting body, quickly passing for 2 to 3 times back and forth on the flame of an alcohol lamp, feeding into a test tube, ensuring that the surface of a culture medium of the test tube is downward at this time, feeding to a position under the isolation culture medium, which is 2 to 4mm away from the isolation culture medium, at the bottom of the test tube by using an inoculating needle, keeping the state and culturing, moisturizing and culturing for 7 to 10d in a state that the temperature is 28 to 30 DEG C and the relative humidity is 70 to 90 percent, and hyphae germinated by the tissue block being upwards butted with the culture medium for survival. The suspended inoculation after the tissue block is moisturized and germinated is adopted, so the success probability reaches over 80 percent. The method aims at wild edible fungi with more attached sundry fungi; and a strain block in the method is a relatively large tissue block usually, can germinate the hyphae easily, is cultured in a way of corresponding to the culture medium in the air, is inoculated to a blank culture medium which is suspended above the tissue block through the germinated aerial hyphae, cannot be polluted easily, and is relatively high in success rate.
Description
Technical field
The present invention relates to a kind of medium and be inverted the method for unsettled method separate tissue wild edible fungus bacterial classification, belong to the microorganism fungus kind separating method.
The research background
Strain separating is the local organization of valuable fruit body, spore or substrate mycelium to be moved receive on the slant tube medium, thereby obtains the pure culture mycelia.The acquisition of microorganism resource has field acquisition and cultivation place to gather two kinds of approach.The collection of open-air sample mainly is applicable to the separation of one-level kind of wild species domestication, genetic breeding, physiological ecological and the various research usefulness of edible mushroom.And in the separation of edible fungus culturing upper level kind, the source of its kind mushroom mainly from cultivation through the acquisition of reserving seed for planting.
Edible fungus species is the important means of production, also is the important experimental material in teaching, the scientific research.The quality of strain quality directly influences the success or failure of cultivation and the height of output, has only good bacterial classification could obtain the product of high yield and high-quality, therefore produces the extremely important link that good bacterial classification is an edible fungus culturing.According to source, reproductive order of generation and the production purpose of bacterial classification, be divided into female kind, original seed to bacterial classification with cultivated species, separate with fruit body, in fruit body appear usually period in a large number.Be preferably in to gather and just separated the same day.Generally should choose tender, solid, dry and comfortable, the anosis fruit body of children is separate object.
No matter be artificial cultivation fruit body or the production of liquid fermentation mycelium, it is crucial obtaining high-quality excellent species.Carrying out strain separating is an important step in the edible fungus culturing.Strain separating must be undertaken by a strict aseptic manipulation program.Strain separating can be divided into spore separation, separate tissue and substrate mycelium and separate three kinds.
Edible fungus species isolated tissue partition method comprises fruit body, sclerotium, and the method that the shoestring interior tissue obtains pure culture all belongs to tissue isolation.In the actual production with maximum be exactly tissue isolation, conventional method for tissue separation is: select good kind mushroom as parting material, behind aseptic water washing, the alcoholic solution surface sterilization with 75%.The blade that cancellation poison is crossed from stem or cap middle part rip cutting, tear, a little block organization of picking cap and stem intersection (match end size) is inoculated on the PDA medium after 3-5 days the white fine hair shape mycelia of generation on the inoculation piece.But separate the problem that wild edible fungus exists at present mainly be: wild edible fungus generally is grown in the field, attaches foreign material outward and assorted bacterium is more, and part suffers worm-eaten again, and the bacterial classification piece of separation very easily pollutes, and is not easy to separate successfully.
Summary of the invention
The object of the present invention is to provide a kind of medium to be inverted the method for unsettled method separate tissue wild edible fungus bacterial classification.
To achieve these goals, technical scheme of the present invention has adopted a kind of medium to be inverted the method for unsettled method separate tissue wild edible fungus bacterial classification, may further comprise the steps: get fruit body; With the adhesive tape parcel, 75% alcohol is cleaned outer surface, vertically cuts fruit body; Cut and get the abundant organizations in the localities' piece of fruit body, on alcolhol burner flame, overdo back and forth fast and send into test tube 2-3 time, this moment, the test tube media surface was downward; Again with transfer needle deliver to test tube bottom isolation medium below; Apart from isolation medium 2-4mm, and keep this kind state to cultivate temperature 28-30 ℃; Preserve moisture under the state of relative moisture 70-90% and cultivate 7-10d, the mycelia that piece of tissue is sprouted can upwards be docked medium and formed work.
Described fruit body brushes away the impurity of mushroom surface earlier with aseptic brush.
Vertically cutting fruit body adopts scalpel to carry out.
Described isolation medium is made up of following raw material: potato 350-450g, fermentation maturity cow dung 90-110g, glucose 15-25g; Potassium dihydrogen phosphate 0.5-1.2g, magnesium sulfate 0.2-1.8g, yeast extract 45-55g; Agar 15-25g, water 1000mL; Streptomycin 50U/mL, penicillin 50U/mL, pH6.5.
The preparation method of said isolation medium is: potato is peeled cut thin skin and add 3000mL poach 30min, cross leaching 2000mL, add the cow dung of fermentation maturity.Little fiery digestion 20min crosses leaching filtrating 1000mL; In this filtrating, add the heating of agar continued, the firepower size does not stop to stir not overflow for good simultaneously; Prevent to be burned, treat that it dissolves fully after, add again glucose, yeast extract,, potassium dihydrogen phosphate, magnesium sulfate, streptomycin and each 50000U of penicillin; Stir, be settled to 1000mL; The medium that has prepared is sub-packed in vitro while hot, and every 5mL is with the tight test tube mouth of space wadding tampon; 7 one, autoclaving, 121 ℃ of temperature; Time 50min, the test tube medium is put into big inclined-plane, covers newspaper and parks 2-4d; The moisture of the media surface that leaves utilizes the sprouting of preserving moisture of edible fungi tissue, is inoculated into naturally on the unsettled medium.
Generally, fruit body of edible fungi can not use 75% alcohol to put on the skin and wipe away, and alcohol is easy to immerse fruit body, causes piece of tissue not sprout; And the present invention adopts adhesive tape parcel fruit body, can disinfect in alcohol to put on the skin and wipe away, and has prevented that effectively the assorted bacterium on fruit body surface from polluting in the space of operation; Antibiotic directly adds isolation medium, has antibacterial activity.The subsidiary assorted bacterium of wild edible fungus is a lot of, adopts piece of tissue to preserve moisture and sprouts the unsettled inoculation in back, and probalility of success reaches more than 80%.The present invention be directed to the outer more wild edible fungus of assorted bacterium that attaches; Bacterial classification piece among the present invention is generally got big piece of tissue; Sprout mycelia and medium easily at a distance from empty corresponding the cultivation, be inoculated into unsettled blank medium above piece of tissue through the aerial hyphae that sprouts; Be difficult for polluting, success rate is than higher.
Description of drawings
Fig. 1 is an inoculation piece position view of the present invention.
Embodiment
The method of present embodiment is: get fruit body, brush away the impurity of mushroom surface with aseptic brush, wrap up with adhesive tape; 75% alcohol is cleaned outer surface, vertically cuts fruit body with scalpel, cuts and gets the abundant organizations in the localities' piece of fruit body; On alcolhol burner flame, overdo back and forth fast and send into test tube 3 times, this moment, the test tube media surface was downward, shown in 1 among Fig. 1; Again with transfer needle deliver to the test tube bottom medium below, apart from medium 3mm, shown in 2 among Fig. 1.And keep this state to cultivate, and 28 ℃ of temperature, relative moisture 80% are preserved moisture and are cultivated 7-10d, and the mycelia that piece of tissue is sprouted can upwards be docked medium and formed work.
The isolation medium of present embodiment is made up of following raw material: potato 400g, fermentation maturity cow dung 100g, glucose 20g, potassium dihydrogen phosphate 1.0g; Magnesium sulfate 1.5g, yeast extract 50g, agar 20g, water 1000mL; Streptomycin 50U/mL, penicillin 50U/mL, pH6.5.
Wherein, the preparation method of isolation medium is: potato is peeled cut thin skin and add 3000mL poach 30min, cross leaching 2000mL, add the cow dung of fermentation maturity.Little fiery digestion 20min crosses leaching filtrating 1000mL; In this filtrating, add each 50000U of glucose, yeast extract, agar, potassium dihydrogen phosphate, magnesium sulfate, streptomycin and penicillin, boil, be settled to 1000Ml to the agar dissolving; The medium that has prepared is sub-packed in vitro while hot, and every 5mL is with the tight test tube mouth of space wadding tampon; 7 one, autoclaving, 121 ℃ of temperature; Time 50min, the test tube medium is put into big inclined-plane, covers newspaper and parks 2-4d; The moisture of the media surface that leaves utilizes the sprouting of preserving moisture of edible fungi tissue, is inoculated into naturally on the unsettled medium.
Claims (5)
1. the method that medium is inverted unsettled method separate tissue wild edible fungus bacterial classification is characterized in that: may further comprise the steps: get fruit body, wrap up with adhesive tape; 75% alcohol is cleaned outer surface, vertically cuts fruit body, cuts and gets the abundant organizations in the localities' piece of fruit body; On alcolhol burner flame, overdo back and forth fast and send into test tube 2-3 time, this moment, the test tube media surface was downward, again with transfer needle deliver to test tube bottom isolation medium below; Apart from isolation medium 2-4mm, and keep this kind state to cultivate temperature 28-30 ℃; Preserve moisture under the state of relative moisture 70-90% and cultivate 7-10d, the mycelia that piece of tissue is sprouted can upwards be docked medium and formed work.
2. medium according to claim 1 is inverted the method for unsettled method separate tissue wild edible fungus bacterial classification, it is characterized in that: described fruit body brushes away the impurity of mushroom surface earlier with aseptic brush.
3. medium according to claim 1 is inverted the method for unsettled method separate tissue wild edible fungus bacterial classification, it is characterized in that: vertically cut fruit body and adopt scalpel to carry out.
4. medium according to claim 1 is inverted the method for unsettled method separate tissue wild edible fungus bacterial classification, and it is characterized in that: described isolation medium is made up of following raw material: potato 350-450g, fermentation maturity cow dung 90-110g; Glucose 15-25g, potassium dihydrogen phosphate 0.5-1.2g, magnesium sulfate 0.2-1.8g; Yeast extract 45-55g, agar 15-25g, water 1000mL, streptomycin 50U/mL; Penicillin 50U/mL, pH6.5.
5. the method for being inverted unsettled method separate tissue wild edible fungus bacterial classification according to claim 1 or 4 described medium; It is characterized in that: the preparation method of said isolation medium is: potato is peeled cut thin skin and add 3000mL poach 30min; Cross leaching 2000mL, add the cow dung of fermentation maturity; Digestion 20min crosses leaching filtrating 1000mL; The medium that has prepared is sub-packed in vitro, with the tight test tube mouth of space wadding tampon, autoclaving, 121 ℃ of temperature, time 50min, the test tube medium is put into big inclined-plane, covers newspaper and parks 2-4d, is inoculated into naturally on the unsettled medium.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105875202A (en) * | 2016-06-13 | 2016-08-24 | 蔡志闯 | Inoculation mechanism, strain containers and method for culturing edible mushrooms |
| CN107667783A (en) * | 2017-10-24 | 2018-02-09 | 翔天农业开发集团股份有限公司 | A kind of sterile cavity method for tissue separation |
| CN116948846A (en) * | 2023-09-19 | 2023-10-27 | 江西农业大学 | Method for separating and purifying colloid bacteria |
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| WO2004075627A1 (en) * | 2003-02-28 | 2004-09-10 | Yonekura, Yasuko | Vital culture of sparassic crispa and method of prparing growth medium |
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| JP2011109941A (en) * | 2009-11-25 | 2011-06-09 | Nihon Kinoko Kk | New variety derived from pleurotus nebrodensis, and method for cultivating mushroom using the same |
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- 2011-12-20 CN CN2011104320931A patent/CN102668874A/en active Pending
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| CN1419809A (en) * | 2001-11-15 | 2003-05-28 | 湛江海洋大学 | Lobay tricholoma strain separation pure culture technigne and cultivation method thereof |
| WO2004075627A1 (en) * | 2003-02-28 | 2004-09-10 | Yonekura, Yasuko | Vital culture of sparassic crispa and method of prparing growth medium |
| WO2005077148A1 (en) * | 2004-02-13 | 2005-08-25 | Toyama, Noriko | Hyphal culture of sparassis crispa and method of preparing composite culture medium |
| JP2011109941A (en) * | 2009-11-25 | 2011-06-09 | Nihon Kinoko Kk | New variety derived from pleurotus nebrodensis, and method for cultivating mushroom using the same |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105875202A (en) * | 2016-06-13 | 2016-08-24 | 蔡志闯 | Inoculation mechanism, strain containers and method for culturing edible mushrooms |
| CN107667783A (en) * | 2017-10-24 | 2018-02-09 | 翔天农业开发集团股份有限公司 | A kind of sterile cavity method for tissue separation |
| CN116948846A (en) * | 2023-09-19 | 2023-10-27 | 江西农业大学 | Method for separating and purifying colloid bacteria |
| CN116948846B (en) * | 2023-09-19 | 2023-12-26 | 江西农业大学 | Method for separating and purifying colloid bacteria |
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Application publication date: 20120919 |