CN1952108A - Long term storage method for large-size fungus species - Google Patents
Long term storage method for large-size fungus species Download PDFInfo
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- CN1952108A CN1952108A CN 200610123387 CN200610123387A CN1952108A CN 1952108 A CN1952108 A CN 1952108A CN 200610123387 CN200610123387 CN 200610123387 CN 200610123387 A CN200610123387 A CN 200610123387A CN 1952108 A CN1952108 A CN 1952108A
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- bacterial classification
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Abstract
The invention disclosed a way of preserving the big fungus strain for a long term. It comprises the following steps: (1) inoculating the strain to the solid medium and cutting the medium into several pieces when the hyphal body nearly covered the flat plate; (2) adding liquid medium and cryoprotectant to the container first, then putting the pieces of medium in the container; covering the lid and preserving it at -70 Deg C. The invention can preserve the strain for a long term without variation; besides, it's very simple and economical.
Description
Technical field
The present invention relates to the culture collection process technical field, particularly relate to the long term storage method of large-size fungus species.
Background technology
The long term storage method of large-size fungus species mainly contains at present: (A) whiteruss preserving process, promptly on cultured inclined-plane, add the aseptic anhydrous liq paraffin of last layer, and paraffin exceeds 1cm place, inclined-plane, places 4-15 ℃ of preservation, and the shelf time is 3-5.The shortcoming of present method is (1) consumption consuming time space; Vitality was very low when (2) bacterial classification of preserving was transferred.(B) nitrogen ultra low temperature preserving process, promptly the mycelium of scraping gently on the plate of making even makes into bacteria suspension, or makes spore suspension with protective material; the glycerine or 10% dimethyl sulfoxide (DMSO) of adding 10%; draw the 0.5-0.8mL suspension and inject ampoul tube, the sealing by fusing mouth of pipe is put in the liquid nitrogen freezing device and is preserved.Though this method energy prolonged preservation bacterial classification, complex operation, apparatus expensive, liquid nitrogen source difficulty and price height, most of research institution does not have condition to use.(the U.S. work of Yang Xin, " edible mushrooms research method ", Chinese agriculture press, 1999; Yellow Nian Lai chief editor, " edible fungi of china encyclopaedia ", Chinese agriculture goes out newspaper office, in May, 1993).
At present, the large-size fungus species preservation mechanism of China's national level mainly contains " China Committee for Culture Collection of Microorganisms microbial strains preservation administrative center (CCGMC) ", " 3 of China Committee for Culture Collection of Microorganisms agricultural microbial strains preservation administrative center (ACCC) and " agriculture microbial strains preservation administrative center edible fungus species branch center "; provincial have more than ten; also have some local bacterial classifications to preserve mechanism; it is costly that these centers or mechanism are used for the bacterial classification of preservation macro fungi every year; a kind of large-size fungus species method for preserving of simple economy if can be provided, and that meaning is very great.
Summary of the invention
The objective of the invention is to overcome the deficiency that exists on the long term storage method of existing large-size fungus species, a kind of long term storage method of simple, economic large-size fungus species is provided.
A kind of long term storage method of large-size fungus species comprises the steps:
(1) will need the bacterial classification of preservation to be inoculated on the solid medium earlier and cultivate, when waiting mycelium to cover with flat board solid medium be cut into some bacterial classification pieces;
(2) in container, add liquid nutrient medium and antifreezing agent earlier, put the bacterial classification piece again into, cover tight bottle cap, preserve down in-70 ℃.
The preferred frozen pipe of said vesse, above-mentioned antifreezing agent preferably glycerine, glycerol concentration preferred 15%.
Compared with prior art, the present invention has following beneficial effect: the preservation for a long time of the method for preserving of large-size fungus species of the present invention, easy and economical, bacterial classification is difficult for producing variation.
Description of drawings
Fig. 1 is that needle mushroom Fve1 bacterial classification is inoculated into the mycelial growth situation on the inclined-plane after preserving 2 years under-70 ℃;
Fig. 2 is that needle mushroom Fve4 bacterial classification is inoculated into the mycelial growth situation on the inclined-plane after preserving 2 years under-70 ℃;
Fig. 3 is that needle mushroom Fve1 bacterial classification is inoculated into the fruiting situation in the liquid nutrient medium after-70 ℃ are preserved 2 years down:
Fig. 4 is that needle mushroom Fve5 bacterial classification is inoculated into the fruiting situation in the liquid nutrient medium after-70 ℃ are preserved 2 years down;
Fig. 5 is that needle mushroom Fve5 bacterial classification is inoculated into the fruiting situation in the solid medium after-70 ℃ are preserved 2 years down;
Fig. 6 is that needle mushroom Fve4 bacterial classification is inoculated into the fruiting situation in the solid medium after-70 ℃ are preserved 2 years down;
Fig. 7 is that needle mushroom Fve1 bacterial classification is inoculated into the fruiting situation in the solid medium after-70 ℃ are preserved 2 years down.
Embodiment
Embodiment 1: the preservation of needle mushroom bacterial classification
1. used substratum is culture medium for golden mushroom commonly used, and their prescription is respectively:
(1) PDSA solid plate substratum: peeling potato 200g, glucose 20g, KH
2PO
43g, MgSO
41.5g, vitamins B
15-10mg, agar 20g, H
2O 1L;
(2) PDSB liquid nutrient medium: peeling potato 200g, glucose 20g, KH
2PO
43g, MgSO
41.5g, vitamins B
15-10mg, H
2O 1L.
2. practise edible mushrooms research centre, wine town for ensaying pin mushroom bacterial strain available from Xishui County, Guizhou city, strain number is Fve1, Fve2, Fve3, Fve4, Fve5.
Concrete steps are as follows:
(1) prepares the solid plate substratum according to a conventional method, in Bechtop, inoculate 0.5cm respectively
25 needle mushroom inclined-plane mothers of size plant in dull and stereotyped middle, and inoculation is placed under 21 ℃ of temperature cultivates;
When (2) one weeks, the back mycelia was just covered with culture dish, utilize scalper to cut apart flat board gently in Bechtop, making becomes 0.5cm
2Square bacterial classification piece; Or the hollow stainless steel column that utilizes design voluntarily is cut into the disk bacterial classification piece of diameter 0.5cm.
(3) get the aseptic frozen pipe of 5 1.8mL, add the liquid nutrient medium (PDSB) of 0.5-1.0mL in Bechtop, adding glycerine afterwards, to make its final concentration be 15%, puts the 0.5cm of 3-8 piece Fve1, Fve2, Fve3, Fve4 and Fve5 respectively into
2Square bacterial classification piece or the diameter disk bacterial classification piece that is 0.5cm, allow liquid nutrient medium all cover the bacterial classification piece.Cover tight bottle cap, write label, in-70 ℃ of Ultralow Temperature Freezers, preserve.Each bacterial classification is preserved 10 pipes.
Embodiment 2: needle mushroom is preserved the detection of bacterial classification
The panel bevel mycelia is observed:
(1) prepares the PDSA substratum routinely, be made into flat board and inclined-plane respectively.
(2) from-70 ℃ of Ultralow Temperature Freezers, take out the needle mushroom bacterial classification every half a year and be inoculated in respectively on the inclined-plane, place 21 ℃ constant incubator to cultivate afterwards.
(3) one week backs are observed, and find that the normal and growth conditions of the mycelia form of bacterial classification well (sees Fig. 1, Fig. 2).
(4) in Bechtop, the same bacterial classification of preserving 2 years down at-70 ℃ is received dull and stereotyped both sides simultaneously, the mycelia of bacterium colony does not produce the antagonism line when contacting, and not variation of bacterial classification is described.
The liquid nutrient medium fruiting experiment:
(1) prepares the PDSB liquid nutrient medium routinely, be sub-packed in the triangular flask of 250mL, the bottled 50mL of each triangle.
(2) in Bechtop, the needle mushroom that embodiment 1 is preserved is inoculated in the triangular flask, places 21 ℃ constant incubator to cultivate afterwards.
After (3) 3 weeks, the bacterial classification of-70 ℃ of preservations all can grow normal sporophore and (see Fig. 3, Fig. 4).
The solid medium fruiting experiment:
Solid culture based formulas: cotton seed hulls 85%, wheat bran 10%, sugar 1%, Semen Maydis powder 3%, lime carbonate 1%.
(1) accurately take by weighing each composition by the solid culture based formulas, with 1: 1.5 material-water ratio, the mixing substratum.
(2) get the polypropylene plastics pocket of 17 * 30cm, carefully put into substratum to 1/2 bag, the substratum degree of tightness is suitable, puts the collar, has filled in tampon.
(3) culture medium bag that installs is placed 121 ℃, 1.5kg/cm
2Down sterilization 1.5 hours of pressure.
(4) treat substratum cooling after, in Bechtop, get above activatory needle mushroom slant strains and meet a 0.5cm
2The bacterial classification piece in culture bag, place 21 ℃ constant incubator to cultivate afterwards.
Mycelia is covered with the bacterium bag after (5) 3 weeks, enters the management of producing mushroom stage, and the management of producing mushroom of needle mushroom is undertaken by the method and the step of " Chinese needle mushroom production " (Guo Meiying chief editor, Chinese agriculture press, 2000).The fruiting situation is seen Fig. 5, Fig. 6 and Fig. 7.
Embodiment 3
Method for preserving of the present invention and existing slant preservation method are compared, specifically see Table 1.
Table 1 the inventive method and conventional long-term preservation method are relatively
| Method | Plant and instrument | The preservation container | 100cm 3The bacterial classification quantity (individual) of preserving | Shelf time | Standing charges | Spawn activity | Preserve operation |
| The whiteruss preservation method | Normal temperature or general refrigerator | 35mL (1.5 * 15.0cm) glass test tube | 3 | 1-3 | The electricity charge | A little less than | Loaded down with trivial details |
| The nitrogen ultra low temperature preservation method | Liquid nitrogen container | Ampoul tube | 30 | For a long time | Consume 1% liquid nitrogen expense every day | By force | Loaded down with trivial details |
| Bacterium piece very low temperature preserving process (the inventive method) | Ultralow Temperature Freezer | 1.8mL the frozen pipe of plastics | 55 | For a long time | The electricity charge | By force | Easy |
Claims (4)
1, the long term storage method of large-size fungus species is characterized in that comprising the steps:
(1) will need the bacterial classification of preservation to be inoculated on the solid medium earlier and cultivate, when waiting mycelium to cover with flat board solid medium be cut into some bacterial classification pieces;
(2) in container, add liquid nutrient medium and antifreezing agent earlier, put the bacterial classification piece again into, cover tight bottle cap, preserve down in-70 ℃.
2, method for preserving as claimed in claim 1 is characterized in that described container is frozen pipe.
3, method for preserving as claimed in claim 1 is characterized in that described antifreezing agent is a glycerine.
4, method for preserving as claimed in claim 1 is characterized in that described bacterial classification is needle mushroom, p mushroom or glossy ganoderma.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610123387 CN1952108A (en) | 2006-11-07 | 2006-11-07 | Long term storage method for large-size fungus species |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610123387 CN1952108A (en) | 2006-11-07 | 2006-11-07 | Long term storage method for large-size fungus species |
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|---|---|
| CN1952108A true CN1952108A (en) | 2007-04-25 |
Family
ID=38058668
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|---|---|---|---|
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102106233B (en) * | 2009-12-29 | 2013-02-27 | 上海市农业科学院 | Method for preserving strains of ectomycorrhizal fungi |
| CN103430773A (en) * | 2013-08-28 | 2013-12-11 | 云南省热带作物科学研究所 | Method for preserving deep brown boletus reticulatus strains |
| CN106085865A (en) * | 2016-08-23 | 2016-11-09 | 宁夏泰瑞制药股份有限公司 | A kind of method for preserving of oblique lid mushroom |
| CN114276934A (en) * | 2021-12-22 | 2022-04-05 | 山东恒信生物科技有限公司 | Preservation method of flammulina velutipes strains |
-
2006
- 2006-11-07 CN CN 200610123387 patent/CN1952108A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102106233B (en) * | 2009-12-29 | 2013-02-27 | 上海市农业科学院 | Method for preserving strains of ectomycorrhizal fungi |
| CN103430773A (en) * | 2013-08-28 | 2013-12-11 | 云南省热带作物科学研究所 | Method for preserving deep brown boletus reticulatus strains |
| CN106085865A (en) * | 2016-08-23 | 2016-11-09 | 宁夏泰瑞制药股份有限公司 | A kind of method for preserving of oblique lid mushroom |
| CN106085865B (en) * | 2016-08-23 | 2019-11-05 | 宁夏泰瑞制药股份有限公司 | A kind of method for preserving of oblique lid mushroom |
| CN114276934A (en) * | 2021-12-22 | 2022-04-05 | 山东恒信生物科技有限公司 | Preservation method of flammulina velutipes strains |
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