CN102242183A - Morel strain identifying method - Google Patents
Morel strain identifying method Download PDFInfo
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- CN102242183A CN102242183A CN2011101624901A CN201110162490A CN102242183A CN 102242183 A CN102242183 A CN 102242183A CN 2011101624901 A CN2011101624901 A CN 2011101624901A CN 201110162490 A CN201110162490 A CN 201110162490A CN 102242183 A CN102242183 A CN 102242183A
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Abstract
The invention belongs to the technical field of microbes, and relates to a morel strain identifying method. The method comprises the following steps of: firstly, observing the mycelia of the strain to be identified on the surface of a culture medium, if the observed mycelia on the surface of the culture medium have the typical morphological characteristics of morel mycelia on the surface of the culture medium, primarily judging that the strain is the morel strain; and further, observing the substrate mycelia, if the observed substrate mycelia have the morphological characteristics of the morel substrate mycelia, determining that the strain is the morel strain. The morel strain identifying method provided by the invention is easy in operation and low in cost, and does not need special instruments and equipment, and the identifying result is easy to judge and reliable.
Description
Technical field
The invention belongs to microbial technology field, relate to a kind of authentication method of morel bacterial classification.
Background technology
Morel be morchella (
Morchella) general designation of fungi, kind surplus the whole world has reported 50, China has reported that kind surplus in the of 10 is (about defining of morchella species, difference is bigger between the different researchers, some scholar only admits 3～6 species titles of morchella), they all are the rare economic epiphytes with important edible and pharmaceutical use.
The morel bacterial classification is meant the Morchella esculenta (L.) Pers mycelium that moves on certain substratum, is important Biological resources, also is the important materials in related scientific research, the teaching.
Utilization to morel mainly contains two kinds of approach.The first is utilized the sporophore of morel, Morchella esculenta (L.) Pers sporophore in the market is mainly derived from field acquisition, and because of source of goods rareness, price is very high always, home market price generally is stabilized in 400～500 yuan of per kilograms, and the world market valency is up to about 200 dollars of per kilograms.Utilize the morel bacterial classification to produce its sporophore by tame method, it is one of mycologist and edible fungus culturing person problem of doing one's utmost to explore for over 100 years more than a hundred years, and, the report of producing Morchella esculenta (L.) Pers sporophore by artificial culture is all arranged both at home and abroad, but the commercialization artificial culture of the morel of being unrealized so far.The morel artificial cultivation technique of having reported all rests on accidental generation of sporophore and the very low level of output, also has sizable gap apart from commercialization production.At present, in many places, still underway about the tame exploration of morel.
Another approach to the morel development and use is to utilize the morel bacterial classification to produce the mycelium product by the method for industrial fermentation.Commercially produced Morchella esculenta (L.) Pers mycelium as seasonings since the sixties in last century abroad.
No matter be the scientific research of being engaged in aspects such as morel artificial culture exploration, still carry out the industrial fermentation production of morel, all need to utilize the morel bacterial classification.Whether the morel bacterial classification, differentiating it really is the morel bacterial classification if being carried out authenticity, be an important process of related scientific research, production and culture presevation unit.
Morel is a kind of fungi that can produce large-scale sporophore, belongs to the macro fungi category, and about the strain identification of macro fungi, traditional method is to make it produce sporophore by artificial culture, identifies according to the feature of sporophore then.Because morel does not produce sporophore substantially under tame condition, just very discrete unit turned out Morchella esculenta (L.) Pers sporophore under very accidental situation, and this makes the method for the morel bacterial classification being identified according to the sporophore feature be difficult to realize.Studying for being engaged in morel, is false as if the bacterial classification of using, and its result of study just can not be correct, and this also is that morel is studied one of major reason that does not make great progress for a long time.For the unit that is engaged in the Morchella esculenta (L.) Pers mycelium fermentative production,, also exist the possibility that causes higher threat if the bacterial classification of using is false.
The development of Protocols in Molecular Biology makes people can utilize the mycelium of fungi to measure its specific dna sequence dna, carries out the strain identification of fungies such as morel then according to its specific dna sequence.But this evaluation based on Protocols in Molecular Biology needs relatively more expensive plant and instrument and professional scientific and technical personnel, and the fund of cost is also more, and many units still can't carry out.
Summary of the invention
The purpose of this invention is to provide a kind of morel strain identification method, overcome the shortcoming of the above two false morels strain identification method, be implemented under the simple and easy condition Rapid identification the morel bacterial classification based on the mycelium morphology feature.
The objective of the invention is to be achieved through the following technical solutions: observe culture of strains primary surface mycelia to be identified earlier, if observed media surface mycelia has the representative configuration feature of morel media surface mycelia, can judge tentatively that this bacterial classification is the morel bacterial classification, further observe its substrate mycelium again, if observed substrate mycelium has the morphological specificity of morel substrate mycelium, promptly this bacterial classification of decidable is exactly the morel bacterial classification.Concrete steps are as follows:
(1) in a culture dish, puts two slide glasss (staying the gap between two slide glasss) side by side, be poured on the slide glass, on every slide glass, all make the plate culture medium of the about 2～3mm of thickness melting and being cooled to 45～50 ℃ PDA substratum; Get the middle part that mycelium is inoculated into every plate culture medium respectively with inoculating needle from the bacterium colony of bacterial classification to be identified, cover the lid of culture dish, be placed in 18～20 ℃ the incubator and cultivate; When the new colony radius that grows when bacterial classification to be identified reaches 1～1.5cm, promptly can be used for the evaluation of following steps on above-mentioned plate culture medium;
(2) one in the described culture dish of step (1) slide glass that carries bacterial classification bacterium colony to be identified is taken out, with lens wiping paper (one side of no substratum) below the slide glass cleaned, distilled water is dripped at the place at colony edge, covered is removed unnecessary water with thieving paper from the suction of cover glass edge;
(3) will put microscopically through the above-mentioned slide glass of handling through step (2) observes, with the morphological specificity of media surface mycelia as judging whether to be the important evidence of morel, if observed media surface mycelia has the representative configuration feature of morel media surface mycelia, can judge tentatively that this bacterial classification is the morel bacterial classification; The representative configuration feature of described morel media surface mycelia is: mycelia has tangible trunk, and longer as the mycelia of trunk, straight and upright, thickness is more even, and barrier film is less, both sides branch estranged; The branch that bears from trunk has two classes, and wherein class branch repeatedly in very short distance is the accumulative dendroid, and it is all shorter that these constitute the dendritic branched sprig of aggregation trees, and thickness is inhomogeneous, and is also not straight; Another kind of branch and trunk plesiomorphism from trunk bears can be described as secondary trunk; Branch characteristic on the secondary trunk is similar to trunk, can produce two class branches, and wherein a class is once more the trunk of one-level, another kind of in very short distance branch repeatedly, be the accumulative dendroid; Though can produce secondary trunk on the trunks at different levels, the secondary trunk number that produces is less, most of branches of generation be short and small and in very short distance repeatedly branched gathering arborizations (see Fig. 1-Fig. 3);
(4) slide glass that another piece in the described culture dish of step (1) is carried bacterial classification bacterium colony to be identified takes out, cut size in the place that bacterium colony is arranged and be about the substratum fritter of 0.3 * 0.3cm, and cut the part (promptly cutting away surperficial one deck substratum that bacterium colony is arranged) of the thick about 1mm in this substratum fritter surface with blade;
(5) cut away the thin slice (tangent plane is surperficial parallel with plate culture medium) that surperficial substratum fritter is cut into thick about 1～2mm with step (4) is described, the substratum thin slice that is cut into is placed water droplet on the slide glass, covered, and the pressing cover slide is squeezed into skim with the substratum thin slice, then slide glass is put microscopically and observed, observed mycelia promptly is a substrate mycelium.If observed substrate mycelium has the morphological specificity of morel substrate mycelium, promptly this bacterial classification of decidable is exactly the morel bacterial classification.The substrate mycelium of morel forms to the substratum growth inside by being the dendritic branched top of aggregation tree on this bacterium culture medium surface mycelia, has following morphological specificity: the mycelia plucked, not straight, color is more shallow than the media surface mycelia look of morel, and branch is many and irregular; As a whole, the mycelia of substrate mycelium of morel and media surface has difference clearly.
In a word, if through above step, observed media surface mycelia and substrate mycelium have the feature of morel media surface mycelia and substrate mycelium respectively, and promptly this bacterial classification of decidable is the morel bacterial classification; If through above step, observed media surface mycelia does not have the morphological specificity of morel media surface mycelia, perhaps observed substrate mycelium does not have the morphological specificity of morel substrate mycelium, and promptly the bacterial classification identified of decidable is not the morel bacterial classification.
Advantage of the present invention is: simple to operate, do not need special different instrument and equipment, and cost is low, and qualification result is judged easily, and qualification result is comparatively reliable.
Description of drawings
Fig. 1～3 are the media surface mycelia of morel;
Fig. 4～6 are the media surface mycelia of non-morel.
Embodiment
Morel strain identification method of the present invention, concrete steps are as follows:
(1) in a culture dish, puts two slide glasss (leaving the gap between two slide glasss) side by side, be poured on the slide glass, on every slide glass, all make the plate culture medium of the about 2～3mm of thickness melting and being cooled to 45～50 ℃ PDA substratum; Get the middle part that mycelium is inoculated into every plate culture medium respectively with inoculating needle from the bacterium colony of bacterial classification to be identified; Cover the lid of culture dish, be placed in 18～20 ℃ the incubator and cultivate; When the new colony radius that grows when bacterial classification to be identified reaches 1～1.5cm, promptly can be used for the evaluation of following steps on above-mentioned plate culture medium;
(2) with one in the described culture dish of step (1) carry bacterial classification bacterium colony to be identified slide glass take out, with lens wiping paper (one side of no substratum) below the slide glass cleaned, distilled water is dripped at the place at colony edge, covered is removed unnecessary water with thieving paper from the suction of cover glass edge;
(3) above-mentioned slide glass is put microscopically and is observed, with the morphological specificity of media surface mycelia as judging whether to be the important evidence of morel; If observed media surface mycelia has the morphological specificity (shown in Fig. 1～3) of morel media surface mycelia, can judge tentatively that this bacterial classification is the morel bacterial classification.The morphological specificity of morel media surface mycelia is: mycelia has tangible trunk, and longer as the mycelia of trunk, straight and upright, thickness is more even, and barrier film is less, both sides branch estranged; The branch that bears from trunk has two classes, and wherein class branch repeatedly in very short distance is the accumulative dendroid, and these constitute, and the dendritic branched sprig of aggregation trees is all short, thickness is inhomogeneous, also not straight; Another kind of branch and trunk plesiomorphism from trunk bears can be described as secondary trunk.Branch characteristic on the secondary trunk is similar to trunk, can produce two class branches, and wherein a class is once more the trunk of one-level, another kind of in very short distance branch repeatedly, be the accumulative dendroid.Though can produce secondary trunk on the trunks at different levels, the secondary trunk number that produces is less, most of branches of generation be short and small and in very short distance repeatedly branched gathering arborizations;
(4) another piece in the described culture dish of step (1) is carried bacterial classification bacterium colony to be identified slide glass take out, cut size in the place that bacterium colony is arranged and be about the substratum fritter of 0.3 * 0.3cm, and cut the part (promptly cutting away surperficial one deck substratum that bacterium colony is arranged) of the thick about 1mm in this substratum fritter surface with blade;
(5) cut away the thin slice (tangent plane is surperficial parallel with plate culture medium) that surperficial substratum fritter is cut into thick about 1～2mm with above-mentioned, the substratum thin slice that is cut into is placed water droplet on the slide glass, covered, and the pressing cover slide is squeezed into skim with the substratum thin slice, then slide glass is put microscopically and observed, observed mycelia promptly is a substrate mycelium; If observed substrate mycelium has the morphological specificity of morel substrate mycelium, promptly this bacterial classification of decidable is exactly the morel bacterial classification.The morphological specificity of morel substrate mycelium is as follows: the mycelia plucked, and not straight, color is more shallow at the mycelia look of media surface than morel, and branch is many and irregular.As a whole, the mycelia of substrate mycelium of morel and media surface has difference clearly.The substrate mycelium of morel is to be the dendritic branched top of aggregation tree on the media surface mycelia by morel to form to the substratum growth inside.
If through above step, observed media surface mycelia does not have the representative configuration feature of morel media surface mycelia, perhaps observed substrate mycelium does not have the morphological specificity of morel substrate mycelium, and promptly the bacterial classification identified of decidable is not the morel bacterial classification.
Respectively the more or less a hundred bacterial classification material that includes the morel bacterial classification has been carried out qualification test with method of the present invention with based on the molecular assay method (contrast) of rDNA internal transcribed spacer district (ITS) sequence, test confirms that two kinds of qualification results are in full accord.See the following form
The qualification result contrast of authentication method of the present invention and Molecular Identification method
Claims (4)
1. morel strain identification method, it is characterized in that, observe the mycelia of bacterial classification to be identified earlier in media surface, if observed media surface mycelia has the representative configuration feature of morel media surface mycelia, can judge tentatively that this bacterial classification is the morel bacterial classification, further observe its substrate mycelium again, if observed substrate mycelium has the morphological specificity of morel substrate mycelium, promptly this bacterial classification of decidable is exactly the morel bacterial classification.
2. morel strain identification method according to claim 1 is characterized in that concrete steps are as follows:
(1) in a culture dish, puts two slide glasss side by side, be poured on the slide glass, on every slide glass, all make the plate culture medium of the about 2～3mm of thickness melting and being cooled to 45～50 ℃ PDA substratum; Get the middle part that mycelium is inoculated into every plate culture medium respectively with inoculating needle from the bacterium colony of bacterial classification to be identified; Cover the lid of culture dish, be placed in 18～20 ℃ the incubator and cultivate; When the new colony radius that grows when bacterial classification to be identified reaches 1～1.5cm, can be used for the evaluation of following steps on above-mentioned plate culture medium;
(2) one in the described culture dish of step (1) slide glass that carries bacterial classification bacterium colony to be identified is taken out, clean below slide glass with lens wiping paper, distilled water is dripped at the place at colony edge, and covered is removed unnecessary water with thieving paper from the suction of cover glass edge;
(3) the above-mentioned slide glass of handling through step (2) is put the mycelia that microscopically is observed media surface,, can judge tentatively that this bacterial classification is the morel bacterial classification if observed media surface mycelia has the representative configuration feature of morel media surface mycelia;
(4) another piece in the described culture dish of step (1) is carried bacterial classification bacterium colony to be identified slide glass take out, cut the substratum fritter of the about 0.3cm * 0.3cm of size in the place that bacterium colony is arranged, and cut the part of the surperficial thick about 1mm of this substratum fritter;
(5) cut away the thin slice (tangent plane is surperficial parallel with plate culture medium) that surperficial substratum fritter is cut into thick about 1～2mm with step (4) is described, the substratum thin slice that is cut into is placed water droplet on the slide glass, covered, and the pressing cover slide is squeezed into skim with the substratum thin slice, then slide glass being put microscopically observes, observed mycelia promptly is a substrate mycelium, if observed substrate mycelium has the morphological specificity of morel substrate mycelium, promptly this bacterial classification of decidable is exactly the morel bacterial classification.
3. morel strain identification method according to claim 1 and 2 is characterized in that the representative configuration feature of described morel media surface mycelia is: mycelia has tangible trunk, mycelia as trunk is longer, and is straight and upright, and thickness is more even, barrier film is less, both sides branch estranged; The branch that bears from trunk has two classes, and wherein class branch repeatedly in very short distance is the accumulative dendroid, and it is all shorter that these constitute the dendritic branched sprig of aggregation trees, and thickness is inhomogeneous, and is also not straight; Another kind of branch and trunk plesiomorphism from trunk bears can be described as secondary trunk; Branch characteristic on the secondary trunk is similar to trunk, can produce two class branches, and wherein a class is once more the trunk of one-level, another kind of in very short distance branch repeatedly, be the accumulative dendroid; Though can produce secondary trunk on the trunks at different levels, the secondary trunk number that produces is less, most of branches of generation be short and small and in very short distance repeatedly branched gathering arborizations.
4. morel strain identification method according to claim 1 and 2, it is characterized in that, the morphological specificity of described morel substrate mycelium is: the mycelia plucked, not straight, color is more shallow than the media surface mycelia look of morel, branch is many and irregular, and the substrate mycelium of morel is to be the dendritic branched top of aggregation tree on the media surface mycelia by morel to form to the substratum growth inside.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106282370A (en) * | 2016-09-20 | 2017-01-04 | 四川省农业科学院土壤肥料研究所 | Morchella esculenta (L.) Pers mating type determines gene identification primer special and fruiting energy force prediction method |
| CN107177514A (en) * | 2017-07-06 | 2017-09-19 | 江油市静远食用菌科技有限责任公司 | A kind of purity is the preparation method of 100% morchella mother culture |
| CN107177663A (en) * | 2017-06-02 | 2017-09-19 | 武汉市农业科学技术研究院蔬菜科学研究所 | A kind of authentication method of hickory chick bacterial strain aging |
| CN111721747A (en) * | 2020-06-30 | 2020-09-29 | 常熟理工学院 | Method for detecting concentration of morchella hypha nucleuses by cell nucleus staining |
-
2011
- 2011-06-16 CN CN2011101624901A patent/CN102242183B/en not_active Expired - Fee Related
Non-Patent Citations (4)
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| 付晓燕: "羊肚菌菌丝体培养及菌丝分化研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106282370A (en) * | 2016-09-20 | 2017-01-04 | 四川省农业科学院土壤肥料研究所 | Morchella esculenta (L.) Pers mating type determines gene identification primer special and fruiting energy force prediction method |
| CN106282370B (en) * | 2016-09-20 | 2020-02-11 | 四川省农业科学院土壤肥料研究所 | Primer special for identifying mating type determinant gene of morchella and fruiting capacity prediction method |
| CN107177663A (en) * | 2017-06-02 | 2017-09-19 | 武汉市农业科学技术研究院蔬菜科学研究所 | A kind of authentication method of hickory chick bacterial strain aging |
| CN107177514A (en) * | 2017-07-06 | 2017-09-19 | 江油市静远食用菌科技有限责任公司 | A kind of purity is the preparation method of 100% morchella mother culture |
| CN111721747A (en) * | 2020-06-30 | 2020-09-29 | 常熟理工学院 | Method for detecting concentration of morchella hypha nucleuses by cell nucleus staining |
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| CN102242183B (en) | 2013-03-20 |
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