CN107674838B - Method for quickly preserving fungi - Google Patents
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- CN107674838B CN107674838B CN201710796139.5A CN201710796139A CN107674838B CN 107674838 B CN107674838 B CN 107674838B CN 201710796139 A CN201710796139 A CN 201710796139A CN 107674838 B CN107674838 B CN 107674838B
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Abstract
The invention relates to a method for rapidly preserving fungi, which adopts an improved filter paper sheet method to preserve fungi, adopts a filter paper sheet with dotted lines to preserve fungi, and has the advantages that hyphae can grow through the filter paper, contact with sufficient nutrition in the growth process, and grow well. The invention improves the operation links of the paper sticking operation and the paper recovery operation, can greatly save time and time, saves labor, has lower pollution rate than the conventional method, and greatly improves the working efficiency. The method saves nearly 87% of paper sheets of one culture dish in the paper sheet pasting step compared with the traditional method, saves about 55% of paper sheets of one culture dish in the paper sheet recycling and storing step compared with the traditional method, greatly improves the working efficiency and lightens the labor intensity. And the method has short time and greatly reduces the pollution rate in the actual operation.
Description
Technical Field
The invention relates to a method for preserving microorganisms, in particular to a method for rapidly preserving fungi.
Background
Fungi are a group of organisms closely related to human life, are an important part of the biological chain as decomposers in the nature, and can cause many diseases of crops, animals and human beings. For example, the blast caused by fungi can cause a large reduction in yield of rice, and in extreme cases even no grain harvest, under meteorological conditions suitable for the blast to occur. Therefore, the study and understanding of the fungi have important significance for developing science and technology, improving the health of people, guaranteeing the grain safety of people and the like. These studies all require the cultivation and preservation of large quantities of fungi.
There are many methods for preserving fungi, including subculture preservation, mineral oil preservation, distilled water preservation, glycerol cryopreservation, low-temperature cryopreservation, freeze vacuum drying preservation, and the like. The subculture preservation method is simple to operate and is generally transferred once in 3-6 months. However, frequent switching can change the biological characters of the fungi which are easy to be changed, for example, the spore-producing capability and the pathogenicity of the rice blast fungi can be changed by multiple subcultures, and the probability of polluting the strains is higher. The mineral oil is stored by covering a layer of mineral oil on a slant culture medium full of fungi, and the strain is transferred once in 1 year generally, so that the characteristics of the strain are not easy to change, but the storage occupies a larger space. The distilled water is stored by placing the mycelia in distilled water and sealing at room temperature or 4 deg.C for several years, but is also inconvenient for storage. The freeze vacuum drying preservation method can be preserved for years, decades or longer, avoids pollution and variation caused by multiple times of transfer, but has high requirements on technology and equipment, and is not suitable for being widely adopted in common laboratories. The hypha or spore attaching carrier of the low temperature freezing preservation method is filter paper, sorghum grains, barley grains or rice straws and the like, and the freezing condition is a refrigerator at minus 20 ℃ and a refrigerator at minus 80 ℃ or liquid nitrogen and the like. The low-temperature drying condition can better preserve the activity of the strain and can be generally adopted under the laboratory condition. As the spore adsorbed on the filter paper needs to be stored, vacuum extraction is needed, the steps are complicated, and hypha is generally stored more. A large amount of strains can be stored by adopting carriers such as sorghum grains, barley grains, rice straws and the like, but the storage process is complex, mildew pollution is easy to occur, and the storage occupied volume is larger. Therefore, the filter paper is a better choice as the dry hypha, and the common method for preserving the strains by freezing at low temperature by using the filter paper is economical and applicable. The operation method comprises cutting the filter paper into 3-5mm small pieces, sterilizing, attaching to solid culture medium, recovering the filter paper after mycelia grow on the surface of the culture medium, drying, packaging into sulfuric acid paper bag, and freezing at low temperature for storage. However, the process of attaching and recovering the filter paper sheet is time-consuming, labor-consuming and easy to pollute, and much time and labor are needed for the preservation of a large amount of rice blast germs and other fungi. With the continuous expansion of research scope and the deepening of research, researchers need a large number of fungus strains as research samples and need to store the fungus strains, so that the use and research in the future are facilitated. However, the preservation of a large amount of strains by conventional preservation methods requires a large amount of time and labor, and the mass preservation of strains cannot be achieved. Therefore, it is highly desirable to develop a method for preserving food in a short time, with high efficiency and less pollution.
Disclosure of Invention
It is an object of the present invention to provide an improved method of preserving fungi with a filter paper sheet.
The invention has the following conception: the current method for storing fungi filter paper is to cut the filter paper into small filter paper with side length of 0.3-0.5cm and stick the small filter paper on the culture medium. The method has the disadvantages that the efficiency of cutting large scraps of paper into small scraps of paper is low, the small scraps of paper are time-consuming and labor-consuming to paste on a culture medium, the pollution is easy, and the recovery of the scraps of paper after hyphae overgrow is time-consuming and tedious. The inventors sought to improve upon filter paper sheet making, decal sheet handling and recovery operations to increase work efficiency and reduce contamination rates.
In order to achieve the aim of the invention, the method for rapidly preserving the fungi adopts an improved filter paper sheet method to preserve the fungi, and comprises the following steps:
1) dividing grid dotted lines at intervals of 0.3cm-0.5cm on a filter paper sheet by using a dotted line cutter, cutting the filter paper sheet into a circle, a square or a polygon matched with the diameter of a culture dish according to the diameter of the culture dish, and forming a round hole with the diameter of 0.3cm-0.5cm or a square hole with the side length of 0.3cm-0.5cm in the center of the cut filter paper sheet to finish the manufacture of the filter paper sheet; sterilizing at high temperature;
2) preparing a culture medium, subpackaging the culture medium in culture dishes after sterilization, pasting the prepared filter paper piece on the culture medium after the culture medium is solidified, placing a hypha block in the center of the culture medium, sealing and placing in an incubator for culture;
3) recovering filter paper sheet after the culture medium is overgrown with mycelia, placing into parchment paper bag, drying, and storing in refrigerator at-20 deg.C.
The whole operation of the method is finished under the aseptic condition. The dotted line knife can be a best KW-triO13939 multifunctional paper knife.
In the foregoing method, step 3) specifically includes: recovering filter paper sheets after the culture medium is full of mycelia, putting the filter paper sheets into a parchment paper bag, sealing the opening, drying the parchment paper bag on a superclean bench, and then drying the parchment paper bag in a dryer at room temperature; taking out the sulfuric acid paper bag after drying, putting the sulfuric acid paper bag into an envelope, putting a proper amount of drying agent into the envelope for long-term storage, putting the envelope into a plastic sealed bag, storing at-20 ℃, and periodically replacing the drying agent.
The fungi of the invention include but are not limited to rice blast, sclerotium rolfsii, rhizoctonia solani, rhizoctonia cerealis and colletotrichum. Magnaporthe grisea is preferred.
The desiccant used in the present invention is allochroic silica gel (HG/T2765.4-2005, Islands Semicidae separation materials Co., Ltd.). Other types of desiccants commonly used in the art may also be used.
The method for preserving the rice blast fungi comprises the following specific steps:
s1, cutting the filter paper sheet into dotted line large paper sheets with the distance of 0.3cm multiplied by 0.3cm by a dotted line cutter, cutting the filter paper sheet into dotted line large paper sheets with the distance of 4cm multiplied by 4cm, and forming a square hole with the diameter of 0.5cm multiplied by 0.5cm in the center of the paper sheet to finish the manufacture of the filter paper sheet;
s2, preparing an oat tomato agar culture medium, subpackaging the culture medium in a culture dish after sterilization, pasting the prepared paper sheet on a culture medium with the diameter of 6cm after the culture medium is solidified, placing the hypha block in the center of the culture medium, sealing and placing in an incubator at 25 ℃ for culture;
s3, recovering paper sheets after the culture medium is fully overgrown by hypha, putting the paper sheets into a parchment paper bag with the size of 8cm multiplied by 12cm, sealing the opening, putting the parchment paper bag on a superclean bench for drying for 2 hours, and then putting the parchment paper bag in a dryer for drying for 7 days at room temperature; taking out the sulfuric acid paper bag after drying, putting the sulfuric acid paper bag into an envelope, putting a proper amount of drying agent into the envelope for long-term storage, putting the envelope into a plastic sealed bag, storing at-20 ℃, and periodically replacing the drying agent.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
according to the rapid fungus preservation method provided by the invention, the fungi are preserved by adopting the filter paper sheets with the dotted lines, and hyphae can penetrate through the filter paper and contact with sufficient nutrition, so that the hyphae grow well. The invention improves the operation links of the paper sticking operation and the paper recovery operation, can greatly save time and time, saves labor, has lower pollution rate than the conventional method, and greatly improves the working efficiency. The method saves nearly 87% of paper sheets of one culture dish in the paper sheet pasting step compared with the traditional method, saves about 55% of paper sheets of one culture dish in the paper sheet recycling and storing step compared with the traditional method, greatly improves the working efficiency and lightens the labor intensity. And the method has short time and greatly reduces the pollution rate in the actual operation.
Drawings
FIG. 1 is a graph showing the comparison of the results of the conventional filter paper sheet preservation method in example 1 of the present invention with the results of the dotted line filter paper sheet preservation method in the present invention (before inoculation). Wherein, A: sticking a small paper sheet with the length of 3-5mm on the oat tomato agar culture medium; b: a4 cm × 4cm dotted-line paper sheet was placed on top of the oat tomato agar medium.
FIG. 2 is a graph showing the comparison of the results of the conventional filter paper sheet preservation method in example 1 of the present invention with the results of the dotted line filter paper sheet preservation method in accordance with the present invention (growth after 10 days of inoculation). Wherein, A: the growth condition of rice blast fungus hypha is 10 days after the rice blast fungus hypha is pasted on a small paper culture medium; b: and (3) growing the rice blast fungus hyphae on a dotted paper culture medium after 10 days.
FIG. 3 is a comparison of the effect of the preservation of Rhizoctonia solani by the conventional filter paper preservation method (A) and the dotted filter paper preservation method (B) in example 2 of the present invention.
FIG. 4 is a graph showing the comparison of the effect of preserving anthrax bacteria by the conventional filter paper sheet preservation method (A) and the dotted filter paper sheet preservation method (B) in example 2 of the present invention.
Detailed Description
1. Paper cutting
The large piece of filter paper is cut into filter paper with a specification of 40cm × 40cm by a paper cutter. Then, the broken lines are drawn on the whole piece of filter paper of 40cm multiplied by 40cm by a broken line paper cutter, firstly, the broken lines are drawn in the horizontal direction at intervals of 0.3cm, then, the broken lines are drawn in the vertical direction at intervals of 0.3cm, and the paper can be cut into small squares of the broken lines of 0.3cm multiplied by 0.3 cm. Then, the resulting mixture was cut into 4cm × 4cm filter paper pieces (the size of the filter paper piece may be determined according to the size of the culture dish used, and a 6 cm-diameter culture dish is used here). Placing 4cm × 4cm filter paper in envelope, and sterilizing at 121 deg.C for 30min or 160 deg.C for 3 hr. Sterilizing, oven drying, and placing in a clean bench.
2. Paper pasting sheet
The medium was prepared, sterilized and dispensed into 6cm diameter petri dishes. After the culture medium solidified, the sterilized 1 piece of filter paper of 4cm × 4cm was picked up with tweezers and lightly pasted on the culture medium. Placing the mycelia in the center of the culture medium, sealing, and culturing in an incubator.
3. Recycled paper sheet
The paper was recovered after the culture medium was saturated with mycelia. The next step is to operate in an ultraclean bench. Opening the culture dish, using forceps to separate the four corners of the paper sheet from the culture medium, then removing the paper sheet from one corner, placing in 8cm × 12cm sulfuric acid paper bag, and sealing. Drying in a clean bench for 2 hr, and drying in a desiccator at room temperature for 7 days. Taking out after 7 days, putting into an envelope, putting a proper amount of desiccant into the envelope for long-term storage, putting the envelope into a plastic sealed bag, storing at-20 ℃, and periodically replacing the desiccant.
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
Example 1 Rapid preservation of Pyricularia oryzae by Filter paper sheet method
1. Paper cutting
A large 1m × 1m filter paper was cut with a paper cutter into filter paper of 40cm × 40cm standard. Then, a dotted line is drawn on the whole piece of filter paper of 40cm multiplied by 40cm by a dotted line paper cutter (a best KW-triO13939 multifunctional paper cutter), firstly, dotted lines with an interval of 0.3cm are drawn in the horizontal direction, then, dotted lines with an interval of 0.3cm are drawn in the vertical direction, and then, the paper can be cut into small squares with a dotted line of 0.3cm multiplied by 0.3 cm. Then, the filter paper was cut into 4cm × 4cm pieces of dotted filter paper (the size of the filter paper piece can be determined according to the size of the culture dish, and a culture dish with a diameter of 6cm was used in this example). To promote subsequent hyphal growth, a small hole of 0.5cm by 0.5cm was cut in the center of a 4cm by 4cm piece of dotted filter paper. Then putting 4cm × 4cm filter paper in envelope, and sterilizing at 121 deg.C for 30min or at 160 deg.C for 3 hr. Sterilizing, oven drying, and placing in a clean bench.
2. Paper pasting sheet
Preparing oat tomato agar culture medium, sterilizing, and subpackaging the culture medium in culture dishes with diameter of 6-cm. After the culture medium is solidified, a sticker is prepared on the culture medium. Burning a tip forceps on the flame of an alcohol lamp for sterilization, then clamping 1 sterilized dotted filter paper sheet of 4cm multiplied by 4cm by the forceps, and lightly sticking the filter paper sheet on a culture medium. Then transferring the rice blast fungi, putting a rice blast fungi mycelium block in the center of the culture medium by using a sterilized forceps, sealing the culture dish by using a Parafilm sealing film, and then putting the culture dish in an incubator at 25 ℃ for culturing.
3. Recycled paper sheet
After 7-10 days, the culture medium is full of mycelia. The next step is to operate in an ultraclean bench. Opening the culture dish cover, using forceps to respectively uncover and separate four corners of the paper sheet from the culture medium, then uncovering the paper sheet from one corner, placing the paper sheet in a sulfuric acid paper bag with the size of 8cm multiplied by 12cm, and sealing the opening. Drying in a clean bench for 2 hr, and drying in a desiccator at room temperature for 7 days. Taking out after 7 days, putting into an envelope, putting a proper amount of desiccant into the envelope for long-term storage, putting the envelope into a plastic sealed bag, storing at-20 ℃, and periodically replacing the desiccant.
Comparison of the results of the conventional filter paper sheet preservation method with the dotted filter paper sheet preservation method of the present invention
See table 1, table 2 and fig. 1, fig. 2.
TABLE 1 Tab time comparison
TABLE 2 comparison of recycled paper sheet times
Wherein, the number of the small paper sheets used in the conventional method is about 40, and the rice blast fungus is inoculated in the center of the culture medium.
It can be seen that the dotted filter paper sheet preservation method of the present invention greatly improves the work efficiency and reduces the labor intensity compared with the conventional filter paper sheet preservation method. And the method has short time and greatly reduces the pollution rate in the actual operation.
The filter paper sheet with the dotted line is used for storing the fungi, and when the filter paper sheet is used, the small paper sheet full of the fungi can be conveniently torn off according to the actual required quantity, so that the strains are activated; if the whole filter paper sheet without broken lines is used for storage, the whole filter paper sheet needs to be cut by scissors when in use, which is very easy to cause pollution and wastes time and labor at the same time.
EXAMPLE 2 Rapid preservation of other fungi by Filter paper
The procedure of example 1 was followed except that the species to be preserved were replaced with Rhizoctonia solani and colletotrichum, and the conventional filter paper sheet preservation method was used as a control. After 3-4 days, the culture medium is full of mycelia. The results are shown in FIGS. 3 and 4, respectively. The results show that the dotted filter paper sheet preservation method is time-saving and labor-saving, and can effectively reduce the pollution rate.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (4)
1. The fungus preservation method is characterized in that the fungus is preserved by adopting an improved filter paper sheet method, and comprises the following steps:
1) cutting a grid dotted line at an interval of 0.3cm-0.5cm on a filter paper sheet by using a dotted line cutter, cutting the filter paper sheet into a circle, a square or a polygon matched with the diameter of a culture dish according to the diameter of the culture dish, and forming a round hole with the diameter of 0.3cm-0.5cm or a square hole with the side length of 0.3cm-0.5cm in the center of the cut filter paper sheet to finish the manufacture of the filter paper sheet; sterilizing at high temperature;
2) preparing a culture medium, subpackaging the culture medium in culture dishes after sterilization, pasting the prepared filter paper piece on the culture medium after the culture medium is solidified, placing a hypha block in the center of the culture medium, sealing and placing in an incubator for culture;
3) recovering filter paper sheet after the culture medium is overgrown with mycelia, placing into parchment paper bag, drying, and storing in refrigerator at-20 deg.C;
the fungus is selected from rice blast (Pyricularia oryzae), Sclerotium rolfsii (sclerotirotium rolfsii), Rhizoctonia solani (Rhizoctonia solani), and Rhizoctonia cerealis (Rhizoctonia solani).
2. The method of claim 1, wherein the entire operation is performed under aseptic conditions.
3. The method according to claim 1, wherein step 3) is specifically: recovering filter paper sheets after the culture medium is full of mycelia, putting the filter paper sheets into a parchment paper bag, sealing the opening, drying the parchment paper bag on a superclean bench, and then drying the parchment paper bag in a dryer at room temperature; taking out the sulfuric acid paper bag after drying, putting the sulfuric acid paper bag into an envelope, putting a proper amount of drying agent into the envelope for long-term storage, putting the envelope into a plastic sealed bag, storing at-20 ℃, and periodically replacing the drying agent.
4. The method of claim 1, comprising the steps of:
s1, cutting the filter paper sheet into dotted line large paper sheets with the distance of 0.3cm multiplied by 0.3cm by a dotted line cutter, cutting the filter paper sheet into dotted line large paper sheets with the distance of 4cm multiplied by 4cm, and forming a square hole with the diameter of 0.5cm multiplied by 0.5cm in the center of the paper sheet to finish the manufacture of the filter paper sheet; sterilizing at high temperature;
s2, preparing an oat tomato agar culture medium, subpackaging the culture medium in a culture dish after sterilization, pasting the prepared paper sheet on a culture medium with the diameter of 6cm after the culture medium is solidified, placing the hypha block in the center of the culture medium, sealing and placing in an incubator at 25 ℃ for culture;
s3, recovering paper sheets after the culture medium is fully overgrown by hypha, putting the paper sheets into a parchment paper bag with the size of 8cm multiplied by 12cm, sealing the opening, putting the parchment paper bag on a superclean bench for drying for 2 hours, and then putting the parchment paper bag in a dryer for drying for 7 days at room temperature; taking out the sulfuric acid paper bag after drying, putting the sulfuric acid paper bag into an envelope, putting a proper amount of drying agent into the envelope for long-term storage, putting the envelope into a plastic sealed bag, storing at-20 ℃, and periodically replacing the drying agent.
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| CN108865890A (en) * | 2018-07-24 | 2018-11-23 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | The long-term preservation method of rice blast bacterial strain |
| CN111849788A (en) * | 2020-08-04 | 2020-10-30 | 青海省农林科学院 | Preservation method of barley stripe disease germs and co-culture filter paper sheet |
| CN112899166B (en) * | 2021-04-06 | 2023-06-06 | 华中农业大学 | Fungus strain preservation method |
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| JP3819862B2 (en) * | 2003-03-17 | 2006-09-13 | 独立行政法人農業生物資源研究所 | How to store tissue of multicellular organisms at room temperature |
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| CN105647904A (en) * | 2016-03-31 | 2016-06-08 | 安徽工程大学 | Method for screening cellulase producing strains and method for producing cellulase by means of fermentation |
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