CN107674838B - Fungal fast storage method - Google Patents

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

Fungal fast storage method

technical field

The present invention relates to a method for preserving microorganisms, in particular to a method for rapid preservation of fungi.

Background technique

Fungi are a class of organisms closely related to people's lives. As a decomposer in nature, they are an important part of the biological chain and can also cause many diseases of crops, animals and humans. For example, rice blast caused by fungi can cause a large reduction in rice yield, and even no grain in extreme cases, when the meteorological conditions are suitable for the occurrence of rice blast. Therefore, research and understanding of fungi are of great significance to the development of science and technology, improving people's health, and ensuring our food security. These studies require the cultivation and preservation of large numbers of fungi.

There are many ways to preserve fungi, including subculture preservation, mineral oil preservation, distilled water preservation, glycerol cryopreservation, low temperature cryopreservation, freeze-vacuum-drying preservation, etc. The subculture preservation method is simple to operate, and is generally transferred once every 3-6 months. However, frequent transfer will change the biological characters of fungi that are prone to mutation. For example, multiple subcultures will change the sporulation ability and pathogenicity of M. Mineral oil preservation is to cover a layer of mineral oil on the sloping medium full of fungi. Generally, it is transferred once a year. The characteristics of the bacteria are not easily changed, but the preservation takes up a lot of space. Distilled water storage is to put the mycelium block into distilled water and seal it at room temperature or 4 ℃. It can survive for several years, but it is also inconvenient to store. The freeze-vacuum-drying preservation method can be stored for several years, decades or longer, avoiding contamination and variation caused by multiple transfers, but it requires high technology and equipment and is not suitable for mass adoption in ordinary laboratories. The mycelium or spore attachment carrier in the cryopreservation method is filter paper, sorghum grain, barley grain or rice straw, etc., and the freezing conditions are -20°C refrigerator, -80°C refrigerator or liquid nitrogen, etc. Low-temperature drying conditions can better preserve the activity of strains, and can also be widely used under laboratory conditions. Since the preservation of the spores adsorbed on the filter paper requires vacuum extraction, the steps are cumbersome, and the mycelium is generally preserved. The use of sorghum grains, barley grains, rice straws and other carriers can preserve a large number of strains, but the preservation process is complicated, mildew pollution is prone to occur, and the preservation takes up a large volume. Therefore, filter paper is a better choice for drying mycelium, and the usual method of cryopreserving strains of filter paper sheets is also more economical and applicable. The operation method is to cut the filter paper into small pieces of 3-5mm, paste them on the solid medium after sterilization, and recover the filter paper after the mycelium has grown to the surface of the medium. Cryopreservation at low temperature. However, the process of sticking the filter paper and recycling the filter paper is time-consuming and labor-intensive and easy to contaminate, and it takes more time and effort to preserve a large number of rice blast bacteria and other fungi. With the continuous expansion of the research scope and in-depth research, researchers need a large number of fungal strains as research samples, and they need to be preserved as strains for future use and research. However, it takes a lot of time and effort to preserve a large number of strains by relying on the previous preservation methods, and even large-scale preservation of strains cannot be achieved. Therefore, it is urgent to develop a method with short time-consuming, high efficiency and less pollution for proper preservation.

SUMMARY OF THE INVENTION

The object of the present invention is to provide an improved method for preserving fungi on filter paper sheets.

The present invention is conceived as follows: the current filter paper method for preserving fungi is to cut the filter paper into small filter paper pieces with a side length of 0.3-0.5 cm and stick them on the culture medium. The disadvantage is that it is inefficient to cut large pieces of paper into small pieces, and it is time-consuming and labor-intensive to stick the small pieces of paper on the culture medium, and it is easy to contaminate. The inventors tried to improve the production of filter paper, the operation of stickers and the operation of recycling paper to improve work efficiency and reduce the pollution rate.

In order to achieve the purpose of the present invention, the fungal fast preservation method provided by the invention adopts the improved filter paper method to preserve the fungus, comprising the following steps:

1) Use a dashed knife to draw a dashed line at an interval of 0.3cm-0.5cm on the filter paper, then according to the diameter of the petri dish, cut the filter paper into a circle, square or polygon that matches the diameter of the petri dish, and cut A round hole with a diameter of 0.3cm-0.5cm or a square hole with a side length of 0.3cm-0.5cm is opened in the center of the good filter paper to complete the production of the filter paper; high temperature sterilization;

2) Prepare the culture medium, pack the culture medium into petri dishes after sterilization, stick the above-made filter paper sheet on the culture medium after the culture medium is solidified, place the mycelium block in the center of the culture medium, and seal it. placed in an incubator;

3) After the mycelium is covered with the medium, the filter paper is recovered, put into a sulfuric acid paper bag, dried and stored in a -20°C refrigerator.

The entire operation of the method of the present invention is carried out under sterile conditions. The dotted line knife can be selected from Kedeyou KW-triO13939 multifunctional paper cutter.

The aforementioned method, step 3) is specifically as follows: after the mycelium is covered with the medium, the filter paper is recovered, put into a sulfuric acid paper bag, sealed, placed on an ultra-clean workbench for drying, and then placed in a desiccator to dry at room temperature; after drying, take out the sulfuric acid Paper bag, put it in an envelope, for long-term storage, put an appropriate amount of desiccant in the envelope, put the envelope in a plastic sealed bag, store it at -20 ℃, and replace the desiccant regularly.

The fungi described in the present invention include, but are not limited to, Rice blast fungus, Rhizoctonia solani, Rhizoctonia solani, Rhizoctonia solani, and anthracnose fungus. Pyricularia oryzae is preferred.

The desiccant used in the present invention is color-changing silica gel (HG/T2765.4-2005, Qingdao Puke Separation Materials Co., Ltd.). Other types of desiccants commonly used in the art can also be used.

The method of the present invention is adopted to preserve the rice blast fungus, and the specific steps are:

S1. Use a dashed knife to draw the dotted lines of the filter paper with an interval of 0.3cm×0.3cm, then cut the filter paper into a large dotted sheet of 4cm×4cm, and open a square with a diameter of 0.5cm×0.5cm in the center of the paper. Holes, complete the production of filter paper;

S2, prepare oat tomato agar medium, after sterilization, divide the medium into petri dishes, after the medium is solidified, stick the above-made paper sheet on the medium with a diameter of 6 cm, and place the mycelium block on the medium The center of the medium, sealed and placed in a 25°C incubator;

S3. After the mycelium is overgrown with the culture medium, the paper is recovered, put into a 8cm×12cm sulfuric acid paper bag, sealed, placed on an ultra-clean workbench to dry for 2 hours, and then placed in a desiccator to dry at room temperature for 7 days; after drying, take out the sulfuric acid paper bag, put it in Put it in an envelope. For long-term storage, put an appropriate amount of desiccant in the envelope, put the envelope in a plastic sealed bag, store it at -20°C, and replace the desiccant regularly.

By the above-mentioned technical scheme, the present invention at least has the following advantages and beneficial effects:

In the method for rapid preservation of fungi provided by the invention, a filter paper sheet provided with a dotted line is used to preserve fungi, the mycelium can penetrate the filter paper, contact sufficient nutrients, and the mycelium grows well. The invention improves the operation of sticking sheets and recycling paper sheets, which can greatly save time and labor, and has lower pollution rate than conventional methods, thereby greatly improving work efficiency. This method saves nearly 87% of paper for each petri dish in the step of sticking the paper compared with the traditional method, and saves about 55% of the paper for each petri dish in the step of recycling and preserving the paper compared with the traditional method, which greatly improves the Improve work efficiency and reduce labor intensity. In addition, the method takes a short time and greatly reduces the pollution rate in actual operation.

Description of drawings

1 is a comparison of the experimental results between the conventional filter paper preservation method and the dotted line filter paper preservation method of the present invention in Example 1 of the present invention (before inoculation). Among them, A: a small paper piece with a length of 3-5mm is attached to the oat tomato agar medium; B: a dashed paper piece with a side length of 4cm×4cm is attached to the oat tomato agar medium.

2 is a comparison of the experimental results between the conventional filter paper preservation method and the dashed filter paper preservation method of the present invention in Example 1 of the present invention (growth after 10 days of inoculation). Among them, A: the growth of the oryzae oryzae mycelium after 10 days on the medium with a small paper sheet; B: the growth of the oryzae mycelium on the medium with a dotted line for 10 days.

FIG. 3 is a comparison of the effects of using the conventional filter paper preservation method (A) and the dotted filter paper preservation method (B) of the present invention to preserve Rhizoctonia solani respectively in Example 2 of the present invention.

FIG. 4 is a comparison of the effects of using the conventional filter paper preservation method (A) and the dotted filter paper preservation method (B) of the present invention to preserve the anthracnose bacteria in Example 2 of the present invention.

Detailed ways

1. Paper cutting

Cut a large piece of filter paper into 40cm×40cm filter paper with a paper cutter. Then use a dotted paper cutter to draw dotted lines on the entire 40cm×40cm filter paper, first draw dotted lines at 0.3cm intervals in the horizontal direction, and then draw dotted lines at 0.3cm intervals in the vertical direction, and the paper can be cut into 0.3cm×0.3cm dashed small square. Then use a paper cutter to cut into 4cm×4cm filter paper pieces (the size of the paper piece can be determined according to the size of the petri dish used, here a petri dish with a diameter of 6cm is used). Pack the 4cm×4cm filter paper in an envelope and sterilize at 121°C for 30min or 160°C for 3 hours. Dry after sterilization, and put it in a clean bench for later use.

2. Sticker sheet

The medium was prepared, and after sterilization, the medium was divided into petri dishes with a diameter of 6 cm. After the medium is solidified, use tweezers to pick up a 4cm×4cm piece of filter paper after sterilization, and gently stick it on the medium. The mycelial block was placed in the center of the medium, sealed and then placed in an incubator.

3. Recycled paper

After the mycelium overgrown the medium, the paper was recovered. The next steps are performed in a clean bench. Open the petri dish, use tweezers to separate the four corners of the paper from the medium, then peel off the paper from one corner, put it in a 8cm×12cm sulfuric acid paper bag, and seal it. Dry on an ultra-clean bench for 2 hours, then in a desiccator at room temperature for 7 days. Take it out after 7 days and put it in an envelope. For long-term storage, put an appropriate amount of desiccant in the envelope, put the envelope in a plastic sealed bag, store it at -20°C, and replace the desiccant regularly.

The following examples are intended to illustrate the present invention, but not to limit the scope of the present 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 all commercially available commodities.

Example 1 Rapid preservation of rice blast by filter paper method

1. Paper cutting

Use a paper cutter to cut a large piece of filter paper of 1m × 1m into a filter paper of 40cm × 40cm. Then use a dotted paper cutter (you can get excellent KW-triO 13939 multifunctional paper cutter) to draw dotted lines on the entire 40cm×40cm filter paper, first draw dotted lines with 0.3cm intervals in the horizontal direction, and then draw 0.3cm intervals in the vertical direction The dotted line can be cut into 0.3cm×0.3cm dotted small squares on the paper. Then use a paper cutter to cut into dotted filter paper pieces of 4cm×4cm (the size of the paper piece can be determined according to the size of the petri dish used, in this example, a petri dish with a diameter of 6cm is used). In order to promote the subsequent growth of mycelium, a small hole of 0.5 cm × 0.5 cm was cut in the center of the 4 cm × 4 cm dotted filter paper. Then put the 4cm × 4cm filter paper in an envelope and sterilize at 121°C for 30min or 160°C for 3h. Dry after sterilization, and put it in a clean bench for later use.

2. Stickers

An oat tomato agar medium was prepared, and after sterilization, the medium was distributed into petri dishes with a diameter of 6-cm. After the medium has solidified, prepare a sticker on the medium. First, burn and sterilize pointed tweezers on the flame of an alcohol lamp, and then use tweezers to pick up a 4cm×4cm dashed filter paper sheet after sterilization, and gently stick it on the medium. Next, transfer the rice blast fungus, place the rice blast fungus mycelial block in the center of the medium with sterilized tweezers, seal the petri dish with Parafilm and place it in a 25°C incubator for cultivation.

3. Recycled paper

After 7-10 days, the hyphae are overgrown with the medium. The next steps are performed in a clean bench. Open the lid of the petri dish, use tweezers to separate the four corners of the paper from the medium, then peel off the paper from one corner, put it in an 8cm×12cm sulfuric acid paper bag, and seal it. Dry on an ultra-clean bench for 2 hours, then in a desiccator at room temperature for 7 days. Take it out after 7 days and put it in an envelope. For long-term storage, put an appropriate amount of desiccant in the envelope, put the envelope in a plastic sealed bag, store it at -20°C, and replace the desiccant regularly.

Comparison of experimental results between the conventional filter paper preservation method and the dotted filter paper preservation method of the present invention

See Table 1, Table 2 and Figure 1, Figure 2.

Table 1 sticker sheet time comparison

Table 2 Comparison of recycling time

Among them, the number of small paper sheets used in the conventional method is about 40, and the rice blast fungus is inoculated in the center of the medium.

It can be seen that, compared with the conventional filter paper preservation method, the dotted filter paper preservation method of the present invention greatly improves work efficiency and reduces labor intensity. In addition, the method takes a short time and greatly reduces the pollution rate in actual operation.

Use the filter paper with dotted line to preserve fungi. When using, you can easily tear off the small piece of paper covered with fungi according to the actual demand to activate the bacteria; if you use the whole filter paper without dotted line to save, when using It is necessary to use scissors to cut the entire filter paper sheet, which is very easy to cause pollution, and is also time-consuming and labor-intensive.

Example 2 Rapid preservation of other fungi by filter paper method

The operation is the same as that in Example 1, except that the preserved strains are changed to Rhizoctonia subtilis and anthracnose, and the conventional filter paper preservation method is used as a control. After 3-4 days, the hyphae are overgrown with the medium. The results are shown in Figure 3 and Figure 4, respectively. The above results show that the dashed filter paper preservation method of the present invention saves time and effort, and can effectively reduce the pollution rate.

Although the present invention has been described in detail above with general description and specific embodiments, some modifications or improvements can be made on the basis of the present invention, which will be obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

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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