Simple reniform worm enlarged culture and reniform worm cyst resuscitation method

Abstract

The invention belongs to the technical field of biology, and particularly relates to a simple reniform worm enlarged culture and reniform worm cyst resuscitation method, which is characterized in that a wheat seedling culture solution is mixed with a reniform worm culture solution of escherichia coli, and the method comprises the following steps: carrying out small-range high-density culture on reniform worm; forming a reniform worm cyst and preserving breeds; resuscitating the reniform worm cyst; the resuscitated kidney-shaped worm can also be subjected to enlarged culture. The method is suitable for culture and cyst resuscitation of various kidney-shaped worms, the culture efficiency of the kidney-shaped worms is greatly optimized, the time required for the kidney-shaped worms to grow to the highest density is shortened, and resuscitation of dormant cysts of the kidney-shaped worms and the recovery speed of population density after resuscitation are both remarkably increased. The method can be applied to researches on morphological classification, cytobiology, ecology and the like of the reniform worm, research on environmental monitoring and treatment of the reniform worm in soil heavy metal pollution and the like, and research on preparation of microbial fertilizers from the reniform worm and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and provides a simple method for expanding and culturing reniformis and reviving reniformis cysts, which can obtain high-density reniformis cells within a corresponding time, and has high stability and simple operation. Background Art

[0002] Renniformes is a single-celled eukaryotic organism belonging to the phylum Ciliophora, class Nephridia, order Nephridia, family Nephridia, genus Nephridia. It has a kidney-shaped body and has a certain array of cilia distributed all over its body. Renniformes is widely distributed and occupies an absolutely dominant niche abundance in the global soil ecosystem.

[0003] When the environment in which the reniform worm lives changes, such as the serious lack of food and water, sudden change in temperature, high population density, large accumulation and excessive metabolic products, the morphology of the worm body will change to a certain extent. The cell surface discharges scales and is surrounded by mucus to form a spherical cyst wall to wrap the cell, so as to resist the harsh environment in the soil, especially the cadmium pollution environment. This physiological process of the reniform worm is called the formation of cysts, which are also called dormant cysts. The cysts of the reniform worm can be stored and transported for a long time. These characteristics make the reniform worm highly adaptable to harsh environments such as pollution. It is a species with a relatively high pollution tolerance index and can be used as a potential eukaryotic microorganism for soil pollution remediation. Many studies have shown that the reniform worm has great potential in the monitoring and control of heavy metal pollution, and also plays an important role in the preparation of microbial fertilizers. Before that, we need to establish an efficient cultivation method and a method for reviving cysts that are conducive to environmental governance and the preparation of microbial fertilizers.

[0004] The methods for high-density culture and rapid recovery of cysts of Renilla reported in the prior art have some defects:

[0005] (1) There is a lack of a simple method for high-density culture of Renilla. The culture scheme commonly used in the literature is to add 1-2 wheat grains to double-distilled water to form a wheat grain culture medium for Renilla culture. It can usually be cultured to the highest density in more than 72 hours. Taking the expanded Renilla as an example, the maximum density is about 4000 / ml. Not only is the culture cycle long, but the maximum population density is also low; the patent application number: 202410479651.7, the invention name "The application of Renilla in controlling nitrogen loss in cadmium-contaminated soil" mentioned that the use of Amoeba sterile culture medium containing 0.3g / LTSB can culture the tooth-spine Renilla (Colpodasteinii) to about 20,000 / mL in about 2 days, but the culture method uses a complex culture medium and the culture time is relatively long.

[0006] (2) There is a lack of methods for rapid recovery of reniform cysts. The current method for recovering reniform cysts after seed preservation is to add wheat grains to them. For example, patent application number: 202111551987.2, invention name "A method for long-term seed preservation and rapid recovery of reniform cysts" mentioned that when reniform cysts are recovered after seed preservation using the air-drying method, the recovery speed is slow, and it takes a long time for the recovered reniform cysts to grow to the highest density.

[0007] As an important species of soil protozoa, Nephridia is an important prerequisite for scientific research as an experimental material and the basis for using it in soil environmental monitoring and management and in the preparation of microbial fertilizers. Therefore, it is very important to establish a simple high-density culture method and a rapid cyst recovery method for it. Summary of the invention

[0008] In view of the problems existing in the prior art, the present invention aims to provide a simple method for expanding the culture of Reniformis and reviving Reniformis cysts, which uses wheat seedling culture fluid mixed with Escherichia coli Reniformis culture fluid to perform simple high-density expansion culture of Reniformis and cyst recovery.

[0009] The present invention is achieved through the following technical scheme, a simple method for expanding the culture of Reniformis and reviving Reniformis cysts, comprising the following steps:

[0010] 1) Prepare 0.1% wheatgrass culture medium, LB culture medium, and 1 mM Tris-HCl buffer;

[0011] 2) Preparation of E. coli culture: E. coli was cultured overnight, centrifuged to remove the supernatant, washed, resuspended in Tris-HCl buffer, and the concentration was measured by spectrophotometer. The concentration was calculated by the formula to be not less than 10 8 CFU / mL of Escherichia coli solution;

[0012] 3) Prepare the culture solution of Renilla: add a certain amount of the Escherichia coli solution obtained in step 2) to the 0.1% wheatgrass culture solution obtained in step 1) to obtain a solution containing 0.1% wheatgrass culture solution and 10 6 CFU / mL of Escherichia coli culture fluid of Renilla;

[0013] 4) Renilla cell culture: A certain amount of Renilla is pipetted into the Renilla culture solution obtained in step 3), and the mixture is cultured in a constant temperature incubator at 15-30°C. The density of Renilla cells is counted every 6 hours to draw a growth curve of the Renilla cell culture and calculate the generation time. When the density of Renilla is 2.2*10 4 -9.8*10 4 Only 100 μg / mL can complete the high-density culture of Renilla in a small area.

[0014] 5) Formation and preservation of reniform cysts: The high-density reniform cysts and culture medium mixture obtained in step 4) was centrifuged at 1500 rpm for 3 min, washed twice with 1 mM Tris-HCl buffer, and then resuspended in 1 mM Tris-HCl buffer to a reniform cyst density of 10 5 / mL; place in a 25℃ constant temperature incubator for 2h to allow all of them to form cysts, and then place in a 4-37℃ constant temperature incubator for seed preservation.

[0015] 6) Resuscitation of Reniformis cysts: The seed-preserved Reniformis cysts were taken out and placed in 0.1% wheat seedling culture solution and placed in a constant temperature incubator at 15-30°C to allow them to revive.

[0016] Wherein, step 1) is a process of preparing 0.1% wheatgrass culture solution: weigh 1g of dried wheatgrass in 200mL ddH2O and boil for 10min. The boiled wheatgrass culture solution is divided into 50mL centrifuge tubes, centrifuged at 3000rpm for 5min, the supernatant is retained, filtered with a 0.22um filter membrane, ddH2O is added to the filtered wheatgrass culture solution to make the volume 1L, and 1% wheatgrass extract is obtained, which is sterilized with high-temperature steam and stored at room temperature for later use. The 0.1% wheatgrass culture solution is obtained by adding 10mL of the 1% wheatgrass culture solution to 90mL of sterilized ddH2O and stored at room temperature;

[0017] Specifically, step 2) is to take out the E. coli DH5α from -80℃, inoculate it in 200mL LB culture medium, and shake it overnight at 37℃. The obtained E. coli culture was divided into 50mL centrifuge tubes, centrifuged at 7000rpm for 10 minutes, and washed twice with 1mM Tris-HCl buffer after removing the supernatant. During the washing, centrifuge at 7000rpm for 5min. Add 10mL 1mM Tris-HCl buffer to the E. coli precipitate, take a small amount of the culture, measure the concentration with a spectrophotometer, and calculate the concentration of the E. coli culture according to the formula to make it reach 10 8 The bacterial solution can be stored at 4°C and used to prepare the Nephrolepis culture solution within one week.

[0018] Wherein, step 3) comprises: the culture solution of Renilla needs to be prepared immediately, and 1 mL 10 8 CFU / mL Escherichia coli solution, mix well and dilute to 100mL to obtain a solution containing 10 6 CFU / mL of Escherichia coli culture medium.

[0019] As a preferred embodiment of the present invention, step 5) is to place the seeds in a constant temperature incubator at 15-30°C for half a year.

[0020] As a preferred embodiment of the present invention, step 6) comprises: taking 10 dormant cysts from step 5) and immersing them in 0.1% wheatgrass culture solution, and placing them in a constant temperature incubator at 25°C for 8 hours, and the cyst recovery rate can reach 100%. The revived reniform worms can be cultured at a high density according to steps 3)-4).

[0021] The advantages and beneficial effects of the present invention over the prior art include:

[0022] 1. It has greatly optimized the culture efficiency of Renilla, shortened the time required for Renilla to grow to the highest density, and greatly increased the maximum density of Renilla growth; the generation time of culture has been significantly reduced.

[0023] 2. The culture medium provided by this method is not only capable of efficiently culturing Renilla, but also significantly improves the recovery rate of dormant cysts of Renilla and the recovery rate of population density after recovery.

[0024] 3. This method can be used as a complete set of methods for high-density culture of Nephridia, long-term seed preservation, and rapid cyst recovery. The required drugs can be reused at different stages, and the operation is easy, providing convenience for scientific researchers. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : A flow chart of the culture and cyst recovery of Renilla provided by the present invention;

[0026] Figure 2 : Growth curve of high density culture of Renilla sphaeroides cells of Example 1;

[0027] Figure 3 : Statistical graph of the recovery rate of the dormant cysts of Renilla bulgaricus of Example 1 after being stored at different temperatures for half a year;

[0028] Figure 4 : The growth curve of the high-density culture of the Nephrops denticulata cells of Example 2;

[0029] Figure 5 : Statistical graph of the recovery rate of the dormant cysts of Nephrops dentata of Example 2 after being stored at different temperatures for half a year. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below with reference to the accompanying drawings and embodiments, but the present invention is not limited thereto.

[0031] Example 1 Cultivation and cyst recovery of Renilla bulgaria

[0032] 1) Preparation of culture medium and buffer: prepare 0.1% wheatgrass culture medium, LB culture medium, and 1 mM Tris-HCl buffer;

[0033] Specifically, the process of preparing 0.1% wheatgrass culture solution by mass percentage is as follows: Weigh 1g of wheatgrass (Lifestream Organic Wheat Grass Powder 250g, purchased from the Amazon website https: / / www.amazon.co.uk / Lifestream-Organic-Wheat-Grass-Powder / dp / B000NRXMOQ) in 200mL ddH2O and boil for 10min. The boiled wheatgrass stock solution is divided into 50mL centrifuge tubes, centrifuged at 3000rpm for 5min, the supernatant is retained, and filtered through a 0.22um filter membrane. ddH2O is added to the filtered wheatgrass stock solution to make the volume 1L to obtain 1% wheatgrass culture solution, which is sterilized by high-temperature steam and stored at room temperature. The preparation of 0.1% wheatgrass culture solution is to add the 10mL of 1% wheatgrass culture solution to 90mL sterilized ddH2O, store at room temperature, and set aside;

[0034] Preparation of LB culture medium: weigh 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl, dissolve in 1 L of ddH2O, stir thoroughly to dissolve, sterilize at high temperature for later use, and store at room temperature;

[0035] The process of preparing 1mM Tris-HCl buffer is as follows: first prepare the mother solution, weigh 121.1g Tris in 800mL ddH2O, stir thoroughly to dissolve, add hydrochloric acid to adjust the pH value to 7.4, dilute the solution to 1L, sterilize with high-temperature steam and set aside to obtain 1M Tris-HCl buffer. 1mM Tris-HCl buffer is 1mL 1M Tris-HCl buffer added to 999mL sterilized ddH2O.

[0036] 2) Preparation of E. coli culture: E. coli DH5α (preserved in the laboratory, purchased from Nanjing Novozyme Biotechnology Co., Ltd., catalog number C502-03) taken out from -80℃ was inoculated into 200mL LB culture medium and cultured overnight at 37℃ in a shaking incubator. The cultured culture was centrifuged at 7000rpm for 10min to remove the supernatant, and washed twice with 1mM Tris-HCl buffer at 7000rpm for 5min. The E. coli was resuspended in 10mL 1mM Tris-HCl buffer and used for spectrophotometer concentration measurement. The concentration of the E. coli culture was calculated to be about 10 according to the formula. 8 CFU / mL.

[0037] 3) Prepare the culture medium of Renilla: Take 1 mL of the 10 8CFU / mL of Escherichia coli culture was mixed evenly with 0.1% wheatgrass culture medium and the volume was fixed to 100 mL to obtain a mixture containing 0.1% wheatgrass culture medium and 10 6 CFU / mL of Escherichia coli culture medium.

[0038] 4) Renilla cell culture: A certain amount of Renilla is taken into the Renilla culture solution obtained in step 3), and the solution is cultured in a constant temperature incubator at 15-30°C. The density of Renilla cells is counted every 6 hours to draw a growth curve of the Renilla cell culture. The growth curve is drawn according to the density at different time points, and the population growth rate of the protozoa is calculated according to the following formula: r = (ln N t -ln N0) / t (t refers to time, N0 = the number of individuals in the population at 0h, N t = the number of individuals in the population at time t), calculating the r value can yield the natural growth equation of the population, and then calculating the generation time according to G = ln 2 / r.

[0039] At the same time, in order to verify the time of cyst formation, the following steps were also performed: the cysts of the cysts to be cultured were placed in a 0.1% wheatgrass culture solution in a 25°C constant temperature incubator one day in advance. On the second day, 10 μL of the cyst solution was taken at each point under an optical microscope for counting using a five-point sampling method to calculate the current cell density of the cysts. The 10 mL of 0.1% wheatgrass culture solution and 10 μL of the cyst solution obtained in step 3) were added. 6 CFU / mL Escherichia coli, Renilla sphaeroides culture fluid and Diplodia sphaeroides, so that the initial density of Diplodia sphaeroides culture is about 100 / mL. Set up three groups of parallel samples, and add a control group without Escherichia coli and only with 0.1% wheat seedling extract as the culture fluid, and culture it in a constant temperature incubator at 25℃. This time is recorded as 0h. After that, the Diplodia sphaeroides are sampled every 6h and the density is calculated. At 120h, 99.95% of the Diplodia sphaeroides have formed cysts, and the counting is stopped at this time.

[0040] 5) Formation and preservation of reniformis cysts: The mixture of reniformis cells and culture medium cultured to a high density obtained in step 4) was collected, centrifuged at 1500 rpm for 3 min, and washed with 1 mM Tris-HCl buffer at 1500 rpm for 3 min. The reniformis was washed twice and then resuspended in 1 mM Tris-HCl buffer to a density of about 10 5 When the treated swollen kidney parasites were placed in a 25°C constant temperature incubator for 2 hours, more than 90% of the swollen kidney parasites formed dormant cysts. They were divided into 7 groups, each group was about 2 mL, approximately 2*10 5The cells were placed in 1 mM Tris buffer and kept in a constant temperature incubator at different temperatures (-20°C, 4°C, 15°C, 25°C, 30°C, 37°C, 55°C) for half a year.

[0041] 6) Recovery of reniformis cysts: A small amount of dormant cysts after half a year of storage are taken from the constant temperature incubators at different temperatures in step 5), and each temperature group has two culture groups, 10 and 50 respectively; a total of 14 groups are placed in 0.1% wheat seedling culture solution, placed in a constant temperature incubator at 25°C for static culture, and the recovery rate of cysts in each group is calculated at different times. The revived reniformis can be cultured in large quantities at high density according to steps 1)-4).

[0042] In this example, the growth curve of the cultured Renilla bulgaricus cells is plotted as follows: Figure 2 As shown in the figure, the culture medium of Renilla inflata was set up with the same three replicates: R1, R2, and R3. All of them grew to the highest density at 33h, with the highest density being 2.2*10 4 / mL. At 36h, more than half of the inflated reniform worms formed cysts, and at 120h, 99.95% of the inflated reniform worms formed cysts. When the highest density was reached, the generation time of inflated reniform worms under this culture method was about 4.24h. It took 42h to culture inflated reniform worms to the highest density using only 0.1% wheat seedling culture medium, and the highest density was about 6000 / mL, and the generation time was about 6.94h.

[0043] In this embodiment, when the number of dormant cysts of expanded kidney-shaped worms stored at different temperatures was 10, all 100% recovered after 8 hours at a storage temperature of 4°C-30°C; when the number was 50, the recovery rate of expanded kidney-shaped worms stored at a storage temperature of 15°C and 25°C after 8 hours was about 60%, and the recovery rate of dormant cysts stored at a storage temperature of 30°C after 8 hours was about 90%. Figure 3 .

[0044] Example 2 Cultivation and cyst recovery of Nephroderma

[0045] In this example, except that the culture object is Nephrops dentata, the other culture steps are the same as in Example 1. The growth curve of Nephrops dentata cell culture is plotted as follows: Figure 4 As shown in the figure, three replicates of the culture medium of Renilla were set up, and they all grew to the highest density in 30 hours, which was about 9.0*10 4 / mL. There is a plateau period of about 12 hours. At 54 hours, about half of the cysts are formed, and at 120 hours, about 90% of the cysts are formed. When the highest density is reached, the generation time of the cysts is about 3.06 hours under this culture method. It takes 36 hours to culture the cysts to the highest density using only 0.1% wheat seedling culture medium, and the highest density is about 3.9*104 / mL, and the generation time is about 4.18h.

[0046] In this embodiment, when the dormant cysts of the tooth-spine nephridia were stored at different temperatures, when the number was 10, the recovery rate was 100% after 8 hours at a storage temperature of 15°C-37°C. When the number was 50, the recovery rate was as high as more than 90% at a storage temperature of 4°C-37°C. Figure 5 .

[0047] The above results show that the culture medium of Renilla made by using wheatgrass culture medium and Escherichia coli can greatly shorten the culture time of Renilla cells and significantly increase the maximum density of Renilla cell culture; the generation time of culture is significantly reduced. The process of dormant cysts reviving after seed preservation at different temperatures shows that cysts can withstand different temperature changes. The operation methods of the entire culture process and revival process are very simple.

[0048] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A simple method for expanding the culture of Renilla and reviving Renilla cysts, characterized in that: The steps include: 1) Cultivate the reniform worms at a high density in a small area; 2) forming reniformis cysts based on the high-density culture of reniformis obtained in step 1 and preserving the cysts; 3) resuscitating the reniform cysts obtained in step 2).

2. The method for simple expansion culture of Reniformis and resuscitation of Reniformis cysts according to claim 1, characterized in that: The step 1) also includes the following steps: 1.1) Prepare 0.1% wheatgrass culture medium, LB culture medium, and 1 mM Tris-HCl buffer; 1.2) Preparation of E. coli culture: E. coli was cultured overnight, centrifuged to remove the supernatant, washed, and resuspended in 1 mM Tris-HCl buffer to make the concentration of E. coli culture not less than 10 8 CFU / mL, and the Escherichia coli bacterial solution is obtained; 1.3) Preparation of reniformis culture solution: Take the Escherichia coli culture obtained in step 1.2 and dilute it 100 times with the 0.1% wheatgrass culture solution to obtain a solution containing 0.1% wheatgrass culture solution and not less than 10 6 CFU / mL of Escherichia coli culture fluid of Renilla; 1.4) Renilla cell culture: A certain amount of Renilla is pipetted into the Renilla culture solution obtained in step 1.3), and the mixture is cultured in a constant temperature incubator at 15-30°C. The density of Renilla cells is counted regularly, and a growth curve of the obtained high-density Renilla cell culture is plotted; when the density of Renilla is 2.2*10 4 -9.8*10 4 Only 100 μg / mL can complete the high-density culture of Renilla in a small area.

3. A simple method for expanding the culture of Reniformis and reviving Reniformis cysts according to claim 2, characterized in that: The preparation method of the 0.1% wheatgrass culture solution in the step 1.1) is as follows: weigh 1g of dried wheatgrass in 200mL ddH2O and boil for 10min; divide the boiled wheatgrass stock solution into portions and centrifuge at 3000rpm for 5min, retain the supernatant, filter with a 0.22μm filter membrane, add ddH2O to the filtered wheatgrass stock solution to make the volume to 1L, obtain 1% wheatgrass culture solution, sterilize with high-temperature steam and store at room temperature; add 10mL of the 1% wheatgrass culture solution to 90mL of sterilized ddH2O to obtain 0.1% wheatgrass extract, and store at room temperature; The LB culture medium in step 1.1) is prepared by weighing 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl, dissolving them in 1 L of ddH2O, stirring them thoroughly to dissolve, sterilizing them at high temperature for later use, and storing them at room temperature; The 1 mM Tris-HCl buffer in step 1.1) is prepared by weighing 121.1 g of Tris in 800 mL of ddH2O, stirring thoroughly to dissolve, adding hydrochloric acid to adjust the pH value to 7.4, diluting the solution to 1 L, and sterilizing it with high-temperature steam for later use to obtain a 1 M Tris-HCl buffer; adding the 1 mL of 1 M Tris-HCl buffer to 999 mL of sterilized ddH2O to obtain a 1 mM Tris-HCl buffer; Alternatively, in the above-mentioned configuration of each liquid, the amount of each substance used is increased or decreased in the same proportion.

4. A simple method for expanding the culture of Reniformis and reviving Reniformis cysts according to any one of claims 1 to 3, characterized in that: The step 2) comprises: formation and seed preservation of reniform cysts: the high-density reniform cysts obtained in step 1) are enriched and then washed, resuspended in Tris-HCl buffer, and placed in a 25°C constant temperature incubator for 2 hours to allow the reniform cysts to form dormant cysts, and placed in a 4-37°C constant temperature incubator for seed preservation.

5. A simple method for expanding the culture of Reniformis and reviving Reniformis cysts according to claim 4, characterized in that: The seed preservation condition of step 2) is to place the seeds in a constant temperature incubator at 15-30° C. for half a year.

6. A simple method for expanding the culture of Reniformis and reviving Reniformis cysts according to claim 4, characterized in that: The step 3) includes: resuscitation of the reniform cysts: taking out the seed-preserving reniform cysts obtained in step 2.1) and placing them in 0.1% wheat seedling culture solution, and culturing them in a 15-30° C. constant temperature incubator to resuscitate them; when the culture time is 6-8 hours, the recovery rate can reach up to 60%-100%, and the resuscitation process is completed.

7. A simple method for expanding the culture of Reniformis and reviving Reniformis cysts according to claim 5, characterized in that: In the step 3), 10 dormant cysts were immersed in 0.1% wheatgrass culture solution and placed in a 25° C. constant temperature incubator for 8 hours, and the cyst recovery rate could reach 100%.

8. A simple method for expanding the culture of Reniformis and reviving Reniformis cysts according to any one of claims 1 to 3, characterized in that: The method further comprises 4) expanding the culture of the revived reniform worms, wherein the culture conditions of step 4) are completely the same as those of step 1).

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