Medium for culturing Balantidium ctenopharyngodoni in vitro, method for preparing the medium and method for culturing Balantidium ctenopharyngodoni in vitro

Abstract

A medium for culturing Balantidium ctenopharyngodoni in vitro, method for preparing the medium and method for culture in vitro are provided, which belongs to technical field of in vitro culture of intestinal protozoa. The formulation of the culture medium includes: 100 ml of Ringer's solution, 0.5 g of yeast extract, 1.0 g of proteose peptone, 3-6 ml of fetal bovine serum, 6-10 ml of horse serum, 300-500 μl of Bacillus licheniformis suspension, 200-300 mg of aseptic starch. Culture steps include: inoculating collected Balantidium ctenopharyngodoni into a prepared medium, filling with nitrogen and sealing, culturing at 15° C. for 48-72 hours, then transferring to another fresh medium, cycling back and forth, proliferating the Balantidium ctenopharyngodoni continuously. Balantidium ctenopharyngodoni are capable of achieving cell division and proliferation in the culture medium, and lays foundation for the physiological and experimental ecology research of the Balantidium ctenopharyngodoni.

Claims

1. A medium formulated for culturing Balantidium ctenopharyngodoni in vitro, comprising: 100 ml of Ringer's solution, 0.5 g of yeast extract, 1.0 g of proteose peptone, 3-6 ml of fetal bovine serum, 6-10 ml of horse serum, 300 -500 μ1 of Bacillus licheniformis suspension having a concentration of 2×10.sup.9-6×10.sup.9 colony forming units (CFU)/ml, and 200-300 mg of aseptic starch; wherein the medium has a pH in the range of 7.0 to 7.5.

2. The medium as recited in claim 1, wherein the Ringer's solution comprises: 6.5 g of sodium chloride (NaCl), 0.14 g of potassium chloride (KCl), 0.12 g of calcium chloride (CaCl.sub.2), 0.2 g of sodium bicarbonate (NaHCO.sub.3), 0.01 g of sodium dihydrogen phosphate (NaH.sub.2PO.sub.4), and 1000 ml of distilled water.

3. A method for preparing a medium formulated for culturing Balantidium ctenopharyngodoni in vitro, comprising steps of: (a) weighing 0.5 g of yeast extract and 1.0 g of proteose peptone, adding 100 ml of Ringer's solution, adjusting pH to 7.0-7.5, autoclaving and then storing at 4° C.; (b) split charging a plurality of sterile anaerobic bottles with the medium formed in the step (a) by a volume of 3-5 ml, and separately adding 9-25 μl of a Bacillus licheniformis suspension to each of the sterile anaerobic bottles, and putting the plurality of the sterile anaerobic bottles into a microbial shaker at 30° C. for culturing until the concentration of Bacillus licheniformis reaches 2×10.sup.9-6×10.sup.9 colony forming units (CFU)/ml suspension; and (c) adding 90-250 μl of fetal bovine serum, 180-500 μl of horse serum and 6-15 mg of aseptic starch to each of the sterile anaerobic bottles containing the medium obtained from the step (b), and then placing the formulated medium in the anaerobic bottles at 15° C. for use.

4. The method as recited in claim 3, wherein an autoclaving temperature in the step (a) is 121° C., and an autoclaving time is 20 min.

5. The method as recited in claim 3, wherein a speed of the microbial shaker in the step (b) is 150 rpm.

6. The method as recited in claim 3, wherein the Ringer's solution is prepared by steps of: (i) sequentially dissolving 6.5 g of sodium chloride (NaCl), 0.14 g of potassium chloride (KCl), 0.2 g of sodium bicarbonate (NaHCO.sub.3), and 0.01 g of sodium dihydrogen phosphate (NaH.sub.2PO.sub.4) in 999 ml distilled water to prepare a solution 1; (ii) preparing a calcium chloride (CaCl.sub.2) solution having a mass concentration of 12%; and (iii) adding 1 ml of the CaCl.sub.2 solution obtained from the step (ii) to the solution 1, mixing uniformly to obtain the Ringer's solution, and storing it at room temperature for use.

7. A method for culturing Balantidium ctenopharyngodoni in vitro, comprising steps of: (1) preparing a plurality of anaerobic bottles sterilized, wherein each of the anaerobic bottles contain 3-5 ml of the formulated medium according to claim 1; (2) dissecting an intestine of a grass carp infected with Balantidium ctenopharyngodoni, collecting the Balantidium ctenopharyngodoni under a stereomicroscope, placing the collected Balantidium ctenopharyngodoni in a sterile petri dish containing an aseptic saline solution, gently washing 2 to 3 times, replacing the aseptic saline solution, then placing the petri dish with washed Balantidium ctenopharyngodoni in a constant temperature incubator at 15° C. and incubating it for a period of time; (3) taking out free Balantidium ctenopharyngodoni from the petri dish obtained from the step (2) under the stereomicroscope, inoculating free Balantidium ctenopharyngodoni into the medium of one of the anaerobic bottles from the step (1), continuously filling nitrogen for 3-5 minutes, sealing the bottle with rubber stopper, and placing the inoculated anaerobic bottles at a constant temperature of 15° C. for culturing for 48-72 h; and (4) transferring all the cultured Balantidium ctenopharyngodoni from step (3) to a second anaerobic bottle filling the second anaerobic bottle with nitrogen for 3-5 minutes, sealing, and culturing in identical conditions recited in the step (3), and repeating transferring and culturing steps every 48-72 hours to proliferate the Balantidium ctenopharyngodoni continuously.

8. The method as recited in claim 7, wherein the aseptic saline solution has sodium chloride (NaCl) at a mass to volume concentration of 0.65%.

9. The method as recited in claim 7, wherein the nitrogen is is greater than 99.99% pure.

10. The method as recited in claim 7, wherein the incubation period in step (2) is in the range of 20-30 minutes.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a growth curve diagram of Balantidium ctenopharyngodoni cultured in a medium in vitro according to an Example 1 of the present invention.

(2) FIG. 2 is a diagram showing a living Balantidium ctenopharyngodoni observed under a microscope after being cultured for 30 days in vitro in the Example 1 of the present invention.

(3) FIG. 3 is a view showing cell division of the Balantidium ctenopharyngodoni which are observed under the microscope after being cultured for 30 days in vitro in the Example 1 of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(4) The precise description of the present invention may be susceptible to various modifications of the present invention without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not to be construed. In fact, it is apparent to those skilled in the art that the various modifications of the embodiments of the invention are possible within the scope of the appended claims.

(5) In order to better understand the invention and not to limit the scope of the invention, all numbers and percentages used in the present application, as well as other values, are to be understood in all instances as modified by the word “about”. Accordingly, unless otherwise stated, the numerical parameters set forth in the specification and the appended claims are an approximation, which may vary depending on the desired properties sought to be obtained. The individual numerical parameters should at least be considered as being based on the reported significant figures and by conventional rounding methods.

(6) In the following examples, the Balantidium ctenopharyngodoni were collected from the grass carp in the Liangzi Lake of Wuhan City, Hubei Province, China, and the grass carp was brought back to the laboratory for anatomy, and isolation of ciliates.

Example 1

(7) According to an Example 1 of the present invention, a medium for culturing Balantidium ctenopharyngodoni in vitro is provide by the preset invention, comprising: 100 ml of Ringer's solution, 0.5 g of yeast extract, 1.0 g of proteose peptone, 3.5 ml of fetal bovine serum, 10 ml of horse serum, 335 μl of Bacillus licheniformis suspension, 300 mg of aseptic starch.

(8) Preferably, formulation ingredients and content of the Ringer's solution comprise: 6.5 g of sodium chloride (NaCl), 0.14 g of potassium chloride (KCl), 0.12 g of calcium chloride (CaCl.sub.2), 0.2 g of sodium bicarbonate (NaHCO.sub.3), 0.01 g of sodium dihydrogen phosphate (NaH.sub.2PO.sub.4) and 1000 ml of distilled water.

(9) The Ringer's solution is prepared by the method as follows, which comprises steps of:

(10) (i) sequentially dissolving 6.5 g of sodium chloride, 0.14 g of potassium chloride, 0.2 g of sodium bicarbonate, and 0.01 g of sodium dihydrogen phosphate in 999 ml distilled water to prepare a solution 1;

(11) (ii) preparing a CaCl.sub.2 solution having a mass concentration of 12%; and

(12) (iii) adding 1 ml of the CaCl.sub.2 solution in the step (ii) to the solution 1, and mixing uniformly to obtain the Ringer's solution, storing at room temperature for use.

(13) In the Example 1 of the present invention, a method for preparing the medium for culturing the Balantidium ctenopharyngodoni in vitro, specifically comprising steps of:

(14) (a) accurately weighing 0.5 g of yeast extract and 1.0 g of proteose peptone, to be put into a conical flask with a volume of 250 ml, adding 100 ml of Ringer's solution, adjusting a pH to 7.0-7.5, sealing the conical flask with foil paper, autoclaving at 121° C. for 20 min and then storing after the medium is cooled to 4° C.;

(15) (b) split charging each of a plurality of 25 ml sterile anaerobic bottles with a 3 ml medium formed in the step (a), and separately adding 10 μl of Bacillus licheniformis suspension to each of the sterile anaerobic bottles, and putting the plurality of the sterile anaerobic bottles into a microbial shaker at 30° C. for culturing until a concentration is 3×10.sup.9 CFU/ml, wherein a rotation speed of the shaker is 150 rpm;

(16) (c) respectively adding 100 μl of fetal bovine serum, 300 μl of horse serum and 9 mg of aseptic starch to each of the sterile anaerobic bottles containing the medium formed in the step (b), and then placing at 15° C. for use, wherein at this time, the medium for culturing the Balantidium ctenopharyngodoni in vitro is completed.

(17) A method for culturing the Balantidium ctenopharyngodoni in vitro, comprising steps of:

(18) (1) taking out a plurality of anaerobic bottles containing the medium for culturing the Balantidium ctenopharyngodoni cultured in vitro;

(19) (2) anesthetizing the grass carp with MS-222 to be put into a dissection tray, rinsing a anus near with sterile water, dissecting the grass carp with a sterile anatomical scissors, taking out viscera, cutting the hindgut to be put into a sterile petri dish with a diameter of 9 cm; opening the hindgut with a small anatomical scissors, and scraping luminal contents of the hindgut with a sterile scalpel to be put into a sterile petri dish with a diameter of 5.5 cm; adding 5 ml of sterile saline solution with a concentration of 0.65% (w/v); standing for 3 min; examining the Balantidium ctenopharyngodoni inhabited in the contents of the hindgut under a stereomicroscope; after finding the Balantidium ctenopharyngodoni, aspirating them with a sterile glass micropipette to another petri dish containing sterile saline with a concentration of 0.65% (w/v); after washing 3 times, replacing with new sterile saline, and placing in a constant temperature incubator at 15° C. for 25 min;

(20) (3) taking out the medium in the constant temperature incubator and the petri dish containing the Balantidium ctenopharyngodoni, and taking 10 active Balantidium ctenopharyngodoni with a sterile glass micropipette to be inoculated to a anaerobic bottle containing the medium for the Balantidium ctenopharyngodoni, sealing the anaerobic bottle with rubber stopper after being continuously filled with nitrogen for 3 minutes, and placing in a constant temperature incubator at 15° C. for 48 hours;

(21) (4) taking out the anaerobic bottle containing ciliates in the constant temperature incubator, pouring out the culture into a sterile petri dish, and transferring all the Balantidium ctenopharyngodoni to another anaerobic bottle containing 3 ml of fresh medium for culturing the Balantidium ctenopharyngodoni in vitro with a sterile glass micropipette under a stereomicroscope, and meanwhile recording a number of the Balantidium ctenopharyngodoni, then filling the anaerobic bottle with nitrogen for 3 min and sealing, then continuing to place in constant temperature incubator at 15° C. for 48 hours;

(22) (5) transferring all the Balantidium ctenopharyngodoni after the constant temperature culture in step (4) in the anaerobic bottle to another anaerobic bottle, and meanwhile recording a number of all the Balantidium ctenopharyngodoni, then filling the anaerobic bottle with nitrogen for 3 min and sealing, then continuing to place in constant temperature incubator at a 15° C. for 48 hours; (6) transferring all the Balantidium ctenopharyngodoni after the constant temperature culture in step (5) in the anaerobic bottle to another anaerobic bottle, repeating transferring and culturing once every 48 hours to make the Balantidium ctenopharyngodoni continuing to multiply, and preparing fresh medium for culturing the Balantidium ctenopharyngodoni in vitro in time during the experiment.

(23) The nitrogen mentioned above is pure nitrogen and a purity of nitrogen is greater than 99.99%.

(24) In the Example 1, the in vitro culture experiment of the Balantidium ctenopharyngodoni continued for one year, and the Balantidium ctenopharyngodoni were observed and counted once every 48 hours. Take a number of the Balantidium ctenopharyngodoni in the first month and generate growth curve, which is as shown in FIG. 1. It can be seen from FIG. 1 that during this one-month period, the Balantidium ctenopharyngodoni in the medium remained strong fertility and vitality, and the growth curve tends to grow continuously.

(25) FIG. 2 and FIG. 3 are respectively two figures showing a living Balantidium ctenopharyngodoni and division of the Balantidium ctenopharyngodoni which are observed under a stereomicroscope after being cultured for 30 days in vitro in the Example 1 of the present invention. As can be seen from FIG. 2 and FIG. 3, the present invention enables the cell division and long-term stable culture of the Balantidium ctenopharyngodoni in the medium cultured in vitro, and maintains vigorous vitality and fecundity during the cultivation process. The present invention has laid a solid foundation for the physiology and experimental ecology of the Balantidium ctenopharyngodoni.

Example 2

(26) According to the Example 2 of the present invention, a medium for culturing Balantidium ctenopharyngodoni in vitro is provide by the preset invention, comprising: 100 ml of Ringer's solution, 0.5 g of yeast extract, 1.0 g of proteose peptone, 3 ml of fetal bovine serum, 6 ml of horse serum, 300 μl of Bacillus licheniformis suspension, 200 mg of aseptic starch.

(27) In the Example 2, formulas and preparing method of the Ringer's solution are identical to the Example 1.

(28) In the Example 2 of the present invention, a method for preparing the medium for culturing the Balantidium ctenopharyngodoni in vitro, specifically comprising steps of:

(29) (a) accurately weighing 0.5 g of yeast extract and 1.0 g of proteose peptone, to be put into a conical flask with a volume of 250 ml, adding 100 ml of Ringer's solution, adjusting a pH to 7.0-7.5, sealing the conical flask with foil paper, autoclaving at 121° C. for 20 min and then storing after the medium is cooled to 4° C.;

(30) (b) split charging each of a plurality of 25 ml sterile anaerobic bottles with a 3 ml medium formed in the step (a), and separately adding 9 μl of Bacillus licheniformis suspension to each of the sterile anaerobic bottles, and putting the plurality of the sterile anaerobic bottles into a microbial shaker at 30° C. for culturing until a concentration is 6×10.sup.9 CFU/ml, wherein a rotation speed of the shaker is 150 rpm;

(31) (c) respectively adding 90 μl of fetal bovine serum, 180 μl of horse serum and 6 mg of aseptic starch to each of the sterile anaerobic bottles containing the medium formed in the step (b), and then placing at 15° C. for use, wherein at this time, the medium for culturing the Balantidium ctenopharyngodoni in vitro is completed.

(32) A method for culturing the Balantidium ctenopharyngodoni in vitro, comprising steps of:

(33) (1) taking out a plurality of anaerobic bottles containing the medium for culturing the Balantidium ctenopharyngodoni cultured in vitro;

(34) (2) anesthetizing the grass carp with MS-222 to be put into a dissection tray, rinsing a anus near with sterile water, dissecting the grass carp with a sterile anatomical scissors, taking out viscera, cutting the hindgut to be put into a sterile petri dish with a diameter of 9 cm; opening the hindgut with a small anatomical scissors, and scraping luminal contents of the hindgut with a sterile scalpel to be put into a sterile petri dish with a diameter of 5.5 cm; adding 5 ml of sterile saline solution with a concentration of 0.65% (w/v); standing for 3 min; examining the Balantidium ctenopharyngodoni inhabited in the contents of the hindgut under a stereomicroscope; after finding the Balantidium ctenopharyngodoni, aspirating them with a sterile glass micropipette to another petri dish containing sterile saline with a concentration of 0.65% (w/v); after washing 3 times, replacing with new sterile saline, and placing in a constant temperature incubator at 15° C. for 20 min;

(35) (3) taking out the medium in the constant temperature incubator and the petri dish containing the Balantidium ctenopharyngodoni, and taking 10 active Balantidium ctenopharyngodoni with a sterile glass micropipette to be inoculated to a anaerobic bottle containing the medium for the Balantidium ctenopharyngodoni, sealing the anaerobic bottle with rubber stopper after being continuously filled with nitrogen for 5 minutes, and placing in a constant temperature incubator at 15° C. for 72 hours;

(36) (4) taking out the anaerobic bottle containing ciliates in the constant temperature incubator, pouring out the culture into a sterile petri dish, and transferring all the Balantidium ctenopharyngodoni to another anaerobic bottle containing 3 ml of fresh medium for culturing the Balantidium ctenopharyngodoni in vitro with a sterile glass micropipette under a stereomicroscope, and meanwhile recording a number of the Balantidium ctenopharyngodoni, then filling the anaerobic bottle with nitrogen for 5 min and sealing, then continuing to place in constant temperature incubator at a 15° C. for 72 hours;

(37) (5) transferring all the Balantidium ctenopharyngodoni after the constant temperature culture in step (4) in the anaerobic bottle to another anaerobic bottle, and meanwhile recording a number of all the Balantidium ctenopharyngodoni, then filling the anaerobic bottle with nitrogen for 5 min and sealing, then continuing to place in constant temperature incubator at a 15° C. for 72 hours; (6) transferring all the Balantidium ctenopharyngodoni after the constant temperature culture in step (5) in the anaerobic bottle to another anaerobic bottle, repeating transferring and culturing once every 72 hours to make the Balantidium ctenopharyngodoni continuing to multiply, and preparing fresh medium for culturing the Balantidium ctenopharyngodoni in vitro in time during the experiment.

(38) The nitrogen mentioned above is pure nitrogen and a purity of nitrogen is greater than 99.99%.

(39) In the Example 2, the in vitro culture experiment of the Balantidium ctenopharyngodoni continued for one year, and the Balantidium ctenopharyngodoni were observed and counted once every 72 hours. During this one-year period, the Balantidium ctenopharyngodoni in the medium remained strong fertility and vitality.

(40) In addition, the applicants respectively observed a living Balantidium ctenopharyngodoni and cell division of the Balantidium ctenopharyngodoni under a microscope after being cultured for 1 year in vitro. The result indicates that the medium prepared by the present invention enables the cell division and long-term stable culture of the Balantidium ctenopharyngodoni, and maintains vigorous vitality and fecundity during the cultivation process.

Example 3

(41) According to the Example 3 of the present invention, a medium for culturing Balantidium ctenopharyngodoni in vitro is provide by the preset invention, comprising: 100 ml of Ringer's solution, 0.5 g of yeast extract, 1.0 g of proteose peptone, 6 ml of fetal bovine serum, 9 ml of horse serum, 335 μl of Bacillus licheniformis suspension, 250 mg of aseptic starch.

(42) In the Example 3, formulas and preparing method of the Ringer's solution are identical to the Example 1.

(43) In the Example 3 of the present invention, a method for preparing the medium for culturing the Balantidium ctenopharyngodoni in vitro, specifically comprising steps of:

(44) (a) accurately weighing 0.5 g of yeast extract and 1.0 g of proteose peptone, to be put into a conical flask with a volume of 250 ml, adding 100 ml of Ringer's solution, adjusting a pH to 7.0-7.5, sealing the conical flask with foil paper, autoclaving at 121° C. for 20 min and then storing after the medium is cooled to 4° C.;

(45) (b) split charging each of a plurality of 25 ml sterile anaerobic bottles with a 3 ml medium formed in the step (a), and separately adding 10 μl of Bacillus licheniformis suspension to each of the sterile anaerobic bottles, and putting the plurality of the sterile anaerobic bottles into a microbial shaker at 30° C. for culturing until a concentration is 2×10.sup.9 CFU/ml, wherein a rotation speed of the shaker is 150 rpm;

(46) (c) respectively adding 180 μl of fetal bovine serum, 270 μl of horse serum and 7.5 mg of aseptic starch to each of the sterile anaerobic bottles containing the medium formed in the step (b), and then placing at 15° C. for use, wherein at this time, the medium for culturing the Balantidium ctenopharyngodoni in vitro is completed.

(47) A method for culturing the Balantidium ctenopharyngodoni in vitro, comprising steps of:

(48) (1) taking out a plurality of anaerobic bottles containing the medium for culturing the Balantidium ctenopharyngodoni cultured in vitro;

(49) (2) anesthetizing the grass carp with MS-222 to be put into a dissection tray, rinsing a anus near with sterile water, dissecting the grass carp with a sterile anatomical scissors, taking out viscera, cutting the hindgut to be put into a sterile petri dish with a diameter of 9 cm; opening the hindgut with a small anatomical scissors, and scraping luminal contents of the hindgut with a sterile scalpel to be put into a sterile petri dish with a diameter of 5.5 cm; adding 5 ml of sterile saline solution with a concentration of 0.65% (w/v); standing for 3 min; examining the Balantidium ctenopharyngodoni inhabited in the contents of the hindgut under a stereomicroscope; after finding the Balantidium ctenopharyngodoni, aspirating them with a sterile glass micropipette to another petri dish containing sterile saline with a concentration of 0.65% (w/v); after washing 3 times, replacing with new sterile saline, and placing in a constant temperature incubator at 15° C. for 30 min;

(50) (3) taking out the medium in the constant temperature incubator and the petri dish containing the Balantidium ctenopharyngodoni, and taking 10 active Balantidium ctenopharyngodoni with a sterile glass micropipette to be inoculated to a anaerobic bottle containing the medium for the Balantidium ctenopharyngodoni, sealing the anaerobic bottle with rubber stopper after being continuously filled with nitrogen for 4 minutes, and placing in a constant temperature incubator at 15° C. for 60 hours;

(51) (4) taking out the anaerobic bottle containing ciliates in the constant temperature incubator, pouring out the culture into a sterile petri dish, and transferring all the Balantidium ctenopharyngodoni to another anaerobic bottle containing 3 ml of fresh medium for culturing the Balantidium ctenopharyngodoni in vitro with a sterile glass micropipette under a stereomicroscope, and meanwhile recording a number of the Balantidium ctenopharyngodoni, then filling the anaerobic bottle with nitrogen for 4 min and sealing, then continuing to place in constant temperature incubator at 15° C. for 60 hours;

(52) (5) transferring all the Balantidium ctenopharyngodoni after the constant temperature culture in step (4) in the anaerobic bottle to another anaerobic bottle, and meanwhile recording a number of all the Balantidium ctenopharyngodoni, then filling the anaerobic bottle with nitrogen for 4 min and sealing, then continuing to place in constant temperature incubator at a 15° C. for 60 hours;

(53) (6) transferring all the Balantidium ctenopharyngodoni after the constant temperature culture in step (5) in the anaerobic bottle to another anaerobic bottle, repeating transferring and culturing once every 60 hours to make the Balantidium ctenopharyngodoni continued to multiply, and preparing fresh medium for culturing the Balantidium ctenopharyngodoni in vitro in time during the experiment.

(54) The nitrogen mentioned above is pure nitrogen and a purity of nitrogen is greater than 99.99%.

(55) In the Example 3, the in vitro culture experiment of the Balantidium ctenopharyngodoni continued for one year, and the Balantidium ctenopharyngodoni were observed and counted once every 60 hours. During this one-year period, the Balantidium ctenopharyngodoni in the medium remained strong fertility and vitality.

(56) In addition, the applicants respectively observed a living Balantidium ctenopharyngodoni and cell division of the Balantidium ctenopharyngodoni under a microscope after being cultured for 1 year in vitro of the present invention. The result indicates that the medium prepared by the present invention enables the split-proliferation cell division and long-term stable culture of the Balantidium ctenopharyngodoni, and maintains vigorous vitality and fecundity during the cultivation process.

Example 4

(57) According to the Example 4 of the present invention, a medium for culturing Balantidium ctenopharyngodoni in vitro is provide by the preset invention, comprising: 100 ml of Ringer's solution, 0.5 g of yeast extract, 1.0 g of proteose peptone, 5 ml of fetal bovine serum, 10 ml of horse serum, 500 μl of Bacillus licheniformis suspension, 300 mg of aseptic starch.

(58) In the Example 4, formulas and preparing method of the Ringer's solution are identical to the Example 1.

(59) In the Example 4 of the present invention, a method for preparing the medium for culturing the Balantidium ctenopharyngodoni in vitro, specifically comprising steps of:

(60) (a) accurately weighing 0.5 g of yeast extract and 1.0 g of proteose peptone, to be put into a conical flask with a volume of 250 ml, adding 100 ml of Ringer's solution, adjusting pH to 7.0-7.5, sealing the conical flask with foil paper, autoclaving at 121° C. for 20 min and then storing after the medium is cooled to 4° C.;

(61) (b) split charging each of a plurality of 25 ml sterile anaerobic bottles with a 5 ml medium formed in the step (a), and separately adding 25 μl of Bacillus licheniformis suspension to each of the sterile anaerobic bottles, and putting the plurality of the sterile anaerobic bottles into a microbial shaker at 30° C. for culturing until a concentration is 4×10.sup.9 CFU/ml, wherein a rotation speed of the shaker is 150 rpm;

(62) (c) respectively adding 250 μl of fetal bovine serum, 500 μl of horse serum and 15 mg of aseptic starch to each of the sterile anaerobic bottles containing the medium formed in the step (b), and then placing at 15° C. for use, wherein at this time, the medium for culturing the Balantidium ctenopharyngodoni in vitro is completed.

(63) A method for culturing the Balantidium ctenopharyngodoni in vitro, comprising steps of:

(64) (1) taking out a plurality of anaerobic bottles containing the medium for culturing the Balantidium ctenopharyngodoni cultured in vitro;

(65) (2) anesthetizing the grass carp with MS-222 to be put into a dissection tray, rinsing a anus near with sterile water, dissecting the grass carp with a sterile anatomical scissors, taking out viscera, cutting the hindgut to be put into a sterile petri dish with a diameter of 9 cm; opening the hindgut with a small anatomical scissors, and scraping luminal contents of the hindgut with a sterile scalpel to be put into a sterile petri dish with a diameter of 5.5 cm; adding 5 ml of sterile saline solution with a concentration of 0.65% (w/v); standing for 3 min; examining the Balantidium ctenopharyngodoni inhabited in the contents of the hindgut under a stereomicroscope; after finding the Balantidium ctenopharyngodoni, aspirating with a sterile glass micropipette to another petri dish containing sterile saline with a concentration of 0.65% (w/v); after washing 3 times, replacing with new sterile saline, and placing in a constant temperature incubator at 15° C. for 25 min;

(66) (3) taking out the medium in the constant temperature incubator and the petri dish containing the Balantidium ctenopharyngodoni, and taking 10 active Balantidium ctenopharyngodoni with a sterile glass micropipette to be inoculated to a anaerobic bottle containing the medium for the Balantidium ctenopharyngodoni, sealing the anaerobic bottle with rubber stopper after being continuously filled with nitrogen for 5 minutes, and placing in a constant temperature incubator at 15° C. for 48 hours;

(67) (4) taking out the anaerobic bottle containing ciliates in the constant temperature incubator, pouring out the culture into a sterile petri dish, and transferring all the Balantidium ctenopharyngodoni to another anaerobic bottle containing 3 ml of fresh medium for culturing the Balantidium ctenopharyngodoni in vitro with a sterile glass micropipette under a stereomicroscope, and meanwhile recording a number of all the Balantidium ctenopharyngodoni, then filling the anaerobic bottle with nitrogen for 5 min and sealing, then continuing to place in constant temperature incubator at 15° C. for 48 hours;

(68) (5) transferring all the Balantidium ctenopharyngodoni after the constant temperature culture in step (4) in the anaerobic bottle to another anaerobic bottle, and meanwhile recording a number of all the Balantidium ctenopharyngodoni, then filling the anaerobic bottle with nitrogen for 5 min and sealing, then continuing to place in constant temperature incubator at a 15° C. for 48 hours; (6) transferring all the Balantidium ctenopharyngodoni after the constant temperature culture in step (5) in the anaerobic bottle to another anaerobic bottle, repeating transferring and culturing once every 48 hours to make the Balantidium ctenopharyngodoni continuing to multiply, and preparing fresh medium for culturing the Balantidium ctenopharyngodoni in vitro in time during the experiment.

(69) The nitrogen mentioned above is pure nitrogen and a purity of nitrogen is greater than 99.99%.

(70) In the Example 4, the in vitro culture experiment of the Balantidium ctenopharyngodoni continued for one year, and the Balantidium ctenopharyngodoni were observed and counted once every 48 hours. During this one-year period, the Balantidium ctenopharyngodoni in the medium remains strong fertility and vitality.

(71) In addition, the applicants respectively observed a living Balantidium ctenopharyngodoni and cell division of the Balantidium ctenopharyngodoni under a microscope after being cultured for 1 year in vitro. The result indicates that the medium prepared by the present invention enables the split-proliferation cell division and long-term stable culture of the Balantidium ctenopharyngodoni, and maintains vigorous vitality and fecundity during the cultivation process.