CN111154652B

CN111154652B - Free-living ciliate protozoan culture medium and application thereof

Info

Publication number: CN111154652B
Application number: CN202010016604.0A
Authority: CN
Inventors: 李继秋; 郭星星; 于子悦; 李钗; 陈新; 林晓凤
Original assignee: South China Normal University
Current assignee: South China Normal University
Priority date: 2020-01-08
Filing date: 2020-01-08
Publication date: 2020-11-27
Anticipated expiration: 2040-01-08
Also published as: CN111154652A

Abstract

The invention discloses a free-living ciliate protozoan culture medium and application thereof, wherein the culture medium comprises the following components in percentage by mass: 80-85% of rice straw powder, 10-12% of tryptone and 1-10% of yeast extract; the culture medium contains the following nutrient components in percentage by mass: 6-8% of water, 13-15% of ash and 16-18% of protein. The culture medium is suitable for culturing various free-living ciliate protozoa with different salinity; rapidly obtain high density protozoa from low density, and can maintain high density state for long term breeding. The product of the invention has the advantages of convenient use, cheap and easily available raw materials, low cost, low price, superior function, convenient popularization and the like.

Description

Free-living ciliate protozoan culture medium and application thereof

Technical Field

The invention relates to a culture medium for free-living ciliates protozoa and use thereof.

Background

Ciliates are taxonomically related to the ciliate phylum of the subgenus of protozoa (Ciliophora), a large group of protozoa with the highest degree of specificity and complexity. The animals in the group become internationally accepted environmental indicators due to important ecological functions and unique biological characteristics. In particular, ciliate protozoa are widely used as model organisms in many research fields such as environmental science, ecology, physiology, and medicine.

In view of this, the study of such animals has long been one of the important areas of concern in the international protozoan community. However, obtaining a high density of ciliate protozoa is a prerequisite and basis for the development of relevant scientific studies.

However, due to the lack of ideal culture medium and proper culture method, high density of protozoan polypide is not obtained rapidly, which is one of the problems restricting protozoan-related research.

Currently, the Protozoan culture Medium commonly used in the laboratory is Carolina Protozoan Pellets Medium (PM, Carolina Biological Supply Company,2700York Road, Burlington, North Carolina 27215) in the United states. However, such culture media are expensive and complicated to purchase, and therefore, there is an urgent need to develop a novel protozoan culture medium which is easy to use, cheap, excellent in function, and easy to popularize.

Chinese patent ZL 201110414123.6 discloses a preparation and culture method of in vitro culture medium for sea pelagic ciliates, comprising the steps of shearing brain tissue and muscle tissue of Paralichthys olivaceus or Scophthalmus maximus, adding sea water, homogenizing to obtain meat paste, and performing heat preservation and water bath to obtain the sea pelagic ciliates culture medium. However, the culture medium of the above patent is exclusively used for the culture of parasitic scuticocida, and is not suitable for the culture of free-living ciliates (e.g., paramecium, tetrahymena, weevil, etc.); in particular, the culture medium of the above patent uses mainly fish body tissues as raw materials, and it is difficult to quantify and standardize the nutrient components of the culture medium, and it also brings insurmountable difficulties for the purification and extraction of ciliate biological materials (e.g., DNA, etc.).

Disclosure of Invention

In order to get rid of the dependence on foreign culture media and solve the problems of the prior ciliate protozoan culture medium, the invention mainly aims to provide a free-living ciliate protozoan culture medium.

It is another object of the present invention to provide the use of the above-mentioned culture medium for culturing free-living ciliates protozoa, which enables a high density of ciliates protozoa to be obtained in a short time.

The purpose of the invention is realized by the following technical scheme:

a free-living ciliate protozoan culture medium comprises the following components in percentage by mass:

straw powder: 80 to 85 percent of

Tryptone: 10 to 12 percent of

Yeast extract (B): 1 to 10 percent;

the free-living ciliate protozoan culture medium contains the following nutrient components in percentage by mass:

6-8% of water, 13-15% of ash and 16-18% of protein.

Preferably, the free-living ciliate protozoan culture medium consists of the following components in percentage by mass:

straw powder: 83.787 percent

Tryptone: 10.809 percent

Yeast extract (B): 5.404 percent.

7.8310% of water, 14.3753% of ash and 16.9961% of protein.

The straw powder is prepared by taking the stem and leaf parts of straw, naturally drying, crushing and sieving with a 100-mesh sieve.

By comparing the difference in the main nutritional components (moisture, ash and protein) between rice straw meal and Pellets Medium, it was found that rice straw meal is lower than Pellets Medium only in terms of the composition of protein, and thus the present invention made the protein content in the Medium of the present invention substantially the same as Pellets Medium by adding and adjusting the ratio of tryptone and yeast extract. Then, experiments prove that the culture Medium of the invention really has better effect than the Pelles Medium.

The culture medium can be used for culturing various kinds of free-living ciliates protozoa;

parasitic ciliates are attached to the surface layers of the bodies or organs of some hosts, and freely-growing ciliates can freely move in water and live in a floating way.

Preferably, the above-mentioned culture medium is suitable for the culture of ciliate protozoa of different salinity,

said different salinity ciliate protozoa comprise freshwater species ciliate protozoa and seawater species ciliate protozoa;

the freshwater species ciliate protozoan is paramecium urensis and/or paramecium caudatum;

the marine ciliate protozoa are paradoxus fanciformes;

specifically, the culture medium is used in combination with Escherichia coli (Escherichia coli) as a bait;

the Escherichia coli is preferably Escherichia coli DH5 alpha strain;

the initial concentration of the Escherichia coli is 10⁶cells/mL。

Specifically, crushing the raw materials of the culture medium, adding water, fully mixing, boiling, sterilizing, cooling to room temperature, inoculating ciliate protozoa in the clear liquid, and culturing;

preferably, medium is added (137.5-550.0) mg per L water;

preferably, the water is distilled water or seawater;

preferably, the ciliate protozoan inoculation density is 10 cells/mL.

Compared with the prior art, the invention has the following advantages and effects:

1. the culture medium is suitable for culturing various free-living ciliate protozoa with different salinity.

2. The product of the invention has the advantages of convenient use, cheap and easily available raw materials, low cost, low price, superior function, convenient popularization and the like.

3. The Medium of the invention has better efficiency than Carolina genomic pellet Medium (Carolina Biological Supply Company) in America when culturing ciliates such as Paramyza multocida, paramecium caudatum, paragonia fancifera and the like. The invention can lead the three ciliates to reach the environmental accommodation capacity within 4 to 6 days, and the environmental accommodation capacity is respectively improved by 33.70 percent, 49.34 percent and 41.58 percent.

Drawings

FIG. 1 is the comparison result of population growth kinetic characteristic parameters of paramecium roridum in different kinds of culture solution;

FIG. 2 is the comparison result of population growth dynamics characteristic parameters of paramecium roridum in different kinds of culture solution;

FIG. 3 is the comparison of the population growth kinetic characteristic parameters of a wandering insect in different species of culture;

wherein a is the environmental tolerance (K) of the different protozoa; b is the intrinsic growth rate (r) of different protozoa; c is the time to maximum growth rate (T) for the different protozoa;

note: the mean of the three replicates were subjected to a Least Squares (LSD) multiple comparison using P >0.05 as the standard, and the treatment groups with insignificant differences were assigned the same english alphabet.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Examples

A free-living ciliate protozoan culture medium SM consisting of rice straw powder, tryptone and yeast extract, with a formulation as shown in Table 1:

TABLE 1 formulation of culture Medium SM

The used straw is taken from conventional rice seeds (nongxiang 32) in double-cropping rice area of Chansha county in Hunan province, the stem and leaf parts of the straw are taken, naturally dried, crushed and sieved by a 100-mesh sieve; the straw powder is mixed with tryptone (OXOID, LP0042), yeast extract (OXOID, LP0021), etc. according to the formula of Table 1, and mixed thoroughly.

The mixed powder was compressed into a tablet having a diameter of 0.7cm, a thickness of 0.5cm and a mass of 0.55g by a tablet press for easy storage and preservation.

The reference Medium was Carolina Protozoan Pellets Medium (PM) available from Carolina Biological Supply Company (2700York Road, Burlington, North Carolina 27215), USA.

The PM and SM nutrient analysis results are shown in table 2:

TABLE 2 analysis of the nutrient composition of the culture media PM and SM

Note: the determination method of the nutrient components adopts GB/T6435 plus 2006, GB/T6438-2007 and GB/T6432 plus 1994 in the detection method standard of the Chinese feed industry.

Culture experiments were performed with 3 ciliate protozoa, paramecium polyclonuca (Parameciun multiconductor), Parameciun caudatus (Parameciun caudatum) and seawater ciliate protozoa, zoophoid servius (Euplotes vannus), purchased from the protozoan laboratory of the university of oceans in china. Escherichia coli (Escherichia coli DH5) was purchased from Invitrogen corporation (Guangzhou, China) and cultured in LB medium and used as a food source for culturing protozoa.

Respectively grinding the tablet culture medium SM and the PM into powder, and respectively adding 550mg of SM and 550mg of PM into 1000ml of Drech's distilled water to prepare SM culture mother liquor and PM culture mother liquor. Then, the mother liquor is diluted by Chen mineral water to prepare 1/4SM culture solution and 1/4PM culture solution according to the requirement of experimental design. Mixing, boiling, sterilizing at 120 deg.C for 20min, cooling to room temperature, and culturing the clear liquid for freshwater species ciliate protozoa (paramecium roridum, paramecium nivea).

Four culture solutions of SM, 1/4SM, PM and 1/4PM for culturing marine ciliate protozoa were prepared by replacing the abovementioned Drechsler's distilled water with artificial seawater. Each liter of artificial seawater contains 27.9720g NaCl, 4.9950g MgCl₂·6H₂O, 0.7992g KCl and 1.0190g CaCl₂Dissolved in distilled water of drochen. Similarly, mixing, boiling, sterilizing at 120 deg.C for 20min, cooling to room temperature, and collecting clear liquid for culturing marine ciliate protozoa (paradoxus fancifolius).

Culturing of any of the various protozoa tested in the experiment (including multiple species)Paramecium, paramecium caudatum, paragonia flabellata) from the same culture organism. Each ciliate protozoan was inoculated into four different types of culture media, PM, 1/4PM, SM and 1/4SM, and Escherichia coli (Escherichia coli) DH5 alpha strain was added as a source of feed for the ciliate protozoan (at a concentration of 10%⁶cells/ml). Performing culture tests in a standard six-hole cell culture plate, wherein the initial inoculation density of the protozoa is 10 cells/ml; the culture experimental system of two freshwater ciliate protozoa is 5ml, the seawater ciliate protozoa, namely, the paradox fanshaped wandering insect, has a fast propagation speed due to small body size, and the experimental system is set to be 3ml for accurate counting; the different treatment groups were incubated at constant temperature 25 ℃ on a shaker (10 rpm/min). Protozoa numbers were recorded every 24h and the culture broth was refreshed. And finishing the culture experiment after the protozoon population to be tested reaches the stable period.

And (3) fitting a protozoan population growth kinetic model according to formula 1 by using SigmaPlut 14.0(Systat Software, Inc), and calculating a population growth kinetic characteristic parameter by combining formula 2. One-way anova was performed on the above parameters using SPSS 24.0(IBM Corporation 2019), and LSD multiple comparisons were performed when there were significant differences in data (P <0.05) under different culture conditions.

Wherein N is the population number (cells/ml), N₀Is the initial population quantity (cells/ml), K is the environmental tolerance (cells/ml), r is the intrinsic growth rate of the population, T is the time (day), and T is the time (day) for the population to reach the maximum growth rate.

The experimental results are shown in fig. 1-3:

FIG. 1(A, B, C) is the comparison result of population growth kinetic characteristic parameters of paramecium polycaryon in different kinds of culture solution.

As seen from fig. 1A, the population growth kinetic parameters of paramecium roridum in 1/4PM culture solution and 1/4SM culture solution have no significant difference in environmental tolerance K (P >0.05), but the K value of paramecium roridum in SM culture solution is significantly higher than that in PM culture solution (P <0.05), the environmental tolerance K value is increased by 33.70%, and both are significantly higher than those in 1/4PM and 1/4SM culture solution (P < 0.05);

as seen from FIG. 1B, there was no significant difference in population growth kinetic parameter intrinsic growth rate r of Parameconium multiformis in 1/4PM, 1/4SM and PM culture solution (P >0.05), but all were significantly lower than r value of Parameconium multiformis in SM culture solution (P < 0.05);

as seen in fig. 1C, paramecium population growth kinetics parameters in 1/4PM, 1/4SM and PM cultures of paramecium spinosum showed no significant difference in time T to reach maximum growth rate (P >0.05), but all were significantly higher than their T value in SM culture (P < 0.05). This indicates that paramecium micrantha has the same growth performance in 1/4PM and 1/4SM culture solution, and as the concentration of the culture medium increases, the paramecium micrantha's growth performance is improved, and has better growth performance in SM culture solution.

FIG. 2(A, B, C) is the comparison result of population growth kinetic characteristic parameters of paramecium caudatum in different culture liquids.

As seen from fig. 2A, the population growth kinetic parameter K of paramecium caudatum at 1/4PM culture solution and 1/4SM culture solution is not significantly different (P >0.05), however, the K value of paramecium caudatum at SM culture solution is significantly higher than that at PM culture solution (P <0.05), the environmental tolerance K value is increased by 49.34%, and both are significantly higher than that at 1/4PM or 1/4SM culture solution (P < 0.05);

as seen from FIG. 2B, there was no significant difference in the population growth kinetic parameter r values of paramecium roridum in 4 culture solutions (P > 0.05);

as seen from fig. 2C, there was no significant difference in T value of population growth kinetic parameter of paramecium in 1/4PM, 1/4SM culture solution (P <0.05), nor in PM and SM culture solution (P >0.05), but T value in the latter two culture solutions was significantly higher than that in the former two culture solutions (P < 0.05). This indicates that paramecium has the same growth performance in 1/4PM and 1/4SM medium, the growth performance of paramecium is improved with the increase of medium concentration, and the paramecium has better growth performance in SM medium.

FIG. 3(A, B, C) is the comparison of the population growth kinetic characteristic parameters of the L.fanciformis in different medium culture solutions.

As seen in FIG. 3A, K value of the fanshaped wandering insect in SM culture was significantly higher than that in PM culture (P <0.05), the environmental tolerance K value was increased by 41.58%, and K value of the fanshaped wandering insect in SM culture was significantly higher than that in 1/4PM or 1/4SM (P < 0.05);

as seen in fig. 3B, there was no significant difference in population growth kinetic parameter r-values of the wandering beetle fanciformes in 1/4PM, 1/4SM and SM cultures (P >0.05), but all were significantly lower than their r-values in PM cultures (P < 0.05);

as seen in FIG. 3C, there was no significant difference in the population growth kinetic parameter T values of the wandering bug in 1/4PM, and SM media (P >0.05), but all were significantly higher than their T values in 1/4SM media (P < 0.05). The result shows that the wandering bug has better growth performance in SM culture solution; with the increase of the concentration of the culture medium, the growth performance of the wandering weevil fanciforme is improved.

To summarize: summary of the experimental results, it was found that 1/4SM broth and 1/4PM broth had the same growth efficacy on free-living ciliate protozoa living in the same salinity habitat. Compared with the PM culture solution, the SM culture solution has higher growth efficiency on the free-living ciliate protozoa living in different salinity habitats.

The results show that the Medium (SM) according to the invention has a better growth efficacy on free-living ciliate protozoa than Carolina Protozoan Pellets Medium (PM) and can be used in laboratory in place of PM.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A free-living ciliate protozoan culture medium is characterized by comprising the following components in percentage by mass:

straw powder: 83.787 percent

Tryptone: 10.809 percent

Yeast extract (B): 5.404%;

7.8310% of water, 14.3753% of ash and 16.9961% of protein.

2. Use of the culture medium of claim 1 for culturing libertian ciliates protozoa, characterized in that:

the free-living ciliate protozoa are paramecium, paramecium caudatum and weevil fantasus;

the culture medium is matched with Escherichia coli for use, and the Escherichia coli is used as a bait.

3. Use according to claim 2, characterized in that: pulverizing the raw materials of the culture medium, adding water, mixing, boiling, sterilizing, cooling to room temperature, inoculating ciliate protozoan to the clear liquid, and culturing.

4. Use according to claim 3, characterized in that:

adding 137.5-550.0mg of culture medium into each L of water;

the water is distilled water or seawater;

ciliate protozoa were inoculated at a density of 10 cells/mL.