CN111088313A - Reagent for rapidly fixing tetrahymena thermophila cell counting and counting method thereof - Google Patents
Reagent for rapidly fixing tetrahymena thermophila cell counting and counting method thereof Download PDFInfo
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- 241000248384 Tetrahymena thermophila Species 0.000 title claims abstract description 48
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 150
- 239000000243 solution Substances 0.000 claims abstract description 71
- 239000007864 aqueous solution Substances 0.000 claims abstract description 21
- 241000223892 Tetrahymena Species 0.000 claims abstract description 17
- 239000000203 mixture Substances 0.000 claims description 5
- 239000008280 blood Substances 0.000 claims description 3
- 210000004369 blood Anatomy 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 abstract description 6
- 210000004027 cell Anatomy 0.000 description 47
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 23
- 229910052740 iodine Inorganic materials 0.000 description 23
- 239000011630 iodine Substances 0.000 description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 21
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 description 12
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 9
- 239000012153 distilled water Substances 0.000 description 5
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- 241000223782 Ciliophora Species 0.000 description 2
- 210000004081 cilia Anatomy 0.000 description 2
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- DKNPRRRKHAEUMW-UHFFFAOYSA-N Iodine aqueous Chemical group [K+].I[I-]I DKNPRRRKHAEUMW-UHFFFAOYSA-N 0.000 description 1
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 1
- 241001518671 Multiformis Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000700141 Rotifera Species 0.000 description 1
- 108010017842 Telomerase Proteins 0.000 description 1
- 241000223257 Thermomyces Species 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
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Abstract
The invention relates to a reagent for rapidly fixing the cell count of tetrahymena thermophila, which is an aqueous solution of ethanol. The method has the advantages that the time for fixing the tetrahymena thermophile cells is rapid, the tetrahymena thermophile cells can be completely fixed within 5min, particularly, the reagent is colorless, and the method has no pollution to instruments. The 75% ethanol solution is a conventional disinfection reagent and is safe to human bodies.
Description
Technical Field
The invention relates to the field of aquaculture, in particular to a reagent for rapidly fixing the count of tetrahymena thermophila cells and a counting method thereof.
Background
Tetrahymena thermophila (Tetrahymena thermophila): belongs to the phylum of protozoa, the phylum of cilia, the class of oligohyma, the order of membranous stomata, the family of tetrahymidae, the genus Tetrahymena, and is a cilium protozoan which lives freely and can be purified and cultured in large quantities. As a single-cell, mobile, eukaryotic cell which is common in natural water, can be artificially purified and cultured in large quantities, and has a relatively large individual (about 50 μm long and 20 μm wide) and no cell wall, the research on cell and molecular biology by using tetrahymena thermophila as an experimental object has been carried out for over 50 years, and a series of breakthrough results are obtained, such as the discovery of actin, the discovery of ribozyme having acquired the prize of Nobel chemistry in 1989, the elucidation of telomere structure (Blackburn,1978) and the discovery of telomerase (Greider, 1985) in Nobel physiology and medicine prize in 2009, and the discovery of histone post-translational modification function (Brownell, 1996), etc. Because the genetic background is clear, the megakaryogenome information is known and is similar to the classification position of ciliate parasites such as fish, namely, the rotifer carterii, the ichthyophthirius multiformis and the like, and the megakaryogenome information is concerned by researchers as an alternative mode organism of the ciliate parasites in drug screening and vaccine research in recent years.
The tetrahymena thermophile, as a model organism, needs to be cultured to reach a certain density, namely a certain amount under a certain volume, no matter in theoretical mechanism research of cell biology and molecular biology or as a substitute experimental object for screening research of vaccines or drugs. Cell counting is an important step in the basic experimental technique or experimental procedure.
Because the tetrahymena thermophila is active and small in size, counting can be observed under a lower power microscope or cell counter (magnified to 50-200 times), and thus fixation or inactivation is required before counting. The conventional agent for inactivating or fixing the tetrahymena thermophila is mainly as follows: trypan blue and iodine solution. Trypan blue is a staining solution widely used for counting living cells, and only enters cells with damaged cell membranes, and the living cells can exclude the dye, so that the number of unstained cells is the number of the living cells. The iodine solution can be prepared by dissolving potassium iodide in small amount of water, dissolving the iodine tablet in potassium iodide solution, and adding water to dissolve iodine completely, wherein the iodine solution comprises 1.0g of Ruogu iodine tablet, 2.0g of potassium iodide, and 300mL of distilled water. The Luogou iodine solution is yellow.
The two reagents for the enumeration of tetrahymena thermophila described above have the following major disadvantages:
1. trypan blue is a dye, is blue and easily pollutes the instrument, and the other disadvantage is that cells are easily agglomerated and cause deviation of counting results.
2. Iodine solution is a solution containing potassium iodide, is a yellow liquid with slight pungent odor, is colored as well, easily pollutes instruments, and is not an ideal reagent because the iodine solution can be absorbed by human skin and causes strong corrosive action on the skin and mucous membranes.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a reagent for rapidly fixing the count of the tetrahymena thermophile cells and a counting method thereof, and particularly, the counting reagent adopted by the invention is a reagent which is free from color, pollution and safety to human bodies, and can be used for rapidly fixing the cells of the tetrahymena thermophile for counting.
In order to achieve the purpose, the invention adopts the following technical scheme:
a reagent for rapidly fixing the cell count of tetrahymena thermophila, wherein the reagent is an aqueous solution of ethanol.
Preferably, the concentration of ethanol in the ethanol aqueous solution is 60-90%.
Preferably, the concentration of ethanol in the ethanol aqueous solution is 65-85%.
Preferably, the aqueous ethanol solution and the tetrahymena thermophila solution have the same volume when mixed
A method of counting an agent for rapid inactivation of tetrahymena thermophila cell count, the method comprising:
s1, sucking the tetrahymena thermophila solution with the required volume and the density to be measured into a sterilized centrifugal tube by using a pipette gun;
s2, adding reagents with the same volume into the centrifuge tube;
s3, lightly mixing the mixture evenly, and sucking inactivated tetrahymena thermophile with the required volume to a blood counting chamber or a cell counting chamber configured by a cell counter after 2min to 5 min;
s4 counts.
Preferably, the reagent is an aqueous solution of ethanol.
Preferably, the concentration of ethanol in the ethanol aqueous solution is 60-90%.
Preferably, the concentration of ethanol in the ethanol aqueous solution is 65-85%.
An ethanol aqueous solution with the ethanol concentration of 60-90 percent is used for rapidly fixing the count of the tetrahymena thermophila cells.
Preferably, the ethanol concentration is 65-85%.
The method has the advantages that the time for fixing the tetrahymena thermophile cells is rapid, the tetrahymena thermophile cells can be completely fixed within 5min, particularly, the reagent is colorless, and the method has no pollution to instruments. The 75% ethanol solution is a conventional disinfection reagent and is safe to human bodies.
Drawings
In FIG. 1, 1B is the morphology of the agent of the present invention observed under an inverted microscope after immobilizing Tetrahymena thermophila Cu428.2 with ethanol preferably at a concentration of 75%; 1A and 1C are sterile water and Lugo iodine solution control respectively;
in FIG. 2, 2B is the morphology of Tetrahymena thermophila B2086.2 observed under an inverted microscope after the reagent of the present invention is immobilized with ethanol preferably at a concentration of 75%; 2A and 2C are sterile water and Luge iodine solution control respectively;
in FIG. 3, 3B shows the form of Cu428.2 under a Count star cell counter after the reagent of the present invention immobilizes the tetrahymena thermophila; FIGS. 3A, 3C, 3D, 3E, and 3F are 30% ethanol, 100% ethanol, sterile water, Rogowski iodine solution, and Trypan blue control, respectively;
FIG. 4 shows the results of counting the culture broth of Tetrahymena thermophila Cu428.2 and B2086.2 diluted 1:1 in ethanol solution of different concentrations according to the present invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings, and it should be noted that the following examples are provided to illustrate the detailed embodiments and specific operations based on the technical solutions of the present invention, but the scope of the present invention is not limited to the examples.
The invention relates to a reagent for rapidly fixing the cell count of tetrahymena thermophila, which is an aqueous solution of ethanol.
Preferably, the concentration of ethanol in the ethanol aqueous solution is 60-90%.
Preferably, the concentration of ethanol in the ethanol aqueous solution is 65-85%.
Preferably, the aqueous ethanol solution and the tetrahymena thermophila solution have the same volume when mixed
A counting method for rapidly fixing a reagent for tetrahymena thermophila cell counting, the method comprising:
s1, sucking the tetrahymena thermophila solution with the required volume and the density to be measured into a sterilized centrifugal tube by using a pipette gun;
s2, adding reagents with the same volume into the centrifuge tube;
s3, lightly mixing the mixture evenly, and sucking inactivated tetrahymena thermophile with the required volume to a blood counting chamber or a cell counting chamber configured by a cell counter after 2min to 5 min;
s4 counts.
Preferably, the reagent is an aqueous solution of ethanol.
Preferably, the concentration of ethanol in the ethanol aqueous solution is 60-90%.
Preferably, the concentration of ethanol in the ethanol aqueous solution is 65-85%.
An ethanol water solution with the ethanol concentration of 60-90 percent is used for counting the cells of the quickly inactivated tetrahymena thermophila.
Preferably, the ethanol concentration is 65-85%.
The advantages of the invention are further illustrated by the following specific examples.
Example 1
Tetrahymena thermophila Cu428.2 in logarithmic growth phase is inoculated, shaking culture is carried out on a Neff culture medium (yeast extract is 2.5g, polypeptone is 2.5g, D-glucose is 5.0g, 0.9g/dL ferric trichloride storage solution is 1mL, distilled water is 1000mL, sterilization is carried out at 121 ℃ for 30min, and storage is carried out at 4 ℃) for 48 hours at 28 ℃ and 100L of culture solution is sucked in a sterilized centrifuge tube under a sterile state, at this time, 100L of 75% ethanol solution of the reagent is added, mixing is carried out for 2min to 5min, 20L of the reagent is sucked immediately and observed under a microscope. Meanwhile, a control group is set, namely 2 sterilized centrifuge tubes are taken, 100L of culture solution is sucked, 100L of sterilized purified water and 100L of sterilized iodine solution are added respectively, the mixture is uniformly mixed for 2min to 5min, 20L of culture solution is sucked and observed under a microscope, and the comparison result is shown in figure 1, wherein in figure 1, 1A is sterile purified water, 1B is 75% ethanol, and 1C is the Rugoji iodine solution.
Tetrahymena thermophila B2086.2 in logarithmic growth phase is inoculated, shaking culture is carried out for 48 hours in Neff culture medium (yeast extract is 2.5g, multivalent peptone is 2.5g, D-glucose is 5.0g, 0.9g/dL ferric chloride storage solution is 1mL, distilled water is 1000mL, sterilization is carried out at 121 ℃ for 30min, and storage is carried out at 4 ℃) at 28 ℃ and 100L of culture solution is sucked in a sterilized centrifuge tube under a sterile state, at this time, 100L of 75% ethanol solution of the reagent is added, mixing is carried out for 2min to 5min, 20L is sucked immediately and observed under a microscope. Meanwhile, a control group is set, namely 2 sterilized centrifuge tubes are taken, 100L of culture solution is sucked, 100L of sterilized purified water and 100L of sterilized iodine solution are added respectively, the mixture is uniformly mixed for 2min to 5min, 20L of culture solution is sucked and observed under a microscope, and the comparison result is shown in fig. 2, wherein in fig. 2, 2A is sterile purified water, 2B is 75% ethanol, and 2C is the Rogowski iodine solution.
According to the observation results of the above-mentioned figure 1 and figure 2, the tetrahymena thermophile cells are fixed by the reagent of the invention in 75% ethanol solution, are pear-shaped or spindle-shaped, and are similar to the forms of the control sterile purified water and the iodine Rogowski solution, and the cell forms of the tetrahymena thermophile Cu428.2 and B2086.2 are not changed by the 75% ethanol solution. While the contrast sterile water moves actively, the contrast Rugoji iodine solution is brownish yellow, and the colors of the tetrahymena thermophila cells and the slide are darker after the cells are dyed, so that the slide is known to be polluted to a certain extent.
Example 2
Tetrahymena thermophila Cu428.2 in logarithmic growth phase is inoculated, and shake culture is carried out for 48 hours at 28 ℃ in Neff medium (yeast extract 2.5g, polypeptone 2.5g, D-glucose 5.0g, 0.9g/dL ferric trichloride stock solution 1mL, distilled water 1000mL, sterilization at 121 ℃ for 30min, preservation at 4 ℃) of 100rpm for standby.
Taking 6 aseptic centrifuge tubes, respectively adding 30% of aseptic centrifuge tube 1, 75% of aseptic centrifuge tube 2, 100% of ethanol solution into aseptic centrifuge tube 3, 100L of sterile purified water into aseptic centrifuge tube 4, 100L of Lugoji iodine solution into aseptic centrifuge tube 5, 100L of trypan blue solution into aseptic centrifuge tube 6, respectively adding 100L of Thermomyces tetragonorrhoeae Cu428.2 culture solution into 6 aseptic centrifuge tubes, uniformly mixing for 2-5 min, and absorbing 20L of culture solution to observe under a Count star cell counter. The results are shown in FIG. 3, where 3A in FIG. 3 is 30% ethanol, 3B is 75% ethanol, 3C is 100% ethanol, 3D is sterile purified water, 3E is Lugol's iodine solution, and 3F is trypan blue solution.
From the results of FIG. 3, we know that the morphology of the fixed cells is similar to that of sterile purified water, and is more dispersed and suitable for counting when the culture solution of tetrahymena thermophila is diluted 1:1 in a 75% ethanol solution (FIG. 3B). Whereas 30% ethanol solution rounded or even broken the cells (fig. 3A); sometimes the cells clumped in 100% ethanol solution (fig. 3C), which was not suitable for counting. The tetrahymena thermophile culture solution is diluted by sterile purified water in a ratio of 1:1, and observation is carried out under a cell counter, so that the tetrahymena thermophile actively swims, a large number of cells escape from illumination, and the number of the cells in the visual field is greatly reduced (figure 3D); the Rogowski iodine solution (FIG. 3E) and trypan blue reagent (FIG. 3F) are colored, and easily cause contamination, especially trypan blue.
The observation under the Count star cell counter shows that: the application of the Count star cell counter to Count, the 75% ethanol solution has more excellent effect than the sterile purified water, the Rogowski iodine solution and the trypan blue solution to treat the tetrahymena thermophile.
Example 3
Tetrahymena thermophila Cu428.2 and B2086.2 in logarithmic growth phase are inoculated at the same time, and shake culture is carried out for 48 hours in Neff culture medium (2.5 g of yeast extract, 2.5g of polypeptone, 5.0g of D-glucose, 1mL of 0.9g/dL ferric trichloride storage solution, 1000mL of distilled water, 30min of sterilization at 121 ℃, and storage at 4 ℃) at 28 ℃ and 100rpm for standby.
Taking 13 sterile centrifuge tubes, respectively adding 100L of 0%, 15%, 30%, 45%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100% ethanol solution and 100% Rogowski iodine solution, respectively, then respectively adding 100L of tetrahymena thermophila culture solution, uniformly mixing for 2min to 5min, sucking 20L, counting under a Count star cell counter, observing and recording cell morphology, and obtaining results shown in Table 1. Multiple comparisons were performed using SPSS 20 software, and the results are shown in fig. 4; the morphology of the tetrahymena thermophila after dilution treatment with ethanol of different concentrations in a ratio of 1:1 is shown in Table 1.
TABLE 1
According to the results in Table 1, the culture solution of tetrahymena thermophila is diluted by ethanol with the concentration of 65-85% in a ratio of 1:1, the fixed form of the tetrahymena thermophila is similar to that of a control, the defects of active movement and difficult counting of cells are overcome, and the method is more suitable for fixing the cells of the tetrahymena thermophila and is applied to counting of a Count star cell counter.
The results in FIG. 4 show that: two thermophilic tetrahymena fixed by ethanol solution with the concentration of 60-90% have no significant difference in counting, and the cell counting of the groups has no significant difference (P >0.05) compared with the 1:1 dilution control of the iodine Rogowski solution; and the difference is obvious (P is less than 0.05) compared with 0%, 15% ethanol, 30% ethanol and 45% ethanol. 4A in fig. 4 shows that 100% ethanol solution immobilizes the tetrahymena thermophila cu428.2 with significant difference (P <0.05) compared to 60% to 90% ethanol solution, and 4B in fig. 4 shows immobilized B2086.2 with insignificant difference (P > 0.05). It is noted that in fig. 4, in the same figure, the letters are identical to indicate no significant difference (P >0.05), and the letters are different to indicate significant difference (P < 0.05).
Therefore, combining the results of Table 1 and FIG. 4, the ethanol solution used to immobilize Tetrahymena thermophila for cell counting is preferably at a concentration of 65% to 85%.
Various corresponding changes and modifications can be made by those skilled in the art according to the above technical solutions and concepts, and all such changes and modifications should be included in the scope of the present invention as claimed.
Claims (10)
1. A reagent for rapid fixation of the cell count of tetrahymena thermophila, characterized in that the reagent is an aqueous solution of ethanol.
2. The reagent for rapid fixed tetrahymena thermophila cell count according to claim 1, wherein the concentration of ethanol in the aqueous solution of ethanol is 60-90%.
3. The reagent for rapid fixed tetrahymena thermophila cell count according to claim 1 or 2, wherein the concentration of ethanol in the aqueous solution of ethanol is 65% to 85%.
4. The reagent for rapid fixed tetrahymena thermophila cell count according to any one of claims 1 to 3, wherein the volume of the aqueous solution of ethanol is the same as that of the solution of tetrahymena thermophila when mixed.
5. A counting method of the reagent for rapid fixed tetrahymena thermophila cell count according to any one of claims 1 to 4, characterized in that the method comprises
S1, sucking the tetrahymena thermophila solution with the required volume and the density to be measured into a sterilized centrifugal tube by using a pipette gun;
s2, adding reagents with the same volume into the centrifuge tube;
s3, lightly mixing the mixture evenly, and sucking inactivated tetrahymena thermophile with the required volume to a blood counting chamber or a cell counting chamber configured by a cell counter after 2min to 5 min;
s4 counts.
6. The counting method of the reagent for rapid fixed tetrahymena thermophila cell count according to claim 5, characterized in that the reagent is an aqueous solution of ethanol.
7. The reagent for rapid fixed tetrahymena thermophila cell count according to claim 6, wherein the concentration of ethanol in the aqueous solution of ethanol is 60-90%.
8. The reagent for rapid fixed tetrahymena thermophila cell count according to claim 7, wherein the concentration of ethanol in the aqueous solution of ethanol is 65% to 85%.
9. An aqueous ethanol solution with an ethanol concentration of 60-90%, which is characterized by being used for rapidly fixing tetrahymena thermophila cells for counting.
10. The aqueous ethanol solution with the ethanol concentration of 60-90 percent according to claim 9, characterized in that the ethanol concentration is 65-85 percent.
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| CN114067315A (en) * | 2021-10-23 | 2022-02-18 | 广州市艾贝泰生物科技有限公司 | Cell counting method, cell counting device, computer device, and storage medium |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4640897A (en) * | 1979-06-28 | 1987-02-03 | Institut Pasteur | Immunoanalysis of basophil-containing blood fraction for diagnosing parasitoses and allergies |
| JP2002277460A (en) * | 2001-02-23 | 2002-09-25 | Abx Sa | Reagent and method for identifying and counting biological cell |
| CN101897328A (en) * | 2010-01-11 | 2010-12-01 | 中国疾病预防控制中心寄生虫病预防控制所 | Method for staining protozoon sample |
| CN101988082A (en) * | 2009-07-31 | 2011-03-23 | 深圳迈瑞生物医疗电子股份有限公司 | Leukocyte classified counting reagent, kit and preparation method thereof and method of leukocyte classified counting |
| CN105326840A (en) * | 2014-07-07 | 2016-02-17 | 广州中医药大学科技产业园有限公司 | Combined medicine containing artemisinin |
| CN108344847A (en) * | 2018-02-05 | 2018-07-31 | 环境保护部华南环境科学研究所 | A method of monitoring water quality toxicity using tetrahymena |
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4640897A (en) * | 1979-06-28 | 1987-02-03 | Institut Pasteur | Immunoanalysis of basophil-containing blood fraction for diagnosing parasitoses and allergies |
| JP2002277460A (en) * | 2001-02-23 | 2002-09-25 | Abx Sa | Reagent and method for identifying and counting biological cell |
| CN101988082A (en) * | 2009-07-31 | 2011-03-23 | 深圳迈瑞生物医疗电子股份有限公司 | Leukocyte classified counting reagent, kit and preparation method thereof and method of leukocyte classified counting |
| CN101897328A (en) * | 2010-01-11 | 2010-12-01 | 中国疾病预防控制中心寄生虫病预防控制所 | Method for staining protozoon sample |
| CN105326840A (en) * | 2014-07-07 | 2016-02-17 | 广州中医药大学科技产业园有限公司 | Combined medicine containing artemisinin |
| CN108344847A (en) * | 2018-02-05 | 2018-07-31 | 环境保护部华南环境科学研究所 | A method of monitoring water quality toxicity using tetrahymena |
Non-Patent Citations (1)
| Title |
|---|
| DAVID J. ASAI&JAMES D. FORNEY: "《METHODS IN CELL BIOLOGY （Volume 62）》", ACADEMIC PRESS * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114067315A (en) * | 2021-10-23 | 2022-02-18 | 广州市艾贝泰生物科技有限公司 | Cell counting method, cell counting device, computer device, and storage medium |
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| CN111088313B (en) | 2024-05-14 |
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