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Reagent for rapidly fixing tetrahymena thermophila for cell counting and counting method of reagent

A technology of Tetrahymena thermophila and cell counting, which is applied in the fields of biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of cell clustering, non-proliferation, skin and mucous membrane corrosion, etc., and achieve the effect of rapid time

Pending Publication Date: 2020-05-01
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 1. Trypan blue is a kind of dye, which is blue and easy to contaminate the instrument. Another disadvantage is that the cells are easy to form agglomerates, which often causes deviations in the counting results.
[0007] 2. Iodine solution is a solution containing potassium iodide. It is a yellow liquid with a slight pungent smell. Therefore, it is not an ideal reagent

Method used

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  • Reagent for rapidly fixing tetrahymena thermophila for cell counting and counting method of reagent
  • Reagent for rapidly fixing tetrahymena thermophila for cell counting and counting method of reagent
  • Reagent for rapidly fixing tetrahymena thermophila for cell counting and counting method of reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Inoculate Tetrahymena thermophila Cu428.2 in the logarithmic growth phase, at 28°C, 100rpm Neff medium (yeast extract 2.5g, polyvalent peptone 2.5g, D-glucose 5.0g, 0.9g / dL trichloride Iron storage solution 1mL, distilled water 1000mL, sterilized at 121°C for 30min, stored at 4°C) Shaker culture for 48 hours, pipette 100L of culture solution into a sterilized centrifuge tube under aseptic condition, at this time, add the reagent 75% ethanol solution 100L, mix for 2min to 5min, draw 20L immediately and observe under the microscope. At the same time, set up a control group, that is, take 2 sterilized centrifuge tubes, draw 100L of culture solution, add 100L of sterilized pure water and iodine solution respectively, mix for 2min to 5min, draw 20L and observe under a microscope. The comparison results are as follows: figure 1 shown, where figure 1 Among them, 1A is sterile purified water, 1B is 75% ethanol, and 1C is Lugol's iodine solution.

[0047] Inoculate Tetrahymena...

Embodiment 2

[0050] Inoculate Tetrahymena thermophila Cu428.2 in the logarithmic growth phase, at 28°C, 100rpm Neff medium (yeast extract 2.5g, polyvalent peptone 2.5g, D-glucose 5.0g, 0.9g / dL trichloride Iron storage solution 1mL, distilled water 1000mL, sterilized at 121°C for 30min, stored at 4°C) and cultivated on a shaking table for 48 hours, ready to use.

[0051] Take 6 sterile centrifuge tubes, add 30% to sterile centrifuge tube 1, add 75% to sterile centrifuge tube 2, add 100% ethanol solution to sterile centrifuge tube 3, add sterile purified water to sterile centrifuge tube 4, Add Lugol's iodine solution to sterile centrifuge tube 5, add 100 L of trypan blue solution to sterile centrifuge tube 6, then add 100 L of Tetrahymena thermophila Cu428.2 culture solution to 6 sterile centrifuge tubes, and mix for 2 minutes To 5min, draw 20L and observe under the Count star cell counter. The result is as image 3 shown, where image 3 3A is 30% ethanol, 3B is 75% ethanol, 3C is 100% et...

Embodiment 3

[0055] Simultaneous inoculation of Tetrahymena thermophila Cu428.2 and B2086.2 in the logarithmic growth phase, at 28°C, 100rpm Neff medium (yeast extract 2.5g, polyvalent peptone 2.5g, D-glucose 5.0g, 0.9g / dL ferric chloride storage solution 1mL, distilled water 1000mL, sterilized at 121°C for 30min, stored at 4°C) for 48 hours on a shaking table, ready to use.

[0056] Take 13 sterile centrifuge tubes, add 0%, 15%, 30%, 45%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100% ethanol solution and Luge Then add 100L of Tetrahymena thermophila culture solution, mix well for 2min to 5min, draw 20L and count under the Count star cell counter, and observe and record the cell morphology. The results are shown in Table 1. Using SPSS 20 software for multiple comparisons, the results are as follows Figure 4 Shown; Wherein the form of Tetrahymena thermophila after the 1:1 dilution treatment of different concentrations of ethanol is as shown in Table 1.

[0057]

[0058]

[0059] Table 1...

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Abstract

The invention discloses a reagent for rapidly fixing tetrahymena thermophila for cell counting. The reagent is an aqueous solution of ethanol. The reagent has the beneficial effects that the time forfixing tetrahymena thermophila cells is short, the tetrahymena thermophila cells can be completely fixed within 5 minutes, and particularly, the reagent is colorless and can do no contamination to instruments. A 75% ethanol solution is a common sterilization reagent and is safe for human bodies.

Description

technical field [0001] The invention relates to the field of aquaculture, in particular to a reagent for rapidly immobilizing Tetrahymena thermophila cell counts and a counting method thereof. Background technique [0002] Tetrahymena thermophila (Tetrahymena thermophila): belongs to the cilia subphylum, Oligohymena, Hymenostomy, Tetrahymenaceae, and Tetrahymena in the protozoa phylum. It is a free-living cilium that can be purified and cultivated in large quantities. protozoa. As single-celled, mobile, common in natural water bodies, can be artificially purified and cultivated in large quantities, relatively large (about 50 μm long, 20 μm wide) eukaryotic cells without cell walls, Tetrahymena thermophila was used as the experimental object. Cell and molecular biology research has lasted more than 50 years, and has achieved a series of breakthrough results, such as the discovery of actin, the discovery of ribozyme, which won the Nobel Prize in Chemistry in 1989, and the dis...

Claims

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Application Information

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IPC IPC(8): C12Q1/06
CPCC12Q1/06
Inventor 潘厚军常藕琴石存斌付小哲王英英
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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