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A kind of staining method of Eimeria tenella protoplasts

A technology of Eimeria cocci and protoplasts, which is applied in the preparation of test samples and other directions, can solve the problem of not being able to retain the complete shape of the immature E. believable effect

Active Publication Date: 2022-07-29
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Purpose of the invention: The purpose of the present invention is to overcome the technical defect that the traditional mechanical method cannot retain the complete shape of the immature E.tenella oocyst wall, and establish a new wall-breaking technology, and then realize the staining of coccidia protoplasts

Method used

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  • A kind of staining method of Eimeria tenella protoplasts
  • A kind of staining method of Eimeria tenella protoplasts
  • A kind of staining method of Eimeria tenella protoplasts

Examples

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Effect test

Embodiment 1

[0032] (1) Take the fresh immature Eimeria tenella oocysts isolated from the cecum after coccidial infection of chickens, use 20% sodium hypochlorite solution, 4 ℃ refrigerator for 10min, and use sterilized water for centrifugal washing 3 times to remove sodium hypochlorite . Add 1.1mol / L sucrose solution to the last sediment, centrifuge at 2000rpm for 10min, float the oocysts, suck the oocysts into another centrifuge tube, and wash with sterilized water 3 times to remove the sucrose.

[0033] (2) placing the purified immature Eimeria tenella oocysts at 4°C and -20°C for 15 minutes, and freeze-thaw cycles three times;

[0034] (3) 37% concentrated hydrochloric acid-95% ethanol aqueous solution (volume ratio of 1:1) was used to treat the immature Eimeria tenella oocysts after freeze-thaw cycles. solution, washed three times with distilled water, and adjusted the concentration of oocysts to 1 × 10 3 pcs / μl;

[0035] (4) Hoechst 33258 dye was added to the Eppendorf tube contai...

Embodiment 2

[0038] (1) Take the fresh immature Eimeria tenella oocysts isolated from the cecum after coccidial infection of chickens, use 20% sodium hypochlorite solution, 4 ℃ refrigerator for 10min, and use sterilized water for centrifugal washing 3 times to remove sodium hypochlorite . Add 1.1mol / L sucrose solution to the last sediment, centrifuge at 2000rpm for 10min, float the oocysts, suck the oocysts into another centrifuge tube, and wash with sterilized water 3 times to remove the sucrose.

[0039] (2) placing the purified immature Eimeria tenella oocysts at 4°C and -20°C for 14 minutes, and freeze-thaw cycles three times;

[0040] (3) 36% concentrated hydrochloric acid-93% ethanol aqueous solution (volume ratio of 1:0.5) was used to treat the immature Eimeria tenella oocysts after freeze-thaw cycles. solution, washed three times with distilled water, and adjusted the concentration of oocysts to 1 × 10 3 pcs / μl;

[0041] (4) Add Hoechst 33258 dye to the Eppendorf tube containing...

Embodiment 3

[0044] (1) Take the fresh immature Eimeria tenella oocysts isolated from the cecum after coccidial infection of chickens, use 20% sodium hypochlorite solution, 4°C refrigerator for 10min, and use sterilized water for centrifugal washing 3 times to remove sodium hypochlorite . Add 1.1mol / L sucrose solution to the last sediment, centrifuge at 2000rpm for 10min, float the oocysts, suck the oocysts into another centrifuge tube, and wash with sterilized water 3 times to remove the sucrose.

[0045] (2) placing the purified immature Eimeria tenella oocysts at 4°C and -20°C for 16 min, and freeze-thaw cycles three times;

[0046] (3) 38% concentrated hydrochloric acid-97% ethanol aqueous solution (volume ratio of 1:1.5) was used to treat immature Eimeria tenella oocysts after freeze-thaw cycles. solution, washed three times with distilled water, and adjusted the concentration of oocysts to 1 × 10 3 pcs / μl;

[0047] (4) Hoechst 33258 dye was added to the Eppendorf tube containing t...

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Abstract

The invention relates to a method for dyeing Eimeria tenella protoplasts, comprising the following steps: (1) taking freshly separated immature Eimeria tenella oocysts, freezing and thawing cycles; (2) using hydrochloric acid-ethanol The oocysts after freeze-thaw cycles are treated, and after the action, centrifugal washing is performed to remove the action solution; (3) dye is added to perform fluorescent staining of Eimeria tenella protoplasts. The invention establishes a wall-breaking technology that preserves the complete shape of the oocyst wall of immature Eimeria tenella, and reports the staining method of the protoplast of Eimeria tenella for the first time. The procedure is simple, the method is credible, and the results are distinct. Easy to observe and judge.

Description

technical field [0001] The invention relates to a method for dyeing parasites, and in particular establishes a wall-breaking technology that preserves the complete shape of the oocyst wall of Eimeria tenella, and provides a coccidia protoplast staining with simple dyeing steps and convenient operation. Law. Background technique [0002] The immature E. tenella oocyst wall is a firm, dense tissue structure that can withstand certain mechanical damage and is difficult to destroy by conventional chemicals, digestive enzymes, washing solutions and hypotonic solutions The oocyst wall structure of coccidia. The oocyst wall has undergone significant structural changes before and after maturation. At this time, for the mature sporulated E. tenella oocysts, the mechanical method of repeated grinding with a grinder must be used to destroy the oocyst wall to a certain extent and allow the mature oocysts to be ground. The sporangia are freed, but the oocyst wall of the coccidia will d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N1/30
CPCG01N1/30
Inventor 李金贵邢涛陈许乐郁星星伯若楠
Owner YANGZHOU UNIV
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