CN114518278A - Morphological staining method for helicobacter pylori - Google Patents
Morphological staining method for helicobacter pylori Download PDFInfo
- Publication number
- CN114518278A CN114518278A CN202210139434.4A CN202210139434A CN114518278A CN 114518278 A CN114518278 A CN 114518278A CN 202210139434 A CN202210139434 A CN 202210139434A CN 114518278 A CN114518278 A CN 114518278A
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- China
- Prior art keywords
- staining
- solution
- helicobacter pylori
- dyeing
- morphology
- Prior art date
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Links
- 241000590002 Helicobacter pylori Species 0.000 title claims abstract description 22
- 229940037467 helicobacter pylori Drugs 0.000 title claims abstract description 22
- 238000007447 staining method Methods 0.000 title claims abstract description 16
- 230000000877 morphologic effect Effects 0.000 title description 4
- 238000004043 dyeing Methods 0.000 claims abstract description 22
- 239000012192 staining solution Substances 0.000 claims abstract description 22
- 239000007788 liquid Substances 0.000 claims abstract description 17
- 238000010186 staining Methods 0.000 claims abstract description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000000243 solution Substances 0.000 claims abstract description 10
- 238000000034 method Methods 0.000 claims abstract description 9
- 238000005406 washing Methods 0.000 claims abstract description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000007789 sealing Methods 0.000 claims abstract description 7
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims abstract description 4
- 230000018044 dehydration Effects 0.000 claims abstract description 4
- 238000006297 dehydration reaction Methods 0.000 claims abstract description 4
- 239000003292 glue Substances 0.000 claims abstract description 4
- 230000007935 neutral effect Effects 0.000 claims abstract description 4
- 239000012188 paraffin wax Substances 0.000 claims abstract description 4
- 239000008096 xylene Substances 0.000 claims abstract description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 27
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 18
- 239000011259 mixed solution Substances 0.000 claims description 14
- 238000003756 stirring Methods 0.000 claims description 13
- 239000007853 buffer solution Substances 0.000 claims description 9
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 claims description 8
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 claims description 7
- 229960000907 methylthioninium chloride Drugs 0.000 claims description 7
- 238000004090 dissolution Methods 0.000 claims description 3
- 239000003086 colorant Substances 0.000 abstract description 4
- 230000008878 coupling Effects 0.000 abstract description 4
- 238000010168 coupling process Methods 0.000 abstract description 4
- 238000005859 coupling reaction Methods 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 3
- 230000001744 histochemical effect Effects 0.000 abstract description 2
- 210000001519 tissue Anatomy 0.000 description 19
- 239000000203 mixture Substances 0.000 description 4
- 241000589989 Helicobacter Species 0.000 description 3
- 230000002496 gastric effect Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- JUQPZRLQQYSMEQ-UHFFFAOYSA-N CI Basic red 9 Chemical compound [Cl-].C1=CC(N)=CC=C1C(C=1C=CC(N)=CC=1)=C1C=CC(=[NH2+])C=C1 JUQPZRLQQYSMEQ-UHFFFAOYSA-N 0.000 description 2
- 208000007882 Gastritis Diseases 0.000 description 2
- 229940052223 basic fuchsin Drugs 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 210000001156 gastric mucosa Anatomy 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 206010017886 Gastroduodenal ulcer Diseases 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 241000282553 Macaca Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 208000008469 Peptic Ulcer Diseases 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 208000023652 chronic gastritis Diseases 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 231100000029 gastro-duodenal ulcer Toxicity 0.000 description 1
- 230000000155 isotopic effect Effects 0.000 description 1
- 238000013048 microbiological method Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
Abstract
The invention relates to the technical field of histochemical staining, in particular to a staining method for the morphology of helicobacter pylori, which comprises the following steps: step 1, preparing a dyeing solution; step 2, after dewaxing and washing the paraffin section tissue, draining excessive water on the section tissue; step 3, dripping prepared staining solution on the sliced tissues, and immersing the sliced tissues by the staining solution; step 4, after staining the tissue slices with the staining solution, washing the tissue slices with running water until the staining solution is not decolored any more; step 5, the fully colored sliced tissues are sequentially subjected to alcohol dehydration, xylene transparency and neutral glue sealing treatment; and 6, observing the sliced tissues subjected to the sealing treatment by using an observation instrument. The dyeing method for the helicobacter pylori morphology facilitates coupling of the dyeing liquid with different colors, enables the coupling liquid with different colors to be more stable, ensures the dyeing color to be bright, is convenient to distinguish, and has the advantages of simple dyeing steps, good dyeing effect and convenient observation.
Description
Technical Field
The invention relates to the technical field of histochemical staining, in particular to a staining method for the morphology of helicobacter pylori.
Background
It is known that helicobacter pylori includes canine helicobacter pylori, gastric helicobacter, helicobacter hei, helicobacter lapenii, and saromorphis, and is a gram-negative bacillus, which is a microbe species known to survive in the stomach of animals such as macaque, rat, pig, dog, etc., and has been successfully isolated from biopsy tissue of gastric mucosa of chronic active gastritis for the first time in 1983, because it is a spiral, microaerophilic bacterium and has very strict requirements for growth conditions.
The gastric helicobacter pylori is closely related to the occurrence of gastroduodenal ulcer, chronic gastritis, gastric cancer and mucosa-associated lymphoma. Currently, the main detection methods of HP include: serological method, isotopic tracing method, cell morphological method, microbiological method and molecular biological method
The existing helicobacter pylori morphological staining method is found in use, so that gastric helicobacter pylori is inconvenient to find and distinguish, detection is complex, a staining agent is unstable, staining is not vivid, and the use limitation is high.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a staining method for the morphology of helicobacter pylori.
(II) technical scheme
In order to achieve the purpose, the invention provides the following technical scheme: a staining method for helicobacter pylori morphology comprises the following steps:
step 1, preparing a dyeing solution;
step 2, after dewaxing and washing the paraffin section tissue, draining excessive water on the section tissue;
step 3, dripping prepared staining solution on the sliced tissues, and immersing the sliced tissues by the staining solution;
step 4, after staining the tissue slices with the staining solution, washing the tissue slices with running water until the staining solution is not decolored any more;
step 5, the fully colored sliced tissues are sequentially subjected to alcohol dehydration, xylene transparency and neutral glue sealing treatment;
and 6, observing the sliced tissues subjected to the mounting treatment by using an observation instrument.
Further, the invention provides a method for preparing the dyeing solution in the step 1, which comprises the following steps:
step a, dissolving EMB dye in 100ml acetic acid buffer solution, then respectively adding eosin and methylene blue, fully stirring and dissolving, and placing the mixed solution into a brown bottle to be protected from light and placed at room temperature for 24 hours for later use;
and b, adding 100ml of chloroform into the mixed solution which is placed for 24 hours, stirring the mixed solution, pouring the mixed solution into a separating funnel, collecting lower-layer liquid after the liquid is layered and stabilized, adding 100ml of chloroform, stirring the mixture, pouring the mixture into the separating funnel, and collecting lower-layer liquid after the liquid is layered and stabilized. Repeating the steps for 3-4 times until no dye is separated out at the upper layer, and finally putting the purified dyeing solution into a brown bottle for later use;
and c, adding 10ml of ethanol into the purified staining solution for complete dissolution, adding an acetic acid buffer solution to the total volume of 1000ml, and completely mixing uniformly, so that the staining solution can be used after being prepared.
Preferably, in the step 3, the staining time of the tissue of the section is 0.5min to 1.5min, and the staining temperature is 20 ℃ to 25 ℃.
Preferably, the pH value of the acetic acid buffer solution in the step a and the step c is 3.6.
Preferably, in the step b, the stirring time is 1-3min after each addition of 100ml of chloroform.
Preferably, the weight ratio of the EMB dye, eosin and methylene blue in the step a is 1: 1:1.
(III) advantageous effects
Compared with the prior art, the invention provides a staining method for the morphology of helicobacter pylori, which has the following beneficial effects:
the dyeing method for the helicobacter pylori morphology facilitates coupling of the dyeing liquid with different colors, enables the coupling liquid with different colors to be more stable, ensures the dyeing color to be bright, is convenient to distinguish, and has the advantages of simple dyeing steps, good dyeing effect and convenient observation.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
A staining method for helicobacter pylori morphology comprises the following steps:
step 1, preparing a dyeing solution;
step 2, after dewaxing and washing the paraffin section tissue, draining excessive water on the section tissue;
step 3, dripping the prepared staining solution on the slice tissue, and immersing the slice tissue by the staining solution;
step 4, after staining the tissue slices with the staining solution, washing the tissue slices with running water until the staining solution is not decolored any more;
step 5, the fully colored sliced tissues are sequentially subjected to alcohol dehydration, xylene transparency and neutral glue sealing treatment;
and 6, observing the sliced tissues subjected to the sealing treatment by using an observation instrument.
Further, the invention provides a method for preparing the dyeing solution in the step 1, which comprises the following steps:
step a, dissolving an EMB dye in 100ml of acetic acid buffer solution, then respectively adding eosin and methylene blue, fully stirring and dissolving, and placing the mixed solution in a brown bottle to be protected from light and placed at room temperature for 24 hours for later use;
and b, adding 100ml of chloroform into the mixed solution which is placed for 24 hours, stirring the mixed solution, pouring the mixed solution into a separating funnel, collecting lower-layer liquid after the liquid is layered and stabilized, adding 100ml of chloroform, stirring the mixture, pouring the mixture into the separating funnel, and collecting lower-layer liquid after the liquid is layered and stabilized. Repeating the steps for 3-4 times until no dye is separated out at the upper layer, and finally putting the purified dyeing solution into a brown bottle for later use;
and c, adding 10ml of ethanol into the purified staining solution for complete dissolution, adding an acetic acid buffer solution to the total volume of 1000ml, and completely mixing uniformly, so that the staining solution can be used after being prepared.
Preferably, in the step 3, the staining time of the tissue of the section is 0.5min to 1.5min, and the staining temperature is 20 ℃ to 25 ℃.
In this example, the dyeing temperature was 20 ℃ and the dyeing time was 1 min.
Preferably, the pH value of the acetic acid buffer solution in the step a and the step c is 3.6.
Preferably, in the step b, 100ml of chloroform is added each time, and then the stirring time is 1-3 min.
In this example, the stirring time was 2 min.
Preferably, the weight ratio of the EMB dye, eosin and methylene blue in the step a is 1: 1:1.
In conclusion, according to the staining method for helicobacter pylori morphology, eosin or basic fuchsin can stain gastric mucosa red when the staining method is used, but eosin or basic fuchsin and methylene blue are mixed due to different chemical properties of the two substances, and one of the two substances can precipitate.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. A staining method for helicobacter pylori morphology is characterized by comprising the following steps:
step 1, preparing a dyeing solution;
step 2, after dewaxing and washing the paraffin section tissue, draining excessive water on the section tissue;
step 3, dripping prepared staining solution on the sliced tissues, and immersing the sliced tissues by the staining solution;
step 4, after staining the tissue slices with the staining solution, washing the tissue slices with running water until the staining solution is not decolored any more;
step 5, the fully colored sliced tissues are sequentially subjected to alcohol dehydration, xylene transparency and neutral glue sealing treatment;
and 6, observing the sliced tissues subjected to the sealing treatment by using an observation instrument.
2. The method for staining the morphology of helicobacter pylori according to claim 1, wherein: the method for preparing the dyeing liquid in the step 1 comprises the following steps:
step a, dissolving EMB dye in 100ml acetic acid buffer solution, then respectively adding eosin and methylene blue, fully stirring and dissolving, and placing the mixed solution into a brown bottle to be protected from light and placed at room temperature for 24 hours for later use;
and step b, adding 100ml of chloroform into the mixed solution which is placed for 24 hours, stirring the mixed solution, pouring the mixed solution into a separating funnel, collecting lower-layer liquid after the liquid is layered and stabilized, adding 100ml of chloroform, stirring the mixed solution, pouring the mixed solution into the separating funnel, and collecting lower-layer liquid after the liquid is layered and stabilized. Repeating the steps for 3-4 times until no dye is separated out at the upper layer, and finally putting the purified dyeing solution into a brown bottle for later use;
and c, adding 10ml of ethanol into the purified staining solution for complete dissolution, adding an acetic acid buffer solution to the total volume of 1000ml, and completely mixing uniformly, so that the staining solution can be used after being prepared.
3. The staining method for the morphology of helicobacter pylori according to claim 2, characterized in that: in the step 3, the staining time of the tissue of the section is 0.5min to 1.5min, and the staining temperature is 20 ℃ to 25 ℃.
4. The staining method for the morphology of helicobacter pylori according to claim 3, characterized in that: the pH value of the acetic acid buffer solution in the step a and the step c is 3.6.
5. The staining method for the morphology of helicobacter pylori according to claim 4, characterized in that: in the step b, 100ml of chloroform is added each time, and then the stirring time is 1-3 min.
6. The staining method for the morphology of helicobacter pylori according to claim 5, wherein: in the step a, the weight ratio of the EMB dye to the eosin to the methylene blue is 1: 1:1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210139434.4A CN114518278A (en) | 2022-02-16 | 2022-02-16 | Morphological staining method for helicobacter pylori |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210139434.4A CN114518278A (en) | 2022-02-16 | 2022-02-16 | Morphological staining method for helicobacter pylori |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114518278A true CN114518278A (en) | 2022-05-20 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210139434.4A Pending CN114518278A (en) | 2022-02-16 | 2022-02-16 | Morphological staining method for helicobacter pylori |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114518278A (en) |
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2022
- 2022-02-16 CN CN202210139434.4A patent/CN114518278A/en active Pending
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