Improved acetic acid carmine reagent as well as preparation method and use thereof

Abstract

The invention discloses an improved acetic acid carmine reagent as well as a preparation method and application thereof, and belongs to the technical field of chemical dye reagent preparation. According to the reagent, glacial acetic acid, distilled water and magenta are uniformly mixed, and then an iron compound, namely ferric hydroxide, is added, so that the dyeing reagent with stable performance is obtained, and the improved acetic acid magenta reagent has the characteristics of being good in dyeing effect, short in dyeing time, easy to color, stable in pH value, free of precipitation impurities, long in shelf life and the like. The improved carmine acetate reagent can be used as a commercial carmine acetate reagent. Meanwhile, the acetic acid carmine reagent is used for dyeing and observing salivary gland chromosomes of third-instar larvae of drosophila melanogaster, and the dyeing effect is better than that of a commercially available acetic acid carmine dyeing solution. The chromosome coloring degree is high, the color difference is obvious, the chromosome is stretched and loosened without hydrolytic fracture, the stability is good, the operability, uniformity and standardization degree in the using process are high, and the method can be widely used.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical dye reagent preparation, and particularly relates to an improved acetic acid magenta reagent and a preparation method and use thereof. Background Art

[0002] Acetomacin staining solution is a commonly used alkaline dye. The staining solution contains high concentrations of acetic acid and magenta, and has an acidic pH. It is a good stain for nuclei and mitochondria, and is often used for fixation and staining of cells. When observing plant cell mitosis, it is necessary to use alkaline staining solutions to stain chromosomes, such as acetic acid magenta or gentian violet staining solution. Acetomacin reagent can also detect whether pollen is in the mononuclear stage.

[0003] With the continuous development of science and technology and the continuous improvement of modernization level, reagents are gradually commercialized and standardized. The staining effect of the currently available acetic acid magenta reagent is not ideal, mainly manifested in poor stability, low chromosome coloring or even no coloring; non-commercially available acetic acid magenta reagents have different formulas and low standardization, mainly manifested in cumbersome preparation processes, inconsistent reagent color, poor stability, low reliability and other problems. Summary of the invention

[0004] The object of the present invention is to provide an improved acetic acid magenta reagent, in which an iron compound, namely ferric hydroxide, is added after glacial acetic acid, distilled water and magenta are evenly mixed, so that not only a dyeing reagent with stable performance is obtained, but also the improved acetic acid magenta reagent has the characteristics of good dyeing effect, short dyeing time, easy coloring, stable pH, no precipitated impurities and long shelf life.

[0005] The present invention is achieved through the following technical solutions:

[0006] An improved acetic acid magenta reagent, the reagent is mainly prepared from the following raw materials in parts by weight:

[0007] 9 parts of glacial acetic acid, 11 parts of distilled water, 0.2 parts of carmine and 0.05 parts of an iron compound;

[0008] The iron compound is iron hydroxide.

[0009] Ferric hydroxide, also known as ferric hydroxide, has the chemical formula Fe(OH) 3 , molecular weight 106.87. Brown cubic crystals or brown flocculated precipitates. Heated to above 500℃, dehydrated to ferric oxide. Insoluble in water, ethanol and ether, freshly prepared is easily soluble in inorganic and organic acids, but difficult to dissolve after aging. Sodium ferrite can be obtained by co-melting with sodium carbonate. It is prepared by the reaction of ferric nitrate or ferric chloride with ammonia water to form a precipitate. It is used as a purifier and absorbent, and is used to make pigments, drugs, and arsenic detoxifiers. Mixed with calcium oxide, it can remove cyanide and hydrogen sulfide in coal gas. Ferric hydroxide is a common iron compound.

[0010] Preferably, the reagent is mainly prepared from the following raw materials in parts by weight:

[0011] 9 parts of glacial acetic acid, 11 parts of distilled water, 0.2 parts of carmine and 0.08 parts of iron compound.

[0012] Preferably, the reagent is mainly prepared from the following raw materials in parts by weight:

[0013] 9 parts of glacial acetic acid, 11 parts of distilled water, 0.2 parts of carmine and 0.2 parts of iron compound.

[0014] Preferably, the reagent is mainly prepared from the following raw materials in parts by weight:

[0015] 9 parts of glacial acetic acid, 11 parts of distilled water, 0.2 parts of carmine and 0.4 parts of iron compound.

[0016] Preferably, the reagent is mainly prepared from the following raw materials in parts by weight:

[0017] 9 parts of glacial acetic acid, 11 parts of distilled water, 0.2 parts of carmine and 0.6 parts of iron compound.

[0018] A method for preparing an improved acetic acid magenta reagent comprises the following steps:

[0019] Select glacial acetic acid according to the corresponding mass fraction, add distilled water, then slowly add magenta and stir evenly, add iron compound and mix, boil, cool and filter to obtain improved acetic acid magenta reagent, and store it in a brown bottle.

[0020] Preferably, the boiling time during the boiling does not exceed 30s.

[0021] Preferably, the improved acetic acid carmine reagent has a shelf life of 1 year at room temperature.

[0022] The invention discloses a method for using an improved acetic acid magenta reagent, comprising dripping the prepared improved acetic acid magenta reagent onto cells and staining the cells for 5 to 15 minutes.

[0023] Compared with the prior art, the present invention has at least the following technical effects:

[0024] The invention provides an improved acetic acid magenta reagent. In the reagent, after glacial acetic acid, distilled water and magenta are uniformly mixed, an iron compound, namely ferric hydroxide, is added, so that not only a dyeing reagent with stable performance is obtained, but also the improved acetic acid magenta reagent has the characteristics of good dyeing effect, short dyeing time, easy coloring, stable pH, no precipitated impurities, long shelf life and the like.

[0025] The improved acetic acid magenta reagent can be used as a commercial acetic acid magenta reagent. At the same time, the above-mentioned acetic acid magenta reagent is used to stain and observe the chromosomes of the salivary glands of the third-instar larvae of Drosophila, and the staining effect is better than that of the commercially available acetic acid magenta dye solution. The main manifestations are high chromosome coloring, obvious color difference, loose chromosome extension without hydrolysis and breakage, good stability, high operability, uniformity and standardization during use, and can be widely used. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a schematic diagram of the staining effect using the reagent of Comparative Example 1, and the scale is 20X;

[0027] Figure 2 This is a schematic diagram of the staining effect using the reagent in Example 1, and the scale is 20X;

[0028] Figure 3 Schematic diagram of the dyeing effect using Example 2, the scale of the diagram is 20X;

[0029] Figure 4 Schematic diagram of the dyeing effect using Example 3, the scale of the diagram is 20X;

[0030] Figure 5 Schematic diagram of the dyeing effect using Example 4, the scale of the diagram is 20X;

[0031] Figure 6 Schematic diagram of the dyeing effect using Example 5, the scale of the diagram is 20X;

[0032] Figure 7 As shown, it is a picture of the dyeing effect of the dyeing solution of specific embodiment 4 after being placed for one year, and the scale of the picture is 20X;

[0033] Figure 8 As shown, it is a picture of the dyeing effect of the dyeing solution of specific embodiment 4 after being placed for one year, and the scale of the picture is 40X. DETAILED DESCRIPTION

[0034] The embodiments of the present invention will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0035] A technical solution of a specific implementation of the present invention is:

[0036] An improved acetic acid magenta reagent, the reagent is mainly prepared from the following raw materials in parts by weight:

[0037] 9 parts of glacial acetic acid, 11 parts of distilled water, 0.2 parts of carmine and 0.05 parts of an iron compound;

[0038] The iron compound is iron hydroxide.

[0039] The reagent is mainly prepared from the following raw materials in parts by weight:

[0040] 9 parts of glacial acetic acid, 11 parts of distilled water, 0.2 parts of carmine and 0.08 parts of iron compound.

[0041] The reagent is mainly prepared from the following raw materials in parts by weight:

[0042] 9 parts of glacial acetic acid, 11 parts of distilled water, 0.2 parts of carmine and 0.2 parts of iron compound.

[0043] The reagent is mainly prepared from the following raw materials in parts by weight:

[0044] 9 parts of glacial acetic acid, 11 parts of distilled water, 0.2 parts of carmine and 0.4 parts of iron compound.

[0045] The reagent is mainly prepared from the following raw materials in parts by weight:

[0046] 9 parts of glacial acetic acid, 11 parts of distilled water, 0.2 parts of carmine and 0.6 parts of iron compound.

[0047] A method for preparing an improved acetic acid magenta reagent comprises the following steps:

[0048] Select glacial acetic acid according to the corresponding mass fraction, add distilled water, then slowly add magenta and stir evenly, add iron compound and mix, boil, cool and filter to obtain improved acetic acid magenta reagent, and store it in a brown bottle.

[0049] The boiling time does not exceed 30s during the boiling.

[0050] The improved acetic acid carmine reagent has a shelf life of 1 year at room temperature.

[0051] The invention discloses a method for using an improved acetic acid magenta reagent, comprising dripping the prepared improved acetic acid magenta reagent onto cells and staining the cells for 5 to 15 minutes.

[0052] Experimental Example 1: Preparation of acetic acid magenta reagent

[0053] Comparative Example 1: Preparation of traditional acetic acid magenta reagent: Take 45 ml of glacial acetic acid, add 55 ml of distilled water, slowly add 1.0 g of magenta, stir evenly and add a rusty iron nail, boil while stirring (boiling time does not exceed 30 seconds), filter after cooling, keep the rusty nail and store in a brown bottle.

[0054] Example 1: Preparation of improved acetic acid magenta reagent: Take 9 ml of glacial acetic acid, add 11 ml of distilled water, slowly add 0.2 g of magenta, and prepare 5 groups. Add 0.05 g of ferric hydroxide respectively, boil (boiling time does not exceed 30 seconds), filter after cooling, and store in a brown bottle.

[0055] Example 2: Preparation of improved acetic acid magenta reagent: Take 9 ml of glacial acetic acid, add 11 ml of distilled water, slowly add 0.2 g of magenta, and prepare 5 groups. Add 0.08 g of ferric hydroxide respectively, boil (boiling time does not exceed 30 seconds), filter after cooling, and store in a brown bottle.

[0056] Example 3: Preparation of improved acetic acid magenta reagent: Take 9 ml of glacial acetic acid, add 11 ml of distilled water, slowly add 0.2 g of magenta, and prepare 5 groups. Add 0.2 g of ferric hydroxide respectively, boil (boiling time does not exceed 30 seconds), filter after cooling, and store in a brown bottle.

[0057] Example 4: Preparation of improved acetic acid magenta reagent: Take 9 ml of glacial acetic acid, add 11 ml of distilled water, slowly add 0.2 g of magenta, prepare 5 groups. Add 0.4 g of ferric hydroxide respectively, boil (boiling time does not exceed 30 seconds), filter after cooling, and store in a brown bottle.

[0058] Example 5: Preparation of improved acetic acid magenta reagent: Take 9 ml of glacial acetic acid, add 11 ml of distilled water, slowly add 0.2 g of magenta, prepare 5 groups. Add 0.6 g of ferric hydroxide respectively, boil (boiling time does not exceed 30 seconds), filter after cooling, and store in a brown bottle.

[0059] Experimental Example 2: Verify the actual use process, dyeing process and effect of the acetic acid magenta reagent prepared in the above comparative example and each embodiment and the improved acetic acid magenta reagent.

[0060] Step 1: Place the fat third-instar fruit fly larvae on a glass slide dripped with distilled water, and observe the two ends of the larvae under a dissecting microscope. The black end is the head of the larvae. After confirming the head, use ophthalmic forceps to move the larvae so that it is horizontal on the glass slide. Hold the ophthalmic forceps in your left hand and gently clamp it at 1 / 4 of the distance from the larvae's head. At this time, the back 3 / 4 of the larvae's body should be kept in the gap of the forceps. Squeeze the 1 / 4 of the body with a little force to make the larvae's head extend forward as much as possible. After repeated squeezing several times, you can see a transparent part behind the larvae's head and the body under the dissecting microscope. Clamp the transparent part with the right forceps, and pull the larvae's body horizontally backward with your left hand, and the larvae's head and body will be separated. Under the dissecting microscope, you can see that the two sides of the head are connected to a transparent object that looks like a banana, and there is a white fat on each side. The second transparent object is the salivary gland. Use a dissecting needle to peel off the fat to get a complete salivary gland.

[0061] Step 2: The obtained salivary glands were transferred to another glass slide, a drop of physiological saline solution was added, and the prepared acetic acid magenta (Examples 1-5 were added to the corresponding groups of glass slides respectively) and the commercially purchased acetic acid magenta reagent (Comparative Example 1) were used for staining, and the staining was performed for 5 minutes respectively. After the staining was completed, the dye solution was absorbed with a filter paper strip, and then a drop of physiological saline solution was added to the glass slide.

[0062] Step 3: Use a filter paper strip to absorb the excess saline solution, then replace another filter paper strip to cover the cover glass, fix it with the fingers of your left hand, and gently tap the cover glass with the other blunt end of the dissecting needle in your right hand to spread and stretch the chromosomes. Place the prepared temporary slide on the stage of the microscope, first observe with a low-power microscope to find the chromosomes that stretch better, then switch to a high-power microscope to observe, find the chromosome with the best staining effect and move it to the center of the field of view, make a record and take a photo.

[0063] The results show:

[0064] 1. Combined with Table 1, Figure 1 This is a schematic diagram of the staining effect using the reagent of Comparative Example 1, and the scale is 20X.

[0065] Figure 1 The results showed that no coloring occurred after staining with the reagent of Comparative Example 1 for 5 minutes; some cells were stained after staining for 30 minutes, but the staining effect was shallow.

[0066] 2. Figure 2 This is a schematic diagram of the staining effect using the reagent in Example 1, and the scale is 20X.

[0067] Figure 2 The results showed that when the reagent of Example 1 was used for staining for 5 minutes, the chromosomes could be clearly seen and the chromosome morphology was good; more chromosomes were stained after staining for 30 minutes; the effect was still good after staining for 1 hour and there was no acid hydrolysis.

[0068] 3. Figure 3 This is a schematic diagram of the staining effect using the reagent in Example 2, and the scale is 20X.

[0069] Figure 3 The results showed that when the reagent of Example 2 was used for staining for 5 minutes, chromosomes could be seen, but the morphology was not very good; when the staining was performed for 10 minutes, the morphology was slightly better than that of 5 minutes.

[0070] 4. Figure 4 This is a schematic diagram of the staining effect using the reagent in Example 3, and the scale is 20X.

[0071] Figure 4 The results showed that after staining with the reagent of Example 3 for 15 minutes, it was clearly seen that the chromosome morphology was good and the staining was a little light.

[0072] 5. Figure 5 This is a schematic diagram of the staining effect using the reagent in Example 4, and the scale is 20X.

[0073] Figure 5 The results showed that the chromosome morphology was clearly visible when the reagent of Example 4 was used for staining for 15 minutes, and the effect was the best.

[0074] 6. Figure 6 This is a schematic diagram of the staining effect using the reagent in Example 5, and the scale is 20X.

[0075] Figure 6 The results showed that after staining with the reagent of Example 5 for 15 minutes, the chromosome morphology was clear and the color was a little dark.

[0076] Table 1 Comprehensive evaluation table of acetic acid magenta reagent

[0077]

[0078] In summary, combined with the above Table 1 and Figure 1-6 It is shown that when the improved acetic acid carmine reagent is used for dyeing for 5 minutes, Example 1 performs well; when the improved acetic acid carmine reagent is used for dyeing for 15 minutes, Example 4 performs well.

[0079] Experimental Example 3: Microscopic observation of the impurity content of the acetic acid magenta reagent prepared in Comparative Example 1 and each embodiment.

[0080] Experimental process: After the prepared staining solutions of comparative example 1 and each embodiment were allowed to stand for 24 hours, 20 μL of the liquid at the bottom of the staining solution was aspirated and the precipitated impurities were observed under a microscope.

[0081] The observation results show that the impurity content of the reagent in Comparative Example 1 is relatively high, reaching 20 impurity particles / 1 μL.

[0082] In each example, Fe(OH) 3 After replacing the rusty iron, the number of impurity particles was less than 3 / 1μL.

[0083] Experimental Example 4: Verifying the stability of the products prepared in each embodiment

[0084] After the prepared dyeing solution of each embodiment was placed at room temperature for a long time for one year, the reagent was used again for dyeing experiments, and the dyeing effect was still good, indicating that the improved reagent is stable.

[0085] like Figure 7 As shown, it is a picture of the dyeing effect of the dyeing solution of specific embodiment 4 after being placed for one year, and the scale of the picture is 20X.

[0086] like Figure 8 As shown, it is a picture of the dyeing effect of the dyeing solution of specific embodiment 4 after being placed for one year, and the scale of the picture is 40X.

[0087] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An improved acetic acid magenta reagent, characterized in that, The reagent is mainly prepared from the following raw materials in parts by weight: 9 parts of glacial acetic acid, 11 parts of distilled water, 0.2 parts of carmine and 0.05 parts of an iron compound; The iron compound is iron hydroxide.

2. The improved acetic acid carmine reagent according to claim 1, characterized in that: The reagent is mainly prepared from the following raw materials in parts by weight: 9 parts of glacial acetic acid, 11 parts of distilled water, 0.2 parts of carmine and 0.08 parts of iron compound.

3. The improved acetic acid carmine reagent according to claim 2, characterized in that: The reagent is mainly prepared from the following raw materials in parts by weight: 9 parts of glacial acetic acid, 11 parts of distilled water, 0.2 parts of carmine and 0.2 parts of iron compound.

4. The improved acetic acid carmine reagent according to claim 3, characterized in that: The reagent is mainly prepared from the following raw materials in parts by weight: 9 parts of glacial acetic acid, 11 parts of distilled water, 0.2 parts of carmine and 0.4 parts of iron compound.

5. The improved acetic acid carmine reagent according to claim 4, characterized in that: The reagent is mainly prepared from the following raw materials in parts by weight: 9 parts of glacial acetic acid, 11 parts of distilled water, 0.2 parts of carmine and 0.6 parts of iron compound.

6. A method for preparing the improved acetic acid magenta reagent according to any one of claims 1 to 5, characterized in that: The steps include: Select glacial acetic acid according to the corresponding mass fraction, add distilled water, then slowly add magenta and stir evenly, add iron compound and mix, boil, cool and filter to obtain improved acetic acid magenta reagent, and store it in a brown bottle.

7. The method for preparing an improved acetic acid magenta reagent according to claim 6, characterized in that: The boiling time does not exceed 30s during the boiling.

8. The method for preparing an improved acetic acid magenta reagent according to claim 6, characterized in that: The improved acetic acid carmine reagent has a shelf life of 1 year at room temperature.

9. A method for using the improved acetic acid magenta reagent according to any one of claims 1 to 5, characterized in that: The modified acetic acid magenta reagent prepared as claimed in claim 6 is added dropwise to the cells and stained for 5 to 15 minutes.

Images