Giemsa dye is composed of Azure II and eosin. Its staining principle and staining results are essentially similar to those of Wright staining. Giemsa Staining Solution has a strong affinity for the cytoplasm and can effectively demonstrate the degree of cytoplasmic basophilia, particularly providing clear staining of azurophilic, eosinophilic, and basophilic granules in blood and bone marrow cells. However, nuclear staining tends to be relatively intense, resulting in less distinct visualization of nuclear structures; therefore, Giemsa Staining Solution is often used in combination with Wright staining solution. Giemsa Staining Solution is mainly prepared from imported Giemsa dye and methanol and can produce clear cellular staining. It is commonly used for staining tissue sections, blood and cell smears, bacteria, chromosome banding preparations, protozoan parasites, and other specimens. Eosinophilic granules contain basic proteins that bind to the acidic dye eosin and are stained pink, and are therefore referred to as eosinophilic substances; nuclear proteins and the cytoplasm of lymphocytes are acidic and bind to the basic dyes methylene blue or azure, resulting in a purple-blue color, and are therefore referred to as basophilic substances; neutral granules are in an isoelectric state and can bind to both eosin and methylene blue, resulting in a light purple color, and are therefore referred to as neutrophilic substances.
This product consists of Giemsa Staining Solution (10×) and phosphate buffer. The two components are mixed at a ratio of 1:9 to prepare the working solution before use; alternatively, they may be used separately, that is, the specimen is first stained with Giemsa Staining Solution and then treated with phosphate buffer, which can also produce satisfactory staining results.
Contents & Storage:
G743377
| Component
| 100 mL | 500 mL
| Storage
|
G743377A
| Giemsa Staining Solution (10×)
| 10 mL
| 50 mL
| Room temperature. Store in the dark.
|
G743377B
| Phosphate Buffer
| 100 mL
| 500 mL
| Room temperature. |
Instructions for Use:
(I) One-Step Smear Staining
1、Preparation of Giemsa working solution:
Mix Giemsa Staining Solution (10×) and phosphate buffer at a ratio of 1:9. Add 1 part of Giemsa Staining Solution (10×) to 9 parts of phosphate buffer and mix thoroughly to obtain the Giemsa working solution. The Giemsa working solution is a ready-to-use reagent and is not suitable for storage; prepare immediately before use.
2、Prepare a blood smear or bone marrow smear using a conventional method. Allow the smear to air-dry naturally, and then fix with methanol for 1–3 min.
3、Place the blood smear or bone marrow smear on a staining rack. Add Giemsa working solution dropwise to completely cover the smear and stain at room temperature for 15–30 min.
4、Rinse slowly with tap water or distilled water from one end of the slide.
5、Dry and examine microscopically.
(II) Two-Step Smear Staining
1、Preparation of Giemsa working solution:
Mix Giemsa Staining Solution (10×) and distilled water at a ratio of 1:4. Add 1 part of Giemsa Staining Solution (10×) to 4 parts of distilled water and mix thoroughly to obtain the Giemsa working solution. The Giemsa working solution is a ready-to-use reagent and is not suitable for storage; prepare immediately before use.
2、Prepare a blood smear or bone marrow smear using a conventional method. Allow the smear to air-dry naturally, and then fix with methanol for 1–3 min.
3、Place the blood smear or bone marrow smear on a staining rack. Add an appropriate amount of Giemsa working solution dropwise to completely cover the smear and stain at room temperature for 10–15 min.
4、Add an equal volume of phosphate buffer, gently agitate the slide, and allow it to stand at room temperature for 5–10 min.
5、Rinse slowly with tap water or distilled water from one end of the slide.
6、Dry and examine microscopically.
(III) Tissue Section Staining
1、Preparation of Giemsa working solution:
Mix Giemsa Staining Solution (10×) and phosphate buffer at a ratio of 1:9. Add 1 part of Giemsa Staining Solution (10×) to 9 parts of phosphate buffer and mix thoroughly to obtain the Giemsa working solution. The Giemsa working solution is a ready-to-use reagent and is not suitable for storage; prepare immediately before use.
2、Immediately place fresh tissue in Regaud fixative and fix for 2 days, replacing the fixative once during this period.
3、Fix in 3% potassium dichromate for 1 day.
4、Wash under running water for 16 h or overnight.
5、Dehydrate and embed according to conventional procedures.
6、Cut sections to a thickness of approximately 5 μm and routinely deparaffinize to water.
7、Wash twice with distilled water for 1 min each time.
8、Place the sections in a staining jar containing Giemsa working solution and stain by immersion for 18–24 h.
9、Rinse briefly with distilled water.
10、Wash with 0.1–0.5% acetic acid for 1–2 min.
11、Rinse briefly with tap water.
12、Rapidly dehydrate three times with absolute ethanol for 5–10 s each time.
13、Clear in xylene and mount with neutral resin.
Precautions:
1、Blood smears or bone marrow smears should be uniform in thickness to avoid affecting the staining results.
2、During smear staining, after Giemsa staining, do not first remove the staining solution or directly rinse the smear forcefully.
3、If the staining is too dark or too light, adjust the staining time or the concentration of the working solution.
4、During smear staining and tissue section staining, pH has a certain influence on the staining results. Slides should be clean and free from acidic or alkaline contamination to avoid affecting the staining results.
5、After dilution, a metallic sheen should be visible on the surface of the staining solution, indicating that the staining solution is active; otherwise, the staining solution may have lost its effectiveness.
6、During tissue section staining, after staining, rapidly rinse with a large amount of 0.1–0.5% acetic acid to prevent surface precipitates from contaminating the sections and becoming difficult to remove.
7、Differentiation with 0.5% acetic acid is commonly used for Giemsa staining of tissue sections and may also be used for cell smears if necessary, but the concentration should be appropriately reduced. When differentiating tissue sections with 0.5% acetic acid, stop the differentiation once the sections appear pink.
8、During Giemsa staining of tissue sections, dehydration with absolute ethanol should be performed rapidly; otherwise, the sections may fade easily.
9、During smear staining and tissue section staining, if rapid results are required, prepare the Giemsa working solution by mixing Giemsa Staining Solution (10×) and phosphate buffer at a ratio of 1:1 and mix thoroughly to obtain the rapid Giemsa staining working solution. Apply the staining solution dropwise to the cell smear or tissue section and heat during staining. After 20–30 s, add fresh staining solution again and repeat this procedure 5–10 times. The remaining steps are the same as described above.
10、The staining solution may be reused, but should not be reused repeatedly. If precipitates are present, filter the solution before use.
11、Regaud fixative: prepare by mixing 3% potassium dichromate and formaldehyde at a ratio of 4:1. Mix thoroughly immediately before use. The fixative loses effectiveness after 1–2 days.
Staining Results:
Smear Staining:
Eosinophilic granules
| Pink
|
| Basophilic granules | Purple-blue |
| Neutral granules | Light purple
|
Tissue Section Staining:
Cell nuclei
| Blue to purple
|
Cytoplasm
| Light blue
|
Chromaffin cell cytoplasm
| Yellow-green
|
Connective tissue
| Light red |
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