How do you prepare Giemsa for staining?
Stock Solution:
- Dissolve 1g of Giemsa (MERCK) powder into 54 ml of glycerin.
- Heat this solution at ~60oC about 1.30h- 2h. Let cool to room temperature.
- Add 84 ml of methanol.
- Let the bottle tightly closed and protected from light for one week.
- Filter the solution and keep protected from light.
How do you make Giemsa stain stock solution?
Stock Solution
- Dissolve 3.8g of Giemsa powder into 250ml of methanol.
- Heat the solution from step 1 to ~60oC.
- Slowly add in 250ml of glycerin to the solution from step 2.
- Filter the solution from step 3.
- The solution needs to stand a period of time prior to use.
How do you make a 10% Giemsa stain?
Therefore, 4.5 mL of Giemsa stock solution should be mixed with 40.5 mL of buffered water to prepare the required amount of 10% Giemsa working solution for staining 15 individual blood films.
What is the composition of the Giemsa stain?
Giemsa’s solution is a mixture of methylene blue, eosin, and Azure B. The stain is usually prepared from commercially available Giemsa powder. A thin film of the specimen on a microscope slide is fixed in pure methanol for 30 seconds, by immersing it or by putting a few drops of methanol on the slide.
What is the function of glycerol in Giemsa stain?
In the early 1900s, Gustav Giemsa designed the Giemsa stain to detect parasites such as malaria and Treponema pallidum in blood smears. He developed a “secret” oxidation process using a unique mixture of methylene azure, methylene blue, and eosin, with glycerol added as a stabilizing agent.
What is Giemsa stock solution?
Giemsa solution contains azure, eosine, methanol and glycerol. Before staining a fixing step with methanol for about 10 min is recommended. Giemsa solution is appropriate for use in all current assays of blood and bone marrow smears.
How do you make 500ml of Giemsa stain?
Preparation of the Giemsa Stain Stock solution (500ml)
- Into 250ml of methanol, add 3.8g of Giemsa powder and dissolve.
- Heat the solution up to ~60oC.
- Then, add 250ml of glycerin to the solution, slowly.
- Filter the solution and leave it to stand for about 1-2 months before use.
What is the optimum pH for staining with Giemsa?
In order to make an accurate diagnosis of malaria, it is essential that blood films be stained with good-quality preparations of Giemsa stock solution and buffered water at pH 7.2.
Which buffer is used with Giemsa stain?
StainRITE® Phosphate Buffer yields satisfactory staining results every time when used with Wright-Giemsa stains.
What are the two methods for Giemsa staining?
The two methods for staining with Giemsa stain are the rapid (10% stain working solution) and the slow (3% stain working solution) methods. The rapid (10% stain working solution) method This is the commonest method for staining 1–15 slides at a time.
What is the component of giemsa stock?
Giemsa stock solution for microscopy Giemsa solution contains azure, eosine, methanol and glycerol. Before staining a fixing step with methanol for about 10 min is recommended. Giemsa solution is appropriate for use in all current assays of blood and bone marrow smears.
What are the steps of gram staining?
The Gram staining process includes four basic steps, including:
- Applying a primary stain (crystal violet).
- Adding a mordant (Gram’s iodine).
- Rapid decolorization with ethanol, acetone or a mixture of both.
- Counterstaining with safranin.
What is the pH of Giemsa stain?
pH 6.8
StainRITE® Wright-Giemsa Stain Phosphate Buffer pH 6.8.
What is the principle of Gram stain?
The basic principle of gram staining involves the ability of the bacterial cell wall to retain the crystal violet dye during solvent treatment. Gram-positive microorganisms have higher peptidoglycan content, whereas gram-negative organisms have higher lipid content.
How is Gram stain prepared?
Dissolve 2.0 g certified crystal violet into 20.0 ml of 95% ethyl alcohol. Dissolve 0.8 g ammonium oxalate into 80.0 ml distilled water. Mix the two solutions together and allow them to stand overnight at room temperature (25°C). Filter through coarse filter paper before use.
How to use Giemsa staining solution?
Follow the aforementioned steps but with the dilute stain of 1:40 dilution (add 0.5 ml stock Giemsa solution to 19.5 ml buffered water) and leave the stain for 90-120 minutes. On microscopic observation, cell organelles, bacteria and, parasites are distinguished based on their morphology and colour;
How to label Giemsa stock solution?
⇒ Label the bottle clearly as Giemsa Stock Solution with the batch number, the name of the person who prepared it, date of preparation and date of expiry, and document in the quality control log-book of your Laboratory.
How do you remove Giemsa stain from slides?
Remove and let air dry. Stain with diluted Giemsa stain (1:20, vol/vol) for 20 min (For a 1:20 dilution, add 2 ml of stock Giemsa to 40 ml of buffered water in a Coplin jar). Wash by briefly dipping the slide in and out of a Coplin jar of buffered water (one or two dips).
How do you make methanol solution for staining?
⇒ Gently pour in about 100 ml of acetone-free methanol through the same funnel, ensuring that all the dry stain is washed into the bottle. ⇒ Re-cap the bottle and shake it in a circular motion for 2–3 minutes to start dissolving the stain crystals in Methanol solution.