Blood smear and Giemsa stain

Summary

This video explains the procedure for performing a blood smear and staining it using the Giemsa method. The process includes preparing the sample, fixing the slide, and staining it with a mixture of methylene blue and eosin. It highlights how different cellular structures, like the nucleus and cytoplasm, are stained in different colors for easy identification under a microscope. The video also covers potential errors, such as improper blood drop size or uneven spreading, and demonstrates the correct technique for optimal results. Finally, it discusses how to preserve the stained sample for long-term storage.

Takeaways

• 😀 Methylene blue and eosin, a combination of basic and acid dyes, are used for differential staining in blood smear samples, allowing for the identification of different cellular structures.
• 😀 The methylene blue stains the acidic components, such as the nucleus, resulting in blue or violet coloration, while eosin stains the basic structures, like the cytoplasm, producing a pink coloration.
• 😀 To prepare a blood smear, a blood sample is placed on a clean glass slide, which is labeled at the frosted end.
• 😀 The blood sample is then spread on the slide using another clean slide held at a 45-degree angle, with the smear being pushed along the slide to produce an even, thin layer.
• 😀 It's important to work quickly and uniformly when making the blood smear to avoid poor-quality results, like deformed red blood cells or uneven distribution.
• 😀 The optimal areas for evaluation in the smear are the 'body' of the slide, where a uniform concentration of erythrocytes is found, as opposed to the 'head' or 'tail' which may contain piles or deformed erythrocytes.
• 😀 Poor smear preparation can result in defects like excess blood accumulation, grooves, or uneven pressure during spreading, leading to poor quality for analysis.
• 😀 A simple protocol for staining includes fixing the sample with absolute methanol, preparing a staining solution, and immersing the slide in the solution for about 10 minutes.
• 😀 After staining, the slides are washed with distilled water and air-dried before microscopic examination.
• 😀 For long-term storage of the slide, an adhesive solution is applied before placing a coverslip to protect the sample.

Q & A

What is the main purpose of differential staining in blood smear preparation?

The main purpose of differential staining in blood smear preparation is to distinguish different cellular structures by using dyes that bind to cellular components based on their chemical affinity. This allows for better visualization of various cell types under a microscope.

What are the two types of dyes used in the staining process described in the video?

The two types of dyes used in the staining process are methylene blue, which is a basic dye, and eosin, which is an acidic dye. Methylene blue stains acidic components like the nucleus, while eosin stains basic structures such as the cytoplasm.

How do methylene blue and eosin work together in the staining process?

Methylene blue (a basic dye) stains acidic components like the nucleus blue or violet, while eosin (an acidic dye) stains basic structures like the cytoplasm a pink color. Together, they help differentiate the various parts of cells in a blood smear.

What is the significance of the different parts of the blood smear (head, body, and tail)?

The head of the smear has a high concentration of erythrocytes, making it less suitable for cell analysis. The body of the smear, where erythrocytes are evenly spread, is ideal for microscope evaluation. The tail, at the end of the smear, is not useful for observation due to the deformation of erythrocytes and the presence of platelets.

What can go wrong if the blood smear is not prepared properly?

If the blood smear is not prepared properly, common issues include an excessively large blood drop, uneven spreading of the blood, areas with too many or too few erythrocytes, or deformed erythrocytes. These issues can affect the quality of the smear and make it difficult to interpret under a microscope.

What steps are involved in the preparation of a blood smear as described in the script?

The preparation steps involve labeling the slide, obtaining a blood sample, placing a drop of blood on the slide, and then spreading it evenly with a second slide. The smear is then left to dry before being stained with methylene blue and eosin for observation under a microscope.

Why is it important to fix the blood smear before staining?

Fixing the blood smear with methanol is essential because it preserves the cellular structures and prevents them from being destroyed during the staining process. It also helps the cells adhere to the slide, preventing them from washing away during the staining procedure.

What is the purpose of using PBS (phosphate-buffered saline) in the staining process?

PBS is used to dilute the staining solution, ensuring that the concentration of dyes is appropriate for differential staining. It also helps maintain the pH of the solution, ensuring that the dyes work effectively without damaging the cells.

What is the final step if the blood smear is to be stored for long-term observation?

If the blood smear is to be stored for long-term observation, an adhesive solution is used to affix a coverslip to the slide. This protects the smear and preserves it for future analysis.

Why is it important not to throw water directly onto the stained sample when washing?

Throwing water directly onto the stained sample can disrupt the staining process and wash away the sample, potentially ruining the slide. It is important to gently wash the slide with distilled water to carefully remove excess stain without disturbing the cells.