How to Prepare a 1 in 10 Dilution of Giemsa Stain

Giemsa stain is considered one of the most significant routine stains in medical laboratories particularly in instances where blood film analysis is performed on a regular basis. It is the routine stain used to diagnose malaria and dirty white blood cells count, bone marrow smears, platelet count, and stain a few bacteria, including Chlamydia.

The majority of laboratories in Kenya and generally all over the world acquire Giemsa stain in concentrated stock solution that should be diluted to form a working solution. The most widespread is 1 in 10 (1:10).

It is important to prepare it properly. With a well-calculated dilution, produces clear visibility of the nuclear stain, the cytoplasm of RBCs, malaria parasites. Weak dilution results in faint smears while overstained dim results and this can undermine diagnosis.

In our guide, we will take you through important steps on how to prepare 1 in 10 dilution of Geisma stain, the materials required, correct measurement, the technique of preparation, time, tips and troubleshooting of the process to have a good staining result.

Understanding the 1 in 10 Dilution Concept

A 1 in 10 (1:10) working solution means: 1 part of stock Giemsa stain + 9 parts of buffer or distilled water = 10 parts total solution.

This ratio does not depend on volume. It does not matter whether you prepare 10 ml or 500 ml but you use the same principle only the measurement scale.

Examples:

10 ml working stain = 1ml Giemsa + 9ml buffer. 

5ml Giemsa + 45ml buffer = 50ml working stain. 

100 ml working stain = 10ml Giemsa + 90ml buffer.

Understanding this makes it easy to scale preparation for routine daily use or batch preparation.

The Equipment and Materials Needed are as follows.

Preparation of the dilution will require:

1. Concentrated Giemsa stock solution.
2. pH of phosphate buffer 6.8 (optimal with blood films; distilled water can be used but can decrease clarity of parasites and nuclei)
3. Clean conical flask (or reagent bottle).
4. Measuring cylinder/serological pipette.
5. Volumetric pipette/micropipette.
6. Glass rod or gentle swirling method of mixing.
7. Option (filter paper), to remove precipitates.
8. Labels/markers of adequate identification. 

Preparation of 1:10 Giemsa Working Solution Step-by-Step Procedure.

The best way to stain it is to follow this process:

1. Choose the volume of total volume you desire to prepare. This depends on workload. In common practice, 50 or 100ml is sufficient to screen malaria.

2. Weigh the volume of required buffer/distilled water. Example of 50ml working stain: Add 45ml of phosphate buffer to a clean container.

3. Add the Giemsa stain stock solution. Measure 5ml stock stain using a pipette and add the stain to the buffer. (Always add stain to buffer never buffer to stain to minimize precipitation)

4. Shake gently- do not shake violently. Wipe slowly or wipe with a clean glass rod. Too much shaking also includes bubbles which influence the quality of staining.

5. Let the mixture rest 10 to 15 minutes before use. This guarantees appropriate dispersion of dye, and enhances smear uptake.

6. Label the bottle with: 

1. Name: Giemsa Working Solution.
2. Dilution: 1:10
3. Date of preparation
4. Expiry/usage period (preferably on the same day to achieve optimum quality)

And your 1:10 Giemsa stain can now be ready to be used in blood films.

Why Phosphate Buffer and not Ordinary Water?

Although distilled water can be used, phosphate buffer pH 6.8 is used to achieve more specific staining, particularly with the malaria parasites. At this pH:

1. Nuclei assume a deep purple color.
2. RBCs appear pale pink/grey
3. Grains of WBC and malaria chromosomes become distinct.
4. Artifacts are greatly minimized.

Tap water may cause the introduction of minerals that alter the staining outcome.

Common Staining Problems and Troubleshooting

It can be difficult to determine why certain stains are not working. This may happen when a stain is intended to be applied to a specific tissue section, yet the stain is not visible or is present in unintended locations.

Problem: Stain too light or pale.  
This normally occurs when the stain is too dilute or when the time of staining is too little.  

Solution: Add stain more or lengthen the staining time until the stain turns out to be of the right intensity.  

Problem: Smear is extremely dark.  
This is usually either due to either an under diluted stain or too long a period of time allowing the stain to be left on the smear.  

Solution: Add the stain properly and reduce the time of staining to prevent over-staining. 

Problem: Background appears to be dirty or granular.  
The dirty or grained background usually shows that the stain has old age or it has precipitated particles.  

Solution: Pre-treatment of the stain should be done to filter the stain and enhance clarity. 

Problem: Parasites are not clear.  
This is normally caused by employing water as opposed to the suggested phosphate buffer in the course of staining.  

Solution: Use phosphate buffer instead of water at a pH of 6.8 to raise density of parasites.

Storage and Handling Tips

• Stain stock Store stain in amber bottle without exposing it to light to maintain its dye.
• Working dilution must not be kept long in a refrigerator; it should be fresh.
• Do not contaminate with dirty pipettes and glassware.
• Periodically filter to eliminate the crystal deposits.

Conclusion

Giemsa is a simple procedure that require accurate preparation of a of 1 in 10 dilution solution. The 1-part stain and 9 parts buffer mixture is the golden rule and this mixture should stand and then used on the same day to observe the parasites well.

Therefore, proper dilution has a direct impact on diagnostic accuracy, particularly in cases of malaria which is a persistent health issue in Kenya and most parts of Africa.

We have all the reagents in our store, follow the links to view the product and make an order within minutes. Our quality products will guarantee you accurate outcome on this procedure.

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