December 22, 2024
Giemsa working solution

Introduction

Giemsa working solution is prepared by diluting stock solution with buffered distilled water in order to stain blood and bone marrow smears:

A freshly prepared Giemsa working solution, made from well-prepared Giemsa stock and diluted with water buffered to pH 7.2 is intended to get optimal staining of malaria blood films. Giemsa stock solution procured for national programmes is standardized to reduce frequent adjustments to SOPs on staining.

Staining with Giemsa stain may be done with a rapid 10% working solution method or a slow 3% working solution method. The rapid method is  mostly used in outpatient clinics and busy laboratories where a quick diagnosis is required for patient care.

The slow method is works in staining for large numbers of slides, especially those obtained during cross-sectional or epidemiological surveys and field research.

Certain facilities may prefer to stain slides individually, even if there are relatively large batches, as this reduces on wastage of the amount of Giemsa stain needed. The volume of working Giemsa stain solution that is needed, particularly for staining individual slides, should be determined precisely to obviate preparation of a large volume that may be used over a long time and possible wasted.

Purpose of preparing Giemsa working solution

Giemsa working solution can be used to stain for malaria blood films. This method is to be modified only with the approval of the national coordinator of quality assurance for malaria microscopy. The methods specified herein are mandatory for all malaria microscopists working in laboratories

Materials required

For 10% Giemsa working solution:
  • Beaker or tube, clean, 5–10-mL capacity;
  • Pasteur pipette and
  • Giemsa stain, transferred and filtered from the stock solution into a 25- or 50-mL bottle;
  • Buffered water, pH 7.2;
  • Whatman filter paper, grade 1.
For 3% Giemsa working solution:
  • Giemsa stain, transferred and filtered from the stock solution into a 25- or 50-mL bottle;
  • A measuring cylinder, clean, 00–500-mL capacity;
  • A Pasteur pipette and Whatman filter paper, grade 1
  • Buffered water, pH 7.2;

Safety precautions

  • Methyl alcohol is highly flammable and highly toxic if inhaled or swallowed. it can cause blindness and even death if taken in any quantity. Do not come into contact and inhalation. When not in use, it must stored in a locked cupboard.
  • Universal precautions including use of relevant personal protective equipment such as gloves, safety glasses and a laboratory coat or gown – must be practised.

Procedure for preparation of 10 ml of 10% of Giemsa working solution

  • Pour 9 ml of buffered water into a beaker or tube.
  • Filter the Giemsa stock solution through with a paper Whatman and transfer to a 25 to 50 ml container
  • Top up 1 ml of Giemsa stock solution. Using a clean, dry pipette, add 1 ml of Giemsa stock solution.
  • Avoid taking the aliquot from the large bottle containing the Giemsa stock solution, to avoid contaminating it.
  • Use stain within 15 min of preparation, and discard unused stain. Prepare the Giemsa working solution just before staining the blood film(s), and use it within a maximum of 15 minutes of preparation. Discard any unused stain.

Procedure for preparation of 10 ml of  3% of Giemsa working solution

  • Pour 97 ml of buffered water into a measuring cylinder
  • Filter the Giemsa stock solution through with paper Whatman and transfer to a 25 to 50 ml container.
  • Using measuring cylinder or a pipette, measure 3 ml of Giemsa stock solution.
  • Avoid taking the aliquot directly from the large bottle containing the Giemsa stock solution, to avoid contaminating it.
  • Use stain within 15 min of preparation, and discard unused stain. Prepare the Giemsa working solution just before staining the blood film(s), and use it within a maximum of 15 minutes of preparation. Discard any
    excess stain.

Note:

  • Do not make up a single batch of Giemsa stain for use or re-use throughout the day or longer.
  • Giemsa stain quickly absorbs water vapor in the air, and, when diluted with de-ionized, distilled or any form or water, it rapidly loses its staining properties, so that slides stain poorly after
  • Pipettes, Measuring cylinders, containers and test tubes must be clean and dry before use
  • Just a short time. The iridescent scum on the surface of made-up Giemsa stain adheres easily to the blood film, making identification of structures difficult.

Calculating the volume of giemsa stock solution and buffered water required for staining individual slides.

 Each blood film requires 3 mL of Giemsa stain working solution. The volume of Giemsa stock solution required for staining individual blood slides can thus be calculated from the formula below:

volume of Giemsa stock solution required per slide =  Giemsa concentration required x 3 mL

 

 

The required volumed of buffered water (pH 7.2) for staining a single slide can be calculated as follows:

volume of buffered water per slide = 3 mL– volume of Giemsa stock required per slide

The 3 mL of Giemsa working solution is prepared by adding the volume of Giemsa stock solution required per slide plus the computed volume of buffered water at pH 7.2 required per slide.. 

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