Giemsa stain is a differential stain that is used to stain the various components of the cells (blood or Aspirated Fluid) and it can be used to study the adherence of pathogenic bacteria to the human cells. Giemsa stain is a mixture of Azure, Methylene blue, and Eosin dye.
It differentially stains the human and bacterial cells which appeared as purple and pink colored bodies respectively. The Giemsa stain is one of the best stains for malaria and other blood parasites and also satisfactory as a routine blood stain to stain the Peripheral blood smear for the examinations of blood film under the microscope.
It is also used to stain the smears prepared by Fine Needle Aspiration Cytology (FNAC). A modification to this stain or a combination of this with other stains like May-Grunewald gives better results in FNAC or FNAB slides.
Nowadays, commercially prepared Giemsa staining solutions are used in most of the laboratories. However, some laboratories still prepare it in their own setup as per their Standard Operating Procedure (SOP).
Here, I’m gonna explain the method of Preparing Giemsa Stain or Giemsa Stock solution in Laboratory. This protocol is correct, however, Do not consider it as the only correct way of preparing Giemsa stain in laboratories as the protocol varies from lab to lab.
Materials Required for Preparing Giemsa Stain
- Giemsa stain powder – 3.8 gm
- Absolute methanol (Acetone free) – 250 ml
- Glycerol – 250 ml
- Methanol-cleaned solid glass beads (3–5 mm in diameter) – 50 pieces
- Spatula or Measuring spoon
- Weighing paper or butter paper
- Graduated cylinder
- Glass or plastic funnel
- Screw-capped, dark or amber glass bottle – 500-ml capacity
- Analytical balance / Weighing scale
Procedure of Preparing Giemsa stain in Laboratory
⇒ Place about 50 glass beads into a dark or amber colored glass bottle.
⇒ Weigh the 3.8 g of Giemsa stain powder on an analytical balance or weighing scale, and pour it into the bottle containing the beads through a funnel.
⇒ 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.
⇒ Add 250 ml of glycerol to the mixture through the funnel and shake again for 3–5 minutes to mix well the contents.
⇒ Add the remaining 150 ml of methanol to the mixture through the funnel, ensuring that the last of the methanol wash the last of the glycerol from the funnel into the staining mixture.
⇒ Tighten the screw-cap on the bottle and continue shaking for 2–3 minutes each about six times on the first day.
⇒ Shake for 2–3 minute each about six times a day for at least 7 days. A shaker may be used; if available otherwise manually shake the contents.
⇒ 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.
⇒ Tighten the screw-cap on the bottle to prevent absorption of water vapors from the air and store in a cool place away from direct sunlight.
Use this stock solution of Giemsa stain in various dilutions as per the purpose of staining, e.g. for blood cell examination in thin smear or for the identification of parasites in thick smear etc.