A Step-by-Step Guide to Giemsa Stain for Malaria

Procedure for Giemsa stain

There are two methods for staining with Giemsa stain. These are:

• The rapid (10% stain working solution)
• The slow (3% stain working solution)

The rapid (10% stain working solution) method

This method is the commonest method staining, it stains 1–15 slides at a time. It is commonly used in outpatient clinics and busy laboratories where a quick diagnosis is needed for patient care. The method is good but needs more stain. The need for speed justifies the additional cost.

The slow (3% stain working solution) method

The slow method is essential for staining larger numbers of slides (≥ 20). It is ideal for staining blood films collected during cross-sectional or epidemiological surveys and field research and for preparing batches of slides for teaching. It is less appropriate when a quick result is needed. The slow method is less expensive than the rapid method because it requires much less stain (3% rather than 10% stain solution).

For the slow (3% stain working solution) method

Giemsa stain (3% solution);

• A small container for Giemsa working stain;
• Absolute methanol, acetone-free;
• Absolute methanol, acetone-free;
• A timer;
• A slide-drying rack;
• A small electric hair-dryer;
• Protective latex gloves, powder-free, disposable and
• Distilled or deionized water buffered to pH 7.2.
• A Pasteur pipette with a rubber teat;
• A small container or beaker for methanol;
• A curved plastic staining tray, plate or staining rack;

• Methanol (methyl alcohol) is highly  flammable and highly toxic if inhaled or swallowed; it can cause blindness and even death if swallowed in any quantity. Avoid contact and inhalation. When it is not in use, it should be 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 the rapid (10%) method

• Prepare 10% Giemsa working solution and put it in a small container.
• Ascertain the amount of 10% Giemsa working solution needed for the number of slides to be stained. Each slide requires approximately 3 mL of stain to cover it.
• With a Pasteur pipette, fix the thin film by carefully dropping methanol onto the thin film only or dip the thin film for 2 s into a small container or beaker containing methanol.
• Avoid contact between the thick film and methanol, as methanol and its vapors will quickly fix the thick film and interfere with haemolysis of the thick film.
• Put the slides on a tray or drying rack and the Let the blood film dry in air on a drying rack or tray.
• Do not let the slide dry in a vertical position with the thin film down, as this may lead to fixing of the thick film by methanol vapor.
• Place slides for staining blood films face down if using a curved staining tray or facing up if using a staining rack.
• Set the timer to 8–10 min.
• At the end of the staining time, gently flush all the stain from the slides by dropping clean water over it.
• Allow the slides to air-dry.
• Pour away the remaining 10% Giemsa solution.

Procedure For the rapid (3%) method

 Drying the thick blood film

• Thick blood smears should be completely dry before being stained. They can be dried faster with warm air from a small hair-dryer. Avoid overheating slides, as they can “heat fix” and thus stain poorly.
• Use of buffered water for rinsing slides
  The pH of the water used for rinsing is important, as acidic water can decolorize the smears. It is therefore recommended that slides be rinsed with the same buffered water that is used for staining and therefore has a pH of 7.2 .

Care of glassware and measuring equipment

• Pipettes, Measuring cylinders, staining troughs and beakers should clean and dry before use.
• The equipment used for Giemsa staining should be rinsed immediately after use in clean water to remove as much of the stain as possible. It should then be soaked for a while in a detergent solution before washing.
• Utensils can be washed with a mild detergent, provided they are rinsed thoroughly in clean water before drying. Any detergent that is left on glass- and plastic-ware can alter the pH of the water and the stain, resulting in poor staining when the equipment is next used.

Note:

• When staining with Giemsa stain (3% or 10% stain working solution), the surface becomes covered with a metallic green scum. Avoid getting it onto blood smears during rinsing, as it can impair examination.
• Blood smears must stained as soon as possible after they are prepared. Storage of unstained slides for a few days in hot, humid conditions before staining will result in auto-fixation, and the thick smear will be rendered useless for microscopy

Staining individual slides with Giemsa stain

• Put the slides individually on the staining rack.
• Making sure they do no touch each other.
• Pour the stain gently onto the slides until they are totally covered. Each slide will require approximately 3 mL of stain. Avoid pouring the stain directly onto thick films.
• Leave the stain on the slides for 45-60 min with 3% Giemsa solution and 10–15 min with 10% Giemsa solution. Internal quality control of your stain will indicate the optimum staining time.
• Apply buffered water to the slides float off the iridescent “scum” on the surface of the stain. Water buffered to 7.2 pH should be poured onto the slides from the thin smear end to avoid undue disturbance and washing-off of the thick films.
• Remove the slides one by one and place them, thick film downwards, in a drying rack to drain and dry, making sure that the thick film does not touch the edge of the rack.

Check out Field stain: Principle, Procedure and Interpretation