Preparation of Giemsa Working Solution and Staining of Malaria Blood Films

The use of Giemsa stain is the most reliable procedure to stain malaria blood film, which is composed of eosin and methylene blue. The eosin component stains the parasite nucleus red, while the methylene component stains the cytoplasm blue. Proper preparation of Giemsa staining of high quality standardized malaria blood thick and thin films on the glass slides to be used in microscopy centers must be observed.

a.  Ensure that the following materials, reagents and supplies are available for proper preparation of stain:

• Giemsa Stock solution 

• Buffered water (ph 7.2) 

• Methanol 

• Pasteur pipettes 

• Graduated cylinder 

• Drying Rack 

• Staining trough 

• Clean tap water 

• Timing clock

b. There are two methods of staining with Giemsa stains: (i) the Rapid (10%) method, and (ii) routine or regular (3%) method;

     b.1.1 Preparation of 10% Giemsa working solution( mass staining)

i. Pour 90 mL of buffered water (ph 7.2) into a 100 mL graduated cylinder;

ii. Using a pasteur pipette, draw 10 mL of Giemsa stock solution. Add the stain to the buffered water in the graduated cylinder;

iii. Cover the top of the graduated cylinder with parafilm. Gently invert the cylinder several times until completely mixed;

iv. Giemsa working solution stain must be discarded through the sink after 24 hours and prepare a fresh working solution again.

     b.1.2 Preparation of 10% Giemsa working solution (individual slide staining)

i. For individual slide staining, each slide needs approximately 3 mL of Giemsa working solution to cover it;

ii. Using a Pasteur pipette add 9 drops of Giemsa stock solution to 3ml of buffered water in a 10 mL graduated cylinder.

     b.1.3 Procedure of Rapid staining of Malaria blood films

i. Allow thick film to dry completely. If rapid results are required, drying maybe hastened by using a blow drier or briefly exposing the slide to gentle heat such as from the microscope lamp. Care should be taken to avoid overheating, otherwise the thick film will be heat-fixed;

ii. Fix the thin film by dipping it briefly in a container with methanol for a few seconds. To permit dehemoglobinization, the thick film should not be fixed; thus avoid methanol or its fumes touch the thick film;

iii. Gently pour the stain on the slide using a pipette on the staining assembly. Alternatively, slides can be placed faced down on a concave staining plate and the stain introduced underneath the slide; (not routinely practice);

iv. Staining Time: It depends on the age of the Giemsa stock solution. It may extend up to 15 minutes if Giemsa stock is freshly prepared;

v. Gently flush the stain off the slide with a clean tap water; do not tip off the stain on the slide and wash, as this will leave a deposit of scum over the smear;

vi. Place the slide on the drying rack, film side downwards, to drain and dry making sure that the film does not touch the edge of the rack.

     b.2 Regular Staining Method (3% Giemsa stain working solution)

This regular method is used for staining larger number of slides such as those collected during surveys or research studies.

b.2.1 Preparation of 3% Giemsa working solution

     i. Pour 97 mL of buffered water (ph 7.2) into a 100 mL graduated cylinder;

     ii. Using Pasteur pipette, draw 3 mL of Giemsa stain. Add the stain to the buffered water in the graduated cylinder;

     iii. Cover the top of the graduated cylinder with parafilm or gently invert the cylinder several times until completely mixed;

     iv. Label the cylinder with contents, date and time prepared and technician’s initial;

     v. Buffered Giemsa stain must be prepared fresh and discarded after 6 hours.

b.2.2 Procedure of Regular Staining of Malaria Blood Film

     i. Fix thin film by briefly dipping it in a container with methanol for a few seconds. Prolong fixation make it   difficult to demonstrate Schuffner’s dots and Maurer’s spots. To permit dehaemoglobinization, the thick film should not be fixed, therefore avoid methanol or its vapor touching the film.

     ii. Place malaria blood film in a staining rack and stain singly, or in batches in staining jar to avoid cross-contamination. Batch staining is advisable only when several malaria blood films are from a single patient.

     iii. Stain films for 30-45 minutes out of the sunlight.

     iv. Pour clean tap water gently into the trough to float off the iridescent scum on the surface of the stain. Alternatively, gently immerse the whole trough in a vessel filled with clean tap water.

     v. Rinse slides briefly and gently under running tap water or by a gentle flow of clean water from a beaker.

     vi. Place the stained slides in a drying rack to drain and dry, film side downwards, making sure the film does not touch the slide rack.

     vii. At all times during preparation and storage, slides should be protected from exposure to dust and insects.

c. Evaluation of Stained Blood Film
  
     c.1 Evaluation of a well-stained blood thin film:

i. The background should be clean and free from debris; the color of erythrocyte is a pale grayish pink;

ii. Leukocytes have deep purple nuclei and well defined granules;

iii. The chromatin of malaria parasite is a deep purplish red and clear purplish blue cytoplasm;

iv. Stippling should show up as schuffner’s dots in erythrocytes containing P. vivax or P. ovale, and Maurer’s spots in erythrocytes containing the larger ring forms of P. falciparum.

     c.2 Evaluation of a well-stained thick film:

i. The background should be clean and free from debris, with a pale mottled-grey color derived from the lysed erythrocytes;

ii. Leukocytes nuclei are a deep-rich purple;

iii. Malaria parasites are well defined with deep-red chromatin pale purplish blue cytoplasm. In P. vivax and P. ovale infections, the presence of schuffner’s stippling in the “ghost” of host erythrocytes can be seen especially at the edge of the film.

     c.3 Staining quality:

i. A malaria blood film that is too pinkish suggests low ph or overstraining;

ii. A malaria blood film that is too bluish or purplish suggests high ph, film is too thick or under-stained.

Source:  Department of Health - Malaria Control Program Manual of Procedures, Guidelines in the Diagnosis and Treatment of Malaria