Goals:
(1) To look at a relatively large amount of blood as quickly as possible.
(2) To remove hemoglobin from red cells which might otherwise obscure the parasites.
(3) To prevent artifacts.
(4) To properly stain the parasites.
Using proper slides:
(1) The slides should be clean and free of grease and dirt.
(2) The slides should be washed in alcohol and then dried prior to use.
Determine the type of blood sample:
(1) If the blood sample does not contain an anticoagulant (for example from a fingerstick) then fibrin may form as the blood dries. It is important to stir these specimens with a small stick while the blood is drying.
(2) Blood collected into an anticoagulant can be applied directly to the slide.
Making the smear:
(1) It is important that the smear not be too thick. You should barely be able to read newsprint through the smear when complete.
(2) Blood can be collected in a capillary tube, the tip of which can be moved in an increasing spiral.
(3) If a capillary tube is not available, then the edge of a slide or an applicator stick can be used.
(4) If the blood smear is too thick then the blood film will probably wash off as a sheet or flake off during staining.
Handling the unstained smear:
(1) Let the slides air dry. If the air is humid, then place the slide in a desiccator.
(2) Protect the smear from excessive heat, sunlight and insects (flies may try to feed on the blood).
(3) Do not heat or fix the smear since this will prevent laking of the hemoglobin out of erythrocytes.
Removing the hemoglobin from the red blood cells (laking):
(1) It is essential to remove the hemoglobin from within the erythrocytes, usually be washing in a hypotonic solution. If the solution is too hypotonic then the red cells may completely lyse, with loss of parasites.
(2) If the smear is fixed, then hemoglobin will not elute out of the red cells and the smear will be impossible to read.
(3) A dilute saponin solution (0.5% to 1%) can be used to treat the smear very briefly (5 seconds) to help lyse red cells, after which the slide is rinsed.
(4) A dilute stain can combine both laking and staining into a single step.
Staining the slide:
(1) The staining solution must be buffered to the proper pH (7.2).
(2) The appropriate stain should be prepared to the proper concentration.
(3) The slide should be stained for the proper length of time, after which it is quickly rinsed then allowed to air dry.
