Preparation of blood smear of Plasmodium spp.
4.1 Main instrument s and equipment
Slides (slides should be clean, free of scratche s and oil stains); Slide boxes; Biomicroscopes (10× eyepieces, 100×oil immersion objectives); Counters.
Note: the used slides should be soaked in detergent solution for 1-2 days, wipe off the old blood films on the slides one by one, rinse them with clean water. After wiping the slides to make them dry and polish, the slides are wrapped in white paper and placed in a plastic bag with every 10 to 20 slides and stored in a dry envi ronment. )
4.2 Main reagents and materials
75% ethanol; Ultra-fine lancet; cotton swab; methanol (analytical purity); Giemsa stai.
solution (solution prepared by lab or commercial reagent); phosphate buffer (pH7.2); xylene (analytical purity); special mirror oil; disposable gloves; sharps container, etc..
4.3 Bloo d c ollection method
The earlobe or the end of the finger in a dults and the big toe or heel in infants can be taken blood from. The operator wears disposable gloves and prepares two clean slides, one as a slide and the second one as a “spreader”. After disinfecting the blood collection site with 75% ethanol cotton swab, waiting for the alcohol to be dry, an Ultra-fine lancet is quickly inserted into the 1-2 mm depth with a gentle pressure using the left thumb, index finger and right middle finger to squeeze out the blood drops. Then hold the middle short side edge of the “spreader” slide with the right thumb and index finger, scrape the blood drops 4-5 μL with the lower left corner of the slide for the thick blood film. Then, use the middle of the same side to scrape the blood drops 1-1.5 μL for the thin blood film.
4.4 Making blood film
4.4.1 Thick blood film
Apply the blood drops in the lower left corner to the center right of the other clean slide which is holding by left hand, and draw a circle from inside to outside to paint a round in a diameter of 0.8-1 cm with the corner of the “spreader” slide as thick blood film. A thickness of 5 to 10 white blood cells in one field under oil immersion objective is suitable.
4.4.2 Thin blood film
Hold the slide by left hand. Push the lower edge of the “spreader” slide flat against the midline of the slide surface to allow the blood to contact with the slide and to run along its edge until about 2 cm width, keeping the “spreader” slide at an angle of 25°-35°, quickly and firmly push the “spreader” slide forward from right to left to form a tongue-like shape as thin blood film. The thickness should be suitable as erythrocytes in contact with each other but no overlapping under a microscopy with 1,000 magnification.(https://www.daowen.com)
4.5 The position of thick and thin blood film on the same slide
The standard blood smear has each thin and thick blood flim in the same slide (Fig. 2-1).
Fig. 2-1 Position of Standard Blood Film
4.6 Blood slide label
After the blood smear is made, it is placed horizontally to laber on the right of slide. Or after it is fully dried, it is labeled with a pencil on the ground glass on one side of the slide or on the root of the tongue-like shape thin blood film.
4.7 Preservation of blood film before staining
After the blood slide is made, put it flat until the thick blood film to dry before staining (pay attention to preventing the blood film from insects licking). The thin blood film must be fixed by methanol before stained with Giemsa stain. The thick blood film must be hemolyzed before stained except for the thick blood film made within one day, which can be stained directly. Blood film placement time should not exceed 48 hours in summer and 72 hours in winter, otherwise thick blood film will be naturally fixed and cannot hemolyze, which affects microscopy examination. Just in case if the blood film cannot be stained in time, the thin film can be first fixed with methanol, and the thick film can be hemolyzed. After drying and wrapping, put it in the dryer and store the dryer in the refrigerator. When using it, put the dryer at room temperature for 1-2 hours and then take it out and stain, following the previously mentioned.
4.7.1 Thin blood film fixation
After completely immersing the thin blood film in methanol solution, take it out immediately, take care to avoid touching the film, and wait for it to dry naturally.
4.7.2 Thick blood film hemolysis
Add a few drops of distilled water or pure water to the dry thick blood film, completely cover it, hemolyze for 3-5 minutes, wait for the blood film to be light gray, and pour the dissolved blood water.