times that can be used for the thin film.
Results
Giemsa stain colors the components of blood as follows: RBCs, pale red; nuclei of WBCs, purple with pale purple cytoplasm; eosinophilic granules, bright purple-red; and neutrophilic granules, deep pink-purple. If malaria parasites are present, the cytoplasm stains blue and the nuclear material stains red to purple-red. Schüffner’s dots and other inclusions in the RBCs will stain red (Fig. 7.4). However, as seen in Fig. 7.4, there can be tremendous variation in color. Nuclear and cytoplasmic staining characteristics of the other blood parasites such as Babesia spp., trypanosomes, and leishmaniae are like those of the malaria parasites (Fig. 7.5 to 7.8). While the sheath of microfilariae may not always stain with Giemsa, the nuclei within the microfilaria itself stain blue to purple. The sheath of Brugia malayi will tend to stain pink, while the sheath of Wuchereria bancrofti may not stain with Giemsa (Fig. 7.9).
Figure 7.4 Plasmodium spp. seen in stained thin blood films. (Left column from top to bottom) Plasmodium vivax: (1) developing ring (note the enlarged RBC, lack of Schüffner’s dots [EDTA blood], and ameboid rings); (2) developing trophozoite (note the enlarged RBC and Schüffner’s dots); (3) mature schizont with approximately 16 merozoites; (4) thick blood film showing developing ring forms; (5) normal WBCs (including polymorphonuclear leukocytes [PMNs], eosinophil, basophil, monocyte, and lymphocyte). (Second column from top to bottom) Plasmodium ovale: (1) young ring form (note the nonameboid ring and Schüffner’s dots that appear earlier than in rings of P. vivax); (2) developing trophozoites (note Schüffner’s dots and fimbriated edges of the infected RBCs); (3) maturing schizont containing merozoites and malarial pigment; (4) thick blood film showing developing schizont; (5) normal PMNs. (Third column from top to bottom) Plasmodium malariae: (1) young ring (note the normal-sized RBC and no stippling); (2) developing trophozoite (note the normal-sized RBC and “band form” configuration of the trophozoite); (3) mature schizont (note the size of the RBC) containing approximately eight merozoites and brownish malarial pigment; (4) thick blood film with mature schizonts containing developing merozoites; (5) normal WBCs (including PMN, eosinophil, basophil, monocyte and lymphocyte). (Last column from top to bottom) Plasmodium falciparum: (1) typical ring forms (note appliqué form at the side of the RBC and multiple rings/cell); (2) typical ring forms (note the ring appearing to be partially outside the RBC—typical for P. falciparum); (3) crescent-shaped gametocyte; (4) thick blood film showing many ring forms; (5) typical PMNs. doi:10.1128/9781555819002.ch7.f4
Figure 7.5 Trypomastigotes. (Left) Trypanosoma brucei gambiense. (Right) Trypanosoma cruzi (note the undulating membrane on both trypomastigotes and the larger kinetoplast in T. cruzi). doi:10.1128/9781555819002.ch7.f5
Figure 7.6 Babesia sp. (Top) Note some of the ring forms are outside the RBCs (box, rare to see in Plasmodium infections). (Bottom) Note the numerous ring forms per RBC; also note the Maltese cross ring formation in the lowermost RBC (arrow). doi:10.1128/9781555819002.ch7.f6
Figure 7.7 Leishmania donovani in an impression smear. Note the numerous small amastigotes containing a nucleus and bar-shaped primitive flagellum. doi:10.1128/9781555819002.ch7.f7
Figure 7.8 Plasmodium falciparum. (Upper row) Note the ring forms (multiple rings per RBC and presence of the “headphone” ring configuration). The photograph at the far right is a good mimic of Babesia organisms, but the rings are not quite as pleomorphic as in Babesia spp. (Lower row) Two examples of P. falciparum gametocytes, two of which appear to be outside the RBC. However, although the RBC membrane is not visible, the gametocyte is intracellular. The image on the far right is an ookinete from the mosquito cycle; this can occur within the EDTA blood specimen if the blood cools and is left standing with the cap removed. In the cooled, aerated blood, the parasites begin the cycle that normally occurs within the mosquito. This artifact can easily be confused with the crescent-shaped gametocyte. doi:10.1128/9781555819002.ch7.f8
Figure 7.9 (Upper) Brugia malayi microfilaria on a blood film stained with Giemsa stain. Note the presence of the sheath (stains pink with Giemsa stain). (Lower) Brugia malayi microfilaria on a thick blood film stained with Giemsa stain (note some of the individual nuclei are visible). (Images courtesy of the CDC Public Health Image Library; lower, photograph by Dr. Mae Melvin.) doi:10.1128/9781555819002.ch7.f9
Wright’s stain is available commercially in liquid form, ready to use, and also as a powder which must be dissolved in anhydrous, acetone-free methyl alcohol before use. Directions for preparing the stain follow.
Reagent
Wright’s Stain
1. Grind 0.9 g of Wright’s stain powder with 10 to 15 ml of methanol (anhydrous, acetone free) in a clean mortar. Gradually add methanol while grinding. As the dye is dissolved in the methanol, pour that solution off and add more methanol to the mortar. Repeat this process until the entire 500 ml of methanol has been used.
2. Store the stain in a tightly stoppered glass bottle at room temperature. Shake the bottle several times daily for at least 5 days.
3. Allow the precipitate to settle, and pour off some of the supernatant fluid into dropping bottles for use. If the stock solution has been disturbed, the supernatant fluid can be filtered
