dried (do not apply heat), it may be stained. The necessity for fixation before staining will depend on the stain selected. Since Giemsa is an aqueous stain, laking of the RBCs occurs during the staining process. However, when Wright’s stain is used, a fixing agent is incorporated into the stain, so that laking of the blood films must occur prior to staining. If using one of the rapid blood stains, read the package insert to confirm laking and/or fixation requirements for the thin and thick blood films.
Figure 7.1 Method for preparation of thin blood film. (A) Position of spreader slide; (B) well-prepared thin film. Arrows indicate the area of the slide (feather edge) used to observe accurate cell morphology. (Illustration by Sharon Belkin.) doi:10.1128/9781555819002.ch7.f1
The instrument-prepared monolayer method or coverslip methods generally do not provide the best morphology for malarial parasites within the RBCs. However, the selection of a slide preparation method can be dictated by personal preference, since a malarial infection can be diagnosed from either type of slide. Potential problems with the preparation and staining of thin blood films can be seen in Table 7.4.
Combination Thick and Thin Blood Films (on the Same Slide)
In some instances (field surveys), it is helpful to prepare slides with both a thick and a thin film on the same slide. With this type of preparation, remember the following.
1. Sufficient time must be allowed for the thick portion of the smear to dry before staining.
2. If Giemsa stain is used, the thin film only must be fixed in absolute methanol before staining.
Combination Thick and Thin Blood Films (Can Be Stained as Either)
To prepare a slide containing blood that can be stained for either a thick or thin blood film, the following method was developed (Fig. 7.2). The specimen usually consists of fresh whole blood collected by finger stick or whole blood containing EDTA collected by venipuncture less than 1 h earlier.
Figure 7.2 Method of thick-thin combination blood film preparation. (a) Position of the drop of EDTA-containing blood. (b) Position of the applicator stick in contact with blood and glass slide. (c) Rotation of the applicator stick. (d) Completed thick-thin combination blood film prior to staining. (Illustration by Sharon Belkin.) Reprinted from reference 9. doi:10.1128/9781555819002.ch7.f2
Visually, the smear should consist of alternating thick and thin portions throughout the length of the glass slide. One should be able to barely read newsprint through the wet or dry film. Also, the film itself should not have any clear areas or smudges, indicating that grease or fingerprints were on the glass.
Detailed Procedure
1. Wear gloves when performing this procedure or preparing any blood films.
2. The procedure depends on the source of the specimen.
A. Blood from a finger puncture is not recommended, since the procedure does not lend itself to “stirring” to prevent fibrin strands.
B. For blood from venipuncture, place a clean 1- by 3-in. glass microscope slide on a horizontal surface. Place a drop (30 to 40 µl) of blood onto one end of the slide about 0.5 in. from the end. Using an applicator stick lying across the glass slide and keeping the applicator in contact with the blood and glass, rotate (do not “roll”) the stick in a circular motion while moving the stick down the glass slide to the opposite end. The appearance of the blood smear should be alternate thick and thin areas of blood that cover the entire slide. Immediately place the film over some small print and be sure that the print is just barely readable.
3. Allow the film to air dry horizontally and protected from dust for at least 30 min to 1 h. Do not attempt to speed the drying process by applying any type of heat, because the heat fixes the RBCs and they subsequently will not lyse (lake) in the staining process.
4. This slide can be stained as either a thick or thin blood film.
5. Label the slide appropriately.
6. If staining with Giemsa is delayed for more than 3 days or if the film is to be stained with Wright’s stain, lyse the RBCs in the thick film by placing the slide in buffered water (pH 7.0 to 7.2) for 10 min, remove it from the water, and place it in a vertical position to air dry. Rapid stains are also acceptable.
Procedure Limitations
1. If the smears are prepared from anticoagulated blood that is more than 1 h old, the morphology of the parasites may not be typical and the film may wash off the slide during the staining procedure. If a tube of blood containing EDTA cools to room temperature and the cap has been removed, several parasite changes can occur. The parasites within the RBCs will respond as if they were now inside the mosquito after being taken in with a blood meal. The morphology of these changes in the life cycle and within the RBCs can cause confusion when examining blood films prepared from this blood (3–8).
A. Stippling (Schüffner’s dots) may not be visible.
B. The male gametocyte (if present) may exflagellate and may resemble spirochetes (Fig. 7.3).
Figure 7.3 (Left) Photograph of exflagellation of the malarial microgametocyte. This may occur when anticoagulated blood is left standing at room temperature for some time prior to smear preparation. The life cycle of the parasite continues in the tube of blood as it would if the parasite had been ingested by the mosquito during a blood meal. (Right) Borrelia in blood. Note that the Plasmodium microgametes can resemble these organisms, particularly if they appear free in the background of the smear. doi:10.1128/9781555819002.ch7.f3
C. The ookinetes of Plasmodium species other than P. falciparum may develop as if they were in the mosquito and may mimic the crescent-shaped gametocytes of P. falciparum.
2. Identification to species, particularly between P. ovale and P. vivax and between the ring forms of P. falciparum and Babesia spp., may be impossible without examining one of the slides stained as a thin blood film. Also, Trypanosoma cruzi trypomastigotes are frequently distorted in thick films.
3. Excess stain deposition on the film may be confusing and make the detection of organisms difficult.
4. If the EDTA blood is 4 to 6 h old prior to blood films preparation, parasites are actually lost from the specimen; the parasitemia may be too low or represent a false negative.
Fixation of Thin Blood Films: Acetone Dip for Thick Blood Films
Thin-Film Fixation. Thin blood films must be completely dry before being fixed in absolute methanol (Giemsa staining). Drying times will
