Preparing Blood Smears
If you are using venous blood, blood smears should be prepared as soon as possible after
collection (delay can result in changes in parasite morphology and staining
Thick smears consist of a thick layer of dehemoglobinized (lysed) red
blood cells (RBCs). The blood elements (including parasites, if
any) are more concentrated (app. 30×) than in an equal area of a thin
smear. Thus, thick smears allow a more efficient detection of parasites
(increased sensitivity). However, they do not permit an optimal
review of parasite morphology. For example, they are often not adequate
for species identification of malaria parasites: if the thick smear is
positive for malaria parasites, the thin smear should be used for species
Prepare at least 2 smears per
- Place a small drop of blood in
the center of the pre-cleaned, labeled slide.
- Using the corner of another
slide or an applicator stick, spread the drop in a circular pattern until it is the size
of a dime (1.5 cm2).
- A thick smear of proper density
is one which, if placed (wet) over newsprint, allows you to barely read the words.
- Lay the slides
flat and allow the smears to dry thoroughly (protect from dust and insects!).
Insufficiently dried smears (and/or smears that are too thick) can detach
from the slides during staining. The risk is increased in smears
made with anticoagulated blood. At room temperature, drying can
take several hours; 30 minutes is the minimum; in the latter case, handle
the smear very delicately during staining. You can accelerate
the drying by using a fan or hair dryer (use cool setting). Protect
thick smears from hot environments to prevent heat-fixing the smear.
- Do not fix thick
smears with methanol or heat. If there will be a delay in staining
smears, dip the thick smear briefly in water to hemolyse the RBCs.
here to view a
video clip for proper preparation of a thick blood smear (Adobe Flash)
Thin smears consist of blood spread in a layer such that the thickness
decreases progressively toward the feathered edge. In the feathered
edge, the cells should be in a monolayer, not touching one another.
Prepare at least 2 smears per
- Place a small drop of blood on
the pre-cleaned, labeled slide, near its frosted end.
- Bring another slide at a
30-45° angle up to the drop, allowing the drop to spread along the contact line of the 2
- Quickly push the upper
(spreader) slide toward the unfrosted end of the lower slide.
- Make sure that the smears have
a good feathered edge. This is achieved by using the correct amount of blood and
- Allow the
thin smears to dry. (They dry much faster than the thick smears, and
are less subject to detachment because they will be fixed.)
- Fix the smears by dipping them
in absolute methanol.
here to view a
video clip for proper preparation of a thin blood smear (Adobe Flash)
Note: Under field conditions, where slides are scarce, national malaria programs (and CDC staff)
prepare both a thick and a thin smear on the same slide. This works adequately if
one makes sure that of the two smears, only the thin smear is fixed.
Procedures for Detecting Microfilariae
- Capillary (fingerstick) blood
Since microfilariae concentrate in the peripheral capillaries, thick and thin smears
prepared from fingerstick blood are recommended.
Anticoagulated (EDTA) venous blood (1 ml) should be concentrated by one of
the following methods:
- Centrifugation (Knotts
- Prepare 2% formaldehyde (2 ml
of 37% formaldehyde + 98 ml H2O).
9 ml of this 2% formaldehyde with 1 ml of patients venous
blood. Centrifuge at 500 × g for 10 minutes; discard
supernatant. Sediment is composed of WBCs and microfilariae
- Examine as temporary wet
thick and thin smears; allow to dry; dip in absolute methanol
before Giemsa staining to enhance staining of microfilariae.
membrane filter (5 µm pore) in filter holder with syringe attachment.
- Mix 1
ml of venous blood (in EDTA) with 10 ml of 10% Teepol®
610 (Shell Co.); allow to stand for several minutes to allow lysis;
transfer to a 10 ml Luer-Loc®
syringe; attach the filter apparatus.
- Force the solution through the
5 µm pore filter, followed by several syringes of water to wash out the remaining blood,
then 1 or 2 syringes full of air to clear excess fluid.
- Prepare a temporary wet mount
by removing the filter and placing it on a glass slide, adding a drop of stain or dye and
- For permanent preparations,
pass 2 to 3 ml of methanol through the filter while it is still in the holder; remove
filter and dry it on a glass slide; then stain it with Giemsa
stain, horizontally (so that the
filter does not wash off the slide); coverslip filter before examining.
information on making blood smears, call the Division of Parasitic
Diseases at (404) 718-4110.
Eberhard ML, Lammie PJ. Laboratory diagnosis of filariasis. Clin Lab