Name: _____________________ Score: ________

Year & Section: __________ Date: ________

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CLINICAL PARASITOLOGY LABORATORY

ACTIVITY 6: BLOOD SMEAR PREPARATION & STAINING FOR BLOOD PARASITES

Principle: Blood smear preparation and staining are procedures used for the recovery and
identification of Plasmodium, trypanosomes, microfilaria, Babesia spp and Leishmania
donovani. Ideally, blood films should be prepared from fresh blood or EDTA-prepared blood
no longer than six hours following specimen collection. In blood films made especially
for the diagnosis of malarial infection, the use of anticoagulants is not recommended
because they may cause distortion of the parasites.

KIND OF SMEAR ADVANTAGES DISADVANTAGES

THIN SMEAR  Little distortion of  Many fields (200-300 OIO
parasite fields) must be examined to
detect parasites when few
before reporting a negative
smear
THICK SMEAR  Often distort parasite  Higher chance of parasite
morphology detection

Giemsa stain is a reliable and preferred stain for blood parasites; however, Romanowsky-
type stains, including Field’s stain, are equally useful for staining blood films.
Wright’s staining is not recommended.

If filariasis is suspected, both diurnal and nocturnal blood samples are drawn to account
for the periodicity of microfilariae. In cases of suspected malaria, if the first
specimens are negative, the patient’s blood should be retested every 6 to 8 hours for at
least three days to account for the periodicity of schizogony.

Materials and Equipment:

1. Giemsa stain (1:20 and 1:50) 7. microscope
2. Absolute methyl alcohol 8. lens paper and oil immersion oil
3. Syringes and lancets 9. masks and gloves
4. EDTA 10. alcohol and cotton
5. Microscope slides 11. test tube racks
6. Staining rack 12. Pasteur pipettes

Procedure:

A. Procedure for Thin Smear Preparation

1. Touch a clean, grease-free slide to a small drop of blood so that the drop is near one end
of the slide.
2. Hold a second spreader slide on edge at 30-degree angle on the slide, which is also holding
the specimen, and draw back into the drop, allowing it to spread along the edge of the
spreader slide.
3. Smoothly and rapidly push the spreader slide forward so that the blood spreads out and
trails in a flat sheet. The amount of blood should be small enough that it is all spread
before the spreader reaches the end of the specimen slide.
4. Allow to air dry and stain as described in the Giemsa staining procedure.
5. Examine the thin film under 100X.
 B. Procedure for Thick Smear Preparation

1. Touch the center clean, grease-free slide to a large drop of blood.

2. Using one corner of a slide or an applicator stick, spread the blood evenly in a circular
film about the size of nickel (1.8 to 2.0 cm in diameter). A thick smear of proper density
is one which, if placed (wet) over newsprint, allows you to barely read the words.
3. Allow to air dry overnight and lake the blood by placing the slide in Giemsa-buffered water
for 10 minutes (laking may be omitted if a 1:50 Giemsa stain is used).Laking causes RBC to
lyse, which causes hemoglobin’s red color to disappear, leaving the blood spot translucent.
4. Stain the thick smear as described in the Giemsa staining procedure.
5. Examine the thick film under 100X.

C. Procedure for Giemsa Staining

THIN SMEAR THICK SMEAR

1. Immerse smear briefly in absolute methyl alcohol (two 1. Flood laked smears in a 1:50
dips). Let the smear air dry. dilution of stock Giemsa for
2. Flood the slide in working Giemsa stain (made by diluting 50 minutes.
stock Giemsa 1:100 with pH 7.0 working buffer) for two 2. Wash by dipping into buffer
hours. The time may be reduced to 45 minutes if a 1:50 for three to five minutes.
dilution is used or 20 minutes if a 1:20 dilution is used. Excessive washing decolorizes
3. Gently rinse the smear by dipping it twice into buffer. the film.
4. Drain and air dry. Do not blot. 3. Air dry.

D. Microscopic Examination

1. Malaria, Babesia spp., trypanosomes and leishmania cytoplasm stains blue, with nuclear
material. Schuffner’s dots stain red. A microfilaria sheath may not stain but the nuclei in
the organism stain blue to purple.
2. RBCs stain pale red; WBCs, purple; eosinophilic granules, bright purple red; and
neutrophilic granules, deep pink-purple
3. Any detected parasites are reported by their scientific name.

Results and Report:

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Thin Film (100x) Thick Film (100X)

Prepared by:
LILY AILEEN B. ALINAO,RMT,MT(ASCP i),MSMT
COURSEWORK FOR PARASITOLOGY LABORATORY: Read and understand the worksheet giving focus on
the principle and the procedures then accomplish as indicated and submit your outputs on
or before 11:59, 20 March 2020

Task 1: Illustrate the procedures. You may use the video in the link provided below.
Follow the prescribed format for reports as indicated in the class guidelines. For the
results (drawing) portion of the worksheet, draw and label what you see in the
microscopic examination shown in this video. This drawing should come at the end of your
procedure illustrations.

https://www.bing.com/videos/search?
q=preparation+of+thick+and+thin+smears+for+blood+parasites&&view=detail&mid=C768D7956EFA76503C51C768D79
56EFA76503C51&&FORM=VRDGAR&ru=%2Fvideos%2Fsearch%3Fq%3Dpreparation%2Bof%2Bthick%2Band%2Bthin
%2Bsmears%2Bfor%2Bblood%2Bparasites%26FORM%3DHDRSC3 (NOTE: The spelling of immersion in this video is
wrong. It is ‘immersion’ and NOT ‘emmersion’.)

Task 2: Draw the appearance of the Hemoflagellates, Plasmodium spp, Babesia spp in a
blood smear under the oil immersion objective. You may refer to internet sources. Make
sure your drawings are labeled.

Task 3: Draw the appearance of the Amoebas & Flagellates in a direct fecal smear and stained
smear at 40X. You may refer to internet sources. Make sure your drawings are labeled.