Veterinary Parasites Laboratory Procedures
Collection of Samples
- Collect fresh feces (rectally for large animals), as free of debris as
- Store in refrigerator in clean, dry container with airtight lid
- If diarrheic feces, examine as soon as possible after collection and do
- Blood can be collected for a smear using a plain syringe and needle, or
an EDTA tube
- Avoid unnecessary agitation to reduce hemolysis of sample
- If blood is to be sent to a laboratory for serology testing, ask the lab
what samples are required
Collection of Intestinal Parasites
1. Direct Fecal Smear
Very good technique for identification of motile parasites commonly used
for diarrheic or mucoid fecal samples. Use feces that is as fresh as possible.
- Place a drop of saline solution on a clean glass slide
- Collect a rectal sample of feces using gloved finger (or touch gloved
finger or toothpick to fresh sample already collected)
- Smear very thinly onto clean slide and cover with coverslip
- May use a stain such as Ziehl-Neelsen, carbol-fuchsin, or Giemsa
(optional-not used often in private veterinary practices)
- Use less light on the microscope for examination due to clear color of
most unstained parasites
Negative results are inconclusive, perform a concentration technique such
as flotation on all samples that are negative direct smear.
Make sure that the sample on the slide is very thin (you should be able to
read print through the sample), if the fecal layer is too thick, it becomes
difficult to identify movement of parasites.
2. Flotation Fluids
Since parasite eggs will sink in water, salt or sugar solutions are used to
concentrate and separate eggs from most fecal debris.
The most commonly used flotation fluids are magnesium sulfate (Epsom
salts), sugar, sodium nitrate, and zinc sulfate. A specific gravity from 1.2
to 1.3 is best for floating most eggs.
Each solution has advantages and disadvantages. Magnesium sulfate is
inexpensive, but if slides have to sit a while before they are read, the fluid
will crystallize and eggs may be distorted. Sugar solution allows slides to be
kept longer before reading, but is sticky and may be more expensive.
Sodium nitrate can be purchased already in solution and therefore saves time
used for mixing, but it is relatively expensive. Zinc sulfate is the best
solution to use for the detection of Giardia cysts because the cysts do
not become distorted as quickly with it.
To prepare flotation fluids:
- For a magnesium sulfate solution of sp.gr. 1.2:
- 8 quarts water
- 10 pounds Epsom salts
- For a sugar solution of sp.gr. 1.27:
- 454 g (one pound) granulated sugar
- 355 ml water (1 ½ cups)
- 6 ml 40% formaldehyde (1 ¼ tsp.)
- Dissolve in a double boiler over heat, then add formaldehyde
- For zinc sulfate solution of sp.gr. 1.18:
- 336 g ZnSO 4 · 7H 2
- 1000 ml distilled water
3. Centrifugation with Magnesium Sulfate Flotation Fluid
Using a centrifuge reduces the number of eggs that rise slowly to the
surface of a flotation setup. Thus reducing the number of false negative fecal
To be used for concentrating protozoan cysts, nematode, cestode and some
arthropod eggs. (Not usually used for an egg which has an operculum)
- Centrifuge tube (12 ml or 15 ml) which has the top ground flat
- Applicator sticks
- Small paper cups
- Floatation fluid (sugar or MgSO 4 solution)
- Square coverslips (preferably plastic)
- In a centrifuge tube, mix a small amount of feces (about 1 tsp.) with
just enough water to soften and mix it well.
- Add flotation solution to achieve a slight reverse meniscus. Place a
coverslip on top.
- Centrifuge 3-5 minutes at speed 3.
- Remove the coverslip, place it on a glass slide and examine
- Wash the non-disposable equipment used
Note: for herbivores or other feces with a lot of debris (e.g. cat feces
with adherent litter), strain feces by mixing with a little water in a paper
cup, add MgSO4, mix, pour through gauze into another cup or
centrifuge tube, then proceed as above.
4. Modified Wisconsin Procedure
(for cattle, horses, dogs, cats, and swine)
- Used to quantify the amount of parasites in an individual animal.
The modified Wisconsin procedure for egg counts by a flotation method is
used to quantify the amount of parasites in an individual animal (cattle,
horses, dogs, cats, swine)
- Weigh out a 2 g fecal sample (5 g for cows) in a paper cup
- Place 10-cc water in the paper cup with the fecal material.
- Stir very well with a spatula and mash the material until it is
completely broken apart
- Pour the mixture through gauze or strainer ( while it is well mixed
) into another cup, stirring the material in the strainer while pouring.
- Press the material remaining in the strainer with the spatula until
- Add a small amount of water to the paper cup just emptied and rinse into
a mixture the material clinging to the sides and bottom, and then pour this
mixture through the material in the strainer, stirring the material in the
strainer while pouring.
- Press the material in the strainer until dry again, then discard.
- Stir the material in the cup that was under the strainer and
immediately pour the contents of the cup into one 15-ml tube. If the
tube is not too full, squirt water down the sides of the cup in sufficient
amounts to remove the material clinging to it and finish filling up the
- Centrifuge the tube at 1500 rpm for 10 minutes (not including the time
required for acceleration and deceleration)
- Decant the tube, being careful tot to pour off the fine material at the
top of the sediment
- Fill the tube ½ full of MgSO4, (sp.gr. 1.2-1.25) and mix
the sediment ant the MgSO4 solution with an applicator stick,
being careful to scrape the sides and bottom of the tube to insure the
removal and mixing of all material.
- Finish filling the tube with flotation fluid
- With a medicine dropper, add MgSO4 to the tube until it is
full enough so that a 22-mm square coverglass can be placed on the top.
(there should neither be an air bubble under the cover slip, nor should the
material overflow so that it runs down the side of the tube)
- Centrifuge at 1500 rpm for 10 minutes (not including time required for
acceleration and deceleration)
- Remove the coverglass by lifting straight upward and place it on a glass
slide. If properly done, there should be a good thickness of material under
- Count the all of the worm eggs under the entire coverglass using a low
power (10x) objective.
- Record the results very carefully giving (a) the specimen number, (b)
the date of collection, (c) the number of worm eggs of each type seen
The count is number of eggs per 2 (or 5) grams of feces.
5. Significance of Eggs Per Gram Counts
- The number of parasite eggs per gram feces is influenced by:
- fecal consistency – the drier the feces, the more concentrated the
parasite eggs within the feces
- total amount of feces produced
- time of day feces collected
- Significant numbers of parasite eggs vary between host species and
- For nematode parasitism:
- 500 EPG = mild infection
- 800-1000 EPG = moderate infection
- 1500-2000 = severe infection
- 300-600 EPG requires treatment
- Lambs – depends upon parasite species, but generally:
- 1000 = moderate and should be treated
- 2000-6000 = severe
- For trematode parasitism (i.e. Fasciola hepatica )
- Cattle 100-200 EPG is considered pathogenic
- Sheep 300-600 EPG is considered pathogenic
6. Fecal Culture
- Baermann technique for fecal sedimentation
- Since strongyle eggs look very similar, identification of the species of
strongylid parasite found requires a culture procedure and identification of
the larvae found.
The following method is a technique for culturing eggs of nematodes of
Strongylida to infective L 3 larvae and then recovering
these larvae for identification. It involves a culture phase, which
minimizes the time for development to the L 3 and one of the many
modifications of the Baermann technique for recovery of the larvae. The
Baermann technique takes advantage of the fact that the L 3 will
migrate out of a fecal mass into a fluid medium but being unable to swim
against gravity will then settle to the bottom of the medium container.
- Examination gloves and specimen containers
- Lab balance
- Dried sterile sphagnum moss ("Nodampoff" sphagnum moss is a
pre-sterilized seed-starting medium available through lawn and garden
- 250 ml glass beakers
- wooden applicator sticks
- 100 x 25 mm disposable petri dishes with covers
- 150 x 25 disposable petri dishes with covers
- cheesecloth or gauze
- rubber bands
- Lugol’s iodine solution
- Centrifuge with centrifuge tubes
- Pasteur pipettes
- Microscope with microslides and coverslips
- Collect at least 10 gm feces rectally from each animal to be
checked using clean gloves and clean specimen containers
(this is important to prevent contamination with free-living nonparasitic
- If the specimen is from cattle, swine or dog , weigh out
a minimum of 10-g sphagnum moss for each specimen to be examined. Place
the moss in a suitable container and mix with sufficient warm tap water to
produce a thick "soup". Allow the moss to become thoroughly saturated and
then squeeze out all excess water.
- Weigh out 5 g feces into a 250 ml beaker and thoroughly break up the
sample with applicator sticks
- If the specimen is from cattle swine or dog , add 15 g
saturated sphagnum moss to the beaker and mix thoroughly. Make sure that
none of the feces is left adhering to the sides of the beaker. Horse,
sheep and goat feces do not require the addition of moss
- Place the feces/moss mixture in a 100 x 25-mm petri dish and
compress lightly . The particles of the material should be in uniform
contact with each other but remain "fluffy" . Cover and incubate at
room temperature for 14 days. (If an incubator is available, horse
feces may be incubated for 7 days t 30 degrees C.) Do not be alarmed if
mold forms on sample during incubation
- Following incubation, remove the cover of the dish and place 2 layers
of cheesecloth or gauze over the top of the dish. Stretch the cloth and
secure with a rubber band. Trim away excess cloth and replace cover.
Invert the dish and tap bottom smartly to dislodge the sample onto the
- Place 2 wooden applicator sticks in a 150 x 25-mm disposable petri
dish and add 100-ml warm tap water. Keeping the 100-mm petri dish
inverted, remove its cover and place it cloth side down onto the
applicator sticks in the 150-mm petri dish. Gently press on the
bottom of the 100-mm dish until a small amount of air escapes from under
the rim and then release. Cover and let stand (Baermannize) at room
temperature for 18 to 24 hours.
- Following Baermannization, remove the 100-mm dish and the wooden
applicator sticks and discard. Add 0.3 ml of Lugol’s iodine solution to
the liquid in the 150-mm dish and agitate gently. Pour the mixture into a
centrifuge tube and centrifuge at 1000 to 1500 rpm for 10 minutes. (If a
centrifuge is not available, the liquid can be poured into an Imhoff
settling cone or similar cone-shaped container and allowed to stand for 10
- Transfer a drop of sediment from the bottom of one of the centrifuge
tubes with a Pasteur pipette to a microslide. Add a coverslip and identify
the L3 larvae present under the Microscope.
- Descriptions of the various larvae are provided in Georgi, Parasitology for
Veterinarians , 6 th ed. (1995), Dunn, Veterinary
Helminthology , 2 nd ed. (1978), or Levine, Nematode
Parasites of Domestic Animals and Man , (1980)
Collection of Blood Parasites
1. Knott's Method
The modified Knott’s method is used for the concentration and identification
- Add 1-ml blood to 10 ml of 2% formalin and mix.
- Centrifuge for 5 minutes at 1000 to 1500 rpm.
- Pour off supernatant fluid. Note: The tube may be inverted on a paper
towel to allow all the liquid to drain.
- Mix sediment with equal volume of 1:1000 aqueous methylene blue.
- Examine as wet mount.
Collection of Muscle Parasites
1. Squash Preparation
Used to identify Trichinella spp cysts within muscle:
- Collect a small amount of fresh muscle
- Choose tissue from either the masseter or diaphragm as these two sites
are most likely to yield positive results
- Place a small amount of tissue on a glass slide
- Cover with a second glass slide
- Press the two slides together using thumb and index finger
- While still holding slides together, tape both ends of the slides together
(scotch tape works well for this)
- Trim away tissue not contained by the two slides
- Examine with a microscope using low power to identify larval cysts within
Copyright © 1997
all rights reserved.
Published by RM Corwin and Julie Nahm,
University of Missouri College of Veterinary Medicine.