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PRACTICAL AND ACCURATE DIAGNOSIS OF CLINICALLY
IMPORTANT PARASITES
Michael W. Dryden, DVM, PhDProfessor of Veterinary Parasitology
Department of Diagnostic Medicine/PathobiologyCollege of Veterinary Medicine
Kansas State UniversityManhattan KS 66506
Techniques:
1. Direct smearEssentially non-diagnostic for most intestinal parasites
May be useful for the diagnosis of protozoal parasites that have motile trophozoite stages
Limitation: small sample size
Techniques 2. Fecal Flotation:• Useful for concentration of parasite material in feces• Based on differential specific gravity of parasite
eggs/cysts, fecal debris and flotation solution. • Flotation solution must have a higher specific
gravity than parasite egg or oocysts.• Specific gravity refers to weight of object compared to
equal volume of water.• Specific gravity of water is 1.000 g/ml and most parasite
eggs are 1.1 - 1.2 g/ml– Flotation solutions should be > 1.24 g/ml
Specific Gravity of Common Parasite Eggs - dogs and cats
• Physaloptera sp. 1.2376
• Taenia 1.2251
• Trichuris vulpis 1.1453
• Toxocara cati 1.1005
• Toxocara canis 1.0900
• Ancylostoma sp. 1.0559
Flotation Solutions• Sugar: 454g / 355ml water ≈1.27 sp. gr.
(add 2ml 37% formaldehyde)
• Sodium Chloride: 400g / 1000ml water ≈ 1.2 sp. gr.
• Magnesium sulfate: 400g / 1000ml water ≈ 1.2 sp. gr.
• Zinc sulfate: 371g / 1000ml water ≈ 1.18 sp. gr.
• Sodium nitrate: 400g / 1000ml water ≈ 1.18 sp. gr.
Fecal Techniques
• Passive – Standing fecal– Commercial kit
• Centrifugation – swinging head– fixed head
Simple vs. Centrifugation
Simple: Standing Fecal Exam
• Mix feces with 3 to 5 ml flotation solution
• Pour into shell vial or centrifuge tube• Place in a supporting rack• Fill with solution and place coverslip• Remove and examine for parasite eggs
or oocysts 5 to 20 minutes later.
Fecal Exam Preparation
1. Put two to five grams (approximate) feces into a small cup.
2. Add a few ml of flotation solution to break up the fecal mass.
Fecal Exam Preparation
3. Strain contents through a tea strainer and then pour into a 12 – 15ml centrifuge tube.
Fecal Exam Preparation
4. Add flotation solution forming a slight positive meniscus.
Fecal Exam Preparation -Standing Fecal
5. Leave undisturbed in a test tube rack for 15 minutes.
6. Then lift coverslipdirectly off tube and put onto microscope slide.
7.Examine entire coverslipat 10X. Use 40X to identify.
8. Record results
• Ovassaay
Centrifugation Technique - Swinging Head• 2 to 5-gm sample placed into cup or beaker and 10 ml
flotation solution added & solution was mixed thoroughly • Mixture poured through a tea strainer or cheese cloth into
a 15-ml centrifuge tube• Four to 5 ml of flotation solution (sp. gr. > 1.2) added to fill
the tube forming a slight slight positive meniscus. • A square 18 mm coverslip placed on tube• Tube placed in centrifuge and balanced• Centrifuge in a swinging bucket centrifuge for 5 minutes at
280xG; then let stand for 10 additional minutes. • Coverslip carefully removed, placed on a microscope slide
and examined systematically under 100x magnification.
Centrifuge
Centrifugation Technique
• Centrifuge in a swinging bucket centrifuge for 5 minutes at 280xG; then let stand for 10 additional minutes.
Fixed Head Centrifuge Technique• Mix feces with 3 to 5 ml flotation solution in a beaker • Pour into centrifuge tube straining through a tea
strainer• Add flotation solution, fill tube to within to 1cm of top. • Place in fixed head centrifuge and centrifuge at
280xg for 5 min.• Remove tube and place in a rack, then fill with
flotation solution forming slight positive meniscus and cover with cover slip.
• Let sit for 10 minutes• Remove and examine for parasite eggs or oocysts
Fecal Techniques - Study
• Centrifugation– swinging head (280g for 5 min.)
• Commercial kit (passive) fecal exam– Ovassay (15 min.)
• 1.1 vs. 1.2 specific gravity solutions
Av.
egg
s re
cove
red
Comparison of Fecal TechniquesOvassay - Centrifugation - Hooks
ovassay centrifuge
average of 3 - 2 gram samples
zinc 1.1 zinc 1.2 sugar 1.20
100
200
Av.
egg
s re
cove
red
Comparison of Fecal TechniquesOvassay - Centrifugation - Rounds
0.3
46.7
ovassay
1
147.7158.3
centrifuge
average of 3 - 2 gram samples
zinc 1.1 zinc 1.2 sugar 1.20
10
20
Av.
egg
s re
cove
red
Comparison of Fecal TechniquesOvassay - Centrifugation - Whips
2.7
ovassay
7.3
11.6
centrifuge
0.00.0 NA
average of 3 - 2 gram samples
Specific Gravity Hydrometers
Specific Gravity Hydrometers• Fisher (www.fisher.com) 1-800-776-7700
Hydrometer 1.2 – 1.425 sp. gr.Cat# 11-522B $18.56
• Cole – Parmer (www.coleparmer.com) 1-800-323-4340General Purpose Hydrometer 1.00 – 1.225 sp. gr.
Cat# P-08291-10 $18.80
• Ward’s Biology (www.wardsci.com) 1-800-962-2660Specific Gravity Hydrometer 1.00 – 2.00 sp. gr.
Cat# 15 W 0887 $10.50
Comparison of Fecal Flotation Techniques
• Sugar vs. salt• Time to examination• Centrifugation vs. standing fecal• Centrifugation vs. standing fecal vs.
commercial kit• Swinging vs. fixed head centrifuge• Dip method
Centrifuge Float 5min Float 10min Float 15min Float 20min0
70
140
210
280
350
Av.
egg
s re
cove
red
Comparison of Fecal TechniquesSugar 5 Min.Centrifugation vs. Float
293
22 17.3 20 21
Toxocara
Specific Gravity = 1.2Results are mean of 3 - 2 gram samples
Centrifuge Float 5min Float 10min Float 15min Float 20min0
60
120
180
240
300
Av.
egg
s re
cove
red
Comparison of Flotation TechniquesNaNo3 5 Min.Centrifugation vs. Float
262.7
46.3 5374.3 76.7
Toxocara
Results are mean of 3 - 2 gram samples Specific Gravity = 1.2
Centrifuge Float 5min Float 10min Float 15min Float 20min0
10
20
30
40
50
60
70
Av.
egg
s re
cove
red
Comparison of Flotation TechniquesSugar 5 Min.Centrifugation vs. Float
52.3
1.7 2.7 1.35.3
Ancylostoma
9
0.7 0.7 0.3 1.7
Trichuris
a a a
Results are mean of 3 - 2 gram samples Specific Gravity = 1.2 a = 2 of 3 samples negative for Trichuris
Centrifuge Float 5min Float 10min Float 15min Float 20min0
10
20
30
40
Av.
egg
s re
cove
red
Comparison of Flotation TechniquesNaNo3 5 Min.Centrifugation vs. Float
24
7.3
13.7
21.719
Ancylostoma
2.30.7 1 1.7
2
Trichuris
Results are mean of 3 - 2 gram samples Specific Gravity = 1.2a = 2 of 3 samples negative for Trichuris
aa
Comparison of Fecal Flotation Techniques
• Sugar vs. salt• Time to examination• Centrifugation vs. standing fecal• Centrifugation vs. standing fecal vs.
commercial kit• Swinging vs. fixed head centrifuge• Dip method
Fixed vs. Swinging Head Centrifuge• Mix feces with 3 to 5 ml flotation solution in a beaker • Pour into centrifuge tube straining through a tea
strainer• Add flotation solution, fill tube to within to 1cm of top. • Place in fixed head centrifuge and centrifuge at
280xg for 5 min.• Remove tube and place in a rack, then fill with
flotation solution forming slight positive meniscus and cover with cover slip.
• Let sit for 10 minutes• Remove and examine for parasite eggs or oocysts
Swinging Fixed Swinging Fixed0
100
200
300
Av.
egg
s re
cove
red
Comparison of Fecal TechniquesFixed vs Swinging Head Centrifuges
49.238.4 35.2 36.2
Toxocara
210
76
137.8
111.8
Ancylostoma
Results are mean of 3 - 2 gram samples Specific Gravity = 1.2
Sugar Sodium Nitrate
5 min. Cent. - Tubes removed & let stand 10 min.
Comparison of Fecal Flotation Techniques
• Sugar vs. salt• Time to examination• Centrifugation vs. standing fecal• Centrifugation vs. standing fecal vs.
commercial kit• Swinging vs. fixed head centrifuge• Dip method
10 min. stand 3 rod dips 10 min. stand 3 rod dips0
100
200
300
Av.
egg
s re
cove
red
Comparison of Flotation Techniques5 Min. Cent. 280xG - Fixed Head
40
1
36
1.5
Toxocara
95
2.5
200
2
Ancylostoma
Results are mean of 3 - 2 gram samples Specific Gravity = 1.2
Sugar Sodium Nitrate
Summary
• Sp. gravity < 1.2 will not recover Toxocara and Trichuris eggs
• Centrifugation is more efficient than Fecalizer, Ovassay or standing fecal.
• After centrifugation leaving coverslips on for an additional 10 minutes increased egg recovery
• Centrifugation in sugar recovered more eggs than sodium nitrate
Summary
• Sodium nitrate recovered more eggs than sugar in the standing fecal flotation.
• Ovassay failed to recover Trichuris in 5 of 12 samples even after 15 minutes.
Centrifuges - Used
• IEC Clinical Centrifuge Model CL, with 6 – place swinging bucket rotor. $250 - $950 – (Older model: not in production, excellent
general purpose centrifuge: make sure it has rotor and shields)
– search under Clinical centrifuge or Table/Bench top centrifuge or IEC clinical centrifuge model CL