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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.


3. Strain contents through a tea strainer and then pour into a 12 – 15ml centrifuge tube.


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


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


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


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


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


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

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


Sugar Sodium Nitrate

5 min. Cent. - Tubes removed & let stand 10 min.

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


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

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