TOPIC 7: CONCENTRATION TECHNIQUES

FLOTATION CONCENTRATION

OUTLINE

A. CONCENTRATION TECHNIQUES
a. Flotation techniques
i. Zinc Sulfate Flotation Technique
ii. Sheather’s Sugar Flotation
Technique
iii. Brine Flotation Technique
b. Sedimentation Techniques
i. Simple Gravity Sedimentation
Technique
ii. Formalin Ether Concentration
Technique
iii. Midi Parasep
iv. Acid Ether Concentration
Technique ●
v. Merthiolate Iodine Formalin
Ether Concentration Technique

Zinc Sulfate Flotation Technique

Concentration Techniques
● Highly recommended in cases of light infection ● original Zinc sulfate flotation procedure was
that may have yielded a negative direct fecal smear developed by Faust in 1938 for the recovery of
reading both helminth eggs and larvae and protozoan
● Design to separate parasites from excess fecal cysts.
debris. ● 33% ZnSO4 (main reagent)
● 2 Methods of Concentration techniques ○ most wide used reagent
○ Flotation Techniques ● Can recover protozoan cysts
○ Sedimentation Techniques
Procedure
Flotation Techniques
1. Transfer about 0.5 teaspoon of stool to a test
● Surface of the preparation tube containing 1-2ml of water and comminute
● Reagents thoroughly then fill the tube to within 2-3 mm of
○ higher SG (1.18-1.20) the top with water. If solid, about 1 gram of stool
● Zinc sulfate, magnesium sulfate, brine, sugar is thoroughly mixed with 10 ml distilled water.
● Cleaner preparation Then transfer suspension to a 15 ml conical
● Ideal for nematode eggs tube.
● Useful for small operculated trematode egg 2. Centrifuge sample at 1500-2500 rpm for 1
○ Opisthorchis minute. Discard the supernatant.
○ Clonorchis 3. Add 1-3 ml zinc sulfate to resuspend the
○ Heterophyids sediment.
● Larger eggs will not float 4. Fill the tube within 2-3 mm of the rim with
○ Fasciola & Fasciolopsis additional zinc sulfate.
● Exposure of parasites to high SG 5. Centrifuge again at 1500-2500 rpm for 1 minute.
○ distortion and shrinkage of protozoan 6. Pipette 1 drop from the surface of the
cysts & thin-walled helminth eggs preparation or get a loopful of the sample from
the surface and place on a clean labeled slide
and cover with a coverslip.
7. Scan the entire coverslip.

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SURNAME / SURNAME / SURNAME | BSMT II

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 TOPIC 7: Concentration Techniques

Report as follows: Procedure

● Helminths: 1. Label vial & slide.

○ # of eggs parasite name/ coverslip
(report separately for mixed infection)
○ no ova seen
● Protozoan cysts:
○ Positive for parasite name & stage
○ None seen
Sheather’s Sugar Flotation Technique
● Sugar boiled in phenol
● Best for the recovery of coccidian oocysts
2. Fill vial with brine half-full.
3. Place half a gram of stool in a vial.
4. Mix the specimens thoroughly to make a
homogenous suspension.
5. Slowly add more brine solution up to the brim of
the vial.
6. Carefully lower a cover slip on top of the vial and
leave it there for 10-15 mins.
7. After 15 minutes, lift the coverslip squarely and
place on a glass slide and examine the entire
coverslip under the microscope.

Report as follows:

● # of eggs parasite name/ coverslip

●
● no ova seen

Sedimentation Techniques

● Bottom of the preparation/ sedimen

●
● Reagents
Procedure
○ lower SG
1. Soften 1 gm of feces with water to a soft.
Simple Gravity Sedimentation Technique
2. Strain the aqueous suspension through a wire
sieve
● stool + tap water → mix→ allow parasites to
3. Mix 1 part aqueous suspension with 2 part of
settle down by gravity
Sheather’s sugar solution
● Less equipment & reagents needed
4. Pour into a centrifuge tube, centrifugation 1500
● Store sediments
rpm for 10 minutes
○ add 10% formalin
5. Pour the supernatant into a meniscus and add a
● Schistosoma eggs will hatch
sufficient solution to bring the meniscus to the
○ add NSS (prevent hatching)
top
● Hasten sedimentation
6. Place a coverslip and wait for 10 minutes
○ add glycerine
7. Examine under microscope
● Advantages:
○ Minimal use of glassware, equipment &
Brine Flotation Technique
reagents
○ Useful in cases of Schistosoma &
● Uses saturated table salt solution
Strongyloides infections
○ NaCl powder - 40 g
○ Sediments are useful for study & future
○ Distilled water - 100 ml
references
● Advantages:
○ Allows settling of cysts, eggs and larvae
○ a. Low cost and simple
● Disadvantages:
● Disadvantages:
○ Time consuming
○ a. Helminth eggs of Hookworm and
○ Lots of fecal debris in the sediments
Schistosoma become badly shrunken
○ b. Not useful for operculated eggs like
Clonorchis, Opisthorchis & Heterophyids

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BEIRA/ BILIRAN/ CASAMAYOR | BSMT II
 TOPIC 7: Concentration Techniques

Midi Parasep

● (Commercially available)

Formalin Ether Concentration Technique ●

● FECT ● Advantages:
● Also known as Ritchie’s Method/ 406 MGL ○ useful in the recovery of helminth eggs
(Medical General Laboratory) Method and protozoan cysts
● Can be performed on formalin- preserved or ○ can be done on formalin-preserved and
PVA-preserved stool PVA-preserved stools
● Reagent: ○ sediments from FECT can be stored for
○ 10% Formalin a long period of time
■ fixative/preservative ● Disadvantages:
○ Ether ○ Loss of parasite in plug of debris
■ dissolve neutral fats ○ Ether is flammable & explosive→
replaced with ethyl acetate (less
efficient)
Procedure
● Composed of two containers
1. To stool about the size of a thumb or a marble,
○ mixing tube (flat bottom)
add 10 ml of 10% formalin in a flat bottom plastic
○ sedimentation tube (conical bottom)
cup.Mix

2. Stand for 30 minutes.

Procedure
3. Strain the fecal suspension through 2 layers of
gauze and collect it in a 10 ml centrifuge tube.
4. Centrifuge for 5 mins @ 1,500 rpm and throw 1. Separate the two tubes.
away the supernatant fluid.
5. Using tap water, wash fecal sediment 3x. (Add
water→ mix→ spin → decant supernatant)
6. With the sediment, resuspend it by adding about
3 ml of 10% formalin. Let stand for 5 minutes.
2. For empty Parasep, unscrew lid and add 6.0 ml
7. Add about 3 ml of ether. Shake vigorously. Spin
of fixative and add 1 drop of surfactant (Apacor
5 mins 1500 rpm.
Triton X solution) to the flat-bottom tube.
8. Ether, fecal debris and formalin are completely
3. Add a pea-sized stool sample then add 2 ml of
decanted using an applicator stick to free the
ethyl acetate.
superficial fecal debris from the centrifuge tube.
4. Mix in thoroughly with the spoon. If the sample is
9. Place 1 drop of sediment on glass slide, place
hard, break it up with the end of the spoon.
coverslip → examine under the microscope

FECT Prep after Centrifugation

5. Vortex or shake to emulsify with the
sedimentation cone pointing upwards.

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BEIRA/ BILIRAN/ CASAMAYOR | BSMT II
 TOPIC 7: Concentration Techniques

6. Invert the Parasep (sedimentation tube at the MIFECT – Merthiolate Iodine Formalin Ether
bottom) and centrifuge at 1200 rpm for 3 mins. Concentration Technique

● Recommended for better identification of

protozoan cysts
● Temporarily stain protozoan cysts
● Sediments can be kept for future use
● Same procedure and reporting as FECT
● Reagents:
○ Merthiolate & Iodine
■ temporary stain
7. Unscrew and discard the filter and mixing tube. ○ Formalin/Formaldehyde
8. Pour off all supernatants. ■ Fixative
9. Pipette 1 drop and place on a glass slide and ○ Ether
cover with coverslip. ■ dissolve neutral fats
10. Scan the entire coverslip systematically.

REFERENCES

Notes from the discussion/learningmats by:

MA’AM JUSIE BERNAL

Silliman University PowerPoint presentation:

Concentration Techniques 21-22

Report as follows:

● Helminths:
○ # of eggs parasite name/ coverslip
■ Mixed helminth infections –
report separately
○ no ova seen
● Protozoan cysts:
○ Positive for parasite name & stage
○ None seen

Acid Ether Concentration Technique (AECT)

● Acid
○ 40% HCl
○ dissolve albuminous material
● Ether
○ dissolve neutral fats
● Same procedure as FECT
● Report for helminths only
● Advantages:
○ Method of choice if stool came from
animals
○ Recommended for detection of
Trichuris, Capillaria, and trematode eggs
especially Schistosoma
● Disadvantages:
○ Ether is flammable & explosive→
replaced with ethyl acetate (less
efficient)
○ Loss of parasite in plug of debris
○ Destruction of cysts

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BEIRA/ BILIRAN/ CASAMAYOR | BSMT II