CMPara: Day 9
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FORMALIN-ETHYL ACETATE FECAL
SEDIMENTATION METHOD
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The purpose of fecal concentration method
is to detect small number of parasites missed
by wet mount
Sedimentation method recovers all protozoa,
eggs, oocysts, and larvae along with debris,
unlike the floatation procedure which is
cleaner
Use one loosely woven straining gauze
Gauze is loosely woven with 8 ply
Using more than one gauze captures
parasites (mucoid stool). False negative
results
Use 15 ml screw cap falcon tube
Strain fecal suspension through gauze
Prepare fecal suspension:
 4g (0.5 teaspoonful) – stool
 10ml – 10% formalin
- Let stand for 30 minutes, mix well or
formalinized or SAF-ed stool: stool
immersed in 10% formalin or SAF and
mixed well
Materials needed:
- Gauze
- Fecal suspension
- Iodine
- Trichrome stain
- Ethyl acetate
- 10% formalin
- 35% formaldehyde
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To rinse fecal suspension, add few mls of
10% formalin on the gauze until falcon tube
is 10 ml filled
Carefully remove and dispose the mounted
gauze and the fecal matter within
10 ml strained fecal suspension
Centrifuge for 10 minutes at 500xg
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If stool is diarrheal (mucoid), skip straining
step. Fix in 10% formalin for 30 minutes and
centrifuge for 10 minutes at 500 x g
If supernatant = clear to light tan: one wash
is enough
If turbid: do a second wash
0.5-1 ml sediment
Decant and discard supernatant fluid
Add 10 ml of 10% formalin to resuspend
sediment
resuspend sediment by mixing well. Use
wooden stick to dislodge sediment
add 4ml of ethyl acetate. Total volume will
be 14 ml in a 15 ml falcon tube
if sediment is very small or original sample
is diarrheal (mucus), do not add ethyl
acetate. Just add 10% formalin, centrifuge,
decant, and examine remaining sediment
close tube tightly. Shake vigorously for 30
seconds
upon mixing, ethyl acetate will extract
debris and fat from feces and leave parasite
at the bottom of the suspension
centrifuge for 10 minutes at 500 x g
there will be 4 layers:
 ethyl acetate
 plug of fecal debris
 10% formalin
 Sediment (parasites)
Free plug of debris with a wooden applicator
stick
Decant and discard all the supernatant fluid
Clean inner walls of tube from traces of
ethyl acetate with cotton swab before
bringing tube into upright position
Presence of ethyl acetate will cause
extensive bubbles under the microscope
Too much or too little sediment will result in
an ineffective concentration sediment
examination
In case sediment is dry, add 1-2 drops of
10% formalin and mix well
Add a drop of sediment on a slide
Cover with a 22x22 mm cover slip
Scan entire coverslip under LPF (100x).
scan 1/3 under HPF (400x)
In a tube, add equal volumes of sediment
and iodine (1:1)
Mix sediment and iodine (1:1), shake well
Add a drop of sediment-iodine mixture on a
slide
Cover with a 22x22 mm coverslip
Scan entire coverslip under LPF (100x).
scan 1/3 under HPF (400x)
Prepare trichrome-iodine wet mount
4 drops of sediment + 4 drops of iodine
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Add 1 drop of trichrome stain
Add a drop of sediment-iodine-trichrome
mixture on a slide
Scan entire coverslip under LPF (100x).
scan 1/3 under HPF (400x)
Sedimentation is recommended:
1. Easiest to perform
2. Recovers broadest range of organisms
3. Least subject to technical error
3.
Store in tightly closed glass bottle for max. 2
years.
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To rinse fecal suspension, add few mls of
10% formalin on the gauze until falcon tube
is 10 ml filled
Carefully remove and dispose the mounted
gauze and the fecal matter within
Unclamp tube, close tightly, and centrifuge
at 500 x g for 10 minutes
Take tube out of centrifuge, observe for
sediment and supernatant
Decant supernatant
Add 2 ml of zinc sulfate
Close tube and mix thoroughly
Dislodge sediment with wooden applicator
Fill tube with zinc sulfate to within 2-3mm
of rim
Mix well and centrifuge at 500 x g for 2
minutes
For maximum detection of parasites,
examine surface film and sediment
Prepare wire loop by bending its circle to
become perpendicular with the shaft of loop
First, start examining surface film within 5
min of stop of centrifuge, otherwise floating
eggs and cysts will sink
Without removing tube from centrifuge,
gently touch surface later by the bent loop
and apply on a clean labeled slide
As an alternative to loop, Pasteur pipette can
be used to remove 1-2 drops from surface
and place on a slide
Add a 22x22 mm coverslip
Examine sediment for heavy eggs
2 layers:
 Zinc sulfate layer
 0.5-1ml sediment
Decant the zinc sulfate layer (supernatant)
Add one drop of sediment onto a glass slide
Apply coverslip
Scan under the microscope at 10x objective.
Turn to 40x to confirm suspected objects. At
least 1/3 of slide should be scanned under
40x
using iodine: add 1-2 drops of D’Antoni’s
iodine to sediment to enhance morphological
details
with the Pasteur pipette, add a drop of
sediment-iodine mix onto a clean labeled
slide
iodine-trichrome stain can also be used as
described in ethyl acetate sedimentation
video
apply coverslip
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ZINC SULFATE FLOTATION METHOD
Purpose: separates protozoan cysts, oocysts, and
eggs from excess debris using zinc sulfate based on
difference in specific gravity
Principle: zinc sulfate has higher specific gravity
than parasites. Parasites rise to surface, whereas
debris sink to the bottom
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Use one loosely woven straining gauze
Gauze is loosely woven with 8 ply
Using more than 1 gauze captures parasites
(mucoid stool). False negative results
Use 15 ml screw cap falcon tube
Strain fecal sample through gauze
Needed:
 Fecal suspension
 Gauze
 10% formalin
 33% zinc sulfate
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Prepare 33% zinc sulfate, ZnSO4:
 33g Zinc Sulfate
 670ml Distilled water
In a flask or beaker with a magnetic stirrer,
dissolve zinc sulfate in distilled water
Adjust specific gravity by adding zinc
sulfate or distilled water using hydrometer.
1.18 for fresh stool and 1.2 for formalinized
stool
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Scan under the microscope at 10x objective.
Turn to 40x to confirm suspected objects. At
least 1/3 of slide should be scanned under
40x
ADVANTAGES:
- More effective for detecting protozoan cysts
such as Giardia duodenalis and amebae
spp., as well as Hymenolepis nana, and
hookworm eggs (S.G < 1.18)
- Detects light helmith eggs: T. trichuria and
E. vermicularis (S.G < 1.18)
- Produces cleaner material (without debris)
than sedimentation technique
DISADVANTAGES:
- Schistosoma and operculated eggs like D.
latum and Fasciolopsis buski are unlikely to
be detected by flotation
- Schistosoma egg is dense. Operculum opens
allowing zinc sulfate in to fill the egg and
sink it to bottom
- Not effective for large trematode eggs, some
tapeworm eggs, and infertile Ascaris
lumbricoides eggs S.G > 1.2)
- Zinc sulfate is a hypertonic solution, causing
lysis of membrane of some parasites leading
to collapse of eggs and cysts such as
Blastocystis spp.
- Unsuitable for trophozoites
- Unsuitable for fatty stool
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Float and sediment should be examined