UNIVERSITI TEKNOLOGI MARA

(BERTAM, PULAU PINANG)

Faculty of Health Sciences
Diploma in Medical Laboratory
Technology
______________________________________

CSI 153
CLINICAL PARASITOLOGY
PRACTICAL 4a & 4b:
FORMALIN-ETHER
SEDIMENTATION CONCENTRATION
TECHNIQUE & ZINC SULFATE FLOTATION
CONCENTRATION TECHNIQUE

NAME : NURUL FATINAJIHAH BINTI ZULIKIPLI

STUDENT ID : 2016625096

IC NUMBER : 981012115026
LECTURER’S NAME : MADAM NURHIDAYAH BINTI
AB. RAHIM

INTRODUCTION

Concentration technique are used to separate the parasite from the fecal debris in a
stool specimen.It is also to increase the chance of discover the presence in a small
amount number.This technique used to detect the parasite when microscopic routine
were failed to detect the eggs of the specimen.Concentration technique was divided
into two techniques which is sedimentation and floatation.

Sedimentation concentration technique used the solutions of lower specific gravity

than the parasitic organisms.This technique takes the advantage of the high specific
gravity of protozoan cyst and helminth egss compared to water.In this experiment,
we used 10% formalin and ether as the solutes to mix with the stool
specimen.Formalin was used in this experiment because the formalin can fixes the
protozoan cyst and helminth eggs so that they are no longer infectious, as well as
prserves their morphology.The purpose of ether in this experiment is an extractor
which is to trap the debris in a layer separates from the parasites.

Floatation concentration technique is a method by using the solution with higher

specific-gravity than the parasitic organism which is can cause the parasitic organism
float.So that in this experiment we used zinc sulfate as the solution.The purpose of
using zinc sulfate as the solution is to separate the parasitic organism from fecal
debris through differences in higher specific gravity.The zinc sulfate concentration
technique provides a cleaner concentration and it is very good for the recovery of
protozoan cyst and most eggs.
 Practical 4a: Formalin-ether sedimentation concentration technique

OBJECTIVE
To concentrate the parasites in the specimen using sedimentation technique for
identification of cyst, egg and larvae.

MATERIALS & EQUIPMENTS

 Wood applicator stick
 2 centrifuges tube
 Centrifuge
 Inoculating loop
 Glass slide
 Cover slip
 Ether
 Distilled water
 Stool specimen
 Gauze cloth
 Formalin
 Saline
 Iodine

METHODS
1. 1 ml of stool specimen was added into a 15 ml centrifugal tube by using a wood
applicator.
2. 3 ml of distilled water was added and the specimen was mixed with water
thoroughly by using a wood applicator.
3. The tube was filled with distilled water until 2 cm from the top of the tube.
4. The mixture was filtered in the tube through gauze layers into a clean container.
5. The tube was rinsed and the filtrate was poured back to the tube.
6. The tube was centrifuged at 2,000 rpm for 1 minute.
7. The supernatant was decanted.
8. 10% formalin was added to the tube and the sediment was mixed with formalin by
using wood applicator.
9. The tube was stood for 10 minutes to preserve the specimen.
10. 5 ml of ether was added.
11. The tube cap was tighten and the tube was shaken vigorously for 30 seconds.
12. The tube was centrifuged at 1,500 rpm for 2 minutes.
13. The plug of debris was free from the top of tube by ringing the sides with an
applicator stick.
14. The supernatant was decanted carefully without disturbing the sediment.
15. A drop of iodine was placed on a glass slide.
16. 1 drop of sediment was taken from the tube by using a disposable Pasteur pipette
and was mixed with the drop of iodine on the glass slide.
17. The mixture on the glass slide was covered with a cover slip.
18. The protozoa cyst, helminth egg and larvae was examined under microscope and
the result was recorded.
19. The entire area under the cover slip was scanned by using objective 10x with
back and forth motion.
20. The suspicious parasite was confirmed by using objective 40x.

RESULTS

Plant fiber

Category Artifact (Plant fiber)

Structure Resemble hookworm larvae
Morphology Cylindrical and brown colour
Motility Non-motile
Reagent Saline

Air bubble

Category Artifact (Air bubble)

Structure Resemble protozan cyst
Round shape and
Morphology
colourless
Motility Non-motile
Reagent Saline
 Crystal

Category Artifact (Crystal)

Structure Charcot-Leyden
Cylindrical shape and
Morphology
colourless
Motility Non-motile
Reagent Saline
 Practical 4b: Zinc sulfate flotation concentration technique

OBJECTIVE
To concentrate the parasites in the specimen using floatation technique for
identification of cyst, egg and larvae.

MATERIALS & EQUIPMENTS

 Wood applicator stick
 2 centrifuges tube
 Centrifuge
 Inoculating loop
 Glass slide
 Cover slip
 Zinc sulfate solution
 Distilled water
 Stool specimen
 Gauze cloth
 Saline
 Iodine

METHODS
1. 1/3 ml of stool specimen was put into a 15 ml centrifugal tube by using a wood
applicator.
2. 1 mlof distilled water was added and the specimen was mixed with water
thoroughly by using a wood applicator.
3. The tube was filled with distilled water until 2 cm from the top of the tube.
4. The tube was centrifuged at 500 x g of 2,000 rpm for 1 minute.
5. The supernatant was decanted.
6. 1 mlof zinc was added to the tube and the sediment was mixed with zinc sulfate
thoroughly.
7. The tube was filled with zinc sulfate.
8. The mixture was filtered in the tube through gauze layer into a clean container.
9. The filtrate was poured back to the tube.
10. The tube was centrifuged at 500 x g or 2,000 rpm for 1 minute.
11. A drop of iodine was placed on a glass slide.
12. 1-2 loopful of sample from the top of the mixture in the tube was taken and was
mixed with the drop of iodine on the glass slide.
13. The mixture on the glass slide was covered with a cover slip.
14. The protozoa cyst, helminth egg and larvae was examined under microscope.
15. The entire area under cover slip was scanned by using objective 10x with back
and forth motion.
16. The susupicious parasite was confirmed by using objective 40x.
RESULTS

Starch granule

Category Artifacts (Starch granules)

Structure Resemble protozoan cyst
Morphology Oval shape and stain red
Motility Non-motile
Reagent Iodine

Air bubble

Category Artifacts (Crystal)

Structure Charcot-Leyden
Round shape and
Morphology
colourless
Motility Non-motile
Reagent Iodine
 Plant fiber

Category Artifacts (Plant fiber)

Resemble hookworm
Structure
larvae
Morphology Brown colour
Motility Non-motile
Reagent Iodine

DISCUSSION

a) Formalin Ether sedimentation concentration technique

From this experiment,the results didn’t show any presence of parasites but it showed
the presence of artifacts that contain similar figure to the parasite.From the figure
above,it shows that artifacts that present were air bubbles,crystal and plant
fiber.Usually,the presence of air bubbles usually confused with the protozoan cyst
while the presence of plant fiber quite similar with the Strongyloides or hookworm
larvae.The presence of crystal,Charcot-Leyden are the breakdown products of
eosinophil cells and may be present in stools or sputum.
There are some advantages and disadvantages from the formalin ether
sedimentation concentration.First of all,the advantage is the speed of this experiment
is very fast which is one sample just only can be processed in 5 minutes.So that,we
can do many slide fromthis experiment in a given time.Other than that,the advantage
is this experiment allows the recovery all the helminth eggs and also protozoan
cyst.This can help us to observe easily under the microscope.Next,formalin ether
sedimentation concentration is one of the experiment that very easy to perform and
have less prone to technical errors.Lastly,the advantage of this experiment is less
risk of infection from the bacteria and also viruses which can give affect to our
results.One of the disadvantage from this experiment is the dangerous of using ether
and formalin.The ether is very flammable while formalin is irritant and both reagents
can give bad affect to us if we didn’t use them properly.Other than that, the
preparation of this experiment maybe contains some debris which is may confuse us
with the parasite during observing under microscope.

Based on this experiment,there were some precautions that we should know to

reduce any errors that may occur.Firstly is when transfer the small amount of ether
and formalin.The volume of ether and formalin which is transferred not correctly may
affect the results.So we have to make sure that we have transferred the volume of
both reagents correctly.Then, the presence of air bubbles is the basic error when
doing this experiements.As the precaution,when placing the cover slip we must let
down the cover slip slowly so the air bubble will not produce.Lastly, before putting the
centrifuge tube into the centrifuge,make sure that the cover of the tube was tighten to
avoid any split during centrifuge.

a) Zinc sulfate floatation concentration technique

The results from this experiments didn’t show any presence of parasites but we can
detect the presence of artifacts such as starch granule,air bubble and plant
fiber.Starch granule sometimes can be seen in stool and the presence of starch
granules when it was partially digested,it stain red.We also usually confuse with the
hookworm larvae when there is the presence of plant fiber or hair fiber.
One of the advantages from this experiment is the procedure will floats most helminth
eggs so that we can observe the presence of helminth eggs under the
microscope.Next, the advantage is this experiment is the simple technique and the
reagents are easily available,chep,not dangerous and easily to prepare.So,we can
save our budget and also save our time.This experiment also can provide clean
concentrate and the reagents have longer shief life.The advantage is we can save
the reagents for longer time and also can make sure that our concentration always
clean.One of the disadvantages from this experiment is procedure will not float some
trematode ova and also some taperworm ova because it is too heavy to float.So that
it may affect our results when observing under microscope.Then,the disadvantage is
when we delay this experiment,we will have the results in distortion.Lastly,the
disadvantage is the specific gravity must be checked frequently to make sure that we
will get the correct results at the end of the experiment.

Based on this experiment,there were some precautions that we have to take

attention to reduce some arrors that occur during the experiment.First of all is there
were the presence of air bubbles when the slide was covered with the cover
slip.So,as the precautions when we place the coverslip, we need to do it from the end
of slide with 45 degree angle and let it down slowly.Then, when we examine the
specimen under microscope,move the glass slide with zig-zag pattern to make sure
the whole slide are observed.The procedure of examine the stool must be done
carefully and properly to get the good results.

CONCLUSION

Last but not least,sedimentation concentration technique used the lower specific
gravity solution such as formalin and ether while floatation concentration technique
used the higher specific gravity solution such as zinc sulfate.The artifacts were
detected from this experiment.
REFERENCES
 Laboratory Manual CSI 153 Clinical Parasitology, Madam Nurhidayah binti Ab
Rahim
 Lecture’s Note-Introduction to Parasitology
 http://www.medilexicon.com/dictionary/19604
 https://microbeonline.com/formal-ether-sedimentation-techniques/
 https://www.google.com/search?
q=advantage+and+disadvantage+of+formalin+ether+sedimentation&rlz=1C1C
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