SPECIMEN AND STOOL FOR INTESTINES NEMATODE AND PROTOZOA

1. Specimen delivery
The correct specimen delivery is very important for the diagnostic value. The
success of macroscopic and microscopic examination for parasite vector and
intermediate host identification depends much on the material preparation.
For examining parasites which need culture, specimens have to be in alive
condition when they arrive in the laboratory. If it is estimated that the
specimen collection takes time, fixative solution can be added to specimen so
it will still have diagnostic value.
Things gto consider in the stool specimen collection :
1) Time and correct collection method
2) The equipment used should be really clean so there wont be a
contamination with other organism, free from soap or desinfectants.
3) A tightly covered container of stout cardboard construction preferably
waxed is recommended.
4) Add the fixative solution for the specimen which does not examine
directly.
5) Label with patients name, age, sex, collecting time, and kinds of
specimen.

Some specimens that are usually used for examining the parasitological
disease are feces, urine, blood, sputum, tissue biopsy, larva and some
arthropod. For examining the intestines parasites, the specimen used is
feces (stool). If a liquid or mushy specimen cannot be examined within half
an hour, or a fully formed one wihin 3 or 4 hours, a prtion of the specimen
should be preserved for subsequent observation. Formalin 5-10% solution
may be used for this purpose.
Bellow is the intestines parasites specimens to prove the diagnosis of
intestines parasite infection in indonesia :
Table. The specimen delivery method to prove the diagnosis of disease caused
by human intestine parasites in indonesia
Kinds of
specimen
parasite
INTESTINE NEMATODE
A.
feces
lumbricoides

T. trichiura

Feces

Examination
method

Fixative
solution

remarks

a. Direct
b. Saturated
NaCl
c. Faust
d. Kato

b. formalin
5%

<
+
<
+

4
1
4
1

hours
year
hours
year

<
+
<
<

4
1
4
4

hours
year
hours
hours /+1

a. Direct
b. Saturated
NaCl
c. Faust
d. Kato

d. -/+
formalin 5%
b. Formalin
5%

Kinds of
parasite
Hookworm

specimen
Feces

E.
vermicularis

Perianal
swabbing

S. stercoralis

Feces

INTESTINE PROTOZOA
E. histolytica
a. intestine
Feces
Amublasis
b. liver
Amubiasis

G. lamblia

Liver abses
Aspirasi

Feces
Duodenal
Solution

E.coli

Feces

Examination
method
a. Direct
b. Saturated
NaCL
c. Faust
d. Kato
e. Harada Mori
a. Anal
swabbing
b. Pita selopan
swabbing
If the
microscopic is
positive and
then cultured
with harada
mori

a.
b.
c.
d.
e.

direct
ritchie
trichrom
direct
ritchie

a.
b.
a.
b.

direct
ritchie
direct
ritchie

a. direct
b. ritchie
c. trichrom

d. -/+
formalin 5%

year

Fixtative
solution
b. Formalin

remarks

d. -/+ formalin
5%
-

< 4 hours
+ 1 year
< 4 hours
< 4 hours /+1
year
< 4 hours
+ 1 day
< 4 hoursd

< 4 hours

a.
b. -/+
formalin
10%
c. PVA
a.
b. -/+
formalin
10%

a.
b. -/+
formalin
10%
a.
b. -/+
formalin
10%
a.
b. -/+
formalin

a.
b.
c.
a.
b.

< 4 hours
+- 1 year
+- 1 year
< 4 hours
+- 1 year

a.
b.
a.
b.

< 4 hours
+- 1 year
< 4 hours
+- 1 years

a. < 4 hours
b. +- 1 year
c. +- 1 year

10%
c. PVA

The purpose of examining the parasitological feces is to help proving the

diagnosis of parasite disease from feces and from other materials from
gastrointestinal.

A. MACROSCOPIC METHOD
In the macroscopic examination, there some points to consider.
1) Feces quantity
The quantity of infants feces is around 100 gram per day, while for grown
up people are around 80-170 gram/day. If someone eats small amount of
food and he/she has a small amount of feces and vice versa, it can be
considered physiologically normal. Liquid and a big amount of feces can
be found in patient who suffers from diarrhea.
2) Feces quality
(A) The color of normal feces is light brown because it contains stercobilin
and urobilin which is produced by intestine bacteria using bile
substance. Physiologically, the feces color also depends on the food
and medicine intake. The pathological changing of feces color may
occur because of, for example, hemorrhaging in proximal intestine
(black like tar, called melena) or caudal (blood red).
(B) Feces consistency
Hard: when the feces is pricked by stick, the stick doesnt insert
into it
Normal: when the feces is pricked by stick the stick will insert into it
and it keeps straight
Mushy: when the feces is pricked by stick the stick will insert into it
but if you let the stick go it will lean.
Rather liquid: when the feces is pricked by stick and let the stick go
it will fall flat to the ground.
Liquid: when the feces is as water
(C) Feces odor
The normal odor of feces is caused by the existance of scatol,indol
and H2S in the feces. On grow-up people who have milk diet and
breast-fed children, their feces doesnt have any odor. In amoebic
dysentery and askariasis, feces smell putrid because of the blood,
while in basilar dysentery, the feces have foul smell because of the
protein decayed.
(D)Feces shape
The pathological shape of feces is various, for example in obstipasi :
feces shape is like rock (coprolithiasis), and on the lower part of the
intestine stenosis patients, the feces is like pencil, like foam on the
dyspepsia and like porridge on diarrhea patient.
(E) The existence of blood or mucus in the feces
There is no mucus or blodd in normal feces. In the acute infection of
E. Histolytic or shigella, there are mucus and blood in the feces. In
chronic infection, theres usually no mucus or blood.
(F) The existence of proglotid (segment)
In the inection of intestine tape worm, proglottid is spontaneous
discharged periodically (e.g. dypilidium or taenia), or after a
treatment with anthelmetica. The identification of worm species can
be easily done by pressing the wet proglotid between two glasses.

B. MICROSCOPIC WORK
There are two kinds of microscopic work
1. Direct examination
- Direct examination using physiological salt
- Direct examination using 2% eosin solution
- Direct examination using 5% iodium solution
2. Indirect examination or concentration method
Indirect examination is done if the feces is too little or the direct
examination shows negative result. Few techniques can be used
such as: Rithcies (1984) sedimentation, direct floating using ZnSO4
or faust et al. (1964) method which is effective to detect cyst. The
floating technique using saturated NaCl can detect worms eggs,
kato-katz method is for count worm eggs per gram feces and
Harada Mori method is to hatch the hook worm and turn it into larva
for species identification.

1. DIRECT EXAMINATION
This kind of examination can be used to ecamine both intestine nematode
and intestine protozoa.
a. Using physiological salt solution
- Put a drop of the solution on the clean and dry glass
- Take a small amount of the feces using the stick and stir using the
solution on the glass, remove the coarse part.
- The cover glass is put slowly on top so that the solution is spread
evenly under the cover glass and there is no air bubble, so it should be
really thin
- Examine under the microscope with medium enlargement (10 x 45),
examine it at least three times.
b. Examination method using eosin salt solution
2% of eosin solution used consists of 2 grams of eosin and 100 ml of
aqueous. The method used is similar to the examination using
physiological salt solution. The slide should be very thin so the color is
pink. If the color is red or orange, it means the slide is too thick.
c. Examination method using iodine/lugol solution
The use of the lugol soulution can cause the vegetative form turn into
round because it dies so it is difficult to do vegetative examination.
Iodium used is 5% which consists of 5 grams of iodium, 10 grams of
kalium iodide and 100 ml of aqueous. The examination technique is similar
with physiological salt solution but the slide is not as thin as in physiological
salt solution examination.

2. INDERECT METHOD FOR EXAMINING INTESTINE NEMATODE

The indirect examination is carried out for egg examination. This method
is divided into two kinds, those are:

a. Qualitative; to find whether the worm eggs exist if the direct

examination fails fo find them.
b. Quantitative, to reveal the amount of eggs in one gram of feces so the
worm infection intensity can be known.

2.a. Qualitative method

There are some methods in indirect qualitative examination, but the
easiest and most effective method is flotation method using saturated solution of
NaCl. This method is based on the density difference of NaCl and eggs. The eggs
will float on the surface of the solution which has bigger density while the feces
will slowly go down at the bottom of the tube. This method can be used to
examine all kinds of worms eggs and cyst except operculated egg, schistosoma
egg, and unfertilized ascaris.
Equipments and materials
-

Feces
Stick (palm leaf rib)
Saturated NaCl solution
Beaker glass 30 ml
Test tube
Object glass
Cover glass

Procedures
1. Fill the test tube with saturated NaCl solution until it is full
2. Put +- 1 gram of feces in the beaker glass
3. Smash the feces using the stick and add with NaCl solution little by little until
it is homogeny and all the saturated NaCl solution is poured into beaker and
mixed very well.
4. The content in beaker is poured into the test tube again until it is full and the
gauze part on the surface is removed using the stick.
5. The cover glass is put on top of the tube so it touches the surface of the
solution. Leave it for 45 minutes.
6. Carefully take the cover glass and put it on top of object glass. Examine it
under microscope with small enlargement (10 x 10)

2.b Quantitative, Kato-Katz Method

This method is used to count the amount of the worm in one gram of
feces:
Equipments and materials
-

An approximately 2.5 x 3 cm of cellophane which has been soaked in

dye solution for 18-24 hours before it is used (the dye solution consists

of: 100 parts of aqueous, 100 part of glycerin and 1 part of 3% green
malachite)
An approximately 3 x 4 cm of filter wire for filtering the feces
Cardboard paper with whole which has certain volume
Stick (palm leaf fib)
10 x 10 cm of wax and filter paper
Object glass
Feces sample

Procedures
1.
2.
3.
4.

Put the filter paper on top of the wax paper

Take the feces using stick and put it on the filter paper
Put the cardboard with hole on the feces so the feces will be filtered
Put the cardboard on the object glass with the holw of the cardboard
on the object glass
5. Press the filter wite with stick and put the feces that has been filtered
on the filter wire into the hole of the cardboard until it is flat on the
cardboard hole surface
6. Lift up the cardboard so the feces will be left on top of the object glass
7. Cover the feces on the object glass with cellophane
8. Put the cellophane with other object glass to flatten the feces under
the cellophane
9. Put the slide upside down on the filter paper; leave it for 20 30
minutes
10.Count the worms eggs
The amount of the worms eggs X 1000/30 the amount of the eggs in one gram
of feces

Type of intestines worm which produces its egg can be correlated with the
amount of adult worm is A. Lumbricoides, T. Trichiura and hook worm. The worm
egg calculation can give a prediction about the infection as follows:

T. trichiura

: 30,000 eggs/ one gram of feces can show infection of hundreds

worm which can cause clinical symptom

Hook worm : 2500 5000 eggs in one gram of feces show a very clear clinical
symptom.

The small amount o hookworm and T.trichiura usually cannot show clinical
symptom but in ascariasiseven only one is found, it can be dangerous because
this parasite can migrate actively to other organs so it can cause a serious
clinical manifestation.

3. Harada Mori Method

This method is used for finding and identifying larva of ancylostoma
duodenale, necator americanus, strongyloides stercoralis, dan
Trychostrongylus sp worms. Using this method, eggs can be developed into
infective larva in the wet filter paper, and then larvae will be found in the
water which is located at the tip of the plastic.
Equipments and materials
-

+- 17 x 3 cm plastic bags
+- 15 x 2.5 cm filter paper
Clean water
Candle flame
Stick
Feces

Procedurs
1. Swab the feces onto the 1/3 part of filter paper
2. Pour the +- 5 ml of water into the plastic bag
3. Put the filter paper that has been swabbed with feces into plastic bag, the
tip of the paper should touch the water but not the feces.
4. Fold the tip of the plastic and staple it and hang it itn the shelf.
5. Leave it for 5 7 days in foom temperature; the culture examines using
these steps
a. cut the bottom tip of the plastic bag and retain the water which
already contains larva in the spinner tube
b. heat the tube with the water in 50 c for 15 minutes until the larva die
but not damage.
c. Spin the tube in a speed of 2500 300 rpm, for one minutes, and take
out the supernatant
d. Take the sediment with pipette, sprinkle it on the object glass and
cover it with cover glass.
e. Examine under microscope using low enlargement

3. Anal Swabbing Method

This method is specially used for finding the enterobius vermicularis worm
egg whic usually intact in the mucosa or skin of perinal region.
Equipments and materials
-

Scotch tape
Tongue depressor
Object glass
Toluol

Procedures
1. Put the scotch tape stretch along a length with the sticky part outside, in
one of the tip of tongue depressor which is held with point finger and
thumb under object glass.
2. Swab the sticky part of the scotch tape on both side of the perianal region
3. Release the scotch tape and tongue depressor and attach the tape to that
object glass
4. Add some drops of toluol between object glass and scotch tape to clear it.
5. Examine under the microscope with small or medium enlargement.

3. FECES ECAMINATION FOR INTESTINE PROTOZOA

Intestine protozoa which is found in feces is know in the form of vegetative
and cyst. The vegetative form is better ecamined in fresh feces because the
typical movement of the protozoa can be seen clearly. In preserved feces, the
vegetative form will die so the movement cannot be seen. The cyst form can last
longer in the feces. E.histolytic in the feces part that contains mucus and blood.
Amoebic dysentery should be differentiate from basilar dysentery.
Amoebic Dysentery
Macroscopic
1. Feces always consist of feces
parts
2. Blood seems infact outside the
feces
3. Mucus is usually clear
Microscopic
1. There is E. Histolytica
2. Leukocyte is rare
3. Erythrocyte is grouping
4. There is charcott leyden crystal
5. There is no or only little amount
of macrophage cell

Basilar Dysentery
Macroscopic
1. Feces often consists of feces and
blood
2. Blood blends completely with
blood
3. Mucus is turbid
Microscopic
1. There is no E. Histolytica
2. There are lots of leukocyte,
grouping
3. Eritrosit tercampur rata dengan
tinja
4. There is no charcott leyden
crystal
5. There are lots of macrophage
cells (karioreksis, kariolisis)

The direct examination is carried out using the similar method as the
method user for examining the intestine nematode, which is using physiological
salt solution, 2% of eosin solution to see the vegetative and cyst and 5% lugol
solution can show cyst clearer.
3.b indirect Examination
The indirect examination is carried out if there is only small amount of
feces or the direct examination shows negative result.

3.b.1 Ritchies sedimentation method (1984)

Equipments and materials
-

Feces
Moist gauze
Centrifuge
Centrifuge tube
Beaker
7.5% formalin solution
Stick
Ether
5% lugol solution

Procedurs
1. Emulsify approximately 1 gm. Of feces in 10 ml. Of normal saline in beaker
glass
2. Filter through two layer moist gauze into a centrifuge tube
3. Centrifuge 45 60 seconds, at 230 rpm, pour off supernatant, resuspend in
fresh saline.
4. Repeat step 3 for 2 3 times, until the supernatant solution is lucid.
5. Add 10 ml. 7.5% formalin, stir and allow to stand for 5 10 minutes
6. Add 3 ml. Of ether or ethyl acetate; shake vigorously
7. Centrifuge 1,5 menutes at 1500 rpm, loosen plug between formalin and
ether layer, decant supernatant
8. Take the sediment with stick, make slide with a drop of 5% lugol solution
9. Examine it under the microscope with medium enlargement (10 x 40)

3.b.2. faust et al. Floating Method (1983)

Equipments and materials
-

Feces
ZnSO4 33% solusion, density 1.18 (331 gram of ZnSO4 + 1000ml
aquadest)
5% of lugol solution
Tube shelves
Stick
Beaker glass
Centrifuge
Centrifuge tube
Corong gelas
Moist gauze

Procedures
1. Emulsify 1 gram of feces with 10 ml. Aquadest in beaker glass

2.
3.
4.
5.
6.
7.
8.

Filter through two layer of moist gauze into the centrifuge tube
Centrifuse 45 60 seconds, 230 rpm, pour off the supernatant
Repeat step 4 for 2 3 times until the supernatant solution is lucid
Add 33% ZnSO4 solution and stir it with stick
Centrifuse 40 60 seconds, 230 rpm
Put the tube in the tube shelf in upright position, leave it for 2 minutes
Take the tube surace using ose carefully, put it in the object glass which has
been added with 5% of lugol solution, cover it with cover glass
9. Examine under the microscope using medium enlargement (10 x 40)

Assignment: perform the feces examination on intestine parasites infection

patient using direct examination and concentration method. Draw the result of
the examination you have done.