Stool Microscopic Examination

Purpose: to examine

Motile parasites such as the larvae of S. stercoralis and trophozoites of E.

hystolytica, G. lamblia, and more rarely, B. coli,
Helminth eggs,
Cysts and oocysts of intestinal protozoa.

Reporting the appearance of faecal specimens

Report the following:

Colour of the specimen,

Consistency, i.e. whether formed, semiformed, unformed, watery.
Presence of blood, mucus, and, or, pus. If blood is present note whether
this is mixed in the faeces. If only on the surface this indicates rectal or
anal bleeding.
Whether the specimen contains worms, e.g. A. lumbricoides (large
roundworm), E. vermicularis (threadworm), or tapeworm segments, e.g. T.
solium, T. saginata.

Microscopic Examination of Faecal Specimens

A. Examination of dysenteric and unformed specimens
1. Using a wire loop or piece of stick, place a small amount of specimen, to
include blood and mucus on one end of a slide. Without adding saline,
cover with a cover glass and using a tissue, press gently on the cover
glass to make a thin preparation.
2. Place a drop of eosin reagent on the other end of the slide. Mix a small
amount of the specimen with the eosin and cover with a cover glass.
3. Examine immediately the preparations microscopically, first using the 10x
objective with the condenser iris closed sufficiently to give good contrast.
Use the 40x objective to identify motile trophozoites, e.g. E. histolytica
amoebae or G. lamblia flagellates.
B. Examination of semi-formed and formed faeces
1. Place a drop of fresh physiological saline on one end of a slide and a drop
of iodine.
2. Using a wire loop or piece of stick, mix a small amount of specimen, about
2mg, with the saline and a similar amount with the iodine. Make smooth
thin preparations. Cover each preparation with a cover glass.
3. Examine systematically the entire saline preparation for larvae, ciliates,
helminth eggs, cysts, and oocysts. Use the 10x objective with the
condenser iris closed sufficiently to give good contrast. Use the 40x
objective to assist in the detection and identification of eggs, cysts, and
oocysts. Always examine several microscope fields with this objective
before reporting No parasites found.
4. Use the iodine preparation to assist in the identification of cysts.
5. Report the number of larvae and each species of egg found in the entire
saline preparation as follows:
Scanty --1-3 per preparation

Few --4-10 per preparation

Moderate number 11-20 per preparation
Many
21-40 per preparation
Very many
over 40 per preparation
C. Non-parasitic structures found in the faeces:
Care must be taken not to report as parasites those structures that can be
normally found in the faeces such as muscle fibres, vegetable fibres, starch cells
(stain blue-black with iodine), pollen grains, fatty acid crystals, soaps, spores,
yeasts, and hairs. Large numbers of fat globules may be seen in faeces when
there is malabsorption. Charcot Leyden crystals (breakdown products of
eosinophils) can sometimes be seen in faeces (also in sputum) in parasitic
infections. They appear as slender crystals with pointed ends.

D. Faecal Concentration Techniques

Sedimentation techniques in which parasites are sedimented by gravity or

centrifugal force, e.g. formol ether concentration method which is the
most frequently used technique because it concentrates a wide range of
parasites with minimum damage to their morphology.

Floatation techniques in which parasites are concentrated by being floated

in solutions of high specific gravity, i.e. zinc sulphate solution that is
denser than the parasites being concentrated. Unlike the formol ether
sedimentation technique, a single floatation technique cannot be used to
concentrate a wide range of parasites because of differences in the
densities of parasites and the damage that can be caused by floatation
fluids to some parasites.

Sedimentation using Formol Ether Concentration Technique

Principle: In the Ridley modified method, faeces are emulsified in formol
water, the suspension is strained to remove large faecal particles, ether or
ethyl acetate is added, and the mixed suspension is centrifuged. Cysts,
oocysts, eggs and larvae are fixed and sedimented and the faecal debris is
separated in a layer between the ether and the formol water. Faecal fat is
dissolved in the ether.
Materials: 10% formol water, diethyl ether or ethyl acetate.
Procedures:
1. Using a rod or stick, emulsify an estimated 1g (pea-side) of faeces in
about 4ml of 10% formol water contained in a screw-cap bottle or tube
made of strong glass, copolymer, or polypropylene.
2. Add a further 3-4ml of 10% formol water, cap the bottle, and mix well
by shaking.
3. Add in 3-4ml of diethyl ether or ethyl acetate.
Caution: ether is highly flammable and ethyl acetate is flammable,
therefore use well away from an open flame. Ether vapour is
anaesthetic, therefore make sure the laboratory is well-ventilated.
4. Stopper the tube and mix for 1 minute. If using a Vortex mixer, leave
the tube unstoppered and mix for about 15 seconds.
5. With a tissue or piece of cloth wrapped around the top of the tube,
loosen the stopper (considerable pressure will have built up inside the
tube).
6. Centrifuge immediately at 750-1000 g (approximately 3000 rpm) for 1
minute.

Formal
ether
sedimentation
concentration
technique,
after
centrifugation.
7. Using a stick or the stem or a plastic bulb pipette, loosen the layer of
faecal debris from the side of the tube and invert the tube to discard
the ether, faecal debris, and formol water. The sediment will remain.
8. Return the tube to its upright position and allow the fluid from the side
of the tube to drain to the bottom. Tap the bottom of the tube to
resuspend and mix the sediment. Transfer the sediment to a slide, and
cover with a cover glass.
9. Examine the preparation microscopically using the 10x objective with
the condenser iris closed sufficiently to give good contrast. Use the
40x objective to examine small cysts and eggs. To assist in the
identification of cysts, run a small drop of iodine under the cover glass.
10.If required, count the number of each species of egg in the entire
preparation. This will give the approximate number per gram of faeces.
Zinc Sulphate Floatation Technique
Principle: The zinc sulphate solution is used which has a specific gravity
(relative density) of 1.180-1.200. Faeces are emulsified in the solution and the
suspension is left undisturbed for eggs and cysts to float to the surface. They
are collected on a cover glass.
Materials: 33% Zinc sulphate solution, test tube.
Procedures:
1. Fill the tube about one quarter full with the zinc sulphate solution. Add
an estimated 1 gram of faeces (or 2ml of fluid specimen). Using a rod
or stick, emulsify the specimen in the solution.
2. Fill the tube with the zinc sulphate solution, and mix well.
3. Stand the tube in a completely vertical position in a rack.
4. Using a plastic bulb pipette or Pasteur pipette, add further solution to
ensure the tube is filled to the brim.
5. Carefully place a completely clean (grease free) cover glass on top of
the tube. Avoid trapping any air bubbles.

6. Leave undisturbed for 30-45 minutes to give time for the cysts and
eggs to float.
Note: Do not leave longer because the cysts can become distorted and
the eggs will begin to sink.
7. Carefully lift the cover glass from the tube by a straight pull upwards.
Place the cover glass face downwards on a slide.
8. Examine microscopically the entire preparation using the 10x objective
with the condenser iris closed sufficiently to give good contrast. Use
the 40x objective, and run a drop of iodine under the cover glass, to
identify the cysts.
9. Count the number of eggs to give the approximate number per gram of
faeces.