Direct examination of faeces and concentration techniques

The clinical and public health reasons for examining faecal specimens for parasites.

In district laboratories the examination of faecal specimens for parasites is requested:

–To identify the parasitic causes of blood and mucus in faeces and differentiate amoebic dysentery from
bacterial dysentery.

–To identify intestinal parasitic infections that require treatment, i.e. those associated with serious
illhealth, persistent diarrhoea, weight loss, intestinal malabsorption and the impairment of development
and nutrition in children. e.g. intestinal schistosomiasis leading to portal hypertension, or chronic O.
viverrini infection leading to cancer of the bile duct.

–To detect serious hookworm infection in patients with severe iron deficiency anaemia, especially in a
pregnant woman or child.

–To assist in the surveillance and control of local parasitic infections caused by geohelminths (soil
transmitted nematodes), and helminths transmitted by the ingestion of infected meat, freshwater fish,
crabs, or shell-fish.

Collection of faecal specimen for parasitic examination

For clinical purposes, a fresh faecal specimen is required.

It should be uncontaminated with urine and collected into a suitable size, clean, dry, leak-proof
container.

The container need not be sterile but must be free of all traces of antiseptics and disinfectants (also a
bedpan if this is first used to collect a specimen from an inpatient).

A large teaspoon amount of faeces is adequate or about 10ml of a f luid specimen.

Several specimens collected on alternate days may be required to detect parasites that are excreted
intermittently, e.g. Giardia.

Unsuitable containers:

Avoid using containers made from leaves, paper, or cardboard (including matchboxes) because these
will not be leakproof, may not be clean, and can result in the faecal contamination of hands and
surfaces.

If no leakproof container is available, a non-leakproof container can be used providing the specimen is
first sealed in a plastic bag. This, however, is not recommended for the routine collection of faeces
because the plastic bag is difficult to label and can be a hazard to laboratory staff.

Important NOTE:

Dysenteric and watery specimens must reach the laboratory as soon as possible after being passed
(within 15 minutes) otherwise motile parasites such as E. histolytica and G. lamblia trophozoites may
not be detected. Other specimens should reach the laboratory within 1 hour of being collected.
Specimens must be labelled correctly and accompanied by a correctly completed request form.
The collection technique will depend on the parasite being investigated, the method that will be used to
examine the faeces, and whether any fixative or preservative is required.

Caution:

Faecal specimens, like other specimens received in the laboratory, must be handled with care to avoid
acquiring infection from infectious parasites, bacteria, or viruses.

Faeces may contain:

–infective forms of parasites such as S. stercoralis, E. vermicularis, T. solium, G. lamblia, E. histolytica,

or C. parvum.

–bacteria such as V. cholerae, Shigella or Salmonella species.

–viruses including hepatitis viruses, HIV, and rotaviruses.

DIRECT EXAMINATION OF FAECES FOR PARASITES Routinely faecal

specimens are examined in district laboratories by direct technique. This involves:

Reporting the appearance of the specimen and identifying any parasitic worms or tapeworm segments.

Examining the specimen microscopically for: –motile parasites such as the larvae of S.
stercoralis and trophozoites of E. histolytica, G. lamblia, and more rarely, B. coli,
–helminth eggs,

–cysts and oocysts of intestinal protozoa.

Need for faecal concentration techniques

The direct examination of faeces is essential to detect motile parasites and is usually adequate to detect
significant helminth infections. Important exceptions are Schistosoma species because only a few eggs
are usually produced even in moderate and severe infections, therefore a concentration technique
should be performed when intestinal schistosomiasis is suspected and no eggs are found by direct
examination.

Concentration techniques may also be required:

–To detect Strongyloides larvae, the eggs of Taenia, cysts of G. lamblia, and to make it easier to detect
small parasites, e.g. small fluke eggs, or the oocysts of intestinal coccidia prior to staining.

–To check whether treatment has been successful.

–To quantify intestinal parasites.

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.

Blood and mucus in faeces

blood and mucus may be found in faeces from patients with amoebic dysentery, intestinal
schistosomiasis, invasive balantidiasis (rare infection), and in severe T. trichiura infections. Other non-
parasitic conditions in which blood and mucus may be found include bacillary dysentery, Campylobacter
enteritis, ulcerative colitis, intestinal tumour, and haemorrhoids.

Presence of pus

This can be found when there is inflammation of the intestinal tract. Many pus cells can be found in
faecal specimens from patients with bacillary dysentery. They can also be found in amoebic dysentery
but are less numerous.

Pale coloured specimens

Pale coloured and frothy specimens (containing fat) can be found in giardiasis and other infections
associated with intestinal malabsorption.

Pale coloured faeces (lacking stercobilinogen) are also excreted by patients with obstructive jaundice.

Microscopical examination of faecal specimens

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. Importance of eosin: Eosin does not
stain living trophozoites but provides a pink background which can make them easier to see.
3 Examine immediately the preparations microscopically, first using the 10X objective with the
condenser iris closed sufficiently to give a good contrast. Use the 40X objective to identify motile
trophozoites, e.g. E. histolytica amoebae or G. lamblia flagellates. Note: The eggs of Schistosoma
species and T. trichiura, and the trophozoites of B. coli can also result in specimens containing blood
and mucus.

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 on the other end.
To avoid contaminating the fingers and stage of the microscope, do not use too large a drop of saline or
iodine.
2 Using a wire loop or piece of stick, mix a small amount of specimen, about 2mg, (matchstick head
amount) with the saline and a similar amount with the iodine. Make smooth thin preparations.
Cover each preparation with a cover glass.

NB: Sample from different areas in and on the specimen or preferably mix the faeces before sampling to
distribute evenly any parasites in the specimen. Do not use too much specimen otherwise the
preparations will be too thick, making it difficult to detect and identify parasites.

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 and 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 identif ication 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.

Non-parasitic structures found in faeces:

Care must be taken not to report as parasites those structures that can be normally found in 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, about 30–40m in
length. Calcium Oxalate crystals. After oxalate is formed, it normally combines with calcium to be
excreted in the stool.

FAECAL CONCENTRATION TECHNIQUES

The following techniques are commonly used to concentrate faecal parasites in laboratories:
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 (also spelt flotation) techniques in which parasites are concentrated by being floated in
solutions of high specific gravity, i.e. solutions that are denser than the parasites being concentrated.
Examples include the zinc sulphate method and saturated sodium chloride method.

Unlike the formol ether sedimentation technique, a single floata tion 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.
The method to used depend on:

–why the technique is being performed, the species of parasites requiring concentration and how will its
morphology is retained by a particular technique.

–the number of specimens to be examined and time available.

–the location, e.g. field or laboratory situation and equipment available.

–experience of staff performing the technique.

–health and safety considerations.

Formol ether concentration technique This is recommendedforuseindistrictlaboratories

becauseitisrapidandcanbeusedtoconcentratea widerangeoffaecalparasitesfromfreshorpreserved
faeces.EggsthatdonotconcentratewellbythistechniquearethoseofFasciolaspeciesandVampirolepis
nanabutconcentrationoftheseparasitesisnotusually required.Riskoflaboratoryacquiredinfectionfrom
faecalpathogensisminimizedbecauseorganismsare
killedbytheformalinsolution.Thetechnique,however,requirestheuseofhighlyflammableetheror
lessflammableethylacetate. When concentrating the oocysts of coccidia, an additional centrifugation
stage is required.

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.

Requirements

–Formol water, 10% v/v. Prepare by mixing 50ml of strong formaldehyde solution with 450ml of
distilled or filtered rain water. –Diethyl ether or ethyl acetate. * *Diethyl ether is a highly
flammable and volatile chemical and therefore its replacement by a less flammable chemical such as
ethyl acetate or acetone is recommended by some workers. When using ethyl acetate, greater care
needs to be taken when discarding the faecal debris layer to prevent remixing with the sediment.
Insoluble particles of ethyl acetate may form under the cover glass. It has an unpleasant odour.
–Sieve (strainer) with small holes, preferably 400–450m in size. The small inexpensive nylon tea or
coffee strainer available in most countries is suitable (can be used many times and does not corrode like
metal sieves).

Procedure

1 Using a rod or stick, emulsify an estimated 1g (pea-size) of faeces in about 4ml of 10% formol water
contained in a screw-cap bottle or tube. Note: Include in the sample, faeces from the surface and several
places in the specimen.

2 Add a further 3–4ml of 10% v/v formol water, cap the bottle, and mix well by shaking.

3 Sieve the emulsified faeces, collecting the sieved suspension in a beaker.

4 Transfer the suspension to a conical (centrifuge) tube made of strong glass, copolymer, or
polypropylene. Add 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, e.g. flame from the burner of a gas refrigerator, Bunsen burner, or spirit lamp.

5 Stopper* the tube and mix for 1 minute. If using a Vortex mixer, leave the tube unstoppered and mix
for about 15 seconds (it is best to use a boiling tube). *Do not use a rubber bung or a cap with a rubber
liner because ether attacks rubber.

6 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).

7 Centrifuge immediately at 750–1000g (approx. 3000 rpm) for 1 minute. After centrifuging, the
parasites will have sedimented to the bottom of the tube and the faecal debris will have collected in a
layer between the ether and formol water.

8 Using a stick or the stem of 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.

9 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.

10 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. Although the motility of
Strongyloides larvae will not be seen, the non-motile larvae can be easily recognized.

11 If required, count the number of each species of egg in the entire preparation. This will give the
approximate number per gram of faeces.

Formol ether oocyst concentration technique2

Follow steps 1 to 6 of the above method. Continue as follows:

7 Centrifuge immediately at low speed, i.e. RCF 300–400g (about 1000rpm) for 1 minute.

8 Using a plastic bulb pipette or Pasteur pipette, carefully remove the entire column of fluid below the
faecal debris and ether and transfer this to another centrifuge tube.

9 Add formol water to make the volume up to 10–15ml. Centrifuge at RCF 750–1000g (about 3000rpm)
for 5–10 minutes.

10 Remove the supernatant. Tap the bottom of the tube to resuspend and mix the sediment. Transfer
the sediment to a slide and examine for oocysts using the 40X objective.

Note: Cryptosporidium and Cyclospora oocysts are best identified in a modified Ziehl-Neelsen stained
smear. (with experience Cyclospora oocysts can be identified in unstained preparations). I. belli oocysts
can be easily identified in an unstained wet preparation.