GHOR ALSAFI LAB.

stool Analysis

A.KAREEM AL-AJOURI 1

Stool Analysis









**. Collection and Preservation of Fecal Specimens:

1. Collection of Fecal Specimens:


Collection of Fecal Specimens

1. Collect stools in a dry, clean, wide-mouth, and leak-proof container. Make sure no
urine, water or other material gets in the container.

2. Label the sample with the:
- Patients name.
- Identification number.
- Date and time of collection.

3. The specimen must be accompanied by a request form from the physician giving:
- Relevant clinical details.
- Recent travel history.
- Which laboratory procedures are to be performed.













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A.KAREEM AL-AJOURI 2
2. Technical Notes on Collection of Fecal Specimens:

Technical Notes on Collection of Fecal Specimens
1. Fresh specimens should be examined, processed or preserved immediately or within a
maximum of one hour of collection. If a number of specimens are received at the same
time, pick out liquid stools and those containing mucus or blood and examine these first
for they may contain motile amoebae that die quickly. (In old specimens, the
morphology of parasites may change, may trophozoites die, and eggs may develop to
larvae).

2. Certain drugs and compounds will render the stool specimen unsatisfactory for
examination. The specimen should be collected before these substances are
administrated, or collection must be delayed until after the effects have passed.
These substances include:
-Barium or bismuth (7-10 days needed for clearance of effects).
-Antibiotics (2-3 weeks needed for clearance of effects).
-Mineral oil.
-Non-absorbable anti-diarrheal preparations.
-Anti-malarial agents.

3. Specimen collection may need to be repeated if the first examination is negative. It is
recommended to collect a specimen every 2-3 days for a total of three specimens. On
occasion, up to six samples may be necessary in a clinically suspect case. (Emission of
parasites is not regular like bacteria and viruses).

4. Confirmation of successful treatment can be made by a further examination of a single
sample on completion of treatment.


3. Preservation:

There are a number of reasons why a lag time may occur from the time of specimen
passage until examination in the laboratory (e.g., the workload in the laboratory or the
transit distance or time for the specimen to reach the laboratory).
To preserve protozoan morphology and to prevent the continued development of some
helminth eggs and larvae, the stool specimen can be placed in preservatives either
immediately after passage (by the patient) or once the specimen is received by the
laboratory.
There are several fixatives available (see table 3.3.3. General Preservatives for Stool
Specimens in Stool Examination for Intestinal Parasites Extra Readings), with the two
most commonly used being 10% formalin and PVA (polyvinyl alcohol). Because 10%
formalin and PVA have complementary advantages, it is recommended that the specimen
be divided and preserved in both types of preservatives.
Preserved specimens can be stored for several months.






GHOR ALSAFI LAB. stool Analysis

A.KAREEM AL-AJOURI 3
1. Formalin 10%:

Formalin 10%
Reagents
Formaldehyde 100 mL
0.85% NaCl 900 mL

Procedure
Formalin 10%:
Dilute 100 mL of formaldehyde with 900 mL of 0.85% NaCl solution.
(Distilled water may be used instead of saline solution).

Note Formaldehyde is normally purchased as a 37% to 40% solution;
however for dilution, it should be considered to be 100%.

Preservation
Add one volume of stools to three volumes of formalin 10%.
Protozoan cysts, coccidian oocysts, helminth eggs, and larvae are well
preserved for long periods.



2. Polyvinyl Alcohol (PVA):

Polyvinyl Alcohol (PVA)
Reagents
1. PVA:
PVA 10 g
Ethyl alcohol, 95% 62.5 mL
Mercuric chloride, saturated aqueous* 125.0 mL
Acetic acid glacial 10.0 mL
Glycerin 3.0 mL

2. * Mercuric chloride, saturated aqueous:
Mercuric chloride (HgCl
2
) 110 g
Distilled water 1,000 mL

Procedure
-Mix the liquid ingredients in a 500-mL beaker.
-Add the PVA powder (stirring is not recommended).
-Cover the beaker with a large petri dish, heavy wax paper, or foil,
and allow the PVA to soak overnight.
-Heat the solution slowly to 75C. When this temperature is reached,
remove the beaker and swirl the mixture for 30 seconds until a
homogenous, slightly milky solution is obtained.

Preservation
Emulsify one part of feces in three parts of PVA solution. This
method will preserve eggs, larvae and trophozoites well but cysts may
show some distortion. Fecal smears made from the feces/PVA mixture
and allowed to dry can be used for staining trophozoites.




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A.KAREEM AL-AJOURI 4

*** Macroscopic and Microscopic Examination of Stools:

1. Macroscopic Examination:

1.1. Consistency:

Fecal consistency varies with diet but certain clinical conditions associated with parasite
presence may be suggested by particular consistencies.
The presence of protozoan trophozoites in stools will depend on the consistency and
passage rate of the feces. Trophozoites are more likely to be found in unformed or liquid
stools and cysts in formed stools.
Stools should be recorded as one of the following:
Hard and dry.
Firm and formed.
Soft and formed.
Soft and unformed.
Liquid and watery.

1.2. Composition:

Stools may contain blood and mucus as evidence of ulceration or colitis due to:
Invasive amoebae (E. histolytica).
Bacillary dysentery (Shigella).
Other inflammatory bowel conditions.
There may be occult blood from gastric ulcers or fresh blood due to hemorrhoids.
Excessive bulky stools may indicate conditions such as giardiasis.

1.3. Color:

Pale yellowish stools are passed in steatorrhoeaic conditions such as giardiasis.
Dark or black stools occur when iron or bismuth is taken or when there is intestinal
hemorrhage.

1.4. Adult Parasites:

The feces may have adult helminths or segments present such as:
Ascaris lumbricoides.
Enterobius vermicularis.
Taenia sp.: Gravid Taenia segments are frequently motile for several days and
may migrate to the top of the container.


GHOR ALSAFI LAB. stool Analysis

A.KAREEM AL-AJOURI 5
2. Microscopic Examination:

The microscopic examination of the stool specimen consists of three separate techniques:
The direct wet smear.
The concentration method.
The permanent stained smear.
Each of these methods is designed for a purpose and forms an integral part of the total
examination.
In addition to normal fecal debris, the microscopic examination of fecal material may
reveal the following:
Trophozoites and cysts of intestinal protozoa.
Oocysts of coccidia and spores of microsporidia.
Helminth eggs and larvae.
Red blood cells (RBCs), which may indicate ulceration or other hemorrhagic
problems.
White blood cells (WBCs), specifically polymorphonuclear leukocytes (PMNs),
which may indicate inflammation.
Eosinophils, which usually indicate the presence of an immune response (which
may or may not be related to a parasitic infection).
Macrophages, which may be present in bacterial or parasitic infection.
Charcot-Leyden crystals, which may be found when disintegrating eosinophils are
present (and may or may not be related to parasitic infection).
Fungi (Candida spp.) and other yeasts and yeast-like fungi.
Plant cells, pollen grains, or fungal spores, which may simulate some helminth
eggs, protozoan cysts, coccidian oocysts, or microsporidial spores.
Plant fibers or root or animal hairs, which may simulate helminth larvae.















GHOR ALSAFI LAB. stool Analysis

A.KAREEM AL-AJOURI 6
2.1. Direct Wet Smear:

Direct Wet Smear
Materials
Microscope slides.
Cover-slips 22 x 22 mm.
Wooden applicator sticks.
Markers.
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Reagents

Saline (0.85% NaCl):
NaCl 0.85 g
Distilled water 100 mL
1. Dissolve the sodium chloride in distilled water in a flask or bottle.
2. Label as 0.85% NaCl.

Lugols Iodine:
Potassium iodide 10 g
Iodine crystals 5 g
Distilled water 100 mL

Stock solution (Lugols Iodine):
- Add the potassium iodide to the distilled water; when dissolved, add iodine crystals.
- Store in a brown bottle, at room temperature, in the dark.

Working solution: (3% Lugols Iodine):
- Dilute a portion of the stock solution 1:5 with distilled water.
- Note: the working solution should have a strong tea color and should be discarded when the color
lightens (usually within 10 to 14 days).
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Method
1. Place 1 drop of 0.85% NaCl on the left side of the slide and 1 drop of Lugols iodine (working
solution) on the right side of the slide.
2. Using an applicator stick, take a small amount of fecal specimen (the amount picked up at the end
of the applicator stick when introduced into a specimen), and thoroughly emulsify the stools in the
saline and iodine preparation (use separate sticks for each). If the stools are formed, take the portion
from well inside the sample and from the surface. If the stools contain mucus or are liquid, take the
portion from the bloodstained mucus on the surface or from the surface of the liquid.
3. Mark the number of the specimen on the slide with a marker.
4. Place a 22-mm cover-slip on each suspension.
5. Systematically scan both suspensions with the 10x objective. The entire cover-slip should be
examined under low power and low light intensity.
6. If something suspicious is seen, the 40x objective can be used for more detailed study. At least
one-third of the cover-slip should be examined with the 40x objective even if nothing suspicious
has been seen.
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Protozoan trophozoites, cysts, oocysts, and helminth eggs and larvae may be seen and
identified.


GHOR ALSAFI LAB. stool Analysis

A.KAREEM AL-AJOURI 7
2.2. Concentration Procedures:

Fecal concentration has become a routine procedure as part of the complete stool
examination for parasites.
Concentration procedures separate parasites from fecal debris and increase the chances of
detecting parasitic organisms when these are in small numbers.
Concentration procedures are divided into flotation techniques and sedimentation
techniques.

** Flotation Techniques:

Most frequently used: zinc sulfate or Sheathers sugar.
Flotation techniques use solutions which have higher specific gravity than the organisms
to be floated so that the organisms rise to the top and the debris sinks to the bottom.
The main advantage of this technique is to produce a cleaner material than the
sedimentation technique.
The disadvantages of most flotation techniques are that the walls of eggs and cysts will
often collapse, thus hindering identification. Also, some parasite eggs do not float.

** Sedimentation Techniques:

Use solutions of lower specific gravity than the parasitic organisms, thus concentrating
the latter in the sediment.
Sedimentation techniques are recommended for general diagnostic laboratories because
they are easier to perform and less prone to technical errors.
Refer to Extra Readings for Formalin-Ethyl Acetate Sedimentation technique.

** Permanent Stained Smears:

The permanent stained smear is recommended for use with every stool specimen
submitted for a routine parasite examination because:
It provides laboratories with a permanent record and a specimen that can be
reexamined as needed.
It can be sent to a reference laboratory when organisms with unusual morphology
are encountered or when identification is difficult.
There are a number of staining techniques available:
The older classical method is the iron hematoxylin method.
For routine diagnostic work, most laboratories select one of the shorter procedures
such as the trichrome stain.
Refer to Extra Readings for the technique.

**. Specialized Stains for Coccidia:

Oocysts of Cryptosporidium parvum, Isospora belli, and Cyclospora sp. may be difficult
to detect without special staining.
These organisms are acid fast and can be identified by acid fast stains (Modified Ziehl-
Neelsen stain).
Refer to Extra Readings for the technique.



GHOR ALSAFI LAB. stool Analysis

A.KAREEM AL-AJOURI 8



**** Other Techniques for Stool Examination:

*. Adhesive Cellophane Tape Method:

The eggs of the pinworm (Enterobius vermicularis) are deposited by the female worm
onto the perianal skin, usually at night.
Eggs or adults are rarely found in stools.
The most widely used diagnostic procedure for pinworm infection is adhesive cellophane
tape.


Adhesive Cellophane Tape Method

The adhesive tape is applied to the perianal area first thing in the morning
before the patient bathes or goes to the bathroom.
The tape is then stretched over a microscope slide for examination.
If an infection is present, eggs and sometimes adult worms of Enterobius
vermicularis will be present on the tape and can be seen under the microscope.
At least three consecutive negative slides should be observed before the
patient is considered free of infection.
Taenia eggs may also be collected in a similar manner.



**. Anal Swabs Method:

If no cellophane tape is available, the anal swab technique can be used.


Anal Swabs Method

Wipe round the anus (but not inside) with a cotton swab.
Dip the anal swab into a test tube containing 0.5 ml (10 drops) of
0.85% NaCl or 10% formalin.
Rinse the anal swab well in the solution.
Draw up the liquid with a Pasteur pipette. Transfer to a slide. Cover
with a coverslip and examine.







GHOR ALSAFI LAB. stool Analysis

A.KAREEM AL-AJOURI 9
** Reporting:

Reporting

The following details should be given when recording the results of a stool
examination:
1. Consistency of the stool (hard and dry, formed, unformed, liquid and watery).
2. Abnormal features seen with the naked eye: (mucus, mucus membranes,
bloodstained mucus, streaks of pus, blood superimposed on the stools).
3. Parasites found by microscopic examination specifying species, stage of
development and quantity.
Species: give the scientific Latin name. Write the first name (the
genus) with a capital letter, e.g. Shistosoma. Write the second name
(the species) with a small letter, e.g. mansoni.
Stage: eggs, larvae, vegetative form, worm segments, etc. When
describing E. Histolytica, always specify whether it contains ingested
red blood cells.
Quantity: occasional (1-2 eggs per slide); a few (3-5); a moderate
number (6-12); many (more than 12).
If no parasites are found, state No ova or parasites seen, and specify
whether this result was obtained by direct examination or by a
concentration method (name method used). Never state categorically:
No parasites.


**. Examples of Reports on Stool Examinations:

Examples of Reports on Stool Examinations
Patient 1 Stools hard and dry.
Few eggs of Trichuris trichiura found by direct microscopy.
Patient 2 Liquid stools, showing bloodstained mucus.
Moderate number of vegetative forms of E. histolytica, a few eggs of
Ancylostoma sp.
Patient 3 Firm, formed stools.
Direct examination: no ova or parasites seen.
Patient 4 Soft, unformed stools.
No ova or parasites seen by direct examination or after concentration
(formalin-ether method).
Patient 5 Semi-liquid, muddy stools.
A few larvae of Strongyloides stercoralis found by direct microscopy.
Patient 6 Stools soft and formed, showing streaks of blood.
Occasional eggs of Shistosoma mansoni present.
Patient 7 Firm, formed stools.
A few segments of Taenia saginata present.
Patient 8 Stool specimen received very small and dried-up.
Direct examination: no ova or parasites seen.


GHOR ALSAFI LAB. stool Analysis

A.KAREEM AL-AJOURI 10
** Quality Control of Stool Examination:

The different stages in the life cycles of parasites found in man makes the provision of a
quality control program for Parasitology difficult.
Microscopic diagnosis of parasites relies ultimately on the skill of the observer to detect
and identify a particular parasite stage.
The emphasis must be on:
Adequate training.
Using freshly prepared and preserved material, particularly from less encountered
species.
Each laboratory should have available a manual covering the required procedures
and aids to identification of parasites.
The number of specimens, time available, choice of the most useful method, cost as well
as the purpose of the test must be taken into account.
Participation into an external quality control program will encourage standardization of
techniques and adequate detection of parasites and accurate reporting.

** Dispatch of Stools for Detection of Parasites:

Stools may be sent to a specialized laboratory for the identification of rare
parasites that are difficult to recognize.
Preservatives used for dispatch of stools are:
10% formalin solution for wet mounting.
PVA for permanent staining.

1. Using 10% Formalin Solution:

In a lid covered bottle, prepare a mixture containing about 1 part of stool to 3 parts of
10% formalin solution.
Crush the stools thoroughly with a wooden applicator.
This method preserves specimens indefinitely if the bottle is tightly closed.

2. Using PVA:

2.1. In a bottle:

Pour about 30 mL of PVA fixative in a container, which should be three-quarters full.
Add enough fresh stools to fill the last quarter of the container.
Break up the stools thoroughly with a wooden applicator.
This method preserves all forms of parasites indefinitely.

2.2. On a slide:

To examine for amoebae and flagellates, place a small portion of the stools on one end of
the slide.
Add to the stool 3 drops of PVA.
Spread carefully with a wooden applicator.
Leave to dry for 12 hours (preferably at 37C).
Slides can be kept for about 3 months.
Slides can be stained on arrival to the specialized laboratory.
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**. Critical Elements of Competence for Evaluation

Ability to perform the technique of stool examination.
Understanding the pre-analytical (specimen collection and storage) and analytical
conditions (procedure and reading).
Ability to identify red blood cells, white blood cells, mucus threads, parasites and other
normal and pathological components of the stool sediment.
Ability to comply with all quality assurance measures of stool examination.
Express skill of using proper terminology in reporting gross and microscopic examination
of stools.