Stool analysis

Medical laboratory
science
Stool analysis
Outline:
• Macroscopic & physical examination of stool (Gross).
• Wet preparation (saline & lugol's iodine).
• Concentration techniques (FECT, flotation)
• Microscopic examination.
Alyazeed hussein, BSc-SUST

For whom stool analysis is required?
• Patient with abdominal pain.
• Patients with diarrhea.
• Patients with anemia.
• Patients with abnormal stool color.
• Too thin or do not grow well.
Specimen Collection for Stool Samples
• Stool samples are collected in clean, dry, leak-proof containers (not required to be sterile!!), since
fecal specimens contain many strains of normally occurring organisms.
• Three consecutive samples are tested!
• Don’t refrigerate the sample. test within 1 hour.
• Never leave stool specimens exposed to the air in containers without lids.
• Patient should avoid medication that affect the result (anti-parasite).
• Patients should be made aware that samples should not be contaminated with water, cleaners or
with urine!!
• Interference substances: Antibiotics, antiseptics, laxatives, mineral oil, soap.

Macroscopic & physical examination of stool
 Color: A normal stool is brown (stercobilin pigment). Normal stool color of infants is yellow-green.
• Abnormal:
• Clay colored (gray white) or pale stool (obstructing jaundice).
• Bright red > lower gastrointestinal bleeding (inflammation, anal fissure, hemorrhoids, tumors).
• Green color > medication and vitamins.
• Black > upper gastrointestinal bleeding (peptic ulcer, stomach carcinoma), or iron medication (anemia).
 Consistency:
• Normal: formed.
• Abnormal: Hard (constipation), semi formed & soft (parasitic infections), loose (diarrhea),
watery (bacterial infections), rice watery (cholera), steatorrhea (Giardiasis).
 pH: Normal: alkaline, varying according to type of diet. Acidic (amoebic dysentery), alkaline (shigellosis).
 Mucus: normally: few undetectable amount mixed with stool, high amount with pus indicate bacterial
infection, mucus with blood indicate amoebiasis.
 Blood: Nil. Blood seen in case of lower GIT bleeding (Bilharziasis, anal fissure, hemorrhoids).
 parasites: Nil. Parasites (worms) seen by naked eye: Ascaris lumbricoides, Enterobius vermicularis, Taenia spp.

Adult worm of Taenia spp.
Gravid (proglottid) segment
Adult worm of E. vermicularis
Adult worms of A. lumbricoidesAlyazeed hussein, BSc-SUST

Stool preservatives:
1. 5%–10% formalin (helminths eggs, larvae and protozoan cysts), concentration
techniques.
2. Polyvinyl alcohol (PVA) for protozoan trophozoites (E. histolytica), cysts and
helminths eggs and larvae.
3. Merthiolate –Iodine Formalin MIF, good for wet preparations and
concentration techniques (preservation and staining).
4. Sodium acetate–acetic acid–formalin (SAF) for protozoan cysts, trophozoites (E.
histolytica) and helminths eggs, larvae. For concentration techniques.
5. Bayer’s solution (modified formalin solution containing copper chloride, glacial
acetic acid, and formalin) for long period.
• Formed stools may be refrigerated for 1–2 days

Parasite stagesMaximum time for
examination
Stool
consistency
Trophozoites, helminth eggs, larvae30 minutesLiquid
Trophozoites, cysts, helminth eggs, larvae1 hourSemisolid
Cysts, helminth eggs, larvae24 hoursFormed
• 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,
Salmonellosis, Intestinal TB, Campylobacter enteritis, ulcerative colitis, intestinal tumor, and
hemorrhoids.
• Pasty stool with high fat content is suggestive of CBD Obstruction, Cystic fibrosis.
• Translucent gelatinous mucous on the surface of the formed stool is found in Spastic Constipation,
colitis.

Stool examination
 Wet preparation:
I. 0.85% Normal saline (NaCl) preparation.
II. 1% Lugol's iodine preparation.
III. Permanent stain for best morphology.
 Concentration techniques, more sensitive than direct wet mount
(detect small numbers of parasites):
I. Formol-ether concentration technique (FECT)
II. Flotation technique (zinc sulphate).

Wet preparation
Materials:
• Cover slips
• 0.85% Sodium chloride
solution (NaCl)
• Lugol’s Iodine Solution
• Wooden applicator
• Fresh stool
• Gloves

Wet preparation normal saline &
Lugol's iodine (Direct smear)
• Place a drop of normal saline (NaCl) or Lugol's
iodine is on a clean microscopic slide. Take a
small amount 2 mg, (matchstick head amount) of
stool with wooden stick. Mix well. Place coverslip,
avoid air bubbles formation!
• Examine under microscope.
• Note that! any part contain mucus, blood and pus
should be included & examined firstly (touch
different parts of stool).
• It is a rapid method for detect ova, cysts and
motile form of parasites, but not sensitive (light
infection).
• Iodine stain cysts, nucleus, glycogen (good
contrast). Note that! iodine kill the parasite
(motile form!).

 Examination of dysenteric and unformed specimens:
• 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.
• 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. Eosin
does not stain living trophozoites but provides a pink background which
can make them easier to see.

Concentration techniques
(concentrate & separate the
parasites from fecal debris)
 Formol-ether concentration technique (FECT): concentrate the parasites in
sediment, formalin for fixation, ether cleans the specimen by dissolving
and float the fat & fecal debris. Recovers eggs, larvae & cysts.
• Procedure:
1. Using a rod or stick, transfer a 1 g (pea-size) of faeces into 7 ml of 10%
formalin and mix well.
2. Sieve (tea strainer or gauze) and collect the suspension in a conical
(centrifuge) tube 15 ml.
3. Add 3 ml of ether, cap the tube then mix well by shaking for 1 minute.
4. Centrifuge at 1500 – 2500 rpm for 5 – 10 minutes.
5. Four layers will be formed, using a stick loosen the layer of fecal debris
from the side of the tube and invert the tube to discard the top three
layers leaving the sediment.
6. Place a drop of sediment and examine microscopically (10X and 40X).

Concentration techniques (cont.)
Flotation technique (33% zinc sulfate): floats parasites free of fecal debris by using
a solution having a specific gravity (1.180-1.200) greater than the parasites, good
for G. lamblia, E. histolytica and eggs of H. nana. Note that! This method not
suitable for operculated and larger eggs since they are too heavy to float, and the
protozoan cysts become distorted (difficult to identify). Ether is flammable!!
• Procedure:
1. In a glass beaker contain (3 – 4 ml) of zinc sulphate solution mix about 1 g of
faeces (or 2 ml if a fluid sample). Transfer the suspension to test tube.
2. Fill the tube with zinc sulphate. Using a Pasteur pipette, add the solution drop
by drop to fill the tube up to the top.
3. Carefully place a clean cover glass on top of the tube.
4. Leave undisturbed for 30 – 45 minutes!!!(don’t leave longer, the cysts become
distorted and the eggs will be being to sink.
5. Carefully remove the cover glass from the tube, place the cover glass face
downwards on a slide. Alternatively, the tube is centrifuged at 2500 rpm for 2
minutes, take a drop from the surface (supernatant).
6. Examine microscopically (10X and 40X). Alyazeed hussein, BSc-SUST

Permanent stains for faecal smears
• Trophozoites and cysts of the intestinal amoebae, flagellates and ciliates can be found and identified best in
permanently stained faecal smears.
1. Trichrome stain (fresh+Schaudinn’s or PVA preserved samples): for trophozoites or cysts of protozoa:
70% alcohol 2 min> 5 min in a mixture of iodine & 70% alcohol> 70% alcohol 2 times> stain in trichrome stain
10 min> acid alcohol 2-3 sec> dip slide in 95% alcohol then 100% alcohol then xylene> mounting medium
(DPX)> coverslip.
2. Iron haematoxylin stain (fresh, PVA or SAF preserved samples):
70% alcohol 5 min> 50% alcohol 2 min> tape water 5 min> haematoxylin stain 10 min> D.W 1 min> picric acid
1 min> tape water 10 min> 70% alcohol+1 drop of ammonia for 5 min> 95% alcohol 5 min> 100% alcohol then
xylene> mounting medium> coverslip.
3. Modified Kinyoun's Ziehl-Neelsen technique (acid-fast stain), for coccidia e.g. C. parvum:
Thin smear, air-dry fixed in methanol for 2-3 min> stain with cold carbol-fuchsin 5-10 min> differentiate in 1%
HCL-ethanol> rinse in tape water> counter stain with 0.25% malachite green or methylene blue 30 sec> rinse
in tape water.

Special technique
• Adhesive tape (cellophane)
method for detecting of
Enterobius vermicularis
(pinworm) eggs:
1. Prepare the tape.
2. Take sample on peri-anal
skin in the morning.
3. Stick tape on microscope
slide.
Cotton swab (dipped in
saline also can be used.
Fingernails.
1 2 3

Microscopic examination

Microscopic examination
• The 10x objective: eggs and larvae of helminths and of ciliates.
• The 40x objective: most of the protozoans.
• Oil immersion+Lugol’s solution smear: differentiation of species.

Fertile egg
Fertile egg (infective stage)
with larvae
Infertile egg Infertile decorticated
egg
Fertile decorticated
eggInfertile egg
Larvae from a broken egg
Ascaris lumbricoides

Trichuris trichiura
Capillaria
philippinensis

Atypical eggs of T. trichiura

Hookworm (Ancylostoma duodenale, Necator americanus)

Hookworm Mite egg

Strongyloides stercoralis

Rhabditiform larvae of S. stercoralis Rhabditiform larvae of Hookworm

Enterobius vermicularis (Pinworm)

Pollen grain

Starch granules

Schistosoma spp
S. mansoni S. japonicum S. intercalatum

Clonorrchis sinensis

Paragonimus westermani

Fasciola spp

Diphyllobothrium
latum
Taenia spp

Diphyllobothrium latumFasciola sppParagonimus westermani

Dipylidium caninum
Hymenolepis diminuta Hymenolepis nana

Dipylidium caninum Plant cell

Unknown artifact

Entamoeba
histolytica (Cysts
and trophozoites)

Entamoeba coli
(Cysts and
trophozoites

Giardia lamblia (Cysts
and trophozoites)

Pentatrichomonas
hominis
Giardia lamblia
Chilomastix
mesnili

Chilomastix mesnili

Blastocystis hominis (Vacuolar
forms)
Isospora belli (Oocysts)
Cryptosporidium parvum (Oocyst)

Miscellaneous & artifacts

Charcot-leyden Triple phosphate Calcium oxalatehematoidin
Crystals

Fat and fatty acid crystals

Plant fibers

Meat fibers
starch

yeast Fungal spore Pollen grainPollen grain
Pollen grain
Pollen grain
Pollen grain
Pollen graindiatoms diatoms diatomsMite eggMoral mushroom
Fungal spore Plant ringPlant cell
Plant cell
unknown

Rotifer

Infection control
Faecal specimens, like other specimens received in the laboratory, must be
handled with care to avoid aquiring infection from infectious parasites, bacteria
and viruses.
• Infective forms of parasites such as S. stercoralis, E. vermicularis, taenia spp, E.
histolytica, G. lamblia, B. coli and C. parvum.
• Bacteria such as Shigella, Salmonella and V. cholerae.
• Viruses such as hepatitis A, E and rotavirus.

In the minds of most laboratory personnel, analysis of fecal specimens
fits into the category of a “necessary evil” and few enjoy such tasks
• This has been a presentation of Alyazeed Hussein.
• Thanks for your attention and kind patience.
• Any questions, additions, or comments?

References
• Clinical Parasitology A PRACTICAL APPROACH, 2nd e
• Medical parasitology 5th e
• Bailey & Scott's DIAGNOSTIC MICROBIOLOGY, 14th e
• CDC.
• A handbook of medical laboratory technology 2nd e
• Fundamentals of the study of urine and body fluids, Springer.
• www.researchgate.com

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