Stool Specimens

Collection

Budi Setiawan
Stool/Feces

Feces consist of:

• undigested food residue
• pigments and bile salts
• Intestinal secretions including mucus
• Leukocytes that migrate from the bloodstream
• epithelium
• Bacteria
• Inorganic materials, especially calcium and phosphate
• Undigested food (in very small amounts)
• Gas
Patients Preparation
• Describe how to collect and what will be checked
• The patient is asked to defecate in a wide-mouthed stool container
• Don't pee in the shelter
• Do not put toilet paper in the tray as this will affect the results
Requirment

1.Collect the stool in a dry, clean, leakproof container. Make sure no urine,
water, soil or other material gets in the container.
2.The image on the right demonstrates the distribution of protozoa in
relation to stool consistency and should be taken into consideration when
specimens are received.
3.Fresh stool should be examined, processed, or preserved immediately. An
exception is specimens kept under refrigeration when preservatives are
not available; these specimens are suitable for antigen testing only.
4. Preserve the specimen as soon as possible. If using a commercial
collection kit, follow the kit’s instructions. If kits are not available, the
specimen should be divided and stored in two different preservatives, 10%
formalin and PVA (polyvinyl-alcohol), using suitable containers. Add one
volume of the stool specimen to three volumes of the preservative.
5. Insure that the specimen is mixed well with the preservative. Formed
stool needs to be well broken up.
6. Insure that the specimen containers are sealed well. Reinforce with
parafilm or other suitable material. Insert the container in a plastic bag.
7. Certain drugs and compounds will render the stool
specimens unsatisfactory for examination. The specimens
should be collected before these substances are
administered, or collection must be delayed until after the
effects have passed. Such substances include: antacids,
kaolin, mineral oil and other oily materials, non-absorbable
antidiarrheal preparations, barium or bismuth (7-10 days
needed for clearance of effects), antimicrobial agents (2-3
weeks), and gallbladder dyes (3 weeks).
8. Specimen collection may need to be repeated if the first
examination is negative. If possible, three specimens passed
at intervals of 2-3 days should be examined.
Preservation of specimens

Necessary when stool specimens cannot be examined within the prescribed

time interval. Various preservatives are available, with the two most
commonly used being 10% aqueous 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 (add one volume of stool to three volumes of the preservative)

Commercial two-vials kits are available for this purpose. Preserved

specimens can be stored for several months.
Specimen Processing

• Examination of fresh specimens permits the observation of motile trophozoites,

but this must be carried out without delay.
• Liquid (diarrheic) specimens (which are more likely to contain trophozoites) should be
examined within 30 minutes of passage (not within 30 minutes of arrival in the
laboratory!)
• Soft specimens (which may contain both trophozoites and cysts) should be examined
within one hour of passage.
• If delays cannot be avoided, the specimen should be preserved to avoid
disintegration of the trophozoites.
• Formed specimens (less likely to contain trophozoites) can be kept for up to one day,
with overnight refrigeration if needed, prior to examination.
Shipment

Shipment of Unpreserved Specimens

• On some occasions, unpreserved stool is requested in order to isolate a
known or suspected pathogen (i.e., culture for microsporidia, PCR testing).
In these cases, the specimen must be placed in a clean container as
quickly as possible and kept under refrigeration until necessary
arrangements are made for pick-up and delivery by an overnight courier.
• The sender must ensure that the specimen remains cold during transport
by using available packing materials such as cold-packs. CDC follows
regulations describing the requirements for proper packaging and
shipment of biomedical materials
Shipment of Preserved Specimens
• The rules for the shipment of preserved specimens are the same as those for
unpreserved specimens except that they do not need refrigeration

Packaging
• Stool specimens must be packaged so as to avoid leakage. The package
should be able to withstand rough handling and passage through routinely
used sorters, conveyer belts, etc
Staining
Modified Acid-Fast Staining Procedure
• This technique is useful for the identification of oocysts of the coccidian species
(Cryptosporidium, Cystoisospora, and Cyclospora), which may be difficult to detect with
routine stains such as trichrome. Unlike the Ziehl-Neelsen Modified Acid-Fast Stain, this
stain does not require the heating of reagents for staining.
Chromotrope Staining Procedure
• This staining method was developed at CDC using various components of the trichrome
staining method to differentiate microsporidia spores from background fecal elements.
Quick-Hot Gram-Chromotrope Staining Procedure
• This is an alternative stain to the chromotrope procedure that is a fast,
reliable, and simple method of staining smears to demonstrate
microsporidian spores in fecal and other clinical specimens.
Trichrome Staining
• It is generally recognized that stained fecal films are the single most productive
means of stool examination for intestinal protozoa.
• The permanent stained smear facilitates detection and identification of cysts and
trophozoites and affords a permanent record of the protozoa encountered. Small
protozoa, missed by wet mount examinations (of either unconcentrated or
concentrated samples) are often seen on the stained smear.
• The Wheatley Trichrome technique for fecal specimens is a modification of Gomori’s
original staining procedure for tissue. It is a rapid, simple procedure, which produces
uniformly well-stained smears of the intestinal protozoa, human cells, yeast, and
artifact material
Trichrome Staining Procedure
Specimen:
• The specimens usually consist of fresh stool or stool fixed in polyvinyl alcohol
(PVA) smeared on microscope slides and allowed to air dry or dry on a slide
warmer at 60°C. Stool preserved in sodium acetate-acetic acid-formalin (SAF)
or some of the one-vial fixatives can also be used.
Reagents
• 70% Ethanol plus iodine: prepare a stock solution by adding iodine crystals to 70% alcohol until you obtain a dark
solution. To use, dilute the stock with 70% alcohol until a dark reddish brown color or strong tea color is obtained.
• 70% Ethanol (twice)
• Trichrome Stain
• 90% Acid Ethanol (90% Ethanol (99,5ml) + Acetid acid-glacial (0,5ml)
• 95% ethanol
• 100% ethanol (twice)
• Xylene or xylene substitute (twice)
Procedure

• For PVA smears, place the slide in 70% ethanol plus iodine for 10 minutes.
• Place slide in 70% Ethanol for 5 minutes.
• Place in second 70% Ethanol for 3 minutes
• Place in Trichrome stain for 10 minutes.
• Destain in 90% ethanol plus acetic acid for 1 to 3 seconds.
• Rinse several times in 100% ethanol.
• Place in two changes of 100% ethanol for 3 minutes each.
• Place in two changes of xylene or xylene substitute for 10 minutes.
• Mount with coverslip using mounting medium (e.g., permount).
• Examine the smear microscopically utilizing the 100× objective.
• Examine at least 200 to 300 oil immersion fields.
Differential Morphology of Protozoa Found in Stool Specimens of Humans: Amoebae-Trophozoites
Differential Morphology of Protozoa Found in Stool Specimens of Humans: Amoebae-Cysts
Differential Morphology of Protozoa Found in Stool Specimens of Humans: Flagellates-
Trophozoites
Differential Morphology of Protozoa Found in Stool Specimens of Humans: Flagellates-Cysts
Differential Morphology of Protozoa Found in Stool Specimens of Humans: Ciliates, Coccidia, and
Blastocystis
Protozoa Found in Stool Specimens of Humans: Amebae
Protozoa Found in Stool Specimens of Humans: Ciliates and Flagellates
Protozoa Found in Stool Specimens of Humans: Coccidia and Blastocystis
Source
https://www.cdc.gov/dpdx/diagnosticprocedures/stool/specimencoll.html