DPDx Diagnostic Procedures for Stool Specimens

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Specimen Collection

Collect the stool in a dry, clean, leakproof container. Make sure no urine, water, soil or other material gets in the container.
This table demonstrates the distribution of protozoa in relation to stool consistency and should be taken into consideration when specimens are received.

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.
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.
Insure that the specimen is mixed well with the preservative. Formed stool needs to be well broken up.
Insure that the specimen containers are sealed well. Reinforce with parafilm or other suitable material. Insert the container in a plastic bag.
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, nonabsorbable antidiarrheal preparations, barium or bismuth (7-10 days needed for clearance of effects), antimicrobial agents (2-3 weeks), and gallbladder dyes (3 weeks).
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 is necessary when stool specimens cannot be examined within the prescribed time interval. Various preservatives are available (see table), with the two most commonly used being 10% aqueous formalin and PVA (polyvinyl-alcohol). If molecular detection (PCR) is required, refer to the molecular diagnosis section to obtain specific information on how to collect, preserve, and ship the specimens.

General Preservatives for Stool Specimens

*Preservative*	*Advantages*	*Disadvantages*
*10% Formalin*	All purpose fixative Easy to prepare Long shelf life Good preservation of morphology of helminth eggs, larvae, protozoan cysts, and coccidia Suitable for concentration procedures and epifluorescence microscopy Suitable for acid-fast, safranin, and chromotrope stains Compatible with immunoassay kits and epifluorescence microscopy	Not suitable for some permanent smears stained with trichrome Inadequate preservation of morphology of protozoan trophozoites Can interfere with PCR, especially after extended fixation time
*MIF* *(merthiolate-iodine-formaldehyde)*	Components both fix and stain organisms Easy to prepare Long shelf life Useful for field surveys Suitable for concentration procedures	Not suitable for some permanent smears stained with trichrome Inadequate preservation of morphology of protozoan trophozoites Iodine interferes with other stains and fluorescence Iodine may cause distortion of protozoa
*LV-PVA* *(low viscosity polyvinyl-alcohol)*	Good preservation of morphology of protozoan trophozoites and cysts Easy preparation of permanent smears stained with such as trichrome (solution both preserves organisms and makes them adhere to slides) Preserved samples remain stable for several months	Inadequate preservation of morphology of helminth eggs and larvae, coccidia, and microsporidia Contains mercuric chloride Difficult and expensive to dispose of Difficult to prepare in the laboratory Not suitable for concentration procedures Cannot be used with immunoassay kits Not suitable for acid-fast, safranin and chromotrope stains
*SAF* *(sodium acetate-acetic acid-formalin)*	Suitable for both concentration procedures and preparation of permanent stained smears Easy to prepare Long shelf life Suitable for acid-fast, safranin, and chromotrope stains Compatible with immunoassay kits	Requires additive (e.g., albumin-glycerin) for adhesion of specimens to slides Permanent stains not as good as with PVA or Schaudinn's fixative
*Schaudinn’s Fixative*	Good preservation of morphology of protozoan trophozoites and cysts Easy preparation of permanent stained smears	Less suitable for concentration procedures Contains mercuric chloride Inadequate preservation of morphology of helminth eggs and larvae, coccidia, and microsporidia Poor adhesion of liquid or mucoid specimens to slides
*Modified PVA* *copper or zinc*	Permanent smears can be made and stained with trichrome Zinc is preferred over copper No mercuric chloride	Staining not consistent Organism morphology may be poor Copper-morphology of cysts and trophozoites is poor Zinc-better morphology but not comparable to LV-PVA
*One-Vial Fixatives* *(such as Ecofix, Parasafe, Unifix, Proto-fix, STF, and others that may be available)*	Concentrate and permanent smear can be made out of one vial Immunoassays can be done on most No mercuric chloride	Certain one-vial fixatives must use certain stains Color difference of stain Staining not always consistent Sometimes more expensive than formalin and LV-PVA

Because 10% formalin and PVA have complementary advantages (see table), 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, one example being the Para-Pak Stool System® (Meridian Bioscience Inc., Cincinnati, OH). Preserved specimens can be stored for several months.

For additional information on stool collection, call the Division of Parasitic Diseases at 770-488-4474.