Introduction to STOOL EXAMINATION
- Specimen Collection — Stool specimens should be collected in a wide- mouthed, clean , leak proof , screw — capped containers.
- Timing- Specimen should be collected before starting antiparasitic drugs and closer to the onset of symptoms.
- Frequency: At least three stool specimens collected on alternative days are adequate to make the diagnosis.
SPECIMENS AND OTHER STOOL:
- Swabs — Useful for detecting eggs of Enterobius vermicularis deposited on the surface of perianal skin.
- Duodenal contents — It is very useful for the detection of small intestine parasites like , Giardia and larva of Strongyloides stercoralis .
- Mucoid bloody stool : acute amoebic dysentery , invasive balantidiasis.
- Dark red stool : indicates upper gastrointestinal tract (GIT) bleeding and a bright red stool is suggestive of bleeding from lower GIT.
- Frothy pale offensive stool containing fat found in giardiasis.
- Adults worms like roundworm , threadworm or segments of tapeworm may be seen.
WHEN TO EXAMINE :
- Liquid stool specimens should be examined within 15-30 minutes, semisolid stools within | hour and formed stools up to 24 hours after collection .
- On prolonged storage , trophozoites may disintegrate , become non- motile and may appear as artifacts .
- Preservatives such as 10% of formalin or polyvinyl alcohol can be used to maintain the morphology of the parasitic cysts and eggs .
- Laboratory diagnosis of parasitic infections is carried out by various methods.
- Morphological identification techniques- stool microscopy and
peripheral blood smear
- Immunodiagnostic methods
- Molecular methods
- Imaging techniques.
DIRECT WET MOUNT (SALINE AND IODINE MOUNT)
- Drop of saline and Lugol’s iodine are placed on two comers of a slide. A small amount of feces is mixed by a stick to form a uniform smooth suspension.
- Cover slip is placed on the mount and examined under low power objective (10X) followed by high power objective (40X).
- Useful in the detection of trophozoites and cysts of protozoa and eggs and larvae
- Motility of trophozoites and larvae can be demonstrated.
- Bile staining property can be appreciated: bile stained eggs appear golden brown and non bile stained appear colorless
- Nuclear details of protozoan cysts and helminthic eggs or larvae
cannot be clearly visualized.
- Nuclear details of cysts , helminthic eggs and larvae are better visualized.
- Helps in species identification.
- Jodine immobilizes and kills paracites, hence motility of protozoan trophozoites and helminthic larvae cannot be appreciated.
- Bile staining property cannot be appreciated.
TYPES OF IODINE, STAINS
- Lugols iodine
- D Antoni’s iodine
- Dobeils iodine
Following structures can be visualized by microscopic examination of stool specimen
- Normal constituents : such as plant fiber, starch cells, muscle fibres , pollen grains .yeast cells .bacterial epithelial cells .fat globules n air bubbles .
- Cellular elements: Pus cells , red cells.
- Charcot Leyden crystals.
- Trophozoites and cysts of protozoa and eggs n larvae of helminths.
Cyst & Trophozoite of Giardia
BILE STAINED EGG
- Trichuris trichura.
- Ascaris lumbricoides.
- Taenia saginata.
EGG THAT FLOAT ON SATURATED SOLUTION OF
COMMON SALT IS
- Fertilized egg of ascaris lumbicoid
- Enterobius vermicularis
- Ankylostoma duodenale
- Trichuris trichura
- Hymenolepsis nana
PERMANENT STAINED SMEAR
- Permanent stained smears are required for accurate diagnosis of intestinal parasites. Commonly used methods are :
- Iron — hematoxylin stain
- Trichrome stain
- Modified acid – fast stain .
- The permanent stained smears helps in the accurate diagnosis of cysts and trophozoites by staining their internal structures .
- If the paracite output is low in feces, direct examination may not be able to detect paracites .then stool specimens need to be concentrated.
- Eggs cysts larvae are recoverd but trophozoites get destroyed.
- Commenly used concentration techniques are:
- Sedimentation techniques.
- Floatation techniques.
- Eggs and cysts settle down at the bottom following centrifugation.
- Formalin – ether concentration technique.
- Formalin – acetone sedimentation technique.
- Formalin_ethyl acetate concentration technique.
- Eggs and cysts float at the surface due to specific gravity gradient.
- Saturated salt solution technique.
- Zinc sulphate floatation concentration technique.
- Sheathers sugar floatation technique.
PRESERVATION OF FECAL SPECIMENS
- Is essential because of following reasons:
- To maintain morphology of cyst .eggs and larvae.
- To prevent further development of helminthic eggs and larvae.
- For teaching purpose.
- For epidemiological analysis.
- Transport of specimen to a referral lab for further identification.
METHODS OF PRESERVATION
- Formalin fixative method
- Sodium acetate formalin fixative method.
- Merthiolate-iodine formalin solution fixative method.
- Schaudinns fluid.
EGG COUNTING(EGG QUANTIFICATION) METHODS
- The intensity of intestinal helminthic infection can be estimated using egg counting in the feces by following methods:
- Direct smear counting method of beaver.
- Katos cellophane tape.
- Stolls method or dilution egg counting method.
- 4 g of feces is mixed thoroughly withS6 ML of N/10 NAOH in a calibrated Stoll’s flask and a uniform suspension is made.
- 0.15 ML of this mixture is transferred to the slide. The slide is kept over 4 mechanical stage.
- Examine under tow power objective and count the total number of eggs(n).
- The no of eggs per grm of feces(N) is calculated by multiplying the count (n) with 100.
- Estimated daily output of eggs is calculated by multiplying the no of eggs ‘gram with the weight of 24 hour fecal sample.
Other Microbiology Notes :-
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