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STOOL EXAMINATIONA COMPREHENSIVE APPROACH

Presented by: Lubna Colour of the specimen.Texture: formed, semi-formed, unformed or fluidPresence of blood, pus, mucus, worms, or tape worm segments?Normal adult faeces appear brown and formed or semi-formed. Infant faeces are yellow, green and semi-formed.

Macroscopic ExaminationAppearancePossible causeUnformed containing pus and mucus mixed with blood.Shigellosis , Campylobacter enteritisUnformed with blood and mucus (acid pH)Amoebic dysenteryUnformed or semi formed often with blood and mucusSchistosomiasisWatery stoolETEC infection, Rotavirus enteritisRice water with mucous flakesCholeraUnformed or watery and sometimes with blood and pus.Salmonella infectionUnformed, pale colored, frothy, unpleasant smelling stools that floats on water (High fat contents)Giardiasis, Other conditions that cause malabsorption e.g. post infective tropical malabsorption.Fluid stools (containing lactose) with pH below 6.Lactase deficiencyUnformed or semi-formed black stools(Positive occult blood)Melaena, Hookworm disease, Iron therapyA. Direct Microscopy:Label a frosted end slide with Patient IDPlace a drop of saline at one end and a drop of iodine at the other end of the slide.Using a wooden applicator stick, emulsify a portion of specimen firstly in the saline and then in the iodine. Apply a cover slip to each preparations. The preparations should be thick enough so as to just read newsprint through them.Examine both wet mounts for the presence of leucocytes, erythrocytes, cysts, ovaReport the results into a log.

Microscopic Examination:B. Stool Concentration Technique:Sedimentation: Formol ether sedimentation.Floatation: Simple Floatation using SalineZnSO4 centrifugal floatation

Procedure for Formol Ether Sedimentation TechniqueAdd 9ml of 5% - 10% formalin to the flat-bottomed tube.Add 3 rounded spoonful of preserved (or 1 spoonful of fresh) stool. For fresh stool specimens, allow 30 minutes for fixation before proceeding to the next step.Add 3 drops of Triton X-100 to the mixed specimens.Add 3ml of ethyl acetate. Pull the vent-straw in the strainer unit out approximately 1 inch.Attach the FPC strainer tightly to the flat-bottomed tube containing the faecal specimen. Shake vigorously for 30 seconds. Pointing the conical end downward, shake the specimen through the strainer into the 15 ml centrifuge tube. Unscrew the FPC strainer with the flat-bottomed tube still attached.Discard the transport tube and strainer in an appropriate manner. Cap the 15ml tube and centrifuge at 500 x g for 10 minutes. After centrifugation, the specimen should appear clearly separated into four layers. See illustration below:

Rim the debris layer using an applicator stick. Pour off the debris and supernatant fluid.With the tube still inverted, use a cotton-tipped applicator stick to clean and remove the remaining debris and ethyl acetate.Return the tube to an upright position and add 2 to 3 drops of 5% to 10% formalin, saline and mix the sediment thoroughly.Prepare slides with a disposable transfer pipette, Coverslip and examine. Use the remaining specimen to examine for coccidian oocysts.Scan the entire Coverslip area systematically using 10 x objectives.Use the 40x objective to examine at least one third of the coverslip area, as well as any areas where suspicious objects may have been seen on LP.Following low power scanning, adding iodine may facilitate the detection of cysts by enhancing morphological detail. Use the 40x objective for this examination. Adding iodine prior to low power scanning should be done cautiously; at lower magnification, some helminth eggs may stain darkly as to appear to be faecal debris.Formol ether sedimentation can be used for detection of all helminthic and protozoan ova and cysts.

RELATIVE SIZE OF PARASITES

Proto-fix Wheatleys Trichrome Stain Set is used for this purpose.Rapid staining procedure for intestinal amoeba and flagellates.

C. Permanent StainingPermanent smears can be prepared from Unconcentrated specimen fixed in Proto-fixA specimen in Proto-fix hat has been concentrated using CONSED concentration procedure.Preparation of smear:Transfer 1-2 drops of the Proto-fix fixed specimen to a slide.May use coated slides like CELLBOND for better adhesion.Lay or hold the slide flat with specimen side up.Gently and evenly spread the sample.Spread the sample out to create thick and thin areas using a chopping motion.Allow to stand for 1-2 minutes. Or until it is dried.

Specimen Collection and Preparation70% Ethanol-1.5 minutes. Drain off excess.70% Ethanol-1.5 minutes. Drain off excess.Trichrome stain-13 minutes. Drain off excess. 90% Acid Ethanol-1 3 seconds. Drain off excess.100% Ethanol- 5 10 seconds. Drain off excess.100% Ethanol-1 minutes. Drain off excess.100% Ethanol-1 minutes. Drain off excess.PRO-Clear-3 minutes. Drain off excess.Mount Cover Slip and examine under Oil Immersion Objective.

Staining ProcedureNuclear Chromatin, Chromatoid Bodies, Ingested RBCs, Bacteria- Purple to red-violetCytoplasm of trophozoites and cysts- Blue tinted with PurpleMacrophages, WBCs, Yeast- Variable- Green to blue to purple or redBackground Material and artifacts blue-green to purple

Expected ResultsMICROMETRY

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