| | Re: How to concentrate stool sample for microscopy?
Target - This procedure is used for the routine recovery of protozoan cysts, helminth larvae, and ova (including operculate and Schistosoma eggs). The eggs of Hymenolepis nana and the cysts of Giardia lamblia and Iodamoeba butschlii do not show a good recovery ratio.
Reagents & Procedure - Transfer from ¼ to 1/2 teaspoon (about 2-4 g) of fresh stool into 10 ml of 5 – 10 % formalin (dissolve in 900 ml di dH2O, 4.0 g NaH2PO4 and 6.5 g Na2HPO, check pH at 7.0 and add 100 ml of formaldehyde [USP - aqueous solution of 37% formaldehyde with sufficient methanol (6-15%) to prevent polymerization]) placed in adapted container with broad mouth. Close tightly top and mix the stool and formalin thoroughly and let the mixture stand for a minimum of 30 min for fixation, that it's stable for several months. Once the sample arrive at the laboratory for analysis, 0.3 ml of a 20% aqueous solution of Triton X-100 was added to the slurry, and the mixture it comes before emulsified and after filtered through wet gauze (no more than two layers of nonsterile gauze) directly into a conical 15 ml centrifuge tube to give the desired amount of sediment (0.5 to 1 ml). Add 3 to 5 ml of ethyl acetate or for the better chemical properties HEMO-DE (Fisher cod.15-182-507A), at the mixture, stopper the tube, invert and shake vigorously for at least 10-30 sec. Hold the tube so the stopper is directed away from your face. After a 15 to 30 sec. wait, carefully remove the stopper. Centrifuge for 10 min at 500 x g or 1200-2500 rpm for 5-10 mins. Four layers will result in the tube after centrifugation : a small amount of sediment containing the parasites (if presents) in the bottom of the tube; a layer of formalin; a plug of fecal debris on top of the formalin layer; and a layer of ethyl acetate at the top. Free the debris plug with an applicator stick and carefully decant the top three layers, leaving the sediment undisturbed. Use a cotton swab to clean the remnants of ethyl acetate from inside of the tube, for don't reading disturb. Remix the sediment with an applicator stick, transfer a drop to a clean slide, add a coverslip (22x22 mm) and observe microscopically for parasites.