321778831-Medical-Parasitology-A-Self-Instructional-Text-6th-Edition.pdf

However some eggshells such as ascaris are im

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However, some eggshells, such as Ascaris, are im- pervious to formalin. 2. Strain the mixture through two layers of damp- ened surgical gauze into a 15-mL glass conical cen- trifuge tube and add enough saline to nearly fill the tube. a. Do not use more than two layers of surgical gauze or more than one layer of the newer “pressed” gauze because thicker layers trap mu- cus that may contain Cryptosporidium spp. oocysts or microsporidia. b. Do not strain any specimen that contains a large amount of mucus. Instead, centrifuge the mix- ture for 10 minutes at 500 g, decant into disin- fectant, and continue the procedure with step 7. 3. Centrifuge the suspension at 500 g (1,500 rpm) for 10 minutes. Decant the supernatant into disinfec- tant. Resuspend the sediment in saline or formalin and recentrifuge if the sample contains excessive CHAPTER 7 Clinical Laboratory Procedures 143 FIGURE 7-2 tahir99-VRG & vip.persianss.ir
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debris. (The rpm values are noted so that workers may more easily adjust speeds to reach the appropri- ate gravity when using common tabletop cen- trifuges, but proper calibration steps should be performed to verify speeds.) 4. Resuspend the washed stool sediment in 7 mL of 10% formalin and add 4 mL of ethyl acetate. Cap the tube with a rubber stopper and shake it vigor- ously for 30 seconds. This step extracts fats from the feces and reduces bulk; do not use if a very small amount of debris is present or if the original speci- men contains much mucus. Carefully remove the stopper away from the face because organic vapor may cause spurting of fecal debris. 5. Centrifuge the tube for 10 minutes at 500 g. Four layers should result (see Figure 7-3). 6. Rim the upper debris layer with an applicator stick and decant the entire supernatant into disinfectant. Invert the centrifuge tube completely in one smooth motion, but do it only once. If excess ethyl acetate is left inside the tube or if the tube is plastic, use a cotton-tipped applicator stick to swab the inside of the tube while the tube is still inverted. Excess ethyl acetate appears as bubbles when the slide prepara- tion is made and may dissolve plastic tubes. 7. Mix together a small drop of fecal sediment and 1 drop of iodine stain on a slide. Add a cover glass and carefully examine the entire preparation micro- scopically for parasites. Also examine an unstained preparation of the concentrate because cysts are refractile and more easily detected unstained and because the morphology of unstained larval forms is more characteristic. Of Note 1. The sedimentation procedure can be used to con- centrate PVA-fixed material as follows. Thoroughly mix the PVA-stool suspension with applicator sticks, add about 4 mL of the mixture to a test tube con- taining 10 mL of saline, and mix well. Filter the mix- ture through gauze as in step 2 and continue the procedure as described. Sodium acetate-acetic acid- formalin (SAF)–preserved specimens can be processed beginning directly at step 2. Cystoisospora belli is usually missed in PVA-preserved concen-
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