if the supernatant fluid after the first wash is light tan or clear. Some prefer to limit the washing to one step (regardless of the clarity or color of the supernatant fluid after centrifugation) to eliminate additional manipulation of the specimen prior to centrifugation. The more the specimen is manipulated and/or rinsed, the more likely it is that some organisms will be lost and accidentally discarded prior to examination of the sediment.
5. Decant and discard the supernatant fluid, and resuspend the sediment on the bottom of the tube in 5 or 10% formalin. Fill the tube half full only. If the amount of sediment left in the bottom of the tube is very small or the original specimen contained a lot of mucus, do not add ethyl acetate in step 6; merely add the formalin, spin, decant, and examine the remaining sediment.
6. Add 4 to 5 ml of ethyl acetate. Stopper the tube, and shake it vigorously for at least 30 s. Hold the tube so that the stopper is directed away from your face.
7. After a 15- to 30-s wait, carefully remove the stopper.
8. Centrifuge for 10 min at 500 × g. Four layers should result: a small amount of sediment (containing the parasites) 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 (Fig. 3.5).
9. Free the plug of debris by ringing the plug with an applicator stick; decant and discard all of the supernatant fluid. After proper decanting, a drop or two of fluid remaining on the side of the tube may run down into the sediment. Mix this fluid with the sediment.
10. If the sediment is still somewhat solid, add 1 or 2 drops of saline or formalin to the sediment, mix, add a small amount of material to a slide, add a coverslip (22 by 22 mm, no. 1), and examine.
11. Systematically scan with the 10× objective. The entire coverslip area should be examined under low power (total magnification, ×100).
12. If something suspicious is seen, the 40× objective can be used for more detailed study. At least one-third to one-half of the coverslip should be examined under high dry power (total magnification, ×400), even if nothing suspicious has been seen. As in the direct wet smear, iodine can be added to enhance morphological detail, and the coverslip can be tapped to see objects move and turn over. The use of iodine is optional.
Results and Patient Reports from Sedimentation Concentration
Protozoan trophozoites and/or cysts and helminth eggs and larvae may be seen and identified. Protozoan trophozoites are less likely to be seen. In a heavy infection with Cryptosporidium spp. or C. cayetanensis, oocysts may be seen in the concentrate sediment; oocysts of C. belli can also be seen. Spores of the microsporidia are too small, and the shape resembles that of other debris within the stool; therefore, they are not readily visible in the concentration sediment. However, special stains can be performed on the sediment for the identification of coccidian oocysts and microsporidian spores.
1. Protozoan cysts may or may not be identified to the species level (depending on the clarity of the morphology).
Examples: Entamoeba coli cysts
Giardia lamblia (G. duodenalis, G. intestinalis) cysts
2. Helminth eggs and/or larvae may be identified.
Examples: Ascaris lumbricoides eggs
Hookworm larvae
3. C. belli oocysts may be identified; however, Cyclospora and Cryptosporidium oocysts are generally too small to be recognized and can be identified using appropriate immunoassays or modified acid-fast staining.
Example: Cystoisospora (Isospora) belli oocysts
4. Artifacts and/or other structures may also be seen and reported as follows.
Note These crystals and cells are quantitated; however, the quantity is usually assessed when the permanent stained smear is examined under oil immersion).
Examples: Moderate Charcot-Leyden crystals
Few RBCs
Moderate PMNs
Procedure Notes for Sedimentation Concentration
1. The gauze should never be more than one (pressed gauze) or two (woven gauze) layers thick; more gauze may trap mucus (containing Cryptosporidium oocysts and/or microsporidial spores).
2. Tap water may be substituted for 0.85% NaCl throughout this procedure, although the addition of water to fresh stool causes Blastocystis spp. cyst (central body) forms to rupture and is not recommended. In addition to the original 5 or 10% formalin fixation, some workers prefer to use 5 or 10% formalin for all rinses throughout the procedure.
3. Ethyl acetate is widely recommended as a substitute for ether (16). It can be used in the same way in the procedure and is much safer. Hemo-De can also be used and is thought to be safer than ethyl acetate (23).
A. After the plug of debris is rimmed and excess fluid is decanted, the sides of the tube can be swabbed with a cotton-tipped applicator stick while the tube is still upside down to remove excess ethyl acetate. This is particularly important if you are working with plastic centrifuge tubes or the plastic commercial concentrators. If the sediment is too dry after the tube has been swabbed, add several drops of saline before preparing the wet smear for examination.
B. If there is excess ethyl acetate in the smear of the sediment prepared for examination, bubbles will be present, which will obscure the material of interest.
4. If specimens are received in SAF, begin the procedure at step 2.
5. If specimens are received in fixative containing PVA, the first two steps of the procedure should be modified as follows.
A. Immediately after stirring the stool-PVA mixture with applicator sticks, pour approximately half of the mixture into a tube (container optional) and add 0.85% NaCl (or 5 or 10% formalin) almost to the top of the tube.
B. Filter the stool-PVA-saline (or formalin) mixture through wet gauze into a 15-ml centrifuge tube. Follow the standard procedure from here to completion, beginning with step 3.
6. Too much or too little sediment will result in an ineffective concentration sediment examination. See the section on Commercial Fecal Concentration Devices later in this chapter.
7. The centrifuge should reach the recommended speed before the centrifugation time is monitored. However, since most laboratories have their centrifuges on automatic timers, the centrifugation time in this protocol takes into account the fact that some time will be spent coming up to speed prior to full-speed centrifugation. If the centrifugation time at the proper speed (10 min at 500 × g) is reduced, some of the organisms (Cryptosporidium and Cyclospora oocysts or microsporidian spores) may not be recovered in the sediment.
Procedure Limitations for Sedimentation Concentration
1. Results obtained with wet smears (direct wet smears or concentration sediment wet smears) should usually be confirmed by permanent stained smears. Some protozoa are very small and difficult to identify as to species with just the direct wet smears. Also, special stains are sometimes necessary for organism identification.
2. Confirmation
