are too small, and the shape resembles other debris within the stool; therefore, they are not readily visible in the concentration sediment.
1. Protozoan cysts may or may not be identified to the species level (depending on the clarity of the morphology).
Example: Giardia lamblia (G. duodenum, G. intestinalis) cysts
2. Helminth eggs and/or larvae may be identified.
Example: Hookworm eggs
3. C. belli oocysts may be identified; however, Cyclospora and Cryptosporidium oocysts are generally too small to be recognized or identified. Subsequent immunoassays or modified acid-fast staining is recommended.
Example: Cystoisospora (Isospora) belli oocysts
4. Artifacts and/or other structures may also be seen and reported as follows.
Note These cells are quantitated; however, the quantity is usually assessed when the permanent stained smear is examined.
Examples: Few macrophages
Moderate PMNs
Procedure Notes for Flotation Concentration
1. The gauze should never be more than one or two layers thick; more gauze may trap mucus (containing Cryptosporidium oocysts, Cyclospora oocysts, and/or microsporidial spores). A round-bottom tube is recommended rather than a centrifuge tube.
2. Tap water may be substituted for 0.85% NaCl throughout this procedure, although the addition of water to fresh stool causes Blastocystis spp. (Blastocystis hominis) 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. If fresh stool is used (nonformalin preservatives), the zinc sulfate should be prepared with a specific gravity of 1.18. If formalinized specimens are to be concentrated, the zinc sulfate should have a specific gravity of 1.20.
4. If specimens are received in SAF or other single-vial preservatives, begin the procedure at step 2.
5. If fresh specimens are received, the standard procedure requires the stool to be rinsed in distilled water prior to the addition of zinc sulfate in step 4. However, the addition of fresh stool to distilled water will destroy any Blastocystis spp. (Blastocystis hominis) cysts present and is not a recommended approach.
6. Some workers prefer to remove the tubes from the centrifuge prior to sampling the surface film. This is acceptable; however, there is more chance that the surface film will be disturbed prior to sampling.
7. Some workers prefer to add a small amount of zinc sulfate to the tube so that the fluid forms a slightly convex meniscus. A coverslip is then placed on top of the tube so that the undersurface touches the meniscus. It is left undisturbed for 5 min. The coverslip is then carefully removed and placed on a slide for examination. This approach tends to be somewhat messy, particularly if too much zinc sulfate has been added.
8. When using the hydrometer (solution at room temperature), mix the solution well. Float the hydrometer in the solution, giving it a slight twist to ensure that it is completely free from the sides of the container. Read the bottom meniscus and correct the figure for temperature, if necessary. Most hydrometers are calibrated at 20°C. A difference of 3°C between the solution temperature (room temperature) and the hydrometer calibration temperature requires a correction of 0.001, to be added if above and subtracted if below 20°C.
Procedure Limitations for Flotation Concentration
1. Results obtained with wet smears (direct wet smears or concentrated specimen wet smears) should usually be confirmed by permanent stained smears. Some protozoa are very small and difficult to identify to the species level with just the direct wet smears. Also, special stains are sometimes necessary for organism identification.
2. Confirmation is particularly important for E. histolytica/E. dispar versus E. coli.
3. Protozoan cysts and thin-shelled helminth eggs are subject to collapse and distortion when left for more than a few minutes in contact with the high-specific-gravity zinc sulfate. The surface film should be removed for examination within 5 min of the time the centrifuge comes to a stop. The longer the organisms are in contact with the zinc sulfate, the more distortion will be seen on microscopic examination of the surface film.
4. 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.
5. If zinc sulfate is the only concentration method used, both the surface film and the sediment must be examined to ensure detection of all possible organisms.
Commercial Fecal Concentration Devices
There are a number of commercially available fecal concentration devices which may help a laboratory to standardize the concentration technique. Standardization is particularly important when personnel rotate throughout the laboratory and may not be familiar with parasitology techniques. These devices help ensure consistency, thus leading to improved parasite recovery and subsequent identification. Some of the systems are enclosed and provide a clean, odor-free approach to stool processing, features that may be important to nonmicrobiology personnel processing such specimens. Both 15- and 50-ml systems are available. It is important to remember that a maximum of 0.5 to 1.0 ml of sediment is needed in the bottom of the tube. Often, when the 50-ml systems are used, there is too much sediment in the bottom of the tube. This problem can be solved by adding less of the fecal specimen to the concentration system prior to centrifugation. Since the sediment is normally mixed thoroughly and 1 drop is taken to a coverslip for examination, good mixing may not occur if too much sediment is used. There also appears to be layering in the bottom of the tubes; again, adding less material to the concentrator at the beginning should help eliminate this problem (Fig. 3.9 through 3.12).
Figure 3.9 (Upper) FPC JUMBO large concentration tubes and connector system (Evergreen Scientific). (Lower) Small concentration tubes and FPC HYBRID connector system (Evergreen Scientific). doi:10.1128/9781555819002.ch3.f9
Figure 3.10 Stool collection vial and funnel used in fecal concentration (Hardy Diagnostics). doi:10.1128/9781555819002.ch3.f10
Figure 3.11 (Top) PARA-SED concentration system with both small and large tubes (Medical Chemical Corp.). (Middle) SED-CONNECT system with collection vials and various reagents (Medical Chemical Corp.). (Bottom) Filter attachment system (Medical Chemical Corp.). Note the screen, which is a substitute for the gauze in the traditional gauze/filter concentration method. See also MICRO-SED. doi:10.1128/9781555819002.ch3.f11
Figure 3.12 (Upper left) MACRO-CON concentration system (Meridian Bioscience). (Upper right) SPINCON concentration system (Meridian Bioscience). (Lower) Funnel used in fecal concentration (Meridian Bioscience). doi:10.1128/9781555819002.ch3.f12
