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Automated Workstation for the Microscopic Analysis of Fecal Concentrates
The FE-5 (Apacor Ltd USA, Brooklyn, NY) is a countertop workstation that automates the microscopic analysis of fecal concentrates (Fig. 3.13). The system automates the aspiration, resuspension, staining or diluting (based on user preference), transfer, presentation, and disposal of fecal concentrates. When the sample button is pressed, within 5 s two samples of fecal concentrate are automatically and simultaneously aspirated from the concentrate tube and transported to the glass dual-flow-cells of the Optical Slide Assembly (Fig. 3.14). Based on user preference, the FE-5 also simultaneously stains or dilutes one of the two samples to be examined. After the microscopic examination of the fecal suspension within the glass viewing chambers, the flow chambers can be purged and cleaned and are ready for the next specimen. The dual-flow-cell Optical Slide Assembly is designed to fit within the stage clips of any standard upright microscope. The Optical Slide Assembly accommodates bright-field, phase-contrast, polarized-light, and other common forms of microscopy. The system can be moved from one microscope to another or can be set up as a semipermanent station for fecal concentrate microscopy. Removal to another microscope just involves removing the Optical Slide Assembly from the microscope stage.
Figure 3.13 Countertop workstation that automates the microscopic analysis of fecal concentrates (Apacor Ltd. USA, Brooklyn, NY). doi:10.1128/9781555819002.ch3.f13
Figure 3.14 Dual-flow-cell Optical Slide Assembly (Apacor Ltd USA, Brooklyn, NY) that fits into the stage clips of any standard upright microscope. doi:10.1128/9781555819002.ch3.f14
The detection and correct identification of many intestinal protozoa frequently depend on the examination of the permanent stained smear with the oil immersion lens (100× objective). These slides not only provide the microscopist with a permanent record of protozoan organisms identified but also may be used for consultations with specialists when unusual morphologic characteristics are found. Considering the morphologic variations that are possible, organisms that are very difficult to identify and do not fit the pattern for any one species may be found. A routine work flow diagram including the permanent stained smear is shown in Fig. 3.15.
Figure 3.15 Work flow diagram for fecal specimens. The total examination includes the direct mount, concentrate, and permanent stained smear (fresh specimen) or the concentrate and permanent stained smear (preserved specimens). doi:10.1128/9781555819002.ch3.f15
Although an experienced microscopist can occasionally identify certain organisms on a wet preparation, most identifications should be considered tentative until confirmed by the permanent stained slide. The smaller protozoan organisms are frequently seen on the stained smear when they are easily missed with only the direct smear and concentration methods. For these reasons, the permanent stain is recommended for every stool sample submitted for a routine parasite examination. It is also important to remember that the fecal immunoassays for specific organisms have proven to be more sensitive than the routine or specialized stains for Giardia lamblia (G. duodenalis, G. intestinalis) or Cryptosporidium spp. (25, 26).
There are a number of staining techniques available; selection of a particular method may depend on the degree of difficulty of the procedure and the amount of time necessary to complete the stain. The older classical method is the long Heidenhain iron hematoxylin method; however, for routine diagnostic work, most laboratories select one of the shorter procedures, such as the trichrome method or one of the modified methods involving iron hematoxylin. Other procedures are available (4, 6–8, 13, 17, 20, 24, 27, 30, 49), and some of them are presented here.
Most problems encountered in the staining of protozoan trophozoites and cysts in fecal smears occur because the specimen is too old, the smears are too dense, the smears are allowed to dry before fixation, or fixation is inadequate. There is variability in fixation in that immature cysts fix more easily than mature cysts, and E. coli cysts require a longer fixation time than do those of other species. It is critical that adequate mixing occur between the fecal specimen and preservative (27–29, 31, 32).
Preparation of Material for Staining
Fresh Material
1. When the specimen arrives, prepare two slides with applicator sticks or brushes and immediately (without drying) place them in Schaudinn’s fixative [(This approach is rarely used because the formulation contains mercury). Preserved stool collected in fecal fixatives (with or without PVA) can be smeared on a slide and allowed to air dry prior to staining.] Allow the slides to fix for a minimum of 30 min; overnight fixation is acceptable. The amount of fecal material smeared on the slide should be thin enough that newsprint can be read through the smear. Smears preserved in liquid Schaudinn’s fixative should be placed in 70% alcohol to remove the excess fixative prior to placement in iodine-alcohol (used for mercury-based fixatives).
2. If the fresh specimen is liquid, place 3 or 4 drops of fixative (Schaudinn’s fixative to which has been added a plastic powder [PVA], which serves as an adhesive to “glue” the fecal material onto the slide) on the slide, mix several drops of fecal material with the PVA fixative, spread the mixture, and allow it to dry for several hours in a 37°C incubator or overnight at room temperature.
3. Proceed with the trichrome staining procedure by placing the slides in iodine-alcohol.
Preserved Material Containing PVA
1. Stool specimens that are preserved in fixative (mercuric chloride, copper sulfate, or zinc sulfate bases) containing PVA should be allowed to fix for at least 30 min. Thoroughly mix the contents of the vial or bottle (fixative/stool/PVA specimen) with two applicator sticks.
2. Pour some of the well-mixed fixative/stool/PVA mixture onto a paper towel, and allow it to stand for 3 min to absorb out the excess PVA. Do not eliminate this step.
3. With an applicator stick (or brush), apply some of the stool material from the paper towel to two slides and allow them to
