DIRECT WET MOUNTS

In addition to having good equipment, the microscopist should be proficient in preparing suitable mounts and in examining these preparations. An audiovisual aid demonstrating proper preparation and examination of smears is available. See section on Training Aids for details.

PREPARATION OF MOUNTS

For direct wet mounts, as well as those from concentrated or cultured material, 3x2-inch slides instead of 3x1-inch should be used. The wide slide affords more space for preparation, permits the mount to be sealed around the edges (fig. 11), and prevents contamination of the microscope or the hands of the observer as might occur if the preparations "ran-over" on narrow slides. Ordinarily, a 22-mm-square coverslip of #1 thickness is used. For oil-immersion examination, #2 thickness is too thick for focusing. Density of the mount is also important. Fecal preparations should be thick enough that fine print can just be read through them (fig 10d). If the mount is too thick, focusing will be difficult and the organisms may be obscured. If the mount is too thin, locating organisms takes longer and may be missed entirely. Mounts should be prepared as follows:

•UNPRESERVED FECES

Place a drop of physiological saline (0.85%) on one end of the slide and a drop of iodine (approximately 1%) on the other end (fig. 10a). Using an applicator stick or toothpick, pick up a very small portion of feces and emulsify it in each solution (fig. 10b). Criteria for proper density should be kept in mind. Large particles should be avoided if possible, since the coverslip will not lie evenly on the slide. Cover the preparations, seal as described below, and examine. If too little fluid has been used and the material does not fill up the coverslip area, additional saline or iodine may be run under the edge. The feces will not be evenly mixed with the diluent, of course, but the preparation can be more easily examined than if vacant spots are present. On the other hand, the coverslip should

not float on the fecal suspension. In this case, a piece of facial tissue or absorbent towel can be used to remove the excess fluid before sealing. Touch the tissue to the edges of the coverslip, but be careful not to remove too much fluid. Iodine preparations will fade rather quickly if exposed to light, so these mounts cannot be left indefinitely before examining. If several preparations are made at one time, a slide folder may be used to hold them.

•SEALING PREPARATION

Sealing the mounts with a mixture of Vaseline-paraffin (vaspar) (approximately 1:1) is recommended as a routine procedure to prevent drying, thus permitting reexamination or examination at a later time. Such preparations may last as long as 2 to 3 days. Sealed mounts prevent the "flow" of the material and can be more easily examined with oil-immersion (as may be necessary to identify small protozoa) than unsealed preparations. Secure the coverslip by first placing a drop of hot vaspar on the four corners of the mount (fig 11). This will anchor the coverslip in place. A cotton swab or camel's hair brush can be used to apply the vaspar. Next, spread a thin layer around the edges of the preparation (fig. 11). The layer of vaspar should cover the edge of the coverslip, sealing it to the slide, but should not be very thick. If the layer is thick enough to form a ridge, the microscope objectives may become coated with the mixture as they are swung into position. Too much of the coverslip should not be covered with vaspar, since a maximum surface of the preparation should be available for examination.

•FORMALIN PRESERVED FECES

If the quantity of Formalin is more than 2 or 3 times that of the feces, let the feces settle and discard the excess before resuspending the material. Place a small drop of iodine near one end of the slide. In preserved specimens, the Formalin replaces the saline used in mounts of fresh feces, and the suspension can be used directly. Thoroughly mix the specimen before taking a sample, since eggs, larvae, and cysts settle to the bottom of the bottle. A drop of material from the top layers of an unmixed positive specimen may not contain organisms. Using 2 applicator sticks or toothpicks held together, remove a drop of the suspension to the slide (fig 10c).

d. Correct density of mounts.

b.

d.

Figure 11.

Sealing Mounts with Vaspar

Smith, M.

If the suspension is thick, it may be necessary to add a small drop of saline to dilute it to a satisfactory density. Add a second portion to the iodine drop and mix. Cover both preparations and seal as described above. The same techniques used for fresh fecal mounts can be used to add additional fluid (saline can be added to the unstained mount) or to remove it. The preparation must be of the correct density for adequate examination.

In making mounts from preserved specimens, a small drop of iodine must be used so that the preparation will not be too thick. Since the iodine will be diluted by the Formalin in the specimen, a slightly stronger concentration than usually used may be needed to obtain adequately stained structures. The need for a more concentrated iodine can be determined by trial. A too-strong solution, however, should be avoided. In the CDC laboratory, Dobell's iodine is used routinely, and a darker, stronger solution is made up for use with formalinized specimens by adding extra iodine crystals to the potassium iodide solution.

•CONCENTRATED SPECIMENS

Preparations from concentrated specimens are made in the same fashion as that described for Formalin preserved material. Pipettes