# Stool Specimens – Microscopic Examination

##### Calibration of Microscopes Using an Ocular Micrometer:

A correctly calibrated microscope is crucial because size is an important characteristic for identification of parasites. This section assumes that an ocular micrometer disk has been installed in one of the oculars and that a stage micrometer is available for calibrating the ocular micrometer. This calibration should be done for each of the microscope’s objectives.

Place the stage micrometer on the microscope stage and focus on the micrometer scale, until you can distinguish between the large (0.1 mm) and the small (0.01 mm) divisions of the scale. Adjust the stage micrometer so that the “0” line on the ocular micrometer is superimposed with the “0” line on the stage micrometer. Without changing the stage adjustment, find a point as distant as possible from the two superimposed “0” lines where two other lines are also exactly superimposed. Determine the number of ocular micrometer spaces, as well as the number of millimeters on the stage micrometer, between the two points of superimposition.

For example: Suppose 48 ocular micrometer spaces (units) equal 0.6 mm. Calculate the number of mm/ocular micrometer space.

0.6 mm x 48 ocular micrometer spaces = 0.0125 mm/ocular micrometer space

Since most measurements of microorganisms are given in µm rather than mm, the value calculated above must be converted to µm by multiplying it by 1000 µm/mm.

For example:

0.125 mm ocular space × 1000 µm/mm = 12.5 µm/ocular micrometer space

Thus in this case, 1 ocular micrometer space (unit) is the equivalent of 12.5 µm.

Follow the above steps for each objective. Calibration readings should be posted on each microscope and the microscope should be recalibrated after every cleaning or changing of objectives or oculars.

##### Wet Mount Preparation:

Figure A

Before preparing a wet mount slide, the microscope should be calibrated. The objectives and oculars used for the calibration procedure should be used for all measurements on the microscope. The calibration factors should always be posted on the side of the microscope.

Protozoan trophozoites, cysts, oocysts, and helminth eggs and larvae may be seen and identified using a wet mount identification technique. To prepare a wet mount, obtain a microscope slide and the stool specimen. Take a small amount of the specimen and place it on a microscope slide. If the stool specimen is still somewhat solid, add a drop or two of saline to the specimen and mix. Ideally, two smears can be prepared on one slide, of which one can be stained with iodine. Thickness of the wet mount should be as Figure A on the right illustrates.

If desired the coverslip(s) can be sealed. A preparation of petroleum jelly and paraffin in a 1:1 ratio can be applied with a cotton tip swab as illustrated in Figure B on the right. It must be heated to approximately 70°C to both mix and use. Sealing the coverslip keeps organisms from moving when using oil immersion objectives and prevents the preparation from drying out. To seal, secure the four corners by placing a drop of hot sealant to anchor the coverslip. Spread a thin layer around the edges. Other suitable sealing preparations can be used if desired.

Systematically scan the entire coverslip area using the 10× objective as illustrated in Figure C on the right. If something suspicious is seen, a higher magnification may be necessary.

CAUTION: Bringing high power objectives too near the edge of the slide will result in the sealant smearing the objective and interfering with the optors.

##### Stained Slide Preparation:

Permanent stained slides are used for identification of protozoan trophozoites and cysts and for confirmation of species. It also permits consultation reference and diagnosis when needed as well as providing a permanent record of organism(s) observed. The microscope should be calibrated before examination begins. Positive microscope slides as well as reference material (plates, photographs, digital images) should be available by the workstation to compare morphological details and organisms. Refer to the staining section of stools for additional information regarding which stains to use.

Normally 3 × 1 slides are used to prepare permanent stained slides. If the specimen is unpreserved, prepare a thin even smear of the material by streaking the material back and forth on the slide with an applicator stick. If necessary dilute feces with saline. For PVA fixed specimens, apply two or three drops of the specimen to the slide and with a rolling motion or an up and down dabbing motion spread the specimen evenly to cover an area roughly the size of a 22 by 22 mm coverslip. For other fixatives, check manufacturers instructions.

After the staining process is complete, systematically examine the smear microscopically utilizing the 100× oil objective. Examine at least 200 to 300 oil immersion fields. Report protozoa seen as either trophozoites and/or cysts as applicable.

##### UV Fluorescence Microscopy Procedure:

The demonstration of Cyclospora oocysts in wet preparations is greatly enhanced by using UV fluorescence microscopy. Despite the age of the specimen or sample, Cyclospora oocysts exhibit intense blue color when observed under a fluorescence microscope (UV excitation filter set at 330-365 nm). If this filter set is not available, a less intense green fluorescence can be obtained with blue excitation (450-490 nm). Under bright-field (differential interference contrast or DIC) microscopy, Cyclospora oocysts appear as refractile spheres (8-10 µm) with a distinct oocyst wall. The utilization of both bright-field (DIC) and fluorescence microscopy provides an efficient and reliable approach to diagnosis. However, it does not provide a permanent stained slide that can be archived.