or loops, depending on the technique employed, are used to transfer material to the slides. Whether or not additional saline must be used will depend on how much fluid the sample contains. Often concentrated material, especially that from sedimentation procedures, will contain grit or large particles. Avoid getting these in the mount if possible, since, if this occurs, the coverslip will not lie evenly on the slide. If particles do get into the preparation, sometimes they can be pushed to one side with the corner of the coverslip or with an applicator stick. (If you hold the applicator stick by the ends and break it, you can usually get a slanted or pointed edge which can be more easily used than the blunt end.) Occasionally, particles can be flattened by pressing gently on the coverslip. If the particle is grit, however, the coverslip may break. When this happens, remove the broken pieces, add another small drop of thoroughly mixed suspension to the material left on the slide, and try again. If the preparation becomes "messy" around the edges of the coverslip, a piece of tissue can be used to clean it. Unless excessive, however, the "messy" edges can simply be covered with the vaspar; there is little, if any, danger of contaminating the vaspar by doing this. If despite everything, the preparation contains particles which cause the coverslip to "rock," add saline or iodine to fill up the space and seal. The density of the preparation will be uneven and may be too thick in spots, but this is preferable to gaps in the mount. •OTHER TYPES OF MATERIAL Wet mounts of urine to be examined for Trichomonas vaginalis or Schistosoma haematobium eggs are usually prepared from the sediment of a centrifuged specimen. (See p. 44) No special stains are needed to demonstrate either organism. Preparations may be sealed or left unsealed if they are examined within a few minutes. Vaginal and urethral material may be mixed with a small drop of saline and examined directly for Trichomonas vaginalis. Neither temporary methylene blue stain nor iodine should be used. Trichomonas trophozoites will lose their characteristic motion within a short time and begin to round up. Also, specimens should not be chilled because this affects the motility, but warm stages are not necessary, since the heat from the microscope lamp will be sufficient. For best results mounts prepared for this parasite should be examined as soon as possible. Wet mounts of aspirated lesion material to be examined for E. histolytica require special treatment, as described on p. 154. Temporary stains such as Quensel's or buffered methylene blue (Nair's solution) may be used to stain the trophozoites. Preparations should be sealed as described above. Duodenal drainage can be examined directly with or without saline. Duodenal aspirates and material obtained by nylon thread should be mixed with a small drop of saline. Temporary stains are of no value with these mounts. Sigmoidoscopic material may be mixed with a drop of saline and examined for amebae trophozoites. Additional preparations with temporary stains such as Quensel's or buffered methylene blue may be useful. Preparations should be sealed as described above. Sputum specimens are also examined as wet mounts. A small portion of the specimen can be mixed with saline, or if the material is thick and viscous, it can be diluted with several volumes of saline in a large tube and shaken vigorously for 30 to 45 seconds. If the specimen is being examined for P. westermani eggs, dilute sodium hydroxide (about 3% to 5%) may also be used to break up the thick portions. Sputum is only rarely examined for amebae trophozoites, but in these cases it must be handled more gently. Mounts with saline and with temporary stains, such as Quensel's or buffered methylene blue, may be made. EXAMINATION Helminths eggs and larvae are large enough to be seen under lowpower magnification (10X objective), although for study of structural details, high-dry magnification (43 or 44X objective) may be needed. Oil-immersion is not necessary and, in fact, may tend to confuse the observer. Cysts and trophozoites of protozoa can be detected with the low-power objective but higher magnifications are necessary for identifying species. Large protozoa, such as Entamoeba coli and Balantidium coli, and species like Giardia lamblia may be easily identified with high-dry magnification, but others, especially small cysts like Endolimax nana or Entamoeba hartmanni may require oil-immersion magnification for accurate identification. Diagnosis of E. histolytica cysts is also more reliable with oil-immersion examination. The light should be reduced for low-power observations, since most organisms will be overlooked with bright light. Illumination should be regulated so that cellular elements in the feces are readily visible. For routine examinations, a systematic scanning, beginning with one corner of the mount and covering the entire preparation, is recommended (fig. 12). This reduces the chances of missing scarce organisms that might be present. Scanning is done with the 10X objective. When organisms are found, the high-dry and oil-immersion objectives can be used for more detailed study. At least one complete saline (or unstained) mount should be searched carefully. An experienced microscopist usually requires from 10 to 15 minutes to do this. The saline or unstained mounts should be examined first, since eggs, larvae, trophozoites (if living), and cysts can be more readily detected in this type of mount than in the iodine preparation. If structures resembling cysts are seen, the iodine preparation should be examined. If no cysts or suggestive structures are found, the iodine mount need not be examined unless the microscopist wishes to do so. In certain instances, final species identification of protozoa can be made; in others, only presumptive diagnoses will be possible. Permanent stained preparations or additional specimens may be needed. Except in rare cases, stained preparations are essential for identifying species of amebae trophozoites. In temporary wet mounts, eggs or larvae may sometimes need to be turned over or reoriented so that the structural details may be seen. This can be done by gently tapping the coverslip with an applicator stick. Occasionally an egg may be lying at an angle or in a different plane and appear unfamiliar. This is particularly true with Trichuris, which may be oriented so that the viewer gets an optical cross-section or oblique view that may be unrecognizable as the familiar Trichuris egg. Schistosoma mansoni eggs also sometimes lie so that the spine does not show, and they too may need to be rolled over to be recognized. Wet mounts of other types of material should be examined in the same manner as described for fecal specimens. SOLUTIONS •IODINE SOLUTIONS Several iodine solutions can be used to stain protozoan cysts satisfactorily. The two described below have been widely used and are simply prepared. The one recommended by Dobell and O'Connor (1921) is a 1% iodine solution that should be prepared fresh about every 10 to 14 days for best results. Lugol's iodine, which must be diluted 1:5 for use, should be prepared fresh about every 3 weeks; the 1:5 dilution should be prepared every 10 to 14 days. PREPARATION 1. DOBELL AND O'CONNOR'S IODINE SOLUTION: Distilled water.. 1 gm 2 gm 100 ml Dissolve potassium iodide in the water. Add the iodine crystals and shake thoroughly. The crystals may not dissolve completely, but the solution should be a definite reddish-brown or the color of medium-strong tea. See fig. 13, page 84, for correct color. Filter or decant into a dropping bottle or other type of dispenser. Replace after 10 to 14 days. Most clinical laboratories do not use 100 ml of iodine solution in a 10- to 14-day period and preparing this amount of stain is wasteful. For practical purposes, the following procedure is suggested. 1. Prepare 500 ml of 2% potassium iodide (10 gm dissolved in 500 ml distilled water). 2. Store this stock potassium iodide solution in a glassstoppered bottle. A brown glass bottle is preferred. The solution will keep for several months, although it may become slightly yellow with age. This does not interfere with its use. Pour a small amount (20 or 30 ml) into a small beaker or Erlenmeyer flask. Add some iodine crystals (about 0.2 or 0.3 gm; the exact amount is not important) and shake until the solution is the proper iodine "color" or strength (fig 13). Replace after 10 to 14 days. Dissolve the potassium iodide in the distilled water. Slowly add the iodine crystals and shake until dissolved. The solution will be a strong reddish-brown. Filter. Store in a tightly stoppered, brown bottle away from the light. Dilute a portion 1:5 with distilled water for use in staining protozoan cysts. TECHNIQUE A small portion of feces or fecal suspension is comminuted in a drop of the iodine solution, mounted with a coverslip, and sealed with heated vaspar. The preparation should be of the correct density for microscopic examination. STAIN CHARACTERISTICS In a correctly stained cyst, the glycogen, if present, appears reddish brown, the cytoplasm appears yellow, and the chromatin stains brown or black. The location of the karyosomes may be more easily determined and the character of the nucleus is more distinct in iodine preparations, but the chromatoid bodies are less visible than in saline mounts. |