PROTOZOA WET PREPARATION -Saline Wet Preparation On preparationsaline.trophozoites and cysts of amoebae and flagellates may be seen. The cyst will appear round or oval, refractile in structure. Amoeba trophozoites will appear round or irregular. Flagellate trophozoites usually appear piriform (elongated,pear-shaped(like a pear)). In feces that have just been excreted (stools no more than 1 hour old), motile/moving trophozoites can still be seen. Motility will be very helpful in identifying species, especially in flagellates. The organism can be identified at low magnification (10x objective), but at strong magnification it is possible to reliably identify cyst or trophozoite structures. With high magnification and dry, you can see motility, inclusion bodies such as erythrocytes andyeastin amoeba trophozoites, chromatin bodies in amoebic cysts and some detailed shapes and structures (sucking disc,spiral grooves or filaments) in trophozoites and flagellate cysts. You will not be able to see details of the nucleus on the preparationsaline. However, it is very necessary to adjust the lighting of the microscope so that the object can be clearly observed. Too much or too little light will affect observations. Don't forget to adjust the focus of the lens to see each layer of the specimen. Remember to systematically inspect each area to avoid missing organisms from observation. -Preparation Buffered Methylene Blue If you see amoeba trophozoites, or structures that lead to amoeba trophozoites, it is best to prepare and examine them with BMB preparations. After 5-10 minutes of staining, the trophozoites sometimes become motile, but often the trophozoites will wrinkle on the BMB preparation (do not confuse wrinkled trophozoites with cysts, do not stain cysts with BMB). In trophozoites, nuclei and inclusions (erythrocytes,yeast) will be painted dark blue, the cytoplasm will be painted light blue. Sometimes, some trophozoites will not stain, so you should look for organisms that stain well. Look for peripheral granules of the nucleus (granules that are around the nucleus), if visible, then they are trophozoites of the Entamoeba species, and you should identify the species. If there are no peripheral granules of the nucleus, the trophozoites are not of the Entamoeba species. -Wet PreparationIodine Wet preparationiodineused to check for amoebic cysts and flagellates. They can be detected at 10x magnification. but they do not appear refractile on preparationsaline. High and dry magnification should be performed to view the characteristics of the cyst and should be measured to ensure correct identification. On preparationiodine, the cytoplasm will be yellow or light brown and the nucleus will be dark brown. In Entamoeba cysts with staining iodine, the arrangement of peripheral chromatin and karyosomes will be visible. (if no peripheral chromatin is visible, then it is not a cyst of Entamoeba). This chromatin will be light yellow in color and may not be very clear. Sometimes young cysts contain glycogen, these will be stained dark brown withiodine. In flagellate cysts with stainingiodine, fibril threads (filament) visible. Specific identification of amoebic and flagellate cysts can usually be made from wet preparationsiodine. However, in certain situations definitive identification cannot be made and permanent staining is required.