LAB4

• Most common errors in the identification of parasites may be due to the following:

  1. Lack of familiarity with the parasites
  2. Cursory examination of the slide
  3. Confusion of parasite with artifacts
• The microscopist should be familiar with the different structures found in the feces such as trophozoites and cysts of Amoeba, helminth eggs and larvae, RBC, macrophages, WBC, fungi, plant cells (pollen grains and spores), epithelial cells, crystals like Calcium oxalate and triple phosphate, bacteria, plant fibers, root hairs and animal cells are similar to helminth ova.
• Direct Fecal Smear (DFS) Direct Wet Mount Technique

  • This is the simplest and moderately efficient procedure for examinations of feces
• Direct Fecal Smear (DFS) or Direct Wet Mount Technique

  • The mixing action of the intestinal tract usually results in an even distribution of parasites in stool.
• Direct Fecal Smear (DFS) or Direct Wet Mount Technique

  • It is highly recommended for the detection of motile trophozoites in liquid, diarrheic and bloody mucoid specimens, however, if the number of organisms are few, this method may be of low sensitivity and insufficient to reveal their presence.
• Direct Fecal Smear (DFS) or Direct Wet Mount Technique

  • The amount of feces used for the direct mount is important. The 2 mg recommended approximates what would form a low cone at the end of a wooded applicator stick.
• Direct Fecal Smear (DFS) or Direct Wet Mount Technique

  • When less than 2 mg of feces is used, the suspension will be too thin and may have blank spaces
• Direct Fecal Smear (DFS) or Direct Wet Mount Technique

  • The use of more than 2 mg results in a suspension that is too thick and parasites may be hidden under fecal debris
• Direct Fecal Smear (DFS) or Direct Wet Mount Technique

  • Temporary stains may be used with wet mount preparations to aid in location and identification of protozoa but are not necessary for eggs and larvae.
• Direct Fecal Smear (DFS) or Direct Wet Mount Technique

  • Dilute iodine solutions are the most commonly used temporary stains and are most useful for recognition of cyst stage; however, they kill and distort the trophozoites.
• Direct Fecal Smear (DFS) or Direct Wet Mount Technique

  • In cysts, the visibility of nuclei is enhanced so that their number and morphological features are more clearly seen.
• Direct Fecal Smear (DFS) or Direct Wet Mount Technique

  • The use of weak iodine solutions is not recommended since they do not stain organisms well.
• Direct Fecal Smear (DFS) or Direct Wet Mount Technique

  • Likewise, the use of an iodine solution that is too strong will stain the organisms so darkly that morphological features cannot be seen
• Direct Fecal Smear (DFS) or Direct Wet Mount Technique

  • Dilute iodine solutions that are recommended are D'Antoni's, Dobell and O'Connor's and Lugol's.
• Kato - Thick Smear Preparation

  • This technique is based on the fact that when a layer of stool is in contact with glycerin-soaked cellophane for a period of time, it becomes clarified and helminth eggs become visible.
• Kato - Thick Smear Preparation

  • The slide should be examined after 10-20 minutes or within one hour after preparation. Allowing the slide to stand for long period of time will cause drying and shells of hookworm ova will become be difficult to see
• Formalin - Ether Concentration Technique (FECT)

  • The use of concentration procedures for fecal examination virtually ensures the detection of even small numbers of organisms that would go undetected if only direct smears of permanent-stained slides were examined.
• Formalin - Ether Concentration Technique (FECT)

  • Concentration procedures generally fall into two categories - flotation and sedimentation.
• Formalin - Ether Concentration Technique (FECT)

  • Sedimentation can be achieved by simple gravity or centrifugation.
• Formalin - Ether Concentration Technique (FECT)

  • Since the sediment will generally contain all the parasites occurring in the stool sample, this procedure has greater diagnostic sensitivity.
• Formalin - Ether Concentration Technique (FECT)

  • An added advantage is that the technique can be readily applied to both fresh and preserved feces.
• Formalin - Ether Concentration Technique (FECT)

  • Consists of four layers:

  1. top layer of ether
  2. plug of debris adhering to the wall
  3. layer of formalin
  4. sediment for examination.
• Formalin - Ether Concentration Technique (FECT)

  • Recently, concern over storage and use of ether, a potentially flammable and explosive material, has led to the use of ethyl acetate as a substitute
• Formalin - Ether Concentration Technique (FECT)

  • Ethyl acetate appears to be somewhat more efficient than ether when specimens contain Taenia or Hymenolepis nana eggs or Giardia cysts since there are fewer tendencies for these eggs or cysts to become trapped in the plug of debris.
• Formalin - Ether Concentration Technique (FECT)

  • However, one disadvantage is that ethyl acetate, when used as a solvent for fresh feces, is not as efficient as ether in extraction of fatty or mucoid materials which may interfere with the microscopic examination of the sample