PRACTICAL

• Direct Fecal Smear (DFS)


  Also known as Direct Wet Mount, a routine method primarily useful in detection of motile protozoan trophozoites
• Direct Fecal Smear


  • Protozoan cysts can be demonstrated using a weak iodine solution as a temporary stain
  • Helminth eggs and larvae can also be detected using this preparation
  • Ideally, DFS preparation should contain approximately 2mg of stool
  • Light infections may not be detected
• Direct Fecal Smear - Procedure

  1. Assess the acceptability of the stool sample

  2. Write the patient's name (or identification number) and the date on the slide

  3. Place a drop of saline on the center of the left half of the slide and place a drop of iodine solution on the center of the right half of the slide

  4. With an applicator stick or match, pick up a small portion of the feces (approx. 2mg) and add it on the drop of saline. Add a similar portion to the drop of iodine. Mix the feces with solutions to form a suspension

  5. Cover each drop with a cover slip

  6. Examine the preparation with the 10x objective, or if needed for identification, the high power objective of the microscope
• World Health Organization Standard Reporting
• Kato-thick and Kato-Katz Technique


  Methods for parasite identification in stool samples
• Egg counting procedures correlate the severity of clinical disease with the intensity of infection or worm burden. It is also done to assess the efficacy of anthelmentics and reduction of worm burden following treatment.
• Kato-thick Technique


  Time-saving, economical and suitable for mass examination. Can be used to identify all common type of helminth eggs. Unsuitable for diarrheic stool and cannot be used to diagnose protozoan cysts and trophozoites.
• Kato-Katz technique


  Used for qualitative and semi-quantitative diagnosis of intestinal helminthic infestations caused by Ascaris lumbricoides, Trichuris trichiura, Hookworms and especially Schistosoma spp.
• WHO has recommended Kato Katz technique in areas with moderate to high transmission rates of soil transmitted helminths (i.e. where the proportion of infected individuals is >20– >50%) or intestinal schistosomiasis (>10–50%).
• When the prevalence of soil transmitted helminths (STH) is <20%, the specificity of this technique makes it less appropriate, and more sensitive tools should be used.
• Reagents and Equipments for Kato-Thick Technique

  • Slides
  • Cellophane (1"x1") soaked in Glycerine-Malachite green solution
  • Microscope
  • Applicator sticks
  • Cover slips
  • Rubber stopper/Cork stopper
  • Forceps
• Kato Thick Technique Procedure

  1. Place stool on slide and cover with cellophane

  2. Press cellophane to spread stool evenly

  3. Leave slide at room temperature for 10-20 minutes

  4. Examine slide within one hour
• If no parasites are observed, report as "No ova/cyst of intestinal parasite found" or "No intestinal parasites seen".
• Stool Consistency and Kato-Thick Technique

  • Hard/Scybalous - X
  • Well-formed - DFS with Kato-Thick
  • Formed - DFS with Kato-Thick
  • Soft - DFS with Kato-Thick
  • Mushy - DFS with Kato-Thick
  • Loose - X
  • Mucoid - X
  • Diarrheic - X
  • Watery - X
• Materials and Equipments for Kato – Katz Technique

  • Glass Slides
  • Cellophane (1"x1") soaked in Glycerine-Malachite green solution
  • Microscope
  • Template (1 mm, 1.5 mm, 0.5 mm)
  • Scraper
  • Wire net (60 - 105 mesh)
  • Filter paper or newspaper
  • Applicator sticks
  • Cover slips
  • Rubber stopper/Cork stopper
  • Forceps
• Kato – Katz Technique Procedure

  1. Sieve stool through wire mesh

  2. Scrape sieved feces onto slide

  3. Place template on slide and fill hole with feces

  4. Remove template, leaving feces cylinder

  5. Cover feces with pre-soaked cellophane strip

  6. Invert slide and press cellophane against feces

  7. Remove slide, leaving cellophane upwards

  8. Allow clearing for 1+ hours

  9. Hookworm eggs clear rapidly, examine within 30-60 mins

  10. 10. Examine slide systematically, report egg counts
• Enterobius vermicularis


  Also known as the human pinworm, a small threadlike worm that infects humans (more commonly in children)
• Enterobiasis or oxyriasis


  Characterized by perianal itching or pruritus ani
• Life cycle of Enterobius vermicularis

  1. Adult female worms migrate down the intestinal tract to exit through the anus to deposit eggs on the perianal skin

  2. Hatched larvae can also enter the anus back into the large intestine causing retroinfection
• Enterobius vermicularis eggs


  Measure 50-60um by 20-30um, elongated, and flattened on one side, giving it the characteristic D-shape appearance
• Embryonated eggs are not commonly found in feces though they may occasionally be found on the surface of the stool, contaminate underwear and beddings, and may be ingested or inhaled
• Scotch tape preparation


  Used to isolate and identify the eggs when a person is suspected of having pinworm infection
• Procedure


  1. Place a strip of clear cellulose tape, adhesive side down on a microscope slide

  2. To obtain a sample from the perianal area, peel back the tape by gripping one end and loop the tape (adhesive side outward) over a wooden tongue depressor that is held firmly against the slide and extend about 2.5 cm beyond it

  3. Press the tape firmly several times against the right and left perianal folds

  4. Once the sample is obtained, remove the slide from tongue depressor and affix the tape sticky side down on the glass slide

  5. Label the slide with patient's name and date of collection

  6. Examine the slide under a microscope using LPO. The eggs can be made more visible by detaching the tape from the slide, adding a drop of xylene or toluene, and again affixing the tape.
• Nematodes


  Roundworms
• Nematode species


  • Ascaris lumbricoides
  • Trichuris trichiura
  • Ancylostoma duodenale
  • Necator americanus
  • Strongyloides stercoralis
• Accurate diagnosis


  • Depends on examination of properly collected and preserved specimens
  • Identification of eggs and larvae in stool serve as an aid in the diagnosis and treatment of an infection
  • Size of larvae, external attributes of eggs (e.g. shape, wall thickness, and presence of mammillated covering) are important characteristics in identification
• Loose/Watery Stool Samples


  1. Write patient name/ID and date on slide

  2. Place drop of saline and iodine solution on slide

  3. Add small portion of stool sample to each drop and mix

  4. Cover each drop with a cover slip

  5. Examine slide under microscope
• Formed Stool Samples


  1. Place 50-60 mg of stool at center of slide

  2. Cover with pretreated cellophane square

  3. Press cellophane gently to spread stool evenly

  4. Leave slide at room temperature for 10-20 minutes

  5. Examine slide under microscope
• Ascaris lumbricoides


  • Corticated fertilized egg
  • Corticated unfertilized egg
  • Corticated and decorticated fertilized eggs
• Strongyloides stercoralis


  • Larva (L1)
• Trichostrongylus spp.


  • Egg
• Enterobius vermicularis


  • Egg