STEP 6: Add three milliliters of ethyl acetate, stopper, invert the tube, and shake
vigorously until thoroughly mixed (about 30 seconds). Release the stopper
with care to avoid spraying the area or self with the specimen.
STEP 7: Centrifuge for two minutes at 2,500 rpm. Four layers will result in the tube
after centrifugation. From top to bottom, the layers are: ethyl acetate, debris
plug, formalin solution, and the sediment containing the parasites (if present).
STEP 8: Free the debris plug with an applicator stick and carefully decant the top three
layers, leaving the sediment undisturbed. Use a cotton swab to clean the
remnants of ethyl acetate from inside of the tube.
STEP 9: Prepare saline and stained wet preparations and examine for parasites.
(4) Merthiolate-Iodine-Formaldehyde Concentration (MIFC). The MIFC
combines fixing, concentration, and staining all in one procedure. Some authors claim
that this procedure is of little value for identification of amoebic trophozoites.
Nevertheless, it is a comprehensive technique and it is widely used.
(a) Stock reagents.
1 Merthiolate-formaldehyde (MF)--Solution A.
Distilled water .............................................. 250.0 ml
Formaldehyde (saturated) ........................... 25.0 ml
Tincture of merthiolate (Lilly 1:1,000)........... 200.0 ml
Glycerin ....................................................... 5.0 ml
Store in a stoppered brown glass bottle. This solution is stable
for months.
2 Lugol's iodine solution (I)--Solution B.
Iodine crystals (powdered) ........................... 5.0 gm
Potassium iodide ......................................... 10.0 gm
Distilled water .............................................. 100.0 ml
Dissolve the potassium iodide in water. Add the iodine crystals
slowly and shake until dissolved. Filter and store in a brown
bottle. This solution is only stable for three weeks.
(b) Working reagent. Add 18.0 milliliters of Solution A to 1.2 milliliters of
Solution B immediately prior to use. For smaller or larger volumes, decrease or increase
both components proportionately. Mixing the two solutions too far in advance causes a
precipitate to form, thus reducing the staining component.
MD0841
2-48
Previous Page
