STEP 4: Let stand for 10 to 15 minutes and scan with the low power of the microscope
for trophozoites and identify under high dry (some authors recommend that the
identification be done with the oil immersion objective).
(d) Interpretation. Trophozoite cytoplasm is stained pale blue while the
nuclei takes on a darker blue color. Living cysts, ciliates, and flagellates do not stain.
Nuclei of Dientamoeba fragilis trophozoites do not stain well.
(6) Buffered Methylene Blue. The Buffered Methylene Blue stain has been
used successfully for staining protozoan trophozoites in wet preparations.
(a) Stock reagents.
1 0.2M acetic acid--solution A.
Glacial acetic acid ........................................ 11.55 ml
Distilled water .............................................. QS to 1,000.00 ml
In a 1,000 milliliter volumetric flask, add the glacial acetic acid to
about 500 milliliter of distilled water and mix well and store in a
stoppered glass bottle.
2 0.2M sodium acetate--solution B.
Sodium acetate (anhydrous NaC2H3O2) ...... 16.4 gm
Distilled water .............................................. QS to 1,000.0 ml
NOTE:
The hydrated salt (NaC2H3O23H2O) can be substituted, but with a quantity of
27.2 grams.
In a 1,000 milliliter volumetric flask, add the salt to about 500
milliliter of distilled water. Mix until dissolved and QS to the 1,000
milliliter mark with distilled water. Mix well and store in a
stoppered brown glass bottle.
3 Methylene Blue powder.
(b) Working solution.
Solution A.......................................................... 46.3 ml
Solution B.......................................................... 3.7 ml
Methylene Blue powder..................................... 3.0 gm
Mix the ingredients well. This solution should be prepared just prior
to use for optimum results.
MD0841
2-44