[Elizabeth_Zeibig]_Clinical_Parasitology__A_Practi(z-lib.org)

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CHAPTER 2 Specimen Collection and Processing
PROCEDURE 2-6
WHEATLEY TRICHROME STAIN
1. Label nine glass Coplin staining jars with the jar number
and contents, as defined below. It is important that the
proper laboratory protocol be followed when labeling
the jars.
2. Using the table below as a guide, transfer the slides
being stained from one jar to the next following the time
lines established below.
3. Following the staining process, seal each slide with a
fixative, such as Permount, and let it dry. An alternative
to a mounting medium is to let the slide air-dry and,
prior to examination, add one drop of immersion oil to
the slide. Let the oil sit on the slide for 10-15 minutes
and then place a no. 1 cover slip onto the oiled smear.
4. Examine each slide under oil immersion.
Trichrome Stain: Suggested Procedure
Reagent
Purpose
Coplin Jar Number
1 2 3 4 5 6 7 8 9
Iodine plus 70% 70% Trichrome 90% 100% 100% Xylene Xylene
70% ethanol ethanol stain acidified ethanol ethanol (substitute) (substitute)
ethanol
alcohol
Removes
mercuric
chloride,
hydration
Removes
iodine,
hydration
Rinse,
hydration
Stain Destain Halts
destaining
Dehydration
Removes
debris,
dehydration
Removes
debris
Time 10 min 5 min 5 min 7 min 5-10 sec Quick rinse 5 min 10 min 10 min
PROCEDURE 2-7
IRON HEMATOXYLIN STAIN (TOMPKINS-MILLER METHOD)
Step No. Reagent Purpose Time (min)
1 Iodine + 70% ethanol Remove fixative 2-5
2 50% ethanol Rinse 5
3 Running tap water Rinse 3
4 4% ferric ammonium sulfate Mordant fixative 5
5 Running tap water Rinse 1
6 Hematoxylin solution (0.5%) Stain 2
7 Running tap water Rinse 1
8 2% phosphotungstic acid Decolorize 2-5
9 Running tap water Rinse 10
10 70% ethanol containing a few drops of saturated
Dehydrate 3
aqueous lithium carbonate
11 95% ethanol Dehydrate 5
12 100% alcohol Dehydrate 5
13 100% alcohol Dehydrate 5
14 Xylene substitute Clearing 5
15 Xylene substitute Clearing 5
16 Mounting fluid (i.e., Permount) (alternative method as Preserve slides
described in Procedure 2-6 can be used)
17 Microscopically examine slides
NOTE: Prepare smears as described in Procedure 2-5. SAF-preserved slides begin with step 2.