REAGENT
SOLUTION
PVA-FIXED
TIME FRESH
STEP 1: Schaudinn's..........................A ................................................................60*
STEP 2: Working Iodine Alcohol......... ................................ 10-20 ........................1
STEP 3: Ethanol, 70% ........................ ................................ 3-5 ............................1
STEP 4: Ethanol, 70% ........................ ................................ 3-5 ............................1
STEP 5: Trichrome Stain .................... ................................ 6-10 ..........................2-8
STEP 6: Acidified Ethanol ..................One Dip **................ One Dip**
STEP 7: Ethanol, 95% ..... .................. ................................ 1-3 Dips............... One Dip
STEP 8: Ethanol, 95% ..... .................. ................................ 5 .......................... Two Dips
STEP 9: Absolute Ethanol .................. ................................ 5 ...............................5
STEP 10: Carbol-Xylene ... .................. ................................ 5 ...............................5
STEP 11: Xylene ............... .................. ................................ 5 ...............................5
*
This step could be shortened to five minutes if the stain is heated to 50 C.
**
This is a very critical step in the procedure. Destaining should be restricted to no
more than 20 seconds. The amount of time that the slide is in contact with the acid
alcohol includes the time in between dishes. For better results, this step should be
carried out for one slide at a time. Mount using a suitable medium.
(3) Chlorazol Black E. Gleason and Healy modified the technique introduced
by Kohn in 1960. This stain, in which fixation and staining is done in a single step, has
shown good results in the clinical laboratory. The procedure is used for fresh specimens
and is not suitable for PVA-fixed smears. The stock stain is stable indefinitely and the
staining time must be determined for each "batch" of stain.
(a) Reagents.
1 Stock stain solutions.
MD0841
2-53