a. Diluent.
Ethanol 90% ........................................... 170.0 ml
Methanol (absolute)................................ 160.0 ml
Acetic Acid (glacial) ................................ 20.0 ml
Liquid phenol .......................................... 20.0 ml
Phosphotungstic acid, 1% ...................... 12.0 ml
Distilled water ......................................... 618.0 ml
Mix the reagents well, remembering to always add acid to
water, not water to acid.
b. Stock stain.
Chlorazol Black E Stain .......................... 5.0 gm
Basic solution ......................................... 1,000.0 ml
Grind the dye in a mortar and add small amounts of the
diluent until a smooth paste is formed. Continue to add more
diluent while mixing until a homogenous mixture is obtained.
Add the remaining diluent. Store in a stoppered brown glass
bottle and allow to "ripen" for four to six weeks. Filter before
use.
2 Working dilutions and times for staining.
STAIN
DILUENT
TIME
DILUTION A
Undiluted
2-3 hours
DILUTION B
20.0 ml
20.0 ml
2-4 hours (24)
DILUTION C
30.0 ml
15.0 ml
2-4 hours
DILUTION D
15.0 ml
30.0 ml
2-24 hours
DILUTION E
10.0 ml
30.0 ml
4-24 hours
Stain two smears with each dilution and determine the
optimum solution, keeping in mind that more than one
solution may be suitable. Practice has shown that solution C
is good for two hours and solution D stains well for four to 24
hours.
MD0841
2-54