[Histonet] Bile stain
Lee & Peggy Wenk
lpwenk <@t> sbcglobal.net
Sun Jun 12 07:01:51 CDT 2005
Depending upon the amount of bilirubin in the specimen, the shades of green
may range from the drab olive to the brilliant emerald. So this may not be a
staining problem. It just may be the state of the bile in the specimen.
That said, I'll include our procedure, which is from the Freida Carson book.
Peggy Wenk, HTL(ASCP)SLS
William Beaumont Hospital
Royal Oak, MI 48073
- - - - - -
BILE - FOUCHET
PREPARED BY: Peggy A. Wenk, BS, HTL(ASCP)SLS
Bile and lipofuchsin appear as brown-yellow pigments. This stain
demonstrates bilirubin (bile pigment) in tissue sections. Red blood cells,
when destroyed, release their hemoglobin. This hemoglobin is broken down
into globin (protein) and heme (iron). When the iron is removed from the
heme portion, the residue is known as biliverdin. The biliverdin is
transported to the liver where it is reduced to bilirubin, and then
converted into bile, where it passes into the gall-bladder, then into the
duodenum. In the cases of biliary obstruction or extensive liver damage,
biliverdin, bilirubin and bile may accumulate in the liver.
Trichloroacetic acid and ferric chloride will oxidize the bilirubin to
biliverdin, to produce a green color. The van Gieson is the counterstain.
Any well fixed tissue, except Zenker.
10% neutral buffered formalin preferred.
Cut routine paraffin sections at 5 um.
Section of tissue with bile
1. Make Fouchet reagent just before use and filter.
Balance, Erlenmeyer flasks, graduated cylinders, magnetic stirrer,
Follow standard safety procedures when preparing stains.
TRICHLOROACETIC ACID is an acid. Add slowly to water. May cause severe skin
burns. May be irritating to eyes and respiratory system.
FERRIC CHLORIDE is a corrosive. May cause skin and eye burns. Can be
ACID FUCHSIN is an irritant.
PICRIC ACID is toxic, highly reactive (4 - NFPA) and an extreme fire hazard
(4 - NFPA).
Keep picric acid moist at all times. If dry around top of jar, wash off dry
particles with running
water before opening. Store in an explosion proof jar.
10% FERRIC CHLORIDE
Ferric chloride (FeCl3?6H2O) 10.0 g
Distilled water 100.0 mL
29% Stock ferric chloride 3.5 mL
Distilled water 6.5 mL
Stir together with magnetic stirrer. Store at room temperature. Stable for 1
Trichloroacetic acid (CCl3COOH) 12.5 g
Distilled water 50.0 mL
10% ferric chloride 5.0 mL
JUST BEFORE USE, carefully add trichloroacetic acid to distilled water. Stir
until dissolved. Add 10% ferric chloride. Filter before use.
1% ACID FUCHSIN
Acid fuchsin (CI 42685) 1.0 g
Distilled water 100.0 mL
Dissolve together. Store at room temperature. Stable for months.
SATURATED AQUEOUS PICRIC ACID
Picric acid (2,4,6-(NO2)3C6H2OH)
Distilled water 100.0 mL
Add picric acid, a gram at a time, to the distilled water, and stir using a
magnetic stirrer. When dissolved, add another gram and stir. Continue to do
this until no more picric acid will dissolve into the distilled water. Store
at room temperature. Stable for months.
See above warning of picric acid.
VAN GIESON COUNTERSTAIN
1% acid fuchsin 5.0 mL
Saturated aqueous picric acid 100.0 mL
Mix together. Store at room temperature. Stable for 4-6 months. May be
reused until weak.
PROCEDURE - Fouchet:
1. Deparaffinize and hydrate sections through graded alcohol to distilled
2. Place slides in freshly filtered Fouchet reagent 5 minutes
3. Rinse in distilled water, 3-5 changes 5-10 seconds each
4. Stain in van Gieson counterstain 1 minute
5. Place slides directly into 95% alcohol, 2 changes 5-10 seconds each
6. Dehydrate through absolute alcohols and clear in xylene.
7. Coverslip using a synthetic mounting media.
Bile or bilirubin shades of green
Cytoplasm, red blood cells yellow
1. Depending upon the concentration of bilirubin, the colors may range from
olive green to emerald green.
2. After the van Gieson counterstain, go directly to 95% alcohol. Placing
the slides in water may decrease the amount of counterstain.
Bancroft JD, Stevens A: Theory and Practice of Histological Techniques, 3rd
ed. New York, NY, Churchill Livingstone, 1990.
* Carson FL: Histotechnology: A Self-Instructional Text, Chicago, IL, ASCP
Sheehan DC, Hrapchak BB: Theory and Practice of Histotechnology, 2nd
edition. Columbus, Ohio, Battelle Press, 1980.
Vacca L: laboratory Manual of Histochemistry. New York, NY, Raven Press,
* Source of procedure.
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Angela
Sent: Thursday, June 09, 2005 3:19 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Bile stain
Does anyone have a good bile stain? Ours turns a drab olive green and our
Dr. would like to see a "bright emerald green".
Thanks in advance for your help.
Angela Bitting, HT(ASCP)
Technical Specialist, Histology
Geisinger Medical Center
100 N Academy Ave. MC 23-00
Danville, PA 17822
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