Special Stains and Staining Techniques:

3 Types: -For particular parts of tissues i.e.

components of connective tissues not readily seen
or well stained by the haematoxylin stain.
-For particular substances, such as iron
and mucin, which do not pick up the
haematoxylin stain.
-For micro-organisms such fungi and
bacteria.
Connective Tiisues:
ollagen and ollagen fibres ! fibrous
insoluble protein found in the connective tissue.
Formed by fibroblasts and may be arranged in
either fine or coarse bundles. an be visuali"ed
using Zenkers fluid or formalin-fixed tissues, in
the case of the latter, the tissue sections will
re#uire moradanting prior to staining $iodine
treatment%.
Van iesons stain ! the simplest method of
differential staining of collagen and other
connective tissue, yet it can probably be called the
most successful. &ven after a hundred years, it is
still used in histological laboratories today.
!eagents:
'eigert(s iron haematoxylin ! stored as
two solutions and mixed in e#ual proportions
immediately prior to use. )t can also be stored for
up to *+ days if stored at ,-.
.an /ieson(s solution ! composed of 0+
m1 02 a#ueous acid 3 0++ m1 saturated a#ueous
picric acid, boiled for 3 minutes, then cooled 3
filtered.
"rocedure:
First, take the section to water, stain with
'eigert(s haematotoxylin for 04-3+ minutes. 5fter
staining, wash with tap water. 6ifferentiate with
acid alcohol, wash 3 blue. 7sing a microscope,
check if the nuclei is dark-black and the
background a paler blue-black color. 8inse well in
distilled water. 5fter rinsing, counter-stain in .an
/ieson(s solution for 4 minutes. Then, rinse well
in distilled water or drain, then rinse in alcohol.
1astly, dehydrate, clear and mount in 69: or
9ermount.
!esults:
;uclei ! brown-black
<ature collagen ! bright red
&lastic fibres ! dark $often black%
=ther tissue elements $smooth muscle
and red blood cells% ! yellow
>ome bile pigments ! green
#assons Trichrome stain ! uses
phosphomolybdic and phosphotungstic acids a
mordant along with a haematoxylin stain
$'eigert(s iron haematoxylin%.
!eagents:
'eigert(s iron haematoxylin
5cetic acid solutions ! 02 3 ?2
solutions acetic acid by mixing appropriate
amounts of glacial acetic acid and distilled water.
9oncean-acid fuchsin solution ! e#ual
volumes of +.42 a#ueous solution of ponceau ?8
and +.42 acid fuchsin in 02 acetic acid.
9hosphorolybdic acid solution ! 03
a#ueous 9hosphorolybdic acid
5niline blue solution ! @oil AB.4 m1 of
distilled water and add ? g aniline blue $water
soluble%. 'hile still hot, add ?.4 m1 glacial acetic
acid. ool and filter.
1ight green solution $?2% ! an be used
in place of aniline blue. ?2 light green in ?2
acetic acidC dilute with distilled water prior to use.
"rocedure:
First, take the section to water, stain with
'eigert(s haematotoxylin for 04-3+ minutes. 5fter
staining, differentiate to blue. 8inse in distilled
water. 5fter rinsing, treat with ponceau-acid
fuchsin solution for ?-3 minutes. 8inse once again
in distilled water. 6ifferentiate in the
phosphorolybdic acid solution for 4-04 minutes at
room temperature. Then, wash well with water.
5fter washing, counter-stain with either aniline
blue or light green solution for 0 minute. 1astly,
wash, dehydrate, clear and mount as desired.
!esults:
;uclei ! blue-black
<uscle, red blood cells, fibrin ! red
onnective tissue ! blue or green
according to the counterstain used.
Connective Tissues:
8eticulin $collagen type )))% ! consist of
a fibrillary extracellular framework in the
connective tissue. These fine branching fibres are
hard to see in haematoxylin!eosia $D3&%
preparations, so they need other techni#ues to be
properly visuali"ed.
Silver $mpregnation #ethod %ordon and
S&eets' ! a popular 3 reliable method. an be
stored many weeks, even at room temperature, in
a clear container. @est made with formalin diluted
with tap water for more reduction of the silver
solution.
!eagents:
=xalic acid ! 42 wEv, a#ueous
Ferric ammonium sulphate!iron alum !
?2 wEv, a#ueous
Formalin ! 0+2 wEv, a#ueous
;eutral red ! 02 wEv, a#ueous!
counterstain
5cidified potassium permanganate
solution ! composed of ,B.4 m1 +.?42 a#ueous
potassium permanganate 3 ?.4 m1 32 a#ueous
sulfuric acid. an be kept as stock solutionsC
composite solution can be kept for several weeks.
5mmoniacal silver nitrate solution ! 4
m1 0+2 a#ueous silver nitrate. 5dd concentrated
ammonia drop by drop rate fre#uent mixing until
before precipitate Fust redissolves. 5dd 4 m1 of
3.02 a#ueous sodium hydroxide and mix. 5
precipitate will form that gradually dissolves upon
the addition of ammonia, drop by drop as before.
>top when there are only a few precipitate
granules remaining. <ake up the final volume to
4+ m1 with distilled water.
"rocedure:
First, take the section to water, then treat
with acidified potassium permanganate solution
for 4 minutes. 'ash off in water for ? minutes.
@leach with oxalic acid solution for
approximately 0 minute or until colorless. 'ash
well in water, and rinse in distilled water. Treat
with iron alum solution for 4 minutes. 'ash well
in several changes of distilled water. Treat with
ammoniacal silver solution for ,-4 seconds with
agitation of the slide. 'ash well in several
changes of distilled water. 8educe in 0+2
formalin in tap water for 3+-*+ seconds with
agitation of the slide. 'ash in water, and rinse in
distilled water. Treat with 42 sodium thiosulpahte
solution for 4 minutes. 'ash in distilled water.
ounterstain if desired, in 02 a#ueous neutral red
for 4 minutes. 1astly, wash, dehydrate, clear and
mount as desired.
!esults:
8eticulum fibres ! black $some pigments
like melanin are also impregnated%
ollagen ! yellow-brown
@ackground ! red if counterstained, clear
if not.
(lastic )i*res:
&lastic Fibres ! are branching fibres of
varying si"es and diameter that consists of protein
elasticin and glycoprotein microfibrils. >pecial
staining is re#uired to visuali"e and identify
changes.
+iegertss !esorcin %)uchsin Stain' ! >everal
variations show remarkable selectivity for elastic
fibres and give the best demonstration of fine
fibres.
!eagents:
=xalic acid solution ! 42 a#ueous
5cidified potassium permanganate
solution ! composed of +.32 a#ueous potassium
permanganate 3 32 a#ueous sulfuric acid. <ix in
e#ual parts immediately prior to use. an be kept
as stock solutions.
5cid 5lcohol ! 6ifferentiator
5mmonia water - ?2 a#ueous
;eutral red ! 02 wEv, a#ueous!
counterstain
'iegerts(s resorcinol fuchsin solution !
6issolve the basic fuchsin and resorcin in the
distilled water, bringing to boil in an evaporating
dish. 'hile boling, slowly add the a#ueous
anhydrous ferrric chloride solution, stirring
continuously. ontinue for approximately 4
minutes. ool and filter into a conical flask taking
care that all the precipitate is collected. 6iscard
the filtrate, dry the flask, and add the dried filter
paper containing the precipitate to the flask. 5dd
the alcohol and heat gently on a hot plate until the
precipitate is dissolved. 8emove the filter paper,
add the concentrated hydrochloric acid, cool and
filter. <ake up the final volume to ?++ m1 by
pouring fresh A42 alcohol through the used filter
paper. Filter before use. This solution keeps well
for months.
"rocedure:
First, take the section to water, then treat
with acidified potassium permanganate solution
for 4 minutes. 'ash well in water and bleach with
42 oxalic acid for approximately 0 minute. 'ash
well in water, and rinse in alcohol. >tain in
resorcin!tuchsin solution for 0-? hours at room
temperature. heck with microscope and stain
until elastic fibres are black. >ave solution. )f
overstained, rinse in ammonia water for 3
minutes. This will remove excess stain.
6ifferentiate in acid!alcohol until the background
is free of stain. 'ash well in tap water.
ounterstain as re#uired with neutral red
stainEeosinE.an /ieson. >ave solution.
overstained, water will remove excess stain.
1astly, wash, dehydrate, clear and mount as
desired.
!esults:
&lastic fibres ! blue-black
@ackground ! red $or according to the
counterstain used%.
Verhoeffs Stain ! a rapid method for staining
elastic fibres a strong black color.
!eagents:
.erhoeff(s solution ! for best results,
make up the solutions the same day they are to be
used.
>olution 5 !
Daematoxilyn 4g, 5bsolute alcohol 0++ m1.
6issolve with the aid of heat. ool and filter.
>olution @ ! Ferric
hloride 0+g, 6istilled water 0++ m1.
>olution $1ugol(s
iodine solution% ! iodine 0g, potassium iodide ?g,
6istilled water 0++ m1.5dd Gm1of solution @
into ?+ m1 of solution 5 and add that to G m1 of
solution .
"rocedure:
First, take the section to alcohol, then
stain with the freshly made .erhoeff(s solution for
04-,4 minutes until the sections are black.
6ifferentiate in ?2 ferric chloride with agitation,
only for a few minutes. heck differentiation by
rinsing in distilled water and examining under the
low power of the microscope. 'ash in water 3 in
alcohol for approximately 4 minutes to remove
the iodine coloration of the background. 'ash
well in water and counterstain in .an /ieson(s
stain for 0-? minutes. 1astly, wash, dehydrate,
clear and mount as desired.
!esults:
&lastic fibres ! black
;uclei ! grey to black
@ackground ! according to counterstain.
Stains for particular su*stances:
>taining of carbohydrates, amyloids,
pigments, and minerals $iron 3 calcium% and
micro-organisms is occasionally done in the
histopathology laboratory for the diagnosis of
specific pathologic conditions.
Car*oh,drates:
Car*oh,drates ! a simple carbohydrate
molecule is a monosaccharide such as glucose that
plays a central role in nutrition but is difficult to
demonstrate within tissues. )t can also be complex
like glycogen, which is a polysaccharide. 5nother
is <ucin, which is a <ucopolysaccharide. The
staining and identification of various types of
carbohydrates have contributed greatly to our
understanding of living structures such as the
liver, heart and etc.
"eriodic acid-Schiff %"-S' Stain ! 5 red or
purple-red color indicates a positive 95> reaction.
95> positive substances include hosts of organic
compounds $amyloid, glycogen, etc.%, micro-
organisms $amoebae, fungi, etc.%, body tissues 3
cells.
!eagents:
9eriodic acid solution ! 02 wEv a#ueous
>chiff(s reagent
Darris(s haematoxylin solution
1ight green counterstain
"rocedure:
First, take the section to distilled water,
then treat with periodic acid solution for 4 minutes
or 0+ minutes for basement membranes. 8inse
well in distilled water. Then, treat with >chiff(s
reagent for 04 minutes. 'ash in running tap water
for 4-0+ minutes to intensify the color reaction.
Then, stain the nuclei with either Darris(s
haematoxylin solution or light green as
counterstain. 5fter counterstaining, differentiate
with blue. 1astly, wash, dehydrate, clear and
mount as desired.
!esults:
95>-positive materials ! magenta $rose
to purple red%
;uclei ! blue or blue-black
-m,loids:
-m,loids ! a starch-like material,
principally a glycoprotein, formed by the
combination of carbohydrate with protein. )n
various pathologic conditions, abnormal #uantities
of glycoprotein may occur in organs and tissues,
leading to amyloidosis with pathologic lesions.
Congo red Stain ! an anionic dye, commonly
used for the staining and demonstration of
amyloids in the microscopic tissue section.
-lkaline Congo red technique ! a progressive
method, re#uiring no differentiation step.
!eagents:
Darris(s alum haematoxylin solution
>odium hydroxide solution ! 02 wEv,
a#ueous
>tock alcoholic sodium chloride !
saturated sodium chloride in G+2 alcohol.
ongo red alkaline solution
"rocedure:
First, take the section to water, then, stain
the nuclei with either Darris(s alum haematoxylin
solution. 5fter staining, differentiate with blue.
Treat with the alcoholic sodium chloride-
hydroxide solution for ?+ minutes, then drain.
>tain with ongo red solutions for ?+ minutes.
8inse in alcohol. 1astly, wash, dehydrate, clear
and mount as desired.
!esults:
5myloid ! orange- red
;uclei ! blue
@ackground ! clear
Toluidine *lue Stain ! is a basic dye which has
been reported to stain many tissue components,
including amyloids, an orthochromatic blue color.
7nder polari"ed light, amyloid is distinguished by
its striking dark red birefringence.
!eagents:
)sopropanol ! 4+2 vEv, a#ueous
Toluidine blue solution 02 solution in
4+2 isopropanol
"rocedure:
First, take the well-paraffini"ed section
to water, removing fixation pigment where
necessary. Then, stain in Toluidine blue solution
for 3+ minutes at 3B-. 5fter staining, blot section
carefully, then place in absolute isopropanol for 0
minute. 1astly, clear and mount as desired.
!esults:
5myloid 3 other tissue components ! an
orthochromatic blue color but under polari"ed
light, amyloid gives a striking dark red
birefringence.