Histochemistry

Techniques
Alcian Blue/PAS
Alkaline Congo Red
Bielschowsky
Bleaching Of Melanin
Bouin
CAB(Chromotrope Aniline
Blue)
Descalcificador
EDTA neutral
Eosin-Phloxine
Fite-Faraco(Wade Fite)
Fouchet
Giemsa(Biopsy)
Giemsa(Cytology)
Giemsa(H. Pylori)
GMS (Grocott)
GMS(basal membrane)
Gomori
Gram
Grimelius
Masson Fontana
Masson Thrichrome
Mayer's Haematoxylin
Modified Giemsa
Mucicarmim
Orcein
Papanicolaou (cytology)
PAS
Perl's
PTAH
Rehydration of air-dried
smears
Resorcin-Fucsin
Safranin O for Cartilage
Safranin(Thrichrome)
Sudan Black(or Oil Red O)
Toluidine Blue(for Mast
Cells)
UNNA(Methyl Green Pyronin)
Van Gieson
Von Kossa
Warthin-Starry
Weil's(Myelin)
Zhiel-Neelsen

Alcian Blue/PAS
1 ) Dewax sections and bring to
water

2) Alcian Blue 5m

3) H2O d

4) Periodic Acid 1% 5m

5) H2O d

6) Schiff's reagent 15m

7) Wash in water

8) Rinse in absolute alcohol

9) Xylene and Mount

Results:

Acid-Mucins-Blue Neutral
Mucins-Magenta Mixtures of the
above-the colour will depend on the
dominant entity and will range from
blue-purple through purple to a
violet or mauve colour

Solutions

A)Alcian blue 1g 3% acetic
acid 100 cm

B)Schiff's reagent Dissolve 1g
basic fuchsin in 200 cm of boiling
distilled water removing the flask
of water from the bunsen just
before adding the basic fuchsin
Allow the solution to cool to 50 C
and add 2g potassium metabisulphite
with mixing Allow to cool to room
temperature then add 2cm
concentrated hydrochloric acid
mix add 2g activated charcoal and
leave overnight in the dark at room
temperature Filter through a N1
Whatman paper when the solution
should be either clear or a pale
yellow colour Store in a dark
container at 4 C

Alkaline Congo Red
1)Sections to distilled water

2)Haematoxylin 3 min

3) Tap water until blue

4) Solution 1 (50ml sodium
chloride Saturated solution + 0.5ml
sodium hidroxid 1%) 20m

5) Solution 2 (50ml Congo
Red/Sodium chloride Saturated
solution + 0.5ml sodium hidroxide
1%) 20 min

6) Wash in alcohol 80%-95%-
100%-Xylene

7) Mount

Results: Amyloid elastic
tissue eosinophil granules-red
nuclei-blue Must use control

Bielschowsky
1 ) Dewax and bring sections to
water

2) Wash well in distilled water
1-2m

3) Place sections in 2% silver
nitrate in the dark at 37 C for
25m

2-3m

5) Reduce in 10% formalin
solution until turn yellow

3-5m

7) Place sections in ammoniacal
silver solution 20-40m

8) Treat with 10% aqueous
formalin solution until ten brown-
yellow

3-5m

10) Tone in 0.2% gold chloride
solution 3-5m

11) Wash well in distilled
water 1-2m

12) Treat with 5% sodium
thiosulphate solution 3-5m

13) Wash in tap water 3-5m

14) Dehydrate clear and mount

Result:

Axous-black

Ammoniacal Silver Solutions:

20% silver nitrate solution
15ml mixed with 10ml Absolute
Alcohol Add strong ammonia to mixed
solution until the initial
precipitate disappears After that
add 5 drops of ammonia and filter
before use

Bleaching Of Melanin
1) Potassium Permaganate 1%-3 m
2) Wash in tap water

3) Oxalic acid 2%-until the
section is clear

Bouin
ac. Pcrico 75ml

formol 25ml

ac. Actico 5ml

CAB(Chromotrope
Aniline Blue)
water

2) Stain in Weigert's
haematoxylin 5m

3) Wash in tap water 5m

4) Stain in 1% Fosfomolibdic
Acid 2m


6) Stain in Chromotrope Aniline
Blue solution for 8m


8) Dehydrate clear and Mount

Results

Nuclei-blue Collagen-blue
Hepatitis-red

Solutions:

Chromotrope 2R 4g

Aniline Blue 1g

0.2N HCL 200ml

Put Aniline Blue in HCL and
warm a little After cool add
chromotrope The PH should be 0.9-
1.3

Descalcificador
Nitric Acid 10ml

Formalin 10ml

H2Od 80ml

EDTA neutral
EDTA disodium salt 250g

Distilled water 1750cm

This solution is often cloudy
It is neutralised to PH 7 by the
addition of approximately 25g of
sodium hydroxide which will also
clear it.

Eosin-Phloxine
1) Eosin (stock solution) 100
ml

2) Phloxine (stock solution) 10
ml

3) Alcohol 95 780 ml

4) Glacial Acetic Acid 4ml

Stock solutions

Eosin y 1g

H2Od 100 ml

Phloxine B 1g

H2Od 100 ml

Fite-Faraco(Wade Fite)
1) Dewax in turpentin 20 m

2) Blot until almost dry

3) Wash in tap water blot dry
5m

4) Stain in fuchsin solution
(from Zhiel 1-filter first) 30m

5m

6) Decolourise in 10% sulphuric
acid until white 5m

5m

8) Stain in haematoxylin (or
methylene blue)

10m 10) Clean in xylene and mount

Results:

M Leprae (and other
mycobacteria)-Red

Nuclei-blue

Solutions:

Kinyoun Solution:

Basic fuchisin 40g

Phenol 80ml Ethanol (95%)
200ml Distilled water 1000ml

Methylene blue 2g

Distilled water 100ml

Fouchet
1) Sections to distilled water

2) Stain in Fouchet's reagent
5m

3) Rinse in distilled water

4 ) Counterstain in Van Gieson
solution

5) Dehydrate through alcohol to
xylene and Mount

Results:

Bile pigments-blue-green
Muscle-yellow

Collagen-red

Solutions: Fouchet reagent: 25%
trichloracetic acid (aqueous) 25ml
10% ferric chloride (aqueous) 25ml
V a n Gieson: Saturated aqueous
picric acid solution 90ml

1% aqueous acid fuchsin
sol.10ml

Giemsa(Biopsy)
water 2) Put slides in May-grunwald
working solution 20 minutes at 60C
3) Put slides in Giemsa working
soluton 40m at 60C

4) Differentiate in acetic acid
(0.5%) 5sec

5) Dehydrate in Alcohol/Acetone
(1:1)

6) Xylene and mount

Results: Red blood cells-yellow
to pink Nuclei-dark blue

Eosinophil granules-red

Solutions:

Working solution (prepare fresh
each time)

Stock May-Grunwald stain 10ml
H2Od 50ml

Stock Giemsa stain 1.75ml

H2Od 50ml

Giemsa(Cytology)
1) Air dry slides (do not fix)

2) Fix in alcohol 95 1m

3) Tap water

4) May-Grunwald Nile (pure) 10m

5) Tap water

6 ) Giemsa Diluted 1/6 in
distilled water 15m

7) Tap water

8) Air dry and mount

Results:

Red blood cells-yellow to pink
Nuclei-dark blue

Eosinophil Granules-red

Giemsa(H. Pylori)
water

2) Put slides in Giemsa 20%
30m


4) Differentiate in alcohol 95

5) Dehydrate and mount

GMS (Grocott)
1)Dewax and bring section to
water

2)Oxidise in 5% chromic acid 1
hour (after 30m prepare solution
(C) and put in oven at 60C)


4) Rinse in 1% sodium
bisulphite

5) Wash in tap water for 3m

6) Rinse well in several
changes of distilled water

7) Place in incubating solution
at 60C in the dark for 1hour

8) Wash well in many changes of
distilled water

for 4m 10) Place section in 3%
sodium thiosulphate for 5m s

11) Wash well in tap water

12) Lightly counterstain in
light green solution 20 seconds



RESULTS:

Fungal hyphae and yeast bodies
- black

Background - pale green

Solutions:

a) 5% solution tebraborate in
distilled water (freshly prepared)

b) 5% silver nitrate 5ml

3% methenamine (hexamine) 100ml

Add the methenamine solution to
the silver nitrate solution a
white precipitate forms but clears
on shaking (solution "a" and "b"
keep well at 4C)

c) Incubating solution

Borax solution "a" 5ml


Methenamine silver "b" 25ml

d) Light green counterstain

Light green 100mg

Acetic acid 0.1ml


GMS(basal membrane)
1 ) Dewax and bring section to
water

2 ) Oxidise in 1% periodic acid
for 15m


4) Rinse in 1% sodium
bisulphite


6) Rinse well in several
changes of distilled water

7) Place in incubating solution
at 60 C in the dark for 1hour

8) Wash well in many changes of
distilled water

for 4m

10) Place section in 3% sodium
thiosulphate for 5m


12) Lightly counterstain in
light green solution 20secod



Results:

Basal Membrane-black
Background-pale green

Solutions:

a) 5% solution tebraborate in
distilled water (freshly prepared)

b) 5% silver nitrate 5ml

3% methenamine (hexamine) 100ml
Add the methenamine solution to the
silver nitrate solution a white
precipitate forms but clears on
shaking (solution "a" and "b" keep
well at 4C)

c) Incubating solution

Borax solution "a" 5ml


Methenamine silver "b" 25ml

d) Light green counterstain

Light green 100mg

Acetic acid 0.1ml


Gomori
1 ) Dewax section and bring to
water

2) Treat with 1% potassium
permangante solution 2m

3) Rise in tap water

4) Bleach in 2% potassium
metabisulphite solution

5) Rinse in tap water

6) Treat with 2% iron alum 5m

7) Wash in several changes of
distilled water

8) Place in silver solution 1m

9) Wash in several changes of
distilled water

10) Reduce in 4% aqueous
formalin solution 4m

11) Wash in distilled water

solution 10m


14) Treat with 2% potassium
metabisulphite solution 1m


thiosulphate solution 1m


1 8 ) Counterstain with van
gieson or light green


Results:

Reticular fibres-black

Nuclei-grey

Other tissue-according to
counterstain

Solutions:

1) 1% potassium permanganate in
distilled water

2) 2% potassium metabisulphite
in distilled water

3) 2% iron alum in Distilled
water

4) 4% formalin(in H2Od)

5) 0.2% gold chloride in
distilled water 6) 2% sodium
thiosulphate in distilled water

7) Silver solution

To 10ml of 10% potassium
hydroxide solution add 40ml of 10%
silver nitrate solution allow the
precipitate to settle and decant
the supernatant Wash the
precipitate several times with
distilled water Add ammonia drop by
drop until precipitate has just
dissolved Add further 10% silver
nitrate solution until a little
precipitate remains. Dilute to 100
ml and filter Store in a dark
bottle

Gram
water

2) Cover the slide with sol 11m
pour off

3) Rinse with sol 2

4) Cover the slide with sol 2
1m

5) Rinse with D W about 5sec

20-60sec

7) Rinse the D W about 5sec

1m

9) Rinse the D W about 5sec

10) Leave to air dry Mounting

Results:

Gram-positive microorganisms-
blue Gram-negative microorganisms-
pink to red

Grimelius
1) Rehydrate sections through
graded alcohols to distilled water

2) Place sections in preheated
silver solution at 60C for 3
hours

3) Remove sections and drain
thoroughly

4) Place sections in freshly
prepared reducing solution at 45C
for 1 minute

5) Rinse sections in distilled
water

6) Examine the sections
microscopically to check
impregnation If the sections are
under-impregnated return to the
silver bath for a further 5-10 m

7) Drain sections and repeat
reduction

8) Rinse in distilled water for
1m


in DPX

Results:

Argyrophil granules-
brown/black Neither insulin nor
somatostatin containing granules
will stain with this method

Solutions:

A)Silver solution 1% aqueous
silver nitrate freshly made 3 cm

Acetate buffer (PH 5.6) 10 cm
Double-distilled water 87 cm
B)Reducing solution

Hydroquinone 1g

Sodium sulphite crystals 5g

Distilled water 100 cm

Masson Fontana
1) Sections to water

2) Treat with silver solution
for 45 m at 37C

3) Wash well in several changes
of distilled water

thiosulphate for 2m


6 ) Counterstain in light green
for 2m 7) Wash in tap water

8) Dehydrate throug graded
alcohol to xylene

9) Mounting

Results:

Melanin-black

Argentaffin-black

Chromaffin-black

Some lipofucsins-black

Nuclei-green

Solutions:

Silver solution:

To 20cm of 10% silver nitrate
add concentrated ammonia drop by
drop until the formed precipitate
almost dissolves A faint
opalescence is evident when the end
point is reached If too much
ammonia is added 10% silver nitrate
can be added drop by drop until the
faint opalescence obtained To this
silver solution add 20cm of
distilled water and filter Stone in
a dark bottle The solution will
last for about four weeks

Masson Thrichrome
water

2) Stain in Weigert's
haematoxylin 5m and differentiate
in HCl 1% in alcohol 70%


4) Stain in acid fuchsin
solution and ponceau de xilidine
solution 1:2 for 5m

5) Treat with phosphomolybdic
acid solution 1min

6) Stain in methyl blue or
light green solution 2m

7) Treat with 1% acetic acid
solution 1m

8) Wash in H2Od Alcohol 100
clear and mount

Results:

Nuclei-blue to black

Cytoplasm muscle and
erythrocytes-red

Collagen-blue or green

Solutions:

1)Weigert's haematoxylin:
A)Haematoxylin 1g

Absolute alcohol 100ml

B)30% ferric chloride 4ml
Hydrochloric acid 1ml


Solution "a" and "b" mixed
immediately before use with 1:1
2)Acid fuchsin solution:

Acid fuchsin 0.5g

Glacial acetic acid 0.5ml


3 ) Ponceau de xilidine
solution: Ponceau de xilidine
(ponceau 2R)1g Glacial acetic
acid 1ml

Distilled water 100 ml

4) 1% phosphomolybdic acid in
distilled water

5) Methyl blue solution:

Methyl blue 2g

Glacial acetic acid 2g


6) Light green solution:

Light green 1g

Glacial acetic acid 1ml


7) 1% acetic acid in distilled
water

Mayer's Haematoxylin
Haematoxylin 1g

Distilled water 1000cm

Potassium or ammonium alum 50g
(12H2O 91.8g)

Citric acid 1.09g

Chloral hydrate 50 g

Sodium iodate 0.2g

1- Dissolve the alum in the
distilled water

2- When complete add the
haematoxilin

3- When all the haematoxilin
has dissolved add the sodium iodate
and let stir for 10m

4- Add the citric acid and stir
for more 10m

5- Add the chloral hydrate and
stir till dissolved

No need to filter.Ready to use.

To test just put a few drops
into warm water they should turn
blue immediately.

Modified Giemsa
water

2) Stain in 2% Giemsa for 30 m

3) Wash in tap water quickly

4) Air dry and bring to xylene

5) Mount

Result: Helicobacter pyloric-
blue Solutions:

Giemsa's stain 2g


Mucicarmim
1)Take sections to water

2)Hematoxylin 5m

3) Tap water

4) Diferenciate in alcohol-Acid
2m

5 ) Mucicarmim solution
(filtrate before use) 30m

6) Distilled water quickly

7) Metanyl yellow 1m

8) Distilled water


Results:

Mucin-dark pink to red
Criptococus capsul-dark pink to red
Nucleo-black Other tissues-yellow
Solutions:

Carmin CI n 75450 1g

Aluminium chloride anidro 0.5g
Distilled water 2ml

Alcohol 50% 100ml

Mix all together and boile for
more or less 2m until turn drak
After cold filtrate and put in
refrigerator Yellow metanil:

Metanil yellow 0.25g


Acetic acid glacial 0.25ml

Orcein
1)Dewax and bring section to
water

2)Treat with acid permanganate
solution 10m


4) Bleach with 2% aqueous
oxalic acid solution until clear


6) Rinse in 70% alcohol

7) Stain in Orcein solution at
room temperature 4hours



Results:

Hepatitis B affected liver
cells-medium brown to black
Background-clear to pale brown

Solutions:

A) Acid permanganate solution
Potassium permanganate 1.5g
Concentrated sulphuric acid 1.5ml
Distilled water 100 ml

B) Orcein solution

Orcein 2g

70% alcohol 100ml

Concentrated hydrochloric acid
2ml

Papanicolaou (cytology)
1) Rinse slides in 95 %
alcohol

alcohol

3) Rinse slides in distilled
water

4) Stain slides in Mayer's
haematoxylin for 3m

5) Wash in tap water to blue
haematoxylin

6) Rinse slides in 95% alcohol

7) Stain slides in OE6 for 3m

8) Rinse slides in 95% alcohol
twice

9) Stain slides in EA50 for 3m

alcohol twice

11) Rinse slides in absolute
alcohol twice

12) Rinse slides in xylene
twice 13) Mount in D P X

Results: Nuclei-blue
Superficial cell cytoplasm-pink
Intermediate and parabasal cell
cytoplasm-blue-green

PAS
1) Section to water

2) 0.5% aqueous periodic acid 5
m

3) Rinse in tap water and
distilled water

4) Stain with Schift's reagent
15m

5) Wash in running tap water
10m

6) Stain in haematoxylin 1min


8) Dehydrate through alcohols
to xylene and mount

Results:

A c i d mucin Neutral mucin-
magenta Nuclei-blue

Solutions:

1) 0.5% periodic acid in
distilled water

2) Schiff's reagent:

Basic fuchsin 1g


Potassium metabisulphite 2g
Hydrochloric acid 1N 2ml (1N=HCl
8.35ml+H2Od 91.65ml)

Activated charcoal 2g

Dissolve 1g basic fuchsin in
200ml of boiling distilled water
removing the flask of water from
the bunsen just before adding the
basic fuchsin Allow the solution to
cool to 50C add 2g potassium
metabisulphite with mixing Allow to
cool to room temperature then add
2ml concentrated hydrochloric acid
mix add 2g activated charcoal and
leave overnight in the dark at room
temperature Filter through a N 1
Whatman paper when the solution
should be either clear or a pale
yellow colour Store in a dark
container at 4C

Perl's
water


3) Transfer section to freshly
prepared incubating solution 10m


5) Counterstain in nuclear fast
red

6) Wash rapidly in tap water

7) Dehydrate through graded
alcohols to xylene

8) Mount

Results:

Ferric iron-blue Nuclei-red
Solutions:

A) Incubating solution:

2% hydrochloric acid 25ml

2% potassium ferrocyanide
25ml (Prepare fresh before use)

B) Nuclear fast red

Aluminium sulphate 5g


Nuclear fast red 100mg

(boil for 5m)

PTAH
(Phosphotungstic Haematoxylin)

water

2) Iron alumen 4% 20m-1hour

3) PTAH solution at 60 1-
2hours

4) Rinse in alcohol 95 (fast)

5) Alcohol 100 xylene and
mount

Results:

Muscle striations neuroglia
fibres fibrin amoebae-dark blue

Nuclei cilia red blood cells-
blue Myelin-light blue

Collagen osteiod cartilage
elastic fibers-deep brownish red
Cytoplasm-pale pinkish brown
Parietal cell-pale blue

Solutions:

P T A H Haematoxylin 0.5g
Phosphotungstic acid 10g H2Od
500ml Potassium permaganate
(0.25%) 25ml Dissolve the
haematoxilin in 100 ml distilled
water and the acid in the remaining
water Mix the two together and add
the potassium permaganate Use after
24 hours

Rehydration of air-dried
smears
1) Put slides in 50% aqueous
solution of glycerine 3m

2) Wash in 95% alcohol 2 times

3) Wash in tap water then in
H2Od

4) Stain in haematoxylin for
3m


6) Stain with eosina or Shorr

7) Wash in 9)5)% alcohol


Resorcin-Fucsin
water

2) Place in working solution
overnight (at least 12 hours)



3-5 m


Results:

Elastin fibre-deep purple
Collagen fibre-red Epithelial cells
and muscles-yellow

Stock Solution:

1)Dissolve 2g crystal violet
and 4g resorcinol in 100ml
deionised water

2)Boil for 3m stirring
constantly with a combined magnetic
stirrer and hotplate

3) Add 15ml freshly prepared
30% aqueous anhydrous ferric
chloride and bring back to the
boil

4) Add 2g basic fuchsin or
pararosaniline (Product P1528;
Sigma Poole Dorset United
Kingdom) in 50 ml distilled water
together with a further 15ml ferric
chloride solution and boil again

5) Cool and filter

6) Retain the filtrate
(solution 1) and the filter paper
with its attached deposit

7) Place this filter paper and
its deposit in 150ml 90% alcohol
and boil on a hotplate for 3m :
then filter once more and retain
both the second filtrate and its
accompanying filter paper with
deposit

8) Make the second filtrate up
to 150 ml with 95% alcohol and
4.5ml concentrated hydrochloric
acid and combine with solution 1

9) Add the second filter paper
together with its deposit

10) Bring to the boil on an
electric hotplate add a further 4g
resorcinol and boil for three m :
filter while hot The filtrate
constitutes the stock solution

Working solution Take 10 ml of
the stock solution and add 40 ml of
50% alcohol Add 1ml concentrated
hydrochloric acid

Safranin O for Cartilage
1) Sections to water

2) Stain with Mayer's
haematoxilina 3) Wash in running
tap water

4) Stain in SafraninO 5m

5) Dehydrate starting on
alcohol 95 and stay 2m on each
solution to differentiate

6) Xylene and mount

Results:

Nuclei - blue Cartilage mucin
mast cell granules - orange to red
Solutions

Safranin O (C I 50240) 0.1g

H2Od 100ml

Safranin(Thrichrome)
water

2) Stain in haematoxylin 3m


4) Stain in eosin 1m

5) Wash in 95% alcohol to 100%
alcohol

6) Stain in SafraninO solution
5m

7) Wash in 100% alcohol

8) Clear and mount

Results: Cytoplasm-pink to
yellowish

Solutions

Safranin O 2g

Absolute alcohol 100 ml

Sudan Black(or Oil Red
O)
1) Put frozen sections in
alcohol 70% 1-2m

2) Stain in black sudan
saturated or oil red O (filtrate
solution first and cover in a petri
dish) 15m

3) Wash in 70% alcohol unitl
almost no colour



6) Rinse in distilled water

7) Mount in glicerine and close
around with nail polish

Results:

Stains unsaturated cholesterol
esters and trigrycerides-blue-black
Some phospholipids appear grey Red
Oil O: hydrophobic lipids and
mineral oils are stained red Some
phospholipids appear pink

Toluidine Blue(for Mast
Cells)
water

2) Cover in Toluidine Blue 0.5%
Solution 30m

3) Wash in tap water quickly

4 ) Differenciate in 0.5%
Glacial acetic acid util only the
cell nuclei and Mast cell are
stained purple control under the
microscopy


6) Air dry Mounting

Results:

Mast cell granules-purple Cell
nuclei-blue

Solution:

0.5% Toluidine blue in H2Od

UNNA(Methyl Green
Pyronin)
1)Dewax and bring sections to
water

2)Rinse in acetate buffer PH
4.8

3) Place in staining solution
25m


5) Rinse fast in acetone

6) Acetone-xylene 1:1 xylene
and mount

Results:

DNA-green-blue RNA-red Some
mucous cells may be stained by the
pyronin

Solution:

Staining solution (Prepare
before use)

Stock 2% methyl green 15cm
(Chloroform washed)

Stock 2% pyronin Y 4cm

Acetate buffer PH 4.8 23cm
Glycerol 14cm

Acetate buffer A Stock solution
0.2M acetic acid (MW 60.05) 1.2cm
glacial acetic acid in 100 cm H2Od

B Stock solution 0.2M sodium
acetate 1.64g sodium acetate
anhydrous (MW 82) or 2.72g sodium
acetate trihydrate (MW 136) in 100
cm of distilled water

Buffer solution PH 4.8

20ml sol A

30ml sol B

50ml H2Od

Van Gieson
water



4) Stain in Van Gieson solution
3m 5) Blot with paper


Results:

Nuclei-blue Collagen-red Other
tissue-yellow

Solution

Van Gieson solution:

Saturated aqueous picric acid
solution 50ml

solution 9ml


Von Kossa
1) Section to distilled water

2) Place in silver nitrate
solution and expose to strong light
60m

3) Wash in 3 changes of
distilled water

4) Treat with sodium
thiosulphate 5m


2m


Results:

Mineralised bone -black
Calcium-golden yellow

Solutions:

1) 1% silver nitrate:

Silver nitrate 1g


2) 2.5% sodium thiosulphate:

Sodium thiosulphate 2.5g


3) Van Gieson:

Saturated aqueous picric acid
solution 50ml

solution 9 ml Distilled water 50
ml

Warthin-Starry
water

2) Buffer solution (a)

3) Solution b at 60 1hour

4) Solution c at 60 3m

5) H2OC at 60

6) Buffer solution


Results:

Spirochactes-black or dark
yellow-brown

Background-pale yellow to brown

Solutions:

A Buffer solution

Sodium acetate 1.64g

Acetic Acid 2.5ml

H2Od 200ml

B Silver solution

Silver nitrate 0.5g

Buffer solution (a) 50ml

C Reduction solution

a)Hydroquinon 300mg

Buffer solution 10ml

Mix 3cc with 45cc of gelatin
(5%) and store at 37%

b) Silver Nitrate 2% at 60
9ml

Mix solution a) and b) before
use

Weil's(Myelin)
water

2) Stain in working solution 20
m

3) Tap water

4 ) Differenciate in 4% iron
alum 8 seconds (until the
background is grey)

5) H2Od

6) Complete differentiation in
Weigert's Borax ferricyanide
solution 1sec

7) H2Od and tap water

8) Eosin 10sec


Results:

Myelin-dark grey to black
Background-pink

Solutions:

Working solution

4% iron alum and 1%
haematoxilin 1:1 Mix just before
use !!!

Weigert's Borax Ferricyanide
Solution

Borax (Di-Sodium Tetraborate)
2g Potassium Ferricyanide 2.5g
(if have 3H2O 2.86g)

H2Od 200ml

Zhiel-Neelsen
1 ) Dewax section and being to
water

2) Wash in D W

3) Stain in Sol 1(dye red) 10m
(filter solution before use)

4) Wash in tap water well

5) Stain in Sol 2(dye blue) 5m
(filter solution before use)

6) Wash in tap water dry

7) Mounting

Results: Bacilli-red

Other tissues-blue

Histochemistry

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Histochemistry

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