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SURGICAL PATHOLOGY - HISTOLOGY Dat e:
STAINING MANUAL - CONNECTIVE TISSUE Page: 1 of 4
COLLA GEN - MA SSON'S T RI CHROME STA I N(T RI )
PURPOSE: Used t o diff erent iat e between collagen and smooth muscle in
t umors, and the increase of collagen in diseases such as cirrhosis.Rout ine st ain f or liver and kidney biopsies.
PRI NCI PLE: As t he name implies, t hree dyes are employed select ively
staining muscle, collagen f ibers, fibrin, and eryt hrocyt es. The general rule
in t richrome staining is that t he less porous t issues are colored by t he
smallest dye molecule; whenever a dye of large molecular size is able to
penet rate, it will always do so at t he expense of t he smaller molecule.
Ot hers suggest t hat t he t issue is st ained first wit h t he acid dye, Biebrich
Scarlet , which binds with t he acidophilic t issue components. Then when
treated with the phospho acids, the less permeable components retain the
red, while t he red is pulled out of t he collagen. At t he same t ime causing a
link wit h t he collagen t o bind wit h t he anil ine blue.
FIXATIVE: Bouin's is preferred, 10% formalin.
TECHNI QUE: Cut paraf f in sect ions 4 m
EQUI PMENT: Rinse glassware in DI water. Coplin jars, 60°C oven or
wat erbat h, microwave
REAGENTS:
Boui n ’ s Fi x at i v e
Sat u rat e d picr ic acid 1 50 0.0 ml
Formaldehyde 500.0 ml
Glacial acet ic ac id 100.0 ml
Mix well, label, dat e and init ial.
Stable for 2 years.
CA UT I ON: Ca r c i n og en i c , i r r i t a nt .
Bi ebr i c h Sc ar l et :
Biebr ich scar let 2.7 gm
Acid f uchsin 0.3 gm
Dist i l led wat er 300.0 ml
Glacial acet ic acid 3 .0 ml
Mix well, label wit h init ial and
date. Solut ion is stable for 6months.
CAUTI ON: Avoi d cont ac t and i nha l a t i on.
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CONNECTIVE TISSUE
MA SSON’ S TRI CHROME Page: 2 of 4
Wei ger t ' s I r on Hem at ox y l i n
St o c k So l u t i o n A :
Hemat oxy lin 5.0 gm
95% alcohol 500.0 ml
Mix well, label wit h init ial and
date. St able for 1 year.
CAUTI ON: Fl ammab l e , avo i d cont ac t and
i n h a l a t i o n .
St o c k So l u t i o n B:
2 9 % f err ic chlor ide 20 .0 ml
Dist i l led wat er 475.0 ml
Hydrochloric acid 5 .0 ml
Mix well, label wit h init ial and
date. St able for 1 year.
CAUTI ON: Co r r os i ve , avo i d con t ac t and
i n h a l a t i o n .
Wei ger t ' s Hem at ox y l i n
Wo r k i n g So l u t i o n
Solut ion A 25 .0 ml
Solut ion B 25 .0 ml
Mix well, solut ion will remain
stable for 3 - 4 days.
Ph os p ho t u ng s t i c /
Ph os p ho m ol y b di c A c i d So l u t i o n:
Phosphotungst ic acid 25 .0 gm
Phosphomolybdic acid 25.0 gm
Dist i l led wat er 1000 .0 ml
Mix well, label wit h init ial and
date. Solut ion is st able for 6
months.
CAUTI ON: Avoi d con t ac t and i nha l a t i on.
A ni l i ne Bl ue:
Anil ine b lue 2.5 gm
Dist i l led wat er 100 .0 ml
Glacial acet ic ac id 1 .0 ml
Mix well, label wit h init ial and
date. Solut ion is st able for 6
months.
Ca ut i o n: A v o i d c o n t a c t a nd i n ha l a t i o n.
1 % A c e t i c A c i d :
Glacial acet ic ac id 10 .0 mlDist i l led wat er 1000 .0 ml
Mix well, label wit h init ial and
dat e. Solut ion is st able f or 1 y ear.
Ca ut i o n: A v o i d c o n t a c t a nd i n ha l a t i o n.
SAFETY: Wear gloves, goggles and lab coat . Avoid cont act and inhalat ion
of dyes and chemicals.
Bouin's cont ains: formaldehyde, a known carcinogen, picric acid canbecome explosive when dry. Toxic by skin absorbt ion. Keep hot uncapped
Bouin’s under t he hood.
Phosphomolybdic, phosphotungsic acid powders, and acet ic acid solut ions
are skin and eye irrit ant s, and st rong corr osives.
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CONNECTIVE TISSUE
MA SSON’ S TRI CHROME Page: 3 of 4
PROCEDURE:
1. *Mordant in Bouin's solut ion, microwave 1 minute, allow to stand 15
minut es.
2. Wash in running t ap wat er to remove t he p icr ic ac id, 5 minutes.
3 . Weiger t ' s work ing hemat oxy lin , 10 minut es.
4 . Blue in running t ap wat er fo r 5 minutes , r inse in d ist i lled wat er.
5 . Biebr ich scar le t fo r 5 minu tes.
6 . Rinse in d ist il led wat er .
7. Phosphotungst ic/ phosphomolybdic acid for 10 minut es, discard
solut ion.
8 . Transfer di rect ly int o Ani line blue for 5 minutes .
9 . Rinse in d ist il led wat er .
10. 1% Acet ic acid for 1 minut e, discard solut ion, r inse in dist i lled wat er.11. Dehydrat e, clear, and coverslip.
*Convent ional met hod: Mordant in Bouin's solut ion, 60 °C f or 1 hour.
RESULTS:
Nuclei b lack
Cy t oplasm , muscle, ery t hrocy t es red
Collagen blue
NOTES:
1. Light green may be subst i tut ed for Ani line blue..
2. 5% phosphotungst ic acid for 5 minut es, must be subst itut ed when
using Light green.
3. When st aining liver biopsies, t he collagen is bett er light blue t han
dark blue.
REFERENCE:
Sheehan D, Hrapchak B, Theory and pract ice of Histot echnology, 2nd Ed,
19 80 , pp 18 9-1 90 , Batt elle Press, Ohio
Bancrof t J, St evens A, Theory and Pract ice of Histological Techniques, 2ndEd, 19 82 , pp 1 31 -13 5, Churchill-Livingst on, NY
Luna L, Manual of Histologic St aining Met hods of t he AFIP, 3rd Ed, 1968, pp
9 4 -9 5 , McGraw-Hil l, NY
Carson F, Histot echnology A Self-Inst ruct ional Text , 1st Ed, 19 90, pp
1 42 -1 4 3 , ASCP, I ll
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CONNECTIVE TISSUE
MA SSON’ S TRI CHROME Page: 4 of 4
Crookham,J, Dapson,R, Hazardous Chemicals in t he Hist opat hology
Laboratory, 2nd ED, 19 91, Anatech
Prepared: By:
Approved: By:Downloaded from WebPath: Internet Pathology Laboratory
htt p:/ / www-medlib.med.ut ah.edu/ WebPath/ webpat h.ht ml
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PROCEDURE CARD
TRI CHROME STA I N, MA SSON'S ( TRI ) - COLLA GEN Page 1 of 2
PROCEDURE:1. *Mordant in Bouin's solut ion, microwave 1 minut e, allow to stand 15 minutes.
2. Wash in running tap wat er t o remove t he picric acid, 5 minutes.
3. Weigert ' s work ing hematoxy lin , 10 minutes.4. Blue in running tap wat er for 5 minut es, rinse in dist i lled wat er.
5 . Biebr ich scar le t f or 5 minu tes.
6 . Rinse in dist i lled wat e r.
7. Phosphotungst ic/ phosphomolybdic acid for 10 minut es, discard solut ion.
8. Transfer di rect ly into Anil ine b lue for 5 minutes.
9 . Rinse in dist i lled wat e r.
10. 1% Acetic acid for 1 minute, discard solution, r inse in dist i l led wat er.
11. Dehydrat e, clear, and coverslip.
*Convent ional met hod: Mordant in Bouin's solut ion, 60 °C fo r 1 hour.
RESULTS:Nuclei black
Cytoplasm, muscle, erythrocytes red
Collagen blue
SAFETY/ PPE: Wear gloves, goggles and lab coat. Keep hot Bouin’s under t he fume hood.
We i g e r t ' s I r o n He m at o x y l i n
St o ck S ol u t i o n A :
Hemat oxylin 5.0 gm
95% alcohol 50 0 .0 ml
Mix well, label wit h init ial and date. Stablefor 1 year.
CAUTION: Fl amm abl e
St o c k So l u t i o n B :
2 9 % f erric chlor ide 2 0 .0 ml
Dist illed wat er 47 5 .0 ml
Hydrochloric acid 5 .0 ml
Mix well, label wit h init ial and date. Stable
for 1 year.
Wo r k i n g So l u t i o n
Solut ion A 2 5 .0 ml
Solut ion B 2 5 .0 ml
Mix well, solution will remain stable for 3 -
4 days.
Bo ui n ’ s Fi x a t i v e
CA UT I ON: Ca r c i n o ge ni c , i r r i t a n t ,
t o x i c .
B i e br i c h Sc a r l e t :
Biebrich scarlet 2.7 gm
Acid fuchsin 0.3 gm
Dist illed wat er 3 00 .0 ml
Glacial acet ic acid 3 .0 ml
Mix well, label with init ial and date. Solut ion
is st able for 6 mont hs.
CAUTI ON: Poss i b l e ca r c i nogen
Ph os p ho t u ng s t i c /
Ph os p ho m ol y b di c A c i d So l u t i o n:
Phosphotungst ic acid 25 .0 gm
Phosphomolybdic acid 25.0 gm
Dist illed wat er 1 0 00 .0 ml
Mix well, label with init ial and date. Solut ion
is st able for 6 mont hs.
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PROCEDURE CARD
TRI CHROME STA I N, MA SSON'S ( TRI ) - COLLA GEN Page 2 of 2A n i l i n e B l u e :
Aniline blue 2.5 gm
Dist illed wat er 10 0 .0 ml
Glacial acet ic acid 1 .0 mlMix well, label with init ial and date. Solution
is st able for 6 mont hs.
1 % A c e t i c A c i d :
Glacial acet ic acid 1 0 .0 ml
Dist illed wat er 1 00 0 .0 ml
Mix well, label with init ial and date. Solutionis st able for 1 year.
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1 % ACETI C A CI D:
Glacial acet ic ac id 10 .0 ml
Dist i l led wat er 1000 .0 ml
Mix well, label wit h init ial and
date. Solution is st able f or 1 year.
Ca ut i o n: Sk i n an d e y e i r r i t a nt
DATE:
TECH:
EXPIRATION:
A NI LI NE BLUE:Aniline b lue 2.5 gm
Dist i l led wat er 100 .0 ml
Glacial acet ic ac id 1 .0 ml
Mix well, label wit h init ial and
date. Solut ion is stable for 6
months.
Ca ut i o n: A v o i d c on t a c t a nd i n h al a t i o n.
DATE:
TECH:
EXPIRATION:
ANI LI NE BLUE
DATE:
TECH:
BI EBRI CH SCARLET:
Biebr ich scar let 2.7 gm
Acid f uchsin 0.3 gm
Dist i l led wat er 300 .0 ml
Glacial acet ic acid 3 .0 ml
Mix well, label wit h init ial and
date. Solut ion is st able for 6
months.
Ca ut i o n: A v o i d c on t a c t a nd i n h al a t i o n.
DATE:
TECH:
EXPIRATION:
BI EBRI CH SCARLET:
DATE:
TECH:
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PHOSPHOTUNGST I C/
PHOSPHOMOLYBDI C A CI D
SOLUT I ON:
Phosphot ungst ic acid 25 .0 gm
Phosphomolybdic acid 25.0 gm
Dist i l led wat er 1000 .0 ml
Mix well, label wit h init ial and
date. Solut ion is stable for 6
months.
Ca ut i o n: A v o i d c on t a c t a nd i n h al a t i o n.
DATE:
TECH:
EXPIRATION:
PHOPHOTUNGST I C /
PHOSPHOMOLYBDI C A CI D:
DATE:
TECH:
WEI GERT' S I RON HEMAT OXYLI N:
St o c k So l u t i o n A :
Hemat oxy lin 5.0 gm
95% alcohol 100 .0 ml
Mix well, label wit h init ial anddate. St able for 1 year.
CAUTION: FLAMMABLE, avoid
cont act and inhalat ion
TECH:
DATE:
EXPIRATION:
WEI GERT’ S I RON HEMAT OXYLI N:
St o c k So l u t i o n B:
2 9 % f err ic chlor ide 20 .0 ml
Dist i l led wat er 475 .0 ml
Hydrochloric acid 5 .0 ml
Mix well, label wit h init ial and
date. St able for 1 year.
CAUTI ON: Co r r os i ve , avo i d con t ac t and
i n ha l a t i o n.
TECH:
DATE:
EXPIRATION:
WEI GERT' S HEMAT OXYLI N
DATE:
TECH:
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