Trichrome variants

In the following charts it is to be understood, unless otherwise specified, that paraffin sections are to be brought to water via xylene and ethanols, and an acid resistant nuclear stain applied prior to staining. Following application of the stain the sections are to be dehydrated with ethanol, cleared with xylene and mounted with a resinous medium.

It should also be noted that most trichrome methods benefit from fixation with picric acid or mercuric chloride containing fixatives. If simple formalin fixation has been used, then treatment with aqueous picric acid or Bouin's picro-acetic-formalin mixture at 56°C for an hour or so will usually be beneficial.

 

 

Terms

Dyes To conserve space, dyes and chemicals are identified by abbreviations. These are explained at the end of this page. With most browsers an explanation will appear if the cursor is placed over the abbreviation for a few seconds.
Variant The individual published method. More complete details are given in individual pages for each method. To access these pages click on the name in this column.
RBC Some trichrome methods incorporate a step for erythrocytes. The commonest colours to use are orange and yellow. Often the dyes are dissolved in ethanol, and may include a polyacid or some other acid.
Plasma In many trichrome methods the plasma stain is the red component. This step usually demonstrates muscle, cytoplasm and fibrin. If the RBC step is omitted, erythrocytes usually stain with this colouration.
Diff Many techniques incorporate a differentiation step. Most commonly this uses phosphomolybdic or phosphotungstic acid. The polyacid removes the plasma stain from collagen. This increases the contrast between the plasma and fibre stains.
Fibre This step incorporates a large molecule dye to stain collagen and bone in a colour that strongly contrasts with the plasma stain. The commonest colours are blue or green. Rarely, yellow is used.
Yellow Yellowsolve techniques use a large molecular weight yellow dye in an anhydrous solvent. The earliest method used absolute ethanol, but the more complex methods use 2-ethoxy-ethanol (cellosolve). These techniques may, or may not, incorporate a polyacid differentiation step.
Chemical Reagents other than dyes and solvents.
Wash A very few methods recommend a final wash in dye solutions. These usually are designed to refresh erythrocyte staining, as this may have been degraded by the end of the method, and to remove excess fibre stain.

 

Multi step methods
Each column in the chart below represents a separate solution. For each solution, all fluids are to be measured in millilitres and all solids in grams. Unless otherwise specified, the solvent is 100 millilitres distilled water. Where another fluid is included, the volume of water should be adjusted to make 100 millilitres total volume of solvent. Where ethanol is specified, absolute (100%) ethanol should be used. In most cases fluids should be diluted first to make the solvent, then the solids added and dissolved in sequence. All solutions should be filtered before being used.

Variant RBC Plasma Diff Fibre Wash Comments
Brillmeyer   AF 0.2   AB 0.5
OG 0.2
PM 1
   
Bensley   AF sat. PM 1 AB 0.5
OG 2.0
   
Crossman   AF 0.3
OG 0.13
AC 1
PM 1 AB 2
AC 2
   
Goldner   AF 0.3
PR 0.7
AC 0.2
OG 2
PM 4
LG 0.2
AC 0.2
   
Haythorne OG 0.8
IA 5
HCl 0.06
Eth 4
AF 0.5   AB 2.5
OG 2.5
PM sat.
   
Heidenhain's
Azan
  AZ 2
AC 1
PM 5 OG 2.0
AB 0.5
AC 7.5
   
Hollande OG sat.   PM 1 LG 0.2   Prestain with 1% BF in 70% Eth. The stain is applied in the order of:– diff, RBC, fibre
Klatskin   PR 1
AC 1
PM 1 AB sat.
AC 2.5
PM 1 Prestain with Harris' hemalum, then treat with 1% PA in 95% Eth. For liver.
Koneff PA 1
OG 0.2
Eth 80
AZ 1
AC 1
PT 5 PT 0.05
OX 2
OG 2
  For pituitary cells
Kricheski   AF 0.25   MB 0.3
OG 0.3
PM 0.3
   
Laidlaw   AF 1 PM 1 OG 0.25
Eth 70
  For acidophil inclusions
Lee-Brown   AF 0.25 PM 1 OG 2.0
MB 0.5
OX 2.0
  A simple modification of Mallory
Lendrum &
McFarlane
PA 1
OG 0.2
Eth 80
AF 0.5
PR 0.5
SS 0.25
AC 1
PM 1 AB 2
AC 1
   
Lendrum
Slidders &
Fraser
PA 2
Eth 95
Dye 0.5
    AC 1
Dye 0.5
  RBC-plasma dyes:-
AF, AE, MR, OG, PL
Fibre dyes:-
SY, SR, BN, DB
Lewis &
Miller
  AF 0.25   MB 0.3
OG 0.3
PM 0.3
  For pituitary cells. This is a modified Kricheski's trichrome, but with much longer staining times.
Lillie   BS 1
AC 1
PM 2.5
PT 2.5
FG 2.5
AC 2.5
   
McFarlane - A   AF 0.8
PA 0.2
AC 2
PA 1
PT 10
Eth 40
AB 2.5
AC 2.5
PA 0.25
PT 2.5
Eth 10
 
McFarlane - B PA 1.0
OG 0.25
Eth 80
AF 0.25
PR 0.25
AC 1.0
PA 1.0
PT 10
Eth 40
AB 2.5
AC 2.5
PA 0.25
PT 2.5
Eth 20
 
Mallory   AF 0.25 PM 1
or
PT 1
OG 2.0
MB 0.5
OX 2.0
   
Masson type   AF 0.35
PR 0.65
AC 1
PM 1 LG 2
AC 2
   
Masson - A   AF 0.35
PR 0.65
AC 1
PM 1 AB sat
AC 2.5
  or PR 1% in AC 1%
or AF 0.5% in AC 0.5%
or AF 1% & PR 1% in AC 1%
Masson - B   AF 0.1 PM 1 AB 0.5
PM 0.5
   
Masson - C   AF 1
AC 1
PM 1 MY sat.    
Masson 44/41   PS 1
AC 1
PT 1 NB 1
AC 1
  For old fibrin. Extended mercuric chloride fixation required. Degrease with trichlorethylene. Refix sections with picro-mercuric-ethanol.
Milligan   AF 0.1 PM 1 FG 0.1
AC 0.2
  Pretreat, 5 min. with
PD 2.25%, HCl 2.5%, Eth 25%.
AB can replace FG.
Möllendorf   EY 1
AC 0.3
PM 2 MB 1    
MSB MR 0.5
PT 2
PS 1
AC 1
PT 1 MB 0.5
AC 1
  For fibrin. Extended mercuric chloride fixation preferred. Several other dyes may be substituted. See method
Obadiah OG 0.5
PT 1
NB 1
AC 1
PT 1 CR 2.5
AC 1
or
PB 1
AC 1
  For very old fibrin. Extended mercuric chloride fixation required. Degrease with trichlorethylene. Refix sections with picro-mercuric-ethanol.
Patay   PR 1 PM 1 LG 0.5
Eth 90
   
Picro-Mallory
short
PA sat
OG 0.2 Eth 80
AF 1
AC 1
PT 1 MB 2
AC 2
  Yellow diff: RBC stain 30,  Eth 70
For fibrin. Requires extended mercury fixation for optimal results.
Picro-Mallory
long
PA sat
LY 0.2
OG 0.2
Eth 80
AC 1
AF 1 or
BS 1 or
LR 0.2 +
AF 0.4
PT 1 MB 1
AC 1
  Stock diff: PA 2.5,  Eth 100,  PT 25
Red diff: stock diff 40,   Eth 20,  DW 40
Blue diff: stock diff 10,  DW 90
For fibrin. Requires extended mercury fixation for optimal results.
Slidders OFG OG 0.5
PT 0.5
Eth 100
AF 0.5 AC 0.5 PT 1 LG 1.5 AC 1.5   For pituitary cells
Weiss   AF 0.04   AB 0.5
OG 0.2
PM 1
   
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Papanicolaou OG 0.5
PT 0.015
Eth 95
LG
BB
EY
PA
LC
Eth
0.22
0.06
0.22
0.17
1 drop
95
or 0.225
0.05
0.225
0.2
1 drop
95
or 0.045
0.05
0.225
0.2
1 drop
95
or 0.1125
0.05
0.225
0.2
1 drop
95

 

One step methods
One step trichrome staining methods use a single staining solution, although some may specify pre or post staining treatments. In the chart below all units given for fluids are in millilitres, and all units for solids are in grams. Each reagent should be dissolved into the solvent specified in the second column. Unless otherwise specified, it is suggested that the solvent be made first, then the solids dissolved in it. Filter each solution before use.

Variant Solvent Chemical Dyes Comments
Cason DW 100 PT 0.5 OG 1.0
AF 1.5
AB 0.5
 
Engel &
Cunningham
DW 100
AC 1.0
PT 0.6 CH 0.6
FG 0.3
Gomori's trichrome modified by adjusting pH to 3.4 with 1N NaOH
Gomori DW 100
AC 1.0
PT 0.6 CH 0.6
FG 0.3
 
Kostowiecki DW 100 PM 1.0 AB 0.06
OG 0.2
Prestain nuclei red.
Ladewig DW 100 OX 2.0 OG 2.0
AF 1.0
MB 0.5
Pretreat with PT 1% 2 min.
McFarlane DW 98
AC 2.0
PT 1.0 PA 0.2
AF 1.0
AB 2.0
Post stain wash in
PA 0.25% & PT 2.5% in 10% Eth
Papanicolaou DW 100 PT 0.112
PM 0.225
OG 0.125
EY 0.21
AF 0.1
AB 0.06
This is not the Papanicolaou method used for cervical cancer screening.
Pollak DW 50
Eth 95% 50
AC 1.0
PT 0.5
PM 0.5
OG 0.25
PR 0.33
AF 0.17
LG 0.15
Add fluids together. Divide into four. Dissolve PM in 1st, PT and OG in 2nd, LG in 3rd, PR and AF in 4th. Add together and filter.
Sweat, Meloan, Puchtler DW 100
HCl 1.0
PM 1.0 CH 0.6
AB 0.6
No nuclear stain
Wallart &
Honette
DW 300
AC 2.0
PM 1.0 AF 1.0
FY 1.0
Add AC to 200 DW. Divide in 2. Dissolve AF in one half, FY in the other. Dissolve PM in 100 DW. Combine 30 of each. Filter.
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Yellowsolve methods
Yellowsolve methods prestain with an aqueous solution of a red dye, some then differentiate with a polyacid. Then a yellow dye in an anhydrous solvent is applied to progressively replace the red dye in tissues other than the target. In the chart below all units given for fluids are in millilitres, and all units for solids are in grams. Unless otherwise specified, the plasma stain and differentiation solvents are 100 mL of distilled water. The yellow solvent is always an anhydrous fluid.

Variant Plasma Diff Yellow Comments
Fuchsin-Miller AF 1.5 PT 1 ML 2.5
EE 100
The target tissue is fibrin. Extended mercuric chloride fixation is required.
Phloxine-tartrazine PH 0.5
CC 0.5
  TA sat.
EE 100
The target tissue is acidophil cell inclusions.
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Other methods
In the chart below all units given for fluids are in millilitres, and all units for solids are in grams. It is suggested that the solvent be prepared first, then the solids dissolved in it. Other pre-made solutions which are required may then be added. Pay particular attention to the comments column as other staining steps may be specified.

Variant Solvent Chemical Dyes Comments
Duprès DW 200 OX 4.0 TB 0.25
OG 4.0
Pretreat with 1% PM for 10 min
TB may be replaced with MG.
Kohashi Eth 50% 35
Gly 40
  EB 0.7
AF (sat aqu) 4
Unna's sol 35
For elastic & collagen pretreat with 0.1% AZ in 1% AC for 15 min. Then in 5% PM for 30-60 min.
Mollier DW 100   NG 1
AC 1
For elastic & collagen prestain with Taenzer and Weigert's iron hematoxylin, then pretreat with 2% AZ in 1% AC for 15-30 min, followed by 5% PM 2-6 hrs.
Paquin
& Goddard
DW 100 PT 0.1 EY 0.07
PH 0.03
OG0.01
For elastic & collagen prestain with iron HX, and post stain with 0.04% AB in 1% AC
Pasini Eth 50% 35
Gly 40
  EB 0.7
AF (sat aqu) 4.0
Unna's sol 35
For elastic & collagen pretreat with 2% PT for 10 min.
Roque HCl 0.02M 100   CH 0.5
AB 2.0
For Mallory bodies & collagen prestain with iron HX, then pretreat with 1% PM for 2 min
Walter Eth 50% 35
Gly 40
  EB 0.7
For elastic & collagen pretreat with 2.5% IA for 16 hours, then with 2% PT for 10 min.
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Abbreviations

Abbr Meaning Abbr Meaning Abbr Meaning
AC
AB
AE
AF
AZ
BB
BF
BN
BS
CC
CH
CR
DB
DW
EB
EE
Eth
Acetic acid
aniline blue
Azo eosin
Acid fuchsin
Azocarmine
Bismarck brown
Basic fuchsin
Benzo new blue GS
Biebrich scarlet
Calcium chloride
Chromotrope 2R
Chicago red
Durazol brilliant blue B
Distilled water
Eosin B
2-Ethoxy-ethanol
Ethanol
EY
FG
FY
Gly
HCl
HX
IA
LC
LG
LY
LR
MB
MG
MR
MY
ML
NG
Eosin Y ws
Fast green FCF
Fast yellow
Glycerol
Hydrochloric acid
Hematoxylin
Iron alum
Lithium carbonate
Light green SF yellowish
Lissamine yellow
Lissamine fast red
Methyl blue
Methyl green
Martius yellow
Metanil yellow
Milling yellow 3G
Naphthol green B
OG
OR
OX
PA
PB
PD
PH
PL
PM
PR
PS
PT
SR
SS
SY
TA
TB
Orange G
Orcein
Oxalic acid
Picric acid
Polar brilliant red BN
Potassium dichromate
Phloxine B
Propalan red 3GX
Phosphomolybdic acid
Ponceau 2R
Ponceau 6R
Phosphotungstic acid
Sirius red F3B
Sodium sulphate
Sun yellow
Tartrazine
Toluidine blue
Incorporated Solutions
Unna DW 50, Eth 100% 25, AC 2.5, Gly 10, OR 0.5, AB 0.5
Taenzer Eth 50% 100, HCl 1.0, OR 0.8
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