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 colors 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 coloration.
- 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 fiber stains.
- Fiber: This step incorporates a large molecule dye to stain collagen and bone in a color that strongly contrasts with the plasma stain. The commonest colors 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 fiber stain.
Multi-step Methods
Each column in the chart below represents a separate solution. For each solution, all fluids are to be measured in milliliters and all solids in grams. Unless otherwise specified, the solvent is 100 milliliters distilled water. Where another fluid is included, the volume of water should be adjusted to make 100 milliliters 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, fiber | ||
| 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 |
| 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 milliliters, 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 | Distilled water 100 | Phosphotungstic acid 0.5 | Orange G 1.0 Acid fuchsin 1.5 Aniline blue 0.5 | |
| Engel & Cunningham | Distilled water 100 Acetic acid 1.0 | Phosphotungstic acid 0.6 | Chromotrope 2R 0.6 Fast green FCF 0.3 | Gomori’s trichrome modified by adjusting pH to 3.4 with 1N NaOH |
| Gomori | Distilled water 100 Acetic acid 1.0 | Phosphotungstic acid 0.6 | Chromotrope 2R 0.6 Fast green FCF 0.3 | |
| Kostowiecki | Distilled water 100 | Phosphomolybdic acid 1.0 | Aniline blue 0.06 Orange G 0.2 | Prestain nuclei red. |
| Ladewig | Distilled water 100 | Oxalic acid 2.0 | Orange G 2.0 Acid fuchsin 1.0 Methyl blue 0.5 | Pretreat with PT 1% 2 min. |
| McFarlane | Distilled water 98 Acetic acid 2.0 | Phosphotungstic acid 1.0 | Picric acid 0.2 Acid fuchsin 1.0 Aniline blue 2.0 | Post stain wash in PA 0.25% & PT 2.5% in 10% Eth |
| Papanicolaou | Distilled water 100 | Phosphotungstic acid 0.112 Phosphomolybdic acid 0.225 | Orange G 0.125 Eosin Y ws 0.21 Acid fuchsin 0.1 Aniline blue 0.06 | This is not the Papanicolaou method used for cervical cancer screening. |
| Pollak | Distilled water 50 Eth 95% 50 Acetic acid 1.0 | Phosphotungstic acid 0.5 Phosphomolybdic acid 0.5 | Orange G 0.25 Ponceau 2R 0.33 Acid fuchsin 0.17 Light green SF yellowish 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 | Distilled water 100 Hydrochloric acid 1.0 | Phosphomolybdic acid 1.0 | Chromotrope 2R 0.6 Aniline blue 0.6 | No nuclear stain |
| Wallart & Honette | Distilled water 300 Acetic acid 2.0 | Phosphomolybdic acid 1.0 | Acid fuchsin 1.0 Fast yellow 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. |
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 milliliters, 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 | Acid fuchsin 1.5 | Phosphotungstic acid 1 | Milling yellow 3G 2.5 2-Ethoxy-ethanol 100 | The target tissue is fibrin. Extended mercuric chloride fixation is required. |
| Phloxine-tartrazine | Phloxine B 0.5 Calcium chloride 0.5 | Tartrazine sat. 2-Ethoxy-ethanol 100 | The target tissue is acidophil cell inclusions |
Other Methods
In the chart below all units given for fluids are in milliliters, 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 | Distilled water 200 | Oxalic acid 4.0 | Toluidine blue 0.25 Orange G 4.0 | Pretreat with 1% Phosphomolybdic acid for 10 min Toluidine blue may be replaced with Methyl green. |
| Kohashi | Ethanol 50% 35 Glycerol 40 | Eosin B 0.7 Acid fuchsin (sat aqu) 4 Unna’s sol 35 | For elastic & collagen pretreat with 0.1% Azocarmine in 1% Acetic acid for 15 min. Then in 5% Phosphomolybdic acid for 30-60 min. | |
| Mollier | Distilled water 100 | Naphthol green B 1 Acetic acid 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% Phosphomolybdic acid 2-6 hrs. | |
| Paquin & Goddard | Distilled water 100 | Phosphotungstic acid 0.1 | Eosin Y ws 0.07 Phloxine B 0.03 Orange G 0.01 | For elastic & collagen prestain with iron hematoxylin, and post stain with 0.04% aniline blue in 1% acetic acid |
| Pasini | Ethanol 50% 35 Glycerol 40 | Eosin B 0.7 Acid fuchsin (sat aqu) 4.0 Unna’s sol 35 | For elastic & collagen pretreat with 2% phosphotungstic acid for 10 min. | |
| Roque | Hydrochloric acid 0.02M 100 | Chromotrope 2R 0.5 Aniline blue 2.0 | For Mallory bodies & collagen prestain with iron hematoxylin, then pretreat with 1% phosphomolybdic acid for 2 min | |
| Walter | Ethanol 50% 35 Glycerol 40 | Eosin B 0.7 | For elastic & collagen pretreat with 2.5% iron alum for 16 hours, then with 2% phosphotungstic acid for 10 min. |
