Crossman’s Trichrome for Muscle and Collagen

By Carmen WongProtocols, Stain Type, Trichrome Staining, Trichrome, Multi-Step

Protocol

Crossman's Trichrome

for Muscle and Collagen

steps

materials

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Materials

Solution A
Material Amount
Acid fuchsin 0.3 g
Orange G 2 g
Acetic acid, glacial 1 mL
Distilled water 100 mL
Thymol a small crystal
Solution B
Material Amount
Phosphomolybdic acid 1 g
Distilled water 100 mL
Solution C
- Option 1
  Material Amount
  Aniline blue 2 g
  Acetic acid, glacial 2 mL
  Distilled water 98 mL
- Option 2
  Material Amount
  Light green SF 1 g
  Acetic acid, glacial 1 mL
  Distilled water 99 mL
Solution D
Material Amount
Acetic acid, glacial 1 mL
Distilled water 99 mL

Material	Amount
Acid fuchsin	0.3	g
Orange G	2	g
Acetic acid, glacial	1	mL
Distilled water	100	mL
Thymol	a small crystal

Material	Amount
Phosphomolybdic acid	1	g
Distilled water	100	mL

Material	Amount
Aniline blue	2	g
Acetic acid, glacial	2	mL
Distilled water	98	mL

Material	Amount
Light green SF	1	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Material	Amount
Acetic acid, glacial	1	mL
Distilled water	99	mL

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Most trichrome stains benefit from picric acid or mercuric chloride fixation.

Protocol

Bring sections to water via xylene and ethanol.
Stain nuclei with an acid resistant nuclear stain such as Weigert’s iron hematoxylin.
Place into solution A for 1 minute.
Rinse with distilled water.
Place into solution B until collagen is decolorised.
Rinse quickly with distilled water.
Place into solution C for 5 minutes.
Rinse with distilled water.
Place into solution D until differentiated.
Rinse with distilled water.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – black
Erythrocytes – yellow
Cytoplasm – red
Collagen – blue or green

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.

March 30, 2023

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Papanicolaou’s Alcoholic Trichrome for Exfoliated Cells

By Carmen WongProtocols, Stain Type, Trichrome Staining, Trichrome, Multi-Step

Protocol

Papanicolaou's Alcoholic Trichrome

for Exfoliated Cells

steps

materials

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This is the Papanicolaou technique commonly used to screen for cervical cancer.

Materials

Ehrlich’s or Harris’ alum hematoxylin or equivalent
Solution A (OG6)
Material Amount
Orange G 5 g
Phosphotungstic acid 0.15 g
Ethanol, 95% 1 L

Material	Amount
Orange G	5	g
Phosphotungstic acid	0.15	g
Ethanol, 95%	1	L

Solution B

Material	Variant
Material	EA 25		EA 36		EA 50		EA 65
Light green SF	2.2	g	2.25	g	0.45	g	1.125	g
Bismarck brown	0.6	g	0.5	g	0.5	g	0.5	g
Eosin Y	2.2	g	2.25	g	2.25	g	2.25	g
Phosphotungstic acid	1.7	g	2	g	2	g	2	g
Lithium carbonate	5	mg	5	mg	5	mg	5	mg
Ethanol, 95%	1	L	1	L	1	L	1	L

Preparation

For each variant:

Dissolve the bismarck brown in 100 mL ethanol, the light green in 450 mL ethanol, and the eosin Y in another 450 mL ethanol.
Combine the three solutions.
Add the acid and lithium carbonate.
Filter.

Tissue Sample

Ethanol-ether fixed smears of exfoliated epithelial cells.

Protocol

Bring smears to water via graded ethanols.
Stain nuclei with alum hematoxylin for 2 minutes.
Blue.
Rinse with 95% ethanol.
Place into solution A for 1 minute.
Rinse with 95% ethanol.
Place into solution B for 2 minutes.
Rinse well 95% ethanol.
Dehydrate rapidly with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – blue
Cells – shades of orange, pink or green

Notes

Although the method calls for smears fixed in an equal parts mixture of ethanol and diethyl ether, smears fixed in 95% or absolute ethanol are completely satisfactory.
Lithium carbonate saturates in water at 1% at room temperature, so 0.5 mL contains 5 mg.
EA 65 is recommended for specimens with large amounts of mucus.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.
Citing:
Papanicolaou, (1942)
Science, v.95, pp.438
Culling C.F.A., (1974)
Handbook of histopathological and histochemical techniques Ed. 3
Butterworth, London, UK.
Bancroft, J.D. and Stevens A. (1982)
Theory and practice of histological techniques Ed. 2
Churchill Livingstone, Edinburgh & London, UK.

March 30, 2023

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Papanicolaou’s Trichrome for Exfoliated Cells

By Carmen WongProtocols, Stain Type, Trichrome Staining, Trichrome, One-Step

Protocol

Papanicolaou's Trichrome

for Exfoliated Cells

steps

materials

print

This is not the Papanicolaou technique commonly used to screen for cervical cancer.

Materials

Ehrlich’s alum hematoxylin or equivalent
Staining solution
Material Amount
Acid fuchsin 1 g
Aniline blue 0.6 g
Eosin Y 2.1 g
Orange G 1.25 g
Phosphotungstic acid 1.12 g
Phosphomolybdic acid 2.25 g
Distilled water 1 L

Material	Amount
Acid fuchsin	1	g
Aniline blue	0.6	g
Eosin Y	2.1	g
Orange G	1.25	g
Phosphotungstic acid	1.12	g
Phosphomolybdic acid	2.25	g
Distilled water	1	L

Tissue Sample

Ethanol fixed smears of exfoliated epithelial cells.

Protocol

Stain nuclei with Ehrlich’s hematoxylin for 2 minutes.
Blue.
Rinse with distilled water.
Place into the staining solution for 2-5 minutes.
Rinse quickly with distilled water.
Dehydrate rapidly with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – blue
Cells – shades of orange, pink or blue

Notes

The method specified that dehydration and clearing should be with dioxane. This reagent is no longer used due to its toxicity. If satisfactory results are not obtained with rapid ethanol dehydration, dehydrants such as t-butanol should be tried.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.
Citing:
Papanicolaou, (1941)
Journal of laboratory and clinical medicine, v. 26, pp. 1200

March 30, 2023

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Dahlia-Iron-Resorcin for Elastic Fibres

By Carmen WongDye Type, Elastic Fibers, Iron Resorcin, Protocols, Stain Target, Stain Type

Protocol

Dahlia-Iron-Resorcin

for Elastic Fibres

steps

materials

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Materials

- Weigert’s iron hematoxylin or equivalent
- Van Gieson’s picro-fuchsin
- Iron-resorcin-dahlia solution
  Material Amount
  Dahlia 2 g
  Resorcin 4 g
  Ferric nitrate, 30% aqu. 50 mL
  0.5% aqu. hydrochloric acid 200 mL
  Methanol 180 mL
  Acetone 20 mL
  Hydrochloric acid, conc. 4 mL

Material	Amount
Dahlia	2	g
Resorcin	4	g
Ferric nitrate, 30% aqu.	50	mL
0.5% aqu. hydrochloric acid	200	mL
Methanol	180	mL
Acetone	20	mL
Hydrochloric acid, conc.	4	mL

Preparation

Bring 200 mL of 0.5% hydrochloric acid in an oversize flask to the boiling point, remove from the heat and carefully add the dahlia.
Return to the boil, and add the resorcin and ferric nitrate.
Continue boiling for 3 minutes, then cool and filter. Discard the filtrate. Dry the filter paper and beaker, then place the precipitate and filter paper back into the flask.
Add the methanol, acetone and hydrochloric acid.
Heat carefully on a hot plate until the precipitate dissolves.

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory.

Protocol

Bring sections to water via xylene and ethanol.
Do a Mallory bleach.
Place into the staining solution for 30 minutes or longer.
Wash with 95% ethanol to remove excess solution.
Differentiate with 1% acid alcohol if necessary.
Wash in water.
Counterstain with iron hematoxylin and van Gieson.
Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

Elastic fibres – blue black
Collagen – red
Cytoplasm – yellow
Nuclei – blue

Notes

Between the 1st and 2nd editions of their book, Bancroft and Stevens changed the solvent for the precipitate to the following:
Solvent Amount
2-methoxyethanol 100 mL
Distilled water 100 mL
Hydrochloric acid, conc. 4 mL
Dry the filter paper and beaker, then place the precipitate and filter paper back into the flask. Add the mixture and heat carefully on a hot plate until the precipitate dissolves.
2-methoxyethanol is also known as ethylene glycol monomethyl ether.
The authors suggest the following dyes as suitable substitutes for dahlia:
- Methyl violet
- Ethyl violet
- Victoria blue
- Thionin
Note that this method uses ferric nitrate instead of ferric chloride.

Solvent	Amount
2-methoxyethanol	100	mL
Distilled water	100	mL
Hydrochloric acid, conc.	4	mL

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Bancroft, J. D. and Stevens, A.,
Theory and Practice of Histological Techniques, Ed. 1 (1977),
Churchill Livingstone, London, UK.
Bancroft, J. D. and Stevens, A.,
Theory and Practice of Histological Techniques, Ed. 2 (1982),
Churchill Livingstone, London, UK.

March 30, 2023

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Dawes’ Trichrome for Fetal & Endocrine Tissue

By Carmen WongProtocols, Stain Type, Trichrome Staining, Trichrome, Multi-Step

Protocol

Dawes' Trichrome

for Fetal & Endocrine Tissue

steps

materials

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Materials

Solution A-1

Material	Amount
Hematoxylin, 10% in 95% ethanol	16	mL
Ethanol, 95%	64	mL
Glycerol	80	mL

Solution A-2

Material	Amount
Ammonium ferric sulphate	24	g
Ferrous sulphate	24	g
Distilled water	100	mL

Solution A-3

Material	Amount
Azophloxine	1.2	g
Chromotrope 2R	0.2	g
Acetic acid, glacial	1.2	mL
Distilled water	160	mL

Preparation of Working Solution A

Make each A solution ensuring all solids are dissolved.
Combine solutions A-1 and A-2 and mix well.
Add solution A-3 to the combined solution.

Solution B

Material	Amount
Phosphomolybdic acid	1.5	g
Aniline blue or Methyl blue	3	g
Acetic acid, glacial	1.5	mL
Picric acid, sat. aqueous	150	mL
Distilled water	160	450

Tissue Sample

5µ paraffin sections of Bouin’s picric-acetic-formalin fixed fetal tissue, or endocrine tissue fixed in the Bouin’s mixture, but without the acetic acid.

Protocol

Bring sections to water via xylene and ethanol.
Place into solution A for 4 minutes.
Wash with tap water.
Place in solution B for 3 minutes.
Wash with distilled water.
Dehydrate quickly with 2 changes of 95% ethanol.
Rinse with absolute ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – black
Muscle – Orange-red
Collagen – Blue

Notes

The author states that both working solutions are stable and can be made in bulk. Multiply the amounts given by 10.
The method is suitable for staining sections in bulk.
The method is suitable as a counterstain for aldehyde fuchsin as well as being a general trichrome.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Dawes, R. L. F., (1972),
A simple connective tissue stain suitable for foetuses and endocrine organs.
Medical Laboratory Technology, v. 29, Nº 1, p.27.

March 30, 2023

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Debiden’s Alum Hematoxylin Variants

By Carmen WongDye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type

Protocol

Debiden's Alum Hematoxylin Variants

steps

materials

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Materials

Material	Variant		Function
Material	1987	1991	Function
Hematoxylin	5 g	5 g	Dye
Potassium alum	100 g	100 g	Mordant
Distilled water	1 L	1 L	Solvent
Glacial acetic acid	20 mL	20 mL	Acidifier
Mercuric oxide, red	2.5 g	–	Oxidant
Javex (5.25% sodium hypochlorite)	–	2 mL	Oxidant

Compounding procedures

Dissolve the alum in the water in a large flask.
Add the hematoxylin. Mix well.
Place in a 60-65°C oven overnight.
Remove from the oven, and add the appropriate oxidizing agent.
Stir until cold. Filter.

Preparation notes

1980

Acetic acid may be added as soon as it is cooled following preparation.
It is usable immediately.
It is preferable to prepare small volumes monthly.

1991

This improves in staining ability if allowed to ripen for a week or two.
If acetic acid is used, it should be added just before use.

Protocol

Bring sections to water with xylene and ethanol.
Place into the staining solution for an appropriate time.
Rinse well with water.
Differentiate with acid ethanol if necessary.
Rinse with water and blue.
Rinse well with water.
Counterstain if desired.
Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – blue
Background – as counterstain or unstained

Notes

These two solutions are modifications of Harris’ hematoxylin and are regressive.
Paraffin sections should be stained for 5 minutes, and cytology smears for 45 seconds.
As with many strong regressive formulae, mucin may be stained blue.
Acid ethanol is 0.5% – 1% hydrochloric acid in 70% ethanol.
Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate etc.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Debiden, D., (1987)
Improved preparation of Harris’ hematoxylin
Histologic, v.18, No.8
Debiden, D., (1991)
A new oxidant for Harris’ hematoxylin
Histologic, v.21, No.2

March 30, 2023

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De Groot’s Alum Hematoxylin

By Carmen WongDye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type

Protocol

De Groot's Alum Hematoxylin

steps

materials

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Materials

Material	Amount	Function
Hematoxylin	2 g	Dye
Ammonium alum	22 g	Mordant
Distilled water	270 mL	Solvent
95% ethanol	650 mL	Solvent
Glycerol	80 mL	Stabiliser
Hydrogen peroxide	7.5 mL	Oxidant
Potassium ferricyanide	0.8 g	–
Calcium chloride	15 g	–
Sodium bromide	7.5 g	–

Compounding procedures

Mix the ethanol, water and glycerol to make the solvent.
Add the peroxide to 15 mL of the solvent.
Add the hematoxylin, and dissolve.
Dissolve the calcium chloride and sodium bromide in 250 mL of the solvent.
Mix with the hematoxylin solution.
Add half the alum, and dissolve.
Dissolve the potassium ferricyanide in 400 mL of the solvent.
Add to the hematoxylin solution.
Dissolve the remaining alum in the remaining solvent.
Add to the hematoxylin solution.

Protocol

Bring sections to water with xylene and ethanol.
Place into the staining solution for an appropriate time.
Rinse well with water.
Differentiate with acid ethanol if necessary.
Rinse with water and blue.
Rinse well with water.
Counterstain if desired.
Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – blue
Background – as counterstain or unstained

Notes

Both hydrogen peroxide and potassium ferricyanide are oxidizing agents. However, it is not clear if potassium ferricyanide is present for this reason.
The strength of hydrogen peroxide is not specified, but the commonest laboratory strength is 30 vols.
Although calcium can mordant hematoxylin, it is not clear if it is present for that reason.
The purpose of the sodium bromide is not clear. It may be present as a preservative, similar to chloral hydrate in some other formulas.
The staining time should be determined by trial.
Acid ethanol is 0.5% – 1% hydrochloric acid in 70% ethanol.
Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate etc.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.

March 30, 2023

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Delafield’s Alum Hematoxylin

By Carmen WongDye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type

Protocol

Delafield's Alum Hematoxylin

steps

materials

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Materials

Material	Amount	Function
Hematoxylin	6 g	Dye
Ammonium alum	90 g	Mordant
Distilled water	600 mL	Solvent
95% ethanol	200 mL	Solvent
Glycerol	150 mL	Stabiliser

Compounding procedures

Dissolve the hematoxylin in 50 mL ethanol.
Dissolve the alum in the water.
Add the two solutions.
Leave, loosely stoppered, in a warm, sunlit place for one week.
Filter, then add the glycerol and the rest of the ethanol.
Leave, loosely stoppered, in a warm, sunlit place for 3 months.
Filter and store in a tightly stoppered container in the dark.
This solution is stable for years.

Protocol

Bring sections to water with xylene and ethanol.
Place into the staining solution for 20 minutes.
Rinse well with water.
Differentiate with acid ethanol if necessary.
Rinse with water and blue.
Rinse well with water.
Counterstain if desired.
Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – blue
Background – as counterstain or unstained

Notes

The solution may be chemically ripened by adding 0.5 g sodium iodate, but such solutions are considered to be inferior in longevity.
Like many strong regressive formulations, cartilage, cement lines and mucins may be blue.
Acid ethanol is 0.5% – 1% hydrochloric acid in 70% ethanol.
Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate etc.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Culling, C.F.A., Allison, R.T. and Barr, W.T.
Cellular Pathology Technique, Ed.4.
Butterworth, London, UK.
Bancroft, J.D. and Stevens A. (1982)
Theory and practice of histological techniques Ed. 2
Churchill Livingstone, Edinburgh & London, UK.

March 30, 2023

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Feulgen Nucleal Reaction for DNA

By Carmen WongAldehydes, Nucleal Reaction, Protocols, Schiff's Reagent Reactions, Stain Target, Stain Type

Protocol

Feulgen Nucleal Reaction

for DNA

steps

materials

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Materials

Hydrochloric acid, 1N
Schiff’s reagent
Light green, 1% aqueous

Tissue Sample

5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Many other fixatives are satisfactory. Fixatives containing strong acids should be avoided as this method depends on the acid hydrolysis of DNA, and acids in some fixatives may pre-hydrolyse the tissue (picric acid in Bouin’s aqueous formal-picric-acetic mixture, for example).

Protocol

Bring sections to water via xylene and ethanol.
Rinse briefly with cold 1N hydrochloric acid.
Place into prewarmed hydrochloric acid for the appropriate time at 60°C.
Rinse briefly with cold 1N hydrochloric acid.
Rinse briefly with distilled water.
Place into Schiff’s reagent for 30-60 minutes at room temperature.
Give three sulphite rinses of about 1 minute each.
Wash well with water.
Optionally, counterstain with light green for 1 minute.
Dehydrate with ethanol, clear with xylene, and mount with a resinous medium.

Expected Results

DNA – red
Background – green

Notes

The appropriate time in hydrochloric acid varies depending on the fixative. The times given below are in minutes, but should be considered a guide only. Trials should be conducted to determine the optimum.
Fixative Time
Bouin Do not use
Carnoy 8
Champy 25
Flemming 16
Formalin (NBF) 10
Formal sublimate 8
Helly 8
SuSa 18
Zenker 5
A modification of this reaction uses more concentrated hydrochloric acid at room temperature. At step 3, place into 5N hydrochloric acid at room temperature for the appropriate time. Then continue on with step 4. This variant is considered to produce darker staining and a smaller loss of DNA.
Fixative Time
Alcoholic fixatives 20 minutes to 2 hours
Formalin containing fixatives 35 minutes to 4 hours
Formalin vapour 2 to 8 hours
Sulphite rinses are now considered unnecessary. They were used originally in the belief that placing directly into water would recolour the Schiff’s reagent and give false positive staining. It is now known that this is not so, providing the Schiff’s reagent is completely washed off. Sulphite rinses consist of:
Material Amount
Potassium metabisulphite, 10% aqu. 5 mL
Hydrochloric acid, 1N 95 mL

Fixative	Time
Bouin	Do not use
Carnoy	8
Champy	25
Flemming	16
Formalin (NBF)	10
Formal sublimate	8
Helly	8
SuSa	18
Zenker	5

Fixative	Time
Alcoholic fixatives	20 minutes to 2 hours
Formalin containing fixatives	35 minutes to 4 hours
Formalin vapour	2 to 8 hours

Material	Amount
Potassium metabisulphite, 10% aqu.	5	mL
Hydrochloric acid, 1N	95	mL

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Pearse, A. G. E., (1968, 1972)
Histochemistry: Theoretical and Applied, Ed. 3
Churchill Livingstone, Edinburgh, London, UK

March 30, 2023

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Duprès’ Magenta for Nuclei

By Carmen WongDye Type, Hematoxylin Alternatives, Protocols, Stain Type

Protocol

Duprès' Magenta

for Nuclei

steps

materials

print

Materials

Stock basic fuchsin

Material	Amount
Basic fuchsin	0.75	g
Ethanol, 95%	20	mL
Distilled water	80	mL

Preparation

Mix the ethanol and water. Add the basic fuchsin.
Heat at 40°C for 48 hours. Cool and filter.

Solution A

Material	Amount
Stock basic fuchsin	5	mL
Aniline water	100	mL
Acetic acid, glacial	1.5	mL
Distilled water	100	mL

Solution B

Material	Amount
Formalin, conc. (40%)	20	mL
Acetic acid, glacial	25	mL
Distilled water	50	mL

Tissue Sample

5 µg paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory.

Protocol

Bring sections to water via xylene and ethanol.
Place into solution A for 1-10 minute.
Rinse with distilled water.
Place into solution B for 5-10 minutes, until differentiated.
Stain with a contrast stain.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – red
Cytoplasm – according to counterstain

Notes

Magenta is an older name for basic fuchsin. Since this is a mixture, it is likely that its component dyes could also be used.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.
Citing:
Duprès, (1935)
Journal number 11425, v. 46, pp. 77

Note: Gray identifies journals by a number from the world list of scientific periodicals, ed. 2, 1934. Unfortunately, the journal listed as number 11425 is not identified by name in the list he provides. In a second reference to the differentiator used in this technique he gives the journal number as 14425. This may be the intended reference:
Duprès, (1935)
Monitore zoologico italiano, v. 46, pp. 77
Florence, Italy.

March 30, 2023

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