Bennett's Alum Hematoxylin

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Materials

Material	Amount	Function
Hematoxylin	1 g	Dye
Potassium alum	90 g	Mordant
Distilled water	1 L	Solvent
Sodium iodate	0.2 g	Oxidant
Chloral hydrate	50 g	Stabiliser
Citric acid	1 g	Acidifier

Compounding procedure

Heat the water. Then add in order:
1. Hematoxylin
2. Sodium iodate
3. Alum
4. Citric acid
5. Chloral hydrate.
Note: Dissolve each ingredient before adding the next.
Cool to room temperature and filter. It may be used immediately.

Protocol

Bring sections to water with xylene and ethanol.
Place into the staining solution for 5-20 minutes.
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

Putt gives no information regarding staining time and characteristics, but the similarity of the formula to Mayer’s (Langeron’s) hemalum suggests it is progressive and selectively nuclear. A staining time of 10-20 minutes should be adequate.
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

F. A. Putt
Manual of Histopathological Staining Methods
John Wiley & Sons, New York, NY., USA

March 30, 2023

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Bencosme’s Alum Hematein

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

Protocol

Bencosme's Alum Hematein

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Materials

Original

Material	Amount	Function
Hematein	2 g	Dye
Potassium alum	133 g	Mordant
Distilled water	920 mL	Solvent
Glacial acetic acid	20 mL	Acidifier

Variant

Material	Amount	Function
Hematein	2.5 g	Dye
Potassium alum	120 g	Mordant
Distilled water	1 L	Solvent
Glacial acetic acid	10 mL	Acidifier

Compounding procedure

Add the alum to the water in a 2 L flask and bring to a boil for 1 minute.
Add the hematein and mix by swirling.
Place an inverted funnel over the flask and simmer for 10 minutes, shaking frequently.
Cool to room temperature and add the acetic acid.
Filter before use and twice weekly.
It may be used immediately.

Protocol

Bring sections to water with xylene and ethanol.
Place into the staining solution for 2 minutes.
Rinse with water.
Differentiate with 0.5% hydrochloric acid in 70% ethanol for one second.
Rinse well with water and blue.
Rinse well with water.
Counterstain with HPS.
Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – blue
Background – shades of red and yellow.

Notes

Note that this solution uses hematein rather than hematoxylin. Hematoxylin could be used, but would require 0.4 grams sodium iodate to be added along with the dye for complete oxidation in the original solution, although 0.3 grams might be more appropriate to extend the life. Comparable amounts would be 0.5 g and 0.4 g, respectively, for the variant formula.
When freshly made this solution stains in 2 minutes. Staining time slowly increases to 10 minutes as the solution ages. Life is about a month.
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

Medical Laboratory Technology, 2nd ed. (1969)
Lynch M.J., Raphael S.S., Mellor L.D., Spare P.D. and Inwood M.J.H.,
W. B. Saunders Co., Toronto, On., Canada

March 30, 2023

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Bensley’s Copper Hematoxylin

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

Protocol

Bensley's Copper Hematoxylin

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Materials

Solution A

Material	Amount	Function
Hematoxylin,	1 g	Dye
Distilled water	100 mL	Solvent

Solution B

Material	Amount	Function
Cupric acetate,	to saturation	Mordant
Distilled water	100 mL	Solvent

Solution C

Material	Amount	Function
Potassium chromate,	5 g	–
Distilled water	100 mL	Solvent

Solution D

Material	Amount	Function
Weigert’s borax-ferricyanide	20 mL	Differentiator
Distilled water	80 mL	Diluent

Compounding procedure

Make each solution separately.

Protocol

Bring sections to water with xylene and ethanol.
Place into solution A for 5 minutes.
Rinse with water.
Dip into solution B.
Rinse with water.
Place into solution C for 2 minutes.
Return to solution A for 1 minute.
Place in solution D until differentiated.
Rinse well with water.
Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

Nuclei and other structures – blue-black

Notes

After step 7, the sections should be intense black. Repeat steps 5-7 if necessary.
Bensley recommended prestaining with Mayers mucicarmine.
Best results are obtained with freshly made solutions

Safety Note

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

References

Bensley R. R. and Bensley, S. H., (1938)
Handbook of Histological and Cytological Technique.
U. Chicago Press, Chicago, USA

March 30, 2023

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Bensley’s trichrome for Muscle and Collagen

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

Protocol

Bensley's trichrome

for Muscle and Collagen

steps

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Materials

Solution A

Material	Amount
Acid fuchsin	To saturation
Aniline water	100	mL

Solution Preparation

Add about 20 g dye to the aniline water.
Shake intermittently for 48 hours. Filter.

Solution B

Material	Amount
Phosphomolybdic acid	1	g
Distilled water	100	mL

Solution C

Material	Amount
Orange G	2	g
Aniline blue	0.5	g
Distilled water	100	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. Formalin fixed tissues may benefit from secondary fixation of sections in Bouin’s fluid.

Protocol

Bring sections to water via xylene and ethanol.
Place into solution A for 10 minute.
Rinse with distilled water.
Place into solution B for 10 minutes.
Rinse quickly with distilled water.
Place into solution C for 60 minutes.
Rinse with 95% ethanol until clouds of dye cease being extracted.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – red
Erythrocytes – orange
Cytoplasm – red
Collagen – blue

Notes

Solution A is Altmann’s acid fuchsin, originally formulated for staining mitochondria.
This method does not specify an acid resistant nuclear stain. Doing so before staining with solution A may improve the technique.

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:
Mallory, F. B., (1938),
Pathological technique.
Saunders, Philadelphia, Pa, USA

March 30, 2023

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Bensley & Bensley’s Impregnation for Reticulin

By Carmen WongMetal Impregnation, Metal Impregnation, Silver, Protocols, Reticulin, Stain Target, Stain Type

Protocol

Bensley & Bensley's Impregnation

for Reticulin

steps

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Materials

Solutions

Material	Var I	Var II
Lugol’s iodine	+	+
Potassium permanganate, 1% aqu.	+	+
Oxalic acid, 5% aqu.	+	+
Sodium thiosulphate, 3% aqu.	+	+
Tannic acid, saturated in ethanol, 95%.	+	–
Strong ammonium hydroxide (s.g. 0.88)	+	+
Ammoniated water	+	–
Silver nitrate, aqu.	1%	2%
Formalin, 20% aqu.	+	+
Yellow gold chloride, 0.2% aqu.	+	+
Sodium hydroxide, 40% aqu.	+	+
Neutral red, 1% aqu.	+	+

Lugol’s iodine

Material	Amount
Iodine	1	g
Potassium iodide	2	g
Distilled water	300	mL

Mix the iodine and potassium iodide in a 500 mL flask. Add 5 mL of the water. When the iodine has dissolved make up to 300 mL with distilled water.

Ammoniacal silver – Var I

Place 20 mL of 1% silver nitrate in a flask. Add 4 drops of 40% sodium hydroxide Add ammonium hydroxide by drops until the precipitate is almost dissolved. Dilute 1:10 with distilled water.

Ammoniacal silver – Var I

Place 20 mL of 2% silver nitrate in a flask. Add 3 drops of 40% sodium hydroxide Add ammonium hydroxide by drops until the precipitate is just dissolved.

Tissue Sample

Bensley & Bensley said that sections of tissue fixed in Zenker, Helly, ethanol or formalin are suitable. Although they commented that Var I gave a complete impregnation of paraffin or celloidin embedded tissue, they recommended Var II for paraffin sections because of their tendency to detach from slides. A section adhesive is recommended.

Protocol

Bring sections to water via xylene and ethanol.
Oxidise in 1% potassium permanganate for 5 min.
Bleach with 5% oxalic acid.
Place in Lugol’s iodine.
Bleach in 3% sodium thiosulphate.
Wash with water.
For Var I:
1. Place in tannic acid solution at 56°C for 5 min.
2. Rinse with ammoniated distilled water.
For Var II:
1. Place in 2% silver nitrate for 16 hrs.
For Var I: Ammoniacal silver solution, VAR I, at 56°C for 10 min. For Var II: Ammoniacal silver solution, VAR II, at room temperature for 30 min.
Wash with water.
Reduce with 20% formalin for 3 min.
Wash with water.
Tone with 0.2% yellow gold chloride.
Wash with water.
Place in 3% sodium thiosulphate.
Wash with water.
Place in neutral red for 1 min.
Wash with water.
Dehydrate with ethanol, clear with xylene and mount in a resinous medium

Expected Results

Reticulin fibres – black
Nuclei – as counterstained
Background – grey or as counterstained

Notes

In the method details above, several steps do not have times given, meaning that the step is required but no other details were given. Common sense should prevail, and the step done for sufficient time to accomplish its obviously intended purpose. If it is a water wash for removal of an excess of the preceding material it would usually be for approximately 1-2 minutes. If it is for toning with gold chloride then see the final note below.
The method details also often specify to “Wash in water” without saying whether distilled or tap water should be used. In many cases it does not matter, but common sense should prevail. If tap water is likely to produce a non-specific precipitate of silver, then use distilled water and, when it specifies to “wash”, give several changes. Tap water varies in quality and individual laboratory’s results may differ due to that. Of course, distilled water could be used throughout, but it is strongly recommended after the silver or gold chloride solutions since these may be affected by tap water contaminants.
Bensley & Bensley said that “the silver carbonate solution of Hortega” could be substituted for their own silver oxide solution in Var I. Hortega gave form several such solutions and the authors do not say which one they meant. These formulas differ mainly by the amounts of 10% aqueous silver nitrate added to 5% aqueous sodium carbonate. All redissolve the resulting precipitate with drops of strong ammonium hydroxide.Hortega’s Ammoniacal silver solutions
- Place 50 mL of 5% sodium carbonate in a flask. Add 12 mL of 10% silver nitrate. Let the precipitate settle, then decant the supernatent. Wash, allow to settle and decant several times. Add ammonium hydroxide by drops until the precipitate is almost dissolved. Dilute to 100 mL with distilled water.
- In addition to the formula above, another adds 12.5 mL silver nitrate, does not decant and wash, but does dilute to 100 mL with distilled water.
- A third adds 20 mL silver nitrate to 80 mL sodium carbonate, does not decant and wash, and does not dilute with distilled water.
- A fourth adds 25 mL silver nitrate to 75 mL sodium carbonate, does not decant and wash, and does not dilute with distilled water.
- A fifth adds 12 mL silver nitrate to 50 mL saturated aqueous lithium carbonate, decants and washes, and dilutes to 100 mL with distilled water.
Ensure that the ammonium hydroxide is fresh and full strength. Keep well stoppered when not in use. After removing the amount required immediately restopper the bottle.
Improperly made ammoniacal silver solutions can affect the quality of the impregnation. There should be a faint, persistent opalescence, with only a faint smell of ammonia.
20% formalin is made by diluting 20 mL strong formalin with 80 mL water.
Bensley & Bensley suggested either Heidenhain’s Azan or 1% aqueous acridine red as counterstains. I have substituted neutral red.
The formula given for Lugol’s iodine is now usually referred to as Gram’s iodine.
Toning is a variable step. Untoned sections give dark brown reticulin fibres on a paler brown background. Many microscopists prefer to tone for about 15 seconds to produce brown-black reticulin fibres on a pale grey-brown background. Others tone longer (a few minutes) to produce black reticulin fibres on a grey background. Longer toning produces purple tones. Tone according to the personal preference of the microscopist reviewing the slides.

Safety Note

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

References

Bensley R. R. and Bensley, S. H., (1938)
Handbook of Histological and Cytological Technique.
U. Chicago Press, Chicago, USA
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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Hine’s Hematoxylin & Eosin for Block Staining

By Carmen WongHematoxylin and Eosin Staining, Protocols, Stain Type

Protocol

Hine's H&E for Block Staining

steps

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Hine’s method is designed to stain blocks of tissue with hematoxylin and eosin (H & E). The blocks are then sectioned, dried and immediately coverslipped. Numerous identical stained sections may be prepared for demonstration and educational purposes.

Materials

Hematoxylin

Material	Amount	Function
Hematoxylin, 1% aqueous	100 mL	Dye
Aluminum sulphate, 5% aqueous	75 mL	Mordant
Lugol’s iodine	25 mL	Oxidant
Acetic acid, glacial	8 mL	Solvent
Glycerol	50 mL	Stabiliser

Eosin

Material	Amount	Function
Ethyl eosin	1 g	Dye
90% ethanol	100 mL	Solvent

Worcester’s fluid

Material	Amount	Function
Distilled water	200 mL	Solvent
Mercuric chloride	14 g	Fixing agent
Formalin, concentrated	22.5 mL	Fixing agent
Acetic acid, glacial	25 mL	Fixing agent

Compounding Procedures

Hematoxylin

Add the ingredients in the order listed.
Filter before use.

Eosin

Dissolve the eosin in the ethanol, and let stand for one month.
For use, dilute with an equal volume of 90% ethanol and filter.

Worcester’s fluid

Dissolve the mercuric chloride in the water.
Immediately prior to use add the formalin and acetic acid.

Protocol

Place fresh tissue blocks in Worcester’s fluid or formalin fixed tissue blocks in formol sublimate. Blocks should be no thicker than 1.5 cm. Fix overnight.
Remove from fixative and place into 70% ethanol for one hour.
Add Lugols iodine to 70% ethanol until dark brown, and complete removal of mercury pigment. Place the tissue into the ethanol and change three times over about 48 hours. The time varies depending on the tissue.
Transfer to fresh 70% ethanol (no iodine) for two hours, then to distilled water for one hour. Trim the tissue to size for processing.
Place the blocks into hematoxylin for seven days.
Wash blocks in running tap water overnight.
Begin dehydration with 70% and 80% ethanols for appropriate periods.
Stain with the working eosin solution for five days.
Dehydrate with absolute ethanol, two changes over five to eight hours.
Blot off excess ethanol and clear in cedarwood oil.
Blot off excess cedarwood oil and place into xylene, two changes for ten minutes each.
Impregnate with paraffin wax under vacuum, three changes of one hour each.
Block out and section.
Remove wax with xylene and coverslip with a resinous medium.

Expected Results

Nuclei – blue
Background – shades of pink

Notes

The method gives good results with all tissue except CNS.
Staining of sections has not deteriorated after 18 years.
Staining of processed tissues has not deteriorated after 18 years.
Bouin’s fluid can replace Worcester’s fluid, but the yellow colouration should be removed by placing in sodium bicarbonate in 50% ethanol (concentration not specified) instead of the iodinated 70% ethanol.
If needed, decalcification can be done with Gooding and Stewart’s fluid after removal of mercury pigment and before staining with hematoxylin.
The hematoxylin and eosin solutions can be re-used.

Safety Note

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

References

Hine, Ian F., (1981)
Block staining of mammalian tissues with hematoxylin and eosin.
Stain technology, v 56, p 119

March 30, 2023

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Böhmer’s Alum Hematoxylin

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

Protocol

Böhmer's Alum Hematoxylin

steps

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Böhmer’s formula is the original alum hematoxylin solution for nuclear staining. It is included for educational and historical reasons as the solution has little use in modern histotechnology.

Materials

Solution A

Material	Variant		Function
Material	Var 1	Var 2	Function
Hematoxylin	3.5 g	8 g	Dye
100% ethanol	100 mL	100 mL	Solvent

Solution B

Material	Variant		Function
Material	Var 1	Var 2	Function
Ammonium alum	0.3 g	0.3 g	Mordant
Distilled water	100 mL	100 mL	Solvent

Compounding procedure

Var 1 is taken from the Microtomist’s Formulary and Guide, and Var 2 from the Microtomist’s Vade-Mecum. The difference in concentration of the hematoxylin may be due to converting an alcoholic logwood extract to grams of dye. In any case, the way it is used makes the differences irrelevant.

Originally, solution A would have been made by soaking logwood chips in ethanol until a suitable concentration of dye was obtained. The solution would then have been allowed to ripen for a long time until it was distinctly deep brown, and filtered before it was used. In a modern variation, simply dissolve the dye in ethanol and leave to ripen, or add a small amount of sodium iodate.

The original called for a few drops of solution A to be added to a small quantity of solution B in a watch glass until the depth of color was judged to be correct. For today’s use, perhaps 5 mL solution A added to 45 mL solution B, more or less, would be satisfactory.

Protocol

Standard Method

Place a small amount of staining solution into a watch glass.
Place frozen sections into the staining solution for an appropriate time.
Transfer sections through at least two changes of clean water.
Mount onto slides.
Dehydrate in ethanol, clear in xylene and mount with a resinous medium

Alternative Method

Bring sections to water via xylene and ethanol.
Place into the staining solution for an appropriate time.
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 appropriate time should be determined by trial, but 10-20 minutes should suffice. The time will depend on the amount of solution A added to solution B. Smaller amounts are likely to take longer to stain with a paler final coloration.
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.
Bolles Lee, A.. Edited by Gatenby, J.B. and Beams, H.W., (1950)
The Microtomist’s Vade-Mecum. 11 ed.,
Churchill, London, UK.

March 30, 2023

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Bosma’s Alum Hematoxylin

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

Protocol

Bosma's Alum Hematoxylin

steps

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Materials

Material	Amount	Function
Hematoxylin, 10% alc.	25 mL	Dye
Ammonium alum	25 g	Mordant
Tap water	865 mL	Solvent
Diethylene glycol	100 mL	Stabiliser
Sodium iodate	0.2 g	Oxidant
Glacial acetic acid	10 mL	Acidifier

Compounding procedure

Place 815 mL hot tap water in a large flask.
Add the ammonium alum, mix well to dissolve and cool to room temperature.
Add the alcoholic hematoxylin solution (ripened).
Add the sodium iodate dissolved in 50 mL cold tap water and mix well.
Add the diethylene glycol and mix well.
Add the acetic acid and mix well. The pH should be 3.1 to 3.3 and the solution may be used immediately.

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

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

Bosma, Rob
A useful hematoxylin without toxic chemicals.
Histologic, V 18, Nº 1, January 1988

March 30, 2023

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Brillmeyer’s Trichrome for Muscle and Collagen

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

Protocol

Brillmeyer's Trichrome

for Muscle and Collagen

steps

materials

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Materials

Solution A

Material	Amount
Acid fuchsin	0.2	g
Distilled water	100	mL

Solution B

Material	Amount
Aniline blue	0.5	g
Orange G	2	g
Phosphomolybdic acid	1	g
Distilled water	100	mL

Tissue Sample

Protocol

Bring sections to water via xylene and ethanol.
Stain with an acid resistant nuclear stain.
Place into solution A for 1 minute.
Drain.
Place into solution B for 2-3 hours.
Wash briefly with distilled water.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

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

Notes

The original technique recommended nuclear staining in Delafield’s alum hematoxylin, with blueing.
Weiss modified this method to shorten the time required.

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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Basic Fuchsin–Picric Acid for Elastic Fibers

By Carmen WongElastic Fibers, Protocols, Stain Target

Protocol

Basic Fuchsin–Picric Acid

for Elastic Fibers

steps

materials

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Materials

Basic fuchsin

Material	Amount
Basic fuchsin	0.5	g
Distilled water	500	mL

Picric acid

Material	Amount
Saturated alcoholic picric acid	12	mL
Acetone	1	L

Tissue Sample

A 10% Formalin variant is suitable. Paraffin sections at 5µ are preferred.

Protocol

Bring sections to water via xylene and ethanol.
Place in basic fuchsin for 1-2 minutes.
Rinse with tap water, then acetone.
Differentiate briefly with picro-acetone until tissue is just decolorised.
Rinse with acetone.
Clear with xylene and mount with a resinous medium

Expected Results

Elastic fibres – red
Other tissues – yellow

Notes

The Brown and Brenn picric acetone usually requires weighing semi-dry picric acid. The amount in the saturated ethanolic solution specified above is very close to that and it is a much safer means of compounding the solution.
If you use the Brown and Brenn variant of the Gram stain, the counterstain solutions from that may be used.
Be careful not to overdifferentiate. Apply picric acetone until most of the red is just removed from the background. This takes only a few seconds. It is easy to overdifferentiate.
This method is not meant as a primary means of staining elastic fibres. It can be done very quickly, about 5-10 minutes, and may be useful when time is limited.
Nuclei are not stained.

Safety Note

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

References

Personal observation, Bryan D. Llewellyn.
Brown, J H and Brenn, L, (1931),
A method for the differential staining of Gram positive and Gram negative bacteria in tissue sections.,
Bull. John Hopkins Hosp., v 48, page 69.

March 30, 2023

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