Category

Stain Type

Kostowiecki’s Trichrome for Muscle, Cartilage and Collagen

By Protocols, Stain Type, Trichrome Staining, Trichrome, One-Step
Protocol

Kostowiecki's Trichrome

for Muscle, Cartilage and Collagen

7
steps
5
materials
print

Materials

  • A red nuclear stain
  • Solution A
    MaterialAmount
    Aniline blue0.06g
    Orange G0.2g
    Phosphomolybdic acid1g
    Distilled water100mL

Preparation of Solution A

  1. Add both dyes to the water.
  2. Bring to the boil for three minutes.
  3. Add the phosphomolybdic acid while hot.
  4. Cool and filter.

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

  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei with a red nuclear stain.
  3. Place into the staining solution until darkly stained, 30-120 minutes.
  4. Rinse with distilled water.
  5. Place in 95% ethanol for 1 minute.
  6. Dehydrate with absolute ethanol.
  7. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  red
  • Muscle  –  orange
  • Cartilage  –  blue
  • Collagen  –  blue-green

Safety Note

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


References

  1. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
    Citing:
    Kostowiecki, (1932)
    Zeitschrift für wissenschaftliche Mikroskopie und für mikroskopische Technik,
    v. 49, pp. 337
March 30, 2023
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Krajian’s Iron Hematoxylin

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Krajian's Iron Hematoxylin

8
steps
6
materials
print

Materials

MaterialAmountFunction
Hematoxylin6 gDye
Ferric ammonium sulfate6 gMordant
Ferric chloride6 gMordant
Potassium iodide6 g–
95% ethanol50 mLSolvent
Distilled water50 mLSolvent

Compounding Procedure

  1. Dissolve the hematoxylin in the ethanol.
  2. Dissolve the other ingredients in the water.
  3. Combine the solutions.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 3 minutes.
  3. Rinse with tap water.
  4. Dip briefly in 1% hydrochloric acid in 70% ethanol.
  5. Wash well in running tap water to blue.
  6. Rinse with distilled water.
  7. Counterstain if desired.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Background  –  as counterstain or unstained

Notes

  • Gray states that this solution is, “an excellent general purpose hematoxylin”.
  • The stability of the solution is not commented on, but it is likely not stable for long.
  • This method is from a technique for micro-organisms, so the staining time may need to be adjusted for sections.

Safety Note

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

References

  1. 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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Krutsay’s Alum & Iron Hematoxylin

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Krutsay's Alum & Iron Hematoxylin

6
steps
6
materials
print

Krutsay’s Alum hematoxylin gives very highly selective staining of nuclei. It may also be easily converted into an iron hematoxylin for use as an acid resistant nuclear stain.

Materials

Alum hematoxylin

MaterialAmountFunction
Hematoxylin1 gDye
Potassium alum50 gMordant
Distilled water1 LSolvent
Hydrochloric acid5 mLAcidifier
Sodium iodate0.2 gOxidant

Iron conversion

MaterialAmountFunction
Iron alum2 gMordant
Hydrochloric acid0.5 mLAcidifer
Distilled water100 mLSolvent

Compounding Procedure

  1. Mix the reagents under Alum hematoxylin together.
  2. Bring to a boil
  3. Cool and filter.
  4. The solution may be used immediately.

Conversion to Iron Hematoxylin

  1. Mix the reagents under Iron conversion together, and dissolve.
  2. For use mix the following:
    • Alum hematoxylin solution – 25 mL
    • Iron conversion solution – 2 mL

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into either staining solution for 5 minutes.
  3. Rinse with water and blue.
  4. Rinse well with water.
  5. Counterstain if desired.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue (alum) or black (iron)
  • Background  –  as counterstain or unstained

Notes

  • This is a progressive solution and is highly selective for nuclei.
  • The iron Alum is (FeNH4(SO4)2.12 H2O)
  • 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

  1. Humason, G. L.
    Animal Tissue Techniques.
    W. H. Freeman and Co., San Francisco, CA, USA
March 30, 2023
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Kleinenberg’s Alum Hematoxylin

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Kleinenberg's Alum Hematoxylin

8
steps
5
materials
print

There are two alternate formulae for Kleinenberg’s Alum hematoxylin.

Materials

Formula I

Hematoxylin

MaterialAmountFunction
HematoxylinsaturatedDye
100% ethanol100 mLSolvent

Alum

MaterialAmountFunction
Ammonium alumsaturatedMordant
70% ethanol100 mLSolvent

Calcium

MaterialAmountFunction
Calcium chloridesaturatedMordant
70% ethanol100 mLSolvent

Compounding Procedure

  1. Prepare each of the solutions.
  2. For use, combine:
    1. Alum solution – 85 mL
    2. Calcium solution – 15 mL
    3. Hematoxylin solution – 1 mL

Formula II

Hematoxylin

MaterialAmountFunction
HematoxylinsaturatedDye
100% ethanol100 mLSolvent

Alum A

MaterialAmountFunction
Ammonium alumsaturatedMordant
Calcium chloridesaturatedMordant
70% ethanol100 mLSolvent

Alum B

MaterialAmountFunction
Potassium alumsaturatedMordant
70% ethanol88 mLSolvent
Alum A solution12 mL–

Compounding Procedure

  1. Prepare each of the solutions.
  2. For use, combine:
    1. Alum B solution – 100 mL
    2. Hematoxylin solution – 3 mL

Protocol

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

Expected Results

  • Nuclei  –  blue
  • Background  –  as counterstain or unstained

Notes

  • Although not stated, the saturated hematoxylin solution should presumably be allowed to ripen.
  • Formula I uses 1 mL of the saturated hematoxylin solution, whereas formula II uses 3 mL, i.e. it is considerably stronger. This may affect the staining times.
  • Alum and calcium chloride combined give aluminum chloride hematoxylin.
  • 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

  1. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
    Citing:
    Cole, A. C., (1884).
    The methods of microscopical research. (formula I), and
    Böhm, A. & Opel, A., (1907).
    Manuel de technique microscopique, Ed. 4, (formula II).
March 30, 2023
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Modified Yellowsolve for General Oversight

By Protocols, Stain Type, Trichrome Staining, Yellowsolve Staining
Protocol

Modified Yellowsolve

for General Oversight

10
steps
8
materials
print

Materials

  • Hemalum
  • Trichlorethylene
  • Solution A
    MaterialAmount
    Phloxine B0.5g
    Calcium chloride0.5g
    Distilled water100mL
  • Solution B
    MaterialAmount
    Luxine pure yellowtosaturation
    2-Ethoxyethanol50mL
    Ethyl phosphate50mL

Tissue Sample

5 µ paraffin sections of neutral buffered formalin fixed tissue are likely suitable. It should be noted, however, that the authors of this method favoured extended fixation in formal sublimate of up to 10 days for fibrin. This fixative is now deprecated due to its mercuric chloride content. With other staining methods, pretreatment of sections with Bouin’s fluid for an hour at 60°C can compensate for the lack of mercury fixation to a large degree.

Lendrum says, “If … the time of differentiation is kept short the result is comparable to the best examples of the old Masson’s erythrosin-saffron technique.” Masson’s erythrosin-saffron is the same technique as the HPS (hematoxylin-phloxine-saffron) using phloxine instead of erythrosin.

Protocol

  1. Dewax sections with xylene.
  2. Place in trichlorethylene for 24 to 48 hours.
  3. Bring sections to water.
  4. Stain nuclei with hemalum, differentiate and blue.
  5. Wash well with water.
  6. Place in solution A for 30 minutes.
  7. Rinse with 2-ethoxyethanol.
  8. Differentiate with solution B, controlling microscopically, until collagen is yellow.
  9. Rinse with 2-ethoxyethanol.
  10. Clear in xylene and mount with a synthetic resinous medium.

Expected Results

  • Nuclei  –  blue
  • Muscle  –  red
  • Background  –  yellow

Notes

  • Stop differentiation when collagen is yellow and muscle is still red.
  • 2-Ethoxyethanol is also known as cellosolve or ethylene glycol monoethyl ether, and has the formula CH3CH2OCH2CH2OH.
  • Trichlorethylene has the formula ClHC=CCl2 or C2HCl3
  • Trichlorethylene should be used in a fume hood.

Safety Note

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

References

  1. Lendrum A C, Fraser D S, Slidders W and Henderson R. (1962)
    Studies on the character and staining of fibrin.
    Journal of clinical pathology, v. 15, p. 401.
March 30, 2023
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Gill’s Aluminum Hematoxylin Variants

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Gill's Aluminum Hematoxylin Variants

6
steps
6
materials
print

Materials

MaterialSolution StrengthFunction
Single (I)Double (II)Triple (III)
Hematoxylin2 g4 g6 gDye
Aluminum sulphate17.6 g70.4 g158.4 gMordant
Distilled water750 mL750 mL750 mLSolvent
Ethylene glycol250 mL250 mL250 mLSolvent
Sodium iodate0.2 g0.4 g0.6 gOxidant
Glacial acetic acid20 mL20 mL20 mLAcidifier

Compounding procedures

  1. Mix the ethylene glycol and water.
  2. Add the hematoxylin, then the sodium iodate.
  3. Add the aluminum sulphate and the acetic acid
  4. Stir for an hour at room temperature.
  5. Filter before use.
  6. The solutions may be used immediately, and are stable for approximately one year.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for an appropriate time.
  3. Rinse with water and blue.
  4. Rinse well with water.
  5. Counterstain if desired.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Background  –  as counterstain or unstained

Notes

  • The three formulations are for progressive staining.
  • The single strength solution is for cytology. Staining time is 2 minutes. The double strength solution is for paraffin sections. Staining time is 3 minutes. The triple strength solution is for paraffin sections. Staining time is 1.5 minutes.
  • 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.
  • The aluminum sulphate should be Al2(SO4)3.18 H2O. Adjust the amount if different.
  • Similarly, the hematoxylin should be anhydrous. If hydrated (C16H14O6.3 H2O) use 2.36 g, 4.72 g and 7.08 g respectively.

Safety Note

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

References

  1. Culling, C.F.A., Allison, R.T. and Barr, W.T.
    Cellular Pathology Technique, Ed.4.
    Butterworth, London, UK.
March 30, 2023
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Gluck’s Impregnation for Reticulin on Zenker Fixed Tissue

By Metal Impregnation, Metal Impregnation, Silver, Protocols, Reticulin, Stain Target, Stain Type
Protocol

Gluck's Impregnation

for Reticulin on Zenker Fixed Tissue

20
steps
12
materials
print

Materials

  • Oxalic acid, 5% aqu.
  • Silver nitrate, 10% aqu.
  • Strong ammonium hydroxide (s.g. 0.88).
  • Sodium hydroxide, 40% aqu.
  • Formalin, 5% aqu.
  • Yellow gold chloride, 0.2% aqu.
  • Neutral red, 1% aqu.
  • Gram’s iodine.
    MaterialAmount
    Iodine1g
    Potassium iodide2g
    Distilled water300mL

    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.

  • Cajal’s Solution
    MaterialAmount
    Strong formalin15mL
    Ammonium bromide2g
    Water85mL

Preparation of Gluck’s Ammoniacal Silver

  1. Place 10 mL of 10% silver nitrate in a flask.
  2. Add 0.5 mL of 40% sodium hydroxide.
  3. Allow to settle, then decant the supernatent.
  4. Wash the precipitate, allow to settle, then decant a few times.
  5. While swirling, slowly add drops of strong ammonium hydroxide until the precipitate just redissolves.
  6. Dilute to 100 mL with distilled water, then add 2 mL pyridine.

Tissue Sample

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

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Rinse well with tap water.
  3. Place in Gram’s iodine for 4-8 hours.
  4. Rinsewith 70% ethanol.
  5. Rinse well with tap water.
  6. Bleach in 5% sodium thiosulphate for 5 minutes.
  7. Wash well with tap water.
  8. Place in Cajal’s solution at 37°C for 24 hours.
  9. Wash with tap water.
  10. Place in Gluck’s ammoniacal silver for 5 minutes.
  11. Rinse with distilled water.
  12. Place in 5% formalin for 5 minutes.
  13. Wash with tap water.
  14. Tone with 0.2% gold chloride solution until grey.
  15. Rinse with distilled water.
  16. Place in 5% sodium thiosulphate for 5 minutes.
  17. Wash well with tap water.
  18. Counterstain with neutral red for 1 minute.
  19. Rinse with tap water.
  20. Dehydrate with ethanol, clear with xylene, and mount with a resinous medium.

Expected Results

  • Reticulin fibres  –  black
  • Nuclei  –  red
  • Background  –  grey

Notes

  • Ensure that both the ammonium hydroxide and sodium hydroxide are fresh and full strength. Keep both well stoppered when not in use. For the ammonium hydroxide, pour sufficient for use from the stock bottle into a beaker, then immediately restopper the stock bottle. Do not return excess ammonium hydroxide to the stock bottle.
  • After making the ammoniacal silver solution but before adding the pyridine, smell the solution to ensure it has only a faint smell of ammonia. If the smell of ammonia is strong it indicates that too much ammonium hydroxide has been added. If so, it is preferable to make the solution again. Improperly made ammoniacal silver solutions can affect the quality of the impregnation.
  • The formalin used to make Cajal’s solution should be neutralised, but do not use buffered formalin. Neutral formalin in this context may be made by keeping strong formalin over marble chips. However, be very careful as the gas given off may increase the pressure inside the container and cause an explosion. Either apply a cap loosely so gas can escape, or use a fermentation lock.
  • 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

  1. 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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Goldman’s Iron Hematoxylin

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Goldman's Iron Hematoxylin

10
steps
8
materials
print

Materials

Solution A

MaterialAmountFunction
Ferric ammonium sulfate4 gMordant
Distilled water100 mLSolvent
Acetic acid, glacial1 mLAcidifier
Sulfuric acid0.12 mLAcidifier

Solution B

MaterialAmountFunction
Picric acid, saturated aqueous100 mLDye and acid
Sulfuric acid0.1 mLAcidifier

Solution C

MaterialAmountFunction
Hematoxylin0.5 gDye
Distilled water100 mLSolvent

Solution D

MaterialAmountFunction
70% ethanol100 mLSolvent
Lithium carbonate, saturated aqueous5 dropsBase

Compounding procedures

  1. Make each solution separately.
  2. The aqueous hematoxylin should be ripened before use.

Protocol

  1. Bring sections to water, removing mercury pigment if necessary.
  2. Place into solution A for 30 minutes to 24 hours.
  3. Wash in running tap water for 10 minutes.
  4. Place in solution B for 3 hours or longer.
  5. Wash in running tap water for 15 minutes.
  6. Place in solution C for 1 hour.
  7. Wash in running tap water for 15 minutes.
  8. Blue with solution D.
  9. Counterstain if desired.
  10. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Basophil cytoplasm  –  grey
  • Background  –  as counterstain or unstained

Notes

  • The stock solutions are stable for some time.
  • The technique was originally intended for the demonstration of protozoa.
  • The method was designed for paraffin sections of material fixed with formalin variants, Bouin’s or Zenker’s fluids.
  • Overstaining occurs only if sections are left in hematoxylin for several hours.
  • Bouin fixed tissue is not as intensely stained as with other fixatives.
  • The time in picric acid (solution B) is necessary, reducing it causes overstaining.

Safety Note

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

References

  1. Lillie, R.D., (1954)
    Histopathologic technique and practical histochemistry Ed.2
    Blakiston, New York, USA.
    Citing:
    Goldman, (1951)
    American Journal of Clinical Pathology,
    v.21, p.198
March 30, 2023
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Goldner’s Trichrome for Muscle and Collagen

By Protocols, Stain Type, Trichrome Staining, Trichrome, Multi-Step
Protocol

Goldner's Trichrome

for Muscle and Collagen

11
steps
9
materials
print

Materials

  • Weigert’s iron hematoxylin or equivalent
  • Solution A
    MaterialVariation 1Variation 2Variation 3
    Ponceau 2R0.7g0.75g–
    Acid fuchsin0.3g0.25g–
    Azophloxine–0.1g5g
    Acetic acid, glacial2mL2mL2mL
    Distilled water1L1L1L
  • Solution B
    MaterialAmount
    Acetic acid, glacial10mL
    Distilled water1L
  • Solution C
    MaterialAmount
    Orange G20g
    Phosphomolybdic acid40g
    Distilled water1L
  • Solution D
    MaterialAmount
    Light green SF2g
    Acetic acid, glacial2mL
    Distilled water1L

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

  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei with Weigert’s iron hematoxylin, or equivalent.
  3. Place into one of the variants of solution A for 5 minutes.
  4. Rinse with solution B.
  5. Place into solution C until collagen is decolorised.
  6. Rinse with solution B.
  7. Place into solution D for 5 minutes.
  8. Place into solution B for 5 minutes.
  9. Blot.
  10. Dehydrate rapidly with absolute ethanol.
  11. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Erythrocytes  –  orange
  • Cytoplasm  –  red
  • Collagen  –  green

Safety Note

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


References

  1. 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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Gridley’s Impregnation for Reticulin

By Metal Impregnation, Metal Impregnation, Silver, Protocols, Reticulin, Stain Target, Stain Type
Protocol

Gridley's Impregnation

for Reticulin

17
steps
9
materials
print

Materials

  • Silver nitrate, 2% aqu.
  • Silver nitrate, 20% aqu.
  • Strong ammonium hydroxide (s.g. 0.88).
  • Sodium hydroxide, 40% aqu.
  • Periodic acid, 0.5% aqu.
  • Formalin, 3% aqu.
  • Yellow gold chloride, 0.5% aqu.
  • Sodium thiosulphate, 5% aqu.
  • Neutral red, 1% aqu.

Preparation of Da Fano’s Ammoniacal Silver

  1. Place 10 mL of 20% silver nitrate in a flask.
  2. Add 0.2 mL of 40% sodium hydroxide.
  3. While swirling, slowly add drops of strong ammonium hydroxide until the precipitate just redissolves.
  4. Make up to 80 mL with distilled water.

Tissue Sample

6 µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. A section adhesive is recommended.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Oxidise with 0.5% periodic acid for 15 minutes.
  3. Rinse well with distilled water.
  4. Sensitise with 2% silver nitrate for 30 minutes.
  5. Rinse well with distilled water.
  6. Treat with da Fano’s ammoniacal silver for 15 minutes.
  7. Rinse with distilled water.
  8. Reduce in 3% formalin for 3 minutes.
  9. Rinse well with tap water.
  10. Rinse with distilled water.
  11. Tone with 0.5% gold chloride solution for 5 minutes.
  12. Rinse with distilled water.
  13. Fix in 5% sodium thiosulphate for 5 minutes.
  14. Wash well with running tap water.
  15. Counterstain with neutral red for 1 minute.
  16. Rinse with tap water.
  17. Dehydrate with ethanol, clear with xylene, and mount with a resinous medium.

Expected Results

  • Reticulin fibres  –  black
  • Nuclei  –  red
  • Background  –  grey

Notes

  • Ensure that both the ammonium hydroxide and potassium hydroxide are fresh and full strength. Keep both well stoppered when not in use. For the ammonium hydroxide, pour sufficient for use from the stock bottle into a beaker, then immediately restopper the stock bottle. Do not return excess ammonium hydroxide to the stock bottle.
  • After making the ammoniacal silver solution, smell the solution to ensure it has only a faint smell of ammonia. If the smell of ammonia is strong it indicates that too much ammonium hydroxide has been added. If so, it is preferable to make the solution again. Improperly made ammoniacal silver solutions can affect the quality of the impregnation.
  • 3% formalin is made by diluting strong formalin with tap water (3 mL strong formalin, 97 mL tap water).
  • Toning is a variable step. Untoned sections give dark brown reticulin fibres on a paler brown background. Although the method specifies 5 minutes toning in 0.5% gold chloride, this is not mandatory. 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.
  • The use of periodic acid followed by an easily reducible silver solution is obviously similar to the Jones’ method for basement membranes and carbohydrates. Note, however, that Gridley’s method applies ammoniacal silver solution at room temperature after sensitising in aqueous silver nitrate at room temperature, then applies formalin as a reducing agent, i.e. it is an argyrophil reaction, while the Jones’ method applies a methenamine silver solution at 56°C for a longer time, without sensitising, and does not use an external reducer, i.e. it is an induced argentaffin reaction. Clearly, in Gridley’s method the periodic acid is being used for the same purpose as the potassium permanganate of the Mallory bleach in other methods.

Safety Note

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

References

  1. 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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