Special Stains Laboratory Report

Table of contents


Staining is a technique that is used to diagnose or study the morphology of abnormal cells such as cancerous cells by highlighting the structural components of a tissue (Bancroft and Gamble, 2008). Staining provides a contrast between different structures in a tissue specimen and allows its examination under a light microscope (Cook, 2006). Haematoxylin and eosin (H&E) is a routine stain that is used to microscopically diagnose a vast majority of specimens in which the haematoxylin stains the nuclei, whereas the eosin is used to stain cytoplasm and other extracellular materials (Bancroft and Cook, 1995). According to Slauson and Cooper (2002) special stains are histochemical stains that react with known substances in the tissue. Mohan (2005) explains that special stains are required in various circumstances, where the pathologist needs to demonstrate certain constituent of the cells or the tissue to confirm the diagnosis by etiologic, histogenic and pathogenic components. This technique is called special because they are not a routine stain that is performed on a tissue specimen, instead they are used in addition to H&E stained sections (Bancroft and Gamble, 2008). Special stains can identify the presence and abundance of any specific class of molecules in a tissue specimen for example periodic acid-Schiff (PAS) reaction is used to identify carbohydrate substances such as glycogen (Slauson and Cooper, 2002). Other examples include Toluidine blue stain which is used to stain mast cell granules, Perl’s stain demonstrates iron in haemochromatosis, Ziehl-Neelsen stains mycobacteria and Giemsa staining is used to identify Helicobacter Pylori and Giardia organisms (Slauson and Cooper, 2002 and Bass et al., 2005)

Masson’s trichrome (MT) and Congo red are the two main special staining methods used in pathology laboratories. Connective tissues consist of cells such as collagen fibres, elastic fibres, and glycosaminoglycans that are scattered within an extracellular matrix (Starr et al., 2011). These cells are distinguished by using a combination of dyes to stain different structures in various different colours (Starr et al., 2011). Masson’s trichrome is used to express collagen in tissues and involves staining with three different sized dyes to stain three diverse tissue densities (Cook, 2006). MT staining produces three distinct colours as the name suggests; nuclei and other basophilic structures are stained black with iron hematoxylin; collagen is stained green or blue depending on aniline light green or aniline blue; and cytoplasm, muscle, erythrocytes and keratin are stained bright red with Biebrich scarlet stain (Young et al., 2006). Since erythrocytes are the densest as they are packed with haemoglobin, and less porous tissues they are stained with the smallest dye molecule, the intermediate cytoplasm and muscles cells are stained by the intermediate sized dye and the collagen is stained with the biggest dye (Bancroft and Gamble, 2008). However, it has also been suggested that the acid dye which is the Biebrich Scarlet, first stains the tissue as it binds to its acidophilic elements (Carson, 2001). Subsequently, the tissue is treated with phosphomolybdic/phosphotungstic acids so that the less permeable components retain the red colour, whereas it is diffused out of the collagen fibers causing it to bind with the aniline blue or aniline light green (Bancroft and Gamble, 2008). Young et al., (2006) describes that in addition to the use MT stain in assessing the degree of fibrosis, it is also used to evaluate portal tract structures such as the bile ducts, arteries and veins in inflamed liver

According to Romhanyi (1971) (cited in Bely, 2006) Congo red is a special stain that is highly specific and a sensitive method for early diagnosis and recognition of amyloidosis. Cook (2006) states that Congo red is used as the preferred method to identify amyloids in most laboratories on formalin fixed, paraffin embedded tissue of patients with amyloidosis. Kiernan (2007) describes that amyloid is an intercellular material that varies in its composition and is deposited in tissues such as heart, muscle, kidneys, spleen, liver and brain, deposits differ in their composition. Rubin and Strayer (2008) explains that Congo red stain has a linear shaped molecule which helps it to bind to the ? pleated sheet structure of the amyloid through non-polar hydrogen bonds, giving it a red colour. Sen and Basdemir (2003) states that Congo red fluorescence (CRF) is another method that examines the amyloid deposits stained with Congo red under polarized light which shows a red-green birefringence and according to Rocken and Eriksson (2009) this is the gold standard for diagnosing amyloid.

During this experiment special stain techniques were used to analyse specific tissue elements


To identify fibroids in uterine tissue section using Masson’s trichrome stain
To identify amyloid in spleen tissue section using Congo red stain
To discuss advantages of special stains
To use special stains to identify important diagnostic features of the tissue
To understand the mechanism used by special stains


Masson’s Trichrome

The formalin fixed and paraffin-embedded uterine tissue section was deparaffinized and rehydrated through 100% alcohol, 95% alcohol, and 70% alcohol. The section was washed in distilled water and then stained in Weigert’s iron hemotoxylin working solution for 10 minutes. It was then rinsed in running warm tap water for 10 minutes and then washed in distilled water. The next step was to stain the uterine tissue section in Biebrich scarlet-acid fuchsin solution for 15 minutes, and then it was washed using distilled water. It was then differentiated in phosphomolybdic – phosphotungstic solution for 15 minutes or until collagen was not red. The tissue section was then transferred directly (without rinsing) to aniline blue solution and stained for 5-10 minutes. Afterwards the tissue section was rinsed briefly in distilled water and differentiated in 1% acetic acid solution for 1 minute. It was then washed in distilled water and dehydrated very quickly through 95% ethyl alcohol, absolute ethyl alcohol (to wipe off Biebrich scarlet-acid fuschin staining) and then cleared in xylene. The section was then mounted with resinous mounting medium. Finally the slide was examined under the light microscope.

Congo Red

The spleen tissue section was deparaffinized and hydrated to distilled water. The section was then stained in Congo red working solution for 10 minutes and rinsed in distilled water. It was then quickly differentiated (5-10 dips) in alkaline alcohol solution and rinsed in tap water. The section was then counterstained in Gill’s haematoxylin for 10 seconds and rinsed in tap water for 2 minutes. Following that, the section was dipped in ammonia water (made by adding a few drops of ammonium hydroxide to tap water and mixing it well) for 30 seconds or until the sections had turned blue. It was then rinsed in tap water for another 5 minutes and dehydrated through 95% alcohol, and 100% alcohol. The section was cleared in xylene and mounted with mounting medium. The slide was then examined under a light microscope.

Fig 1: Normal uterine tissue stained with Masson’s trichrome viewed under 10 x 10 microscopic magnification

Fig 2: Fibroid uterine tissue stained with Masson’s trichrome viewed under 10 x 10 microscopic magnification

The microscopic slide (Fig. 1) shows a normal uterus tissue that was stained with Masson’s trichrome, which showed the nuclei stained black, smooth muscle stained red and the collagen fibres stained blue. Figure 2 shows a uterus tissue specimen stained with Masson’s trichrome that revealed excessive amount of collagen stained in blue, smooth muscle stained red and nuclei stained black.

Fig 3: Spleen tissue stained with Congo red showing amyloid deposits under 10?10 microscopic magnification

Fig. 4 Spleen tissue stained with Congo red adapted from Stevens and Lowe, (2000)


Norwitz and Schorge (2006) states that fibroids also referred to as Leiomyomata, are benign tumours of the myometirum of the uterus that is mainly composed of smooth muscle and extracellular fibrous material such as the collagen, Fibroids do not invade surrounding tissues or organs and they can occur in different locations within the uterus (Lark, 1996). The symptoms include heavy periods, frequent urination, constipation, bloating and backache (Tulandi, 2003)

Masson’s trichrome was used to stain the uterus tissue (Fig. 1) which showed the collagen stained in blue, smooth muscle and erythrocytes stained red and the nuclei appeared black. MT stain revealed that the normal uterus tissue (Fig. 1) had a small amount of collagen present whereas the other uterus tissue (Fig. 2) showed an elevated amount of collagen stained in blue which suggested fibrosis. MT is an advantageous technique in medicine as it allows comparing the degree of fibrosis before and after the treatment biopsies to show if the treatment has been effective and successful. It is a routine stain for kidney and liver biopsies and this can be used on paraffin fixed sections as well as on frozen sections. Kambic et al., (1986) describes that there are different types of collagen and their organization is better shown using Sirius red with polarized light. Sirius red is a hydrophilic dye, in which type I collagen appears orange or red whereas type III collagen appears green (Kumar, 2005).

Congo red was used to stain a spleen tissue section where figure 3 showed amyloid deposits in pink and nuclei in blue. Fig. 4 revealed a high amount of amyloid deposits in pink which suggested amyloidosis that is the disorder caused by abnormal deposition of intracellular or extracellular insoluble amyloid which changes the normal tissue function (Stevens and Lowe, 2000).

Special stains is an important tool for pathologists as it allows to the microscopically view and identify cells, tissues and microorganisms, providing an alternative to immunohistochemistry, flow cytometry and various other diagnostic techniques.


  1. Bancroft, J. D. & Gamble, M. (2008). ‘Theory and practice of histological techniques’. [Online]. (6th ed). Philadelphia, PA : Churchill Livingstone/Elsevier. Available from: http://books.google.co.uk/books?id=Dhn2KispfdQC&printsec=frontcover&dq=Theory+and+practice+of+histological+techniques&hl=en&ei=ifGETZKEPcWwhAfSvPW_BA&sa=X&oi=book_result&ct=result&resnum=1&ved=0CC8Q6AEwAA#v=onepage&q&f=false. [Accessed 7th March 2011].
  2. Bancroft. J. D. & Cook, H. C. (1995). ‘Manual of histological techniques and their diagnostic application’. Edinburgh [u.a.] : Churchill Livingstone
  3. Bass, P., Burroughs, S. & Way, C. (2005). ‘Systematic pathology : a clinically-orientated core text with self assessment’. Edinburgh : Elsevier Churchill Livingstone.
  4. Bely, M. (2006). ‘Histochemical differential diagnosis and polarization optical analysis of amyloid and amyloidosis’ TheScientificWorldJournal. 6, p.154-168.
  5. Carson, F. L. (2001). ‘Histotechnology : a self-instructional text’. (2nd ed). Chicago : ASCP Press.
  6. Cook, D. J. (2006). ‘Cellular pathology : introduction to techniques and applications’. (2nd ed). Bloxham: Scion Publishing Limited. http://onlinelibrary.wiley.com/doi/10.1046/j.1440-1827.2003.01513.x/abstract
  7. Kiernan, J. A. (2007). ‘Histological and histochemical methods’. (4th ed). Cold Spring Harbor (N.Y.) : Cold Spring Harbor Laboratory Press.
  8. Mohan, H. (2005). ‘Essential pathology for dental students’. [online]. (3rd ed). New Delhi : Jaypee Brothers. Available from: http://books.google.co.uk/books?
  9. id=HmkTtLyxXF8C&printsec=frontcover&dq=essential+pathology+for+dental+students&hl=en&ei=xRCCTaiHMs24hAezp6G8BA&sa=X&oi=book_result&ct=bookthumbnail&resnum=1&ved=0CDAQ6wEwAA#v=onepage&q&f=false. [Accessed: 11th March 2011]
  10. Odze, R. D. & Goldblum, J. R. (2009). ‘Surgical pathology of the GI tract, liver, biliary tract and pancreas’. [Online]. (2nd ed). Philadelphia, PA : Saunders/Elsevier. Available from: http://books.google.co.uk/books?id=8ITX093f1j0C&pg=PA1143&dq=Masson%27s+trichrome+staining+in+liver&hl=en&ei=OB2GTYuVLYuqhAfJ_6m8BA&sa=X&oi=book_result&ct=book-thumbnail&resnum=2&ved=0CDgQ6wEwAQ#v=onepage&q=Masson%27s%20trichrome%20staining%20in%20liver&f=false. [Assessed 16th March 2011]
  11. Rocken, C. & Eriksson, M. (2009).
  12. ‘Amyloid and amyloidoses’ Der Pathologe. 30, (3), p.182-192.
    Rubin, R. & Strayer, D. S. (2008).
  13. ‘Rubin’s Pathology : clinicopathologic foundations of medicine’. [Online]. (5th ed). Philadelphia [u.a.] : Lippincott Williams & Wilkins. Available from: http://books.google.co.uk/books?id=kD9VZ267wDEC&pg=PA990&dq=mechanism+of+congo+red+stain&hl=en&ei=ADGFTcSQDZGJhQf-n5iuBA&sa=X&oi=book_result&ct=book-thumbnail&resnum=4&ved=0CEUQ6wEwAw#v=onepage&q=mechanism%20of%20congo%20red%20stain&f=false. [Assessed 7th March 2011].
  14. Sen, S. & Basdemir, G. (2003). ‘Diagnosis of renal amyloidosis using Congo red fluorescence’ Pathology international. 53, (8), p.534-538.
    Slauson, D. O. & Cooper, B. J. (2002).
  15. ‘Mechanisms of disease : a textbook of comparative general pathology’. [Online]. (3rd ed). St. Louis, MO. [u.a.] : Mosby. Available from : http://books.google.co.uk/books?id=vRhtM0UMUh4C&pg=PA5&dq=special+stains&hl=en&ei=-vSETaMeqJKEB83RwccE&sa=X&oi=book_result&ct=book-thumbnail&resnum=1&ved=0CCoQ6wEwADgK#v=onepage&q=special%20stains&f=false. [Accessed 4th March 2011].
  16. Young B., Lowe, J. S, Stevens, A. & Heath, J. W. (2006).
  17. ‘Wheater’s functional histology : a text and colour atlas’. (5th ed). Edinburgh : Churchill Livingstone
    Tulandi, T. (2003).

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