Characterization of the histologic appearance of normal gill tissue using special staining techniques

Abstract

Anatomic pathologists are familiar with stains used in light microscopy to identify cells, storage products, tissue deposits, and pathogens. Assessment of the surrounding tissue with special stains may reveal aspects of interest for the tissue or the species. We illustrate the expected staining characteristics of normal rainbow trout gill tissue with routine hematoxylin and eosin and 18 other histochemical stains.

Introduction

The histologic appearance of most fish tissues is generally similar to that of well-characterized homologous organs in terrestrial animals. Gill tissue, however, represents an anatomically unique and histologically distinct feature of fish. The gross anatomy and histology of fish gills has been well-characterized.^4,6,7 Briefly, teleost fish gills are made up of arciform, osseous structures in the pharyngeal region. These arches support 2 rows of paired filaments, also termed primary lamellae. Each primary lamella is referred to as a hemibranch, and together the paired hemibranches on each gill arch are termed a holobranch. Most teleost fish species have 4 holobranches on each side of the head, though the medial most holobranch is often directly attached to the internal surface of the branchial cavity. Primary lamellae project laterally from gill arches, with folds of secondary lamellae projecting out perpendicularly from the primary lamellae. The secondary lamellae are perfused with blood from lamellar capillaries, which are supplied by afferent filamental arteries and drained by efferent filamental arteries. The secondary lamellae provide the primary interface for gas exchange and regulation of ions, plasma pH, nitrogen excretion, and water to and from the environment.^3,15,18

The lamellar epithelium of the gill is composed of several types of cells. Greater than 90% of the gill epithelium is comprised of a single layer of thin, non-keratinizing, squamous or cuboidal epithelial cells, termed pavement cells (PVCs).¹⁷ These cells cover the vast majority of the lamellar surface. The apical membranes of PVCs have microvilli that increase the surface area of the epithelium to aid in gas exchange, ion uptake, and acid-base transport.⁴ Pavement cells may also help anchor mucus to the surface of the epithelium.⁴ A second major cell type within the lamellae is the pillar cell. Pillar cells are modified endothelial cells that are unique to fish gills. The nucleus of this cell type is polymorphic and centrally located within the cell. The bodies of pillar cells span the lumen, or lacuna, of the secondary lamellae, and the ends of the plasma membrane flare out at the points of contact with the interstitial connective tissue to form thin flanges that extend to neighboring pillar cells.⁷ Pillar cells surround collagen pillars that are anchored to the internal surfaces of epithelial basement membranes. The structure and location of the pillar cells allow free movement of blood within the lamellae by preventing the luminal blood-filled capillaries from collapsing.

Other important cell types within the lamellae include mucous cells, chloride cells, and occasionally mononuclear inflammatory cells and eosinophilic granular cells. Mucous cells are large, ovoid cells that have a flattened nucleus and abundant cytoplasm. Their cytoplasm contains large quantities of mucus secretory granules. Mucous cells are typically sparse in healthy gills, and their location is highly variable. They can be found on the efferent lamellar edge most commonly, but also the afferent lamellar edge, interlamellar space, base of the lamellae, or the outer margin of the lamellae. The mitochondrial-rich chloride cells are large, ovoid cells that are concentrated along afferent edges of filaments and interlamellar spaces, and occasionally on lamellar surfaces.¹⁷ In general, given their high density of mitochondria, chloride cells are thought to supply ATP for ion transport, specifically hydrogen excretion, sodium uptake, and acid-base regulation.³ In saltwater teleost fish, chloride cells have been shown to function in chloride elimination, whereas in freshwater fish they function in chloride uptake.⁶

Gills undergo pathologic changes similar to other tissues, in that edema, inflammation, hyperplasia, and necrosis are common responses to adverse stimuli.^5,12,15 As a result of direct exposure to the external aquatic environment, gills are especially vulnerable to traumatic, toxic, infectious, parasitic, and environmental insults.^{2,11,13,14,16,18} Although gill tissue has a limited repertoire of pathologic responses, documented lesions include: chloride cell hyperplasia, mucous cell hyperplasia, lamellar cell degeneration and necrosis, epithelial lifting, lamellar or filament fusion, lamellar adhesions, and epithelial cell hypertrophy and hyperplasia.¹⁸ Special staining techniques have been used clinically to aid in describing and diagnosing histologic lesions in fish.⁸ However, we are unaware of any literature that characterizes the normal histologic appearance of gill tissue with the more commonly used light microscopic histochemical special stains. Therefore, we documented the normal histologic appearance of freshwater teleost (i.e., bony fish) gill tissue, specifically looking at the primary and secondary lamellae, after processing with a variety of special stains. Although minor differences in the anatomy and location of various structures may exist between fish species, the rainbow trout gill serves as a model for fish in general. Our report can serve as a reference for pathologists, clinicians, and researchers who may use these stains in histologic evaluations of fish gill tissue, and help identify and confirm various gill components.

Materials and methods

Adult (15–25 cm, 240–280 g) rainbow trout (Oncorhynchus mykiss) were obtained from 8 groups of 1–2-y-old fish being raised at a local cold-water hatchery. Two fish from each group were arbitrarily netted, sedated with sodium bicarbonate buffered MS-222 (150 mg/L; Western Chemical, Ferndale, WA), and euthanized by cervical separation. The second gill arch (i.e., counting from the outside inward) from both the left and right branchial areas of each fish were immediately excised by severing its dorsal and ventral cartilaginous attachments, and placed in 10% neutral-buffered formalin. After 48 h in fixative, one gill arch from each group was submitted to the Histopathology Laboratory of the Virginia-Maryland College of Veterinary Medicine (Blacksburg, VA) for processing and hematoxylin and eosin (H&E) staining, whereas the other 3 gill arches from that group were held in reserve. Of the 8 groups of fish, one was judged as representative of normal gill tissue with minimal disease or artifact. Gill tissues from fish in this group were sectioned at 5 µm along a plane that was parallel to the long axis of the filaments, and each section was stained with either routine H&E or 1 of 18 differential stains using standard techniques (Table 1).^1,6 For each of the histologic stains, color digital images were obtained of representative gill structures (BX41 microscope, DP70-BSW camera, Olympus, Center Valley, PA). Images were captured (cellSens software package, Olympus). The resulting images were divided into 2 groups based on the stain’s usage for structural components or infectious etiologies (Table 2).

Table 1.

Special histologic stains and their use.

Stain	Positive controls	Staining target	Counter stain
Structure
Alcian blue	Glycosaminoglycans, glycoproteins	Mucus	Nuclear fast red
Argyrophil stain (Churukian–Schenk)	APUD cells	Adrenal chromaffin cells, pancreatic endocrine cells, enterochromaffin cells, thyroid cell cells, pituitary cells	Nuclear fast red
Fontana–Masson	APUD cells	Neuroendocrine tumors, melanin	Nuclear fast red
Perl Prussian blue iron	Hemosiderophages	Intracytoplasmic iron	Nuclear fast red
Reticulin	Reticular fibers	Collagen type III	Hematoxylin
Toluidine blue	Heparin granules	Mast cells	None
Trichrome	Collagen	Collagen	None
Verhoeff elastic	Elastin	Artery	Van Gieson
Von Kossa	Calcium	Calcium	Nuclear fast red
Infectious
Acid-fast	Mycobacteria	Mycobacteria, cryptosporidia, Isospora, microsporidia	Methylene blue
Fite stain	Atypical acid-fact mycobacteria	Mycobacterium spp.	Methylene blue
Giemsa	Bacteria, myxosporidia	Bacteria, myxosporidia	None
GMS	Polysaccharides	Fungi	Light green
Gram	Bacteria	Positive blue and negative pink	Neutral red
Luna	Microsporidia	Microsporidia	None
Mucicarmine	Mucin	Mucus, Cryptococcus	Metanil yellow
PAS	Glycogen	Glycogen, basement membrane, phospholipids	Hematoxylin
Steiner	Spirochetes	Spirochetes, non-filamentous bacteria	Nuclear fast red

APUD = amine precursor uptake and decarboxylation; GMS = Grocott methenamine silver stain; PAS = periodic acid–Schiff.

Table 2.

Gill staining characteristics of various histologic stains.

Stain	Pavement cell	Chloride cell	Mucous cell	Pillar cell	Chondrocytes
Structure
Alcian blue
Cytoplasm	Pink	Blue	Blue	Pink	Light purple
Nucleus	Red	Red	Inapparent	Red	Red
Argyrophil
Cytoplasm	Yellow-brown	Yellow-brown	Yellow-brown	Yellow-brown	Yellow-brown
Nucleus	Dark brown	Dark brown	Dark brown	Dark brown	Colorless
Fontana –Masson
Cytoplasm	Light pink	Light pink	Light pink	Light pink	Light pink
Nucleus	Red	Red	Red	Red	Red
Perls Prussian blue
Cytoplasm	Pink	Pink	Pink	Pink	Light pink
Nucleus	Blue	Blue	Dark pink	Blue	Pink
Reticulin
Cytoplasm	Pink	Pink	Gray	Pink	Gray
Nucleus	Dark pink	Dark pink	Inapparent	Dark pink	Pink
Toluidine blue
Cytoplasm	Light blue	Light blue	Purple	Light blue	Dark purple
Nucleus	Blue	Blue	Inapparent	Blue	Blue
Trichrome
Cytoplasm	Red-purple	Red-purple	Red-purple	Red-purple	Light blue
Nucleus	Dark blue	Dark blue	Dark blue	Dark blue	Blue
Verhoeff elastic
Cytoplasm	Gray-purple	Gray-purple	Gray-purple	Gray-purple	Pink
Nucleus	Purple	Purple	Purple	Purple	Dark purple
Von Kossa
Cytoplasm	Gray-pink	Gray-pink	Gray-pink	Gray-pink	Pink
Nucleus	Red	Red	Red	Red	Dark pink-to-red
Infectious
Acid-fast
Cytoplasm	Decolorized	Decolorized	Decolorized	Decolorized	Purple
Nucleus	Light blue	Light blue	Light blue	Light blue	Light blue
Fite
Cytoplasm	Decolorized	Decolorized	Decolorized	Decolorized	Dark purple
Nucleus	Blue	Blue	Blue	Blue	Dark purple
Giemsa
Cytoplasm	Light pink	Light blue-gray	Light blue-gray	Light pink	Blue
Nucleus	Blue	Blue	Blue	Blue	Purple
GMS
Cytoplasm	Blue-green	Blue-green	Black	Blue-green	Light brown
Nucleus	Blue-green	Blue-green	Not detected	Blue-green	Blue-green
Gram
Cytoplasm	Colorless	Colorless	Colorless	Colorless	Light red-to-pink
Nucleus	Dark pink-to-red	Dark pink-to-red	Dark pink-to-red	Dark pink-to-red	Dark pink
Luna
Cytoplasm	Light blue	Light blue	Light blue	Light blue	Colorless
Nucleus	Dark blue	Dark blue	Dark blue	Dark blue	Dark blue
Mucicarmine
Cytoplasm	Yellow-brown	Yellow-brown	Yellow-brown	Yellow-brown	Pink
Nucleus	Dark brown	Dark brown	Dark brown	Dark brown	Brown
PAS
Cytoplasm	Blue-green	Blue-green	Dark pink	Blue-green	Pink
Nucleus	Blue-green	Blue-green	Inapparent	Blue-green	Blue-green
Steiner
Cytoplasm	Gold	Gold	Gold	Gold	Gold-to-brown
Nucleus	Gold	Gold	Gold	Gold	Dark gold

GMS = Grocott methenamine silver stain; PAS = periodic acid–Schiff.

Results

Gill tissues stained with H&E (Fig. 1A) were characterized by the following: cytoplasm of the PVCs, chloride cells, pillar cells, and mucous cells stained eosinophilic and had a granular appearance (Fig. 1B). Nuclei of these cells stained darkly basophilic. Collagen matrix stained basophilic, and the chondrocytes had light basophilic cytoplasm with a dark basophilic nucleus. Erythrocytes had homogeneously eosinophilic cytoplasm, and their nuclei were darkly basophilic.

Figure 1.

A. Normal gill tissue of a rainbow trout (Oncorhynchus mykiss). H&E. Original objective 40×. B. Higher magnification depicting pillar cells (P), pavement cells (PVC), mucous cells (M), chloride cells (C), and erythrocytes (E). H&E.

The 9 structural stains had the following staining characteristics for normal gill tissue (Fig. 2).

Figure 2.

Nine histochemical stains for structural components applied to gill tissue of the same rainbow trout depicted in Figure 1. A. Alcian blue; B. Argyrophil (Churukian–Schenk); C. Fontana–Masson; D. Verhoeff elastic; E. Trichrome; F. Reticulin; G. Von Kossa; H. Toluidine blue; and I. Perl Prussian blue iron. Original objective 40×.

Alcian blue stain (Fig. 2A)

PVCs, pillar cells, and erythrocytes stained pink with a red nucleus. Mucous cells had faint blue cytoplasm with an inapparent nucleus. Chloride cells had blue cytoplasm and a red nucleus. Chondrocytes had red nuclei and purple cytoplasm.

Argyrophil stain (Churukian–Schenk; Fig. 2B)

PVCs, chloride cells, pillar cells, and mucous cells stained very light yellow-brown, looking almost decolorized, with dark-brown nuclei. Cytoplasm of erythrocytes was yellow-brown, again almost decolorized, and nuclei were brown. Chondrocytes were yellow-brown to colorless and had colorless nuclei.

Fontana–Masson stain (Fig. 2C)

PVCs, chloride cells, pillar cells, and mucous cells had cytoplasm that stained light pink to red, and nuclei stained red. Erythrocytes, collagen matrix, and chondrocytes stained similarly. Scattered black granules consistent with melanin were noted in some areas of the tissue (Fig. 4D).

Verhoeff elastic stain (Fig. 2D)

PVCs, chloride cells, pillar cells, and mucous cells had gray-purple cytoplasm that had a granular appearance with dark-purple nuclei. Erythrocytes had decolorized cytoplasm with dark-purple nuclei. Collagen matrix stained pink, and the chondrocytes had granular, pink staining cytoplasm and dark purple–stained nuclei.

Trichrome (Gomori one-step stain; Fig. 2E)

PVCs, chloride cells, pillar cells, and mucous cells had red and purple cytoplasm and a granular appearance with dark-blue nuclei. Erythrocytes had dark-pink to red cytoplasm and red-to-purple nuclei. Collagen matrix stained light blue to light gray, and chondrocytes had light-blue to light-gray cytoplasm with blue-to-purple nuclei.

Reticulin stain (Fig. 2F)

PVCs, chloride cells, and pillar cells had light-pink cytoplasm and darker pink nuclei. Mucous cells had gray cytoplasm with a slightly granular appearance, and their nuclei were not discernible. The erythrocytes had gray-pink cytoplasm and pink nuclei. Collagen matrix within the gill filament stained gray-to-black, and chondrocytes had gray-to-colorless cytoplasm with pink nuclei.

Von Kossa stain (Fig. 2G)

PVCs, chloride cells, pillar cells, and mucous cells had gray-pink cytoplasm and red nuclei. Erythrocytes had decolorized cytoplasm and red nuclei. Collagen matrix appeared decolorized or light pink, and chondrocytes appeared pink-to-decolorized with dark-pink to red nuclei.

Toluidine blue stain (Fig. 2H)

PVCs, chloride cells, and pillar cells had light-blue to colorless cytoplasm that appeared granular, and blue nuclei. Mucous cells had purple granular cytoplasm and non-discernible nuclei. Erythrocytes had decolorized cytoplasm and blue nuclei. Collagen matrix stained light blue, and chondrocytes had dark-purple cytoplasm and blue nuclei.

Pearl Prussian blue stain (Fig. 2I)

PVCs, chloride cells, and pillar cells had light-pink cytoplasm and blue nuclei. Mucous cells had pink cytoplasm and darker pink nuclei. Erythrocytes had pale-blue cytoplasm with darker blue nuclei. Collagen matrix stained pink, and chondrocytes were light pink with pink nuclei.

The 9 stains used to detect infectious agents had the following staining characteristics for normal gill tissue (Fig. 3).

Figure 3.

Nine histochemical stains for infectious agents applied to gill tissue of the same rainbow trout depicted in Figure 1. A. Acid fast; B. Fite; C. GMS; D. Mucicarmine; E. Gram; F. Steiner; G. Giemsa; H. PAS; and I. Luna. Original objective 40×.

Acid-fast bacteria stain (Fig. 3A)

Pavement cells, chloride cells, mucous cells, and pillar cells were all decolorized when stained. Erythrocytes were also decolorized to light blue with blue nuclei. Collagen matrix stained light blue, and chondrocytes stained purple with a dark-blue nucleus.

Fite stain (Fig. 3B)

PVCs, chloride cells, pillar cells, and mucous cells had cytoplasm that decolorized, and nuclei stained blue. Erythrocytes stained similarly, although some of the nuclei stained pink-to-purple as well. Collagen matrix stained light blue, and chondrocytes stained dark purple with dark-purple nuclei. Eosinophilic granular cells (EGCs) were accentuated, and their granules stained light blue.

Grocott methenamine silver stain (GMS;Fig. 3C)

PVCs, chloride cells, pillar cells, erythrocytes, collagen matrix, and chondrocytes all stained blue-green, with their nuclei staining slightly darker than the cytoplasm. Mucous cells contained dark-gray to black granules within the cytoplasm; nuclei were indiscernible (Fig. 4B). There was a slight brown hue to chondrocyte cytoplasm.

Mucicarmine stain (Fig. 3D)

Cytoplasm of the PVCs, chloride cells, pillar cells, and mucous cells stained yellow-brown and had a granular appearance; nuclei stained dark brown. Erythrocyte cytoplasm stained homogeneously yellow with light- to dark-brown nuclei. Collagen matrix stained yellow, and chondrocytes had pink cytoplasm and brown nuclei.

Gram stain (modified Brown and Brenn;Fig. 3E)

Cytoplasm of PVCs, chloride cells, pillar cells, mucous cells, and erythrocytes was colorless, and nuclei were dark pink to red. EGCs had dark-pink cytoplasmic granules. Collagen matrix stained light pink, and chondrocytes had light-red to pink cytoplasm and dark-pink nuclei.

Steiner stain (microwave method; Fig. 3F)

Lamellar cells were a homogeneous gold color (both cytoplasm and nuclei), with the nuclei staining slightly darker. Erythrocytes stained similarly. Collagen matrix stained yellow-gold, and chondrocytes had gold-to-brown cytoplasm, which had a granular appearance, and darker gold nuclei. A few blue-black granules were scattered throughout portions of the gill section, perhaps depicting melanin because they were negative for iron.

Giemsa stain (Fig. 3G)

PVCs and pillar cells had very light-pink cytoplasm and blue nuclei. Chloride cells and mucous cells had light-blue to gray cytoplasm and blue nuclei. Erythrocytes had dark-pink cytoplasm and blue-to-purple nuclei. Collagen matrix stained pink, chondrocyte cytoplasm stained blue, and their nuclei stained darker purple. EGCs were accentuated with pink granules and blue-to-purple nuclei (Fig. 4C).

Figure 4.

Four histochemical stains for specific cell types of gill tissue of the same rainbow trout depicted in Figure 1. A. Mucous cell (M) with PAS; B. Mucous cell (M) with GMS; C. Eosinophilic granular cell (EGC), chloride cell (C), pavement cell (PVC), and pillar cell (P) with Giemsa; D. Melanin (m) granules with Fontana–Masson. Original objective 100×.

Periodic acid–Schiff (PAS) stain (Fig. 3H)

PVCs, chloride cells, and pillar cells had blue-green cytoplasm and nuclei. Cytoplasm of the mucous cells was dark pink (magenta) and had a granular appearance; the nucleus was indiscernible (Fig. 4A). Erythrocytes had blue-green cytoplasm and nuclei. Collagen matrix stained green, chondrocyte cytoplasm stained pink with a granular appearance, and their nuclei stained dark blue-green.

Luna stain (Fig. 3I)

The cytoplasm of the PVCs, chloride cells, pillar cells, and mucous cells stained colorless to light blue with dark-blue nuclei. Erythrocytes stained light to dark blue with dark-blue nuclei. Collagen matrix stained colorless to light blue, and chondrocytes had dark-blue nuclei in colorless matrix.

Discussion

H&E staining is probably the staining technique used most commonly in routine histologic investigations. It is a basophilic and acidophilic stain. Hematoxylin (a basic dye) is a positively charged combination of hematin dye and aluminum salts. Aluminum salts bind to the tissue, and hematin dye, in turn, binds to the salts causing certain parts of the cell (e.g., the nucleus and parts of the cytoplasm containing RNA) to stain purple (i.e., basophilic). Eosin (an acidic dye) is negatively charged and stains basic cellular structures, such as the cytoplasm, red or pink (i.e., eosinophilic). In general, light microscopists are familiar with H&E staining of tissues; therefore, H&E was used as a standard for comparison for the other histologic stains. There were no unexpected findings with H&E.

Because pathologists and researchers may not be as familiar with gill tissue as with mammalian lungs, we applied 18 light microscopic stains to gill tissue in addition to routine H&E. Such an examination can confirm normal staining characteristics of the tissue, differentiate cell types, and perhaps uncover the presence of expected or unexpected components of the tissue. Because the gills in our study were initially evaluated to be normal, the use of stains for infectious organisms was not expected to identify such agents, and despite applying 18 light microscopic special stains to the gill tissue, unexpected findings were not identified.

We did not demineralize gills in our study because they were soft enough to be sectioned without artifact. However, sometimes gill tissue from larger or older fish may need to be demineralized with EDTA or other substances (i.e., weak organic acid) to allow sectioning without histologic artifact or prevent damage to microtome knives. Alternatively, the cartilaginous supporting base of the gill can be trimmed from the gill tissue prior to embedding. Demineralization does not generally change the staining characteristics of gill tissues except for mineralized material such as the gill arch and supporting cartilaginous structures, which will appear eosinophilic and basophilic, respectively.

For the structural stains, alcian blue stains are used to demonstrate acid glycosaminoglycans and sialomucins (glycoproteins) in tissues. At pH 2.5, both sulfated and carboxylated acid glycosaminoglycans and glycoproteins are stained blue.¹ If the pH is reduced to 1.0, the technique becomes specific for sulfated acid glycosaminoglycans and glycoproteins, and compounds that are only carboxylated will not stain blue.¹ In both cases, background tissues are stained pink-to-red using a nuclear fast red counterstain technique. Alcian blue staining can be combined with hyaluronidase treatments to eliminate staining of connective tissues such as hyaluronic acid, chondroitin sulfate A, or chondroitin sulfate C, while preserving stain uptake in glycoproteins. Alcian blue techniques may also be combined with PAS and hematoxylin staining to differentiate acidic and neutral polysaccharides, in which case, acidic mucosubstances would stain blue with alcian blue, neutral mucins would stain magenta with the PAS reaction, and some substances would react with both techniques and stain purple. As might be expected, the mucous cells and the chondroid elements of gill tissue were highlighted by this stain.

Churukian–Schenk argyrophil staining is used to demonstrate certain cells that can absorb silver but require an external reducing agent to produce a visible metallic form.¹ This is in contrast to argentaffin cells, which can both absorb and reduce silver without the requirement for an external reducing agent.¹ Typically these stains are used to demonstrate amine precursor uptake and decarboxylation (APUD) cells, including adrenal chromaffin cells, pancreatic endocrine cells, enterochromaffin cells in the gastrointestinal tract, thyroid “C” cells in mammals, and some pituitary cells. In addition to demonstrating these neuroendocrine cells, argyrophil stains have also been used to detect neuroendocrine (carcinoid) tumors, which produce secretory cytoplasmic granules that result in diffuse cytoplasmic argyrophilia. The Churukian–Schenk technique involves incubating slides in silver solution followed by a reducing solution, and counterstaining with nuclear fast red. Argentaffin and argyrophil granules stain dark brown to black, cell nuclei stain red, and background tissues stain pale yellow-brown. As would be expected, this stain did not specifically detect such cells in the gill tissue.

Fontana–Masson stain is an argentaffin staining technique.¹ Argentaffin substances absorb silver in solution and do not require an external reducing agent to convert the silver to a visible metallic form. Argentaffin substances include melanin granules, cytoplasmic granules of carcinoid tumors, and neurosecretory granules in APUD cells. The reaction occurs in a silver nitrate solution, and toning with gold chloride removes the yellow background before counterstaining with nuclear fast red. Melanin and argentaffin substances stain black, nuclei stain pink, and background tissues stain light pink. The black granules noted in the gill tissue are consistent with melanin.

Perl Prussian blue stain is utilized to detect processed ferric iron, which is stained bright blue.¹ The counterstain is nuclear fast red. In the normal gill, where there was no detectable iron, the stain revealed minimal light-blue components against a light-pink background. This stain may be useful in environmental or toxicologic studies in which environmental contaminants are being evaluated.^2,11,13,14

Reticulin stain is generally used to demonstrate reticular fibers, specifically collagen type III fibers, which are found primarily in liver, lymph nodes, spleen, and kidneys.¹ The counterstain is hematoxylin. The main function of these fibers is to provide support. Reticular fibers will appear black and cell nuclei will appear red when stained using this technique, and are demonstrated as fine fibers in the matrix of the gill filament.

Toluidine blue is a basic thiazine metachromatic dye with high affinity for acidic tissue components.¹ Alkaline solutions of toluidine blue are commonly used for staining semi-thin (0.5–1 μm) sections of resin-embedded tissue. At high pH (~10), the dye binds to nucleic acids and all proteins. Like the Giemsa stain, toluidine blue is often used to detect heparin in the cytoplasmic granules of mammalian mast cells. It may also be used to stain proteoglycans and glycosaminoglycans in cartilage. The strongly acidic macromolecular carbohydrates of cartilage will appear blue or metachromatic when stained with toluidine blue, as demonstrated by cartilage accentuation.

Gomori one-step trichrome stain is used to differentiate connective tissue, muscle, and collagen fibers.¹ Generally, trichrome stains consist of nuclear, collagenous, and cytoplasmic dyes in mordants such as phosphotungstic or phosphomolybdic acid. The staining procedure is a one-step system that combines the cytoplasmic and connective-fiber stain in a phosphotungstic acid–acetic acid solution. Tissue sections are treated with Bouin solution to intensify the final coloration. Nuclei are then stained with Weigert iron hematoxylin, followed by chromotrope 2R (stains cytoplasm and muscle fibers) and fast green FCF or aniline blue (counterstain that highlights collagen fibers). Once staining is complete, nuclei will appear dark blue to black, cytoplasm and muscle fibers will appear red, and collagen will appear blue. The gill arch collagen is light blue to gray.

Verhoeff elastic stain is useful for demonstrating atrophy of elastic tissue fibers.¹ Tissue samples are stained with a regressive hematoxylin that contains iodine and excess ferric chloride. Differentiation is accomplished as the excess ferric chloride breaks down the tissue–mordant dye complex. The dye will be attracted to the excess ferric chloride in the differentiating solution, and will be removed from the tissue. Elastic tissue has the strongest affinity for the iron–hematoxylin complex and will retain the dye longer than other tissue elements. Elastic fibers and nuclei of the tissue sample will stain black, collagen will stain red, as in the gill tissue, and other background tissue elements will stain yellow following Van Gieson counterstain.

Von Kossa stain is used to detect calcium salt deposits in tissue.¹ The staining technique involves treating tissue sections with silver nitrate solution. The calcium is reduced by strong light and is replaced with silver deposits, which is visualized as metallic silver. Histologically, calcium salts will appear black. Cell nuclei will take on a red color, and cytoplasm will appear pink. No calcium salts were detected in gill tissue.

For the infectious stains, acid-fast staining operates on the principle that the capsule of acid-fast organisms will take up carbol-fuchsin stain and resist acid decolorization.^1,6 The section is counterstained with methylene blue. Organisms with a waxy lipoid capsule will stain bright red with carbol-fuchsin. Other organisms and tissues will decolorize with acid–alcohol, as in the case of the gill, and will stain light blue with methylene blue counterstain. Acid-fast techniques are useful for the demonstration of mycobacteria and nocardia in fish, and can also demonstrate Cryptosporidium spp., Isospora spp., and microsporidian spores. This stain does not demonstrate gill architecture particularly well.

Similarly, Fite stain is a technique used to demonstrate acid-fast organisms.^1,6 It is useful for the demonstration of atypical non-tubercular Mycobacterium spp., particularly Mycobacterium leprae or other aquatic mycobacteria. M. leprae appears to resist uptake of stain after deparaffinization with conventional xylene techniques. In the Fite stain, a xylene–peanut oil solution is used to remove paraffin, and the acid-fast staining characteristic of the M. leprae organism is maintained. This technique is considered inferior for the demonstration of other typical Mycobacterium spp. As with other acid-fast techniques, acid-fast organisms will stain red with carbol-fuchsin; the background will decolorize with acid–alcohol and counterstain light blue with methylene blue. As expected, specificity of the Fite stain was not demonstrated in the gill tissue, except for the EGCs.

Giemsa staining procedures are Romanowsky-type techniques combining acidic and basic dyes (i.e., eosin and methylene blue, respectively) to produce a wide range of colors in tissues and blood smears.^1,6 These are “all-purpose” stains used routinely for assessment of nuclear and cytoplasmic features, and a modified version (i.e., Diff-Quick) is commonly used by clinicians in practice. Nuclei of cells and bacterial organisms are stained blue. Cytoplasm is stained variably pink, gray, or blue depending on the cell type, and this variability of staining is demonstrated well throughout the gill tissue. Because Giemsa stain can highlight myxozoans or amoebae, it is often utilized in evaluation of diseased fish to help identify such organisms in tissue sections; polar capsules of myxozoan spore stages stain basophilic. Eosinophilic granular cells were accentuated by the stain in our trout gills, where these cells had pink cytoplasm.

The Grocott methenamine–silver nitrate (GMS) stain is used for the demonstration of fungal organisms.^1,6 An oxidation step is performed with chromic acid, after which only substances with high concentrations of polysaccharides remain reactive with the methenamine–silver solution. Fungal cell walls are rich in polysaccharides, and thus react with methenamine–silver to form metallic silver even after the oxidation step. A light-green solution is used for counterstaining. Fungi stain black, mucin stains dark gray or taupe, and background tissues stain light green. The latter 2 stain characteristics were demonstrated well by mucous cells and connective tissue of the gill. GMS stain will demonstrate infection of the gill and/or skin with the common opportunistic pathogens Saprolegnia spp. or Aphanomyces spp.

Gram staining techniques are well-characterized and widely used for the differentiation of gram-negative or -positive bacterial organisms.^1,6 Crystal violet and iodine are applied sequentially, followed by decolorization with acetone. This decolorization step disrupts the lipopolysaccharide layer exterior to the cell wall in gram-negative organisms, allowing the crystal violet–iodine complex to escape. The intact peptidoglycan layer in gram-positive bacteria prevents crystal violet–iodine complexes from escaping. A fuchsin dye such as neutral red is used to counterstain gram-negative organisms. The result is blue staining of gram-positive organisms and red staining of gram-negative organisms. Background tissues will generally be yellow, and nuclei will generally stain light red, as in the gill tissue. Several significant bacterial gill diseases are caused by gram-negative bacteria such as Aeromonas spp. and Flavobacterium spp.; gram-positive gill pathogens include Mycobacterium spp. and Nocardia spp.

Luna stain has primarily been used for identifying elastic fibers, mast cells, and eosinophils in mammals, and has been promoted as a selective stain for the detection of microsporidia in zebrafish.^1,6,10 Background tissue stains a uniform light blue; elastic fibers, mast cells, eosinophils, and microsporidian spores stain brick red.¹⁰ Other fish tissues that stain red include erythrocytes, eosinophilic granular cells, bone, and the lens of the eye.

Mucicarmine stain is primarily used to stain mucin, a substance produced by epithelial and connective tissue cells.^1,6 Excess mucin is often secreted by epithelial cells in certain types of inflammatory processes. The rose-red dye in mucicarmine staining solution gets its color from the aluminum–carminic acid complex known as carmine. The binding of these 2 chemicals creates a positive charge that can attract and bind to acid mucins in tissue. Mucin usually stains a deep rose, cell nuclei stain black, and other tissue elements stain yellow. Gill arch cartilage was enhanced by this stain. The stain is useful for staining encapsulated fungi.⁹

PAS stain is often used to demonstrate glycogen.^1,6 Tissue sections are oxidized by periodic acid, which results in formation of aldehyde groupings. The aldehyde groups are then detected using the Schiff reagent. Once the reagent is added, a colorless and unstable dialdehyde compound forms and then is transformed to the final colored product by restoration of the quinoid chromophoric grouping. Hematoxylin is the counterstain. Glycogen, neutral mucosubstances, basement membranes, collagen fibers, glycolipids, and phospholipids stain pink, red, or purple. Mucous cells were specifically well demonstrated in gill tissue with this stain.⁸

Steiner stain is a silver staining solution that is widely used to detect spirochetes and other non-filamentous bacteria.^1,6 The bacteria absorb the silver from the solution with the aid of a reducing agent. Once the reducing agent is added, the silver is transformed to a visible metallic state. Spirochetes, Flavobacterium spp., and other non-filamentous bacteria stain dark brown to black; everything else stains bright yellow to light brown. The counterstain is nuclear fast red. This stain did not highlight any specific components of gill tissue.

Given that many pathogenic organisms can infect gill tissue because of its interface with water and role in oxygenation, stains for infectious agents can be useful. Fish are susceptible to infection by Mycobacteria spp., hence acid-fast and Fite stains for typical and atypical mycobacterial species are useful in diagnostic fish practice. Giemsa stains can be utilized to identify bacteria and protozoan parasites in section, and the tissue Gram stain helps distinguish gram-negative from gram-positive organisms. Luna stain has been described for detection of microsporidian organisms. Steiner stain is utilized for unique bacterial organisms. PAS, GMS, and mucicarmine stains can all be utilized in the identification of fungi, which can be primary or secondary pathogens of the gill tissues of fish.

Given that gill filaments contain connective tissue and cartilage, the staining characteristics of Masson trichrome demonstrating blue collagen against a background of red tissues is not unexpected. Increased connective tissue in gills using a similar stain, picrosirius red, has been demonstrated.⁸ Additionally, the presence of minimal reticulin fibers in the gill arch is not unexpected, and similarly, iron deposits were not expected with Perl Prussian blue staining. Other than in blood vessels, elastin was not present in the gill arch. Despite seeing some granular pigment, calcium salts were not identified by Von Kossa staining. Von Kossa staining has been reported to demonstrate gill chloride cells, although not prominent in our study.⁸ Toluidine blue staining is principally used in mammals to identify mast cells, a type of cell yet undescribed in fish. Additionally, given that there are no known resident APUD cells in gill tissue, it was not unexpected that Fontana Masson and argyrophil stains were negative.

As would be expected, many structures in fish have similar staining characteristics with structural stains used more commonly in mammals. The Fontana–Masson, trichrome, Verhoeff elastin, alcian blue, and reticulin stains may have the most relevance for examining melanocytes, connective tissue, arteries, cartilage, and reticular fibers of hepatic sinusoids, respectively, more closely in fish. Some of the most useful stains for identifying common pathogens of fish gills include the Fite acid-fast stain for atypical mycobacteria. Additionally, PAS or GMS stains will highlight fungal hyphae such as Saprolegnia in histologic section; Luna stain will accentuate microsporidia; Giemsa stain can often identify bacterial colonization. The Gram stain is important to then further classify pathogenic bacteria as either gram-negative or -positive, along with their shape, when culture is not available.

Knowing the expected staining characteristics of fish gill tissue is important for diagnosticians and researchers alike. We have provided a comprehensive assessment of 18 stains for structural components and infectious agents in addition to routine H&E of normal gill tissue from the rainbow trout. We hope this report can act as a reference and illustrated guide for appropriate fish gill staining and structural abnormality and pathogen detection.