pigment, lipids, and other substances in the glands of the external auditory meatus of man

PIGAIENT, LIPIDS, AND OTHER SUBSTANCES IN THE GLANDS O F T H E EXTERNAL

AUDITORY MEATUS O F MAN

WILLIA4M MOSTAGSA, CHARLES R. NOBACK AND FREDERICK G. ZAK

Long Island College of Medicine, Department of Anatonzy, Brooklyn, X c t u Pork, and Laboratories, Division of Patholo,qy, 17;loitnt Binai Hospital, New Pork

Ar~io ld Biologzral Laboratory, Rrown Universi ty , Providence, m o d e Is land,

ELEVEN FIGURES

INTRODUCTION

Cerumen, or ear wax, is a poorly understood pigmented, lipoidal and protein-containing mixture which is secreted by the glands of the external auditory meatus. Two types of glands are present in tlie cartilaginous portion of the cx- ternal auditory meatus : (1) sebaceous glands, located superficially under the epidermis and (2) apocrine ceruminous glands, lying deeper in the connective tissue. Little is known about either of these glands. Furthermore, practically nothing is known about the nature of the pigmented substance secreted by the ceruminous glands.

TTe haw studied these glands by the application of some cytocliemical methods. Evidence will be presented that the lipid substances of cerumen are secreted by the sebaceous as well as by the ceruminous glands. The pigment which is secreted by the ceruminous (sells has been studied in situ and in the residual cerumen in the external auditory meatus. It will be shown that the pigment appears to resemble ceroid, a pigment usually associated with espeTirnenta1 nutritional cirrhosis in the liver, but that it is not ccroid (Pappenlicimer and Victor, '46).

409

410 W. A'lONTAGNA, C. K. NOBACK A S D F. G. ZAIi

MATERIAL AND METHODS

The materials for this investigation were obtained at autopsy, 10 hours 01- more having elapsed betweeii death and iiecropsy. Tissues were obtained from 15 female and 8 nialc Caucasian subjects whose ages ranged from 14 months to 64 years; and one Negro female and one Japanese male. Since the qualitative studies of this paper reveal no appreciable differences iii sex, age, or race, 110 reference will be made to these.

For general histological observations paraffin sections, 3 p in thickness, of tissues fixed in 10% neutral formalin, were stained in heniatoxylin axid eosin, in iron hematoxylin, and in Ifasson's triacid stain. Cytoplasmic basophilia was studied by staining sectioiis of niatcrials fixed in 10% formalin with a 0.02% solution of toluidin blue or with eosin-methyleiie blue. Control sections were incubated, before staining, in a 0.1% solution of ribonuclease buffered to pH 6.7. For tliymonucleic acid, deparaffiiiized sections were hydrolyzed in normal HC1 and then treated with the Schiff reagent. Goniori's ( '37) am- inoiiiacal silver nitrate method was used to blacken the pigment granules.

Glycogen was studied by applying the periodic acid method of Hotchkiss ('48) to tissues fixed in acetone, since tissues fixed in picric-alcohol-form01 were not available.

Lipids were studied in tissues fixed in 10% neutral formalin or in Raker's ( '44) formal-calcium and formal-calcium- cadmium. Frozen sections, 10 p in thickness, were stained with Sudan IV, Sudan black, and other oil-soluble dyes (Lillie, '48), and with Nile-blue sulfate. Plastnals were revealed by treating frozen sections hydrolyzed with mercuric chloride with the Schiff reagent. For the study of cholesterol, sections were treated with the Liebermann-Burchard test ; other sections were treated with digitonin and subsequently with the Liebermann-Burchard reaction after the modification of Everett ('47). For the identification of fatty acids, frozen sections were placed in a 1.5% solution of calcium salicylate in 10% formalin and subsequently stained with the Fischler

GLAKDS O F EXTERXAL AUDITORY MEATUS 411

technique as modified by Mallory ( '38) and Lillie ( '48). Baker's ( '46, '47) acid haematein and pyridine extraction tests for phospholipids were applied to tissues fixed in formal- calcium. Unstained frozen sections were observed under polarized light f o r birefringence, and under near-ultraviolet light for autofluorescence.

In the ceruminous glands we have observed the reaction of the above mentioned tests with pigment granules. We further studied the character of the pigment by treating frozen sections with acetone, alcohol, ether, carbon disulfide, alcoholic and aqueous potassium hydroxide, sodium hydroxide, and 3% hydrogen peroxide. To establish whether the pigment is a carotinoid or a chromolipoid, frozen sections were treated with strong sulfuric acid or with an iodine-potassium iodide solution (Lison, '36). Paraffin sections were stained with carbol fuchsin and differentiated in acid-alcohol.

Cerumen removed from patients with impacted cerumen plugs was extracted with ether-alcohol. The extract was dried over a steam bath. The dried solute was then re-extracted with ether, and the ether-soluble substance was tested with the Liebermann-Burchard test for cholesterol. The original insoluble residue was further washed with hot alcohol, dried, and placed in warm distilled water to dissolve the pigment. The aqueous solution was filtered and dried over a steam bath. The solubility and fluorescence of the dry pigment were studied.

Phosphatases were studied in tissues fixed in acetone, fol- lowing the methods of Gomori ('41a, '41b) and Wolf et al. ('43). Tipase was demonstrated by the method of Gomori ( '46).

THE GLANDS O F THE EXTERNAL AU1)ITORY MEATUS

The adult external auditory meatus of man is approximately 26 mm in length (Symington, 18%). The outer region has a framework of elastic cartilage, which is continuous with that of the pinna. A shallow area near the tympanum has a bony

412 W. MONTAGNA, C. R. NOBACK AND F. G . ZAK

framework. Auricular glands are found in the cartilaginous tube while the bony portion is usually aglandular.

Beneath the epithelium of the concha above the tragus and antitragus, clusters of large sebaceous glands open onto the surface either directly, or indirectly through hair follicles by dilated orifices visible with the naked eye. These glands are especially large at the level of the tragus and the orifice of the meatus. Toward the inner portion of the cartilaginous meatus the sebaceous glands become gradually smaller and finally disappear in the innermost portion of the tube. I n the concha, at the level of the tragus, numerous coiled merocrine sudoriparous glands are found beneath the sebaceous glands. At the orifice of the meatus the coiled tubules are replaced by the large alveolar, apocrine, ceruminous glands. These glands are large throughout the cartilaginous meatus and are particularly abundant in the roof of the tube. All of the glandular elements are embedded in a dense fibrous tissue which contains fine elastic fibers as described by Kolmer ( '27).

The sebaceous glands form a superficial glandular bed just beneath the epithelium of the external auditory meatus (fig. 1). These glands rarely reach the deeper layers of the connective tissue but they are not restricted to the level of the outer one-third of hair follicles. The apocrine ceruminous glands are found in the deeper connective tissue layer, often resting upon the perichondrium of the cartilaginous tube (fig. 1). Their long ducts usually open directly onto the surface of the tube but occasionally they open into sebaceous ducts in agreement with Simonetta and Magnoni ('37). The ceruminous ducts, especially in older individuals, usually become dilated into spherical utriculi near the surface of the auditory canal. These utriculi may be 1 or 2 mm in diameter and contain concentrically stratified keratinized concretions. These dilatations are more abundant in the innermost portion of the cartilaginous tube where the sebaceous glands are either very small or absent. Occasionally similar utriculi are found in the ducts of the sebaceous glands.

GLANDS O F EXTERNAL AUDITORY MEATUS 413

The ceruminous glands are found in aggregates of 6 or more. The component tubules of each glandular cluster usually have the same appearance. The lumina may be narrow in the glands of some clusters and very broad in those of others.

The epithelium of the ceruminous glands resembles that of apocrine tubules elsewhere in the body. The epithelial cells are tall columnar (20 to 25 p) in those tubules which possess a comparatively narrow lumen, while in the distended tubules they are flat (2 to 5 p ) . The spherical nucleus of ceruminous cells usually rests at the base of the cell, and contains one or more large nucleoli. Nearly all the cells, whether they are tall or flattened, contain, supranuclearly, variable amounts of brownish-yellow pigment granules. Pigment is usually abundant in the tall columnar cells, but occasionally even these cells may contain small amounts of it. Cells which apparently are in the process of secretion protrude into the lumen as conical or spherical blebs (fig. 3). These blebs often contain pigment granules and lipid droplets.

The ceruminous cells rest on a layer of myoepithelial cells, resembling the situation described in the sudoriparous glands by Bunting et al. ( '48). These cells lie upon a thick, hyalin basement membrane whose surrounding loose connective tissue is richly vascularized.

DISTRIBUTION O F L I P I D SUBSTANCES

Suclaw, IV, SudaB black, a,md other oil-soluble dyes. Frozen sections were stained with Sudan IV, Sudan black, and a number of oil-soluble dyes (for techniques see Lillie ('48). Sudan black, oil-red 0 and oil-blue N1 gave the most clear-cut results.

The stratified squamous epithelium which lines the surface of the external auditory meatus shows an interesting distribution of lipid droplets. The stratum corneum usually appears to be completely infiltrated with lipids. The basal cells of the

The oil-soluble dyes were received from National Aniline Division, Allied Chemical and Dye Corporation.

414 W. MONTAGNA, C. It. PTOBACK AND F. G . ZAH

stratuiii germinativuni show many small dust-like lipid droplets, which are larger and more abundant in the cells of the outer root-sheath of hair follicles.

Often, solid epithelial buds o r cords are found proliferating from the surface epithelium and from the outer root-sheath of hair follicles and growing into the surrounding connective tissue. These cell-masses are interesting principally because their component cells enclose lipid droplets whose distribution suggests that of the sebaceous glands. The small peripheral cells of the epithelial proliferations contain only small lipid granules, while progressively toward the center the granules become larger. The central cells contain large droplets like typical sebaceous cells. TJ7e are not certain if these structures represent “de novo” formation of sebaceous glands from the surface epithelium and from the outer root-sheath.

The distribution of lipids in the sebaceous glands (fig. 6) is essentially similar to that already described in sebaceous glands of other locations in the rat and dog (Montagna and Noback, ’47; Montagna and Parks, ’48). All of the cells in mature sebaceous acini are laden with lipid droplets; in less mature acini the peripheral cells contain small lipid particles.

Near their orifices the ducts of some sebaceous and ceruminous glands form utriculi which resemble those described by David (’32) and Crew and Mirskaia (’31) in the skin of hairless mice. The concentrically stratified concretions which are contained in these dilatations stain deeply with Sudan dyes and oil-soluble dyes (fig. 2).

Varying amounts of sudanophilic droplets are found in the ceruminous cells (figs. 4 and 5). The cells of the actively secretory tubules contain coarse sudanophilic droplets in the distal portion and many delicate granules near the nucleus (cf. Bunting et al., ’48, f o r the apocrine sudoriparous glands). These cells often contain so many lipid globules that in sections where the lumen of the tubules is not shown, they resemble sebaceous cells. In some apparently active tubules the columnar cells show only fine particles, such as are found in the flat epithelium of tlic dilated tubules. When sections are

GLANDS OF EXTERNAL AUDITORY MEATUS 415

immersed in acetone or alcohol and subsequently treated with Sudan black, the larger lipid droplets are dissolved but the delicate granules around the nucleus still stain clearly black.

Although the pigment granules in the ceruminous cells stain intensely with oil-soluble dyes, they are dyed most clearly with Sudan black. Immersion of sections in lipid solvents or inclusion in paraffin does not abolish their sudanophilia.

Nile-bZue sulfate. The use of Nile-blue sulfate (Smith, '08, '11) for the selective demonstration of neutral fats and fatty acids has been severely criticized by Kaufmann and Lehmann ( '26). Controversy exists concerning its cytochemical validity. Lison ('36) states that a rose color-reaction indicates the presence of triglycerides and a blue color-reaction signifies nothing of a specific nature. Cain ('47) in an excellent com- prehensive reinvestigation of the potentialities of Nile-blue sulfate has confirmed some of Smith's early findings. Nile blue is an oxazine dye. In aqueous solutions spontaneous oxidation of ovaziiie forms small amounts of a pink dye, oxazone, which stains fatty substances indiscriminately like oil-soluble dyes, while the blue oxazine combines with fatty acids to form blue ' ' soap-like compounds ' ' with them (Smith, '11). Thus the pink coloration imparted to neutral fats by oxazone can be considered specific only after the action of the blue oxazine which renders fatty acids and other lipids blue (for details see Smith, '11, and Cain, '47). Since substances other than lipids may also stain blue, interpretation of materials stained blue by oxazine must be made cautiously by studying comparable sections treated with Sudan black.

In frozen sections treated with Nile blue the stratum corneum of the surface epithelium of the auditory tube is bright pink, as are masses of residual cerumen which adhere to the surface. In the solid epithelial buds described above the larger lipid droplets in the central cells are clearly pink, while the smaller sudanophilic globules in the peripheral cells stain blue. In the sebaceous glands, the sebum and all of the large sebaceous cells are full of pink droplets. In highly active glands where the peripheral acinar cells are large and lipid-

416 W. MONTAGNA, C. R. NOBACK AND F. G. ZAK

engorged, the lipid droplets stain pink. I n less mature glandular acini where peripheral cells are smaller, the delicate lipid particles which they contain stain principally blue, perhaps indicating the presence of fatty acids in agreement with Melczer and Deme ( '42). The concretions contained in the ductular utriculi stain uniformly pink.

In the cells of the ceruminous glands there is a close corre- lation between the amount of sudanophilic droplets and the abundance of pink-staining globules. When sudanophilic droplets are abundant in cells, most of them stain pink, but others stain deep blue. I n ceruminous tubules where lipids are less abundant, most droplets stain blue. It appears, then, that at least some of the lipids secreted by the ceruminous glands contain triglycerides, and that perhaps the blue- staining droplets contain fatty acids.

Fischler Wtethod f o r fa t ty acids. Since this method, even as modified by Mallory ( '38) and Lillie ( '45), has not yet been submitted to a critical test, it would be unwise to claim its specificity f o r fatty acids. The results here presented, there- fore, may be of questionable value.

In the sebaceous glands, the new scbum and the degenerating sebaceous cells show a positive blue-black reaction while the old sebum remains nearly colorless. In the sebaceous cells, the large sudanophilic globules are colorless, but they often show a blue-black film along their peripheries (compare with phospholipids). The black particles do not always correspond with the lipid particles which stain blue with Nile blue. All of the sebaceous cells contain small black particles scattered in the cytoplasm. They are not particularly abundant at the periphery of the acini where Melczer and Deme ( '42) report the presence of fatty acids (compare with Nile-blue sulfate).

When sections fixed in f ormalin containing calcium salicylate are immersed in dilute hydrochloric acid or in ether- alcohol previous to treatment with Fiscliler test, the black particles are still present. In sections immersed in ether- alcohol there is a diminution of the black lipid particles, but in

GLANDS O F EXTERNAL AUDITORY M E A T U S 417

sections immersed in a mixture of ether-alcohol and hydrochloric acid, the color reaction is absent.

Baker's acid haernatein test f o r phospholipids. Baker ( ,46, '47) and Cain ('47) give evidence that the acid haematein test reveals phospholipids in tissues.

In sections treated with Baker's method, positive black or blue particles are found in the cytoplasm of sebaceous cells, while the sudanophilic lipid droplets remain colorless. The distribution of these positive elements in the acinar cells appears to correspond to mitochondria, as described by Nicolas at al. ('14a), and to both mitochondria and Golgi bodies, as interpreted by Ludford ( ' 2 5 ) . I n the peripheral acinar cells, which contain very small sudanophilic particles, acid haematein reveals small black dots around the nucleus and in the supranuclear cytoplasm, resembling Bowen's ( '29) figures of the Golgi element. In the more centrally located cells, the black cytoplasmic particles are more abundant and are in the form of granules and rodlets. While the large lipid droplets are not stained by acid haematein, they are always surrounded by a delicate black ring (cf. Ludford, '25, and Bowen, '29, for the Golgi bodies in sebaceous cells). In the mature cells which are about to undergo sebaceous degeneration, and in the degenerating sebaceous cells in the center of the acinus, the strands of cytoplasm which remain between the enlarged lipid droplets are completely blackened. In addition, this net- work of cytoplasm contains coarse, black granules and rodlets (fig. 7). The sebum in the center of the gland contains some black-staining substances which diminish in staining reaction toward the exit of the sebaceous ducts.

In the ceruminous glands, positive black rounded particles and rodlets are found especially in those active columnar cells which contain lipid droplets (fig. 8). These particles and rodlets have no definite orientation, their distribution being similar to that of the mitochondria in sudoriparous glands described by Nicolas et al. ( '34b).


None of the material described is colored with acid haematein in sections ~ h i c h have been treated with Baker's pyridine- extraction test. This implies that they contain phospholipids.

Liebermam-Burchard t e s t for cholesterol. When dried ether extracts of whole cerumen are dissolved in chloroform and acetic anhydride and concentrated sulfuric acid are added to the cliloroform solution, the solution beconies reddish, then blue, and finally bluish green in color. This is a specific color test for cholesterol and cholesteryl esters.

When modifications of this test (Kay and Whitehead, '37; Everett, '47) are applied to frozen sections of the external auditory tube, a positive green color reaction appears in the sebaceous glands. The green color develops in the sebum and in the degenerating sebaceous cells, while in the other large sebaceous cells only a pale bluish-green color is apparent. In the peripheral cells there is no reaction. I n sections treated with digitonin according to Everett's ('47) method, the reaction was identical to that in the untreated sections. When the digitonin-treated sections were immersed in acetone or chloroform before the application of the Liebermanii-Burchard test, no reaction occurred. This suggests that there is no free cholesterol in the sebum, and that the steroids present are cholesteryl esters. The ceruminous glands are negative to the test.

Plasntal reactioiz. The results obtained with this test tend to be inconsistent in spite of careful standardization of the procedure. If sections are treated immediately after section- ing, the reaction is usually weak; if they are allowed to stand for a few hours, they give a stronger reaction. I n sebaceous glands the sebum assumes a magenta color, the mature sebaceous cells give a weaker reaction, and the peripheral cells are negative. The ceruminous cells which contain abundant lipid droplets also give a strong reaction, but those which are practically devoid of lipid are negative (cf. Bunting et al., '48, fo r the sudoriparous glands).

Birefr ingence and autofluorescewe. Only the sebum in se- baceons ducts exhibits any appreciable amount of birefrin-

GLA4NDS O F EXTERNAL AUDITORY MEATUS 419

gence. The sebaceous cells and the new sebuni in the center of the glands are isotropic. Even in very active sebaceous glands which contain large amounts of sebum, anisotropicity is scant. Since these glands contain cholesterides, as evi- denced by the Liebermann-Burchard tests, one would expect abundant birefringence. However, cholesterol may not always be present in tissues in birefringeiit form (cf. Okey, '44, who found that high concentrations of cholesterol in the liver when in solution with fat may not show birefringence). These glands contrast with the sebaceous glands of the rabbit, rat, cat, and dog, which are brightly anisotropic.

We noted no birefringence in the ceruminous glands, even in those laden with lipid droplets, in contrast with the observations of Kolmer ('27) and Policard ('28) who find birefringent crystals in these glands. The keratinized utriculi interposed at the exits of many sebaceous ducts and ceruininous ducts, however, are strikingly anisotropic. These concretions glow with a white light intermingled with hues of yellow and purple.

Under ultraviolet light, the sebum in sebaceous ducts usually fluoresces with a bright yellow light, while the rest of the sebaceous glands emits a dull grayish-yellow light. The keratinized utriculi also fluoresce with a bright yellow light.

I n the ceruminous tubules the lipid globules emit a dull yellow or gray light but the pigment granules fluoresce with an intensely bright orange light (fig. 11). The fluorescence of the pigment granules is not appreciably dimiuished in sections which have been immersed in acetone, ether, carbon disulfide, or other solvents. Prolonged immersion in pyridine partially removes the fluorescent pigment.

THE PIGMENT

Several tests were applied to the ceruminous glands to de- termine the nature of their pigment granules. As we have already described above, the pigment is highly sudanophilic, resists lipid solvents, and fluoresces with a bright orange light. In frozen sections placed in strongly oxygenated water, dilate


hydrochloric acid, potassium hydroxide, potassium bichromate, chromic acid, acetone, chloroform, or carbon disulfide, the physical nature of the pigment is not visibly altered. In sections left in water at 3°C. for two weeks, the pigment granules were unaffected. Application of strong sulfuric acid to sections did not affect the pigment. Immersion of sections in a solution of iodine and potassium iodide gave no visible reaction. (Lison, '36, states that the last two tests, when they are applied to carotinoids, evoke specific deep blue-to-violet color reactions.) The properties just outlined appear to coin- cide with those of chromolipoids (Lison, '36). I n paraffin sections treated with several silver impregnation methods, the pigment either stained weakly o r not at all. When treated with Gornori's ( '37) ammoniacal silver nitrate method for reticulum, the pigment granules stain clearly black (figs. 9 and 10). The property of reducing ammoniacal silver nitrate is characteristic of melanin while chromolipoids do not ex- hibit this property. The pigment granules do not give an iron reaction. With methylene blue, toluidin blue, and Nile blue, they stain a greenish color. With carbol fuchsin and acid- alcohol they show acid-fastness.

When whole fresh cerumen is smeared on a slide and observed under ultraviolet light, the cerumen flakes fluoresce with a bright reddish-yellow light. Immersion of these smears in ether does not diminish the orange fluorescence. When a drop of water is added to the smear under observation with ultraviolet light, the yellow color is gradually dissipated and the water assumes a greenish fluorescence which does not fade after prolonged exposure (cf. Popper, '41, f o r fluorescence of vitamin A). The pigment in whole cerumen, then, unlike that in ceruniinous cells, is soluble in water.

When samples of whole cerumen are extracted with ether- alcohol, only a trace of the cerumen pigment enters in solution, imparting a pale yellowish tinge. T h e n the filtered ether- alcohol solution is then dried and re-extracted with ether, the ether solution is colorless, while the ether-insoluble substance


has a pale straw color. This residue is a viscous, pungent- tasting substance which is soluble in alcohol but not in acetone.

The dried residue of ether-alcohol extracted whole cerumen, is a coarse brownish mass. It is insoluble in acetone, alcohol, and other solvents, but the pigment enters readily into solution with water. The brown aqueous filtrate fluoresces with a green light. The aqueous filtrate when dried over a steam bath leaves a yellow-to-brown residue which fluoresces with a golden-brown light, like the fluorescence of ceroid described by Popper et al. ('44). This solute appears to be the ceruminous pigment.

When a drop of water or of concentrated sulfuric acid is added to flakes of pigment under ultraviolet light, one can see, as the flakes dissolve, the change of color from reddish-brown in the dry pigment to green in the water or acid solution.

PHOSPHATASES AND LIPASE

Fragments of 4 external auditory tubes were fixed in ice-cold acetone, imbedded in paraffin, and studied for phosphatases and lipase. Weak reactions were generally obtained. These findings are of little value because, although these enzymes are not very labile, post-mortem changes may have altered their abundance and distribution.

In the case of alkaline phosphatase, good positive reaction was observed principally in the endothelial cells of arterioles surrounding the glands and in the papillae of hair follicles. Either these structures possess a greater amount of the enzyme, or the enzyme here is more resistant to autolytic changes. The basal cells of the Malpighian layer of the surface epithelium of the canal give a mild positive reaction. I n the sebaceous glands, only the peripheral cells show some enzyme activity. The large central cells show none. I n the ceruminous glands, enzyme activity is strong in the myoepithelial cells, and in the basal portion and the nuclei of the lining cells. The apices of the ceruminous cells display little alkaline phosphatase activity, but the residual content of the tubules becomes black to brown. In control sections incubated

422 11'. MONTAGNA, C. R. NOBACK -4ND F. G. ZAK

in a mixture without the substrate, only parts of the residual cerumen ;ire blackened.

With acid phosphatase techniques, our results were erratic. Some enzyme activity is usually present in the sebaceous and ceruminous glands. Experience gained in handling this technique with comparable tissues in laboratory animals, suggests that fresher tissues might yield better results.

As with the phosphatases, lipase activity in the glands of the external auditory meatus is scant. Coarse brown granules, indicative of enzyme activity, are formed in the stratified squamous epithelium of the surface of the tube. I n the sebaceous glands, lipase activity is evident principally in the sebum and in the larger sebaceous cells whether they are undergoing degeneration or not. A few brown granules are formed in the active ceruminous glands, but in the epithelium of the dilated tubules none are seen.

BASOPHILIA

For the study of cytoplasmic basophilia, paraffin sections were stained either with eosin-methylene blue o r with a 0.02% aqueous solution of toluidin blue. The strongest basophilia is observed in the basal cells of the surface epithelium of the external ear tube, and in the outer root-sheath of hair follicles. The epithelial buds growing from the surface epitheliuni and from the hair follicles (described above) show intense cytoplasmic basophilia. Those sebaceous acini which have lipid accumulation and cellular enlargement only in the center, show cytoplasmic basophilia in the peripheral cells. The enlarged sebaceous cells have a delicate blue reticulum between the lipid droplets. I n more mature aciiii, basophilia is mini- mal, and is restricted to cellular boundaries and interglobular cytoplasmic reticulum. I n degenerating sebaceous cells, basophilia is absent. In the ceruminous glands, the tall coluninar cells are blue at the base, but their apices are usually colorless. In dilated tubules, the flattencd epithelial cells arc generally strongly basophilic.

GLAKDS O F EXTICENAL AUDITOSY MEATUS 423

Control sections digested in ribonuclease, or immersed in normal hydrochloric acid before staining, no longer reveal the cytoplasmic basophilia described, but the nuclear basophilia remains unchanged.

Nuclear basophilia was studied in sections stained with the Feulgen nucleal reaction. Like cytoplasmic basophilia, nuclear basophilia is most intense in the basal layer of the stratified squamous epithelium and in the outer root-sheath of hair follicles, as well as in the growing epithelial buds. I n the sebaceous glands, the nuclei of the peripheral cells are compact and darkly stained ; those of lipid-laden cells are pale and granular. In degenerating cells the nucleal reaction is faint or absent. With this method no nuclear fragments are revealed in the sebum. In the ceruminous cells, the nucleal reaction is very strong in the myoepithelial cells and in the flattened epithelial cells. In the tall columnar cells loaded with pigment and lipid droplets, the nucleus stains a light pink color. I n control sections not hydrolyzed in hydrochloric acid prior to the application of the Schiff reagent, the nucleal reaction is absent.

GLYCOGEN

Sections of external auditory tube fixed in acetone were treated with the periodic acid method of Hotchkiss ( '48) for glycogen. With this technique many elements recolor the Schiff reagent ; only those substances which in control sections are dissolved out by saliva are interpreted as glycogen. In the lining epithelium of the tube, small glycogen granules are found in the stratum corneum and in the basal part of the cells of the Malpighian layer. In the stratified squamous epithelium of the ducts of the sebaceous and ceruminous glands, the enlarged, and keratinized, surface cells are often loaded with glycogen droplets. In the basal one-half of some hair follicles, the cells of some outer root-sheaths are heavily laden with glycogen. In the hair bulb, only an occasional cell shows delicate glycogen granules. The epithelial buds, which appear to be young sebaceous acini, contain glycogen in the central cells. In sebaceous glands, delicate granules are seen

424 W. MONTAGNA, C. R. NOBACIi AND F. G. ZAK

either surrounding the nucleus or scattered among the lipid droplets of mature sebaceous cells which are about to break down. The sebum may show traces of glycogen, especially when it contains detectable cellular debris. I n the ceruminous glands, glycogen droplets are scattered in the cytoplasm among pigment granules. Pigment also recolors the Schiff reagent but it stains purplish even after salivary digestion. The clear content of the ceruminous tubules, like the pigment, stains purple and is not digested by saliva. Not all the tubules show the same amount of glycogen inclusion; it is abundant in some and absent in others.

DISCUSSION

The normal external auditory tube is covered by a thick layer of yellow-brown, bitter-tasting, waxy cerumen. Naka- shima ( ’33), in a thorough qualitative and quantitative study of the cerumen of Japanese subjects, reports finding 12.3% fat, 2.48% cholesterol, 0.85% fatty acids, 1.94% cerotic acid, 43.23% protein-containing substances, 3.46% ash content, and traces of a pigmented bitter-tasting substance (it is not clear from Nakashima’s paper whether this represents one or two substances). It is doubtful that the composition of the cerumen of Caucasians is identical with that of Japanese, since Naka- shima states that the cerumen of Japanese is usually whitish even when found in impacted plugs. I n white subjects, on the other hand, state cerumen is usually dark-brown to black. (In sections of the specimen of the one Japanese male, pigment granules are scarce in the ceruminous glands but they are abundant in the glands of Caucasians.)

The biological properties of cerumen are also poorly understood. One would surmise that a substance secreted so copiously should be of some significance. Yet, other than the obvious property of acting as a lubricant, or as a protective adhesive which collects foreign particles, no function is known. Policard ( ’28) suggests that cerumen, in virtue of its pungent odor and taste, may repel insects. Early beliefs that cerumen possesses bactericidal properties are not upheld by


the work of Tenaglia ('26) who found acid-fast bacilli in cerumen of patients recovering from tuberculosis ; Balzano ('34, cited from Pirodda, '37) states that rather than bactericidal, cerumen is itself a good culture medium for micro- organisms. Pirodda ('37) studied the effect of cerumen on the growth of cultures of Staphylococcus, Streptococcus, Diplococcus, Corynebacterium, etc. His work shows little significant difference between the growth of cultures which contained whole cerumen, or ether extracts of cerumen, and control cultures grown in pure media.

Cerumen is the combined accumulation of the secretory products of sebaceous and ceruminous glands, Kolmer ( '27) and Policard ('28) state that the lipid substances of cerumen are secreted wholly by the sebaceous glands, while the ceruminous glands, although their cells contain birefringent crystals, do not contain or secrete lipid substances. By the application of methods for the study of lipids, we have demonstrated that the ceruminous glands as well as sebaceous glands contain and secrete lipid substances.

The sebaceous glands secrete principally triglycerides and cholesteryl esters, as well as some plasmal, "Fischler- positive" material, and phospholipid. Many ceruminous tubules are lined by cells which are at times so full of lipid droplets that they resemble sebaceous cells. Some of these lipid droplets appear to contain neutral fats, while others contain substances which stain the blue oxazine base of Nile blue and which are blackened by treatment with the Fischler test. Some plasmals and phospholipids are also localized in the ceruminous cells. The Tiebermann-Burchard test shows that steroids probably are not contained in the ceruminous cells. Birefringent lipids are persent only in the sebum near the orifices of sebaceous ducts and in the keratinized utriculi. None of the glandular substances is birefringent. The lipids of the ceruminous tubules are isotropic (post-mortem changes may be responsible for the lack of anisotropicity) . The sebum and the utriculi fluoresce with a yellow light, but the other glandular elements are practically nonfluorescent. The pig-

426 W. MONTAGNA, C. 1:. NOBACK AXD F. G. ZAK

meiit granules in the ceruniinous cells, however, emit a bright orange-red light.

The pigment granules iii the ceruminous cells are insoluble in water, alcohols, acetone, ether, chloroform, benzene, carbon disulfide, potassium bichromate, chromic acid, hydrogen peroxide, dilute acids, and dilute alkalies. They are sudanophilic and stain clearly, even after extraction or after inclusion in paraffin. They are acid-fast, recolor leucofuchsin, and are not digested by saliva. They stain blue-green with Nile-blue sulfate, and green with methylene blue and toluidin blue, and are not stained by hematoxylin. When treated with strong sulfuric acid or with the iodine-potassium iodide test fo r caroteiioids (carotenoids give a violet reaction to these 2 tests) the pigment gives no reaction. The pigment is not ordinarily argyrophilic, but it reduces ammoniacal silver nitrate (Gom- ori's method). Under ultraviolet light, the pigment granules fluoresce with an orange-red light even after inclusion in paraffin.

The pigment from whole cerumen is insoluble in lipid solvents, but soluble in water. Dried, water-extracted pigment fluoresces with a rich reddish-brown color. In solution with water it has a greenish fluorescence.

The properties just outlined make it apparent that the cerumen pigment is different from most of the nascent biological pigments. It is not melanin. The latter is nonfluorescent (Hamperl, '34), does not stain with lipid dyes, reduces silver nitrate readily, and is easily bleached with oxidizing agents. It is not porphyrin since porphyrins are extractable with ether (Dobriner and Rhoades, '40). It is not hemosiderin since it contains no iron ( Connor, '28). The properties mentioned eliminate carotenoids (lipochromes) , which are soluble in lipid solvents, are reduced quickly with reducing agents, and give a specific violet color-reaction with sulfuric acid, or iodine-potassium iodide tests. Although in general the pigment has the properties of chromolipoids, these must also be ex- cluded since they are not supposed to reduce ammoniacal silver nitrate (Lison, '36). Tt does not appear to be hemo-

GLANDS O F EXTEBNhL AUDITORY MEATUS 427

fusciii, which stains readily with basic aniliiie dyes but is not sudanophilic after paraffin imbedding (Endicott and Lillie, '44). I n some respects the pigment resembles ceroid, having in common with it some of the properties outlined above. The fluorescent light of ceroid, as described by Endicott and Lillie ('44), differs from that of cerumen pigment in that ceroid displays a ' ' greenish-yellow fluorescence which changes slowly to a yellowish-white fluorescence" (Popper et al., '44, describe the fluorescence of liver ceroid as a golden-brown color). Furthermore, these authors state that many ceroid globules stain a purple color with Nile-blue sulfate, while cerumen pigment always stains a greenish-blue. In addition, ceroid, unlike the pigment in secreted cerumen, is not water soluble. These appear to be significant enough differences to classify cerumen pigment separately from ceroid.

The pattern of distribution of ribonucleoprotein in tbc sebaceous glands is consistent with that described in the glands of the rat (Montagna and Noback, '47). In the ceruminous glands cytoplasmic basophilia is similar to that of the apocrinc sudoriparous glands (Bunting et al., '48; Montagna and Parks, '48). The presence of ribonucleoproteins in cytoplasm is apparently related to protein synthesis (Caspersson et al., '41 ; Brachet, '47). Since whole cerumen contains large amounts of protein substances (Nakashima, '33), there is a suggestion here that some of them are produced i,n the ceruminous glands.

Our results show weak to medium-strong alkaline pliospha- tase activity in the ceruminous glands, while the sebaceous glands show practically none. Very little acid phospliatase activity is noted. Lipase activity is evident, but not marked, in the sebaceous glands. These facts do not imply that enzyme activity in these glands is scant or absent. 111 an unpublished work with comparable material from the cat and dog, wc find that in material obtained from freshly killed animals there is abundant enzyme activity in the glands, while in material fixed several hours after the death of the animals the results

428 ITr, MONTAGNA, C. R. NOBACB AXD F. G . ZAK

are erratic. It is likely, then, that post-mortem changes have altered or denatured enzyme activity in these glands.

Glycogen is revealed in small amounts principally in the ceruminous glands. These tests were carried out in sections of tissues fixed in acetone since we had no material fixed in Rossman’s fluid. The results obtained, for this reason, may not represent an accurate distribution of glycogen in these glands.

I. Both the sebaceous and the ceruminous glaiids of the external auditory meatus contain lipid substances. The cells of sebaceous glands contain neutral fats, cholesteryl esters, some “Fischler-positive” substances, phospholipids (possibly mitochondria, or Golgi, or both), and plasmals. The cells of the ceruminous glands contain some globnles of neutral fat, fatty acids ( 1 ) revealed by Nile-blue sulfate and the Fiscliler method, granules and rodlets of phospholipids (apparently mitochondria) , and some plasmals.

2. Neither the ceruininous nor the sebaceous glands reveal birefringent crystals. The old sebum in the sebaceous ducts, however, is anisotropic. Scbum fluoresces with a yellow light. The pigment granules in the ceruminous gIands fluoresce with a bright orange-red light.

3. Pigment in the ceruminous cells is insoluble in a number of lipid solvents, water, acid, and alkali. It does not bleach with reducing agents. It is sudanophilic even in paraffin sections. It stains green with toluidin blue, mcthylcne blue, and Nile-blue sulfate. It is acid-fast and recolors leucofuclisin when treated for glycogen but is not digested by saliva. I t does not give the iron reaction and it reduces arnmoniacal silver nitrate. 4. Pigment in residual cerumen possesses essentially the

same properties as the granules within the ceruminous cells, with the exception that it is soluble in water and in sulfuric acid and it is not sudanophilic. Dried water-extracted pigment is a brownish substance which fluoresces with a reddish or golden-brown light. In solution with water it fluoresces

SUMMARY

GLANDS O F EXTERNAL AUDITOllP MEATUS 429

with a green light. Although certain properties of the pigment substance resemble those of ceroid, it is not identical with ceroid.

5. Some alkaline phosphatase and lipase are demonstrable in the glands of the external auditory tubc. Little or no acid phosphatase activity is observed. Since tlie tests were carried out in post-mortem specimens, their validity may be questioned.

6. I n the ceruminous glands, much cytoplasmic basophilia is present in the epithelial cells. This basophilia is removed by ribonuclease and appears to represent ribonucleic acid.

7. A small amount of glycogen is present in sebaceous and ceruminous glands.

LITERATURE CITED

BBKER, J. R. 1944 The structure and chemical coniposition of tlie Golgi ele-

1946 The histochemical recognition of lipine. Quart. J. Micro. Sci.,

1947 Further remarks on the histochemical recognition of lipine.

ment. Quart. J. Micro. Sci., 85: 1-71.

87: 441-471.

Quart. J. Micro. Sci., &!: 463-465. BALZANO 1934 Cited from Pirodda ( ’ 3 7 ) . BOWEN, R. H. 1929 The cytology of glnndulnr secretion (cont.). Quart. Rev.

BRACHET, J. 1947 Embryologic chimique. Masson & C’“, Paris. BUNTING, H., G. B. WISLOCKI AND E. W. DRNPSEY 1948 Chemical histology of

human eccrine and apocrine sweat glands. Annt. Rec., 100: 61-77. CAIN, A. J. 1947 The use of Xile blue in the examination of lipoids. Quart. J.

Micro. Sci., 88: 383-392. 1947 An examination of Baker’s acid haematein test for phospho-

lipines. Quart. J. Micro. Sci., 88: 467-478. C ~SPERSSON, T., TI. LANDSTROWHYDEN AND L. AQTJILLONIUS 1941 Cytoplasma-

nukleotide in Eiweissproduzierenden Druscnzellen. Chromosoma, 2 : 111-131.

CONNER, C. L. 1928 Studies on lipochromes. IV. The nature of the pigments in certain organs. Am. J. Path., 46: 293-308.

CREW, A. E., AND L. M I R s K a I A The character “haivless” in the mouse. J. Gen., 25: 17-24.

DAVID, L. T. 1932 The external expression and comparative dermal histology of hereditary hairlessness in mammals. Zcit. Zellf orsch. Mikro. Annt., 14: 616-719.

DOBRINER, K., AND C. P. RHOA4DES The porphyrins in health and disease. Phpsiol. Rev., $0: 416-468.

Biol., 4 : 484-519.

1931

1940

430 W. MONTAGNA, C. R. NOBACIC AND F. G. ZAK

ENDICOTT, K. M., AND R. I). LILLIE 1944 Ceroid, the pignieiit of dietary cirrhosis of rats. Its ch:ir:icteristics and its differentiation from hemofusein. Am. J. Path., 20: 149-153.

EVERETT, J. 0’. Hoinioiial factors responsible for tlepositioii of cholesterol in the corpiis luteuui of the rat. Fhidocriii., 41: 364-377.

GOXORI, G. 1937 Silrer impregnation of reticulum iii paraffin sections. Am. J. Path., 13: 993-1001.

The distribution of phospliatase in normal organs and tissues. J. Cell. and Comp. Physiol., 1’7: 71-83.

Distribution of acid phosphatascs i n the tissues under normal and under pathologic conditions. Arch. Path., 32 : 189-199.

Distribution of lipase in the tissues under normal and under pathologic conditions. Arch. Path., 41 : 121-129.

HAMPERL, H. 1934 Die Fluoresceiizmikroskopie meiischlicher Gembe. Virch. Arch. Path. Anat. Physiol., 8112: 1-51.

HOTCHKISS, R. D. A rnicrochemical reaction rcsnlting in the staining of polysaccharide structures in fixed tissue preparations. Arch. Eiochem.,

1926 Rind die in der histologischen Technik gebriiuchlichen Fettdifferenzirruiigsmethoden spezifisch? Virch. Arch.

K ~ Y , TV. W., AND R. WHITEHEAD 1937 Fa t ty substances; from The Miero- scopist ’s Vade-Rlecum, 10th ed., chap. 28, pp. 278-285, Blakiston, Philadelphia.

1927 Gehororgan. From Molleiidorff ’s Handb. Mikr. Anat. Mensch., Bd. 3, T. 1.

1947

-___ l941a

1941b

1946

-~

1948

36: 131-141. K a u r n i a x ~ , C., AND E. LEHXANN

Rcc., 79: 17-38.

KOLIIER, W.

LILLIE, R. D. 1948 Histopathologic technic. Blakiston, Philadelphia. LISON, L. 1936 Histochimie Aiiiniale. Gauthier-Villars, Paris. LUDFORD, €1. J. 1925 Thc cytology of tar tumors. Proc. Roy. Soc. London, 1.9:

MALLORY, F. R. 1938 Pathological technique. W. B. Saundcrs, Philadelphia. MFLCZER, N., ASD S. DEME

55 7-5 7 7.

1942 Beitr6ge zur Tatigkeit der mtmxhlichcn Talg- driisen; histologisch nachneisbare chemisehe, VerAnderung wahrend der Talgerzcugnng. Dermatologica, S6 : 24-36.

MONTAGNA. W., ~ N D C. R. NOBACK 1947 Histochemical ohserrations on the sebaceous glaiids of the rat. Am. J. Anat., X1: 39-62.

MONTIGNA. W., ~ N D H. F. PARKS A histochemical study of the glands of the anal sac of the dog. Anat. Rec., 100: 297-318.

NAKASHIU 4, 8. 1933 pbr r die chemisehe Zusammensetzung ilrs Cerumens. Zeit. Physiol. Cliem., 816: 105-109.

NrroL-is, ,J., C . R i e G 4 m IVTD &I. FAVRE Sur lcs mitochondrirs des glandcs s6bacPes de l’homme et siir la signification g6ii6ralc d r ccs organites du protop1:ismn. XVTJth 111. Congr. &fed., Sec. 13., l k m . and Syph., pp. 101-104.

____ 1914b Sui la fine structure des glandes sudoriparrs de l’hommc, particiili8rement en ce qui conceriic les mitochoiidries a t les phhomitiies de s6cr6tion. XVIItli Int . Congr. Med., See. 13, Derin. and Syph.,

1948

1914:i

pp. 105-109.

GLANDS OF EXTERNAL AUDITORY MEATUS 431

OKEY, R. Cholesterol injury in the guinea pig. J. Biol. Chem., 156: 179-

PAPPENHEIXER, A. M., AND J. VICTOR “Ceroid” pigment in human tis-

I’IRODDA, A. 1937 Ha il cerumc potere battericida 9 L ’Oto-Rino-Laring., It.,

POLICARD, A. 1928 Precis d ’histologie physiologiqne. Paris. POPPER, H. 1941 Histologic distribution of vitamin “ A ” in human organs

under normal and pathologic conditions. Arch. Path., 31 : 766-802. POPPER, H., P. GYORGY AND H. GOLDBLATT 1944 Flnoresccnt material (ceroid)

in experimental nutritional cirrhosis. Arch. Path., 37 : 161-168. SIMUNETTA, B., A N D A. MA4GN0NI L o suiluppo delle ghiandolc webacee P

eeruminose del eondotto uditivo esterno nell’ uomo. Arch. It. Anat. e di Embriol., 39: 245-261.

On the simultaneous staining of neutral f a t and fatty acid by oxazine dyes. J. Path. Bact., 18: 1-4.

1911 The staining of f a t by Nilc-blue sulfate. J. Path. Eact., 2 5 : 53-55,

SYMINGTON, J. 1885 The external auditory meatus in the child. J. Anat. and Physiol., 19: 280-285.

TENAGLIA, G. 1926 Bacilli acido-resistcnti nel cerume auricolare. Pensiero Med.,

WOLF, A., E. KABAT AND w. NEWMAN 1943 Histochemical studies on tissue enzymes. 111. A study of the distribution of acid phosphatases with special reference to the nervous system. Am. J. Path., 19: 432-429.

1944 190.

sues. Am. J. Path., 22: 395-412. 1946

7: 171-182.

1937

SMITH, J . L. 1908

1 5 : 384-386.

PLATE 1

EXPLANATION OF FIGVREB

1 Section of external auditory meatus of a girl 2 years of age. Hematoxyliu and eosin. This shows the distribution of the sebaceous glands, superficially, and the ceruminous tubules below them. 5 X ocular, 10 X objective.

2 Frozen section showing an utrieulus of a cernminous duct contaiuing a keratinized concretion, stained with Studan black. Male, 60 years of age. 10 X ocular, 10 X objectire.

3 Ceruminous tubule in a frozen section stained with Sudan black. Observe that the ceruminous cells protrude into the lumen as conical or spherical proccsses. The black particles about the unstained nuclei are the pigment granules. Other than pigment, sudanophilic particles are scant. Female, 24 years old. 1 2 X ocular, 47.5 X objective.

Frozen section stained with Sudan black, showing clusters of ccruuiinous tubules. Note that the glands within the same cluster appears to show the same amount of sudanophilia. Male, 48 years of age. 10 X ocular, 10 X objective.

Ceruminous tubule from figure 4. Note distribution of sudanophilic globules. 12 X ocular, 47.5 X objective.

Sebaceous glands stained with Sudan black. Male, 37 years old. 10 X ocular, 10 X objective.

4

5

6

432

G I A N D S OF EXTERNAL AUDITORY &fEATUS W. PIONTAGNA, 0. R. NOBACK AND F. C. ZAK

PLATE 1

433

PLATE 2

EXPLANATION O F FIGURER

7 Sebaceous cells treated with the arid haematein test for phospholipids. The black positive particles are distributed between large colorless lipid droplets. Male, 42 years old. 10 X ocular, 90 X objective.

8 Cells of ceruminous tubules treated with acid haematein. Slightly retouched. Male, 42 years old. 10 X ocular, 90 X objective.

9 Ceruminous tubules in tissue fixed in formalin and treated with Gomori’s ammoniacal silver nitrate method. Note general distribution of pigment granules in ceruminous glands. Male, 43 years old. 10 X ocular, 1 0 X objectire.

1 0 11 Cerurninous tubules in a frozen section rieived under ultrariolet light.

Enlarged detail from figure 9. 10 X ocular, 90 X objective.

Slightly retouched. 10 X ocular, 10 X objective.

434

GLAKDS O F EXTERNAL AUDITORY MEATVS \V. MONTARN4. C R. N O B A C K I N D F. (1 ZAK

PLATE 2

435

pigment, lipids, and other substances in the glands of the external auditory meatus of man

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