3D fiber deposited polymeric scaffolds for external auditory canal wall.

The external auditory canal (EAC) is an osseocartilaginous structure extending from the auricle to the eardrum, which can be affected by congenital, inflammatory, and neoplastic diseases, thus reconstructive materials are needed. Current biomaterial-based approaches for the surgical reconstruction of EAC posterior wall still suffer from resorption (biological) and extrusion (synthetic). In this study, 3D fiber deposited scaffolds based on poly(ethylene oxide terephthalate)/poly(butylene terephthalate) were designed and fabricated to replace the EAC wall. Fiber diameter and scaffold porosity were optimized, leading to 200 ± 33 µm and 55% ± 5%, respectively. The mechanical properties were evaluated, resulting in a Young's modulus of 25.1 ± 7.0 MPa. Finally, the EAC scaffolds were tested in vitro with osteo-differentiated human mesenchymal stromal cells (hMSCs) with different seeding methods to produce homogeneously colonized replacements of interest for otologic surgery. This study demonstrated the fabrication feasibility of EAC wall scaffolds aimed to match several important requirements for biomaterial application to the ear under the Tissue Engineering paradigm, including shape, porosity, surface area, mechanical properties and favorable in vitro interaction with osteoinduced hMSCs. This study demonstrated the fabrication feasibility of outer ear canal wall scaffolds via additive manufacturing. Aimed to match several important requirements for biomaterial application to ear replacements under the Tissue Engineering paradigm, including shape, porosity and pore size, surface area, mechanical properties and favorable in vitro interaction with osteo-differentiated mesenchymal stromal cells.

Exposure to non-ionizing electromagnetic fields emitted from mobile phones induced DNA damage in human ear canal hair follicle cells.

The aim of this study was to investigate effect of radiofrequency radiation (RFR) emitted from mobile phones on DNA damage in follicle cells of hair in the ear canal. The study was carried out on 56 men (age range: 30-60 years old)in four treatment groups with n = 14 in each group. The groups were defined as follows: people who did not use a mobile phone (Control), people use mobile phones for 0-30 min/day (second group), people use mobile phones for 30-60 min/day (third group) and people use mobile phones for more than 60 min/day (fourth group). Ear canal hair follicle cells taken from the subjects were analyzed by the Comet Assay to determine DNA damages. The Comet Assay parameters measured were head length, tail length, comet length, percentage of head DNA, tail DNA percentage, tail moment, and Olive tail moment. Results of the study showed that DNA damage indicators were higher in the RFR exposure groups than in the control subjects. In addition, DNA damage increased with the daily duration of exposure. In conclusion, RFR emitted from mobile phones has a potential to produce DNA damage in follicle cells of hair in the ear canal. Therefore, mobile phone users have to pay more attention when using wireless phones.

Sensory neurons in the human jugular ganglion.

The jugular ganglion (JG) contains sensory neurons of the vagus nerve which innervate somatic and visceral structures in cranial and cervical regions. In this study, the number of sensory neurons in the human JG was investigated. And, the morphology of sensory neurons in the human JG and nodose ganglion (NG) was compared. The estimated number of JG neurons was 2721.8-9301.1 (average number of sensory neurons ±â€¯S.D. = 7975.1 ±â€¯3312.8). There was no significant difference in sizes of the neuronal cell body and nucleus within the JG (cell body, 1128.8 ±â€¯99.7 µâ€¯m2; nucleus, 127.7 ±â€¯20.8 µâ€¯m2) and NG (cell body, 963.8 ±â€¯225.7 µâ€¯m2; nucleus, 123.2 ±â€¯32.3 µâ€¯m2). These findings indicate that most of sensory neurons show the similar morphology in the JG and NG. Our immunohistochemical method also demonstrated the distribution of ion channels, neurotransmitter agents and calcium-binding proteins in the human JG. Numerous JG neurons were immunoreactive for transient receptor potential cation channel subfamily V member 1 (TRPV1, mean ±â€¯SD = 19.9 ±â€¯11.5 %) and calcitonin gene-related peptide (CGRP, 28.4 ±â€¯6.7 %). A moderate number of JG neurons contained TRPV2 (12.0 ±â€¯4.7 %), substance P (SP, 15.7 ±â€¯6.9 %) and secreted protein, acidic and rich in cysteine-like 1 (SPARCL1, 14.6 ±â€¯7.4 %). A few JG neurons had vesicular glutamate transporter 2 (VGLUT2, 5.6 ±â€¯2.9 %) and parvalbumin (PV, 2.3 ±â€¯1.4 %). SP- and TRPV2-containing JG neurons had mainly small and medium-sized cell bodies, respectively. TRPV1- and VGLUT2- containing JG neurons were small to medium-sized. CGRP- and SPARCL1-containing JG neurons were of various cell body sizes. Sensory neurons in the human JG were mostly free of vasoactive intestinal polypeptide (VIP), tyrosine hydroxylase (TH) and neuropeptide Y (NPY). In the external auditory canal skin, subepithelial nerve fibers contained TRPV1, TRPV2, SP, CGRP and VGLUT2. Perivascular nerve fibers also had TRPV1, TRPV2, SP, CGRP, VIP, NPY and TH. However, PV- and SPARCL1-containing nerve endings could not be seen in the external auditory canal. It is likely that sensory neurons in the human JG can transduce nociceptive and mechanoreceptive information from the external auditory canal. Theses neurons may be also associated with neurogenic inflammation in the external auditory canal and ear-cough reflex through the vagus nerve.

Comparison of changes in mitochondrial bioenergetics between keratinocytes in human external auditory canal skin and cholesteatomas from normoxia to hypoxia.

Cholesteatoma has attracted many studies seeking to uncover its nature and the pathogenesis of related diseases. However, no researchers have explored the mitochondrial bioenergetics of cholesteatoma. The aim of this study was to investigate the energy demand and differential mitochondrial respiration profiles between keratinocytes in external auditory canal (EAC) skin and cholesteatoma samples cultured in normoxic (20% O2) and hypoxic (5% O2) conditions. Enhanced cellular proliferation of both types of keratinocytes was found in hypoxia compared to normoxia. In 20% O2 conditions, cholesteatoma keratinocytes exhibited less mitochondrial mass, lower ATP levels, and significantly lower basal oxygen consumption rate (OCR) and reserve capacity compared to normal skin keratinocytes. In contrast, in hypoxic conditions, cholesteatoma keratinocytes showed markedly higher levels in maximal OCR and reserve capacity, as well as lower proton leak OCRs, compared to normal skin keratinocytes. Hypoxia induced the reverse mitochondrial bioenergy profile from that in normoxia between these two types of keratinocytes, implying that an adaptive change of mitochondrial respiration to oxygen fluctuations may develop in cases of cholesteatoma. Such adaptation in response to hypoxic conditions may play a role in explaining the pathogenesis of acquired cholesteatoma.

Ear canal keratinocyte culture: clinical perspective.

HYPOTHESIS: Autologous epidermal sheets obtained by cultivating keratinocytes of the external auditory meatus can be used to repair cutaneous defects of the ear canal. The Rheinwald and Green method has been used to know whether the produced epidermal layer preserves its specificities after the culture. BACKGROUND: Using a split-thickness skin graft during a functional ear atresia surgery does not allow for the restitution of external auditory canal self-cleaning. Some authors cultivated external auditory meatus keratinocytes and showed migration capacities of these colonies. METHODS: Samples of preauricular skin and of the bony part of the external auditory canal were harvested from 10 patients. Keratinocytes were extracted and cultured until an epidermal sheet was obtained. The output, the keratinocyte plating efficiency, and the production delay were measured during the culture. Culture product sections and biopsy sections were examined using optical microscopy after standard coloration and indirect immunohistochemistry. RESULTS: Nine epidermal layers from 10 biopsies were obtained in each group. A significant difference between external auditory meatus and preauricular keratinocyte plating efficiency was highlighted. The average production delay of 23 cm2 external auditory canal and preauricular epidermal layers was 21 days. There was no difference in the cytokeratine expression between external auditory canal and preauricular skin, nor between external auditory canal and preauricular culture products. All cultures expressed the cytokeratine 5 characteristic of stratifying epithelium. CONCLUSION: The Rheinwald and Green keratinocyte culture method allows the production of ear canal-stratified epidermal sheets, which can be used for external ear reconstruction.

Impression Smear Agreement with Acetate Tape Preparation for Cytologic Sampling.

Cutaneous cytologic sampling techniques are used to detect bacteria, yeast, and inflammatory cells for diagnosis and therapeutic monitoring. Studies have examined slide evaluation techniques, ear swab cytology staining methods, and observer variations; few studies compare common clinical sampling techniques. The primary aim of this study was to measure detection of microorganisms and neutrophils by impression smear compared to acetate tape preparation; comparison of agreement between two acetate tape staining methods was a secondary aim. Thirty lesions consistent with superficial pyoderma were sampled via impression smear and acetate tape preparation. Acetate tape preparations were either stained with modified Romanowksy stain solutions two and three or solution three alone. Impression smears were stained in the standard manner. Bacteria, yeast, and neutrophils were evaluated using a semi-quantitative scale [0-4]. Quantities were aggregated and compared using Cohen's kappa to measure agreement between methods. When impression smears were compared to acetate tape, the lowest agreement occurred for neutrophils, with impression smears detecting more neutrophils. Comparison of acetate tape staining methods had the highest agreement for yeast detection. Sampling technique and staining method did not differ for detection of bacteria. Impression smears detected more neutrophils, and yeast detection appeared equivalent for acetate tape staining methods.

Comparison of 4 fixation and staining methods for the cytologic evaluation of ear canals with clinical evidence of ceruminous otitis externa.

BACKGROUND: Swab cytology of ear canals is one of the most useful and rapid methods to assess the presence of external ear infections. Smears are generally stained with rapid Romanowsky-type stains, with or without prior heat fixation. OBJECTIVES: The aim of this study was to compare 4 different methods of fixation and staining of ear swab cytology samples from dogs. METHODS: Eight dogs with otitis externa were selected from a dermatology referral population. A cotton swab was used to obtain ceruminous material from 12 ear canals. Four smears of each swab were prepared on glass slides (randomly identified as A, B, C, or D) and air-dried for cytologic examination. Samples marked A were stained with Dip Quick (Jorgensen Laboratories Inc, Loveland, CO, USA) after heat fixation; samples marked B were stained without heat fixation; samples marked C were heat-fixed and dipped only in the counterstain (the blue reagent) of Dip Quick; and samples marked D were dipped only in the counterstain, without heat fixation. Ten high-power fields (hpf; X100 oil immersion objective) in each slide were evaluated by 2 observers, and total numbers of keratinocytes, yeast, bacteria, and neutrophils were counted. Statistical comparison was performed using an ANOVA model applied after verifying the normal distribution of the data, and using nonparametric sign tests and Wilcoxon signed rank tests. RESULTS: No statistically significant differences were observed in the numbers of keratinocytes, yeast, bacteria, or neutrophils among the 4 staining methods (P > .05), although significant interobserver differences were found. CONCLUSION: We conclude that heat fixation does not improve the quality of ceruminous ear swab samples for cytologic evaluation, and propose a 1-step dip in the blue reagent alone as a rapid method of staining samples from canine ear canals.

Histophysiological observations on the external auditory meatus, middle, and inner ear of the Weddell seal (Leptonychotes weddelli).

The external auditory meatus, middle, and inner ear of the deep-diving Weddell seal (Leptonychotes weddelli) were studied with light microscopic, histological, and histochemical techniques in order to contribute to the open discussion on the orientation of this seal in the darkness of the deep Antarctic seas. The external auditory meatus is characterized by a well-developed venous plexus, single apocrine ceruminous, and numerous holocrine sebaceous glands and an incomplete tube of elastic cartilage. The tympanic membrane is comprised of two layers of radially and concentrically arranged collagen fibers and by elastic fibers which are concentrated in the outer part of the ear drum. The tympanic cavity is lined by a pseudostratified prismatic ciliated epithelium with goblet cells; a plexus of wide venous vessels marks the subepithelial lamina propria. The cochlea is about 10 mm high and forms about two and a half turns. The richly pigmented stria vascularis is well vascularized, while the cell-rich prominentia spiralis contains only single small blood vessels. The organ of Corti contains one row of inner and three rows of outer hair cells. Cells of Hensen, Claudius, and Boettcher are present. The basilar membrane is of comparatively uniform simple structure and is composed of abundant glycoproteins, proteoglycans, collagenous fibers, and the loose tissue of the tympanal layer. The spiral ligament is built up by abundant proteoglycans and a complex system of radial and concentric collagen fibers; close to the osseous wall of the bony cochlea it contains fine elastic fibers. The inner zone of the osseous wall of the cochlea strikingly contains hyaline cartilage. The thin lamina spiralis ossea is covered by a limbus spiralis with interdental cells secreting the lamina tectoria, which has a fibrous texture and contains glycoproteins and negatively charged components.

Canal wall reconstruction with Mimix hydroxyapatite cement: results in an animal model and case study.

OBJECTIVE/HYPOTHESIS: To assess Mimix hydroxyapatite cement for its applicability in canal wall reconstruction using the gerbil as a canal wall model. A case is presented to illustrate a novel technique of canal wall reconstruction using Mimix on the basis of the findings of our animal research. STUDY DESIGN: This was a preclinical study. METHODS: Ten Mongolian gerbils were implanted with Mimix, with the left side used to simulate mastoid obliteration and the right side used to simulate canal wall reconstruction. Pre- and postsurgery auditory-evoked brainstem responses were used to assess ototoxicity, and hematoxylin-eosin staining of histologic sections was used to assess inflammatory and foreign-body response and new bone formation. RESULTS: Rapid wound healing was achieved with each of the nine animals evaluated, with no erythema, edema, or drainage. Inspection of the ear canal at the time of sacrifice revealed no signs of otitis media and no middle ear effusions. Microscopic examination showed no inflammatory response or foreign-body reaction, good mucosalization on the side of the implant facing the bulla, and minimal fibrosis adjacent to the skin. Eight of nine specimens showed new woven bone ingrowth at the bone implant interface, with active osteoblasts and viable lacunae cells. There were no apparent fractures in the implanted material. CONCLUSIONS: Mimix hydroxyapatite cement is biocompatible and suitable for canal wall reconstruction in the animal model. The characteristics of this cement, namely its ability to set quickly in a moist environment, offer advantages over previously used cements for canal wall reconstruction.

The human external auditory canal, secretory system--an ultrastructural study.

Human external auditory canal skin, with a special emphasis on the secretory system, was studied by light, transmission electron and scanning electron microscopy. Two types of secretory glands were observed: modified apocrine (ceruminous) and sebaceous. The sebaceous secretory cellere homogeneous; on the other hand, modified apocrine secretory cells contained heterogeneous secretory granules. They were ither dark granules or light granules. Evidence to support both the apocrine as well as the eccrine mode of secretion was noted in the modified apocrine gland. This finding is partly in agreement with early reports based on light microscopy with suggested only an apocrine mode and data based on transmission electron microscopy which showed only the eccrine mode. Significance of the secretion by the external ear canal and its role in a local immune defense system is discussed.

Epidermal differentiation in the human external auditory meatus.

The differentiation of epidermis in the various parts of the human ear canal was documented on the basis of cytokeratin (Ck) expression patterns. Immunohistochemistry was performed on cryostat sections of normal meatal skin using a comprehensive panel of monospecific Ck antibodies representing the main lines of epithelial differentiation. The epidermis of the cartilaginous part showed a Ck profile characteristic of normal skin type differentiation. The deep meatal skin, including the tympanic membrane, showed a peculiar type of differentiation: in addition to epidermal Cks, hyperproliferation-associated Cks 6, 16, and 17 were expressed in the suprabasal cells, while the simple epithelia cell marker Ck 19 was found in the basal cells. The presence of hyperproliferative Cks in the deep meatal skin could only partly be related to areas of proliferative activity. Keratinocytes, which express markers of hyperproliferation, are migratory. Therefore, their presence in the meatal skin is likely to be related to the peculiar pattern of keratinocyte migration, the purpose of which is to keep the meatus free from desquamation products.

Increased activity of the diastrophic dysplasia sulfate transporter in otosclerosis and its inhibition by sodium fluoride.

HYPOTHESIS: This study investigates the function of the diastrophic dysplasia sulfate transporter (DTDST) in otosclerotic bone and the effect on it of sodium fluoride (NaF). BACKGROUND: Otosclerosis is a localized bone dystrophy with increased bone turnover. DTDST is implicated in the regulation of the bone turnover. MATERIALS AND METHODS: Primary cultures of cells were obtained from the stapes and external auditory canal (EAC) of 26 patients with otosclerosis and from nine control patients. Sulfate uptake was quantified under basal conditions and with NaF. The NaF signaling pathways were investigated using forskolin and verapamil. RESULTS: The relative initial rates of sulfate uptake and the apparent Vmax values were: otosclerotic stapes > EAC > control stapes = control EAC. The sulfate uptake by the otosclerotic stapes was correlated with the loss of sensorineural hearing. The amounts of DTDST mRNA (RNase protection assay) in the four subgroups did not differ. NaF (10(-6)M, 1 hr) inhibited sulfate uptake by the otosclerotic stapes and EAC cells but not by control samples. CONCLUSION: The authors believe that whether the increased DTDST activity is a cause or an effect of otosclerosis, it appears to be a specific target for NaF treatment.

Structure and development of vestibular hair cells in the larval bullfrog.

Structure and development of hair cells in vestibular sensory organs of the larval bullfrog were examined with scanning electron microscopy. The larval vestibular sensory epithelia resembled those of the adult frog. Based on morphology of the ciliary tufts, seven hair cell types were identified. One of them, the type A hair cell, appears to be the morphogenetic precursor of other hair cell types. The size of the stereocilia of type A hair cells is comparable to the surrounding microvilli. The distribution of immature type A hair cells suggests that the periphery of the sensory epithelia is the principal growth zone and the site of formation of new hair cells. However, a far greater number of type A hair cells were found in high frequency sensitive sensory organs (sacculus, amphibian and basilar papillae) than low frequency sensitive vestibular sensory structures (canal cristae, utriculus and lagena). This phenomenon may suggest that the time period required for the maturation of type A hair cells to their ultimate hair cell types in the low frequency sensitive vestibular organs is shorter than in the high frequency sensory structures. It is also possible that the low frequency sensitive vestibular organs may have completed their morphogenetic development in the early larval stages, while morphogenesis of hair cells in the high frequency sensory structures continues throughout the lifetime of a bullfrog.

Stratified squamous epithelium in relation to the tympanic membrane: its development and kinetics.

The pathways of auditory epithelial migration on the human tympanic membrane and their rate of movement were investigated by Hopkins rod photography of dye markings. The origin of these pathways was determined in both the human and the mouse by studying the development of the stratified squamous epithelium of the tympanic membrane and external auditory meatus from earliest embryonic life to maturity. Two pathways of migration are present. In one, epithelium moves from the region covering the tip of the handle of the malleus upwards to the lateral process and then posterior-superiorly with all dye on the pars flaccida to its posterior superior edge. In the second, dye moves centrifugally and radially outwards from the edges of the handle and pars flaccida regions to the annulus. Rate of movement can be determined approximately only and by reference to anatomical landmarks. The first pathway was traced embryologically to migration possibly commencing in the fundus of the primordial first branchial groove. The second pathway has its source in the growth of the meatal plate. A study of the development of the early meatal plate in the mouse suggests that movement of epithelium over the pars tensa region could be the result of a "pulling" effect of mitotically active cells in a generation center at the edge of the tympanic membrane resulting from negative contact inhibition.

Auditory epidermal cell migration. VII. Antigen expression of proliferating cell nuclear antigens, PCNA and Ki-67 in human tympanic membrane and external auditory canal.

A location of proliferating cells was investigated in eight human normal tympanic membranes (TMs) and external auditory canals (EACs) by an immunohistochemical method using two different types of antibodies for nuclear antigens in proliferating cells: anti-PCNA monoclonal antibody, and anti-Ki-67 polyclonal antibody. Four specimens prepared for cryostat sections were immunostained by both antibodies. Another four were fixed in 4% formaldehyde solution, embedded in paraffin wax and were reacted only with anti-PCNA antibodies. The expression pattern of Ki-67 was basically the same as of PCNA. In the pars tensa (PT), immunoreactivities were expressed in the nuclei of basal layer cells and cells just overlying the basal layer of epidermis both in the handle of the malleus (HM) and annular regions. In the intermediate region of the PT, no immunoreactivity was found basically, apart from a few labelled cells observed in the upper-third of the superior quadrant. In the pars flaccida (PF) and in both the osseous and cartilaginous regions of the EAC, positive cells were also situated in the basal layer and the deeper aspect of the suprabasal layers without any specific distributing pattern. It was certified that the generation centre of epidermal cells (keratinocytes) in the PT was located in both the HM and annular regions, and that stem cells in the PF and the EAC were uniformly scattered in the basal layer and the deeper aspect of the spinous layer. According to these findings, the migratory patterns of auditory epidermal cells in the human TM and EAC were discussed.

An ink impregnation study of the migratory skin in the external auditory canal of the guinea-pig.

If the tympanic membrane or attic skin is wounded with a fine needle dipped in ink, ink particles are introduced into the epidermis and underlying tissue. These particles are subsequently taken up by cells in the epidermis and dermis. In this experiment the distribution of ink within the skin of ear canal was studied in nineteen guinea-pigs, one to ten days after wounding. Examination of the intact canal reveals that ink becomes distributed along a precise line from the wound to the point of desquamation. On sectioned tissue, the ink in this line is found to be mainly intracellular, initially in the epidermis, and subsequently in the upper dermis. When considered with other evidence, these results indicate that migration probably occurs in the deeper layers of the epidermis, and that it stops at the junction of the deep and superficial parts of the ear canal.

Effect of 17 beta-estradiol on diastrophic dysplasia sulfate transporter activity in otosclerotic bone cell cultures and SaOS-2 cells.

OBJECTIVE: Diastrophic dysplasia sulfate transporter (DTDST) is involved in the regulation of bone turnover, and its activity in otosclerosis has been shown to be abnormally high. Taking into account the role of estrogens in the progression of otosclerosis, the possible effect of estrogens on DTDST was investigated in otosclerotic bone cell cultures and in SaOS-2, a human osteoblastic cell line. MATERIAL AND METHODS: Primary bone cell cultures of stapes and external auditory canal (EAC) bone were obtained from 33 patients with otosclerosis and 18 control patients undergoing cerebellopontine angle tumor surgery. These cultures were assessed in parallel with SaOS-2 cells. Estrogen receptors (ERs) were detected using reverse transcriptase polymerase chain reaction. DTDST activity was assessed by sulfate uptake at baseline and after 24 h of incubation with 17 beta-estradiol at concentrations ranging from 10(-12) to 10(-6) M. RESULTS: Stapes and EAC cultures predominantly expressed mRNA of ER alpha, while ER beta expression was predominant in SaOS-2 cells. In stapes and EAC cultures no modification of DTDST activity was observed with 10(-8) M 17 beta-estradiol. In SaOS-2 cells, DTDST activity was inhibited by 17 beta-estradiol (93.5+/-9.21 vs 83.6+/-8.83 pmol/mg protein/5 min, n=29; mean of differences=10.0+/-3.22, paired t-test, p<0.01). CONCLUSION: DTDST activity is regulated by estrogens in SaOS-2 cells, but not in primary cell cultures from stapes and EAC. This difference in the regulation mechanisms may be related to the type of estrogen receptor expressed.

Development of the human external ear.

External ear development is a lengthy and complex process that extends from early embryonic life until well into the postnatal period. Initial development of the auricle and external auditory canal during the fourth and fifth weeks of gestation is closely associated with anatomical changes involving the pharyngeal arch apparatus of the human embryo. The auricle and external canal are well formed by the time of birth but do not attain their full size and adult configuration until about 9 years of age. Sebaceous and modified apocrine glands, which are responsible for cerumen production, begin their development at about 5 months gestation in association with hair follicles in the outer portion of the external canal. Although they appear anatomically mature before birth, these glands do not reach full functional capacity until puberty.

Cytologic evaluation of otic exudates.

The multifactorial nature of otitis externa requires accurate etiologic information to ensure therapeutic success. The collection and preparation of cytologic samples of otic exudates are simple to perform, and information of immediate diagnostic and therapeutic value can be attained. Evidence of epidermal hyperplasia and increased glandular secretory activity suggests a noninfectious cause. Large numbers of microorganisms and infiltrating leukocytes confirm the presence of infection. The presence of ear mites, particularly O. cynotis, is always significant. Abnormal epithelial cells that appear singly or in sheets and clusters support the diagnosis of neoplasia; the extent of cell differentiation determines malignancy. Inflammatory cells and acantholytic keratinocytes from vesicular otic lesions suggest autoimmune skin disease.

Otic cytology in health and disease.

Accurate characterization of the primary cause and perpetuating factors is essential for successful management of ear disease in dogs and cats. Cytology is a simple, rapid, and practical diagnostic test that should be performed routinely on any and all patients presented for clinical signs consistent with otitis externa. In combination with clinical signs, otoscopic evaluation, and diagnostic testing of primary disease, serial cytology enhances the ability of veterinarians to diagnose secondary infections, monitor progression of disease, evaluate response to therapy, and make appropriate management decisions. Cytologic specimens should be evaluated for the presence, numbers, and characteristics of three key features: yeast, bacteria, and leukocytes. More than five yeast organisms or more than 25 bacteria per high-powered field is suggestive of significant microbial activity warranting therapeutic intervention. The presence of leukocytes, particularly with phagocytized bacteria, indicates "true infection" rather than overgrowth; if suppurative discharge is present, systemic therapy is needed. Cytology combined with culture and susceptibility is the best method for identification of bacterial overgrowth and infection; however, if only one test can be performed, always choose cytology. Culture results assist in the selection of appropriate antibiotic therapy, but cytology determines whether systemic antibiotics are indicated, which organisms are most significant, and when therapy can be discontinued.