ORIGINAL ARTICLE Location of Bacterial Biofilm in the Mucus Overlying the Adenoid by Light Microscopy Birgit Winther, MD; Brian C. Gross, MD; J. Owen Hendley, MD; Stephen V. Early, MD Objective: To determine the location of bacteria and tion protocol. One adenoid that was missing the surface biofilm in adenoid tissue and in mucus overlying the epithelium was excluded from further evaluation. adenoid. Main Outcome Measure: Identification of bacteria by light microscopy. Design: Adenoids removed in 1 piece were oriented to the cephalic and caudal ends. Mucus was fixed by the Results: Bacteria in large numbers were present in the gradual addition of Carnoy fluid. Consecutive histo- mucus overlying the surface of all 9 adenoids; bacteria were logic sections were stained with periodic acid–Schiff for not found in the parenchyma of the adenoids below the visualization of the exopolysaccharide matrix, Giemsa for epithelial surface. Bacterial biofilms were present on 8 of visualization of bacteria and cells, and fluorescent in situ the 9 adenoids. Sessile (attached) biofilm was present on hybridization with a universal probe for visualization of the caudal end of only 1 adenoid. Multiple planktonic (un- bacteria. attached) biofilms were present on 7 adenoids, always in areas not subject to mucus flow. Biofilms were most com- Setting: Department of Otolaryngology–Head and Neck mon on the caudal portions of adenoids. Surgery, University of Virginia. Conclusions: Bacteria of the adenoid reside in secre- tions on the surface and in crypts. Biofilms, predomi- Participants: We obtained adenoids from children 10 nantly planktonic, were present on 8 of 9 adenoids ex- years or younger who had chronic adenotonsillitis or ob- cised because of hypertrophy. Whether biofilms have a structive sleep apnea. Twenty-seven adenoids were used role in the causation of adenoid hypertrophy is not known. to develop the fixation method. We examined histologic sections from 9 of 10 adenoids fixed using the final fixa- Arch Otolaryngol Head Neck Surg. 2009;135(12):1239-1245 HE ADENOID HAS STRUC- face of adenoids; some bacteria were ture and function that are present in the secretion and on the sur- similar in many ways to face of the excised adenoids in histologic those of a lymph node.1 Mu- sections after formalin fixation. In the ini- cus from the nose flows tial phase of the present study, bacteria Tacross the surface of the adenoid, which were not found in formalin-fixed tissue in is in the midline on the posterior wall of the adenoid parenchyma or on the ad- the nasopharynx. Adenoid hypertrophy/ enoid surface with fluorescent in situ hy- hyperplasia, common in young children, bridization (FISH) using a universal bac- may lead to adenoidectomy. One justifi- terial probe.3 cation for removal is the concept that For the present study we hypoth- chronic infection of the adenoid may lead esized that the bacteria of the adenoid re- to hypertrophy, necessitating removal to side in the mucus overlying the adenoid, clear the infection. Aerobic bacteria in large which is often lost during routine fixa- quantities can be cultured easily from the tion. The approach to the hypothesis was surface of the adenoid, but bacterial in- facilitated by the work of Swidsinski et al,4 Author Affiliations: fection in the substance (parenchyma) of who used Carnoy fluid on biopsy samples Departments of the adenoid has not been demonstrated. of colon mucosa to demonstrate bacteria Otolaryngology–Head and Neck Ivarsson and Lundberg2 demonstrated the in the mucus overlying colonic epithe- Surgery (Drs Winther, Gross, and Early) and Pediatrics presence of many bacteria and polymor- lium. The use of Carnoy fluid, based on 5,6 (Dr Hendley), University of phonuclear leukocytes (PMNs) with in- previous work, was crucial in preserva- Virginia School of Medicine, gested bacteria in Giemsa-stained touch tion of bacteria in the mucus layer on mu- Charlottesville. preparations of secretions from the sur- cosal surfaces because the mucus layer was (REPRINTED) ARCH OTOLARYNGOL HEAD NECK SURG/ VOL 135 (NO. 12), DEC 2009 WWW.ARCHOTO.COM 1239 ©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 means for identification of bacteria consisted of FISH with A a universal bacterial probe. Bacterial biofilms are defined as aggregates of bacte- ria “embedded in a matrix of extracellular polymeric substances.”7 The 2 types are sessile biofilm, which is adherent to a surface, and planktonic biofilm (sludge flocs), which is roughly spherical in shape and is not attached to a surface. The community of bacteria in bio- film is surrounded by an extracellular substance com- monly composed of polysaccharides, protein, and nucleic acid.8 For the purpose of locating bacteria and biofilm in this study, consecutive histologic sections from each paraffin block of adenoid tissue were stained with periodic acid–Schiff (PAS) for visualization of the exopolysaccharide matrix, Giemsa for visualization of bacteria and cells, and FISH for specific detection of bacteria. METHODS ADENOID SPECIMENS B Thirty-seven adenoids removed in 1 piece (Figure 1A) were used. The indications for adenoid removal were chronic ad- enotonsillitis or obstructive sleep apnea. The use of the speci- mens was exempt from the need for informed consent because they were considered discarded material by the Institutional Review Board for Health Sciences Research at the University of Virginia; no identifying information on the patients was avail- able other than age (Յ10 years). We used 27 adenoids to develop a protocol for the reten- tion of bacteria and mucus on the adenoid surface so that the relationship of bacteria to the adenoid surface could be as- sessed. The histologic findings on 9 of 10 adenoids fixed using C Nasopharyngeal lumen the final protocol are described in the “Results” section. One F F adenoid that was missing the surface epithelium was excluded F F from further evaluation. F C I C I V I C V V V DEVELOPMENT OF FIXATION PROTOCOL V Fixative M Figure 1. Adenoid removed from a child 10 years or younger with chronic We used Carnoy fluid as a fixative throughout. Carnoy fluid adenotonsillitis or obstructive sleep apnea. A, Gross appearance of the adenoid contains ethanol, chloroform, and acetic acid. The ethanol con- removed in 1 piece and oriented to the cephalic/caudal ends. Lines indicate 3 centration is 60%,3-5 but the acetic acid concentration may be cross-section cuts (cephalic, middle, and caudal). B, Whole mount of a cross 30% with 10% chloroform6 or 10% with 30% chloroform.9 Three section, stained with periodic acid–Schiff (PAS). The adenoid has opened up adenoids were cut in half; half was fixed with the 10% acetic like a fan, revealing interfold spaces. C, Schematic of a cross section of adenoid. Each fold (F) consists of a vascular core (V), surrounded by lymphoid acid formulation and the other half with the 30% acetic acid follicles (not shown on the schematic; seen on the PAS-stained section in B). formulation. Fixation with the 30% acetic acid version re- Five folds are depicted. The epithelium on top of the folds forms the sulted in better preservation of the mucus on the surface. There- nasopharyngeal surface of the adenoid. The horizontal cut through the adenoid fore, Carnoy fluid with 60% ethanol, 30% acetic acid, and 10% after fixation allows the folds to separate from each other like a fan, revealing chloroform was adopted as the standard fixative; tissue was fixed the interfold space (I). This space is very narrow before the cross section is for 2 hours. cut. It is lined by adenoid epithelium and may contain cells in fluid or mucus. Adenoid crypts (C) are narrow epithelium-lined cul de sacs that may open onto nasopharyngeal surface or the interfold wall. Crypts may extend very deeply Application of Fixative into the fold tissue. The vascular core of each fold contains blood vessels and is surrounded by a “string of pearls” that consists of primary and secondary Adenoids placed in fixative in a bottle did not have mucus with lymphoid follicles covered by specialized adenoid epithelium.1 Mucus-secreting glands (M) (stained with PAS in B) are seen in the base of the folds. Glandular bacteria on the surface because the mucus had washed off be- ducts open onto the epithelial surface at the base of the interfold spaces. fore fixation. Subsequently, adenoids were placed with the cut (posterior) surface on filter paper in a Petri dish; fixative was added to the dish so that the mucus on the nasopharyngeal sur- 4 lost during formalin fixation. In the present work, we face might be fixed in place by acetic acid fumes before the level used Carnoy fluid to fix surface mucus in place to allow of fixative was high enough to cover the tissue. Adenoids fixed visualization of bacteria on the adenoid. The definitive in this way for 2 hours before being cut into smaller pieces for (REPRINTED) ARCH OTOLARYNGOL HEAD NECK SURG/ VOL 135 (NO. 12), DEC 2009 WWW.ARCHOTO.COM 1240 ©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 embedding had flakes of fixed mucus, but the fixed mucus was RESULTS often dislodged during cutting. Sectioning the adenoids be- fore fixation caused cells from the interior of the adenoid to be squeezed out onto the surface. ANATOMY OF THE ADENOID Finally, Carnoy fluid was added gradually during a 30- IN CROSS SECTION minute period to the adenoid on the filter paper. At 30 min- utes, the surface with overlying mucus was fixed, but the in- Each adenoid specimen was removed as 1 piece and terior of the adenoid was not. The tissue was cut into smaller placed on filter paper with the cephalic/caudal orienta- pieces that were placed in cassettes without sponges before being returned to Carnoy fluid for the remaining 90 minutes re- tion (Figure 1A).