Hematoxylin and Eosin Staining for Detecting Biofilms: Practical and Cost-Effective Methods for Predicting Worse Outcomes After Endoscopic Sinus Surgery

Hong, Sang Duk; Dhong, Hun‐Jong; Chung, Seung‐Kyu; Kim, Hyo Yeol; Park, Jun-Oh; Ha, Sang Yun

doi:10.3342/ceo.2014.7.3.193

Cited by 10 publications

(13 citation statements)

References 14 publications

(27 reference statements)

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“…Recent reports have advocated for the use of the hematoxylin and eosin (H&E) stain over the Gram stain for detection of biofilms/bacteria in tissues [ 24 , 25 ]. However, while existing patterns of inflammation elucidated via H&E can lead to some insight on the infection status of a wound, individual bacteria are not easily detected with H&E staining alone [ 26 ].…”

Section: Resultsmentioning

confidence: 99%

“…Histologically, ex vivo detection of bacterial biofilms has been completed with expensive and time consuming techniques such as electron microscopy and confocal microscopy [ 33 , 34 ]. Light microscopy has been pursued for biofilm localization in conditions such as rhinosinusitis [ 24 , 25 , 35 ]. However, in the case of burn wounds, non-specific staining of connective tissue, cellular debris, and other proteins present within the eschar occurs due to the absorption of the crystal violet dye used in the Gram stain [ 15 ].…”

Section: Resultsmentioning

confidence: 99%

“…Moreover, the majority of wound infections encountered are polymicrobial. Clinically, H&E is the standard staining technique used by pathologists, and recently, has been used to identify bacteria in both planktonic and biofilm forms within wounds [ 24 , 25 ]. H&E staining has several attributes such as affordability and quick turnaround, as well as the ability to highlight both cells (hematoxylin) and connective tissue (eosin).…”

Section: Resultsmentioning

confidence: 99%

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An optimized staining technique for the detection of Gram positive and Gram negative bacteria within tissue

et al. 2016

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BackgroundBacterial infections are a common clinical problem in both acute and chronic wounds. With growing concerns over antibiotic resistance, treatment of bacterial infections should only occur after positive diagnosis. Currently, diagnosis is delayed due to lengthy culturing methods which may also fail to identify the presence of bacteria. While newer costly bacterial identification methods are being explored, a simple and inexpensive diagnostic tool would aid in immediate and accurate treatments for bacterial infections. Histologically, hematoxylin and eosin (H&E) and Gram stains have been employed, but are far from optimal when analyzing tissue samples due to non-specific staining. The goal of the current study was to develop a modification of the Gram stain that enhances the contrast between bacteria and host tissue.FindingsA modified Gram stain was developed and tested as an alternative to Gram stain that improves the contrast between Gram positive bacteria, Gram negative bacteria and host tissue. Initially, clinically relevant strains of Pseudomonas aeruginosa and Staphylococcus aureus were visualized in vitro and in biopsies of infected, porcine burns using routine Gram stain, and immunohistochemistry techniques involving bacterial strain-specific fluorescent antibodies as validation tools. H&E and Gram stain of serial biopsy sections were then compared to a modification of the Gram stain incorporating a counterstain that highlights collagen found in tissue. The modified Gram stain clearly identified both Gram positive and Gram negative bacteria, and when compared to H&E or Gram stain alone provided excellent contrast between bacteria and non-viable burn eschar. Moreover, when applied to surgical biopsies from patients that underwent burn debridement this technique was able to clearly detect bacterial morphology within host tissue.ConclusionsWe describe a modification of the Gram stain that provides improved contrast of Gram positive and Gram negative microorganisms within host tissue. The samples used in this study demonstrate that this staining technique has laboratory and clinical applicability. This modification only adds minutes to traditional Gram stain with reusable reagents, and results in a cost- and time-efficient technique for identifying bacteria in any clinical biopsy containing connective tissue.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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An optimized staining technique for the detection of Gram positive and Gram negative bacteria within tissue

et al. 2016

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“…Light microscopy (LM) is a basic imaging technique that is useful for providing the visual identification of biofilm presence and also has significant prognostic value [ 13 ]. It can be used for quantitative assessment of biofilm biomass, being easy and low cost to perform [ 14 , 15 ].…”

Section: Microscopy Techniques Applied To Biofilm Imagingmentioning

confidence: 99%

Microscopy Methods for Biofilm Imaging: Focus on SEM and VP-SEM Pros and Cons

Relucenti

Familiari

Donfrancesco

et al. 2021

Biology

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Several imaging methodologies have been used in biofilm studies, contributing to deepening the knowledge on their structure. This review illustrates the most widely used microscopy techniques in biofilm investigations, focusing on traditional and innovative scanning electron microscopy techniques such as scanning electron microscopy (SEM), variable pressure SEM (VP-SEM), environmental SEM (ESEM), and the more recent ambiental SEM (ASEM), ending with the cutting edge Cryo-SEM and focused ion beam SEM (FIB SEM), highlighting the pros and cons of several methods with particular emphasis on conventional SEM and VP-SEM. As each technique has its own advantages and disadvantages, the choice of the most appropriate method must be done carefully, based on the specific aim of the study. The evaluation of the drug effects on biofilm requires imaging methods that show the most detailed ultrastructural features of the biofilm. In this kind of research, the use of scanning electron microscopy with customized protocols such as osmium tetroxide (OsO4), ruthenium red (RR), tannic acid (TA) staining, and ionic liquid (IL) treatment is unrivalled for its image quality, magnification, resolution, minimal sample loss, and actual sample structure preservation. The combined use of innovative SEM protocols and 3-D image analysis software will allow for quantitative data from SEM images to be extracted; in this way, data from images of samples that have undergone different antibiofilm treatments can be compared.

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“…Hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), and Brown and Brenn Gram staining have been recently proposed as practical, cheap and reliable methods for detection of bacterial biofilms in different infection foci (Akiyama et al, 2003;Bulut et al, 2014;Davis et al, 2008;Hochstim et al, 2010;Oates et al, 2014;Toth et al, 2011;Winther et al, 2009;Zhang et al, 2009). The detection of biofilm by these practical and cost-effective staining methods has been used for a quantitative assessment of biofilm biomass (Bakke et al, 2001;Bulut et al, 2014;de Carvalho & da Fonseca, 2007) and could have a significant prognostic value (Hong et al, 2014).…”

Section: Microscopy Methodsmentioning

confidence: 99%

Critical review on biofilm methods

Azeredo

Azevedo

Briandet

et al. 2016

Critical Reviews in Microbiology

736

679

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Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a negative impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiology, structure and composition. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, composition and physiology are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a critical perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms. ARTICLE HISTORY

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Hematoxylin and Eosin Staining for Detecting Biofilms: Practical and Cost-Effective Methods for Predicting Worse Outcomes After Endoscopic Sinus Surgery

Cited by 10 publications

References 14 publications

An optimized staining technique for the detection of Gram positive and Gram negative bacteria within tissue

An optimized staining technique for the detection of Gram positive and Gram negative bacteria within tissue

Microscopy Methods for Biofilm Imaging: Focus on SEM and VP-SEM Pros and Cons

Critical review on biofilm methods

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