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

Abstract

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.

A tractable, simplified ex vivo human skin model of wound infection

“…Reepithelialization from the wound border (black arrows) across the wound bed (purple arrows), and percentage reepithelialization of noninfected (CTRL) and S. aureus ‐ infected (SA) wounds. (B) Wounds stained with modified Gram stain . Bacterial aggregation is indicated by orange arrows.…”

The Role of Gram Staining in Staphylococcal Scalded Skin Syndrome

“…If Gram staining is performed, the usual findings compatible with S. aureus would yield Grampositive cocci in clusters [2]. With a high degree of suspicion, finding the bacteria would provide additional evidence to start empiric therapy.…”

Diabetes alters the involvement of myofibroblasts during periodontal wound healing

“…The collagen matrix was visualized using the Sirius red (Abcam) staining method coupled to polarized light microscopy (Junqueira, Bignolas, & Brentani, 1979). Gram staining of tissue sections was performed following a previously published method (Becerra, Roy, Sanchez, Christy, & Burmeister, 2016). A numerical code was given to each histological sample.…”