RESEARCH : Fluorescence Microscopy–Based Protocol for Detecting Residual Bacteria on Medical Devices

Abstract

Standard methods are needed to reliably and efficiently assess bacterial contamination of processed medical devices. This article demonstrates a standard operating procedure (SOP) for fluorescence microscopy–based detection of residual bacteria on medical devices (BAC-VIS). BAC-VIS uses a 4',6-diamidino-2-phenylindole (DAPI) stain with fluorescent microscopy to quickly and cost-effectively detect bacterial contamination of processed medical device parts. The BAC-VIS protocol was optimized and achieved greater than 80% staining efficiency and a signal-to-noise ratio of more than 20 using four representative organisms. The SOP was first validated for use on a buildup biofilm model, accessory channels of contaminated clinically used devices, and inoculated endoscope end caps and O-rings. The buildup biofilm model was used to evaluate BAC-VIS after repeated treatment of adherent bacteria with three common high-level disinfectants: glutaraldehyde, ortho-phthalaldehyde, and peracetic acid. Next, BAC-VIS was used to assess clinically used endoscope parts that cultured positive for Gram-negative bacteria. DAPI-stained cells were found on all culture-positive devices, especially in grooves and imperfections on the surface. Finally, BAC-VIS was used to detect bacteria on inoculated endoscope device components. The results showed potential for BAC-VIS to be a valuable tool for industry and academic/medical researchers for investigations of contaminated medical devices. Results obtained using BAC-VIS can increase understanding of the role of design in cleanability, wear, and prevention of contamination and may lead to improvements in materials and design that could make processed endoscope use safer for patients. Of note, this protocol is not for detecting bacteria on scopes or scope parts that will be put back into clinical use.

Systematic enhancement of microbial decontamination efficiency in bone graft processing by means of high hydrostatic pressure using Escherichia coli as a model organism

“…10,[66][67][68] Therefore, future research will analyze the effects of HHP treatment in these Grampositive bacteria, including relevant clinical isolates. Through adaptation of existing in vitro models of biofilm formation, 69,70 the inactivation of planktonic bacteria as well as mature biofilm can be studied.…”

“…Therefore, future research will analyze the effects of HHP treatment in these Gram‐positive bacteria, including relevant clinical isolates. Through adaptation of existing in vitro models of biofilm formation, 69,70 the inactivation of planktonic bacteria as well as mature biofilm can be studied. It is conceivable that further adjustments to the HHP treatment involving additives to the medium or physical methods like sonication, extensive rinsing, immersion in disinfectants, or combinations of these methods are necessary.…”

Dissolvable alginate hydrogel-based biofilm microreactors for antibiotic susceptibility assays

“…To determine if bacterial growth in the microreactor environment was comparable to liquid culture, we compared growth rates of microbes cultured in tubes to cultures in microreactors. Escherichia coli RP437/pRSH103, P. aeruginosa PAO1/pTdK-GFP, and Staphylococcus aureus AH2547/pCM29 were cultured in tubes or microreactors for 24 h. These strains have previously been reported to form biofilm in the literature [ 26 , [38] , [39] , [40] , [41] , [42] ]. Subsequently, samples from tube cultures or samples released through hydrogel dissolution of microreactors were plated to measure viability by colony forming units (Log10(CFU/mL)).…”

“…Biofilms usually form when microbes reach a critical density and produce extracellular polymeric substances through quorum sensing [ 19 , 20 ]. Besides biofilms on hard surfaces such as teeth and medical instruments, in vivo studies have shown that biofilms are also found in small clumps in tissues and often in microcrevices on/around medical devices [ [21] , [22] , [23] , [24] ]. Biofilms can even form in synovial fluid when cells nucleate due to high viscosity [ 25 ].…”

The utility of lighted magnification and borescopes for visual inspection of flexible endoscopes

“…[25][26][27] Researchers from the Food and Drug Administration found gram-negative bacteria too numerous to count in gastroscopes and determined that cells often reside in grooves or other channel defects, which "may be favorable sites for buildup biofilm formation." 28 The clinical implications of such defects have been described by outbreak investigators. One investigation determined that multidrug-resistant Pseudomonas aeruginosa and Klebsiella pneumoniae were harbored in a bronchoscope channel with physical defects and retained soil observed during borescopy.…”