DETECTION OF VIABLE BACTERIA CELLS BY BIOLUMINESCENCE: A BIOENERGETIC APPROACH ¹

Abstract

The cellular ATP content of fourteen freshly harvested bacteria including Bacillus, Campylobacter, Citrobacter, Escherichia, Lactobacillus, Listeria, Pediococcus, Pseudomonas, Salmonella, Streptococcus and Yersinia, was determined using a luciferin-Iuciferase bioluminescence approach. Incubation of bacteria with carbonyl cyanide meta-chlorophenyl hydrazone (CCCP), a membrane protonophore, prior to cell breakage substantially lowered the bioluminescence signals indicating a decrease of cellular ATP content. The addition of CCCP afrer cell breakage had no detectable effect on the ATP levels. This differential effect of CCCP was not observed using heat-killed bacteria, i.e., the ATP content was not affected by CCCP incubation. The CCCP egects on cellular ATP level were detectable in bacterial suspensions with concentrations ranging from 106 to Id CFU/mL. Upon cold storage, the ATP content, but not the population of viable bacteria, decreased. The ATP content could be paaially restored by the addition of glucose. The ATP content restored by the addition of glucose was also sensitive to CCCP treatment. These results demonstrated that viable bacterial cells can be differentiated from dead cells by their responses to membrane protonophores.Mention of brand or firm names does not constitute endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned.

LUMINESCENT METHODS TO DETECT VIABLE AND TOTAL ESCHERICHIA COLI O157:H7 IN GROUND BEEF†

“…So, the effects of a B‐PER were tested. We used B‐PER in our previous studies to release cellular ATP from bacteria (Tu et al. 2004).…”

Future of portable devices for plant pathogen diagnosis

“…3a). 52,53 The final stage of immunological pathogen diagnosis is to detect the binding signal by integrating immunological recognition with numerous detection platforms including antibody-directed epifluorescence microscopy, 54 electrochemiluminescence, 55 fiber-optic sensing 56,57 and surface plasmon resonance. 58 Despite progress on immunological biosensors, their applications in on-site pathogen detection are very slow particularly for plant pathogens.…”

Application of ATP-based bioluminescence technology in bacterial detection: a review

“…Deininger et al 15 proposed a method for the rapid assessment of E. coli contamination in beach water using ATP luminescence. Three years later, Tu et al 16 used this method to determine the cellular ATP content of 14 freshly harvested bacteria, which proved the feasibility and practicability of the bioluminescence method. Compared with fluorescence, bioluminescence does not require external illumination and has a very low background signal, making it suitable for continuous long-term real-time monitoring in live cell cultures with single cell resolution.…”