Bacteria are everywhere. They are in the water, they are in the ground and they are all over you. The vast majority of them are harmless, but certain bacteria produce polysaccharides — complex carbohydrates that tend to aggregate, enabling the bacteria that made them, other nearby bacteria and loose particulate matter to cluster into a sticky, sludgy substance called biofilm.Several species of bacteria develop over time into a sticky substance called biofilm, which disrupts paper manufacturing (left) and fouls up pipes and other structures (right). Courtesy of Angeles Blanco, Complutense University of Madrid, Spain.Slime-forming bacteria are especially problematic in industries such as paper and cardboard processing, where they create masses of biofilm that coat pipes, befoul manufacturing equipment and infiltrate the products themselves, reducing their quality.Treating biofilmTypically, biofilms are treated with antimicrobial agents, but doing so requires knowledge of which bacteria are present among the organisms, saccharides, and other organic and inorganic material that comprise the film. Not addressing the correct bacteria could lead to the overuse of improper biocides. Unfortunately, identifying the organisms that comprise any given biofilm is time-consuming and labor-intensive, usually involving cultivation of biofilm samples in the laboratory prior to analyzing them.Now, however, researchers at Complutense University of Madrid in Spain have combined two well-established methods to develop an analytical technique that permits identification of bacterial species in biofilms. In addition, they used the technique to observe which antibacterial methods are most effective against the organisms typically found in paper mills.The scientists, led by Angeles Blanco of the university’s chemical engineering department, began with fluorescence in situ hybridization — a technique in which fluorescent probes targeted to specific segments of the bacteria’s DNA are excited by laser irradiation and then located via microscopy as they emit fluorescence.As described in the April 2008 issue of Applied Microbiology and Biotechnology, the investigators used two common probes — fluorescein isothiocyanate (FITC) and the iso-thiocyanate derivative Cy3 — to evaluate how effectively four oligonucleotide probes targeted various bacterial species. They added FITC and Cy3 to slime samples taken from the wet end of a board paper machine that used 100 percent recovered paper as its raw material.They illuminated the samples with a 488-nm argon-ion laser or a 550-nm laser, then looked for the subsequent fluorescent emissions with a Bio-Rad confocal microscope. The FITC emitted at 520 nm; the Cy3, at 565 nm.The researchers found that a probe based on enterobacterial repetitive intergenic consensus (ERIC) sequences — which are found in all Enterobacteriaceae species — was successful in all enterobacteria but did not label any other species. Furthermore, they found that probes based on KPN-1 satisfactorily labeled Klebsiella pneumoniae, K. oxytoca and Raoultella planticola. They also observed that adding lysozyme as a permeabilizer increased fluorescence after hybridization did occur.But although fluorescence in situ hybridization showed which probes did or did not successfully bind to their targets, the technique does not indicate whether the bacteria samples are viable — the primary test as to when a biocide is effective. Therefore, the investigators tested several bactericidal treatments, using flow cytometry to test for viability.They used four biocides, a dispersant and an enzyme preparation — all commercially available from the same manufacturer — and tested the effects of 100 ppm of each after 0, 2, 8 and 24 h. They used a flow cytometer made by Becton Dickinson (now BD Biosciences) of San Jose, Calif., along with a 488-nm laser to illuminate bacterial cells stained with propidium iodide and with a green fluorescent nucleic acid stain from Molecular Probes Inc. (now part of Invitrogen Corp. of Carlsbad, Calif.). Analysis of the population size and complexity of each sample quantified cellular viability after each of the six treatments.The researchers observed that two of the biocides were highly effective against all strains of bacteria, one biocide was effective against only one strain, and the fourth was effective against none. Likewise, the dispersant and the enzymatic treatment had mixed results. Because enzymatic treatments are considered environmentally friendly, the investigators tried doubling the amount used on one biofilm sample, finding that it reduced all enterobacteria by 85 percent and essentially eliminated Klebsiella strains.According to Blanco, the group will continue to model the effects of bacterial control products and work to develop online optical techniques that permit real-time analysis of biofilm formation.Contact: Angeles Blanco, Complutense University of Madrid; e-mail: ablanco@quim.ucm.es.