In vitro laser ablation of laboratory developed biofilms using an Nd:YAG laser of 532 nm wavelength

Abstract

We studied the laser ablation of laboratory-developed biofilm on titanium and glass surfaces. Specifically, Pseudoalteromonas carrageenovora, a marine biofilm forming bacterium was used to generate laboratory biofilm. Two fluences, 0.05 and 0.1 J/cm(2) and three durations of irradiation, 30 s, 5 min, and 10 min were tested using an Nd;YAG laser of 532 nm wavelength (in the green light area). Nonirradiated coupons with biofilm served as control. The biofilm removal efficiency increased with the increase in laser fluence and duration of irradiation. The maximum biofilm area cover on control coupons of glass and titanium was 62.5 and 76.0%, respectively. Upon irradiation with fluence 0.1 J/cm(2) for the very short duration of 30 s, this reduced to 5.6 and 12.4% and at 10 min to 2.17 and 0.7% on glass and titanium coupons, respectively, while the controls did not show any reductions (62.5 and 76.0% respectively, for glass and titanium coupons). The biofilm TRC (Total Resuscitated Cells) reduction during this period was even more prominent than the area cover, indicating that the remaining biofilm portions on coupons after irradiation were largely composed of dead bacterial cells. The TRC in the irradiation chamber medium for short durations of irradiation showed a significant increase, indicating that the laser irradiation removed live bacteria from the biofilm. The re-growth of the resuscitated cells showed they could grow like the control cells but with a significant lag. The laser's efficiency in the removal of biofilm was better seen on titanium coupons than on glass. Our results showed that a low-power pulsed laser irradiation could be used to remove biofilm formed on hard surfaces.

Critical review on biofilm methods

“…It has also proven useful for quantitative biofilm analysis, especially to confirm findings obtained by quantitative (viable count, CV staining) or other imaging (light microscopy, SEM) techniques (Ansari et al, 2013;Chatterjee et al, 2014;Li et al, 2015;Salunke et al, 2014). Among the characteristics of the sample surface examined, height and roughness analyses from AFM images allow quantification of biofilm biomass in terms of thickness and EPS amount, respectively (Ansari et al, 2013;Chatterjee et al, 2014;Danin et al, 2015;Li et al, 2015;Mangalappalli-Illathu et al, 2008;Nandakumar et al, 2004;Qin et al, 2009;Sharma et al, 2010).…”

Recolonization of laser‐ablated bacterial biofilm

“…Our earlier studies showed that low-power laser irradiation from an Nd:YAG laser resulted in a substantial bacterial, microalgal, and invertebrate larval mortality (Nandakumar et al, 2002a -c). In addition, observations carried out on biofilms also showed that laser irradiation could significantly remove biofilms formed on hard surfaces within a very short period of time (Nandakumar et al, 2003b). According to our earlier studies, laser irradiation from a pulsed laser with fluence 0.1 J/cm 2 removed more than 90% of natural biofilm formed on coupon surfaces.…”

“…They were used as received. Coupon preparation was carried out as explained in Nandakumar et al (2003b).…”

“…Biofilm developed on the coupons after 6 h of exposure was used for the study. A detailed description of the formation of biofilm is given in Nandakumar et al (2003b).…”

Removal of biofilms using carbon dioxide aerosols

“…Many techniques have been developed to control or remove biofilms -electrical fields (Blenkinsopp, Khoury, & Costerton, 1992), laser irradiation (Nandakumar, Obika, Utsumi, Ooie, & Yano, 2004), vibration (Bott, 2000), micro-jets (Bayoudh, Ponsonnet, Ben Ouada, Bakhrouf, & Othmane, 2005), chlorine (Chen & Stewart, 2000) and the like -but all of these techniques have disadvantages. Chlorine is an excellent biocide and relatively cheap, but its oxidation products are generally toxic and may even be carcinogenic (Bott, 2000).…”