title: Laser sterilization of microorganisms
Creators: name: Ward, Glenn David
Publisher: name: University of Glasgow
Year: 1997
Subjects: subject: Biomedical engineering. lasers in medicine, sterilization.

description: The effect of light from a variety of laser sources was investigated in a microbicidal capacity under a number of different conditions. Initially, Escherichia coli, Serratia marcescens, Staphylococcus aureus, Deinococcus radiodurans, Micrococcus luteus, Bacillus subtilis, B. stearothermophilus, Candida albicans and Saccharomyces cerevisiae were exposed to near infrared light, from a high-powered, 1064 nm, Neodymium:Yttrium Aluminium Garnet (Nd:YAG) laser, as lawned cultures on agar plates. The effect of the different Nd:YAG laser operation parameters (pulse repetition frequency, pulse energy, pulse width and beam diameter) were also investigated on lawned cultures of E. coli. A system of quantifying the effect of the laser light on microorganisms lawned on agar plates, which utilised the non-uniform energy distribution of the laser beam, was developed and the different organisms and laser parameters were compared. Nd:YAG laser light killed all microorganisms mentioned above, producing clear areas within the microbial lawns. For each microbial species, a dose/response curve was obtained of area of clearing versus energy density (Jcm-2). The energy density that produced an inactivated/sterilized area (IA) equal to 50 percent of the beam area was designated the IA50-value and was plotted. Average IA50-values were all within a three fold range and varied from 1768 Jcm-2 for Serratia marcescens to 4489 Jcm-2 for vegetative cells of Bacillus stearothermophilus. The differences in sensitivity could not be attributed to cell shape, size, pigmentation, or Gram reaction. The PRF, pulse energy and beam diameter all affected the sterilization capability of Nd:YAG laser on a lawned culture of E. coli. The average IA50-value at a PRF of 10 Hz was 2800 Jcm-2 which was decreased to 2200 Jcm-2 as the PRF was increased to 30 Hz. Similarly, an increase in the pulse energy from 10 J to 30 J enhanced the sterilization efficiency of the Nd:YAG laser on E. coli lawned on agar surfaces with a reduction in the average IA50-value from 2700 Jcm-2 to 1850 Jcm-2 respectively. Variation in the beam diameter was shown to have a large effect on the threshold energy density (the lowest ED required to sterilize an area in the bacterial lawn). Laser light at a beam diameter of 2.75 cm had a threshold energy density of 1400 Jcm-2 which was reduced to 800 Jcm-2, with a beam diameter of 1.25 cm. There was not such a notable effect on the IA50-values with only 200 Jcm-2 separating both of these beam diameters. The pulse width of Nd:YAG laser light did not affect the bactericidal ability of laser light. At the lowest energy densities where sterilization was achieved for the majority of organisms (around 2000 Jcm-2), no effect was observed on the nutrient agar surface, but as the energy density was increased, a depression in the agar surface was formed, followed by localised melting of the agar. Also around the periphery of the cleared areas microcolonies were frequently observed which indicated sub-lethal damage to the microbial cells. Preliminary investigations indicate that these colonies contain auxotrophic organisms.