In the absence of laser light, L case/ and S. sobrinus were most susceptible to TBO following exposure for one hour. Although AIPCS2 exhibited low toxicity
towards the four target bacteria L easel and A. naeslundii were most susceptible
The screening assay indicated that L case/ was the least susceptible to light- activated killing following sensitisation with TBO and that S. mutans was the most susceptible. However, S. mutans was less susceptible to killing when sensitised by AIPCS2 and exposed to GaAIAs laser light and L. case/ was most
susceptible. The screening assays also suggest that 25 pg/ml TBO and 0.44 J of HeNe laser light were sufficient for killing of all four target bacteria as was 10 pg/ml AIPCS2 and 1.18 J of GaAIAs laser light. However, this may not represent
number of bacteria. Irradiation of the bacteria alone and exposure of the bacteria to the sensitiser alone did not have any detectable effect on bacterial viability. Bedwell et al (1990) used a similar screening technique to test phthalocyanine against Helicobacter pylori and found an energy density of only 1.5 J/cm^ to be successful with a concentration of only 20 pg/ml. However the light source was a copper vapour pumped dye laser at a wavelength of 675 nm (the absorption peak of the sensitiser) whereas in this study the wavelength of laser light used was 660 nm which is not perfectly matched to the absorption peak of the sensitiser and may explain why slightly higher energy doses and sensitiser concentrations are required for successful killing.
Substantial kills (approximately 10^ cfu) of all four target bacteria were achieved with both sensitiser/laser combinations. Variation of the light dose applied to the sensitised bacteria had an effect on the kills attained. Increasing the light dose resulted in an increase in the bactericidal effect. Variations in susceptibility were observed and, on the basis of light dose per cell, S.
sobrinus appeared more susceptible than S. mutans to HeNe laser light and L.
case/ and A. naeslundii were the least susceptible. S. mutans was more susceptible to GaAIAs laser light following sensitisation with AIPCS2 under the
conditions employed in this study. However, direct comparisons are difficult due to unavoidable differences in initial cell density.
Okamoto et al (1992) have also demonstrated killing of sensitised strains of S.
mutans various sensitisers using a HeNe laser as the light source. Initially
they studied various strains of mutans streptococci by pouring suspensions of the bacteria over mitis salivarius agar plates and then exposing these to light from a 6 mW HeNe laser. Inhibitory zones were present in all cases although
when crystal violet and trypan blue, which are found in mitis salivarius agar, were extracted, no effect on the bacteria was observed. Following this a range of dyes were tested on S. sobrinus using mitis salivarius agar plates with both crystal violet and trypan blue removed. 10 of the 17 dyes tested were found to be effective including toluidine blue O, crystal violet and bromophenol blue. Quantitative experiments then demonstrated that S. sobrinus, but not E. coli,
could be killed on exposure to light from a 30 - 40 mW HeNe laser following sensitisation with crystal violet at a concentration of 8 pg/ml. Similar energy densities to those used in this study were required for killing of S. sobrinus. In an early study MacMillan et a! (1966) demonstrated the effective killing of seven species of bacteria following sensitisation with toluidine blue and irradiation with light at a wavelength of 632.8 nm. In the presence of toluidine blue alone, the viability of the cells remained unaffected. In 1977 I to at a! also demonstrated the killing of yeast cells (Saccharomyces cerevisiae) with various sensitisers, of which toluidine blue was one of the most effective, and recently Paarderkooper at a! (1992) have successfully treated the yeast Kluyvaromycas
marxianus with toluidine blue and light as well as with a chloroaluminium
phthalocyanine sensitiser (Paarderkooper at a/, 1995). Wilson at a! (in a number of studies) have also shown TBO and AIPCS2 to be successful
photosensitisers of a wide range of both oral and non-oral bacteria at energy densities comparable to those found succesful in this study with similar concentrations of sensitiser.
For clinical convenience, an ideal photosensitiser would rapidly sensitise the bacteria to light-induced killing. Both AIPCS2 and TBO meet this requirement by
effect of TBO and HeNe laser light on Candidia albicans (Wilson and Mia, 1994) it was seen that the numbers killed increased with increasing pre irradiation time between 1 and 3 minutes but increasing the pre-irradiation time to greater than 3 minutes did not increase the kill. However, in another study concerned with the photosensitised killing of S. aureus, increasing the time the sensitiser was in contact with the bacteria before irradiation did not effect bacterial killing (Wilson and Pratten, 1995).
This in vitro work has shown that cariogenic organisms can be sensitised by a low concentration of photosensitiser to killing by low doses of low power laser light within clinically-acceptable times.
CHAPTER FOUR - EFFECT OF pH ON THE LETHAL PHOTOSENSITISAION OF CARIOGENIC BACTERIA