The overall goal of the following procedure is to experimentally validate the enhancement of the efficacy of gentamicin by visible light on Pseudomona aerogen in solution. This is accomplished by first growing out the experimental bacterial culture. In the second step, the bacteria are illuminated in the presence or absence of antibiotic treatment.
Finally, the samples are collected from the culture over a 24 hour period, and then the colony forming units within the cell culture at each time point are quantified. Ultimately, the effect of the illumination on bacterial susceptibility to elimination by antibiotic treatment can be assessed. The implications of this technique extend toward the in vivo therapy of infections as illumination enhances the effectiveness of the antibiotic treatment.
The Demonstration of the procedure will be done by Anna Resnick. A master student in my laboratory Begin by growing out the bacterial culture of interest in the appropriate growth medium. Here the gram-negative P aerogen strain, PAO one is being cultured in luria broth at 37 degrees Celsius free teen hours.
Next, centrifuge the cells for five minutes at about 3, 145 Gs and room temperature. Then remove the snat and resuspend the bacteria in 10%Laria broth regrow the cells for another two hours in Lauria broth at 37 degrees Celsius to allow the culture to reenter the stationary phase. Then divide the bacterial suspension into two groups, adding Gentamycin to one of the groups.
Now place each of the cultures onto magnetic stirs and turn them on. Next, split either a continuous wave nd YAG laser or alternatively a Q switched pulsed nd YAG laser into two optical paths to illuminate the cultures when the illumination has begun. Start removing 20 microliter samples from the control and antibiotic treated groups about every two hours for 24 hours.
The next day serially dilute the acquired samples. For example, for this experiment, the bacteria were diluted eight times by a factor of 10 in 200 microliter inoculation volumes. Then plate the dilution on luria broth, agar plates, and incubate the samples overnight at 37 degrees Celsius the next morning.
Determine the number of colony forming units or CFU per plate for each treatment, and then calculate the log reduction in CFU as illustrated here. Note that you denotes the colony forming unit values at each time point precisely at the time of measurement. CFU denotes the colony forming unit and C denotes the CFU found in the control sample at the start time.
Further, the units of the CFU per milliliter equals the number of colonies times the dilution factor divided by the inoculation volume. Here a schematic presentation of the laser-based setup is shown either a continuous wave NDA laser or a Q switched laser is split into two paths using an optical 50 50 beam splitter. Both samples are positioned on a stir.
This image shows the experimental setup in which the laser is split between two tubes in order to eliminate both tubes Under identical conditions. One culture is treated with antibiotics and the other the control is not. The bacteria are then exposed to the lasers for 24 hours.
This graph shows the effective illumination on the growth of p aerogen with a continuous wave laser light or a Q switch laser respectively in the presence or absence of antibiotic treatment in the control, no light exposure samples. There was no reduction in cell viability regardless of whether or not the cultures received antibiotics suggesting that the bacteria are resistant to gentamicin, nor did the treatment with the laser alone affect cell growth. However, in the cultures treated with both the laser as well as the antibiotic bacterial viability was reduced by several orders of magnitude.
The most prominent effect was measured after 24 hours in which the combination of either continuous wave or Q switch laser reduced the viability of the bacterial cells by eight orders of magnitude compared to the antibiotic alone or light alone control groups. These data suggest a solution for the treatment of the antibiotic-resistant bacteria. Thus, the dots in this figure represent the light scattering points for facilitating dual administration of diffused laser illumination and antibiotic treatment through a catheter placed into the bacteria infected tissue.
This final figure of the absorption spectrum around 532 clearly demonstrates that both the bacterial strain p aerogen and the antibiotic gentamicin are transparent at this wavelength, making this wavelength a viable choice for the combination treatment, The presented technique and paved the way for a searchers in the field of microbiology and medicine to explore the efficacy of this technique. Also for biofilms and organ systems. Don't forget that working with pseudomona orgen microorganisms can be extremely hazardous, so proper sterile technique and the sterilized environment and equipment should always be used while performing this procedure.
