Investigating the Effects of Terahertz Radiation on Bacillus subtilis
Jillian P. Giles1,2, Brittany J. Raitt3, Cecil S. Joseph1,2, Mark E. Hines3, Robert H. Giles1,2
1Submillimeter
Wave Technology Laboratory, University of Massachusetts Lowell, 175
Cabot St., Lowell, MA 01854
2Department
of Physics and Applied Physics, University of Massachusetts Lowell,
1University Ave., Lowell, MA 01854
3Department
of Biological Sciences, University of Massachusetts Lowell,
1 University Ave., Lowell, MA 01854
Abstract
Medical and security sensing applications of Terahertz (THz) imaging are currently
being developed. As a result, there is a need to further investigate the effects of THz
radiation on biological systems. In this study, a 94 GHz mechanically tuned Gunn Oscillator
was used to irradiate Bacillus subtilis at 94 GHz. The bacteria were cultured in trypticase
soy broth (TSB) and placed in polystyrene 96 well plates. The samples where irradiated
during the exponential growth phase for 1, 2, and 24 hours. Both the experimental and
control plates were kept at room temperature (~25°C) and were monitored for the
duration of the experiment using thermocouples interfaced with a computer via Labview
software. By evaluating the absorption of each well at 600nm immediately before and after
irradiation, the population density within each well was assessed. Following this, the
metabolic activity of each well was measured after irradiation by adding tetrazolium dye,
XTT, to the wells and evaluating the absorption of each well at 490nm after 2 hours of
incubation.
1. Introduction
1.1 Applications of Terahertz Radiation
Biomedical and security applications of terahertz imaging technologies are
currently being developed. In the biomedical field, characteristic terahertz spectra are used
to identify different polymorphic forms of drugs, terahertz pulsed systems are used to
investigate materials and biological samples, and terahertz pulsed imaging is used to detect
tooth enamel thickness and differentiate between tumor and healthy human tissue [1]. The
security applications of terahertz radiation include the detection of concealed weapons,
plastic explosives, chemicals, and dangerous biological agents [2, 3]. Because little is
known about the effects of low power, non-ionizing terahertz radiation on biological
systems, further research is needed to better determine safety standards for the use of
these new terahertz technologies.
Dynamics and Fluctuations in Biomedical Photonics IX, edited by Valery V. Tuchin,
Donald D. Duncan, Kirill V. Larin, Martin J. Leahy, Ruikang K. Wang, Proc. of SPIE Vol. 8222,
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1.2 Overview of Previous Work
It has been suggested that terahertz radiation may impact cell division in biological
organisms [4], thus there is a need to investigate the effects of terahertz radiation on
biological systems. A study examining the genetic and epigenetic effects caused in human
peripheral lymphocytes by low-power, continuous-wave (CW) 0.1 THz radiation led to the
conclusions that long exposure increases the chance of developing cancer [5]. In contrast, a
study investigating the genotoxic effects of 120 to 140 GHz radiation in human peripheral
blood lymphocytes found no chromosomal damage or alteration of cell cycle kinetics [6].
Another study investigating the cellular and molecular response of human dermal
fibroblasts to 2.52 THz radiation at a power density of 84.8 mW/cm2 concluded the effects
exhibited were primarily thermal effects [7]. These research groups, along with several
others, ascertained differing results as to whether or not low power THz radiation has
negative effects on mammalian cells.
Along with examining the effects THz radiation has on mammalian cells, several
other studies have been conducted on the effects this radiation has on bacterial organisms.
A study investigating the effects of 41-43 GHz radiation on Escherichia coli found no
significant differences in the growth rate and absorption spectrum between non-irradiated
and irradiated cells [8]. Similarly, a study examining the response of E. coli in logarithmic
phase to 99 GHz CW radiation concluded that cell viability, colony characterization, and
metabolic activities were not affected by 1 or 19 hour exposures to this radiation [9]. While
these studies found no negative effects exhibited by irradiated E. coli cells, several other studies
found THz radiation may interrupt cell-to-cell communication [10]. and alter the genome
conformational state, processes of DNA and protein synthesis, and the rate at which cells divide
[11].
Based on the differing results obtained by the aforementioned research groups, there is a
need to establish a research protocol for irradiating biological molecules because a large number
of irradiation parameters affect the results obtained experimentally. In developing a research
protocol that effectively addresses the various irradiation parameters, other researchers will be
able to confirm and expound upon prior research.
1.3 Project Overview
The current radiofrequency safety standards given by the International Council on
Non-ionizing Radiation Protection (ICNIRP) [12], the Institute of Electrical and Electronics
Engineers (IEEE) [13], and the U. S. Federal Communications Commission (FCC) [14] are
based on avoiding the short-term, harmful health effects induced by THz radiation on
psychophysical perceptions, such as excitation of nerves and muscles, and on biological
tissues, such as large increases in thermal temperature. Due to the lack of studies
performed using THz radiation, these international radiofrequency standards were
deduced from the spectral regions adjacent to the THz spectral region (0.1 – 10 THz). The
international safety standards for the ICNIRP, IEEE, and FCC are listed in Table 1 on the
following page.
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Biomedical and security applications of terahertz imaging technologies currently
being developed are exposing people to low-power THz radiation for relatively long
periods of time. The international radiofrequency safety standards do not take into account
the possible exposure side effects that low-power THz radiation may have on biological
molecules, such as low-frequency bond vibrations, crystalline phonon vibrations,
hydrogen-bonding stretches, and torsion vibrations. Consequently, there is a need to
further investigate the effects that low-power THz radiation has on biological systems to
better determine safety standards for operating new THz technologies.
The objective of this project was to develop an experimental protocol to investigate
the effects induced in B. subtilis from exposure to low-power 94 GHz radiation for 1, 2, and
24 hours. The power density used was 1.3 mW/cm2 and the temperature of the B. subtilis
cultures was monitored throughout the experiment to ensure exposure to the radiation did
not increase the temperature by more than 1 degree Celsius. Therefore, the effects being
investigated in this experiment were non-thermal.
Table 1: Radiofrequency safety standards given by ICNIRP, IEEE, and FCC.
Occupational Exposure
General Public Exposure
ICNIRP Guidelines
2-300 GHz, 50 mW/cm2
2-300 GHz, 10mW/cm2
IEEE MPE Limits
3-300 GHz, 100 W/m2
2-100 GHz, 10 W/m2
100-300 GHz, increases from
10-100 W/m2
FCC MPE Limits
1.5-100 GHz, 5 mW/cm2
1.5-100 GHz, 1mW/cm2
2. Experimental Setup
2.1 Optical Path Design
The source used to irradiate the biological samples in this experiment was a
Millitech 94GHz Mechanically Tuned Gunn Oscillator. This source was capable of providing
up to 10 mW of power and had a bandwidth of 1GHz. A conical horn was used to shape the
mode of the beam emitted from the source. An optical system was designed to direct the
beam emerging from the source onto the bottom of a polystyrene, 96 well plate (Costar)
containing the biological samples.
The measured beam waist of the terahertz beam emerging from the conical horn
attached to the source was 5.6 mm. This beam propagated 76.2 mm to an 8.89 cm focal
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length, 7.62 cm diameter, off-axis parabolic mirror that focused the beam onto the
biological sample plate. At the sample plate, 13.97 cm from the off-axis parabolic mirror,
the beam waist was 11mm and the power measured was 5.4 mW. Based on measurements
of the Gaussian beam profile and the power measured at the sample plate, the power
density exerted on the sample was approximately 1.3mW/cm2. Figure 1 shows a schematic
of the optical system.
Figure 1: Schematic of optical system.
2.3 Sample Preparation
Bacillus subtilis, obtained from Ward’s Natural Science in freeze dried form, was
grown overnight from 100 μL of frozen culture in 30 mL of trypticase soy broth (TSB). In
the morning, 100 μL were removed, mixed with 30 mL of TSB, and placed in a 37° C
incubator with vigorous shaking. The bacteria were allowed to grow until the culture
measured an optical density of approximately 0.070 at 600nm in the spectrophotometer.
The growth curve for the B. subtilis was performed prior to the start of the experiment to
ensure the cells were at the beginning of the logarithmic phase when irradiated.
At the appropriate optical density, 100 μL of culture was pipetted into each of the
middle 60 wells of two 96 well plates. Both plates were read in the SpectraMax microplate
reader at 600nm using SoftMax Pro software. The experimental plate was then placed on
the irradiating setup and the control plate was placed nearby. The experimental plate was
irradiated for 1, 2, or 24 hours. The temperature of the experimental plate and the control
plate was monitored for the duration of the exposure by inserting thermocouples into well
B6 of each plate.
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3. Results
The population growth of the exposed B. subtilis cells was analyzed by measuring
the optical density of each well in the experimental and control plates at 600nm before and
after irradiation. At 600nm, the amount of absorption was proportional to the number of
cells in the well. The average optical density of the experimental wells was then compared
with the average optical density of the control wells. The intensity graphs in Figure 2 depict
the optical density of each well at 600nm before and after two hours of irradiation. The
average optical density of the experimental wells before and after irradiation was 0.059
and 0.086, respectively. The average optical density of the control wells before and after
irradiation was 0.060 and 0.085, respectively. Using a two-tailed t-test, the difference in the
average optical density between the experimental wells and the control wells after
irradiation was found to be insignificant.
The metabolic activity of the exposed B. subtilis cells was also analyzed. This was
done by adding 50 μL of the tetrazolium dye, XTT, to each well after the experimental plate
had been irradiated. The XTT used, obtained in powdered form, was mixed with phosphate
buffered saline (1mg/mL) and then activated with menadione (mixed with 95% ethanol in
1mM concentration) in a ratio of 12.5 parts XTT to 1 part menadione. After adding the
activated XTT to each well, the plates were then incubated for two hours at 37°C with mild
shaking. A positive outcome meant that the bacterial cells in the well were able to
metabolize the XTT, as indicated by a color change in the suspension. The XTT dye
absorbed best at 490nm, so quantitative analysis of metabolic activity involved measuring
the optical density of each well at 490nm and comparing the average optical density of the
experimental wells with the average optical density of the control wells. The intensity
graphs in Figure 3 depict the optical density of each well at 490nm after the experimental
plate had been irradiated for two hours, and the XTT dye had been added to each well and
allowed to incubate. The average optical density of the experimental wells was 0.957 and
the average optical density of the control wells was 1.101. Using a two-tailed t-test, the
difference in the average optical density between the experimental wells and the control
wells was found to be insignificant.
The temperature of the experimental plate and the control plate was monitored for
the duration of the exposure by inserting thermocouples into well B6 of each plate. Figure 4
is a graph of the temperature of the control plate and the temperature of the experimental
plate during a two hour exposure period. The blue represents the temperature of the
control plate and the red represents the temperature of the experimental plate. As can be
seen, the temperature of the experimental plate was approximately the same as the control
plate. Therefore, any effects observed in the experimental plate were not caused by an
increase in temperature.
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Experimental Plate
Absorption before radiation
Absorption immediately after radiation
Mean OD600nm: 0.059
Control Plate
Absorption before radiation
Mean OD600nm: 0.086
Absorption immediately after radiation
Mean OD600nm: 0.060
Mean OD600nm: 0.085
Figure 2: Optical density of each well at 600nm before and after 2 hours of irradiation.
Experimental Plate
Control Plate
Absorption after XTT added and incubated for 2 hours
Absorption after XTT added and incubated for 2 hours
Mean OD490nm: 0.957
Mean OD490nm: 1.101
Figure 3: Optical density of each well at 490nm after experimental plate had been
irradiated and the XTT dye had been added to each well and allowed to incubate.
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Temperature During Exposure
24.3
24.2
Temperature (°C)
24.1
24
23.9
23.8
23.7
23.6
23.5
23.4
23.3
12:00:00 AM
12:28:48 AM
12:57:36 AM
1:26:24 AM
1:55:12 AM
2:24:00 AM
Time
Control
Experimental
Figure 4: Temperature of the experimental plate and the control plate during 2 hours
exposure period.
4. Discussion
There was a total of 13 exposure trials: 1 one hour, 9 two hours, and 3 twenty-four
hours. The results obtained for exposing B. subtilis to low-power 94 GHz radiation for 1, 2,
and 24 hours revealed no statistically significant differences in population growth and
metabolic activity between the irradiated and the control cells. The room temperature
fluctuations may have affected the results obtained between trials, and therefore the
exposure setup will be moved into an incubator to regulate the environmental temperature
for further exposure experiments. The structure of the bacterium B. subtilis may also have
affected its response to 94GHz radiation, so further investigation into the affects this
radiation has on other bacterial organisms, such as Staphylococcus aureus and Escherichia
coli, will be conducted. There is special interest in E. coli’s response to 94 GHz due to its
large structural differences in comparison with B. subtilis. Future work also includes using
other irradiation frequencies, such as 584 GHz and 1.4 THz, to investigate the response of
bacterial cells to THz radiation.
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5. Conclusions
An optical system was designed to irradiate biological samples, placed in 96 well
plates, at 94 GHz. Initial measurements indicate that the irradiation of B. subtilis at 94GHz
for 1, 2, and 24 hours with a power density of 1.3 mW/cm2 does not affect metabolic
activity or population density. Further investigation with different bacterial specimens and
irradiation parameters is required to determine the affects THz radiation has on biological
systems.
6. Acknowledgements
The authors would like to thank the student researchers at the Submillimeter-Wave
Technology Laboratory, Chapin Johnson, Brian Soper and ThuQuynh Dinh, for their
assistance in conducting this research.
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