Download The effects of disinfecting treatments of UV and heat on the growth

The effects of disinfecting treatments of UV and heat s on the growth level of
Escherichia coli
John Baker
May 9, 2007
Senior Seminar, 401
Tables of Contents
Abstract
1
Introduction
2
Methods and materials
9
Results
14
Discussion
19
Acknowledgment
22
Literature cited
23
Abstract
This study was undertaken to determine the effectiveness between Ultraviolet irradiation
(UV) and heat stress on Escherichia coli species. I hypothesized that the UV treatment would
cause greater mortality rate and inhibit the growth of the colony continuing 24 hours after
exposure. Sixty samples were tested upon, of which 20 were exposed to a black light emitting
wavelength of 365 nm at four times of exposure of 5, 10, 15, or 20 minutes. Twenty samples
were administered to a heat treatment were placed at 50º C ±1º C for four times of exposure at 5,
10, 15, or 20 minutes. The remaining 20 samples were used as a control. The absorbance was
measured for each sample before, after, and 24 hours after exposure to the treatment using a
spectrophotometer. A comparison between the differences in absorbance before and after
exposure to UV and heat treatment were assessed. A two-way analysis of variance (ANOVA)
test revealed no significant differences in the bacteria absorbance due to interaction (p = 0.471),
treatment (p = 0.084), or lengths of exposure (p = 0.071).
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Introduction
There are numerous illnesses associated with being exposed to bacteria. A common type
of bacteria that can inflict humans with a variety of complications is Escherichia coli O157:H7, a
strain that can cause bloody diarrhea, abdominal cramps, and hemolytic uremic syndrome, a
severe and sometimes fatal condition in which red blood cells are destroyed and the kidney is
unable to function properly and fails (United States EPA, 2006). These bacteria are highly
infectious and occupy areas to which humans are exposed. It is present in uncooked ground
beef, unclean surfaces, contaminated drinking sources, and edible plants (United States EPA,
2006). The method of killing bacteria in the household involves antibacterial sprays; however,
their usefulness and ability to kill microorganisms may not be effective. Even worse, these
antibacterial sprays can be harmful to users, since the vapors contain traces of chemicals that can
cause effects such as nausea and vomiting for the user. However, treatments such as heat and
ultraviolet radiation are becoming widely accepted in the sewage industry to kill bacterium
coliforms, without compromising the water with harmful chemical substances (Yoon et al.,
2004). UV irradiation has been utilized in European sewage treatment facilities for years, but
since its introduction domestically, it is now becoming the norm in treating contaminated waters
(Yoon et al., 2004). It is known that bacteria exposed to irradiation of 300 nanometers (nm) will
often die, mutate, or grow more slowly (Ramabhandran and Jagger, 1976).
Ramabhandran and Jagger (1976) tested the inhibition of growth of E. coli samples by
exposure to a narrow spectrum of ultraviolet wavelength at 320 nm. They proposed that
irradiation of the tRNAs (transfer ribonucleic acid) of the bacteria would reduce the absorption
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of amino acid below the normal amount needed for proper body function, leading to an inhibition
in E. coli growth (Ramabhandran and Jagger, 1976). They tested this hypothesis using two
strains of E. coli: K-12 and Br/r, and exposed them to two General Electric T- 15 BLB, black
light blue, which produces the 320 nm wavelength (Ramabhandran and Jagger, 1975). Both E.
coli K12 and Br/r samples were inoculated on Petri dishes and kept in an incubator at
temperatures of 37° C and 30°C, respectively. Each sample was irradiated for 10 minutes using
the BLB set at 320 nm. To determine the amino acid intake of the tRNAs of the E. coli K12 and
Br/r after irradiation, 100 microliters (μl) of both samples were taken from the Petri dish, mixed
with 100 μl 2 M formic acid, and centrifuged to remove dead cell debris. Afterwards, the
remaining substances were placed on chromatography paper and exposed to medical x-rays for
1-2 weeks, which showed if the amino acid intake was lower than normal by measuring the
distance it moved from the origin placed on the paper (Ramabhandran and Jagger, 1976). The
results showed that the amino acid intake of the tRNAs in both K12 and Br/r samples were less
than the amount needed for proper function, and growth rates inhibited. However, inhibition was
found to be only temporary as the Br/r sample was observed to experience gradual growth and
achieved normal growth in the range of 50-100 minutes (Ramabhandran and Jagger, 1976).
Furthermore, the inhibition in the K12 sample was observed to be shorter than the Br/r observed,
with normal growth occurring in 20 minutes. The occurrence of the samples obtaining normal
growth patterns was attributed to the length of time to the treatment as the duration was not
sufficient to induced permanent damage, and favorable conditions outside of the treatment was
able to stimulate tRNA to regain normal function (Ramabhandran and Jagger, 1976). The
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treatment administered by Ramabhandran and Jagger (1976) was limited because there was only
one length of exposure. If several times of exposure were used, it would be able to differentiate
any effects UV irradiation may possess or suited for the disruption of proper functioning of E.
coli to maintain stability, and overall increased inhibition.
Webb and Brown (1982) conducted a similar experiment on K12 AB2480, a radiationsensitive variant E. coli, from exposure to black light blue (BLB) and monochromatic light. The
BLB produced wavelengths in the mid-UV range, while the monochromatic light produced
wavelengths of near-UV (Webb and Brown, 1982). They examined the effects of irradiation by
BLB and monochromatic wavelengths for two specific purposes: which wavelength had a greater
influence in reducing the E. coli population; and to observe if biological abnormalities would
occur in the samples if the cyclobutylpyrimidine dimers (CPD), a mechanism in many bacterium
species that allows cell repair caused by UV radiation, was induced with UV radiation for 30
minutes, the highest exposure time. The BLB test was conducted using twelve 4-W light bulbs
surrounding a 5 cm square chamber in which a 1 cm Pyrex vessel of E. coli K12 sample was
placed. The vessel was constantly circulated with 15% ethanol in order to maintain a constant
temperature throughout irradiation (Webb and Brown, 1982). The monochromatic lamp was set
up differently from the BLB experiment as the lamp was filtered by absorption papers of CuSO4
and CoSO4, which reduced the original wavelengths into the range of 380-400 nm (Webb and
Brown, 1982). The samples were then irradiated for a length of 30 minutes in each treatment.
The results compared mortality rate of the E. coli between the BLB and monochromatic
treatments. They found that mortality was higher in the BLB treatment, than samples exposed to
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the monochromatic treatment between the ranges of 20-30 minutes. However, at 30 minute
exposure time, nearly the entire population of the E. coli was killed in both BLB and
monochromatic treatments. Their secondary findings showed that the E. coli that survived the
irradiation in both treatments had higher than normal counts of cyclobutypyrimidine dimers.
Due to a sudden increase in dimers, there were abnormalities present in the E. coli sample
genetic structure, with damages including DNA single-strand breaks, alkalilabile bonds, and
pyrimidine glycols (Webb and Brown, 1982). This experiment shows that the effect of two
different wavelengths was capable of producing detrimental and significant mortality rate in E.
coli. However, BLB was more effective in killing E. coli at shorter duration of time then
monochromatic. It is in the best consideration that I implement a black light in order to obtain
results between treatments that are most, assumed, effective in killing E. coli.
Induced heat treatments on E. coli species in the food industry is a concern for all
businesses that sells or serve beef and poultry, considering that they would suffer if an E. coli
outbreak were to occur and affect their consumers. Yuk and Marshall (2003) investigated the
effects of heat shock and heat-adapted E. coli resistance, lipid membranes, and verotoxin. They
cultivated enterohemorrhagic E. coli 0157:H7 from the American Type Culture Collection (Yuk
and Marshall, 2003). The control cultures were grown on slant agar plates using Trypticase soy,
and placed in an incubator for a total of 36 hours (two-intervals of 18 hours) at 37º C (Yuk and
Marshall, 2003). The heat shocked working cultures were created by measuring an E. coli
population of 10 8 CFU/ml (colony forming units), and exposing them to temperatures of 42º C
and 45º C in temperature-controlled water baths. The heat-shocked cells were then transferred in
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closed bottles, and placed in pre-warmed water at a temperature of 57º C for duration of 18
minutes. To determine the membrane composition after exposure, the samples were centrifuged
for 5 minutes at 3,800 x g, the gravitation force exerted by the machine onto the test tubes, and
transferred into test tubes with the addition of distilled water, 150 ml methanol, and 45 grams of
NaOH (Yuk and Marshall, 2003). The result shows that the heat shocked samples had higher
resistance to the treatment than the controlled samples, because they were able to acclimate in
temperatures that exceeded their optimal growth range but not detrimental (Yuk and Marshall,
2003). For the lipid membrane composition in the heat shocked samples, they did not fine an
increase or decrease in fatty acids, or injury to the integrity of the cell. This could be attributed
to the fact that the treatment influenced has also extended to the cell membrane of the E. coli,
resulting in resistance to treatments that solely affects or penetrates the membrane. The findings
of Yuk and Marshall (2003) exemplifies that resistance to heat stress could be achieved in
bacteria, however, the means of acquiring the resistance was pre-disposed through prolonged,
and, non-detrimental environmental conditions.
The use of UV light has become a popular and ecologically friendly component in
eliminating waste and converting contaminated sewage water into reusable water, which could
aid countries facing water shortages. In South Korea, Yoon et al. (2004) conducted an
experiment to determine if UV irradiation is an effective disinfecting treatment of E. coli
coliforms, bacilli that inhabit intestinal tract of humans and mammalians, and if the treated water
could then be use for agricultural purposes. This study was performed in Yangpyoung, Korea, at
a sewage treatment facility. The effluent (sewage discharge) was transferred from the facility
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pipes into a separate tank via pipes created by Yoon et al. (2004). The separate tank was fitted
with two chambers, with one chamber used to hold the lamps for irradiation, and the other
chamber used for observation (Yoon et al., 2004). They tested if effluent flow rate affected the
amount of coliforms being irradiated; flow rates of 10, 20, 30, and 40 m³/ day were used. They
used three lamp intensities of 17, 25 and 40 florescent watts to see which was most efficient in
the disinfection of the coliforms (Yoon et al., 2004). The data measured and compiled
determined whether flow rate, intensity of the lamp, or the exposure time had the most influence
on the disinfection of the E. coli coliform. The results showed that none of the flow rates had
any impact on E. coli coliform exposure, but the intensity of the lamp contributed the most in
reducing coliform numbers. The amount of coliform removal was different among the lamp
intensities: intensities of 17 and 25 watts had removal of 100 MPN (most probable number)/100
ml of colonies in a 60 second irradiation, however, the 40 watt intensity had complete coliform
removal (Yoon et al., 2004). Yoon et al. (2004) determined that intensity of 40 watts at short
exposure time was more lethal to coliforms than lower intensities for longer exposure times
(Yoon et al. 2004). Although the results show that UV irradiation was effective in disinfecting
E. coli in the effluent, residuals of dissolved organic material (DOM), total suspended solids
(TSS), and other variables that are also reside in effluent were not affected by UV irradiation,
and can affect the treatment by reducing the intensity of the radiation. Since
cyclobutylpyrimidine dimers allows the bacteria to repair cellular damage by UV radiation,
coliforms that are weakened by UV irradiation can repair themselves compromise the water for
agricultural use (Yoon et al., 2004). However, Yoon et al. (2004) believes that the disinfected
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water that contained some numbers of E. coli coliforms could still be used for agricultural
purposes, because the E. coli weakened stage, during which could be quickly transferred to an
agricultural field. Since agricultural fields are exposed to UVA radiation from the sun, it would
be lethal enough to kill the remaining weakened coliforms in the water. It sounds reasonable, but
it is speculation and has not been tested. This study is very useful, as the data supports the Yoon
et al. (2004) hypothesis that ultraviolet radiation, a cheap and environmentally friendly resource,
can be used to sanitize sewage discharge for agricultural purposes. It is also very helpful in my
research, as UV irradiation should have a pronounced detrimental effect on E. coli population
over a short amount of time.
A study by Fiksdal and Tryland (1999) observed the severity of cell injury in E. coli after
applying three different types of stresses: UV light, heat, and starvation. The first two stressors
are important as they are the most used conventional means of disinfecting E. coli in sewage
facilities, and are potential alternatives to antibacterial sprays in used in household kitchens. The
main stock for this experiment was obtained from a sewage facility and was grown on tryptic soy
agar (Fiksdal and Tryland, 1999). A 15 watt germicidal lamp was used for irradiation with a
dose of 254 nm. The E. coli was placed in Petri dishes and then irradiated for lengths of 30
seconds to 3 hours (Fiksdal and Tryland, 1999). The heat stress experiment was performed with
the E. coli samples placed in water-baths at 63° ± 1° C for a period of 0-30 minutes (Fiksdal and
Tryland, 1999). They found that UV irradiation of 254 nm at low doses caused little cellular
damage to the E. coli sample. However, prolonged exposure to 254 nm showed that the E. coli
sample experienced cellular damage and many of the internal structures of the sample were
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unable to work properly or failed completely (Fiksdal and Tryland, 1999). The results of the
heat stress experiment were similar to those found in the UV irradiation experiment, however,
the damages and internal structures were much more severely damaged after a one-time exposure
of 20 minutes. Fiksdal and Tryland (1999) compared the UV irradiation and heat stress
experiment data with one another and found that both were able to suppress damage and growth
in E. coli. The only difference between the two treatments was that it required longer doses of
UV irradiation to have the same lethality in E. coli sample as those exposed in the heat stress
experiment of 20 minutes. The development of my research hypothesis followed Fiksdal and
Tryland’s (1999) own experiment, in that I compared the effectiveness between UV and heat
treatment on E. coli samples.
E. coli is a bacteria that is a health concern because it can be found in undercooked food
or a contaminated water supply. Although heat is a conventional means of killing E. coli,
ultraviolet radiation has become increasingly popular and is being considered as an alternative
means of killing E. coli in the food industry. My hypothesis was that exposure of E. coli to a
black light of near-UV 365 nm would result in greater mortality and a reduced growth rate than
the heat stress treatment. My experiment exposed E. coli to three treatments; UV irradiation,
heat, and a control, which was used to compare the survival and growth of E. coli.
Methods
Preparing E. coli
The bacteria studied were Escherichia coli K12, as this strain is non-pathogenic, while
retaining physical and chemical characteristics similar to natural pathogenic E. coli (Thomas and
Favre, 1980). E. coli K12 was purchased from Carolina Biological Supply Company. However,
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due to culturing problems I substituted the purchased stock to an E. coli stock culture that had
been prepared by another Saint Martin’s University biology student. Using this culture, I created
a stock from which I created 60 working samples. The nutrient broth, manufactured by Ward’s,
was used as a medium in the population growth of E. coli. It was prepared by mixing 8 grams
for every liter of water. Each test tube was filled with 15 ml as it allowed for each culture to be
tested for three separate spectrophotometer, spectronic model 333183, readings, as a single
cuvette required approximately 4 ml. These samples, prior to inoculation, were autoclaved,
Tuttnauer model 2540E, at 121° C for 15 minutes at 15 psi. A micropipette was used to pipette
50 µl of the stock culture into each working sample. Between each inoculation, the stock tube
was shaken to eliminate E. coli clumping.
Each sample was exposed to one of three treatments: UV irradiation, heat stress, or
control (Table 1). The UV treatment exposed the samples to radiation at a wavelength of 365 nm.
This treatment was to test my hypothesis that at this wavelength, variables of proximate distance
and time of exposure would result in counts of higher mortality rate and decreased ability for the
bacteria to repair itself.
The heat treatment involved using a temperature-control oven. The working
samples were exposed to 50° C, and the results were compared against the results found in the
UV treatments in terms of which had the highest mortality of E. coli.
The control was exposed to the fluorescent lights and a room temperature of 22°C of the
laboratory room 402, at Saint Martin’s University. This was used to determine the population
growth in normal conditions, and to further differentiate the influence each treatment, heat or
10
UV, had on the mortality rate of the E. coli samples.
Each treatment exposed E. coli samples to 4 lengths of exposure, with 5 working samples
in each time frame (Table 1). The total number of samples was 60 across all three treatments.
Since 5 working cultures were exposed per length of exposure, I replicated this experiment three
times for a total of 180 samples (Table 1).
Table 1. Five test tubes of E. coli samples were exposed to 1 of 3 treatments: UV irradiation,
heat stress, or control. These were exposed for a certain length of time of 5, 10, 15, or 20
minutes for each treatment.
Treatments
5 mins
10 mins
15 mins
20 mins
UV irradiation
5
5
5
5
Heat
5
5
5
5
Control
5
5
5
5
After inoculation, the 60 working cultures were placed in an incubator for 48 hours at 37°
C, as this is the optimum temperature to promote growth and obtaining adequate population
density for E. coli (Webb and Brown, 1982). The spectrophotometer was calibrated prior to
measuring the absorbance rate of the samples. The machine was zeroed to 0%, and the
wavelength was set to 686 nm. This wavelength was chosen because it is the standard in
measuring optical density of bacteria (Brown, 2005). A blank cuvette consisting of sterilized
nutrient broth was set in the spectrophotometer, and the transmittance of light was set to 100%.
After calibration, a cuvette filled with 4 ml of the working sample, before and after exposure to
treatment, was placed in the spectrophotometer. However, since dead bacteria are able to absorb
11
light, measuring the absorbance rate did not enable me to know whether the bacteria were alive
or dead. To avoid this problem, the working samples, after exposure, were untouched for a
period of 15 minutes for settling to occur. I pipetted the top layer of the sample into the cuvette
for testing, assuming that the dead bacteria had settled to the bottom of the test tube (Professor
Gregory Garman, personal communication, February 2007).
Ultraviolet Irradiation
Twenty working samples were exposed to a wavelength of 365 nm using a black light.
This wavelength was chosen because its effects on E. coli have not been extensively researched
compared to other wavelengths such as 254 nm (Oguma et al., 2005). I used the UV machine,
spectroline model CC-80, in room 408 of Saint Martin’s University laboratory. I was able to
leave the test tubes in a confined and protective setting, with the UV light exposing the entire
culture. After exposure, the absorbance rate of each sample was measured using the
spectrophotometer. The samples were placed in the incubator for 24 hours at 37° C to observe if
the growth would be inhibited or increased, which would test whether the effects of inhibition
were temporary. I measured the absorbance rate of the samples after 24 hours using the
spectrophotometer. This was repeated for a total of four
times at lengths of 5, 10, 15 or 20 minutes of UV exposure.
Exposure to Heat
Twenty working cultures were exposed to a 50° C environment using a Boekel Scientific
oven. The temperature was fixed and maintained to ±1° C. After exposure to the treatment, I
measured the absorbance rate of the samples using the same model spectrophotometer. The
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working samples were then transferred to the incubator for 24 hours at 37° C to test for inhibited
growth. After 24 hours, the samples were measured using the spectrophotometer. This
treatment was repeated for a total of four times at lengths of exposure of 5, 10 minutes, 15
minutes or 20 minutes.
Control
The control consisted of 20 working cultures that were situated in an environment
consisting of fluorescent lights and a room temperature of 22.1 ±1° C, in laboratory room 402 of
Saint Martin’s University. The absorbance rate for each sample was measured before and after
treatment. The samples were placed in the incubator for 24 hours at 37° C to observe inhibited
growth and measured for absorbance using a spectrophotometer. This was conducted for
exposure of 5, 10, 15, or 20 minutes of exposure.
A positive control experiment was established at the end of the primary experiment. The
positive control was the autoclave. Ten samples of E. coli, through the same exact procedure as
stated above, were inoculated and autoclaved at 15 psi, 121º C, for 15 minutes. After
autoclaving was done, the samples’ absorbances were measured. Approximately 1 ml of the
each treated sample was re-inoculated into 10 ml of nutrient broth via pipette. These were
placed for 24 hours in the incubator at 37º C and after 24 hours, the samples absorbances were
measured to test for growth of the bacteria.
Statistical Analysis
I analyzed the data using a two-way Analysis of variance (ANOVA), comparing the three
treatments and length time of exposure against one another as the two main factors (Minitab,
13
2005). If differences were seen among the independent factors, confidences levels were
examined to compare treatments.
Results
In Figure 1, comparisons of the three treatments showed positive growth after exposure to
treatments within all lengths of exposure. The growth range for the control from 5 minutes was
0.0042 to 0.0622 at 10 minutes of exposure. The range in absorbance at 15 minutes is 0.0105 to
0.535 at 20 minutes (Figure 1). Comparison of the absorbance in the control showed that
absorbance increased the highest during 10 and 20 minutes length of exposure, while growth at 5
and 15 minutes length of exposure had the lowest absorbance growth. The samples after
exposure to the heat treatment experienced positive growth after exposure to all four lengths of
time. The absorbance, at 5 minutes of exposure, ranged from 0.196 to 0.0206 at 10 minutes of
exposure. The absorbance at lengths of exposure from 15 to 20 minutes, ranged from 0.0382 to
0.0246 (Figure 1). The absorbance at 5 minutes was the lowest of the four lengths of exposure,
and the absorbance growth for samples increased gradually at 10 and 15 of exposure; however, at
the absorbance decreased at 20 minutes of exposure. In the UV treatment, the absorbance had
showed positive growth during lengths of exposure of 10, 15, and 20 minutes. The absorbance
ranged at 5 minutes of exposure at 0 to 0.04 at 10 minutes, and at 15 minutes the absorbance
ranged from 0.0286 to 0.0274 at 20 minutes (Figure 1). The absorbance in the UV treatment had
shown the highest absorbance at 10 minutes of exposure with longer exposure times showing
gradual decrease in absorbance at 15 and 20 minutes of exposure.
14
0.1
0.08
Absorbance
0.06
0.04
Control
Heat
UV
0.02
0
five
ten
fifteen
twenty
-0.02
-0.04
Exposure time to treatment (min)
Figure 1. Mean absorbance of E. coli samples exposed to control, heat, and UV treatment over
four lengths of exposure. Each bar represents 5 samples from a total of 60 samples measured.
Error bar represent standard error of the mean.
15
Figure 2 shows 10 samples absorbance in the range of 0.107 to 0.382; sample 2 showed
higher than normal absorbance perhaps due to a white, thin film present in the cuvette which
may have amplified the absorbance.
0.45
0.4
0.35
Absorbance
0.3
0.25
0.2
0.15
0.1
0.05
0
0
2
4
6
8
10
12
10 sam ples before exposure to treatm ent
Figure 2. Absorbance of ten samples before exposure to positive treatment. Each point
represents an E. coli sample.
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Figure 3 shows that after exposure to the treatment, the absorbance range for sample 1
through sample 10 0.182 to 0.249. Comparison of Figure 2 to Figure 3 indicates an increased in
absorbance, and the position of the points to be held in a similar fashion.
0.3
0.25
Absorbance
0.2
0.15
0.1
0.05
0
0
2
4
6
8
10
12
10 sam ples after exposure to treatm ent
Figure 3. Absorbance of ten samples before exposure to autoclave treatment at 121° C, 15 psi,
for 15 minutes. Each point represents an E. coli sample.
17
Figure 4 shows the first 5 samples ranged in absorbance from 0 to 0.02, and the other 5
samples have absorbance still in the range of 0.177 to 0.219.
0.25
Absorbance
0.2
0.15
0.1
0.05
0
0
2
4
6
8
10
12
10 samples 24 hours after exposure to treatment
Figure 4. Absorbance of ten samples over a period of 24 hours at 37° C in an incubator. Each
point represents an E. coli sample.
Table 1 shows the result of a two-way ANOVA test on the absorbance of the treatments
and time of exposure the samples were exposed. There was no significant interaction between
treatment and time (F = 0.95; d.f. = 6; p = 0.471). The factor which consisted of the control,
heat, and UV, were shown to have no differences between one another on the mortality rate of E.
coli (F = 2.61; d.f. = 2; p = 0.084). Also, there was no significant difference due to times of
exposure for the E. coli samples (F = 2.50; d.f. = 3; p = 0.071).
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Table 1. Interaction is the treatments and lengths of exposure had on E. coli samples. Treatment
represents the control, heat, or UV irradiation influenced on the samples. Time refers to four
lengths of time at 5, 10, 15, or 20 minutes of exposure to each treatment.
Source
Interaction
treatment
time
Error
Total
DF
6
2
3
48
59
SS
0.0041039
0.0037681
0.0054164
0.0346856
0.0479740
MS
0.0006840
0.0018841
0.0018055
0.0007226
F
0.95
2.61
2.50
P
0.471
0.084
0.071
Discussion
Contrary to my hypothesis, there was no significant difference between the effects of UV
irradiation and heat treatment on E. coli. Furthermore, there was no difference in E. coli
mortality among different lengths of exposure (Fig. 1). In contrast to my expectations, the
bacteria absorbance after exposure to either treatment increased. However, the absorbance in the
samples after 24 hours of incubation was observed to had decreased. This pattern was observed
to be inherent in both samples exposed to either UV or heat treatment.
The increased in absorbance after treatment could be attributed to the spectrophotometer.
It was assumed that the spectrophotometer was displaying the absorbance of both living and
killed bacteria cells transferred from the test tube to the cuvette. To test this basis, a secondary
test using an autoclave as a positive control was conducted. Ten samples were exposed to
mortality induced temperatures of 121° C via the autoclave on the same E. coli species used in
the primary experiment. The absorbance measured after exposure to the treatment was found to
have increased (Fig. 3). The samples were then inoculated into new nutrient broth and incubated
for 24 hours; it was speculated that measurements from the spectrophotometer should be zero on
the account that the bacteria cells were killed during treatment and unable to replicate. The
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findings of the first five samples were expected, as the readings ranged in the values of 0.00%0.02%. The absorbances of the remaining 5 samples were in the ranged of 0.177 to 0.219;
similar to the absorbance measured prior to inoculation into new test tubes (Fig. 4). The
absorbance in the first five samples was 0% because the bacteria cells were killed during the
treatment, and the cell membrane deteriorated, leaving no intact cells to be measured. However,
the absorbance found in the remaining 5 samples may have occurred because the cell membrane
did not deteriorate unlike those in the first 5 samples, and bacteria cells were intact for
measurement for the spectrophotometer. It can be assumed that the spectrophotometer inability
to distinguish between dead and alive bacteria cells is viable on the basis that readings should
have been zero or near zero since the autoclave temperatures were lethal for E. coli species.
The increased in absorbance for the samples could have been attributed to the changing
environmental condition. As the temperature intensified during each treatment, metabolic rates
for the E. coli samples would experience accelerated metabolic rates. During the allotted
exposure time, the original E. coli population would undergo simultaneous spontaneous
generations, resulting in additional bacteria cells which could have led to the absorbance
increasing after exposure to the treatment (Professor Gregory Garman, personal communication,
April 2007). This behavior is likely to occur during stressful environmental conditions for many
species of bacteria. The bacterium would have a heightened metabolic rate and then experience
a free-radical burst, a condition in which the bacteria commit suicide by releasing spores to
create new generations, which would increased the overall population and is known to make the
bacterium resilient to the detrimental condition they were produced in (Xiaoyun et al., 2004). As
20
a consequence, the E. coli species were in stressful conditions that were beneficial and stimulated
colony growth and the ability to spawn resistant generations
Although absorbance increased after exposure to either treatment, there were decreased in
absorbance after 24 hours of incubation seen in samples exposed in both UV and heat treatment.
This decreased could have occurred because the doses of the treatments were effective in causing
long-term damage to the cellular membrane of the E. coli from which repairs were improbable,
and death within 24 hours (Hazan et al., 2004). It should also be re-addressed that bacteria that
were killed during the treatment had intact cell membranes that were able to be measured by the
spectrophotometer. The injury to the cell membrane in some individuals of the colony
deteriorated within the 24 hours incubation period, while the cell membrane of other individuals
of the colony cell membrane did not deteriorate within the allotted time.
Due to these results, my hypothesis is currently inconclusive. I am not able to accept or
refute it due to the complications that occurred during my experiment and obscured the data. It
is in my best interest in future replications of this experiment not to rely on the
spectrophotometer, and I recommend using agar plates to examine and culture treated samples.
This would allow me to assess physical characteristics brought on the bacteria due to exposure to
the treatment, and I could count colonies through visual observation. A number of disinfecting
methods should be included such as several light intensities, and comparison of black light to
monochromatic light to determine if one is more effective than the other. Different species of
bacteria should be use to test whether UV or heat treatment is much more effective in killing.
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Acknowledgements
I am happy to thank Dr. Mary Jo Hartman and Dr. Margaret Olney for their support
during my research, analysis of the data, and the extensive editing of my paper. I appreciated
Cheryl Guglielmo help during my research in the handling and assisting of the lab equipment. I
would like to thank Dr. Gregory Garman for assisting me in my discussion on the effects of heat
on the metabolic rate of E. coli and Dr. Greg Milligan for providing the spectrophotometer
during my research. I would like to extend my gratitude to the SMU senior seminar class who
were supportive during my research and helpful in the editing of my paper.
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