The Effect of Heat on the Survival of Bacillus megaterium and
Bacillus subtilis Bacteria.
2/24/97
Karen Robins
Title
The Effect of Heat on the Survival of Bacillus megaterium and Bacillus
subtilis Bacteria.
Introduction
I am trying to determine at what temperature bacteria dies. After working
with bacteria for a few weeks, I believe that bacteria will die at 100°C. The
reason why I think this is because 100°C is the point of boiling and its the
point that I've always been told that bacteria dies off at. For example,
whenever there has been water contamination, people have said that you should
boil water and then it will be safe to drink.
Methods and Materials
To start my experiment, I took four liquid nutrient broth cultures that were
in medium sized test tubes and were resting in a medium sized beaker. The broth
was of a yellow color and was filled up about halfway in each test tube. Each
test tube had a metal cap or a piece of aluminum foil covering it to make sure
that the nutrient broth would not be contaminated. I then took a source culture
of megaterium. The source culture was a petri dish that contained nutrient agar,
which looks like a clear jello substance. Spread out on and around the agar is
the ivory colored substance of Bacillus megaterium. With both the test tubes and
source culture nearby, I lit the Bunsen burner and stuck my metal wire loop
through the flame for a few seconds until the entire loop gave off a red hot
glow, which let me know that any substance that had previously been attached to
the loop was now burned off. I stuck the loop through the flame up to handle
part to be extra cautious. I was now ready to transfer bacteria. I opened the
top part of the agar plate part way because I didn't want to contaminate the
source culture with substances from the air. I then took my sterile loop and
scraped over the areas where the bacteria was growing. I pulled my loop out and
closed the petri dish. With my bacteria infected loop in one hand, I opened the
top to one of the nutrient broth test tubes and with my other hand, stuck my wire
loop into the broth and swirled my loop around for a few seconds to mix the
bacteria and broth. After I pulled my loop out, I quickly replaced the top of
the test tube to avoid contamination. I also labeled the test tube with the type
of bacteria, the date, and my name. I then put my wire loop through the Bunsen
burner flame again to get rid of any excess bacteria so it would again be
sterile. I repeated this process three more times. So in total, I had four test
tubes with megaterium in nutrient broth. I put all these test tubes into a
beaker and placed the beaker into the incubator at 30°C.
The next day I checked on my test tubes and discovered that there didn't
appear to be any growth because I didn't see any sediment. I then took a source
culture of Bacillus subtilis and repeated the transferring process, using new
broth cultures that would then be incubated. After a few days when I had
observed growth in both types of bacteria test tubes, I took the cultures out of
the incubator. I then took out a hot plate and placed a beaker of water on it
with a thermometer in the water. I heated the water to 50°C. Then for ten
minutes, I heated a test tube of megaterium and a test tube of subtilis. When
the ten minutes was up, I removed both test tubes and placed them in a beaker. I
took out a new agar dish and drew a line down the middle with a marker to have
both the subtilis and megaterium substances on the same plate but separated. I
then lit the Bunsen burner again and heated my wire loop thoroughly until it was
red hot. I opened the top to one of my heated test tubes and stuck my sterile
loop into the broth and stirred it around a bit to make sure that it would be
coated with the broth/bacteria substance. I opened one side of the new agar dish
part way and stuck my loop, with the substance on it, inside and swirled it
around in an "S" shape on the surface of the agar. After I had sufficiently
covered the area, I took my loop out of the dish and closed the lid quickly to
avoid any other substances coming in. I took my loop and put it through the
Bunsen burner flame until it was red hot and I was sure that all other substances
were killed. I then labeled the outside of the petri dish with the name of the
substance, the date, my name, and the temperature it was heated at and the amount
of time it spent at that particular temperature. I repeated the process with the
other substance that was heated at 50°C for ten minutes. I then took the petri
dish with both substances on it and placed it in the incubator at 30°C. After a
day, I would take the petri dishes out of the incubator and record results. This
would mean seeing how well the colony grew or if the culture grew at all. I
would then put the dishes back in the incubator again to see if further growth
might occur. After two days, I would permanently take the dishes out of the
incubator and leave them in my drawer. I then would repeat the process again
except that this time I would use new broth cultures with the two different types
of bacteria that I had made. I would put the two types of bacteria/broth test
tubes in a beaker of water for ten minutes on a hot plate with a thermometer to
measure the temperature One set would be at 75°C and another set on a separate
hot plate and beaker of water at 100°C. I then would repeat the entire process
over and over again at the three different temperatures of 50°C, 75°C, and 100°C
but this time I would change the length of time that the test tubes would be
heated for. The time variables would be ten, twenty, and thirty minutes. I made
one full set at ten minutes. I only heated the 100°C for twenty minutes. I made
two full sets for thirty minutes.
Results
The first part of my result process was in the growing of the transferred
bacteria from the source culture to the nutrient broth. What I first found was
that one day of incubation was not enough time for the transferred bacteria to
grow. I did not realize this and thought that the megaterium was not going to
grow because I did not see any sediment. So I tried subtilis as another bacteria
to use for my experiment because in our previous lab experiment, subtilis had
grown quite nicely. I waited over the weekend, for both types of bacteria to
grow. When I had growth in test tubes from both types of bacteria I started my
experiment.
The results of my streak test varied because I heated the test tubes at
different temperatures for different lengths of time. For ten minutes at 50°C, I
recorded a large amount of growth with the megaterium and a moderate amount of
growth for the subtilis. At 75°C for ten minutes, I found no growth on the
megaterium side and a moderate amount of growth on the subtilis side. At 100°C
for ten minutes, the megaterium had some distinct colonies and the subtilis had a
moderate amount of growth. For twenty minutes, I only did a streak test at
100°C. The megaterium had a small amount of growth and the subtilis had seven
distinct colonies. I did two streak tests for thirty minutes. The first test I
discovered that at 50°C, the subtilis and megaterium both had abundant growth.
At 75°C, the subtilis had a large amount of growth while the megaterium had only
a small streak. At 100°C, there were six individual colonies on the megaterium
side, while the subtilis had no growth. The second test for thirty minutes, I
found that at 50°C, the megaterium had a large amount of growth in one big smear.
The subtilis had a small amount of growth. At 75°C, the megaterium had a
moderate amount of growth in individual colonies. The subtilis had two medium
size colonies. At 100°C, there was no growth in either the subtilis or
megaterium.
The table below shows on a scale of 0-2, how much growth I recorded. The X
represents where I did not perform the experiment. The table is divided into the
three different temperatures with the different amounts of time that I heated the
bacteria for. The bar graph on the following page is the average amount of
growth for each separate bacterium, which was calculated from my data table, at
the three different temperatures.
Discussion
Throughout the experiment, I noted a number of things that did not go the way
I had hoped. First, because I was using heat in my experiment, I couldn't work
with the agar dishes because the agar and the plastic dish would melt from the
high temperatures. Instead I had to work with the liquid broth cultures that
were stored in glass test tubes. By using the broth cultures, I had to transfer
bacteria from petri dishes to the test tubes. I then had to wait for the
bacteria to grow. The glass test tubes don't have as much air circulating in
them and since bacteria need air to grow, the rate of growth was slower than
growing bacteria in the agar dishes.
During the process where I had to heat up the test tubes, the temperatures
during the allotted time weren't always consistent. I mean that sometimes the
hot plate would get too hot and 50°C would creep up to 55°C and I would have to
turn the hot plate off and wait for the temperature to come back down to 50°C.
Then when I took samples of the bacteria from the test tubes to grow on the agar
dish, I would have to incubate the dishes. After one day of incubation, I would
have results and I would record them. Then the next day when some dishes that I
hadn't found results for from the first day, would now suddenly have results. So
I would have to change or fine tune my data. Also I found contamination in some
of the dishes. The contamination was either from water or another type of
bacteria. So even though, I tried to be really careful, in a few of my dishes I
wasn't always so successful.
I believe that my results say something about the variables of heat and time
when put together in the right combination. I found a pattern in my results. I
found that the longer I heated the bacteria for, the longer it took for the
bacteria to grow if it did. Also the higher the temperature that heated the
bacteria for, then it was more likely I would get individual colonies instead of
a big smear.
Conclusion
From this experiment I discovered many different things about the high
resistance of bacteria to heat. First of all, at the great temperature of 100°
there still was growth even though, I only found one bacterium that resisted
growth only once out of the two trials. I can't conclude officially that
bacteria can be killed off at the boiling point. I can say that bacteria do have
high resistance to heat, but their growth rate is not as abundant at the higher
temperatures . I mean that the growth process takes longer and there are less
colonies that appear on the streak plate.
Further investigations that I might suggest to enhance this experiment are to
heat bacteria for longer amounts of time and at a variety of different
temperatures. Also using other types of bacteria, such as Escherichia coli and
Sacinae lutea, and comparing results to see which bacteria is the least resistant
to heat.
And this is the second paragraph.
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