BACTERIA AND TEMPERATURE
In addition to being factor to consider in microbial growth, temperature is also
used as a means of limiting bacterial growth or killing bacteria to pasteurize or sterilize.
Chilling bacteria slows growth down significantly, and subjecting bacteria to high enough
heat kills them. Clearly, the normal growth temperature of the bacteria plays a role,
because pyschrophilic bacteria would prefer to be in the refrigerator and thermophilic
bacteria would prefer to be at temperatures that could be used to prevent the growth of or
kill other bacteria. In this experiment, we are testing the ability of some mesophilic
bacteria to withstand heat treatment at different temperatures. These bacteria all have an
optimal growth temperature somewhere between room temperature and 50 C, so we
expect heat treatment to either temporarily stop their growth or to kill them. Also, the
entire experiment takes place within 30 minutes time, so even if the bacteria could grow
at the temperature used, there is little time for them to grow.
High temperatures wreak havoc on large molecules such as proteins and nucleic
acids. Many weak bonds, such as hydrogen bonds, begin to fall apart as the molecules
jiggle rapidly from the increase in heat. When such molecules lose their three
dimensional shape, they stop working, and often aggregate and tangle irreversibly. Thus
the cell dies.
The rate at which a population of bacteria dies depends on the bacterium, the
temperature, and the time exposed to that temperature. The most useful measure of how
long it will take to kill a population of bacteria is called the decimal reduction time, or
sometimes just the D value. The D value is the length of time it takes to kill 90% of the
population at a given temperature.
Let's suppose the D value for Escherichia coli at 70 C is one minute. That means
every 1 minute E. coli is exposed at that temperature, only 10% of the cells remain alive.
If you graph this, you will see that this is not linear but logarithmic. If there are 1,000
cells to start with suspended in liquid medium in a tube, after 1 minute 90% will be killed
leaving 10% or 100 cells. After a second minute, 10% or 10 cells will be left. After the
third minute, 1 cell, and after four minutes, no cells will be left. Thus to kill all the E.
coli in this tube of medium, you could heat it at 70 C for 4 minutes. Bacteria would die
quicker at a higher temperature, so the D value at 100 C would be less, that is, 90% of
the cells would be killed in a shorter time at 100 C than at 70 C. Note that the D value
does not depend on the number of bacteria; whether there be hundreds or millions, the D
value tells you how long it takes to kill 90% of the cells. That still means that the more
cells there are, the longer it takes to be sure all of them are dead.
When preparing media to grow bacteria or glassware to put them in, we want to be
sure that these things are sterile, devoid of any life, to prevent contamination. The
treatment we use, usually heat, must be sufficient to kill the most hard to kill microbes
present (those that have the highest D values). These are always endospores. Endospores
are resting cells with very tough walls that confer heat resistance. Thus our heat
treatments must be intense enough to destroy all endospores as well as "vegetative"
bacteria. Dry heat at 170 C for 2 hours (an oven) will sterilize glassware. Dry heat is
not useful for liquid medium; the liquid would only heat to 100 C until all the water
boiled away, then gunk left behind would heat to 170 C. For liquid-containing items we
use an autoclave. Ever use a pressure cooker? The principle is identical. The material
being sterilized is subjected to steam under pressure; steam can reach higher temperatures
than water, and the temperature rises to 121 C. Wet heat is much more effective in
killing cells and spores than dry heat, and exposures of 15-30 minutes to hot steam (under
pressure) is enough to sterilize most things.
Sometimes we use heat to kill some organisms without necessarily killing all of
them. The most obvious example is pasteurization, in which a beverage is heated at 72
C for 15 seconds. This is long enough to kill a number of disease-causing bacteria that
can be found in milk, but does not sterilize it. The remaining organisms, which are called
thermoduric because they are relatively heat tolerant, can still cause a beverage such as
milk to spoil, so that milk must be refrigerated. We don’t generally regard thermophiles
as thermoduric since they not only withstand high temperatures but they grow at high
temperatures. We think of some mesophiles as being thermoduric, able to tolerate a brief
exposure to heat but unable to successfully grow at high temperatures.
The Exercise
In this exercise, we are going to examine the heat tolerance of three different
bacteria. At least one of them will be a species of Bacillus and will thus be a sporeformer. The three bacteria will be divided up around the class, and the data from all the
sections of this course will be pooled, averaged, and returned to you.
You will be working in with your lab partner. Each pair will have a tube with a
liquid culture of a bacterium. Your entire lab bench will be assigned a temperature, for
example 50, 70, or 95 C. If you have been assigned the near boiling water (95 C), you
should set up a coffee can and tripod like you did for the
acid fast and spore staining techniques. Each group will
have a Petri plate of growth medium. With a wax pencil or
Sharpie, you should mark on the bottom of the plate so that
0'
it will be divided into 5 wedge shaped areas and label them
as shown below.
15 '
Using aseptic technique, obtain a loopful of bacteria
30 '
and streak it onto the section of plate you labeled 0' as
shown. Be careful about inoculating the correct sections of
5'
the plate. When you turned the plate upside down you
2'
labeled it clockwise; viewed from the top of the right-side
up plate, the labels are now counter-clockwise!
Now incubate your tube (with the cap on loosely) in
the water bath assigned to your bench. After 30 seconds,
quickly remove the cap and take out a loopful of culture.
Streak that onto the section marked 30". Continue timing the incubation, and after
another 1 minute and 30 seconds for a total time of 2 minutes, remove another sample.
Streak that loopful of cells on the proper section and continue until the culture has been
treated for a total time of 15 minutes. Avoid removing the tube as this would allow the
culture to cool and interrupt the timing. If you do need to remove the tube from the water
bath, avoid holding the tube by the cap since the tube itself may slide out and break.
Those doing the boiling water may need to steady the tube with a test tube holder while
removing loopfuls of cells. At the end of the experiment, the plates will be incubated
until the next class period.
After the plates have been incubated, you will judge the amount of growth in each
section using the following criteria:
Score
Description
5
Maximum growth, characteristic of untreated cells
4
Confluent growth, less than maximum, but more than a score of "3"
3
Small, very crowded colonies
2
More than 6 colonies (but less than a score of "3")
1
One to six colonies
0
No growth at all
 Since each survivor produces one colony, the amount of growth covering the plate is
not as important as the number of colonies. This is important to remember, because some
bacteria will produce very large colonies when there are few colonies and the cells have
room to spread out. Four very large colonies may be a lot of growth, but should still only
get a score of "1"!
You will be asked to record your data and write it on the board or pass it into your
instructor. The results from all sections of the class will be pooled, and you will receive a
handout with the final results.