as normal flora
Escherichia coli, a gram-negative bacilli, is a common
bacteria found in most organisms. In fact, E. coli is
common in the intestinal tract of humans. In this section,
evidence will be given to support why E. coli can be
found in humans.
picture of the gram-negative rods of E. coli (http://www.nirgal.net/graphics/e_coli.jpg)
Before we begin our study into why E. coli behaves the
way it does in humans, let us first turn to what type of
environment E. coli likes best. In this discussion, we
will refer to lab work performed over the course of two weeks in
order to understand more about these bacteria.
Deciphering how an organism moves in its environment gives us an
insight into how the organism works. A way to test for the
motility of an organism is to use semi-solid agar tubes and a
straight wire needle to inoculate the media with the organism.
After using aseptic technique, a researcher would inoculate
E. coli into an agar tube by carefully driving the straight
wire into the middle of the media and then back out in the same
fashion. After incubation, one would analyze the results by
looking for movement away from the stab line. If the media gives
a uniform cloudiness, then the organism is motile; if the media
is clear with a persistent stab line, then the organism is
non-motile. E. coli gives a positive result for motility.
both pictures above, the motile organism is located on the left.
Notice how growth in the tubes on the right only have growth
along the stab line. This type of growth visually
indicates that the organism stabbed is nonmotile.
The use of oxygen by an organism gives scientists a way to
classify organisms based on the idea of whether the organism is
an aerobe, anaerobe, microaerophile, or facultative anaerobe. A
microorganism that must have free oxygen to grow is aerobe, so
these organisms will grow in a standard incubator. An organism
that is killed by oxygen is classified as an anaerobe, so
absolutely no free oxygen can be present for these organisms.
How can one get rid of free oxygen though?
In order for anaerobes to grow, scientists must flush out all
the atmospheric oxygen and allow other compounds in such as
nitrogen or carbon dioxide. One common practice used to
cultivate anaerobes is a Gas-Pak Anaerobic system. This jar is
made anaerobic with the use of a "gas pak" which houses hydrogen
and carbon dioxide. The hydrogen produced from the gas pak and
the oxygen found in the jar combine to form water, which is
evident on the side of the container as condensation. The
environment inside the Gas-Pak system in now comprised of carbon
dioxide, which allows anaerobes to grow.
This is a picture of the Gas-Pak
jar used in Microbiology lab. Note the methylene blue indicator
strip used to tell when the air inside the chamber is oxidized
(blue) or reduced (white).
Microaerophiles grow in limited concentrations of oxygen. In
order for the best growth of microaerophiles, one must only have
a small percentage of oxygen left in the container. A common
practice used to grow these organisms is to assemble a candle
jar. The plates of organisms are placed in the bottom of a glass
jar, then a candle is placed on top of the plates, light the
candle, and then seal the jar tightly. In time, the flame will
burn off most of the oxygen in the jar and leave approximately
six to seven percent oxygen in the environment.
This is a picture of a candle jar
used in Microbiology lab with four plates inside containing
various organisms including E. coli.
Facultative anaerobes can grow with or without oxygen, so these
organisms are present in a standard incubator, candle jar, or an
anaerobic jar. E. coli is a facultative anaerobe because
growth was evident in every environment tested (standard
incubator, candle jar, and anaerobic jar).
The three plates above represent each of the three ways we
exposed bacteria to various oxygen conditions in the lab. The
picture on top shows how E. coli (specimen in top
left) and another organism grew in an aerobic environment. The
middle picture shows all four organisms growth after being
exposed to the environment in the candle jar (E. coli is
top right on the plate). The bottom picture shows the plate
exposed to the anaerobic environment in the Gas-Pak jar (E.
coli is top left).
When growing organisms it is vital to know what is needed to see
growth, does E. coli need only a carbon source, amino
acids, nitrogen, or just minimal media? In order to find out,
one must expose this organism to a variety of complexities of
media to see where E. coli grows best.
Most specimens in nature are heterotrophs meaning they require
an organic form of carbon because they cannot make it
themselves. Autotrophs, such as plants, utilize carbon dioxide
as their carbon source and therefore make their own food.
Organisms that require only a carbon source to grow are said to
be prototrophs. Those that need more than carbon, such as amino
acids, are said to be auxotrophs. The term fastidious implies
the organism needs much more than carbon and amino acids.
During our analysis, we used four types of media including
minimal media, minimal media with dextrose/glucose, minimal
media with dextrose/glucose and Casamino acid, and minimal media
with dextrose/glucose, Casamino acid, and yeast extract. Nothing
grew on the minimal media plate, so E. coli is not an
autotroph, but E. coli did grow on all the rest of the
plates. The result is E. coli is a prototroph, so it
needs a carbon source to grow. E. coli can use simple
carbon sources, such as glucose, and nitrogen sources, ammonium
sulfate is preferred, for all its survival needs (Gyles 4).
An important growth factor that was not tested in laboratory was
the ability of E. coli to absorb iron from the
environment. This is critical for invasive E. coli
because the organism competes with its host for limited iron (Gyles
7). The invasive and harmful strains of E. coli carry
various toxins, which will be discussed in another section of
the website, that enhance its ability to cause more damage to
The effect of temperature can be detrimental to an organism
because of the enzymes and other heat-sensitive chemicals used
to keep the organism alive. The effect of heat on an organism
depends on a variety of things including age, amount of
organism, the type of heat and the duration of heat. There are
two types of heat: dry and moist. Dry heat kills specimens by
dessication or dehydration. Moist heat kills organisms by
denaturing proteins (i.e. enzymes), cell membranes, and nucleic
acids (Lab manual 177). Common practices to determine how
temperature affects microorganisms are thermal death time (TDT)
and thermal death point (TDP). Thermal death time is the
shortest amount of time it takes to kill a specimen, so
temperature is kept constant. Thermal death point is lowest
temperature at which a certain species is killed in 10 minutes
(177). Most organisms have an optimal temperature range for
their best growth, E. coli happens to fall in the range
between 20 to 50 degrees Celsius. Microorganisms in this range
are said to be mesophiles. Those capable of growing at a
temperature range below 20 degrees are said to be psychrophilic.
Microorganisms growing at temperatures above 50 degrees are said
to be thermophilic.
Using thermal death time method, E. coli was subjected to
a water bath at 55 degrees Celsius for sixty minutes. A single
loop of culture was taken at time 0, 5 minutes, 10 minutes, 20
minutes, 30 minutes, and 60 minutes. The loop of E. coli
was quadrant streaked on an agar plate and then incubated. After
analyzing the results, E. coli withstood 55 degrees for
at least 60 minutes. No growth was evident on the 60-minute
plate, so E. coli cells died between 30 minutes and 60
minutes. To achieve a better estimate on the exact time of cell
death, one would need to analyze date between the time of 30 and
60 minutes. One might quadrant streak a loop of culture at every
ten minutes between 30 and 60 minutes to get a better estimate.
In this picture, one sees all the
six plates from the thermal death time procedure (T0: top left,
T5: top middle, T10: top right, T20: bottom left, T30: bottom
middle, and T60: bottom right). Notice the growth on the plates,
T60 had condensation on the lid so it gives the impression there
is growth, but those are only water droplets.
Finding which range of temperatures E. coli grows best
one needs a variety of temperatures or rather a variety of
incubator temperatures. One plate went into the following
incubator temperatures: 5, 25, 37, and 55 degrees Celsius. E.
coli grew at 22 and 37 degrees Celsius; therefore it is a
mesophile. Remembering that E. coli is found in humans
explains why E. coli grows best at these temperatures
because the human body has a temperature in this range.
picture shows the growth of E. coli in the incubators
that stayed constant at 22 and 37 degrees Celsius. The plate at
22 degrees is on the left and E. coli is grown at the
bottom right quadrant. The plate on the right is the plate
incubated at 37 degrees and E. coli can be seen at the
top right quadrant of the plate.
chemical makeup of the environment can lead to little or no
growth of a microorganism as well. If the environment contains a
great deal of salt, the organism's cells may shrivel up (plasmolysis)
because of the hypertonic environment. If the environment
contains less than the concentration of salt/sugar inside the
cell, the individual cells will take on water and lyse (plasmoptysis).
The amount of salt or sugar can cause great effects on
individual cells of a specimen. Osmotolerant microorganisms can
withstand certain ranges of osmotic conditions. Organisms that
require high salt concentrations are said to be halophilic, and
those that require high sugar concentrations are saccharophilic.
From the previous experiments, we noted that most organisms
enjoy a happy medium of temperatures and the same is said for
pH. Most microorganisms thrive at a near neutral pH between 6
and 8. However, there are always exceptions to the rule.
Acidoduric organisms can thrive (not multiply) in acid,
acidophiles can survive and grow in low pH, and alkalophiles can
thrive in high pH.
Using a variety of media including various ranges of salt,
sugar, and pH, we tested how well E. coli grew on each
media. No E. coli growth was evident on the plates
ranging in salt concentrations (5%, 10%, and 20%), no growth on
various sugar concentrations (10%, 20%, and 50% dextrose). At
the various pH levels 5, 7, and 9, E. coli grew on all
plates with the best growth at pH of 7. Knowing that the normal
pH of the body is between 5 and 7, it should not be surprising
that E. coli grows best at a neutral pH.
From the information and experiments covered in depth
previously, one sees why E. coli is common in humans.
Research shows that the pH and temperature for E. coli to
grow is similar to the temperature and pH of the human body. Our
bodies also contain a great deal of supplements for E. coli
to grow on as well including sugars, nitrogen, amino acids, etc.
Oxygen is readily available in the human body but there are
places such as in the intestinal tract where oxygen is hard to
come by except for oxygen from the blood. Being a facultative
anaerobe, E. coli is able to adapt to any situation. This
ability to adapt to many situations frightens many scientists
because it shows the versatility of this tiny organism.
+For source information, please
click here or refer to the references.html page.