Escherichia coli 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.

A 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.

In 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 hosts.

     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.

     This 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.

     The 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.

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