Sunday, March 10, 2013

Time Out for Pathology

E. coli Growth Inhibition As A Result Of Temperature Changes

            The use of Escherichia coli in laboratory settings has helped vastly increase the pool of knowledge regarding bacterial growth requirements and metabolic pathway utilization. For the binary fission used by prokaryotes to increase population size, sufficient nutrients for the production of building blocks along with an adequate energy source are required. E. coli is an organism that can synthesize from a carbon source all of their requirements for growth such as amino acids, pyrimidines, purines, and other necessary nutrients. (Todar, 2012) While there are different metabolic processes by which a cell can produce precursor metabolites and energy, some are much more efficient than others. When an optimal carbon source is not available, cells can still generate the macromolecules needed for growth but the rate of growth is slowed. Even when the ideal conditions for maximum cell growth are present, inevitably the growth rate will slow as resources are depleted. Once the limiting resource contributing to cell growth has vanished, cell breakdown will begin and eventually the cell population will enter the death phase. There are other environmental factors that can also adversely affect the rate of cell growth, such as pH, oxygen availability, the specific nutrients available, and temperature (Schussler, et all, 2012). Temperature is an important variable concerning almost all chemical reactions. Especially regarding organisms that have acclimated to the environment inside the human body, a lower temperature can cause an inhibited or even a lack of cell growth.
            To gain concrete knowledge of the optimal temperature for cell growth, an experiment was designed to provide data regarding the growth differential of E. coli at cooler temperatures rather than the assumed optimal temperature of 37 degrees Celsius, the internal temperature of the human body. A strain of wild type, nonpathogenic E. coli was grown in glucose as the organism to be subjected to experimental conditions. For the experiment, a growth medium of M9 was used. Glucose was added as a variable to elicit growth of the organism in both experimental temperatures, and as a control the organism was also grown in the M9 culture without any glucose. The two experimental flasks consisted of M9, glucose, and the E. coli strain. Both were agitated during the growth phase. One flask was cultured at 25 degrees Celsius and the other at 37 degrees Celsius, each for 90 minutes total. The control of M9 without glucose but also with the E. coli strain was also agitated and grown at 25 degrees Celsius, also for 90 minutes. The experimental cultures were duplicated so that each M9, glucose, and E. coli specimen replicated so there were two separate flasks for each temperature variable. Every flask was tested for culture population using the optical density calculated by a spectrophotometer. All the experimental and control flasks had the absorbance level measured at the beginning of the experiment to establish a baseline, and again after 30 minutes, at 60 minutes, and at 90 minutes. The density of the E. coli population was reflected in higher absorbance levels measured of the optical density by the spectrophotometer, providing a linear progression of cell growth and in certain populations, consequent die off, over a total of 90 minutes.
            The results of the experiment demonstrated that the control of M9 and E. coli with no glucose did not really grow. The averaged optical density from the multiple control flasks was around 0.6 at baseline and stayed the same at each 30-minute increment; this was to be expected because there was no carbon source for the organism to utilize for growth. The cultures of E. coli grown with M9 and glucose at 25 degrees Celsius did not demonstrate very much growth, with an averaged baseline of 0.6 that rose only to 0.825 at its highest measurement which was at 60 minutes. The following measurement at 90 minutes showed the culture population began decreasing, with an average absorbance value of 0.7. The most growth was seen, as anticipated, in the culture grown at 37 degrees Celsius. The baseline measurement of optical density was actually below 0.5 but over the consecutive 30-minute intervals, the culture growth increased to an optical density of 0.2, significantly higher growth than that seen when the culture was grown at 25 degrees Celsius.
            This study does confirm that E. coli grows best at a warm 37 degrees Celsius, as compared to a cooler room temperature level such as 25 degrees Celsius. The incorporation of agitation, which should provide optimal growth of a bacterial species by maximizing the rate of reactions in metabolic processes, did ensure that both of the experimental cultures had the benefit of maximum possible growth, thereby equalizing that particular variable. The control culture of M9 and E. coli, which did not show any population increase at all, demonstrated that the glucose added to the cultures was necessary for the possibility of any kind of growth. The E. coli cultures grown at25 degrees demonstrated only barely discernible growth and even entered a die-off phase much more quickly than would be expected, based on the extensive growth seen by the same organism at 37 degrees Celsius, suggesting that chemical reactions were so slowed by the 12-degree difference that not even the limiting resource was used up before the bacterial population lost what little ability it had for growth. Because the cooler temperature of 25 degrees Celsius severely inhibited the maximal growth potential for E. coli, it would be a logical conclusion that most enteric flora which has acclimated to the internal human body would be unable to demonstrate much growth of any kind if faced with a variable such as a slightly cooler environment. There are some bacteria species that do thrive in more hostile or extreme environments, but those are not organisms that have evolved residing in the human body such as E. coli. This experiment is a valid reaffirmation of previously acquired knowledge regarding the abilities and limitations of prokaryotes that live in close association with the human body. Additional study similar to this one could include a wider range of temperatures instead of just two differing temperatures, and also testing different strains of E. coli, especially pathogenic strains such as E. coli 0157:H7, foodborne strain that causes severely debilitating illness.
Schussler E, Hudson, J., Rowe E., Lemieux M., and Naswa S.(2012) Cell Growth and Metabolism. University of Tennessee, Knoxville. Organization and Function of the Cell, Biology 140 Laboratory Manual. 17-23.
Todar K. Online Textbook of Bacteriology, University of Wisconsin [internet] 2008-2012 [cited 2013 March 6] Available from:

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