Growth Dynamics of E. coli in Varying Concentrations of Nutrient Broths, pH, and in the Presence of an AntibioticDvora Sze go, Elysian Preston Darcy Kmiotek, Brian Libby Department of Biology Rensselaer Polytechnic Institute Troy, NY 12180 Abstract The purpose in this experiment of growth dynamics of E. coli in varying media was to determine which media produces the maximum number of cells per unit time. First a control was established for E. coli in a 1.
0 x nutrient broth. This was used to compare the growth in the experimental media of 0. 5 x and 2. 0 x, nutrient broths; nutrient broths with an additional 5. 0 mM of glucose and another with 5. 0 mM lactose; nutrient broths of varying pH levels: 6.
0, 7. 0, and 8. 0; and finally a nutrient broth in the presence of the drug / antibiotic chloramphenicol. A variety of OD readings were taken and calculations made to determine the number of cells present after a given time.
Then two graphs were plotted, Number of cells per unit volume versus time in minutes and Log of the number of cells per unit volume versus Time growth curve. The final cell concentration for the control was 619, 500 cells / m L. Four media, after calculations, produced fewer cells than that of the control, these were: Chloramphenicol producing 89, 3 01 cells / ml ; glucose producing 411, 951 cells / m L; lactose producing 477, 441 cells / m Land finally pH 6. 0 producing 579, 557 cells / m L. The remaining four media, after calculations, produced cell counts greater than the control: 2 X with 1, 087, 009 cells / m L; 0. 5 X with 2, 205, 026 cells / m L; pH 8 with 3, 583, 750 cells / m L and finallypH 7.
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0 with 8, 090, 325 cells / m L. From these results the conclusion can be made that the environment is a controlling factor in the growth dynamics of E. coli. This was found through the regulation of pH and nutrient concentrations. In the presence of the drug / antibiotic , chloramphenicol, cell growth was minimal. Introduction E.
coli grows and divides through asexual reproduction. Growth will continue until all nutrients are depleted and the wastes rise to a toxic level. This is demonstrated by the Log of the number of cells per unit volume versus Time growth curve. This growth curve consists of four phases: Lag, Exponential, Stationary, and finally Death. During the Lag phase there is little increase in the number of cells.
Rather, during this phase cells increase in size by transporting nutrients inside the cell from the medium preparing for reproduction and synthesizing DNA and various enzymes needed for cell division. In the Exponential phase, also called the log growth phase, bacterial cell division begins. The number of cells increases as an exponential function of time. The third phase, Stationary, is where the culture has reached a phase during which there is no net increase in the number of cells. During the stationary phase the growth rate is exactly equal to the death rate.
A bacterial population may reach stationary growth when required nutrients are exhausted, when toxic end products accumulate, or environmental conditions change. Eventually the number of cells begins to decrease signaling the onset of the Death phase; this is due to the bacteria’s inability to reproduce (Atlas 331-332).
The equation used for predicting a growth curve is N = N 0 eat. N equals the number of cells in the culture at some future point, N 0 equals the initial number of cells in the culture, k is a growth rate constant defined as the number of population doublings per unit time, t is time and e is the exponential number. The k value can be easily derived by knowing the number of cells in a exponentially growing population at two different times. K is determined using the equation k = (ln N-ln N 0) /t, where ln N is the natural log of the number of cells at some time t, ln N 0 is the natural log of the initial number of cells and t is time.
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This equation allows one to calculate the numbers of cells in a culture at any given time. The reciprocal of k is the mean doubling time, in other words, the time required for the population to double, usually expressed as cells per unit volume. (Edick 61-62) Temperature is the most influential factor of growth in bacteria. The optimal temperature of E. coli is 37 C, which was maintained throughout the experiment. Aside from temperature, the pH of the organisms environment exerts the greatest influence on its growth.
The pH limits the activity of enzymes with which an organism is able to synthesize new protoplasm. The optimum pH of coli growing in a culture at 37 C is 6. 0-7. 0. It has a minimum pH level of 4. 4 and a maximum level of 9.
0 required for growth. Bacteria obtains it nutrients for growth and division from their environment, thus any change in the concentration of these nutrients would cause a change in the growth rate (Atlas 330).
Drugs/Antibiotics are another very common tool in molecular biology used to inhibit a specific process. Chloramphenicol, used in this experiment, inhibits the assembly of new proteins, yet it has no effect on those proteins which already exist ().
The growth dynamics of E. coli were evaluated in individual media trials.
By using only one variable the results can be directly correlated to that particular variable. For example in this experiment the temperature was held at a constant 37 C, and the variables were the broths which the E. coli we reusing to grow. The k values needed to be determined in order to provide an accurate projection of cell growth, by providing a constant initial cell count. The purpose of this experiment was to determine the effects of varying media, and compare which media produces the maximum number of cells per unit time.
Methods and Materials The initial step of this experiment was to establish a control of E. coli in a nutrient broth with a concentration designated as 1. 0. A variety of media were established, there were nutrient broths with concentrations of 0.
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5 x and 2. 0 x, nutrient broths with additional an 5. 0 mM of glucose and another with 5. 0 mM lactose. There were also nutrient broths of varying pH levels: 6. 0, 7.
0, and 8. 0. The last of the medium contained drugs / antibiotics , a very common to olin molecular biology used to inhibit a specific process, chloramphenicol 200 mg / ml . Each solution had a corresponding blank used to zero the spectrophotometer. These blank consisted of the medium before inoculation with E. coli.
Beginning with approximately 50 ml of each of these inoculated solutions, 3. 0 ml of each was pipette d out and placed into a cu vet, if care is used, to speed up this process, the sample may be poured into the cu vet. After the aliquots of each sample had been transferred to a cu vet the OD was measured at 600 nm. The solutions were then placed in an incubator or water bath with forks, to maintain a constant temperature of 37 degrees Celsius.
Every 15 minutes thereafter for a 150 minute time period 3. 0 ml of each solution was removed and the OD 600 was measured and recorded. The samples are not to remain out of the water bath for an extended period of time. If a spectrophotometer was not available the sample was placed in an ice bath, the cells were chilled in 2-3 minutes and thus no grow could occur. However all moisture was wiped off the outside of the cu vet with a Kim wipe before placing it in the spectrophotometer, as water will cause serious damage to the instrument. To prevent cells from settling at the bottom of the cu vet, the samples were gently swirled to ensure that the cells are evenly distributed throughout the cu vet, then the reading was taken as quickly as possible.
The k values were determined for each time interval of all experimental media by taking the natural log of the number cells at time t minus the natural log of the number of cells at t-15 minutes and dividing by 15 minutes. Beginning with an initial cell count equal to that of the control, these k values we reused with the growth equation to calculate the number of cells in each media at each time inter val. These calculations were necessary in order to accurately compare the growth in each medium. If this procedure was not followed the results are likely to be misinterpreted. This was because the initial cell counts for each sample were different. Graphing the numbers obtained directly from the experiment showed misleading final cell counts.
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A table was also made of 1/k, the mean doubling time. The k used in this calculation was derived using the initial and final cell counts and dividing by the entire time period. Finally graphs were made of the number of cells per mL versus Time in minutes and the Log number of cell per mL versus Time in minutes, which produces the traditional growth curve. Results The first part of the experiment was to determine the cell number using the optical densities and multiplying them by 1. 5 x 106. These numbers were then used as raw data to calculate the k values of each time interval for each media.
Using these k values cell counts were calculated for all media beginning with an initial count of 70, 500 cells. These results were graphed, plotting the number of cells per mL versus the time in minutes. (Graphs 1, 3, 5) These graphs show the growth dynamics of E. coli in the varying media. The control atti me 150 minutes produced a final cell count of 796, 500 cells / m L. After doing the necessary calculations to determine k values and thus make all of the graphs begin at one standard point the graphs were plotted.
Through these graphs (1, 3, 5) it was visible that four media produced fewer cells than that of the control, these were: Chloramphenicol producing 89, 301 cells / m L; glucose producing 411, 951 cells / m L; lactose producing 477, 441 cells / m L, and finallypH 6. 0 producing 579, 557 cells / m L. The remaining four media produced cell counts greater than the control: 2. 0 x with 1, 087, 009 cells / m L; 0. 5 x with 2, 205, 026 cells / m L; pH 8. 0 with 3, 583, 750 cells / m L and finally pH 7.
0 with 8, 090, 325 cells / m L. The Log number of cells was plotted versus Time (Graphs 2, 4, 6).
Thesis the form of the traditional growth curve. By observing these graphs it can be told that the same media which produced greater final cell counts also produced a greater final value on the growth curve. The average k values for each media were found and the values for 1/k, the mean doubling time, were computed (Chart 5).
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These results clearly exhibit the effect media has on the growth dynamics of E.
coli. The control sample had an average doubling time of 69 minutes while pH 7. 0 doubled in only 30. 4 minutes and chloramphenicol has a calculated doubling time of 381 minutes. Discussion This study confirmed our hypothesis that varying the media will produce different effects on growth rate. By graphing the number of cells per mL versus Time in minutes it can be seen which of the media provided the best environment for cell growth.
The graphs of the Log number of cells versus Time produces the traditional growth curves. The results supported the hypothesis stating that E. coli has the best growth rate at a pH 7. 0 with a final cell count of 8, 090, 325 cells / m L, however the pH of 8. 0 producing 3, 583, 750 cells / m L was found to produce a greater number of cells than that of a pH of 6. 0 579, 557 cells / m L.
The change in nutrients also had a great affect on the cell production (the control produced a final cell number of 619, 500 cells / m L).
The 0. 5 x nutrient broth produced 2, 205, 026 cells / m L while the 2. 0 x nutrient broth only produced 1, 087, 009 cells / m L. Although these are both higher than the control sample, it is interesting to note that the 0. 5 x broth actually produced more cells than the 2.
0 x broth. This shows that more isn’t necessarily better. There are fewer cells in both the lactose enhanced medium and the glucose enhanced medium samples than in the control. This may be due to the fact the E.
coli is able to ferment both glucose and lactose producing complex end products (Benson 153).
In the presence of chloramphenicol, the drug / antibiotic , the growth rate reaches the stationary phase at time 120 when there are 57, 000 cells / m L (experimental).
This is due to the fact that chloramphenicol inhibits the assembly of new proteins, yet has no effect on those proteins which already exist. Therefore, in the presence of chloramphenicol, translation was inhibited preventing the cells from growing and dividing (Atlas 371).
The growth curve produced by graphing the Log number of cells / ml versus time in minutes were found to be incomplete. The expected reasoning for this is due to the fact that the experiment was not run for a sufficient amount of time.
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By observing these graphs it can be told that the same media which produced greater final cell counts also produced a greater final value on the growth curve. This is because all that was done to convert these numbers was to take the log of the cell number, so should there be any error it will be in all three sets of graphs. The final step of computing the 1/k values provided the knowledge of which of the media more closely resembled that of an optimal environment, the data obtained from this experiment showed that the media at apH of 7. 0 most closely resembled this ideal environment of 37 C, a pH between 6. 0-7. 0, a rich nutrient concentration and no antibiotics present.
In this environment the cells growth rate exceeds that of the cells death rate and the cells are able to continue for a longer period of time in the log stage of the cell growth cycle. Any error in the findings are more than likely due to human error. It could be due to the samples remaining out of the water bath for an extended period of time and a spectrophotometer not being available, therefore additional cell growth could have occurred. Or if the sample had been placed in the ice bath, the water on the outside of the cu vet may not have been thoroughly wiped off therefore causing error in the OD 600.
The final possibility for this error is that the cells in the sample may have settled to the bottom of because the reading was not taken fast enough. In conclusion the k values should be constant throughout each individual media, but should differ between the various media. The results from this experiment showed the k values to fluctuate slightly. Also, the results of this experiment showed, after calculations, that in the 0. 5 x nutrient broth more cells were produced than in either the 1. 0 x and 2.
0 x broths, this could be due to the fact that the cells were growing at a slower rate but they were not dying as fast or producing as many toxins as in the 1. 0 x broth or the 2. 0 x broth. This is only a hypothesis but is supported by the lab manual which says, ‘This suggests and has been substantiated experimentally, that the waste products produced by the bacteria are significant factor in the limitation of population size’ (Edick 64).
The pH change was also a contributor to the number of cells which were produced in a specific media. The pH 7. 0 produced a significantly larger number of cells than that of 6. 0 and 8. 0, this is more than likely due to it being the established optimal pH for the growth of E. coli.
As stated in the previous paragraphs the broth which contained chloramphenicol produced significantly fewer number of cells than any other medium. This was due to the fact that the antibiotic / drug inhibits translation and prevented the cells from growing and dividing. This experiment could be examined further through the use of different nutrient enhanced media, media containing induce lac operon’s, temperatures changes, and different drugs / antibiotics at different concentrations. References Atlas, Ronald M.
1995. Principles of Microbiology, St. Louis, MO: Mosby-Yearbook Inc. Benson, Harold J.
1994. Microbiological Applications 6 th edition, Dubuque, IA: Wm. C. Brown Publishers. Edick, G. F (1992).
Escherichia coli: Laboratory Investigations of Protein Biochemistry, Growth and Gene Expression Regulation, 3 rd edition; RPI.