ns Of Nutrient BrothGrowth Dynamics of E. coli in Varying Concentrations of Nutrient Broths, pH, andin the Presence of an AntibioticDvora Szego,Elysia PrestonDarcy Kmiotek,Brian LibbyDepartment of BiologyRensselaer Polytechnic InstituteTroy, NY 12180AbstractThe purpose in this experiment of growth dynamics of E. coli in varying mediawas to determine which media produces the maximum number of cells per unit time.

First a control was established for E. coli in a 1.0x nutrient broth. This wasused to compare the growth in the experimental media of 0.5x and 2.0x, nutrientbroths; nutrient broths with an additional 5.0mM of glucose and another with5.0mM lactose; nutrient broths of varying pH levels: 6.0, 7.0, and 8.0; andfinally a nutrient broth in the presence of the drug/antibiotic chloramphenicol.

A variety of OD readings were taken and calculations made to determine thenumber of cells present after a given time. Then two graphs were plotted, Numberof cells per unit volume versus Time in minutes and Log of the number of cellsper unit volume versus Time growth curve. The final cell concentration for thecontrol was 619,500 cells/mL. Four media, after calculations, produced fewercells than that of the control, these were: Chloramphenicol producing 89,3 01cells/ml; glucose producing 411,951 cells/mL; lactose producing 477,441 cells/mLand finally pH 6.0 producing 579,557cells/mL. The remaining four media, aftercalculations, produced cell counts greater than the control: 2X with 1,087,009cells/mL; 0.5X with 2,205,026 cells/mL; pH 8 with 3,583,750 cells/mL and finallypH 7.0 with 8,090,325 cells/mL. From these results the conclusion can be madethat 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 thepresence of the drug/antibiotic, chloramphenicol, cell growth was minimal.

IntroductionE. coli grows and divides through asexual reproduction. Growth will continueuntil all nutrients are depleted and the wastes rise to a toxic level. This isdemonstrated by the Log of the number of cells per unit volume versus Timegrowth curve. This growth curve consists of four phases: Lag, Exponential,Stationary, and finally Death. During the Lag phase there is little increase inthe number of cells. Rather, during this phase cells increase in size bytransporting nutrients inside the cell from the medium preparing forreproduction and synthesizing DNA and various enzymes needed for cell division.

In the Exponential phase, also called the log growth phase, bacterial celldivision begins. The number of cells increases as an exponential function oftime. The third phase, Stationary, is where the culture has reached a phaseduring which there is no net increase in the number of cells. During thestationary phase the growth rate is exactly equal to the death rate. Abacterial population may reach stationary growth when required nutrients areexhausted, when toxic end products accumulate, or environmental conditionschange. Eventually the number of cells begins to decrease signaling the onsetof the Death phase; this is due to the bacteria’s inability to reproduce (Atlas331-332).

The equation used for predicting a growth curve is N=N0ekt. N equals thenumber of cells in the culture at some future point, N0 equals the initialnumber of cells in the culture, k is a growth rate constant defined as thenumber of population doublings per unit time, t is time and e is the exponentialnumber. The k value can be easily derived by knowing the number of cells in aexponentially growing population at two different times. K is determined usingthe equation k=(ln N-ln N0 )/t, where ln N is the natural log of the number ofcells at some time t, ln N0 is the natural log of the initial number of cellsand t is time. This equation allows one to calculate the numbers of cells in aculture at any given time. The reciprocal of k is the mean doubling time, inother words, the time required for the population to double, usually expressedas cells per unit volume. (Edick 61-62)Temperature is the most influential factor of growth in bacteria. Theoptimal temperature of E. coli is 37C, which was maintained throughout theexperiment. Aside from temperature, the pH of the organisms environment exertsthe greatest influence on its growth. The pH limits the activity of enzymeswith which an organism is able to synthesize new protoplasm. The optimum pH ofE coli growing in a culture at 37C is 6.0-7.0. It has a minimum pH level of 4.4and a maximum level of 9.0 required for growth. Bacteria obtains it nutrientsfor growth and division from their environment, thus any change in theconcentration of these nutrients would cause a change in the growth rate (Atlas330). Drugs/Antibiotics are another very common tool in molecular biology usedto inhibit a specific process. Chloramphenicol, used in this experiment,inhibits the assembly of new proteins, yet it has no effect on those proteinswhich 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 thatparticular variable. For example in this experiment the temperature was heldat a constant 37C, and the variables were the broths which the E. coli wereusing to grow. The k values needed to be determined in order to provide anaccurate 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 MaterialsThe initial step of this experiment was to establish a control of E.coliin a nutrient broth with a concentration designated as 1.0. A variety of mediawere established, there were nutrient broths with concentrations of 0.5x and2.0x, nutrient broths with additional an 5.0mM of glucose and another with5.0mM 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 toolin molecular biology used to inhibit a specific process, chloramphenicol200mg/ml. Each solution had a corresponding blank used to zero thespectrophotometer. 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 pipetted out and placed into a cuvet, if care is used, tospeed up this process, the sample may be poured into the cuvet. After thealiquots of each sample had been transferred to a cuvet the OD was measured at600nm. The solutions were then placed in an incubator or water bath with forks,to maintain a constant temperature of 37 degrees Celsius. Every 15 minutesthereafter for a 150 minute time period 3.0 ml of each solution was removed andthe OD600 was measured and recorded. The samples are not to remain out of thewater bath for an extended period of time. If a spectrophotometer was notavailable the sample was placed in an ice bath, the cells were chilled in 2-3minutes and thus no grow could occur. However all moisture was wiped off theoutside of the cuvet with a Kimwipe before placing it in the spectrophotometer,as water will cause serious damage to the instrument. To prevent cells fromsettling at the bottom of the cuvet, the samples were gently swirled to ensurethat the cells are evenly distributed throughout the cuvet, then the reading wastaken as quickly as possible.

The k values were determined for each time interval of all experimental mediaby taking the natural log of the number cells at time t minus the natural logof the number of cells at t-15minutes and dividing by 15 minutes. Beginningwith an initial cell count equal to that of the control, these k values wereused with the growth equation to calculate the number of cells in each mediaat each time interval. These calculations were necessary in order to accuratelycompare the growth in each medium. If this procedure was not followed theresults are likely to be misinterpreted. This was because the initial cellcounts for each sample were different. Graphing the numbers obtained directlyfrom the experiment showed misleading final cell counts. A table was also madeof 1/k, the mean doubling time. The k used in this calculation was derivedusing 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 minutesand the Log number of cell per mL versus Time in minutes , which produces thetraditional growth curve.

ResultsThe first part of the experiment was to determine the cell number usingthe optical densities and multiplying them by 1.5 x 106. These numbers werethen used as raw data to calculate the k values of each time interval for eachmedia. Using these k values cell counts were calculated for all media beginningwith an initial count of 70,500 cells. These results were graphed, plotting thenumber of cells per mL versus the time in minutes. (Graphs 1,3,5) Thesegraphs show the growth dynamics of E. coli in the varying media. The control attime 150 minutes produced a final cell count of 796,500 cells/mL. After doingthe necessary calculations to determine k values and thus make all of the graphsbegin 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 thecontrol, these were: Chloramphenicol producing 89,301 cells/mL; glucoseproducing 411,951 cells/mL; lactose producing 477,441 cells/mL, and finallypH6.0 producing 579,557 cells/mL. The remaining four media produced cell countsgreater than the control: 2.0x with 1,087,009 cells/mL; 0.5x with 2,205,026cells/mL; pH 8.0 with 3,583,750 cells/mL and finally pH 7.0 with 8,090,325cells/mL. The Log number of cells was plotted versus Time(Graphs 2,4,6). Thisis the form of the traditional growth curve. By observing these graphs it can betold that the same media which produced greater final cell counts also produceda greater final value on the growth curve. The average k values for eachmedia were found and the values for 1/k, the mean doubling time, were computed(Chart 5). These results clearly exhibit the effect media has on the growthdynamics of E.coli. The control sample had an average doubling time of 69minutes while pH 7.0 doubled in only 30.4 minutes and chloramphenicol has acalculated doubling time of 381 minutes.

DiscussionThis study confirmed our hypothesis that varying the media will producedifferent effects on growth rate. By graphing the number of cells per mLversus Time in minutes it can be seen which of the media provided the bestenvironment for cell growth. The graphs of the Log number of cells versus Timeproduces the traditional growth curves.

The results supported the hypothesis stating that E.coli has the bestgrowth rate at a pH 7.0 with a final cell count of 8,090,325 cells/mL, howeverthe pH of 8.0 producing 3,583,750 cells/mL was found to produce a greater numberof cells than that of a pH of 6.0 579,557 cells/mL. The change in nutrientsalso had a great affect on the cell production (the control produced a finalcell number of 619,500 cells/mL). The 0.5x nutrient broth produced 2,205,026cells/mL while the 2.0x nutrient broth only produced 1,087,009 cells/mL.

Although these are both higher than the control sample, it is interesting tonote that the 0.5x broth actually produced more cells than the 2.0x broth. Thisshows that more isn’t necessarily better. There are fewer cells in both thelactose enhanced medium and the glucose enhanced medium samples than in thecontrol. This may be due to the fact the E.coli is able to ferment both glucoseand lactose producing complex end products(Benson 153). In the presence ofchloramphenicol, the drug/antibiotic, the growth rate reaches the stationaryphase at time 120 when there are 57,000 cells/mL(experimental). This is due tothe fact that chloramphenicol inhibits the assembly of new proteins, yet has noeffect on those proteins which already exist. Therefore, in the presence ofchloramphenicol, translation was inhibited preventing the cells from growingand dividing (Atlas 371).

The growth curve produced by graphing the Log number of cells/ml versustime in minutes were found to be incomplete. The expected reasoning for this isdue to the fact that the experiment was not run for a sufficient amount of time.

By observing these graphs it can be told that the same media which producedgreater final cell counts also produced a greater final value on the growthcurve. This is because all that was done to convert these numbers was to takethe log of the cell number, so should there be any error it will be in all threesets of graphs. The final step of computing the 1/k values provided theknowledge of which of the media more closely resembled that of an optimalenvironment, the data obtained from this experiment showed that the media at apH of 7.0 most closely resembled this ideal environment of 37C, a pH between6.0-7.0, a rich nutrient concentration and no antibiotics present. In thisenvironment the cells growth rate exceeds that of the cells death rate and thecells are able to continue for a longer period of time in the log stage of thecell growth cycle.

Any error in the findings are more than likely due to human error. Itcould be due to the samples remaining out of the water bath for an extendedperiod of time and a spectrophotometer not being available, thereforeadditional cell growth could have occurred. Or if the sample had been placed inthe ice bath, the water on the outside of the cuvet may not have been thoroughlywiped off therefore causing error in the OD600. The final possibility for thiserror is that the cells in the sample may have settled to the bottom of thecuvet because the reading was not taken fast enough.

In conclusion the k values should be constant throughout each individualmedia, but should differ between the various media. The results from thisexperiment showed the k values to fluctuate slightly. Also, the results of thisexperiment showed, after calculations, that in the 0.5x nutrient broth morecells were produced than in either the 1.0x and 2.0x broths, this could be dueto the fact that the cells were growing at a slower rate but they were not dyingas fast or producing as many toxins as in the 1.0x broth or the 2.0x broth.

This is only a hypothesis but is supported by the lab manual which says, “Thissuggests and has been substantiated experimentally, that the waste productsproduced by the bacteria are significant factor in the limitation of populationsize” (Edick 64). The pH change was also a contributor to the number of cellswhich were produced in a specific media. The pH 7.0 produced a significantlylarger number of cells than that of 6.0 and 8.0, this is more than likely due toit being the established optimal pH for the growth of E. coli. As stated in theprevious paragraphs the broth which contained chloramphenicol producedsignificantly fewer number of cells than any other medium. This was due to thefact that the antibiotic/drug inhibits translation and prevented the cells fromgrowing and dividing. This experiment could be examined further through the useof different nutrient enhanced media, media containing induce lac operons,temperatures changes, and different drugs/antibiotics at differentconcentrations.

ReferencesAtlas, Ronald M. 1995. Principles of Microbiology, St. Louis, MO: Mosby-YearbookInc.

Benson, Harold J. 1994. Microbiological Applications 6th edition, Debuque, IA:Wm. C.

Brown Publishers.

Edick, G.F (1992). Escherichia coli: Laboratory Investigations of ProteinBiochemistry,Growth and Gene Expression Regulation, 3rd edition; RPI.