ns Of Nutrient BrothGrowth Dynamics of E. coli in Varying Concentrations of Nutrient Broths, pH, and

in the Presence of an Antibiotic

Dvora Szego,

Elysia 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 1.0x nutrient broth. This was

used to compare the growth in the experimental media of 0.5x and 2.0x, nutrient

broths; nutrient broths with an additional 5.0mM of glucose and another with

5.0mM 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/mL. 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/mL; lactose producing 477,441 cells/mL

and finally pH 6.0 producing 579,557cells/mL. The remaining four media, after

calculations, produced cell counts greater than the control: 2X with 1,087,009

cells/mL; 0.5X with 2,205,026 cells/mL; pH 8 with 3,583,750 cells/mL and finally

pH 7.0 with 8,090,325 cells/mL. 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=N0ekt. N equals the

number of cells in the culture at some future point, N0 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 N0 )/t, where ln N is the natural log of the number of

cells at some time t, ln N0 is the natural log of the initial number of cells

and t is time. 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 37C, 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

E coli growing in a culture at 37C 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 37C, and the variables were the broths which the E. coli were

using 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 0.5x and

2.0x, nutrient broths with additional an 5.0mM of glucose and another with

5.0mM 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 tool

in molecular biology used to inhibit a specific process, chloramphenicol

200mg/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 pipetted out and placed into a cuvet, if care is used, to

speed up this process, the sample may be poured into the cuvet. After the

aliquots of each sample had been transferred to a cuvet the OD was measured at

600nm. 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 OD600 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 cuvet with a Kimwipe 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 cuvet, the samples were gently swirled to ensure

that the cells are evenly distributed throughout the cuvet, 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-15minutes and dividing by 15 minutes. Beginning

with an initial cell count equal to that of the control, these k values were

used with the growth equation to calculate the number of cells in each media

at each time interval. 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. 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 at

time 150 minutes produced a final cell count of 796,500 cells/mL. 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/mL; glucose

producing 411,951 cells/mL; lactose producing 477,441 cells/mL, and finally

pH6.0 producing 579,557 cells/mL. The remaining four media produced cell counts

greater than the control: 2.0x with 1,087,009 cells/mL; 0.5x with 2,205,026

cells/mL; pH 8.0 with 3,583,750 cells/mL and finally pH 7.0 with 8,090,325

cells/mL. The Log number of cells was plotted versus Time(Graphs 2,4,6). This

is 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). 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/mL, however

the pH of 8.0 producing 3,583,750 cells/mL was found to produce a greater number

of cells than that of a pH of 6.0 579,557 cells/mL. The change in nutrients

also had a great affect on the cell production (the control produced a final

cell number of 619,500 cells/mL). The 0.5x nutrient broth produced 2,205,026

cells/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 to

note that the 0.5x broth actually produced more cells than the 2.0x 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/mL(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.

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 a

pH of 7.0 most closely resembled this ideal environment of 37C, 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 cuvet may not have been thoroughly

wiped off therefore causing error in the OD600. The final possibility for this

error is that the cells in the sample may have settled to the bottom of the

cuvet 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 0.5x nutrient broth more

cells were produced than in either the 1.0x and 2.0x 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 1.0x broth or the 2.0x 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 operons,

temperatures changes, and different drugs/antibiotics at different

concentrations.

References

Atlas, Ronald M. 1995. Principles of Microbiology, St. Louis, MO: Mosby-Year

book

Inc.

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

Wm. C.

Brown Publishers.

Edick, G.F (1992). Escherichia coli: Laboratory Investigations of Protein

Biochemistry,

Growth and Gene Expression Regulation, 3rd edition; RPI.