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

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