The effect of chloramphenicol on E Coli cell growth 1 Running head: THE EFFECT OF CHLORAMPHENICOL ON E COLI CELL GROWTH The Effect of Chloramphenicol on E Coli Cell Growth The effect of chloramphenicol on E Coli cell growth 2 Abstract.
Escherichia Coli is a gram negative bacterium found in the intestines of most warm blooded animals. We hypothesized that because the antibiotic inhibits protein synthesis, its addition into an E Coli cultur would cause growth to be arrested. In order to confirm the effect of chloramphenicol on E Coli growth we compared the increase in cell concentration of two cultures of E Coli over a four hour time period. One of the cultures was treated with chloramphenicol after the first hour. The concentration of the E Coli in the culture with chloramphenicol soon stopped increasing and after three and a half hours actually began to slightly decline. In contrast, the untreated bacteria experienced continued logarithmic growth. From these results we concluded that chloramphenicol does in fact inhibit protein synthesis in E Coli and act as an effective growth inhibitor. The effect of chloramphenicol on E Coli cell growth 3 Introduction

This study documented the effect of chloramphenicol on E Coli cell growth.
Chloramphenicol is an antibiotic used in third world countries to treat infections stemming from both gram positive and gram negative bacteria. It treats infections by binding to the 50S subunit of a bacterial ribosome, thus blocking the tRNA-accepting site of the mRNA complex and effectively stopping transpeptidation (chloramphenicol). Ultimately, chloramphenicol blocks bacterial growth by disabling the formation of peptide bonds between amino acids. Chloramphenicol has this effect on bacteria when present in concentrations between 1 and 10 µg/ml. However, in strains of resistant bacteria it may take up to 1000ug/ml for chloramphenicol to have an effect. Countless studies, including one done by Cavalli and Maccaro have already proven that it halts growth in E Coli by inhibiting protein synthesis (Brock 35). The goal of our study was to once again independently confirm this phenomenon. When untreated with antibiotics and left undisturbed, E Coli Cultures tend to grow in a predictable and logarithmic manner. They undergo four major stages of growth: a phase of enlargement, a phase of logarithmic multiplication, a stationary phase, and a phase of logarithmic death (Edick). These phases are also commonly known as phase I, II, III, IV respectively. Phase I is a time of protein synthesis and cell growth and during which there is no division taking place. DNA is first transcribed into RNA. Then mRNA molecules begin translation by attaching themselves to ribosomes. Meanwhile tRNA molecules with bases complementary to those of ribosome-attached mRNAs are dispersed in the cytoplasm. As individual tRNA molecules attach their mRNA sequence in the ribosome, they each bring with them an amino acid which is then added to the growing polypeptide chain through peptide bond formation. Then another tRNA molecule binds to the next corresponding codon and the process continues to repeat itself until a complete protein has been formed (Reece). By inhibiting peptide bond formation by binding to the 50s subunit of the ribosome Chloramphenicol effectively brings this process to a standstill and halts protein synthesis. When a critical number of proteins have formed in this manner, E Coli cultures enter growth phase II, the phase of logarithmic multiplication. During this phase bacteria undergo asexual binary fission. Growth is rapid at first while there is still an unlimited supply of nutrients and waste products have not yet begun to accumulate. As waste products accumulate and the nutrient supply begins to be used up, net growth halts as cell death becomes equal to cell formation. This stationary phase usually lasts for an extended period of time until finally nutrient supply falls below a critical point. The culture then enters a stage of logarithmic death which is the polar opposite of stage two. The number of live cells in the culture decreases slowly at first but then pick up speed as more and more cells become unable to deal with the nutrient concentration around them(Edick). By interfering with phase one of the E Coli growth curve, chloramphenicol causes the bacteria to exhibit a growth curve that differs from this standard pattern. Materials & Methods
The effect of chloramphenicol on E Coli cell growth 4 Materials The two E Coli cultures were cultivated a broth composed primarily of beef heart and treated gelatin. A trace amount of E Coli was added to this medium to begin growth of the main culture. The flask containing the broth was then shaken and stored at 37 degrees Celsius for 20 hours until it became a stationary culture. A few hours before the onset of the experiment, the ecoli culture was used to create the two cultures later used in the experiment. One milliliter of ethanol was then added to both the chloramphenicol and the control culture. Growth Assay The cultures were then left to grow at 37 degrees Celsius. Every thirty minutes over a time period of four hours samples of culture were read using a spectrophotometer that had been blanked and set to a wavelength of 600 nm. After one hour, an appropriate amount of chloramphenicol to give a final antibiotic concentration of 200 µg/ml was dissolved in ethanol and then added to the culture. These O.D. values were then converted to cell density values using the knowledge that OD1.000 = 1 x 106 cells/ml. The resulting growth curves where then documented.
Table I- Lists all E Coli concentrations collected in cells per milliliter. The table also
includes the difference in cell concentration between the two cultures at any moment in
time in order to accentuate the contrast over time.
Figure I- compares the number of cells per milliliter of the treated experimental culture
with that of the control. OD reading were taken over a four hour period of time was
graphed and readings were taken every thirty minutes. Concentrations in this graph were
derived from OD readings and the formula OD6001.000 = 1 x 106 cells/ml.
Chloramphenicol had an easily recognized effect on E Coli cell concentration, causing cell growth to deviate from the standard growth curve after thirty minutes and prematurely reach a stationary phase During the last half hour of the experiment, measured cell concentration in the culture treated with chloramphenicol even declined by 2.5 x 10 4 cells per ml as the culture prematurely entered the cell of logarithmic death. In contrast the control culture exhibited logarithmic growth and did not appear to reach stationary phase until three and a half hours into the experiment. At no point did the measured cell concentration actually decline. As stated earlier, E Coli concentration probably stopped increasing after the addition of chloramphenicol because of its role in inhibiting protein synthesis. Protein synthesis is an indispensible part of the cell cycle without which cells cannot replicate The effect of chloramphenicol on E Coli cell growth 5 themselves. This is especially true during DNA replication for which proteins such as primase, helicase, topoisomerase, and polymerase are necessary (Becker & Kleinsmith & Hardin, 2006). It is important to note that the control culture not treated with chloramphenicol did not exhibit ideal exponential growth. This trend was most likely due to the fact that the E Coli did not have ideal growth conditions to start with due to constraints on nutrient concentration and growing space. According to our data, there was a thirty minute time lapse between the addition of chloramphenicol to the experimental culture and the beginning of stationary phase. Indeed, during this thirty minute interval there was no appreciable difference in cell growth between the experimental and control cultures. One possible explanation may be that not all intercellular growth requires protein synthesis per se since much of it involves the continued growth of existing proteins (Ben-Shaul). Thus when cells were initially treated with chloramphenicol, OD values continued to increase as a result of growth of preexisting protein chains. When these cells reached their maximum size however and began preparing for division, progression through the cell cycle was prevented by lack of protein synthesis and then protein concentrations began to stabilize. Another possible explanation is the accumulation of free nucleotides in the cytoplasms of the E Coli cells. According to previous studies, the nuclei of E Coli bacteria treated with sub-lethal concentrations of chloramphenicol progressively grew in size over time. When placed in a fresh medium, bacteria resumed with cell division and their nuclei shrank back to normal. This increase was attributed to an increase in the number of free nucleotides. This increase was hypothesized to result from the fact that chloramphenicol blocks the incorporation of free amino acids into large complexes but does not inhibit their formation per se (Brock 38). The cause of the decline in measured protein concentrations for the experimental culture after three and a half hours is likewise debatable. Generally speaking, spectrophotometers do not differentiate between live and dead cells. Because they measure protein concentrations, any affect in cell condition should leave the OD value unchanged (Edick). Thus, the acceleration in cell death which naturally accompanies the onset of phase IV should not have resulted in a decrease in OD readings (the basis of the cell concentration values used in this experiment). Even cell damage caused by chloramphenicol should not have caused this anomaly. However, previous studies have shown that cells growing in sub-lethal concentrations of chloramphenicol do tend to exhibit abnormal bar-like shapes or L forms as their proteins cluster closer together (Brock 38). By causing cells to shrink, this clustering could have been responsible for the drop in OD readings. Another explanation could be cell lysis. By inhibiting protein synthesis in E Coli cells, chloramphenicol effectively halts the formation of new lysosomes. Thus treated E Coli cells are no longer able to digest waste products which instead accumulate in the cells. As a result, osmosis causes water molecules to flow enter the cell eventually causing the cell to become hypotonic. Cell membranes then burst. These lysed cells cannot be detected with a spectrophotometer and thus appear to "disappear". Thus measured cell concentration decreases. Chloramphenicol has also been shown to break down the ribosomes of bacteria treated with it for an extended period of time. This could also be responsible for the decline in OD values (Gupta, 1975). The effect of chloramphenicol on E Coli cell growth 6 Many questions still remain answered by our experiment even after chloramphenicol's role in inhibiting cell growth has been clearly shown. For one, we only tested the effect of chloramphenicol on one concentrati E Coli It is still unclear whether an E Coli concentration optimally favorable for growth would counteract the inhibiting properties of chloramphenicol. Also, previous studies have shown that bacteria tend to be more susceptible to chloramphenicol when grown in a temperature above 37 degrees Celsius (Brock). Further experimentation could be done to determine whether this increased sensitivity would entail a shorter time lag between treatment with chloramphenicol and the onset of the stationary phase or whether it would entail an earlier and steeper decline in measured OD values or some combination of both scenarios. The experiment could also be repeated with varying pHs to see if chloramphenicol's ability to bind to the 50S RNA subunit is impaired at certain levels of acidity. Our study was also limited in that we only measured concentration for four hours. The full affect of chloramphenicol on logarithmic decay would have been an interesting addition. In summary, our study was successful in that it conclusively proved that chloramphenicol limit's cell growth but could still be expanded upon in order to get a more nuanced understanding of its affect on the E Coli cell cycle. The effect of chloramphenicol on E Coli cell growth 7 References
Becker, Kleinsmith, Hardin. (2006), The World of the Cell. Ch 18; Pearson Education. Ben-Shaul, Y. (1969), Effect of Chloramphenicol on the Growth, Size Distribution, Chlorophyll Synthesis and Ultrastructure of Euglena Gracilis, 627-644: Tel Aviv University. Accessed 15 Mary 2008. Brock, T. (1996), Chloramphenicol, 32-39: Indiana University. Accessed 19 May 2008. "Chloramphenicol." Encyclopedia Brittanica Online. Academic Search Premier. EBSCO. Edick, G. (2008), An Introduction to Research Methods in Cell and Molecular Biology, p 64: Department of Biology Rensselaer Polytechnic Institute. Gupta, R. (1975), Killing and Lysis of Escherichia coli in the Presence of Chloramphenicol: Relation to Cellular Magnesium, 748-749: American Society for Microbiology. Accessed 19 March 2007.

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