Optimal Conditions for the Growth of E Coli

Optimal Conditions for the Growth of E. Coli Sarah M. Don

Biology EEI Semester 4, 2008 Mrs Gibson

Contents 1 Introduction 1.1 Ecology . . . . . . . 1.2 Cell Structure . . . . 1.3 Plasmid . . . . . . . 1.4 Virulence . . . . . . 1.5 Environmental Stress 1.6 Resistance Mutation 1.7 Antibiotics . . . . . . 1.8 Laboratory Species . 1.9 Objective . . . . . .

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2 Hypotheses

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3 Independent Variables

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4 Dependent Variable

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5 Basic Test and Control Set-ups

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6 Controlled Variables

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7 Materials and Equipment

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8 Procedure

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9 Results

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10 Discussion 10.1 Salt . . . . . . . 10.2 Glucose . . . . . 10.3 pH . . . . . . . . 10.4 Temperature . . . 10.5 Antibiotics . . . . 10.6 Further Research

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11 Conclusion 12 Appendix A 12.1 Dilution of E. Coli . . . . . . . . 12.1.1 Aim . . . . . . . . . . . . 12.1.2 Equipment and Materials 12.1.3 Procedure . . . . . . . . .

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1

Introduction

Escherichia Coli (E. Coli) is part of the common microflora in the large intestine, causing no harm to the host. Because it is a fast growing prokaryote, it is widely used in laboratories for growing cultures for testing and experimentation. By identifying the optimal growth conditions and environmental tolerances for the K-12 strain, E.coli may be more efficiently cultured for laboratory experimentation.

1.1

Ecology

As E. coli is part of the common microflora in the large intestine, it is accustomed to a pH of 7-8 and body temperature of 37o C. As glucose is absorbed in the small intestine, the E. coli would be used to low concentrations. However, as glucose (C6 H12 O6 ) is its energy source, if excess glucose were available for consumption, it would be expected that the E. coli would utilise it and grow at a faster rate. Salt (NaCl) is absorbed in the colon, so the amount of salt that the E. coli is exposed to depends on how much salt is consumed by the host organism. However, because of the mechanism of osmosis, extremely high levels as well as complete absence of salt could be lethal to E. coli bacteria.

1.2

Cell Structure

The shape of the E. Coli bacterium is cylindrical, and it is covered in fimbriae (flagellum-like structures protruding from the cell membrane that propel the bacterium through its medium). (Wikipedia, 2008) E. coli is also gram-negative, which means that its cell wall is composed of a layer of peptidoglycan, opposed to the phospholipid bilayer of gram-positive bacteria. (MedicineNet, 1999)

Figure 1: E.coli bacterium (Ussery, 2001)

E. coli bacteria are prokaryotic, meaning that they do not have a nucleus. Instead, their DNA is continuous chromosome in the shape of a ring which is called a plasmid. Because the plasmid is suspended in the cytoplasm, it is very easy for the E. Coli bacteria to take up extraneous DNA and RNA fragments and add them to their genome. This makes E. Coli an ideal laboratory specimen for studying genetic mutation and conducting recombinant DNA experiments.

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1.3

Plasmid

Figure 2: E.coli plasmid (Mathews, 1996)

The Plasmid of the K-12 strain of E.coli is one long looped chromosome that consists of 4580 genes. 1374 of these genes code for enzymes.(Venter, 2008) When environmental conditions such as temperature and pH are altered, the shape of the active site of enzymes changes. If the active site of an enzyme is morphed too much, it may not be able to perform its function.

Figure 3: Enzyme working normally (Wikipedia, 2008)

As shown in Figure 3, the enzyme has a particular shape that a substrate fits into. However, if environmental factors such as temperature or pH are altered and the shape of the active site changes, the enzyme is said to be ‘denatured’. While the enzyme is denatured it does not function. This 3

can have a detrimental affect on the health of E.coli bacteria. In this investigation, the permissible temperature and pH ranges for growth were identified.

1.4

Virulence

E.coli is able to behave like a virus by misleading a body cell to engulf it by endocytosis. Once the E.coli has entered the cytoplasm of the host cell, it can add part of it’s genome to the host cell’s. It can also act as a parasite once in the host cell by using the host cell’s resources and producing toxins which eventually kill the host cell. However this is not characteristic of the K-12 laboratory strain.

1.5

Environmental Stress

Environmental change and stress is the most prevalent cause of evolution and mutation in the plasmid of E.coli bacteria. Wild-type bacteria have a wide variety of genes so when environmental change occurs (within permissible parameters), some of the E.coli may survive and their offspring inherit the gene(s) that allowed them to survive. When E. coli bacteria become stressed, they exchange sections of DNA and reproduce quickly to improve chances of survival.

1.6

Resistance Mutation

When bacteria are exposed to antibiotics, there are three windows of affect that depend on the concentration of the antibiotic. The first window is when the concentration of the antibiotic is so low that none of the bacteria are affected. The next window is described as when only some of the bacteria survive and reproduce with their offspring inheriting the gene that provided resistance to that type and concentration of antibiotic. The third window is when the concentration of antibiotic is so high that all the bacteria die. This mechanism works best when the E.coli bacteria are wild and have a wide selection of gene types. When strains such as K-12 are produced and sold commercially for laboratory use, all the bacteria in one sample have been taken from the same colony and are very genetically similar. This reduces the chance of such a distinct natural selection process.

1.7

Antibiotics

There are different types of antibiotic that use different mechanisms of action to kill their target bacteria. For example, Penicillin G and Ampicillin are both kinds of β-lactam antibiotics which contain β-lactam rings, which is a chemical compound that inhibits the cell wall synthesis of grampositive1 bacteria.(Wikipedia, 2008) Another kind of antibiotic are those that inhibit protein synthesis such as tetracycline, chloramphenicol and streptomycin (see Figure 4). There are also bacteriostatic 2 antibiotics such as sulphatriad, which inhibit the production of folic acid which is necessary for DNA and RNA synthesis. Different attributes of E.coli can be learned by observing its response to different types of antibiotic, as in this investigation. 1

Gram-negative bacteria have a cell wall made of a layer of peptidoglygan sandwiched between two bilayers of phosopholipids. However, gram-positive bacteria have a cell wall made of only one bilayer of phospholipids on the inside of the cell wall and a layer of peptidoglycan on the outside, making the membrane somewhat vulnerable to biochemical attack. E.coli, however, is classified as a gram-negative bacteria. 2 Bacteriostatic as opposed to bactericidal - antibiotics that slow or stop the growth without actually killing the bacteria. (Wikipedia, 2008)

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Figure 4: Inhibited enzyme (Wikipedia, 2008)

1.8

Laboratory Species

E. coli K-12 is the most common strain of E. coli for laboratory use. However, because it is cultured commercially, it has certain attributes that differ from the wild type. Such differentiation includes the loss of resistance to other flora in the gut, and the toxins that such co-existing bacteria produce. As E. coli K-12 is used for many kinds of biological experimentation (mainly recombinant DNA experiments), it is important to know the optimal conditions for growth and the bacteria’s tolerances to certain environmental factors and introduced substances. In order to control as many variables as possible when conducting recombinant DNA experiments, it is ideal to culture E. coli in its optimal growth conditions so as to not induce stress, causing mutation and reproduction of that mutation.

1.9

Objective

The objective of this extended experimental investigation was to identify the optimal growth conditions for culturing the K-12 laboratory strain of E.coli. The bacteria’s idea conditions and tolerances to certain environmental factors (salt concentration, glucose concentration, pH and temperature) as well as introduced substances (antibiotics) were tested.

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Hypotheses 1. As the salt concentration increases, the amount of E.coli growth over 24 hours decreases. 2. As the glucose concentration increases, the amount of E.coli growth over 24 hours also increases. 3. The optimal pH for E.coli growth is 7.0. 4. The E.coli is not tolerant of any of the antibiotics. 5. The optimal temperature for E.coli growth is 37o C.

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Independent Variables 1. In the salt test, the concentration of salt was altered. 2. In the glucose test, the concentration of glucose was changed. 3. In the pH test, the pH was altered. 4. In the antibiotic test, the type of antibiotic was changed. 5. In the temperature test, the temperature was the independent variable.

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Dependent Variable

For the salt, glucose, pH and antibiotic tests, the measured variable was the radius around the test discs that was clear of E.coli growth after 24 hours. For the temperature test, the measurable variable was the number of colonies that grew in a 24 hour period.

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5

Basic Test and Control Set-ups

Figure 5: E. coli tolerances test plate setup

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6

Controlled Variables

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7 • • • • • • • • • •

8

Materials and Equipment Bunsen burner Heat proof tile Filter paper Hole punch Esky Tripod Water bath Forceps 6 thermometers 2 incubators

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• • • • • • • • • •

10% NaCl solution 5.0% NaCl solution 2.0% NaCl solution 1.0% NaCl solution 0.1% NaCl solution 10% C6 H12 O6 solution 5.0% C6 H12 O6 solution 2.0% C6 H12 O6 solution 1.0% C6 H12 O6 solution 0.1% C6 H12 O6 solution

Freezer ice pack 10 sterile agar plates Inoculating loops 0.1M NH3 solution 0.1M CH3 COOH solution 0.1M HCl solution 0.1M NaOH solution Distilled water (H2 O) Antibiotic test ring E. Coli solution diluted to 103 /10mL

Procedure 1. 18 discs were punched out of filter paper with the hole punch. 2. An inoculating loop was used to evenly spread the E. Coli on one agar plate labelled as NaCl. 3. The forceps were passed through the bunsen flame. 4. One of the filter paper discs was quickly passed through the flame to sterilise it, while being sure not to burn the paper. 5. The disc was then dipped in the 10% NaCl solution and placed on the agar plate labelled as NaCl. 6. Steps 3-5 were repeated for the 5.0%, 2.0%, 1.0% and 0.1% solutions. 7. A control disc with no NaCl solution was also added to the agar plate labelled as NaCl (see Figure 5). 8. Steps 1-6 were repeated to make a test plate of the five different concentrations of C6 H12 O6 solution and control (see Figure 5). 9. Steps 1-6 were repeated to make a test plate of the pH solutions; NH3 , CH3 COOH, HCl, NaOH, H2 O, as well as the control disc (see Figure 5).

10. Steps 1-3 were repeated and an antibiotic test ring was placed on the agar (see Figure 5). 11. The four test plates were placed in an incubator set at 37o C for 24 hours. 12. The remaining E. Coli was diluted to 103 cells in 10mL of nutrient broth (see Appendix A). 13. Step one was repeated to make up 6 test plates for the temperature test. 14. One test plate was placed in a refrigerator set at 6o C, another in an esky at 19o C as shown in Figure 5, another in a warm cupboard at 35o C, another in an incubator set at 37o C, another partially submerged in a water bath set at 50o C and the last at 45o C suspended above the water bath. 15. The extent of E. Coli growth in each test plate from the five tests was observed after 24 hours. 9

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Results Table 1: Salinity Test Results Concentration of NaCl (%) Radius of inhibition zone (mm) 10 0.0 5.0 0.0 2.0 0.0 1.0 0.0 0.1 0.0 0.0 0.0

Table 2: Glucose Test Results Concentration of C6 H12 O6 (%) Radius of inhibition zone (mm) 10 0.0 5.0 0.0 2.0 0.0 1.0 0.0 0.1 0.0 0.0 0.0

Table 3: pH Test Results pH Radius of inhibition zone (mm) 0.0 1.0 2.4 0.0 7.0 0.0 11.6 1.0 14.0 2.0 None 0.0

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Figure 6: Affect of pH of E.coli

Table 4: Temperature Test Results Temperature ( C) No. of Colonies Average Diameter of Colonies (mm) 6 0 15-20 0 35 10 8 37 11 10 45 6 10 50 4 5 o

Figure 7: Affect of Temperature of E.coli

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Table 5: Antibiotic Test Results Antibiotic Concentration (µg) Radius of inhibition zone (mm) Ampicillin 10 0.0 Chloramphenicol 25 5.0 Penicillin G 25 0.0 Streptomycin 10 7.0 Sulphatriad 200 0.0 Tetracycline 25 6.0

10 10.1

Discussion Salt

None of the concentrations of salt inhibited the growth of E.coli. This indicates that K-12 E.coli is able to tolerate added salt of up to 10% concentration. However, the nutrient agar that the E.coli test plates were made with already contained a certain unknown concentration of salt. If the nutrient agar had not contained any salt at all, then E.coli inoculated where the control (0%) and the discs with lower concentrations of salt were placed may have died by osmosis. Thus the results that concerned the lower concentrations of salt were not entirely reliable. However, the higher concentrations may have still been viable although not as a higher boundry. So the hypothesis, that as the concentration of salt increases the growth of E.coli decreases, was rejected because for all the concentrations of salt tested, there was similar E.coli growth during the test period. To further improve this experiment, a nutrient broth containing no salt at all would be used so that the affect of having 0% salt concentration could be accurately tested. Much higher salt concentrations would also be tested. If more extreme concentrations of salt were used then the the E.coli cells growing in the extreme high and low concentration areas would die by osmosis. The E.coli in extremely low concentrations of salt would gain too much water or burst, and the E.coli in extremely high concentration of salt would lose too much water in an effort to reduce the concentration gradient of salt inside and outside the bacteria cells.

10.2

Glucose

None of the concentrations of glucose had any affect on the growth of E.coli. It was expected that as the concentration of glucose increased, the growth of E.coli also increased. However, the variation between the concentrations of glucose may have been too small for a noticeable increase in E.coli growth as the concentration of glucose increased. Also, even the maximum concentration of glucose tested, 10%, may have been too low to affect the growth of E.coli to an observable extent. The nutrient agar would have also contained a certain amount of glucose, so the lower concentration glucose test discs did not provide meaningful results. To further improve this experiment, much larger concentrations of glucose would be used. Also, a different nutrient agar containing no glucose would be used so that a true 0% glucose test disc could be used and more meaningful results may be obtained. If the tested concentrations of glucose were more extreme, then the E.coli growing where there was no glucose would find it very difficult to survive. The E.coli growing in higher concentrations of glucose would absorb the extra glucose provided and turn it into energy, allowing all its chemical process to occur faster and thus grow and reproduce more quickly. 12

10.3

pH

There was a very clear relationship between pH and the size of the inhibition zone, which was able to be represented graphically as in Figure 6. E.coli appeared to be more tolerant of low pH than of high pH. As shown in Figure 6 and Table 3, the E.coli was able to tolerate an acid of pH 2.4 more easily than a base of pH 11.6. However, the result that the E.coli could tolerate a pH of 2.4 was considered anomalous. The reason why E.coli is not able to tolerate extremely alkaline and acidic environments is because many of the enzymes that are part of important processes in the E.coli bacterium are very pH-sensitive. When the change in pH is so extreme, enzymes in E.coli become denatured, and are prevented from doing their job. Depending on the enzyme, becoming denatured could cause all sorts of interruptions to biochemical processes, however, inhibition of enzymes leads to death of the E.coli. Enzymes typically only have a very small window of tolerance to pH variation, so for the E.coli to have survived in environments of pH 2.4 - 7.0 is very unlikely. There was probably a fault in the pH 2.4 solution (acetic acid) or test disc. As the result of E.coli tolerance to pH 2.4 was considered anomalous, the hypothesis, that 7.0 is the optimal pH for E.coli growth was accepted. The radii of the inhibition zones around the test discs were very small compared to those of the antibiotic test. This could be due to the concentration of the varying pH substances being too low to show the full affect of the pH. The results were not considered to be anomalous, however, because they do show a direct correlation between the pH and inhibition of the E.coli. To further improve this experiment, higher concentrations of each of the varying pH solutions would be used so that the effect that the pH has on the E.coli is much clearer. Also, more varying pH solutions would be tested. The difference between a pH of 2.4 and 7.0 is quite large, as is the difference between pH 7.0 and 11.6. The maximum tolerable pH of E.coli may not have been 7.0, but pH 11.6 was the next tested pH after 7.0, and 11.6 was too basic for the E.coli to survive. So, all that can be concluded about the maximum pH is that it is between 7.0 and 11.6. Further pH values would need to be tested in order to obtain a more accurate estimate of the maximum pH for E.coli growth. Similarly, the minimum pH for E.coli growth may be somewhere between pH 0.0 and 2.4, however further pH values would need to be tested to find the actual minimum pH for E.coli growth. Furthermore, pH 2.4 may have been an anomalous result. However, because there were no other tested pH values between 2.4 and 7.0, it is not certain that the pH of 2.4 is the true minimum pH that E.coli can tolerate.

10.4

Temperature

Temperature, like pH, affects the activity of enzymes. The results show a direct correlation between the temperature and E.coli growth as shown in Table 4 and Figure 7. The hypothesis, that the optimal temperature for E.coli growth is 37o C (body temperature), was accepted. The most and largest colonies were present on the temperature test plate that was placed in the incubator set at 37o C. As with the case of varying pH, when the temperature is changed to a temperature outside the tolerable range of an enzyme, the enzyme becomes denatured and cannot function. This is what was happening in the E.coli bacteria that were on test plates in the refrigerator and esky. The E.coli on the test plates in 45o C and 50o C were able to survive, but as the temperature increased from 37o C to 50o C, the size and number of colonies decreased. The hypothesis, that the optimal temperature for E.coli growth is 37o C was accepted. The results are reliable because precise laboratory techniques were used and the E.coli was diluted just enough so 13

that the colonies were clearly distinct and countable. All the temperature test plates remained sterile throughout the experiment as no other bacterial or fungal colonies were present at the end of the 24 hour growth period. This experiment could be improved by testing a greater number of temperature intervals so that an ideal temperature range for E.coli growth could be more precisely identified.

10.5

Antibiotics

The E.coli responded to 3 of the 6 types of antibiotic. Therefore the hypothesis, that the E.coli is not tolerant of any of the antibiotics, was rejected. The E.coli was not affected by either Penicillin G or Ampicillin. This is because E.coli is a gramnegative bacteria and produces β-lactamase which an enzyme that breaks down the β-lactam rings of the penicillins. Because E.coli produces β-lactamase, it is able to prevent the β-lactams from the penicillin from destroying its cell membrane. Sulphatriad also did not inhibit the growth of E.coli. As discussed in the introduction, Sulfatriad is a bacteriostatic antibiotic that suspends the production of folic acid in all bacteria. However, because the E.coli was growing on nutrient agar, it is possible that the agar contained enough folate for the folic acid production process to be bypassed. Therefore the E.coli continued to grow. The chloramphenicol, tetracycline and streptomycin were all able to kill the E.coli bacteria. These three antibiotics inhibit the activity of enzymes that are instrumental in DNA and RNA synthesis. So the conclusion from the antibiotic test is that E.coli has β-lactamases and therefore cannot be killed by β-lactam antibiotics. Also, there was folate in the nutrient broth so the sulphatriad had no effect on the E.coli, and the best way to kill E.coli is to treat it with antibiotics that attack the DNA and RNA synthesising enzymes. To further improve this experiment, various other kinds of antibiotics with different mechanisms of action could be tested.

10.6

Further Research

There is further relevant research pertaining to the topic of optimal growth conditions and tolerances of E.coli bacteria. Research into E.coli’s compatibility with fungi and other bacteria would be helpful to explain anomalous results when other bacterial or fungal colonies are present in the test plates. Also, the growth rate of the E.coli under certain conditions (such as the variables manipulated in the experiments in this investigation) could be tested.

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Conclusion

The original hypotheses of the salt, glucose and antibiotic tests were rejected, and the hypotheses of the pH and temperature tests were accepted. Further improvements could be made to all the experiments to gain more accurate results. However, from the results obtained in this extended experimental investigation, E.coli growth is not affected by low concentrations of salt or glucose, 37o C is the optimal temperature and 7.0 is the optimal pH for E.coli growth, and the antibiotics that interrupt DNA and RNA synthesis are lethal to E.coli bacteria.

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References [1] APUA (2007) How Antibiotics Work - the Mechanism of Action, Allience for the Prudent Use of Antibiotics, U.S.A, http://www.tufts.edu/med/apua/Miscellaneous/mechanisms.html (10/09/08) [2] Brana, H. et al. (1981) ”The Mechanism of Resistance to Streptomycin in Escherichia coli. Function Analysis of the Permeability Barrier of Cells Harbouring the R1 drd -19Km− Plasmid”, Folia Microbiol., vol.26, pp.345-350. [3] Bowater, L. (2004 ”Characterization of a temperature-sensitive DNA ligase from Escherichia coli ”, Microbiology, vol.150, pp.4171-4180. [4] Chopra, I., Roberts, M. (2001) ”Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance”, Microbiolgy and Molecular Biology Reviews, vol.65, no.2, pp.232-260. [5] Gibson, L. (2008) Personal correspondence. [6] Hillen, W. (1996) ”Tetracyclines: antibiotic action, uptake, and resitance mechanisms”, Archives of Microbiology, vol.165 no.6 pp.359-369. [7] Jardetzky, O. (1963) ”Studies on the Mechanism of Action of Chloramphenicol”, The Journal of Biological Chemistry, vol.238, no.7, pp.2498-2507. [8] Korolik, V. (2008) Personal correspondence. [9] Livermore, D. M. et al. (1986) ”Behaviour of TEM-1 β-lactamase as a resistance mechanism to ampicillin, mezocillin and azlocillin in Escherichia coli ”, Journal of Antimicrobial Chemotherapy, vol.17, no.2, pp.139-146. [10] Mathews, C. K. (1996) Biochemistry, Benjamin Cummings Publishing Company, California, U.S.A. [11] MedicineNet (1999) Definition of Gram-negative, MedicineNet http://www.medterms.com/script/main/art.asp?articlekey=9586 (03/08/08)

Inc.,

[12] MicrobeWiki (2008) Escherichia, MicrobeWiki, http://microbewiki.kenyon.edu/index.php/Escherichia (13/09/08) [13] Southern Biological (2006) Bacterial and Fungal Cultures, Southern Biological, Victoria, Australia. [14] Thomas, G. (2005) E. coli K-12 - model NOT menace in schoolwork, Microbiology On-line, http://www.microbiologyonline.org.uk/ecoli.htm (12/09/08) [15] Ussery, D. (2001) Analysis of E. coli Promoters, Introduction to Bioinformatics, http://www.cbs.dtu.dk/staff/dave/MScourse/Lekt.17.04.2001.html (14/09/08)

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[16] Venter, J. C. (2008) Summary of Escherichia coli, Strain K12, version 12.1, SRI International, Marine Biological Laboratory, DoubleTwist Inc., The Institute for Genomic Research, J. Craig Venter Institute, University of California at San Diego, UNAM, and Macquarie University, http://biocyc.org/ECOLI/organism-summary?object=ECOLI (16/09/08) [17] Virual Medical Centre (2008) Anti-bacterials (Antibiotic Medicine), Virual Medical Centre, http://www.virtualmedicalcentre.com/treatments.asp?sid=72 (10/09/08) [18] Wikipedia (2008) Escherichia Coli, Wikipedia, http://en.wikipedia.org/E.coli (24/08/08) [19] Wikipedia (2008) Enterobacteriaceae, Wikipedia, http://en.wikipedia.org/Enterobacteriaceae (14/09/08)

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Appendix A

12.1

Dilution of E. Coli

12.1.1

Aim

12.1.2

Equipment and Materials

• • •

1.0mL syringe 10mL syringe 6 sterile containers

12.1.3

• • •

54mL nutrient broth Permanent marker 1 single colony of E. coli in 10mL nutrient broth (approx. 109 cells)

Procedure

1. The work bench was sterilised by wiping the surface with alcohol. 2. 9.0mL of nutrient broth was measured into each sterile container with the 10mL syringe. 3. Each of the sterile containers containing 9.0mL of nutrient broth was labelled starting with 108 , then 107 , until the last container was labelled as 103 . 4. With the 1.0mL syringe, 1.0mL of the initial E. coli solution was added to the sterile container marked as 108 so that the the E. coli in the new solution was diluted by a factor of 10 and thus contained approximately 108 cells. 5. The solution marked as 108 was swirled gently. 6. 1.0mL of the solution marked as 108 was added to the container marked as 107 so that the new solution contained approximately 107 E. coli cells. 7. The solution marked as 107 was swirled gently. 8. This process was repeated until the E. coli had been diluted by a factor of 106 and the solution labelled 103 had approximately 103 cells of E. coli. 9. Only the final solution (103 ) was used for culturing the E. coli.

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