Optimal Conditions for the Growth OfE

Optimal Conditions for the Growth ofE. Coli Sarah M. Don Biology EEI Semester 4, 2008 Mrs Gibson Contents 1 Introduction 2 1.1 Ecology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.2 CellStructure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.3 Plasmid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.4 Virulence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1.5 EnvironmentalStress. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1.6 ResistanceMutation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1.7 Antibiotics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1.8 LaboratorySpecies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1.9 Objective. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2 Hypotheses 6 3 Independent Variables 6 4 Dependent Variable 6 5 Basic Test and Control Set-ups 7 6 Controlled Variables 8

7 Materials and Equipment 9 8 Procedure 9 9 Results 10 10 Discussion 12

10.1 Salt. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 10.2 Glucose. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 10.3 pH. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 10.4 Temperature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 10.5 Antibiotics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 10.6 Further Research. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

11 Conclusion 15 12 Appendix A 18

12.1 Dilution of E. Coli. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 12.1.1 Aim. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 12.1.2 Equipment and Materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 12.1.3 Procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

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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 37oC. As glucose is absorbed in the small intestine, the E. coli would be used to low concentrations. However, as glucose (C6H12O6) 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 CellStructure

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 alsogram-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 aplasmid. 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.

2 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

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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 ofβ-lactam antibiotics which containβ-lactam rings, which is a chemical compound that inhibits the cell wall synthesis of gram- positive1 bacteria.(Wikipedia, 2008)

Another kind of antibiotic are those that inhibit protein synthesis such as tetracycline, chloram- phenicol and streptomycin (see Figure 4). There are alsobacteriostatic2 antibiotics such as sulpha- triad, 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.

1Gram-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.

2Bacteriostatic as opposed to bactericidal - antibiotics that slow or stop the growth without actually killing the

bacteria. (Wikipedia, 2008) 4

Figure 4: Inhibited enzyme (Wikipedia, 2008) 1.8 LaboratorySpecies

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 condi- tions 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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2 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 37oC.

3 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.

4 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 7