Cytopathic Effects as a Result of Infection of E-coli

Cytopathic Effects as a Result of Infection of E-coliAnalysis of cytopathic effects as a result of cloudion of Escherichia coli by T4 phage using spectrophotometry and face assayAbstractT4 bacteriophage is a double stray DNA computer computer computer virus that infects Escherichia coli and is an important tool in research and study of genetics. It contains astir(predicate) 168,800 base pairs of DNA. It has an icosahedral capsid as its head that contains its nucleic acid and has a tail structure create of tail fibres, eccentric, base p posthumouss and coat. These different structures of T4-phage play important roles when invading the server kiosk (in this case E.coli). T4-bacteriophage infects the legions cadreular ph cardinal by lytic pathway where the force cells be employ to produce virus components and it results in the lysis of armament cell releasing the virus components to the external membrane. This try plans to exa momente the cytopathic effects of the T4 -phage on E.coli using a spectrophotometer. Also, plaque assay is carried out in evidence to tally the original concentration of phage in the solution. The spectrophotometer variant increased for both turn back and T4-infected closes as the eon of post infection increased. The highest reading was 0.192 for statement at 120 min pi.IntroductionViruses are small infectious agents and are about 10-30nm in size. Strelkauskas, et al (2010) describes viruses as obligate intracellular parasites which means they need a soldiers cell in order to survive and replicate. The viruses are classified depending on their morphology, chemical composition and mode of facts of life. They are dual-lane into two groups found on the nucleic acid they contain DNA or RNA. They are sub-divided into further groups correspond to their symmetry of capsid where the genetic material is enclosed within an envelope or without an envelope (naked). Also, the cast of the nucleic acid (single-stranded or dou ble stranded and linear or circular) play a role in the classification of viruses (Gelderblom, 1996). A fully assembled infectious virus is cognize as a virion and its structure is based on two important things the ability to survive in harsh conditions until it can infect the master of ceremonies cell and to be able to shed the protective barrier in order to enter a host cell.Bacteriophages are viruses that infect bacterial cells and were first observed in 1915 and 1917 by Twort and dHerelle. T4 bacteriophage is a member of T-even phages and infects Escherichia coli (E.coli) bacteria. It is considered as one of the complex viruses because of its genome which consist of 274 open reading frames and 40 of these reading frames encodes for structural proteins (Leiman, et al, 2003). T4- bacteriophage has been extensively studied since 1940s and plays a major role in cash advance of modern genetics and molecular biology. Some of the early essential ideas of genetics including the basis of genetics code, mRNA, ribo virtually and the codon down came from studies using T4-bacteriophages (Miller et al, 2003). T4- bacteriophage contains double stranded DNA as its nucleic acid which is enclosed within a protective coat know as capsid. Its capsid is icosahedral in shape (shown in bod 1). It also has tail part which is make up of coat, sheath, tail fibres and base plates. The tail part is useful when recognising a host cell followed by penetration to the host cell thus make infection.Bacteriophages call for two types of replication strategies lytic cycle and lysogenic cycle (Baker, et al, 2011). Lysogenic infection in E.coli is exemplified by bacteriophage lambda where the viral DNA replication takes place without destroying the bacterial cell. Here, the virion infects the host cell and rather of triggering to a greater extent virus reproduction, it recombines with the host genome causing no damage to the cell which is known as prophage. These viruses can remain i nactive within the bacterial cell for years and only cause infection beneath authentic circumstances. T4-bacteriophage is a virulent virus that causes lytic infection to E.coli. The first step in this cycle is adsorption where the virus recognises the cell receptors on the surface of the host cell and attaches itself to the host using its tail fibres. Once attached to the host cell, the virus must penetrate through the cell membrane of E.coli. The base plate of the virus comes in contact with the cell wall of the host cell causing con fleshational change in structure of the virus. This allows the sheath to contract and the core is pushed through the wall into the cell membrane where the viral DNA is injected into the host cell (Todar, 2008). DNA viruses have the same genome configuration as the host cell hence the replication run apply in host cell can be used for viral replication. T4- bacteriophage transcribes nucleases that poop out down the bacterial DNA which is used to pr oduce more viral genome. The newly made viral nucleic acid and structural proteins are assembled together to form virulent viruses. The lysozymes produced by the bacteriophages as a late viral protein lyses the cell wall of the E.coli causing it to burst open hence releasing virulent virus which have to potential to infect other cells. The lifecycle of a T4-bacteriophage takes about 25-35 minutes to finish (Mayer, 2010).Figure 1 The left picture shows the electron microscopic image of T4- bacteriophage and the right shows the model of the T4-bacteriophage.The head of the virus contains a capsid organize by icosahedral structure that holds the phages double stranded DNA. The collar connects the head and the tail structures. The tail consists of core, sheath, base plate and tail fibres which are involved in recognising host cells and then attaching phage to specific receptors on the host surfaces (Todar, 2008).The virus-infected bacterial cell generally shows some changes in their ph e nonype such as altered shape, detachment from the substrate, cell lysis, membrane fusion, membrane permeability, inclusion bodies and apoptosis. These changes are known as the cytopathic effects of a virus (Cann, 2005). In this experiment, the lysis of E.coli is examined using a spectrophotometer which determines the absorbance of the agricultures to indicate the maturation of the bacterial cell within the flasks. Also, it is important for clinical and research virologists to know the number of infectious virus particles in a sample which is known as the titre. The plaque assay gives the most accurate results when find out the titre of the phage. T4- bacteriophages can be grown on bacterial lawn. Infected E.coli cells are lysed so, they will form visible plaques on the agar plate which are counted to calculate the titre of the virus.The experiment aims to firstly infect the bacterial cells with T4-bacteriophage in order to indirectly observe the cytopathic effects of virus inf ection by observe changes in bacterial cell growth, compared to an uninfected (control) culture. An absorbance reading is taken using a spectrophotometry as the cells in the culture grow and divide making the culture opaque and thus increasing the absorbance of the culture. Also, the experiment plans to assess the progress of T4-phage production during the infection of E.coli by taking samples of extracellular virus at regular prison term intervals post infection. Furthermore, the virus from for each one samples were quantified using plaque assays to demonstrate the progress of virus amplification that occurred during the mathematical abut of infection in the bacterial cells.Materials and methodsE.coli was used as the host cell of T4-bacteriophages in the following experiments. bill the changing cell density of cultures using a spectrophotometer Two cultures were made the control culture (C) consists of salmagundi of LB broth and E.coli whereas the T4- infected culture (T) wa s made by mixing LB broth, E.coli and 100l of bacteriophage. The immediate absorbance readings at 0 seconds were measured in the spectrophotometer at waveduration 600nm and LB broth solution was used as a blank. The flasks containing the two cultures were placed in the orbital shaker covered with foil lids passim the readings of absorbance. During the 1 hour incubation, 10 l of the T4-infected sample was placed in a C-chip Haemocytometer and the bacterial cells were counted under the microscope which was used to calculate the multiplicity of infection (MOI). After 1 hour incubation, the absorbance readings were taken at 15 minutes interval until the cultures had been monitored for at least 2 hours.Harvesting virus sample At 40 min post infection, 1 ml of each of the cultures were taken out and placed into sterile microfuge tube. The tubes were centrifuged at 6,500 rpm for 5 min and 750 l of supernatant was removed from the T4-infected tube. It was then incubated over night and anot her(prenominal) sample was collected after 23.30 hours post infection.Preparation for plaque assay A serial dilution of 10-7, 10-8 and 10-9 were made from the overnight T4-infected sample and dilution of 10-4, 10-5 and 10-6 were made from the 40 min p.i. T4-infected sample by using sterile M9 medium. 1 ml of E.coli culture was transferred to each of the two bottlefuls containing 0.1 ml of the each diluted sample. The bottles were placed in urine bath at 37C for 15 minutes for bacterial infections to begin. One small bottle of molten soft agar was taken from 42C bath and poured into labelled agar plates and every bit mixed with the bottle containing E.coli and diluted sample. This process was followed for all 6 agar plates. Then, the plates were incubated at 37C overnight after they were set completely.Plaque counts After the incubation intent, the agar plates were examined and the number of plaques formed on each plate were counted and recorded.ResultsThe absorbance readings take n from the spectrophotometer at 600 nm of both the control and the T4-infected cultures is shown in table 1. At 0 min pi, both the cultures had similar readings (0.05 and 0.054 for C and T respectively) as they were just made. After one hour incubation blockage (60 min pi), on that point was increase in the absorbance for both of the cultures. However control had higher absorbance reading than T4-infected (0.106 and 0.064 respectively). Overall, the absorbance readings of both the cultures increases with the time except for 90 min pi in T4-infected where in that respect is a decrease in absorbance reading by 0.028. The readings recorded for control is higher than T4-infected at each time period and the highest reading is at 120 min pi in the control culture (0.192). put off 1 absorbance reading of the cell cultures at 600nmFigure 2 Absorbance reading of control and T4-infected cultures against timeCalculating the Multiplicity of infection (MOI)- the number of virions (pfu) per ce llT4-titre = 2.74 x 1010 pfu/mlCell count in T4-infected using C-chip Haemocytometer = 2808 cells per grid.Number of cells per mL = 2808 x 10,000 = 2.808107 cells/mLNumber of cells per flask = 2.808107 x 23.1 (total volume) = 6.48 x 108 cells/flaskMOI = (0.1x titre)/cells per flask = 2.74109/ 6.48108 = 4.22 pfu/cell.Table 2 class data of the plaque counts of T4-infected E.coliThe growth curve of the T4-infected E.coli is shown in rule 3. A growth curve of a virus normally shows the eclipse period, latent period, rise period and the burst size. At 0 min pi, there is high number of extracellular cells (2.66 x 109 pfu/ml) as the virus has not been taken up by the E.coli cells. As the time increase to 20 min pi, the curve levels fell down due(p) to penetration of viruses into the cell (as shown in figure 3). This phase is known as eclipse phase where the input virus begins to uncoat so, no infectious virus is detected. Latent period covers the period from the time of disappearance of infecting virus (eclipse phase) to re-appearance of it in E.coli. The rise phase is when there is a graduated increase in T4-phage titre as viral replication occurs and new cells are formed. Then the virus T4-tire levels off towards the end as cell lysis take place releasing the newly formed virus particles. However, the rise phase (shown is figure 3) doesnt not rise gradually. The T4-titre rises until 60 min pi (2.845x 109) and instead of levelling off, there is a huge record in the virus titre (8.05 x 107) at 80 min pi.In addition, the T4-virus sampled after overnight incubation (23.30 hours post infection), the dilution factors 10-7, 10-8 and 10-9 resulted in plaque counts of 12, 50 and 59 respectively. However, the plaques were only observed in one plate of each dilution and in the other plates no plaque were observed (Table 3 in appendix).DiscussionAlbrecht, et al (1996) states Infection caused by cytocidal viruses is normally associated with alterations in cell morphology, c ell physiology and sequential biosynthetic events. The changes in cell morphology can sometimes be perceptible which is known as cytopathic effects and they can be rounding of infected cells, administration of syncytia, and appearance of atomic/cytoplasmic inclusion bodies. T4- bacteriophage usually causes death of host cell after replication causing cytopathic effects. A spectrophotometer is used to detect these cytopathic effects of T4-phage in E.coli. The spectrophotometer can only detect opacity due to the presence of bacterial cell and cannot detect virus as they are very small. So, as there is growth in cells in the culture, they become more opaque increasing the absorbance reading of the culture. The T4-infected culture didnt have as much growth as the control as its absorbance readings are lower in all of the time period (as shown in figure 2).T4-phages can only replicate within the host cell therefore it must be grown in a bacterial cell. As shown in figure 3, at time 0 m in pi, the amount of virus titre is the highest (2.66 x 109 pfu/ml). This is because the process has just begun as the T4-phage injects its ds DNA into the host cell after cell contact which is known as adsorption. Then, the virus titre write downs down to 2.45 x108 pfu/ml at 20 min pi as it is attached to the host cell by receptor binding in order to penetrate into the host cell. At 40 min pi, the virus titre level begins to rise as the virus soak up to replicate within the host cell. The virus titre is suppose to increase till a certain time period and will gradually level off as the virus cell replication increases and after the host cell is filled with viral components, cell lysis occur releasing the newly formed virus phages to infect the surrounding bacteria. However, as shown in figure 3 this does not happen. The virus titre rises till post infection 60 min but there is a huge drop in virus titre after (8.05 x 107pfu/ml).There are errors in some of the results of the experi ment resembling the decrease in absorbance reading of T4-infected at 90 min pi and the huge drop in virus titre at 60 min pi. Also, an overnight sample was taken from T4-infected culture at 23.30 hours post infection and plaque assay was carried out with dilution factors of 10-7, 10-8 and 10-9. There is no plaque formation in one of the two plates in each dilution (shown in table 3 in appendix). These errors in results can be due to various factors such as contamination in the samples due to sad sterilise technique, insufficient mixing and pipetting errors when making serial dilutions.Also, in plaque assay, the morphology of plaque depends on various environmental factors like the phage, the host and the growth conditions (Maloy, 2002). The size of the plaque is proportional to the efficiency of adsorption, the length of latent phase and the burst size. Also, the phages are affected by various physical and chemical factors like temperature, acidity, ions, etc. The other error can be made when run the plaques in plaque assay. The counting is subjective to the counter as different counter would get different results for the same plate. The colonies were concentrated so it would be hard to differentiate between single colonies as they grow very close together. For more accurate counting of the plaque, the plates with 10-200 plaques would be chosen as there were three dilution factors of plates to choose from and anything with 200 plaque counts were not counted. Also, the experiments could be repeated more than once and for longer period of time when monitoring T4-infected E.coli so, average could be taken out which would give more accurate and reliable results.In conclusion, T4-bacteriophages are virulent infecting E.coli cells by lytic pathway and this can be measured by examining the cytopathic effects using spectrophotometer. Also, plaque assay of T4-infected E.coli helps to determine the virus titre concentration of virus in a sample. T4- bacteriophages a re a model being to study and are involved in advancement of modern genetics and molecular biology. Also, they could be used to treat bacterial diseases as the theory states that phages can selectively kill the host cell without negative the human cells. Some potential applications that are being considered include adding phage suspension to grafts in order to control skin infections and intravenous fluids for blood infections (Talaro, et al, 2007).Word count 2,763ReferencesAlbrecht, T., Fons, M., Boldogh, I., et al. Effects on Cells. In power S, editor. medical Microbiology. 4th edition. Galveston (TX) University of Texas Medical Branch atGalveston 1996. Chapter 44.Available from http//www.ncbi.nlm.nih.gov/books/NBK7979/Baker, S., Griffiths, C. and Nicklin, J. (2011). BIOS Instant Notes in Microbiology. 4th edn. raw York Taylor Francis Ltd.Cann, A.J. (2005).Principles of molecular Virology. 4th ed. United States Elsevier Academic Press. 210-211.Gelderblom HR. Structure and Clas sification of Viruses. In Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX) University of Texas Medical Branch at Galveston 1996. Chapter 41.Available from http//www.ncbi.nlm.nih.gov/books/NBK8174/Leimana, P.G., Kanamarua, S., Mesyanzhinovb, V.V., Arisakac, F., Rossmanna, M.G. (2003). Structure and morphogenesis of bacteriophage T4.Cellular and Molecular Life Sciences. 60 (1), 23562370.Maloy, S. (2002).Phage plaques.Available http//www.sci.sdsu.edu/smaloy/MicrobialGenetics/topics/phage/plaques.html. Last accessed 30 March 2014.Miller, E S., Kutter, E., Mosig, G., Arisaka, F., Kunisawa, T., Rger6, W. (2003). Bacteriophage T4 Genome.Microbiology and molecular Biology Review. 67 (1), 86-156.Strelkauskas, A., Strelkauskas, J. and Moszyk-Strelkauskas, D., 2010. Microbiology, a clinical approach. New York Garland Science.Talaro, K.P. (2007).Foundations in Microbiology Basic Principles. 7th ed. Phillipines McGraw-Hill. 160-181.Todar, K., 2012. Bacteriophage. Online. Availa ble at http//textbookofbacteriology.net/phage.html.. (Accessed 30 March 2014).Appendix Table 3 T4 virus sampled after overnight incubation (23.30 hours pi)