Analysis of cytopathic effects as a result of infection of Escherichia coli by T4 bacteriophage using spectrophotometry and plaque assay Essay

Analysis of cytopathic effects as a consequence of infection of Escherichia coli by T4 bacteriophage utilizing spectrophotometry and plaque assay

Abstraction

T4 bacteriophage is a dual isolated Deoxyribonucleic acid virus that infects Escherichia coli and is an of import tool in research and survey of genetic sciences. It contains about 168,800 base brace of DNA. It has an icosahedral mirid bug as its caput that contains its nucleic acid and has a tail construction formed of tail fibers, sheath, base home bases and coat. These different constructions of T4-phage drama of import functions when occupying the host cell ( in this instance E.coli ) . T4-bacteriophage infects the host cell by lytic tract where the host cells are used to bring forth virus constituents and it consequences in the lysis of host cell let go ofing the virus constituents to the external membrane. This experiment programs to analyze the cytopathic effects of the T4-phage on E.coli utilizing a spectrophotometer. Besides, plaque check is carried out in order to find the original concentration of phage in the solution. The spectrophotometer reading increased for both control and T4-infected civilizations as the clip of station infection increased. The highest reading was 0.192 for control at 120 min pi.

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Introduction

Viruss are little infective agents and are about 10-30nm in size. Strelkauskas, et Al ( 2010 ) describes viruses as “obligate intracellular parasites” which means they need a host cell in order to last and retroflex. The viruses are classified depending on their morphology, chemical composing and manner of reproduction. They are divided into two groups based on the nucleic acid they contain: Deoxyribonucleic acid or RNA. They are sub-divided into farther groups harmonizing to their symmetricalness of mirid bug where the familial stuff is enclosed within an envelope or without an envelope ( naked ) . Besides, the constellation of the nucleic acid ( single-stranded or dual stranded and additive or round ) play a function in the categorization of viruses ( Gelderblom, 1996 ) . A to the full assembled infective virus is known as a virion and its construction is based on two of import things ; the ability to last in rough conditions until it can infect the host cell and to be able to cast the protective barrier in order to come in a host cell.

Bacteriophages are viruses that infect bacterial cells and were foremost observed in 1915 and 1917 by Twort and d’Herelle. T4 bacteriophage is a member of T-even phages and infects Escherichia coli ( E.coli ) bacterium. It is considered as one of the complex viruses because of its genome which consist of 274 unfastened 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 function in promotion of modern genetic sciences and molecular biological science. Some of the early indispensable thoughts of genetic sciences including the footing of genetic sciences codification, messenger RNA, ribosome and the codon have came from surveies utilizing T4-bacteriophages ( Miller et al, 2003 ) . T4- bacteriophage contains dual isolated Deoxyribonucleic acid as its nucleic acid which is enclosed within a protective coat known as mirid bug. Its mirid bug is icosahedral in form ( shown in figure 1 ) . It besides has tail portion which is made up of coat, sheath, tail fibers and base home bases. The tail portion is utile when recognizing a host cell followed by incursion to the host cell therefore doing infection.

Bacteriophages have two types of reproduction schemes: lytic rhythm and lysogenic rhythm ( Baker, et Al, 2011 ) . Lysogenic infection in E.coli is exemplified by bacteriophage lambda where the viral DNA reproduction takes topographic point without destructing the bacterial cell. Here, the virion infects the host cell and alternatively of triping more virus reproduction, it recombines with the host genome doing no harm to the cell which is known as prophage. These viruses can stay hibernating within the bacterial cell for old ages and merely do infection under certain fortunes. T4-bacteriophage is a deadly virus that causes lytic infection to E.coli. The first measure in this rhythm is surface assimilation where the virus recognises the cell receptors on the surface of the host cell and attaches itself to the host utilizing its tail fibers. Once attached to the host cell, the virus must perforate through the cell membrane of E.coli. The basal home base of the virus comes in contact with the cell wall of the host cell doing conformational alteration in construction of the virus. This allows the sheath to contract and the nucleus is pushed through the wall into the cell membrane where the viral DNA is injected into the host cell ( Todar, 2008 ) . Deoxyribonucleic acid viruses have the same genome constellation as the host cell ; hence the reproduction procedure used in host cell can be used for viral reproduction. T4- bacteriophage transcribes nucleases that interrupt down the bacterial DNA which is used to bring forth more viral genome. The freshly made viral nucleic acid and structural proteins are assembled together to organize deadly viruses. The muramidases produced by the bacteriophages as a late viral protein lyses the cell wall of the E.coli doing it to split unfastened hence let go ofing virulent virus which have to potential to infect other cells. The lifecycle of a T4-bacteriophage takes about 25-35 proceedingss to complete ( Mayer, 2010 ) .

http://textbookofbacteriology.net/phagestructure.jpg

Figure 1: The left image shows the negatron microscopic image of T4- bacteriophage and the right shows the theoretical account of the T4-bacteriophage. The caput of the virus contains a mirid bug formed by icosahedral construction that holds the phage’s double stranded DNA. The collar connects the caput and the tail constructions. The tail consists of nucleus, sheath, base home base and tail fibers which are involved in recognizing host cells and so attaching phage to specific receptors on the host surfaces ( Todar, 2008 ) .

The virus-infected bacterial cell by and large shows some alterations in their phenotype such as altered form, withdrawal from the substrate, cell lysis, membrane merger, membrane permeableness, inclusion organic structures and programmed cell death. These alterations are known as the cytopathic effects of a virus ( Cann, 2005 ) . In this experiment, the lysis of E.coli is examined utilizing a spectrophotometer which determines the optical density of the civilizations to bespeak the growing of the bacterial cell within the flasks. Besides, it is of import for clinical and research virologists to cognize the figure of infective virus atoms in a sample which is known as the titer. The plaque check gives the most accurate consequences when finding the titer of the phage. T4- bacteriophages can be grown on bacterial lawn. Infected E.coli cells are lysed so, they will organize seeable plaques on the agar home base which are counted to cipher the titer of the virus.

The experiment aims to foremost infect the bacterial cells with T4-bacteriophage in order to indirectly detect the cytopathic effects of virus infection by supervising alterations in bacterial cell growing, compared to an clean ( control ) civilization. An optical density reading is taken utilizing a spectrophotometry as the cells in the civilization grow and divide doing the civilization opaque and therefore increasing the optical density of the civilization. Besides, the experiment programs to measure the advancement of T4-phage production during the infection of E.coli by taking samples of extracellular virus at regular clip intervals post infection. Furthermore, the virus from each samples were quantified utilizing plaque checks to show the advancement of virus elaboration that occurred during the procedure of infection in the bacterial cells.

Materials and methods

E.coli was used as the host cell of T4-bacteriophages in the undermentioned experiments.

Measuring the altering cell denseness of civilizations utilizing a spectrophotometer:Two civilizations were made ; the control civilization ( C ) consists of mixture of LB stock and E.coli whereas the T4- septic civilization ( T ) was made by blending LB stock, E.coli and 100?l of bacteriophage. The immediate optical density readings at 0 seconds were measured in the spectrophotometer at wavelength 600nm and LB broth solution was used as a space. The flasks incorporating the two civilizations were placed in the orbital shaker covered with foil palpebras throughout the readings of optical density. During the 1 hr incubation, 10 µl 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 cipher the multiplicity of infection ( MOI ) . After 1 hr incubation, the optical density readings were taken at 15 proceedingss interval until the civilizations had been monitored for at least 2 hours.

Reaping virus sample:At 40 min station infection, 1 milliliter of each of the civilizations were taken out and placed into unfertile microfuge tubing. The tubings were centrifuged at 6,500 revolutions per minute for 5 min and 750 µl of supernatant was removed from the T4-infected tubing. It was so incubated over dark and another sample was collected after 23.30 hours post infection.

Preparation for plaque check:A consecutive dilution of 10-7, 10-8and 10-9were made from the nightlong T4-infected sample and dilution of 10-4, 10-5and 10-6were made from the 40 min p.i. T4-infected sample by utilizing unfertile M9 medium. 1 milliliter of E.coli civilization was transferred to each of the two bottles incorporating 0.1 milliliter of the each diluted sample. The bottles were placed in H2O bath at 37°C for 15 proceedingss for bacterial infections to get down. One little bottle of liquefied soft agar was taken from 42°C bath and poured into labelled agar home bases and equally assorted with the bottle incorporating E.coli and diluted sample. This procedure was followed for all 6 agar home bases. Then, the home bases were incubated at 37°C overnight after they were set wholly.

Plaque counts:After the incubation period, the agar home bases were examined and the figure of plaques formed on each home base were counted and recorded.

Consequences

The optical density readings taken from the spectrophotometer at 600 nanometer of both the control and the T4-infected civilizations is shown in table 1. At 0 min pi, both the civilizations had similar readings ( 0.05 and 0.054 for C and T severally ) as they were merely made. After one hr incubation period ( 60 min pi ) , there was addition in the optical density for both of the civilizations. However control had higher optical density reading than T4-infected ( 0.106 and 0.064 severally ) . Overall, the optical density readings of both the civilizations increases with the clip except for 90 min pi in T4-infected where there is a lessening in optical density reading by 0.028. The readings recorded for control is higher than T4-infected at each clip period and the highest reading is at 120 min pi in the control civilization ( 0.192 ) .

Absorbance ( 600 nanometer )

Time ( min pi )

Control

T4-infected

0

0.057

0.054

60

0.106

0.064

75

0.116

0.075

90

0.147

0.047

105

0.151

0.058

120

0.192

0.153

Table 1: optical density reading of the cell cultures at 600nm

C:Usersadmin_2Pictures2014-03-31 ss 002.jpg

Figure 2: Optical density reading of control and T4-infected civilizations against clip

Calculating the Multiplicity of infection ( MOI ) – the figure of virions ( pfu ) per cell

T4-titre = 2.74 x 1010pfu/ml

Cell count in T4-infected utilizing C-chip Haemocytometer = 2808 cells per grid.

Number of cells per milliliter = 2808 ten 10,000 = 2.808×107cells/mL

Number of cells per flask = 2.808×107x 23.1 ( entire volume ) = 6.48 ten 108cells/flask

MOI = ( 0.1x titer ) /cells per flask = 2.74×109/ 6.48×108= 4.22 pfu/cell.

Time ( min pi )

Dilution factor

Average plaque count ( from both home bases )

Dilution factor

Average plaque count ( from both home bases )

T4 titer ( pfu/ml )

Log10titer

0

10-6

266

2.66 x 109

9.425

20

10-5

162

10-5

328

2.45 x108

8.389

40

10-6

184

1.84 x 109

9.265

60

10-6

284.5

2.845x 109

9.454

80

10-5

80.5

8.05 x 107

7.906

100

10-5

169.5

10-5

127.5

1.46 x 108

8.164

120

10-5

122

1.22 x 108

8.806

Table 2: category informations of the plaque counts of T4-infected E.coli

C:Usersadmin_2Pictures2014-03-31 ss 001.jpg

Figure 3: growing curve of T4-infected E.coli against clip

The growing curve of the T4-infected E.coli is shown in figure 3. A growing curve of a virus usually shows the occultation period, latent period, rise period and the explosion size. At 0 min pi, there is high figure of extracellular cells ( 2.66 x 109pfu/ml ) as the virus has non been taken up by the E.coli cells. As the clip addition to 20 min pi, the curve degrees fell down due to incursion of viruses into the cell ( as shown in figure 3 ) . This stage is known as occultation stage where the input virus begins to uncoat so, no infective virus is detected. Latent period covers the period from the clip of disappearing of infecting virus ( eclipse stage ) to re-appearance of it in E.coli. The rise stage is when there is a gradual addition in T4-phage titer as viral reproduction occurs and new cells are formed. Then the virus T4-tire degrees off towards the terminal as cell lysis take topographic point let go ofing the freshly formed virus atoms. However, the rise stage ( shown is figure 3 ) doesn’t non lift bit by bit. The T4-titre rises until 60 min pi ( 2.845x 109) and alternatively of levelling off, there is a immense bead in the virus titer ( 8.05 x 107) at 80 min pi.

In add-on, the T4-virus sampled after nightlong 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 severally. However, the plaques were merely observed in one home base of each dilution and in the other plates no plaque were observed ( Table 3 in appendix ) .

Discussion

Albrecht, et Al ( 1996 ) states Infection caused by cytocidal viruses is usually associated with changes in cell morphology, cell physiology and consecutive biosynthetic events. The alterations in cell morphology can sometimes be noticeable which is known as cytopathic effects and they can be rounding of septic cells, formation of syncytia, and visual aspect of nuclear/cytoplasmic inclusion organic structures. T4- bacteriophage normally causes decease of host cell after reproduction doing cytopathic effects. A spectrophotometer is used to observe these cytopathic effects of T4-phage in E.coli. The spectrophotometer can merely observe opacity due to the presence of bacterial cell and can non observe virus as they are really little. So, as there is growing in cells in the civilization, they become more opaque increasing the optical density reading of the civilization. The T4-infected civilization didn’t have every bit much growing as the control as its optical density readings are lower in all of the clip period ( as shown in figure 2 ) .

T4-phages can merely retroflex within the host cell therefore it must be grown in a bacterial cell. As shown in figure 3, at clip 0 min pi, the sum of virus titer is the highest ( 2.66 x 109pfu/ml ) . This is because the procedure has merely begun as the T4-phage injects its Ds Deoxyribonucleic acid into the host cell after cell contact which is known as surface assimilation. Then, the virus titer drops down to 2.45 x108pfu/ml at 20 min pi as it is attached to the host cell by receptor binding in order to perforate into the host cell. At 40 min pi, the virus titer degree begins to lift as the virus start to retroflex within the host cell. The virus titer is suppose to increase till a certain clip period and will bit by bit level off as the virus cell reproduction additions and after the host cell is filled with viral constituents, cell lysis occur let go ofing the freshly formed virus phages to infect the environing bacteriums. However, as shown in figure 3 this does non go on. The virus titer rises till station infection 60 min but there is a immense bead in virus titer after ( 8.05 x 107pfu/ml ) .

There are mistakes in some of the consequences of the experiment like the lessening in optical density reading of T4-infected at 90 min pi and the immense bead in virus titer at 60 min pi. Besides, an nightlong sample was taken from T4-infected civilization at 23.30 hours post infection and plaque check was carried out with dilution factors of 10-7, 10-8 and 10-9. There is no plaque formation in one of the two home bases in each dilution ( shown in table 3 in appendix ) . These mistakes in consequences can be due to assorted factors such as taint in the samples due to hapless sterilise technique, deficient commixture and pipetting mistakes when doing consecutive dilutions.

Besides, in plaque check, the morphology of plaque depends on assorted environmental factors like the phage, the host and the growing conditions ( Maloy, 2002 ) . The size of the plaque is relative to the efficiency of surface assimilation, the length of latent stage and the explosion size. Besides, the phages are affected by assorted physical and chemical factors like temperature, sourness, ions, etc. The other mistake can be made when numbering the plaques in plaque check. The numeration is subjective to the counter as different counter would acquire different consequences for the same home base. The settlements were concentrated so it would be difficult to distinguish between individual settlements as they grow really near together. For more accurate numeration of the plaque, the home bases with 10-200 plaques would be chosen as there were three dilution factors of home bases to take from and anything with & gt ; 200 plaque counts were non counted. Besides, the experiments could be repeated more than one time and for longer period of clip when supervising T4-infected E.coli so, norm could be taken out which would give more accurate and dependable consequences.

In decision, T4-bacteriophages are deadly infecting E.coli cells by lytic tract and this can be measured by analyzing the cytopathic effects utilizing spectrophotometer. Besides, plaque check of T4-infected E.coli helps to find the virus titer – concentration of virus in a sample. T4- bacteriophages are a theoretical account being to analyze and are involved in promotion of modern genetic sciences and molecular biological science. Besides, they could be used to handle bacterial diseases as the theory states that phages can selectively kill the host cell without damaging the human cells. Some possible applications that are being considered include adding phage suspension to grafts in order to command skin infections and endovenous fluids for blood infections ( Talaro, et Al, 2007 ) .

Word count: 2,763

Mention

Albrecht, T. , Fons, M. , Boldogh, I. , et Al. Effectss on Cells. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston ( TX ) : University of Texas Medical Branch at

Galveston ; 1996. Chapter 44.Available from: hypertext transfer protocol: //www.ncbi.nlm.nih.gov/books/NBK7979/

Baker, S. , Griffiths, C. and Nicklin, J. ( 2011 ) .BIOS Instant Notes in Microbiology. 4Thursdayedn. New York: Taylor & A ; Francis Ltd.

Cann, A.J. ( 2005 ) .Principles of Molecular Virology. 4th erectile dysfunction. United States: Elsevier Academic Press. 210-211.

Gelderblom HR. Structure and Classification of Viruses. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston ( TX ) : University of Texas Medical Branch at Galveston ; 1996. Chapter 41.Available from: hypertext transfer protocol: //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 ) , 2356–2370.

Maloy, S. ( 2002 ) .Bacteriophage plaques.Available: hypertext transfer protocol: //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. , Ruger6, 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 attack. New York: Garland Science.

Talaro, K.P. ( 2007 ) .Foundations in Microbiology: Basic Principles. 7th erectile dysfunction. Phillipines: McGraw-Hill. 160-181.

Todar, K. , 2012. Bacteriophage. [ Online ] . Available at: & lt ; hypertext transfer protocol: //textbookofbacteriology.net/phage.html. . & gt ; ( Accessed 30 March 2014 ) .

Appendix

Dilution assayed

Plaques on home base 1

Plaques on home base 2

Average figure of plaques

10-7

12

12

10-8

50

50

10-9

59

59

Table 3: T4 virus sampled after nightlong incubation ( 23.30 hours pi )

1

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