Methods for isolation, purification, and propagation of. H7 nctc 12900 and atcc 43895 were isolated from sheep and cattle faecal samples at the evergreen state college, as per their standard protocol raya et al. Here, we describe the methods for isolation, purification, and propagation of campylobacter jejuni bacteriophages from samples expected to contain high number of phages such as chicken feces. For pols labs there are two options described in the phage discovery guide for extracting bacteriophage from your environmental sample. Phage choice, isolation, and preparation for phage therapy. Feb 25, 2020 phage genomic dna was extracted using a modified zinc chloride phage precipitation protocol described by santos. A widely used method for determining phage concentration in a sample takes advantage of this lytic activity. Use of mko7 helper phage for isolation of singlestranded phagemid dna protocol. Transfer 1 ml of the phage lysate 1 x 108 pfuml into a 5 ml or 15 ml tube provided by the user. An optimized enrichment technique for the isolation of. This article contains protocols for the isolation of phages from environmental samples, enrichment of phages targeting host bacteria of interest, and induction of.
Phage on tap a quick and efficient protocol for the. Serially dilute phage samples to 102, 103, and 104 in phage buffer with cacl 2 see toolbox. Overlay soft ts and tsbw agar were prepared by adding half of the amount of agar used for hard underlay agar. A simple, reproducible and costeffective procedure to. Add proteinase k to 100 gml and incubate at 65c for 45 min. Phage plaque assays the plaque assay is a method for titering bacteriophage. Isolation of phage via induction of lysogens springerlink.
Optimizing protocols for extraction of bacteriophages prior. Since the codiscovery of phages in 1915 by frederick twort and 1917 by felix dherelle, their potential as subjects of laboratory research has been exploited to great effect. Isolation and characterization of lytic bacteriophages. Once the phage particles are assembled, they are released from the bacteria, often by lysing the host and bursting out, killing the host cell in the process. We started our study by testing five different methods to isolate phage dna from faecal samples described in. Procedure for the isolation and purification of a phage i. Using the norgen phage dna isolation kit objective extract phage genomic dna using the norgen phage dna isolation kit. Fusobacterium nucleatum is a periodontal pathogen that has been directly associated with the development and progression of periodontal disease, a widespread pathology that affects the support tissues of the tooth. Abstract methicillin resistant staphylococcus aureus mrsa is a major human pathogen responsible for several life threatening conditions. Antibody phage display methods and protocols robert. Bacteriophage is studied widely to improve the safety of foods and prevent food borne diseases of bacterial aetiology, as well as to reduce the use of antibiotics in livestock. Isolation, culture, and characterization of bacteriophages.
The growth or spread of the new viruses is then restricted or limited to the neighbouring cells by the gel. How to prepare a soil sample and enrich for phages by adding a bacterium of choice. You can quantitate the number of phage in a sample plating dilutions you can either spread your dilution on one plate and use five plates for five dilutions or spot your dilutions in circles on the plate. This step should be performed at this point in the. Dnase i and rnase i were added to final concentrations of 100. Methods and protocols, second edition, expert researchers explore the latest in this cuttingedge technology, providing an invaluable resource that will guide readers in the design and execution of experiments based around antibody phage display.
Therefore, the high campylobacter specific phage isolation rate observed in this study confirms the suitability of method 6 for phages isolation. Collaborator, nathan verberkmoes oak ridge national labs, worked with tucson marine phage lab to develop more sensitive proteomics assays for viral isolates which we are now also using for environmental viral concentrates. Detection of bacteriophages in environmental samples. Pdf isolation of bacteriophages from environmental. Plate phage tube dilution 1 a none 2 b none 3 b 101 4 b 102 5 b 103 6 b 104 7 b 105 8 b 106 9 control no phage none 20 incubate the plates overnight at 37 c. Whether youre new to phage research or have been doing it for some time like myself almost a decade, this book and its accompanying volume 2 are a must for any phage researcher. Underlay hard ts and tsbw agar were prepared as previously described. These are available as convenient pdf files online at customer. Schematic of phage isolation from filterfeeding organisms. Arthrobacter phage banana was serial diluted in pb and plated on agar plates as described in sections 4.
Phage dna isolation kit product insert norgen biotek. A rapid and simple protocol for the isolation of bacteriophages from. Isolation and characterization of bacterial host strains. Working with phage display technology detailed description of the materials, methods and space needed for those who want to start phage work may be found in specialized textbooks barbas et al. To the remainder, make the solution 10 mm excess edta, 0. The method, which has gained a renewed interest because of increasing frequency of infections by multidrugresistant bacteria, has potential benefits, but much research remains before the method is fully developed and can be considered an.
Most bacterial cells carry prophage genomes either integrated into the host dna or present as repressed plasmids. In this video, you will see that both bacteriophage extraction protocols begin the same way. A bacteriophages host range can determine the phages utility for phage therapy. Transmission electron microscopy showed that the virion is composed of an icosahedral head and a. Isolation and characterization of bacteriophage against.
The basic protocols used with phage studies can also be obtained in these books, whereas. Virus purification and concentration iron chloride precipitation of viruses from seawater protocol at protocols. Plating out phage 04182011 making a plate lysate 07122011 phage dna extraction modified wizard method 07122011 phage dna extraction by phenol. This book should prove to be a valuable resource to even the experienced phage biologist and an essential reference book for postgraduate students and scientists new to the field of phage biology.
Isolation and purification of bacteriophages how do you isolate a bacteriophage phage and obtain a pure phage preparation. Isolation and characterisation of bacteriophages against shigella flexneri soo zheng may bacteriophages are viruses that parasitise on bacteria. Isolation of bacteriophages from environmental sources, and creation and functional screening of phage dna libraries chapter pdf available october 20 with 5,957 reads how we measure reads. Procedure for the isolation and purification of a phage. Phage overlay assay for quantification of infectious bacteriophage particles francis, freddy. Isolation and characterization of phages infecting bacillus. The goal of this method is to enrich your sample for phages capable of infecting your desired host. Some phages will infect only a few strains narrower host range, others will infect many strains of the same species broader host range, and a few phages can infect more than one species polyvalent host range.
The overall steps are 1 liberation of phages from the sample material. Norgens phage dna isolation kit provides a rapid method for the isolation and purification of total dna. This protocol describes a technique to recover viruses from natural waters using ironbased flocculation and largeporesize filtration, followed by resuspension of. This one requires about 5 ml of sample, and utilizes a commercial dna isolation kit. Standard isolation protocols do not seem to select for any particular host range. Typically 10 ml of a hightiter phage lysate is used for dna extraction. Host range, instead, is usually characterized after the phage is isolated, with isolation typically accomplished on a single strain of bacteria 2,3,7, although some groups favor using a mix of multiple host strains for phage isolation 810. In this protocol phages can be isolated from virtually any source by using bacteria to fish them out. Pdf isolation of bacteriophages from environmental sources. Prepare serial dilutions of the bacteriophage suspension distribute 0. Each plaque indicates an initial infection with one phage followed by lysis of neighboring bacteria in the lawn. At the crux of phage display technology is the ability to express peptide or protein sequences as fusions to the coat proteins of a bacteriophage.
The protocol described here allows for the selection of phages carrying random dna deletions, and, in addition, the subsequent isolation of those mutations affecting the lysislysogeny functions. The enriched isolation is different from the direct isolation because the enriched isolation protocol is designed to add a step for bacteriophage replication before. Centrifuge as above and transfer the supernatant again. This is achieved by plating a phage suspension using the double agar method, and a susceptible host strain, to obtain plaques and further purifying the phage contained within the plaque. Seven of the selected plaques putative phages still showed. The method, which has gained a renewed interest because of increasing frequency of infections by multidrugresistant bacteria, has potential benefits, but much research remains before the method is fully developed and can be considered an important contribution to the fight against.
You can visualize plaques if you spot the phage on a lawn of actively growing compatible bacteria. Nagra, manveer department of microbiology and immunology, university of british columbia the historical applications of plaque assays included isolation of virus and enumeration of viral particles in a solution. Request pdf phage choice, isolation, and preparation for phage therapy phage therapy is the use of bacteriophagesviruses that use bacteria as their host cellsas biocontrol agents of bacteria. Phage display technology applications and innovations. Phage genomes represent a vast gene pool from which bacteria can draw upon for rapid evolution. Isolation of bacteriophages from environmental sources, and creation and functional screening of phage dna libraries chapter pdf available october 20 with 5,957 reads how we. Isolation and characterization of bacteriophages with. Turbidity reduction method was performed as per protocol of harrigan and mccance 9 with slight modifications. Isolation of a novel bacteriophage specific for the. In this technique, the phage from the sample is mixed with bacteria and soft agar.
Viral isolation the phage assay cappuccino and sherman, 2001 was used to determine if phage were present in the sewage sludge suspension. You will be able to use one plate to get your result. All isolated phages formed clear plaques with the strains used for isolation. Sediment sample plus the overlying water were collected march, 20 into sterile jars, capped on site and preserved in cooled boxes for transportation to the molecular laboratory in jomo kenyatta university of agriculture and technology jkuat. Efficient isolation of campylobacter bacteriophages from.
These are available as convenient pdf files online at. Detection of bacteriophages in environmental samples protocol. As a rule of thumb, 1 ml of phage lysate with a titer of 1 x 10 10 contains about 0. Isolate a new mycobacteriophage from an environmental sample. In the cell, the virus nucleic acid either converts all cell machinery over to the manufacture of new virus particles, causing lysis of the bacteria hence a lytic or virulent bacteriophage, or else comes into equilibrium with the genetic material of the host a temperate bacteriophage by physically inserting its genome into the bacterial chromosome and becoming a prophage. Background there are various ways to yield clean dna from a high titer phage lysate. Phage display technology is an in vitro screening technique for identifying ligands for proteins and other macromolecules. Protocol practical method for isolation of phage deletion. No lysis plaques were observed from seawater samples fig. Collect environmental samples in 1550 ml screwcap conical tubes. Characterization of a newly isolated phage infecting. Transform phagemid vector into appropriate f strain cj236 for kunkel mutagenesis.
The basic method of phage isolation has remained unchanged since it was. Optional dnase treatment if phages are enriched in the cultural media with a host, dnase i treatment is necessary in order to avoid host genomic dna contamination in the phage dna elution. Isolation and characterization of bacteriophages with lytic. We started our study by testing five different methods to isolate phage dna from faecal samples described in table 1. Determine whether a putative plaque is due to phage or artifact such as an air bubble. When one phage particle adsorbs to a susceptible cell, penetrates the cell, replicates and release new phage particles which infect other bacteria in the vicinity of the initial host cell. A scalable, efficient and cheap method for phage isolation from faeces. Its full of easy to follow procedures with hintsnotes that will help understand the purpose of some of the steps undertaken. A total of 30 welldefined lysis plates were selected and separated from the rest fig. After identification by manual curation,114 contigs were classified as.
Request pdf phage choice, isolation, and preparation for phage therapy phage therapy is the use of bacteriophagesviruses that use bacteria as their host cells. Phage dna isolation kit product insert norgen biotek corp. More or less lysate up to 20 ml may be used depending on the phage stock titer and the expected genome size of the phage. How to prepare and test a soil sample for the presence of phages. Methods and protocols pulls together the vast body of. Chapters present a wide range of methods of isolating recombinant. Liquid or solid samples can be collected from virtually anywhere, particularly where decay is occurring. Isolation of bacteriophage turbidity reduction and streak plate technique were used as preliminary methods for screening of lytic activity of phages. Host range, instead, is usually characterized after the phage is isolated, with isolation typically accomplished on a single strain of bacteria 2,3,7, although some groups favor using a mix of multiple host strains for phage isolation 8,9,10. Phage therapy involves clinical treatment of bacterial infections with phages bacteriophages. Save 50100 l of the phage as a high titer stock for future endeavors. This is done by removing the endogenous bacteria from your sample and. Only few phage extraction protocols from soil samples have been.
373 1063 543 1487 386 828 790 1502 1134 32 1280 799 815 954 454 188 37 358 1281 1397 363 1036 1198 410 1340 1338 419 797 920 403 225 164 1504 1399 1249 458 1292 1047 1360 630 1331 679 1369 1113 1197