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Preparation of stocks of bacteriophage lambda by plate lysis and elution
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Objectives

 

To prepare the stocks of bacteriophage by plate lysis and elution method.

 

Theory

 

Bacteriophages the viruses that infect bacteria were discovered independently by Frederick W.Twort and Felix d’Herelle in 1915 and 1917 respectively. The word bacteriophage is first coined by D’Herelle which means ‘bacteria eater’. Phages are commonly composed of a nucleic acid core and protein coat which covers the nucleic acid inside it. Bacteriophages exist in two forms: Lytic and lysogenic. Phages, in lytic cycle, can multiply within the host cell and eventually cause its death by lysis.  While in lysogenic cycle, phage can stay within the host cell without any harm by integrating its DNA to the host genome.

 

Lytic cycle of bacteriophage


Preparation of phage stocks is a vital step in Phage biology. In a bacterial culture, the phage suspension multiplies by taking bacterial cell machinery and lyse the cells eventually by releasing new phage particles. As a result, the medium contains a high concentration of phage particles. Lysed bacterial cell debris and unlysed cells are removed by centrifugation. Chloroform is also used along with phage dilution buffer to lyse the remaining cells and finally getting a solution of phage called phage lysate. Like preparation, conservation of bacteriophage stock is also a crucial step in phage biology. The most applicable method for long term storage of phage stock is placing them in SM buffer at 4oC. SM buffer or SM phage diluents which contains gelatin, Chloroform and Mg2+ are used for routine manipulation of phage stocks. The gelatin used in SM buffer helps to stabilize the phage particles while storage. Chloroform maintains the sterility of phage stock by hindering bacterial growth without causing any harm to phage
 

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