Cultivation and Quantification of Bacteriophages

CULTIVATION AND QUANTIFICATION OF BACTERIOPHOGR INTRODUCTION Bacteriophage are viruses that can infect and destroy bacteria Bacteriophage is also known as phage (virus) Virus that affack bacteria were observed by Twort and herelle in 1915 →Bacteriophages are composed of DNBIRNA as theis genetic maternal in either Circular/lineas form, as a single/double Standed molecule →They occur widely in nature and can readily be isolated fram feces and Sewage sample Isolation of Bacteriophages: Separation of phages from Sample -Isolation of phages can be done by taking sample which 18 assome to have the Virus and filtering out in a filles [Chamberland) that can hold on all the bacterial Cells but fliters, only the particies which are domalies than bacteria filterate contains viruses, use that vine filterate for Cultivation of Vious. filleuken fer pare siges the other mitranganlime. Cultivation of Bacteriophages The cultivation of bacteriophages is done in different stepas Step-1: obtaining the bacteriophages te dont & Sample in elthes flitering con getting at such sample (like sewage water). Step-2: Amplification or multiplication of phages Amplificatiso can be done by mixing with Sanyia (Phage) host bacteria (E.coli) Into liquid media and allowit grow for 24 hours of 31°c → As the tests dividing and growing the viruses altuches and then multiply increasing number of phages. Step-3:-Now, the increased number of phages came isolated le, Separation of phages from cell debris. by centrifugation and filteration with o-au filters. →The filterate will have a pure phage suspension Step-4:- The obtained pure suspension of phage 16 diluted serially is to folds) dilutions →This to fold dilution stared separately at required temperature ie., 37℃ ie, Take a test tubes containing 0.9ml of nutrient Broth (0) Trypton broth and add olmi of phoge Stack Suspension to ist tube mix and transfer tmi Sample to and tube like wise upto ath tube. Step-5:- one side serially diluted phages are ready on another side we have grow the culture ie, liquid media bacterial cultcore(asm) →Now mix together in Serially cliluted phages and bacterial culture and Incubated for so minutes. Step-6:- Incubation step is important as the phages attester to the bacteria at this tone After Incubation period, the mixed Culture i-e, Sample containg virve + bacterial cultuar has to be mixed with soft Agar. Step-7: Now transfer (ar) pour this on to hard Agar plate and Sparad →After this, plates are incubated and observe for proque Plague: A plaque is a visible structure formed attrs introducing a viral sample medium. to a cell culture grown on some nutrient QUANTIFICATION OF PHAGES:- It refers to counting on enumerating the placque in plates. →The titre of phage suspension can be determined by number of plaques that formed from given Volume of suspension. phage titre unit is expressed as plaque forming unil (PFU) per mi ie, Pfu/ml The number of effecting viable proges - Quantification of phages are done by PLAQUE ASSAY Plaque Assay- It is a well established method for measuring virus Concentrations as it refers to infectious dose The assay relies on determining the number of plaque foaming Unit (PFU) Created in a monolayer of Virus infected Celss. PFU-Plaque forming units measures infectious particies in a given sample counting the number of pfu on a series of plates. PFU- No. of Phages Valume plated x Dilution factor