Microbiology 251-11 Lab 5-6

Microbiology 251-11 - WEEK 5 February 8 10 Bacterial Transformation Effect of Washing on Hand Flora Nasal Flora Exercisesto Complete Pure Culture

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WEEK 5: February 8 - 10 Bacterial Transformation, Effect of Washing on Hand Flora, Nasal Flora Pure Culture Isolation Check your streak plate to see if isolated colonies were obtained and if only one type of colony appears on each plate. Again, if not, how could your technique be improved? * * * Titration of Bacteriophage Results Count the plaques formed by the phage by making a mark with a marking pencil on the bottom of the plate over each plaque and counting as you mark. Count only one set of replicate plates. You should calculate the plaque forming units per milliliter (PFU/ml) of the original sample based on the average number of plaques on the two plates. Multiply the direct count by the dilution used to obtain the PFU per ml of stock culture. Stock concentration (PFU/ml) = # plaque/volume plated (ml) x 1/concentration plated For example, if you counted 46 plaques on one of your 10 -3 plates and 40 on the other, you would average the two, yielding 43 x 10 3 or 4.3 x 10 4 PFU/ml of original stock. Report your calculation to your TA who will determine an average for the class and communicate that number back to the students. There is also a demonstration of virus-infected and non-infected cell cultures. The virus kills the cells leaving gaps in the tissue culture monolayer, with infected cells curling up, demonstrating what is called cytopathic effect (CPE). Note the difference between the 'lawn' of infected cells versus the uninfected. This CPE is analogous to the holes in the bacterial cell lawn caused by the phage, except that since the virus is released from a lysed host cell into the liquid media, the released viruses may infect cells anywhere in the monolayer, not just the adjacent cells as in your phage plates. * * * Bacterial Transformation MCRO 251 Lab Manual Exercises to Complete New Exercises 28
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Introduction Bacterial transformation is a form of horizontal gene transfer accomplished through the uptake of ‘naked’ DNA by certain bacteria. Following uptake, this exogenous DNA is either maintained as an episomal plasmid (such as the plasmid in today’s lab) or it may be incorporated into a homologous site in the host bacterium’s chromosome. Either results in a recombinant organism. Some species of bacteria are naturally able to take up and incorporate such foreign DNA via transformation at some point in their cell cycle and are referred to as ‘competent’. Other bacteria may be made competent through chemical or physical treatment which allows them to take up the exogenous DNA. Most E. coli are not naturally competent. In today’s exercise, we will use E. coli DH5 α , a strain which has had competence conferred on it by a pre-treatment with a concentrated solution of calcium, such that it is made porous to exogenous DNA During the lab today you will combine the competent cells and the plasmid DNA in a tube, and expose the suspension to changes in temperature (4°C 42°C 4°C) allowing the exogenous DNA to be transported through the cell envelope. Following uptake, the plasmid will replicate inside the host cell. As in most
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This note was uploaded on 04/12/2011 for the course MCRO 251 taught by Professor Lorrainecramer during the Spring '09 term at UNC.

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Microbiology 251-11 - WEEK 5 February 8 10 Bacterial Transformation Effect of Washing on Hand Flora Nasal Flora Exercisesto Complete Pure Culture

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