AEDnet Exchange Programme PLEASE ENSURE YOU COMPLETE ALL SECTIONS OF THIS FORM Please complete the form and return to [email protected]along with evidence ofthe duration of the secondment (e.g. copies of flight tickets) and a short written reportoutlining your findings whilst at the host University. Researcher details: First Name: Mykola Last Name: Keleberda Address: 56, ap. 198, Peremogy ave, Kharkiv, Ukraine Post code: 61202 Contact Number: 00380685828904 Email Address: [email protected]Gender: Male Date of Birth: 14/05/1974 Nationality: Ukrainian Passport Number: ER399419 Details of Secondment:
Transcript
AEDnet Exchange ProgrammePLEASE ENSURE YOU COMPLETE ALL SECTIONS OF THIS FORM
Please complete the form and return to [email protected] with evidence ofthe duration of the secondment (e.g. copies of flight tickets) and a short written reportoutlining your findings whilst at the host University.
Host University: Welsh School of Pharmacy and Pharmaceutical Sciences
Cardiff University – Professor Les Baillie
Redwood Building, King Edward VII Avenue,
Cardiff CF10 3XF, Wales, UK
Dates of secondment: 13/02/2017 - 11/03/2017
Description of activities and results
The secondment was from 13/02/17 to 11/03/17. Our Ukrainian delegation was hosted by the laboratory of Prof. Les Baillie at the Welsh School of Pharmacy and Pharmaceutical Sciences of the Cardiff University. Our research group in Ukraine is working on phages isolation from soil of different burial sites. The methods used by research group in Cardiff will allow us to produce high titer stocks of bacteriophages quickly and in higher volume. The main aim of the training was to characterize bacteriophages and for the further application as antibacterial agents in phage therapy and environmental decontamination. I set up soil microcosms with the spores of the Stern strain B. anthracis 34F2. I also learn how to purify a solution of phages that were previously unknown to me. I was trained on phage isolation from soil and phage enumeration. I was introduced to the method that shows the effect of germinants on spore reduction in soil and the principle of adding germinants to soil to trigger the conversion of spores to vegetative form.As a result of my secondment I have learned multiple techniques involving the purification and enumeration of bacteriophages. I would like to thank Prof. Baillie and his colleagues for the very informative training that will be very useful in my work in Kharkiv.
Methods
Phage purification
Serial dilutions of phage were created (from 10-1 to 10-9)in 5ml LB broth. 1ml of each dilution was removed into separate tubes. 4.5ml of LB broth was added to host bacteria (grown for 16 hours) on slopes and vortexed to resuspend bacteria in broth, and 100µl of host bacteria was added to the 1ml dilutions. 4ml of TSB soft agar (0.5%) was added to each dilution and poured onto a TSB agar plate, and incubated for 16-20 hours. Single plaques were then picked using a sterile pipette tip, placed into 1ml of broth, and left for 15-30 minutes at room temperature to let phages diffuse into the broth.
Serial dilutions were created from the picked plaque broth and the process above was repeated for a total of three times to ensure phages was purified.
Phage propagation and enumeration
Bacteriophage propagation in broth was performed by inoculating fresh TSB with 50 μl of an overnight culture of the bacterium spore and 50 μl bacteriophage which was then incubated at 37°C and 180 RPM overnight. Following overnight incubation Vortex mix. Add a drop of chloroform at a ratio of 1:1000 chloroform: broth and Vortex mix again. Leave the suspension for 5 minutes and then centrifuge at 5,000 x g for 20 minutes to pellet the bacterial debris. The supernatant is then filtered using a double stacked syringe filter (0.45um and 0.22um filter joined together). The resulting phage suspension was stored at 4°C.
Filtered supernatant was then serially diluted (from 10-1 to 10-9) and 1ml was removed from each tube into separate sterile tubes. 100µl of host bacteria (1x 108) was added and 4ml of TSB top agar (0.5%). PFU were counted to determine the titre of the phage.
Bacteriophage isolation from soilEnvironmental phage isolations were performed by adding 25 g of soil to 25 ml of TSB followed by the addition of 10 ml of mid-log phase culture of the Sterne strain of Bacillus anthracis. The resulting suspension was mixed thoroughly and was incubated overnight at 37°C with shaking at 125 RPM.
Each incubated mixture was centrifuged at 5,000 x g for 20 mins and the supernatant fraction was filtered using syringe filter of pore sizes 0.45 μm and then 0.22 μm. Each filtrate was screened for the presence of bacteriophages against B. anthracis Sterne using the agar method described below.
To recover the phage a plug of agar which included the plaque was taken andsuspended in 500 µl phage buffer to which a small drop of chloroform was added to lyse any remaining bacterial cells. The suspension was vortexed and then left overnight at 4°C to allow any phage particles to diffuse out of the agar. The samples were centrifuged at 5,000 x g for 10 minutes and the supernatant fraction decanted into a sterile microcentrifuge tube.
This solution was then retested for the presence of phage using the agar method and if plaques were seen, the extraction was repeated a minimum of three times to ensure that the phage preparation was pure. If plaques were not seen after an initial round of purification and testing against B. anthracis, no further testing was performed.
Bacteriophage plaque morphology: The plaque morphology of each phage was determined using an agar overlay method. The phage preparation at a range of dilutions was added to overnight cultures of the propagation strain of bacteria in 5ml molten TSA agar. The agar was poured over the surface of a TSA plate and allowed to dry. The plate was then incubated overnight at 37°C for 18 – 24 hours and examined for the presence of plaques.
