Deliverable 1 1 A standardised method for the isolation of B anthracis specific bacteriophages

Introduction: One of the aims of this project is to harmonise and standardize methods across the network so that results obtained from different laboratories can be compared. We have developed a B.anthracis specific bacteriophage isolation method for the recovery of phages from B.anthracis contaminated animal burial sites (WP1.1). The method shown below is a composite of the approaches developed by researchers from Kafkas University in Turkey (KAU), the Eliava Institute in Georgia (EIG) and Cardiff University in the UK (CU). Training in this combined method has already been provided to researchers from the National Center for Disease Research in Georgia.

Composite B.anthracis specific Bacteriophage isolation and production methods

Bacteriophage isolation from soil
Environmental 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 and
suspended 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.

Bacteriophage propagation
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.

Bacteriophage enumeration (drop count method to determine PFU/ml): A version of the Bacteriophage agar propagation method was used.
1. Prepare a fresh culture of the bacteria (inoculate fresh media with 1ml of overnight culture of bacteria – usually will result in exponentially growing bacteria after 6 hr)
2.Prepare 8 eppendorfs filled with 900 μl of sterile PBS/water
3.Serial dilute bacteriophage in the 8 eppendorfs ensuring vortexing between each dilution using 100 μl neat bacteriophage culture
4.Add 0.1ml of exponentially growing bacterial host to 4.9ml of 50% TSA (or TSB with 0.7% agar-agar): pour agar as an overlay onto set TSA plate. Prepare two agar plates in total, allow to dry and divide into four sections.
5.Drop 10 μl of each bacteriophage dilution in triplicates onto one of the four sections.
6.Allow the drops to dry on the plate, then incubate overnight.
7.Phage titre determined as above (multiply by dilution and amount spotted to determine PFU/ml).

Spore production methods
The spore production method is adapted from a method developed by Curtis Thorne (J. Virol. 2(7):657.1968) The method is as follows;
1 Prepare an overnight broth and Incubate broth at 37oC
2 overnight with shaking at 250RPM
3 Remove broths from incubator and centrifuge at 3800 x g for 10 min at 20oC
4 Discard the supernatant and re-suspend in 10 mL Sterile distilled water
(SDW)
5.Innoculate 1 mL of this suspension into 50 mL NBYS broth. Incubate at 28 oC for 3 days with shaking at 250 RPM
6.Following incubation transfer broth to centrifuge tubes and centrifuge at 3800 x g for 15 min at 4oC.
7.Discard supernatant and re-suspend in 30 mL SDW
8.Store in the fridge at 4oC for 3 days
9. Remove suspensions from the fridge and centrifuge at 4oC for 15 min at 3800 x g.
10. Discard the supernatant and re-suspend pellet in 30 mL SDW
11. Heat shock suspensions at 65oC for 30 min
12. A 1 mL aliquot of the suspension will then be removed and perform pre and post heat shock viable counts. Aliquots are also looked at under x100 magnification phase contrast to see phase bright numbers.

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