Checked broth cultures in the 37C shaker incubator. pRK2013 and pMarA grew to a visible turbidity. However, the P. larvae 9545 culture inoculated in BHI Poly 60 did not have visible growth. This is contrary to what has been determined previously, that P. larvae 9545 is resistant to polymyxin antibiotic. The P. larvae inoculated into BHI only broth did have visible growth. Below is the comparison image of both P. larvae cultures. Both cultures were inoculated from a single colony of P. larvae on MYPGP only agar and incubated at 37C 225rpm overnight.
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| P. larvae 9545 in BHI broth. Left is BHI only, Right is BHI Poly 60 |
Setting up liquid tripartite conjugation:
1600 ul of each overnight culture was transferred to a 2 ml microfuge tube and centrifuged at room temperature, 13,000 rpm, for 1.5 minutes to pellet. Supernatant was discarded and the pellet was re-suspended in 1000 ul of ddH2O. Pellet was washed three times to remove any excess antibiotics from the original broth media.
The final pellet was re-suspended in 1000 ul of BHI only broth. A 1:1:1 mixture (200 ul of each) was made using each of the three culture; P. larvae, pMarA, and pRK2013. The mixtures were made in a 1.5 microfuge tube. Triplicate tubes were incubated at room temperature, 30C, and 37C. These tubes were NOT shaking and will remain incubating overnight.
Setting up solid tripartite conjugation:
The solid conjugation makes use of the 1:1:1 mixture that has been previously made. Sterile filter discs were aseptically transferred to MYPGP agar. A volume of 50 ul of the mixture was inoculated onto the disc on the agar. Triplicate plates were incubated at room temperature, 30C, and 37C overnight. Plates were not inverted.
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| Inoculate 50 uL of mixed culture to filter disc |
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| Aseptically transfer sterile filter disc to agar |
//EWW
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