PFD50 with Dicty and Vancomycin

Today I am using the Dicty predation assay to observe the cytotoxicity of several strains of bacteria when vancomycin in present on the media. I will be using eight Stenotrophomonas maltophilia strains from the ATCC: BAA2423, BAA84, 700, 13270, 17660, 13637, 51, 49; Escherichia coli B/r; Klebsiella aerogenes; Burkholderia cenocepacia K56-2. The purpose of this experiment is to see if there is an increase in the cytotoxicity of the bacteria in the presence of vancomycin. If so, we will see an increase in the PFD50 (plaque forming unit) when vancomycin is present in the media. Klebsiella and E.coli will be used as positive controls. I will be using the following protocol.

Dictyostelium PFD₅₀ Protocol

Materials

• Cultures of selected bacteria
• S. malt BAA2423, BAA84, 700, 13270, 17660, 13637, 51, 49 
• Escherichia coli B/r 
• Burkholderia cenocepacia K56-2 
• Klebsiella aerogenes  
• Dictyostelium discoideum grown on E.coli B/r
• 2.0mL eppendorf tubes
• p200 pipette with tips
• p1000 pipette with tips
• Sterile loops
• Sterile spreaders
• Sterile water
• p20 multichannel pipette with tips
• 96-well plate
• SM/2 +Vanco²⁵ plates
• SM/2 plates

Protocol

1. Make overnight cultures of bacteria strains by suspending a colony in 3mLs of LB in a 15 mL centrifuge tube and place tubes in 37°C shaking incubator for ~16-20 hours
2. From overnight cultures, pipet 100µL of bacteria solution onto one SM/2+Vanco²⁵ plate and one SM/2 plate. Spread the solutions evenly with a sterile spreader to create a bacterial lawn and let dry in the hood for 30 minutes.
3. Pipette 1mL of sterile water into a 2.0mL eppendorf tube and suspend 4-5 dicty fruiting bodies.
4. Serial dilute tube 1:10 to 10^-7 in 2.0mL eppendorf tubes

• Add 900µL sterile water to seven 2.0mL tubes and transfer 100µL to the subsequent tube (change pipette tips in between transfers and mix each suspension thoroughly).
5. In a 96 well plate, fill one row with the 8 dilutions of Dictyostelium with 300µL, and refill the wells when needed.
6. With a multichannel pipet, allocate 4 rows of 2 µL of the dilutions onto each plate.
7. Let the plate sit upright for at least 30 minutes to prevent the spots from running tßogether.
8. Create a titer by pipetting 100µL of the overnight culture of Klebsiella onto 15 SM/2 plates.
9. Add 10 µL of the 10^-1 to 10^-6 dilution to a set of 3 plates and spread the liquid out evenly (3 replicates for an accurate titer).
10. Observe the plates every day for one week for plaque formation.
11. Before titer plates are overgrown, calculate the PFU for available plates.

//BEE