Cockroach survival assay with Stenotrophomonas maltophilia and vancomycin

The purpose of my experiment that I am working on today is to observe the toxicity of Stenotrophomonas maltophilia when the antibiotic vancomycin is introduced. We have seen an increase in biofilm formation at sub-inhibatory concentration of vancomycin in several S. maltophilia isolates, as well as increased cytotoxicity by the amoeba Dictyostelium discoideum.

I struck out four of our laboratory strains of S. maltophilia (BAA 2423, 13270, G, and K) onto LB plates a few days ago. I used these plates yesterday to make overnight cultures that I used today, as directed in the following protocol that I used today.

Protocol for Cockroach Survival Assay

Materials

• Bacterial cultures
• Stenotrophomonas maltophilia isolates BAA 2423, 13270, G, and K
• LB broth media
• LB + Vancomycin100 broth media
• 70 Cockroaches ~1 inch 
• Insulin Syringe with 31G needle
• Repeat micropipetter 
• 2 mL Eppendorf tubes
• p1000 Pipettes and tips

Procedure

1. From fresh plates of bacteria, make an overnight culture using 10 ml of LB broth in a sterile 50mL centrifuge tube.  Suspend a colony of the bacteria in the media and place in a 37°C shaking incubator.
2. Transfer 1mL of the overnight liquid culture into a 2mL eppendorf tube. 
3. Make 2 replicates of each isolate for +/- vancomycin
4. Centrifuge cultures (2.5 min at 10,000g) into a pellet
5. Discard supernatant and resuspend one pellet with 1mL LB broth and the other with 1mL LB + vancomycin100. 
6. Incubate at 37°C for 2 hours.
7. Vortex and load an insulin syringe with resuspended bacterial culture and inject 25μL into each roach in a set of 10. Repeat with each bacterial culture.
8. Create control group by injecting 10 roaches with 25μL LB+Vancomycin100. 
9. Place 3-4 roaches of the same group into petri plates and store in 37°C incubator. Observe daily for survival.

I will check the roaches every 24 hours for one week and record any deaths*.


RESULTS (number of roaches alive out of 10)

Group                         Day1   Day2    Day3    Day4    Day5    Day6    Day7  %survival

Control (LB+Vanco)    10         9          9          9           9          9          9          90%

2423                               8          6         5           5          5           5          5          50%

2423+Vanco                  6          3         2           1          0           0          0          0%

13270                            4          0          0           0          0           0          0          0%

13270+Vanco               8          8          7           7          6           6          6          60%

G                                   2          2          2           2          2           2          2          20%

G+Vanco                      2          1          1           1          1           0          0          0%

K                                   3          0          0           0          0           0          0          0%

K+Vanco                      5          2          1           1          1           1          1          10%

 

*roaches seemed very dehydrated with very little hemolymph. Hemolymph from dead roaches after 24 hours of injection was plated onto LB agar plates and put in 37degC incubator overnight. Bacterial colonies were observed on all plates.  This was done by sterilizing the ventral thoracial-coxal joint area of hindleg with 70% isopropyl alcohol, and then piercing with a 16G1 1/2 gauge needle to remove hemolymph with a sterile inoculating loop.

//BEE