Cockroach survival assay with Stenotrophomonas maltophilia and vancomycin

Today I will be repeating the roach survival assay with several changes to the previous protocol.  I will be using a 1:10 dilution of the overnight culture I prepared yesterday to inject the roaches. We want to see if there will be similar results with a lower concentration of cells. I will also be using the Escherichia coli B/r strain and Klebsiella aerogenes in addition to the three S.maltophilia strains used previously (BAA84, BAA2423, and 13270).  I will be using the "drop plate" method for the titer plates because it has been recommended for more accurate results.

Protocol for Cockroach Survival Assay 

Materials 

• Bacterial cultures  
• Stenotrophomonas maltophilia isolates BAA 2423, BAA 84, and 13270 
• Escherichia coli B/r
• Klebsiella aerogenes
• LB broth media  
• LB + Vancomycin100 broth media 
• 150 Cockroaches ~1 inch  
• Insulin Syringe with 31G needle 
• Repeat micropipetter  
• 2 mL Eppendorf tubes 
• p1000 Pipettes and tips
• 37°C incubator (shaking and stationary)

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 for ~16-20 hours. 
2. Transfer 1mL of the overnight liquid culture into a 2mL eppendorf tube.  
• Make 2 replicates of each isolate for +/- vancomycin 
3. Spin down cultures (2.5 min at 10,000g) into a pellet 
4. Discard supernatant and resuspend one pellet with 1mL LB broth and the other with 1mL LB + Vancomycin100.  Repeat for each isolate.
5. Let cultures incubate for 2 hours at 37°C
6. Dilute all cultures 1:10 by transfering 100μL into 900μL of the same media and antibiotic concentration.
7. Load an insulin syringe with resuspended bacterial culture and inject 25μL into each roach in a set of 10 using a repeat micropipetter (set at 2.5 where 1 Div= .01 mL). Repeat with each bacterial culture +/-vancomycin. 
• Make sure roaches have been incubated at 37°C for at least 24 hours before injection. 
• For E.coli B/r and K.aerogenes, use both diluted and undiluted cultures because there is no data for undiluted survival.
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 for survival for one week.
10. Create a titer for each strain by serial diluting  the final cultures 1:10 to the -7 dilution and using the drop plate method.

• This can be done by filling 7 1.5mL eppendorf tubes with 900μL sterile PBS and transferring 100μL to the subsequent tube (change pipette tips in between transfers and mix each suspension thoroughly). Repeat for each isolate used.
• From the -4 to -7 dilutions, pipette 5 spots of 10μL onto an LB plate divided into 4 quadrants, as shown by image. 
• Repeat for each dilution for each bacterial strain used. 
• Let plates sit upright until spots abosrb into plate.
• Invert and place in 37°C incubator overnight. (Final dilutions with be -6 to -9)
• Count colonies in the 3-30 range and determine CFU/mL for injection cultures. 

RESULTS (number of roaches alive out of 10)

Group                               Day1  Day2  Day3  Day4  Day5  Day6  Day7  %survival

Control (LB+Vanco)         10      10      10        10      10        10       10          100%

BAA2423                            10        7        7          5         5          5         5            50%

BAA2423+Vanco               8         6        5         4         4          4         4            40%

BAA84                                 8         5        4          4         3         2         2            20%

BAA 84+Vanco                  6         4        3          3         1          1          1            10%

13270                                   6         4        3          3         3         3         3            30%

13270+Vanco                     9         5        4          4         3          3         3            30%

E.coli B/r (10^-1)              9         3        2          2         2         2         2            20%
E.coli B/r+Van(10^-1)     10       6        5          3         2          2         2            20%
E.coli B/r (undilute)          2        1         1          1         1          0         0            0%
E.coli B/r+Van(undilute) 5        0        0         0         0          0         0            0%
K.aerogenes (10^-1)          4        4         4         4         4         4          4            40%
K.aer+Van (10^-1)             3        2        2          2         2         2          2            20% 
K.aer (undilute)                 2        2         2          2         2         2         2            20%
K.aer+Van(undilute)         1        1         1           1         1          1          1            10%

CFU/25μL (dosage per roach):
BAA 2423                           1.07x10^6
BAA 84                                1.35x10^6
13270                                  1.10x10^6
E.coli B/r (10^-1)            8.50x10^5
E.coli B/r (undilute)       8.50x10^6
K.aerogenes (10^-1)        3.00x10^5
K.aerogenes (undilute)   3.00x10^6

//BEE