LD50s of Listeria monocytogenes in Blaptica dubia roaches

Last Friday (10/24/14) I started an experiment for the high-throughput study of Listeria monocytogenes virulence using Blaptica dubia roaches.  I had previously determined the LD50 values of several mutants of Listeria from Dr. Teresa Bergholz lab in Galleria mellonella (wax worms). I using the roaches for add information about the isolates and to further develop the roach model.

The following protocol was used on Friday.

1. 2 days before: Request cultures from Becky and streak E.coli DH5alpha on an LB plate
2. 1 day before: Create overnight (20 hour) cultures in BHI broth and DH5alpha in LB broth and incubate at 37deg C, shaking >225rmp
3. Day of Injections: Serial dilute 10-fold to 10^-7.
• Inject a set of 8 roaches for each dilution 10^-1 to 10^-5.Wipe roach with isopropyl alcohol before injection
• Using sharpened pipette tips, pierce the 2^nd to last segment of the abdomen on the dorsal side.
• Inject 20μl of bacterial solution.
• Place 4 roaches in a sterile plastic petri dish for storage.
4. Create a control group by injecting 8 roaches with PBS in the same way.
5. Make a titer by dividing a plate into 4 quadrants, and spotting five 20μl drops of each 10-fold dilution on BHI plates (LB for E. coli).
6. Store roaches at 37°C for 10 days—record any deaths.
7.  Calculate LD₅₀ of each strain using titer and survival of roaches.

Sharpening of extra-long gel loading tips.

Recording of roach survival

//BEE

Francisella LD50 in Blaptica dubia roaches

from 10/17/14

Unfortunately due to complications with the media, spot titer plates did not produce individual colonies to determine CFU/dose for the three Francisella strains being tested. Because of this, the LD50 cannot be determined.

Francisella plates

However, the spot plates for Burkholderia K56-2 strain and E.coli DH5alpha were able to be read, and the PBS control showed no signs of bacterial contamination.

E.coli, K56-2, and PBS plates

Roaches in 37 deg C incubator

The roaches are being tracked for survival and will be observed for 6 more days.











//BEE 

LD50 of Francisella in Wax Worms

In addition to finding the LD50 values Francisella mutants in the Dubia roaches, I have also been repeating the work done by Ben to find the LD50s in the Greater Wax moth. I used the same protocol that was used for finding the LD50s of Listeria for Dr. Bergholz (Listeria LD50 protocol using wax worms).

A few modifications were made to accommodate for the fastidious nature of Francisella, which will not grow well in broth cultures. Mueller-Hinton plates with 2% Isovitalex were used to make 48 hour plates of each Ft culture. A loop of bacteria was suspended in 1ml of sterile PBS to form a bacterial suspension, which was then diluted with PBS.

//BEE

Francisella LD50 in Blaptica dubia roaches

Today I started an experiment for the high-throughput study of Francisella tularensis virulence using Blaptica dubia roaches. The purpose of this study is to ultimately develop a novel insect model that can be more applicable to long term studies.

The following protocol was used today.

1. Request blinded strains from dawn on Modified Mueller Hinton plates with appropriate times.
2. Suspend a colony of each bacterial strain in 1mL PBS.
3. Serial dilute 10-fold to 10^-6.
• Inject a set of 10 roaches for each dilution 10⁰ to 10^-5.Wipe roach with isopropyl alcohol before injection
• Using sharpened pipette tips, pierce the 2^nd to last segment of the abdomen on the dorsal side.
• Inject 20μl of bacterial solution.
4. Create a control group by injecting 10 roaches with PBS in the same way.
5. Make a titer by dividing a plate into 4 quadrants, and spotting five 20μl drops of each 10-fold dilution on Mueller-Hinton plates with 2% isovitalex.
6. Store roaches at 37°C for 10 days—record any deaths.
7.  Calculate LD₅₀ of each strain using titer and survival of roaches.

//BEE 

Lab Work Update

For the past month I have been working with Dr. Teresa Bergholz's lab. They have supplied me with Listeria monocytogenes and several mutants strains, and I have tested their virulence in Galleria mellonella (wax worms) to determine the lethal dose 50% (LD50). The following protocol was used for the 8 isolates being tested. A total of three replicates were done to find the final LD50s.

Listeria monocytogenes LD50 Protocol

1. Obtain inoculated plates from Becky
2. Give to Dawn for blinding of overnight broth cultures in BHI media 
• Incubate at 37°C shaking for 20 hours 
3. Remove 1 mL of bacterial suspention into a 2.0 mL epp tube 
4. Wash cells to remove BHI media and resuspend with PBS 
• Centrifuge at 13,000g for 1 minute in 2.0mL epp tube, wash twice. 
5. Make serial 1:10 dilutions of each overnight culture to 10-7 using 2.0 mL epp tubes and PBS as diluent 
6. Create a spot plate titer on BHI plates to determine CFU/mL of each dilution 
• Using 5x10μL spots for -4 to -7 dilutions 
7. With a Insulin syringe, inject 30μL into each worm 
• Set of 10 wax worms for each dilution of each mutant 
8. Create a control group by injecting 10 worms with PBS 
9. Place worms in labeled petri plates and securely store them at 37°C for 9 days, observe and record day and number of deaths and pupate 
10. Calculate LD50 for each mutant of Listeria using F020_LD50Gm excel file

Data was entered into the excel file and stored on my OneDrive account (Listeria Survival Data in wax worms) and found the following data:

Isolate    Rep 1          Rep 2         Rep 3         Average         St Dev
TB7        6.53E+03    3.39E+06    1.12E+04    1.14E+06    1.95E+06
TB10      4.18E+04    1.30E+03    1.13E+04    1.81E+04    2.11E+04
TB18      2.74E+04    1.62E+04    1.16E+04    1.84E+04    8.13E+03
TB19      4.77E+04    2.47E+03    1.10E+04    2.04E+04    2.40E+04
TB25      2.66E+05    5.56E+04    1.24E+05    1.48E+05    1.07E+05
TB26      7.07E+04    2.62E+04    6.18E+04    5.29E+04    2.35E+04
TB27      1.13E+04    1.53E+03    6.83E+03    6.55E+03    4.89E+03
TB133    4.18E+04    7.42E+04    8.25E+04    6.62E+04    2.15E+04
DH5a     3.39E+06    6.60E+04    1.47E+05    9.79E+05    1.61E+06
                                   3.13E+05   

Table can be viewed in the excel file here:Listeria LD50 Values     

//BEE