I used four different temperature variables: room temp (~20C), 30C, 37C, and 40C. Groups of roaches were acclimated to each temp 7 days prior to initial injections (t=0). They were provided with ample food and water to ensure roaches were healthy and viable before injections.
A 48-hour chocolate agar plate of F.t. LVS struck from -80C freezer stock was used. Due to the time it takes to inject the large amount of roaches needed for this experiment, there was concern regarding the viability of F.t. LVS during the entire experiment. A previous experiment indicated that viable F.t. LVS cells may decrease by one log within 5 hours of being suspended in ddH20, which would skew or LD50 values to be higher with later groups. Therefore, new dilution sets were prepared before each temperature variable for injections in the 5 groups of 10 roaches (as described in the protocol below).
1. Prepare 4 sets of 1:10 dilution tubes with sterile PBS.
2. Suspend on loop of F.t. LVS into PBS and serial dilute 1:10 to the -6 dilution.
3. Inject 20 microL of each dilution (-1 to -5) into a set of 10 roaches.
a. Wipe roaches with alcohol prior to injections.
b. Inject with a micro-step repeat pipette, and confirm volume prior to dilutions.
c. Inject roaches the second to last segment of the abdomen on the dorsal side, under the armor plate.
d. After injected, store roaches in oversize petri plates--make sure to tape shut to prevent escape.
4. Make titer spot plate using dilutions -1 to -6 on chocolate agar plates, with 10micrL spots; incubate at 37C.
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| Sample spot-plate, different dilutions were plated in the 4/14 experiment |
a. Follow same procedure used in step #3.
6. Store roaches at appropriate temperature and check daily for survival. Remove and discard dead roaches.
7. Repeat steps #3-5 for each of the temperature variables.
//BEE
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