Red Flour Beetle Pilot

From 9-3-14.

Checked the condiment cups containing the red flour beetles (RFB) under the three different treatment groups (Corn Flour, Wheat Flour, and a 50:50 mix). This study began eight days ago and they have since been incubating at room temperature in a box protected from light. I changed out the food (flour) in the twelve mason jars containing our RFB colonies.

How to change food in RFB jars

1. Transfer entire jar contents to a new clean container for transfer
2. Tilt the container on its side at almost a 45 angle (picture below):

3. After a short while (<1 minute) about 90% of the RFB will  gather at one end of the tilted container (picture below):

4. While the jar is being tilted, add fresh flour into the empty jar that once housed the RFB using a spoon. Fill the jar about 1/4 full with flour.
5. Using a spoon, carefully scoop up the gathered RFB in the tilted container and transfer them to the jar containing fresh flour.
6. Close the jar and label the side with the passage number, the date, initials, and also the type of flour that was used (corn or wheat).
7. Combine all the left over flour and remaining RFB from the tilted containers and freeze them overnight. Discard them after 24 hours.

Three of the RFB jars were passaged into corn flour, three into wheat flour, and three into a mixture of 50:50 corn:what flour. Jars were placed into a box protected from light and stored at room temperature.

RFB/Bacteria Pilot:

A sterile 96 well plate had its wells filled about 1/4-1/2 way with corn flour using a sterile scoopula. Only about half the wells will be used in this experiment due to the difficulty of transferring and containing the RFB once inside the well. 5 uL of bacterial spores were added to the tops of the flour present in specific wells. Each treatment was repeated eight times (entire column). This is a quick and dirty way to gage the effect these spore forming bacteria have on the RFB. Below is a table illustrating which bacteria (and dilution) was added to each column. The spot was not allowed to dry and it appeared to be a single water droplet in the flour.

Column 1	Column 3	Column 5	Column 7	Column 9
Control (no bacteria added)	5 uL of 10^-1 P. larvae Spore Stock D	5 uL of 10^-4 P. larvae Spore Stock D	5 uL of 10^-1 B. thuringiensis Spore Stock	5 uL of 10^-4 B. thuringiensis Spore Stock

After the flour was spiked with spores, a single RFB was added to each using a scoopula. It was challenging to keep the RFB inside the wells and still have enough of an opening to add additional ones. Static electricity would actually pull the RFB into wells or stick to the covers, so they would almost "jump" from well to well. The cover was used to keep the RFB from crawling out, but it was easy to accidentally  crush them. Regardless, all the treatment columns were filled with live and healthy RFB. A couple pictures of what the plate looked like with the flour and RFB is seen below:

The plate was incubated at 30C (optimal temperature for the bacteria) in the walk in incubator. The RFB survival will continue to be monitored in order to gage the lethality of these strains of bacteria on them.

//EWW