From 9-11-14.
Discovered that there was a lot of humidity in the two 500 mL containers housing the wax worms stored at 30C. There was visible condensation on the inside of the containers and it smelled of moisture. A number of the wax worms were turning black (dead). I split the two 500 mL containers into two additional containers, transferring half of each to a new container. I removed the dead larva and returned all the containers to the 30C.
Transferred the wax worm food container that was in 35C into the 30C incubator because I am not able to see any early instar larva. I believe that the increased temperature was responsible for the wax worms not emerging.
//EWW
Friday, September 26, 2014
Chlorine Dioxide Pilot Study
From 9-17-14.
Last week:
Performed colony counts of the three P. larvae spore stocks growing on MYPGP agar in 37C 5%CO2.
Will use equation below to calculate CFU/ml of my P. larvae spore stocks.
My spore stocks are resuspended in 500 uL ddH2O so,
Stock F - 3.56x10^6 CFU/ 500 uL
Stock G - 3.12x10^6/ 500 uL
Stock H -1.36x10^7/ 500 uL
Retrieved 500 gram of Mueller-Hinton Broth from USDA. Stored in chemical cabinet in Fisher Lab.
//EWW
Last week:
Performed colony counts of the three P. larvae spore stocks growing on MYPGP agar in 37C 5%CO2.
| Stock: | Stock F | Stock G | Stock H |
| Dilution: | 10^-3 | 10^-3 | 10^-4 |
| 20 | 22 | 7 | |
| 18 | 10 | 5 | |
| 12 | 10 | 2 | |
| 17 | 21 | 9 | |
| 22 | 15 | 11 | |
| Avg CFU: | 17.8 | 15.6 | 6.8 |
Will use equation below to calculate CFU/ml of my P. larvae spore stocks.
Determining CFU/mL from colony count formula/equation:
[# colonies counted / volume pipetted onto plate in uL] * [ 1 / serial dilution made (ie 10^-2)] * [1000 uL / 1 mL] = CFU/mL
My spore stocks are resuspended in 500 uL ddH2O so,
Stock F - 3.56x10^6 CFU/ 500 uL
Stock G - 3.12x10^6/ 500 uL
Stock H -1.36x10^7/ 500 uL
Retrieved 500 gram of Mueller-Hinton Broth from USDA. Stored in chemical cabinet in Fisher Lab.
//EWW
Wednesday, September 17, 2014
Red Flour Beetle Pilot
From 9-14-14.
9-16-14
Checked survival status of the RFB under the five different treatment groups again. This has been five days of incubation for the beetles.
Repeated the RFB/P. larvae lethality assay except using larval RFB instead of adult RFB. The larva were worm like and not red/brown in color. A picture of the larva can be found here. 5 ul of bacterial spores were added to each well as before, except this time it was allowed to dry for an hour in a flow hood. A single RFB larva was added to each well. Pictures of the set up are shown below:
The plate was incubated at room temperature protected from light. The larva will continue to be monitored and survival will be determined depending on if they pupate into adults. This may take up to a week.
9-16-14
Checked survival status of the RFB under the five different treatment groups again. This has been five days of incubation for the beetles.
Control
|
P. larvae 10^-1
|
P. larvae 10^-4
|
B. thru 10^-1
|
B. thru 10^-4
|
|
Alive (n=8)
|
6
|
6
|
7
|
5
|
5
|
Some of these number counter what was previously recorded. This may be an error on my part - counting some RFB as dead when they were in fact alive. This is still very much a pilot experiment, and I will have to come up with a better method to calculate alive/dead status in the future!
Repeated the RFB/P. larvae lethality assay except using larval RFB instead of adult RFB. The larva were worm like and not red/brown in color. A picture of the larva can be found here. 5 ul of bacterial spores were added to each well as before, except this time it was allowed to dry for an hour in a flow hood. A single RFB larva was added to each well. Pictures of the set up are shown below:
The plate was incubated at room temperature protected from light. The larva will continue to be monitored and survival will be determined depending on if they pupate into adults. This may take up to a week.
//EWW
Chlorine Dioxide Pilot Study
From 9-11-14.
9-16-14
The three P. larvae spread plates have been incubating at 37C 5% CO2 for seven days. Spores from these plates were collected using the method afore described. There were fewer visible colonies that previously seen before after seven days of incubation of P. larvae. Regardless, spore preps were performed and the final spore pellet was resuspended in 500 uL of sterile ddH2O.
Performed the drop plate method on MYPGP to quantify CFU/mL in the three P. larvae spore stocks. Also performed this method on LB only using the B. thuringiensis spore stock. P. larvae plates were incubated at 37C 5%CO2 and B. thuringiensis plates were incubated at 30C.
Today
B. thuringiensis plates were checked after about 18 hours and had countable colony formations.
My stock spores are resuspended in 500 uL of ddH2O so,
9-16-14
The three P. larvae spread plates have been incubating at 37C 5% CO2 for seven days. Spores from these plates were collected using the method afore described. There were fewer visible colonies that previously seen before after seven days of incubation of P. larvae. Regardless, spore preps were performed and the final spore pellet was resuspended in 500 uL of sterile ddH2O.
Performed the drop plate method on MYPGP to quantify CFU/mL in the three P. larvae spore stocks. Also performed this method on LB only using the B. thuringiensis spore stock. P. larvae plates were incubated at 37C 5%CO2 and B. thuringiensis plates were incubated at 30C.
Today
B. thuringiensis plates were checked after about 18 hours and had countable colony formations.
B. thur Spore Stock Dilution
|
||
10^-8
|
10^-9
|
10^-10
|
15
|
1
|
0
|
20
|
2
|
1
|
21
|
2
|
0
|
18
|
3
|
0
|
14
|
2
|
0
|
17.6
|
2
|
0.2
|
The bold underline numbers are the averages from each dilution that was plated out.
Determining CFU/mL from colony count formula/equation:
[# colonies counted / volume pipetted onto plate in uL] * [ 1 / serial dilution made (ie 10^-2)] * [1000 uL / 1 mL] = CFU/mL
My stock spores are resuspended in 500 uL of ddH2O so,
Concentration of the B. thuringiensis is 3.52x10^11 CFU/500 uL
//EWW
Sunday, September 14, 2014
Red Flour Beetle Pilot
From 9-11-14.
Checked survival of the RFB under stereo microscopy after three days of incubating at 30C. The RFB that were still moving were considered alive and those that were not moving were considered dead. The RFB are quite lively and the only difficulty in determine alive/dead status is actually finding it in the small amount of flour in the 96 well plate.
Below is a table describing the survival of the RFB under the 5 different treatments:
Below is a figure describing the information in the table above:
From this data it would appear that P. larvae has little to no effect on the RFB, however the B. thur looks like it has a lethal effect. However, the P. larvae spore stock was in a relatively low concentration to begin with (~10^6 CFU) whereas the B. thur was very high (>10^8 CFU). So, the RFB were exposed to a higher level of B. thur than they were P. larvae. Perhaps if they were given a higher concentration of P. larvae we could see a lethal effect over this time period.
The goal of this study was to determine if the bacteria had an effect on the RFB, and it was successful so far. It appears that both P. larvae (albeit slightly) and B. thur have an effect on RFB survival. The 96 well plate will be continued to incubate at 30C and the survival will be continued to be monitored.
//EWW
Checked survival of the RFB under stereo microscopy after three days of incubating at 30C. The RFB that were still moving were considered alive and those that were not moving were considered dead. The RFB are quite lively and the only difficulty in determine alive/dead status is actually finding it in the small amount of flour in the 96 well plate.
Below is a table describing the survival of the RFB under the 5 different treatments:
|
|
Control
|
P. larvae 10^-1
|
P. larvae 10^-4
|
B. thur 10^-1
|
B. thur 10^-4
|
|
Amount
alive (n= 8)
|
7
|
6
|
6
|
3
|
5
|
Below is a figure describing the information in the table above:
The goal of this study was to determine if the bacteria had an effect on the RFB, and it was successful so far. It appears that both P. larvae (albeit slightly) and B. thur have an effect on RFB survival. The 96 well plate will be continued to incubate at 30C and the survival will be continued to be monitored.
//EWW
Thursday, September 11, 2014
The Waxworm & the P. larvae
From 9-8-14.
Received a number of containers in the mail containing wax worms. Most of them were tracked for Bridget E.'s project, however I was allowed to have a single container for my future wax worm studies. Below is a picture of half the wax worms that Bridget E. is maintaining.
I will continue to monitor the progress of these wax worms as they turn into wax moths. As the moths lay eggs I will transfer them to the prepared Gerber diet as well.
//EWW
Received a number of containers in the mail containing wax worms. Most of them were tracked for Bridget E.'s project, however I was allowed to have a single container for my future wax worm studies. Below is a picture of half the wax worms that Bridget E. is maintaining.
I divided up the container of wax worms that I received into three different containers seen below. Wax paper was tightly folded and hung into the containers from the cap of the container. The wax worms are mostly in good health (>95%) and are quite lively. The containers were incubated at 35C in the Fisher Lab incubator.
//EWW
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.
2. Tilt the container on its side at almost a 45 angle (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.
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 transfer2. 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):
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
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