ClO2 Study - P. larvae Spores on Wood

From glass on 5-8-15.

Performed ClO2 exposure to P. larvae 9545 on pine wood chips.

A 100 uL volume of Spore Stock #14 was added to the top of a small pine chip purchased from Tractor Supply. The volume was allowed to evaporate and adhere to the chips (placed in petri plates) in the biological safety cabinet for one hour. The volume was rather large for the size of most of the chips. After the one hour time, some spots could be seen under the chips on the petri plate. It appears the 100 uL volume was enough to fully saturate the pine chip and some soaked through to the bottom of the petri plate. I hope I did not lose too much of the spores on the plates.

Three pine chips with adhered spores were transferred to modified anaerobic chambers. A volume of 50 mL of ddH2O was also added to the chambers to increase humidity. Dry weight concentrations of ClO2 reagents were mixed in PCR tubes and place within the chambers.

Total dry weights of reagents:

0 mg

25 mg

50 mg

100 mg

After the reagents were combined in the PCR tubes and mixed the tube was placed within the chamber (while open) and the chamber was sealed using the cover. The metal stir bar was used again inside the chambers in order to mix the gas more effectively. The chambers were protected from light and incubated at room temperature for six hours.

Unfortunately, the magnetic stir bar was not longer spinning in the 100 mg container after six hours. The bar apparently spun out of control and knocked over the petri plate containing the pine chips and the PCR tube containing the reagents. This prevented a good mix of the two reagents, and likely I will not be able to use the results from this treatment group. The ppm of chlorine gas was checked in each container after six hours.

ppm of Cl in each container after six hours:

mg	ppm Cl
25	15
50	20

Temperature inside the hood: 28.3 C

The determined ppm inside the containers were about half of what they were when using the glass cover slips. The reduced ppm of Cl in the containers could be due to the presence of the pine chips. It could be that they are serving as a sink for the gas similar to how water does.

The pine chips were removed from the modified anaerobic chambers and re-suspended in 1 mL of sterile ddH2O in 50 mL conical tubes. The tubes were vortexed for 30 seconds and left to incubate at room temperature for 5 minutes. The volume was diluted and plated onto MYPGP agar. After about five days I will be able to count colonies and determine CFU/mL of the spores subjected to ClO2 treatment.

ND Isolate Spore Stocks

The P. larvae spores extracted from ND isolated (from honey) were determined today once the colonies were countable on the MYPGP agar. Below are the calculated CFU/mL concentration of these spores stocks. The stocks are stored in 4C and will be used in the near future for the ClO2 study.

ND Stock #	CFU/mL
2a -A	1.56E+06
2a -B	1.03E+06
4a -A	7.30E+04
4a -B	3.50E+04
5a -A	3.25E+06
5a -B	2.25E+06
22a -A	5.00E+06
22a -B	3.75E+06
55a-A	7.50E+05
55a-B	7.75E+05

//EWW

Wax Worm/P. larvae LD50

From 5-6-15.

While the wax worms are continuing to pupate into wax moths and lay eggs I am continuing my investigation into how to use bee's wax in place of the oats in the LD50 experiment.

Since the 96 well polystyrene plates with the wax in them would need to be heated to too high of a temperature in order to melt the wax I am moving on to other methods.

My initial plan was to melt the bee's wax inside of a larger container and then quickly pipet the liquid wax to the wells. A small block of bee's wax was placed inside an Eppendorf tube and PCR tube (seen below) and heated in a water bath at 60C for 1 hour. However, at this temperature the wax did not melt and still appeared as the first picture below. By increasing the temperature to 70-80C the wax melted in almost 30 seconds after being placed inside the water. The melted wax inside the tubes is also seen below.

Solid bee's wax inside Eppendorf and PCR tubes

Melted bee's wax after incubating at 70-80C for 30 seconds in a water bath

Unfortunately, I was not able to pipet the wax as it cooled almost instantly as soon it was sucked up into the pipet (really neat!). I was not able to eject the wax into the wells since it was solidified and stuck inside the pipet. I could possibly try heating the pipets themselves, but instead I moved on to another method.

Small slabs of bee's wax was cut using a razor blade as seen below and transferred to a well in a 96 well polypropylene PCR plate (also seen below).

Cut bee's wax to be put into 96 well plate

96 well polypropylene PCR plate

The 96 well plate was placed inside the same plate heater previously used with the polystyrene plate, except this time water was added inside the plate holder to help equalize the heating process. At 60C the wax did not melt, but when pushed to ~70C the wax melted fairly quickly to the bottom of the well. This process seemed to work very well, except I will need to do more with standardizing the amount of wax that is added to each well as some have visibly more than others due to the amount initially added.

There were some air bubbles below the wax in some of the wells when the wax wasn't initially pushed all the way to the bottom of the well. Also, as soon as the plate of melted wax was removed from the water bath the wax solidified (almost) instantly. The wax was still soft enough to easily poke a hole through it with a toothpick. Now that I have a method to melt the wax, I need to determine a way to incorporate the spores without destroying them in the process (due to heat).

//EWW

ClO2 Study - P. larvae Spores on Glass

From 5-5-15.

I performed diagnostic PCR on the five P. larvae isolates using the AFB primers using new PCR super mix. However, the ran gel was completely blank except for the ladder! Even my positive control, P. larvae 9545, did not have a band appear. I continue to have issues with my PCR reactions.

I don't believe there is an issue with my DNA template, since my positive control has been used successfully in the past. However, perhaps the DNA has began to degrade and is no longer viable as a template for the reaction. But that doesn't explain why none of the P. larvae isolates amplified.

There may be an issue with the PCR supermix. We just received it in the mail and I have never successfully used it before. Perhaps I am not mixing the reaction correctly, however I did heavily scrutinize the instructions and am fairly confident the reaction was set up appropriately. I can ask my lab mates who have also been using the new supermix if they've been having any issues with it.

Maybe there is an issue with the thermocycler itself? As mentioned, this is not the first time I've had issues with PCR recently. I double checked my parameters for the AFB reaction and it is still set up appropriately. But, perhaps the thermocycler is having an issue with getting to the temperatures that are listed or maintaining that temperature.

I will further investigate these possible phenomenons as a possible source of my PCR problem.


//EWW

Wax Worm/P. larvae LD50

From 4-19-15.

All of the early instar wax worms I have had in the 96 well plates were frozen and disposed of. After monitoring each well's survival for over two weeks I was not able to discern any sort of pattern, trend, or consistency in the type and concentration of spores they were exposed to.

The wax worms were exposed to B. thuringiensis and P. larvae spores on a single piece of oat. An image of the setup can be seen here. Based on my results using this setup it may not be the most effective way to expose the wax worms to spores. After 1 week the early instar wax worms had eaten about 1/2 of the oat and after 2 weeks those that were still alive had consumed most of the oat. The well may have no longer contained any oat, but it contained an almost equivalent amount of frass (insect feces). This could not be a very healthy environment for the wax worms.

Results & Discussion
The only big drop off in survival of the wax worms in the negative control (no spores) was seen on Day 1. Since this was only 24 hours after they were transferred to the wells their deaths can likely be attributed to handling mistakes (crushing with forceps). The deaths at Day 1 was significantly reduced in the 96 well plates that were started last, likely due to my improved skill with the forceps to transfer the wax worms without injuring them. Death in other treatment groups was also seen on Day 1, however those treatment groups seemed to experience more rates of death than the negative control after that day (which was expected).

I will not report on the exact day/deaths of each wax worm exposed to the different treatment groups as the results were highly inconclusive. There didn't seem to be much difference in the rates of death in wax worms exposed to the highest concentration of P. larvae spores (1000 spores) and those exposed to the lowest (1 spore). More surprisingly, there was not a difference in the wax worms that were exposed to B. thur (a known insect pathogen) spores compared to the other treatment groups. B. thur was used as a positive control, yet there was no difference in survival when compared to the negative control!

It is my belief that the reason for these sporadic results is due in large part to the method at which the wax worms are being exposed to the spores. After one week they only eat a portion of the oat and it is likely that the portion that is ingested doesn't even contain the spores that it was inoculated with. It is also likely that once the wax worm actually get around to ingesting the spores on the oat they may be at an instar that is able to fend off the disease. Although no research has been done to know at what instar the wax worms are/aren't susceptible to P. larvae, if at all. Additionally, since only a volume of 3 uL was added to the oat, the spores were only present on part of one side of the oat. It would be ideal to more evenly distribute the spores on the food source for the wax worm's ingestion, however that might not be possible with the oats. For these reasons, I would like to move on to a different food source in this study.

Using Wax and Honey

All of my wax worms in my colony are currently pupating, so I am unable to continue with the early instar LD50 study at this time.So, I worked on creating an alternative method (as opposed to the oats) for exposed wax worms to the spores. I would like to use either bee's wax or honey to replace the oat in this experiment. With either of these two types of diet I could much easier homogenize the spores and it would be a more natural route of exposure for the wax worms.

I would still like to use the 96 well plates, but I need to devise a way to coat the bottom of the wells with either wax or honey, preferably the wax. I am a bit concerned with the early instar wax worms drowning in the honey if there is too large a volume present. A small shaving of bee's wax (used a razor to cut) was transferred to a number of wells using a forceps. Additionally, a small volume of honey was also transferred to a number of wells. The 96 well plate was placed in a heated plate warmer in order to heat the plate and melt the wax/honey to the bottom of the well.

Results were varied, but over all not very promising. The plate needed to be heated to nearly 100 C to melt the wax into place. I am concerned I am getting near the temperature required to melt the polystyrene plate (something that has happened before using the same plate heater). I am also concerned with how the spores will handle such high temperatures. I will continue to fine-tune the wax process, perhaps by heating a batch of wax first and then transferring the hot wax via a pipette to the wells.

//EWW

ClO2 Study - P. larvae Spores on Glass

From ClO2 Study on 4-28-15.
From ND P. larvae isolates on 4-25-15.

The five ND P. larvae isolates were grown on Columbian Blood Agar slants for 10 days at 37C in order to generate spores. The spores were extracted today using the method previous described . The stocks were diluted and plated onto MYPGP agar in order to determine CFU/mL of the stocks. There appears to be significantly varied amounts of spores depending on the isolate. I was only able to recover a very small spore pellet in a couple of the isolates, but a rather large pellet in others. I will know exact CFU after they grow on the plates.

Spore stocks created for:

dd1-5a

dd5-4a

dd8-55a

dd11-2a

dd12-22a

Spore stocks stored in 4C with other spore stocks until use. These spores will be used in the ClO2 study.

//EWW

Wax Worm Maintenance

From 3-31-15.

Made more wax worm diet as previously done using the following ingredients once again:

Ingredients Needed:

16 oz of Gerber baby food (or equivalent), whole wheat or multigrain

50 g wheat bran or crushed bran flakes

120 mL sterile ddH2O

170 mL Glycerin or Glycerol

100 g Table sugar

800 uL Amphotericin B (to protect food from mold)

Caleb D. assisted with making the diet. The created diet was stored in a tupperware container at room temperature until use.

All of the wax worms are currently nearing the pupation stage or about to emerge as wax moths. Their containers have been modified to include wax paper around the edges near the lid to later collect their eggs.

//EWW

Sporulation on Blood Agar

From 4-28-15.

I was able to count the number of colony forming units (CFU) from the MYPGP plates that had the P. larvae spore stocks diluted on it. The CFU/mL was calculated based on the number of colonies that were counted.

Stock #	Source	CFU/mL	CFU/mL
19	Slant	1280000	1.28E+06
20	Slant	620000	6.20E+05
21	Slant	740000	7.40E+05
22	Slant	620000	6.20E+05
23	Slant	830000	8.30E+05
24	Slant	1000000	1.00E+06
25	Slant	580000	5.80E+05
26	Slant	760000	7.60E+05
27	Slant	350000	3.50E+05

The spore stocks, along with their dilutions are still in the 4C fridge until use.

//EWW