P. larvae CFU/larva Pilot

Follow up from 7-27-14.

Very few colonies grew from the drop plates on MYPGP and LB Only that had been incubating at 37C 5%CO2 for three days. There were approximately 2 colonies per 10 uL drop (5 drops total) from the No P. larvae CFU/mL homogenized larva on MYPGP and about 1 colony per 10 uL drop from the 1000 CFU/mL P. larvae homogenized larva. This was surprising since I expected there to be more colonies in general, and definitely more growth from the 1000 CFU than the No CFU samples.
This trend was also seen in on the LB Only plates.

The colonies that grew don't appear to be P. larvae based on visual morphology. They appear to be more rounded and dimpled than the flat P. larvae colonies. The colors are fairly similar however.

I created genomic lysates of several colonies that grew from both treatments and from both plates and will amplify them using the AFB-F/R primers to determine if they are P. larvae.

//EWW

Abx Screening of P. larvae

Made a batch of MYPGP agar + several antibiotics in order to determine the sensitivities of P. larvae to them. This experiment is a preliminary to an upcoming allelic exchange.

Antibiotics

Kanamycin 50 ug/ml

Ampicillin 100 ug/ml

Erythromycin 5 ug/ml

Polymyxin 60 units

(A stock of each antibiotic is stored at 4C)

250 mL of each MYPG-Abx agar was created. A four day old broth culture of P. larvae in MYPGP+thiamine broth (grown at 37C 5%CO2) was diluted ten fold down to 10^-3. Drop plates were preformed on each antibiotic agar for each P. larvae dilution. 

Plates were incubated at 37C in the static incubator (not inverted) overnight and then transferred to the 37C 5%CO2 incubator for three days. The growth on each of the plates are shown below:

 

 

It would appear that P. larvae is

Polymyxin resistant, but sensitive to erythromycin, ampicillin, and kanamycin. A few of the colonies that grew on the No Abx control and from the Polymyxin plates were converted to genomic lysates and will be amplified via PCR using the 16SrRNA P. larvae identification primers, AFB-F/R.

//EWW

P. larvae CFU/larva Pilot

Goal: determine the lethal CFU/larva of P. larvae.

For this pilot study, three previously dead honey bee larva around the 3rd/2nd instar will be homogenized and drop plated onto MYPGP and LB agar. Larva were from the LC50 study on 7-19-14.

Three dead larva from the 1000 CFU/mL treatment group and three from the 0 CFU/mL (control) treatment group were transferred via inoculation loop to 1 mL of sterile ddH2O. Larva were homogenized using the sterile plunger of a syringe.

Five 10 uL volumes of each homogenized samples were plated onto MYPGP and LB only agar.

 

Plates were incubated at 37C not inverted. It will be interesting to see what and how much growth is observed.

Note: I am continuing to have contamination issue with MYPGP agar from the P. larvae germination study.

//EWW

P. larvae LC50

Received 144 first instar honey bee larva from the USDA. Larva were transferred from the Jenter box using a small brush to a 48 well plate containing 2 uL of BAD. Larva were transferred via Styrofoam container to the NDSU lab were they were transferred to 2 uL of P. larvae spiked BAD on 48 well plates.

Treatments:

1000 CFU/mL P. larvae

400 CFU/mL P. larvae

No  CFU/mL P. larvae

Larva were incubated at 35C in anaerobic chambers. Tomorrow the larva will be transferred from their P. larvae spiked BAD to a new 48 well plate containing 10 uL of BAD. Survival will be monitored via microscopic observation and they will continually be fed increasing amounts of BAD.

Day 1 - 2 uL BAD

Day 2 - 10 uL BAD

Day 3 - 20 uL BAD

Day 4 - 30 uL BAD

Day 5 - 40 uL BAD

Day 6 - 50 uL BAD

Day 7 - None (stop feeding)

//EWW

P. larvae germination in BAD medium assay

Diluted Spore Stocks D and E in a ten fold serial dilution down to 10^-4. Performed blot plates on MYPGP agar to determine CFU/mL of each stock. Plates were incubated at 37C not inverted.

Repeated germination assay using optical density of Spore Stocks D and E.

The data from this assay is on an Excel file and it is far too time consuming to transfer the graphs from those results onto this blog. I also do not wish to simply copy and paste all the raw data as the tables will become skewed after transfer.

Also repeated CFU/mL germination assay using Spore Stocks D and E. Plates incubated at 37C not inverted.

//EWW

Waxworm Notes

I plan to use waxworms (Galleria mellonella) in future experiments with P. larvae including, but not limited to, LC50 determination. Below are some notes related to waxworms and waxworm in vitro rearing recovered from the internet (online) and Ben M. notes. Ben M. was the undergraduate who previously reared waxworms for the Fisher Lab (Bug Man).

Places to buy waxworms online: www.wormman.com/cat_wax.cfm. Or Ebay.

Eggs hatch around 4-6 days

Larva mature around 6-7 weeks (highly influenced by temperature
-mature larva will crawl (wander) to top of container

Sick or dead waxworms will turn black (do not confuse with "burnt" colored waxworms which are pupating)

Moths emerge about 1-2 weeks after pupation begins

WAXWORM LIFE CYCLE

Total life cycle: 57.6 days (46-69 days)

Egg - 9.5 days

1st instar - 4.3 days

2nd instar - 3.9 days

3rd instar - 2.9 days

4th instar - 2.7 days

5th instar - 2.8 days

6th instar (18 mm)- 3.4 days

7th instar - 2.7 days

Pupa (12 mm)- 25.4 days 

Moth

Received two small containers of mature waxworms from Dr. Fisher. Containers were transferred to plastic 0.5 L media bottles with three small holes punched into the covers. One of the containers were incubated at 35C (bee incubator) and another at 30C in the walk in incubator.

I will continue to monitor them for pupation. Once they pupate into moths they will lay eggs and I plan to harvest them.

//EWW

P. larvae germination in BAD medium assay

Checked MYPGP plates containing the dilutions for the germination assay and counted the colonies that formed.

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

Results:

BHI
	No Heat	Heat
CFU/ml	1.11x10^6	5.5x10^4
H2O
	No Heat	Heat
CFU/ml	9.3x10^5	8.9x10^5
BAD
	No Heat	Heat
CFU/ml	2.25x10^6	1.65x10^6

The results indicate that P. larvae spores germinate in the BHI + 1ug/ml Thiamine media, but do not in the H2O and BAD medias. The CFU/mL concentration of P. larvae that was observed growing on the MYPGP agar decreased after the sample had been heated to 65C for 15 minutes in the BHI treatment group. This indicates that vegetative cells that had germinated from the P. larvae spores were destroyed at such high temperatures (only the spores are so heat resistant). This results makes sense since BHI is what P. larvae is brothed in for growth.

The CFU/mL concentrations remained relatively the same in the H2O and BAD treatment groups.This indicates that the P. larvae spores did not germinate in those medias since it appears that CFU didn't decrease due to the heating like seen in the BHI treatment groups. 

I would like to repeat these experiments in the future with different spore stocks to confirm these findings.

//EWW

P. larvae LC50

Checked status of each honey bee larva via microscopy. Transferred alive larva to a new 48 well plate and added 10 uL of BAD next to them (touching them so they are on the parameter).

Dead as of 24 hours:

Control (no P. larvae spores) -4

1000 CFU/mL P. larvae - 3

400 CFU/mL P. larvae - 3

200 CFU/mL P. larvae -4

There were relatively few deaths after 24 hours (out of 24 for each treatment group). The deaths observed this soon are likely due to damage during transfer with the paint brush. Interestingly, all the larva in well A1 for all the treatment groups is dead- that was the first larva I transferred for each group.

Larva continue to incubate a 35C in the anaerobic chambers. I will monitor status again tomorrow and provide additional BAD.

//EWW

P. larvae germination in BAD medium assay

Drop plates from 7-17-14 continue to incubate at 35C. No contamination has developed like seen before on the MYPGP agar. In the future all MYPGP agar will be poured and dried inside a fume hood for sterility.

Yesterday:

Read a paper about germination determination of P. larvae in an artificial media by using optical density changes. I performed a quick pilot study to determine if OD could even be quantified on a spectrophotometer using the thick BAD. The Beckman spectrophotometer in Fisher Lab was able to detect the OD and it wasn't beyond its scale.

Proceed with a time course study of germination. Diluted Spore Stock A in a 1:5 dilution in BAD in a 96 well plate. Using the Pruess Lab incubated spectrophotometer Laura N. created a program that would incubate the plate for 2 hours at 35C and take OD580 readings every 5 minutes.

I am unable to attach the raw data that is in an Excel file, but below is a quick graph of the data without standard deviations. Standard deviations remained rock solid between each of the two replicates in BAD. The variation between the two is likely due to minor pipetting error as it is difficult to accurately pipet the viscous BAD.

It appears that OD decreased over time when P. larvae is incubated in BAD. Below is the same assay performed in H2O and BHI+1ug/mL thiamine instead of BAD.

It appears OD increases (albeit slightly) over time when P. larvae spores incubate in H2O and remains relatively the constant in BHI. I plan to repeat this OD germination experiment again with more replicates and different spore stocks to see if these trends remain consistent.

According to Alvarado, 2013 a decrease in OD over two hours indicates that the P. larvae spores are germinating. This would mean that the P. larvae spores germinate in my BAD as evident by the decrease in OD over the two hours.

I would like to repeat this OD experiment using different Spore Stocks to confirm these results.

//EWW

P. larvae LC50

Retrieved first instar honey bee larva from USDA hives today (9 am). Did not receive larva yesterday as planned because there was a significant lack of larva present in the Jenter box. The larva that was retrieved this morning were from a Jenter box placed in the hive on Tuesday afternoon at 3pm (~65 hours ago).

A total of 96 first instar honey bee larva were removed from the Jenter box via paint brush and transferred to 48 well plates that contained 2 uL of Bee Artificial Diet (BAD).

48 well plates containing larva were transferred to NDSU Fisher Lab immediately after in a Styrofoam container. Larva were then transferred and split into 4 different 48 well plates, 24 larva to each plate (one for each treatment group).

Treatment groups

(from 7-17-14 dilutions that had since been incubated at 4C)

200 CFU/mL P. larvae spores 

400 CFU/mL P. larvae spores 

1000 CFU/mL P. larvae spores 

No CFU/mL P. larvae spores 

A 2 uL volume of the treatment group was pipetted right next to (touching) the larva. There are 24 replicates for each treatment group. The 48 well plates were placed inside the anaerobic chamber and incubated at 35C. Completion time was about 10 am. Larva will incubate for 24 hours after which they will be examined for survival and transferred to fresh BAD for the remainder of their lives.

//EWW

P. larvae LC50

I am poised to receive a large amount of first instar honey bee larva tomorrow morning (8am). The Jenter boxes were placed in the hive at ~3pm on Monday, and now early Friday they will be collected.

Prepared P. larvae spore dilutions in BAD today (~5pm) from Spore Stock A (3.05x10^6 CFU/mL).

Spore Stock A --> 1:1000 (1 uL +999 uL BAD) --> 3050 CFU/mL

3050 CFU/mL --> 20:61 ( 328 uL + 672 uL BAD) --> 1000 CFU/mL (this will be a treatment)

1000 CFU/mL --> 2:5 (200 uL + 300 uL BAD) --> 400 CFU/mL (this will be a treatment)

1000 CFU/mL --> 1:5 (100 uL + 400 uL BAD) --> 200 CFU/mL (this will be a treatment)

The last treatment group will contain no P. larvae spores and will serve as a control. There will be a total of 4 treatment groups. Each spore dilution was stored at 4C overnight until use.

//EWW

P. larvae germination in BAD medium assay

Checked the two MYPGP agar plates from the batch created yesterday that had been incubated overnight at 37C and there was no visible contamination at all this time! It would seem that pouring the plates in the fume hood prevented the contamination that I have been seeing in my previous two batches of MYPGP agar. 

Mysterious blob contamination. 

Blood agar (gamma hemolysis)

MacConkey agar didn't have any growth at all. The plate was re-streaked from the contaminant and incubated at 37C overnight.
Both the indole test and the blood agar test today implicate Klebsiella spp. as being the contamination culprit. 


Moving forward, I performed the BAD germination assay again as well as re-plated the samples from 6-10-14 that had since been stored at 4C.

Repeated procedure from 6-10-14 using Spore Stock A (3.05x10^6 CFU/mL P. larvae spores). Used the MYPGP agar from the most previous batch that had not been found to be contaminated. Drop plates were incubated at 37C overnight not inverted.

Three treatment groups: H2O, BHI+Thiamine (1ug/mg), B.A.D.

Briefly, the Spore Stock A was diluted ten fold down to 10^-4. Each of the ten fold dilutions (10^-1 to 10^-4) were diluted 1:5 in each of the treatment groups listed above in eppendorf tubes. Tubes were incubated at 35C shaking at 150rpm for 2 hours. Samples were blot plated to calculate CFU/mL and also diluted ten fold down to 10^-2 for each dilution. The incubated samples were then heated in a water bath at 65C for 15 minutes and the plating/diluting method was repeated.

//EWW

P. larvae Project Pilot

The remaining honey bee larva that had pupated has now emerged as adult bees! Picture below:

This means that I now have 9 adult bees from the pilot study that began on 6-27-14. Originally there were 22 larva, 18 of which survived transfer. There was 81.1% survival rate to pupation (Link) and about 50% survival rate to adult bees (not including those lost in transfer). Considering I didn't closely monitor the larva after pupation and had basically left them for dead, 50% is phenomenal. Bees will be disposed of properly in the near future.

//EWW

P. larvae germination in BAD medium assay

I performed a Gram stain of the mysterious blob and it revealed a large number of Gram negative (pink) rod shaped bacterium. This was what was believed, but still doesn't necessarily narrow down the possibilities. The reason I performed a Gram stain and am so interested in identifying what the contaminant is is because I was worried it was my P. larvae that was the culprit.

Gram Stain Procedure

60 seconds Crystal Violet
60 seconds Iodine
10 seconds Ethanol
30 seconds Safranin

There were a few areas of the Gram stain that appeared to have Gram positive (purple) rods, but due to their clumping and vicinity they were likely really Gram negative. Regardless, I created crude genomic lysates of three separate contamination zones and performed the AFB PCR amplification. The results (not pictured) did not implicate these mysterious contamination blobs to be P. larvae.

Discussed with Dr. Gibbs and Heather V. about the contamination issues with my MYPGP agar. Upon inspection of the contamination, the culprit was believed to be Klebsiella spp. due to the slimey nature and morphology.

I isolation streaked a loop full of a mysterious blob onto a MacConkey and Blood agar retrieved from the Gibbs lab. Plates incubated at 37C overnight. The results of this will further identify the bacteria.

Performed Indole and oxidase tests:

Oxidase Test: Top right = mystery blob, top left = Klebsiella aerogenes, bottom right = E. coli B/r

Indole Test: Mystery blob,  Klebsiella aerogenes,  E. coli B/r

Mystery blob was found to be Indole negative and Oxidase positive. However, I am not too confident about the oxidase test (I have never very good luck with performing or concluding that test). 

Indole : Positive = no color change

            Negative = color change to red

Oxidase: Positive = purple color change

              Negative = no color change

Klebsiella aerogenes is:

Catalase positive

Indole negative

Non-motile

Oxidase negative

Note: not all Klebsiella spp. display these tendencies.

I will check the MacConkey and Blood agar plates tomorrow and further conclude what this mystery bacterium is.

Made up a new batch of MYPGP agar using Fisher Lab reagents (0.5 Liter), this time the plates were poured in the fume hood after it had been thoroughly sterilized. Two of the plates were placed in the 37C incubator and will be checked tomorrow for contamination. The remaining plates were stored in the cold room. 

//EWW

P. larvae Project Pilot

There were four honey bee larva that emerged as adult bees! Surprisingly, these four were located on the 24 well plate that was incubated at 35C not in the anaerobic chamber! Those were the ones that I had marked for death and looked like they were going to die soon. The larva that was hand selected from the 24 well plate that looked healthy and transferred to filter paper on a petri plate have not emerged as adult bees yet. In fact, they look closer to death than the others left on the 24 well plate. Images of the adult bees are seen below:

Conclusion: The larva not in the anaerobic chamber and left on the 24 well plate emerged as adult bees and the others that were transferred to filter paper and left in the chamber still have not. 

//EWW

P. larvae germination in BAD medium assay

Double checked the MYPGP agar plates from the new batch that were incubated overnight at 37C 5%CO2. They are seen in the two images below:

 

The same type of contamination that seen in the previous batch of MYPGP agar was also seen in this batch! Where is it coming from? This problem will need to be rectified before I can continue with the germination assay.

I will Gram stain the bacteria that is growing on the agar. I will also attempt to re-make the agar using new sources of reagents.

//EWW

P. larvae PCR Identification

Repeated P. larvae PCR Identification procedure using the lysates frozen at -20C.

Key: 

Top Lanes- AFB primers
1	2	3	4	5	6	7	8	9	10

Code	Sample
2	Spore Stock A
3	Spore Stock B
4	Spore Stock C
5	Plate B (7-5-14)
6	Plate C (7-5-14)
7	Drop Plate - BHI Treatment (7-3-14)
8	Drop Plate - H2O Treatment (7-3-14)
9	Drop Plate - B.A.D. Treatment (7-3-14)

Lanes 1 and 10 contained the Hi-Lo DNA Markers

My suspicions of the first time I amplified these samples was true, all spore stocks did indeed amplify using these P. larvae specific primers (Stocks A, B, and C). More interestingly, Sample 9 did not amplify, indicating that it is not P. larvae. This sample was from a colony on the drop plate of P. larvae incubated with Bee Artificial Diet (BAD) in the experiment to see if P. larvae can germinate in that medium. There was definitely bacteria that grew on the MYPGP agar, but apparently it wasn't P. larvae... Maybe it was another bacteria, or maybe there was human error when selecting a colony. Regardless, it was very interesting that that particular sample didn't test positive for P. larvae.

//EWW

P. larvae Project Pilot

A few of the initial honey bee larva from 6-21-14 that are getting close to emerging as adult bees. Most of the larva did begin to pupate, however succumbed to death when left on the 24 well plates during that life stage. Their death is likely due to being left on their own feces after the defecation stage. Literature review revealed that typically the larva is transfer to filter paper (Kim wipes) on a petrie plate, as seen below. It may be too late to save most of the larva as they look unhealthy (dark in color). 

I did not monitor or tend to these larva very closely because I do not plan on using the larva for any experiments once their pupation stage begins. That any survived to adult bees is surprising considering I left them alone on the plate with their feces and didn't transfer them to a cleaner environment. 

//EWW

P. larvae germination in BAD medium assay

Confirmed  that the remaining MYPGP batch of agar also had the same contamination issue after viewing a sample of the plates incubated at 37C overnight. I am not 100% sure that the new batch of MYPGP agar is also free of contamination. The plates that were incubated at 37C appeared to be contamination free, however the plates incubated at 37C 5%CO2 appeared to have a thin layer of bacteria on it...

To confirm that the new batch of MYPGP agar is free of contaminants, two plates were incubated at 37C 5%CO2 (one inverted and the other not). I will continue to monitor the plates tomorrow after 24 hours incubation.

//EWW

P. larvae LC50 Pilot

Observed the remaining surviving honey bee larva under dissection microscopy. None of them appear to have eaten any of the BAD food provided to them, nor have they appear to have grown in size. Additionally, they don't appear to move at all when view via microscopy. This likely indicates they are all dead, and is also likely they were dead yesterday.

The 24 well plate will be continued to be incubated at 35C and the larva will be monitored in case I have mistakenly declared them dead. The larva was not fed any additional BAD today.

In the future, I will likely transfer the larva directly to the 24/48 well plate at the USDA onto BAD and then transfer them to the NDSU Van Es Lab.

//EWW

B. dubia LD50 P. larvae Pilot

A cockroach has died in the 200 CFU/mL P. larvae spore treatment group. It was frozen at -20C and will be disposed of tomorrow. I will continue to monitor survival. This is the third day after the experiment had begun and now one roach has died in each of the treatment groups.

Treatment-alive after 72 hours
200 CFU/mL - 2
10,000 CFU/mL - 2
Control (no spores) - 2

//EWW

P. larvae LC50 Pilot

Checked honey bee larva via microscopy. It appears that the larva that was alive yesterday is still alive today. It was difficult to determine if they truly were still alive. The didn't appear to be degraded (only a circulatory system present) like the others that were definitely dead the second day. However, they didn't ungulate as the confirmed alive did either. Perhaps they are already dead, but preserved in the large amounts of BAD present.

The surviving larva were fed an additional 30 uL BAD (pipetted on top of them). This makes 9 surviving larva. I will continue to monitor their survival by microscopy as well as comparing the sizes of tomorrow to today. I will also make note if any of their BAD food has decreased from their wells because they have been eating it. They surviving larva do appear to be a bit larger than those that had died already.

//EWW

P. larvae PCR Identification

Made another 1% gel (0.5 grams + 50mL TAE buffer) in the Fisher Lab electrophoresis rig. Used the teaching lab 10-well comb because the Fisher Lab doesn't own any combs of that size.

Loaded samples from 7-9-14 that were amplified using the AFB primer sets. (2 uL EZ Vision + 15 uL sample, loaded 7 uL into each well). Ran the gel at 120V for 30 minutes. Upon imaging, there were absolutely no bands that appeared on the gel and even the DNA marker wasn't very apparent.  For some reason I had nearly a blank gel.

The amplicons had been stored at -20C for 4 days. I am confident that the samples were loaded successfully into the wells, that they were thoroughly mixed with the loading dye,  and that they ran the correct way across the gel. I am not sure what caused the previously visible amplicons to no longer appear on the gel. My only conclusion is to repeat the amplification and try again (soon!).

In the future, I will re-PCR amplify each of the samples using the AFB primers and visualize via gel electrrophoresis. DNA lysates from each sample have been stored at -20C.

//EWW

B. dubia LD50 P. larvae Pilot

Surprisingly there was one dead cockroach from the 10,000 CFU/mL P. larvae spore treatment group. I would sterilize the outside of the roach (bleach) and grind it up for PCR analysis using the method described here, but unfortunately there was also a dead roach in the control group!

I will continue to monitor their survival, if the remaining two roaches in the 10,000 CFU/mL group die in the near future and the control group remains alive then perhaps I will further investigate the cause of death. For now, the dead roaches were frozen at -20C and will be disposed of tomorrow morning.

Treatment-alive after 48 hours
200 CFU/mL - 3
10,000 CFU/mL - 2
Control (no spores) - 2

//EWW

P. larvae germination in BAD medium assay

Created a new batch of MYPGP agar plates (1 liter). Incubated one plate at 37C overnight to see if the same contamination issue arises again. I also placed a few MYPGP agar plates from the previous contaminated batch in the 37C and 37C 5%CO2 just to confirm that all the plates from that batch are contaminated and should be thrown out.

For this batch of MYPGP agar I used 1 gram of sodium-pyruvate from the media kitchen, because we are running low in the Fisher Lab stock. The first time I made MYPGP agar I also used the sodium-pyruvate from the media kitchen and had no contamination issues. That was the only variation between the previous two batches of MYPGP agar, even though I don't think the reagent was the problem.

//EWW

P. larvae germination in BAD medium assay

Still investigating what caused the mysterious globs to appear on my MYPGP agar and what it is. On 7-11-14 (24 hours ago) I incubated plate of MYPGP agar that hadn't been inoculated with anything into the 37C incubator. Today, that plate was examined and is seen below:

Clearly there is a contamination issue with my MYPGP agar plates. The exact same mysterious globules appeared on the plate and even some drying was observed. I have never had any problem with the previous two batches of MYPGP agar, nor have I ever had any contamination issues with my pouring method or that incubator in particular.

I checked the two streak plates of the mystery goop plated on an old MYPGP plate from a previous batch and LB agar. The plates have been incubating overnight at 37 and are seen below:

The mystery substance proliferated on both medias, but formed completely different colonies. Growth on the MYPGP agar produced similar colonies to what was struck on it (because they were both MYPGP). Growth on the LB produced white colonies that appeared almost dry on the agar. They appear to be a pure culture in both cases and they were both viewed under microscopy. In both cases, large amounts of rod shaped bacteria can were observed, a Gram stain was not performed, however they are most likely Gram negative due to some sort of contamination. 

Checked the initial drop plates where the contaminates were first identified and have since been transferred to the 37C 5%CO2. They are seen below:

 

The once very hydrated and goopy looking colonies are now dried out (albiet still somewhat goopy) once they had been transferred to the 5%CO2. I am not able to count the 10uL drops that were placed on the plates.

I am not sure how this contamination happened, but the remaining MYPGP agar from the batch will need to be disposed of and a new batch of plates created. Perhaps the flask used to pour the media was contaminated, or the plates/media were left open too long. Maybe the incubator isn't as clean as it should be? Maybe this batch of plates was poured thicker or thinner than usual? Not sure.

This entire experiment will need to be repeated using the new batch of MYPGP agar. I still have the dilutions created after this last attempt and I may repeat their blotting in an attempt to count CFU/mL. 

//EWW

P. larvae LC50 Pilot

Check survival of remaining larva under dissecting microscope. Two of the confirmed alive larva are now dead and two of the questionably alive larva are also dead now. The remaining surviving larva were fed 20 uL of BAD and continued to incubate at 35C in the anaerobic chamber at >90% humidity.

I noticed the pools of BAD that the larva were on were unusually dry, perhaps the humidity needs adjusting? I will continue to monitor survival and feed the remaining larva.

I performed the first step in creating new spore stocks using the spread plates from 7-5-14 that were created from Spore Stocks B and C. Those plates have been incubating at 37C 5%CO2 for 7 days. Plates containing nearly a complete lawn of bacteria. The bacteria appeared on the agar at a time appropriate for P. larvae as well as contained uniform colonies similar to what P. larvae should look like (flat, white, well defined colonies).

15 mL of sterile ddH2O was added to each plate at 5 mL intervals, plate was gently rubbed with a sterile spreader, and the solution was pipetted and transferred to 50 mL conical tubes. These two solutions, named Spore Stocks D (from the stock B plate) and Spore Stock E (from the stock C plate) were stored at 4C for the time being and are awaiting further processing. Spores will be extracted using the modified spore extraction method from 6-24-14.

//EWW

B. dubia LD50 P. larvae Pilot

No deaths observed in any of the treatment groups.  In hindsight, I should have had a BAD only control, but at this point there really isn't a point as this study was a shot in the dark to begin with. If I start seeing death in the experimental treatment groups then I will repeat this this experiment.

Roaches continue to incubate at 37C. I will continue to monitor their survival in the coming week.

//EWW

B. dubia LD50 P. larvae Pilot

Fed P. larvae spores to Blaptica dubia cockroaches to determine the effect. Mostly I wanted to see if it was possible to feed the dubia roaches the spore stocks diluted in the Bee Artificial Diet (BAD). 

Hand fed dubia roaches with 5 uL of P. larvae spores diluted in BAD from 7-10-14 P. larvae LC50 Pilot study. Juvenile dubia roaches were fed 5 uL of 10,000 CFU/mL and 200 CFU/mL of P. larvae spores in BAD using a P20 pipet in triplicate. Roaches were placed into petri plates and incubated at 37C. A control plate was also set up. Pictures below show me feeding the dubia roaches.

Younger roaches were attempted to be fed the spores, but did not ingest the BAD as readily as the juveniles did (or at all). I doubt I will see any death in this experiment due to the fact these were not new born roaches and P. larvae typically only affects insects with compromised immune systems such as those in the first instars. That is the case in honey bees however, who knows what will be observed in the next few days. Mostly, I wanted to see how practical or possible it is to feed the dubia roaches substances via pipet. 

It should be noted that the BAD was by no means forced down the roaches throat. In fact, the BAD disappeared from the end of my pipet tip almost as quickly as it was pushed out right next to the roach's mandibles. 

//EWW

P. larvae LC50 Pilot

Checked honey bee larva that was placed on the 5uL drop of 200 CFU/mL from 7-10-14 (24 hours ago). I viewed each second instar larva on the dissecting microscope/stereo microscope. It was surprisingly easy to identify if the larva was still alive.

If dead: will appear only to be a circulatory system (can see "veins"), dried up, or very tiny.
If alive: will appear cream colored, glistening in the scope, and will ungulate slightly if watched closely.

Unfortunately, a large number of the 50 larva were confirmed dead after 24 hours. For most of the dead larva appeared to only be a translucent bag containing a circulatory system.

There were only 8 confirmed alive larva and three questionably alive larva after 24 hours. That is between a 16-22% survival rate, which is horrible. It is incredibly likely that the large amount of death was due to transfer and the larva drying out.

Regardless, the remaining surviving larva were transferred to a new 24 well plate onto 10 uL of BAD using a camel hair paint brush. The larva was incubated in the anaerobic container at 35C. I will continue to monitor their growth, continue to feed them, and check their survival each day for eight more days.

I will have to rethink my transfer method for this experiment. Likely I will transfer the larva directly to the 24 well plate from the Jenter box at the USDA and then bring them back to NDSU to infect with P. larvae spores. Hopefully next week I will obtain more honey bee larva.

//EWW

P. larvae germination in BAD medium assay

Checked blot plates stored non-inverted in the 37C static incubator from 7-10-14. The 10 uL drops had soaked into the MYPGP agar by now and look good, however, there was development of large slimly droplets on top of the agar and not always on top of where the 10 uL drops were placed. These mysterious blobs were seen on almost all of the plates (including both BAD and H2O treatment groups). Pictures of the blobs are shown below.

This was a new batch of MYPGP agar made two days before use. They were visibly darker than those previously made. It is possible that an ingredient was or wasn't added at the correct concentration. I will double check my notes to see what amounts of each ingredient was added for this batch.

Some of the plates showed signs of dehydration or cracking of the agar (right image). This is odd since they have only been incubating for 24 hours and they weren't thinner than a usual agar plate that can be incubated for weeks before they begin to dry out. I have no idea what those mysterious blobs are from. Could it be possible that they are some sort of component of the media that pooled during incubation? Or some sort of contaminant?

The blobs are creamy in color and have an almost sticky texture, similar to the BAD that was used. However, the blobs appeared on plates from the H2O treatment, the ones that didn't even have BAD!

I isolation streaked out the mysterious blob onto LB agar and an old plate of MYPGP agar from a previous batch and incubated them at 37C. I also pipetted up some of blob (about 50 uL) and froze it at -20C just in case.

The blot plates were all transferred to the 37C 5%CO2 incubator and carefully inverted.

//EWW

P. larvae LC50 Pilot

Followed LC50 SOP from 7-2-14. Diluted Spore Stock A (3.05x10^6 CFU/mL) to create the 4 different experimental treatment groups (200, 400, 1000, and 10,000 CFU/mL) by following the dilution scheme shown in the image below. Each dilution was made in BAD as diluent. I will write up a more fluid and easy to follow procedure in the future.

Convoluted diluted scheme

It was somewhat difficult to accurately pipet the BAD at first, but once you allow enough time for the fluid to fill the pipet tip the volumes are what they should be. Tip: pipet slowly and leave the tip in the fluid longer than you would for less viscous liquids.

The honey bee queen from the "Woodchipper" hive at the USDA was placed in the Jenter box four days ago (Monday) and was freed the following day. Since then the Jenter box remained in the hive until it was brought back to the USDA today. 

Unfortunately, there weren't very many hatched larva after four days (despite literature review indicating that was when the first instar hatch). I was only able to retrieve 50 first instar larva from the USDA, less than a third of what I had hoped for. 

1        Larva were transferred from the Jenter box using camel hair paint brushed onto a petri plate                   containing a thin lawn of BAD.

2        Petri plates were wrapped in moist cloth and placed inside a Styrofoam container for transfer to               NDSU Van Es Fisher Lab.

3       Larva were then transferred from the petri plate to 24 well plates (one per well) using the same                type of paint brush (the thinner and shorter the brush bristles, the better for this experiment).

4       Because so few larva were obtained, only one treatment group was applied. 5 uL of the 200                  CFU/mL P. larvae spore-spiked BAD was added on top of each first instar larva

                    Note: 200 CFU/mL was chosen because literature review determined that was the                                         relative LC50 amount

5.     Three 24 well plates containing infected larva were incubated at 35C in the anaerobic container              (90% humidity) until tomorrow when the larva will be transferred to clean BAD on a new plate.

I am not very confident about the survival rate due to transfer this time. The petri plate containing the BAD was very dry in some spots containing the larva. In the future I will transfer the larva directly to the 24 well plates at the USDA and then transfer them to the NDSU lab. The small amount of Royal Jelly present on them that was placed there by the worker bees after hatching should be enough to sustain them long enough for transfer and prevent them from drying out.

It is disappointing there were so few hatched larva after four days. In the future I will retrieve the Jenter box early on the fifth day and select the smallest larva for my study. 

I will continue to monitor the survival of the larva.

//EWW

P. larvae germination in BAD medium assay

Repeated BAD germination experiment with several modifications.

1. Diluted Spore Stock A (3.05x10^6 CFU/mL) ten fold down to 10^-4.
           (100 uL spore stock + 900 uL ddH2O in eppendorf tubes for ten fold serial dilution.)
2. Added 100 uL of each spore stock dilution from 10^-1 to 10^-4 to 400 uL of BAD and also to 400 uL       of ddH2O in eppendorf tubes.
3. Incubated the tubes at 35C (same temperature that the honey bee larva is incubated at) shaking at                 150rpm for 2 hours.
4. Perform drop plate method to determine CFU/mL via colony counts.
          (plate five 10 uL drops onto MYPGP agar for each sample)
5. Further dilute each sample in a 10 fold dilution down 10^-2 further and repeat step 4.
          (100 uL sample + 900 uL ddH2O, repeat twice)
6. Transfer 200 uL of each original sample from both the BAD and H2O treatment groups that had been           incubating to new eppendorf tubes and heat them in a 65C water bath (Gibbs lab) for 15 minutes.
7. Repeat steps 4 and 5 using these samples.
8. Incubate all MYPGP agar plates at 37C not inverted for 24 hours.
9. Transfer plates to 37C 5%CO2 and invert them. Incubate an additional 48 hours and then perform colony     counts and compare.

If the P. larvae spores do not germinate in the BAD, I expect to see relatively the same CFU on both the heated and not heated samples in the BAD treatment compared to the H2O treatment group. If P. larvae spores are able to germinate in the BAD, then I expect to see lower CFU in the heated samples than the not heated samples in the BAD group compared to the H2O group.

Actual dilution on the plates includes the initial serial dilution from the stock (step 1) the 1:5 dilution from each treatment creation (step 2) and the 1:100 dilution from the drop plate (step 4). The samples were further diluted in step 6 as well and should also be factored in while determining CFU/mL in the future.

//EWW

P. larvae PCR Identification

Created a 1% agarose gel ( 0.5grams of agarose + 50 mL of TAE buffer) with two 10 well lanes. Loaded DNA markers and PCR amplicons from 7-8-14. 10uL of DNA sample + 2 uL of EZ Vision Loading Dye were loaded into wells. The markers that were used was Hi-Lo DNA Marker from Minnesota Molecular (Link). The base pair sizes per band of the Hi-Lo markers is shown below:

Hi-Lo DNA Marker

Gel was ran at 130 Volts for 30 minutes. The image of the gel is shown below: (it is technically a picture of a picture of a picture!)

Key: 

Top Lanes- AFB primers
1	2	3	4	5	6	7	8	9	10
Bottom Lanes- ERIC primers
1	2	3	4	5	6	7	8	9	10

Code	Sample
2	Spore Stock A
3	Spore Stock B
4	Spore Stock C
5	Plate B (7-5-14)
6	Plate C (7-5-14)
7	Drop Plate - BHI Treatment (7-3-14)
8	Drop Plate - H2O Treatment (7-3-14)
9	Drop Plate - B.A.D. Treatment (7-3-14)

Lanes 1 and 10 contained the Hi-Lo DNA Markers

Note: The gel electrophoresis rig from the teaching lab was used. There were some difficulties with setting/cooling the gel (it seemed to take quite a bit longer than it should have) and with loading (due to depth perception difficulty in using an unfamiliar rig and possibly removing the comb before the gel was completely cooled). 

The top lanes were PCR amplicons using the AFB primer sets and the bottom lanes contained amplicons using the ERIC primer sets (7-8-14). There wasn't enough space on the gel to put the negative control (DH5a). Several of the amplicons were positive in the top lanes and the band sizes seen correlate very closely to what was expected (1106bp). As mentioned, there was some difficulties with loading and cooling the gel, this was especially evident when I loaded the first few wells in the top lane. It is incredibly odd that the Spore Stock B sample (in lane 3 of the top) did not amplify with the AFB primers, but the plate that was inoculated using Spore Stock B did amplify (in lane 5 of the top)... More interestingly, that there was not P. larvae from the Drop plates in the BAD treatment group (in lane 9 of the top). This may mean that the antimicrobial capabilities of the royal jelly component of BAD killed any present bacteria.

The bottom lanes did not amplify at all in any of the samples using the ERIC set of primers. This most likely means that the PCR parameters need to be adjusted for this set specifically. Because the AFB primer set did amplify, it would be expected to have some amplification using the ERIC set as they are both identifying/genotyping designed primers for P. larvae.

Future:

I am planning on re-running this gel using the remaining amplicons as there was so much trouble with loading and cooling of the gel. It would also be worth looking into other isolated colonies of the BAD treatment group from the "P. larae germination in BAD medium assay" study.

//EWW

P. larvae germination in BAD medium assay

Checked drop plates that were diluted further on 7-5-14. Images are below from the three treatment groups. Not a lot could be concluded by these plates. The MYPGP plate had been incubating at 37C 5%CO2 for four days. The cultures that the samples were diluted from were stored in the refrigerator a few days before use, so it wasn't ideal conditions to begin with. The dilution protocol will be adjusted for future repeats of this experiment.

 

Growth could be observed at the 10^-3 dilution. I wont even calculate the actual dilution seen on the plate as this experiment will be repeated soon with more appropriate dilution schemes and stricter time points. The above MYPGP plates were discarded. This experiment will be repeated tomorrow, assuming I can find the time.

//EWW

P. larvae PCR Identification

Retrieved two primer sets for identifying and genotyping P. larvae via PCR from Integrated DNA Technologies (IDT). Sales Order #10506260.

Primer sets:

Name	Sequence	PCR-product size	Reference
AFB-F	5'-CTTGTGTTTCTTTCGGGAGACGCCA-3'	1106 bp	Dobbelaere et al., 2001 Link
AFB-R	5'-TCTTAGAGTGCCCACCTCTGCG-3'
ERIC1R	5´-ATGTAAGCTCCTGGGGATTCAC-3´	Several amplicons	Versalovic et al., 1994 (Link)
ERIC2	5´-AAGTAAGTGACTGGGGTGAGCG-3´

Primers were reconstituted by adding nuclease free sterile ddH2O relative to their nm concentration.

Reconstituting PCR Primers

Name	nm conc	Volume added
AFB-F	31	310 uL
AFB-R	23	230 uL
ERIC1R	27.3	273 uL
ERIC2	28.5	285 uL

Crude genomic DNA lystaes were created from Spore Stocks A-C, culture plates of Spore Stocks B- C, and isolated colonies from the P. larvae germination in BAD medium assay (H2O, BHI, BAD treatment groups). Total of 8 different samples. Followed Crude Genomic DNA Lysate for PCR protocol to create DNA lysate.

Set up PCR reactions of each sample using the two different primer sets. Created master mixes for each reaction for a final volume of 25uL per reaction.

Master Mix Set-up (per reaction)

21 uL of Platinum PCR SuperMix (Link)

1 uL of forward PCR primer

1 uL of reverse PCR primer

2 uL of crude genomic DNA

25uL TOTAL VOLUME

Created PCR thermocycler parameters via literature review and named the program "AFB". This AFB program will be used for both AFB-F/AFB-R and ERIC1R/ERIC2 sets of primers.

AFB Thermocycler Parameters

1. 95C for 15 minutes

2. 93C for 1 minute

3. 55C for 30 seconds

4. 72C for 1 minute

5. Repeat 2 - 4 x29

6. 72C for 5 minutes

7. 20C for infinite

PCR amplicons were stored at -20C.

//EWW