P. larvae Project Pilot

Checked on how honey bee larva were progressing after they are no longer being fed BAD (images below). A number of larva appear to have completed their defecation stage and begun to straighten out. They will begin to pupate soon.

Only retrieved 22 honey bee larva from the USDA on 6-27-14. This means they have been growing for 8 days and on the 9th day is typically when pupation begins. Today, 18 of the larva appear to be in good health still and are growing at a progressive state. There were 5 dead larva that seemed to have stalled in growth practically right after they were brought to the NDSU lab. The dead did not progress to the 2nd or 3rd instar life stage. There death is likely due to handling when they were placed onto the 24 well plates.

Rate of survival:
With the initial deaths due to handling/transfer: 81.8%
Without including those deaths the survival is 100%, so far.

Typically, from what I've observed after rearing the larva at the USDA, the majority of larval death comes from during the initial transfer due to rough handling and after the pupation stage begins (likely due to the absence of BAD containing royal jelly).

I will continue to monitor the larva growth as they begin to pupate.

Plated 100 uL of the 10^-1 Spore Stocks B and C on MYPGP agar. Incubated inverted in the static 37C no CO2.

//EWW

P. larvae germination in BAD medium assay

The purpose of this experiment is to determine if P. larvae is able to germinate in the Bee Artificial Diet (BAD).

Used the ten fold serially diluted Spore Stock A that was created on 6-27-14 that had since been stored at 4C. Added 100 uL of the 10^-1 to 400 uL of BAD in eppendorf tubes. Repeated for dilutions 10^-2 to 10^-4.
For controls, added 100 uL of each dilution to 400 uL of BHI+thiamine and 100 uL of each dilution to 400 uL of H2O.

Incubated all samples at 35C shaking at 150 rpm for 2.5 hours.

Performed drop plate procedure per Link for each dilution under each treatment onto MYPGP agar. (note: 10 uL per drop, repeated five times). Incubated plates not inverted at 35C 5% CO2. Remaining samples were stored at 4C.

I am not sure how many colonies I will actually be able to identify in the BAD treatment group, as the media is fairly cream colored and slightly viscous.

Perhaps I should have made dilutions from each sample, but since they were already diluted before being diluted even further with different treatments and only incubated for 2.5 hours, it may not be necessary...

//EWW

P. larvae Project Pilot

Added 50 uL of BAD to all honey bee larva in the 24 well plates. This is the last time I will feed them before they begin to pupate. After the bee larva has finished their 6th instar feeding life stage they will have a large defecation stage and then begin to pupate, creating a very thin cocoon. The bee larva will be left in the 24 well plates and incubated at 35C during the entirety of their pupation stage. These larva have been incubating since June. Image of what they look like in the 24 well plates is shown below:

The larva will begin to straighten out once all the food is consumed, defecate, then begin to pupate into honey bees.

Attempted to look at Spore Stocks under confocal light microscopy, but didn't see many spores. There was a substantial amount of cellular debris seen in Spore Stock A and relatively few spores.
I need to find a microscope with phase contrast capabilities, but I don't think any microscope in Van Es has that feature.

Inoculated 100 uL of the 10^-1 dilution of Spore Stocks B and C onto MYPGP agar. Plates were incubated at 35C 5%CO2 not inverted. I will attempt to isolate spore stocks from these samples 6-7 days from now.

//EWW

P. larvae germination in BAD medium assay SOP

Below is the yet tentative protocol :

Germination Determination of P. larvae in Bee Artificial Diet

This procedure is to determine if P. larvae is able to germinate in the Bee Artificial Diet (BAD) before it is fed to the larval honey bees.

1. Perform P. larvae Spore Extraction protocol.
2.  Perform 10 fold serial dilution of heat killed Spore Stock.
3. Inoculate 100 uL of each dilution into 400 uL of BAD in Eppendorf tubes.

Inoculate 100 uL in 400 uL of sterile ddH2O and 400 uL of BHI broth with 1ug/ml of Thiamine as negative and positive controls, respectively.

4. Incubate at 35C for 2 hours.
5. Perform 10 fold serial dilution of each sample and perform drop plat protocol to quantify CFU/mL.

//EWW

P. larvae LC50 SOP

Below is the tentative protocol for determining the LC50 of P. larva during the first instar of honey bee larva.

Lethal Concentration (LC50) Assay of P. larvae in A. mellifera (Honey Bee) Larva

Protocol to infect 1^st instar larva with P. larvae

At USDA

1. Trap queen honey bee inside Jenter box. Place box inside bee hive and leave queen trapped for 24 hours. (Monday)
2. Remove cap cover of Jenter box so that the queen can escape. Place box back into bee hive. Leave box inside hive for 72 more hours. (Tuesday)
3. Remove Jenter box from hive and transfer to USDA building covered by a moist cloth inside a Styrofoam container. (Friday)
4. Carefully transfer honey bee larva from the Jenter box to 10 uL of Bee Artificial Diet (BAD) in 24 cell culture plates using a camel hair paint brush. Transfer one larva to one 10 uL puddle of BAD in one of each of the wells.

Note: Try to select only the smallest larva for 1^st instar development stage.

5. Transfer bee larva in a Styrofoam container to the Fisher lab in Van Es at NDSU.

At NDSU

6. Dilute small portion of BAD with different concentrations of P. larvae spores from Spore Stocks stored in 4C to create the following five treatment groups:

Treatments 200 CFU/mL 400 CFU/mL 1000 CFU/mL 10,000 CFU/mL No CFU/mL (control)

7. Pipette 5 uL of each treatment group into 24 well plates and carefully transfer bee larva from Step 5 to each of the newly created BAD-P. larvae puddles (one larva per 5 uL). Incubate plate at 35C for 24 hours.

Note: Ideally, n = 100 for each treatment group.

8. Check survival of each bee larva using the dissecting microscope. Live larva will slightly ungulate and appear to be a light white/cream color. If dead- remove from experiment, record weight, and free at -20 for future use. If alive – move to Step 9.
9. Transfer experimental treatment group larva to a new 24 well plate containing 10 uL of un-inoculated BAD. Add 10 uL to control larva. Incubate at 35C for 24 hours.
10. Continue to check survival for a total of nine days. Continue to provide larva with increasing amounts of BAD as described in the table below:

Day	BAD provided
1	5 uL
2	10 uL
3	20 uL
4	30 uL
5	40 uL
6	50 uL
After 6 days, cease BAD

//EWW

P. larvae Project Pilot

Added 40 uL of BAD to all honey bee larva in the 24 well plates. It appears that the majority of larva had consumed most if not all the present BAD. Added no additional BAD to the eggs/larva that was in the 96 well plates (they do not look like they are doing well still). All larva is still being incubated at 35C. Two pictures of larva growing in the 24 well plates is shown below:

Larva is growing at a steady pace (size is increasing). 

Checked P. larvae colony forming plates that were inoculated on 6-27-14. Plate counts reveal relatively no additional CFU on any dilution. Lawns of bacteria are not present on any other dilution plates that weren't presently seen. They have been incubating for five days now at 37C 5%CO2. I was able to get colony counts from Stocks A on the 10^-5 plate = there were 32 colonies present. Picture of the serial dilutions are seen below. The plates were discarded today.

This means there are 32x10^5 CFU/mL or 3.2x10^6 CFU/mL. This concurs with the 1.448 10^6 CFU/mL determined from the 10^-4 plate on 6-30-14. 

Spot/Drop plates created on  6-30-14 we observed and colony counts were performed. Spore Stocks A, B, and C are shown below. 10 uL of the 10^-1 to 10^-4 serial dilution were inoculated onto MYPGP agar. They have been incubating for two days. 

The colonies were difficult to count the exact amount. Not all dilution colonies were counted, only those that had identifiable single colonies that could be attempted to be counted (~10 - 40 colonies). Again, each dilution was plated in five replicates. The relative CFU counted, average of the five replicates, and standard deviations are listed below for each of the three Spore Stocks A-C.

Spore Stock	Replicate CFU	Average	Std
A (from 10^-3)	22	22.6	4.219004622
	18
	20
	24
	29
B (from 10^-3)	20	21.2	5.357238094
	29
	16
	17
	24
C (from 10^-2)	70	68	8.366600265
	60
	70
	80
	60

The calculated CFU/mL as per the formula laid out on 6-30-14 is displayed below.

Spore Stock	CFU/mL
A	2.26x10^6
B	2.12x10^6
C	6.8x10^5

These calculated CFU/mL roughly concur with what was calculated for Spore Stocks A and B. 

//EWW

P. larvae Project Pilot

Added 30 uL of BAD to all honey bee larva in the 24 well plates. It appears that the majority of larva had consumed most if not all the present BAD. Added 10 uL to the eggs/larva that was in the 96 well plates (they do not look like they are doing well). Two pictures of larva growing in the 24 well plates is shown below:

Moved the spot plates from 6-30-14 from the static 37C incubator to the 37C 5%CO2 incubator and inverted them. The P. larvae grows better with the CO2 concentration.

Began to isolate spores from 1:100 dilution of P. larvae on MYPGP plate from 6-21-14 (10 days of incubating at 37C 5%CO2), but stopped after the first centrifugation. Barely any pellet was seen (if any) and little culture was recovered from the agar plate. This may be due to the fact the plate had been incubated for 10 days. It is recommended to only incubate the plates 6-7 and then recovered spores as that is peak spore production. Discarded the sample. I will only attempt to harvest spores after the 6-7 days of incubation in the future.

//EWW