P. larvae Project Pilot

Added 20 uL of BAD on all honey bee larva growing on the 24 well plate. They appear to have grown in size and some have even consumed all the food present in their wells. It would appear that the larva isn't dead after all and is no longer stalled in growth (if it ever was). 

Acquired 24 more honey bee larva/eggs from the USDA hives. I am not quite sure if they are eggs or first instar larva, nor if they are still viable as they were from a honey comb stored in their incubator for a few days. The honey comb was going to be thrown away, so I just grabbed a few more honey bee samples. The 24 larva/eggs were placed on/near 10 uL BAD in a 96 well plate.

All honey bee larva was transferred from the Innova 4400 shaker incubator into the Yamato IC600 (same as seen here: Link). The reason for this transfer was because it was unnecessary to incubate the larva in a shaker incubator when not needing to use the shaking capabilities and because the shaking incubator was not protected from the light and the effect of light on honey bee larva has not yet been fully explored. Now the larva will incubate in the dark in a static incubator at 35C. Images of new incubator seen below:

New incubator for honey bee larva (35C)

New incubator for honey bee larva inside. Note: anaerobic chambers placed inside.

Checked P. larvae colony forming plates that were inoculated on 6-27-14. They have been incubating for three days and colony counts were attempted today. Not all the plates looked very good. I was able to get colony counts from Stocks A and B, but not Stock C. 

CFU plates from top to bottom Stocks A-C

Plates 10^-3 colony counts can and were performed for Stocks A and B. However, this was not possible for Stock C as there was a lawn like bacterial growth...

Stock C 10^-3 dilution. Unable to perform colony counts.

This bacterial lawn like phenomenon was not limited only to Stock C. It was observed in several other dilution plates of Stock A and B as well. Seen below are two dilution plates of Stock B where the lawn of bacteria is observed. I am not quite sure what caused this. It is highly possible that the bacteria seen on these lawn plates are not P. larvae and that it is a contaminant. 

 

Colony counts were performed on spore Stocks A and B regardless. Images below:

Spore Stock A 10^-3 dilution

Spore Stock B dilution 10^-3

Spore Stock A = 1448 colonies on 10^-3 dilution plate

Spore Stock B = 1248 colonies on 10^-3 dilution plate

Determining CFU/mL from colony count formula:

[# colonies counted / volume pipetted onto plate in uL] * [ 1 / serial dilution made (ie 10^-2)] * [1000 uL / 1 mL] = CFU/mL

Meaning:

Spore Stock A = 1448 x 10 ^3 CFU/mL = 1.448 10^6 

Spore Stock B = 1248 x 10^3 CFU/mL = 1.248 x 10^6

Obviously, replicates would need to be performed to confirm this concentration.

Performed spot plates to quantify colony forming units as per Link to confirm what was seen in Spore Stocks A and B. And also to determine the concentration of Spore Stock C. Plated five 10 uL drops of the 10^-1 to 10^-4 ten fold dilution series created on 6-27-14 that have since been stored at 4C. Drop plates were created of each of the three spore stocks and incubated at 37C not inverted.

Example: Drop plate of Spore Stock A.

Special note: I went out to the USDA honey bee hives again this morning to put in the Jenter Boxes and I was stung in the left middle finger through the glove. This marks the fourth time I have been stung. First time was in the right ear, second on the right cheek the same day, and third on the left side of my neck.

//EWW