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.

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P. larvae Project Pilot

Observed growth of honey bee larva in the 24 well plates and they don't appear to have grown in size from the day before... nor have they eaten any of the 10uL of BAD. It could be they have stalled their growth, or they are simply dead. Maybe it was a lack of oxygen flow or humidity levels weren't ideal. I did not add any more BAD too the larva today and will continue to monitor their progress. I will try to find another chamber to incubate and humidify the larva instead of the anaerobic chamber.

I will try to recover more larva from the USDA hives and incubate them in Van Es in the near future as well. I need to figure out what is stalling/killing the larva in my incubator set up ASAP!


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P. larvae Project Pilot

Added 10 uL of BAD to each bee larva present on the 24 well plates. The bee larva seems to have increased in size. Survival seems to be very high as well. It is difficult to determine if the larva are dead at this stage because they are so small and don't turn a dark color.

Day 2 Larva

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P. larvae Project Pilot

Retrieved 24 first instar honey bee larva from USDA hive. Transferred each larva to 10 uL of BAD in a well of a 24 well plate (see below). Transferred using camel hair paint brush.

The well plate was put inside an AnaeroPack container (Link and image below). The larger side container was filled with 15.5% glycerol in water to keep up humidity.

Anaerobic container was placed into an Innova 4400 shaker incubator set at 35C with no shaking (Link). I will monitor survival of the larva to see if they can survive in my environment. If they can survive until pupation, then I will introduce P. larvae to the next batch of honey bee larva I receive.

Isolated another stock of P. larvae spores that had been incubating at 37C 5%CO2 on MYPGP for 10 days (Colony concentration was similar to the image seen on this Link on the top left). Followed the same modified procedure as 6-24-14 (Link). There was significantly less of a viable pellet this time as opposed to 6-24-14. Maybe there are less spores present after 10 days compared to the previous seven days of incubation. Or maybe I did not successfully remove all the cell debris and vegetative cells the previous day. Regardless, I created a 10 fold serial dilution of the spore stock created today ("Stock C") and the two spore stocks created on 6-24-14 ("Stocks A and B"). Diluted down to 10-7 in sterile ddH2O. Plated 100 uL of each dilution onto MYPGP agar and incubated at 37C 5%CO2. Will perform colony counts to determine the concentration of my spore stocks.

Made 1 Liter of MYPGP for colony counts (Recipe). Note: did not add the 10% Glucose as per the recipe due to its temporary unavailability. Stored around 20 plates in the 4C walk in fridge.

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P. larvae Project Pilot

Performed P. larvae spore isolation and purification. I did not exactly follow the protocol from 6-24-14 (Link), I made several modifications discussed below. Used a P. larvae plate inoculated on 6-17-14 onto MYPGP (picture seen Link, top left picture). The plate had been incubating at 37C 5%CO2 for 7 days. This experiment was mostly to familiarize myself with the process.

Modified Paenibacillus larvae Spore Extraction from Agar

11. Wash the surface of the agar with 5 mL of sterile ddH₂O. Use a sterile spreader to gently rub off the colonies into suspension. Pipet off and collect the 5 mL of sterile ddH₂O.
12. Repeat the wash step two more time and combine all the wash volumes in a 50 mL conical tube (total of 15 mL)
13. Split the 15 mL volume into 15 1mL aliquots in 1.5 eppendorf tubes
14. Centrifuge the eppendorf tubes at 16,000 x g, 1 minute, at 4°C.
15. Carefully pipet off the supernatant. Very carefully attempt to remove the "white fluffy" precipitate that is covering the darker and more compact pellet.
16. Suspend the pellet in 1 mL of cold sterile ddH₂O.
17. Repeat steps 5-7 five additional times.Gradually consolidate the number of eppendorf tubes to just two (ie transfer the suspended pellets to another tube containing a pellet and suspend that one as well).
18. Suspend the pellet in 1 mL of cold sterile ddH₂O.
19. Heat eppendorf tubes at 65°C for 15 minutes in water bath. (Heating will kill vegetative cells).
20. Repeat wash steps 5-7 five times.
21. Suspend pellet in 500 uL of sterile ddH2O.
22. Store at 4°C.

I will eventually perform a serial dilution of the spores I've collected and plate them on MYPGP agar to perform colony counts. I need to get an accurate appraisal of how many spores are present in order to use them to calculate the lethal concentration (LC50) in honey bee larva.

Quick microscopic examination after step 1 revealed very low numbers of spores. In the future I will incubate plates for 10 days to allow for an increased spore production. I will also harvest from plates with nearly a lawn of bacteria. In the near future I will perform another microscopic examination of my purified spores to gauge their concentration.

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P. larvae Project Pilot

Checked plates containing P. larvae. Will be ready to attempt to harvest spores tomorrow if I have enough time in between my bee rearing duties and I am able to procure adequate equipment.

          

P. larvae on MYPGP                               P. larvae on Chocolate Agar

Below are images of P. larvae that was plated onto MYPGP agar. Cultures were diluted in sterile ddH2O from broth culture of P. larvae. Note: 100 uL broth culture + 900 uL ddH20 = 1:10 dilution, plate 100 uL of that onto agar + 1:100 dilution.

       

P. larvae diluted 1:100 on MYPGP          P. larvae diluted 1:1000 on MYPGP 

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Paenibacillus larvae Spore Extraction from Agar

Paenibacillus larvae Spore Extraction from Agar

Fisher Lab. 2014.

Materials:

Broth culture of P. larvae	MYPGP Agar	37°C with 5% CO2
P200 pipet	P200 sterile pipet tips	65°C water bath
Pipettor	5 & 50 mL sterile pipets	Refrigerated centrifuge
Cold sterile ddH2O	50 mL conical tube	4°C refrigerator
Sterile spreaders

1. Create a 1:10 dilution from a two day broth culture of P. larvae in sterile ddH₂O. Plate 100 µL of diluted culture onto MYPGP agar using spreader.
2. Incubate plate at 37°C with 5% CO₂ for 6-7 days. (Plates with 50 to 5000 colonies is ideal)
3. Wash the surface of the agar with 5 mL of sterile ddH₂O. Use a sterile spreader to gently rub the surface of the agar to loosen the spores from the surface. Pipet off and collect the 5 mL of sterile ddH₂O.
4. Repeat the wash step two more time and combine all the wash volumes in a 50 mL conical tube (total of 15 mL)
5. Centrifuge the wash volume at 12,000 x g, 15 minutes, at 4°C.
6. Discard the supernatant.
7. Suspend the pellet in 30 mL of cold sterile ddH₂O.
8. Repeat steps 5-7 four additional times.
9. Suspend the pellet in 5 mL of cold sterile ddH₂O.
10. Store at 4°C.

Quantify Spore Concentration

11. Heat spore stock at 65°C for 15 minutes in water bath. (Heating will kill vegetative cells).
12. Serial dilute a portion of spore stock in sterile ddH₂O.
13. Plate 100 µL of each dilution onto MYPGP agar. Incubate at 37°C with 5% CO₂ for 6-7 days.

Optional: perform direct microscopic counting of spore stock. This will result in an overestimation – heat resistant spore counts are ~6% of direct microscopic counts.

Protocol adapted from de Graaf, et al. 2012. Standard methods for American foulbrood research. International Bee Research Association. Journal of Apicultural Research. DOI 10.3896/IBRA.1.52.1.11.

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P. larvae Project Pilot

P. larvae on MYPGP agar seems to be growing fine. Will be a few more days yet before I can attempt to isolate spores. Retrieved another plate with fungal contaminants on the growing honey bee larva (see below). Attempted to isolate and streak out white fungal species onto Nutrient agar. Incubated plate at 35C (same temperature that it was discovered at).

Fungal contaminant on 5th instar honey bees. White fluffy fungal mold completely engulfed a larva.

Checked plates of contaminants from yesterday (see below). Appears that near-pure fungal colonies are growing. I will continue to incubate at 35C. 

Streak plates of the fungal contaminants that were killing the honey bee larva from 6-21-14. 

Absolutely no growth on any of the BAD (Bee Artificial Diet) plates from 6-20-14 at any of the 25C, 35C, 37C temperatures. I will continue to monitor for growth, however, I am fairy confident that the diet that is being fed to the honey bees is free of contaminants, at least the contaminants that would be detected in the parameters I've been looking at.

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P. larvae Project Pilot

Diluted P. larvae 1:10 and 1:100 in sterile ddH2O. Plated 100 uL of each dilution onto MYPGP agar. Incubate 37C 5% CO2.

Laura N. had a few extra motility agar plates left over from her Steno motility assay. Inoculated a single isolated colony of P. larvae into the center of motility agar. Incubated at 35C, 37C, and 25C.

Having a issue with mold/fungal growth in the pupating larva stage of the in vitro bee rearing project (see below). Larva is beginning to succumb to fungal growth and dying off in significant quantities. Filter papers used for humidity/moisture content was likely too saturated. Will remedy in future. Attempted to isolate fungal growth- incubated at 35C on Nutrient Agar.

(Contaminated Honey Bee larva)

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P. larvae Project Pilot

Made freezer stocks of overnight broth culture of P. larvae grown in BHI+Thiamine:

           800 uL broth culture + 200 uL 80% glycerol    Stored in -20C in Room 114

Plated 100 uL of overnight broth culture onto MYPGP agar in triplicate. Incubated at 37C 5% CO2. Will use these plates for spores in 6-7 days if they aren't overcrowded with growth. I will determine how much growth is present in 2 days and then potentially dilute out the broth culture and plate that. Literature review indicates that too many colonies on a single plate can actually decrease sporulation.

Performed Gram stain of broth culture. Heat fixed a loop full of broth culture onto slide.
           1. Crystal Violet ~1 minute
           2. Iodine ~30 minutes
           3. Alcohol <10 seconds
           4. Safranin ~30 seconds
Gram stain revealed Gram Positive (almost blue in color) rods.

Helped Laura N. make motility agar (Tryptone + Agar + NaCl) today as well. She may have extra plates for which I can gauge the motility of P. larvae.

Brought a small sample of the Bee Artificial Diet (BAD) that we have been feeding the bees at the USDA. Plated 100 uL of BAD onto Nutrient Agar and incubated at 37C, 35C, and 25C. Stored remaining BAD sample at 4C.

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P. larvae Project Pilot

Growth observed on MYPGP and Chocolate Agar. More growth on Chocolate agar observed.

Brothed an isolated colony of P. larvae from chocolate agar into 10 ml of BHI broth with 1ug/ml of Thiamine. Incubated at 37C 5% CO2 concentration. Will make freezer stocks tomorrow. Will also perform Gram stain to confirm Gram positive is present.

Continued to incubate P. larvae plates until sporulation occurs in another ~4 days.

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P. larvae Project Pilot

The overall goal of this experiment is to determine LD50 for P. larvae at the first and second instar in honey bee larva. This will likely require around 100 bee larva to accomplish. We have a number of other possible directions to head with this project, but the LD50s will be the start. Right now, I am focusing on culturing, creating freezer stocks, and isolating spores from P. larvae. As only the spores are invective to the bees, that is what I'll need.

Paenibacillus larvae arrived in the mail today from Microbiologics. ATCC number 9545

Struck out onto Chocolate Agar and MYPGP Agar and incubated at 37C 5% CO2. P. larvae grows very well in bee hemolymph, but is otherwise difficult to grow in media. Growth on MYPGP seemed to result in higher sporulation, per literature review.

Agar information can be located at the Coloss Honey Bee Research Association (Link).
"

 MYPGP agar (per litre):

• 10 g Mueller-Hinton broth (Oxoid CM0405)
• 15 g yeast extract
• 3 g K₂HPO₄
• 1 g Na-pyruvate
• 20 g agar
•  Autoclave at 121°C / 15 min.
•  Add 20 ml 10 % glucose (autoclaved separately).

"

Incubation of the P. larvae plates will likely take 6-7 days before spore collection. 

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Transferring pBE110 to S17-1

Last week attempted to transfer the purified pBE110 plasmid into E. coli S17-1 and E. coli DH5a through electroporation. Made electrocompetent E. coli cells (per May 21, 2014 method) and transformed the pBE110 DNA into it. Let incubate at 37C for 1 hour then plated 200 ul onto LBTet15 and incubated overnight.

Results: No colonies from pBE110 formed on DH5a plates. Colonies formed on pUC19 positive control for the S17-1, but not the DH5a. Had some colonies form on S17-1 pBE110 transformants (see picture below).

                                                         
                   Wild type S17-1 on LB only                            S17-1 pBE110 transformants

Colonies do not look like E. coli. Most likely contaminates. Brothed isolated colony of transformants into LBTet15 broth. Incubated at 37C LBTet15 for 48 hours.

After 48 hours nothing grew in the broth. How unfortunate.

Future plans with this approach may need to be re-evaluated.

I haven't been able to update my online lab notebook as much lately as I have been working on a P. larvae project at the ARS USDA building a lot. I will likely not be posting any of the collaboration data that I have been doing over there at the federal building, but will report online about my work with the bacteria.


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RNA Extraction

DeeptiT. of the T.Bergholz lab performed the DNase treatment to the extracted RNA and then quantified each on the nanodrop.

Isolate	260	280	260/280	260/230	ng/ml
B24	47.2	24	1.99	2.22	1887.1
B84	58	29.4	1.97	2.19	2320.2

We decided not to examine the RNA samples on the Bioanalyzer to check if the RNA was indeed good quality. It was determined that this RNA Extraction procedure does indeed work well for S. maltohpilia and will be included in a possible future methods publication. We currently have no use for RNA from S. maltophilia, but it is still useful to know of a method that works for its extraction that doesn't cost a lot of money (as opposed to using a kit) should we ever be in need of a large quantity of RNA.

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