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.

//EWW

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)

//EWW

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.

//EWW

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.

//EWW

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. 

//EWW

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.


//EWW

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.

//EWW