Wax Worm/P. larvae LD50

Goal: Create and LD50 curve of P. larvae spores using early instar wax worm larvae. This information will be important in determining if wax worms can be used as surrogates to the honey bee larvae. This information will also be used in a future grant proposal. 

The P. larvae spore stock G (1-31-15) that has since been stored at 4C in ddH2O was serially diluted 1:5 in Bee Artificial Diet (BAD) made last summer. The spores were diluted down to 1:3125. 

To set up the inoculation, the wax worms will be attempted to be fed the spores through the BAD. For this, small plastic containers (seen above) with covers will be used. About 150 mg of wax pellets were added to the center of each container using a metal scoopula. A volume of 100 uL of the diluted spores in BAD was added to the top of the wax pellets and mixed together using a sterile toothpick (picture seen below).

Mixing in the BAD/spores with a tooth pick

A total of 10 wax worms were added to each container. There were duplicate containers made of each spore dilution (2 biological replicates with 10 technical replicates each). Negative controls included a "no diet", where the wax worms were placed only on the wax pellets, and a "no bacteria", where the wax worms were introduced only to the BAD with no spores. Bacillus thuringiensis spores (a known insect pathogen) was used as a positive control (diluted 1:5 and 1:25).

10 WW to a single container

The containers were placed inside a tupperware container and placed in the walk in 30C incubator (where the wax worms were originally taken from). I will check the survival of each of the containers over the course of a week and hopefully be able to put together an spore ingestion LD50 if this is indeed a viable method of determining.


//EWW

Chlorine Dioxide Pilot Study

From 2-24-15.

I was able to count the Bacillus thuringiensis colonies that grew on the dilution plates overnight. The actual spore counts are stored on an Excel file. I did not add it to this post since there was formatting issues and it was very difficult to read.

Equation used to calculate concentration of Spore Stocks:

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

Results:

	Concentration of ClO2
	None	25 mg	50 mg	100 mg
CFU/mL	353333	24000	10666	3266

A one to two log reduction in spores was observed using the three low concentrations of ClO2 gas reagents. The set up used in this experiment (50 mL conical tube) was not ideal for ensuring that the two reagents are mixed thoroughly, but the main purpose of this pilot study was to better familiarize myself with the spore handling and quantification methods- which I now feel comfortable enough with to begin using the modified anaerobic chambers and different surfaces other than glass cover slips... Just as soon as I increase my P. larvae spore yields.

//EWW

Detection of P. larvae in Local Honey

From 2-21-15.

I observed growth in dd1-8a, dd5-Q, dd8-63a, and the roach trachea isolates that were inoculated in LB broth, however limited or no growth was observed with the dd11-2a and dd12-22a isolates, This is very interesting as they both grew in BHI previously.

In order to further investigate this phenomenon, I inoculated an isolate colony of each into 3 mL of TSB broth and incubated the tubes at 37C 225 rpm. 

//EWW

Chlorine Dioxide Pilot Study

From 2-21-15.

Performed chlorine dioxide gas exposure to the Bacillus thuringiensis spores on the glass cover slips in the 50 mL conical tubes as performed previously (1-31-14).

Three different chlorine dioxide concentrations were weighed out using the analytical balance in the Pruess Lab for a combined mass of: 25 mg, 50 mg, and 100 mg. The two components to generate the gas, Part A and Part B, are combined in a 1:1 ratio in the 50 mL tube to generate the gas. So, in order to get the combined mass, a 12.5 mg, 25 mg, and 50 mg masses were weighed out for each of the two components.

Parts A and B were combined in the 50 mL conical tubes, the two mix at the bottom. Quickly after the two are combined, the glass cover slips containing the dried spores were aseptically added to the 50 mL tube. Due to the size of the cover slip and the narrowing of the tube the glass never comes in direct contact with the two components at the bottom. After the addition of the cover slip, the tube is closed and sealed with Parafilm.

The tubes were transferred to the small shaking incubator protected from light. The tubes were gently rocked using the shaker, but no additional heat was applied. The tubes were incubated at room temperature, gently rocking, and protected from light for six hours.

After six hours, the glass cover slips were removed from the 50 mL conical tubes inside a fume hood and transferred to a new 50 mL tube containing 1.0 mL of sterile ddH2O. During this aseptic transfer, the glass cover slips were all broken using the forceps. This process will allow the glass containing the dried spores to come in better contact with the water and promote re-suspension. The tubes containing the broken glass were vortexed for 10 seconds and allowed to incubate at room temperature for 5 minutes.

Broken glass cover slips in 1 mL H2O inside a 50 mL tube

The re-suspended contents of each tube was diluted 1:10 in sterile ddH2O down to 10^-8. 10 uL volumes of each dilution was plated onto LB only agar in five replicates. The plates were incubated at 37C overnight.

//EWW

Chlorine Dioxide Pilot Study

From 2-3-15.

Goal: Perform chlorine dioxide pilot study using the 50 mL conical tubes as before except I will be using Bacillus subtilis and Bacillus thuringiensis spores that I have recently extracted using the heating/washing procedure.

Sterile glass cover slips were aseptically transferred to petri plates in the fume hood and a volume of 25 uL of spores from either Bacillus spp. were added to the center of the glass side. The slides were left in the fume hood for 30 minutes or until the spores had evaporated onto the slides.

//EWW

Detection of P. larvae in Local Honey

From 2-7-15.

I am having some difficulty getting the honey isolates dd1-8a, dd5-Q, dd8-63a, dd11-2a,dd12-22a, and the roach trachea isolate to grow from -80C. They grew on MYPGP agar at 37C without an issue, however they are not growing in broth media.

For the second time, the honey isolates did not grow in BHI broth media. Isolated colonies were inoculated into 10 mL of BHI broth media and incubated at 37C shaking at 225 rpm. Even after three days there was no visible growth observed. I have been able to get growth performing this method in the past with these isolates, however that was with BHI broth media I had prepared myself. It is possible that the BHI media I am currently using is from a different source and results in no growth of my isolates.

Regardless, I have inoculated isolated colonies of each of the isolates into 10 mL of LB broth media and incubated them at 37C shaking 225 rpm. If I do not observe growth after 24 hours I will begin to suspect there is an issue with the bacteria themselves and perhaps not an issue with the media.

//EWW

Wax Worm Maintenance

From 1-3-15.
From 7-22-14.

Eggs from the two wax worm containers seen on 1-3-15 has since been split into numerous other containers with food in order to increase the size of the colony for future use. Those original two containers have had their eggs transferred for the most part and are both ready to be frozen down to kill any remaining eggs. However, some of the few remaining eggs have recently hatched and are visible in the containers. So, rather than simply freezing the containers, I will perform a crude preliminary experiment on them.

Goal: Determine if the addition of only corn syrup or only honey to the newly hatched wax worms will be enough nutrients to progress them to adulthood.

Two new containers had either ~50 mL of corn syrup or ~50 mL of honey added to the bottoms of them (pictures seen below).

The early instar wax worms along with the debris from the original two containers were transferred to the corn syrup and honey containers (pictures seen below).

The containers were transferred to the 30C walk in incubator with the rest of the colony. None of the earlier transferred eggs appear to have hatched yet and will continued to be monitored. Once a suitable amount of wax worms have hatched and grown they will be used in a LD50 study using P. larvae spores.

//EWW

P. larvae Biofilms

From 11-2-14.

Goal: Determine if P. larvae is capable of forming biofilms. We will begin by investigating whether or not it can form substantial biofilms on a 96 well polystyrene plate.

I will be inoculating P. larvae ATCC 9545 and E. coli B/r in a 10-fold serial dilution of a 96 well plate in BHI broth. Overnight broth cultures of the two bacteria will be used. Below are two images of the P. larvae plates for a visual reference of what it should look like on MYPGP agar.

P. larvae 9545 on MYPGP agar grown at 37C for 3 days

P. larvae 9545 on MYPGP agar grown at 37C for 3 days

Isolated colonies of P. larvae and E. coli were inoculated into 10 mL of appropriate liquid media.

P. larvae - BHI + Poly60

E. coli B/r - LB only

Plate set up, Rows A-D are P. larvae, Rows E - H are E. coli

A 10-fold serial dilution was made in quadruplicate of each culture on the 96 well plate down to 10^-7. The plate was covered and incubated at 37C. A crude crystal violet assay will be employed to gauge biofilm formation.

//EWW

Listeria & Cockroaches

I injected 20 juvenile Dubia cockroaches with 10 uL of a GFP-tagged Listeria broth culture for Dr. Fisher.

I received a broth culture of a Listeria spp. from the Teresa Bergholz lab with the label 881 TB 351 on it. I was told this was a GFP-tagged Listeria strain. I pelleted 2 mL of the broth culture and re-suspended it in 2 mL of PBS.

A 100 uL syringe and needle was used to inject 10 uL of the re-suspended Listeria culture into the bottom side of 20 dubia roaches. Injects were at the second or third tegumen from the anus of the roach. Injects were as shallow (close to the surface) as they could be.

Two containers with 10 injected roaches each were place in the 37C incubator. A BHI plate with 10 ug/mL ERT was also streaked with the Listeria and incubated at 37C as well.

//EWW

Detection of P. larvae in Local Honey

From 1-31-15.

The P. larvae isolates that were isolated from local honey sources were transferred from the -20C freezer to the -80C freezer into the Fisher/Doetkott referrence strain box number 4.5. The isolates dd1-8a, dd5-Q, dd8-63a, dd11-2a,dd12-22a, and the roach trachea isolates were also stored in this box in the -80C freezer. Most of these isolates are not in duplicate or triplicate, but I plan to make triplicates of dd1-8a, dd5-Q, dd8-63a, dd11-2a, and dd12-22a once the cultures grow in the 37C shaker incubator. 

The -80C freezer is pretty unorganized, perhaps acquiring a freezer box rack of some kind would help to organize all the Fisher lab boxes. I will try to locate of these racks.

//EWW

Chlorine Dioxide Pilot Study

From 1-31-15.

Counted colonies that had formed on my MYPGP agar plates from the recent chlorine dioxide exposure study. Colony counts are listed below:

		Dilutions & CFU Counts
Spore Stock	ClO2 Reagents	10^-0	10^-0	10^-0	1:2	1:2	1:2
H	None	164	152	188	80	68	62
		155	148	194	66	76	84
		190	168	156	78	72	84
		136	178	168	88	62	58
		168	166	162	74	58	66
	Avg:	162.6	162.4	173.6	77.2	67.2	70.8
	Std:	19.67	12.28	16.58	8.07	7.29	12.38
	Avg of all:			166.20			71.73
	Std of all:			16.17			9.79
H	10mg	29	34	41	14	12	9
		27	29	38	14	13	16
		23	26	45	17	9	12
		34	25	37	13	15	15
		28	32	44	15	12	12
	Avg:	28.2	29.2	41	14.6	12.2	12.8
	Std:	3.96	3.83	3.54	1.52	2.17	2.77
	Avg of all:			32.80			13.20
	Std of all:			6.96			2.31
H	100 mg	No colonies formed			No colonies formed
		Dilutions & CFU Counts
Spore Stock	ClO2 Reagents	10^-0	10^-0	10^-0	1:2	1:2	1:2
I	None	400	333	386	188	234	244
		414	376	376	222	200	220
		398	440	364	228	192	190
		380	434	358	244	182	188
		422	422	388	222	208	188
	Avg:	402.80	401.00	374.40	220.80	203.20	206.00
	Std:	16.16	45.55	13.22	20.43	19.73	25.22
	Avg of all:			392.73			210.00
	Std of all:			29.97			21.82
I	10 mg	16	12	10	7	8	7
		12	16	17	4	8	5
		11	15	13	5	9	10
		19	20	13	7	6	11
		20	21	16	8	6	9
	Avg:	15.60	16.80	13.80	6.20	7.40	8.40
	Std:	4.04	3.70	2.77	1.64	1.34	2.41
	Avg of all:			15.40			7.33
	Std of all:			3.52			1.95
I	100 mg	No colonies formed			No colonies formed

Calculating CFU:
Spore Stock H = 84,000 CFU/mL (8.4x10^4)
Spore Stock I = 32,000 CFU/mL (3.2x10^4)
---------------------------> pelleted & re-suspended in 500 uL ddH2O
Spore Stock H = 1.68x10^5 CFU/mL
Spore Stock I = 6.4x10^4 CFU/mL
---------------------------> 50 uL of spore stocks added to glass cover slip
Cover slips exposed to ClO2 gas
---------------------------> re-suspend cover slip spores in 1000 uL of ddH2O
---------------------------> 10 uL volumes plated onto MYPGP agar

Equation used to calculate concentration of Spore Stocks:

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

For Spore Stock H:
           32.8/166.2 CFU x 100% = 19.7% survival of spores
                        Meaning that the 10 mg concentration of ClO2 reagent was ~80% effective at killing   the spores

For Spore Stock I
         15.4/392.73 CFU x 100% = 3.92% survival of spores
                     Meaning that the 10 mg concentration of ClO2 reagent was ~96% effective at killing the spores

Unfortunately, my original spores stocks were at a relatively low concentration to begin with (around 10^4 CFU), and this limited what we could see at the 100 mg concentration since we could only see about a 3 log killing rate and nothing more. Realistically, the spore stocks should be around 10^8 or 10^9, so I will have to work on improving my ability to recover and purify P. larvae spores.


/EWW