P. larvae growth & Biofilm Pilot Study

Goal: Perform a rudimentary growth curve of P. larvae 9545 compared to several other bacteria strains. Also, determine the ability of P. larvae to form biofilms relative to closely related species. The growth curve will be performed using the continual plate reader located in the Pruess lab, and biofilm quantification will be performed using the crude crystal violet method.

Inoculated a single colony of bacteria into 5 mL of LB broth and incubated at 37C shaking at 225 rpm overnight. Each inoculation was performed in duplicate, resulting in two independent cultures for each of the six bacteria listed below.

Bacteria inoculated for study
P. larvae 9545
P. alvei 33A1
P. dendritiformus 30A1
B. cereus 6A5T
B. thuringiensis 4Q1
E. coli  B/r

#######################################################################
Friday October 31, 2014

Set up growth curve.

198 uL of LB broth was added to each well of a sterile 96 well plate. Added 2 uL overnight broth culture to each well, resulting in a 2:200 dilution (1:100). The cover was removed from the plate and a transparent adhesive cover was placed on top of it to prevent evaporation for the duration of the experiment. The plate was placed in the automatic plate reader in Pruess lab where it's optical density at 600 nm would be determined every hour for a total of 48 hours at room temperature would be calculated.

Plate set up:
There are two biological replicates of each strain, with eight technical replicates of each biological replicate.

Column 1 : P. dendritiformus

Column 2 :P. dendritiformus

Column 3 : P. alvei

Column 4 : P. alvei

Column 5 : P. larvae

Column 6 : P. larvae

Column 7 : B. cereus 

Column 8 : B. cereus 

Column 9 : B. thuringiensis

Column 10 : B. thuringiensis

Column 11 : E. coli

Column 12 : E. coli

//EWW

Chlorine Dioxide Pilot Study

From 10-28-14.

I was unable to count any colonies from the two treatment groups on the MYPGP plate from two days ago. There were no colonies observed on the 10^0 dilution for both the control and experimental (ClO2) treatment groups. This likely indicates that the samples were diluted out beyond observation. Only 50 uL of broth culture was affixed to a glass cover slip, and then that was re-suspended in 10 mL of ddH2O, that is a 1:200 dilution right away. Also, the broth culture consisted of mostly vegetative cells, which likely died during the drying process. It is not terribly odd that no colonies were observed in my control treatment when considering that fact.

In the future, if I am interested in repeating this experiment for TOTAL cells, what I could do, and should have done this time, was centrifuge down the broth culture to a pellet and then re-suspend the pellet in a smaller volume as to concentrate my sample before affixing to the cover slip. That way I'd have a higher initial concentration and the effects of diluting and die off will be less devastating.

In the mean time, the experiment was repeated as before, except this time P. larvae 9545 spores were used in place of the broth culture. Specifically 50 uL of Spore Stock E was added to each cover slip. Spores were dried onto the cover slip as before and exposed to ClO2 at the same concentration (86 mg of each component). Incubated for six hours at room temperature slightly shaking on a shaking incubator.




//EWW

Chlorine Dioxide Pilot Study

From 10-18-14.

Want to determine if the chlorine dioxide gas is able to kill P. larvae at all. This pilot probably should have been performed right away at the start of this project, as the effect of ClO2 on Bacillus spp has been evaluation, but it has never been done on Paenibacillus. So, I will first adhere complete P. larvae 9545 cells (vegetative and spores) onto a sterile glass cover slip and expose it to an arbitrarily high concentration of ClO2 gas for six hours in order to gauge its effect. I should hopefully see a decline in CFU in the groups exposed to the gas.

Required Materials

P. larvae 9545 broth culture in BHI+thiamine (incubated for 2 days at 37C)

Glass cover slips (used for microscopy)

50 mL conical tubes

P200 + sterile tips

Petri plates

Sterile forceps

Chlorine Dioxide Gas components 

1. Wash several glass cover slips in ethanol and transfer them to a glass petri dish

2. Allow cover slips to dry (ethanol evaporates) and autoclave using dry cycle

3. Aseptically transfer cover slips to a petri dish using a forceps (flamed for sterility)

4. Carefully add 50 uL of P. larvae broth culture onto each cover slip

5. Allow broth culture to dry onto cover slip (takes about 1 hour in the fume hood)

6. Aseptically transfer cover slips containing bacteria to 50 mL conical tubes

7. At the bottom of the experimental tubes, mix each ClO2 components shortly before adding the cover slip

8. Transfer tubes to a shaking incubator protected from light - gently rotate shaker at room temperature for six hours

9. Aseptically transfer the cover slip to a new 50 mL conical tube
10. Add 10 mL of sterile ddH2O to the tube and vigorously vortex for 10 seconds
11. Ten fold serially dilute the now re-suspended bacteria

12. Plate onto MYPGP agar using the drop plate method
13. Incubate plates at 37C overnight not inverted, invert plates after 24 hours and allow to continue incubating for an additional 24 horus
14. Count CFU and determine concentration

//EWW

Stocks from Bacillus Genetic Stock Center

From 10-24-14.

Made freezer stocks of the majority of the Bacillus Stock Center isolates. Overnight cultures were combined with glycerol in a freezer tube and stored at -80C in the orange spore stock box.

800 uL of overnight broth culture + 200 uL of 80% glycerol

Freezer stocks made:

4Q1 - Bacillus thuringiensis subsp. israelensis ONR60A
30A1 - Paenibacillus dendritiformus subsp. dendron T168
30A2 - Paenibacillus dendritiformus subsp. charalis C168
33A1 - Paenibacillus alvei III3DT-1A
33A2 - Paenibacillus alvei III2E
40A1- Brevibacillus laterosporus ATCC 9141
40A2 - Brevibacillus latersporus ATCC 6457
40A6 - Brevibacillus latersporus NRRL B-4189

Freezer Stock Manifest

I was not able to make a freezer stock of 26A3, Brevibacillus formosus SS 8604, as it did not grow on the NYSM agar. Currently it is still located on two discs at 4C. I may in the near future attempt to culture this bacterium again.

//EWW

Stocks from Bacillus Genetic Stock Center

From 10-23-14.

Bacterial growth is visible around many of the discs on the agar media. A small portion of the bacteria was isolation streaked around the disc where growth was not observed. Plates were inverted and incubated at 37C overnight.

Interestingly, there appears to be either contamination or odd pure culture colony formation seen in two of three cultures plated on NYSM agar.

Two plates of NYSM agar that had questionable growth on them.

NYSM agar plate containing P. alvei 33A1

NYSM agar plate containing P. alvei 33A1

The third NYSM agar plate that was inoculated that had Brevibacillus formosus 26A3 on it did not show any growth yet. I might try to culture the two Paenibacillus strains on MYPGP agar instead of the NYSM to see the results.

//EWW

Stocks from Bacillus Genetic Stock Center

From 10-8-14.

Prepared media in order to make freezer stocks of the remaining spores from the Bacillus Stock Center shipment. The spores have since been stored at 4C.

Nutrient Agar and Nutrient Broth was prepared.
NYSM Agar was prepared as well (Link).

ATCC medium: 2302 NYSM medium 

Nutrient agar...............23.0 g 

Yeast extract................0.5 g 

MnCl2 ........................9.9 mg 

CaCl2 . 2H2O ................10.3 mg 

MgCl2 . 6H2O ...............203.3 mg 

Distilled water..............1.0 

Autoclave at 121C for 15 minutes. 

Media was autoclaved for 20 minutes.

Strains:

4Q1 - Bacillus thuringiensis subsp. israelensis ONR60A
26A3 - Brevibacillus formosus SS 8604
30A1 - Paenibacillus dendritiformus subsp. dendron T168
30A2 - Paenibacillus dendritiformus subsp. charalis C168
33A1 - Paenibacillus alvei III3DT-1A
33A2 - Paenibacillus alvei III2E
40A1- Brevibacillus laterosporus ATCC 9141
40A2 - Brevibacillus latersporus ATCC 6457
40A6 - Brevibacillus latersporus NRRL B-4189

Media plated on for each strain:

4Q1 - LB agar
26A3 - NYSM agar
30A1 - LB agar
30A2 - LB agar
33A1 - NYSM agar
33A2 - NYSM agar
40A1- Nutrient agar
40A2 - Nutrient agar
40A6 - Nutrient agar

Discs were aseptically transferred to the center of their appropriate agar media and were saturated with a small amount of appropriate broth media. Plates were incubated at 37C not inverted for 24 hours.

//EWW

Strains for Sequencing

From 10-17-14.

Digested gDNA with EcoR1 using EcoR-HF with Cutsmart buffer from NEB. Digests performed at 37C for 25 minutes. Digested and undigested DNA was mixed with EZ loading dye and loaded into a 1% agarose gel. Gels were ran at 140 V for 1 hour.

Gel image results:

Digest Gel 1

Lane 1	Lane 2	Lane 3	Lane 4	Lane 5	Lane 6	Lane 7	Lane 8
HiLo	1 cut	1	4 cut	4	5 cut	5	9 cut

Digest Gel 2

Lane 1	Lane 2	Lane 3	Lane 4	Lane 5	Lane 6	Lane 7	Lane 8
HiLo	6 cut	6	7 cut	7	8 cut	8	9

Key

1	P. larvae 9545
4	4D1
5	6A5T
6	13A1
7	13A4
8	4AA1
9	13A82

It appears that all gDNA was successfully digested into unique bands and that the gDNA alone was one band. This indicates that the gDNA is what we think it is - gDNA. In the very near future, about four of these samples will sent for sequencing.

//EWW

Chlorine Dioxide Pilot Study

From 10-8-14.

Repeated chlorine dioxide water pilot from last week. This time a small fan (Ultra-Mini Fan from Go Travel) was added to each anaerobic container and the two chemical components were combined in a small sachet instead of a PCR tube as performed before. Additionally, a humidity and temperature detector was added to the last container in each treatment group. Temperature and humidity was measured at each of the six time points for each treatment group.

Ultra-Mini Fans from Go Travel that were used in this experiment

Each anaerobic chamber was set up similar to the picture below. The fans were afixed the bottom of the chamber using double sided tape so they would not fall over and remained at a fixed location throughout the experiment. Only the six hour treatment had the temperature/humidity gauge in it.

Anaerobic plate set up for this experiment

Below are better images of the nozzle that was put into the covers of the anaerobic chambers. Each chamber had a single nozzle placed in them by drilling a small hole and gluing the nozzle into place.

Below are the raw data results from the Cl2 gas column gauge. Also included is the temperature and humidity at each time point. Because of how the experiment was set up, 200 mL of gas was removed only once from each container.

Raw Data	10/18/14
	Hour 1	Hour 2	Hour 3	Hour 4	Hour 5	Hour 6
No Water	75	60	50	50	45	40
Temp	22.8 C	22.7 C	22.5 C	22.2 C	22.1 C	22.2 C
Humidity	30%	30%	30%	31%	31%	31%
Water	100	95	95	80	70	50
Temp	22.6 C	22.4 C	22.1 C	22.1 C	22.2 C	22.0 C
Humidity	98%	98%	98%	94%	92%	90%
Correction Factor of 0.25
	Hour 1	Hour 2	Hour 3	Hour 4	Hour 5	Hour 6
No Water	18.75	15	12.5	12.5	11.25	10
Water	25	23.75	23.75	20	17.5	12.5

//EWW

Strains for Sequencing

Strains from Bacillus Stock Center and P. larvae 9545 were prepped to be sent for sequencing at Cornell University Institute of Biotechnology.

Bacillus strains were incubated in 10 mL LB broth at 37C shaking at 225rpm for ~24 hours and P. larvae cultures (performed in triplicate) were incubated at 37C 225 rpm for four days. Cultures had their genomic DNA (gDNA) isolated using a DNEasy Blood and Tissue kit and subsequently purified and concentrated to 100 uL using a Zymo Genomic DNA Clean & Concentrator. gDNA purity and concentration was quantified using a Nano Drop located in the McEvoy Lab.

Results:

Strains	ng/ul	mg/100 ul	260/280	260/230
P. larvae 9545	59.6	5.96	1.92	2.15
P. larvae 9545	58.5	5.85	1.9	2.24
P. larvae 9545	53.9	5.39	1.89	2.08
B. thur 4D1	55.6	5.56	1.94	2.26
B. cer 6A5T	17.5	1.75	1.99	2.08
L. spha 13A1	78	7.8	1.93	2.37
L. spha 13A4	107.1	10.71	1.92	2.35
B. thur 4AA1	75.5	7.55	1.93	2.37
L. spha 13A82	64.9	6.49	1.88	2.3

Next, gDNA will be digested using EcoR1 enzyme and visualized on a gel before they are sent for sequencing.

//EWW

Chlorine Dioxide Pilot Study

From 9-26-14.

Performed chlorine dioxide gas pilot study at the USDA ARS BRL building. This was performed as a collaboration with the Animal Metabolism Unit a the USDA. We will be generating chlorine dioxide gas in anaerobic chambers. Gas is generated more efficiently at higher humidity, however water serves as a sink for generated gas. So, we wanted to determine the effect of having a water source in the anaerobic container while generating chlorine dioxide gas. For this pilot, we used modified anaerobic chambers equipped with a nozzle attached and sealed to the cover of each container. Experiment was performed in a laminar flow hood in a room with closed windows and no lights on.

Gas concentrations were measured using chlorine gas detection tubes (seen below) in conjugation with a GasTec (Link) pump. At each time point the glass detection tube was broken on each end and attached to the nozzle on the cover and a volume of 100 mL was pulled into the tube twice, for a total of 200 mL.

Six modified anaerobic chambers were utilized, with three containing a 100 mL beaker containing 50 mL of ddH2O. 86 mg of each component A and B to generate chlorine dioxide gas were combined together in a PCR tube, This amount was calculated to generate 125 ppmV of chlorine gas after six hours. closed and mixed briefly for 10 seconds, and the cap was opened and the tube was placed in cryofreeze tube that was in a small beaker to prevent tipping. The lid of the anaerobic chamber was closed. Gas concentrations were measured every hour for six hours in each of the containers.

Raw Data (ppm)
125 ppmV	Hour
	1	2	3	4	5	6
Box 1	90	95	90	95	80	75
Box 2	95	100	95	90	80	80
Box 3	80	80	80	75	70	60
Box 4	85	90	85	80	70	60
Box 5	90	90	90	80	70	65
Box 6	75	80	75	75	75	70

Boxes 3, 4, and 6 contained 50 mL of water.

Chlorine dioxide gas will be fully generated after six hours. The biggest issue with the results that were collected is that we are essentially diluting the concentration each time we take a 200 mL volume from the container. The generation rate of gas also needs to be taken into account. Further calculations of the raw data need to be performed in order to fully gauge the generation rate of gas between the two treatment groups.

In the future, only one 200 mL gas sample will be taken from a container, and not multiple times. This will require the use of multiple containers, one for each time point. Additionally, a small fan will be added to the container during the generation of gas to aid in mixing and generation of chlorine dioxide gas.

//EWW

The Waxworm & the P. larvae

From 10-6-14.

None of the wax worms appear to have died in either of the two treatment groups. Additionally, they appeared to have at least doubled in size in just the last two days. I will continue to monitor them for survival.

//EWW

Stocks from Bacillus Genetic Stock Center

From 10-7-14.

The Bacillus spp. all displayed growth around the disc on the LB agar in the 30C incubator. An inoculating loop was used to streak for isolation on the agar plate around the disc. The plates will continue to incubate at 30C overnight.

//EWW

Abx Screening of P. larvae

From 10-6-14.

Checked for growth on the MYPGP abx plates that have since been incubating at 37C 5%CO2.

If there was a visible bacteria on the plate (usually in a lawn) it was determined to be resistant to that particular antibiotic. If there was no visible bacteria on the agar, then the bacterium was susceptible to that particular antibiotic and was unable to grow because it had been killed.

Results:

Abx	E. coli B/r	P. larvae 9545
Amp100	S	S	S	S
Eryth5	R	S	S	S
Kan50	S	S	S	S
Poly60	S	R	R	R

S - sensitive

R- resistant

These P. larvae antibiotic results confirm what was previously done on 7-31-14. P. larvae 9545 is resistant to polymyxin, but sensitive to ampicillin, erythromycin, and kanamycin.

This information will be important for an upcoming allelic exchange that will be performed on P. larvae.

//EWW 

Stocks from Bacillus Genetic Stock Center

From 10-6-14.

Made freezer stocks of the five E. coli strains that were incubated overnight in LB Abx at 37C 225 rpm.

800 uL of broth culture + 200 uL of 80% Glycerol in a freezer tube

Made freezer stocks all in triplicate. Place in orange tube box and wrote "Fisher Spore Box" on it. The box was stored in the -80C freezer on the top shelf.

Plated several of the spore forming bacteria that also arrived from the Bacillus Genetic Stock Center.

Spores were soaked into small discs present in wrapped aluminum foil. These small discs were transferred to LB agar using sterile forceps (flamed to sterilize). Three drops of LB media were carefully added to the disc that was present on the LB agar. Plates were incubated at 30C in the walk in incubator, not inverted.

Strains that were plated onto LB

4D1 Bacillus thuringiensis 

4AA1 Bacillus thuringiensis

6A5T Bacillus cereus

13A1 Lysinibacillus sphaericus

13A4 Lysinibacillus sphaericus

13A82 Lysinibacillus sphaericus

//EWW

Red Flour Beetle Pilot

From 10-6-14.

Counted the amount of RFB that had pupated into adults by digging through the small amount of flour present in each 96 well using an inoculating loop.

Results:

Nearly all the RFB had successfully pupated. Literature reviews show that B. thur is not pathogenic to RFB and P. larvae's effect is still unknown. Perhaps the low concentration of P. larvae used was not sufficient to kill the RFB.

The only RFB that didn't successfully pupate was found in the B. thur 10^-1 treatment group. All the P. larvae and control groups had successfully pupated into adults.

In the future, I'll perform a germination/sporulaiton study in the flour itself. I will spike flour with a concentration of P. larva and create a slurry. After drying, the flour will be incubated and then a portion will be heated to determine germination of P. larvae.

//EWW

Abx Screening of P. larvae

From 7-31-14.

Plated 50 uL of a broth culture of P. larvae onto several MYPGP + Abx plates in order to screen for sensitivities. These were the plates that were previously made for the experiment performed at the end of July that have since been stored in the 4C walk in fridge.

Bacterial broth was spread plated onto the agar using a sterile cell spreader and a rotating circular spinner. Plates were incubated at 37C 5%CO2 inverted.

E. coli B/r was also plated on all the abx MYPGP plates as a control to see if the abx are still functioning properly.

Antibiotics used:

Kanamycin 50 ug/ml

Ampicillin 100 ug/ml

Erythromycin 5 ug/ml

Polymyxin 60 units

//EWW

The Waxworm & the P. larvae

From 9-26-14.

Copious amounts of wax worms were visible in the gerber diet container that was incubated at 30C. They all appear close to the wandering life stage and are large in size. Their visible growth appears to have happened suddenly, as I was not able to see younger instars in the food half a week ago when it was last viewed.

Bottom view of wax worms and gerber diet after 9-8-14.

A large amount of "silk" had been formed on the top of the food, making it difficult to identify the wax worms. Wax worms were left at room temperature in the lab overnight until I decide what to do with them.

Top view of wax worms and gerber diet after 9-8-14.

My containers housing the wax moths appeared to have succumbed to moisture. There was visible condensation on the insides of most of the containers. The wax paper present in the container is visibly moist and sagging. A number of the wax worms have turned black indicating their death and the containers smell of moisture and rot. 

Regardless, a number of the wax moths are still alive and moving, so the containers were left at 30C until further notice. I will periodically check for egg formation.

Crude P. larvae waxworm pilot study:

50 uL of BHI + 1mg/L thiamine broth was added to the bottom of six wells of a 12 well plate. Additionally, 50 uL of a broth culture of P. larvae was added to the remaining six wells of the 12 well plate. On top of the added liquid, a small portion of the gerber diet was added. The youngest instar waxworm was added to each of the wells of the 12 well plate and the cover taped shut. This plate set up was repeated in duplicate. The two plates were incubated in the 30C walk in incubator. I just want to see if the P. larvae will have a lethal effect on the wax worms. Of course, the wax worms were older than I would have liked (as P. larvae has been shown to be an opportunistic pathogen to early instar honey bees) and I don't know the concentration of the P. larvae that was added, but you have to start somewhere!

//EWW

Red Flour Beetle Pilot

From 9-17-14.

Froze the 96 well plate containing the RFB and bacteria-spiked flour. I will determine which RFB pupated into adults and which did not after exposure to the bacteria. It will be easier to determine if the RFB pupated after they are dead and I can move the flour away if they are difficult to find.

//EWW

Stocks from Bacillus Genetic Stock Center

Received a number of E. coli and Bacillus spp. from the Bacillus Genetic Stock Center in the mail on September 24, 2014. They have since been stored at 4C until then.

Today, media was created in order to inoculate broth cultures of the five E. coli strains.

E. coli Strains
ECE10 (MM294(pBS42))- on LB Cm5ug/ml
ECE32 (MC1061(pHP13)) - on LB Cm5ug/ml
ECE90 (TG1(pDG641)) - on LB Amp100ug/ml
ECE93 (TG1(pDG780)) - on LB Amp100ug/ml
ECE 98 (TG1(pDG1515))- on LB Amp100ug/ml

A single isolated colony (sometime multiple if they were small) from each E. coli were resuspended in 10mL of LB broth +Abx and incubated at 37C at 225 rpm.

Antibiotic broth media

Stock Ampicillin was at 100 mg/ml. So, 10 ul was added to 10 mL of LB broth in three 50 ml conical tubes.

Stock Chloramphenicol was at 50 mg/ml. So, 2.5 ul was added to 10 mL of LB broth in two 50 ml conical tubes.

E. coli B/r was subsequently inoculated in Abx media as well to serve as a negative control.

Cultures will incubate overnight and freezer stocks will be made.

There were a number of B. spp. also included with the order, however the media required to re-suspend the spores is not prepared yet and will have to wait until a later date.

//EWW