Tripartite Conjugation in P. larvae

From 12-3-14.

Transposon insert, pMarA, was from this paper. This process was successful in Bacillus spp., however, it has not been attempted in P. larvae ATCC 9545. It is highly likely that this process should work in P. larvae, which is why I am attempting it.

Ran into a few complications regarding antibiotics for the tripartide conjugation between P. larvae, pMarA, and pRK2013. Either the bacteria's resistances are not what I originally thought they were based on literature review and previous experiments, or our antibiotic stocks are no longer functioning properly. I believe the latter to be the case as upon further inspection some of the antibiotics (like ampicillin) were created in April of this year. It is now December.

This experiment will be done to determine the antibiotic resistance/susceptibilities of each of the bacteria that will be used in the tripartide conjugation. From plate cultures, bacteria were inoculated into 10mL of BHI broth spiked with antibiotic concentrations:

Bacteria	Abx	Growth?
pMarA	Kan50 Amp100	Yes
pMarA	Poly60	No
pRK2013	Kan50	Yes
pRK2013	Amp100	No
pRK2013	Poly60	No
P. larvae	Poly60	NO
P. larvae	Kan50 Amp100	No

Visual growth was observed after 24 hours, shaking at 225 rpm at 37C, on those that are marked as "Yes" growth. It is odd that P. larvae was not resistant to Polymyxin at a concentration of 60 ug/ml, because it was shown to be able to grow under such conditions on 10-8-14. It is my understanding that the polymyxin antibiotic used in this experiment was the exact same tube used in the previous experiment. It is known that P. larvae is a slower grower than the other E. coli strains and that may be the reason no growth was observed in the P. larvae tubes. For that reason I will continue to incubate those two tubes and observe them at a later date.

Another reason that P. larvae didn't grow under the Polymyxin condition may be because it was an older plate culture that had been stored at 4C for a few days. However, viable spores should have still be present even if there was no present viable vegetative cells. Perhaps the spores are unable to germinate in the presence of Polymyxin, which it is ordinarily resistant to as a vegetative cell? This might be a route for a quick pilot study in the future...

Obviously, a negative control for each bacterial strain would have been appropriate in this experiment, however the lab is on short supply on conical tubes and there was a limited supply of BHI broth available at the time. In essence, I did have negative controls, just not in the traditional sense. I was fairly confident that the bold antibiotics used in the table above would result in bacterial growth since it had been observed previously.

The antibiotic resistances was confirmed in both pMarA and pRK2013, Kan50Amp100 and Kan50, respectively. It is also good that both pMarA and pRK2013 are sensitive to Polymyxin, since that is what will be used as a counter selection for P. larvae. Below is a visual display of how the tripartide conjugation will be set up using suspension:

Broth tripartide conjugation visual display

I will also attempt a solid conjugation using filter discs on agar plates... If I can locate where our remaining sterile filter discs are.

//EWW

The Waxworm & the P. larvae

From 11-11-14.

Made up another batch of wax worm food from the gerber baby food. Transferred young larva from the colony to five small containers. There was about 12 wax worms and about 10 grams of food in each container. Wax paper was hung from the cap of each container down to the food as done before.

The wax worms were not in the best of health due to overcrowding in their previous containers. Any wax worms that didn't get transferred were frozen in the -20C freezer overnight and disposed of the next day.

The five wax worm containers were placed within another container and incubated in the walk in 30C incubator. I will continue to monitor their growth, but in the future the colony will remain a smaller size due to a lack of a need for them at this current time.

//EWW

Detection of P. larvae in Local Honey

From 12-22-14.

Screened the remaining suspected P. larvae isolates from Dawn D. Created crude genomic lysates of each isolate and performed diagnositc PCR using the AFB primers.

Results:

Not the best image, but the bands (or lack of bands) can be clearly seen. This is actually a photo of a photo of a photo due a corrupt saved file of the image on my USB drive. So, this image is of the printed out picture of the gel.

Top Row
HiLo Marker	18	19	20	21	22	23	24	25	Positive
Bottom Row
HiLo Marker	26	27	28	29	30	31	Neg1	Neg2

Positive control was P. larvae ATCC 9545. Neg1 was the E. coli strain pMarA ran in this experiment, and Neg2 was the E. coli strain ran in the previous experiment. 

Lab Code	EW Code
dd1-6a	18
dd1-8a	19
dd5-J	20
dd5-K	21
dd5-L	22
dd5-M	23
dd5-M	24
dd5-N	25
dd5-Q	26
dd8-55a	27
dd8-63a	28
dd8-86a	29
dd11-2a	30
dd12-22a	31
DH5a	Neg1
pMarA	Neg2

Discussion:
Of all the isolates from Dawn D. that had been screened using the diagnostic PCR AFB primers, only one did not test positive. That one isolate was from the previous study on 12-22-14. The negative result was from isolate dd1-7a (EW code 02). All other isolates were confirmed as being Paenibacillus, specifically P. larvae because the AFB primers amplified a conserved region of the 16S rRNA only found in P. larvae. A complete list will be compiled of all the confirmed isolates and provided to Dawn D. for future studies. It is my understanding that freezer stocks will be made of the positive isolates, at -80C, to be used in future studies.

It was surprising to find that so many honey providers in the area have P. larvae present in their honey. However, I am uncertain on exactly how many isolates were initially screened by Dawn D. that proved to be both Gram positive and Oxidase positive before they were given to me for diagnostic PCR analysis. Also, there wasn't a relatively large presence of the bacteria in any honey source, and it could be that there is just always a low presence of the bacteria in any bee hive. Perhaps that is a route for a future study.

//EWW

Detection of P. larvae in Local Honey

Goal: Identify P. larvae isolates from local honey providers in North Dakota and the Minnesota area. These isolates will be used for future studies as wild type strains of P. larvae. In order to protect the providers of the honey samples, the businesses will not be identified.

Dawn D., lab technician of the Fisher lab, has acquired 12 honey samples (Lab code for honey providers is dd1 to dd12) from a number of states in the country. These honey samples were diluted and cultured on MYPGP agar. Suspected P. larvae isolates were further isolated onto MYPGP. Suspected isolates were then Gram stained and oxidase tested to help confirm their identity as possibly being Paenibacillus.

Of the many isolates recovered, a handful were confirmed to be Gram positive and oxidase positive indicating potential Paenibacillus larvae isolates. The lab code was developed by Dawn D, however my own code was created for simplicity:

Lab Code	EW Code
dd1-5a	01
dd1-7a	02
dd5-4a	03
dd8-55a	04
dd8-63a	05
dd8-67a	06
dd8-70a	07
dd8-78a	08
dd8-82a	09
dd8-83a	10
dd8-85a	11
dd8-85a	12
dd8-86a	13
dd8-91a	14
dd8-95a	15
dd8-99a	16
dd8-119a	17

The code dd# is referring to one of the 12 honey sources, and the last part is referring to a specific isolate that was identified that source. The above list is only a portion of the isolates confirmed to be potential Paenibacillus.

Confirmation of identification of the suspected isolates as being P. larvae was performed by using diagnostic primers developed according to Development of a fast and reliable diagnostic method for American foulbrood disease (Paenibacillus larvae subsp. larvae) using a 16S rRNA gene based PCR (Dobbelaere, 2001). Primer 1 and Primer 2 were used according to the paper (referred to as AFB primers). PCR parameters were as dictated by the paper as well.

AMRESCO Ready PCR Mix, 2x was used for the amplification reaction. A master mix was created and 2 uL of crude genomic lysates were added for each PCR reaction.

Results of PCR:

Top Row
HiLo Marker	01	02	03	04	05	06	07	08	Positive
Bottom Row
HiLo Marker	09	10	11	12	13	14	15	16	17

The bottom row amplicons were mixed with EZ vision loading dye, whereas the top row was not. This explains the difference in band intensities between the top and bottom rows. Positive control was Paenibacillus larvae ATCC 9545 and the negative control was E. coli pMarA, however I ran out of room on this gel to run the negative control. I will the negative control on the subsequent gel. HiLo DNA marker was used.

Interestingly, all the isolates, except for one, tested positive as being P. larvae. The only isolate that didn't amplify using the AFB primers was EW 02 (dd1-7a). I will continue to screen the remaining suspected Paenibacillus isolates.

//EWW

Tripartite Conjugation in P. larvae

The goal of this project is to develop a mutagenesis procedure in P. larvae 9545 that will randomly insert transposons into its genome. This particular method has been shown to be effective in other Bacillus spp..

December 2nd, 2014

Brothed:
P. larvae 9545 into 10 mL of BHI + thiamine (target genome)
EC pMarA into 10 mL of BHI Amp100 and Amp100 + Kan50 (transposon)
EC pRK2013 into 10 mL of BHI Kan 50 (helper plasmid)

//EWW

The Waxworm & the P. larvae

From 10-8-14.

Checked status of wax worms in the two 12 well plates that were either exposed to P. larvae or BHI broth. Most of of them were dead by now (first exposed them 36 days ago). The plates have been incubating at 30C for that time period.

There were 12 replicates of each treatment (P. larvae & BHI), with six on each of the two 12 well plates. I determined which life stage each of the wax worms were at when they died.

Results: Life Stage Treatment Larva Pupae Moth BHI 3 2 7 P. larvae 1 5 6

Interestingly, one of the larvae (wax worm) and one of the moths was still alive in the BHI treatment group. None were alive in the P. larvae treatment group. I am continually baffled by the seemingly sporadic longevity of these insects. Both treatments resulted in similar amounts of moth life stage development wax worms after 36 days. More wax worms died during the larva stage in the BHI group, which is odd, but likely due to handling issue and not a reflection of the BHI broth itself.

In the future, I'd like to perform this experiment with earlier instar wax worms and an increased concentration of P. larvae. This experiment was performed as very much a crude pilot study as I had an abundance of wax worms at the time and also a broth culture of P. larvae on hand. It is interesting to see that a higher concentration of P. larvae may be needed to see an effect on the wax worm's survival, if there is one at all.

//EWW

Chlorine Dioxide Pilot Study

From 10-28-14.

Determined CFU from ClO2 killing effect on P. larvae pilot that was performed recently. MYPGP plates have been incubating at 37C inverted since then (11 days).

There were no quantifiable CFU on any of the MYPGP plates that were treated with ClO2 gas. However, there were a number of colonies from the control treatment group.

Determining CFU/mL from colony count formula/equation:

[# colonies counted / volume pipetted onto plate in uL] * [ 1 / serial dilution made (ie 10^-2)] * [50 uL / 1 mL] = CFU/mL

Control Treatment, No ClO2
	Replicate 1		Replicate 2		Replicate 3
	10^-2	10^-3	10^-2	10^-3	10^-2	10^-3
	25	3	24	1	22	3
	33	2	25	0	26	2
	31	0	25	2	25	3
	22	3	33	3	25	5
	21	2	19	4	29	2
Avg	26.4	2	25.2	2	25.4	3

50 uL of P. larvae spore stock was added to a cover slip. This volume was resuspended in 5 mL of ddH2O after treatment (1:100), and then subsequently diluted 1:10.

P. larvae spores remaining after no ClO2 treatment:
1.28x10^4 CFU/50 uL 

Meaning,
P. larvae spores remaining after ClO2 treatment:
< 500 CFU/50 uL

There was far less CFU (beyond what I could determine) in the experimental treatment group.



From 10-18-14.

Repeated pilot to determine if water and high humidity has an effect on the generation of chlorine dioxide gas. The experiment was repeated, with one difference - the electric fans were replaced by metal stir bars and a stir plate (picture below). This experiment took place in Van Es Hall Room 114, Fisher Lab, as opposed to the previous experiments which were performed at the USDA ARS building.

Metal stir bar plates were acquired from the T. Bergholz lab. Each of the plates were identical Barnstead Thermolyne Cimarec brand hot plates. The hot plate function was not used during this experiment. The metal stir function was set to "4" on the dial for each during the experiment. 

Above is the anaerobic plate set-up. In order to prevent the metal stir bar from losing its magnetism to the plate and flying off damaging either the beaker of water or the sachet containing the ClO2 reagents a petri plate lid was affixed to the bottom of the chamber using double sided tape. A metal stir bar was placed within the petri plate cover. Unfortunately, there was a very fine line between efficient and consistent stirring and it being too fast or not enough to move the bar. This resulted in the dial being set to "4". There wasn't much air movement in my opinion due to this problem.

The sachets that were used were larger than the ones previously used as I was unable to find the more narrow ones. The larger sachet could have resulted in less mixing of the ClO2 reagents.

Only two modified anaerobic chambers were used in this pilot experiment. One containing a 50 mL beaker with 50 mL of ddH2O and the other just containing the beaker with no water added. This will determine if there is indeed a difference in ClO2 generation based on increased humidity as previously discussed. Chamber were set up next to each other on the Fisher lab bench. Humidity and temperature gauges were also added to each of the two containers. Reagents A and B were combined in the sachet and mixed briefly for 10 seconds by shaking the sachet. The sachet was placed in the side of the chamber and cover was added to the top, pinching the top of the sachet.

Chambers were allowed to incubate at room temperature, while stirring, for six hours. Chambers were protected from direct light by placing a large box over the tops of the two chambers during the entire time frame. Chlorine gas concentration was measured using the GasTech tool as before.

Results:

H2O experimental treatment after 6 hours:

79% humidity

25.6C

45 Cl conc

No H2O control treatment after 6 hours:

27% humidity

26.4C

45 Cl conc

Humidity was higher in the treatment group containing the 50 mL of ddH2O, which is to be expected with the additional water. Temperatures remained relatively similar after six hours in both treatment groups. Additionally, so did the quantified concentration of chlorine in each container. This amount was also similar to previous experiments that have been performed including the one that used the electric fan as a means to mix the gas more efficiently. This is unfortunate, as it appears that the addition of the metal stir bar did not have an effect on the dissemination of ClO2 gas. This value of 45 is far below the calculated concentration of gas that was supposed to be present relative to the amount of reagents added. I am beginning to wonder if there isn't a problem with circulating gas, but rather an issue with our calculations.

In the future I would like to determine the quantifiable concentration of ClO2 with increasing amounts of reagents that have been mixed in order to determine if there is indeed a gradient as their should be. I'll likely mix increasing amounts of Reagents A and B in anaerobic containers without any stirring mechanism and let the reaction occur for six hours. After which I'll measure the amount of gas in each chamber.

Interestingly, the electric fans used on 10-18-14  to mix the gas now seems to have a dramatically reduced functioning capacity. The reviews for the fan claimed that it would function for over nine hours with fresh batteries, however after four hours while being exposed to ClO2 gas the fans died. The fans were removed from the gas and fresh batteries were added, and now the fans only function for two hours before they stop. It would appear that the ClO2 gas effected the functionality of the fans, likely a corrosion issue due to the salt formation. For this reason, we likely wont be able to use any sort of electronic, battery powered, mixing tools in the future. I wonder what effect the gas is having on the temperature and humidity gauges that are also in the anaerobic chambers....


//EWW

P. larvae growth & Biofilm Pilot Study

From 10-30-14.

Acquired growth curve data from automatic plate reader in Pruess lab. Proceded to perform crude biofilm quantification using crystal violet stain.

Followed protocol used by B. Eklund located here with a few modifications listed below (namely the use of antibiotics was not employed):

Materials

• Trypticase soy broth
• Cultures of bacteria on plates
• 15ml conical tubes
• Pipet aid with 5ml sterile glass pipet
• Shaker, 37°C
• 96 well plate, sterile
• Sterile distilled water
• 0.1% crystal violet
• p200 pipet and tips
• 95% ethanol
• 96 well plate reader

Procedure

1. Create an overnight culture of selected bacteria strains by adding 3ml TBS into each 15ml conical tube, add a loop of bacteria, and vortex well until bacteria is thoroughly suspended.
2. Place tubes in 37°C shaker incubator overnight.
3. Add 3mls of 2xTSB into new 15ml conical tubes labeled appropriately. From overnight cultures, pipet 30µl from culture into new media to create a 1:100 dilution.
4. Add 100µl of inoculated media into each designated well, final volume of all wells should be 200μL
5. Incubate plates in the electronic plate reader in the Pruess lab set to 37°C for 48 hours. Set the machine to take the OD reading every 2 hours at 600 nm.
6. Carefully remove the culture from each inoculated well.
7. Add 200 uL of 0.1% crystal violet to each well.
8. Let plate sit in the crystal violet for 15 minutes at room temp.
9. Remove the crystal violet and wash each well with sterile distilled water three times to remove excess crystal violet and any unattached cells.
10. Let the plate dry for 15 minutes in the hood.
11. Add 200 µl 95% Ethanol to each well, let sit for at least 5 minutes; vortex gently if necessary.
12. Read plate at 600 nm with plate reader.

Biofilm Results:

	P. dendritiformus		P. alvei		P. larvae		B. cereus		B. thuringiensis		E. coli B/r
	1	2	3	4	5	6	7	8	9	10	11	12
A	0.421	0.598	0.163	0.146	0.137	0.14	0.116	0.159	0.116	0.115	0.317	0.323
B	0.406	0.418	0.159	0.146	0.12	0.147	0.171	0.144	0.14	0.128	0.292	0.21
C	0.432	0.514	0.155	0.147	0.161	0.139	0.16	0.123	0.143	0.147	0.346	0.251
D	0.478	0.484	0.155	0.134	0.129	0.134	0.134	0.11	0.136	0.154	0.212	0.209
E	0.523	0.488	0.151	0.13	0.141	0.119	0.136	0.126	0.131	0.146	0.231	0.221
F	0.621	0.672	0.152	0.156	0.118	0.124	0.128	0.107	0.137	0.131	0.321	0.231
G	0.549	0.472	0.157	0.168	0.135	0.137	0.12	0.135	0.135	0.137	0.222	0.213
H	0.336	0.556	0.168	0.155	0.187	0.133	0.132	0.125	0.144	0.114	0.239	0.226

Growth Curve Results:

	Avg
	1 2	3 4	5 6	7 8	9 10	11 12
	P. den	P. alvei	P. larvae	B. cereus	B. thur	E. coli
0	0.1146875	0.1123125	0.1171875	0.1361875	0.1258125	0.1141875
1	0.109625	0.1019375	0.111875	0.1351875	0.1219375	0.10825
2	0.11025	0.1008125	0.1105	0.1379375	0.121375	0.1084375
3	0.1100625	0.1000625	0.10975	0.1470625	0.12275	0.1099375
4	0.1095625	0.099625	0.10925	0.1658125	0.1265	0.1105625
5	0.10925	0.0993125	0.109	0.20025	0.1355625	0.1140625
6	0.109	0.09925	0.10875	0.248625	0.1524375	0.1205
7	0.1089375	0.099	0.1086875	0.302375	0.1749375	0.128
8	0.109	0.0989375	0.108625	0.35375	0.201125	0.1355
9	0.109375	0.098875	0.108625	0.4006875	0.2369375	0.144
10	0.10975	0.0989375	0.1085625	0.43725	0.2765625	0.1535625
11	0.1103125	0.0989375	0.1084375	0.467	0.3139375	0.1608125
12	0.1110625	0.099	0.1085625	0.484625	0.3569375	0.171125
13	0.1116875	0.099	0.1085625	0.49575	0.379	0.1831875
14	0.11275	0.0990625	0.1085625	0.5043125	0.3969375	0.196
15	0.1143125	0.09925	0.1086875	0.5115	0.4115625	0.2083125
16	0.11675	0.0994375	0.108875	0.518125	0.4153125	0.22075
17	0.12075	0.1	0.109	0.52175	0.4099375	0.233875
18	0.1250625	0.1006875	0.109375	0.5208125	0.409625	0.24775
19	0.12725	0.1016875	0.1096875	0.5193125	0.4060625	0.2603125
20	0.1303125	0.103125	0.11	0.51775	0.404375	0.27175
21	0.135125	0.1055	0.1104375	0.5208125	0.402125	0.2835
22	0.1406875	0.1081875	0.1111875	0.5278125	0.3991875	0.2933125
23	0.14725	0.1121875	0.111875	0.5313125	0.3931875	0.297625
24	0.1548125	0.1189375	0.113125	0.5385	0.3885625	0.30225
25	0.1653125	0.1263125	0.1140625	0.5408125	0.385625	0.307
26	0.1835	0.135125	0.1154375	0.5480625	0.38025	0.31025
27	0.20025	0.1459375	0.117125	0.5544375	0.3798125	0.314
28	0.20975	0.16025	0.119	0.55625	0.3735625	0.3175625
29	0.2175	0.17375	0.1213125	0.5593125	0.3684375	0.321375
30	0.2249375	0.1925625	0.1240625	0.5629375	0.3679375	0.325125
31	0.234125	0.21175	0.1273125	0.5666875	0.3618125	0.3273125
32	0.24125	0.229	0.130875	0.56925	0.357875	0.3301875
33	0.2491875	0.2513125	0.1350625	0.5728125	0.3539375	0.3320625
34	0.2571875	0.2655	0.139875	0.576125	0.3513125	0.334875
35	0.2681875	0.2849375	0.145375	0.57925	0.345375	0.3374375
36	0.2801875	0.3023125	0.151125	0.582375	0.345875	0.3394375
37	0.294125	0.31175	0.1571875	0.5850625	0.339625	0.3411875
38	0.3096875	0.324875	0.16375	0.5853125	0.337625	0.343375
39	0.3225625	0.3345625	0.1705	0.5846875	0.3350625	0.3456875
40	0.336125	0.340375	0.177375	0.5854375	0.331875	0.3480625
41	0.3505	0.3425625	0.1843125	0.587125	0.3283125	0.350125
42	0.36425	0.34425	0.1915	0.5896875	0.325875	0.352875
43	0.3765	0.3438125	0.1986875	0.5928125	0.323625	0.3550625
44	0.387125	0.3436875	0.205875	0.5973125	0.3223125	0.35775
45	0.3986875	0.3460625	0.212875	0.60225	0.3200625	0.3596875
46	0.408375	0.3481875	0.2201875	0.6073125	0.3164375	0.3621875
47	0.4179375	0.3508125	0.2271875	0.6119375	0.31625	0.364625
48	0.4291875	0.3545625	0.2336875	0.619875	0.315	0.3669375

//EWW

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