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