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