While bacman is gearing up to protect people from arsenic , we decided to pay a visit to the gfp expressing bacteria. It s no doubt that measuring flourescence sounds exciting but even more appealing was the opportunity to be a part of a large community and share data.
So we prepared ourselves to answer the Intelab question : Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?
And then we started : Day 1 : Transforming E coli DH5 alpha cells with the required biobr/icks. Day 2 : Checking plates . Some biobr/icks didn’t give enough colonies. Day 3 : Transforming bacteria with 1.5 uL of biobr/icks. Checking plates. We had beautiful and ample colonies this time. Day 4 : Performing all calibr/ation protocols with ludox , silica beads , fluorescein. Day 5 : Performed the cell measurements. Day 6 : Repeated cell measurements. Day 7 : Performing CFU protocol. Day 8 : Counting colonies and updating excel files.
Results
LUDOX CL-X
H2O
Replicate 1
0.049
0.027
Replicate 2
0.052
0.029
Replicate 3
0.065
0.024
Replicate 4
0.054
0.026
Arith. Mean
0.055
0.027
Corrected Abs600
0.029
Reference OD600
0.063
OD600/Abs600
2.211
Fluoroscence Raw Readings
Hour 0
Neg. Control
Pos. Control
Device 1
Device 2
Device 3
Device 4
Device 5
Device 6
LB + Chlor (blank)
Colony 1, Replicate 1
3172
4940
7332
4129
3103
11501
9596
3409
3096
Colony 1, Replicate 2
3052
4405
6672
4129
3096
11512
11568
3705
3093
Colony 1, Replicate 3
3062
4513
8277
4535
3313
13552
9811
3612
3561
Colony 1, Replicate 4
2939
4559
8165
4393
3302
12578
10198
3595
3501
Colony 2, Replicate 1
2694
4122
9256
4855
3459
11239
7266
3883
3138
Colony 2, Replicate 2
2569
3211
8933
4591
3636
11017
6864
3606
3429
Colony 2, Replicate 3
3512
3431
9091
4728
3668
10965
7255
3981
3510
Colony 2, Replicate 4
1739
3625
8312
4983
3606
11556
7160
3682
3439
Hour 6
Neg. Control
Pos. Control
Device 1
Device 2
Device 3
Device 4
Device 5
Device 6
LB + Chlor (blank)
Colony 1, Replicate 1
3452
52293
20259
33054
3866
30129
19804
11665
3751
Colony 1, Replicate 2
3411
48522
18326
31173
3667
27049
14295
12347
3677
Colony 1, Replicate 3
3662
48774
18739
32604
3646
26488
16048
11921
3409
Colony 1, Replicate 4
3646
56393
21454
31670
3704
28766
16617
10720
3600
Colony 2, Replicate 1
3618
35310
19407
67038
3680
21001
20176
11145
3641
Colony 2, Replicate 2
4077
34070
21004
69337
4237
18555
19423
10521
3911
Colony 2, Replicate 3
3509
34721
20843
62524
3723
17359
16708
11012
3134
Colony 2, Replicate 4
3466
36496
17450
61077
3757
18339
16887
10866
3084
Abs600 Raw Readings
Hour 0
Neg. Control
Pos. Control
Device 1
Device 2
Device 3
Device 4
Device 5
Device 6
LB + Chlor (blank)
Colony 1, Replicate 1
0.085
0.088
0.085
0.087
0.08
0.087
0.084
0.08
0.073
Colony 1, Replicate 2
0.086
0.082
0.087
0.09
0.09
0.088
0.088
0.088
0.079
Colony 1, Replicate 3
0.08
0.08
0.078
0.084
0.083
0.078
0.081
0.085
0.068
Colony 1, Replicate 4
0.082
0.085
0.079
0.078
0.085
0.08
0.077
0.087
0.069
Colony 2, Replicate 1
0.081
0.082
0.076
0.076
0.08
0.078
0.081
0.079
0.068
Colony 2, Replicate 2
0.08
0.074
0.076
0.079
0.077
0.076
0.076
0.077
0.063
Colony 2, Replicate 3
0.067
0.075
0.08
0.083
0.076
0.078
0.077
0.072
0.069
Colony 2, Replicate 4
0.072
0.069
0.068
0.07
0.075
0.072
0.074
0.077
0.071
Hour 6
Neg. Control
Pos. Control
Device 1
Device 2
Device 3
Device 4
Device 5
Device 6
LB + Chlor (blank)
Colony 1, Replicate 1
0.325
0.334
0.092
0.393
0.39
0.162
0.098
0.352
0.064
Colony 1, Replicate 2
0.319
0.316
0.091
0.382
0.383
0.152
0.081
0.372
0.062
Colony 1, Replicate 3
0.346
0.329
0.09
0.388
0.382
0.145
0.087
0.368
0.062
Colony 1, Replicate 4
0.344
0.349
0.096
0.386
0.388
0.155
0.082
0.341
0.059
Colony 2, Replicate 1
0.353
0.318
0.076
0.531
0.379
0.137
0.129
0.346
0.056
Colony 2, Replicate 2
0.368
0.311
0.082
0.533
0.409
0.133
0.132
0.334
0.059
Colony 2, Replicate 3
0.338
0.312
0.076
0.527
0.385
0.149
0.14
0.355
0.076
Colony 2, Replicate 4
0.338
0.328
0.084
0.514
0.387
0.156
0.137
0.349
0.076
Observations
The test device 3 doesn’t show a great increase in fluorescence though its absorbance is increasing. Its expression level thus must be low.
The test device 2 has shown difference florescence and absorbance for the two difference colonies. This could be indication of improper growth in one colony.
The test device 1 has shown very low growth after 6 hrs. This could be due to slow growth of bacteria in set conditions and time.
Conclusion
At the end of interLab we learnt not only how difficult it might be to standardize data and yet how important this process is. We were glad to be a part of the interLab study and contribute in some way what was required in the effort to standardize data.
End result
Team IISER Kolkata is happy and satisfied with interLab.
