Team:NTU-Singapore/InterLab

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InterLab Study

 Introduction

The purpose of this InterLab measurement is to try to reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of optical density (OD).

Experiments

 Two orthogonal approaches were used as follows:

    1. Converting between absorbance of cells to absorbance of a known concentration of beads;
    2. Counting colony-forming units (CFUs) from the sample.

 Calibration 1: OD600 Reference Point

We first used LUDOX CL-X (45% colloidal silica suspension) to determine the correction factor to covert Abs600 to OD600. Four replicates of the LUDOX and H2O were measured to determine the corrected absorbance and will be used as correction factor for OD600 of bacterial culture.

Table 1. Absorbance measurement of LUDOX and H2O and correction factor table.

Samples
LUDOX CL-X
H2O
Replicate 1
0.096
0.079
Replicate 2
0.097
0.079
Replicate 3
0.097
0.079
Replicate 4
0.096
0.079
Arithmatic Mean
0.096
0.079
Corrected Abs600
0.017
Reference OD600
0.063
OD600/Abs600
3.684

 Calibration 2: Particle Standard Curve

Serial dilutions of monodisperse silica microspheres were prepared in 96 well plate and Abs600 were measured. The measurement will be used to construct a standard curve of particle concentration, which uses the Abs600 measure-ments to estimate the number of cells.

Figure 1. Graph of particle standard curve in linear scale.

Figure 2. Graph of particle standard curve in logarthmic scale.

 Calibration 3: Fluorescence Standard Curve

Serial dilution of fluorescein was prepared in a 96-well plate and the fluorescence was measured at 485 nm excitation and 525 nm emission. The measurement will be used to construct a standard curve of the fluorescence at different fluorescein concentrations which can convert the cell-based reading to an equivalent fluorescein concentration.

Figure 3. Graph of fluorescein standard curve in linear scale.

Figure 4. Graph of fluorescein standard curve in logarthmic scale.

 Cell Measurement

On Day 1, E. coli K-12 DH5-α was used to transform with devices from distribution kit following the recommended pro-tocol. On Day 2, two colonies from each of the plates were picked and were inoculated in 5 mL of LB medium with chloramphenicol overnight at 37°C and 220 rpm. On Day 3, the overnight cultures were diluted by 1:10 dilution. Abs600 were then measured and the cultured further diluted into Abs600 of 0.02 in a final volume of 12 mL of LB medium with chloramphenicol in a 50 mL falcon tube. 500 μL samples of the diluted cultures at 0 hours were pipetted into 1.5 ml microtubes (on ice) and the remained cultures were incubated at 37°C and 220 rpm for 6 hours. Later, another 500 μL samples of the incubated cultures were pipetted into 1.5 ml microtube (on ice) before all the samples were being transferred into 96 well plate and measured in plate reader of the Abs600 and fluorescence.

Table 2.  Abs600 raw readings at zero hour.

Hour 0
Negative Contol
Positive Control
Device 1
Device 2
Device 3
Device 4
Device 5
Device 6
Blank
Colony 1, Replicate 1
0.0906
0.0912
0.0901
0.0927
0.0917
0.0899
0.0922
0.0915
0.0888
Colony 1, Replicate 2
0.0902
0.0913
0.0895
0.0925
0.0918
0.0901
0.0914
0.0920
0.0888
Colony 1, Replicate 3
0.0907
0.0906
0.0899
0.0904
0.0904
0.0899
0.0892
0.0903
0.0885
Colony 1, Replicate 4
0.0907
0.0899
0.0896
0.0947
0.0906
0.0901
0.0909
0.0904
0.0881
Colony 2, Replicate 1
0.0897
0.0899
0.0903
0.0930
0.0935
0.0907
0.0911
0.0923
0.0886
Colony 2, Replicate 2
0.0893
0.0909
0.0920
0.0915
0.0903
0.0893
0.0889
0.0911
0.0884
Colony 2, Replicate 3
0.0898
0.0900
0.0904
0.0912
0.0908
0.0899
0.0901
0.0903
0.0883
Colony 2, Replicate 4
0.0898
0.0907
0.0892
0.0912
0.0910
0.0898
0.0908
0.0897
0.0883

Table 3.  Abs600 raw readings at six hour.

Hour 6
Negative Contol
Positive Control
Device 1
Device 2
Device 3
Device 4
Device 5
Device 6
Blank
Colony 1, Replicate 1
0.3568
0.5789
0.4595
0.1942
0.2120
0.5303
0.1435
0.4008
0.0888
Colony 1, Replicate 2
0.3783
0.6175
0.4476
0.1990
0.2324
0.6441
0.1498
0.4503
0.0883
Colony 1, Replicate 3
0.3634
0.6198
0.4345
0.2022
0.2438
0.5487
0.1542
0.4308
0.0876
Colony 1, Replicate 4
0.3576
0.6262
0.3999
0.2047
0.2256
0.6474
0.1548
0.4302
0.0883
Colony 2, Replicate 1
0.3181
0.6395
0.4633
0.2659
0.2301
0.5547
0.1415
0.3600
0.0876
Colony 2, Replicate 2
0.3194
0.5946
0.4852
0.2693
0.2276
0.5798
0.1409
0.3469
0.0878
Colony 2, Replicate 3
0.3037
0.6617
0.4977
0.2630
0.2445
0.6076
0.1429
0.3445
0.0882
Colony 2, Replicate 4
0.3158
0.5979
0.5198
0.2583
0.2385
0.5624
0.1423
0.3412
0.0885

Table 4.  Fluorescence raw readings at zero hour.

Hour 0
Negative Contol
Positive Control
Device 1
Device 2
Device 3
Device 4
Device 5
Device 6
Blank
Colony 1, Replicate 1
157.178
136.637
138.135
137.935
135.459
136.525
151.241
136.672
121.16
Colony 1, Replicate 2
156.191
135.774
134.593
139.643
131.590
130.852
150.068
127.833
122.640
Colony 1, Replicate 3
143.262
123.210
133.450
138.785
121.965
129.367
134.375
131.285
121.701
Colony 1, Replicate 4
136.576
120.414
129.576
132.096
118.703
120.904
129.443
126.387
118.962
Colony 2, Replicate 1
148.573
128.531
131.145
138.077
128.285
128.560
142.344
129.976
128.251
Colony 2, Replicate 2
142.612
125.763
135.822
131.942
120.345
125.954
131.168
125.691
125.363
Colony 2, Replicate 3
142.852
126.543
130.409
137.826
125.659
134.329
135.663
135.497
123.677
Colony 2, Replicate 4
141.356
120.340
130.495
139.587
125.113
131.185
133.978
127.237
117.807

Table 5. Fluorescence raw readings at six hour.

Hour 6
Negative Contol
Positive Control
Device 1
Device 2
Device 3
Device 4
Device 5
Device 6
Blank
Colony 1, Replicate 1
205.913
1331.539
1806.157
549.313
159.453
1656.044
257.231
340.370
125.968
Colony 1, Replicate 2
224.410
1309.594
1758.518
540.421
170.163
1652.895
270.258
310.506
127.470
Colony 1, Replicate 3
224.478
1381.396
1780.017
545.329
170.036
1715.484
275.204
297.621
123.568
Colony 1, Replicate 4
190.569
1354.688
1777.020
566.134
159.311
1662.665
282.622
342.703
131.254
Colony 2, Replicate 1
193.166
1288.940
1829.988
621.227
147.919
1642.740
238.437
330.906
121.437
Colony 2, Replicate 2
190.624
1380.807
1784.891
588.461
136.893
1645.495
238.984
330.819
120.125
Colony 2, Replicate 3
176.735
1431.043
1847.053
576.866
155.330
1674.707
240.363
380.889
118.102
Colony 2, Replicate 4
161.863
1349.904
1844.492
627.817
159.844
1721.881
264.774
364.899
124.668

 Net Abs600 Measurement

Grow rate of the cells were measure using Abs600. From Figure 4 below, device 5 show the slowest growth rate as the increase in Abs600 was the lowest.

Figure 5. Graph of Net Abs600 at zero hour.

Figure 6. Graph of Net Abs600 at six hour.

 Net Fluorescein a.u. Measurement

Based on the plate reader we used, the positive control, device 1 and 4 showed fluorescein reading at significantly higher values than the rest of the samples after 6 hour of incubation.

Figure 7. Graph of Net Fluorescein a.u. at zero hour.

Figure 8. Graph of Net Fluorescein a.u. at six hour.

 uM Fluorescein/OD600 Measurement

From Figure 10, the negative control, device 3 and 6 showed uM fluorescein/OD at values significantly lower than other samples, while device 1 show the highest value of uM fluorescein/OD.

Figure 9. Graph of uM Fluorescein/OD600 at zero hour.

Figure 10. Graph of uM Fluorescein/OD600 at six hour.

 MEFL / Particle Measurement

From Figure 12, again, the negative control, device 3 and 6 showed MEFL/particle at value significantly lower than that of other samples, while device 1 showed the highest value.

Figure 11. Graph of MEFL/Particle at zero hour.

Figure 12. Graph of MEFL/Particle at six hour.

Conclusion

Devices 1 showed the highest fluorescein reading, while the net Abs600 indicated the cells only grew at moderate rate compared to others. Devices 1 also showed the highest value of uM fluorescein/OD and MEFL/particle at 6 hour of incubation. This might indicate that growth of cells with device 1 was inhibited. On the other hand, device 4 showed the highest Abs600 and the second highest fluorescein reading after device 1. Value of uM fluorescein/OD and MEFL/ particle after 6 hours of incubation in device 4 also only came after device 1. Therefore, we can conclude that device 4 showed the best fluorescein while maintaining growth rate.