Difference between revisions of "Team:Nanjing-China/InterLab"
| Line 684: | Line 684: | ||
<p>This procedure can be used to calibrate OD600 to colony forming unit (CFU) counts, which are directly related to the cell concentration of the culture, i.e. viable cell counts per mL. This protocol assumes that 1 bacterial cell will give rise to 1 colony.<br /> | <p>This procedure can be used to calibrate OD600 to colony forming unit (CFU) counts, which are directly related to the cell concentration of the culture, i.e. viable cell counts per mL. This protocol assumes that 1 bacterial cell will give rise to 1 colony.<br /> | ||
For the CFU protocol, we counted colonies for our two Positive Control (BBa_I20270) cultures and two Negative Control (BBa_R0040) cultures.</p> | For the CFU protocol, we counted colonies for our two Positive Control (BBa_I20270) cultures and two Negative Control (BBa_R0040) cultures.</p> | ||
| − | <div class="word-note" align="center"><img src="https://static.igem.org/mediawiki/2018/ | + | <div class="word-note" align="center"><img src="https://static.igem.org/mediawiki/2018/c/ce/T--Nanjing-China--interlab-5.png" width="70%" /> |
<p><font size="-1">Figure 8. Colony Forming Unit Calculations for Each Culture</font></p></div> | <p><font size="-1">Figure 8. Colony Forming Unit Calculations for Each Culture</font></p></div> | ||
<p align="left">Based on the assumption that 1 bacterial cell gives rise to 1 colony, colony forming units (CFU) per 1mL of an OD600 = 0.1 culture can be calculated as follows:</p> | <p align="left">Based on the assumption that 1 bacterial cell gives rise to 1 colony, colony forming units (CFU) per 1mL of an OD600 = 0.1 culture can be calculated as follows:</p> | ||
Revision as of 11:27, 16 September 2018
InterLab
Our team Nanjing-China used E. coli K-12 DH5-alpha to conduct the InterLab Study. The instrument used during our measurements is Tecan Infinite M1000 Pro plate reader which could read both fluorescence and absorbance from the top of the plate. It has variable temperature settings and pathlength correction, which can be disabled.
Calibration Protocols
We used black plates with transparent bottom for the calibration measurements, which had flat-bottomed wells.
Calibration 1: OD600 Reference point - LUDOX Protocol
|
LUDOX CL-X |
H2O |
Replicate 1 |
0.0506 |
0.0372 |
Replicate 2 |
0.050799999 |
0.0353 |
Replicate 3 |
0.0517 |
0.033100002 |
Replicate 4 |
0.050500002 |
0.034600001 |
Arith. Mean |
0.051 |
0.035 |
Corrected Abs600 |
0.016 |
|
Reference OD600 |
0.074 |
|
OD600/Abs600 |
4.669 |
|
Figure 1. OD600 Reference point
We used LUDOX CL-X as a single point reference to obtain a ratiometric conversion factor to transform our absorbance data into a standard OD600 measurement.
Calibration 2: Particle Standard Curve - Microsphere Protocol
Figure 2. Particle Standard Curve
Figure 3. Particle Standard Curve (log scale)
We prepared a dilution series of monodisperse silica microspheres and measure the Abs600 in our plate reader. The size and optical characteristics of these microspheres were similar to cells, and there was a known amount of particles per volume. This measurement would allow us to construct a standard curve of particle concentration which could be used to convert Abs600 measurements to an estimated number of cells.
Calibration 3: Fluorescence standard curve - Fluorescein Protocol
Figure 4. Fluorescein Standard Curve
Figure 5. Fluorescein Standard Curve (log scale)
We prepared a dilution series of fluorescein in four replicates and measure the fluorescence in a 96 well plate in our plate reader. By measuring these in our plate reader, we generated a standard curve of fluorescence for fluorescein concentration. We would be able to use this to convert our cell based readings to an equivalent fluorescein concentration.
Cell measurement protocol
After completing all three of the calibration measurements, we started performing the cell measurements. We use E. coli K-12 DH5-alpha strain, the same plates and volumes that we used in our calibration protocol, as well as the same settings (e.g., filters or excitation and emission wavelengths) that we used in our calibration measurements for the sake of consistence.
Fluorescence 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 |
502 |
1077 |
507 |
1112 |
490 |
1514 |
759 |
623 |
477 |
Colony 1, Replicate 2 |
517 |
1177 |
530 |
1184 |
524 |
1683 |
842 |
656 |
500 |
Colony 1, Replicate 3 |
492 |
1129 |
506 |
1118 |
489 |
1520 |
760 |
632 |
486 |
Colony 1, Replicate 4 |
506 |
1202 |
556 |
1234 |
517 |
1709 |
760 |
664 |
499 |
Colony 2, Replicate 1 |
488 |
1102 |
621 |
1368 |
508 |
1307 |
821 |
662 |
498 |
Colony 2, Replicate 2 |
513 |
1165 |
635 |
1443 |
534 |
1444 |
979 |
706 |
483 |
Colony 2, Replicate 3 |
490 |
1077 |
601 |
1360 |
508 |
1361 |
939 |
683 |
495 |
Colony 2, Replicate 4 |
510 |
1135 |
621 |
1493 |
535 |
1380 |
972 |
601 |
480 |
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 |
564 |
2645 |
2277 |
2692 |
517 |
6432 |
4002 |
1131 |
465 |
Colony 1, Replicate 2 |
569 |
2742 |
2536 |
2759 |
585 |
6076 |
4277 |
1114 |
452 |
Colony 1, Replicate 3 |
592 |
2672 |
2449 |
2844 |
565 |
5808 |
4207 |
1094 |
453 |
Colony 1, Replicate 4 |
582 |
2718 |
2577 |
2927 |
590 |
6399 |
4286 |
1126 |
468 |
Colony 2, Replicate 1 |
506 |
2577 |
3731 |
2690 |
548 |
7511 |
4772 |
1171 |
466 |
Colony 2, Replicate 2 |
570 |
2772 |
4227 |
2903 |
603 |
7621 |
5307 |
1230 |
475 |
Colony 2, Replicate 3 |
558 |
2662 |
3997 |
2762 |
575 |
7373 |
5061 |
1243 |
486 |
Colony 2, Replicate 4 |
572 |
2756 |
4160 |
2997 |
625 |
7555 |
5278 |
1299 |
473 |
|
|
|
|
|
|
|
|
|
|
Figure 6. Fluorescence Raw Plate Readings
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 |
0.0604 |
0.0583 |
0.0401 |
0.0594 |
0.0596 |
0.0565 |
0.0489 |
0.0613 |
0.0390 |
Colony 1, Replicate 2 |
0.0604 |
0.0654 |
0.0395 |
0.0601 |
0.0598 |
0.0583 |
0.0499 |
0.0603 |
0.0380 |
Colony 1, Replicate 3 |
0.0634 |
0.0602 |
0.0429 |
0.0633 |
0.0615 |
0.0586 |
0.0528 |
0.0649 |
0.0416 |
Colony 1, Replicate 4 |
0.0527 |
0.0541 |
0.0365 |
0.0560 |
0.0570 |
0.0538 |
0.0436 |
0.0550 |
0.0338 |
Colony 2, Replicate 1 |
0.0667 |
0.0670 |
0.0440 |
0.0763 |
0.0654 |
0.0535 |
0.0535 |
0.0621 |
0.0391 |
Colony 2, Replicate 2 |
0.0704 |
0.0687 |
0.0420 |
0.0743 |
0.0647 |
0.0551 |
0.0545 |
0.0622 |
0.0391 |
Colony 2, Replicate 3 |
0.0643 |
0.0651 |
0.0413 |
0.0698 |
0.0623 |
0.0591 |
0.0532 |
0.0610 |
0.0387 |
Colony 2, Replicate 4 |
0.0601 |
0.0619 |
0.0369 |
0.0658 |
0.0596 |
0.0495 |
0.0477 |
0.0652 |
0.0448 |
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 |
0.2471 |
0.2457 |
0.1465 |
0.2426 |
0.2047 |
0.0992 |
0.2124 |
0.2152 |
0.0384 |
Colony 1, Replicate 2 |
0.2451 |
0.2410 |
0.1536 |
0.2435 |
0.2155 |
0.0936 |
0.2201 |
0.2046 |
0.0380 |
Colony 1, Replicate 3 |
0.2467 |
0.2410 |
0.1541 |
0.2535 |
0.2139 |
0.0920 |
0.2125 |
0.2025 |
0.0412 |
Colony 1, Replicate 4 |
0.2391 |
0.2315 |
0.1500 |
0.2437 |
0.2051 |
0.0885 |
0.2081 |
0.1980 |
0.0327 |
Colony 2, Replicate 1 |
0.2345 |
0.2556 |
0.2119 |
0.2481 |
0.2537 |
0.2062 |
0.2615 |
0.2253 |
0.0389 |
Colony 2, Replicate 2 |
0.2604 |
0.2684 |
0.2357 |
0.2631 |
0.2746 |
0.1958 |
0.2798 |
0.2302 |
0.0386 |
Colony 2, Replicate 3 |
0.2532 |
0.2640 |
0.2202 |
0.2516 |
0.2643 |
0.1922 |
0.2676 |
0.2323 |
0.0410 |
Colony 2, Replicate 4 |
0.2525 |
0.2607 |
0.2241 |
0.2612 |
0.2682 |
0.1844 |
0.2632 |
0.2281 |
0.0336 |
Figure 7. Abs600 Raw Plate Readings
Protocol: Colony Forming Units per 0.1 OD600 E. coli cultures
This procedure can be used to calibrate OD600 to colony forming unit (CFU) counts, which are directly related to the cell concentration of the culture, i.e. viable cell counts per mL. This protocol assumes that 1 bacterial cell will give rise to 1 colony.
For the CFU protocol, we counted colonies for our two Positive Control (BBa_I20270) cultures and two Negative Control (BBa_R0040) cultures.
Figure 8. Colony Forming Unit Calculations for Each Culture
Based on the assumption that 1 bacterial cell gives rise to 1 colony, colony forming units (CFU) per 1mL of an OD600 = 0.1 culture can be calculated as follows:
- Count the colonies on each plate with fewer than 300 colonies.
- Multiple the colony count by the Final Dilution Factor on each plate.
Address
Life Science Department
#163 Xianlin Blvd, Qixia District
Nanjing University
Nanjing, Jiangsu Province
P.R. of China
Zip: 210046
