Revision as of 02:56, 13 October 2018

InterLab

Interlab Study

It is a good opportunity for our team to participate in the project which aims to improve the measurement tools available to both the iGEM community and the synthetic biology community as a whole. And we are glad to cooperate with many other teams from around the world to solve the same problem in synthetic biology. This year's iGEM InterLab study is about could we use CFU/ml to replace the absorbance in fluorescence/od measurement.

To accomplish the goals, we use measure the fluorescence and absorbance with SpectraMax i3x plate reader. In additionally we need to get the competent DH5α and transforme the plasmids into cells. And this wiki will present the workflow and the results we get. We separate the wiki into 3 segments , and the first part is transformation of the plasmids into DH5α.

Transformation

According to the requirements of iGEM, we transformed all the parts (BBa_R0040, BBa_I20270, BBa_J364000, BBa_J364001, BBa_J364002, BBa_J364007, BBa_J364008, BBa_J364009) into competent DH5α. And we successfully get sufficient colonies in all the plates. Therefore we select 2 colonies in each plate to cultivate them In LB medium containing chloramphenicol for overnight with 37℃, 220rpm.

Calibration 1: OD600 Reference point - LUDOX

Through this calibration, we could transform the absorbance (Abs600 ) data from plate reader into a comparable OD 600 measurement in the spectrophotometer, which will help a lot during the following experiments. By doing so, we get the data following.

	LUDOX CL-X	H2O
Replicate 1	0.056	0.039
Replicate 2	0.059	0.039
Replicate 3	0.058	0.040
Replicate 4	0.056	0.040
Arith. Mean	0.057	0.040
Corrected Abs600	0.018
Reference OD600	0.063
OD600/Abs600	3.549

Table 1: We get the ratiometric conversion factor of 3.549 in our plate reader after measuring the wells adding 100ul Ludox and water.

Calibration 2: Particle Standard Curve - Microsphere

Because the monodisperse silica microspheres are similar to the cells in size and optical characteristics during measuring the absorbance in 600nm. It is necessary to construct the particle standard curve and in this way we could change the absorbance into estimated cells.

Number of Particles	2.35E+08	1.18E+08	5.88E+07	2.94E+07	1.47E+07	7.35E+06	3.68E+06	1.84E+06	9.19E+05	4.60E+05	2.30E+05	0
Replicate 1	0.542	0.347	0.202	0.124	0.080	0.059	0.050	0.047	0.043	0.041	0.040	0.042
Replicate 2	0.531	0.344	0.187	0.116	0.077	0.057	0.048	0.042	0.041	0.041	0.040	0.038
Replicate 3	0.794	0.319	0.177	0.105	0.076	0.062	0.049	0.046	0.043	0.042	0.040	0.039
Replicate 4	0.609	0.314	0.193	0.106	0.076	0.057	0.048	0.046	0.043	0.042	0.040	0.042
Arith. Mean	0.619	0.331	0.190	0.113	0.077	0.059	0.049	0.045	0.043	0.042	0.040	0.040
Arith. Std.Dev.	0.122	0.017	0.011	0.009	0.002	0.002	0.001	0.002	0.001	0.001	0.000	0.002
Arith. Net Mean	0.579	0.291	0.150	0.073	0.037	0.019	0.009	0.005	0.002	0.001	0.000

Table 2 :the raw data we get after diluting a series of monodisperse silica microspheres in the 96 cell well plates.

Figure 1：The Particle Standard Curve with monodisperse silica microspheres (log scale)

Figure 2 : The Particle Standard Curve with monodisperse silica microspheres (line scale)

The standard curve are straight line at line scale but not at log scale. It may caused by along with the increasing dilution factor, the instrumental error and pipetting error could bring more huge effect in data and it will present more dramatically in log scale.

Calibration 3: Fluorescence standard curve – Fluorescein

We also need to measure a standard curve for fluorescence of Fluorescein which can be used to correct our cell-based readings to an equivalent Fluorescein concentration. Following the instructions, we could obtain following results for the serial dilution of Fluorescein in phosphate buffered saline

Fluorescein uM

10.00

5

2.5

1.25

0.625

0.313

0.156

0.078

0.039

0.0195

0.0098

0

Replicate 1

4.833E+04

2.670E+04

2.013E+04

1.019E+04

4.769E+03

2.687E+03

1.385E+03

6.670E+02

3.450E+02

1.630E+02

6.800E+01

2.000E+00

Replicate 2

4.477E+04

3.233E+04

1.903E+04

1.032E+04

4.521E+03

2.770E+03

1.147E+03

5.660E+02

2.560E+02

1.950E+02

1.200E+02

4.000E+00

Replicate 3

4.932E+04

3.348E+04

2.032E+04

1.298E+04

6.118E+03

2.550E+03

1.468E+03

5.660E+02

2.710E+02

1.730E+02

7.900E+01

5.000E+00

Replicate 4

4.846E+04

3.634E+04

2.116E+04

1.180E+04

4.451E+03

3.568E+03

1.445E+03

7.330E+02

3.010E+02

1.310E+02

6.800E+01

8.000E+00

Arith. Mean

4.772E+04

3.221E+04

2.016E+04

1.132E+04

4.965E+03

2.894E+03

1.361E+03

6.330E+02

2.933E+02

1.655E+02

8.375E+01

4.750E+00

Arith. Std.Dev.

2.012E+03

4.042E+03

8.766E+02

1.323E+03

7.808E+02

4.586E+02

1.471E+02

8.192E+01

3.925E+01

2.660E+01

2.472E+01

2.500E+00

Arith. Net Mean

4.771E+04

3.221E+04

2.016E+04

1.132E+04

4.960E+03

2.889E+03

1.357E+03

6.283E+02

2.885E+02

1.608E+02

7.900E+01

Table 3: The raw data after measuring the fluorescence (excitation light: 485 nm, emission light 530nm) in plate reader.

Figure 3 : The fluorescein standard curve in line scale (excitation light:485nm, emission light 530nm)

Figure 4 : The fluorescein standard curve in log scale (excitation light:485nm, emission light 530nm)

The standard curve are straight line in both the line and log scale. So the standard curve may be credible after we use it in following experiment and the analyzing results.

Cell measurement

The fourth segment is the results of absorbance 600nm and fluorescence of transformed cells. According to the protocol we get the data and comparing them with the results in CFU/ml to get our conclusion whether CFU/ml could replace the absorbance in fluorescence/od in laboratory works.

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	8136	12501	16278	14892	9048	11885	11704	10121	9438
Colony 1, Replicate 2	8934	12193	15878	14098	8884	12074	11650	10111	9750
Colony 1, Replicate 3	8658	12142	15435	14105	8925	12015	11719	9945	9940
Colony 1, Replicate 4	8898	12121	16042	13753	9422	12022	11406	10105	9443
Colony 2, Replicate 1	8838	12257	14105	15215	9315	13763	14827	9855	9797
Colony 2, Replicate 2	8420	12662	13753	15391	9273	13825	15043	9858	9515
Colony 2, Replicate 3	8517	12629	15215	14978	9463	13933	14695	9483	9337
Colony 2, Replicate 4	8113	12936	15391	15182	9035	14550	15303	10083	9624

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	0.2	0.23	0.121	0.216	0.271	0.204	0.251	0.236	0.044
Colony 1, Replicate 2	0.194	0.243	0.124	0.22	0.267	0.195	0.235	0.241	0.046
Colony 1, Replicate 3	0.185	0.242	0.126	0.23	0.259	0.182	0.243	0.241	0.041
Colony 1, Replicate 4	0.196	0.238	0.125	0.235	0.258	0.198	0.232	0.231	0.041
Colony 2, Replicate 1	0.226	0.265	0.121	0.24	0.283	0.185	0.241	0.233	0.043
Colony 2, Replicate 2	0.222	0.253	0.123	0.26	0.302	0.196	0.231	0.231	0.043
Colony 2, Replicate 3	0.226	0.252	0.124	0.244	0.289	0.226	0.233	0.225	0.043
Colony 2, Replicate 4	0.218	0.261	0.125	0.246	0.296	0.222	0.231	0.226	0.044

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	0.062	0.049	0.051	0.052	0.056	0.047	0.06	0.055	0.041
Colony 1, Replicate 2	0.06	0.047	0.052	0.052	0.057	0.048	0.063	0.051	0.04
Colony 1, Replicate 3	0.063	0.048	0.051	0.053	0.056	0.048	0.07	0.052	0.042
Colony 1, Replicate 4	0.07	0.049	0.056	0.052	0.058	0.048	0.067	0.051	0.044
Colony 2, Replicate 1	0.067	0.053	0.055	0.058	0.059	0.046	0.066	0.046	0.041
Colony 2, Replicate 2	0.066	0.052	0.051	0.062	0.06	0.049	0.069	0.055	0.043
Colony 2, Replicate 3	0.069	0.054	0.053	0.053	0.059	0.048	0.063	0.047	0.042
Colony 2, Replicate 4	0.067	0.053	0.051	0.051	0.059	0.048	0.064	0.049	0.041

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	10832	26586	11469	36302	11679	65637	31750	16588	9903
Colony 1, Replicate 2	11396	27771	12384	36210	12366	57468	32740	17149	10228
Colony 1, Replicate 3	11623	27875	12080	37821	12440	53989	32033	17207	10542
Colony 1, Replicate 4	11544	28119	12230	35304	12461	56895	31155	17143	10570
Colony 2, Replicate 1	11754	30339	11623	33540	15737	67747	31675	15916	10791
Colony 2, Replicate 2	12659	34078	11544	35802	13098	65635	34026	16744	10459
Colony 2, Replicate 3	12801	33879	11754	37368	12987	65949	34497	17233	10963
Colony 2, Replicate 4	12642	33499	12659	38675	13067	67308	35003	16936	10695

Table 4: The raw data we get from the plate reader after measuring the samples from 0h and 6h during cultivation of the transformed cells.

After synthesis the raw data that we get from the calibration protocol and cell measurement protocol, there are also some forms after analyzing. (for easily to compare the difference between the devices, there are some diagrams only present after cultivating 6h, because we think some data getting from samples in 0h is less valuable)

Unit Scaling Factors:
OD600 / Abs600	3.55
uM Fluorescein / a.u.	1.25E-04

Table 5: The unit scaling factors of our standard curve in calibration experiment.

	Net Abs 600
Hour 0:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	0.021	0.008	0.010	0.011	0.015	0.006	0.019	0.014
Colony 1, Replicate 2	0.020	0.007	0.012	0.012	0.017	0.008	0.023	0.011
Colony 1, Replicate 3	0.021	0.006	0.009	0.011	0.014	0.006	0.028	0.010
Colony 1, Replicate 4	0.026	0.005	0.012	0.008	0.014	0.004	0.023	0.007
Colony 2, Replicate 1	0.026	0.012	0.014	0.017	0.018	0.005	0.025	0.005
Colony 2, Replicate 2	0.023	0.009	0.008	0.019	0.017	0.006	0.026	0.012
Colony 2, Replicate 3	0.027	0.012	0.011	0.011	0.017	0.006	0.021	0.005
Colony 2, Replicate 4	0.026	0.012	0.010	0.010	0.018	0.007	0.023	0.008

Hour 6:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	0.156	0.186	0.077	0.172	0.227	0.160	0.207	0.192
Colony 1, Replicate 2	0.148	0.197	0.078	0.174	0.221	0.149	0.189	0.195
Colony 1, Replicate 3	0.144	0.201	0.085	0.189	0.218	0.141	0.202	0.200
Colony 1, Replicate 4	0.155	0.197	0.084	0.194	0.217	0.157	0.191	0.190
Colony 2, Replicate 1	0.183	0.222	0.078	0.197	0.240	0.142	0.198	0.190
Colony 2, Replicate 2	0.179	0.210	0.080	0.217	0.259	0.153	0.188	0.188
Colony 2, Replicate 3	0.183	0.209	0.081	0.201	0.246	0.183	0.190	0.182
Colony 2, Replicate 4	0.174	0.217	0.081	0.202	0.252	0.178	0.187	0.182

Table 6: The net absorbance 600nm.

Figure 5 : The net absorbance in 600nm of transformed cells after 6h cultivation.

Net Fluorescein a.u.
Hour 0:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	-1302.00	3063.00	6840.00	5454.00	-390.00	2447.00	2266.00	683.00
Colony 1, Replicate 2	-816.00	2443.00	6128.00	4348.00	-866.00	2324.00	1900.00	361.00
Colony 1, Replicate 3	-1282.00	2202.00	5495.00	4165.00	-1015.00	2075.00	1779.00	5.00
Colony 1, Replicate 4	-545.00	2678.00	6599.00	4310.00	-21.00	2579.00	1963.00	662.00
Colony 2, Replicate 1	-959.00	2460.00	4308.00	5418.00	-482.00	3966.00	5030.00	58.00
Colony 2, Replicate 2	-1095.00	3147.00	4238.00	5876.00	-242.00	4310.00	5528.00	343.00
Colony 2, Replicate 3	-820.00	3292.00	5878.00	5641.00	126.00	4596.00	5358.00	146.00
Colony 2, Replicate 4	-1511.00	3312.00	5767.00	5558.00	-589.00	4926.00	5679.00	459.00

Hour 6:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	929.00	16683.00	1566.00	26399.00	1776.00	55734.00	21847.00	6685.00
Colony 1, Replicate 2	1168.00	17543.00	2156.00	25982.00	2138.00	47240.00	22512.00	6921.00
Colony 1, Replicate 3	1081.00	17333.00	1538.00	27279.00	1898.00	43447.00	21491.00	6665.00
Colony 1, Replicate 4	974.00	17549.00	1660.00	24734.00	1891.00	46325.00	20585.00	6573.00
Colony 2, Replicate 1	963.00	19548.00	832.00	22749.00	4946.00	56956.00	20884.00	5125.00
Colony 2, Replicate 2	2200.00	23619.00	1085.00	25343.00	2639.00	55176.00	23567.00	6285.00
Colony 2, Replicate 3	1838.00	22916.00	791.00	26405.00	2024.00	54986.00	23534.00	6270.00
Colony 2, Replicate 4	1947.00	22804.00	1964.00	27980.00	2372.00	56613.00	24308.00	6241.00

Table 7: The net Fluorescence data

Figure 6 : The net fluorescein of transformed cells after 6h cultivation (excitation light:485nm, emission light 530nm)

uM Fluorescein/OD
Hour 0:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	-2.180	13.460	24.047	17.431	-0.914	14.338	4.193	1.715
Colony 1, Replicate 2	-1.434	12.269	17.953	12.738	-1.791	10.213	2.904	1.154
Colony 1, Replicate 3	-2.146	12.902	21.465	13.311	-2.549	12.158	2.234	0.018
Colony 1, Replicate 4	-0.737	18.829	19.333	18.940	-0.053	22.667	3.000	3.325
Colony 2, Replicate 1	-1.297	7.207	10.818	11.204	-0.941	27.886	7.073	0.408
Colony 2, Replicate 2	-1.674	12.293	18.624	10.872	-0.500	25.254	7.475	1.005
Colony 2, Replicate 3	-1.068	9.644	18.786	18.029	0.261	26.929	8.970	1.027
Colony 2, Replicate 4	-2.043	9.703	20.274	19.540	-1.150	24.740	8.680	2.017

Hour 6:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	0.209	3.153	0.715	5.396	0.275	12.246	3.710	1.224
Colony 1, Replicate 2	0.277	3.131	0.972	5.250	0.340	11.146	4.187	1.248
Colony 1, Replicate 3	0.264	3.032	0.636	5.074	0.306	10.833	3.740	1.172
Colony 1, Replicate 4	0.221	3.132	0.695	4.482	0.306	10.373	3.789	1.216
Colony 2, Replicate 1	0.185	3.096	0.375	4.060	0.725	14.101	3.708	0.948
Colony 2, Replicate 2	0.432	3.954	0.477	4.106	0.358	12.678	4.407	1.175
Colony 2, Replicate 3	0.353	3.855	0.343	4.618	0.289	10.563	4.355	1.211
Colony 2, Replicate 4	0.393	3.694	0.852	4.870	0.331	11.181	4.570	1.206

Table 8: The um fluorescence/OD data

Figure 7 : The ratio of fluorescein/OD in 0h (excitation light:485nm, emission light 530nm)

Figure 8 : The ratio of fluorescein/OD after cultivation for 6h (excitation light:485nm, emission light 530nm)

Discussion: According to the net fluorescence per od value, the order of the strengths of the different promoters is estimated based on the average of two colonies in each group. The conclusion is that "device 4">"device 5">"device1" approximately equal to "positive control" >"device 2">"device 6">"device 3">"negative control". The results of the comparison with the official RFP were basically the same, while the positive and negative controls and the repetition of the two colonies indicated that the results were credible.

Unit Scaling Factors:
Particles / Abs600	4.00E+08
MEFL / a.u.	7.51E+08

Table 9 : The unit scaling factors of our standard curve in calibration experiment.

MEFL / particle
Hour 0:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	-1.17E+05	7.20E+05	1.29E+06	9.32E+05	-4.89E+04	7.67E+05	2.24E+05	9.17E+04
Colony 1, Replicate 2	-7.67E+04	6.56E+05	9.60E+05	6.81E+05	-9.58E+04	5.46E+05	1.55E+05	6.17E+04
Colony 1, Replicate 3	-1.15E+05	6.90E+05	1.15E+06	7.12E+05	-1.36E+05	6.50E+05	1.19E+05	9.40E+02
Colony 1, Replicate 4	-3.94E+04	1.01E+06	1.03E+06	1.01E+06	-2.82E+03	1.21E+06	1.60E+05	1.78E+05
Colony 2, Replicate 1	-6.93E+04	3.85E+05	5.78E+05	5.99E+05	-5.03E+04	1.49E+06	3.78E+05	2.18E+04
Colony 2, Replicate 2	-8.95E+04	6.57E+05	9.96E+05	5.81E+05	-2.68E+04	1.35E+06	4.00E+05	5.37E+04
Colony 2, Replicate 3	-5.71E+04	5.16E+05	1.00E+06	9.64E+05	1.39E+04	1.44E+06	4.80E+05	5.49E+04
Colony 2, Replicate 4	-1.09E+05	5.19E+05	1.08E+06	1.04E+06	-6.15E+04	1.32E+06	4.64E+05	1.08E+05

Hour 6:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	1.12E+04	1.69E+05	3.82E+04	2.89E+05	1.47E+04	6.55E+05	1.98E+05	6.55E+04
Colony 1, Replicate 2	1.48E+04	1.67E+05	5.20E+04	2.81E+05	1.82E+04	5.96E+05	2.24E+05	6.67E+04
Colony 1, Replicate 3	1.41E+04	1.62E+05	3.40E+04	2.71E+05	1.64E+04	5.79E+05	2.00E+05	6.26E+04
Colony 1, Replicate 4	1.18E+04	1.67E+05	3.71E+04	2.40E+05	1.64E+04	5.55E+05	2.03E+05	6.50E+04
Colony 2, Replicate 1	9.89E+03	1.66E+05	2.01E+04	2.17E+05	3.87E+04	7.54E+05	1.98E+05	5.07E+04
Colony 2, Replicate 2	2.31E+04	2.11E+05	2.55E+04	2.20E+05	1.92E+04	6.78E+05	2.36E+05	6.28E+04
Colony 2, Replicate 3	1.89E+04	2.06E+05	1.84E+04	2.47E+05	1.55E+04	5.65E+05	2.33E+05	6.48E+04
Colony 2, Replicate 4	2.10E+04	1.98E+05	4.56E+04	2.60E+05	1.77E+04	5.98E+05	2.44E+05	6.45E+04

Table 10: MEFL/particle

Figure 9 : The ratio of fluorescein/particle in 0h (excitation light:485nm, emission light 530nm)

Figure 10 : The ratio of fluorescein/particle after cultivation for 6h (excitation light:485nm, emission light 530nm)

Discussion : MEFL/particle also show the same results that device 4 have the stongest promotor and device 3 have the weakest promotor. And the decreasing promotor strength is "device 4">"device 5">"device1" approximately equal to "positive control" >"device 2">"device 6">"device 3">"negative control", which corresopnd to the results in flourescence per od .

CFU/ml data in Neg.Control and Pos.Control.

We dilute the neg.control and pos.control into the absorbance into 0.1, and we finish the following dilution according to the protocol. The protocol is easy to understand and there presents the data we record during experiment.

Neg.Control Relplicate 1	Neg.Control Relplicate 2	Neg.Control Relplicate 3	Pos.Control Relplicate 1	Pos.Control Relplicate 2	Pos.Control Relplicate 3	LB+Cam
0.148	0.154	0.153	0.142	0.158	0.145	0.042
0.157	0.165	0.159	0.143	0.135	0.139	0.041

Table 11: The original absorbance of neg.control and pos.control after dilution in plate reader

	Colony numbers
Final Dilution Factor	Neg.Control Relplicate 1	Neg.Control Relplicate 2	Neg.Control Relplicate 3	Pos.Control Relplicate 1	Pos.Control Relplicate 2	Pos.Control Relplicate 3
8*10^4	542	2300	1992	285	404	261
8*10^5	174	211	143	25	40	46
8*10^6	33	9	35	5	3	1
Final Dilution Factor	Neg.Control Relplicate 1	Neg.Control Relplicate 2	Neg.Control Relplicate 3	Pos.Control Relplicate 1	Pos.Control Relplicate 2	Pos.Control Relplicate 3
8*10^4	992	1488	660	267	209	221
8*10^5	140	288	79	16	22	22
8*10^6	8	53	16	2	13	1
Final CFU/ml	Neg.Control Relplicate 1	Neg.Control Relplicate 2	Neg.Control Relplicate 3	Pos.Control Relplicate 1	Pos.Control Relplicate 2	Pos.Control Relplicate 3
colony 1	2.01E+08	1.69E+08	1.97E+08	2.28E+07	3.20E+07	2.88E+07
colony 2	1.12E+08	3.27E+08	3.92E+07	2.14E+07	1.67E+07	1.77E+07

Table 12: The colony numbers in plates

Figure 11 : The colony numbers per milliliter in 0.1 absorbance culture (600nm)

we selectively choose some data to calculate the final CFU/ml . The selective standard is according to the plate colony numbers which is between 30-300. After analyzing the final CFU/ml in the negative and positive control, we found that although the origin absorance are similar, there are some difference between the CFU/ml. we are not sure whether there exists pipetting errors during dilution process . But all the data we got shows that the expressing GFP consistantly in Pos.Control may affect the growth of bacteria .

Final conclusion

We could conclude that there are some relationships between the fluorescence/OD and fluorescence per particle, So it is definitely that we could use particle of cells to replace the Absorbance of cells. However, there is no obvious relationship between the particles of cells and the CFU/ml in culture. Because CFU/ml presents the numbers of survival cells and the bacteria which have relatively strong vitality. Compared with positive control, negative control obviously have a higher chance to express antibiotic resistant gene instead of the unnecessary GFP. So when transformed cells grow on the plates, there are more chances to survival in the antibiotic plates for cells who express more less unnecessary proteins. Of course, there may exists some pipetting errors during dilution process, but the data we obtain from the experiment show that we may can’t normalize colony-forming units (CFUs) instead of OD during fluorescence measurements.