Virginia Tech/bacteria model

The first step to modeling the spread of an epidemic is to model the population itself.

If one does not understand how the population grows, then how is it possible to understand the spread of a disease through that population? It is very easy to grow E. coli; it only needs some media, a warm environment, and some aeration. The problem is generating the growth curve. It is possible to measure the OD600 over a period of time, but the team wanted to generate data with number of bacteria.

Generating the Calibration Curve

The team decided to generate OD600 vs Time, OD600 vs CFU/mL, and CFU/mL vs Time curves. It was also decided to grow two different strains of bacteria C600 and LE392 in both LB and TNT media.

This was accomplished using the following procedure: ATCC C600 and LE392 were regenerated from a previous overnight culture. This was done by adding 250 uL of culture into 10 mL of LB broth and allowing it to grow for 2 hours at 37oC and 220 rpm.

1. Four 50 mL Corning Tubes were labeled: "LE392 in TNT," "LE392 in LB," "C600 in TNT," and "C600 in LB."

2. 30 mL of each media was added to the proper tube and each tube was then inoculated with 500 uL of the appropriate liquid culture (LE392 or C600).

3. A 500 uL aliquot was taken from each tube and labeled T=0 (Time) and the appropriate strain and media.

4. The 4 Corning Tubes were placed in a shaker/incubator at 37oC and 220 rpm with the caps lightly screwed on so that air could reach the cultures and allowed to grow.

5. An OD600 measurement was taken and recorded from the aliquots taken at Time = 0.

6. Aliquots were taken an measured every 30 minutes for four hours (i.e. T=0, T=0.5, T=1.0 ... T=4.0). (Only 100 uL of the 500 uL was used to make the measurement)

7. After each measurement, the aliquots were placed in the refrigerator at 4oC.

8. Once all the measurements were completed, dilutions were made from each of the remaining aliquots for C600 in LB media.

9. The dilutions were as follows: 1:100, 1:1000, 1:10000, and 1:1000000 for the last two time intervals.

10. 20 uL of the first three dilutions were plated on LB media for the first three time intervals.

11. 20 uL of the second two dilutions were plated on LB media for the next four time intervals.

12. 20 uL of the last two dilutions were plated on LB media for the last two time intervals.

13. The plates were allowed to grow overnight at 30oC

After producing a calibration curve to convert OD600 measurements to total number of cells, it was decided to use the new plate reader to measure growth of LE392 in a single well. The new plate reader allows for temperature control, shaking, and can take numerous readings very quickly. Thus, it is possible to follow the growth of LE392 in 96 wells without a considerable amount of work. The only problem was the small volume in each well made it easy for evaporation to occur, but this was compensated for by adding sterilized MilliQ waterpart way through the experiment.

The following protocol was used to grow and measure the OD600 of LE392:

  1. 280 uL of LB media containing 10 mM MgSO4 was put in each well of a 96 well plate.
  2. 20 uL of LE392 ON culture was added to the follow wells:
  3.
       	1 	2
     	3
     	4
     	5
     	6
     	7
     	8
     	9
     	10
     	11
     	12
     A 	0 uL
     	0 uL 	0 uL 	0 uL 	0 uL 	0 uL 	0 uL 	0 uL 	0 uL 	0 uL 	0 uL 	0 uL
     B 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL
     	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL
     C 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL
     D 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL
     E 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL
     F 	20 uL 	20 uL 	20 uL
     	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL
     G 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL
     H 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL 	20 uL
  4. The cells were placed in the plate reader and maintained at 37oC with occasional shaking.
  5. The OD595 was read every 5 minutes for 25 cycles.
  6. After the 25th cycle, 20 uL of autoclaved Milli Q water was added to each well.
  7. The cells were placed in the plate reader and maintained at 37oC with occasional shaking.
  8. The OD595 was read every 5 minutes for 25 cycles.
  9. After the 25th cycle, 65 uL of autoclaved Milli Q water was added to each well.
 10. The cells were placed in the plate reader and maintained at 37oC with occasional shaking.
 11. The OD595 was read every 5 minutes for 80 cycles (overnight... it is known that the wells will eventually dry up but hopefully enough data will be collected prior to this occuring)