They took the E. coli and B. subtilis and placed them in specific media for each step the in process of replication. They used the process of microscopy to help them see the specimens, since they were unable to see them with their naked eye. They also used flow cytometry to help them to identify and sort through the cells and their DNA by staining them with a fluorescent dye and detecting the fluorescence through a illumination microscope. This helped them to calculate the number of replication forks per cell, along with the cells age and size at initiation. The researcher did an excellent job describing each media and staining technique that they used during their experiments. For example, they explained exactly how much nutrient agar each specimen was placed in during the initial growing phase. The researchers also made sure to use precise measurements and calculations for each step in their experiments. They were so meticulous that, if need be, a student or professor could repeat the experiment for their own research. The only thing that should have been taken into consideration is the temperature at which the specimens were obtained and grown.
An example of this would be the temperature of the room/lab that they conducted the research. This could have played a huge role in whether or not the size of the bacteria would affect the amount of DNA needed to replicate. The findings were presented in such a way that one could understand the outcome just by looking at the graphs, charts and tables that were presented alongside of the documented writing. The findings were reported without bias. This is due to the evidence that was collected during each step of the experiment. It is very difficult to report something with bias when the reports show/explain what actually occurred. The overall outcome of the study was that, regardless of size, DnaA needs to build up to a certain amount in order to trigger initiation in E. coli, which was thought to be the case in the beginning. In contrast, small B. subtilis cells could initiate replication with amounts of DnaA that were 30% less than the natural cells.
As a result, while DnaA is speed limiting for initiation in both organisms, the mechanisms controlling its activity may vary in different bacteria. In conclusion, the research study was done to the highest notch. The researchers covered each spectrum that was needed to prove the outcome; which was that each bacterium needs DnaA in order to initiate replication, but it does not matter the size of the cell because each one is different. This research could definitely be helpful, especially when you want to know how fast it takes bacteria to grow and replicate, when it comes to finding a drug to treat a bacterial infection or disease. The only thing that should be taken into consideration or documented next time is the temperature of the room or location that the experiments were conducted. Besides that, the research study was completed and explained to the utmost highest standard.
Demarre G, Chattoraj DK (2010) DNA Adenine Methylation Is Required to Replicate Both Vibrio cholerae Chromosomes Once per Cell Cycle. PLoS Genet 6(5): e1000939. doi:10.1371/journal.pgen.1000939
Hill NS, Kadoya R, Chattoraj DK, Levin PA (2012) Cell Size and the Initiation of DNA Replication in Bacteria. PLoS Genet 8(3): e1002549. doi:10.1371/journal.pgen.1002549