User:Kinkreet/Fluorescence dilution

Background
Proliferation and growth of a bacterial population can be determined by plating cells or tissue lysates on a solid medium and counting the number of colony-forming units (cfu) after a set time; alternatively, growing the cells in liquid culture and measuring the turbidity of the culture at set time points. However this method assumes homogeneity of the population, that all bacteria proliferate and grow at uniform rates. This is, however, not always the case. Genetically-identical cells in the same colony or population can have heterogeneity in gene expression; this is likely to be due to bifurcation. This phenomena is known as bistability. Bistability has been observed inBacillus subtilis and Escherichia coli. . Heterogeneity may also arise due to feedback regulatory mechanisms or noise. A nice demonstration of heterogeneity can be found in Streptomyces coelicolor, which can display all stages of the life cycle (from mycelial growth and spore formation) in a single colony. A better way to characterize growth and proliferation is to use live-cell microscopy, where one can follow a cell and distinguish between different sub-populations. However, this is often time-consuming and involve a large subjective input, unless a specialized software is developed; furthermore, the number of cells that can be followed is limited, often insufficient for statistical analysis.

Fluorescence dilution
Instead, one can use fluorescence dilution in alongside flow cytometry. Bacteria are first transfected with a plasmid containing an inducible gene encoding for a fluorescent protein. The inducer is added and this induces the expression of the gene and production of the protein, resulting in the bacteria becoming fluorescent. Once enough fluorescence is obtained, the inducer is removed, so the cell will not produce anymore of the fluorescent protein; however, any fluorescent proteins will remain in the bacteria until it is degraded. When the bacteria divide, the fluorescent protein are shared between the daughter and parent cell, and so each division results in bacteria which are half as fluorescent as their parents. The level of fluorescence of each cell can be measured and recorded using flow cytometry.

Using green fluorescent protein (GFP), Roostalu was able to monitor the cell division ofEscherichia coli; he observed uniform growth in populations in the exponential growth phase, whereas no growth was observed in populations in the stationary phase. By putting the stationary phase E. coli into fresh media, two types of populations emerged, those that did not divide at all, and those that did. The ones that did not divide was not dead, but lay dormant as 'persister cells' ; when ampicillin was added to the media, the non-dividing cells was able to survive and retains their viability, whereas the dividing cells did not. In this way, Roostalu was able to identify and sort out heterogeneity of cells for further study.

Limitations
Helaine created fluorescent plasmid constructs to be transfected into Salmonella, and found the fluorescence intensity to be too low after 10 cycles of cell division. Therefore, fluorescence dilution is only a better alternative to cfu counts provide bacterial numbers do not exceed roughly one thousand-fold the original number.