Autonomous Growth of Isolated Single Listeria monocytogenes and Salmonella enterica Serovar Typhimurium Cells in the Absence of Growth Factors and Intercellular Contact

The aim of this study was to observe growth of isolated single bacterial cells in the absence of growth factors and intercellular contact. In order to exclude stochastic uncertainties induced by dilution series, a new micromanipulation method was developed to ensure explicit results under visual control. This was performed with particular care for production of single prokaryotic cells and subsequent investigation of their autonomous growth. Over 450 single isolated Listeria monocytogenes and Salmonella enterica subsp. enterica serovar Typhimurium cells in lag, log, and stationary growth phases were investigated by this method, which included thoroughly washing the cells. The proportion of living cells within the initial cultures was compared to the proportion of positive samples after enrichment of the separated single cells. This resulted in P values of ≥0.05 using the chi-square test for statistical analysis, indicating no significant difference, and clearly demonstrates reproduction of isolated single bacterial cells without the need for growth factors or intercellular contact. Ease of handling of the apparatus and good performance of the cleaning procedures were achieved, as was validation of the method, demonstrating its suitability for routine laboratory use.The possibility of independent growth of isolated single prokaryotic cells has been discussed recently and remains controversial (15). Undoubtedly, cell-to-cell communication plays a key role in the establishment and development of bacterial communities. Both physical and chemical factors influence the organization of biofilms, sporulation, and resuscitation of bacterial populations. The analogy of the chemical factors to eukaryotic pheromones, as well as their role in bacterial cell division, has been postulated (34). These facts are evident and have been well investigated, and, in summary, the necessity of intercellular contacts, population effects, and intrinsic growth factors such as resuscitating promoting factor (rpf) is generally supposed (24, 36). Nevertheless, when it comes to cell division and growth of low inocula of prokaryotic cells, some questions appear to remain open. Considering evolutionary developments, asexual reproduction is a prerequisite for survival of single organisms in the environment and one of the necessities for the success of the prokaryotic kingdom. Taken the other way, asexual reproduction also suggests the possibility of independent growth of isolated single bacterial cells. The scientific community remains divided over this possibility, not least because of the possible heterogeneity of bacterial cultures in terms of the physiological status of every single cell and the effect of a Poisson distribution in highly diluted cultures, which influence experimental setups and results (17).If one viable prokaryotic cell is to be shown to be capable of generating a population of daughter cells, a prerequisite is its isolation and physical manipulation as an individual cell. Simple serial dilution protocols, such as the most probable number (MPN) method, achieve this task, but they suffer from a lack of certainty that the diluted solution indeed contains only one viable cell, free from any adherent growth factors. In such cases the accuracy of the data obtained by multiple dilution procedures becomes uncertain regarding the Poisson distribution of highly diluted bacterial cell (≤10 CFU/ml) suspensions (11, 32).Combined microinjection and micromanipulation methods are alternative applications. These are widespread routine techniques that have been developed recently for large eukaryotic cells (14, 38). However, application of these methods to prokaryotic cells leads to new physical conditions defined by the technical equipment secondary to the size of the handled cells (≤1 μm). These include limitations of fluorescence microscopy, general microscopic analysis, and photographic documentation. Over the past 30 years several attempts to improve the management of single prokaryotic cells using micromanipulation techniques have been made (10). The core issue of these studies has been isolation of single microbial cells from mixed populations under direct visual control (9, 13).In contrast, the aim of this study was to develop a method capable of serial manipulation of single prokaryotic cells under visual control. This was accomplished to produce series of single prokaryotic cells and subsequently to investigate their autonomous growth. The growth of single Listeria monocytogenes and Salmonella enterica subspecies enterica serovar Typhimurium cells was investigated in highly diluted buffer systems without facultative growth factors being transferred from the primary enrichment. In this study over 450 single cells isolated from different growth phases (lag phase, log phase, and stationary phase) were manipulated using a novel single bacterial cell manipulation (SBCM) technique.