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1  Nanyang Technological University, Singapore



Drinking water quality is critical for all countries while a more challenging problem in many developing countries. In the worldwide well accepted standard for drinking water quality, setup by U.S. Environmental Protection Agency (EPA), a single harmful bacterium cannot be allowed in tap water. However, the EPA recommended standard testing method is still based on the conventional approach of antibody-bacterium conjugation. An immunological detection method, such as ELISA, operated in a biological lab, needs a skillful operator with a cycle taking about 24 hours. It certainly cannot meet the requirements of today’s fast changing living environment. Especially currently we are very concern about food, water and air poisoning and contamination. Thus rapid identification of bacterial species in single bacterium level becomes very crucial.


The measurement of physical parameters, the length, width, the ratio of them and the refractive index of bacteria species were reported by Liu et al. with the approach of refractometry [1]. Our presentation reports latest efforts in investigation of optical scattering patterns of single bacterium of three species, namely E. coli, Shigella flexneri, and Bacillus subtilis under acoustic focusing. These optical scattering patterns were obtained with a customized a prototype system including a customized laser illumination module and a scattering pattern recording module. Representative scattering patterns of a single bacterium flowing through a microfluidic channel are displayed in Fig. 1. As their average physical dimensions still have some differences though having overlapping, their scattering patterns also have differences. But low signal to noise ratio and size non-uniformity of bacteria of the same species, as well as waterborne impurity microbial from 0.8 mm to 3 mm make the optical patterns not reliable for bacterial identification.


We have also measured four strains of E. coli with their scattering patterns are displayed in Fig. 2 which show almost identical scattering patterns. Thus, it is concluded that bacteria species and strains cannot be identified reliably from only physical parameters mentioned above. These parameters can enrich our knowledge to bacteria species in different time points of their life cycle, but not enough to distinguish them in single bacterium level.


To address this problem, we have proposed a fluo-scattering detection system with a schematic drawing is shown in Fig. 3. The approach combines the fluorescence detection of immuno-labeling of bacterium with specific antibody, like what illustrated in Fig. 4, with the optical scattering module to increase the identification efficiency. Fig. 5 shows our preliminary result of differentiation of E. coli (O114) labeled with SyBr gold and 2 mm polystyrene beads. While Fig. 6 shows differentiation of Bacillus spore with unknown waterborne impurity microbial.


With more fluorescence detection channels are built in the fluo-scattering detection system, it has high potential to be able to differentiation 3 to 4 species/strains of bacteria.