The Separation, Bioflim Formation And An Immunomagnetic Bead Detecting Technique For Pseudomonas Fluorescens

Psychrophile is a class of bacteria which could grow between 4℃ to 25℃. Pseudomonas fluorescens is one of psychrophile causing the spoilage of milk through producing protease and esterase during cold storage, and difficult to be inactivated by high-temperature. P. fluorescens could easily form biofilm on the surface of food processing equipment, which is difficult to be removed off and could lead to food pollution through persistent spreading bacteria. In the experiment, the strains of P. fluorescens were obtained from raw milk, the qualitative observation was carried out and effect of silver nanoparticles was examined for the formation of P. fluourcens biofilms, and an IMB-ELISA method was developed. The main findings are as follow:1. To separate the P. fluorescens strain from raw milk, the colony were cultured on the LB plate through the streaking method. Eight strains, which could emit fluorescence under the UV rays, were identified as the suspected P. fluorescens with the physiological and biochemical characteristics. 4 strains were confirmed as P. fluorescens through 16 S r DNA sequencing. Two New Zealand white rabbits were immunized with subcutaneous and intramuscular injections of the inactivated bacteria of P. Fluorescens. The surem was separated and purified by bitter-ammonium sulfate method. The results demonstrated that serum antibody concentration was 2.566 mg/m L and antibody titers were all 1:12800 for No.1 and No.2 rabbits.2.In the experiment, the qualitative observation was carried out for the formation of P. fluourcens biofilms by using crystal voilet staining. The results showed that the bacteria could aggregate on the silicone piece and formed the initial biofilm after 12 hours culture. After 48 hours, the biofilm became relatively dense. The effects of Na Cl concentration, temperature, p H, bacterial initial concentration and appropriate carbon and nitrogen source on the growth of biofilm were evaluated for P. fluourcens. When Na Cl concentration was 0.05 mol/L-0.1 mol/L, culture temperature was 25°, p H value was 7.5 and initial concentration was 8log CFU/m L, P. fluourcens could form biofilms more easily. The appropriate carbon and nitrogen source were respectively glucose and soybean protein for P. fluourcens to form biofilms as soon as possible.3. The experiments were carried out to explore the effect of silver nanoparticles on the formation of P. fluourcens biofilms on ethylene-vinyl acetate(EVA) pieces by colony counts, crystal voilet staining, Scanning Electron Microscopy(SEM) and Confocal Laser Scanning Microscope(CLSM). The biofilm stained by crystal violet was examined with a digital microscope to calculate the biofilm coverage rate(BCR) by NIS-Element BR. The mortality of bacterial was calculated by Image Structure Analyzer(ISA) software for the biofilm observed with CLSM. The results showed that the bacteria could aggregate on the control and EVA contained 0.4% silver nanoparticles and respectively formed the grown biofilm after 12 h and 48 h culture, but the biofilm on the control EVA had more bacterial count and BCR than that of silver nanoparticles(P<0.05). At the 24 t h h, the bacteria could form the dense film including the bacteria body and its secretion in the microscope vision of 10000 times on the control EVA, but there only were dispersive bacteria on silver nanoparticle EVA. The bacterial mortality in the film on silver nanoparticle EVA was significantly higher than that of control group at 24 t h, 48 t h hour(P<0.05).4. Nano-iron oxide beads of 10 nm average particle size were prepared by coprecipitation, of which magnetic saturation was 51.061emu/g. The magnetic beads had good dispersion to meet the requirements of biological applications. The above antiserum was coupled with magnetic beads to establish IMB-ELISA method for monitoring P. fluorescens. The results showed that the IC50 of ELISA method was 106.6CFU/m L and IMB-ELISA was 104.2CFU/m L. In contrast with traditional ELISA, IMB-ELISA has higher sensitivity, lower cross-reaction to other pathogens and higher specificity. It is an effective method for rapid and accurate detection of P. fluorescens.