Applications Of Nano-Flow Cytometry In Spore Detection And Effect Evaluation Of Sporicidal Methods

Endospore(spore)forming bacteria can form dormant spores in response to extreme environmental conditions such as nutrient deficiencies.They are highly resistant to heat,high pressure,radiation,and a variety of toxic chemicals.In one hand,some spores may cause disease threaten the production and daily life of human beings,and make the complete killing of bacterial spores a huge challenge in the pharmaceutical,food,biodefense industry,and medical hygiene.On the other hand,some spore-forming bacteria are probiotics,and the spores produced by them have the advantages of exdended shelf life and resistance to gastric acid,which are ideal biological agents in the medical field.Therefore,the detection and analysis of spores has important theoretical and practical significance for food and environmental sanitation,quality control of spore biological preparations,and the development of disinfectants.The existing methods of killing bacterial spores are generally high temperature sterilization and long-term soaking with high-concentration chemical agents.These methods will cause changes in the color,flavor,and nutritional content of the food,and will also cause certain damage to the quality of the sterilized items,and even cause serious pollution to the environment.Thus,it is very important to use a milder sporicidal method.The existing mild sporicide method is to kill spores after germinating.Meanwhile,it is urgent needed to find a better way to evaluate the sporicidal activity accurately and quickly by distinguishing the three different states of ungerminated spores,germinated live spores and germinated dead spores at the same time.The traditional detection methods of spores mainly include plate couting,phasecontrast microscopy,spectrophotometry(OD600),and chemical and biological sensors,etc.,which are time-consuming,inaccurate,and susceptible to external environments.Besides,the above methods could not distinguish the three different states of spores mentioned above at the same time.Using a laboratory-built nano-flow cytometer(nFCM)for multiparameter analysis upon fluorescent staining of bacterial cells,we developed a sensitive,rapid and quantitative method that enables the detection of spores in different states.This dissertation consists of the following sections.The first chapter introduces the relationship between spores and human beings,the physiological process and resistance characteristics of spores,the existing methods of killing spores,and the application of flow cytometry in bacterial analysis.The research plan and main contents of this dissertation are put forward in the end.Chapter two describes the development of a method for the rapid detection of spore physiological process.Bacillus subtilis was used as the model bacteria and the SYTO 9 green-fluorescent nucleic acid dye was used to label the bacterial genome.We detected the side scatter and fluorescence signal of single bacteria by the nFCM to monitor the ratio of vegetative cells(or germinated spores)and spores.The results of nFCM were in good agreement with those of the plate counting,with a correlation coefficient of R2>0.99.This method can be extended to other spore-producing bacteria,such as Bacillus cereus,Bacillus pumilus and Bacillus licheniformis.In chapter three,the nFCM was upgraded to two exciatation lasers and used for the exploration of the mild sporicidal disinfection method.Besides SYTO 62 nucleic acid labeling,calcein-AM was used to label the living cells.Using Bacillus subtilis as the model system,the three different states of spores:ungerminated spores,germinated live spores,and germinated dead spores can be well distinguished from each other.The results of nFCM were consistent with the plate counting,with the correlation index R2>0.99,but reduced the analysis time from two days to fifty minutes.Then,this newly developed method was applied to examine the mild sporicidal effect in two sporekilling scenarios,namely,heating after germination and hydrogen peroxide treatment after germination.The sporicidal activity under different conditions was tested,which holds great potential in guiding the optimization and selection of spore killing methods.Chapter four is the summary and outlook.The results of this dissertation are summarized,the parts that need improvement are discussed,and the future application of this newly-developed spore detection method in more fields is prospected.