Spores of Gram-positive bacteria such as Bacillus and Clostridium cause huge economic losses to the food industry. In food products, spores survive under food preservation conditions and subsequent germination and outgrowth eventually causes food spoilage. Therefore efforts are being made to eliminate or inactivate these bacterial spores in foods. In this regard food industry uses different preservation methods such as thermal-treatment, weak acids, antimicrobial compounds etc. Complete thermal inactivation of microorganisms in food products has become less popular due to the adverse effects on food quality and flavour. To fulfill the consumer’s demand the “hurdle preservation” approach is favoured to control the outgrowth of these bacteria in foods. The spore germination and outgrowth is often very heterogeneous. Heterogeneity makes it difficult to pinpoint which phases of germination and outgrowth are specifically affected upon a given preservation treatment. Therefore, the work presented in the thesis was focused on to the development of an image analysis tools for the measurement of germination and outgrowth of spores as well as the internal pH of the cells at single spore/cell level. To develop a closed air-contaning chamber for actual experimentation with aerobic bacterial spore formers and finally to study the inhibitory effect of weak organic acid, heat and tea compounds on different phases of germination and outgrowth of the spores as well as the effect of sorbic acid and acetic acid on the internal pH of B. subtilis cells at single cell level.