Successful rapid start-up or re-start of biological filters, defined as containers and media for the growth of nitrifying bacteria that oxidize ammonia to nitrite and nitrite to nitrate, depends on a wide variety of biological, chemical and physical factors. In many situations operators resort to adding mixtures of nitrifying bacteria, in various forms, to the system to inoculate the bio-filter and jump-start the nitrification process with little success. While much study and attention is given to the engineering aspects of the aquaponic systems and the physical aspects of the bio-filtration components the actual nitrifying bacteria and their preferences are often considered as little more than a black box. Research using a suite of molecular methods, that are culture-independent, have allowed much knowledge to be gained about how nitrifying bacteria react, positively and negatively, to the prevailing physical and chemical culture conditions. Presented here will be the major factors affecting the establishment of nitrifying bacteria during the start-up phase with or without adding an ammonia solution to feed the bacteria, how changes in basic water chemistry caused by fish in the system may degrade conditions for nitrifying bacteria and considerations for promoting nitrifying bacteria to rapidly and successfully start-up the biological filter.