Peptidoglycan is the major structural component of the bacterial cell wall; essential for viability and shape determination. This crucial function makes peptidoglycan synthesis an important target for antibiotics such as penicillin. In order to develop novel antimicrobials, especially against drug resistant Staphylococcus aureus it is important to understand peptidoglycan dynamics at the molecular level. S. aureus makes peptidoglycan primarily during septation and divides on 3 orthogonal planes resulting in cell wall sectoring, with each cell having material of different ages. However, how peptidoglycan is biosynthesised at the molecular level is still not understood. In order to approach the issue of peptidoglycan synthesis I have used a combination of novel probes and fluorescence microscopy. A range of fluorescent probes, based on analogues of peptidoglycan constituents were used in order to allow imaging. I have used 2 new microscopy approaches, structured illumination microscopy (SIM) and stochastic optical reconstruction microscopy (STORM). Previously unresolved features of peptidoglycan synthesis were revealed. Firstly, peptidoglycan synthesis occurs as a zone across the septum, secondly, there is an apparent split in the septum at the mother cell wall-septum interface and finally synthesis is not confined to the septum but occurs throughout the cell wall. As well as molecular level information my study has given cellular level insights. The dynamics of peptidoglycan insertion showed progression of synthesis during a division cycle and the inheritance of material over several generations. It was observed that within a population there is a degree of heterogeneity in peptidoglycan insertion both at the cellular and population level. The existence of transiently non-growing cells within larger exponentially growing population alluded to the presence of potential persisters, able to withstand environmental assault. My work has led to a reassessment of models of growth and division of S. aureus from the molecular to population level.