SERS is currently being explored as a rapid method for identification of bacteria but variation in the experimental procedures has resulted in considerable variation in the spectra reported for a range of bacterial species. Here, we show that mixing bacteria with a conventional citrate-reduced silver colloid (CRSC) and drying the resulting suspension yield highly reproducible spectra. These signals were due to intracellular components released when the structure of the bacteria was disrupted during sample preparation. This reproducibility allowed us to examine the effects of variables that do not arise in SERS of simple solutions but are relevant in studies of bacteria. These included growth phase and biological variation, which occurred when the same bacterial isolates were cultured under nominally identical conditions on different days. It was found that even under optimal standardized conditions the effect of differences in experimental parameters such as growth phase was very large in some bacterial species but insignificant in others. This suggests that it is important to avoid drawing general conclusions about bacterial SERS based on studies using small numbers of samples. Similarly, discrimination between bacterial species was straightforward when a small number of isolates with distinct spectral features were investigated; however, this became more challenging when more bacterial species were included, as this increased the possibility of finding different species of bacteria with similar spectra. These observations are important because they clearly delineate the challenges that will need to be addressed if SERS is to be used for clinical applications.