Teichoic acids (TA) are linear phospho-saccharidic polymers and important constituents of the cell envelope of Gram-positive bacteria, either bound to the peptidoglycan as wall teichoic acids (WTA) or to the membrane as lipoteichoic acids (LTA). The chemical composition of TA varies greatly but the presence of both WTA and LTA is highly conserved, hinting at an underlying fundamental function that is distinct from their numerous specific roles in diverse organisms. We report here the observation of a periplasmic space in the Gram-positive Streptococcus pneumoniae by cryo-electron microscopy of vitreous section. The thickness and appearance of this region change upon deletion of genes involved in the attachment of teichoic acids, supporting the role of TA in the maintenance of a periplasmic space in Gram-positive bacteria as their main universal function. Consequences of these mutations were further examined by super-resolved microscopy (dSTORM), following metabolic and fluorophore coupling by click-chemistry in pulse and pulse-chase experiments. This novel labeling method also enabled the titration the actual amount of TA per cell and to determine the ratio of WTA to LTA, to follow the change of TA length during growth phases, and to discover that a mutant devoid of LTA accumulates the membrane-bound TA precursor, that may compensate the absence of LTA. Most importantly, the possibility to follow TA during cell fractionation led to the discovery that LTA containing membranes sediment at low centrifugal forces, allowing easy separation. The existence of a periplasmic space in Gram-positive has long been debated. The finding that compromising the attachment of teichoic acids changes the appearance and thickness of the periplasm in the pneumococcus indicates a role of these polymers in the maintenance of this space between the membrane and the cell wall. Using metabolic labeling and electrophoresis showed that LTA-containing membranes are easily sedimented. This finding casts doubts on previous results, since most LTA were likely unknowingly discarded in these studies. Our method of TA analysis opens a new era in the investigation of these important and poorly known bacterial polymers and their role in the periplasmic space of Gram-positive organisms.