In 1962 Cole and Hahn published in Science an unassuming sounding paper entitled Cell wall replication in Streptococcus pyogenes. The authors asked the question: do strep cells synthesize their cell wall by intercalating new parts at different sites on their surface or does this take place at one site only? The question was not trivial. Cocci do not have a clearly defined mid-cell region as do rod cells. Furthermore, some rod-shaped bacteria were thought to use an intercalation mechanism for cell division. So the authors kept an open mind and laid out the following premise: If bacterial cell wall could be differentially labeled during growth to distinguish portions of different relative ages, answers to some of these questions might be forthcoming.
To begin, they made use of the fairly novel (well, actually “modern” would be a better term, as it was invented in 1942) development of fluorescent antibodies to differentially label portions of the wall during cell division. They used antibodies against the type-specific M-protein. The antibodies do not affect the growth of strep cells, so they can be used to track the synthesis of new cell wall in an actively growing culture. The authors used their antibodies in different ways, with the same results. In the example shown in the figure, they pre-labeled the cells with fluorescent antibody, washed them, then exposed them to non-florescent antibody for various times. Under the UV microscope, the original halves of the cell walls appeared as fluorescent “X’s”, which are in fact two back-to-back “C’s” representing the fluorescently-labeled older cell wall.
The authors’ concluded from this relatively simple and straightforward experiment that: ….in Streptococcus pyogenes, new cell wall is not diffusely intercalated with the old, but its formation is instead initiated equatorially along a circumference which is the site of the next cross-wall formation. Not only is this a definitive result but it provides an exciting picture, don't you think?
Cole, R., & Hahn, J. (1962). Cell Wall Replication in Streptococcus pyogenes: Immunofluorescent methods applied during growth show that new wall is formed equatorially Science, 135 (3505), 722-724 DOI: 10.1126/science.135.3505.722