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Evolution is endlessly creative, presenting exceptions to nearly every classification rule. We just described a neat breakdown of bacteria into monoderms (one membrane, thick cell wall, positive Gram stain) and diderms (two membranes, thin cell wall, negative Gram stain). But some cells, like these Mycobacterium marinum, defy such easy classification. Mycobacteria are diderm, with an inner and an outer membrane, and a cell wall. But they have unique molecules (named mycolic acids in their honor) in the outer membrane. These acids interfere with Gram staining, yielding an intermediate result between positive and negative. And their cell wall comprises three layers of sugars, each with a unique molecular composition. The middle layer is the familiar peptidoglycan.
These cells illustrate another important point: we cannot always see everything that is there. In this case, we are missing an additional layer outside the outer membrane called the capsule. The capsule, present in many bacteria (mostly diderm, but also some monoderm), is formed by an “extracellular polymeric substance,” or EPS: long chains of sugars, sometimes linked to the outer membrane and sometimes free-floating. These sugars help the cell attach to surfaces and offer an extra layer of protection, trapping water to prevent desiccation and making it more difficult for hydrophobic molecules like detergents to get through to disrupt the membrane(s). It also makes it more difficult for viruses to reach the cell, and for eukaryotic predators like macrophages to eat it. The capsule is unstable and therefore often lost during sample preparation for cryo-ET.