When fresh whole leaves of six different species of forage legumes were suspended in an artificial rumen medium and inoculated with rumen bacteria, bacterial adhesion and proliferation were noted at the stomata, and penetration of the stomata by these bacteria was documented by electron microscopy. The invading bacteria adhered to surfaces within the intercellular space of the leaf and produced very extensive exopolysaccharide-enclosed microcolonies. After some of the legume leaf cell walls were disorganized and ruptured by bacterial digestion, these cells (notably, parenchyma and epidermal cells) were invaded by bacteria, with subsequent formation of intracellular microcolonies. However, other cells were neither ruptured nor colonized (notably, stomata guard cells and vascular tissue). At all stages of the digestion of intact legume leaves, the rumen bacteria grew in microcolonies composed of cells of single or mixed morphological types, and a particular ecological niche was often completely and consistently occupied by a very large microcolony of cells of single or mixed morphological types.The bacterial digestion of cut grass leaves and of cell walls prepared from various grasses has been documented by several groups (1,2,9,13,14). Brazle and Harbers (3) studied the digestion of air-dried alfalfa hay by scanning electron microscopy, but no detailed studies of the sequence of events in the bacterial digestion of fresh whole legume leaves have been reported.Various bacteria have been shown to adhere to plant cell walls to produce "pits" by their cellulolytic activity (1, 2, 5). The cell walls of different plant tissues have shown sharp differences in the extent to which they are colonized and digested by bacteria (1, 2, 5), and workers in the United Kingdom (9,13,14) and in the southern United States (1, 2) have found that morphologically different cellulolytic bacteria predominate in the digestion of plant materials in their particular geographic regions. Studies of the adhesion of groups of rumen bacteria to their polymeric substrates have shown that amylase producers adhere to starch (11) but not to cellulose and that cellulose decomposers adhere to cellulose (17, 18) but not to starch. Thus, we expect that plant material will be heavily colonized by various types of bacteria soon after it is introduced into the rumen; therefore, it is not surprising that Forsberg and Lam (10) found 75% of the adenosine triphosphate of the bacteria in the rumen contents to be associated with food particles.Because transmission electron microscopy of ruthenium red-stained sections allows both bacteria and their exopolysaccharide products to be seen throughout leaf tissues during digestion, we incubated intact legume leaves with rumen bacteria and examined them at intervals using this method. We have shown (7) that the rumen bacterial population is made up of three distinct subpopulations-the rumen fluid bacteria, the food particle-associated bacteria, and the bacteria adherent to the rumen epithelium. To obtain a good represen...