Colonization of the gastrointestinal tract by bacteria of the normal flora was followed by bacteriological and special histological techniques in mice from several colonies. These histological techniques were designed to preserve the intimate associations that become established between particular strains of microorganisms and the epithelium of the mucosa of certain areas of the gut. The findings were as follows:

1. The various strains of bacteria of the normal flora became established in the different areas of the guts of infant mice according to a definite time sequence.

2. The first types of bacteria that could be cultured from the gut were lactobacilli and Group N streptococci. Within the first day after birth, these bacteria colonized the entire digestive tract and formed layers on the stratified squamous epithelium of the nonsecreting portion of the stomach and of the distal esophagus.

3. The bacterial types that appeared next were coliforms and enterococci. From about the 9th to the 18th day after birth, these bacteria could be cultured in extremely high numbers from the cecum and the colon. Histological sections of those organs taken during the first 2 or 3 days of that interval revealed microcolonies of Gram-positive cocci in pairs and tiny Gram-negative rods embedded in the mucous layer of the epithelium. The microcolonies were well separated from the mixture of digesta and bacteria that occupied the center of the lumen; they may have consisted of the coliforms and enterococci mentioned above; but this possibility remains to be proved.

4. Histological sections also revealed that, at about the 12th day after birth, long, thin Gram-variable rods with tapering ends were present, side by side, with the small Gram-negative rods and Gram-positive cocci in the mucous layer. By the 15th day after birth, the fusiform bacteria formed thick layers in the mucus, and seemed to be the only bacteria remaining in that location. It has not yet been possible to enumerate these tapered rods by culture methods, but as judged by visual appearances in the histological sections, they seemed to outnumber all other bacteria in the cecum and the colon by a factor of as much as 1000.

It must be stressed that these bacterial layers are readily disrupted and even washed away by conventional histological techniques; their discovery was largely due to the use of the special histological techniques described in the text.

The bacteriological and histological findings described here constitute further evidence for the hypothesis that symbiotic associations exist between microorganisms and animals, and that a very large percentage of the bacteria in the gastrointestinal tract constitutes a true autochthonous flora.

The constant occurrence of several distinct associations of bacteria with the special histological structures of the animal host renders obsolete the notion that the intestine constitutes a chemostat in which the bacterial populations are randomly mixed. For a full understanding of the ecology of the normal microflora, it is necessary to think of body surfaces as distinct microenvironments in which virtually pure cultures of a few species of microorganisms interact with their host and the adjacent microbial populations. Experiments based on this hypothesis are admittedly difficult to design, but on the other hand studies based on the assumption that microorganisms exist as mixtures in the gastrointestinal tract will be only of limited value and may often be misleading.

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