In vivo distribution of Helicobacter felis in the gastric mucus of the mouse: Experimental method and results

We describe a method that permits the collection of very small samples (2 n l) from precisely defined positions within the gastric mucus of anesthetize d mice. This method was used to study the in vivo local distribution of bac teria within the mucus of Helicobacter felis-infected mice. A total of 200 samples from 40 mice were analyzed. Each sample was microscopically analyze d, within less than I min, as a native preparation. To avoid changes in bac terial location within the mucus after collection and to improve the counti ng accuracy, bacterial motility was blocked by adjusting the pH inside the collecting pipette to 4.5. The mucus in a collected sample was subdivided i nto three layers, an epithelial layer (the first 25 mu m of mucus from the tissue-mucus interface), a luminal layer (the last 25 mu m to the mucus-lum en interface), and the remaining central mucus layer. The volume of the ana lyzed segments in the sample was between 4 and 9 pi. The concentration of b acteria inside the epithelial mucus layer was 3,400 per nl, but it aas only 50 per nl inside the central mucus layer. The mean distance of H. felis to the epithelial surface was 16 mu m. A total of 75% of all H. felis bacteri a resided in the mucus zone between 5 and 20 mu m from the tissue surface, with no bacteria closer than 5 mu m to the epithelial surface. This method permits the study of factors determining the density of colonization and di stribution of bacteria along chemical gradients with a high precision.