Schreiber S, Stüben M, Josenhans C, Scheid P, Suerbaum S
Institut für Physiologie, Ruhr-Universität Bochum, D-44780 Bochum, Germany.
Infect Immun. 1999 Oct;67(10):5151-6. doi: 10.1128/IAI.67.10.5151-5156.1999.
We describe a method that permits the collection of very small samples (2 nl) from precisely defined positions within the gastric mucus of anesthetized mice. This method was used to study the in vivo local distribution of bacteria within the mucus of Helicobacter felis-infected mice. A total of 200 samples from 40 mice were analyzed. Each sample was microscopically analyzed, within less than 1 min, as a native preparation. To avoid changes in bacterial location within the mucus after collection and to improve the counting accuracy, bacterial motility was blocked by adjusting the pH inside the collecting pipette to 4.5. The mucus in a collected sample was subdivided into three layers, an epithelial layer (the first 25 micron of mucus from the tissue-mucus interface), a luminal layer (the last 25 micron to the mucus-lumen interface), and the remaining central mucus layer. The volume of the analyzed segments in the sample was between 4 and 9 pl. The concentration of bacteria inside the epithelial mucus layer was 3,400 per nl, but it was only 50 per nl inside the central mucus layer. The mean distance of H. felis to the epithelial surface was 16 microm. A total of 75% of all H. felis bacteria resided in the mucus zone between 5 and 20 micron from the tissue surface, with no bacteria closer than 5 micron to the epithelial surface. This method permits the study of factors determining the density of colonization and distribution of bacteria along chemical gradients with a high precision.
我们描述了一种方法，该方法允许从麻醉小鼠胃黏液内精确界定的位置采集非常小的样本（2纳升）。此方法用于研究感染猫幽门螺杆菌的小鼠黏液内细菌的体内局部分布。共分析了来自40只小鼠的200个样本。每个样本在不到1分钟内作为天然制剂进行显微镜分析。为避免采集后黏液内细菌位置发生变化并提高计数准确性，通过将采集移液器内的pH值调节至4.5来阻断细菌运动。采集样本中的黏液被细分为三层，上皮层（从组织 - 黏液界面起的前25微米黏液）、腔面层（到黏液 - 腔界面的最后25微米）以及其余的中央黏液层。样本中分析片段的体积在4至9皮升之间。上皮黏液层内细菌的浓度为每纳升3400个，但中央黏液层内仅为每纳升50个。猫幽门螺杆菌到上皮表面的平均距离为16微米。所有猫幽门螺杆菌细菌中，共有75%位于距组织表面5至20微米的黏液区域，且没有细菌距离上皮表面小于5微米。该方法允许高精度地研究决定细菌沿化学梯度定殖密度和分布的因素。
