Development of a freeze substitution technique to examine the structure ofLactobacillus casei GR-1 grown in agar and under batch and chemostat culture conditions

Abstract

A morphological examination was undertaken ofLactobacillus casei GR-1 by a freeze substitution technique developed to prevent condensation upon fixation and to preserve extracellular material surrounding the cell wall. The strain was cultured for 24 h in 5% CO2 at 37°C initially in brain heart infusion agar supplemented with 2% yeast extract, and the cells formed a short, electron-dense, tightly bound capsule observed under electron microscopy. The cell wall structure was resolved in most cases. Batch cultures were then established by use of pooled human urine with and without addition of lactose and glucose. Examination of the bacteria demonstrated less compact, but more fibrous extracellular material surrounding the cells in a less uniform fashion. The lactobacilli were then grown under nitrogen-and carbonlimited conditions in a chemostat continuous culture system. The nitrogen-limited cells formed a tightly bound, uniform, and electron-dense capsule, while the capsule of the carbon-limited cells appeared longer, more fibrous, but less electron dense in nature. The results indicate that nutrient conditions affect the morphology of lactobacillus and verify that the freeze substitution technique is a useful method to analyze the structure of bacterial cell surfaces. The importance of nutritional changes in the microbial ecology of the urogenital tract can be uncovered by examining these organisms with different culture techniques combined with freeze substitution and electron microscopy.