Ngo Thi N A, Naumann D
Robert Koch-Institute, Nordufer 20, 13353 Berlin, Germany.
Anal Bioanal Chem. 2007 Mar;387(5):1769-77. doi: 10.1007/s00216-006-0829-z. Epub 2006 Oct 26.
Microorganisms rarely occur as individual cells in nature and are, instead, organized in complex multicellular communities such as colonies, fruiting bodies, or biofilms. Interest in the natural microbial life-style has increased during the last decade and a whole plethora of techniques has been used to gain insight into the development, structure and composition of diverse microbial communities. We have developed a technique for investigating the spatial heterogeneity of microbial growth in macro-colonies which essentially entails excision of the colonies with the underlying agar, freezing and subsequent cryotoming of the colonies, then FTIR microspectroscopic mapping of the cryosections. Colonies from Legionella, Bacillus, and Candida strains were chosen as model systems of multi-cellular communities to evaluate the technique. The results obtained indicate pronounced cell population heterogeneity even in relatively young colonies cultivated under laboratory conditions. Spectral data obtained from different positions within, e.g., a colony of Legionella bozemanii 120 h old indicated that levels of the storage material poly-beta-hydroxybutyric acid were significantly higher in cells at the surface of the colonies than in those growing at the bottom next to the agar surface. Similarly, in a 24-h-old macro-colony of Bacillus megaterium significantly more of the capsular compound polyglutamic acid was detected in upper layers than in deeper layers of the colony. Results demonstrate that FTIR microspectroscopy can be an useful tool for investigation of the spatial heterogeneity of cell growth within microbial macro-colonies. It is suggested that the method also can be adapted to the analysis of more complex multicellular communities, for example fruiting bodies, biofilms, or colonies growing under natural conditions.
在自然界中，微生物很少以单个细胞的形式出现，而是以复杂的多细胞群落形式存在，如菌落、子实体或生物膜。在过去十年中，人们对自然微生物生活方式的兴趣有所增加，并且已经使用了大量技术来深入了解各种微生物群落的发育、结构和组成。我们开发了一种用于研究大型菌落中微生物生长空间异质性的技术，该技术主要包括切除带有下层琼脂的菌落、冷冻并随后对菌落进行冷冻切片，然后对冷冻切片进行傅里叶变换红外光谱（FTIR）显微成像。选择来自军团菌、芽孢杆菌和念珠菌菌株的菌落作为多细胞群落的模型系统来评估该技术。获得的结果表明，即使在实验室条件下培养的相对年轻的菌落中，细胞群体也存在明显的异质性。例如，从120小时龄的博兹曼军团菌菌落内不同位置获得的光谱数据表明，菌落表面细胞中的储存物质聚-β-羟基丁酸水平明显高于琼脂表面附近底部生长的细胞。同样，在24小时龄的巨大芽孢杆菌大型菌落中，上层检测到的荚膜化合物聚谷氨酸明显多于菌落的深层。结果表明，FTIR显微光谱可作为研究微生物大型菌落内细胞生长空间异质性的有用工具。有人认为，该方法也可适用于分析更复杂的多细胞群落，例如子实体、生物膜或在自然条件下生长的菌落。
