Investigating the heterogeneity of cell growth in microbial colonies by FTIR microspectroscopy

Abstract

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.

Surface-enhanced Raman scattering (SERS) revealing chemical variation during biofilm formation: from initial attachment to mature biofilm

“…The low repeatability of SERS spectra for biofilm samples might be mainly caused by the natural chemical heterogeneity of bacterial colonies and biofilms. Similar variations were also reported in previous studies by others on biofilm heterogeneity by applying different methods, including CLSM [9], FTIR [14], normal Raman [17], and SERS [29], which strongly suggested that the biofilms have a heterogeneous structure with non-uniform distribution of various chemicals. The chemical heterogeneity in biofilms can be attributed to the microscale heterogeneity in solute chemistry that is present within the biofilm matrix [41], such as the concentration gradients of oxygen, nutrients, and metabolic products.…”

Responses of unsaturated Pseudomonas putida CZ1 biofilms to environmental stresses in relation to the EPS composition and surface morphology

“…With the aging of biofilms, cells in the biofilm experienced several stresses, such as cellular aging, nutrient limitation and competition among each other and the EPS were also varied dependently. At the early growth phases of biofilm (12 and 24 h), the large amount of extracellular carbohydrate and DNA may be required for the initial establishment of a biofilm (Whitchurch et al 2002;Jackson et al 2004 carbohydrates, proteins and even DNA were accumulated, and these macromolecules possibly could be served as carbon, nitrogen, and phosphorus sources during nutrient depletion (Steinberger and Holden 2004;Ngo Thi and Naumann 2007;Wang et al 2007). Reduction of cell size is considered as a starvation response of many bacteria (James et al 1995).…”

“…1b). After the maturation of biofilm, these carbohydrates, proteins and even DNA could possibly serve as carbon, nitrogen, and phosphorus sources during nutrient depletion (Steinberger and Holden 2004;Ngo Thi and Naumann 2007;Wang et al 2007). …”

Recent trends in non-invasive in situ techniques to monitor bacterial colonies in solid (model) food

“…Thi et al (2003) assessed an improvement of this technique by coupling the FT-IR spectrometer with a light microscope and obtain microscopic spectra that provide information on the number, size and shape of the micro-colony cells. Furthermore, this technique was extended in Thi and Naumann (2007) to study the spatial heterogeneity of cell growth within macro-colonies. Cells were grown in agar under optimal conditions in order to get macro-colonies with a diameter of about 3–5 mm.…”