Bacterial Colonies in Solid Media and Foods: A Review on Their Growth and Interactions with the Micro-Environment

Abstract

Bacteria, either indigenous or added, are immobilized in solid foods where they grow as colonies. Since the 80's, relatively few research groups have explored the implications of bacteria growing as colonies and mostly focused on pathogens in large colonies on agar/gelatine media. It is only recently that high resolution imaging techniques and biophysical characterization techniques increased the understanding of the growth of bacterial colonies, for different sizes of colonies, at the microscopic level and even down to the molecular level. This review covers the studies on bacterial colony growth in agar or gelatine media mimicking the food environment and in model cheese. The following conclusions have been brought to light. Firstly, under unfavorable conditions, mimicking food conditions, the immobilization of bacteria always constrains their growth in comparison with planktonic growth and increases the sensibility of bacteria to environmental stresses. Secondly, the spatial distribution describes both the distance between colonies and the size of the colonies as a function of the initial level of population. By studying the literature, we concluded that there systematically exists a threshold that distinguishes micro-colonies (radius < 100-200 μm) from macro-colonies (radius >200 μm). Micro-colonies growth resembles planktonic growth and no pH microgradients could be observed. Macro-colonies growth is slower than planktonic growth and pH microgradients could be observed in and around them due to diffusion limitations which occur around, but also inside the macro-colonies. Diffusion limitations of milk proteins have been demonstrated in a model cheese around and in the bacterial colonies. In conclusion, the impact of immobilization is predominant for macro-colonies in comparison with micro-colonies. However, the interaction between the colonies and the food matrix itself remains to be further investigated at the microscopic scale.

Keywords: Growth; bacterial colony; cheese; diffusion limitation; porosity; spatial distribution.

Figure 1

Representation of the colony and its surrounding “living space” (the area within which the colony is active) with the two respective radii R_col and R_bnd; d is the distance between two neighboring colonies. Adapted from Malakar et al. (2002a) and Wimpenny (1992).

Figure 2

Representation of two situations of neighboring colonies. (A) When the production of lactic acid of one colony does not impact on its neighbors and (B) when the production of lactic acid of one colony does impact on its neighbors. Adapted from Malakar et al. (2002a) and Wimpenny (1992).

Figure 3

Growth/no growth regions of Salmonella Typhimurium in TSB (tryptic soy broth) at 20°C as a function of pH and NaCl concentrations, with gelatine concentrations of 0 and 50 g/l. Adapted from Theys et al. (2010).

Figure 4

Growth/No growth regions of Listeria monocytogenes in broth (solid line) and in agar (dotted line) medium at 25°C as a function of pH and a_w, (modified by increasing the NaCl concentration). Adapted from Koutsoumanis et al. (2004).

Figure 5

Simplified model illustrating the spatial variations in the specific growth rate (μ) within a growing bacterial colony of a facultative anaerobe, such as Salmonella Typhimurium. Adapted from McKay et al. (1997).

Figure 6

pH profile through a 2-day old colony of Salmonella Typhimurium, inoculum density 1 cell/ml, initial pH 7.0, glucose at 1% (w/v). Solid squares indicate points where actual measurements were taken. Solid lines indicate pH isopleths which represent an approximation of where the pH gradients may lie. The green area shows colony location. Adapted from Walker et al. (1997).

Figure 7

pH profiles measured using a pH-sensitive fluorophore (C-Snarf-4) and confocal microscopy for a colony (radius = 65 μm) growing in a model cheese throughout acidification: 19 h (), 24 h (), 26 h (), and all measurements from 42 to 72 h (red bold line, ). Adapted from Jeanson et al. (2013).

Figure 8

CO₂ and O₂ concentration profiles with depth at 24 h (♦) and 48 h (■) after inoculation with Lactobacillus paracasei CI3 in MRS 0.1% agar. A MIMS (membrane inlet mass spectrometric) probe was inserted through column of growth. Adapted from Tammam et al. (2001).

Figure 9

O₂ concentration profiles under the rind of Cheddar cheese at 2 days (♦), 9 days (■), and 15 days (▴) of maturation. Adapted from Tammam et al. (2001).

Figure 10

Schematic diagram of the three culture conditions for bacterial cells and their main characteristics; planktonic culture conditions are the most studied.

Figure 11

Schematic representations of the two concepts of interactions between the colony and the matrix; arrows show the diffusing molecules.

Figure 12

Theoretical relation (black line) for two different spatial distributions, 1 and 2, between the ratio of the exchange surfaces (S₁/S₂) and the ratio of inoculation levels (I₁/I₂); (Δ) experimental data either manually measured or obtained from image analysis of confocal microscopy images from Jeanson et al. (2011) and from Le Boucher et al. (2013).