What makes a colony of bacteria

The aim of this review is to describe the growth of colonies by pointing out when and how it differs from the planktonic growth. We particularly discuss the occurrence of variability at the microscopic scale of the physiological states inside the colony, of pH inside and around colonies and of oxygen around the colonies.

The diffusion of substrates within the matrix and the access of bacteria to the substrates is also a major concern for the bacterial activity. The second objective is to build concepts on the different situations when growth of bacteria is impacted by the growth in colonies or not, depending on the initial level of population and two other concepts on the different ways of interacting with a food matrix, i. Finally, experimental exploration of these two concepts will be examined in model cheese.

Furthermore, a large table assembles the main parameters of growth and size of colonies for different experimental culture conditions studied with several bacterial species Table 1. Table 1. Summary of the main studies about growth kinetics and distribution size and neighboring distances of bacterial colonies, with details of experimental conditions, and main conclusions. As early as the 60's, Pirt, of the University of London, had started to take into account the immobilization of bacteria in the predictive growth models Pirt, More recently the 90's, Wimpenny, from the University of Wales, started to study the consequences for bacteria by growing as colonies.

Wimpenny et al. They determined several characteristics of the behavior in colonies comparing with planktonic growth, such as growth rates under different conditions, and pH gradients within and around colonies of different sizes.

Before the research on this topic stopped at the University of Wales, Wimpenny collaborated with Brocklehurst Walker et al. Brocklehurst and his group Parker et al. This system has become the ideal tool to study submerged colonies in gelatine and agar media, which was associated with a non-destructive and in situ microscopic examination.

It comprises a 2 mm thick frame in a PVC sleeve shown to be permeable to gas. The inoculated medium solidifies inside the frame and the immobilized cells develop as colonies within the formed solid gel. Subsequently, Brocklehurst collaborated with Malakar Wageningen University, Netherlands who worked on pH microgradients, introducing imaging techniques Malakar et al.

Van Impe studied mostly large pathogen bacterial colonies grown in agar or gelatine media and used micro-electrodes to measure pH. Other research groups have recently compared planktonic and immobilized bacterial growth using molecular techniques to study the difference of gene expression Knudsen et al. Microcalorimetry has been recently used to study the carbon metabolism at different inoculation levels Kabanova et al.

The techniques used to study the immobilized bacterial colonies are described in a recent review Lobete et al. Imaging fluorescent techniques have allowed the observation of colonies within an opaque matrix such as model cheese. We also investigated the role of the size of colonies during ripening by combining omics techniques Le Boucher et al. The growth of colonies has been studied using a qualitative approach and several publications have described how a bacterial colony grew on and within a solid matrix, how they were distributed depending on the inoculation level, and how neighboring colonies interacted with each other either from the same or different species.

Since the first studies, it has been demonstrated that the growth of bacterial colonies on the surface is a concentric pattern Wimpenny, Cell division starts from the initial immobilized cell, with the colony expanding progressively at the periphery thus following a concentric pattern Wimpenny, ; Pipe and Grimson, In the exponential growth phase, the number of cultivable cells is linearly correlated to the Log colony volume Wright et al.

Image analysis techniques have thus been proposed to replace the time-consuming plating techniques. The height of a bacterial colony growing on a surface of a medium was modeled as a function of the glucose concentration of the medium. Indeed, the glucose concentration is low on the top of the colony. It has been suggested that the growth of bacteria and the development of pH profiles in and around the colony were determined by the local presence, and diffusion of glucose, in the medium beneath the colony Wimpenny, This was the main reason offered to explain why the growth of immobilized cells may be different from that of planktonic cells.

It has been demonstrated that most of the mathematical models based on a laboratory broth overestimate the bacterial growth in milk, and even more so its growth in cheese-like media Theys et al.

In conclusion, all the studies on bacterial colony growth have suggested that the growth of colonies growth rate, final size, and shape was determined by local concentration of substrates and thus by possible limitations of the diffusion of substrates or end-products in solids McKay et al. When considering the dimensions of a colony, there are two radii of particular importance: the colony radius from the center of the colony to its periphery R col , and the boundary radius from the center of the colony to the limit of its influence on the medium R bnd Malakar et al.

Furthermore, the larger the radius R bnd , the greater the distance for the substrate to diffuse to reach the colony. Figure 1. Adapted from Malakar et al. The spatial distribution of bacterial colonies is defined by the size of colony and the distances between neighboring colonies. The theoretical distances between colonies were first estimated assuming that i all the cells in the inoculum gave rise to a colony and ii that they were randomly distributed Poisson l a w.

These two assumptions were confirmed from experimental data obtained by confocal image analysis. It was also demonstrated that the final population was always the same regardless of the inoculation level Jeanson et al. As a consequence, the size of colonies was negatively correlated to the level of inoculation, that is, the lower was the inoculation level, the larger the colonies.

A separate study Kabanova et al. The values of distances and radii measured were slightly smaller than those reported by Jeanson et al. However, the latter used isothermal microcalorimetry which is based on dynamic measurements of heat flow rate.

In the agar medium, the shape of colonies was lenticular; this may explain the difference between the measured and the calculated values. Moreover, the L. Table 2. Size of colonies calculated by microcalorimetric method or measured from micrographs as a function of different inoculation levels of two different species of lactic acid bacteria grown in agar, milk gels, or in model cheese.

For a given inoculation level, the variation of the radii of bacterial colonies followed a Normal distribution centered on the mean radius. Indeed, considering that a colony arises from a single cell, the asynchrony of division of any bacterial culture Kreft et al. Some immobilized cells start their division later than others but all cells stopped to grow at the same time.

As a result, different numbers of divisions may occur in neighboring colonies Koutsoumanis and Lianou, If the distance between two neighboring colonies denoted as d is greater than R bnd , one can consider that there is no interaction between the colonies, but if it is closer one can consider that some level of interaction exists Figure 2 and Table 1.

This applies whether the neighboring colonies comprise the same strain or are formed from different strains or species. Interactions between different species may be in the form of competition for the same substrate Thomas and Wimpenny, b or of inhibition because of production of metabolites such as a bacteriocin like nisin Thomas and Wimpenny, a or lactic acid Antwi et al. This review focuses on the few studies on colonies taking into account the distances between the inhibiting and the affected colonies.

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. A strain of Salmonella enterica subsp. Enteritidis thereafter inhibited a strain of Pseudomonas fluorescens , while a strain L. These results were then confirmed in another study Thomas et al. The Listeria strain was inhibited either by nisin from a nisin-producer Lactococcus strain or, to a lesser extent, by lactic acid production from a non nisin-producer strain.

The growth of bacteria as colonies is subjected to several constraints that are absent in planktonic cultures, such as a necessary diffusion of substrates through the solid matrix, with potentially limited access to the substrates.

Predictive growth models for bacteria have mainly been based around parameters taken from planktonic cultures and led to the observation that they were not applicable for modeling immobilized growth Pipe and Grimson, ; Skandamis and Jeanson, Attention was thus given to understand when and how immobilized growth differed from planktonic growth, especially under the stressful conditions of the food environment.

In this section, two aspects of the consequences of immobilization of bacteria are presented: i their responses to conditions of stress and ii on the micro-heterogeneity of the micro-environment inside and around the colonies. The environment existing in food products rarely provides optimal conditions for the growth of microorganisms.

The main factors affecting the bacterial growth in food are temperature, pH, NaCl concentration, water activity a w and substrate concentration. Increasing the NaCl or sucrose concentrations also decreases the a w and increases the osmotic pressure, with combined negative effects.

Several studies have modified these parameters to determine the conditions leading to growth and no growth conditions comparing planktonic and immobilized bacterial growth. Most of these studies have focused on pathogenic species, aiming at predicting or preventing their growth in food. The experimental details and results from the most cited studies in the literature are listed in Table 1.

The growth of a strain of Salmonella enterica subsp. Typhimurium thereafter in gelatine medium was compared to its growth in broth, at different conditions of pH and NaCl Brocklehurst et al.

The results show that S. The maximum viable cell counts were less affected by a low value of a w reduced by high sucrose and NaCl concentrations if the colony was submerged rather than on the surface.

An explanation could be that the substrates are only accessible through the small area of the underside of surface colonies, whilst it is accessible all around the colony on a bigger area when submerged.

By comparing a strain of S. Typhimurium growing as submerged colonies or in planktonic culture, it was shown that the a w was the most influential parameter on the growth rates Theys et al. However, decreasing a w by increasing NaCl concentration was relatively more harmful to the growth of colonies, because of the combined effect on osmotic pressure, than by increasing gelatine concentrations of the media Theys et al. In agreement with the latter, a lower growth rate of growth was observed in planktonic cultures than in submerged colonies of a strain of S.

Typhimurium and the growth in colonies increased its sensitivity to the inhibition exerted by oregano oil Skandamis et al. Surprisingly, the growth rate of submerged colonies of S. Typhimurium was found lower in broth than in agar medium, but lower in gelatine medium than in broth Walker et al. Furthermore, when the growth rate was not affected by the immobilization of bacteria, the lag phase was increased in comparison to planktonic growth Knudsen et al.

Figure 3 is an example of the detrimental effect of immobilization of bacteria on their growth when under severe conditions such as low pH and high concentration of NaCl. Figure 3. Adapted from Theys et al. Similarly, a strain of L. However, in this study, a w had been decreased by increasing the NaCl concentration, the harmful effects of both the NaCl and a low a w were thus combined.

Figure 4. Adapted from Koutsoumanis et al. Under the same conditions, a strain of L. The conclusion from all these results is that the growth of bacteria in colonies differs from the planktonic growth, i below a specific inoculation level depending on the species or the strain of bacteria and ii especially in stressful conditions because of narrower boundaries of conditions conducive to growth.

The heterogeneity in and around the colonies results from different aspects of the bacterial activity: growth rates or lysis , substrate consumption and metabolic activity.

The potential existence of microgradients within and around the colony would suggest that the environmental conditions pH, oxygen, redox potential, etc.

The metabolic action, either with respect to the consumption of substrates or the production of end-products, is likely to create microgradients of concentration that cause the heterogeneity of bacterial activity inside the colony. Firstly, the studies about the heterogeneity of growth and metabolic activity inside colonies are discussed. Then, with the technical evolution from micro-electrodes to the recent imaging techniques, the possible existence of microgradients in the environmental parameters inside and around the colony is discussed.

These parameters include the pH, resulting from production of lactic acid, and oxygen concentration, resulting from its consumption by bacteria. Two types of heterogeneity within the colony have been shown: i a gradient of growth rates or metabolite production from the center to the periphery of the colony arising because of the concentrical growth pattern Wimpenny, , and ii a random heterogeneity due to random differences of division or gene expression between cells Mikkelsen et al.

Different aspects of the heterogeneity can be observed: morphology, growth rates, or metabolic activity metabolite pattern. The spatial heterogeneity of colony growth, between active growth for the periphery cells and maintenance activity for the central cells where glucose was scarce, was modeled for Bacillus Kreft et al. Typhimurium McKay et al. Soon after the formation of the colony 13 h , the growth rate at its periphery was twice that of the center, demonstrating that the periphery of a large colony was the region of maximum metabolic activity Figure 5 and Table 1.

The growth slowed down in the center of the colony due to the accumulation of lactic acid possibly combined with the depletion of glucose or carbon sources. Figure 5. Adapted from McKay et al. Metabolic heterogeneity has been described by the observation of gradients in lysis activity, as well as gradients of metabolite production or enzyme activity within the colony. An intense lysis of cells was observed in the center of colonies of Vibrio cholerae by using a vital stain of the cells Wimpenny, The same conclusion was drawn from using Fourier transform infrared FT-IR microspectroscopic mapping of large colonies of Bacillus megaterium obligate aerobes and Legionella bozemanii microaerophiles.

For smaller colonies, results are less clear. For example, the adenylate pool which includes ATP has been shown to be affected by the growth in submerged colonies of S. Typhimurium Walker et al. The authors suggested that the variation of adenylate production through incubation time, in comparison with broth culture, could be due to an heterogeneity within colonies but this heterogeneity has never been proved.

Identification at early stage of growth of bacterial colonies was possible using a new highly sensitive and non-destructive technique, chromatic confocal microscopy Drazek et al. Finally, the variability of phenotype randomly occurs when a sub-population develops under stressful conditions, either in colonies or in planktonic cultures.

This phenomenon was observed under acid stress conditions for small colonies of L. Heterogeneity of division and shape was observed in small colonies of L. In small colonies of six different strains of L. The dead cells were randomly distributed until 38 h, and were then concentrated in the center of the colony at h Ryssel et al. For this reason, small colonies are homogeneous because all cells exhibit the same growth state. The production of lactic acid from bacteria has often been suggested to be the reason why growth stops, due to the accumulation of lactic acid in and around colonies.

However, the question remained if there were also pH microgradients around small colonies or in food such as cheese. Using micro-electrodes, the first pH profiles were performed only on large colonies because of the poor resolution of the technique. Typhimurium in agar gels, Wimpenny et al. In contrast, colonies of S. Using ratio-imaging fluorescence, pH microgradients were observed in and around submerged colonies of L.

Figure 6. 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. In order to confront the observations in agar and gelatine to a real food medium, pH was measured at the microscopic level in a model cheese and in real commercial cheeses. Using ratio-imaging fluorescence, local pH was measured during the acidification of colonies of L. Regardless of the observed colony size, no pH microgradients could be observed around colonies Figure 7.

Furthermore, in the same model cheese, the same strain of L. These results are in agreement with those described above and observed in a gelatine medium for colonies of L. The accumulation of lactic acid around the colonies has been suggested as the main explanation for the lower growth rate in renneted milk gels when compared with that in liquid milk Stulova et al.

The simplified composition no fat, no NaCl and the homogeneous structure of the model cheese Jeanson et al. Figure 7. Although one might not necessarily see the importance of colonial morphology at first, it really can be important when identifying the bacterium. Features of the colonies may help to pinpoint the identity of the bacterium. Different species of bacteria can produce very different colonies.

In the above picture of a mixed culture, an agar plate that has been exposed to the air and many different colony morphologies can be identified.

Nine obviously different colonies are numbered: some colony types recur in various areas of the plate note 3 and 4. Not only are pigment differences seen, but also size, edge, pattern, opacity, and shine. Two circles have been drawn around merging colonies, where the species of the 2 colonies are different. Trying to pick a bit of one of those adjacent colonies increases the chances of picking up another mixed culture, consisting of the 2 species that were merged together.

Measure with a millimeter rule. Magnified edge shape use a dissecting microscope to see the margin edge well. For more information on bacterial colony picking and colony picking lab equipment, contact Hudson Robotics today.

What is a Bacterial Colony? See all Posts. Published On: December 15th, Bacterial Colony Definition and Overview A bacterial colony is what you call a group of bacteria derived from the same mother cell. Why Grow Bacterial Colonies?

When done manually in a lab, the colony picking protocol is somewhat tedious and looks something like this in a simplified manner: An agar plate is studied to identify a suitably isolated bacterial colony to pick. Once selected, the colony is picked up using a toothpick, pipette tip or inoculation loop and transferred to a colony picking cell culture medium, which could be liquid or agar. Medium is then incubated overnight to encourage further bacterial growth.

The resulting colonies are then tested to determine if the end goal has been obtained — either the colony successfully produces a product, or a unique bacteria is found with unique therapeutic or other commercial properties.