How do colonies on the surface of the

However, S. It grows very rapidly at all temperatures into medium sized colonies. Your ability to isolate and see the three organisms on your plate is dependent on appropriate incubation conditions. Your plates will be incubated at room temperature for 48 hrs. You should be able to identify the three organisms based on colony size and pigment.

There are several methods of streaking for isolation. The vast majority of our students have been most successful with the quadrant method of streaking which is described below. Microbiology Resource Center. Introduction There are approximately 10, named species of microbes. There are two main ways to isolate organisms. Streaking for isolation on an agar plate The pour plate method Streaking for isolation on an agar plate involves the successive dilution of organisms until you have the cells at a low enough density that single cells are physically isolated spatially to give rise to recognizable individual colonies.

Overview You will be given a broth sample containing three organisms, Staphylococcus xylosus, Serratia marsescens, and Escherichia. Materials 1 mixed-culture in tryptic soy broth TSB tube containing Staphylococcus xylosus, Serratia marsescens, and Escherichia.

Label your plate with your name, date, section, and organism. Refer to the aseptic technique protocol. Be sure that you have adequately mixed your broth tube so the organisms are uniformly suspended in the broth. Recap your TSB tube. You can do the next part with your plate on the lab bench or holding it in your hand.

You decide which works best. Lightly drag your loop back and forth across the surface of the agar. Refer to Figure 1. The more you drag the more bacteria you deposit. Do not squeeze the teat bulb of the pipette after it is in the broth as this could cause bubbles and possibly aerosols. Gently release the required volume of inoculum onto the centre of the dish. Replace the lid. Remove the cap with the little finger of your left hand.

Microbiology teacher resources Society for General Microbiology — source of Basic Practical Microbiology, an excellent manual of laboratory techniques and Practical Microbiology for Secondary Schools, a selection of tried and tested practicals using microorganisms.

Microbiology online MiSAC Microbiology in Schools Advisory Committee is supported by the Society for General Microbiology see above and their websites include more safety information and a link to ask for advice by email. Search Search. You are here: Practical Biology Standard techniques Making a pour plate.

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. Adapted from Jeanson et al. Oxygen O 2 is one of the most important parameters for determining the behavior of bacterial growth. For example, for facultative anaerobes such as S. On the other hand, for aerobes such as Pseudomonas putida , the top layer of the colony was the zone of the most intense cell division Reyrolle and Letellier, Oxygen gradients were first measured inside a colony of B. It has been measured mainly on large surface colonies because O 2 is present over the whole surface of the colony.

The O 2 concentration decreases with depth moving within the colony and also in depth through the medium below and around the colony in all directions Wimpenny, However, Tammam et al.

Instead, these authors developed in situ mass spectroscopy measurements to investigate the concentrations of O 2 and CO 2 concentrations in MRS agar inoculated with a strain of L. They showed that in the aerobic zone, there was a gradient of O 2 concentration through a 5 mm depth in agar after 24 h of inoculation whilst gradients of CO 2 concentration occurred in the same zone but through a 20 mm depth Figure 8.

Figure 8. A MIMS membrane inlet mass spectrometric probe was inserted through column of growth. Adapted from Tammam et al. For the first time in Cheddar cheeses, these authors also investigated the evolution of the concentrations of O 2 and CO 2 at depth just below the rind Tammam et al.

After 15 days, no O 2 could be measured at a depth of 4 mm Figure 9. The small colonies of lactococci, observed within the curd by confocal microscopy, were suggested as responsible for the consumption of O 2 leading to the decrease of the redox potential known in Cheddar cheese manufacture, for example Caldeo and McSweeney, The CO 2 concentration was also directly linked to the heterofermentation of lactococci colonies, which produced up to 16 mM of CO 2 after days of ripening at a depth of 15 mm.

A chemically reducing environment, i. However, in contrast to pH, local variation of the redox potential around colonies has never been investigated at the microscopic scale.

Figure 9. In conclusion, it seems clear that heterogeneity can occur within and around the colonies of bacteria with respect to several parameters directly linked to the bacterial metabolic activity. However, the size of the colonies, and thus the inoculation level, is a major factor determining heterogeneity and the existence of such microgradients. To sustain the growth of bacteria in colonies, substrates have to diffuse from the solid food matrix to the colony. At the same time, end-products have to diffuse away from the colony to the matrix, especially if they inhibit bacterial growth such as lactic acid.

The existence of diffusion limitations is the first hypothesis put forth to explain slower growth of the cells in the center of the colony and the microgradients arising in and around the colony. This paradigm has been widely used by different groups to explain their results Brocklehurst et al. Even if microgradients of pH and O 2 have been measured, to our knowledge, microgradients of redox potential, inhibitors, or substrates have not, and their existence is still to be shown.

Furthermore, some of these studies initially suggested diffusion limitations of the substrates, but then concluded, in the case of numerous and small colonies in favorable growth conditions, that there were no mass transfer limitations of substrates and lactic acid Stecchini et al. For instance, Malakar et al. They obtained a mean diffusion coefficient of 2. Therefore, Malakar et al. This conclusion is questionable though, because others studies such as those conducted by Ribeiro et al.

These results demonstrated that the diffusion coefficient of glucose was dependent on the gel microstructure because it decreased with the pore size of the gel network. On the other hand, the diffusion rate of glucose and a small protein insulin-like growth factor was shown to be independent of the pore size of the gel with an increased concentration of agar Stecchini et al.

Finally, the little number of studies on diffusion in gels does not allow clear conclusions on the limiting effect of diffusion of substrates or inhibitors. In cheese, diffusion of small molecules water, NaCl, lactose has been studied while knowledge on diffusion of macro-molecules lacks of data Floury et al.

The major conclusion was that the dextrans which are flexible and charge-neutral molecules as large as kDa were able to diffuse through the model cheese as well as the milk proteins which are rigid and charged molecules. However, the milk proteins were more hindered in the cheese protein network than dextran molecules of similar hydrodynamic radii Silva et al. From these studies, it remains very difficult to draw specific conclusions about the potential effects of diffusion limitations of substrates or end-products on bacterial growth and metabolic activity.

Indeed, these diffusion rates have now to be compared to enzymatic reaction rates in immobilized conditions, which are, to our knowledge, still unknown and difficult to determine experimentally. We can only suggest that diffusion within the model cheese matrix is probably not the most limiting factor for the growth of cells at the periphery of colonies where the concentration of the substrates is very high.

However, one can wonder what happens to the molecules, especially large molecules, upon reaching the center of the colony. In other words, is the colony porous enough to large molecules, either to penetrate the colony or to be expelled from the colony when released after bacterial lysis?

This section outlines the consequences of the immobilization of bacteria in colonies on their growth and metabolic activity in order to identify general principles and theoretical concepts of importance for fermented food products. When immobilized as colonies in a solid matrix, bacteria experience multiple constraints on their growth pattern: they develop as colonies and diffusion limitations may limit their access to the substrates.

Micro-colonies have previously been defined as colonies displaying a radius R col as small as 1. However, all these studies were either focused on micro- or on macro-colonies but never integrated data on both. The present overview of literature led to the conclusion that micro- and macro-colonies were two different conditions of growth depending on a threshold of size, determined by the initial level of population.

Figure 10 illustrated the two conditions of colonies along with the planktonic form of culture for comparison, defined as follows:. Figure Schematic diagram of the three culture conditions for bacterial cells and their main characteristics; planktonic culture conditions are the most studied.

The threshold between micro-colonies and macro-colonies is determined by the inoculation level above which growth in optimal conditions resembles to planktonic growth. The hypothesis of diffusion limitations around colonies seems relevant for macro-colonies but not for micro-colonies as the growth rate of bacteria is then comparable to that in the exponential phase of planktonic growth McKay and Peters, ; Malakar et al. On the other hand, if there is heterogeneity of growth rates inside the macro-colonies, the hypothesis is that some of the substrates, most likely the larger molecules, do not reach the center of the colony so that those cells cannot access such substrates.

These hypotheses lead to the question: are colonies porous to large molecules? Schematic representations of the two concepts of interactions between the colony and the matrix; arrows show the diffusing molecules.

The exchange between the micro-environment and the colony is thus that of each of the cells and depends neither on the size of the colony, nor on their number. This concept is close to the planktonic condition in term of interaction of bacteria with the medium. Thus, for a given number of bacteria, the total exchange area is then determined by the size and the number of colonies, and is of major importance in governing the activity of the colonies within the matrix.

The exchange surface overall exchange surface per unit of medium volume increases with the number of colonies as their size decreases Jeanson et al. In the case of two different spatial distributions, labeled 1 and 2, the terms S 1 and S 2 represent two different exchange surfaces resulting from the two different inoculation levels I 1 and I 2.

We assume that i the packing density of cells and the volume of individual cells inside the colonies are equal for both spatial distributions; ii the inoculation level is equal to the number of colonies one cell gives one colony. The low precision of the experimental measurements may explain this difference.

These concepts are theoretical but may be of great value in food processing. As described above, milk proteins and dextrans molecules up to kDa can diffuse within in a model cheese, but are these large molecules able to also diffuse in to the colony?

A first study explored the resistance to diffusion exerted by cells of E. We thus investigated the porosity of a bacterial colony to molecules of different sizes Floury et al. Indeed, dextran molecules as big as kDa larger than milk proteins can diffuse through a bacterial colony but their diffusion coefficient could be limited by their size. We observed the same results with strains of Lactobacillus rhamnosus and L. In conclusion, it was clearly demonstrated that the diffusion behavior of macromolecules through bacterial colonies immobilized in a model cheese did not depend so much on the size of the diffusing solute molecules, but mainly on their physicochemical properties Floury et al.

In cheese, carbon sources such as lactose are soluble and can diffuse freely as in agar or gelatine medium. On the contrary, nitrogen-based substrates are mostly caseins which are bound up in the network and cannot diffuse, except for a minor proportion of free caseins. Assimilable nitrogen substrates are peptides produced from the activity of bacterial cell-wall proteases.

In the case of colonies embedded within cheese, only the cells on the periphery can theoretically access the caseins in the network. Taking cheese as an example, this raises the questions: i how does the spatial distribution of colonies influence the bacterial metabolism and ii how do the cells at the center of the colony access the nitrogen substrates, i.

In order to explore this hypothesis, we measured the influence of two different spatial distributions of micro-colonies of L. The inoculations levels, respectively, 1.

As a consequence, small colonies cheeses contained higher amounts of amino acids and some of the peptides than big colonies cheeses. Nevertheless, the increase in concentration of metabolites between small and big colonies cheeses ranged from 1. The results obtained with the small and big colony cheeses are in agreement with the observations on the porosity of colonies. The relatively small proportion of cells in the periphery could produce enough peptides for the cells of the whole colony.

Another study performed in a renneted milk gel showed that the overall concentration of total free amino acids was 1. Even if rennet increased the hydrolysis of caseins into peptides, this result supports the idea that peptides diffuse inside the colony where they are further degraded into amino acids by intracellular aminopeptidases.

However, the growth rates were subsequently lower in the milk gels than in milk during the second exponential phase when the bacterial strain had to synthesize its own cell-wall protease to sustain growth Stulova et al. The hypothesis given by the authors was that there was an accumulation of lactic acid around colonies. This result also supports the argument of a limited access to caseins, leading to the slower growth rate , but to a free access to the peptides.

Most likely, the colony acts as a selective filter depending on the properties of the diffusing molecules with a greater preference for flexible and neutral molecules regardless of their size. The objective of this review was a comprehensive understanding based on published literature of the impact of bacterial growth as colonies in a food context.

Finally, the spatial distribution emerges as the most crucial parameter in determining whether the immobilization of bacteria has an impact or not. The conclusions differ widely: i if colonies are small and numerous micro-colonies , the implications of growing in colonies rather than as free planktonic growth are minor; ii whereas if colonies are large and relatively few in number macro-colonies , the implications of such immobilization become significant, mostly in terms of a relatively lower growth rates and their lower resistance when under conditions of stress.

In the case of bacterial contamination or indigenous microflora, the initial population is low and colonies thus develop as macro-colonies. It is thus important to increase the understanding on the behavior of pathogenic bacteria in solid matrices in order to improve the predictive growth models in solid foods. In the case of LAB in fermented foods, the inoculation levels are high and one can wonder if the growth in micro-colonies really impacts on the growth and the metabolic activity of bacteria in foods by comparison with that as planktonic growth.

Moreover, interactions and even communication between colonies, like quorum sensing, is still unexplored in solid food media Skandamis and Nychas, The newly available imaging techniques may open a great field of research in this respect.

SJ: design and wrote the review manuscript. JF: expert in the diffusion of molecules in cheese and porosity of colonies; improved the review manuscript. VG: expert in proteolysis by bacteria; improved the review manuscript.

SL: initiated the topic in the lab; improved the review manuscript. AT: head of the research group; design and extensively improved the review manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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