Scientific Publications - Work Done by Microbiology Reader Bioscreen C

Lett Appl Microbiol. 2004;38(3):181-184

A comparison of the traditional method  of counting viable cells and  a quick microplate method for monitoring  the growth characteristics  of Listeria monocytogenes

K. Horáková, M. Greifová, Z. Seemannová, B. Gondová and G.M. Wyatt

ABSTRACT

Aims: To determine: (i) the growth parameters (specific growth rate, lag time, asymptotic amount of growth, generation time and time for maximum growth rate) of Listeria monocytogenes in different broths by standard cultivation methods and (ii) whether a microplate method in conjunction with a standard nondedicated plate reader could be adapted to routine assay.

Methods and Results: Growth curves were determined from cell numbers in a standard tube method at 2 h intervals by serial dilution and plating, and in a microplate method by absorbance measurements. Growth curves were fitted with a modified Gompertz function.

Conclusions: The microplate method was similar to the standard cultivation methods in accuracy, required less chemical reagents, and considerably reduced the time required for analyses. This work also illustrates that growth characteristics of bacteria are not necessarily constant, and depend on the methodology used.

Significance and Impact of the Study: It is not the intended purpose of this paper to present all the data for the media tested but instead to illustrate the success of the microplate method for studying growth kinetics compared to a standard cultivation method and system precission. The method will be of considerable benefit to laboratories unable to afford dedicated workstations.

INTRODUCTION

For Listeria monocytogenes, it has been well-documented that both in vivo virulence (Buncic et al. 2001) and expression of virulence factors, such as transcriptional activator protein (PrfA), internalin, listeriolysin, phospholipases, metalloprotease and actin polymerization proteins (Leimeister-Wachter et al. 1992; Conte et al. 1994; Buncic and Avery 1996; Renzoni et al. 1997), are influenced by bacterial growth phase and temperature.

The efficiency of rapid methods such as ELISA for the detection of Listeria may also be affected by the characteristics of the culture environment, and little information on this subject is available. The objectives of this study were to determine: (i) the growth parameters (specific growth rate, lag time, asymptotic amount of growth, generation time and time for maximum growth rate) of L. monocytogenes in different broths by standard cultivation methods and (ii) whether a microplate method could be adapted for growth studies to reduce the time and media requirement and while still maintaining the accuracy and reproducibility of growth parameter measurements.

MATERIALS AND METHODS

Bacterial strains and enrichment media

The cultures used in this study were L. monocytogenes 88/049 and 86/010 from the collection at the Institute of Food Research (Norwich, UK). Stocks of these cultures were kept on Oxford agar at +4°C.

Growth kinetics of the chosen Listeria strains were measured in variety media, including brain-heart infusion broth (BHI), Listeria enrichment broth (LEB), UVM formulation of Listeria enrichment broth (UVM1), Fraser broth without ferric ammonium citrate (Fraser; Merck, Darmstadt, Germany), glucose tryptone yeast extract broth (GTK) and nutrient broth no. 2 (NB2; Imuna, ariské Michalany, Slovakia). All these media were prepared and handled according to the manufacturer's guidelines.

Growth curves

The strains were cultured in BHI broth for 16 h at 37°C. For the standard cultivation method, from a 18-h culture grown in BHI, the inoculum was prepared by decimal dilution to a final concentration of 10³ CFU ml ¹. The cultures were incubated stationary at 37°C. Initial number of bacteria was 5  10³ CFU per test tube. At each time point (2-h intervals), samples were serially diluted and plated onto Oxford agar plates to estimate culturability. The plates were incubated at 37°C for 24 h before counting colonies.

For the microplate method both L. monocytogenes strains (inoculated with initial concentrations of 10³-10⁷ CFU ml ¹) were prepared in eight different broth media and added (200  l) to each well of a 96-well plate. Microplates were incubated at 37°C and optical density at 630 nm was determined at 2-h intervals by using a microplate reader (Humareader, Human, Germany).

Curve fitting

Growth curves were fitted with a modified-Gompertz function as described by Gibson et al. (1988). This function has the form:

where N _(t) is the density of population in time t, A is the value of the lower asymptote, C is the difference of values of upper and lower asymptote, M is the time at which the exponential growth rate is maximal and B is the slope of tangent to the curve at M.

In our experiments, we used relative population N _(t)/N ₍₀₎ or A _(t)-A ₍₀₎ for densimetric assay. Following this, the parameter A in eqn (1) is equal to zero.

Equation (1) then changes to:

RESULTS

Growth curves were generated by using optical density values and/or culturability. Growth parameters (specific growth rate, lag time, asymptotic amount of growth, generation time and time for maximum growth rate) were calculated by fitting the growth curves using a modified Gompertz equation (Gibson et al. 1988). Growth parameters obtained by standard cultivation method for L. monocytogenes strains in various growth media are presented in Table 1.

The correlation of values obtained by the standard cultivation and microplate methods are presented in Fig. 1. For the correlation of growth rate, the time when the growth rate reached the highest value was chosen (maximal growth rate). At identical initial inocula (10³ CFU ml ¹ for both methods) these times were prolonged for the microplate method compared with the standard method in each monitored culture media. The most marked difference, 5 h, was observed in UVM medium (Fig. 1).

All further experiments were made by the microplate method. At first, the influence of the density of the inocula between the range 10³-10⁷ CFU ml ¹ on the growth character of two L. monocytogenes strains was studied (Table 2). The effect of inoculum level on growth rate varied with strain of L. monocytogenes and culture medium (Table 2). The maximum concentration of cells was reached at all initial inocula, but at times it was of a reverse proportion, that were inversely related to the inoculum density (Fig. 2).

According to the specific growth rate and mean generation time the efficiency of the growth media for L. monocytogenes strains were in the order BHI > LEB > GTK >Fraser > UVM > NB2 (Table 2).

In addition to its effect on growth rate, the influence of inoculum on the maximum optical density reached was determined. Changes in maximum optical density were similar for both strains of L. monocytogenes (BHI > LEB >GTK > UVM > Fraser > NB2) in the range O.D. 1·1-0·5.

FIGURES

Fig. 1 Correlation of time to reach maximum specific growth rate (M) for Listeria monocytogenes 88/049...

Fig. 2 Effect of inoculum level on growth curves for Listeria monocytogenes strain 88/049 () 10³, () 1...

 
Table 1 Growth parameters obtained by standard cultivation method for Listeria monocytogenes strains i...

 
Table 2 Effect of inoculum level on _m (specific growth rate) of Listeria monocytogenes in various grow...

DISCUSSION

Whilst enrichment of samples in Listeria-selective media is widely used in conjunction with antibody-based Listeria diagnostic assays, the detection of Listeria spp. in an ELISA format will depend not only on cell number, but also on the expression of cellular antigen. The latter factor may be affected by the choice of medium used for the isolation and growth of cells from suspect food samples prior to testing. We have developed an L. monocytogenes-specific ELISA using antibodies raised to the virulence protein internalin B (L. Karamonova, M. Blazkova, L. Fukal, P. Rauch, M. Greifova, K. Horakova, M. Tomaska, P. Roubal, G.M. Brett and G.M. Wyatt, unpublished data), and an increased amount of this protein in culture could improve the detection limit of the assay.

Assessing the characteristics of culture media and appropriate growth conditions can be tedious and time-consuming using traditional microbiological methods. The 96-well microtitre plate-based assay is a format which allows simultaneous testing of a large number of cultures, and which can be mechanized. Although specialized and/or dedicated versions of the micro-well format, such as the Bioscreen Microbiology Workstation (Thermo Labsystems Oy, Helsinki, Finland) have been used to measure growth characteristics of Listeria (Wu et al. 2000), these are expensive. However, many laboratories will already possess the standard 96-well microplate spectrophotometer used for ELISA end-point determination, and this instrument is an obvious alternative to the specialized machine for determining growth of bacteria. We therefore used such a reader for assessing the growth characteristics of potential culture media for our L. monocytogenes ELISA system.

The described microplate method was found to be suitable for studying growth of different L. monocytogenes strains in various culture media. From a range of standard culture media tested, GTK gave the fastest growth rate for L. monocytogenes, although several other media were almost as good (Table 1).

The microplate method was similar to the standard method in accuracy of determination, required less chemical reagents, and considerably reduced the time required for analyses. It will be of considerable benefit to laboratories unable to afford the dedicated workstations.

ACKNOWLEDGEMENTS

The authors are very grateful to the European Union for financial support of this work (INCO COPERNICUS project ERBIC 15-CT98-0902).

REFERENCES

•    Buncic, S. and Avery, S.M. (1996) Relationship between variations in pathogenicity and lag phase at 37°C of Listeria monocytogenes previously stored at 4°C. Letters in Applied Microbiology 23, 18-22.

•    Buncic, S., Avery, S.M., Rocourt, J. and Dimitrijevic, M. (2001) Can food-related environmental factors induce different behaviour in two key serovars, 4b and 1/2a, of Listeria monocytogenes? International Journal of Food Microbiology 65, 201-212.

•    Conte, M.P., Longhi, C., Petrone, G., Polidoro, M., Valenti, P. and Seganti, L. (1994) Listeria monocytogenes infection of Caco-2 cells: role of growth temperature. Research in Microbiology 145, 677-682.

•    Gibson, A.M., Bratchell, N. and Roberts, T.A. (1988) Predicting microbial growth: growth responses of Salmonella in a laboratory medium as affected by pH, sodium chloride, and temperature. International Journal of Food Microbiology 6, 155-178.

•    Leimeister-Wachter, M., Domann, E. and Chakraborty, T. (1992) The expression of virulence genes in Listeria monocytogenes is thermoregulated. Journal of Bacteriology 174, 947-952.

•    Renzoni, A., Klarsfeld, A., Dramsi, S. and Cossart, P. (1997) Evidence that PrfA, the pleitropic activator of virulence genes in Listeria monocytogenes, can by present but inactive. Infection and Immunity 65, 1515-1518.

•    Wu, Y., Griffiths, M.W. and McKellar, R.C. (2000) A comparison of the Bioscreen method and microscopy for the determination of lag times of individual cells of Listeria monocytogenes. Letters in Applied Microbiology 30, 468-472.

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