Chapter 1 General Introduction
2.2 Materials and Methods
2.2.1 Bacterial strains, growth media and sub-culture
Escherichia coli O157:H7 strain 2988 (Shu & Gill, 2002) and Salmonella enterica serovarTyphimuriumATCC1772 (Gill et al., 2001) were kindly provided by Bioactives Research New Zealand Ltd. (Auckland, New Zealand). Listeria monocytogenes Scott A ATCC49594, Staphylococcus aureus ATCC25932 and Vibrio parahaemolyticus (local isolate) were held in this laboratory. The stock cultures were stored at -80oC.
The stock bacterial strains were sub-cultured aerobically in Brain Heart Infusion (BHI) broth (CM225, Oxoid, Hampshire, UK) or on BHI agar (BHI broth supplemented with 1.5% agar from Sigma, St. Louis, MO, USA), except Vibrio parahaemolyticus which was sub-cultured and assayed aerobically in Trypticase Soy Broth (TSB; CM129, Oxoid, Hampshire, UK) supplemented with 3% sodium chloride (TSBS) (Heinis et al., 1977) or TSBS agar (TSBS broth supplemented with 1.5% agar from Sigma, St. Louis, MO, USA).
For subculture, each of the test strains was streaked onto an agar plate and incubated for 24 h at 37oC. A single colony of each strain was re-streaked onto a fresh agar plate and incubated for 24 h at 37oC. An isolated colony was used to inoculate 10 mL of broth medium and then incubated for 16 h at 37oC. The resulting cultures were used as stock for further use.
2.2.2 Enumeration of bacterial samples
The concentrations of bacterial samples were enumerated by plate count on BHI or TSBS agar plates. A drop plate method was employed for enumeration of bacteria throughout this study, following the descriptions of Herigstad et al. (2001) and Chen et al. (2003) with modifications. Briefly, 25 µL of bacterial suspension was loaded into the first well of each row in a 96-well plate (Nunc, Roskilde, Denmark) pre-loaded with 225 µL of sterile 0.1% peptone (BactoTM, Becton Dickinson, MD, USA) water per well. Serial dilutions (10 fold) were made using a multi-channel pipette (CAPP, Odense, Denmark) by transferring 25 µL from column i into medium in the next column {i + 1} after mixing 10 times. This process was repeated until the last dilution. Pipette tips were changed between dilutions. Thereafter, three replicates of 25 µL from each of the selected dilutions were plated onto an agar medium using a single-channel pipette. Plates were allowed to dry, and then placed into an incubator for 24 - 48 hours at 35oC. Colonies were enumerated and colony forming units per mL (CFU/mL) were calculated as:
The number of bacteria
= number of colonies x dilution factor x 1000 x 25-1 (CFU/mL).
The count was recorded as the mean of three replicates.
2.2.3 Preparation of the inocula
For preparation of the inocula, each of the test pathogens was sub-cultured on BHI agar plates and BHI broth, except V. parahaemolyticus which was grown and enumerated on TSBS agar plates or broth. One hundred microlitres of the resulting culture was then used to inoculate 10 mL of fresh broth medium which was incubated at 37oC for 16 h. After 16 h, the culture was distributed in 1 mL portions in 2 mL sterile tubes and stored at -80oC. A frozen aliquot was thawed in a water bath (37oC) for 10 min and then serially diluted in pre-warmed 0.1% (w/v) peptone water. The diluted bacterial suspension was enumerated by standard plate count using the drop plate method described in Section 2.2.2 on standard plate count agar (CM463, Oxoid, Hampshire, UK) or TSBS agar (TSBS, supplemented with 1.5% agar).
2.2.4 Preparation of growth media
The media used in the experiments were buffered BHI and TSBS in 0.2 M phosphate buffer (bBHI and bTSBS, respectively) at a final pH of 6.8 and 6.3, respectively, after an overnight equilibration under CA (see Section 2.2.5). The bBHI and bTSBS were made by constitution of the required amounts of BHI and TSBS media powder in 0.2 M phosphate buffer at initial pH values of 7.6 and 6.6, respectively, which were predetermined to give pH values of 6.8 and 6.3, respectively, after autoclaving and equilibration under CA.
2.2.5 CA system and growth conditions
The determination of growth curves was performed using 600 mL glass jars specially constructed for this purpose, provided with a central opening which was sealed with a silicone septum. The jars were filled with 100 mL of media and autoclaved (121oC, 15 min). The jars were connected in series with sterile tubing and isolated from each other using sterile 0.2 µm filters (Millex®-FG, Millipore, Billerica, USA) (Figure 2.1).
Figure 2.10 The controlled atmosphere system for the evaluation of its effect on growth of foodborne pathogens. The compressed gases were premixed through a tubing system and
introduced into individual jars, which were separated by filters (Millex®-FG). The
inoculation and collections of samples were carried out aseptically by syringes through a septum on each of the jars.
The CA was composed of pure compressed nitrogen and CO2 pre-mixed at a controlled concentration of 40% CO2 and 60% N2, with a residual concentration of O2 less than 40 ppm. The pre-mixed gases were filtered through a 0.2 µm filter (Millex®-FG) and
introduced to the jars at a flow rate of 40 mL/min to produce a CA environment. The gas composition was monitored with a Novatech CO2/O2 analyser (Model 1673-5,
Novatech Controls Pty Ltd, Cheltenham, Victoria, Australia). The jars in the system were filled with bBHI or bTSBS at initial pH values of 6.8 and 6.3, respectively, after equilibration. The controls consisted of jars with bBHI or bTSBS at pH 6.8 and 6.3, respectively, with free exchange of air without CA. The trials were carried out in triplicate for each condition at 20oC for all 5 pathogens and at 7o
2.2.6 Inoculation
C for L. monocytogenes, the only one of the five organisms known to grow well at this temperature.
The enumerated stock cultures were thawed and diluted in 0.1% peptone water to a concentration of approximately 1 x 105 CFU/mL and used as inocula. Each jar was inoculated with 1 mL of inoculum to give an initial concentration of 1 x 103
2.2.7 Sampling
CFU/mL. The inoculation was carried out aseptically using a spinal needle (Becton Dickinson & Co., Frankin Lakes, NJ, USA) through the silicone septum of the opening on the lid of each jar.
Samples were taken aseptically using a spinal needle (Becton Dickinson & Co., Frankin Lakes, NJ, USA) through the silicone septum of the opening on the lid at intervals of 4 to 6 hours after mixing by swirling. A plate count of the sample was carried out immediately as described in Section 2.2.2.
2.2.8 Fitting of growth data to predictive model
The growth data of bacteria in broth were subjected to analysis using Gompertz Solver software (Version 1.0, Agricultural Research Service, USA). A predicted growth curve was then produced and derived growth parameters were obtained for a given data set based at a lowest sum of squares error (SSE). The model is described below:
)]
(
[
)
/
(CFU
mL
A
Ce
e
B
t
M
Log
=
+
−
−
−
where e is the base of the natural logarithm (approximately = 2.71828) and A, B, C and M are the parameters that define the shape of the sigmoidal curve. A represents the initial (minimum) CFU/mL value for the growth curve data, B the slope of the
regression line at the inflection point for the curve, C the difference between the minimum and maximum CFU/mL values and M the midpoint of the regression line used for the parameter B estimate. The t represents inoculation time in hours following inoculation.
With bacterial growth data, the parameter values for A, B, C and M can be found that make the sigmoidal curve most closely fit the observed data. Therefore, the kinetic parameters, including the exponential growth rate (EGR), lag phase duration (LPD), and the maximum population density (MPD) can be calculated from the parameter estimates (A, B,C and M) as described by Buchanan & Phillips (1990):
The exponential growth rate (EGR) = BC/e The lag phase duration (LPD) = M-(1/M) Maximum population density (MPD) = A + C
The observed and predicted growth data were plotted using SigmaPlot software (Version 8.0, SPSS Inc., Chicago, USA).
The predicted growth kinetics parameters under CA and non-CA conditions at different pH values and temperatures were analysed by one-way analysis of variance (ANOVA) using Minitab Software (release 14, PA, USA).