The 23rd Annual Meeting of the Thai Society for Biotechnology
“Systems Biotechnology: Quality & Success”
O-VI-8
Effect of Oxford selective agents on colony growth of
Listeria and non-Listeria strains
Ruamporn Liamkaew, and Aluck Thipayarat
Department of Food Engineering, Faculty of Engineer, King Mongkut’s University of Technology Thonburi, Bangkok,
10140, Thailand.
Abstract
The colony expansion of Listeria ivanovii, Escherichia coli and Salmonella anatum was
evaluated on OXA selective agar with various selective agent concentrations. The digital imagery of
the colony expansion was processed to estimate the number of pixels of the colony-forming area by
using image analysis technique. A kinetic model (i.e., logistic model) was applied to estimate growth
characteristics of bacterial colony. All bacterial strains showed equivalently optimal growth
characteristic when grown on the OXA without selective agents. The growth profile of Listeria
colonies was improved when the concentration of OXA selective agents was minimal. Higher
concentrations of the selective agent were able to effectively suppress the growth of both competing
bacteria and provided better selectivity toward L. innocua. The optimal concentration of OXA
selective agents was 6%. At this concentration, L. innocua was able to grow very well and distinctly
showed the unique black halo while the colony growth of E. coli and S. anatum was inactivated
effectively.
Keywords: colony growth, image analysis, Listeria, oxford agar, selective agents
Listeria
detection
process
manufacturing purposes.
Introduction and Objective
This research gears toward studying the
colony growth of L. innocua on solid agar
substrate.This bacterium was used as a model
microorganism representing the pathogenic L.
monocytogenes. E. coli and S. anatum were
included to represent competitive bacteria
commonly found in food processing and used to
study the improvement of isolation step in
Listeria detection process. Many foods can be
contaminated by Listeria spp., including raw
vegetables, raw milk, fish, poultry, process
chicken and beef (1). Most universal standards
and microbiological manuals refer to Oxford
agar as a popular selective agar media to detect
and enumerate Listeria spp. (2).
The selective agents in the Oxford medium
allow simultaneous suppression of background
flora (e.g. Escherichia coli and Salmonella spp.)
but promote the growth of Listeria spp.
However, the selective agents not only inhibit
non-Listeria organisms, but are also detrimental
to some degree to Listeria spp. (3). Hence, most
applied inhibitors prolong growth of Listeria in
agar culture and delay the detection process due
to the slow colony expansion. In this research,
the influence of selective agent concentrations
on colony growth of Listeria and non-Listeria
strains was studied in order to optimize the
for
food
Materials and Methods
2.1 Bacterial strains
Listeria innocua, Escherichia coli and
Salmonella anatum used in this experiment were
obtained from Department of Medical Science,
Thailand. Frozen stocks of bacterial strains were
cultured in Tryptic Soy Broth for 24 h at 37C
and prepared to reach ~102 CFU/ml. Bacterial
culture (20 l) were inoculated on Oxford media
and incubated at 37C for 48h.
2.2 Media preparation
Oxford agar (OXA) media was prepared
according to the FDA’s Bacteriological
Analytical Manual (BAM) method. The
concentrations of selective agents were varied at
12.5%, 6%, 3% and 1.5% of the conventional
OXA media recipe. The OXA medium without
selective agent (OXA+0%SA) was used as a
positive control in this experiment.
2.3 Kinetics growth of bacterial colony
The rate of development of microbial
growth was captured and evaluated to monitor
colony expansion. The colony area was
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The 23rd Annual Meeting of the Thai Society for Biotechnology
“Systems Biotechnology: Quality & Success”
O-VI-8
Table 1Bacterial colony images after 48 hour of
incubation using various concentrations of selective
agents on Oxford agar
calculated in pixels using ImageJ 1.44 (Wayne
Rasband, USA). The colony image was digitized
using a digital microscope equipped with 1.3 MP
CCD (DinoLite Model: AM413ZT, Taiwan).
Media
Results and Discussion
Colony areas of L. innocua, E. coli and S.
anatum were monitored under different selective
stresses in the Oxford-based agar (Figure 1).
Double-dilution of the selective inhibitors was
carried out starting from 12.5% to 0% and the
colony area expansion was facilitated by the
decrease of inhibitor concentrations. Not only
the lag time was shortened but also the colony
grew to a larger size. The colony measurement
showed the colony area was doubled after 48 h
of growth on medium without inhibitors.
OXA+
100%SA
OXA+
6%SA
OXA+
1.5%SA
OXA+
0%SA
L.
innocua
E. coli
S.
anatum
References
Posfay-Barbe KM, Wald ER. Listeriosis.
Semin Fetal Neonatal Med. 2009;14(4): 228233.
2. Pinto M, Burri S, Mena C, Almeida G,
Carneiro L, Teixeira P, Gibbs PA.
Comparison of Oxford A, PALCAM and gar
and Listeria monocytogenes blood agar for
the recovery of L. monocytogenes from
foods and environmental samples. FOOD
CONTROL. 2001;12:511-514.
3. Jacobsen CN. Short communication The
influence of commonly used selective agents
on the growth of Listeria monocytogenes.
INT J FOOD MICROBIOL. 1999; 50:221226.
1.
Figure 1. Colony area growth of L. innocua under
various inhibitory stress using Oxford-based agar
The distinction between the control (the
conventional Oxford agar) and the modified
recipes was lead-time to detect Listeria colonies.
The Listeria colonies of most modified Oxford
agars was detected as early as 12 h after
inoculation; however, that of the standard
Oxford required up to 24 h. The comparison of
the colony images of Listeria colonies after
incubation for 48 h displayed the effect of
Oxford inhibitors (Table 1). The reduction of
Oxford inhibitors to 6% was very effective to
promote colony growth of L. innocua while
discouraging growths of competing strains (i.e.,
E. coli and S. anatum). At the 1.5% treatment
and lower, the modified OXA was not selective
for L. innocua growth and S. anatum showed
more resiliency than E. coli toward the
application of Oxford inhibitors.
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