This study investigated the effect of varying concentrations of LB nutrient on the growth of Escherichia coli and Lactobacillus plantarum biofilms. Co-cultures and single cultures of each bacteria were grown at LB concentrations of 20g/L, 40g/L, and 60g/L. Results showed L. plantarum grew best at 40g/L while E. coli favored higher concentrations. The co-culture mimicked L. plantarum growth at lower concentrations but shifted to resemble E. coli growth at 60g/L, indicating nutrient levels influence bacterial competition. This suggests probiotic effectiveness may decrease under conditions of high nutrient availability that favor pathogenic bacteria like E. coli.

1. Assessing the effects of variable LB nutrient concentration
on Escherichia coli and Lactobacillus plantarum growth in
solid agar
A study in the co-culture growth dynamics of bacterial biofilms
MINA KODSI
Rensselaer Polytechnic Institute
Department of Biology
Troy, NY 12180
EPEC’s are a pathogenic strain of the common Escherichia coli bacterium naturally found in the lower
intestine of mammalian species. This pathogenic strain is known for its ability to secrete toxins into the
epithelial cells lining the intestine walls – altering host cell monolayer resistance, macromolecular per-
meability, and morphology. The objective of this experiment was to determine the ideal nutrient con-
centration that enables Lactobacillus plantarum to outgrow Escherichia coli when grown in co-culture
biofilms. Co-cultures of E. coli and L. plantarum and single cultures of both bacterial species were cul-
tivated under 3 LB nutrient concentrations (20g/L, 40g/L, and 60g/L) for 24 hours. Cellular growth dy-
namics were analyzed using Coomassie Blue staining, and ImageJ analysis. Percent cover-age values
were obtained for each culture and plotted to identify cross-trends between the bacteria growing in
isolation and together in a co-culture. At an LB nutrient concentration of 40g/L, covering 71% of the
plate it was cultivated in, the co-culture exhibited its highest plate coverage, and exhibited growth pat-
terns similar to that of the single culture of L. plantarum. This concludes that L. plantarum’s capability
in outgrowing E. coli is maximized at low to medium nutrient concentrations. This study also supports
the idea that LB nutrient is greatly influential on the growth dynamics exhibited in L. plantarum and E.
coli biofilms.
INTRODUCTION
Escherichia coli is a gram-negative rod shaped bacte-
rium that is naturally found in the gut of most mammalian
species. While most strains of E. coli are harmless and
exhibit beneficial symbiotic effects in food digestion,
pathogenic strains such as, Enteropathogenic E. coli
(EPEC) are a highly invasive. EPEC bacterium secretes
toxins, such as shiga toxins (also found in Shigella bacte-
rium), that cause inflammation in epithelial host cells lin-
ing the intestinal wall3
. The diffusion of these toxins into
the epithelial cells of the intestine is due to E. coli’s ability
to form bacterial biofilms across epithelial host cells.
Helpful bacterial strains, known as probiotics, prevent
and aid in the digestion of pathogenic strains of bacteria,
like EPEC. Lactobacillus plantarum is a highly effective
probiotic agent, and recently the 299V strain of L. planta-
rum was recognized as the most impactful probiotic
strain available. Like E. coli, L. plantarum can form bac-
terial biofilms across the epithelial cells lining the intes-
tine2
.
L. plantarum’s ability to form large-surface area biofilms
is why it effectively prevents pathogenic E. coli toxins
from diffusing across the apical membrane of the intesti-
nal wall and into the epithelial cells. Thus, these toxins
are unable to alter monolayer resistance, macromolecu-
lar permeability, and host cell morphology in the epithelial
cells; changes that cause the symptoms associated with
EPEC infection3
. However, the efficacy of this probiotic
is largely dependent upon L. plantarum’s capability to
outgrow E. coli in the formation of bacterial biofilms. This
capability can vary greatly due to several growth factors
including; nutrient availability, moisture levels, medium
permeability, availability of growth space, temperature,
and waste production1
. Our purpose therefore, was to
analyze one of the most significant cross-species growth
factors that impacts biofilm formation and cellular growth
dynamics exhibited in both bacterial species: nutrient
availability.
We cultivated co-cultures of E. coli and L. plantarum and
single cultures of both bacterial species under various LB
nutrient concentrations for 24 hours. Percent of plate
coverage was obtained by use of ImageJ analysis, and
the values were plotted to identify cross-trends in the
bacterial growth dynamics exhibited in all three cultures
at the three different LB nutrient concentrations. Our
results concluded that L. plantarum’s capability in out-
growing E. coli is maximized in low to medium LB nutri-
ent concentrations.
PURPOSE OF RESEARCH
The purpose of this study was to analyze the influence of
LB nutrient concentration on the colony plate coverage
exhibited in both pure cultures and co-cultures of L.
plantarum and E. coli. We hope to identify trends that will
maximize L. plantarum’s ability to outcompete E. coli in
co-culture biofilms– possibly leading to more effective
probiotic defense against pathogenic bacterial infections.

2. 2
MATERIALS AND METHODS
In order to examine the growth of these bacterial species
– in isolation and together – we prepared 9 solid agar
plates. The nutrient used in all cultures was Lysogeny
Broth (LB) and all cultures were prepared in identical
dishes to reduce extraneous variables. Cultures were all
inoculated with both strains of bacterium from standard
culture tubes of each. A total of 3 different cultures were
prepared at each of the 3 nutrient concentrations.
Preparation:
100mL of LB agar was prepared at each of the following
concentrations: 20g/L, 40g/L, and 60g/L. After prepara-
tion, they were placed in 3 separate flasks and auto-
claved. Finally, each flask (concentration) was used to
pour 3 identically sized petri dishes; one dish inoculated
with E. coli, one dish inoculated with L. plantarum, and
one dish inoculated with both bacterial cultures. These
dishes were then left to grow for 24 hours in a 37 °C
incubator.
Staining & Imaging:
To observe the general colony growth patterns of both
bacteria strains, we stained the cultures with Coomassie
blue stain without disrupting their growth in the agar me-
dium. Each of the cultures were covered with Coomass-
ie stain and left for 90 seconds. The dishes were then
rinsed from the stain using distilled water and then im-
aged using a stereomicroscope for colony growth analy-
sis with ImageJ.
RESULTS
Images were obtained using stereomicroscopy (Figure 1)
and analyzed with ImageJ software to create a plot of
nutrient concentration vs. plate coverage of the colony
(Figure 2).
Pure Cultures:
We observed a similar trend in both bacterial strains that
showed a higher rate of growth with greater plate cover-
age as the nutrient concentration increased. As we
doubled the concentration from 20 to 40g/L, the isolated
culture of E. coli exhibited a minor increase in growth
size. However, under the same increase of nutrient con-
centration, the isolated culture of L. plantarum exhibited
a major increase in growth, growing 53% larger than its
initial size as the concentration doubled. Surprisingly,
when the concentration was tripled from 20g/L to 60g/L,
E. coli did exhibit a significant change in growth – in-
creasing by more than 60% of its initial size at 20g/L.
However, L. plantarum did not increase in size signifi-
cantly, but rather exhibited a much more modest 10%
increase in plate coverage.
Co-cultures:
Initially, at 20g/L, the co-culture exhibited similar growth
patterns to both pure cultures. However the co-culture
deviated from the expected trend in growth, and showed
a peak growth in size at 40g/L. The size decreased,
however, by 10% when the concentration was increased
to 60g/L. Furthermore, while the co-culture seemed to
grow very similarly to the isolated culture of L. plantarum
from 20 to 40g/L, at concentrations higher than 40g/L,
the growth pattern seemed to mimic that of the isolated
culture of E. coli. Possible implications of this will be
mentioned in the discussion.

3. 3
Figure 2: Plot of colony plate coverage vs. LB nutrient concentration for the 9 cultures
Culture area analysis using the ImageJ program was done by calculating area values for each dish and comparing this value to the area of the
bacterial colony outgrowth on each dish. Percent coverage values for each culture were then calculated from these values and graphed (above).
Values indicate how much of the dish each culture was able to spread out and cover within a 24 hour period – indicating the relative ease with
which each bacteria strain was able to spread and, in vivo, to form biofilms. Notice a domination of the co-culture growth by L. plantarum at the
40g/L concentration and likewise for E. coli at 60g/L.
E. coli dominates
L. plantarum dominates

4. 4
DISCUSSION
Our study proved very successful in examining the
cross-species growth dynamics of both E. coli and L.
plantarum. We discovered that nutrient availability is one
of the major factors affecting E. coli and L. plantarum
competition.
Pure Culture Analysis:
From both our quantitative data obtained by ImageJ
analysis (Figure 2) and qualitative data seen in the culture
images (Figure 1), we were able to draw significant con-
clusions regarding the growth patterns of E. coli and L.
plantarum in regards to nutrient concentration. In re-
gards to E. coli, we noticed that this strain of bacterium
tends to favor high concentrations of nutrients. At higher
concentration levels (60g/L), E. coli sees a major in-
crease in growth (more than 60%) while lower concen-
trations (20 and 40g/L) did not seem to have a significant
impact on E. coli colony size. On the other hand, L.
plantarum seems to be on the other side of the equation,
favoring low to medium concentration levels (as seen in
the 53% increase in size from 20 to 40g/L). This infor-
mation allows us to draw to the conclusion that diets
high in calories or carbohydrates may increase chances
of infection, as E. coli gains the advantage in out com-
peting L. plantarum within epithelial cells lining the lower
intestines.
Co-Culture Analysis:
Even more astounding results were discovered when we
compared the co-culture growth to both pure cultures.
At the lower concentrations, 20 and 40g/L, the co-
culture followed a growth pattern almost identical to L.
plantarum. This could be further evidence of L. planta-
rum’s maximum efficiency and dominance over E. coli
and low to medium nutrient concentrations. However,
as the concentration increased to 60g/L, the co-culture
showed a stark change towards the growth pattern of E.
coli. These results help to further our conclusion that E.
coli gains an advantage over probiotics like L. plantarum
at high nutrient concentrations.
CONCLUSION:
Thus far we can conclude that variable nutrient concen-
trations can indeed affect the competition of E. coli with
probiotic agents. At nutrient concentrations higher than
40g/L, there was a clear dominance in the co-culture by
E. coli, while L. plantarum remained the dominant bacte-
rium at low to medium nutrient concentrations. Further
research can examine growth dynamics of co-cultures
when grown within various LB broth concentrations. A
study using SDS-PAGE to determine what protein sig-
nals are altered in both L. plantarum and E. coli when
grown in co-culture biofilms at various nutrient concen-
trations may also be done.
Future studies may examine the environment of the in-
testine in terms of nutrient availability in both: intestines
inhabited by E. coli naturally found in the lower intestine
and intestines with pathogenic E. coli strains. If compari-
son between the two environments affirms E. coli
achieves dominance in high nutrient environments, new
dietary measures could be implemented as a means of
treatment and prevention of EPEC infection in the intes-
tine.
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"Luria Broth (LB) and Luria Agar (LA) Media
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2. Mangell, Peter, et al. “Lactobacillus plantarum
299v inhibits Escherichia coli-induced intesti-
nal permeability." Digestive diseases and sci-
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3. Papoff, Paola, et al. "Gut microbial transloca-
tion in critically ill children and effects of sup-
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