Comparison of Bacteria on Surfaces of Iron Rich and Iron Poor Vegetables
Jasmine Whitaker, Lakiyah Campbell, Scott Harrison, Wiley Hitchcock, Joseph Whittaker, Mary Smith and Jian Han
Biology Department, College of Arts and Sciences, North Carolina Agricultural and Technical State University
Abstract
Background
Iron is an important mineral essential for the human body in order
to perform functions such as oxygen delivery, energy production,
and DNA synthesis. Adequate iron levels in the body ensure
healthy organ and muscle function, and provide energy required to
perform daily activities.

Iron can be obtained from various dietary resources. Table 1
shows the iron contents for serving amounts of different
vegetables (1 cup = 240 mL; USDA 2004).
Table 1. Iron content in some vegetables
Food
Amount
Iron content
(mg)
Spinach (cooked)
1 cup
6.43
Peas (frozen)
1 cup
2.43
Mustard Green
1 cup
1.3
Green Beans (canned)
1 cup
1.21
Broccoli (frozen)
1 cup
1.12
Rutabaga (cubes)
1 cup
0.7
Broccoli (raw)
1 cup
0.64
Cucumber (sliced)
1 cup
0.3

Iron an essential micronutrient bacteria need to survive, and must
acquire from their environments or hosts (Skaar 2010). Foodborne bacterial pathogens have been found on both iron-poor and
iron-rich vegetables as shown in Table 2. There may be some
association between the type of pathogen and availability of iron
for different types of vegetables. Only 6 of the 19 reports of Gram
positive pathogens (32%) occurred on iron-poor vegetables while
19 of 39 reports of Gram negative pathogens (49%) occurred on
iron-poor vegetables.
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1A
Table 2. Iron-poor and iron-rich vegetables from which Gram negative
and Gram-positive pathogens have been isolated and reported. Ironpoor: <0.5 mg per cup. Iron-rich: ≥ 0.5 mg per cup. Adapted from Buck
et al 2003 and USDA 2004.
bacteria per measured sample (7,774). This was almost
300% more than that found for the iron-moderate vegetable
rutabaga. There may be additional factors other than iron
influencing abundance of bacteria on vegetables.
Iron-poor vegetables Iron-rich vegetables
Pathogen
Gram Status
Aeromonas
alfalfa sprouts,
Gram negative
cauliflower, lettuce
asparagus, broccoli,
celery, pepper, spinach
Campylobacter
jejuni
lettuce, mushroom,
Gram negative
potato
parsley, pepper,
spinach
Escherichia coli
O157:H7
alfalfa sprouts,
Gram negative
cabbage, lettuce
celery
alfalfa sprouts,
cabbage, cauliflower,
Gram negative eggplant, endive,
lettuce
artichokes, beet greens,
celery, mung bean
sprouts, parsley,
pepper, spinach,
tomato
Salmonella
Conclusion
 The iron-rich vegetable, mustard green, contained the most
Shigella
Gram negative celery, lettuce
Vibrio cholerae
Gram negative cabbage, lettuce
Bacillus cereus
alfalfa sprouts,
Gram positive
cucumbers
 Bacteria on iron-rich vegetables showed more variety. They
contained both Gram positive and Gram negative bacteria
with various arrangements and morphologies. Gramnegative bacteria predominated in the iron-poor vegetable,
cucumber, consistent with our analysis of reports of foodborne pathogens where Gram-negative bacteria were more
frequently found on iron-poor vegetables.
Bacteria in Four Types of Vegetables
1B
parsley, scallions
soybean sprouts
10000
9000
Number of bacteria colonies
Iron is required by cellular organisms for energetic function and
other critical pathways. We investigated whether bacterial growth and
variety is influenced by the relative presence of iron in different
grocery store vegetables. In this study, the iron-rich vegetables were
mustard green, the iron-poor vegetables were cucumber, and the ironmoderate vegetables were broccoli and rutabaga. Bacterial densities
on the surface of these vegetables were measured by colony
counting, and bacteria from these colonies were examined further by
Gram staining. We found mustard green to have the most bacteria.
Bacteria from the iron-rich and iron-moderate vegetables were both
Gram positive and negative, while bacteria on cucumber were
predominantly Gram negative. The abundance and variety of bacterial
strains on vegetables therefore appears to be affected by the
availability of iron, and this finding serves to motivate further
investigation of bacteria across a wider range of plants based on
nucleic acid sequence analysis, direct microscopic counting, and
quantification of iron availability.

Results
Background cont.
8000
 Future
7000
studies will include broader sampling of different
vegetables, nucleic acid sequence analysis of bacteria,
direct microscopic counting, and quantification of iron
availability on vegetable surfaces and tissues.
6000
5000
4000
3000
2000
1000
References
0
cucumber
rutabaga
broccoli
mustard
green

U.S. Department of Agriculture, Agricultural Research Service. 2004.
USDA National Nutrient Database for Standard Reference, Release
17.
Nutrient
Data
Laboratory
Home
Page,
http://www.nal.usda.gov/fnic/foodcomp

Holt JG, Bergey DH, and Krieg NR. (1994) Bergey's Manual of
Determinative Bacteriology. Lippincott Williams & Wilkins. ISBN#
0683006037.
Hypothesis

Ryan K, Ray CG, Ahmad N, Drew WL, and Plorde J. (2004) Sherris
Medical Microbiology (4th ed). McGraw Hill. pp232-233.
The hypothesis of this study is that the amount of iron in
vegetables will influence the types of bacteria on those
vegetables.

Buck JW, Walcott RR, and Beuchat LR. (2003) Recent Trends in
Microbiological Safety of Fruits and Vegetables. Plant Health
Progress PMN.

Skaar EP. (2010) The battle for iron between bacterial pathogens and
their vertebrate hosts. PLoS Pathog 6(8): e1000949.
Clostridium
botulinum
cabbage, mushrooms pepper
Gram positive
Listeria
monocytogenes
cabbage, cucumber,
eggplant, lettuce,
Gram positive
mushrooms, potato,
radish
Staphylococcus
Gram positive alfalfa sprouts, lettuce carrot, parsley, radish
tomato
Types of Vegetables
Figure 1 Comparison of bacteria colonies for different vegetables. 1A: bacterial
colonies on a nutrient agar plate. 1B: Mean number of bacterial colonies from
sampling different types of vegetables in all stores. Each error bar represents the
standard error of the mean.
2A
2C
2B
2D
 Materials and Methods

Vegetables were collected from three chains of grocery stores:
four Harris Teeter stores, four Food Lion stores, and three WalMart stores. Vegetables selected for the study were broccoli,
cucumber, mustard green and rutabaga. Two samples of each
vegetable were selected from each store and total of 88 samples
were collected in this study.

The surface of the vegetables were swabbed with a sterile cotton
Q-tip pre-dipped in phosphate buffer saline. Samples were stored
in LB broth at 4ºC overnight.

Ten µl was taken from the stock and, with 1:100 dilution, plated on
a nutrient agar plates. Plates were incubated at 37°C for 48 hours.
After incubation, the bacterial colonies were quantified.

Representative colonies of different morphologies were selected
and analyzed by Gram stain (Holt et al. 1994; Ryan et al. 2004).
Digital images were made at 1000x magnification with a light
compound microscope using the software, Axio Vision Rel 4.8.
2E
2F
Figure 2 Bacteria identified by Gram stain. 2A: gram positive, long rods (bacilli), in
chain arrangements from bacteria found in broccoli sample. 2B: gram positive, short
rods, sporadically arranged bacteria found from mustard green samples. 2C: gram
negative, rounded spheres (cocci) as well as short rods (bacilli) isolated from
rutabaga samples. 2D: gram negative, short rods (bacilli) with rounded ends; bacteria
isolated from rutabaga. 2E: gram positive, spheres (coccus), and tightly clustered
bacteria found from broccoli samples (possibly genus Staphylococcus). 2F: gram
positive, slightly elongated spheres (cocci), and tightly clustered bacteria found from
mustard greens samples (possibly genus Staphylococcus).
Acknowledgements
This study is supported by Interdisciplinary Training for Undergraduates
in Biological and Mathematical Sciences (UBM) Program (Grant
0634598 and 1029426 from the National Science Foundation)