Transcript Analysis of Lunch Meat Microbial Contamination

Analysis of Lunch Meat Microbial
Contamination
Experiment and Analysis by Donald Kline,
Jr
Problem of providing safe food
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Throughout history, people have been faced
with the challenge of not just providing food,
but keeping it safe from contamination
Problems of having safe
meat
• Meat is especially susceptible to disease
because it came from a living animal with
natural flora like E. coli in addition to
diseases the animal could have contracted
during its life
• The nutrients in meats provide can provide
an excellent growth
source for microbial life
Pathogens
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Pathogens in food are the leading cause of
death in some undeveloped countries, with a
death toll of around two million people
annually
Medical care for pathogens costs over a
billion dollars per year worldwide
Common contaminants in meat
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E. Coli
Salmonella
Clostridium botulinum
Campylobacter jejuni
Listeria monocytogenes
Clostridium perfringens
Staphylococcus aureus
Historical ways people kept meat safe
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Until 1860, it was thought that food became spoiled
through spontaneous generation
Drying using the sun is the oldest form of food
preservation
Salting can be an effective means of preserving meat
Smoking and heating were other common ways to
keep food safe
Meats usually could not be kept safe from
contamination very long
Common ways to keep meat safe
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Keep the meat in a hypertonic environment
Sterile packaging
Cooking
Refrigeration
Salting
Freeze-Drying
Canning and Bottling
Preservatives
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A preservative is used to prevent spoilage in foods
from microbial growth and contamination
Common chemical preservatives are calcium
propionate, sodium nitrate, sodium nitrite, sulphites,
disodium EDTA, formaldehyde, glutaraldehyde, and
methylchloroisothiazolinone
Natural preservatives include salt, sugar, vinegar,
and diatomaceous earth. Citric and ascorbic acids
can also assist in the preservation of certain foods.
Preservatives in this experiment
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Virginia Brand ham contained water, sugar, salt,
dextrose, potassium chloride, sodium phosphate,
sodium erythorbate, sodium nitrite, and sugar
Giant Eagle contained the following preservatives:
water, salt, sugar, dextrose, sodium phosphates,
sodium erythorbate, and sodium nitrite.
Russer ham contained water, salt, dextrose, sodium
nitrate, sodium phosphate, and sodium erythorbate.
E. Coli
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E.Coli is a pathogen that
is found in the lower
intestines of warm
blooded animals
Represents prokaryotic
cell model in this
experiment
There are almost 73,000
cases of infection and 61
deaths per year in the
United States
Yeast
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Type of yeast used in this experiment was
Saccharomyces cerevisiae
Yeasts also contribute to food spoiling by
producing waste products when they
metabolize food
Yeasts contribute to making many foods we
use today
They represent the eukaryotic cell model in
this experiment
Purpose and Hypothesis
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Determine how different brands of sterilized
lunchmeat’s preservatives and ingredients
would affect the survivorship of yeast and E.
coli cells
The null hypothesis was that the lunch meats
would not significantly affect survivorship of
S. cerevisiae and E. coli cells
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Materials
96 LB agar plates( 1.5 % tryptone, .5 % yeast extract, 1% NaCl, 1.5 % agar)
LB media (1 % tryptone, 5 % yeast extract, 1% NaCl)
72 YEPD agar plates(1% yeast extract, 2% peptone, 2% dextrose, 1.5% agar)
Sterile dilution fluid (10mM KH2PO4, 10mM K2HPO4, 1mM MgSO4, .1mM CaCl2,
100mM NaCl)
Klett spectrophotometer
Sterile pipette tips and Micropipettors
Vortex
Incubator
Sidearm flask
Spreader bar
Ethanol
20 mL Sterile capped test tubes
E.coli B
Saccharomyces cerevisiae
Giant Eagle Old Fashioned Ham, Russer Cooked Ham,
and Dietz and Watson Virginia Brand Ham
Hole puncher
Metric Scale and weigh boat
Micro burner
Procedure
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E. coli B and Saccharomyces cerevisiae were grown over night in
a sterilized media
A sample of the overnight cultures were added to separate fresh
LB (bacteria) and YEPD (yeast) in a sterile sidearm flask.
The cultures were incubated at 37°C (bacteria) and 30°C (yeast)
until a density of 50 Klett spectrophotometer units was reached.
These represent cell densities of approximately 108 (bacteria) and
107 (yeast) cells per mL.
The cultures were diluted in sterile dilution fluid to a concentration
of approximately 105 cells per mL.
The hams were sliced, massed to .4 grams for each brand of ham
and sterilized separately by soaking in 95% ethanol. After the
ethanol was evaporated, the hams were placed in 15mL sterile
polystyrene conical tubes.
100uL of the 105 cells/mL cell suspensions were pipetted directly
onto the surface of the ham pieces.
Procedure 2
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The tubes were allowed to incubate at room
temperature for the following time periods: 0, 45, 90,
and 135 minutes (bacteria) and 0, 30, and 60 minutes
(yeast).
One mL of SDF was pipetted onto the ham/cell mixture
and the tubes were gently vortexed.
After vortexing to evenly suspend cells, 100uL of the
cell suspension was transferred to 9.9mL of SDF and
then the SDF was vortexed and 100 uL aliquots were
then spread onto either LB agar or YEPD agar.
The plates were incubated for 24 hours at 37°C
(bacteria), or 48 hours at 30°C (yeast).
The resulting colonies were counted. Each colony is
assumed to have arisen from one cell.
100 uL
100 uL
100 uL
108 cells/mL
(bacteria) or 107
cells/mL (yeast)
1mL of
SDF
105 cells/mL
for both yeast
and bacteria
105 cells/mL
with ham
103 cells/mL
102 cells
Colonies
E. coli Survivorship
1400
1200
1000
800
600
400
200
0
VA
GE
Ru
Co
0
45
90
Tim e (m inutes)
135
p Values for Bacteria
Control
Virginia
G. Eagle
Russer
0 min Bac
0.026904 p › .05
p › .05
p › .05
45min Bac
1.35E-10 p › .05
p ‹ .01
p ‹ .01
90min Bac
1.1E-12 p ‹ .01
p ‹ .01
p › .05
135min Bac
6.83E-13 p › .05
p ‹ .01
p › .05
Conclusions for Bacteria
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Only Giant Eagle Ham had a significant
negative impact on E. coli survivorship.
Russer Cooked Ham appeared to have the least
negative impact on E. coli survivorship. The
variation was not statistically significant.
Giant Eagle Old Fashioned Ham appeared to
have the most negative impact on bacterial
survivorship
Yeast Survivorship
500
Colonies
400
VA
300
GE
200
Ru
Co
100
0
0
30
Time (minutes)
60
Data Chart of p values for yeast
Control
Virginia
G. Eagle
Russer
0 min Yeast
0.0258 p › .05
p ›.05
p › .05
30min Yeast
0.327624 p › .05
p › .05
p › .05
60min Yeast
0.013356 p › .05
p ‹ .05
p › .05
Yeast Conclusions
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Giant Eagle ham had a statistically
significant negative impact on yeast
survivorship, while Russer cooked ham
and Virginia Brand Ham did not
Russer Cooked Ham appeared to have the
least negative impact on yeast
survivorship. The variation was not
statistically significant.
Limitations and Extensions
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In the process of sterilizing the hams, it might have
affected the composition of the preservatives
The moisture content of the hams might have
varied due to the sterilizing and drying processes
Some hams might retain the bacteria or yeast on
the surface longer than others
Infuse the meats directly into agar to allow longer
exposure times for the cells
Sterilize ham with gamma irradiation
Use different models of bacteria and eukaryotic cell
models
Sources
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Centers for Disease Control and Prevention
(CDC)
Food and Drug Administration (FDA)
Partnership for Food Safety Education
(PFSE)
World Health Organization (WHO)
Acknowledgement to Professor John Wilson
University of Pittsburgh Biostatistician for his
advice and statistical analysis