BY ZACHARY MODISPACHER
11TH GRADE
CENTRAL CATHOLIC
HIGH SCHOOL
• Chicken is one of the most consumed meats in the world, though can
pose health risks (salmonella).
• Salmonella was thought only to infect the eggs after they are laid; new
studies show it is capable of infecting the eggs before they are laid.
• Studies show salmonella also infects chickens that are bred for their
meat, increasing the chances of contracting this disease from an
undercooked or raw chicken.
• Knowledge of proper cooking techniques, destroying the disease, has
improved the overall health of the world population.
• Is heat the only way to kill salmonella, or are there possible alternatives
such as citric acid?
INTRODUCTION
• Citric acid is a weak organic acid that is
normally crystallized at room
temperature into white grains.
• It is found in many sources in citrus
fruits like lemons, oranges, and limes,
and 20% of it goes into food production.
• It was first discovered in 1784 and more
than a million tons of it are produced
throughout the world.
• It has a ph of 2, and is a carboxylic acid,
yet it is slightly stronger than carboxylic
acids due to the fact that its anon can be
stabilized.
CITRIC ACID
C6H8O7.
• Gram negative, non spore forming, rod
shaped, endobacteria found in many
meats, but predominantly in chicken.
• Can be found in many warm and cold
blooded animals and while it can be
airborne is mainly transmitted by tainted
food.
• Can survive for weeks outside of a living
body and cannot be destroyed by being
frozen; they do however perish after
being heated to 60 degrees Celsius (140
Fahrenheit) for 12 minutes.
• 142,000 cases of salmonella infection in
the United States a year, and from 1990
to 2005 there have been 1316 deaths due
to the disease.
SALMONELLA
•Salmonella is the most common cause of food poisoning.
•Infection occurs after eating food that has not been thoroughly cooked.
•Symptoms usually appear within 12 to 72 hours and include:
headache
fever,
diarrhea,
nausea and vomiting.
•The condition usually clears but antibiotics may be
required, and those with weakened immune systems
may be more impacted.
SALMONELLA and the WORLD HEATH
•Salmonella is a pathogen and other food-
borne illnesses are pathogens
•A gram negative and gram positive model
were used as surrogates.
•E coli is a gram negative rod shaped
bacterium that is normally found in the
lower intestine of warm blooded
organisms.
•Staph is a gram positive bacterium that is
normally found on the skin and respiratory
tracts of humans, and is a major cause of
skin infections.
E COLI/STAPH
•E. coli infections come from contact with the feces or
contaminated water or food.
•Infections can be eliminated if the meat is cooked to
160°F (71°C)
•Infections associated with poor hygiene in poorer under
developed nations.)
•Symptoms include: Bloody diarrhea.
Stomach cramps.
Nausea and vomiting.
•.
ECOLI and the WORLD HEATH
• 2 types of staph infections
• Cellulitis is a bacterial infection of the skin and underlying fat tissue.
• Folliculitis is an infection of the pilosebaceous follicle (the hair and
oil gland) and is the most common form of staph skin infection.
•About 25% of people normally carry staph (in the nose, mouth, etc.)
•The infection often begins with a little cut, which gets infected with
bacteria.
•Infections range from a simple to antibiotic-resistant infections to
flesh-eating infections.
•Elderly, diabetics, and those with a weak immune system are more
susceptible to this type of skin infection.
STAPH and the WORLD HEALTH
•To determine at what levels of concentration citric acid is able to
completely destroy all colonies of e coli and staph present on the
medium.
•To test if the citric acid is able to destroy the colonies within the
amounts that are safe for human consumption after the bacteria have
been removed.
•To determine if citric acid is an acceptable substitute for heat when
the chicken is being prepared for human consumption and heat is not
available.
PURPOSE
•The citric acid will not be able to reduce the bacterial cell count to
an acceptable level for human consumption for both the bacteria
and the acid.
•The acid concentrations tested will not be able to eliminate the
bacteria so that there are no colonies present on the surface of the
agar.
NULL HYPOTHESIS
90 Agar gel medium plates
Citric Acid Solution
Escherichia coli, strain DH5 Alpha
Staphylococcus epidermidis
(Staph)
Filtered spring water
Spreader bar
Various pipettes
16 sanitized tubes
Incubator
Vortex
Camera
Graph paper & pencil
LB media (0.5% yeast extract, 1%
tryptone, 1% sodium chloride).
MATERIALS
Procedure I 1. E. coli and Staph was grown overnight in sterile LB media.
2. A sample of the overnight culture was added to fresh media in a sterile sidearm flask.
3. The culture was placed in an incubator (37°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 108 cells/mL.
4. The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL.
5. Prepare 10% Citric Acid Solution (stock solution) for use in experiment.
6. Sterilized Citric Acid Solution was mixed with the appropriate amount of SDF to create citric acid concentrations of 10%, 1%, and 0.1%.
Procedure I Continued 6. 100 µL of cell culture was then added to the citric acid
solutions, yielding a final volume of 10 mL and a cell density of approximately 103 cells/mL.
7. The solutions were vortexed and allowed to sit at room temperature for 15 minutes.
8. After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the tubes and spread on a set of regular LB plates.
9. The plates were incubated at 37 C for 24 hours.
10. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.
1. Sterilized Citric Acid was infused into the LB agar media and used to create the LB agar plates.
2. E. coli and Staph was grown overnight in sterile LB media.
3. A sample of the overnight culture was added to fresh media in a sterile sidearm flask.
4. The culture was placed in an incubator (37°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 108 cells/mL.
5. The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL.
PROCEDURE II
6. 100 µL of cell culture was then added to an SDF solution of 9.9mL, yielding a final volume of 10 mL and a cell density of approximately 103 cells/mL.
7. After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the solution and spread on the pre-prepared LB plates.
8. The plates were incubated at 37 C for 24 hours.
9. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.
Procedure II Continued
STAPH PLATES The following two (2) slides are a sampling of the Staph plates that were
cultivated over two days, they are infusion in the lower far left, 1% in the
lower left, 0% in the lower right, 0.01% in the lower far right, and the
0.1% is in the top left. The plates were cultivated for an extra day due to
the colonies being to small for a total count to be taken after the first day.
STAPH PLATES II
E COLI PLATES
To the left are the 0% in top and 0.01% in bottom, and to the right
is the 0.1% in top, 1% in middle, and infusion at the bottom.
STAPH GRAPH
428
386.6
304.1
131.4
11
0
50
100
150
200
250
300
350
400
450
0% 0.01% 0.10% 1% INFUSION
NU
MB
ER
OF
CO
LON
IES
CONSENTRATIONS
STAPH P-Value= 3.80E-19
ECOLI GRAPH
256.6
203.6 213.4
23.1
252
0
50
100
150
200
250
300
0% 0.01% 0.10% 1% INFUSION
NU
MB
ER
OF
CO
LON
IES
CONSENTRATIONS
E COLI P-Value= 9.09E-08
E COLI DUNNETT’S TEST 0.01%
CITRIC ACID
1.6
T CRIT
NOT
SIGNIFICANT
0.1%
CITRIC ACID
1.3
T CRIT
NOT
SIGNIFICANT
1%
CITRIC ACID
7
T CRIT
SIGNIFICANT
INFUSION
CITRIC ACID
0.01
T CRIT
NOT
SIGNIFICANT
STAPH DUNNETT’S TEST 0.01%
CITRIC ACID
1.78
T CRIT
NOT
SIGNIFICANT
0.1%
CITRIC ACID
5.4
T CRIT
SIGNIFICANT
1%
CITRIC ACID
12.9
T CRIT
SIGNIFICANT
INFUSION
CITRIC ACID
18.1
T CRIT
SIGNIFICANT
CONCLUSION While the variable was able to significantly decrease the survivorship of
colonies at certain concentrations, it was not able to fulfill the objectives of
completely destroying the bacteria and being safe for human consumption.
The concentrations of 0.1%, 1%, and infusion for the staph; and the
concentration of 1% for the e coli was significantly able to decrease the
amount of surviving colonies.
The first NULL must be accepted as the citric acid was not able to destroy the
bacteria at the levels that would not be hazardous to the health of any human
who would consume it.
The second NULL must also be accepted as the citric acid was not able to
fully destroy the bacteria on any of the concentrations that were used in this
experiment.
LIMITATIONS and EXTENSIONS
• The sample may not have been evenly spread out on the
surface of the plate to facilitate even growth.
• Only one exposure time used in liquid pulse.
• Only a model used not necessarily similar to meat.
Extensions
• Using other model organisms found in meat, such as
salmonella.
• Using more replicates.
• Testing other types of acids.
Sources and Acknowledgments
Thanks to Mr. Mark Krotec for the laboratory assistance in
preparing the colonies.
Thanks to Mark Krotec for also agreeing to sponsor the
experiment.
http/ Wikipedia.org
http/ CDC. Gov/ E. coli O157:H7 Infection
http/ CDC. Gov/ Salmonella Infection