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