BY ZACHARY MODISPACHER 11 TH GRADE CENTRAL CATHOLIC HIGH SCHOOL

BY ZACHARY MODISPACHER 11 TH GRADE CENTRAL CATHOLIC HIGH SCHOOL

INTRODUCTION 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?

CITRIC ACID C 6 H 8 O 7. 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.

SALMONELLA 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 and the WORLD HEATH 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.

E COLI/STAPH Salmonella is a pathogen and other foodborne 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.

ECOLI and the WORLD HEATH 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.

STAPH and the WORLD HEALTH 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.

PURPOSE 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.

NULL HYPOTHESIS 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.

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 MATERIALS Camera Graph paper & pencil LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride).

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 10 8 cells/ml. 4. The culture was diluted in sterile dilution fluid to a concentration of approximately 10 5 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 10 3 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.

PROCEDURE II 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 10 8 cells/ml. 5. The culture was diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/ml.

Procedure II Continued 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 10 3 cells/ml. 7. After vortexing to evenly suspend the cells, 100 µl aliquots were removed from the solution and spread on the preprepared 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.

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.

NUMBER OF COLONIES STAPH GRAPH 450 428 STAPH P-Value= 3.80E-19 400 386.6 350 300 304.1 250 200 150 131.4 100 50 0 0% 0.01% 0.10% 1% INFUSION CONSENTRATIONS 11

NUMBER OF COLONIES 300 ECOLI GRAPH E COLI P-Value= 9.09E-08 250 200 256.6 203.6 213.4 252 150 100 50 0 23.1 0% 0.01% 0.10% 1% INFUSION CONSENTRATIONS

0.01% CITRIC ACID E COLI DUNNETT S TEST 1.6 T CRIT NOT SIGNIFICANT 0.1% CITRIC ACID 1% CITRIC ACID INFUSION CITRIC ACID 1.3 T CRIT 7 T CRIT 0.01 T CRIT NOT SIGNIFICANT SIGNIFICANT NOT SIGNIFICANT

STAPH DUNNETT S TEST 0.01% 1.78 NOT CITRIC ACID T CRIT SIGNIFICANT 0.1% CITRIC ACID 1% CITRIC ACID INFUSION CITRIC ACID 5.4 T CRIT 12.9 T CRIT 18.1 T CRIT SIGNIFICANT SIGNIFICANT 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