The die of food poisoning globally, and anyone, including

The Effects of Less Hazardous Chemical Substances, Such as Crisco Vegetable Oil, Heinz Distilled White Vinegar, and Diamond Crystal Table Salt, on Escherichia coli. Growth.Tiffany Tsai, Faika TabiaRL3 2017bAbstractMany manufacturers utilize dangerous chemicals as food preservatives to prevent or retard spoilage of foods caused by bacteria such as Escherichia coli.. In our study, we wanted to determine whether less hazardous chemicals such as Crisco vegetable oil, Heinz distilled white vinegar, and Diamond Crystal table salt could be used as alternatives to prevent bacterial growth. We tested this with the sterile filter paper method, in which a set number of filter papers are soaked in each substances and are then placed in each of the four chambers of the agar plates, which are covered with bacteria. After 24 hours of incubation, the zone diameter for each filter paper was measured and recorded. The data showed that while all experimental groups and control groups experienced growth in zone diameter,  Heinz distilled white vinegar had greatest effect on retardation of bacterial growth,  and the control group, which consist of dry filter paper, had the least significant growth. The vinegar and the control group yielded a p-value of less than 0.1 (p < 0.1), meaning the two had significantly different effects on bacterial growth.  As we hypothesized, vinegar was the most effective substance of the three to kill bacteria.IntroductionEscherichia coli, is a specific type of bacteria that resides in the gut of humans and animals, and pathogenic strains are one of the main causes of food poisoning (Marler, 2017). It is  a type of bacterium among the numerous bacteria and viruses that cause the greatest number of illnesses, hospitalizations, and deaths in the United States (foodsafety.gov staff, 2009). Foodborne diseases, which are maladies caused by food poisoning, are carried by food contaminated with bacteria, viruses, parasites, or toxins, and is a serious problem in America today. An estimated 48 million Americans become sick, 128,000 are hospitalized, and 3,000 die from foodborne diseases each year in the United States (Scallan, Hoekstra, et al. 2011). Therefore, we must control and improve consumer safety with manufactured food products. The American Association of Poison Control Centers (AAPCC) treated over 2.2 million individuals for food poisoning around the country (aapcc.org staff, 2017). Every year, 351,000 people die of food poisoning globally, and anyone, including toddlers, children, teenagers, adults and the elderly, is susceptible (Sifferlin, 2015). Also, the Centers for Disease Control and Prevention (CDC), has estimated that over 1,000,000 cases of foodborne diseases can be traced to salmonella infections which occur annually. Any person can be susceptible to foodborne illness at any given time. Effects of food poisoning include but are not limited to abdominal cramps, diarrhea, vomiting, mild fever, loss of appetite, weakness, nausea, and headaches (Selner, 2017). In an effort to kill and prevent growth of bacteria that cause food contamination, some manufacturers utilize food preservatives to prevent or retard spoilage caused by chemical changes (The Encyclopædia Britannica, 2015). However, these practices may cause human exposure to overconsumption of dangerous chemicals. For example, sulfates, a chemical which one commonly uses to preserve various fruits, may cause side effects upon ingestion in the form of headaches, palpitations (a rapid, strong, or irregular heartbeat ), allergies, or even cancer (Sharma, 2015). Other chemicals, such as nitrates and nitrites, are commonly used as curing agents in meat products. When consumed, they are converted into nitrous acid, which is suspected to cause stomach cancer (Sharma, 2015). Another class of chemicals, benzoates, are used in food as antimicrobial preservatives, and have been suspected to cause allergies, asthma and skin rashes (Sharma, 2015).     Common preservatives that may be less hazardous for ingestion are Crisco vegetable oil, Heinz distilled white vinegar, and Diamond Crystal table salt. By testing the effect of these common substances on E. Coli, we could determine whether either decreases the growth rate of E. Coli. In our study, we wanted to determine whether less hazardous chemicals could be used to prevent bacterial growth and thus preserve food. We hypothesized that if less hazardous chemicals, such as Crisco vegetable oil, Heinz distilled white vinegar, and Diamond Crystal table salt, are placed in a agar plate alongside with bacteria, then the growth of bacteria on the agar plate with vinegar would be retarded the most because the chemicals would prevent bacterial growth. We tested this with the sterile filter paper method, in which a set number of filter papers are soaked in each substances. Then, for each substance, one filter paper is placed in each of the four chambers of the agar plates, which are covered with bacteria. After 24 hours of incubation, the zone diameter for each filter paper is measured and recorded in centimeters. This data shows how susceptible the E. coli is  to each substances. Therefore, the aim of our study was to find healthier alternatives for food preservation by determining whether less hazardous chemicals could be used to prevent bacterial growth. We found that vinegar is the most effective substance of the three to kill bacteria.Materials and MethodsPreparation of Agar Plates:    In preparation of the agar plates, the bottom of each of the eight 100mm x 15mm agar plates (Petri Dishes with solid growth medium) were labeled with four "chambers": Control ( dry filter paper- no substance), Oil, Table Salt, and Vinegar with a Sharpie (Figure 1.) Four agar plates were separated into one trial and the other four were part of the second. After gloves were put on, a sterilized cotton swab was dipped into E. Coli. from an overnight starter culture and sweeped in a gentle motion, evenly over each of the agar plates. Each of the agar plates' covers was opened, covered in bacteria, and then quickly shut again to prevent contamination. Preparation of Substances into Sterile Tubes:In preparation of the substances into sterile tubes, 10.0 ml distilled water was placed into a sterile test tube using a sterile pipette. Twenty ml of Diamond Crystal: All Purpose table salt was poured into a sterile test tube, and the mixture was stirred using the pipette. The test tube was then closed with the cap and placed on a test tube rack for two minutes so that the salt could dissolve. After two minutes, eight Antibiotic Sensitivity  sterile disks was placed inside using a sterilized forceps, to prevent contamination. 24.0 mL of Kirkland Signature Canola oil was then placed into another sterile test tube and eight sterile disks were placed inside using the same sterilized forcep. Then, 24.0 mL of Heinz distilled white vinegar was poured into a third sterile test tube, and using the sterile forceps, eight sterile disks were placed inside. Both test tubes with oil and vinegar were closed and placed on the test tube rack. The disks were kept inside the test tubes for 10 minutes to allow the liquids to be absorbed by the disks, which was timed using a stopwatch. The eight sterile disks remained untouched. These are the control of the experiment. (Figure. 2) Placing Disks onto Agar Plates and Observing Results: Into order to place the discs onto agar plates, they were first prepared. After 10 minutes of substance being absorbed by the sterile disks in the test tubes, using new sterile forceps, all the disks were removed from within the test tubes and blotted lightly on sterile filter paper to get rid of excess matter. Note that a separate sterile forcep was used to touch each substance, so that substances would not mix and potentially alter the results of the experiment. The disks, based on the substance they absorbed, were placed flat onto the agar plates in correspondence with the labels. The other eight remaining disks, with no substance absorbed by them were also placed onto the agar plates on their corresponding chamber: "Control: No Substance" The lids of the plates were placed quickly over the latter after every disk put within it to keep everything as sterile as possible. The eight agar plates were placed back into the incubator for another 24 hours at 37? . After 24 hours, observations were conducted. Using a metric ruler, the zone of inhibition of each of the 4 disks in the eight agar plates were measured (Figure. 3).ResultsIn this experiment, the effect of different substances on bacterial growth was assessed by subjecting the filter papers soaked with each substance, Crisco vegetable oil, Heinz distilled white vinegar, and Diamond Crystal table salt, in an agar plate alongside with bacteria. After incubation, the zone diameter for each filter paper was measured and recorded. The data showed Heinz distilled white vinegar to have the greatest effect on bacterial growth, with an average of 1.875 cm zone diameter (See Table 1).  In contrast, the control group with no substance had an average of 0.76625 cm zone diameter (See Table 1). We conducted 2 trials with 4 samples of each substance, with a total sample size of 8 (n=8). The student t- test results showed our data to have a p value less than 0.01 (p < 0.01) (See Table 2).Table 1. The effect of different substances, Crisco vegetable oil, Heinz distilled white vinegar, and Diamond Crystal table salt, on bacterial growth as opposed to the control of no substance. The zone of inhibition diameter of the filter papers in each condition was measured after 24 hours of incubation. The data of the 2 trials conducted were averaged and the mean, standard deviation, variance, and the standard error of the mean were calculated.  We conducted 2 trials with 4 samples of each substance, thus a total sample size of 8 (n=8). The student t- test results showed our data to have a p value less than 0.01 (p < 0.01) (See Table 2).SubstancesCrisco vegetable oil (cm)Heinz distilled white vinegar (cm)Diamond Crystal table salt (10% solution)(cm)No Substance (Control)(cm)Mean of Means1.051.8751.250.76625Standard Deviation0.2927700220.7126410.35856860.068855439Variance0.0857142860.50785710.12857140.004741071Standard Error of The Mean0.1035098340.25195660.12677310.024344074Table 2. The Student t- test was conducted between the values of samples of Heinz White Vinegar and No Substance control. The t- test was conducted to test the significance of our results. Statistical ComponentHeinz White Vinegar and No SubstanceSample size (n)8Degrees of Freedom (df)6t-value4.380161079p-value>0.01Figure 1. The effect of different substances, Crisco vegetable oil, Heinz distilled white vinegar, and Diamond Crystal table salt, on bacterial growth as opposed to the control of no substance. The zone diameter was measured in centimeters. Vinegar experienced a drastic growth in diameter while the control of no substance experienced a less significant growth. The sample size is 8 (n=8). The p- value is less than 0.01 (p< 0.01). The error bars are the standard error of the mean between the substances in each trial.DiscussionIn this experiment, the effect of different substances on bacterial growth was assessed. We hypothesized that if less hazardous chemicals, such as Crisco vegetable oil, Heinz distilled white vinegar, and Diamond Crystal table salt, are placed in an agar plate alongside with bacteria, then the growth of bacteria would be retarded because the chemicals would prevent bacterial growth. Our results showed that Heinz distilled white vinegar to have the greatest effect on bacteria growth, with an average of 1.875 cm zone diameter and all substances used resulted in retardation of bacterial growth (See Table 1 and Figure 1).  In contrast, the control group with no substance had an average of 0.76625 cm zone diameter (See Table 1). The results do support our hypothesis in that all the substances showed to have effect in retarding  the growth of bacteria, while the experiment with no substances added had little to no impact. This suggests that vinegar is more effective ar killing bacteria than other substances tested such as salt water and oil. The drastic effect of vinegar on bacteria growth showed that the use of less harmful chemicals can indeed preserve foods from bacteria as well. The student t- test results showed our data to have a p-value less than 0.01 (p<0.01) (See Table 2). The critical value gathered from the results between the vinegar and the control group was 4.380161079 and with a 6 degree of freedom.(See Table 2) This led to the p-value of less than 0.01. The p-value means that the probability that my differences are due to chance is less than 1% (p<0.01), thus I conclude that the differences are due to the manipulation of independent variables and our data are statistically significant.   The null hypothesis of this experiment would be that  if less hazardous chemicals, such as Crisco vegetable oil, Heinz distilled white vinegar, and Diamond Crystal table salt, are placed in a agar plate alongside with bacteria, then the growth of bacteria on each agar plate would be the same. We are 99% confident that the null hypothesis is untrue and we would expect to see the same results 99% of the time. To our surprise, although not significant, there is a change in bacterial growth with the control sample, a zone diameter of 0.76625cm (See Table 1). We predicted there to be no effect because no substances were placed on the filterpapers of the control group. Possible sources of error could be that the filter papers were left exposed to the air for too long, causing contamination, or bacteria resists some components of the filter paper.In conclusion, our experiment showed that vinegar is an effective substance for killing bacterias. These findings are consistent with and extend the results of  other early studies. Previous studies have found vinegar (6% acetic acid solution), to be a mycobactericidal disinfectant and efficiently kills bacteria after 30 minutes of exposure. (Cortesia C C, 2014). In addition, another study has found that under an appropriate temperature, the combined use of vinegar and sodium chloride is remarkably effective for the preservation of bacterial poisoning (Entani E1 & Asai M, 1998).There are several limitations and possible sources of errors in the process of conducting this experiment. The condition of the equipments provided by the school are not guaranteed sterilized and the temperature of the lab room was not under our control. Some test tubes were left out for a long period of time, so there was a potential for contamination. The amount of time we had to conduct the experiment was limited, within a week we conducted two trials, each having to incubate overnight. Under the time limit, we were not able to allow the bacteria to incubate for a longer amount of time, which could have influenced the outcome of our results.To further investigate the effect of vinegar on bacterial growth, experiments should be conducted on testing and comparing the effect vinegar with varying pH levels on bacterial growth. These experiments should test the bacterial growth overtime in order to have a better understanding. A further application for this research is testing for "How does manipulating the pH levels of vinegar affect bacterial growth?"ReferencesAmerican Association of Poison Control Centers (2017). Retrieved from http://www.aapcc.org/Cortesia C, Vilchèze C, Bernut A, et.al (2014). "Acetic acid, the active component of vinegar, is an effective tuberculocidal disinfectant." mBio, volume 5(2),00013-14. Doi:10.1128/mBio.00013-14. Retrieved fromhttp://mbio.asm.org/content/5/2/e00013-14.abstract       Entani E1, Asai M, Tsujihata S, Tsukamoto Y, Ohta M. (1998) "Antibacterial actionvinegar against food-borne pathogenic bacteria including Escherichia coli O157:H7." Nakano Central Research Institute of Nakano Vinegar Co J Food Prot, 61(8), 953-9.Retrieved fromhttps://www.ncbi.nlm.nih.gov/pubmed/9713753Foodsafety.gov staff. (2009). "Bacteria and Viruses" foodsafety.gov. Retrieved from https://www.foodsafety.gov/poisoning/causes/bacteriaviruses/index.htmlMarler B. (2017) "E.Coli Food Poisoning" FoodBorne Illness Retrieved from http://www.foodborneillness.com/ecoli_food_poisoning/Scallan E, Hoekstra RM, Angulo FJ, (2011) "Foodborne Illness Acquired in the United States—Major Pathogens." Emerging Infectious Diseases, 17(1),7-15. doi:10.3201/eid1701.P11101. Retrieved fromhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3375761/Selner M., Yu W., and Watson K., (2017) "Food Poisoning" Healthline Retrieved from https://www.healthline.com/health/food-poisoning#outlook9 Sharma S.(2015) "Food Preservatives and their harmful effects." International Journal of Scientific and Research Publications, 5(4), 2250-3153 http://www.ijsrp.org/research-paper-0415.php?rp=P403889The Editors of Encyclopædia Britannica. (2015) "Preservative" Encyclopædia Britannica, Retrieved fromhttps://www.britannica.com/topic/preservative