The purpose of this study was to determine if commercial fruit and vegetable washes are effective at removing bacteria from the surface of fruits and vegetables. The first part tested three commercial fruit and vegetable washes (Environne Fruit and Vegetable Wash™, Fit Fruit and Vegetable Wash™, and Veggie Wash™) against water in an Escherichia coli zone of inhibition test at 5˚ C and 37 ˚ C. A two-way ANOVA was used to compare the zones of inhibition for each cleaner (water, Veggie Wash™, Environne™, and Fit Fruit and Vegetable Wash™) and the two temperatures at which the plates had been incubated (37˚ C and 5˚ C). A significant interaction between cleaner effectiveness and temperature was found with a one-way ANOVA, and a t-test was performed to compare the means of the two temperatures (37˚ C and 5˚ C) to each other. The test showed a significantly higher inhibition of bacterial growth at 37˚ C than at 5˚ C. A one-way ANOVA showed the cleaners were not significantly different from one other except water, which had larger zones of inhibition than Fit Wash™ and Veggie Wash™.
In the second part of this study, there were four different cleaning methods (unwashed, dish towel, water and a vegetable brush, and Fit Fruit and Vegetable Wash™). Twenty non-organic apples and 20 organic apples were cut into fourths and each quarter was cleaned with one of the four cleaning treatments. The quarters were swabbed and placed in nutrient broth and incubated at 37˚ C. Half of the samples were incubated for 24 hours, and the other half for 48 hours. The optical density of the samples was measured using a spectrophotometer.
A two-way ANOVA was used to compare the optical density of organic versus non-organic apples incubated for 24 hours. There was no significant interaction between organic or non-organic and the four cleaning methods, so the individual factor effects were examined independently with a one way ANOVA. The first factor was cleaning method, and the test showed no significant difference among cleaning with no treatment, a dish towel, water and a vegetable brush, and Fit Fruit and Vegetable Wash™. The next factor was being organic or non-organic, and at the 95% confidence interval, non-organic apples had a higher spectrophotometer reading of bacteria than organic apples. Another two-way ANOVA was used to compare the optical density of organic versus non-organic apples incubated for 48 hours. There was no significant interaction between organic or non-organic and cleaning methods, so the individual factor effects were analyzed further. The two factors were the four cleaning methods and the two apple types (organic vs. non-organic apples), and there was no significant difference among any of them.
Grocery stores are a convenient place for people to buy food. As technology becomes more advanced, more foods are available to buy. A group of food items that has been readily available for purchase at the supermarket is produce. Fruit and vegetables of all sizes, colors, shapes, and flavors can be purchased. Most of the produce items are not individually wrapped, but rather on the shelf, exposed to air, dirt, and people’s hands. The fruit may have been picked from an orchard with insect problems, the pesticides used on an orchard may have been too concentrated, or the truck used for transportation may have had fecal matter in it. There are many variables that can lead to the surfaces of produce being unclean, or even unsafe. People may think about these factors as they buy produce from the grocery store or farmer’s market. However, fruits are not necessarily “clean” at the grocery store, and they should be cleaned before being eaten.
Fruits and vegetables are usually sprayed with pesticides before being picked and put out on the market (Carnevale et al., 1991). Pesticides are used to kill insects that may consume the fruit, and some pesticides are used to keep fruits free from bacteria (Carnevale et al., 1991). During the transport process, many different things may come in contact with the fruit. People’s hands (which could have encountered anything), boxes and crates, which have come in contact with several other objects, and other factors could play a role in contamination (Sapers, 2001). By the time the fruit arrives on the shelf at the supermarket, it could be covered by a number of organisms.
There have been several stories in the news of Escherichia coli outbreaks in the food industry. In 1999, there was an outbreak of E. coli in New York from water consumption of tap water, ice, snow cones, and lemonade (Chartan, 1999). The outbreak was one of the largest in the United States, where 1000 people were sickened and two people were killed. In New York, about 200 cases of E. coli infection occur every year, but this outbreak happened at a fair in the last week of August of 1999 (Chartan, 1999). In September of 2006, about 200 people were infected with E. coli from spinach, and three people died (Sander, 2006). Many people overreact to news stories about food contamination and they stop buying produce for awhile. There is a potential danger for contamination from E. coli and other harmful bacteria in fruit and vegetables (Sapers, 2001).
Bacteria and pesticides are two dangerous organisms or substances that can be on the surfaces of fruits and vegetables. There are many chemicals used in agriculture, including those used for fertilizing, controlling pests, enriching nutrients, and making livestock healthy (Carnevale et al., 1991). If the chemicals are not used correctly or at correct levels, there can be risks to human health (Carnevale et al., 1991).
Ripley (2000) conducted a study between 1991 and 1995 in Ontario, Canada, looking at pesticide residues on fruits and vegetables. Samples of 1,536 vegetables and 802 fruits were tested for several pesticides through the Food Inspection Branch of the Ontario Ministry of Agriculture, Food and Rural Affairs (Ripley, 2000). The samples were not washed before analysis, and about one third of the samples had no pesticide residues at all. The other 60% had one or more pesticides found on them, and more fruits than vegetables had traces of pesticides. The pesticides seen the most often were captan, dithiocarbamate (DTC) fungicides, endofulfan azinphos-methyl, phosmet, parathion, and iprodione (Ripley, 2000). All of the pesticides were found in very low levels, most of them at concentrations below the Canadian limit of 0.1 μg/g (Ripley, 2000). The study also found that only 3.2% of vegetables and 3.1% of fruits had amounts of pesticides that violated maximum residue limits (Ripley, 2000). However, those regulations are not necessarily violated in the United States, because Canada has stricter maximum residue limits. Produce items most frequently violating the limits were lettuce, celery, peaches, and apricots. This study shows that pesticides are found on the surface of fruits and vegetables, and there is a possibility that unhealthy levels can be found in produce. This is one reason why cleaning is imperative.
Fresh fruits and vegetables are a public health concern. Some pieces of produce not only contain dirt and pesticides, but also potentially harmful bacteria. The number of foodborne disease outbreaks in the United States has risen from 0.7% in the 1970s to 6% in the 1990s (Johnson et al., 2006). Much of the fresh produce that comes into the US is imported, and the globalization involved introduces even more food safety risks. In 2000-2002, there were many states that had Salmonella enterica outbreaks from Mexican cantaloupe, in 1997 there were outbreaks of hepatitis A from Mexican strawberries, and in 1996 there were 1500 cases of cyclosporiasis from contaminated imported raspberries (Johnson et al., 2006).
The Food and Drug Administration (FDA) in the U.S. is responsible for keeping imported produce safe, but they have no jurisdiction over other countries. In 1998, the FDA teamed up with the U.S. Department of Agriculture (USDA) and wrote the Guide to Minimize Microbial Food Safety Hazards for Fresh Fruit and Vegetables. It is a set of voluntary guidelines for all people involved in the produce industry in hopes of decreasing the risk of pathogenic microbiological contamination (Johnson et al., 2006).
Part of decreasing the risk of contamination is examining each step in food production and transport. One concern comes from Enterococcus species that have become possible pathogens. They are ubiquitous and inhabit the gastrointestinal (GI) tract of animals and humans (Johnson et al., 2006), and can be spread from water or manure slurry to crops and soil, and then spread back to humans from the crops. This vicious cycle can be harmful to humans. The study Johnson and her colleagues performed in 2006 looked at Mexican produce and the microbial load they carry in the transport process. Samples were taken from the southern United States, and eleven different types of produce were sampled. From the packing sheds, 466 samples were either labeled “bin,” “wash tank,” “rinse,” “box,” or “conveyer belt,” depending where each item was collected from (Johnson et al., 2006). With sterile gloves, two sets of 400-600 grams of each type of produce were put in sterile bags on ice and shipped to a laboratory for testing and analysis. At the packing site where the fruit was obtained, every piece of produce was swabbed and then the swab was put in ten ml of letheen broth, sent to the lab, and evaluated within 24 hours. In the lab, the produce samples were tested for total aerobic bacteria, total coliforms and Escherichia coli using specific petrifilms, and total Enterococcus using special agar (Johnson et al., 2006).
Another part of the study was the detection of pathogens. Johnson et al. (2006)took 25 gram produce samples and analyzed them for Salmonella, L. monocyogenes, and E. coli 0157:H7 (2006). For E. coli testing, the samples were put in 225 ml of broth at 37˚ for 24 hours and then plated on agar (Johnson et al., 2006). A commonly studied strain of E. coli is the O157:H7 strain, which can cause severe diarrhea in humans. At least two colonies were also screened to detect the O:157 antigen, and so a latex reagent kit was used. After the data had been collected, statistical tests were performed, including a one-way analysis of variance (ANOVA), Tukey comparisons, geometric means, and standard deviations (Johnson et al., 2006).
The produce items were separated into four categories: leafy greens, herbs, melons, and vegetables; 310 of the samples were domestic, and 129 were Mexican imports. In the domestic group, all groups of produce had low microbiological levels, and they did not change significantly during the packing process. The only exception was cantaloupe, which had a dramatically significant increase of coliforms of almost 2 log10 CFU/g, (p<0.05) (Johnson et al., 2006). E. coli and Enterococcus levels also increased the same amount. It is to be noted though that during processing at the packing shed, degradation (decomposing and rotting) was observed by Johnson et al. (2006)in the cantaloupe. This was important, because the cantaloupe levels increased significantly, and the authors of the study thought that the degradation had something to do with it.
When the produce items were compared, almost every test showed higher microbial loads in domestic than in Mexican imports. The geometric mean total aerobic bacteria, E. coli, and Enterococcus levels were all significantly higher (p<0.05) on domestic herbs than Mexican herbs (Johnson et al., 2006). However, Mexican herbs had an increase (p<0.05) of E. coli between the time the herbs entered the shed and when they left the shed for distribution (Johnson et al., 2006).
Johnson et al. (2006) found no E. coli O157:H7, Salmonella, or Shigella in any of the produce samples that were tested. Overall, the quality of produce from both groups was excellent. The issue of food contamination not only affects food safety, but also food loss issues. About 30% of produce has to be thrown away between harvest and consumption because of microbial spoilage (Johnson et al., 2006). Since fruit and vegetables can be purchased year round, from all over the world, there is always a risk of bacterial contamination. The results of this study are of limited usefulness, because the sample size for Mexican imported produce was very small. This related to my study because it deals with bacteria found on produce. Although E. coli is not normally found on apples and other produce items, there is still possibility of an outbreak.
Because of the recent concerns with fruits and vegetables being microbiologically safe and the outbreaks that have been caused by E. coli, Salmonella, and Hepatitis A virus (Sapers, 2001), produce needs to be cleaned and disinfected before being ingested. Harmful substances can cling to the surface of produce, and then be ingested, causing illness. The original reasoning behind washing was to remove dirt on the surface. We now know there is the potential danger of bacteria, pesticides, and other microorganisms on produce items. Thus, we need better, more efficient cleaning methods, because by cleaning the fruits, illness by potential microbes and viruses can be avoided.
Sapers (2001) suggests that the solution to cleaning fruit should not be better cleaning methods, but rather improving techniques to avoid contamination before the fruits and vegetables are put on the market. The earlier in the life cycle the bacteria cling onto food, the harder it becomes to remove it later in life. If a fruit or vegetable becomes contaminated, it needs to be washed and decontaminated as soon as possible. At any point in the process between production and distribution, produce can become contaminated with feces from animals or humans.
Sapers (2001) contaminated apples with harmless strains of E. coli. After thirty minutes, bacteria were removed by washing with water, and the results showed that 90% of the bacteria were removed (Sapers, 2001). But, when the scientists extended the exposure time to 24 hours, almost every bacterium clung to the apple and washing the fruit had no effect (Sapers, 2001). The most common places to find bacteria on produce are in pores, cracks, or divots. Bacteria also are found in the stem area of the fruit. Sapers (2001) concluded that the highest numbers of bacteria on apples are found around the stem and calyx (a floral structure where the sepals are collectively located, this can be found opposite to the stem), areas that are frequent missed when washing the fruit. Also, when fruits and vegetables are being packed and processed, there is a chance that bacteria can be passed through cuts or punctures from the handlers or machinery. Even worse, the handlers of the fruit may pass on bacteria from their own hands, clothes, or mouths.
There are many pieces of commercial washing equipment used to rid dirt from produce; unfortunately, it is unknown whether bacteria are being removed in the process. Sapers’ (2001) experiments washed apples with different liquids, including hot and cold water, chlorine, hydrogen peroxide, detergents, and an experimental sanitizing agent (Sapers, 2001). There are many commercial washers available to wash fruit, such as U-bed, flat-bed brush washers, reel washers, pressure washes, immersion pipeline washers, and many more (Sapers, 2001). The problem with the large washers is that they cannot focus on each individual piece of fruit, especially in areas such as the stem and calyx. In the lab, many cleaning agents remove more bacteria than commercial washers. Along with a good cleaning system, one must also have good cleaning chemicals.
Chlorine is used most frequently as a sanitizing agent for produce. It removes most of the bacteria, but it can cause mutations or carcinogenic reaction products by reacting with organic residues (Sapers, 2001). Ozone is a sanitizing agent that is approved by the FDA (Sapers, 2001), and it can be used instead of chlorine. It is ineffective on pears, but it works on other fruits and vegetables. Another product, chlorine oxide, is effective in removing E. coli from exposed apples (Sapers, 2001). New methods for decontaminating and sanitizing produce are being researched, and up to this point, no method is 100% effective.
Scientists are coming up with new ways to use sanitizing agents, one of which is hydrogen peroxide. Hydrogen peroxide treatment takes up to an hour and can cause damage to small, soft fruits such as raspberries, but is highly effective in its vapor form (Sapers, 2001). Using other anti-microbial sanitizing products in their vapor phases is extremely useful in removing bacteria. Sapers (2001) showed that bacteria can cling to fruits, and that there are many ways to remove the microbes. This was useful to my study, because I tested non-pathogenic strains of E. coli on apples, and looked at which fruit wash was most effective in removing bacteria. However, I did not use large commercial washers to wash my apples, only a sink in the laboratory.
Even if fruits and vegetables are washed after being purchased at the grocery store, there is still a potential for contamination. High numbers of bacteria can be found inside refrigerators. Studies have shown that foodborne illnesses are three times higher in private homes than in commercial businesses (Jackson et al., 2007). Improper cleaning, lack of maintenance, and incorrect storage of food are some factors that can increase the risk of contamination of food, such as produce. Bacteria can spread from unwashed raw foods or contaminated hands and surfaces. From there, bacteria can cling to the inside of a refrigerator, which can lead to a longer exposure time for contamination (Jackson et al., 2007). Most people do not set the refrigerator temperature to the correct setting in their home, which can produce bacterial growth. Jackson et al. (2007) stated that when refrigerators are set too high, growth of organisms and pathogens such as Staphylococcus aureus, Salmonella spp., Listeria monocyogens, and Yersinia enterocolitica can occur. Bacteria can also be transferred onto food from utensils, sponges, and towels. Jackson et al. (2007) looked for Campylobacter spp., E. coli O157:H7, L. monocytogenes, S. aureus, Salmonella spp., and Y. enterocolitica on the inside of refrigerators to examine the prevalence of these pathogens.
Their study examined 342 randomly selected households. The refrigerator from each household was swabbed with a sponge at its base, shelves and side, and the sponge was chilled and taken back to the lab. Within six hours, each sponge was soaked in 250 ml of sample stock solution, recovered for four hours, diluted in Maximum Recovery Dilutent, and then placed on four plates containing different types of agar (Jackson et al., 2007). The samples were incubated for 24-48 hours at temperatures of 25-37 ˚ C (depending on the type of agar), and the results detected certain types of bacteria. Parts of the sample stock were also put in broth solutions and incubated to detect the presence of other bacteria.
E. coli O157:H7, Campylobacter spp., and Salmonella spp. were not found in any of the refrigerators. S. aureus was found most frequently, in 6.4% of refrigerators tested (Jackson et al., 2007). L. monocytogens was found in 1.2% of refrigerators, Y. enterocolitica in 0.6%, and non-quantified amounts of Proteus mirabilis, Klebsiella spp., Enterobacter loacae, Enterobacter agglomerans, and Pseudomonas spp. were found in some refrigerators (Jackson et al., 2007).
Jackson et al.’s 2007 study related to my study because they were looking for E. coli in household refrigerators. In part one of my experiment, I measured the zones of inhibition of E. coli at a refrigerator temperature setting (approximately 5˚ Celsius) and compared growth rates to those at body temperature (37˚ Celsius). According to Jackson et al. (2007), E. coli are rarely found in the food chain except on carcasses, meat products, and raw food surfaces, where they can survive at refrigerator temperatures. Another way in which their study related to mine is that it showed that refrigerators contain bacteria that can contaminate foods. This is yet another reason to clean fruits and vegetables before ingesting them.
Procter and Gamble manufactures a product called Fit Fruit and Vegetable Wash™ (Krieger et al., 2003), and claim the product is 98% better than water at removing pesticides on fruits and vegetables (Krieger et al., 2003). The ingredients in Fit™ wash are water, oleic acid, glycerol, ethyl alcohol, potassium hydrate, sodium carbonate, citric acid, and grapefruit oil (Krieger et al., 2003). The purpose of an experiment done by Krieger et al. (2003) was to see if Procter and Gamble’s label was misleading. In the first part of the experiment, apples were soaked in captan, a fungicide. Homemakers were asked to wash the fruit as they would at home, and the apples were cleaned with either tap water, tap water plus Fit Wash™, or not cleaned at all.
In the second part of the experiment, the apples were soaked in a mixture of captan and methomyl (an insecticide). The apples were washed using the directions on the bottle of Fit Fruit and Vegetable Wash™. One capful of Fit™ was added to two liters of water in a bowl that comes with the product. Fruit was soaked for 30 seconds, then rinsed with the diluted Fit™ solution four times for 30 seconds each, and finally rinsed for 5 seconds. After draining, the apples were bagged and transported to the lab.
According to Krieger et al. (2003), the lab technicians were not biased, and they reported results in μg pesticides/g fruit (parts per million, or ppm). In the first part, produce washed with Fit™ plus water had an average of 3.7 ppm of captan, and produce washed with water had 4.1 ppm (Krieger et al., 2003). ANOVA and Tukey tests, determined that Fit™ did not have a significant difference (p=0.78) in removing pesticides compared to water (Krieger et al., 2003). In the second part, more captan was removed than methomyl. The wash with Fit™ removed more methomyl than water, but less total residue. All of the washes lowered the amount of captan on the fruit.
Krieger et al. (2003) explained that all three methods of cleaning, even the un-cleaned apples, had safe and tolerable pesticide levels, as determined by the FDA. The claim that Procter and Gamble make about their product being 98% more effective than water is not statistically correct, according to Krieger et al. (2003). The only way in which Fit Wash™ works “better” than water is by removing wax from surfaces. There are other invalid claims made by fruit and vegetable washes, including Environne Fruit and Vegetable Wash™, Fresh Wash™, Harvest Wash™, and Veggie Wash™. Krieger et al. (2003) studied on those products too and concluded that none of them were more effective than water in removing pesticides and dirt from produce. If each wash removed as much captan as claimed compared to water, then the products would have had to remove up to 808% of total residue on the fruit (Krieger et al., 2003). Using water to remove pesticide residue from surfaces of fruits is, in fact, extremely effective according to Krieger et al.’s 2003 experiment. When the apples were washed with water, 81% of the captan was removed; Fit Wash™ removed 90% of the captan (Krieger et al., 2003). Their experiment is the closest to what I did in my senior seminar project, except that I looked at bacteria instead of pesticides. Pesticides can only be quantified with special equipment, and St. Martin’s University did not have such equipment available.
Since my study had several parts, I had a hypothesis for each part. In part one, I hypothesized that there would be no significant difference in the zone of inhibition of E. coli when treated with water compared to fruit and vegetable washes. I came to this conclusion from Krieger et al.’s 2003 study, where there was no difference between Fit Wash™ and water. I also hypothesized that there would be a higher average inhibition of bacterial growth at 37˚ C than at 5˚ C, because in Jackson et al.’s 2007 study, no E. coli grew at refrigeration temperatures, and 37˚ C is an optimal temperature for some bacteria to grow. In the second part of my study, I hypothesized there would be no difference in the total amount of bacteria present on apples after being cleaned with water, with a kitchen dish towel, or with Fit Fruit and Vegetable Wash™, again from Krieger et al.’s 2003 study. I also hypothesized that there would be more total bacteria on non-organic apples than the organic apples. I think this because I have observed in grocery stores that organic apples are more expensive and more on the outside of the produce section, where less people are likely to shop, and so less bacteria is likely to spread to their surfaces. I also hypothesize that there would be more total bacteria on apples incubated for 48 hours than those incubated for 24 hours. I chose this because I think more bacteria will grow if given a longer incubation period.
Preparing agar plates
The first part of this study was a microbiological experiment to test the antimicrobial effectiveness of fruit and vegetable washes in inhibiting the growth of E. coli (Leavitt et al., 1997). Nutrient agar (Fisher) was mixed with distilled water, and put into a large beaker. Following the manufacturer’s instructions, 23 grams of agar were added to every liter of water. Each beaker contained 500 ml of water, so a total of three beakers were mixed. The beakers were covered with aluminum foil, labeled, and slowly swirled. Then the agar solutions were sterilized at 121˚ C for 20 minutes at 17 psi in a Tuttnauer autoclave 2540E.
Once the agar had been autoclaved, it was cooled to room temperature and poured into Petri plates. The Petri plates were placed in stacks of five, and starting at the bottom of the pile, the lid was lifted off, and agar was poured to cover approximately 75% of the plate’s surface. The next plate was filled the same way. This process was repeated until all of the plates were poured. The agar in the plates was allowed to polymerize, and then the plates were placed into a plastic bag, closed with tape, and put into a refrigerator at 5˚ C for storage.
Culturing Escherichia coli
To grow E. coli, nutrient broth (Ward’s) was prepared to culture the E. coli bacteria. Thirty grams of nutrient broth powder were added for each 1000 ml of water. The solution was heated and stirred with a magnetic bar until the powder had dissolved. Twelve milliliters of nutrient broth was poured into four test tubes, the lids were placed on very loosely, and then sterilized in the autoclave at 121˚ Celsius for 15 minutes at 17 psi. After being autoclaved, the lids on the test tubes were tightened and stored in the refrigerator for future use.
A non-pathogenic strain of E. coli (Ward’s) was purchased for my study. The bacteria was in powder form, so to culture it, the directions on the package were followed. Using a sterile pipette, 0.5 ml of liquid media was added to the cryovial of E. coli. To mix the bacteria and media together, another sterile pipette was used to draw the liquid up and down several times. Once it had been mixed, 1 ml was added to a pre-autoclaved test tube of nutrient broth. The cap was loosened, and the tubes were put into the VWR Scientific Inc. incubator at 37˚ Celsius. After 24 hours, the test tube did not show any bacterial growth. It was thought that the nutrient broth used may have been ineffective, so an additional experiment was conducted. It turned out that the incubator had been 3˚ C below optimal growth temperature for E. coli (37˚ C), so that was most likely the reason why the bacteria did not originally grow effectively.
Testing different nutrient broths for most effective E. coli growth
Because the test tube did not show any bacterial growth, an experiment was conducted to see if the nutrient broth used was ineffective. Four different types of broth powder were selected and mixed with water according to their labels. They were two different bottles of nutrient broth from Culture Media & Supplies, Inc., another type of nutrient broth from Ward’s, and BBL™ Trypticase™ soy broth (Becton, Dickinson, & Company). Four different beakers, one for each type of broth, had 100 ml of distilled water and powder broth added to it. Each beaker was heated with a magnetic stir rod inside until the powder was dissolved. Then, 12 ml of each type of broth was poured into each of eight test tubes and autoclaved at 121˚ C for 15 minutes at 17 psi. After being autoclaved, 500 µL of the E. coli was added to each, and tubes were incubated for 48 hours at 37˚ C. The Trypticase™ soy broth showed the greatest bacterial growth (it had the highest turbidity), and so those solutions were used for the zone of inhibition tests.
Zone of inhibition test
For the zone of inhibition test, there were four different treatments: water (as a control), Environne Fruit and Vegetable Wash™, Fit Fruit and Vegetable Wash™, and Veggie Wash™. Each fruit and vegetable wash was purchased in the Olympia or Tacoma, Washington area. Twenty-five plates were used for each treatment.
The workspace was disinfected with alcohol. The eight tubes of E. coli in nutrient broth pooled. 250 µL of E. coli was pipetted onto the center of the plate. A bent glass rod was sterilized over a Bunsen burner and used to spread bacteria evenly (Gallant-Behm et al., 2005).
A pair of forceps were sterilized over a Bunsen burner and used to pick up a sterilized filter disc, dip it halfway into one of the four treatments, and placed on a Petri plate (Brown, 2005). Four discs were placed on each of the 100 Petri plates, for a total of 400 filter discs. For each of the four treatments, half of the plates were placed in an incubator at 37˚ Celsius for 96 hours, and the other half were placed in a refrigerator at 5˚ Celsius for 96 hours. The reason why they remained incubated for 96 hours was because after checking the plates at 24 and 48 hours, there were no visible zones of inhibition, so additional time was given for bacterial growth. The refrigeration temperature was chosen because it is close to what the US Department of Agriculture calls a “safe” temperature for storing food (4˚ C) (Durant, 2001). The other temperature (37˚ C) was chose because it is body temperature, where food is stored after being ingested.
After 96 hours, the zones of inhibition were measured with a millimeter ruler, by measuring the diameter of the area where E.coli’s growth was inhibited (Horita et al., 2005). As seen in Figure 1, the zone of inhibition is the clear area around the filter disc where the bacteria growth has been inhibited.
Figure 1. The zone of inhibition method. The small tan circle in the middle is the filter disc. The white area, where the small, thin arrow is pointing, represents the zone of inhibition (Tendencia, 2004).