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This experiment was carried out to investigate and show the effects of different thawing methods of food on the growth of bacteria. Corn suspension was prepared using serial dilution and introduced onto the agar plate by spread plate method. The degree of bacterial contamination of the defrosted food was represented by the number of bacterial colonies formed on the agar. After 48 hours of incubation, the number of bacterial colonies found on the agar was measured and recorded. Aseptic technique was used throughout the experiment to improve accuracy of the results as it could prevent contamination. The data collected was analyzed by using Kruskal-Wallis test. The null hypothesis was rejected. The results supported the experimental hypothesis; there are significant differences in the growth of bacterial colony on the agar by using different thawing methods. The best method to defrost the food is using refrigerator.
RESEARCH AND RATIONALE:
Freezing is a modern convenience for storing and preserving food.1 When food is brought to a temperature of -18°C (0°F), the growth of the microorganisms is halted and the activity of the enzyme is greatly reduced. Besides, freezing turns the water naturally present in foods to ice crystals. Since liquids expand when they freeze, these crystals can disrupt the food's fragile cell structure. The more quickly food is frozen, the smaller the ice crystals will be and the less damage they will do to the food structure.
Defrosting the food which is also known as thawing is a procedure to remove the food from frost or ice. 2 The food is warmed so that it can be prepared and served. Frozen food should be defrosted in either very hot or cold conditions, where microorganisms will not thrive. Extreme heat will penetrate and defrost vegetables very quickly whereas at low temperature, the activity of the bacteria is slowed down and eventually stopped.
The experiment is aimed to advise people based on investigation results on the safest way to defrost food by comparing the effect of different methods of defrosting the food on the growth of the bacteria.
In this experiment, frozen corns were used as it was safer than to use meat. Bacteria present in the uncooked meat might contain pathogens that were harmful to the person carrying out the experiment. 5 However, the frozen corns could not fully and perfectly represent the different classes of food as a whole. Thus, the results might differ for other classes of food and further works could be done by carrying out on the experiment with other classes of food.
There is a significant amount of bacterial growth in the defrosted food corresponding to the different methods of defrosting.
There is no significant difference in bacterial growth on the agar plate corresponding to the different methods of defrosting.
Manipulated: Different methods of defrosting the frozen corns
Responding: The number of bacterial colonies formed on agar
Constant: Temperature of incubation, duration of incubation, thickness and composition of agar, volume and concentration of solution.
Petri dishes, label stickers, 20uL micropipette, pairs of sterilized forceps, pestle and mortar, weighing balance, Bunsen burner, test tubes, test tube rack, sterile glass rod, vortex machine, incubator, autoclave.
70% ethanol, sterile distilled water, molten nutrient agar, antiseptic solution (Dettol), frozen corns, sterile 1cm3 syringe, sterile 10cm3 syringe. (565 words)
A trial experiment was carried out to determine the volume and dilution of the solution required for the experiment and the suitability of the methods and procedures.
In this trial, two volumes were chosen, 10 µL and 20µL. A micropipette was used to measure the corn suspension to be introduced to the agar. Spread plate method was used to spread the dilution evenly over the agar by using the sterile glass rod. Besides, a serial dilution was carried out to determine the most appropriate dilution for all the 8 samples. 5cm3 of sterile water was added to 5g of the corn sample. The corns were crushed by using pestle and mortar. The stock solution was vortexed using a vortex machine. The samples were diluted to different concentrations to produce a small number of colonies on each agar plate. Each 10µL and 20µL of the dilutions was spread on the agar plates. These were repeated with the different samples. The agar plates were incubated for 48 hours. The number of bacterial colonies formed on the agar was counted.
The agar plates were labeled with alphabets for ease of work.
A - Defrosted by using the microwave
B - Defrosted under the sunlight
C - Defrosted by using the conventional oven
D - Defrosted at room temperature
E - Defrosted in a pail of cold water
F - Defrosted in the refrigerator
G - Frozen corns (positive control)
H - Sterile distilled water (negative control)
10µL of corn suspension
Number of Bacterial Colony
20µL of corn suspension
Number of Bacterial Colony
From the results obtained from the trials, 20µL of the dilutions and the concentration of the dilution (10-2) were selected. 20µL was chosen as some of the stock solution may remain on the glass rod and resulted in less bacterial colonies formed on the agar plate. Concentration of the dilution (10-2) was appropriate as agar plates that had 30 to 300 colonies were statistically valid 3 and suitable for counting. It was because a plate with less than 30 colonies was less accurate and reliable and would result in higher percentage error. However, a plate with more than 300 colonies would be too difficult to be seen clearly and counted. For the agar plate with less than 30 colonies, the data was recorded as too few to count (TFTC) and for the plate with more than 300 colonies, the data was indicated as too numerous to count (TNTC). 3
REAL EXPERIMENT PROCEDURES:
Preparing Agar Plates
The mouth of the conical flask containing the sterile molten agar was flamed by using a Bunsen burner.
In the laminar flow chamber, the lid of the Petri dish was lifted as little as possible when pouring the agar to the plate.
The agar was poured until half of the Petri dish and no air bubbles were trapped in the agar.
The agar was set aside to solidify at room temperature.
Steps 1 to 4 were repeated for the remaining 23 Petri dishes.
The Petri dishes were labeled and the replicates were differentiated by 1, 2 and 3 respectively.
Preparing Inoculum Using Serial Dilution
5g of the corns were weighed by using the electronic weighing balance.
5ml of sterile distilled water was added to the corns.
The corns were crushed as finely as possible by using pestle and mortar.
The corn suspension was vortexed by using a vortex machine.
9cm3 of sterile distilled water was added to each of the two test tubes by using a 10cm3 syringe.
1cm3 of the corn suspension was transferred by using a 1cm3 syringe to the first test tube labeled A (i). This was the 10-1 solution. The solution was mixed well.
1cm3 of the solution from the 10-1 solution was transferred to the second test tube labeled A (ii). This was the 10-2 solution that would be used in the experiment.
Steps 1 to 7 were repeated for the corn samples defrosted in different condition and sterile distilled water.
Introducing Inoculum to the Agar Plate
20µL of the solution from test tube labeled A (ii) was transferred to the agar plate by using a micropipette.
The lid of the Petri dish was lifted as little as possible to prevent entry of other bacteria from surroundings.
The solution was spread evenly onto the surface of agar by using a sterile glass rod. This was known as the spread plate method.
Steps 1 to 3 were repeated with different replicates and the corn samples of different method of defrosting.
The Petri dishes were placed inverted in the incubator at 30°C for 48 hours.
The number of bacterial colony formed on the agar was counted and tabulated.
RISK ASSESSMENT AND SAFETY PRECAUTIONS:
Laminar flow chamber was used in the experiment to prevent contamination of the agar. Aseptic technique was used throughout the experiment to prevent bacterial contamination in the agar and the place conducting the experiment. 70% ethanol was used to wipe the table top before and after conducting the experiment. Hands were cleaned by using Dettol before handling the sterilized apparatus to avoid the apparatus from being contaminated. These measures were taken to improve the accuracy of the results. Gloves were worn throughout the experiment. Glass wares such as test tubes and glass rod were handled with care. Sterile apparatus and distilled water were used to terminate any other bacteria that might be present. Petri dish lids were lifted as slight as possible when pouring the agar and introducing the inoculums. The mouth of the flask containing agar was flamed to ensure that there was no contamination due to any other bacteria. The micropipette was used with care. When sucking up the solution, the knob should not be released too fast as air bubbles could form in the tip. This would reduce the real solution needed for the experiment and lead to inaccuracy of the results. The used teats of the micropipette were discarded into the biohazard disposal container containing disinfectant solution. The Petri dishes were sent for autoclaving before disposal at the end of the experiment.
Ways of Defrosting Corns
Number of bacterial colony units formed
I chose Kruskal-Wallis test as a mean to analyze the data. It was the nonparametric analysis of variance by ranks.4 It was because this test was used to compare more than three samples to determine if they came from equal populations.
Ways of Defrosting Corns
Number of Bacterial Colony Formed
Sum of Data
Mean of Data
Sum of Rank
Mean of Rank
Median of Data
Defrosted in the microwave
Defrosted under sunlight
Defrosted in the oven
Defrosted at room temperature
Defrosted in the refrigerator
Defrosted in a pail of cold water
Sterile distilled water
Î± = 0.05
Degree of Freedom, df:
df = k-1, where k= number of groups
df = 8-1 = 7
Kruskal-Wallis test value / Chi Square value, H:
Where H = Kruskal-Wallis test value/ Chi Square value
N = Total number of observations in all samples
Ti2 = Square of the sum of rank of each group
n = total number of observations in each group
The significance level, Î± = 0.05. The H value calculated according to the formula is 22.68. The degree of freedom calculated using the formula is 7. Thus, critical value, Ï‡2 was obtained from the Chi-square table. It was found out that the when the degree of freedom is 7, the Ï‡2 is 14.0671 at 5% significance level. (Refer to appendix 1 for the Chi-square table.)
The H value that was calculated was 22.68 which was greater than Ï‡2 = 14.0671. Therefore, null hypothesis was rejected.
A bar chart of number of bacterial colonies against ways of defrosting corns was plotted. Error bars were displayed on the bar chart to show the overall distribution of the data. The error bars did not overlap each other indicating that the means are statistically different.
From the results that were obtained from the Kruskal-Wallis test, it clearly showed that different methods of defrosting the food had different effects on the growth of the bacteria as the null hypothesis was rejected and there was enough evidences to support that the different methods of defrosting would result in at least one set of differences in the growth of bacteria.
From table 3, it was clearly shown that the best method to defrost the frosted food was to defrost it in the refrigerator. It was because this method only recorded 71 bacterial colonies when compared to the rest. It was because at low temperature, the microorganisms would not thrive and thus would not lead to food spoilage. Although it was the slowest method but it was the safest method to defrost the food. It was because the food was still in good condition and remained safe to be consumed.
This was followed by defrosting in a pail of cold water that recorded with a mean of 92 bacterial colonies with a difference of 21. This method was faster than refrigerator thawing but required more attention. The defrosted food must be stored in a plastic bag and placed in cold water, changing the water every 30 minutes so it would continue to thaw. It was because cold water would maintain the surrounding temperature at a relatively low temperature as compared to room temperature. It could help to slow down the bacterial growth and thus decreased the bacterial contamination of the food.
However, the least preferable method of defrosting food would be to defrost it in the microwave which was the most convenient and common way used by the people nowadays. This method recorded the largest amount of bacterial colony formed on the agar, with a mean of 226. While in the process of defrosting, outer layer might become warm and begin to cook. Microbes would thrive on defrosted food as danger zone of temperature was reached and the food was not cooked immediately after defrosting.
Frozen corns were used as the positive control to show that there were even bacteria present in the frozen food initially and the bacterial growth was due to the presence of the bacteria in the food. Sterile distilled water was used as the negative control to show that introduction of sterile distilled water would not cause any bacteria growth. This showed that agar itself will not promote the bacteria growth and the aseptic techniques were carried out appropriately to ensure that there was no contamination from the surroundings.
Justifications of the use of the materials and apparatus:
In this experiment, the volume of the dilution needed is 20µL. Micropipette was used to measure accurately and dispense the dilution on the agar. Petri dishes were chosen to set the agar and allow the growth of bacteria. Petri dishes were transparent and we could observe the progress of the experiment and record the number of bacterial colony formed on the agar easily.
Potential errors during the experiment
Errors may have occurred due to bacterial infection because the mouth of the agar bottle was not flamed enough. Besides, the corns might not be crushed finely enough and the suspension were not concentrated enough for the bacteria to grow.
Ways to ensure the reliability and precision of the results
A few steps were taken to make sure that the results obtained from the experiment were accurate and reliable. Firstly, the agar was poured at around 50°C. It was because at this temperature, the agar was not too hot to be handled and would cause less condensation in the Petri dishes making the calculation of the bacterial colony less accurate.
Secondly, the Petri dishes were placed inverted inside the incubator to prevent water vapour from dripping onto the agar. If the water dropped on the growing bacteria, it would be contaminated and lead to inaccuracy of the results.
Thirdly, none of the apparatus that were to be kept sterile were removed or brought out of the laminar flow chamber throughout the experiment. It was to prevent bacterial contamination from the surrounding.
There was still some inevitable contamination although precautions had been taken and aseptic techniques were carried out. It may be due to the opening of the lid of the Petri dish during introduction of dilution to the agar. Microorganisms in the surrounding might enter and lead to contamination. Furthermore, the different parts of corn might have different levels of susceptibility to bacterial growth, leading to differences in the amount of bacterial growth.
Further work could be carried out by using different types of food that was defrosted, not only vegetables but also meat safely in a more advanced laboratory. This could show that which type of food is more prone to contamination by bacteria. Furthermore, food contamination was caused by many factors. Only one factor was studied in this experiment. Other factors, for example, the relationship between method of thawing and the method of cooking can be studied too.
Based on the results obtained and the statistical analysis, it can be concluded that different ways of defrosting the food have different effect on the growth of bacteria. Null hypothesis was rejected. Statistical analysis showed that there were significant differences in number of bacterial colony formed on the agar at 5% significance level. The best method was chosen, that was to defrost the frozen food in the refrigerator.
EVALUATION OF SOURCES:
Source 1, 4 and 9 are published books by the well-known authors that have been reviewed and up to date. Thus the information is reliable and accurate.
Source 2 is an online dictionary from Cambridge University press. It is the most popular online dictionary and thesaurus for learners of English. So, the definition of defrosting is correct and acceptable.
Source 3 is a website created by the Northern Illinois University (NIU). The information is written and provided by the NIU Department of Biological Sciences thus I t is trustable and acceptable.
Source 5 and 7 is a website specifically designed for providing examples of biology practical by The Nuffield Foundation & Society of Biology. The information provided had been reviewed before published.
Source 6 is a website that provides hundreds of free text and video-based lectures on statistics. There are professionals that published the lecturers online so the information should be factual.
Source 8 is a journal that obtained from the library database available in the college. The journal article used was written by the scholars. It had gone through numerous revisions before it is published.
Appendix 1: Chi-Square Table
Table from http://statisticslectures.com/chisquaretable.php
Appendix 2:C:\Users\User\Documents\cindy\crop ori.jpg
Diagrams showing one set of the results from the real experiment on methods of defrosting in the refrigerator. Diagram at the top was taken before the experiment and diagram at the bottom was taken after 48 hours incubation. (3448 words)