Quantification of Microbes in Food

Abstract

Microorganisms can be found in many food products, but detection and quantification of these microbes could be more easily approximated by standard plate counts of colony-forming units of diluted bacteria sets in plate count agar. It was then hypothesized that the fresh food products would have greater CFU’s per gram than the desiccated or frozen food products. Each group obtained one of the six food products and measured out 20 grams of food and performed various dilutions of their food product with sterile water blanks into three empty petri plates with plate count agar added to each plate and incubated. Group 1 had raisins and calculated 280,000 CFU/20 grams, group 2 had grapes and calculated 9,000 CFU/20 grams, group 3 had fresh blueberries and calculated 1,550,000 CFU/20 grams, group 4 had frozen blueberries and calculated 50,000 CFU/20 grams, group 5 had fresh broccoli and calculated 3,400,000 CFU/20 grams, and group 6 had frozen broccoli and calculated 1,900,000 CFU/20 grams. This partially proved the hypothesis that most of the fresh foods had higher CFU’s per 20 grams than the frozen foods, yet was not the same way for raisins, which had a higher CFU count than the fresh grapes.

Introduction

 Microorganisms, such as bacteria, can inhabit many environments and can withstand a wide variety of environmental stresses to be able to thrive and reproduce in large numbers, and can form mutualistic or parasitic relationships with other organisms (Bashan, 2016). One of the places where these microorganisms can inhabit is within the food that we eat. The presence of bacteria in food is common and tends to not pose an immediate risk to people’s health, and they can play a major role in fermentation of popular food items around the world, such as alcoholic beverages and dairy products (Nishino, Matsuda, & Yamazaki, 2018). However, there is still a possibility that large amounts of bacteria can overgrow and negatively affect the quality of food items. To make sure that food is properly prepared, packaged, and stored to prevent this overgrowth of bacteria is the implementation of quality control procedures, including testing of the number of bacteria in any given sample of food. One method of bacterial quantification is a standard plate count of colony-forming units, or CFU’s, on a plate count agar through serial dilutions to determine obtain a plate count with the highest number of colonies which are easily countable. The purpose of this experiment is to determine an approximate number of colony-forming units present on each of a small variety of common food products. The hypothesis derived prior to this experiment is that the fresh food groups will have more CFU’s per gram overall on their plates than the dried or frozen food groups, because the fresh food groups may not have been monitored or processed as thoroughly as the frozen food groups so it may not be properly clean from microorganisms, and the desiccation and freezing temperatures may inhibit the growth of microorganisms, which can also lower the CFU count on the non-fresh food groups (Hadawey, et al, 2017).

Materials and Methods

A total of six food samples were obtained for this experiment from each group, which included raisins, fresh grapes, frozen blueberries, fresh blueberries, frozen broccoli, and fresh broccoli. Three empty petri plates were then obtained for each group and labeled with the corresponding group information and plated dilution, which is either a 1:100, 1:1,000, or 1:10,000 dilution, respectively. One water blank that contained 180 mL of sterile water was obtained and 20 grams of each group’s food sample was added to each water blank and blended for five minutes to provide the 1:10 dilution of each food sample. A 1.1 mL pipette was then obtained and used to distribute 0.1 mL of the blended mixture into the plate labeled 1:100. A 99 mL sterile water blank was obtained, and 1.0 mL of the diluted material was transferred via the pipette into the water blank to create a 1:1,000 dilution of each food sample. Then 1.0 mL of the diluted material was distributed from the 99 mL water blank into the plate labeled 1:1,000. Then 0.1 mL of the diluted material was distributed from the 99 mL water blank into the plate labeled 1:10,000. A bottle of plate count agar was then obtained and poured into each petri dish and solidified and incubated for 24 hours before counting the CFU’s for each plate.

Results

The results for each group’s CFU count was taken by first counting the highest countable CFU plate from one of the three petri plates and taking the final dilution by dividing the original dilution by the plating factor, which was 0.1 for all plates, and multiplying the CFU and the final dilution. Group 1 had raisins and counted 28 CFU’s on their chosen plate, had a final dilution of 10,000, and their calculated CFU’s per 20 grams of food was 280,000 CFU/20 grams. Group 2 had fresh grapes and counted 9 CFU’s on their chosen plate, had a final dilution of 1,000, and their calculated CFU’s per 20 grams of food was 9,000 CFU/ 20 grams. Group 3 had fresh blueberries and counted 155 CFU’s on their chosen plate, had a final dilution of 10,000, and their calculated CFU’s per 20 grams of food was 1,550,000 CFU/20 grams. Group 4 had frozen blueberries and counted 50 CFU’s on their chosen plate, had a final dilution of 1,000, and their calculated CFU’s per 20 grams of food was 50,000 CFU/20 grams. Group 5 had fresh broccoli and counted 34 CFU’s on their chosen plate, had a final dilution of 100,000, and their calculated CFU’s per 20 grams of food was 3,400,000 CFU/20 grams. Lastly, group 6 had frozen broccoli and counted 19 CFU’s on their chosen plate, had a final dilution of 100,000, and their calculated CFU’s per 20 grams of food was 1,900,000 CFU/ 20 grams.

Table 1: The comparison of fresh food products and the food products that were either frozen or desiccated, based on their number of colony-forming units per 20 grams.

Conclusion

In conclusion, the frozen foods had greater quantities of CFU’s per gram than their fresh counterparts, but the raisins had a greater quantity of CFU’s per 20 grams than the fresh grapes. The food item with the highest CFU’s per 20 grams was the fresh broccoli, with 3,400,00 CFU/20 grams, whereas the food item with the lowest CFU’s per 20 grams was the fresh grapes, with 9,000 CFU/20 grams, which could be determined by the chart with comparisons of the numbers of CFU’s per 20 grams of food. This indicates that the hypothesis was mostly valid, with the exception the grapes, which had a lower CFU’s per 20 grams than the treated raisins and the lowest CFU/20 grams count of all the food groups in the experiment. Another piece of data which stands out on the graph is that the frozen broccoli, while it still had fewer CFU’s than its fresh counterpart, had the next highest CFU’s per 20 grams. The fresh broccoli and blueberries which had higher CFU counts than their frozen counterparts proved the hypothesis on how the freezing of food slowed down bacterial growth. However, the raisins still had a higher CFU count than the fresh grapes, and this could be explained by potential experimental errors and limitations which occurred. Some of these errors and limitations include contamination from numerous sources, unknown ages or treatment of the fresh and treated food products, including the blender and the water blanks not truly being sterile, inability of growth of microbes which cannot be easily cultured to provide a more accurate CFU count, and inaccurate pipettes which may not have been as efficient as expected. To combat the issue of contamination from this experiment, there could have been more efficient, air-tight blenders specifically made for labs to prevent spills or limiting outside contamination, as well as the use of better disinfectants for cleaning and obtaining treated food products with a longer expiration date. The data, for the most part, still shows the effectiveness of treating products to inhibit the growth of bacteria and other microbes, and although many of these microbes are either harmless or beneficial to us, there is still the chance of pathogens or overgrowth of other bacteria being present in food products, and the use of standard plate counts on plate count agar can help to more easily predict the presence and quantity of these microbes to develop different strategies to halt excess microbial growth to make food safer for consumption.

References

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• Hadawey, A., Tassou, S. A., Chaer, I., & Sundararajan, R. (2017). Unwrapped food product display shelf life assessment. Energy Procedia,123, 62-69. Retrieved from doi:10.1016/j.egypro.2017.07.233
• Nishino, T., Matsuda, Y., & Yamazaki, Y. (2018). Separation of viable lactic acid bacteria from fermented milk. Heliyon,4(4). Retrieved from doi:10.1016/j.heliyon.2018.e00597