Bacterial Enumeration Lab Report
Enumeration of Bacterial Contamination in Hamburger Meat from Unknown Sources
C
March 6, 2012
The importance of bacterial enumeration has become even more apparent in recent years due to the increasing numbers of harmful bacteria found in meat products. This process is the key to understanding the populations of microorganisms that contaminate the food supply. Much of the bacteria in meat has been shown to be resistant to multiple drugs; so disease-causing microbes are becoming an increasing threat to public health. Many studies indicate that this drug resistance may be caused by the large amounts of antibiotics being administered to cows that haven’t been infected. For instance, an increase in tetracycline use has been shown to result in the development of tetracycline-resistant E. coli amongst cows (Sharma et. al., 2008). However, bacterial contamination may also be due to indecent food handling procedure. Although the FDA has strict …show more content…
laws concerning the handling of meat, it is unclear as to whether packagers and distributers actually follow these regulations. High levels of contamination in beef have been linked the improper sanitization of cutting boards in the boning process (Widders et. al., 1995).
The intent of this study was to quantify the bacteria found in several samples of hamburger meats taken from different, unknown sources. If improper or varying handling techniques are the cause of bacterial contamination, then the quantities of bacteria found in the different samples of meat will have fluctuating enumeration. Using both spread plates and pour plates, different dilutions of each hamburger sample were allowed to incubate in nutrient rich agar for one week before a bacterial enumeration was performed. It is anticipated that the average counts for each unknown hamburger sample will be comparable in number, regardless of where each was obtained.
Materials and Methods
Each hamburger sample was prepared in a suspension by blending two grams of the meat with 18ml of 0.75% NaCl. This mixture was homogenized for 20 seconds and apportioned into 1 ml culture tubes. Another five culture tubes were prepared with 4.5 ml of 0.75% NaCl and used to make five more 10-fold serial dilutions of the 10-1 hamburger suspension. Four more 10-fold serial dilutions were made for each of the spread plates containing nutrient rich agar. The unknown solutions were spread across each plate using a sterile glass hockey stick. 1.0 ml of the hamburger solution was transferred aseptically to each of the four empty pour plates. Molten, nutrient rich agar was poured into each plate, enough to cover the bottom completely, and swirled gently in a “figure 8” motion. This resulted in dilutions of 10-3, 10-4, 10-5, and 10-6 for both types of plates. All eight plates were left to incubate at room temperature for one week (Shand, 2012).
After one week of incubation, the plates were observed and the number of colonies in each were counted and recorded. Plates containing less than 30 colonies were marked as NSS, which stands for “Not Statistically Significant”, and plates containing more than 300 colonies were marked as being TNTC, or “Too Numerous To Count”. The titer for each plate was calculated by taking the count of bacteria and diving it by the plate’s dilution value, with the units being colony forming units per gram (CFU/gm). Each viable count was used to calculate the average titer for each of the different unknown solutions (Shand, 2012).
Results
In spread plates, viable count assays taken for the sample unknowns showed a high of 2.46x107 CFU/gm found in Unknown E and a low of 2.44x105 CFU/gm found in Unknown C (Table 1). The highest average titer was 3.6x107 CFU/gm, which was found in Unknown E, and the lowest average was 8.9x105 CFU/gm, found in the Unknown C (Figure 1). Unknown A had an average titer of 9.1x106 CFU/gm, which was the second highest average in the spread plates (Figure 1). Unknowns B and D had the most similar averages, with titers of 1.77x106 CFU/gm and 1.82x106 CFU/gm (Table 1).
Overall, pour plates produced lower counts than the spread plates, with the highest average being 1.19x107 CFU/gm found in the Unknown E and the lowest being 1.84x105 CFU/gm, which was found in Unknown D (Table 1).
Unknown A produced a titer of 1.82x106 CFU/gm, the second highest average in the pour plates, followed closely by Unknown B, which had an average of 1.22x106 CFU/gm (Figure 1).
Discussion
It was hypothesized that the amount of bacterial contamination in several samples of meat will have variable assays due to the different methods of meat handling. Using bacterial enumeration, the quantities of bacteria in unknown samples from different sources were counted and averaged after being allowed to grow for one week. Results supported the prediction, as Table 1 shows that the average titers for each unknown hamburger solution varied, with bacterial counts ranging from 1.84x105 CFU/gm to 3.6x107 CFU/gm. Fluctuating averages were found in both the spread plates and pour
plates. After the samples were grown in nutrient rich agar, bacterial counts of each sample were taken. For each Unknown solution, the spread and pour plates resembled relatively close counts, with the pour plates having a smaller average titer in each case. Unknown sample E showed the highest amount of contamination, with averages of 3.6x107 CFU/gm for the spread plate and 1.19x107 CFU/gm for the pour plate. Unknown D showed an unusually large difference in enumerations of the pour and spread plate, which could have been due to an error in preparing the pour plate. The varying bacterial enumerations point towards food handling differences playing a role in the amount of contamination in meat. The samples with high counts of contamination could have been processed in poor conditions with unsanitary machinery, or simply due to improper handling. Further study may show that the high number of bacteria could also be due to the region the meat was from or due to varying levels of antimicrobials used on the cows that the meat came from. Improving the sanitization techniques of facilities in which meat is processed has been shown to decrease the levels of bacteria significantly (Widders et. al., 1995). Future experimentation could include a study of the conditions the meat was handled in compared to the numbers of bacteria, before and after improved sanitation methods are installed. Information gathered could help determine the most influential contributor to meat contamination.
Works Cited
Shand, R.F. (2012). Fundamental techniques and experiments in microbiology. Flagstaff, AZ: Northern Arizona University.
Sharma, R., K. Munns, T. Alexander, T. Entz, P. Mirzaagha, L.J. Yanke, M. Mulley, E. Topp, T. McCallister (2008). Diversity and Distribution of Commensal Fecal Escherichia coli Bacteria in Beef Cattle Administered Selected Subtherapeutic Antimicrobials in a Feedlot Setting. Agriculture and Agri-food Research Center. Alberta, Canada. Widders, PR; Coates, K J; Warner, S; Beattie, J C; Morgan, IR; et al. Australian Veterinary Journal72. 6 (1995): 208-211.
C
March 6, 2012
The importance of bacterial enumeration has become even more apparent in recent years due to the increasing numbers of harmful bacteria found in meat products. This process is the key to understanding the populations of microorganisms that contaminate the food supply. Much of the bacteria in meat has been shown to be resistant to multiple drugs; so disease-causing microbes are becoming an increasing threat to public health. Many studies indicate that this drug resistance may be caused by the large amounts of antibiotics being administered to cows that haven’t been infected. For instance, an increase in tetracycline use has been shown to result in the development of tetracycline-resistant E. coli amongst cows (Sharma et. al., 2008). However, bacterial contamination may also be due to indecent food handling procedure. Although the FDA has strict …show more content…
laws concerning the handling of meat, it is unclear as to whether packagers and distributers actually follow these regulations. High levels of contamination in beef have been linked the improper sanitization of cutting boards in the boning process (Widders et. al., 1995).
The intent of this study was to quantify the bacteria found in several samples of hamburger meats taken from different, unknown sources. If improper or varying handling techniques are the cause of bacterial contamination, then the quantities of bacteria found in the different samples of meat will have fluctuating enumeration. Using both spread plates and pour plates, different dilutions of each hamburger sample were allowed to incubate in nutrient rich agar for one week before a bacterial enumeration was performed. It is anticipated that the average counts for each unknown hamburger sample will be comparable in number, regardless of where each was obtained.
Materials and Methods
Each hamburger sample was prepared in a suspension by blending two grams of the meat with 18ml of 0.75% NaCl. This mixture was homogenized for 20 seconds and apportioned into 1 ml culture tubes. Another five culture tubes were prepared with 4.5 ml of 0.75% NaCl and used to make five more 10-fold serial dilutions of the 10-1 hamburger suspension. Four more 10-fold serial dilutions were made for each of the spread plates containing nutrient rich agar. The unknown solutions were spread across each plate using a sterile glass hockey stick. 1.0 ml of the hamburger solution was transferred aseptically to each of the four empty pour plates. Molten, nutrient rich agar was poured into each plate, enough to cover the bottom completely, and swirled gently in a “figure 8” motion. This resulted in dilutions of 10-3, 10-4, 10-5, and 10-6 for both types of plates. All eight plates were left to incubate at room temperature for one week (Shand, 2012).
After one week of incubation, the plates were observed and the number of colonies in each were counted and recorded. Plates containing less than 30 colonies were marked as NSS, which stands for “Not Statistically Significant”, and plates containing more than 300 colonies were marked as being TNTC, or “Too Numerous To Count”. The titer for each plate was calculated by taking the count of bacteria and diving it by the plate’s dilution value, with the units being colony forming units per gram (CFU/gm). Each viable count was used to calculate the average titer for each of the different unknown solutions (Shand, 2012).
Results
In spread plates, viable count assays taken for the sample unknowns showed a high of 2.46x107 CFU/gm found in Unknown E and a low of 2.44x105 CFU/gm found in Unknown C (Table 1). The highest average titer was 3.6x107 CFU/gm, which was found in Unknown E, and the lowest average was 8.9x105 CFU/gm, found in the Unknown C (Figure 1). Unknown A had an average titer of 9.1x106 CFU/gm, which was the second highest average in the spread plates (Figure 1). Unknowns B and D had the most similar averages, with titers of 1.77x106 CFU/gm and 1.82x106 CFU/gm (Table 1).
Overall, pour plates produced lower counts than the spread plates, with the highest average being 1.19x107 CFU/gm found in the Unknown E and the lowest being 1.84x105 CFU/gm, which was found in Unknown D (Table 1).
Unknown A produced a titer of 1.82x106 CFU/gm, the second highest average in the pour plates, followed closely by Unknown B, which had an average of 1.22x106 CFU/gm (Figure 1).
Discussion
It was hypothesized that the amount of bacterial contamination in several samples of meat will have variable assays due to the different methods of meat handling. Using bacterial enumeration, the quantities of bacteria in unknown samples from different sources were counted and averaged after being allowed to grow for one week. Results supported the prediction, as Table 1 shows that the average titers for each unknown hamburger solution varied, with bacterial counts ranging from 1.84x105 CFU/gm to 3.6x107 CFU/gm. Fluctuating averages were found in both the spread plates and pour
plates. After the samples were grown in nutrient rich agar, bacterial counts of each sample were taken. For each Unknown solution, the spread and pour plates resembled relatively close counts, with the pour plates having a smaller average titer in each case. Unknown sample E showed the highest amount of contamination, with averages of 3.6x107 CFU/gm for the spread plate and 1.19x107 CFU/gm for the pour plate. Unknown D showed an unusually large difference in enumerations of the pour and spread plate, which could have been due to an error in preparing the pour plate. The varying bacterial enumerations point towards food handling differences playing a role in the amount of contamination in meat. The samples with high counts of contamination could have been processed in poor conditions with unsanitary machinery, or simply due to improper handling. Further study may show that the high number of bacteria could also be due to the region the meat was from or due to varying levels of antimicrobials used on the cows that the meat came from. Improving the sanitization techniques of facilities in which meat is processed has been shown to decrease the levels of bacteria significantly (Widders et. al., 1995). Future experimentation could include a study of the conditions the meat was handled in compared to the numbers of bacteria, before and after improved sanitation methods are installed. Information gathered could help determine the most influential contributor to meat contamination.
Works Cited
Shand, R.F. (2012). Fundamental techniques and experiments in microbiology. Flagstaff, AZ: Northern Arizona University.
Sharma, R., K. Munns, T. Alexander, T. Entz, P. Mirzaagha, L.J. Yanke, M. Mulley, E. Topp, T. McCallister (2008). Diversity and Distribution of Commensal Fecal Escherichia coli Bacteria in Beef Cattle Administered Selected Subtherapeutic Antimicrobials in a Feedlot Setting. Agriculture and Agri-food Research Center. Alberta, Canada. Widders, PR; Coates, K J; Warner, S; Beattie, J C; Morgan, IR; et al. Australian Veterinary Journal72. 6 (1995): 208-211.