Patrick Sheehan MB 354 1 Isolation and identification of Enteric Bacteria in uncooked meat Abstract The purpose of this experiment was to identify opportunistic pathogens present in uncooked meat that are members of the Enterobacteriaceae family To isolate the potential pathogens a serial dilution was performed From here 4 individual and distinct CFU s were selected and transplanted onto selectivedifferential media in order to allow for further testing By exposing the suspected pathogen to a variety of tests to ensure that the bacteria in question is an Enteric bacteria the suspected bacteria was finally ran through an Entropluri Test in order to identify the isolate based on the results of the test Introduction Several different types of bacteria are known to exist in a symbiotic relationship with humans by making their homes in the large and small intestines of their host A classic example of this is the Enterobacteriaceae family which is a family of gram negative bacteria that are commonly found in the intestines of mammals However a small portion of this family are known to be either opportunistic pathogens cause disease under certain human conditions or recognized human pathogens A famous example of one of these is Escherichia coli which is one of the leading causes of foodborne illnesses in the world as it can very easily spoil meat and other food products By isolating bacteria found on a provided food sample chicken breast this experiment highlights the importance of proper hygiene and food handling because of how it demonstrates how easily food can be contaminated with potentially harmful bacteria Methods A sample of uncooked chicken breast was provided to pull samples from after the meat had been left at room temperature overnight to allow for sufficient bacterial growth to occur both on the surface and inside of the meat A one gram sample was taken from the breast and placed in solution of PBS and allowed to sit for 10 minutes After the PBS had successfully permeated the membrane of the sample of chicken the solution was aggressively vortexed in order to successfully ensure through mixing This mixed solution was taken and then used to perform a 10 fold serial dilution from a concentration of chicken of 10 2 to 10 7 of PBS in a 1 0 mL solution Keen Starting from the 10 7 fold dilution each fold of dilution was then taken and plated onto one of three selective differential media agars These plates totaling in 18 were then incubated at 37 C for 48 hours to allow for the formation of a variety of CFUs to form on the provided media After the incubation period was completed details of the CFU s were taken and recorded Additionally the total number of colonies per plate was also recorded Six of the most promising colonies were then fishtailed streaked onto plates consisting of EMB HE and LB agars and then allowed to incubate at 37 C for an additional 48 hours to allow for formation of isolated subcultures After this second incubation period was preformed all remaining isolates that were able to accumulate biomass Four of the original six underwent gram staining to test for enteric Isolates All four isolates were successfully determined to be gram negative which allowed for all four isolates to be tested down the line In order to achieve this all four remaining isolates were taken and then restreaked onto an LB plate This LB plate was then incubated at 37 C for an additional 48 hours Patrick Sheehan MB 354 2 At the conclusion of this 3rd incubation period all remaining isolates were taken to undergo an Oxidase test Three of the four isolates tested Oxidase negative and therefore were able to continue with the remainder of the tests Isolate four was selected to continue with all further tests A sample of this isolate was taken and used to inoculate an EnteroPluri Test system following standard protocol A second sample of isolate four was taken and used to inoculate a tube containing brilliant green bile and MUG broth tubes All tubes and the EnteroPluri Test system were allowed to incubate at 37 C for 48 hours to allow for the formation of Biomass in each of the testing tubes The results of the EnteroPluri Test and both the brilliant green bile and MUG tubes were then capable of being observed and recorded as sufficient Biomass had accumulated in all of them The final stage of testing was to perform the Indole and Voges Prokauer VP test Since both of these test can only be performed in the EnteroPluri Testing tube after sufficient accumulation of biomass has formed they had to be performed after the incubation period was over At the conclusion of the Indole and VP tests the information that was gathered allowed for the successful identification of a pathogenic bacteria taken from the chicken breast sample Results Table 1 7 10 6 10 5 10 4 10 10 3 2 10 EMB 7 97 300 TNTC TNTC TNTC HE 2 33 100 TNTC TNTC TNTC LB 6 66 150 TNTC TNTC TNTC Table 1 displayed above shows the results of the total number of CFU s that were successfully able to grow from each fold of the serial dilution from all 3 media plates As expected the dilution that yielded the least amount of CFUs was the 10 7 dilution while the 10 2 dilution yielded the greatest number of CFUs Dilutions from plates in the 10 4 to 10 2 dilution range were labeled as TNTC meaning too numerous to count As they expressed such a large amount of biomass that it was deemed unnecessary to individually count every CFU present Image 1 Patrick Sheehan MB 354 3 Image 1 displayed above shows the actual images of the 3 separate selective differential medias that were used to plate each fold of the dilutions during the serial dilution test The image in the left hand column displays the 10 2 and 10 3 dilutions The middle column displays the 10 4 and 10 5 dilutions Finally the right hand column displays the 10 6 and 10 7 dilution It can be clearly seen that the number of CFUs displayed in each image is accurately recorded and displayed in Table 1 Image 2 Patrick Sheehan MB 354 4 Image 2 displayed above displays the gram stain results from the 4 sub cultured isolates that were successfully able to grown on the selective differential media It can observed that all four of these gram stain tests have cells that are pink in color which indicative of gram negative bacteria Image 3 Image 3 displayed above displays the gram stain results from the 2 sub cultured isolates that were unable to accumulate biomass on the selective differential medias It can be