Ground Meat Bacteria Test

According to the FDA's "Ground Beef and Food Safety" article, when meat is ground, more of the meat is exposed to the harmful bacteria. Bacteria multiply rapidly in the "Danger Zone" - temperatures between 40 and 140 °F. To keep bacterial levels low, the ground beef must be stored at 40 °F or less. It must be used within 48 hours, or freezed. This project is for the purpose of investigation of the kind of bacteria that can be found on the ground meat after the safety period is expired.

The ground beef was bought at a local supermarket. The package was thawed in the refrigerator. Part of the meat was cooked, and the rest was left for another 48 hours. Then a smear was taken and incubated for 24 hours at 32 °C.

Observation. Under light microscope (600X magnification) the cultured bacteria are short, motile rods.

Gram stain: Negative.

Oxidase test. This test is useful to put our bacteria into one of the large groups of gram-negative rods: 1) Aeromonas, Pseudomonas, Vibrio group (oxidase positive) or 2) Enterobacteriaceae group (oxidase negative). Our organism is oxidase negative.

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The Enterobacteriaceae are a ubiquitous group of bacteria consisting of enteric pathogens (Salmonella and Shigella) and many others capable of causing serious infections. The differentiation tests in this group begin with separation of bacterial genera based on their ability to ferment lactose.

Lactose fermentation. For this test, a sample from the original plate was inoculated into a petri dish with MacConkey agar. The plate was incubated for 48 hours at 32 °C.

The negative lactose fermentation test brings into the picture a large group of bacteria which includes Edwardsiella tarda, Morganella morganii, Proteus,Providencia, and Salmonella (except S. enterica subsp. salamae (Group II),S. enterica subsp. arizonae (Group IIIa), S. enterica subsp. indica (Group VI), and S. bongori Group V which are capable to ferment lactose.). This group also includes Serratia, Shigella, and Yersinia genera. These will be further separated based on the results of indole test.

Indole test. Organisms which possess the enzyme tryptophanase, will break tryptophan amino acid to produce indolic metabolites of indole: skatole (methyl indole) and indoleacetic acid. When Kovac's reagent (pale yellow) is added, the indole reacts producing red color. Organisms which do not possess the enzyme, will produce no color change upon addition of the reagent. This test is important in separation within the Enterobacteriaceaefamily. As indicated by no change of color at surface of the broth, our organism is indole negative.

Picture of indole-negative  bacteria in a test tube
Indole-negative test indicated by no color change at surface

Motility is an important characteristic of many enteric bacteria. In our case it helps in separation of Erwinia-Salmonella-Serratia (motile) fromShigella-Yersinia (nonmotile). Our organism is motile.

Citrate test is used in identification of many members ofEnterobacteriaceae. The positive test is represented by the development of a deep blue color throughout the medium within 24 to 48 hours.

Photo of gram-negative bacteria growing on Simmons citrate agar; positive result is indicated by deep blue color of the medium.
Citrate-positive colonies on Simmons citrate agar (indicated by the deep blue color of the medium)

The results of the citrate test excluded S. serotype Typhi, S. serotypePullorum, S. serotype Paratyphi A, S. serotype Gallinarum, and S. serotype Choleraesius (all citrate negative).

Vogues-Proskauer test. In our case, this test is useful to separateSalmonella (all negative) from Serratia (positive). Our organism is VP-positive which is represented as red color in the upper part of the broth:

Photo of test tube with VP-broth indicating a positive reaction by the formation of red color at surface.
Vogues-Proskauer-positive test indicated by red color in the upper part of the broth

Gelatin hydrolysis. Since some Serratia species show very low percentage of positive reactions in VP test, it makes sense to run some additional tests to provide a more solid evidence supporting Serratia (all positive except S. fonticola) against Salmonella (all negative) from . One of such tests is gelatin hydrolysis. Gelatin is a protein which is solid at 22 - 25 °.C. If the bacterium makes the enzyme gelatinase, the gelatin is hydrolyzed and becomes a liquid. The arrows indicate the areas where bacteria showed most activity transforming solid medium into liquid:

Photo of gelatin hydrolysis-positive  bacteria; positive result is indicated by solid medium transformed into liquid.
Gelatin hydrolysis positive test indicated by liquid areas in the medium

Hydrogen sulfide (TSI) test is one of the most important tests in separation Salmonella (positive ) from Serratia (all negative).

Photo of a test tube with bacteria tested negative for hydrogen sulfide production.
Hydrogen sulfide (H2S) negative test indicated by the absence of black color in the medium

Ta-da! Looks like the leftover raw ground meat became a home to bacteria tentatively identified as Serratia. Certain species are known to be pathogens of plants, animals, and humans.

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