When applying biology to real world cases, it takes numerous tests to be able to identify exactly what a type of bacteria an unidentified bacterium is, whether it is a bacterium that is causing a person to be ill or a bacterium that is being studied in a laboratory. Often times a bacteria is taken from a contaminated sample that may contain more than one type of bacteria and therefore the specific bacteria that is to be identified has to be isolated. This can often be done by streaking the sample to single colonies. The individual colonies can then be put through several different tests that allow one to identify distinctive aspects of the bacteria. Some examples of these tests are a gram-stain and an API test. These tests allow one to figure out what properties a bacteria strain contains, such as gram negative or gram positive with the gram staining test, or what bacteria an unknown is based on positives and negative of the API test.
The results of API tests, will show extremely accurately what a bacteria is based on previous tests performed. One such bacteria than can be identified is Salmonella choleraesuis. There have been many studies done where the particular strain of Salmonella needed to be identified so that a vaccine could be found. In one such study, the strain of Salmonella Choleraesuis had to be identified in order to then use live Salmonella choleraesuis to reduce the seroprevalence and the number of Salmonella carrier pigs at slaughter (Schwarz et al, 2011). By being able to identify the specific strain of the species Salmonella, scientists were about to prevent Salmonella from being passed on through pigs because of the Salmonella choleraesuis vaccine. Identification of a specific bacteria can help scientists make so many improvements in getting rid of deadly bacteria.
Materials and Methods
This lab was performed over several different days in order to utilize several different techniques in identifying the unknown bacteria. A tube that contained a red species S. marcescens and a white unknown bacteria was streaked to single colonies onto an agar plate using an inoculating loop. The plate was placed in the 37° incubator for 24 hours. Once incubation had occurred the single colonies were used for the different microbiology lab techniques. The first of these techniques used was to determine what the morphology of the unknown bacteria was. The second of these tests was a gram stain. This test was done in order to determine if the unknown bacteria was gram negative or gram positive. The third technique used was a hanging drop slide, which was prepared using another single colony and a light microscope at 100x resolution, in order to observe the bacterias motility.
The fourth test used was an oxidase test, which was also performed using another single colony of the unknown bacteria. This was done by placing the bacterial colony on filter paper and adding drops of oxidase reagent. The filter paper was then observed to see if it changed blue or not, in order to see if the bacteria produced cytochrome c oxidase. The final test used in the experiment was an API test. To begin the API test, a solution with bacteria and 5 mL of sterile saline, had to be made with a turbidity the same as the McFarland No. 3 (BaSO4) standard. This was done by adding loopfuls of bacteria to the saline solution, mixing the solution on the vortex, and then comparing the turbidity to the McFarland No. 3 standard, until the tubes were both at the same cloudiness.
This created solution was then used in the API test by adding specified amounts to each of the microtubes on the API strip. For each of the microtubes whose names were not underlined or boxed, the tubes were filled to where the microtubes met the capsule. In the microtubes whose names were underlined, the microtubes were slightly underfilled, and then the capsule was filled with mineral oil in order to create and anaerobic environment. The last of the microbes were the ones whose names were boxed. In each of these the microtube and the capsule were filled all the way up with the bacteria. The API test strip was then placed in the 37°C incubator for 20 hours.
After this time, observations were made about each of the different microtubes based on a given summary of results chart for the API test. A select number of microtubes had to have a few reagents added such as 10% ferric chloride, Barritts A and B solutions, Kovacs reagent, nitrate test reagent, zinc, and hydrogen peroxide, in order to see how the specific microtubes reacted at that time. Once the API test was performed, the negatives and positives found for each of the microtubes was used to identify the unknown bacteria using an identification table that showed the positives and negatives of many known bacterias.
In the first part technique used in the lab, the morphology of the unknown bacteria colonies was found to be circular form, convex elevation, and entire margin. The second technique used, gram-staining, produced pink bacteria, which had the appearance of elongated cocci. The pink color indicated that the unknown bacteria was gram-negative. In the third technique, the hanging drop slip, there was not true motility and instead only Brownian movement. In the fourth technique, the oxidase test, the filter paper did not turn blue. In the final test, the API test, the results (Fig. 1) shows that the unknown bacteria being studied was Salmonella species, or more specifically, Salmonella choleraesuis.
(Fig. 1) The API strip results after 20 hours of incubation at 37°C Discussion
Through the use of the API test, the results most accurately showed that the bacteria was Salmonella species. This is what the bacteria was, however, it was a specific kind of Salmonella, S. choleraesuis. In the results of the tests, there were four different microtubes that contained different results than in the identification table. In each of the these cases, the API test showed positive results when the table showed negative results. Just because these didnt match exactly, didnt mean that the experiment didnt work right. Each organism is different and because of this will have different reactions at times. In each of the cases, there was a small percentage that the results would be different than the identification table said it would be. With that, the Salmonella species all together was able to be identified better because, in general the different strains of Salmonella each come from the same species. There could also have been slight error when the microtubes were filled up.
There may have been times when the tubes were overfilled or underfilled or air bubbles may have gotten into the tubes changing the results. Overall, the results were fairly accurate since the Salmonella species was able to be identified. The other tests used in the experiment were also useful in helping identify that S. choleraesuis was the unknown bacteria. It can be shown through previous tests that S. choleraesuis and Salmonella strains have similar characteristics to those found from the techniques used in this lab. In several cases it is shown that Salmonella strains are gram-negative (Kempf et al, 2012). The fact that S. choleraesuis fit the characteristics of previous studies helps show that bacteria was found correctly when preforming the different techniques.
Agbor, T. A., & McCormick, B. A. (2011). Salmonella effectors: Imortant players modulating host cell function during infection. Cell Microbiology , 13 (12), 1858-69.
Schwarz, P., Kich, J. D., Kolb, J., & Cardoso, M. (2011). Use of an avirulentlive Salmonella Choleraesuis vaccine to reduce the prevalence of Salmonella carrier pigs at slaughter. Vet Rec , 21 (169), 553.