Being able to identify an unknown microorganism is an important part of modern medicine. For example, knowing what pathogen is infecting a patient’s body allows a doctor to choose the correct antibiotic so the patient can be treated. These series of tests are learned in the microbiology and this report examines all the techniques and skills needed to identify a bacterium. Before beginning the tests, a pure culture is created using aseptic technique, so that the biochemical tests will be performed on the unknown bacteria only and not possible contaminants. The first test in the identification process is used to identify whether the bacterium is Gram-positive or Gram-negative. This shows us whether the bacterium has a thick or thin peptidoglycan cell wall. Gram-negative and Gram-positive bacteria have very different treatment plans if the bacterium is pathogenic. After the Gram stain, one must test for positive or negative lactose and glucose fermentation by using EMB agar, MacConkey agar, lactose fermentation broth, glucose fermentation broth, and TSIA medium. The MR-VP test is used to determine how much acid a glucose fermenter produces and also tests whether the bacteria produces 2,3 butylene glycol and acetoin from glucose. The test using SIM medium tests for hydrogen sulfide production, motility, and indole production. The citrate test determines whether or not the bacterium can survive using citrate as its only carbon source. Finally, the urease test determines whether or not the bacterium can produce the enzyme urease. Based on these tests, the student can match the results of each test to the table provided in the microbiology lab book.
The first step in identifying a bacterium is to generate a pure culture. To do this, I made a streak plate from the broth given to me from the instructor. Then, I performed the first test on the unknown bacteria which was the Gram stain. I prepared a clean slide for the Gram stain by putting a single drop of water on the slide. Then, using aseptic technique, I transferred a small amount of solid unknown bacteria number ten from the pure culture on the slide creating a smear. After this, I heat fixed the bacteria to the slide using an open flame. Then I began the staining process by first putting crystal violet, the primary stain, on the slide for thirty seconds then rinsing it off. Next, the mordant, iodine, is added for one minute. Iodine intensifies the chemical bond between the bacteria and the crystal violet stain. After it was rinsed, ethyl alcohol was added for ten seconds. This is the differentiating step in the Gram stain. Ethyl alcohol removes the crystal violet from Gram-negative bacteria while it does not remove the crystal violet from Gram-positive bacteria. In the final step I added safranin, the counterstain, for thirty seconds. The counterstain stains any colorless cells red. The finished Gram stain of unknown bacteria number ten was then examined under a microscope. I observed red rods under the microscope which led me to the conclusion that it was a bacillus Gram-negative bacterium. Next, I transferred cells from the pure culture created to a nutrient agar slant. From the nutrient agar slant, I inoculated EosinMethylene Blue (EMB) agar. This agar is selective and differential. It selects for Gram-negative bacteria because it inhibits Gram-positive bacteria. Its differential property is a lactose fermentation test. Rapid lactose fermenters have a metallic green or purple color while non-lactose fermenters do not. The inoculated EMB agar was incubated for one week. When examined, the unknown bacteria grew and appeared to have a purple metallic color. I interpreted these observations and concluded that the unknown bacteria ten was in fact a rapid lactose fermenter. The MacConkey agar was also inoculated from the nutrient agar slant. MacConkey agar is selective and differential as well. It selects for Gram-negative bacteria by inhibiting the growth of Gram-positive bacteria because of the crystal violet and bile salts in the medium. The differential property of MacConkey agar is the color change that occurs when the pH of the medium is lowered. The pH indicator is neutral red and turns a dark pink when acid is produced which indicates that the bacterium is a lactose fermenter. Non-lactose fermenters have colorless colonies. After I incubated the MacConkey agar for one week, I examined it. The bacteria grew and the colonies were a dark pink. I interpreted these observations to mean the unknown bacteria ten was a lactose fermenter. Next, I inoculated two Durham Fermentation tubes testing lactose fermentation in one and glucose fermentation in the other. I incubated them for one week. Both tubes after one week were yellow meaning acids were produced. The lactose fermentation tube also showed there was gas production. This was noted because there was a bubble in the inverted test tube of the lactose fermentation Durham tube. Next, I inoculated the MR-VP broth from the nutrient agar slant. This was incubated for one week and then split the broth into two tubes. The Methyl Red test is used to see that although more than one species can ferment glucose, E. coli specifically produces much more acid and turns a dark red color. A negative reaction will turn yellow or orange. When I added Methyl Red to the broth, it turned an orange/red color. I interpreted that to be negative. The Voges-Proskauer test checks for the production of 2,3 butylene glycol and acetoin from glucose. The broth will turn pink or red for a positive result and yellow if it is negative. I added the reagents to the second tube containing broth and waited twenty minutes. The tube appeared red near the top half of the tube. I concluded that the Voges-Proskauer test was positive. Next, I inoculated a TSI agar slant by streaking the slant and stabbing the slant using bacteria from the nutrient agar slant. The Triple Sugar Iron Agar (TSIA) test checks for the production of hydrogen sulfide, and it shows whether the bacteria can ferment glucose and lactose/sucrose. If positive for production of hydrogen sulfide, the butt of the tube will be black. If negative for all sugars, the whole tube will be red. If positive for glucose only, the slant will be red and the butt will be yellow, and if positive for all sugars it will be completely yellow. I incubated the inoculated TSI agar slant for one week. Upon examination, the entire tube was yellow. I came to the conclusion that unknown bacteria ten was negative for hydrogen sulfide production and positive for the fermentation of glucose and lactose or sucrose. Next, I inoculated SIM medium by stabbing the medium with a needle containing unknown bacteria ten from the nutrient agar slant. SIM (Sulfide Indole Motility) medium incorporates three tests in one medium. It tests for the production of hydrogen sulfide by checking whether the enzyme cysteine is produced. It also tests for indole production by adding reagents which will turn the broth pink/red with a positive reaction. Motility is also tested which is indicated by a spreading of the microbe’s growth away from the initial stab. After incubating the SIM medium for one week, I observed there to be no black production and also no color change after the reagents were added. I also observed the spreading of the bacterium in the tube away from the stab. I interpreted this to mean that unknown bacteria number 10 is hydrogen sulfide negative, indole production negative, and positive motility. Next, I inoculated the citrate agar slant with bacteria from the nutrient agar slant. The citrate utilization test determines whether the bacterium can survive using only citrate as its carbon source. If bacteria growth occurs, the agar will turn a rich blue color. After incubating the citrate agar slant for one week, I observed the slant to be bright blue. I interpreted this to mean that unknown bacteria number ten can, in fact, utilize citrate as its only carbon source.