The Great Unknown

The Great Unknown: #1
Lydia P. Seagraves
The University of Texas at Arlington

Abstract
Students were given an unknown bacterium and were asked to identify it based on a number of biochemical tests. In this experiment, unknown #1 was to be identified. After a series of tests, the unknown bacterium was identified as Klebsiella Pneumonia. Identifying bacterium is an important skill as a future medical professional to determine diseases and illnesses and recommend the appropriate medications and plans of action.

The Great Unknown: #1
To narrow down which tests should be performed in order to identify the bacteria, a gram stain must be conducted. Bacterium can be either gram-positive or gram-negative and will be determined by a staining technique. Gram-positive bacterium has a thick peptidoglycan layer, which holds on to the crystal violet stain causing the stain to look purple. Gram-negative bacterium has a thin peptidoglycan layer and stain a red color. After determining the gram stain, it was still unclear whether the bacterium was gram positive or gram negative due to the contamination of the broth. One gram-positive and two gram-negative tests were performed to narrow down the results.
The first test, mannitol salt agar (MSA), was a gram-positive test. According to the Microbiology Lab Handout, MSA has a high salt concentration which makes it selective as organisms may or may not be able to tolerate the salt concentrations. The mannitol of the MSA makes it differential as acid may or may not be produced from fermentation. If there is no growth or acid presence on the MSA plate the results are MSA- (Stout, 2018, pg. 43).

The tryptic sugar iron agar (TSIA) test and the eosin methylene blue test (EMB) were the two gram positive tests conducted to clarify whether the bacteria was gram positive of gram negative. According to the Microbiology Lab Handout, TSIA tests for the production of gas, sulfur, and how the bacteria uses the sugars. Bacteria that ferment glucose present a red slant and yellow butt in the test tube. Bacteria that ferment glucose and an additional sugar present a yellow slant and a yellow butt in the test tube. If there is no color change, no sugars have been fermented. If there is sulfur production, there will be a dark black color in the medium. Bubbles in the medium means that gas has been produced. A yellow color change means that the waste products from fermentation have lowered the pH. A red color means no sugars have been fermented causing the pH to be raised (Stout, 2018, pg. 45). The EMB test is a selective test in that only gram-negative bacteria can grow on its medium, according to the Microbiology Lab Handout (Stout, 2018, pg. 42). If there is growth on the EMB plate, it can be confirmed that the bacterium is gram-negative. If the bacteria receive energy via lactose fermentation, the growth will be a, “bright metallic green or purple color and/or have dark centers on the medium…” (Stout, 2018, pg. 42) If the bacteria are not lactose fermenting, it will not have color. Color differences are a result of how much acid has been produced. If the dyes react with lactose fermenters in the acidic environment, the growth will be dark purple.

Other tests conducted were the SIM (sulfur, indole, motility) test, methyl red (MR) test, voges-proskauer (VP) test, citrate test, urease test, and MacConkey test. The SIM test detects sulfur, tryptophan, and if the organism moves. A positive result for sulfur is similar to the TSIA test as it turns black because sulfur has been reduced by enzymes such as cysteine desulfurase or thiosulfate reductase. A positive result for tryptophan is a red color that settles at the top of the medium after adding Kovac’s reagent (a negative result is a brown color). Kovac’s reagent has a substance called aminobenzladehyde and when added to the SIM media produces quinoidal if it is a positive result. Motility is observed if the medium is cloudy around where the medium was stabbed (Stout, 2018, pg. 44).
The MR and VP tests are the same broth, but different reagents are added to each test to determine different factors. The MR tests whether there is acid presence by adding methyl red. If there is acid presence, the MR broth will turn red, if not, it will turn yellow. If the MR tests positive (red) the pH is around 4.4. If negative the pH is at about 6.2. Adding methyl red to the MR broth tests for pH if mixed acid fermentation has occurred (Stout, 2018, pg. 43). For the VP test, Barritt’s reagent A and B are added to test for the presence of 2,3 butanediol which is a product of glucose fermentation. A positive test for VP results in a pink color change, while a negative presence results in no color change. If the VP test is positive (red) acetoin and diacetyl have reacted with guanidine nuclei (Stout, 2018 pg. 45).

According to the Microbiology Lab Handout, the citrate test tests for the enzyme citrate permease. Citrate permease allows bacteria to use citrate as their main energy source. A positive result turns the once green medium to blue. If the medium turns blue, citrate permease has raised the pH because it is converting materials into energy (Stout, 2018, pg. 41).
The urease test tests for the presence of the urease enzyme. A positive result can take anywhere from 24 hours to 8 days. A positive result will become pink, while a negative result will not change color (Stout, 2018, pg. 45). According to the lab manual, urease tests for the speed of urea hydrolysis. Urea contains potassium phosphate, if the bacteria can overcome the organism, the pH will have been raised and turned to pink from anywhere between 24 hours (fast urea hydrolysis) to 8 days (slow urea hydrolysis) (Leboffe, 2010, pg. 187)Method
To begin, an unknown broth of a bacteria was given to identify. A quad-streak was conducted and put in the incubator for 24 hours. The quad-streak was conducted to isolate colonies for the gram staining process.

Next, a gram stain was conducted to determine whether the bacterium was gram-positive or gram-negative. Students conducted a gram stain to narrow down which biochemical tests should be performed. In this experiment, the unknown bacterium was contaminated from the broth used in the quad-streak making the results of the gram stain ambiguous. To determine if the bacteria was gram-positive or gram-negative the MSA, EMB, and TSIA tests were conducted.
The first test conducted was the TSIA test. According to the lab manual, Microbiology: Laboratory theory and application, using aseptic techniques, the stabbing needle was flamed for sterilization purposes, using the needle, the bacteria was taken from the quad-streak plate and stabbed and spread on top of the TSIA medium. The results were observed after 24 hours (Leboffe, 2010, pg. 206).

The next test was the MSA test. The loop was flamed, and bacteria was taken from the quad-streak plate and inoculated onto the MSA medium. There was no special plating technique used (such as a t-streak or quad-streak). The bacteria were spread over the MSA medium, incubated for 24 hours, and was observed (Leboffe, 2010, pg. 137).

Next was the EMB test. Again, the loop was flamed, and bacteria was taken from the quad-streak plate and inoculated onto the EMB medium. There was no special plating technique used. The bacterium was spread over the EMB medium, incubated for 24 hours, and then was observed (Leboffe, 2010, pg. 142).

The next test was the MR test. The loop was flamed, and bacteria was taken from the EMB plate. The bacterium was inoculated into the MR broth and incubated for 48 hours. After 48 hours, 8 drops of the methyl red reagent were added to the broth and results were observed. (Leboffe, 2010, pg. 161).

For the VP test, the loop was flamed, and bacteria was taken from the EMB plate. The bacterium was inoculated into the VP broth and incubated for 48 hours. After 48 hours, 15 drops of Barritt’s reagent A were added to the broth and 5 drop of Barritt’s reagent B were added to the broth and observed (Leboffe, 2010, pg. 163).

For the citrate test, the loop was flamed, and bacteria was taken from the EMB plate. The bacterium was inoculated onto the top of the citrate (not stabbed), and observed after incubating for 24 hours (Leboffe, 2010, pg. 175).

For the urease test, the loop was flamed, and bacteria was taken from the EMB plate. The bacterium was inoculated into the urea broth. The test was incubated and observed once a day for 7 days (Leboffe, 2010, pg. 187)
For the SIM test, the stabbing needle was flamed, and bacteria was taken from the EMB plate. The bacterium was then stabbed into the SIM and incubated for 24 hours. After 24 hours, 5 drops of Kovac’s reagent was added to the medium and observed (Leboffe, 2010, pg. 204).

Lastly, the MacConkey test was conducted. The loop was flamed, and bacteria was taken from the EMB plate. There was no special plating technique used. The bacterium was spread over the EMB medium, incubated for 48 hours, and then was observed (Leboffe, 2010, pg. 140).

Results
The EMB, TSIA, and MSA were conducted first to determine whether the bacterium was gram-positive or gram-negative. For the MSA test, there was no growth or scant growth. If there had been growth on the MSA plate, the bacteria would most likely be gram positive. The TSIA test was conducted to see if the bacteria produced sulfur or not in order to determine if it is gram positive or gram negative. Most gram-negative bacteria do not produce sulfur including this unknown. EMB selects only for gram-negative bacteria. Since there was growth, observed as purple and pink with black centers, it can be concluded that the bacterium is gram-negative. Refer to the introduction of the paper for explanations of test results outlined below.

The TSIA test showed a yellow slant and a yellow butt (glucose was fermented), bubbles (meaning that gas had been produced), and there was no black (no sulfur).
After determining that it was gram-negative, the EMB plate was used to inoculate all other gram-negative tests starting with MR test. After adding 8 drops of methyl red to the broth, the broth turned yellow, meaning that the bacteria cannot produce mixed acid fermentation.
After adding 15 drops of Barritt’s reagent A and 5 drops of Barritt’s reagent B to the VP broth, there was no observable color change. The unknown bacterium does not use 2,3 butanediol as a precursor for glucose fermentation.
After 24 hours of incubation, the citrate test turned from a bright green to a bright blue. The bacterium does contain the enzyme citrate permease to make energy.
For the SIM test the results showed that there was no production of sulfur or indole. There was some motility of the unknown bacteria, but not much so the results were marked as weak to none (wk/-).
The MacConkey test showed pink edges around where the bacteria had been spread. According to the lab manual, Microbiology: Laboratory theory and application, this means that the unknown bacteria is a lactose fermenter (Leboffe, 2010, pg. 140).

The urease test resulted in no color change. The bacterium does not contain the enzyme urease.
TSIA: MSA: EMB:
VP: Citrate: Urease:
MR: MacConkey: SIM
Discussion
According to the lab handout, EMB and MacConkey may not always match to their theoretical results (Stout, 2018, pg. 28) However, EMB and MacConkey tests both match their theoretical results. Because it is not a determining factor, no bacteria were ruled out or confirmed.
The TSIA test ruled out Proteus mirabilis, Pseudomonas aeruginosa, and Salmonella typhimurium. The TSIA had a yellow slant and a yellow butt (A/A) which means the bacteria ferments glucose on both of the sugars. The fermentation of sugars lowers the pH and causes the color to change yellow (Stout, 2018, pg. 45).
MR ruling out Enterobacter aerogenes. E. aerogenes is negative for acid, however the results show that the unknown bacterium is also negative for acid fermentation. This could be another result of the bacteria being contaminated from the broth, making it hard to differentiate
The VP test confirmed that the bacteria was also not E. aerogenes. The unknown bacterium does not have 2,3 butanediol. At this point, the unknown bacteria could be Escherichia coli or Klebsiella pneumoniae.
The citrate test ruled out the possibility of our unknown to be E. coli. The unknown bacteria had a positive result for the citrate enzyme (it turned from green to blue).
The SIM test confirmed that our bacteria could not be E. coli and E. aerogenes. The results for the SIM test could only be matched to K. pneumoniae. E. aerogenes was a possibility, but there needs to be more motility.
K. pneumoniae fits the results of all the other tests except the urease test. According to the lab manual, Microbiology: Laboratory theory and application, the urease test for K. pneumoniae should turn out pink after approximately 8 days (Leboffe, 2010, pg. 187). Since the urease test was only observed for 7 days, it is possible that the color could have changed to a pink. Although this test was not observed for the correct length of time, the unknown can still be concluded as K. pneumoniae because of the other biochemical test results.

References
Cowan M.K. (2012). Microbiology: A system’s approach (3rd ed.). New-York: McGraw-Hill
Stout et Al. (2018) Microbiology lab notebook handout. University of Texas at Arlington.

Leboffe, M.J., ; Pierce, B.E. (2010). Microbiology: Laboratory theory and application (3rd ed.). Englewood, CO: Morton Pub.