Eric M. Simpson
Dr. Jeffrey C. Burne
Biology 1134
Unknown Bacteria Project
On June 22, 2011 , I selected unknown bacteria number seven. Prior to this date, I had repeatedly reviewed the safety rules for the lab. Throughout the semester, I have
diligently adhered to these rules and have, to the best of my ability, followed aseptic and sterile techniques as prescribed. Preparation for the performance of the laboratory tests included reading the assigned chapters in advance, and usually, re-reading in an attempt to understand what I had just read. As questions arose, I have repeatedly reviewed the laboratory manual, the textbook, gone on-line, and on numerous occasions have investigated the recommended references from the library. In lab practice, I was extremely protective of the equipment I used; the microscope was never slid, mishandled, left out or unclean after any usage. I always labeled my tubes and plates with my name, the date, the test, the disease number, and the culture medium. I was careful to ensure that handling of the trays, tubes and plates caused no further contamination of the environment or the other classmates. I cleaned my area of operations as needed; however, this was a team-effort, and quiet often, this was performed by my rather proficient “tablemates”. In an effort to aid in the appropriate identification of my unknown bacteria, I began accumulating considerable notes while recording the results of all tests performed.
diligently adhered to these rules and have, to the best of my ability, followed aseptic and sterile techniques as prescribed. Preparation for the performance of the laboratory tests included reading the assigned chapters in advance, and usually, re-reading in an attempt to understand what I had just read. As questions arose, I have repeatedly reviewed the laboratory manual, the textbook, gone on-line, and on numerous occasions have investigated the recommended references from the library. In lab practice, I was extremely protective of the equipment I used; the microscope was never slid, mishandled, left out or unclean after any usage. I always labeled my tubes and plates with my name, the date, the test, the disease number, and the culture medium. I was careful to ensure that handling of the trays, tubes and plates caused no further contamination of the environment or the other classmates. I cleaned my area of operations as needed; however, this was a team-effort, and quiet often, this was performed by my rather proficient “tablemates”. In an effort to aid in the appropriate identification of my unknown bacteria, I began accumulating considerable notes while recording the results of all tests performed.
On June 22, upon receiving my bacteria, I recorded its number in my notes. After receiving number seven, I performed a subculture, a streak plate, and a gram stain of my unknown bacteria. Performance of the subculture into nutrient broth took priority. I knew that—per the syllabus— in order to ensure that tests performed were on live bacteria, this subculture was to be the first step of every class; therefore, it became habitual to perform a subculture into nutrient broth as the first step of the class (as directed on a weekly basis). I also wanted to ensure that further testing did not risk contaminating my specimen. Next, in an effort to create a pure culture, or “clone”, I used the broth of the original bacteria culture to make a streak plate. This plate, I interpreted the next class day as having significant growth over the first quadrant, some growth over the second quadrant, and individualized colonies of growth consisting of several small (pinkish) round “clusters” spread mostly over the third quadrant and minimally growing into the fourth quadrant.
I interpreted my Gram stain results as being gram-negative cocci. The bacteria were very small and round, and not appearing to be in groups, pairs, clusters or chains. They also stained pink on repeated tests on this first day of testing. I know that some bacteria—such as the acid-fast bacteria Mycobacterium and pathogenic species of Nocardia—do not absorb the Gram stains. Informed that we would not have any acid-fast bacteria, I concluded that I had gram-negative cocci bacteria. I repeated the Gram stain test during the next two classes, and on each occasion, I interpreted the tests as revealing gram-negative results. Since then, I have read that some bacteria test gram-positive when young, and then test gram-negative while they are “budding individuals” (Bergey’s Manual of Determinative Bacteriology).
On June 27, after performing the subculture of my bacteria and reading and interpreting the streak plate, I initiated subcultures of my bacteria with four sugar broths. The results of these tests varied slightly. On June 29, I determined that the lactulose broth had changed to having a yellow color, and noted this as being positive for the production of acidic byproducts. The acidic byproducts caused the pH indicator, phenol red, to change color from red to yellow, indicative of a drop in the pH, and thus, an increase in the acidity of the subculture broth. The lactulose test tube also contained a Durham Durham
Also on June 27, I initiated a SIM test. The SIM test is actually three unrelated tests (sulfide/indole/mobility) conducted within one test tube. To aid in the differentiation between bacteria—as is the purpose of all of the tests performed—these tests were conducted to determine three features of the unknown bacteria. As usual, during the performance of these differentiation tests, the prepared tube must be inoculated with the bacterium. In this case, the medium was “stabbed” quickly about halfway down with the inoculating needle. Then, after allowing time for sufficient growth to occur at the appropriate temperature, results were interpreted.
The SIM test results were interpreted on June 29. The SIM medium contains peptones and sodium thiosulfate as substrates, and ferrous ammonium sulfate as the hydrogen sulfide indicator. Cysteine is a component of the peptones used in the medium. “Cysteine, in the presence of cysteine desulfurase, loses its sulfur atom through the addition of hydrogen from water to form hydrogen sulfide gas” (Harley 157). Also produced, is ammonia and pyruvic acid. The sulfide test was the first test interpreted. I looked for a black precipitant along the stab line. This would have occurred if the bacteria had caused a production of hydrogen sulfide, because the hydrogen sulfide would have combined with the ferrous ammonium sulfate. I did not observe any black precipitate, so I concluded that my bacteria were negative for the production of hydrogen sulfide in the SIM medium.
Next, I observed for motility. This motility would have been represented by growth of the bacteria away from the stab line. If there had been growth away from the stab line, I would have observed a growth pattern than resembled “an upside down Christmas tree”. I observed growth, but in doubt about the extent of the growth, I interpreted this growth to be the result of an imperfect stab into the medium. My interpretation of this test is that my bacteria are nonmotile.
The final test on the SIM medium was conducted to determine whether my bacteria contain the enzyme tryptophanase. Tryptophanase breaks down the amino acid tryptophan. Tryptophan is in nearly all proteins. If tryptophanase is in my bacteria, it will hydrolyze tryptophan causing the production of indole, pyruvic acid and ammonia. Unlike the pyruvic acid and ammonia, indole is not utilized by the bacteria. Therefore, it accumulates, and its presence can be detected in the SIM medium after the addition of Kovac’s reagent. Kovac’s reagent reacts with the indole causing a bright red compound. If this color change occurs after the addition of the Kovac’s reagent, the test is interpreted as being positive for indole. If a color change to red does not occur on the surface of the medium, this indicates that tryptophan was not hydrolyzed, and the test is negative for indole. My bacteria apparently did not hydrolyze tryptophan; I did not witness a color change within my test tube. My test was negative for indole.
On June 29, in addition to interpreting the SIM tests, two stains were performed on my bacteria. A capsular stain, which I repeated during an extra evening class, revealed that my bacteria did not appear to be encapsulated. Using oil immersion under the high-power lens, after performing the stain according to the instructions posted on the Vista website, I found that my bacteria were negative for capsules. In other words, I did not observe what would have appeared as “halos” around my bacteria. “In medical microbiology, demonstrating the presence of a capsule is a means of determining the organism’s virulence, the degree to which a pathogen can cause disease” (Tortura, et al 71). I did not expect to find encapsulated bacteria, because we were instructed that we would not be using extremely pathogenic organisms.
Another staining method performed on June 29 was an endospore stain. I used the Schaeffer-Fulton method to determine if endospores were present in my bacteria. The presence of endospores, coupled with their location and size, helps in the differentiation and identification of bacteria. According to our textbook, “certain gram-positive bacteria…form specialized ‘resting’ cells called endospores” (Tortora et al 96). If I had found endospores, I would have questioned the results of my Gram stain, for my Gram stain results indicated that I have gram-negative bacteria. My test results for endospores were negative.
In addition to interpreting the results of the sugar tests, the streak plate and the SIM medium and performing two stains on the unknown bacteria, June 29 was the day that we initiated tests to determine catalase activity, gelatinase activity, the EMB and the ENDO cultures and the phenylalanine deaminase culture. These last tests were interpreted a week later, on July 6. Each test is discussed individually as follows:
Some bacteria contain flavoproteins that reduce oxygen, and in doing so, produce hydrogen peroxide. Hydrogen peroxide is extremely toxic to cells because it is an oxidizing agent and can “destroy cellular constituents very rapidly” (Harley 178). Obligate aerobes, which require oxygen, and facultative anaerobes, which grow in the presence or the absence of oxygen, have either catalase or peroxidase that will catalyze the destruction of H2O2. In the process, two molecules of hydrogen peroxide are converted by the enzyme catalase, to two molecules of water and one molecule of oxygen (O2). By adding a fresh supply of H2O2 to an inoculated tryptic soy agar slant culture, catalase can be detected by noting the development of bubbles. The absence of bubbles indicates that catalase is not present in the bacteria. I added a few drops of the reagent hydrogen peroxide to the top of my inoculated slant tube and observed minute bubbles of free oxygen gas on the top of the medium. Therefore, my test was positive for catalase.
In the gelatinase test, I tested for the ability of my bacteria to hydrolyze gelatin. This test is very important because the presence of gelatinase in a bacteria is indicative of a very pathogenic bacteria. A bacteria with gelatinase has the potential of causing tissue damage throughout the body. If the bacteria can break down gelatin, it can break down collagen. Collagen is in connective tissue and connective tissue is found throughout our body. Collagen is also in bones and cartilage. If the proteolytic enzyme gelatinase had been present in my bacteria-inoculated nutrient gelatin test tube, liquefaction would have occurred within the tube after a period of incubation. If liquefaction was noted, it would have required a brief period of refrigeration of the test tube to confirm that the liquefaction did not resolidify, and thus, that the results were correct as originally interpreted. The lab manual states, “Nutrient gelatin may require up to a 14-day incubation period for positive results” (Harley 174). Based on our directions and test sequence (and disregarding the manual’s notice), since no liquefaction occurred, I determined that my bacteria had been negative for the enzyme gelatinase.
Review of the DIFCO Manual alerted me to the purposes of the ENDO and the EMB tests. “EMB…is used for isolating and differentiating lactose-fermenting from lactose-nonfermenting gram-negative enteric bacilli…are pH indicators, as well as inhibitors of microorganisms other than gram-negative bacilli” (DIFCO 257). “ENDO…is used for confirming the presence of coliform organisms…indicator to differentiate lactose fermenting and lactose non-fermenting” (DIFCO). Information that is much more detailed was revealed throughout the passages about these agars. Upon reading this material, I realized that the color of the colonies could help to differentiate the type of bacteria. My colonies were colorless, so I considered the possibility that I may have Salmonella until I read that Salmonella is a rod-shaped bacterium. Since EMB agar inhibits the growth of most gram-positive bacteria (exceptions are staphylococci, streptococci and yeast), and my plate has growth, I was reassured that I may have a gram-negative bacteria. If my “Z” pattern of growth on the EMB agar plate had had a blue tinge to it, I would have suspected that my bacteria fermented lactose. I did not observe a blue tinge to the pattern of growth, so I concluded that my bacteria did not ferment lactose. If the “Z” pattern on the ENDO plate had contained red colonies of growth, I would have suspected that acetaldehyde was produced, that “the aldehyde…[was] fixed by the sodium sulfite”, and that the fuchsin had formed the red colonies. Non-lactose fermenting bacteria produce “clear, colorless colonies”. My colonies were clear and colorless, therefore, in light of these two tests, I concluded that my bacteria do not ferment lactose.
The testing on July 6 was concluded upon the completion of the phenylalanine deaminase test. This test helps to differentiate among enteric bacteria, and was performed to determine if my bacteria produces phenylalanine deaminase. This enzyme catalyzes the removal of the amine group NH3+ from phenyalanine. The end products of this reaction include organic acids, water and ammonia. The small Petri dish that I had previously inoculated with a “Z” pattern had growth evident on it. To perform the test, 3-5 drops of acidic ferric chloride was added to the top of the growth. If the bacteria had reacted with the phenylalanine agar and produced phenlypyruvic acid (one of the organic acids produced) the acidic ferric chloride would have reacted with it and a color change to green would have occurred. That did not occur with my bacteria. Therefore, I concluded that my bacteria was negative for the ability to oxidatively degrade (deaminate) phenylalanine.
As a class, we were informed that all of our bacteria are negative for oxidase. This “test distinguishes between groups of bacteria based on cytochrome c oxidase activity” (Harley 189). The lab manual states that the family Enterobacteriaceae are oxidase negative. Although we did not get to test for oxidase, we were provided with a demonstration of an oxidase-positive result. The Pseudomonas bacteria displayed a purple color change after drops of the oxidase reagent were added. If there had not been oxidase in the bacteria, there would have been either no color change, or a light-pink coloration of the colonies.
July 6 was also a day for initiating more tests. The second motility test was one of these. I also inoculated a tube containing thioglycollate broth, one containing urea broth, two containing MRVP broth and one with citrate broth. On July 11, my second motility test confirmed my first impression of the prior motility test. There did not seem to be growth away from the stab line. So again, I concluded that my bacteria are nonmotile.
On July 11, I furthermore concluded that I had been successful at cultivating facultatively anaerobic bacteria. This was determined because my thioglycollate broth had growth throughout the tube, with the densest growth occurring in the upper half of the tube. If the growth had been limited to just along the top of the tube, I would have concluded that the bacteria were obligate aerobes. If, on the other hand, the growth had occurred only along the bottom of the tube, I would have concluded that my bacteria were strict obligate anaerobes, for they grow only in the absence of oxygen.
The urease activity test was next. This was performed to determine if my bacteria contains the enzyme urease. Urease breaks the nitrogen and carbon bond in amide compounds such as urea. This results in the formation of ammonia, carbon dioxide and water. The hydrolysis of urea produces an alkaline environment as the ammonia accumulates in the medium that contains a pH indicator such as phenol red. As the alkalinity increases, the pH indicator (phenol red) causes a color change to a deep pink or purplish red color. This color change is an indicator that the bacteria can degrade urea by using urease. This color change is a positive test result. No color change would be a negative test result. My test resulted in a bright pink coloration of the medium—indicative of a urea hydrolysis; thus, it was a positive result.
The IMViC test was the last test to perform on the 11th day of July. The “I” part had already been completed when I did the indole test. Remaining was the methyl red (M), the Voges-Proskauer (V) and the citrate (C) tests. The “MR” test determines whether bacteria catabolized glucose for their energy in a fermentation process which causes a pH change because the resulting products are mixed acids. The pH indicator in the medium will detect an increase in the acidity caused by the production of mixed acids such as lactic acid, acetic acid and formic acid. In this use of methyl red as a pH indicator, the test results are positive if the indicator turns red and negative if the indicator turns yellow. My test resulted in a change to a yellow color, therefore my bacteria is not a mixed acid fermenter.
The “VP” test, like the “MR” test of the IMViC tests, uses MRVP broth as a medium, and it is a fermentation test. It also tests for the fermentation of glucose, but the purpose is to determine if butanediol, instead of mixed acids, is produced. In theory, these two tests are always opposite of each other. In reality though, one must know that if test results seem skewed, to trust the “MR” test because it is 90% reliable, as opposed to the “VP” test, which is only 70% reliable. Testing required adding 40% KOH and a 5% solution of alpha-naphthol in absolute ethanol (Barritt’s reagents A and B) to the previously inoculated and cultured medium. Development of a red color in the medium 15 minutes after adding the reagent represents a positive result. Absence of redness is indicative of a negative result. My tests resulted in having to abide by the “reliability rule” because I received a negative methyl red test result and a negative Voges-Proskauer test result. Therefore, I concluded that the “MR” test was correct and that I had received a negative methyl red test result.
The citrate utilization test was the last of the IMViC tests. This test determines whether bacteria can use citrate as a sole source of carbon for energy. If citrate permease is available, it will facilitate use of citrate and result in the production of pyruvic acid, oxaloacetic acid and carbon dioxide. Simmons citrate agar slants contain sodium citrate for carbon, NH4+ for nitrogen and bromothymol blue as the pH indicator. Since oxygen is necessary for the use of citrate, this test is performed on slants. Sodium carbonate is formed as carbon dioxide combines with sodium when bacteria oxidize citrate. The resulting alkalinity raises the pH and the indicator turns blue. This color change to blue represents a positive citrate test. Citrate-negative cultures stay green and show no growth in the medium. My test tube contents changed from green to blue; therefore, I had a positive citrate test result.
On July 11, I started the nitrate reduction test, the antibiotic and the antiseptic sensitivity tests, and the last of the sugar tests (that I have already reported on). In addition, I was informed that the lipase test result is positive; however, on the 13th, I was further advised to ignore the previous test report, and that the lipase test is actually negative. The test for lipase is a lipid hydrolysis test. Lipase is an enzyme that hydrolyzes triglycerides and water into glycerol and free fatty acids. The bacteria, in metabolism processes such as glycolysis, the citric cycle and the beta-oxidation pathway, can then use these products. Phospholipids are in all cells; therefore, this test is important because of the significance of the potential harm that bacteria could cause if they can hydrolyze lipids. This test is performed on a medium consisting of spirit blue agar. If the bacteria possess lipase, the agar will change from a lavender color to a clear color because of the pH indicator in the medium detects acid production. This would be a positive reaction. A negative reaction is reflected by no change in color. Since it was negative, my test would have resulted in no change in color.
Initiated on July 11 and completed on July 13, the nitrate reduction test was performed to determine if my bacteria has the enzyme nitrate reductase. Bacteria that obtain energy through chemical oxidation, chemolithoautotropic bacteria, use inorganic compounds as their electron donors and carbon dioxide as their primary energy source. Bacteria that require organic compounds for their energy source, chemoorganoheterotrophs, use nitrate as their “terminal electron acceptor during anaerobic respiration” (Harley 214). With the use of the enzyme nitrate reductase, nitrate is reduced to nitrite in this process. Some bacteria can use other enzymes to reduce the nitrite even further. Most enteric bacteria reduce nitrate. My bacteria was positive for nitrate reductase, evidenced by a reaction of the reagents sulfanilic acid and naphthylamine which caused a color change to red. If a negative result had occurred, zinc oxide would have been utilized to rule-out a false negative.
At last, the final tests were completed on my bacteria. Two plates of Mueller-Hinton agar were prepared, allowed to harden and inoculated with my final subculture broth. Both plates were arranged in a clock-like pattern with five test items on each. One plate was prepared with the antiseptics that I had chosen. The other plate held five antibiotics. With the exception of one, the antiseptics were all “duds”. Of the five items in the antiseptic group, only Equate Foaming Hand Wash Wash.
In the Kirby-Bauer sensitivity antibiotic test, Penicillin (P10) and Vancomycin (VA30) showed no signs of inhibiting the growth of my bacteria. Since gram-negative bacteria have a low susceptibility to PCN, this was further confirmation that my bacteria are gram-negative. Kanamycin (K30) had the largest zone of inhibition; it represented about a fourth of the available surface of the plate. Chloramphenicol (C30) had a zone of inhibition extending around it about twice the size of the pill fragment itself. The Novobiocin (NB30) had a small zone about the size of the pill itself surrounding its edges.
In conclusion, unfortunately my test results did not lead me “incontrovertibly to the identity of the Genus of” (Dr. Burne’s syllabus) my bacteria. I have spent many hours going from library to library, book to book, but I may not have what it takes to find the answer to the project. The algorithms, or “flowcharts” as they are known, usually lead me to a choice between Veillonella or Neisseria. I have found others occasionally, but I always find something that excludes them from being a real possibility. I know you told us that Neisseria would not be an option and I believe that I also heard you say “probably not” when someone asked if Veillonella is one of the unknown bacteria. Although I have not even narrowed it down to a genus, I feel that I have not wasted my time, for I have learned a lot while participating whole-heartedly in this brain-straining exercise. If effort counts towards anything, and if I could only prove how much of my time and energy has been expended towards this goal, then I believe you would realize that I have earnestly tried to find the answer to this puzzling problem. Well, be it what it is, this is the best that I can do at this time, because my time has run out, and now it is time to turn this in to turnitin.com, print out a hard-copy, and get ready for school. But before I go, I submit this bacterium as being the closest that I could find. It matches most of the significant tests performed. Yersinia enterocolitica is a pleomorphic gram-negative bacillus that belongs to the family Enterobacteriaceae. It is non-lactose fermenting, glucose-fermenting, does not produce hydrogen sulfide, oxidase-negative, most isolates reduce nitrates, urease-positive, facultatively anaerobic and “motile at 25 [degrees] C. and nonmotile at 37 [degrees] C.” (Khan web).
Works Cited
Garrity, George. Bergey’s Manual of Systematic Bacteriology. 2nd Edition. Vol. 2. Part B. New York
Harley, John. Laboratory Exercises in Microbiology. 8th Edition. New York
Khan, Zartash Zafar MD. “Yersinia Enterocolitica.” Medscape Reference. Web. 18 July 2011 . http://emedicine.medscape.com/article/232343-overview
Tortora, Gerard J., Berdell R. Funke, and Christine L. Case. Microbiology: an Introduction. 10th Edition. San Francisco , CA
Zimbro, Mary Jo, et al. Difco & BBL Manual: Manual of Microbiological Culture Media. 2nd Edition. Sparks , Maryland
WOW! IS ALL THIS REALLY TRUE? GREAT WORK, SON.
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