Identification of Unknown Bacteria:
Isolation from Windowsill of Microbiology Laboratory
Klebsiella

Introduction
The bacterium, Klebsiella, which was isolated from the windowsill of the microbiology lab, was a gram-negative rod. The purpose of this lab was to isolate and identify the genus of a bacterium from the environment, in this case, a windowsill. Isolation and identification techniques are important aspects of microbiology. Applications include clinical diagnoses, evaluation of water quality, and discovery of new microbial species. In this particular experiment, a bacterium from the windowsill was identified. This technique would be useful if a certain bacterium was known to be a pathogen, and it was required to check to see whether it was present. If the proper tests are chosen and conducted aseptically, the bacterium can be identified to the genus stage in this particular laboratory. Additionally, the bacterium identified should be one which is possibly found in a free-living environment or one that can be found in soil and water samples since the windowsills belong to a biological laboratory.

Methods and Results
The isolation was made by first inoculating an agar plate with a wet swab of the windowsill. The spread method used was the T streak and, after incubation at 37*, resulted in isolated colonies. A colony that was fairly big and isolated from other colonies was chosen to be examined. Half of the colony was used to make a slide. A gram stain was performed, and it was deduced that the bacterium was gram negative. The stain also revealed that the bacterium was a short, fat rod. The rest of the colony was used to inoculate another agar plate by way of the T streak method. After incubation, the plate looked as if it contained bacterial colonies of one type of morphology. The yellow, mucoid colonies were rather large indicating that the bacterium is fast growing. A colony from this new streak was confirmed to be gram negative rod by another gram stain as well as through the potassium hydroxide (KOH) test. The procedure used can be found in BIO 103 Lab Manual (Vilgalys, 2001). Because the culture, once the 3% KOH was added, became mucilaginous, the gram-negative label was confirmed. A final isolation streak was made to make sure that the colonies were axenic. Additionally, a plate was made to be used as a back up if a plate were to become contaminated.
After a pure culture was created, a couple more preliminary tests were taken. The motility and oxygen tolerance tests were chosen because they would narrow down the possibilities of the identity of the bacterium quickly (Holt, 1984). The tests were conducted as described in the BIO 103 Lab Manual (Vilgalys, 2001). The motility test after 48 hours showed that the bacterium was non-motile. The controls showed that there was no contamination between the strips. Additionally, the known motile yielded a positive result, and the known non-motile gave a negative result. The controls added confidence to my diagnosis. The oxygen tolerance tests showed that the organism was a facultative anaerobe. The Gas Pak system showed growth on both the plate that was in the anaerobic pak and the plate that was exposed to oxygen. These results were confirmed by the growth in trypticase soy agar deep, which was consistent throughout the tube, showing that the bacterium can grow with or without oxygen.

The next step was to determine what genera of bacteria, which are gram-negative, facultative anaerobic rods, are most likely to be found on a windowsill of a microbiology laboratory. As it turns out, this type of bacterium is very common (Holt, 1984). The possibilities included a wide array from the Enterbacteriaceae family, Cardiobacterium, or Eikenella (Holt, 1984). The tests used to discriminate amongst these possibilities are described in greater detail in the BIO 103 Lab Manual (Vilgalys, 2001). The first test conducted was the oxidase test, due to its large discriminating ability in this situation. Since the bacterium produced a negative result, the test eliminated Cardiobacterium and Eikenella.

After determining that the bacterium belonged to the Enterbacteriaceae family, tests, which take advantage of the fact the different bacteria synthesize and utilize different enzymes, had to be considered to differentiate amongst the genera. A problem that was encountered during the determination of what tests to conduct was that many of the species within the genera show variation amongst themselves. For example, some species of Klebsiella produce butanediol during fermentation, others do not. Therefore, it was very difficult to choose a test that would eliminate half of the possibilities. However, some tests were immediately disregarded as options because they would have not eliminated any of the candidates (Holt, 1984).
The procedure began with the cysteine desulfurase test. No black precipitate was formed eliminating Leminorella and Salmonella as possibilities (Holt, 1984). The citrate utilization test was then conducted and a royal blue color appeared, indicating that citrate can be used as a source of carbon for this bacterium (Vilgalys, 2001). The positive result eliminated Shigella, Tatumella, and Yersinia (Holt, 1984). The bacterium was then tested to see whether butanediol was produced during fermentation. The fact that the solution turned red in about twenty minutes after the alpha naphthol and potassium hydroxide was added showed a positive result leaving Enterobacter, Klebsiella, Serratia and Proteus as possibilities (Holt, 1984). The next test determined whether the bacterium was able to convert urea to ammonia and carbon dioxide by producing urease (Vilgalys, 2001). The slant turned a slight pink, neither yielding a strong positive or negative. Because Proteus yields a strong positive, it was eliminated (Koneman, et al, 1992). Since Klebsiella is known to hydrolyze urea very slowly, it seems to be the strongest candidate for the bacteriumís identity (Koneman, et al., 1992).

More research was done on Klebsiella, Serratia, and Enterobacter. Since Enterobacter is rarely non-motile, it was eliminated as a possibility. However, there was no test available in this laboratory to help in discriminating between Klebsiella and Serratia (Holt, 1984). A lysine test was conducted to see whether Enterobacter could be confidently eliminated, but the test was negative indicating that it should remain as a slight possibility (Holt, 1984). The acid/gas and the phenylalanine tests were conducted to confirm that it could be either Klebsiella or Serratia (Holt, 1984). The acid/gas test showed that the bacterium yielded both acid and gas upon utilization of glucose, which was consistent with the genera (Holt, 1984). On the other hand the phenylalanine test was a weak positive. Both of these bacteria should have yielded a negative. However, with further investigation the phenylalanine test was found to be inconsistent, and, when a known Serratia species was tested in the lab, it yielded a weak positive when it should have been negative.

Discussion and Conclusion
I am very confident that the bacterium I isolated from the windowsill belongs to the Enterobacteriaceae family because they are characterized as having large, mucous colonies, all fermenting glucose, and all lacking the cytochrome oxidase enzyme (Delost, 1997). Furthermore, the family exists free-living in the environment (Shimeld, 1999). These qualities all characterize the bacterium isolated from the windowsill. I am also confident that the bacterium is either Klebsiella or Serratia because the tribe that they belong to is the only one that tests positive to butanediol production (Delost, 1997). I am only fairly confident that the bacterium is Klebsiella because no test clearly proved it. However, the fact that Serratia is usually motile and that Klebsiella yields a pink color for the urease test shows some indication that the bacterium belongs to the Klebsiella genus (Koneman, et al., 1992). Furthermore, Serratia usually produces a red pigment (Koneman, et al., 1992).

For future experiments, I would definitely keep the same general format for identifying an unknown. I would begin with tests that can eliminate a large portion of the possibilities and then look for more specific tests. However, the problems in this investigation arose due to the fact that the possible genera for the bacterium had many similar characteristics and not enough tests were available to make a confident decision. If further tests could have been performed, I would have attempted to grow the bacteria on MacConkey agar. This medium is known for its ability to differentiate among gram-negative bacilli (Delost, 1997). When grown on MacConkey agar, Klebsiella appear as pink, mucoid colonies and Serratia as clear colonies (Delost, 1997). This test would have yielded a result that could have clearly differentiated between the two strong candidates for the unknown.

In any case, although the bacteria from these genera can cause disease in humans, Klebsiella and Serratia are usually nonpathogenic in healthy humans (Shimeld, 1999). When these bacteria do infect a person with a weak immune system, they can cause diseases such as pneumonia and other lung diseases; meningitis, the inflammation of the membranes surrounding the brain and spinal cord; and endocarditis, the inflammation of the inner tissue of the heart (Delost, 1997). The fact that these bacteria have a high probability of existing on such seemingly benign locations as the windowsill show the need for people to wash their hands throughout the day to prevent contracting such diseases as caused by these bacterium (Koneman, et al., 1992).
 

References
Delost, M.D. 1997. Introduction to Diagnostic Microbiology. Mosby, St. Louis, MO.

Holt, J.R. (ed.). 1984. Bergeyís Manual of Systematic Bacteriology, 1st edition, Volume 1. Williams & Wilkins, Baltimore, MD.

Koneman, E.W.; Allen, S.D.; Janda, W.M.; Schreckenberger, P.C.; Winn, W.C. 1992. Color Atlas and Textbook of Diagnostic Microbiology. J.B. Lippincott Company, Philadelphia, PA.

Shimeld, L.A. 1999. Essentials of Diagnostic Microbiology. Delmar Publishers, Albany,
NY.

Vilgalys, R. 2001. Laboratory Exercises for BIO 103 General Microbiology. Duke University, Durham, NC.
 

Identification of Unknown Bacteria:
Given Unknown #85
Citrobacter freundii

Introduction
The bacterium, Citrobacter freundii, which was isolated from the given unknown #85, was a gram-negative rod. The purpose of this lab was to isolate and identify this bacterium from a solution, which contained three different species of bacteria. Isolation and identification techniques are important aspects of microbiology. Applications include clinical diagnoses, evaluation of water quality, and discovery of new microbial species. If the proper tests are chosen and conducted aseptically, then the isolated and identified bacterium should be one of the three bacteria that was contained in the original solution.

Methods and Results
The isolation was made by first inoculating an agar plate with solution #85, which contained the three bacterial species (Vilgalys, 2001). The spread method used was the T streak and, after incubation at 37*C, resulted in isolated colonies. A colony, which was fairly big and very isolated from other colonies, was chosen to be examined. Half of the colony was used to make a slide. A gram stain was performed and it was deduced that the bacterium was gram negative. The stain also revealed that the bacterium was a long, slender rod. The rest of the colony was used to inoculate another agar plate by way of the T streak method. After incubation, the plate looked as if it contained bacterium colonies of one type of morphology. The milk-white colonies were rather small indicating that the bacterium is slow growing. A colony from this new streak was confirmed to be gram negative rod by another gram stain as well as through the potassium hydroxide (KOH) test. The procedure used can be found in the BIO 103 Lab Manual (Vilgalys, 2001). Because the culture, once the 3% KOH was added, became mucilaginous, the gram-negative label was confirmed. A final isolation streak was made to make sure that the colonies were axenic. Additionally, a plate was inoculated in order to be used as a back up if the sample were to become contaminated or lost.

After a pure culture was created, a couple more preliminary tests were taken. The motility and oxygen tolerance tests were chosen because they would narrow down the possibilities of the identity of the bacterium quickly. The tests were conducted as described in the BIO 103 Lab Manual (Vilgalys, 2001). The motility test after 48 hours showed that the bacterium was motile. The controls showed that there was no contamination between the strips. Additionally, the known motile yielded a positive result and the known non-motile gave a negative result. The controls added confidence to my diagnosis. The oxygen tolerance tests showed that the organism was a facultative anaerobe. The Gas Pak system showed growth on both the plate that was in the anaerobic pak and the plate that was exposed to oxygen. These results were confirmed by the growth in trypticase soy agar deep, which was consistent throughout the tube, indicating that the bacterium can grow with or without oxygen.

Further differentiation was based upon the presence or absence of different enzymes. A summary of the sequence of procedures can be found in Figure 1 at the end of the report. All diagnostic procedures used in this report can be found in the BIO 103 Lab Manual (Vilgalys, 2001). The list of possibilities for gram-negative, motile, facultative anaerobic rods was fairly extensive, so a test was chosen which would eliminate about half of the options. A reliable test which fit this description was the cysteine desulfurase test (Holt, 1984). The heavy black precipitate showed that the bacterium synthesizes this particular enzyme and liberates hydrogen sulfide. From the list of possibilities provided by the laboratory, the options that remained were Proteus vulgaris, Serratia marcescens, Salmonella typhimurium, and Citrobacter freundii.

At this point, more specific tests had to be conducted that would either confirm or eliminate each of the bacterium one at a time. The tryptophanase test, which determines whether the bacterium produces indole, resulted in Kovacís reagent not revealing a red layer. This negative result eliminated P. vulgaris, which does produce indole (Holt, 1984). The next test conducted determined whether or not during fermentation butanediol is produced. The test must be conducted carefully because instead of testing for butanediol, which is neutral and hard to detect, the intermediate acetoin is measured (Vilgalys, 2001). When 40% KOH was added to a portion of the incubated solution, there was no indication of a color change towards red or pink. This negative result eliminated S. marcescens (Holt, 1984). Finally, the last test chosen to differentiate between S. typhimurium and C. freundii was the lysine decarboxylase test. Both the control, which was the broth without lysine, and the broth with the lysine turned yellow. The control provided a confirmation for the test because the fact that the control turned yellow implied that the procedure was conducted properly. This indication of a negative result showed that the bacterium could be identified as being C. freundii (Holt, 1984).

Discussion and Conclusion
The tests conducted show with great confidence that the bacterium isolated was C. freundii. The controls as well as the clear results from reliable tests support this statement. Furthermore, C. freundii was one of the bacteria, which was initially used to create solution #85. Even though this bacterium closely resembles species in the Escherichia and Salmonella genera, the tests chosen clearly eliminated those possible species (Shimeld, 1999). These distinguishing tests are important because these species are known to be pathogenic. The doctor bases his or her antibiotic treatment depending on what species is found in the blood, urine, or feces sample. Although not particularly fast, the tests chosen in this report were reliable and could identify the bacterium as C. freundii in an accurate manner. In this way the doctor would prescribe the antibiotic carbenicillin rather than sulfamethoxazoletrimethoprim for E. coli or S. typhimurium (Shimeld, 1999). The success of the identification was mainly due to the fact that preliminary tests enabled me to focus on a few possibilities. Then due to the fact that I had experience doing this kind of testing I was able to choose tests that would eliminate species accurately. The only problem encountered was that midway through the testing the morphology of the colonies that were grown changed indicating that the streak plate created was contaminated. A new streak plate was made using a colony from an old culture known to be pure. All tests were redone and confirmed as being consistent with the previous findings.

 References
Holt, J.R. (ed.). 1984. Bergeyís Manual of Systematic Bacteriology, 1st edition, Volume 1. Williams & Wilkins, Baltimore, MD.

Shimeld, L.A. 1999. Essentials of Diagnostic Microbiology. Delmar Publishers, Albany,
NY.

Vilgalys, R. 2001. Laboratory Exercises for BIO 103 General Microbiology. Duke University, Durham, NC.
 
 
 

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