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Microorganisms exist in great abundance in virtually every conceivable environment. While these microscopic life forms play a large role in our daily lives, they also live in mixed communities where interaction between species occurs. This allows for each species to carry out unique functions where every member has its place. Some can only grow in the presence of oxygen, while others cannot tolerate it to any extent. Microorganisms can be differentiated and classified by their apparent relatedness to one another. In the past, a great deal of emphasis was put on the morphological and physiological characteristics of microbes in classification. This is a problem however, as some of the phenotypic characteristics traditionally used in bacterial classification do not always correlate well with the groups established on the basis of relatedness (Krieg, 1986). In the last few decades, great progress has been made in the knowledge of the structure, physiological characteristics, and the nucleic acid composition of microbes (Willey et al. 2008). One of the ways in which classification has dramatically improved in efficiency is through bioinformatics. This field utilizes mass spectrometry to map the interconnections and correlations between DNA sequences and protein sequences in an effort to understand cellular function at the molecular level and differentiate among the many taxa of microbes (Demirev et al. 1999). While the use of such technology is advantagous, it can be complex and expensive, which is why it is our objective to classify an isolate using the classical method of differentiating taxa by their physiology and morphology. To do this we are to perform a wide array of analysis and tests that can be carried out without the use of extensive technology.
The following is based on the Materials and Methods sections in the Winter 2008 Biol 203: Microbiology Lab Manual by Dr. Keith Egger.
To start we serially diluted samples of forest soil and coarse woody debris, and prepared slants, deeps, broths, and streak plates using the diluted samples. The enumeration and growth patterns were observed and recorded. 4 isolates were sub-cultured and labelled to be tested. Over a period of 4 weeks, a series of observations and tests were performed on the 4 chosen isolates as follows:
Morphology of the colonies were examined using a compound microscope and characterized by form, elevation, margin, appearance, optical property, colour, texture, and diameter. Individual cell morphology was characterized by cell shape, cell arrangement, Gram stain, and dimensions at a specific magnification. Next the biochemical and nutrient cycling tests were performed, starting with the starch hydrolysis test which involved the addition of iodine. Motility was then tested for by using Sulfide, Indole, and Motility deeps and observing growth away from initial stab lines. Ammonification was tested for using peptone broths and Nessler’s reagent, while the nitrification was tested for with ammonium sulfate, Trommsdorf’s reagent, and H2SO4. Nitrate reduction was then tested for by adding reagents and observing changes in colour. Finally, the breakdown of hydrogen peroxide with catalse was tested for by adding hydrogen peroxide. The microbes preferred living environment was tested for by exposing the 4 bacterial isolates to an array of temperatures, pH conditions, and osmotic pressures. Optimal temperature was tested for by testing each of the isolates at 4, 10-15, 22, and 54 degrees celcius. pH conditions tested were 3, 5, 7, and 9 while osmotic pressure was tested by exposing the isolates to concentrations of 0%, 0.5%, 2%, and 5% NaCl.
The unknown specimen labelled Isolate #2 was found to be rod shaped, with slightly rounded ends clustered in very short chains or occuring singly. Table 1.0 shows that colonial morphology was undulate in elevation and entire in margin, and the appearance was very dull. All colonies were opaque in their optical nature, and all appeared a uniform dark red colour. The texture of the colonies was rough in both appearance and contact. While the length of each colony was not consistant, an average colony diameter of 6 mm was observed. Individual cell morphology showed that again the length was not constant even between cells, instead the size varied greatly between 1 and 10 micrometers, with a generally constant width of about 0.6 micrometers. The Gram stain test showed that isolate was clearly gram positive, as the colour of the cells were a deep purple when the Gram stain test was done.
Bacterial isolate #2 did not demonstrate the ability to hydrolyze starch, and showed no motility whatsoever (Table 1.0). No signs of ammonification or nitrification were observed, as both tests proved to produce negative results. Nitrate reduction was observed, as the addition of reagent A and B in the denitrification test produced a pale red colour. The catalase test proved negative, as no bubbles were formed with the addition of hydrogen peroxide.
When testing the optimal conditions at which growth occured for bacterial isolate #2 we found the most growth was produced under 54 degrees celcius, with the second most growth occuring at 22 degrees celcius. The relative difference in growth between the two temperatures suggests an optimal temperature around 40 degrees celcius. When testing pH conditions, the most abundant growth was observed at a pH of 5, while pH 7 achieved a fairly large growth (Table 1.0). The optimal pH for this microorganism is therefore slightly below 6. When exposed to varying concentrations of NaCl, bacterial isolate #2 was not successfull in conditions of high osmotic pressure. The largest growth occured at 0% and 0.5% NaCl, some growth at 2%, and virtually no growth occured at 5% NaCl.
By performing the tests described above, and comparing the results to literature, I have been able to identify my unknown bacterial isolate as a species from the broad genus Lactobacillus. This genus is comprised of over 100 species, none of which I could match my unknown isolate to using obvious characteristics (Wykapedia). The determination of the Gram stain and cellular shape of the unknown isolate was instrumental in first determining what group of microorganisms the unkown fell into. Lactobacillus are usually small (between 2 and 5 micrometers), with entire margins, convex, smooth, glistening, and opaque with an occasional brick red colour sometimes forming rough colonies. (Kandler and Weiss, 1984). All but the convex shape of the colony supports my hypothesis, which was probably due to misinterpretation. Also while the colour observed and roughness of texture matched the expected colour found in the literature, it is also stated that it is in rare cases that a pale red/rough colony is demonstrated by Lactobacillus.
The biochemical and nutrient cycling test results for the unkown bacterial isolate were similar to the physiological test results in that most but not all observed characteristics matched those found in the literature for the genus Lactobacillus. Of the tests that supported my hypothesis, the lack of starch hydrolysis suited Lactobacillus, as distinct starch degredation leading to clearing zones is only observed in a few species (Kandler and Weiss, 1984). The second biochemical test also supported my hypothesis as no signs of motility or H2S production were observed, and these characteristics are incredibly rare in all Lactobacillus species (Kandler and Weiss, 1984). Tests for ammonification and nitrification were incluclusive, as no literature was found to compare to experimentally determined results. The last biochemical test that supported my hyptothesis was the catalase activity test. This is true because when hydrogen peroxide was added to the isolate, a very small amount of bubbles were produced. While Lactobacillus species do not contain catalase, some strains decompose peroxide by a pseudocatalase, which is likely the case for our unknown. The nitrate reduction test proved to be the most contradicting to the hypothesis that the unkown isolate was Lactobacillus. This was because the addition of reagent A and B during the test produced a pale red solution, characteristic of denitrification, while the literature states that nitrate reduction in Lactobacillus is highly unusual (Kandler and Weiss, 1984). This could be due to the fact that if present, nitrate reduction usally only occurs when terminal pH is poised above 6.0, which was most likely the case in our trial (Kandler and Weiss, 1984).
Two of the three tests for environmental effects on growth supported our hypothesis, while the third did not act to prove or disprove, due to a lack of comparable literature. The incubation of our unknown isolate at varying temperatures produced significant support for the hypothesis as the optimal growth conditions were between 30-45 degrees celcius. This matches the literature value and best fits the unkown isolate into the category of thermophile, whose somewhat rare members can grow successfully at high temperatures such as the 53 degrees celcius tested (Willey et al. 2008). The growth temperatures of Lactobacillus species range between 3 and 55 degrees celcius, with an optimum occuring between 30 and 40 degrees celcius (Kandler and Weiss, 1984). Similarly, the slightly acidic conditions preferred by Lactobacillus (between 5.5-6.2) were matched experimentally achieving an optimum slightly below 6 and helped to prove the hypothesis (Kandler and Weiss, 1984).
Lactobacillus are considered apathogenic, and are mostly found in dairy products, grain products, meat and fish products, water, sewage, beer, wine, fruits and fruit juices, pickled vegetables, sauekraut, silage, sour dough, and mash. Lactobacillus can be considered facultative anaerobes, as they usually grow best under reduced oxygen habitats (Kandler and Weiss, 1984). While most prefer mesophilic to thermophilic conditions, strains of some species grow even at low temperatures close to freezing point. This characteristic, along with their acidophilic nature, allows Lactobacillus to reduce pH to below 4.0, thus preventing growth of most competitors and making them vital inhabitants of the human digestive tract (Kandler and Weiss, 1984). Lactobacillus is a very important contributer to food technology, as they function in the production and spoilage of fermented vegetable feed, food (e.g sourkraut, mixed pickles), and drinks (e.g beer, wine, juices).
Other tests that would have been advantageous in determining the type of unkown would have been a test for lactate production, as at least half of the end product carbon is lactate (Kandler and Weiss, 1984). While this would have been incredibly effective for this specific genus, it would not be an effecient test to perform, as this is a characteristic almost only found in Lactobacillus. Another test which would be very useful would be to test for spore formation, as it is similar to the Gram stain test in that is it useful for general seperation of groups of microorganisms. Some limitations of the tests performed include the time constraint, as incubation was usually necessary for at least 48 hours between trials. Also, not all phenotypic that are shared between strains correlate well with relatedness groups. This means that if we are to use the method of analysing microorganisms by shape, size and other physiological characteristics we are limited to the ones that do actually represent relatedness between groups.
Some possible sources of error in this study include the reliance upon interpretation of vague results for a large number of tests. An example of this can be seen in the test for ammonification, as it was very difficult to differentiate a pale yellow from a lack of colour change. Another possible source of error could be improper lab technique, such as failing to keep pH tubes completely clean. This mistake could have drastically changed the results of the experiment. A definite possible source of error in this lab was contamination. While using aseptic technique and making sure everything was kept sterile, cultures were still exposed to the air for brief periods, which could have allowed the introduction of a foreign species.
Overall the objectives of this lab were met successfully, as the goal was to properly isolate and identify an isolate strain using cited literature, which was done without any problems.