M.luteus ai
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School
University of Nairobi**We aren't endorsed by this school
Course
BIOL 228
Subject
Biology
Date
Dec 29, 2024
Pages
15
Uploaded by CountElephantMaster1176
UniversityClassAssignmentBIOL_228_Formal_Lab_Report_on_the_Identification_of_Mi…90969 45988Document DetailsSubmission IDtrn:oid:::1:3108498848Submission DateDec 9, 2024, 3:29 PM UTCDownload DateDec 9, 2024, 3:29 PM UTCFile NameBIOL_228_Formal_Lab_Report_on_the_Identification_of_Micrococcus.docxFile Size36.1 KB13 Pages2,271 Words13,345 CharactersPage 1 of 15 - Cover PageSubmission ID trn:oid:::1:3108498848Page 1 of 15 - Cover PageSubmission ID trn:oid:::1:3108498848
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1 BIOL 228: Formal Lab Report on the dentification and Characterization of Micrococcus luteusfrom Environmental Sample Student’s NameInstitution’s AffiliationCourse Code and Section Instructor’s NameDate Page 3 of 15 - AI Writing SubmissionSubmission ID trn:oid:::1:3108498848Page 3 of 15 - AI Writing SubmissionSubmission ID trn:oid:::1:3108498848
2 BIOL 228: Formal Lab Report on the Identification and Characterization of Micrococcus luteusfrom Environmental Sample Microbial identification is a key factor in microbiology since the identification of microorganisms is the first step toward understanding their roles in different environments, health, and industries. Bacteria are diverse and their identification is based on morphological, physiological and biochemical tests. In this particular project the aim was to purify, identify and describe an unknown bacterium originating from a given environmental source. Correct identification of bacteria is crucial for the use in clinical diagnostics, environmental testing and food analysis. The unknown bacterium in this study was isolated at room temperature and was subjected to Gram staining which confirmed it as a Gram positive cocci. According to Bergey’s Manual of Systematic Bacteriology, the bacterium is Micrococcus luteus, a bacterium that is found in soil, water and air. The following is the experimental procedures, results, and analysis that enabled the identification made in this report. Materials and Methods Initial Sampling and Culture Conditions Swabbing was done in a mock environment and at room temperature. The sample was spread on nutrient agar and grown to get single isolated colony. Appearance of the colonies was observed and recorded based on color, size and the surface texture. Gram Staining A streak of the unknown bacterium was made and was then stained using Gram staining to identify its cell wall. Under the microscope, the cells were purple, meaning that they were Gram positive. This was done by noting their morphology as cocci in clusters that is characteristic of Micrococcus species. Page 4 of 15 - AI Writing SubmissionSubmission ID trn:oid:::1:3108498848Page 4 of 15 - AI Writing SubmissionSubmission ID trn:oid:::1:3108498848
3 Biochemical Testing To ascertain the identification, several biochemical tests were carried out. These tests assist to evaluate the metabolic potential of the bacterium and also to come up with its identification. The following tests played a crucial role: Catalase Test: The bacterium was also positive for catalase; this is an enzyme that will decompose hydrogen peroxide into water and oxygen. This reaction was evident by the formation of bubbles when hydrogen peroxide was applied. The catalase test is very important in differentiation between Micrococcus and other Gram positive cocci such as Streptococci which do not produce catalase. This positive result indicated that the bacterium was an aerobic organism as Micrococcus luteusis a strictly aerobic organism. Oxidase Test: The bacterium also tested oxidase positive due to purple coloration when the oxidase reagent was applied. This result indicates that cytochrome c oxidase, an essential enzyme in the electron transport chain of aerobic organisms, is present. This test also supported the identification of Micrococcus luteusas this bacteria is known to contain this enzyme. Mannitol Salt Agar (MSA) Test: On MSA the bacterium failed to ferment mannitol and this set it apart from other organisms such as Staphylococcus aureuswhich is able to ferment mannitol. The failure to ferment mannitol, while still able to grow on the medium was another positive attribute in support of Micrococcus luteusas a non-fermentative organism. Indole Test: The Indole test was carried out in order to identify the presence of indole from tryptophan. The bacterium did not produce indole, so species that can perform this reaction were excluded. This negative result in turn served to eliminate some of the possibilities of enteric bacteria that otherwise produce indole. Page 5 of 15 - AI Writing SubmissionSubmission ID trn:oid:::1:3108498848Page 5 of 15 - AI Writing SubmissionSubmission ID trn:oid:::1:3108498848
4 Citrate Utilization Test: In this test, the bacterium gave positive reaction which indicated that it could grow on citrate as the only source of carbon. Micrococcus luteuscan be distinguished from other bacteria because it can grow on citrate. The positive result for citrate utilization affirmed the identification of the unknown bacterium as Micrococcus luteus. Agar Tests: When cultured on other types of media, namely Nutrient Agar, the bacterium produced smooth, round, yellow colonies. This also supported the morphological observations made at the beginning of the investigation. The colony color, growth rates and lack of hemolysis on blood agar were typical of Micrococcus luteusand ruled out other bacterial species that are likely to produce different colony colors and or exhibit hemolysis. Narrowing Down the Identification Process The genus Micrococcusis known for its aerobic, non-motile, catalase-positive, and oxidase-positive characteristics. The results from the biochemical tests helped narrow the bacterium’s identity further, specifically identifying it as Micrococcus luteusbased on the following key features: Yellow pigmentationon nutrient agar, a key characteristic of Micrococcus luteus. Gram-positive cocciarranged in clusters or tetrads, as observed under the microscope. Catalase-positiveand oxidase-positivereactions, which support its aerobic metabolism. Negative result for mannitol fermentation, which eliminated other potential candidates, such as Staphylococcusspecies. Page 6 of 15 - AI Writing SubmissionSubmission ID trn:oid:::1:3108498848Page 6 of 15 - AI Writing SubmissionSubmission ID trn:oid:::1:3108498848
5 Inability to produce indole, which helped exclude certain enteric and other Gram-negative bacteria. These characteristics fit well with the description of Micrococcus luteusfound in Bergey’s Manual and further strengthened the conclusion that the unknown bacterium was indeed Micrococcus luteus. Results Morphological Observations Colony Morphology: Colonies appeared yellow, round, and opaque. Cell Morphology: Gram-positive cocci observed under the microscope were arranged in clusters, characteristic of Micrococcusspecies. Table 3: Biochemical Test Results Test Result Interpretation Catalase Test Positive (bubbles formed) Indicates the presence of catalase enzyme, which breaks down hydrogen peroxide into oxygen and water. Oxidase Test Positive (purple color change) Indicates the presence of cytochrome c oxidase, an enzyme in the electron transport chain. Mannitol Fermentation Negative (no color change on MSA) Indicates the inability to ferment mannitol, helping distinguish it from Staphylococcus aureus. Page 7 of 15 - AI Writing SubmissionSubmission ID trn:oid:::1:3108498848Page 7 of 15 - AI Writing SubmissionSubmission ID trn:oid:::1:3108498848
6 Indole Test Negative (no red layer after Kovac's reagent) Indicates the bacterium does not produce indole from tryptophan, ruling out certain enteric bacteria. Citrate Utilization Positive (growth on citrate agar) Indicates the ability to use citrate as the sole carbon source. This is a typical trait of Micrococcus luteus. OxidaseDark purple color Positive (presence of cytochrome c oxidase) CatalaseBubble formation Positive (catalase enzyme activity) Mannitol Salt AgarGrowth, no color change Non-fermenter of mannitol Simmons CitrateGrowth, color change to blue Positive (citrate utilization) TSINo growth or fermentation Negative (no sugar fermentation) MotilityNo movement observed Non-motile Oxygen RequirementHeavy growth in aerobic condition Obligate aerobe Summary of Key Identification Features for Micrococcus luteusCharacteristic Observation/Result Colony Color Yellow Gram Reaction Positive (purple) Cell Shape Cocci (spherical) Page 8 of 15 - AI Writing SubmissionSubmission ID trn:oid:::1:3108498848Page 8 of 15 - AI Writing SubmissionSubmission ID trn:oid:::1:3108498848
7 Cell Arrangement Clusters or tetrads Catalase Test Positive (bubbles) Oxidase Test Positive (purple color change) Mannitol Fermentation Negative (no color change on MSA) Indole Production Negative (no red color) Citrate Utilization Positive (growth on citrate agar) Pigmentation (on agar) Yellow (due to carotenoid production) Based on these results, the bacterium was identified as Micrococcus luteus. Discussion The identification of the unknown bacterium as Micrococcus luteuswas done through a number of steps including Gram staining, colony morphology and biochemical tests, and by consulting the Bergey’s Manual of Systematic Bacteriology. These approaches cumulatively offered sufficient data to support the identification of the bacterium, and to distinguish it from other similar species. Although successful, the identification process also revealed some aspects that, if subjected to more molecular analyses, could improve the accuracy of the results. Morphological Observations The first lead to the identification of the unknown bacterium was the colony morphology. The bacterium produced typical bright yellow colonies on nutrient agar, which is a typical feature of Micrococcus luteus. This species is known to form yellow-pigmented colonies, which are as a result of synthesis of carotenoid pigments. These pigments are Page 9 of 15 - AI Writing SubmissionSubmission ID trn:oid:::1:3108498848Page 9 of 15 - AI Writing SubmissionSubmission ID trn:oid:::1:3108498848
8 involved in the colour and also have a function of shielding the bacterium by absorbing light and thus preventing oxidative damage. Its round morphology, noncapsulated surface, and transluscent property were also consistent with the description of Micrococcus luteusin Bergey’s Manual.When viewed under the microscope further, the bacterium was found to be Gram-positive. The cells were small, round cocci grouped in clusters or tetrads, a characteristic of the Micrococcusgroup. These cocci, which are stained purplish by Gram staining, are an essential feature of the morphology that was useful for the differentiation of Micrococcus luteusfrom other Gram-positive cocci like Staphylococcus or Streptococcus that form clusters or chains, respectively. Microscopic Examination Characteristic Observation Gram Stain Positive (purple) Shape Cocci (spherical) Arrangement Clusters or tetrads Size 0.5-1.0 μm in diameter Surface Smooth Biochemical Testing Biochemical Testing After morphological identification, additional biochemical tests were carried out to get a more precise identification. The first observable outcome was from the catalase test Page 10 of 15 - AI Writing SubmissionSubmission ID trn:oid:::1:3108498848Page 10 of 15 - AI Writing SubmissionSubmission ID trn:oid:::1:3108498848
9 where the formation of bubbles occurred when hydrogen peroxide was used. Catalase enzyme decomposes hydrogen peroxide into water and oxygen, a feature typical for aerobic organisms including Micrococcus luteus.This result was important because it ruled out the bacterium from genera that do not produce catalase including the Streptococcus genus. The oxidase test also came out positive because when the reagent was added to the sample, its colour turned dark purple. This implied that bacterium has cytochrome c oxidase which is an enzyme present in the electron transport system of aerobic bacteria. This result was expected because Micrococcus luteusis an aerobic organism and it requires oxygen to perform its metabolic activities. The identification was also aided by the Mannitol Salt Agar (MSA) test. Micrococcus luteus does not ferment mannitol and as a result there was no color change on the MSA plate. This is different from other catalase positive cocci, for instance Staphylococcus aureuswhich is able to ferment mannitol. The failure to metabolise mannitol coupled with the ability to grow on MSA enabled the distinction of Micrococcus luteusfrom similar species. Last of all, the test for Simmons citrate was positive as the bacterium was able to ferment citrate without any other supplement. This ability to grow on citrate media also strengthened the identification of Micrococcus luteussince this species is known to metabolise many types of carbon sources. Narrowing Down the Identification Process At this point, the nature of the bacterium was getting defined a little more clearly. The genus Micrococcusis widely described by the following features: aerobic, non-motile, and positive for the catalase test. Also, the negative result on mannitol fermentation test and positive result on citrate utilization test also supported the identification of Micrococcus Page 11 of 15 - AI Writing SubmissionSubmission ID trn:oid:::1:3108498848Page 11 of 15 - AI Writing SubmissionSubmission ID trn:oid:::1:3108498848
10 luteus. Another thing that supported this species was the yellow coloration of the colonies which is characteristic of this species. In microbiology, the various species of Micrococcusare very closely related in most respects, including metabolic requirements and production of catalase. However, the presence or absence of certain enzymes such as those that ferment mannitol can be used to eliminate some of the species. Micrococcus luteusdoes not ferment sugars such as mannitol, which was useful in ruling out the other Micrococcus species which may have different metabolic characteristics.Limitations and Future Experiments While I successfully identified Micrococcus luteususing conventional microbiological methods, it had its own drawbacks. The biochemical tests used in this study are suitable for distinguishing between genera and species. However, they may sometimes be inconclusive or easily misunderstood. For example, two species of Micrococcus may have identical biochemical characteristics, and additional methods must be employed to isolate the organism. To avoid such limitations, other molecular methods including PCR and 16S rRNA gene sequencing could be used. These methods are more selective, work with specific genetic markers and give more accurate identification by comparing the bacterial DNA with the reference databases. PCR methods are particularly useful when the morphology and biochemistry of the bacterium are inconclusive or when there is uncertainty of contamination in the sample. Species level identification could, however, be done with higher resolution using 16S rRNA sequencing which is a molecular method used in bacterial identification. In addition to the molecular methods, other assays for enzymatic activities could be done. For instance, a nitrate reduction test would determine if the bacterium has the ability to Page 12 of 15 - AI Writing SubmissionSubmission ID trn:oid:::1:3108498848Page 12 of 15 - AI Writing SubmissionSubmission ID trn:oid:::1:3108498848
11 reduce nitrate to nitrite which is true for some Micrococcus species. This would help to improve the identification and it may also bring out a difference between Micrococcus luteus and other related species. One more area of further research would be to explore the aspects of the ecological versatility of Micrococcus luteus. Because it can live in soil, air and water, change in environmental factors like temperature, pH, or salinity when the bacterium is exposed to these elements can explain how the bacterium survives. This type of research may be helpful for other branches of environmental microbiology as well as for investigation of the bacterial behavior in the context of changes in their environment. Relevance of Findings The identification of Micrococcus luteushas significance in several aspects particularly in microbial and biotechnological analyses. It is not pathogenic to human beings and thus safe to be used in experiments for microbial physiology and biochemistry. Micrococcus luteusis very versatile with regards to the environment it can grow in, and is thus well suited for studies of stress resistance in microorganisms. Besides, because it can produce many metabolic enzymes, it has been used in several industries such as bioremediation and synthesis of antibacterial agents. ConclusionIn concluisoon, it is clear that morphological, biochemical tests, and manual references are very helpful in identification of unknown bacteria such as Micrococcus luteus.This experiment was able to demonstrate characteristics of Micrococcus luteusincluding Gram positive cell wall, oxidative metabolism and colony pigmentation. This study can be extended in future by using more accurate molecular techniques in the analysis to improve the results. Page 13 of 15 - AI Writing SubmissionSubmission ID trn:oid:::1:3108498848Page 13 of 15 - AI Writing SubmissionSubmission ID trn:oid:::1:3108498848
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13 References Bergey, D. H., & Holt, J. G. (2000). Bergey’sManual of Systematic Bacteriology. Springer. Biology 228 Laboratory Manual. (2024). Okanagan College. Campbell, N. A., & Reece, J. B. (2008). Biology (8th ed.). Pearson Benjamin Cummings. Pechenik, J. A. (2007). A Short Guide to Writing About Biology (6th ed.). Pearson Education, Inc. Page 15 of 15 - AI Writing SubmissionSubmission ID trn:oid:::1:3108498848Page 15 of 15 - AI Writing SubmissionSubmission ID trn:oid:::1:3108498848