Identification and Analysis of Bacillus megaterium
April 19, 2019.
Identification and Analysis of Bacillus megaterium
Introduction
Background Information
The experiment was conducted to identify bacterial species based on the gram stain reaction, morphology, and arrangement of the isolated colonies in the growth medium. Identifying the bacterial morphology, arrangement and gram reaction were pivotal to narrowing down the bacterial species and carrying specific tests that would help identify the specific bacteria. For instance, the MR/VP test was used to determine the fermentation pathway that the unknown bacteria used to utilize glucose while starch hydrolysis test identified whether the bacterial species of interest could hydrolyze amylase and amylopectin using alpha-amylase and oligo-1,6-glucosidase enzymes; thus, differentiating the genera Bacillus and Clostridium. Therefore, the utilization of specific tests and procedures were instrumental in bacterial identification and analysis.
Purpose Objective
The primary goal of the experiment was to identify unknown bacteria using specific guidelines and procedures of identifying and differentiating bacterial species.
Materials and Methods
Materials
Various materials were critical for the successful identification of the unknown. A Gram Stain was used in elucidating the Gram reaction, arrangement, and morphology of the bacterial colony. A spore test kit was instrumental in determining whether the bacteria sporulated. A starch agar helped in identifying whether the bacteria could hydrolyze glucose in the agar. Methyl Red and Voges Proskauer stains were elemental in identifying the species of bacilli bacteria that are gram-positive and exhibits a rod shape.
Procedure
The first step was describing the bacterial colony using the terms provided in the laboratory handout by indicating as side A or B. The second step involved a Gram Stain reaction procedure, determination of bacterial morphology, and the arrangement observed. In the third step, the instructor confirmed the Gram reaction before proceeding with the inoculation steps. The following step was to follow the directions on the appropriate flow chart from lab notes and manual to identify the appropriate tests to carry out. Additional tests (spore staining, starch hydrolysis test, and Methyl Red/ Voges Proskauer (MR/VP) test), inoculation on starch agar, and Voges–Proskauer (VP) test was done for Gram-positive rods that were identified during the procedure. My lab partner inoculated the identified gram-negative rods on an API 20 E strip and motility agar isolated. Finally, all the reactions used to test for the unknown gram-positive rods were recorded on a log sheet to aid in the writing of a lab report.
Results
The initial findings included observations of the colony in the growth media and recorded as Side A and B. Colony description are as follows:
| Description | Side A | Side B |
| Form | Circular | Circular |
| Surface Appearance | Smooth | Smooth |
| Elevation | Flat | Convex |
| Internal Structure | Finely Granular | Amorphous |
| Optical Characteristics | Translucent | Translucent |
| Hemolysis | Beta | Alpha |
| Gram Reaction | Gram Positive | Gram Negative |
| Morphology | Rods | Rods |
| Arrangement | Chains | Random |
This experiment pursued the Gram-positive rods whereby, additional stains and tests were used to determine the unknown. The first test was spore staining whereby the bacteria tested positive, showing the ability to grow spores. The outcomes observed included green spores and pink rod-like structures. The second test was MR/VP staining which tested negative affirming that the bacteria was B. megaterium and not B. cereus or B. subtilis which both test positive for VP stain and negative for MR test (Sneath, Mair, Sharpe, & Holt, 1986). Finally, the Gram-positive bacteria grew on starch agar after inoculation; thus, tested positive for starch hydrolysis test. There were clear zones observed in the growth medium after the addition of iodine.
Discussion and Conclusions
Analysis of Data
The bacterial colony description was beta-hemolytic meaning that the organism lyses red cells entirely using hemolysins O and S (cytotoxins) produced by the bacteria. The flat characteristic described the side view elevation of the colony while smooth implies that the margins of the colony are entire. Gray defined the bacterial pigmentation during growth in the medium. Translucent appearance described the opacity of the colony meaning that it was almost clear but distorted (Kysela, Randich, Caccamo, & Brun, 2016). Finally, a Gram-positive reaction meant that the bacteria had peptidoglycan on the cell wall while rod-shaped and chain-like structures defined the morphology and arrangement of the bacteria respectively (Seltmann & Holst, 2013).
- megaterium is an endospore-forming bacterial species, and that is why it tested positive for the spore stain test. According to Gupta, Zhou, Bailey, and Christie (2015), the spores are essential for the bacteria in responding to adverse environmental conditions because they are characteristically highly resistant and dormant; hence, facilitate bacterial survival. MR test was used to determine whether the bacteria fermented succinic, lactic, and acetic acids after glucose supplication. The test was negative because B. megaterium produced less concentration of these acids during glucose fermentation; hence the pH remained high. More so, VP helps determine that the bacteria did not produce acetoin for conversion to diacetyl during anaerobic respiration.
Noteworthy, amylopectin and amylase are large molecules and cannot pass through the bacterial cell wall, necessitating the secretion of the two enzymes that break the starch molecules into simpler glucose subunits that are capable of entering the glycolytic pathway directly. Interpreting the results of a starch hydrolysis test requires the addition of iodine into the starch agar whereby it will react with the starch to form a dark-brown color (Hansen, Wayment, Klein, & Godfrey, 2018). More so, the hydrolytic reaction creates clear zones around the bacterial growth; hence, a positive test for the bacterial species.
Critique of the Experiment
The experiment was straightforward with minimal limitations. The procedural steps were crucial to identifying the unknown bacteria. However, I will include additional tests to define specific bacteria based on research information accurately.
Clinical Significance of Bacillus megaterium
Many studies have concluded that B. megaterium is non-pathogenic and does not elicit harmful outcomes to environmental and human health. More so, it is rare that this bacterial species causes critical clinical conditions. However, in their study, Loong et al. (2017) explained that the bacteria might have severe ramifications despite its dismissal as an insignificant contaminant. The researchers also cited other studies that implicated the bacteria in the formation of a brain abscess, ovarian mass torsion, food-borne illnesses, and sepsis. Also, the bacteria can attach and invade human erythrocytes, and its isolates have been identified to contain toxin-encoding genes associated with food poisoning.
In conclusion, the experiment utilized defined procedures to define the bacterial colony isolates and carry out tests to identify the unknown. The identification of gram-positive rods prompted for additional tests including MR/VP, spore staining, and starch hydrolysis test. The experiment concluded that the unknown organism was Bacillus megaterium because it is the only bacteria in the genus Bacilli that tests negative for MR/VP.
References
Gupta, S., Zhou, K. X., Bailey, D. M. D., & Christie, G. (2015). Structure–function analysis of the Bacillus megaterium GerUD spore germinant receptor protein. FEMS Microbiology Letters, 362(24). https://doi.org/10.1093/femsle/fnv210
Hansen, C., Wayment, B., Klein, S., & Godfrey, B. (2018). Iodine–Starch test for assessment of hyperhidrosis in amputees, evaluation of different methods of application*. Disability and Rehabilitation, 40(25), 3076–3080. https://doi.org/10.1080/09638288.2017.1367965
Kysela, D. T., Randich, A. M., Caccamo, P. D., & Brun, Y. V. (2016). Diversity Takes shape: understanding the mechanistic and adaptive basis of bacterial morphology. PLOS Biology, 14(10), e1002565. https://doi.org/10.1371/journal.pbio.1002565
Loong, S. K., Teoh, B. T., Johari, J., Khor, C. S., Abd-Jamil, J., Nor’e, S. S., … AbuBakar, S. (2017). Penicillin-susceptible, oxidase-negative, nonhemolytic, nonmotile bacillus megaterium in disguise of Bacillus anthracis [Research article]. https://doi.org/10.1155/2017/2578082
Seltmann, G., & Holst, O. (2013). The Bacterial Cell Wall. Springer Science & Business Media.
Sneath, P. H. A., Mair, N. S., Sharpe, M. E., & Holt, J. G. (Eds.). (1986). Bergey’s Manual of Systematic Bacteriology (Vol. 2). Baltimore: Williams & Wilkins.
