The Use of Biochemical Strategies in the Classification of Unknown Microorganisms

Introduction

The object of this investigation was to classify an unknown microorganism that was assigned to us. We used staging properties, cell morphology, cell arrangement and biochemical test results in order to achieve our goal. A flowchart was provided as a guide to aid in the choice of test and process of elimination in order to arrive at a conclusive identification.

Methods

The flowchart applied as a guide indicated that the initial step in identifying the microorganism was to preform a Gram stain. The Gram stain is utilized in distinguishing between gram-positive or gram-negative cell walls, as well as the microorganism cellular morphology. Bacteria stain inversely due to chemical and physical variances in their cell walls, corresponding to Brief Microbiology: Theory and Application. Bacterial cell walls are complex arrangements comprised of layers of peptidoglycans among other composites. Gram-negative bacteria are comprised of a slim layer of peptidoglycan enclosed by a superficial layer of lipoproteins, phospholipids, and lipopolysaccharides, however numerous layers of peptidoglycans are characteristic of gram-positive bacteria. Crystal violet is readily accepted by both gram-positive and gram-negative cell walls. Iodine combines with the crystal violet stain in the cytoplasm to form CV-I, a molecule bulkier than the crystal violet that was first inserted into the cell. The cell wall of the gram-positive bacteria is desiccated by the alcohol decolorizing agent. The decolorizing agent disbands the outer lipopolysaccharide layer of gram-negative cells causing the CV-I macromolecule to be rinsed out past the thin layer of peptidoglycan. In gram-positive cell walls however, the CV-I cannot be washed out through the copious layers of peptidoglycans. The counterstain safranin makes the decolorized bacteria appear red (55).

The second step on the flowchart was the Endospores test. The equipment needed to carry out this test was not immediately available so this step was skipped. The third step was the Catalase test. An organism’s capability to synthesize catalase is observed via the catalase test. According to Brief Microbiology: Theory and Application, catalase is an enzyme that decontaminates the cell by transforming hydrogen peroxide formed in the ETC to water and molecular oxygen. Anaerobic respirers reduce inorganic molecules such as nitrate to nitrogen gas or sulfate to hydrogen sulfate gas while oxygen is reduced to water or other compounds by aerobic respirers. Aerobic and anaerobic bacteria can be differentiated via this testing approach. Bacteria that yield catalase can be effortlessly distinguished using standard store-grade hydrogen peroxide. Oxygen gas bubbles form immediately following the addition of hydrogen peroxide to a catalase-positive culture. The lack of bubbles is indicative of a catalase-negative organism. (257)

Methyl Red and Voges-Proskauer was the fourth step. Methyl Red and Voges-Proskauer Broth is a combination medium used for both MR and VP tests. The liquid consists of only peptone, glucose, and a phosphate buffer. These tests are constituents used to discriminate between members of the family Enterobacteriaceae and separate them from other Gram-negative rods. Furthermore, organisms adept of performing a mixed acid fermentation are detected through use of this analysis. The mixed acid fermentation constructed by such organism overwhelms the phosphate buffer in the medium resulting in a reduction in the pH. Adding of the dye methyl red indicator substantiates mixed acid fermentation. This addition succeeds incubation. Methyl red appears red at pH 4.4 and yellow at pH 6.2. There exists an assorted shade of orange amongst these two pH values. Brief Microbiology: Theory and Application affirms that the only true indication of a positive result is a red color. Orange represents a negative or inconclusive reaction while yellow represents a negative reaction. (251)

Results

The primary testing procedure, the Gram stain, denoted a Gram-positive rod shaped unknown bacterium. This was concluded based on morphology and purple characteristic of the organism. The presence of bubble indicated a positive catalase test. The MRVP test indicated a positive result due to the red color of the broth following the addition of hydrogen peroxide. The classification of the unidentified microorganism was concluded to be Bacillus subtilis.

Discussion

Each assessment performed using the provided flowchart confirmed the identity of the unknown microorganism to be Bacillus subtilis. The primary experiment, the Gram stain, commenced the identification of the unknown bacteria by classifying it as rod-shaped and gram-positive. The following positive result concerning the catalase test, as outline by the flowchart, further distinguished the bacteria as a catalase-positive organism. The positive MRVP results indicated bacterial mixed acid fermentation, which lead the conclusion and identification of the microorganism to be Bacillus subtilis.

Conclusion

This investigation was carried out to distinguish a microorganism of unknown identity in a controlled laboratory setting throughout a two week period. A variety of differential testing and staining methods were utilized during this process. Knowledge of these procedures was acquired through out the microbiology course. Each week, interpretations were completed and documented to further distinguish characteristics of the microorganism. Microscopic observations as well as differential test results on various agars and broth culture were documented and presented via flowchart. A better understanding concerning the classification of bacteria based on reactions with different media and observable characteristics is valuable knowledge with an endless amount of possible applications.