Identification of Unknown Microorganisms�Sonya Singh – Dr. Abreeotta Williams�Converse University�
Microorganisms are generally microscopic such as bacteria, protozoa, algae, fungi, and sometimes viruses (1). Bacteria are single-celled and ubiquitous. Some are pathogenic; however, most are relatively harmless or even helpful. Bacteria tend to have three main shapes: bacilli, cocci, and spirilla (2). When identifying a species both traditional and non-traditional methods may be used. Traditionally, staining has proven to be a reliable technique as gram staining remains the most common. While gram staining is used to indicate a peptidoglycan layer (gram positive), there are other stains used to indicate such characteristics. Endospore staining exposes the presence of sporadic cells, simple staining is used to observe the structure and shape of the cell, and negative staining to observe bacteria that can be damaged by heat-fixing as well as distinguishing shape and presences of flagellum. Staining serves as a preliminary technique when elimination can be used as a basis (3).
Non-traditional methods can be faster and less intensive such as the sequencing of genetic traits and investigating biochemical reactions through tests such as assay. (4). An assay, specifically the Remel MICRO-ID is fifteen biochemical tests that are used to differentiate bacteria based on the microbial degradation of certain substrates within the organism. By observing the effects of these chemicals on the organism at a cellular and molecular level we can further analyze functions of the cell and biological processes that can help identify it (5).
Sequencing is the process of determining the nucleic acid sequence of the entire genome of an organism. As each organism contains their own unique DNA sequences, these can be compared in a database to use and identify certain strains of bacteria (6). This investigation aims to correctly identify an unknown organism Using both traditional and non-traditional identification methods a strain of Corynebacterium can be identified. By Lehmann and Neumann in 1896, Corynebacterium was defined as a non-motile, animal pathogenic or parasitic species of bacteria.
The initial sample was collected outside of the Petri School of Music at Converse College from the outdoor garden fountain on June 6th, 2021. Sterile cotton swabs were first soaked in distilled water and used to collect the sample off the surface. The was streaked on Luria Agar plates. The plated samples were placed in the incubator for 48 hours at 38°C. Samples were allowed to sit in open air at room temperature. Samples then underwent simple staining, gram staining, endospore staining, catalase test, DNA extraction, DNA sequencing, and Remel MICRO-ID Assay
The bacterial colonies grown on the LB Media presented large growth of white/yellow-colored colonies that tinted red after some time. After the simple stain, the organism presented a rod-shaped structure (bacilli). The organism did not present endospores as the organism was stained pink from the safranin. In addition to this the gram staining resulted in the organism showing gram positive. After adding the hydrogen peroxide, the organism reacted by bubbling thus indicating it is catalase positive. The 15 biochemical tests performed in the assay showed the organism to be nitrate reductase negative, phenylalanine deaminase negative, hydrogen sulfide positive, indole negative, ornithine decarboxylase positive, lysine decarboxylase negative, malonate utilization positive, urease positive, esculin hydrolysis positive, 𝛽-galactosidase negative, arabinose fermentation positive, adonitol fermentation negative, inositol fermentation negative, and sorbitol fermentation negative. The DNA sequencing yielded no match, thus is inconclusive.
By analyzing all the data collected from the tests, it was assumed that the organism is a strain of Corynebacterium. A simple stain was done to look at the structure and morphology of the microorganism. The organism can be identified as bacilli due to its elongated rod shape. Gram staining differentiates gram positive bacteria from gram negative. Structure differentiates gram positive and gram negative cells. Gram positive bacteria have a thick peptidoglycan layer, gram negative bacteria have a thin peptidoglycan layer with a larger outer membrane. A gram staining procedure is followed to find if the bacteria is positive or negative. Adding Iodine creates a complex with the crystal violet, so the organism appears purple. If positive, the crystal violet-iodine crystal complex should stick in the peptidoglycan layer indicating its presence. As the gram staining test resulted in the organism staining purple, it can be deducted that the organism is gram-positive. The endospore staining detects the presence of endospores. A catalase test is a biochemical test performed on aerobic organisms such as Corynebacterium. As organism bubbled with the addition of hydrogen peroxide, the organism is catalase positive. This ruled out Lactobacillus and Arcanobacterium as they are catalase negative. The organism was previously thought to be Listeria monocytogenes as it is a gram-positive, catalase positive, non-sporadic, rod-shaped organism. However, its colony morphology presented a blue green color while the unknown organism was white or red. With the assay, the organism presented nitrate reductase negative while Mycobacterium is a strong nitrate positive organism. Actinomyces is also catalase negative and urease negative while the assay came back urease positive. Listeria Monocytogenes was also negative for H2S and urease negative, but it is indole positive. After narrowing it down through staining and comparing the assay results of the microorganism, Corynebacterium was inferred, however, the hypothesis is neither accepted or refuted as the sequencing was inconclusive. Further work will be required to identify the microorganism. To make identification more precise, acid-fast stains or more detailed assays could be performed. Once identified, the bacteria’s composition could be observed and tested for industrial uses.
Introduction
Methods
Results
Discussion
Literature Cited
Acknowledgements
Thank you, Dr. Williams and Dr. Erturk!