Unknown Bacteria Lab Report Introduction
Identification of unknown bacteria involves systematic and carefully selected procedures that apply strategic techniques to decipher the types of bacteria inherent in an unknown bacterial culture. According to Rhoads, Wolcott, Sun, and Dowd (2012), bacteria are categorized into species according to their metabolic and physical characteristics. The primary purpose of this lab report is to provide different lab techniques to assist students who are experiencing problems in identifying unknown bacteria. The reason for identifying the unknown bacteria is to assist students in deciphering different bacteria using different biochemical tests. The knowledge derived from the lab report is crucial in the medical environment for making some antibiotics to be used in treating patients as well as understanding the sources of a given disease. Thus this project is crucial as it provides the concept of identification of species on characteristic gene sequence and also is crucial for students as it will help them understand the molecular and biochemical differences in bacteria.
The process of unknown bacteria identification starts with observation of the general bacteria characteristics, including linkages, shapes cell, and wall. By applying different tests and experiments such as PR fermentation, Citrate test, Phenylethyl alcohol agar (PEA), Gram Stain, Gelatinase test, Catalase, Voges-Proskauer (VP), Methyl Red (MR) and Eosin Methylene Blue agar (EMB), one is able to identify unknown bacteria using either molecular or biochemical methods. Gram Stain is a laboratory process of detecting the presence of bacteria in samples obtained from a suspected area, as cited in Rhoads et al., (2012). Gelatinase test is carried out to identify whether an organism can produce proteolytic enzymes that are responsible for liquefying gelatin. The hydrolysis of gelatin indicates the presence of the gelatinases. The Catalase test identifies and differentiates “staphylococci (Catalase-positive” from “streptococci (Catalase-negative).” The catalase is generated by bacteria respiring using oxygen and safeguards them from oxygen metabolism toxic by-products.
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Methyl Red test refers to a biochemical test conducted on bacteria to test whether an organism can produce mixed-acid fermentations, usually from glucose. The Methyl Red test is a part of the IMViC tests. Notably, the IMViC serials involve citrate test, Voges Proskauer test, methyl red test, and indole test (Ryan & Ray 2004). The principle of the Methyl Red Test revolves on the idea that some of the bacteria have the capability of performing mixed acid fermentation of glucose in the MR-VP medium. PR Fermentation applies a fermentation medium (usually phenol red broth) with a single carbohydrate to differentiate between positive and negative enteric bacteria. A citrate test is applied to test whether an organism can utilize citrate as an energy source. According to Ryan and Ray (2004), “EMB is a differential medium for isolating fecal coliforms. Eosin Y and Methylene blue inhibit the growth of Gram-positive organisms” (p. 25). The “Phenylethyl alcohol agar” (PEA) test is conducted on blood samples to measure Gram-Positive Organism levels in the blood (Malanovic & Lohner 2016). PEA test is a medium of cultivating Gram-positive organs by inhibiting the develoment of Gram-negative organisms through interference with synthesis of DNA.
Voges-Proskauer (VP) test, on the other hand, is conducted to detect acetoin in bacterial broth culture. VP tests whether an organism can generate “acetyl methyl carbinol” as an outcome of fermentation of glucose. Once “acetyl methyl carbinol” is detected, it is turned into diacetyl through a strong alkali with 40% KOH and oxygen. According to Malanovic and Lohner (2016), the procedure for carrying out the VP test is as follows:
Allow the medium to equilibrate before inoculation to room temperature.
Lightly inoculate the medium using the organisms obtained from a pure culture.
Incubate the medium aerobically for 24 hours at 37˚C.
Aliquot 2mls of the broth to a test tube.
Add about six drops of 5% alpha-naphthol and mix well.
Adding two drops of 40% KOH and mix well.
Observation of pink-red color within 30 minutes.
The most effective unknown bacteria identification method is biochemical identification. In the biochemical identification, the first step involves the using a single colony to streak the EMB-lactose agar plate to decipher whether the gram is negative or positive. The gram-negative bacteria are protected from the cell wall and grow on the plates, while gram-positive bacteria are prevented from growth by the EMB dye. In the next step, dry oxidase test tubes are used, and small amounts of the unknown bacteria are pipetted from liquid culture. In case the bacteria have enzymes, substrates of the enzymes on the slides are turned into a purple product where a spot is observed. The compartment with an unknown bacterium is inoculated whereby the test tubes placed in an incubator at 37˚ C. the incubation is carried out overnight, and an examination on each compartment is conducted to identify the media color as well as the production of gas. A comparison of the compartments is made to identify whether the colors indicate the presence of positive or negative enzymes. The identification of unknown bacteria is successful after evaluating the outcomes of the Oxi/Ferm or Enterotube test.
References
Colco, R (2005). "Gram Staining". Current Protocols in Microbiology
Malanovic, N., & Lohner, K. (2016). Antimicrobial peptides targeting gram-positive bacteria. Pharmaceuticals , 9 (3), 59.
Rhoads, D. D., Wolcott, R. D., Sun, Y., & Dowd, S. E. (2012). Comparison of culture and molecular identification of bacteria in chronic wounds. International journal of molecular sciences , 13 (3), 2535-2550.
Ryan K.J., Ray C.G. (editors) (2004). Sherris Medical Microbiology (4th ed.). McGraw Hill
