Identification of an unknown microorganism using a classical approach PDF

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report on approach for identification of unknown bacteria with practical tests...

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Running head: IDENTIFICATION OF AN UNKNOWN MICROORGANISM

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Identification of an unknown microorganism using a classical approach

BIOL2330 S02 Mansi Mansi Kwantlen Polytechnic University Unknown#16

100364284, BIOL2330, Dawne MacLeod, 26 November 2019

IDENTIFICATION OF AN UNKNOWN MICROORGANISM

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Abstract The study was done to identify a certain unknown bacterium by classical approach performing biochemical tests. A dichotomous key was prepared for eight of the different bacteria which involved series of tests. The colony characteristics of the bacteria were also observed by growing bacteria on agar plate. The gram stain technique was used to determine if the bacterium was gram-negative or gram-positive and then catalase test and oxidase test was performed which lead to the result that the unknown#16 was Staphylococcus aureus. The consistency of the results was seen according to literature observations whether the results of the tests were correct in relation to the purpose of each test. The gram stain gave positive reaction and the bacterium was coccus. The oxidase test resulted as negative and the catalase test resulted as positive and the bacteria grew without oxygen which shows it was facultative anaerobe. Introduction The purpose of the lab study was to use different techniques and chemical tests to identify an unknown bacterium. This study is important for knowing specific disease-causing agent in medicine industry or microorganisms to be used in food industry. The colony characteristics and the results of stain techniques and biochemical tests were used to identify the unknown bacterium. Each of the seven bacteria have different characteristics and will give different results to chemical tests. The gram-negative bacteria were alcaligenes faecalis, Enterobacter aerogenes and Escherichia coli whereas the gram-positive bacteria were Enterococcus faecalis, Staphylococcus aureus, Micrococcus luteus and Bacillus subtilis (Willey, et.al., 2018). A dichotomous key was prepared which involved a series of steps to get to a specific bacterium. For example, gram stain was performed for unknown and if it gives negative result

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with red colour, the next step was indole test and if the indole test results are positive then the unknown was determined to be Escherichia coli. Among the gram-positive bacteria, only Bacillus subtilis is rod-shaped and motile while the other three are coccus and non-motile. Enterococcus faecalis is catalase negative while the others are catalase positive (Harley, 2017). Moreover, Micrococcus luteus is an obligate aerobe which means it can only grow in presence of oxygen and is oxidase positive whereas Enterococcus faecalis, Bacillus subtilis and Staphylococcus aureus are facultative anaerobes which can grow in presence or absence of oxygen but only Enterococcus faecalis and Staphylococcus aureus are oxidase negative as Bacillus subtilis is variable in oxidase test (Harley, 2017). Staphylococcus aureus and micrococcus luteus are both found in water, soil and on skin, Bacillus subtilis is found in intestines and soil and Enterococcus faecalis in urinary tract (Harley, 2017). However, among the gram-negative bacteria, Alcaligenes faecalis is an obligate aerobe and oxidase positive while Enterobacter aerogenes and Escherichia coli are facultative anaerobes and oxidase negative (Harley, 2017). All of them are motile and catalase positive (Harley, 2017). Alcaligenes faecalis is indole negative and is found freely in environment whereas Escherichia coli is indole positive and is found in environment and in intestines (Harley, 2017). Enterobacter aerogenes is indole negative and is found in intestines, soils and wastes (Harley, 2017). Materials and methods Firstly, the unknown bacterium was grown on an agar plate and the colony characteristics were observed. The gram stain was used to make a slide for our unknown bacterium. The smear of

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bacteria was heat fixed on the slide and then placed on staining rack. The smear was stained with crystal violet for 30 seconds and then rinsed with distilled water for 5 seconds and then the slide was covered with Gram’s iodine mordant for 1 minute and rinsed with distilled water, after that decolourizer was added for 15 to 30 seconds and then again rinsed with distilled water (Harley, 2017). At last, slide was counterstained with safranin for 60 to 80 seconds and then rinsed with distilled water and bibulous paper was used to dry the slide and then the slide was seen under the microscope (Harley, 2017). The microorganisms seen were stained blue to purple colour which means the known was gram-positive and was non-motile and coccus. After knowing that it is gram positive and coccus, catalase test was performed. On the colony formed on agar plate, drops of hydrogen peroxide were added. the bubbles formed which indicates a positive catalase test. Then, oxidase test was performed in which the colony was collected on a cotton swab and oxidase reagent drops were added. The colour did not change to blue which shows a negative oxidase test. Results The unknown#16 bacterial colony was observed, and the form of colony was circular with convex elevation, entire margin, dull appearance, translucent and smooth texture (Table1). The gram stain was performed on the smear of bacteria and gave positive result as the microorganism appeared blue under oil immersion (Table 2). The cell shape was coccus and the arrangement were grape-like clusters in majority (Table 2). The catalase test was performed which gave positive result, then oxidase was done which gave negative results and the bacteria was determined as Staphylococcus aureus (Table3).

IDENTIFICATION OF AN UNKNOWN MICROORGANISM

For more clarification, oxygen classification was done by growing unknown bacteria on GasPak plate in GasPak anaerobic system. There was growth of bacteria which shows that the bacteria were facultative anaerobes as it grew even in the absence of oxygen (Table 3). Table1: The table represents the colony characteristics of the unknown#16 bacterium Form

Circular

Elevation

Convex

Margin

Entire

Appearance

Dull

Optical property

Translucent

Pigmentation

Nonpigmented

Texture

Smooth

Table2: The characteristics of bacterium cell Cell arrangement

Majority were Grape-like clusters and some singular or in pairs

Cell shape

Coccus

Cell size

0.1µm

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Table3: The table represents the chemical tests and their results performed on unknown #16 Test

Reagent

Purpose

Observations

Result

Gram stain

Crystal violet, iodine,

To determine if

The bacterium

Gram-

alcohol, safranin

bacterium is gram-

was seen blue

positive

positive or gram-

in colour under

negative

oil immersion

To determine the

Bubbles

Catalase test

Hydrogen peroxide

Positive

production of catalase formed on colony Oxidase test

Oxidase reagent

To determine

No colour

Negative

(Tetramethyl-p-

presence of

change

phenylenediamine)

cytochrome c oxidase

Oxygen

GasPak plate and

To determine if

Growth of

Facultative

classification

GasPak Anaerobic

oxygen is required or

bacterial

anaerobe

system

not

colony on plate

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Flowchart for the series of tests Unknown#16

Gram stain

Gram-positive coccus Enterococcus faecalis Staphylococcus aureus Micrococcus luteus

Catalase test (Positive) Micrococcus luteus Staphylococcus aureus

Oxidase test (Negative) Staphylococcus aureus Discussion The purpose of the study was to identify an unknown bacterium using a series of tests and techniques and it was accomplished as the unknown bacteria was determined as Staphylococcus aureus which was correct, and the experiment was successful. Staphylococcus aureus is a grampositive coccus shaped bacterium which is non-motile and a facultative anaerobe which means it can grow with or without oxygen and it is catalase positive (Foster, Ti, 1996). It is gram-positive which means it has thick layer of peptidoglycan which does not allow crystal violet stain to escape. Thus, it stains blue by gram stain technique. This bacterium is found in environment such

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as dust, water, soil and in food, skin and nasal passages. It is responsible for causing infections in humans and other mammals which makes the study of this bacterium important. However, the ability of Staphylococcus aureus to form biofilms make the treatment with antibiotics difficult for any infections caused by them (Foster, Ti, 1996). The identification of this bacteria is necessary as it is also responsible for causing infections in humans and animals such as bovine mastitis in cattle (Li, et.al., 2017). Also, these bacteria can form biofilms which make them resistant to antibiotics and host immune system (Li, et.al., 2017). After performing certain tests, the unknown bacterium was determined to be Staphylococcus aureus. The gram stain gave a gram-positive reaction which indicated that the bacterium was gram-positive (Table3). In the first week, the bacteria were grown in a TSA tube for performing the biochemical tests next week. The catalase test was positive which shows that catalase enzyme is present in this bacterium and the oxidase test was negative and shows that the cytochrome c oxidase is not present in the bacteria (Table 3). Moreover, the bacteria grew in absence of oxygen (Table 3). The results of the tests were consistent as Staphylococcus aureus is a facultative anaerobe, catalase positive, gram-positive bacterium. So, the classical approach to identify an unknown microorganism gave correct results. Staphylococcus aureus is known to be present in form of grape-like clusters or chains. Most of the cells were in grape-like clusters when viewed under microscope and some of them were present as single or in pairs (Table 2). The cell size was 0.1µm approximately and the shape was coccus (Table 2). The colony characteristics were also observed for the bacteria growth on agar plate and the results were circular form, convex elevation, entire margin, dull appearance, translucent property and nonpigmented as the colony was white in colour (Table 2).

IDENTIFICATION OF AN UNKNOWN MICROORGANISM

References Foster, T. (1996, January 1). Staphylococcus. Retrieved from https://www.ncbi.nlm.nih.gov/books/NBK8448/. Harley, J.P. (2017). Laboratory exercises in microbiology (tenth ed.). New York, United States of America: McGraw-Hill Education Li, T., Lu, H., Wang, X., Gao, Q., Dai, Y., Shang, J., & Li, M. (2017). Molecular characteristics of Staphylococcus aureus causing bovine mastitis between 2014 and 2015. Frontiers in Cellular and Infection Microbiology, 7, 127. doi:10.3389/fcimb.2017.00127 Willey, J. M., Sherwood, L., &Woolverton, C. J. (2018). Prescott’s microbiology (10th ed.). Vancouver, B.C.: McGraw-Hill Education.

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