Staphylococcus epidermidis & Escherichia coli

 

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Staphylococcus epidermidis & Escherichia coli

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There are several motivations for knowing the identity of microorganisms. The motives can range from knowing the causative agent of a disease in an individual, knowing the appropriate microorganism for making certain antibiotics and foods. The purpose of doing this practical exam is to reflect what we have learned during the summer semester in the microbiology laboratory.

This research was done by applying all of the methods that have I learned in the microbiology laboratory class for the identification of an unknown bacterium. In the research I will perform biochemical test to determine the unknown microorganisms, by performing biochemical test. I will be gaining much more insight and experience of how microorganisms are differentiated, and isolated to determine diseases in humans and animals.
Staphylococcus epidermidis & Escherichia coli
Material and Methods

Grain stain
Day one of the practical exam, I had chosen unknown test tube sample number 14. The first procedure that took place was a Gram Stain from the unknown sample, and then I also inoculated the agar plates listed below. Day two after the plates were incubated and grown, the morphology was observed and recorded. A second Gram stain was performed from the growth of the Phenylethyl Alcohol agar plate, which only permits gram positive growth. Then, biochemical test listed below were performed from an isolated colony on both MacConkey agar plate and Phenylethyl Alcohol agar plate. Note all of these tests were performed by the methods listed in the microbiology laboratory manual by McGraw Hill education.

Staphylococcus epidermidis & Escherichia coli

Staphylococcus epidermidis & Escherichia coli
Inoculated the following plates with unknown # 14:
·    Blood Agar Plate
·    MacConkey Agar Plate
·    Phenylethyl Alcohol Agar Plate
·    Nutrient Agar Plate
·    Mannitol Salt Agar Plate

Biochemical Test performed on unknown # 14:
·    Triple Sugar Iron Agar
·    SIM Medium (Sulfur, Indole and Motility)
·    Simmon’s Citrate Medium
·    Urease Test
·    Methyl Red and Voges-Proskauer Tests
·    Nitrate Reduction Test
·    Phenol Red Broth with Glucose
·    Bile Esculin Test
·    Catalase Test
Staphylococcus epidermidis & Escherichia coli
Results and Interpretation:

A. Description of colonies on each plate
·    Blood Agar Plate – I observed colonies with partially clear zones with light green growth, indicating alpha hemolysis. The organism formed shiny small circular colonies, which have raised elevation, smooth entire margins that are convex.
·    Mannitol Salt Agar Plate – The organism did not ferment the mannitol, since the media remained red. The organism formed tiny circular colonies, which have entire margins that are slightly raised.
·    MacConkey Agar Plate – The organism has fermented lactose, forming pink smooth tiny circular colonies as a result of acid production.
·    Phenylethyl Alcohol Agar Plate – The organism has formed tiny circular white colonies.
·    Nutrient Agar Plate – The organism has formed tiny circular translucent colonies.

Staphylococcus epidermidis & Escherichia coli
B. Results of biochemical reactions from MacConkey Agar Plate

Staphylococcus epidermidis & Escherichia coli

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Gram Stain: Gram negative rods

Triple Sugar Iron Agar description – The organism fermented the glucose, lactose and/or sucrose and produced acid as a byproduct, which lowered the pH and turned the slant and butt yellow. Adding, I also observed the medium lifted from the bottom of the tube indicating gas was produced by the organism, with no H2A production.

Triple Sugar Iron Agar Tests    Results
Glucose Fermentation    Positive
Lactose and/or Sucrose Fermentation    Positive
Peptone Utilization    Negative
Production of H2S    Negative
Production of Gas    Positive

SIM Medium (Sulfur, Indole and Motility) Test     Results
Indole    Positive
Motility    Positive
Production of H2S    Negative

Simmon’s Citrate Medium Test    Result
Utilization of Citrate    Negative

Urease Test     Result
Urea Hydrolysis    Negative

Nitrate Test     Result
Nitrate Reduction     Positive

Methyl Red Test     Result
Mixed acid Fermentation     Positive

Vogues-Proskauer Test     Result
2,3 Butanediol Fermentation     Negative
Staphylococcus epidermidis & Escherichia coli
Presumption ID: Following several biochemical tests, it was concluded that first organisms in unknown test tube 14 was Escherichia coli.
Justification – After performing the gram stain to determine the unknown was a gram negative rob microorganism, the organism was inoculated into several agar test tubes and the test results lead me to my conclusion. First, in the Triple Sugar Iron test the organism was able to ferment glucose, lactose and/or sucrose, producing acid and lowering the pH changing the butt and slant yellow, with displacement of medium indicating gas production. However, did not produce H2S. Secondly, The organism was positive for motility, indole production, mixed acid fermentation and nitrate reduction. The organism had negative results for urea hydrolysis, 2,3 butanediol fermentation and utilization of citrate. Adding, on the MacConkey agar plate the organism produced pink growth. In the Blood Agar plate I observed colonies with partially clear zone demonstrating beta hemolysis, Madappa (2016) stated in a laboratory study that E.coli is beta-hemolytic on blood agar. After reviewing all my test results and characteristic of previous laboratory tests on E.coli, it has leaded me to conclude that the unknown is E.coli.

Staphylococcus epidermidis & Escherichia coli

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C. Results of biochemical reactions from Phenylethy Red Agar Plate

Gram Stain: Gram Positive Staphylococci

Bile Esculin Test     Result
Bile Esculin hydrolysis    Negative with growth

Phenol Red Broth with Glucose    Result
Glucose Fermentation     Positive

Catalase Test    Result
Catalase Production     Positive

Mannitol Agar Plate     Result
Mannitol Fermentation    Negative with growth

Presumption ID: Following several biochemical tests, it was concluded that second organism in unknown test tube 14 is Staphylococcus epidermidis.
Justification – After performing the gram stain to determine the unknown was gram positive staphylococci the organism was inoculated into several agar test tubes and agar plates, and the test results lead me to my conclusion. In the laboratory manual it states the catalase test is use to differentiate staphylococci from streptococci, since staphylococci produces catalase. The organism was positive for catalase production. The Mannitol Salt agar result helped me conclude that my organism was S. epidermidis, because it was able to grow in the high salt concentration however, was not able to ferment the Mannitol and produce acid.

Staphylococcus epidermidis & Escherichia coli

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