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Unknown Lab Report | How to Identify E. coli | Microbiology

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Unknown #117 and Alternate #9

 Josephine Vitale

May 5th, 2015

Microbiology Lab



Correctly identifying microorganisms is an important and imperative process. The reasons for its importance stem from the fact that microorganisms are all around us, ever changing, affecting everyone and everything they come in contact with. Identifying microorganisms can help with the prevention and treatment of disease and sickness, benefit our ecosystem, and completely change the larger species they surround, despite their microscopic size (1). The tools and techniques used while dealing with microorganisms can make or break one’s ability to separate and identify different species with accuracy. The study taken place was carried out using the laboratory methods and tools practiced throughout the spring semester in microbiology. The ultimate goal was to correctly identify two unknown microorganisms given by the professor using the knowledge and skills learned in microbiology lab and lecture.


Materials and Methods

An unknown labeled as 117 was given out by the lab instructor, as well as directions for any tools, instruments, and methods to be used to correctly identify the unknowns.

The first necessary method was to streak the unknown onto a nutrient agar plate using the Streak Plate method learned using “Lab Manual for General Microbiology”. This method was carried out by streaking a nutrient agar plate in four different directions, in hopes of isolating two different unknowns. The streak plate method utilizes an inoculating loop, sterilized by a Bunsen burner provided, to decrease chance of contamination on the nutrient agar plate (2). Once the unknown was streaked correctly, the agar plate was then inoculated in a 35 degrees Celsius inoculation chamber for 48 hours. This temperature and time was chosen using knowledge that bacteria need time and an optimal growth temperature to grow colonies (2).

After 48 hours of time, the nutrient agar plate was taken out of the inoculation chamber and was investigated using the naked eye to find two separate species growing. The first round of streak plating was not effective and another streak plate was completed and inoculated again for 120 hours before investigation. After the second attempt at inoculation, two species were seen with the naked eye. The lab instructor provided information that one species was a gram-positive bacteria and the other species was a gram-negative. Therefore, the next necessary step for correct identification of each unknown was to complete a gram staining procedure described on page 67 of the lab manual (2).  The gram-staining procedure correctly identified the gram-negative species as rod shaped for further testing.


This list identifies all tests performed on the gram-negative unknown.

  1. Simmons Citrate Test
  2. Urea Test



Table 1 lists the test, purpose of test, reagants or media, observations, and results


Table 1. Gram-negative unknown tests

Gram Stain


To identify Gram reaction of the bacterium Crystal violet, Iodine, Alcohol, Safranin Pink rods Gram-negative rods
Simmons Citrate


To determine if bacterium can utilize citrate for carbon and energy. Green-colored Citrate slant No color change Negative Citrate test
Urea To determine if bacterium can produce the enzyme “urease” Urea broth No color change Negative Urea test








The two initial streak plates created for identifying the gram-positive bacteria both came back ineffective. The inaccuracy stemmed from procedural flaws when gram-staining with a microscope. An alternate and pre-isolated unknown was then provided by the instructor. Therefore, the first necessary step was to streak plate and inoculate the unknown before another gram-staining procedure could be completed. Once a gram-stain procedure was completed, the gram-positive unknown was identified as rod-shaped. Only two organisms, Bacillus cereus and Bacillus subtilis, were rod-shaped bacteria in the unknown list provided by the instructor. Therefore, because of time constraints due to procedural flaws earlier on, only one deciding test was done on unknown #9. The Methyl Red test was carried out using the instructions provided by our lab book on page 34 (2).



This list identifies the test performed on the gram-positive unknown.

  1. Methyl Red Test


Table 2 lists the test, purpose of test, reagants or media, observations, and results


Table 2. Gram-positive unknown tests

Gram Stain To identify Gram reaction of the bacterium Crystal violet, Iodine, Alcohol, Safranin Purple rods Gram-positive rods
Methyl Red To identify mixed acid fermentation when supplied glucose 1 Methyl Red-Voges Proskaur broth


5 drops of methyl red

No color change after methyl red was applied Negative Methyl Red test











Unknown B- Bacillus subtilis

Both unknown organisms provided by the instructor were identified using specific tests and interpreting the results of these tests. Unknown A, Escherichia coli, was gram-stained to determine the bacterial shape. When it was determined that it was a gram-negative rod due to its tube-shaped features and red staining, further testing needed to be done to eliminate potential organisms. The first test completed was the Citrate test. This test had a negative test result, which immediately eliminated Klebsiella pneumoniae, Enterobacter aerogenes, and Pseudomonas aeruginosa. The final two organisms to be decided between were Escherichia coli and Proteus vulgaris. These two organisms have opposite test results in the Urea test. Therefore, a urea test was completed and analyzed. The urea test came back a negative test result, indicating that Escherichia coli was the unknown gram-negative bacteria.

Unknown B, Bacillus subtilis, was first gram-stained to determine the shape of the bacterium. Once it was determined that it was a rod-shaped bacterium, only two possible options were left: Bacillus subtilis and Bacillus cereus. One test that would differentiate the two because of opposite testing results was the methyl red test using the MRVP broth tube and methyl red drops. This procedure was carried out as practiced earlier on in the semester during lab. Once the methyl red drops were added to the MRVP broth tube and no color change occurred, it was determined that Bacillus subtilis was the gram-positive bacteria, an alternate unknown provided by the instructor.

Throughout the experiment, procedural complications and flaws did set back results in a timely manner. For example, the first two streak plates made were ineffective at separating the two unknowns from each other. This issue could be an indication that the streak plate could have been done more carefully or that there was a contamination of sorts on the agar plate. Also, when gram-staining the gram-positive bacteria, it was first thought to be a cocci-shaped organism. Once these results were discussed with the instructor as incorrect, gram-staining had to be redone.

One of the unknowns found, Escherichia coli, is a very vast and diverse species of bacteria. Escherichia coli is quite a unique bacterium due to the fact that some strands can cause a lot of harm, disease, and sickness to animals and humans, while some strands are a part of a healthy flora inside the body (3). E. coli in the gastrointestinal tract can be very beneficial for overall G.I. health. Having a healthy level of E. coli in your flora can be advantageous because of its digestive properties. Like any other enzyme in the intestines, E. coli aids your body with healthy digestion of food (4). Also, E. coli is a component of suppressing bad bacteria from inhabiting the intestines and reproducing.  A healthy amount of E. coli is very important for having an overall healthy flora and digestion.  A major reason this specific strand of E. coli could potentially cause the body harm is if it were to travel to other parts of the body, such as the urinary system. This could cause painful infections such as urinary tract infections (3). Another way E. coli can cause more harm than good is if the specific strand of this bacteria has no beneficial reason to be in the body. One strand in particular, O157:H7, is a harmful strand that contaminates water and food that humans or animals could ingest (4). This strand can cause stomach tenderness leading to severe diarrhea and vomiting. The treatment of E. coli includes regulating fluid and essential mineral intake, as well as dialysis or blood transfusion in more severe cases. Therefore, just like our instructor says about many things discussed in class: Too much of a good thing can sometimes cause more harm than good.





  1. “Importance of Microorganisms in the Ecosystem”. Adnan, Amna. Biotech Articles Online. Published 07-09-2010
  2. “Lab Manual for General Microbiology”. Virginia McDonald, Mary Thoele, Bill Salsgiver, Susie Gero. Revised 04-2011.
  3. “Fact Sheet: E Coli”. The Oregon Health Services. Revised 03-2011.
  4. “Benefits and Dangers of The Bacterium Escherichia Coli”. Published 06-09-2012.





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