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Bacillus subtilis | Microbiology Unknown Lab Report

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Unknown Lab Report


Unknown Number 113

Kristopher Klein

April 30, 2013

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BIO: 203, 604

Spring 2013



There are numerous reasons for identifying an unknown microbe.  Whenever a patient has a bacterial infection, testing must be done in order to find the correct antibiotic to treat them.  Before properly assessing and treating the patient, the microorganism(s) have to be identified.  This process is also useful in the production of antibiotics on a larger scale.  Using the methods that have been learned so far in the microbiology lab the unknown bacterium was identified.


Materials and Methods:

The lab instructor assigned a vial labeled 113.  This vial contained two unknown bacteria, one Gram negative and one Gram positive, that required identification.  The process of identification was achieved by utilizing procedures learned during the present semester.  Procedures were followed as stated in the laboratory manual by McDonald, Thoele, Salsgiver, and Gero (1), unless otherwise noted.

Since the unknown sample contained two unidentified bacteria, the first step was to isolate each bacterium into pure cultures.  This was done by making an isolation streak from the mixed culture onto a nutrient agar plate using the process described in the manual.  This step is imperative because the bacteria need to be separated and isolated before they can be identified.  After the nutrient agar plate was incubated and grown, the presence of two separate bacteria was clearly visible.

Using the isolation streak technique, each bacterium was isolated onto separate nutrient agar plates, one was labeled “A” and the other labeled “B”.  After a few days of incubation and growth, culture “A” was clearly isolated, but culture “B” was not.  A Gram stain was performed on culture “A”.  This test is crucial and must be done correctly because it not only reveals whether the stain is positive or negative, but the shape of the bacteria as well.  Plate “A” was determined to be Gram-negative rods.  Culture “B” was inoculated onto Mannitol Salt Agar because this media is selective for Gram-positive bacteria.  The MSA allowed isolation of the Gram-positive bacteria and the growth was transferred onto a nutrient agar plate into a pure culture.  A Gram stain showed the bacteria were Gram-positive rods.

After the Gram stains were determined, specific biochemical tests were performed.  The biochemical tests performed were chosen based on the identification table that was given from the lab instructor.  The flow charts on the following pages list these tests and results for the Gram-positive and Gram-negative bacteria.


All of the following tests were performed on the Gram-negative bacterium:

  1. Urea
  2. Simmon’s Citrate
  3. Eosin-Methylene Blue
  4. Casein

All of the following tests were performed on the Gram-positive bacterium:

  1. Casein
  2. Glycerol
  3. Maltose



After determining Unknown “A” was a Gram-negative rod, a Urea test was performed, next a Simmon’s Citrate tube was inoculated, followed by an Eosin-Methylene Blue Agar, and a Milk agar.  The following table and flowchart convey the results.


Table 1: Biochemical Tests for Gram-negative Unknown

Gram stain Determine the gram reaction of the bacterium Crystal Violet, Iodine, Alcohol Safranin Pink rods Gram-negative rods
Urea Detects the enzyme urease, which breaks down urea, producing an alkaline pH Urea broth Little to no color change Negative for urease
Simmon’s Citrate To determine if the bacteria will produce citrate permease which allows them to take in the citrate and convert it to pyruvate Simmon’s Citrate Agar Changed from green to blue towards the top of the tube Positive citrate test
Lactose To determine if the bacterium will ferment lactose with acid production Eosin-Methylene Blue Agar Color change where streaked to a purple color Positive lactose fermenter with weak acid production
Casein To determine if the organism produces casease which hydrolyzes the milk protein casein Milk Agar No change Negative casein test

Flowchart *Removed due to formatting problems

After initial tests concluded Unknown “B” was a Gram-positive rod, a Casein test was performed, followed by a Glycerol test, and a Maltose test.  Below illustrates the results in table and flowchart form.


Table 1: Biochemical Tests for Gram-positive Unknown

Gram stain To determine the Gram reaction of the bacteria Crystal violet, Iodine, Alcohol, Safranin Purple rods Gram-positive rods
Casein To determine if the organism produces casease which hydrolyzes the milk protein casein Milk agar Clearing around the area of growth Positive casein test
Glycerol To determine if the bacterium will ferment glycerol with acid production Glycerol broth Color change from red to yellow Positive for glycerol fermentation with acid production
Maltose To determine if the bacterium will ferment maltose with acid production Maltose broth Little to no change Negative maltose test



Flowchart * Removed due to formatting issues



The biochemical tests performed on the unknown Gram-negative bacterium worked systematically to narrow down the possibilities and eventually eliminate every organism except the correct one.  The Gram stain showed the unknown labeled “A” was a Gram-negative rod.  This alone did not narrow down the field because every possible Gram-negative bacterium was rod shaped.  The Urea test was negative showing the unknown did not emit the enzyme urease.  Three choices fit this profile.  The Simmons Citrate test was positive, removing one of the choices.  The lactose test, using the Eosin-Methylene Blue Agar, and the casein test were done at the same time.  The results of these tests revealed the correct one out of the remaining two.  After the incubation period, the casein test was negative and the lactose test was positive.  The results of the tests confirmed the unknown Gram-negative bacterium was Enterobacter aerogenes.

            The true identity of the unknown labeled “B” proved a little more challenging.  The Gram-stain helped eliminate three out of the five challengers immediately because it was a Gram-positive rod.  The other possibilities were cocci shaped.  A casein test was performed because only one of the remaining two produced the enzyme casease, which would show clearing on the Milk Agar.  This test was positive.  From the results of this test the unknown bacterium should have been Bacillus cereus.  Confirmation from my lab instructor showed it was incorrect.  The next tests performed were a Glycerol test and a Maltose test.  Both of the results should have been consistent as far as the fermentation of either carbohydrate.  Both should be positive or both should be negative.  The results were positive for Glycerol fermentation, but negative for maltose fermentation.  Due to the process of elimination, I knew my Gram-positive bacterium was Bacillus subtilis, since it was not Bacillus cereus.

I was able to determine the identity of the Gram-negative bacterium from the biochemical tests conducted.  However, the same cannot be said for the other half of the unknown #113.  Two out of the three tests performed on the Gram-positive bacterium showed opposite results from what they should have been.  The only explanation for the inconclusiveness of the tests is contamination of the sample at some point after the gram stain was performed.

Of both bacteria discovered, I chose Bacillus subtilis to highlight in the following section.  Bacillus subtilis, known also as the hay bacillus or grass bacillus, is a Gram-positive, catalase-positive bacterium (2).  A member of the genus Bacillus, B. subtilis is rod-shaped, and has the ability to form a tough, protective endospore, allowing the organism to tolerate extreme environmental conditions (3).  Although this species is commonly found in soil, more evidence suggests that B. subtilis is a normal gut commensal in humans.  A 2009 study compared the density of spores found in soil (~106 spores per gram) to that found in human feces (~104 spores per gram).  The number of spores found in the human gut is too high to be attributed solely to consumption through food contamination.  Soil simply serves as a reservoir, suggesting that B. subtilis inhabits the gut and should be considered as a normal gut commensal (4).  As a model organism B. subtilis is commonly used in laboratory studies directed at discovering the fundamental properties and characteristics of Gram-positive spore-forming bacteria.  In particular, the basic principles and mechanisms underlying formation of the durable endospore have been deduced from studies of spore formation in B. subtilis.  It can divide symmetrically to make two daughter cells, or asymmetrically, producing a single endospore that can remain viable for decades and is resistant to unfavorable environmental conditions such as drought, salinity, extreme pH, radiation and solvents.  The endospore is formed at times of nutritional stress, allowing the organism to persist in the environment until conditions become favorable.  Prior to the process of sporulation the cells might become motile by producing flagella, take up DNA from the environment, or produce antibiotics.  These responses are viewed as attempts to seek out nutrients by seeking a more favorable environment, enabling the cell to make use of new beneficial genetic material or simply by killing of competition (2).  B. subtilis is only known to cause disease in severely immunocompromised patients, and can conversely be used as a probiotic in healthy individuals.  It rarely causes food poisoning (5).  The beneficial effects of B. subtilis spores on the balance of the intestinal microflora are the rationale for its general use as a probiotic preparation in the treatment or prevention of intestinal disorders (4).



1.) McDonald, Virginia, Mary Thoele, Bill Salsgiver, and Susie Gero. Lab Manual for General Microbiology. N.p.: St. Louis Community College at Meramec, 2011. Print.

2.) Madigan, Michael T., John M. Martinko, and Thomas D. Brock. Brock Biology of Microorganisms. Upper Saddle River, NJ: Pearson Prentice Hall, 2006. Print.

3.) Nakano, Michiko M., and Peter Zuber. “ANAEROBIC GROWTH OF A “STRICT AEROBE” (BACILLUS SUBTILIS).” – Annual Review of Microbiology, 52(1):165. N.p., Oct. 1998. Web. 28 Apr. 2013.

4.) Hong, Huynh A., Reena Khaneja, and Simon Cutting. “Bacillus Subtilis Isolated from the Human Gastrointestinal Tract.” ScienceDirect.com. Research in Microbiology, Mar. 2009. Web. 28 Apr. 2013.

5.) Oggioni, Marco R., Gianni Pozzi, and Pier E. Valensin. “American Society for Microbiology Journal of Clinical Microbiology.” Recurrent Septicemia in an Immunocompromised Patient Due to Probiotic Strains of Bacillus Subtilis. American Society for Microbiology, Jan. 1998. Web. 28 Apr. 2013.


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