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Unknown Microbiology Report Sample

By at July 18, 2014 | 8:24 am | Print


Unknown Number 110

Penny Pitman

General Microbiology BIO 203

Spring 2014



It is essential in medicine to be able to identify different microorganisms for diagnosing the cause of various diseases, and determining the action for treatment. This study was done using the methods of sterile technique and biochemical tests that have been practiced throughout the semester in the microbiology laboratory.  The purpose of this study was to identify the two different bacterium from a mixed culture, one Gram positive and one Gram negative.


The lab instructor gave out a test tube labeled number 110 with two unknown bacterium. The sterile techniques and methods were followed using the laboratory manual by McDonald et. Al (2).  The first procedure performed was applying a series of streaks onto a nutrient agar with a sterile inoculating loop using the quadrant streak method, and placing it in the incubator at 37 degrees Celsius to grow for two days. The plate was studied and one distinct colony was identified.  A Gram stain was performed on the isolated colony.  The Gram stain procedure was carefully followed according to the steps in the laboratory manual.  Gram positive purple cocci bacteria were identified using a microscope. The Gram negative were unable to be isolated from the nutrient agar.

In order to get the Gram negative to grow, another test was performed taking a sample from the original tube using the sterile inoculating loop, and streaking a MacConkey agar placing it in the incubator at 37 degrees Celsius. All plates were labeled, and tests noted in the journal. The Gram negative bacterium was still not growing on MacConkey agar so another inoculation was done using an EMB agar.  Still unable to get results using the EMB agar, the lab instructor gave an isolated tube labeled alternate A5.

Table 1 and 2 list the tests, purpose, reagents, observations and results for each bacterium.  All of the following tests were performed on these unknowns:

Gram Positive

  1. Gram Stain
  2. Urea
  3. Catalase
  4. Nitrate

Gram Negative

  1. MacConkey
  2. EMB
  3. Nitrate
  4. Indole
  5. Urea



The unknown number 110 was streaked on a nutrient agar plate, and a Gram stain was performed.  The results were Gram positive cocci.  The Gram stain for Gram negative was taken from alternate A5 with the results of Gram negative rods. Table 1 and 2 lists all of the biochemical test, their purpose and results.  The results are also shown in flow chart form.




TABLE 1                                            GRAM POSITIVE TESTS









Gram Stain To determine the gram reaction of the bacterium Crystal violet, Iodine, Decolorizer, Safranin Purple clusters Gram positive cocci
Urea To determine if urease hydrolyzes urea PH indicator phenol red No color change Negative urea test
Catalase To determine if catalase is present H202 No Bubbles present Negative Catalase test
Nitrate To test for nitrate reductases, from nitrates to nitrites Reagents A&B Zinc No color change after reagents A&B or Zinc Negative Nitrate test


TABLE 2                                            GRAM NEGATIVE TESTS









MacConkey To select for enteric bacteria and inhibit gram+ growth PH indicator Neutral Red No color changeNo growth Negative MacConkey test
EMB To select for enteric bacteria and inhibit gram+ growth Dyes Eosin & Methylene blue No growthOr color change Negative EMB test
Nitrate To test for nitrate reductases, from nitrates to nitrites Reagents A&B Zinc Immediate color change after reagents A&B Positive Nitrate test
Indole To determine the ability of an organism to split indole from tryptophane Kovacs No red ring on the top Negative indole test
Urea To determine if urease hydrolyzes urea PH indicator phenol red Broth turned bright pink Positive Urea Test






Gram stain


Gram positive cocci


                                               Cocci                                              Rods

                        Staphylococcus aureus                                   Bacillus subtilis

                        Staphylococcus epidermidis                           Bacillus cereus

                        Enterococcus faecalis           


  Urea (Negative)


Positive                                       Negative

Staphylococcus epidermidis   Staphylococcus aureus

                                                Enterococcus faecalis


Catalase (Negative)


    Positive                                      Negative  

Staphylococcus aureus           Enterococcus faecalis

                           To Confirm


Nitrate (Negative)


   Positive                                      Negative

Staphylococcus aureus           Enterococcus faecalis





Gram stain


Gram negative Rod


Nitrate (Positive)


Positive                                     Negative

 Klebsiella pneumonia / Enterobacter aerogenes                  Escherichia Coli

Proteus vulgaris / Pseudomonas aeruginosa


Indole (Negative)

Positive                                 Negative

Proteus vulgaris                     Enterobacter aerogenes / Klebsiella pneumonia

Pseudomonas aeruginosa




Urea (Positive)


Positive                                        Negative

                                    Klebsiella pneumonia                    Enterobacter aerogenes

                                                                                            Pseudomonas aeruginosa




The results of the Gram positive bacterium after all biochemical tests were performed was identified as Enterococcus faecalis. A Gram stain discovered that the bacteria were cocci shaped which narrowed the results down to three different bacteria.  The first biochemical test performed was a negative Urea test which left two bacteria. The last test was a negative Catalase test that led to the only Gram negative bacteria left which was E. faecalis.  A Nitrate test was performed to confirm the result of E. faecalis. This result was confirmed by Lab instructor.  There were no problems encountered with finding this conclusion.

The results for the Gram negative bacterium after tests were performed was identified as Klebsiella pneumonia. There were problems in the beginning getting an isolated culture for the Gram negative bacterium. Due to problems isolating a pure culture on the original nutrient agar, two different agar plates were inoculated using a sterile swab with the original broth, and spread onto the EMB and MacConkey agars.  This was to try and get the Gram negative bacterium to grow and inhibit the Gram positive.  There was still no growth on both tests. The Lab instructor gave an isolated Gram negative tube labeled alternate A5. A Gram stain was performed and identified as Gram negative rods.  The first biochemical test performed was a Simmons Citrate test with a negative result.  The lab instructor said that the test result was wrong and to try another test. This led to the Nitrate test with a positive result that narrowed it down to four bacteria. A SIMs test was performed to determine if bacteria can reduce sulfur which produces hydrogen sulfide to determine the ability of an organism to split indole from tryptophan, and to check for motility.(2) The result was negative on all three tests, which left three bacteria. The final test performed was a positive Urea test with the conclusion of K. pneumonia as the Gram negative bacteriumThis result was confirmed by the Lab instructor.

K. pneumonia, is a member of the Enterobacteriaceae family that is normally found in the intestinal, respiratory, and urogenital tracts of our body. The bacterium K. pneumonia was  named after Edwin Klebs, a 19th century German microbiologist.(3) Common manifestations that occur with an infection in the lungs include flu like symptoms, with fever, cough, and possible thick bloody mucous. Those who are most at risk are the elderly and sick patients within a clinical setting who are receiving treatment for other conditions such as breathing machines, intravenous catheters and those taking antibiotics for an extended period.(1)  K. pneumonia has a capsule around the cell surface which provides resistance to many antibiotics and other defense mechanisms. The most effective treatment has been with the use of cephalosporin’s, and aminoglycosides. (3)


  1. Klebsiella pneumoniae in Health Care Settings. (2012, August 27). Retrieved April 25, 2014, from Center For Disease Control and Prevention.
  2. McDonald, Virginia et al. Lab Manual for General Microbiology (BIO 203)
  3. Obiamiwe Umeh, M., & Editor, C. (2013, May 29). Klebsiella Infections. Retrieved April 27, 2014, from Medscape.



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