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How to Identify Enterobacter aerogenes and Bacillus cereus | Microbiology Unknown Report

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Sommar Spiniolas

May 5, 2015

BIO 203: Microbiology




Bacteria are ubiquitous, which makes it important to be able to identify them. They are found on our skin, in our bodies, in the soil, in our food, in the air, and in water. (4) Many of these bacteria are harmless, or even helpful in fighting other bacteria, or to help the body function optimally. Others however, are pathogenic to humans and animals. It is necessary to be able to differentiate between those that are harmful, and those that are beneficial, for health reasons. This study was done using all of the techniques learned so far in the microbiology laboratory course in order to identify two unknown bacteria.



On April 2, a broth tube containing two unknown bacteria labeled 105 was received from the lab professor, Professor Snaric. The tube contained one gram positive bacterium, and one gram negative bacterium, which both needed to be identified. All of the procedures used to identify the unknowns were performed by following the lab manual for general microbiology unless otherwise noted. (2) This includes following proper aseptic technique on each step.

After receiving the tube of unknowns, a streak plate was done on nutrient agar. After incubating the streak plate at 37* Celsius for five days, it was determined that a fresh one needed to be done. Another nutrient agar plate was streaked, this time using the quadrant streak method according to the lab manual. After two days of incubation at 37*C, one unknown was isolated and transferred to a separate nutrient agar plate using the quadrant streak method in an attempt to grow a pure colony. This will be referred to as Unknown A. Once again, a fresh streak plate was done from the broth in order to grow the other unknown. After five days of incubation at 37*C, there were clearly two distinct colonies on the fresh streak plate, and Unknown A was growing well. The second unknown was then transferred to a nutrient agar plate. This one will now be referred to as Unknown B. At this time, both bacteria were gram stained according to the lab manual (2), in order to determine their identity as gram positive or gram negative bacteria. Once they were identified, both unknowns were placed back into the incubator at 37*C.

The following day a Methyl Red test was performed on Unknown A, according to the lab manual, to test for acid that is produced during glucose fermentation. A Methyl Red broth was inoculated with the unknown, using a loop that had been flame sterilized. A control tube of Methyl Red was not inoculated. They were both incubated at 37*C. Two days later, Methyl Red drops were added to both the control and the inoculated tubes. After results were seen, an oxidase test was performed to test for the enzyme cytochrome oxidase, which catalyzes the transfer of electrons to oxygen to form water. A filter paper was soaked with Oxidase Reagent, and a sterile wooden applicator was used to smear a sample of Unknown A onto the wet disc. After results were seen, a milk agar plate was then obtained and a casein test was performed according to the lab manual. A quadrant streak of Unknown A was done on the milk agar, and it was placed in the incubator at 37*C for two days when results were then determined.

For Unknown B, a Methyl Red test was also performed as described above. After the two day incubation, results were obtained. A casein test, as described above, was then the performed on Unknown B. The milk agar was placed in the incubator at 37*C for two days when the results were then determined.

All of the following tests were performed on Unknown A:

  1. Methyl Red
  2. Oxidase
  3. Casein

All of the following tests were performed on Unknown B:

  1. Methyl Red
  2. Casein



The following tables detail the tests performed for both of the unknowns. The purpose of the test, the media and reagents used in the test, the observations made during the test, and the results of the test, are clearly outlined. The flow charts will show the path taken to narrow down the bacteria based on the test

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results, and then finally to eliminate all choices but one, which is the unknown’s identity.



Gram Stain To determine the gram reaction of the bacterium Crystal violet, Iodine, Alcohol, Safranin Very small reddish rods Gram negative rods
Methyl Red To detect acid as an end product of glucose fermentation MRVP broth/Methyl Red drops After adding methyl red, broth turned from yellow to a darker yellow Negative Methyl Red test
Oxidase To test for the presence of the enzyme cytochrome oxidase Filter paper/Oxidase reagent No color change on disc Negative Oxidase test
Casein To test for the presence of the enzyme casease Milk Agar Agar remained white and opaque Negative Casein test






Gram stain


Gram negative rod


Methyl Red test (negative)


                                            Positive                            Negative

                               Escherichia coli                            Enterobacter aerogenes

                               Klebsiella pneumonia                 Pseudomonas aeruginosa

                                   Proteus vulgaris                                                 

                                                                                     Oxidase test (negative)


                                                                         Positive                                Negative

                                                       Pseudomonas aeruginosa     Enterobacter aerogenes


                                                                                             Casein test (negative)

 Enterobacter aerogenes

Unknown A-Enterobacter aerogenes





Gram Stain To determine the gram reaction of the bacterium Crystal violet, Iodine, Alcohol, Safranin Purple rods Gram positive rods
Methyl Red To detect acid as an end product of glucose fermentation MRVP broth/Methyl Red drops After adding methyl red, broth changed from yellow to a deep cherry red Positive Methyl Red test
Casein To test for the presence of the enzyme casease Milk Agar Agar had a clear ring around the unknown Positive Casein test







Gram Stain


Gram positive rod


Methyl Red test (positive)


Positive                                         Negative

                              Bacillus cereus                                    Bacillus subtilis


Casein test (positive)

Bacillus cereus


UNKNOWN B-Bacillus cereus




After several tests, it was concluded that Unknown A was Enterobacter aerogenes, and Unknown B was Bacillus cereus. This was verified by the professor. The only issue encountered was overgrowth of bacteria after the first streak plate. This was due to not using the proper quadrant streak technique. The quadrant streak technique was followed from then on with success. Both unknowns grew well once they were isolated on nutrient agar. Once the gram staining was complete, and the gram positive and gram negative were identified, testing went quickly. The first test chosen for the gram negative bacterium was the Methyl Red test because it would narrow down the bacterium to either two or three possibilities. It successfully narrowed it down to two, E. aerogenes and P. aeruginosa. An oxidase test was then performed to identify the bacteria. It showed a negative result meaning it was E. aerogenes. A casein test was performed to verify this conclusion. It was also negative, which supported the conclusion of E. aerogenes. The gram staining of the gram positive bacterium revealed purple bacillus, narrowing it down to B. cereus and B. subtilis. A Methyl Red test was chosen in order to identify the bacterium. It was a positive result meaning it was B. cereus. A casein test was performed in order to verify this conclusion. It was positive which supported B. cereus as the unknown.

  1. aerogenes is a gram negative rod shaped bacteria from the Enterobacteriaceae family. They live in soil, water, sewage, dairy products, and are part of the normal flora of both humans and animals. (4) They can be motile, with peritrichous flagella, or non-motile. They can grow both aerobically and anaerobically, making them facultative anaerobes. They can ferment glucose as well as other sugars, and reduce nitrate to nitrite.
  2. aerogenes is a nosocomial opportunistic pathogen. It, along with Enterobacter cloacae, are the most isolated bacteria found in infected hospital patients. Most of these infections are due to an accidental transfer of bacteria during surgery, or prolonged use of venous or urethral catheters in long term hospital patients. It is attributed to gastrointestinal, respiratory, and urinary tract infections. (1) It can also be found in wound, bloodstream, and central nervous system infections. Enterobacteriaceae account for almost fifty percent of septicemia cases. Because of the severity of these infections, it is extremely important to try to prevent them by using proper aseptic techniques during surgery and other procedures.

An increasing problem with this bacteria is its drug resistance. There have been outbreaks in ICUs in multiple countries due to a strain that is multi-drug resistant. Research shows it is resistant to penicillins and cephalosporins, (3) due to the production of the enzyme beta-lactamase. Many are also resistant to tetracycline, streptomycin, and other aminoglycosides.





  1. Fraser, Susan L., MD and Christian P. Sinave, MD. “Enterobacter Infections.”  Medscape (2014).    4 May 2015.
  2. McDonald, Virginia, Mary Thoele, Bill Salsgiver, and Susie Gero. Lab Manual for General Microbiology-BIO 203.  Louis Community College at Meramec, 2011.  Print.
  3. Thiolas, Aurélie et al. “Successive Emergence of Enterobacter Aerogenes Strains Resistant to Imipenem and Colistin in a Patient.”  Antimicrobial Agents and Chemotherapy4 (2005): 1354–1358.  PMC.  Web.  4 May 2015.
  4. Tortora, Gerard J., Berdell R. Funke, and Christine L. Case. Microbiology: An Introduction.  10th  San Francisco: Pearson Benjamin Cummings, 2010.  Print.



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