Unknown Lab Report, Microbiology | Enterobacter aerogenes

MICROBIOLOGY UNKNOWN LAB REPORT

by Cheryl A. Glaeser

 Enterobacter aerogenes and Enterococcus faecalis

 

INTRODUCTION:

            Bacteria serve many different roles in people’s lives today.  It is important to identify bacteria and differentiate pathogenic and non-pathogenic organisms.  By identifying the organism present in infectious diseases, appropriate antibiotics and duration of therapy can be chosen.  Understanding specific bacterial characteristics can influence these choices greatly.  Beneficial bacteria can be just as important as they are used for the production of food materials, nutritional supplements, and in the production of antibiotics to fight disease.  The concepts and techniques learned in the microbiology laboratory class served as a foundation for the identification of unknown bacteria.




MATERIALS AND METHODS:

The professor of the microbiology lab distributed unknowns to all of the students.  The unknown specimen for this study was number 107.  The techniques learned in this lab were employed to aid in the identification of this unknown.  The course laboratory manual by McDonald et. al¹ served as the foundation for the procedures employed, unless stated otherwise.

The unknown was plated on a nutrient agar plate with aseptic technique using the isolation streak method as described in the lab manual.  The plate was incubated at 37 degrees Celsius until the following lab period.  At that time, the bacterial growth was noted and its characteristics were recorded.  A Gram stain was performed on a sample of the colonies that had grown.  This Gram stain demonstrated Gram-negative rods and Gram positive cocci.  The fact that two organisms appeared from the apparent isolate on the nutrient agar proved that the bacterial isolation was unsuccessful.  This failure led to re-plating the unknown broth onto another nutrient agar plate.  This plate was incubated similarly as the first.

The following laboratory period one colony form was noted growing on the nutrient agar plate after incubation.  The Gram staining technique was again performed on the isolate and proved to be Gram negative rods.  The instructor confirmed that this was the staining characteristic of one of the bacteria present in the unknown broth.  The isolate was then plated on a Mannitol Salt Agar plate (MSA) to inhibit Gram negative bacterial growth.  This plate was left to incubate at 37 degrees Celsius until the next lab period.

The next lab, one colony type was noted growing on the MSA agar.  There was a yellow color change in the agar indicating fermentation of Mannitol producing acid.  A Gram stain was performed on the bacterial isolate indicating Gram positive cocci were present.

The next step was to identify the two different bacteria that were isolated in the previous steps.  The biochemical tests performed on the Gram negative rod isolate were chosen from the unknown chart that the professor handed out to the class.

Table 1 lists the test, purpose, reagents, observations and results.

The following tests were performed on this unknown:

1. Indole
2. Simmons Citrate
3. Methyl Red
4. Voges-Proskauer
5. Nitrate

The biochemical tests performed on the Gram positive cocci again were chosen from the unknown chart handed out in lab.

Table 2 lists the tests, purpose, reagents, observations and results.

The following tests were performed on this unknown:

1. Indole
2. Simmons Citrate
3. Nitrate
4. Urea

RESULTS:

TABLE 1: Gram Negative Rod Unknown

TEST	PURPOSE	REAGENTS	OBSERVATIONS	RESULTS
Indole	To determine an organisms ability to convert Tryptophan to Indole	SIM mediaTryptone broth	No color changeMotility present	Negative Indole test
Simmons Citrate	To determine if an organism can use Citrate as its sole carbon source	Citrate slant (green)	Color changed from green to blue	Positive Simmons Citrate test
Methyl Red	To determine an organisms ability to produce acid from glucose metabolism	MRVP brothMethyl Red 5-10 drops	Performed twice1. Color changed from yellow to red2. No color change-stayed yellow	1. Positive MR test2. Negative MR test
Voges-Proskauer	To determine if an organism produces acetyl methyl carbinol	MRVP brothBarritt’s A & B	Performed twice1. Improper technique applied-discarded2. Color change from yellow to pink	1. No result2. Positive VP test
Nitrate	To determine an organisms ability to reduce nitrate to nitrite or other nitrogenous compounds	Nitrate reagents A & BPowdered Zinc	Color change from yellow to red	Positive Nitrate test

 

 

 

 

TABLE 2: Gram Positive Cocci Unknown

TEST	PURPOSE	REAGENTS	OBSERVATIONS	RESULTS
Indole	To determine an organisms ability to convert Tryptophan to Indole	SIM mediaTryptone broth	No color changeMotility present	Negative Indole test
Simmons Citrate	To determine if an organism can use Citrate as its sole carbon source	Citrate slant (green)	No color change	Negative Simmons Citrate test
Nitrate	To determine an organisms ability to reduce nitrate to nitrite or other nitrogenous compounds	Nitrate reagents A & BPowdered Zinc	Non-specific color change-peach color	False positive
Urea	To determine an organisms ability to hydrolyze urea using the enzyme urease	Urea broth	No color change	Negative

 

FLOW CHART UNKNOWN #107 

**Removed due to Formatting Problems. 

 

Unknown #1: Enterobacter aerogenes                   Unknown #2: Enterococcus faecalis

 




DISCUSSION / CONCLUSION:

The identification of bacteria to the level of species is important for several reasons.  Difficulties inherent in the thought processes and techniques necessary to identify the unknown bacteria in Unknown #107 were evident throughout this exercise.  The issues encountered included the thoughtful choice of appropriate tests and the technical ability to perform the tests correctly.

The first bacterium identified was a Gram negative rod after its isolation on nutrient agar.  Technical difficulties were encountered in the isolation of the bacteria and the Gram staining processes.  The initial isolation did not lead to the two unknowns that were expected.  After isolating a pure culture, selective testing aided in the identification of the bacterium present.  The Indole test was used to separate the Gram negative rods into those that can convert the amino acid Tryptophan to Indole and those that cannot.  The unknown Gram negative rod present in this culture gave a negative Indole test result thus eliminating E. coli and P. vulgaris.  The Simmons Citrate test was chosen, but did not help in further reducing the possibilities for the unknown, because all three remaining possibilities tested similarly.  The Methyl Red test was performed, but technical issues gave an initial false positive test for the unknown.  After guidance from the professor, the Methyl Red test was performed a second time giving the expected negative result.  This indicated that the unknown did not produce acids as a byproduct of glucose metabolism.  The negative result eliminated K. pneumoniae from the list of remaining possibilities.  The Voges-Proskauer test demonstrated a positive result indicating that Enterobacter aerogenes was the unknown bacterium.  The positive test indicated that the unknown, E. aerogenes does not produce acetyl methyl carbinol.  The Nitrate test was not necessary, but gave the positive result that would be expected.

Without the guidance of the professor, a proper identification would not have been possible.  By repeating the Methyl Red test, a proper identification was achieved.

Similarly, several biochemical tests were applied to the second isolate, Gram positive coccus.  The Mannitol test was initially chosen and a positive result eliminated Staphylococcus epidermidis from the group of three possible bacteria given as unknown choices.  The color change from red to yellow indicated that the unknown could grow in high salt concentrations and was a mannitol fermenter.  The Simmons Citrate test was chosen to further define the possibilities, but this test was not helpful as a negative test result would be expected for both of the remaining bacterial choices.  This indicates that both S. epidermidis and E. faecalis do not use citrate as their sole carbon source.  The Nitrate test gave a false positive result which would have led to the incorrect identification of Staphylococcus aureus as the unknown instead of Enterococcus faecalis, the true unknown.  The Urea test was negative, but this would be expected for both of the remaining choices as neither can hydrolyze urea using the enzyme urease.  The problems encountered with the identification of the Gram positive coccus were due to both technical and test selection issues.  This demonstrates the importance of proper technique in performing the selective tests for bacterial identification.  Even if proper technique is used, if improper tests are chosen, bacterial choices cannot be eliminated.

The importance of identifying the genus and species of bacteria is demonstrated by the second unknown.  The antibiotic selection for S.Aureus may be vastly different than for E. faecalis.  Enterococcus faecalis is one of the bacteria present in normal flora of both humans and animals.  It is a Gram positive coccus that grows in chains.  Of the more than 17 species in the genus Enterococcus, only a few cause clinical disease.  E.faecalis is one of the pathogenic species that is very important in nosocomial (hospital-borne) infections due to its potential for resistance to multiple antibiotics.²  It is important to differentiate Staphylococcus from Enterococcus as each has its own pattern of resistance to antibiotics.  Vancomycin Resistant Enterococcus (VRE) and Methicillin Resistant Staphylococcus aureus (MRSA) have become hot button issues in the war against bacterial infections, especially in the hospital environment.³  Although most Staphylococcus and Enterococcus species are sensitive to Vancomycin^4,5, it may not be necessary for all species and can be reserved for more serious resistant infections.  Furthermore, E. faecalis may often be resistant to the group of antibiotics called cepahlosprins, but many Staphylococcus species including some S. aureus may be sensitive.^4,5  This comparison is only one example of the importance of applying the concepts of bacterial identification in a real world setting.  The concepts used to identify the unknowns in this laboratory activity have a very important place in society today.

 

ADDITIONAL EXAMPLES OF MICROBIOLOGY UNKNOWN LAB REPORTS

 

REFERENCES:

Fraser, Susan L., MD. “Enterococcal Infections .” Enterococcal Infections. Ed. Burke A. Cunha, MD. N.p., 28 Sept. 2012. Web. 29 Apr. 2013.

Furuno, Jon P., Eli N. Perencevich, Judith A. Johnson, Marc-Oliver Wright, Jessina C. McGregor, J. Glenn Morris, Sandra M. Strauss, Mary-Claire Roghman, Lucia L. Nemoy, Harold C. Standiford, Joan N. Hebden, and Anthony D. Harris. “Methicillin-resistant Staphylococcus Aureus and Vancomycin-resistant Enterococci Co-colonization.” Centers for Disease Control and Prevention. N.p., Oct. 2005. Web. 29 Apr. 2013.

Lab Manual for General Microbiology. St. Louis:

Rayner, C., and WJ Munckhof. “Antibiotics Currently Used in the Treatment of Infections Caused by Staphylococcus Aureus.” National Center for Biotechnology Information. U.S. National Library of Medicine, Dec. 2005. Web. 29 Apr. 2013.

Scott, IU, RH Loo, HW Flynn, Jr., and D. Miller. “Endophthalmitis Caused by Enterococcus Faecalis: Antibiotic Selection and Treatment Outcomes.” National Center for Biotechnology Information. U.S. National Library of Medicine, Aug. 2003. Web. 29 Apr. 2013.