MICROBIOLOGY UNKNOWN LAB REPORT
by MEGAN KARR
DECEMBER 6TH, 2012
INTRODUCTION
There are multiple reasons why knowing how to identify different microorganisms is useful. “Microorganisms are essential to our very existence. They are ubiquitous, found in common environments such as soil, water, and air” (4). Knowing the difference between different microorganisms can not only help identify a disease, but also what’s causing a disease in a patient, and what antibiotic can help treat that disease. Microorganisms are also in all foods, and knowing the different microorganisms can help identify when food is past its prime. To identify an unknown bacteria the study was done by applying all of the methods that have been used so far during the microbiology laboratory class.
MATERIALS AND METHODS
An unknown test tube labeled number 105 was given to the student by the professor. The test tube contained a mixed culture of two unknown bacteria, one a gram-positive and one a gram-negative. Methods that have been used throughout the semester to identify specific bacteria were applied to the unknown. The methods and procedures were followed as the laboratory manual for microbiology stated, unless stated otherwise.
On day one, the first objective was to complete an isolation streak of the mixed culture onto nutrient agar. The goal of this test was to correctly isolate the two different bacteria. The nutrient agar was incubated at 37 degrees Celsius for 48 hours. Upon observation, on day two the nutrient agar plate appeared to be tinted a light green color, which is comparable to the pigment of the agar completed in exercise #10 on the lab manual, which led to the belief that the gram negative bacteria was Pseudomonas aeruginosa. To confirm these beliefs, a new isolation streak of organism A’s pure colony was created.
On day three a gram stain of the pure colony was completed following the steps for gram stain on page 67 of the lab manual. Going forward the gram negative bacteria was labeled Bacteria A and the gram-positive bacteria was labeled Bacteria B and this is how each bacterium will continue to be referred to. The gram stain revealed a gram negative organism with a rod shape. These results did not narrow down the options since all of the gram-negative organisms were rod-shaped. A gelatin agar test was completed to test bacteria A’s ability to hydrolyze gelatin through the action of the extracellular enzyme gelatinase. The gelatin in this medium is used to solidify the agar. If the medium liquefies, the result is positive. The result of the gelatin agar test for bacteria A came back negative which ruled out Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogenes, and Proteus vulgaris. This left Pseudomonas aeruginosa as the gram negative bacteria.
On day four a Kligler Iron agar test was completed to test for fermentation of glucose. The Kligler Iron agar test resulted in a negative reaction for fermenting glucose which backed up the gelatin agar test results, meaning organism A was Pseudomonas aeruginosa (1).Please refer to the information shown in table 1 and flowchart A.
All of the following tests were performed for Bacteria A:
1) Gelatin Agar test
2) Kligler Iron test
3) Glucose test
4) Oxidase test
Bacterium B was unable to be isolated during the original isolation streak completed on day one. Two more isolation streaks were completed, both with the same results. A Mannitol Salt Agar (MSA) test was completed on day two using the original mixed culture 105 in efforts to isolate only bacterium B. The student tried this test because MSA is a selective agar for gram positive bacteria. On day three the results of the MSA plate were checked and no bacteria was noted. This most likely occurred due to a bad streak of bacteria or a contaminated original culture. The professor gave the student a brand new pure colony of gram positive bacteria. This will still be noted as Bacteria B going forward.
A gram stain was completed and the results were a gram positive rod, which ruled out Staphylococcus aureus, Staphylococcus epidermidis, and Enterococcus faecalis, and leaving Bacillus cereus, and Bacillus subtilis as possibilities. A nitrate test was done next to confirm results of the gram stain. The Nitrate test determines if the bacteria can anaerobically reduce nitrate. The nitrate test confirmed the gram stain, leaving the choices for bacteria B to be Bacillus cereus, or Bacillus subtilis. A gelatin test was completed and the results were positive, which backed up the results of the gram stain and the nitrate test. A Methyl Red test was then completed to test for the production of a mixture of acids due to glucose fermentation. This test had a positive result which ruled out Bacillus subtilis, leaving Bacillus cereus to be bacteria B (1). Please refer to the information shown in table 2 and flowchart B.
The following tests were performed for Bacteria B:
1) Nitrate test
2) Gelatin test
3) Methyl Red test
RESULTS
Table 1:
| Gram Negative |
Shape |
Gelatin |
Glucose |
Oxidase |
|
| E. coli |
Rod |
Negative |
Acid |
Negative |
|
| K. pneumoniae |
Rod |
Negative |
Acid |
Negative |
|
| Enterobacter aerogenes |
Rod |
Negative |
Acid |
Negative |
|
| Proteus vulgaris |
Rod |
Negative |
Acid |
Negative |
|
| Pseudomonas aeruginosa |
Rod |
Positive |
Negative |
Positive |
| Gram Positive |
Shape |
Nitrate |
Gelatin |
Methyl Red |
|
| Bacillus cereus |
Rod |
Positive |
Positive |
Positive |
|
| Bacillus subtilis |
Rod |
Positive |
Positive |
Negative |
|
| S. aureus |
Cocci |
Positive |
Negative |
Positive |
|
| S. epidermidis |
Cocci |
Positive |
Negative |
Positive |
|
| Enterococcus faecalis |
Cocci |
Negative |
Negative |
Positive |
Table 2:
FLOWCHART A
UNKNOWN BACTERIA A
↓
Gram Stain
↓
Gram negative Rod
↓
Gelatin test (positive)
↙ ↘
Positive Negative
Pseudomonas aeruginosa Escherichia coli
↓ Klebsiella pneumoniae
Glucose (negative) Enterobacter aerogenes
↙ ↘ Proteus vulgaris
Positive Negative
Escherichia coli Pseudomonas aeruginosa
Klebsiella pneumoniae ↓
Enterobacter aerogenes Oxidase test (positive)
Proteus vulgaris ↙ ↘
Positive Negative
Pseudomonas aeruginosa Escherichia coli
↓ Klebsiella pneumoniae
↓ Enterobacter aerogenes
↓ Proteus vulgaris
|
Unknown Bacteria A– Pseudomonas aeruginosa |
FLOWCHART B
UNKNOWN BACTERIA B
↓
Gram Stain
↓
Gram Positive Rod
↓
Nitrate test (Positive)
↙ ↘
Positive Negative
B. cereus Enterococcus faecalis
B. subtilis
S. aureus
S. epidermidis
↓
Gelatin test (Positive)
↙ ↘
Positive Negative
B. cereus S. aureus
B. subtilis S. epidermidis
↓
Methyl Red test (Positive)
↙ ↘
Positive Negative
B. cereus B. subtilis
|
Unknown B– Bacillus cereus |
↓
DISCUSSION/CONCLUSION
After several tests and experiments it was determined that Bacteria A was Pseudomonas aeruginosa and Bacteria B was Bacillus cereus. After performing the gram stain for bacteria A and determining the unknown was a gram negative rod, the organism underwent a multitude of biochemical tests to narrow down the exact bacteria. All of the biochemical tests went well for bacteria A. Bacteria B proved difficult to isolate, and the student needed to obtain a new pure culture of a gram positive bacterium and start the identifying process over. After performing the second gram stain for bacteria B, and determining the unknown was a gram positive rod, the organism underwent a multitude of biochemical tests to further determine the exact bacteria. This set of biochemical tests for bacteria B went well and no problems were encountered.
Pseudomonas aeruginosa is a pathogen that can be found in the bodies’ respiratory tract, and urinary tracts. This bacterium can reside in any human being and not be detected, as long as the person is healthy. When a person becomes ill, the immune system begins to focus all of its energy onto the illness, Pseudomonas aeruginosa makes an appearance. This is considered an opportunistic bacterium. Pseudomonas aeruginosa can cause urinary tract infections, respiratory infections, ear aches, and lung problems. Pseudomonas aeruginosa can be difficult to treat as it is resistant to many antibiotics, meaning the bacterium will fight off antibiotics that are meant to defeat it. (2)
As of 1993, Bacillus cereus was not being considered a pathogen, but it is now recognized as one. This bacterium is known as the cause of food poisoning, diarrhea, and non-gastrointestinal infections. Bacillus cereus produces what is known as beta-lactamase, which causes it to be resistant to beta-lactam antibiotics. (3)
REFERENCES
- Lab Manual for General Microbiology, St. Louis Community College at Meramec. McDonald, Thoele, Salsgiver, Gero. April 2011
- “Pseudomonas Infection.” (2011): 1. Web. 1 Dec 2012. <https://www.webmd.com/a-to-z- guides/pseudomonas-infection-topic-overview>.
- “Bacillus cereus.” (1993): 1. Web. 1 Dec 2012. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC358292/>.
- Meade-Callahan, Maura. “Microbes: What they do and how antibiotics change them.” (2001): n.pag. Web. 1 Dec 2012. <https://www.actionbioscience.org/evolution/meade_callahan.html>.
