Microbiology Unknown Lab Report
by Nicole Deckard
Introduction
Having an unknown bacterium in the body could be a normal thing or it could be a serious problem. The knowledge to find out if the bacteria is part of your normal flora or a pathogen is something that lab technicians do on a daily bases. Knowing what an unwanted pathogen is will ensure that doctors can treat a patient effectively and without causing harm to the effected patient. This lab will help the student understand how to test and come to a conclusion to what the unknown pathogen that given to them is. This lab will also help to understand why the bacteria in our lives are so important to everyday life.
Materials and Methods
Unknown number 112 was given to the student by the professor. The unknown was inoculated with two different bacterium, one gram positive and one gram negative. On day one, the objective was to grow the bacterium as a mixed culture on a nutrient agar plate. The student followed steps to keep the lab sterile using sterile technique. Once the nutrient agar plate was inoculated the plate was put in an incubator set at 37 degrees Celsius for 48 hours. On day two, the plate was examined to show some green and white colonies of bacteria growth. The next objective was to grow the bacteria as a pure culture. The student took one colony of each bacterium to make two separate nutrient agar plates. The plates where incubated at 37 degrees again for 48 hours. This step of incubation was performed for all the tests in this experiment. Some of the tests incubated longer because of the weekend, but the required time is 48 hours. And please note, that the days are labeled as work days not actual 24 hour days. The pure cultures were examined on day three. The two different plates were labeled as “A” and “B”. Both plates were then Gram stain to determine further test. The tests selected by the student are listed below.
Test for A Test for B
1. Mannitol Salt Agar test 1. Simmons Citrate
2. Catalase Test 2. Voges Proskauer Test
3. Nitrate Test 3. Methyl Red test
4. H2S test 4. Mannitol Salt Agar test
5. Gelatin Test
Results:
Table 1
|
Gram Positive |
Shape | Mannitol test | Catalase test | Nitrate test | H2S test |
|
|
|||||
|
Bacillus cereus |
rod |
Negative |
Positive |
Positive |
Positive |
|
Bacillus subtilis |
rod |
Negative |
Positive |
Positive |
Positive |
|
Staphylococcus aureus |
cocci |
Negative |
Positive |
Positive |
Positive |
|
Staphylococcus epidermidis |
cocci |
Positive |
Positive |
Positive |
Positive |
|
Enterococcus faecalis |
cocci |
Positive |
Negative |
Negative |
Negative |
Table 2
| Gram Negative | Shape | Simmons Citrate | Voges Proskauer test | Methyl Red test | Mannitol | Gelatin test |
| Escherichia coli | Rod | Negative | Negative | Positive | Positive | Negative |
| Klebsiella Pneumoniae | Rod | Positive | Positive | Positive | Positive | Negative |
| Enterobacter aerogones | Rod | Positive | Positive | Negative | Positive | Negative |
| Protous vulgaris | Rod | Negative | Negative | Positive | Negative | Negative |
| Pseudomonas aeruginosa | Rod | Positive | Negative | Negative | Negative | Positive |
FLOWCHART
UNKNOWN A
↓
GRAM STAIN
↓
Gram Positive Cocci
↓
Mannitol Test
↙ ↘
Positive Negative
Staphylococcus aureus Stahylococcus epidermidis
Enterococcus faecalis
↓
Catalase
↙ ↘
Positive Negative
Staphylococcus aureus Enterococcus faecalis
↙
Nitrate (Negative)
Enterococcus faecalis
↓
H2S (Negative)
Enterococcus faecalis
FLOWCHART
UNKNOWN B
GRAM STAIN
↓
Gram Negative rod
↓
Simmons Citrate
↙ ↘
Positive Negative
Klebsiella pneumonia Escherichia Coli
Enterobacter aergenes Protous vulgaris
Pseudomonas aeruginosa
↓
Voges Proskauer Test
↙↘
Positive Negative
Klebsiella pneumonia Pseudomonas aeruginosa
Enterobacter aergenes
↓
Methyl Red (Negative)
Pseudomonas aeruginosa
↓
Mannitol (Negative)
Pseudomonas aeruginosa
↓
Gelatin (Positive)
Pseudomonas aeruginosa
Discussion/Conclusion
All tests were conducted as detailed in the Lab Manual for Microbiology. This is the manual use though the semester in the student’s lab class. The results were read by the student to narrow down the possibilities of what the unknown could be.
The Gram positive bacterial was labeled as “A”. This bacterium, when examined under the microscope, was found to be cocci in shape. This ruled out Bacillus cereus and Bacillus subtilis as possible answers, as they are rod shaped bacteria. This left Staphylococcus aureus, Staphylococcus epidermidis, and Enterococcus faecalis as possibilities for the unknown. On day four, the bacteria were inoculated on a Mannitol Salt Agar (MSA) plate. A MSA is used to test for acid.
The results shown on day five that the agar had turned yellow indicating a positive for this test. This ruled out S. aureus as the unknown. Hydrogen peroxide is the testing agent for the catalase test, if the bacterium bubbles; it indicates the release of oxygen gases. When tested, the bacteria didn’t bubble therefore this test was negative, making S. epidermidis an invalided choice for the unknown. Leaving the only choice to be E. faecalis. Further testing was done to ensure this was the correct unknown. A nitrate test determines if a bacteria is capable of reducing nitrate to nitrite. This test tube turned red after the addition of zinc, resulting in a negative result. A Sulfide Indole Motility tube, also known as SIM tube, was then inoculated. This test for bacteria that produce thoisulfate reductase (H2S) shown by the appearance of black in the tube. When read on day seven, the tubes had turned did not turn black giving a negative result. Further concluding that the bacteria was indeed E. faecalis. This information is shown on table 1 and flowchart A.
The Gram negative bacterial was labeled as “B”, and when examined under the microscope the shape was rod. This did not eliminate any of the five possibilities because they were all rod shaped bacteria. Day four, the Simmons citrate test was started. Results from the Simmons test were read on day five. The tube had turned blue indicating that the production of citrate permease was present. This eliminated Escherichia Coli and Protous vulgaris, leaving Klebisella pneumonia, Enterobacter aergones, and Pseudomonas aeruginosa as the possible choices for the unknown. Then a Voges Proskauer test was performed to indicate if acid produced from glucose was present. This test was read to show no signs of red which indicated a negative result, therefore making P. aeruginosa the only choice.
To further evaluate, the student used a methyl red test on which was read on day six as positive. This was retested because it elimated P. aeruginosa. The retest showed no signs of red after nitrate agents 1& 2 were used which indicating no reduction of nitrates. This confirmed the conclusion that the unknown was P. aeruginosa. Yet, since the student had a false result on one test, two more tests were started on day seven. The MSA test which is primarily used on gram positive bacteria, provided a negative result for P. aeruginosa. The Gelatin test, which tests for the ability to hydrolysis gelatin which was confirmed when the gelatin liquefies within the tube. Further confirming that P. aeruginosa was the unknown bacteria for the Gram negative bacteria. This information is shown in table 2 and Flow chart B.
Enterococcus faecalis was once classified as a group D streptococcus until 1984. This bacterium is part of a human’s normal flora. It is very dangerous when it leaves the intestinal tract. E. faecalis infections in a nosocomial setting are life threating. It causes urinary tract infection, bacteremia, and wound infections. It is a MDR infection, meaning it is a multi-drug resistant infection. This includes Vancomycin. Once this optimistic infection is spread, person to person or within the person, it is very hard to kill. It is also spread by consumption of contaminated food. Risk factors include having an intravascular or urinary catheter and being on a broad spectrum antibiotics.
Pseudomonas aeruginosa is found in water, soil, and on humans. Is bacterium can live in aerobic and anaerobic conditions. This is an opportunistic bacterium. Most people are not harmed by this pathogen, it is when the person is in the hospital for a long period of time this bacterium takes its chance to infect. It can live on a wide variety of nutrients, it can also live for 6 hours up to 16 months on inanimate, dry hospital surfaces. Nosocomial infections include urinary tract infections, pneumonia and bacteremia. P. aeruginosa is the fourth most common nosocomial isolated pathogen, making up 10% of all nosocomial infections. Some infections can be complicated and life threating in patients with a lowered immune system, especially those with a low white blood cell count. It is also a common infection with intravenous drug-users. P. aeruginosa is linked with cases of cystic fibrosis. Testing a patients to diagnose a Pseudomonas aeruginosa infection is done by testing the blood, which is a normally a sterile site. It forms a solid fluorescent green media when stored at temperatures of 20 degrees to 42 degrees Celsius. Most cases of P. aeruginsa are treatable and curable.
