UNKNOWN LAB REPORT
Michelle Olms
There are many reasons why scientists try to find the identity of microorganisms. These reasons span from understanding infectious diseases and immune functions to understanding human and microbe relationships (why we need certain microbes for survival). The main reason why these scientists do this is so that they have a better understanding of the microbial world and how microbes react to their environment. This study was done by applying all of the scientific methods that have been learned so far in the microbiology lab. The purpose of this study was to closely identify unknown bacteria.
A tube of unknown bacteria labeled as number 102 was given out by the lab professor. The methods that have been learned thus far for identifying bacteria have been applied to this unknown. Procedures were followed as stated in the course laboratory manual by McDonald et.al (1), unless otherwise noted. First, the unknown bacterium was inoculated onto a Nutrient Agar plate using the streak plate method described in the lab manual. This procedure was done so that the unknown bacteria could form isolated colonies on the plate. Once 2 different isolated colonies were formed on the plate, the morphology of the colonies was observed and recorded and those colonies were isolated onto new Nutrient Agar plates. Gram stains were then performed on these 2 quality controlled bacteria in order to distinguish them apart.
After determining the reactions of the gram stains, specific biochemical tests, chosen from the unknown identification tables in the lab manual, were performed. Since one of the colonies from unknown #102 was determined to be a Gram negative rod (bacillus), a urea test was performed and the organism was inoculated into a urea tube. The results from the test were observed and recorded and a casein test was performed in which the bacteria were inoculated onto a Milk Agar plate. The results from this test were observed and recorded. A mannitol test was later performed in which the bacteria were inoculated onto a Mannitol Salt Agar plate. Afterwards, a nitrate test was performed in which the bacteria were inoculated into a nitrate tube. The last test that was performed was the Simmon’s Citrate test. With this test, the bacteria were inoculated into a Simmon’s Citrate tube.
The other colony, from unknown #102, was determined to be Gram positive rods (bacillus). From this determination, a mannitol test was performed and the organism was inoculated onto a Mannitol Salt Agar plate (MSA). After the results from the test were observed and recorded, a urea test was performed in which the bacteria were inoculated into a urea tube. Next, a Simmon’s Citrate test was performed in which the bacteria were inoculated into a Simmon’s Citrate tube. A casein test was later performed in which the bacteria were inoculated onto a Milk Agar plate. Lastly, a nitrate test was performed in which the bacteria were inoculated into a nitrate tube. All of these tests were performed by the methods listed in the lab manual by McDonald et al. (1). Table 1 lists the test, purpose, reagents, and results.
All of the following tests were performed on this unknown:
- Urea test
- Mannitol
- Simmon’s Citrate
- Nitrate
- Casein
Gram Negative Tests
|
Test |
Purpose |
Reagents |
Observations |
Results |
Interpretations |
|
Gram stain |
To determine the Gram reaction of the bacterium | Crystal Violet, Iodine, Gram Decolorizer (Alcohol), Safranin Red | Pink rods | Gram negative rods (bacillus) | Organism is Gram negative |
| Urea test | To determine the presence of urease (breaks down urea) | None | Peach | Negative | Organism does not show a presence of urease (did not break down urea) |
| Casein test | To determine the production of casease (hydrolyzes casein) | None | Clearing | Positive | Organism produced casease (hydrolyzed casein) |
| Mannitol test | To determine the ability of a bacterium to ferment mannitol | None | Red | Negative | Organism did not ferment mannitol or produce an acid |
| Nitrate test | To determine the ability of an organism to produce nitrate reductase (reduce nitrates to nitrites) | Reagent A and Reagent B | After adding Reagent A, color changed from white/clear to red | Positive | Organism produced nitrate reductase and reduced nitrate to nitrite |
| Simmon’s Citrate | To determine the ability of an organism to produce citrate permease and use citrate as a sole source of carbon | None | Color changed from green to blue | Positive | Organism is able to utilize citrate as a carbon source and produce citrate permease |
Gram Positive Tests
|
Test |
Purpose |
Reagents |
Observations |
Results |
Interpretations |
| Gram stain | To determine the Gram reaction of the bacterium | Crystal Violet, Iodine, Gram Decolorizer (Alcohol), Safranin Red | Purple rods | Gram positive rods (bacillus) | Organism is Gram Positive |
| Mannitol test | To determine the ability of a bacterium to ferment mannitol | None | Red | Negative | Organism did not ferment mannitol or produce an acid |
| Urea test | To determine the presence of urease (breaks down urea) | None | Peach | Negative | Organism does not show a presence of urease (did not break down urea) |
| Simmon’s Citrate | To determine the ability of an organism to produce citrate permease and use citrate as a sole source of carbon | None | Color changed from green to blue | Positive | Organism produced citrate permease and used citrate as a sole source of carbon |
| Casein test | To determine the production of casease (hydrolyzes casein) | None | Clearing | Positive | Organism produced casease and hydrolyzed casein |
| Nitrate test | To determine the ability of an organism to produce nitrate reductase (reduce nitrates to nitrites) | Reagent A and Reagent B | Red | Positive | Organism is able to produce nitrate reductase and reduce nitrates to nitrites |
FLOWCHART
*Removed due to formatting issues.
Unknown Gram Positive – Bacillus cereus
The test results of both the Gram negative and Gram positive bacteria led to the identification of each of these species due to the reactions that each organism showed in each of the tests above. For the first colony that was isolated from the unknown tube, the gram stain test determined that the bacteria were Gram negative rods. Additional tests (Urea, Mannitol, Nitrate, Simmon’s Citrate, and Casein) resulted in different and unique reactions from the Gram negative bacteria. In the Urea test, the bacteria showed a negative reaction and did not change color or produce urease. With the Mannitol test, the bacteria also showed a negative reaction by not producing an acid or fermenting mannitol. However, the Nitrate, Simmon’s Citrate, and Casein tests all showed a positive reaction because the bacteria changed colors (from clear to red (Nitrate), from green to blue (Simmon’s Citrate)) and showed a clearing (Casein).
These particular reactions helped me to narrow down my choices as to what genus and species of bacteria this could be. Each test helped me eliminate certain choices and further confirm my hypothesis. After recording the bacteria’s reaction in the Casein test, I correctly identified that the Gram negative bacteria was Pseudomonas aeruginosa. There were no problems in my experiments with the Gram negative bacteria.
Pseudomonas aeruginosa is a Gram negative bacterium that is commonly found in soil and ground water. It is a member of the Gamma Proteobacteria class of bacteria and belongs to the bacterial family Pseudomonadaceae (Todar). This bacterium is an opportunistic pathogen that is more likely to infect patients who are already very sick (immunocompromised) than healthy patients. This unique organism can cause a wide range of infections such as burns, wounds, urinary tract infections, blood infections, abscesses, and meningitis (Tortora, 308). P. aeruginosa is a major cause of both healthcare-associated infections and chronic lung infections in people with cystic fibrosis. This bacterium is also a grave concern to cancer patients and burn victims (ehagroup.com). Most infections are susceptible to third generation cephalosporins (ceflazidime), carbapenems (imipenem and meropenem), aminoglycosides (gentamicin, tobramycin), and colistin (HPA.org). P. aeruginosa is very resistant to many antibiotics.
With the second colony that was isolated from the unknown tube, the gram stain test determined that the bacteria were Gram positive rods. Additional tests (Urea, Mannitol, Nitrate, Simmon’s Citrate, and Casein) resulted in different and unique reactions from the Gram positive bacteria as well. In the Urea test, the bacteria showed a negative reaction and did not change color or produce urease. With the Mannitol test, the bacteria also showed a negative reaction by not producing an acid or fermenting mannitol. However, the Nitrate, Simmon’s Citrate, and Casein tests all showed a positive reaction because the bacteria changed colors (from clear to red (Nitrate), from green to blue (Simmon’s Citrate) and showed a clearing (Casein).
These particular reactions helped me to narrow down my choices as to what genus and species of bacteria this could be. Each test also helped me eliminate certain choices and further confirm my hypothesis. After recording the bacteria’s reaction in the Casein test, I correctly identified that the Gram positive bacteria was Bacillus cereus. Some problems were encountered during my experiments with the Gram positive bacteria. With the gram stain test, it was difficult to determine if the bacteria was Gram positive rods or Gram positive coccus. In the Simmon’s Citrate test, the reacton from the Gram positive bacteria showed a small negative reaction in certain parts of the tube (the color remained blue).
Cited Sources
1. McDonald, Virginia, Thoele, Mary, Salsgiver, Bill, and Gero Susie. Lab Manual for General Microbiology. Meramec. 2011. Print.
2. Todar, Kenneth. “Pseudomonas.” Pseudomonas. N.p., n.d. Web. 01 Dec. 2013. <http://textbookofbacteriology.net/pseudomonas.html>.
3. Tortora, Gerard J., Berdell R. Funke, and Christine L. Case. Microbiology: An Introduction. 11th ed. Boston: Pearson, 2013. 308. Print.
4. “Pseudomonas Aeruginosa.” HPA.org. Health Protection Agency, n.d. Web. 02 Dec. 2013. <http://www.hpa.org.uk/Topics/InfectiousDiseases/InfectionsAZ/PseudomonasAeruginosa/>.
5. “What Is Pseudomonas Aeruginosa?” Pseudomonas Aeruginosa. Environmental and Public Health Consultants, n.d. Web. 02 Dec. 2013. <http://www.ehagroup.com/resources/pathogens/pseudomonas-aeruginosa/>.
