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UNKNOWN LAB REPORT
Unknown Number 120, Alternate 3
May 5, 2015
Professor Jay Snaric
Introduction to Microbiology
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Although naked to the eye, microorganisms play a key role in the lives of every human being. In the health field, while microorganisms are responsible for causing diseases, they are also actively involved in the making of antibiotics. Without identifying the microorganisms that caused the disease, it would be impossible to isolate an antibiotic that would successfully treat the patient. Similarly, in the food business, microorganisms are involved in managing food safety and maintaining a high quality of food. In this scenario, it is vital that the correct bacteria are identified and used when cooking and keeping supplies and utensils free of harmful microorganisms. This study involves utilizing all of the methods and procedures covered in the microbiology laboratory class in order to learn the process of correctly identifying the two unknown bacteria.
MATERIALS AND METHODS
At the beginning, a test tube labeled 120 was handed out by the lab instructor. In order to isolate the two unknown bacteria from the single test tube, an isolation streak plate needed to be performed. This procedure, as outlined in the lab manual written by McDonald et al., carefully pulls bacteria along the four sides of a nutrient agar plate to try to clearly isolate the two microbes (10). After forty-eight hours of incubation at thirty-seven degrees Celsius, it appeared as if only one type of bacterium grew on the streak plate. Although only one bacterium had grown, it was in the best interest of the study to keep working, and then return to the other bacterium once it had been determined whether it was gram positive or gram negative. Moving forward, another isolation streak plate was performed using just the one bacterium that appeared on the original streak plate. Once incubated, the nutrient agar came back pure, thus allowing a procedure called a gram stain to be performed. Described in the lab manual by McDonald et al., the gram stain was performed using the microbes from the inoculated agar plate and came back as gram positive cocci (67). Then, a urea test and a nitrate test were both performed on the gram positive cocci. The urea test requires urea broth tubes, while the nitrate test requires nitrate broth tubes, nitrate reagent A, nitrate reagent B, and powdered zinc. Both tests are further outlined in the laboratory manual (McDonald et al. 36). Once these tests were completed, the identity of the unknown bacterium was revealed.
Gram Positive Methods
|Gram Stain||To differentiate bacterial cells based on their cell wall||Crystal violet, Iodine, Alcohol, Safranin||Purple balls||Gram Positive cocci|
|Urea Test||To determine if bacteria had made urease, thus broke down urea||None||Urea broth remained tan color (unchanged)||Negative Result|
|Nitrate Test||Tests for the reduction of nitrate||Nitrate Reagent A, Nitrate Reagent B, Powdered zinc||Add reagents, No color change, added zinc, no color change||Positive Result|
When isolating the bacteria at the very beginning on the isolation streak plates, only the gram positive bacterium was visible. At this point in the study, the gram negative bacteria were needed to grow, so EMB agar and MacConkey agar were chosen to be inoculated. These specific agars were chosen because they inhibit the growth of gram positive bacteria and select for the growth of gram negative bacteria. After incubation, there were visible purple spots on the EMB agar so a gram stain was performed with those microbes and gram negative and gram positive bacteria appeared. Due to this, the lab instructor handed out an alternate number three. Using the alternate number three, a nutrient agar plate was streaked, incubated, and then gram stained. The gram stain confirmed that alternate number three was gram negative rods. Then, a urea test, a sulfur reduction test, and an indole test were performed. The urea test requires urea broth tubes, the sulfur reduction test requires a SIM tube, and an indole test requires a SIM tube and indole reagent. All three of these tests are described in the laboratory manual (McDonald et al. 19,36). The results of these tests confirmed the identity of the second unknown bacterium.
Gram Negative Methods
|Gram Stain||To differentiate bacterial cells based on their cell wall||Crystal violet, Iodine, Alcohol, Safranin||Pink rods||Gram negative Rods|
|Urea Test||To determine if bacteria had made urease, thus broke down urea||None||Urea broth turned bright pink||Positive result|
|H2S Test||To determine if sulfur had been reduced||None||Tube had a black clump||Positive result|
|Indole Test||To determine if indole had been produced||Indole reagent||Top of test tube appeared red||Positive result|
Starting with the first unknown, a gram stain, urea test, and nitrate test were performed. The gram stain revealed that the unknown was a gram positive cocci bacterium. In addition, the urea test was negative and the nitrate test was positive. The second unknown was a gram negative rod and that bacterium had three separate tests performed. Specifically, the urea test was positive, the H2S test was positive, and the indole test was positive. In conclusion, the gram positive bacterium was Staphylococcus aureus, and the gram negative bacterium was Proteus vulgaris.
Biochemical Tests – Gram Positive
|Gram Stain||Crystal violet, iodine, alcohol, safranin||23OC(room temp)||Purple balls||Gram positive cocci||Organism has cell walls made of thick peptidoglycan|
|Urea||Urea broth||37OC||Urea broth remained tan colored (unchanged)||Negative result||Bacteria do not produce urease, so urea is not broken down|
|Nitrate||Nitrate broth, nitrate reagent A, nitrate reagent B, powdered zinc||37OC||Add reagents, no color change, added zinc, no color change||Positive result||Nitrate has been fully reduced|
Biochemical Tests – Gram Negative
|Gram stain||Crystal violet, iodine, alcohol, safranin||23OC(room temp)||Pink rods||Gram negative rods||Organism has a thinner layer of peptidoglycan and an outer lipid membrane|
|Urea||Urea broth||37OC||Urea broth turned bright pink||Positive result||Urea is broken down by urease|
|H2S||SIM tube||37OC||Black clump appeared in tube||Positive result||Organism produces hydrogen sulfide|
|Indole||SIM tube, indole reagent||37OC||Top of test tube appeared red||Positive result||Organism produces indole and is a bi-product of amino acid metabolism|
FLOW CHART – Removed due to formatting issues.
Through multiple tests and procedures, the identity of both unknowns were determined. Once the gram stain revealed that the gram positive bacterium was cocci, the bacteria Bacillus cereus and Bacillus subtillis could be excluded because they are rod shaped. The first test performed for the gram positive bacteria was the urea test. This test yielded negative results and thus eliminated Staphylococcus epidermidis. Then, the nitrate test confirmed that Enterococcus faecalis was not the unknown, and rather Staphylococcus aureus was.
When the urea test was performed on the gram negative bacteria, the results were positive, which eliminated the bacteria Escherichia coli, Enterobacter aerogenes, and Pseudomonas aeruginosa. Next, the sulfur reduction test determined that the unknown is not Klebsiella pneumoniae, leaving only one option, which is Proteus vulgaris. The indole test also confirms that the unknown bacterium is Proteus vulgaris.
All of these tests executed led to the correct identities of both unknowns. The only problem that was encountered throughout the process, was when the gram stain was performed using the bacteria from the EMB agar and both gram negative and gram positive bacteria grew. Using the EMB agar, only gram negative bacteria was supposed to grow, but because both types of bacteria grew, an alternate bacteria was given by the instructor.
Staphylococcus aureus is a gram positive, facultative anaerobic bacteria. It is normally found in the nose and on the skin of 25%-30% of healthy adults and 25% of people in the hospital [“Staph Infection (Staphylococcus aureus)”]. Usually, this bacteria is not harmful when it is outside the body; however, once the bacteria enters the body, disease and infection follows. While skin infections are most commonly caused by S. aureus, this bacteria can also cause pneumonia, food poisoning, toxic shock syndrome, and blood poisoning (“Staphylococcal”). Some symptoms and signs of a localized staph infection include pus, tenderness, pain, redness, swelling, and drainage. In specific cases, staph infections are contagious until they are resolved. Infections could be spread through personal hygiene items like razors and bandages. Certain people, such as newborns, breastfeeding women, and people with chronic conditions such as diabetes, cancer, vascular disease, lung disease, and weakened immune system, are at a greater risk of infection. Depending on the type of staph infection, there are different modes of treatment. Minor skin infections may be treated with antibiotic ointment, while other staph infections may be treated with oral, or IV antibiotics [“Staph Infection (Staphylococcus aureus)”].
Staphylococcus aureus can also cause food poisoning if food is not refrigerated properly or if someone accidently contaminates the food. The incubation period is one to six hours and the symptoms may include nausea, vomiting, diarrhea, loss of appetite, severe abdominal cramps, and fever. Typically, the food poisoning lasts twenty-four to fourty-eight hours. Steps such as washing hands before handling food, keeping kitchens and serving areas sanitized, and avoiding handling food if you have an infection or cut can be taken to prevent food poisoning (United).
Methicillin resistant Staphylococcus aureus (also known as MRSA) is a strain of bacteria that is resistant to antibiotics that are usually used to treat staph infections. There are two main forms, one that is health-care associated, and one that is community associated. MRSA is more difficult to treat than typical staph infections and can affect the bloodstream, lungs, heart, bones, and joints [“Staph Infection (Staphylococcus aureus)”].
There are several ways to reduce one’s risk of developing a staph infection. First and foremost, washing hands with soap and water helps defend against germs and Staphylococcus aureus. Also, one should keep wounds covered and avoid sharing personal items such as razors and towels. For women, it is important to change tampons frequently because S. aureus can cause toxic shock syndrome if tampons are left in for extended periods of time. Lastly, one should wash clothing and sheets in hot water to remove all bacteria (“Staph Infections” Mayo). Staphylococcus aureus is all around us, but with proper knowledge and awareness, disease and infection can be avoided.
McDonald, Virginia, Mary Thoele, Bill Salsgiver, and Susie Gero. Lab Manual for General Microbiology. Saint Louis:STLCC-Meramec, 2011. Print.
“Staph Infection (Staphylococcus Aureus).” Medicinenet.com. Medicinenet, Inc, 4 Mar. 2015. Web. 29 Apr. 2015.
“Staph Infections.” Mayo Clinic. Mayo Foundation, 11 June 2014. Web. 29 Apr. 2015.
“Staphylococcal Infections.” MedlinePlus. U.S. National Library of Medicine, 2 Oct. 2014. Web. 29 Apr. 2015.
United States. U.S. Dept. of Health and Human Services. “Staphylococcus.” Foodsafety.gov. n.d. Web. 30 Apr. 2015.