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DISCUSSION/CONCLUSION:

How did the test results lead to the identification?

            Different bacteria can live and flourish in certain environments, while others will die or never grow in the very same environment due to their dissimilar characteristics.  These tests help determine which bacteria can grow in certain situations.  For both bacteria, the Gram stain was a necessity in order to determine if the microbe was Gram Positive or Gram Negative.  Once the Gram Positive bacteria was successfully visible under the microscope, it was clear that it was cocci.  Knowing this information helped to cross two bacteria (both rods) off the unknown list.  From there, the Mannitol test helped narrow down the choices even more.  This particular Gram Positive microbe was able to ferment Mannitol and produce acid giving it a positive result.  This positive result narrowed the choices down from three to two (S. aureus and E. faecalis).  The only test that would allow for differentiation between these two bacteria was the Nitrate reduction test.  This test came back negative, which meant this microbe could not reduce nitrate to nitrite or any other nitrogenous compounds.   Only one Gram Positive bacteria given cannot reduce nitrate, which lead to its identification: E. faecalis.

The other Gram stained slide was viewed under the microscopes and pink/reddish rods were clearly visible making it the Gram negative bacteria.  This Gram stain, however, did not eliminate any of the Gram Negative bacteria because they were all rods.  The first test was the Simmons Citrate test.  This test determines whether or not a microbe can use citrate as its sole carbon source.  It was a positive result, meaning it could in fact use citrate as its only carbon source.  This test narrowed down the choices from five to three bacteria (K. pneumoniae, E. aerogenes, and P. aeruginosa). The Urea test was then conducted.  This test is used to identify bacteria capable of hydrolyzing urea using the enzyme urease.  The test came back negative, which helped to eliminate K. pneumoniae, since that particular bacteria produces urease. Casein was the last test used in order to identify the Gram Negative bacteria. After incubation, there was a clearing in the milk agar, which proved that this bacteria could hydrolyze the milk protein casein using the enzyme casease.  The only bacteria that was able to do that was P. aeruginosa.

 




Was it the correct identification?

            After showing the professor the results that lead to the identification of these bacteria (E. faecalis and P. aeruginosa), it was confirmed the correct microbes were found.

What problems were encountered?

            The only problem that was encountered was during the original streak plate.  After two days of incubation, it was hard to isolate two different bacteria into two separate nutrient agar plates due to overgrowth.  In order to avoid contamination or future problems, another streak plate was done and more carefully.  After the second try, two separate bacteria were growing and it was possible to successfully isolate them both.

Pseudomonas aeruginosa

Pseudomonas aeruginosa wasn’t successfully isolated until 1882.  In a publication entitled when “On the Blue and Green Coloration of Bandages”, Carle Gessard reported the growth of the organism from cutaneous wounds of two patients with bluish-green pus (3).  P. aeruginosa is a gram negative, rod shaped bacteria.  It is a strictly aerobic microbe, which means it needs oxygen to grow and survive.  It is noted for its ability to live in versatile environments, cause nosocomial infections in susceptible individuals and it’s resistance against antibiotics.

P. aeruginosa is an important microbe to be aware of because it has an ability to survive with minimal nutrients and can tolerate a variety of physical conditions. This allows P. aeruginosa to be prevalent in both hospital settings, as well as, in our overall community. While in the hospital, P. aeruginosa can be found on a variety of equipment.  Such equipment includes: respiratory machines, antiseptics, soaps, mops, etc. (3).  While out in the community, P. aeruginosa can be found in swimming pools, hot tubs, humidifiers, and even vegetables. While P. aeruginosa is still prevalent in our community, it is in hospitals that this bacteria can cause major infection.  It is considered an opportunistic pathogen, which means it usually does not pose a problem, however, it can become pathogenic.  When a person enters a hospital, it is normal for their immune system to be compromised.  When an individual becomes immunocompromised their ability to fight off an opportunistic pathogen like P. aeruginosa becomes more difficult.

While P. aeruginosa normally does not cause illness in a healthy person, it can cause major problems for an individual in the hospital with a compromised immune system.  It is the most common pathogen isolated from patients who have been hospitalized longer than 1 week (4). Some common nosocomial infections caused by P. aeruginosa include: pneumonia, urinary tract infections, blood stream infections, as well as, infection of surgical sites.  Nosocomial P. aeruginosa infections can be dangerous because the individual is already under either physical or mental stress (or both), but another frightening fact is that P. aeruginosa is resistant to many antibiotics.

P. aeruginosa can develop resistance to antibiotics, either through the acquisition of resistance genes on mobile genetic elements (i.e., plasmids) or through mutational processes that alter the expression and/or function of chromosomally encoded mechanisms. (3) While these drug resistant abilities are positive aspects for P. aeruginosa, it is very dangerous for individuals fighting infection. The drug resistance makes treating this pathogen even more difficult and vastly limits avenues in which doctors can take to help treat patients with this infection.  The question then becomes, if there is an overuse of the antibiotics that do work, will P. aeruginosa become resistant to those too?

REFERENCES:

(1) McDonald, Virginia (et al.). Lab Manuel for General Microbiology. Saint Louis Community College

at Meramec. 2011

(2) Lab Review for Practical Microbiology.

(3) Lister, Philip D. (et al). “Antibacterial-Resistant Pseudomonas aeruginosa: Clinical Impact and

Complex Regulation of Chromosomally Encoded Resistance Mechanisms.”

www.ncbi.nlm.gov/pmc/articles. American Society of Microbiology. 2009. April 28, 2015.

(4) Lessnau, Dieter-Klaus, MD. “Pseudomonas aeruginosa Infections.”

www.emedicine.medscape.com/article/226748. WebMD LLC. 2014. April 28, 2015.

UNKNOWN LAB REPORT

Unknown Number 120, Alternate 3

Emily Specter

May 5, 2015

Professor Jay Snaric

Introduction to Microbiology

Spring 2015

INTRODUCTION

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

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

 




RESULTS

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

Biochemical Tests – Gram Negative

FLOW CHART – Removed due to formatting issues.

DISCUSSION/CONCLUSION

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.

Works Cited

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.

Microbiology

Unknown Lab Report

Kelly Downar

Spring 2015

Introduction

Identifying a specific bacterium from a wide variety of bacteria is important in trying to help a sick patient take the correct preventative measures needed. It is essential in knowing the right bacteria causing the problem, so that the person can be treated in the correct way and the correct antibiotics are administered. In this particular study an unknown was given by the professor. The procedures taken to figure out, and isolate the two separate bacteria were taught thus far in Microbiology lab class. Doing specific tests on the two bacteria’s will give you the ultimate answer at the end as to what the bacteria is, so correct measures can be taken. Once the correct identity of the bacteria is know, more information on the specific bacteria can take place.

 




Materials and Methods

Mr. Snaric gave each student a specific unknown labeled with different numbers on them that contained both specimen. Unknown labeled 104 was given to me in lab by the instructor. Numbers of different methods were used to identify the specific bacteria given. The procedures following the various different methods can be found in our lab manual.

The first procedure that took place was to take the mixed culture of bacteria, and do a streak plate. The streak plate is done on a nutrient agar plate to try and isolate a pure culture of each unknown. (pg.10 Lab Manual) Alternate number 4 and 6 were given by the professor due to overgrowth on the original streak plate. Separate streak plates are done on the nutrient agar using the quadrant streak method. This would get the bacteria to grow as isolated pure colonies in order to run tests on them.

Once growth appeared for my alternates it was then time to figure out which alternate number was gram negative, and which was gram positive. In order to figure this out you have to do a Gram stain. This is where a small scrap of each sample of bacteria, is placed on the slide using all the specific dyes. The dyes used in order are Gram Crystal Violet, Gram Iodine, Gram Decolorizer, and Gram Safarin. The exact procedure followed is in the lab manual. (pg.67 Lab Manual) Once this is done the microscope is used to identify the color and shape, which will tell  whether the bacteria is gram positive or negative.

Each bacterium is now isolated and alternate number 4 is gram negative, and other tests are now needed to identify the exact bacteria.  The next procedure performed on alternate 4 was the Simmons Citrate. A citrate agar slant is inoculated with the bacteria on the surface of the test tube. Once that is done it is inoculated, and the results are checked the next day. This is used to determine if the bacteria that produce the enzyme citrase breakdown citrate. (pg.36 Lab Manual)  The next procedure performed on alternate 4 was the Urea test. For this urea tubes are used that contain a broth known as urea. The citrate agar slants is inoculated, and then incubate it. If the ph indictor turns a bright magenta color then it indicates the breakdown of urea by the particular bacteria. (pg.38 Lab Manual) The last procedure was the indole and Hydrogen Sulfide test. This was done with the Sim tube that is a liquid medium. The Sim tube is inoculated, and then incubated.  The “I” in Sim stands for indole. To test for indole you add Kovac’s reactant as stated in the lab procedure. This reactant brings indole to the top of the test tube. (pg.36 Lab Manual)  The “S” in Sim stands for sulfur reduction. The Sim tube is inoculated, and then incubated to find the results a day later. These tests gave the answer to which bacteria alternate number 4 was.

The other alternate given was number 8. From doing the Gram stain it was found that is it positive, and is rod shaped. For the gram stain you follow the procedures in the lab manual , and then look at the results under the microscope.(pg.67 Lab Manual) Knowing that it gave two options of bacteria to go with, and run tests on. The first procedure was the Casein, and a milk agar was used for this. It is to be inoculate with the bacteria, and then incubated. Clearing around the inoculated area was to be observed, as stated in the lab manual. (pg.28 Lab Manual) The next procedure was Methyl Red which the MR-VP test tubes are used. It is a broth based test tube that essential is used to test whether or not glucose is fermented. (pg.34 Lab Manual)  These were the only procedures done for this because I had it narrowed down to two from the beginning, so not as many test needed to be run. This gave the answer to alternative number 8.

All the following tests were performed on Alternate 4:

• Simmons Citrate
• Urea
• Indole
• Hydrogen Sulfide

All the following tests were performed on Alternate 8:

• Casein
• Methyl red

Results

Simmion Citrate test- Purpose: Is the ammonium phosphate in the test tube broken down? – If so then it produces a blue color on the surface, otherwise if not it stays its normal green color.

Urea test- Purpose: Does the enzyme urease break down urea? If so then the broth turns magenta pink, otherwise it stays the same color it normally is.

Indole test- Purpose: Does the enzyme trytophanase degrade the amino acid tryptophan into indole? If so then when the Kovac’s Reagent is added it will bring the indole to the surface of the test tube producing a red color making the result positive, otherwise no red color is produced.

Hydrogen Sulfide test- Purpose: Does the hydrogen sulfide react with ferrous sulfate to produce a black precipitate? If black is produced in the tube then the test would be positive, otherwise if not then a negative result.

Casein test- Purpose: Does the bacteria produce the enzyme casease which hydrolyzes the milk protein casein? If so and there is clearing around the bacteria then it indicates a positive result, otherwise would be negative.

Methyl Red- Purpose: Is a mixture of acids produced as a result of glucose fermentation? If so the broth will turn red indicating a positive result, otherwise it will be a negative result.

 




Discussion/ Conclusion

The only problem encountered was in the beginning when given the initial number 104. When a streak plate was done on 104 to try and isolate the two different bacteria’s there was always overgrowth. They were never able to isolate on their own, so two different alternate numbers were given by the professor.

Alternate number 4 from doing a Gram stain was figured out to be gram negative rods. First the Simmons Citrate test was performed it did not turn blue on the surface, so I knew the results were negative. The citrate test tube stayed it’s normal green color. Next was the Urea test, which came back negative because it didn’t turn the bright magenta color. The urea stayed the color it normally is in the test tube. With those two tests being done it narrowed it down to two options, which were Proteus vulgaris and Escherichia Coli. Next were the Hydrogen sulfide and indole test. The indole test gave me a positive result because the surface of the test tube turned red. The Hydrogen sulfide test on the other hand was negative because it didn’t turn the test tube a black color. Proteus vulgaris would be positive for both urea and hydrogen sulfide. Leaving to believe that alternate number 4 gram negative rod is E.coli.

Alternate number 8 when a Gram stain was done came out to be gram positive rod. That being known it only left two options Bacillus cereus or Bacillus subtilis. The first test was the Casein , which came out to be a positive result because there was clearing around the bacteria. Next was the Methyl Red test this also gave a positive result showing that it does produce a mixture of acids as a result of glucose fermentation, which turns the broth a red color. This concluded that alternate number 8 gram positive rod is Bacillus cereus because both these tests were positive. If both the test were negative then it would have been Bacillus subtilis.

Background Information on Alternate number 8

Bacillus cereus is a large gram positive, rod shaped bacteria that produce toxins and is capable of producing endospores. “B. cereus is mesophilic, growing optimally at temperatures between 20°C and 40°C, and is capable of adapting to a wide range of environmental conditions”, as stated in Bacillus cereus article. There are two illnesses that this particular bacterium can cause. “The first illness gives you diarrhea, whereas the second one gives you vomiting on top of diarrhea”, as stated in Bacillus cereus. The duration of this illness can last up to 24 hours at the most. As in most illness you are to get rest and drink lots of fluids, just like it is essential to do with this illness. This bacterium is found in many foods especially certain foods that sit out for long periods of time at room temperature. Their main habitat is soil. “Many sources of outbreak for B. cereus is said to be rice, meat loaf, turkey loaf, and many vegetables.” (Bacillus Species)