MICROBIOLOGY UNKNOWN LAB REPORT
Unknown Number 118
Jennifer Mills
April 30, 2013
Microbiology, BIO:203.604
Spring/2013
INTRODUCTION
In the past, it has been vital to distinguish the identities of microorganisms in the world. Knowing their identity has aided in diagnosing numerous diseases and has discovered the most beneficial treatment. The purpose of this study was to identify a Gram-positive and a Gram-negative bacterium from a mixed culture. The methods that were previously studied and practiced in the Microbiology laboratory class were applied in order to identify two unknown bacteria.
MATRIALS AND METHODS
The lab instructor gave out a test tube labeled number 118, which consisted of two unknown bacteria, one Gram-negative and one Gram-positive. Sterile techniques were followed while performing precise instructions as stated in the referenced Laboratory Manual.
The first procedure was performed to isolate a pure culture from the mixture in the test tube onto a solid media. A sterile inoculating loop collected bacteria from the test tube with the unknown, and streaked a series of zigzag lines along two nutrient agar plates, using the Quadrant Streak Method. These plates were incubated for two days to allow the bacteria to grow. Both plates were studied, noting their characteristics, which were recorded in a journal. One distinct colony grew and a Gram stain was performed on the isolated colony. The Gram stain procedure was carefully followed according to the referred Laboratory Manual. Gram-negative rod-shaped bacteria were identified using the microscope. The glass slide and nutrient agar plate were labeled Gram-negative and were then stored in the refrigerator. Gram-positive bacteria did not grow. After determining the Gram-negative reaction, specific tests were performed.
In order to identify the Gram-positive bacteria, a sample from the original test tube was streaked on a Mannitol Salt Agar plate and placed in the incubator at 37 Degrees Celsius. There was only one type of bacteria that grew. This was isolated and a Gram stain was performed. Both of the plates were labeled and stored in the refrigerator. Gram-positive cocci-shaped bacteria were identified using the microscope. Several biochemical tests were chosen based on the identification table which was given by the lab instructor. These tests and results were recorded on the flow chart and the tables on the following pages for the Gram-negative and Gram-positive bacteria.
Tables one and two list the tests, purposes, reagents, observations and results.
All of the following tests were performed on these unknowns:
Gram Positive
- Mannitol Salt Agar
- Urea
- Catalase
Gram Negative
- Simmons Citrate
- Mannitol Salt Agar
- Gelatin
- Galactose
RESULTS
Unknown number 118 was streaked on a nutrient agar plate. A Gram stain was performed. It was determined that it was a Gram-negative rod. Gram-positive did not grow. In order to identify the Gram-positive bacteria, a sample from the original test tube was streaked on an MSA plate. A Gram stain was performed which identified Gram-positive cocci. Table 1 and Table 2 list all of the biochemical tests, purposes, reagents, observations, and results. The results are also displayed in a flowchart.
TABLE 1. Gram Negative (-) Tests
|
TEST |
PURPOSE |
REAGENTS |
OBSERVATIONS |
RESULTS |
|
Gram stain |
To determine the gram reaction of the organism |
Crystal violet, Iodine, Decolorizer, Safranin |
Pink |
Gram-negative rods |
|
Simmons Citrate |
To determine if an organism is able to utilize citrate as carbon source |
Citrate slant (green) |
Color change from green to blue |
Positive Simmons Citrate |
|
Galactose |
To determine the fermentation of galactose |
pH indicator phenol red |
No color change |
Negative galactose fermenter |
|
Mannitol Salt Agar |
To determine if it ferments mannitol |
pH indicator phenol red |
No color change |
Negative mannitol fermenter |
|
Gelatin |
To determine if it hydrolyzes gelatin |
Gelatin tube |
It turned to liquid after refrigeration |
Positive gelatin test |
|
TEST |
PURPOSE |
REAGENTS |
OBSERVATIONS |
RESULTS |
|
Mannitol Salt Agar |
To determine if it ferments mannitol |
pH indicator phenol red |
Medium changed from red to yellow |
Positive Mannitol fermenter |
|
Gram stain |
To determine the gram reaction of the organism |
Crystal violet, Iodine, Decolorizer, Safranin |
Purple cocci |
Gram-positive cocci |
|
Urea |
To determine if urease hydrolyzes urea |
pH indicator phenol red |
No color change |
Negative urea test |
|
Catalase |
To determine if catalase is present |
H2O2 |
Bubbles are present |
Positive catalase test |
DISCUSSION/CONCLUSION
The result of the tests for Gram-negative led to the identification of Pseudomonas aeruginosa. A Gram stain discovered that the bacteria were rod-shaped. A Simmons Citrate test was performed and the positive result narrowed it down to three bacteria. After a Gelatin and a Galactose test were performed, the only bacterium that remained was Pseudomonas aeruginosa. A negative result on an MSA plate verified this result. This was the correct identification because all of the other Gram-negative were eliminated. There were no problems encountered in finding this conclusion.
The result of the tests for Gram-positive led to the identification of Staphylococcus aureus. A sample from the unknown bacteria was streaked on an MSA plate. A Gram stain was performed which verified Gram-positive cocci. This was a positive result for mannitol fermentation which narrowed it down to two bacteria. A negative urea test was performed, which also narrowed it down to the same two bacteria. A positive catalase test verified that the bacteria in the unknown would have to be S. aureus. This was the correct identification because all of the tests performed, identified S. aureus as an unknown Gram-positive bacterium. The only problem I encountered was during the isolation streak, Gram-positive could not be isolated on a nutrient agar plate. However, it did grow on an MSA plate and was isolated on a nutrient agar plate.
S. aureus is a bacterium that is frequently found in the respiratory tract and on the skin. It is a common cause of skin infections, diseases and food poisoning, and is not always pathogenic (Tolan, 2011). Sometimes, disease-associated strains produce toxins that promote serious infections in the body. These toxins have proteins that activate antibodies which cause resistance. This emergence of resistance has led to MRSA (Methicillin Resistant S. aureus), and is a worldwide problem.
S. aureus was first discovered in 1880 by Sir Alexander Ogston, a surgeon in Scotland. He saw this bacterium in pus inside an abscess in a knee joint. Twenty percent of the human population are carriers of S aureus, which can be found on the skin and inside the nasal passages. S. aureus is the most common species to cause Staphylococcus infections. It can cause many illnesses, from minor skin infections to life-threatening diseases such as meningitis, pneumonia, osteomyelitis, endocarditis, toxic shock syndrome, and sepsis (Todar, 2012). It is estimated that some 500,000 patients in American hospitals contract a staphylococcal infection each year. It is often the cause of postsurgical wound infections and one of the five most common causes of nosocomial infections (Todar, 2012).
REFERENCES
- McDonald, Virginia, et al. Lab Manual for General Microbiology (BIO 203)
- Todar, Kenneth. “Son of Citation Machine.” Son of Citation Machine. Todar, 2012. Web. 19 Apr. 2013.
- Tolan, Robert W., MD. “Staphylococcus Aureus Infection.” Staphylococcus Aureus Infection. WebMD LLC., 22 Nov. 2011. Web. 19 Apr. 2013.
