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Microbiology Unknown Lab Report
by Courtney Wiedemer
Throughout the duration of the microbiology class, many techniques were taught for culturing, transferring, viewing, and testing metabolic capabilities of microorganisms. The techniques were taught to allow students to be able to correctly identify an unknown microorganism by applying the skills learned throughout the semester. The capability of being able to identify an unknown bacterium allows for many advances in healthcare. It allows doctors to be able to identify the pathogen causing a specific disease within a patient, how to treat the patient, and which antibiotics the microorganism is most susceptible to.
MATERIALS AND METHODS
A test tube filled with broth was handed out by the laboratory instructor. The tube was labeled 101 and contained a mixed culture of two unknown bacterium to be identified; one Gram-positive and one Gram-negative bacterium. The techniques taught during the course were applied to the identifying of the two unknown bacterium. The procedures ran during the course of identification of the two unknowns were followed according to the directions described within the reference course laboratory manual.
The first procedure needed to be done was to streak the unknown onto a Nutrient Agar plate, using the streak method discussed in the lab manual. This procedure needed to be done to grow the unknowns on a solid medium. The plate was incubated for 48 hours. The morphology of the colonies grown on the plate was observed and recorded. At this point, the mixed culture of the unknowns was streaked onto a Mannitol Salt Agar, which inhibits the growth of Gram-negative bacteria, and MacConkey agar, which inhibits the growth of Gram-positive bacteria. The streak method used was the isolation method described in the lab manual. The plates were incubated for 48 hours. After 48 hours the morphology of the colonies grown on each plate was observed and recorded. A Gram stain was then performed for each plate to confirm the correct bacteria grew on the plates. Further isolation needed to occur; there for a Nutrient Agar plate was streaked, using the isolation streak method in the lab manual, of each bacterium. The plates were then incubated. Gram stains were then performed to confirm no contamination. Specific biological tests were then performed. The biological tests were chosen from the table given to the students from the lab instructor. The tests were chosen on the basis of splitting the Gram-positive and Gram-negative bacteria groups in halves until a single microorganism was identified and confirmed. The biochemical tests were performed by the methods discussed in the lab manual.
All of the following tests were performed during the identification of the unknowns:
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- Methyl Red
- MacConkey Agar
- Mannitol Salt Agar
Table 1 lists the test, purpose, reagents and results for the Gram-positive bacteria. Table 2 lists the test, purpose, reagents and results for the Gram-negative bacteria. Chart 1 is a flow chart depicting the tests and in what order they were performed during identification of the Gram-positive unknown. Chart 2 is a flow chart depicting the tests and in what order they were performed during the identification of the Gram-negative unknown.
Table 1 ( Staphylococcus epidermidis)
|Mannitol Salt Agar||Inhibit the growth of Gram-negative bacteria and identify if bacteria ferments mannitol||Mnitol||Medium turned yellow; positive result for mannitol fermentation|
|Methyl Red||Identify if bacteria ferments glucose and produces a mixture of acids||Methyl red|
GlucoseBroth turned red; positive result for glucose fermentation and mixture of acidsCatalaseDetect the production of the enzyme catalaseHydrogen peroxideColony bubbled; positive result for catalaseUreaDetect the presence of ureasePhenol redBroth turned pink; positive result for urease
Table 2 ( Pseudomonas aeruginosa)
|MacConkey agar||Inhibits the growth of Gram-positive bacteria and identify if bacteria ferments lactose||Neutral red||Colonies were colorless; negative for lactose fermentation|
|Urea||Detect the presence of urease||Phenol red||Broth did not turn pink; negative for urease|
|Voges-Proskauer||Detect production of acetoin from glucose fermentation||Barritt’s A and B||Broth was green, did not turn pink; test was negative for glucose fermentation|
|Nitrate||Detect production of nitrate reductases and the reduction of nitrates to nitrites||Reagent A and B||Broth turned red after addition of A and B; positive reaction for nitrates to nitrites|
|Oxidase||Detect the production of the enzyme oxidase||Oxidase reagent||Bacteria turned purple within 30 seconds; positive for oxidase|
Chart 1 (Staphylococcus epidermidis)
Gram positive cocci
Bacillus cereus Enterococcus faecalis
Methyl Red (positive)
Bacillus subtilis Bacillus cereus
Bacillus cereus Staphylococcus epidermidis
Chart 2( Pseudomonas aeruginosa)
Gram negative rod
Klebsiella pneumonia Escherichia coli
Enterobacter aergenes Proteus vulgaris
Proteus vulgaris Escherichia coli
Proteus vulgaris Pseudomonas aeruginosa
After several differential tests, it was determined that unknown 101 contained Pseudomonas aeruginosa and Staphylococcus epidermidis. After isolating two separate pure cultures, a Gram stain was performed, determining one bacterium to be a Gram-negative rod and the other to be Gram-positive cocci. The organisms were grown on Nutrient Agar plates for the use in inoculating the biochemical tests used to determine the identity of the unknowns. All of the biochemical tests were performed as directed in the lab manual.
The tests ran on the Gram-negative unknown (Pseudomonas aeruginosa) were as followed: MacConkey Agar, Voges-Proskauer, Oxidase, Nitrate, and Urea. The observations after incubation on the MacConkey Agar showed colorless colonies, which indicated the microorganism does not ferment lactose. The proceeding tests were ran to split the Gram-negative bacteria into equal halves. Voges-Proskauer was the first biochemical test ran; the results were negative for glucose fermentation. The nitrate test followed; the results were positive for the reduction of nitrites to nitrates. Elimination continued with the Urea test, which the result was negative for urease. These tests eliminated the bacteria until Pseudomonas aeruginosa was the only microorganism left. An Oxidase test was then ran to confirm the bacteria. The test was positive for oxidase, therefor concluded that the unknown as Pseudomonas aeruginosa. The lab instructor confirmed this was the correct Gram-negative microorganism for the mixed culture of unknown 101. Table 2 contains the tests and the recorded results for Pseudomonas aeruginosa.
The tests ran on the Gram-positive unknown (Staphylococcus epidermidis) were as followed: Mannitol Salt Agar, Methyl Red, Catalase, and Urea. The observations after the incubation on the Mannitol Salt Agar concluded that the bacterium ferments mannitol. The tests that followed were run to eliminate Gram-positive bacteria until there was only one left. The Catalase test was the first test ran; the result was positive for catalase. This concluded that the Gram-positive unknown was Staphylococcus aureus. The lab instructor identified Staphylococcus aureus as the wrong Gram-positive unknown. The lab instructor suggested that the Mannitol Salt Agar gave a false positive and that other tests should be ran. Methyl Red was the next biochemical test to be done, which the result was positive for glucose fermentation and mixed acids. The Urea test was the final test. The results were positive for urease, which eliminated all but Staphylococcus epidermidis as the Gram-positive unknown microorganism for the mixed culture of unknown 101. The lab instructor confirmed this was correct. Table 1 contains the test and the recorded results for Staphylococcus epidermidis.
Staphylococcus epidermidis belongs to the genus Staphylococcus. It is typically found colonizing the skin and mucosa of humans. S. epidermidis is part of the humans’ normal flora. Most strains of S. epidermidis are nonpathogenic, but can be pathogenic in the hospital environment. S. epidermidis is a facultative anaerobe. It is mainly spread by skin to skin contact or medical instruments during procedures. S. epidermidis causes infections such as meningitis, urinary tract infection, conjunctivitis, and endocarditis. People who have compromised immune systems or internal prosthetic devices are more at risk to developing infections caused by S. epidermidis. The most potent antibiotics used in treating S. epidermidis are vancomycin, linezolid, daptomycin, gentamicin and rifampin. If the infection is caused due to the prosthetic devices, they must be removed and replaced.
“Staphylococcus Epidermidis â Coagolase Negative Staphylococci.” Staphylococcus Epidermidis. N.p., n.d. Web. 05 Dec. 2012.
McDonald, Virginia, Mary Thoele, Bill Salsgiver, and Susie Gero. Lab Manual for General Microbiology.