UNKNOWN LAB REPORT
Unknown Number 110
Penny Pitman
General Microbiology BIO 203
Spring 2014
INTRODUCTION
It is essential in medicine to be able to identify different microorganisms for diagnosing the cause of various diseases and determining the action for treatment. This study was done using the methods of sterile technique and biochemical tests that have been practiced throughout the semester in the microbiology laboratory. The purpose of this study was to identify the two different bacterium from a mixed culture, one Gram-positive and one Gram-negative.
MATERIALS AND METHODS
The lab instructor gave out a test tube labeled number 110 with two unknown bacterium. The sterile techniques and methods were followed using the laboratory manual by McDonald et. Al (2). The first procedure performed was applying a series of streaks onto a nutrient agar with a sterile inoculating loop using the quadrant streak method and placing it in the incubator at 37 degrees Celsius to grow for two days. The plate was studied and one distinct colony was identified. A Gram stain was performed on the isolated colony. The Gram stain procedure was carefully followed according to the steps in the laboratory manual. Gram-positive purple cocci bacteria were identified using a microscope. The Gram-negative were unable to be isolated from the nutrient agar.
In order to get the Gram-negative to grow, another test was performed taking a sample from the original tube using the sterile inoculating loop and streaking a MacConkey agar placing it in the incubator at 37 degrees Celsius. All plates were labeled, and tests were noted in the journal. The Gram-negative bacterium was still not growing on MacConkey agar so another inoculation was done using an EMB agar. Still unable to get results using the EMB agar, the lab instructor gave an isolated tube labeled alternate A5.
Tables 1 and 2 list the tests, purpose, reagents, observations, and results for each bacterium. All of the following tests were performed on these unknowns:
Gram Positive
- Gram Stain
- Urea
- Catalase
- Nitrate
Gram Negative
- MacConkey
- EMB
- Nitrate
- Indole
- Urea
RESULTS
The unknown number 110 was streaked on a nutrient agar plate, and a Gram stain was performed. The results were Gram-positive cocci. The Gram stain for Gram-negative was taken from alternate A5 with the results of Gram-negative rods. Tables 1 and 2 list all of the biochemical test, their purpose, and their results. The results are also shown in flow chart form.
TABLE 1 GRAM POSITIVE TESTS
| TEST |
PURPOSE |
REAGENTS |
OBSERVATIONS |
RESULTS |
| Gram Stain | To determine the gram reaction of the bacterium | Crystal violet, Iodine, Decolorizer, Safranin | Purple clusters | 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 | H202 | No Bubbles present | Negative Catalase test |
| Nitrate | To test for nitrate reductases, from nitrates to nitrites | Reagents A&B Zinc | No color change after reagents A&B or Zinc | Negative Nitrate test |
TABLE 2 GRAM NEGATIVE TESTS
| TEST |
PURPOSE |
REAGENTS |
OBSERVATIONS |
RESULTS |
| MacConkey | To select for enteric bacteria and inhibit gram+ growth | PH indicator Neutral Red | No color changeNo growth | Negative MacConkey test |
| EMB | To select for enteric bacteria and inhibit gram+ growth | Dyes Eosin & Methylene blue | No growthOr color change | Negative EMB test |
| Nitrate | To test for nitrate reductases, from nitrates to nitrites | Reagents A&B Zinc | Immediate color change after reagents A&B | Positive Nitrate test |
| Indole | To determine the ability of an organism to split indole from tryptophane | Kovacs | No red ring on the top | Negative indole test |
| Urea | To determine if urease hydrolyzes urea | PH indicator phenol red | Broth turned bright pink | Positive Urea Test |
FLOWCHART
UNKNOWN #110
Gram stain
Gram positive cocci
Cocci Rods
Staphylococcus aureus Bacillus subtilis
Staphylococcus epidermidis Bacillus cereus
Enterococcus faecalis
Urea (Negative)
Positive Negative
Staphylococcus epidermidis Staphylococcus aureus
Enterococcus faecalis
Catalase (Negative)
Positive Negative
Staphylococcus aureus Enterococcus faecalis
To Confirm
Nitrate (Negative)
Positive Negative
Staphylococcus aureus Enterococcus faecalis
FLOWCHART
UNKNOWN #110
Gram stain
Gram negative Rod
Nitrate (Positive)
Positive Negative
Klebsiella pneumonia / Enterobacter aerogenes Escherichia Coli
Proteus vulgaris / Pseudomonas aeruginosa
Indole (Negative)
Positive Negative
Proteus vulgaris Enterobacter aerogenes / Klebsiella pneumonia
Pseudomonas aeruginosa
Urea (Positive)
Positive Negative
Klebsiella pneumonia Enterobacter aerogenes
Pseudomonas aeruginosa
DISCUSSION / CONCLUSION
The results of the Gram positive bacterium after all biochemical tests were performed was identified as Enterococcus faecalis. A Gram stain discovered that the bacteria were cocci shaped which narrowed the results down to three different bacteria. The first biochemical test performed was a negative Urea test which left two bacteria. The last test was a negative Catalase test that led to the only Gram negative bacteria left which was E. faecalis. A Nitrate test was performed to confirm the result of E. faecalis. This result was confirmed by Lab instructor. There were no problems encountered with finding this conclusion.
The results for the Gram negative bacterium after tests were performed was identified as Klebsiella pneumonia. There were problems in the beginning getting an isolated culture for the Gram negative bacterium. Due to problems isolating a pure culture on the original nutrient agar, two different agar plates were inoculated using a sterile swab with the original broth, and spread onto the EMB and MacConkey agars. This was to try and get the Gram negative bacterium to grow and inhibit the Gram positive. There was still no growth on both tests. The Lab instructor gave an isolated Gram negative tube labeled alternate A5. A Gram stain was performed and identified as Gram negative rods. The first biochemical test performed was a Simmons Citrate test with a negative result. The lab instructor said that the test result was wrong and to try another test. This led to the Nitrate test with a positive result that narrowed it down to four bacteria. A SIMs test was performed to determine if bacteria can reduce sulfur which produces hydrogen sulfide to determine the ability of an organism to split indole from tryptophan, and to check for motility.(2) The result was negative on all three tests, which left three bacteria. The final test performed was a positive Urea test with the conclusion of K. pneumonia as the Gram negative bacterium. This result was confirmed by the Lab instructor.
K. pneumonia, is a member of the Enterobacteriaceae family that is normally found in the intestinal, respiratory, and urogenital tracts of our body. The bacterium K. pneumonia was named after Edwin Klebs, a 19th century German microbiologist.(3) Common manifestations that occur with an infection in the lungs include flu like symptoms, with fever, cough, and possible thick bloody mucous. Those who are most at risk are the elderly and sick patients within a clinical setting who are receiving treatment for other conditions such as breathing machines, intravenous catheters and those taking antibiotics for an extended period.(1) K. pneumonia has a capsule around the cell surface which provides resistance to many antibiotics and other defense mechanisms. The most effective treatment has been with the use of cephalosporin’s, and aminoglycosides. (3)
REFERENCES
- Klebsiella pneumoniae in Health Care Settings. (2012, August 27). Retrieved April 25, 2014, from Center For Disease Control and Prevention.
- McDonald, Virginia et al. Lab Manual for General Microbiology (BIO 203)
- Obiamiwe Umeh, M., & Editor, C. (2013, May 29). Klebsiella Infections. Retrieved April 27, 2014, from Medscape.
