Enterococcus faecalis (E. faecalis) | Unknown Micro Lab Report

 UNKNOWN LAB REPORT

Rio Hall

Microbiology   BIO:203.604

Spring Semester/2013

INTRODUCTION

The ability to determine an unknown microorganism and isolate more than one unknown microorganism from a mixed culture is beneficial to humankind for multiple reasons.  Predominantly, healthcare professionals need to ascertain the specific cause of an illness in order to be able to properly treat it with the most effective antibiotic.  Microorganisms can make people ill, however, most of the microorganisms on Earth are beneficial and provide symbiotic relationships that are essential to the ecosystems on the planet.  A few examples of advantageous microbial use are: waste management (i.e. specific organisms, like Alcanivorax (1) that can eat away an oil spill such as the one we had in the Gulf in 2010), food supply (2), and chemical uses, such as making polymers from genetically-modified Escherichia coli (3).  This study was carried out by application of procedures learned in the microbiology laboratory course for the purpose of the identification of two unknown bacteria.

 




MATERIALS AND METHODS

An unknown, labeled as number 108, was given out by the lab instructor.  Procedures followed used previous lab exercises from the course laboratory manual by McDonald et al. (4), unless otherwise noted, to discern the metabolic characteristics of the bacteria.

The first procedure was to streak a nutrient agar plate with mixed culture # 108.  After Gram stains were performed, a mixed culture still existed, so a second nutrient agar plate was streaked with the mixed culture.  Upon the second attempt, a second Gram stain was done and the two bacteria were still not isolated.  The mixed culture was incubated at 35 degrees Celsius on MacConkey and EMB agar in order to inhibit the growth of the Gram-positive organism.  The growth on the MacConkey and EMB plates were Gram stained as a mixed culture.

Since an isolated culture had not yet been achieved, a third isolation streak was performed from the mixed culture onto a nutrient agar plate; this time carefully streaking the bacteria into four quadrants attempting to procure an isolated colony.  Finally, Gram stains showed two distinct colonies: Gram-positive cocci and Gram-negative rods; the unique colonies were grown in the incubator on separate nutrient agar plates and Gram stained to verify isolation.

The Gram positive cocci were grown on a Mannitol Salt Agar plate and displayed a positive result for mannitol fermentation by turning the phenol red in the medium to yellow.  This step narrowed down the unidentified Gram positive bacteria from five choices to two.  A final Blood Agar test displayed a negative result by showing partial to zero hemolysis of red blood cells indicating that the Gram positive unknown was Enterococcus faecalis.

Simmons Citrate was inoculated by a single stab with the Gram negative rods and showed a positive result by the medium turning from green to blue.  Two Gram negative bacteria were eliminated using this step.  Next, a Methyl Red test was completed and yielded a negative result, thereby eliminating yet another organism.  The final determinant was a Voges Proskauer test, which had a negative result as indicated by the tube turning yellow not red.  The Gram negative bacteria is Pseudomonas aeruginosa.

All of the following tests were performed on this unknown:

1. Simmons Citrate
2. Methyl Red
3. Voges Proskauer
4. Mannitol Salt Agar
5. Blood Agar

• MacConkey and EMB tests were also performed, but did not prove to be determinative.

RESULTS

TABLE 1: Physiological and Biochemical Results