New York City College of Technology

Identification of Unknown Bacteria

in Mixed Culture

Maria Alba

BIO 3302-E178

Prof. Haque

05/14/2018

Introduction:

Microbiology is the study of microorganisms; Bacteriology, a branch of microbiology specializes in the study of bacteria. The purpose of this experiment is to apply the knowledge gained during the semester in Microbiology lab, to identify the bacteria in a mixed culture. Each student was given a mixture of two different bacteria inside. The tube used in this experiment was number seventeen. Inside the tube was one gram positive and one gram negative organism. The organisms we learned during the semester were Bacillus subtilis, Clostridium sporogenes, Enterobacter aerogenes, Enterococcus faecalis, Escherichia coli, Micrococcus luteus, Mycobacteria smegmatis, Pseudomonas aeruginosa, Proteus vulgaris, Serratia marcescens, Staphylococcus aureus, and Staphylococcus epidermis.

Materials and Methods:

The first step in determining the unknown bacteria was the Gram stain. Though my staining was not properly done, and some stain was left on the slide; After looking under the microscope I was able to see some pink and some purple cells; this helped me determine that unknown #17 contains both Gram Positive and gram-negative bacteria with a cocci and rod morphology that can be seen in clusters or in chains. After the Gram stain, the next step in identifying the unknown bacteria was the use of selective media. For this part I used the Phenylethyl Alcohol Agar dish, MacConkey Agar Dish, PEA, and the Blood Agar dish as well as the Nitrate reduction test and TSI slant.

Results:

To further confirm the presence of a Gram-Positive bacterium I used the PEA dish which as seen in figure 4 shows the growth of colonies; This medium inhibits the growth of Gram Negative bacteria and allow Gram Positive bacteria, further confirming the presence of a Gram-Positive bacterium. To then identify the Gram-positive bacteria, I used the Mannitol Salt Agar dish which as shown in figures 2 and 3 show growth of colonies with yellow spots; This agar helped me narrow down my research to Staphylococcus aureus and Streptococcus faecalis. The last determining factor was the use of the Blood Agar dish. Figure 6 shows Beta Hemolysis which is a reaction caused by Staphylococcus aureus.

To help me further confirm the presence of Gram negative bacterium MacConkey agar; the results of the MacConkey agar show certain growth of bacteria in pink, as seen in figure 6; This helped further confirmed the presence of a Gram-Negative bacteria, as this agar inhibits the growth of Gram Positive bacteria while allowing the growth of Gram Negative Bacteria, the presence of pink in the dish helped me narrow down my research to Escherichia coli. To further confirm this hypothesis, I used the Nitrate reducing test this test helps us identify bacteria such as Pseudomonas aeruginosa and other Gram – enteric bacteria; the results for this test was positive as shown in figure 7, a red color is present which confirms the reduction of nitrate. To differentiate between these two, I used a TSI slant, as shown in figure 8 the results for this test were acid production with an acid butt, both which are seen in Escherichia coli.

In conclusion the observations provided above helped me identify the unknowns in test tube #17 as Staphylococcus aureus and Escherichia coli.

3

5

7

1

2

6

4

8

1. Gram stain of bacteria under microscope.

2. Unknown bacteria in Mannitol Salt Agar Dish.

3. Unknown bacteria in Mannitol Salt Agar Dish.

4. Unknown bacteria in PEA Dish.

5. Unknown bacteria in Blood Agar dish.

6. Unknown bacteria in MacConkey agar Dish.

7. Unknown bacteria in TSI slant.

8. Unknown bacteria in Nitrate.

Bibliography

1) Obenauf, S. and Finazzo, S. (2013). Microbiology fundamentals. New York: McGraw Hill.