Identification of Human Streptococcal Pathogens

Background

This laboratory exercise illustrates the medical significance of streptococci. The Bile Esculin test is used to isolate and identify bacteria that are able to hydrolyze esculin in the presence of bile. The test is commonly used to identify Group D streptococci and enterococci which produce a positive test result (Cappuccino 441).

The bile esculin agar is a selective and differential medium containing bile salts that inhibit the growth of Gram-positive bacteria with the exception of Group D streptococci and enterococci. The bile esculin agar media also contains nutrients, esculin, and ferric citrate. When an organism hydrolyzes esculin, the media turns dark brown or black after the incubation period. This hydrolysis of esculin produces esculetin and dextrose. The dark brown or black color indicates the presence of phenolic iron complex which is the product of the reaction of esculetin and ferric citrate (austincc.edu).

Group D streptococci are associated with urinary tract infections, bacteremia, and endocarditis (Patterson).   Group D streptococci and enterococci include E. faecalis, E. faecium, and S. bovis (austincc.edu)

Materials and Methods

The materials for this laboratory experiment include: Bile Esculin agar slants, test tubes, sterile water, sterile cotton swabs, Bunsen burner, inoculating loop, adhesive tape, marking pen.

The procedure consists of the following steps.

Using the adhesive tape and marking pen, identify one Bile Esculin slant as “Throat” and the other slant as “Hand”.

Use a glassware making pen to label “Throat” a test tube containing sterile water. Use a sterile cotton swab to rub the back of the throat of a test subject to obtain a specimen of the organisms living in that area. Inoculate the “Throat” test tube with the specimen to obtain a uniform suspension.

Use a sterile inoculating loop to transfer a sample of the “Throat” specimen to the Bile Esculin slant labeled “Throat”.

Use a glassware marking pen to label “Palm” a test tube containing sterile water. Use a sterile cotton swab moistened with sterile water to rub the palm of the hand of a test subject to obtain a specimen of the organisms living in that area. Inoculate the “Palm” test tube with the specimen to obtain a uniform suspension.

Use a sterile inoculating loop to transfer a sample of the “Palm” specimen to the Bile Esculin slant labeled “Palm”.

Both slants are to be at 37 degrees Celsius for a period of 48 hours.

After the incubation period is completed, remove both slants from the incubator.

Results

The slant labeled “Throat” showed a positive Bile Esculin test result indicated by a black color.  The slant labeled “Palm” showed a negative Bile Esculin test result indicated by a yellow color.  Figure 1.1 shows the results of both tests.

Figure 1.1 Bile Esculin slants showing results for “Throat” and “Palm” cultures

Conclusions

The test result of the Bile Esculin test for the “Throat” culture indicates the presence of Group D streptococcus or enterococcus. Further tests, such as Pyrrolidonyl Arylamidase (PYR) Differential should be conducted to determine if the organisms are genus Enterococci (PYR positive) or Group D Streptococci(PYR negative) (medialabinc.net).

It also would be interesting to examine the results of culturing the organisms on blood agar to determine the type of hemolytic reaction. Group D streptococci usually show alpha or gamma hemolysis.

 

The test result of the Bile Esculin test for the “Palm” culture indicates the absence of Group D streptococcus or enterococcus.

Review Questions

1. What is the mechanism of the Bile Esculin test?
   Bile Esculin agar contains bile salts that inhibit the growth of most gram positive organisms. Enterococci and Group D streptococci are not inhibited. The agar also contains nutrients, esculin, and ferric citrate. Some organisms are able to hydrolyze esculin into esculetin and dextrose. When esculetin reacts with the ferric citrate, a dark brown or black iron complex is formed (austincc.edu).
2. Why is it important medically to distinguish between the enterococci and non-enterococci?Organisms belonging to the genus Enterococcus are responsible for several opportunistic infections in humans. “The enterococci have become the second most common bacterium isolated from nosocomial urinary and wound infections, and the third most common cause of nosocomial bacteremia” (Kaiser).The organisms of the genus Enterococcus have become the source of a large number of nosocomial infections. In addition, the organisms have developed resistance to multiple antibiotic drugs (Palmer).
3. Why can some streptococci produce secondary sites of infection?Some streptococci produce secondary sites of infection when they invade the blood (bacteremia) and travel to their preferred secondary sites of infection (e.g., cerebrospinal fluid, heart valves, and joint spaces). The mechanism for blood invasion can occur via the alveoli in the lungs (Todar).
4. The streptococci are known to be fastidious organisms that require an enriched medium for growth. How would you account for the fact that a medium enriched with blood (blood agar) is the medium of preference for growth of these organisms?The nutritional requirements of fastidious organisms are complex. They need specific nutrients in their diet. Blood agar is a medium rich in nutrients (Lammert 168).

 

 

Works Cited

Austincc.edu. “Bile Esculin Test.” Austincc.edu. Austin Community College, n.d. Web. 5 April 2014.

Cappuccino, James G. Microbiology: a laboratory manual – 10^th ed. Glenview, IL: Pearson, 2014. Print.

Kaiser, Gary. “Isolation and identification of streptococci and enterococci.” ccbcmd.edu. 2012. Web. 5 April 2014

Lammert, John. Techniques in Microbiology. San Francisco, CA: Pearson Education Inc., 2007. Print.

Media Lab Incorporated. “Case Studies in Clinical Microbiology.” medialabinc.net. n.d. Web. 5 April 2014.

Palmer, Kelli, et. al. “Comparative Genomics of Enterococci.” asm.org. American Society for Microbiology. 21 February 2012. Web. 5 April 2014.

Patterson, Maria Jevitz. “Chapter 13 Streptococcus.” nih.gov. National Center for Biotechnology Information, 1996. Web. 5 April 2014.

Todar, Kenneth. “Online Textbook of Bacteriology.” textbookofbacteriology.net. n.d. Web. 5 April 2014

Last updated on 7-April-2014 at 2:42 PM