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Staphylococcus epidermidis | Microbiology Lab Report

By at January 19, 2014 | 9:14 am | Print

 UNKNOWN LAB REPORT

Unknown Number 108

Caroline Coley

Microbiology

Fall/2013

 

INTRODUCTION 

Microorganisms exist everywhere in nature.  Bacteria, viruses and fungi have been a leading cause of death in the history of mankind.  They have contributed to the shaping of societies and world history.  The relationship between microbes and the human body is a very important topic to study.  These microorganisms affect us in both a beneficial and harmful way and it is necessary to understand how this works.  This study used the techniques and procedures outlined in the MacDonald et al. Lab Manual (1) to identify unknown bacteria.




MATERIALS AND METHODS:

The lab instructor distributed a test tube of unknown bacteria labeled 108.  The instructor informed students that each tube contained a Gram positive and Gram Negative bacteria.  Methods from the McDonald et al. Lab Manual (1) have been used to identify the unknown bacteria.  The first procedure accomplished was an isolation streak on nutrient agar.  This isolation streak was an attempt to isolate the two separate bacterial colonies so further tests could be accomplished for identification.  The four-quadrant streak method described in the lab manual was used for this procedure.  Once the streak method had been performed, the nutrient agar was incubated at 37 degrees Celsius for 48 hours.  After incubation, the nutrient agar plate was examined to determine if isolated growth had been obtained (1).

Once examined, it was determined that only one species of bacteria had been isolated.  The appearance of the growing colony was a cloudy, milky white.  This growth was then used to inoculate a new nutrient agar plate with the streak method to ensure isolation of one bacterium.  This nutrient agar plate was placed in the incubator at 37 degrees Celsius for five days.

After five days of incubation, the isolation streak plate was determined to be growing the same cloudy, milky white bacteria as the sample it was retrieved from.  This bacterium was then Gram stained using the procedures from the McDonald et al. Lab Manual (1).  This Gram Stain was then observed at 100X using a compound light microscope and immersion oil.  It was determined to be Gram-negative rods.  With this discovery, the original sample was inoculated on to Mannitol Salt Agar (MSA).  This was done in an attempt to grow the Gram-positive bacteria from the original sample.  This was done using the streak method and incubated at 37 degrees Celsius for 48 hours.  After determining the Gram reaction, specific biochemical tests were accomplished to determine the identity of the Gram-negative rods.  The tests accomplished were from the Unknown Chart distributed by the lab instructor.  Each test was performed in accordance with the lab manual by McDonald et al. (1).

All of the following tests were performed on this unknown:

  1.  Simmons Citrate Slant
  2.  Mannitol Salt Agar (MSA)
  3.  Urea
  4.  Indole via SIM tube
  5.  Sulfur Reduction via SIM tube
  6.  Methyl Red
  7.  Voges-Proskauer
  8.  Mannitol Broth
  9.  MacConkey’s Agar
  10.  Eosin-Methylene Blue
  11.  Nitrate Broth

Table 1 lists the test, purpose, reagents and results for the Gram-negative bacteria.

After 48 hours of incubation, the MSA plate that was inoculated with the original sample from tube 108 was interpreted.  This plate demonstrated cloudy growth and yellow agar where the growth was present.  Since MSA is a selective media, which selects for Gram-positive bacteria, a Gram stain was accomplished on this growth to determine if it was the Gram-positive bacteria.  The Gram stain demonstrated Gram-positive cocci.  Bacteria was taken from the MSA plate and inoculated onto a Nutrient Agar plate to grow an isolated colony.  This plate was then incubated at 37 degrees Celsius for five days.  Growth occurred on the nutrient agar and this new growth was also Gram stained.  The growth was also determined to be Gram-positive cocci and this was the sample used to accomplish the remaining tests.

All of the following tests were performed on this unknown:

  1. Mannitol Salt Agar (MSA)
  2. Urea
  3. Methyl Red
  4. Voges-Proskauer
  5. Mannitol Broth
  6. Nitrate
  7. Catalase   

Table 2 lists the test, purpose, reagents and results for the Gram-positive bacteria. 

 

RESULTS:

TABLE 1. Gram Negative Test Results

 

TEST

PURPOSE

REAGENTS

OBSERVATIONS

RESULTS

Gram Stain

To determine the Gram reaction of the bacterium based on cell wall

Crystal violet, Iodine, Alcohol, Safranin

Pink Rods

Gram Negative Rods

Mannitol Salt Agar (MSA)

To determine the ability of the bacterium to ferment Mannitol

None

No color change from red to yellow

Negative Mannitol fermentation

Simmon’s Citrate Agar

To determine the ability of bacterium to use Citrate as sole source of Carbon

None

No color change from green to blue

Negative result for production of citrate permease

Urea

To determine the ability of the bacterium to reduce urea with the enzyme urease

None

No color change

Negative result for production of the enzyme urease

Indole

To determine the ability of the bacterium to reduce tryptophan to indole with the enzyme tryptophanase

Kovac’s Reagent

Red color at the surface of the tube

Positive indole test

Sulfur Reduction

To determine if bacterium can reduce sulfur to hydrogen sulfide gas with the enzyme thiosulfate reductase

None

No black precipitate in tube

Negative result for sulfur reduction

Methyl Red

To determine the final products of glucose fermentation

Methyl Red

Broth turns red color

Positive result indicates presence of a mixture of acids

Voges-Proskauer

To determine if acetoin is produced from pyruvic acid during glucose fermentation

VP reagent A, VP Reagent B

No color change

Negative result for acetoin production

Mannitol Broth

To determine if bacterium ferments mannitol

None

Color change from red to yellow

Positive result for mannitol fermentation

MacConkey’s Agar

To determine if bacterium ferments lactose

None

Brick red growth colony

Positive result for lactose fermentation

Eosin-Methylene Blue Agar (EMB)

To determine if bacterium ferments lactose and the strength of acid end products

None

Green metallic growth

Positive result for lactose fermentation with strong acid end products

Nitrate

To determine if bacterium reduces nitrate to nitrite

Nitrate Reagent A, Nitrate Reagent B, Zinc

No color change after adding Nitrate Reagent A & B, no color change after adding Zinc

Positive result for nitrate reduction

 

 

 

FLOWCHART FOR GRAM NEGATIVE BACTERIA
*Removed due to formatting issues

Gram Negative Unknown – Escherichia coli

TABLE 2. Gram Positive Test Results

 

TEST

PURPOSE

REAGENTS

OBSERVATIONS

RESULTS

Gram Stain

To determine the Gram reaction of the bacterium based on cell wall

Crystal violet, Iodine, Alcohol, Safranin

Purple Cocci

Gram Positive Cocci

Mannitol Salt Agar (MSA)

To determine the ability of the bacterium to ferment Mannitol

None

Color change from red to yellow

Positive result for lactose fermentation

Urea

To determine the ability of the bacterium to reduce urea with the enzyme urease

None

No color change

Negative result for production of the enzyme urease

Methyl Red

To determine the final products of glucose fermentation

Methyl Red

Broth turns red color

Positive result indicates presence of a mixture of acids

Voges-Proskauer

To determine if acetoin is produced from pyruvic acid during glucose fermentation

VP Reagent A, VP Reagent B

Color change to pink/red color

Positive result for acetoin production

Mannitol Broth

To determine if bacterium ferments mannitol

None

No color change

Negative result for mannitol fermentation

Nitrate

To determine if bacterium reduces nitrate to nitrite

Nitrate Reagent A, Nitrate Reagent B, Zinc

After addition of Reagent A & Reagent B, color change to pink/red

Positive result for reduction of nitrate

Catalase

To determine the production of the enzyme catalase

None

Bubbling after bacterium added to hydrogen peroxide

Positive result for the enzyme catalase

 

 

 

 

 

 

 

 

 

 

 





DISCUSSION/CONCLUSION:

The Gram-negative bacteria were identified after some initial confusion.  The first two tests accomplished were Simmon’s Citrate and Mannitol Salt Agar (MSA).  Both tests displayed a negative result after incubation.  This led to initial mis-identification as these results pointed to the bacteria Proteus vulgaris.  Further testing of urea and sulfur reduction did not support the Proteus vulgaris hypothesis.  The lab instructor discussed the validity of the Mannitol Broth over the MSA plate.  The Gram-negative bacteria was inoculated into Mannitol Broth and incubated.  This resulted in acid production as opposed to the negative acid production on the MSA plate.  This new data pointed to Escherichia coli. This led to further testing ending in the correct conclusion.  One test for the Gram-negative bacteria was not consistent with the Unknown Chart results.  The nitrate test was performed twice and both times a positive result was achieved.  According to the lab Unknown Chart, E. coli should exhibit a negative reaction for nitrate reduction.  Internet research suggests that E. coli does reduce nitrate and should exhibit a positive result to the nitrate test (2).    

The Gram-positive bacteria were difficult to isolate in the beginning.  The original sample number 108 was inoculated onto an MSA plate since this media selects for Gram-positive bacteria.  After incubation, the MSA plate displayed isolated colonies and turned yellow demonstrating Mannitol fermentation.  Relying on the results from this MSA plate and a positive Nitrate result led to an initial deduction of Staphylococcus aureus.  Mannitol broth was then inoculated with the Gram-positive bacteria and a negative result was achieved.  This led to further testing of Methyl Red and Catalase.  The results from these tests concluded that the Gram-positive bacteria were Staphylococcus epidermidis.  One test was not consistent with the Unknown Chart results for Staphylococcus epidermidis.  Urea was tested on two separate occasions and both results were negative.  Other students had issues with Urea tests, so it is possible that there was something wrong with the Urea.

Staphylococcus epidermidis is a gram-positive spherical bacterium that form irregular clusters.  Staphylococci are commonly found on the skin and in the mucous membranes of humans and other mammals.  S. epidermidis is the species most commonly isolated from human epithelia according to the Microbiology textbook (3).  It is most frequently found on the head, in the nares and axillae.  S. epidermidis belongs to a group of coagulase-negative Staphylococci, which is distinguished by a lack of the enzyme coagulase.  It is a highly diverse species with 74 different species types.  The species is specially equipped to survive the harsh conditions of the skin and mucosae (4).  S. epidermidis is a microorganism that demonstrates commensalism with the human body.  This is a symbiotic relationship in which one organism benefits while the other is unharmed (3).

S. Epidermidis was previously viewed as a common commensal microorganism that produced little harm to its human host.  Research now shows it to be an important opportunistic pathogen that is a common cause of nosocomial infections.  S. epidermidis is commonly the cause of infections based on medical devices.  Since this microorganism mainly lives on the skin, it commonly enters the body through insertion of an indwelling medical device such as a catheter.  These infections are rarely life threatening, but they are difficult to treat.  This results in a heavy burden on the medical community (4).

S. epidermidis are often found in biofilm on the surface of indwelling medical equipment such as catheters.  Biofilm is a microbial community that usually forms in a slimy layer.  Biofilm associated infections are difficult to treat and eradicate.  Biofilms are often resistant to antibiotics and host defenses (5).  Most nosocomial infections are due to biofilm on medical equipment (3).

For a pathogen to survive in the human body, it must be able to evade host defenses.  The first defense upon entering the epithelium is the innate immune system.  This consists of neutrophils phagocytizing and ingesting bacteria.  S. epidermidis produces exopolymers that help it evade this phagocytosis.  A few strains of S. epidermidis also produce an exotoxin that has the ability to lyse human neutrophils.  The human body has a difficult time clearing long lasting S. epidermidis infections even though the acquired immune system produces antibodies against this species (4).

Specific S. epidermidis strains have developed antibiotic resistant genes.  The most common resistance seen in hospitals is Methicillin resistance.  This is the most common antibiotic used against Staphylococcus infections.  Some strains have also developed resistance to other antibiotics.  Methicillin has been replaced by Vancomycin to treat these infections and there are strains that have become resistant to this antibiotic as well.  The frequency of these antibiotic resistances points to an increasingly problematic overuse of antibiotics (4).             

 

REFERENCES 

1. McDonald, V., Thoele, M., Salsgiver, B., & Gero, S. (2011). Lab manual for general microbiology. St Louis, MO.

2. nitrate reduction test results following growth in nitrate broth. (2012, Nov 1). Retrieved from http://www.microbelibrary.org/library/2-associated-figure-resource/3762-nitrate-reduction-test-results-following-growth-in-nitrate-broth

3. Tortora, G., Funke, B., & Case, C. (2013). Microbiology an introduction. (11 ed.). New York, NY: Pearson Education Inc.

4. Otto, M. (2009). Staphylococcus epidermidis – the “accidental” pathogen. Nature Reviews. Microbiology, 7(8), 555-567. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2807625/

5. Wang, R., Kahn, B., Cheung, G., & Bach, T. (2011). Staphylococcus epidermidis surfactant peptides promote biofilm maturation and dissemination of biofilm-associated infection in mice. The Journal of Clinical Investigation, 121(1), 238-248. Retrieved from http://www.jci.org/articles/view/42520

 

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