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Staphylococcus Epidermidis Unknown Lab Report | Microbiology

By at June 27, 2014 | 9:29 am | Print

Introduction:

It is important to understand and identify microorganisms for a variety of reasons. In a clinical setting, it is important to learn the cause of a patients illness and or symptoms, and how to treat it. And even within a non-professional setting, such as at home cooking, understanding why food-borne illnesses are caused and how to prevent them and treat them, falls back onto the understanding of how these microorganisms work, what they are, and how they’re doing it. Microbiology discusses these examples, and many more that are relatable to everyday life. This particular study was conducted by using all of the methods learned this past semester in order to understand and identify unknown bacterium. 

 




Materials and Methods:

In General Microbiology, the class had been assigned an “unknown bacteria” assignment. Each student received two unknown bacteria that were to be isolated. The instructor gave an unknown test tube labeled as #122. Using the many methods that were learned from the laboratory manual by McDonald at St.Louis Community College, would allow for the identification of the said unknowns. Considering the test tube given contained two different unknown bacteria, the first initial task was to isolate the mixed culture in order to gain a pure culture. Isolation can be done by performing a streak plate, in which the bacterium is streaked out onto a nutrient agar plate in several different directions in hopes of creating two distinct isolated colonies. After creating two streak plates and incubating them at 37 degrees Celsius, there was positive growth on both plates, and well isolated colonies. Following isolation, conducting a Gram Stain on both was necessary to see if only one bacteria appeared under the microscope, to ensure a successful isolation and no contamination. Gram stains involve the use of a Bunsen Burner, inoculating loops, grams crystal violet, grams iodine, alcohol, and grams safranin all used to set the organism to a slide viewable under the microscope, and to stain it which allows for a better visual and distinguishes between a Gram + and a Gram – bacterium. Knowing whether the bacteria are Gram + or Gram – is extremely important, the difference between the two is in their cell membranes. Gram + stains a purple color and contains a thick peptidoglycan layer, whereas Gram – stains a pink or red color and contains lipopolysaccarides, Gram – bacteria tend to be more pathogenic. Once each colony had been Gram stained, viewing them under the microscope revealed that they were in fact successfully isolated with no contamination, and also revealed their shape and + or – information. Bacteria (A) Gram Stained showed a purple color, and cocci shaped cells. Bacteria (B), a red/pink color with rod shaped cells.

Bacteria (A) had been narrowed down to 6 different bacteria, based off of the “Unknown Chart” that was given in class. The test that was conducted following the Gram Stain was a Nitrate Test. This test looks for the reduction of nitrates to nitrites. The materials needed were the Nitrate Test broth tube, reagents A & B, zinc, and of course the addition of the bacteria to the broth. A red color occurred and revealed that Bacteria (A) had reduced nitrates to nitrites, a positive result. This result lead to a Mannitol Salt Agar (MSA) test. MSA is selective for Gram + bacteria, which was suitable for Bacteria (A). The test subjects bacteria to a high salt concentration, in order to inhibit other growth and results in acid production if bacteria ferments mannitol. The tube and plate used both came back positive after Bacteria (A) was added to both, and allowed for one final test to reveal the unknown bacteria. The final test conducted was a Urea Test, which detects the enzyme urease which breaks down urea. The materials needed were the Urea broth, and Bacteria (A). When urease is present and the urea is broken down, the test changes from a neutral color, to a neon pink color. The urea test results were negative, however, they should have been positive. The test could have been conducted incorrectly, or a lack of growth caused a misinterpreted test. Professor Snaric had provided the needed/missing information regarding the Urea test and revealed that Gram + Bacteria (A) was in fact Staphylococcus Epidermidis.

Bacteria (B) had also been narrowed down to a select few following the Gram stain. A Nitrate test was initially conducted for the Gram – Bacteria (B) and the results showed a positive result for the reduction of nitrates to nitrites. This lead to a Mannitol Salt Agar (MSA) test, that had also been positive for acid production due to mannitol fermentation. The Nitrate and Mannitol tests lead to a test known as Simmons Citrate. Simmons Citrate distinguishes bacteria based on whether or not they use Citrate as their sole carbon source. The materials needed for this particular test were the Simmons Citrate Agar, unknown Bacteria (B), and a sterile inoculating loop to spread the bacteria onto the agar. The test had been incubated for about 2-3 days and came back with a color change of green to blue, another positive result. These three tests conducted began to raise more questions than answers and was not eliminating any possibilities for Bacteria (B). After a day of organizing possible tests that would result in answers instead of questions, the next test conducted was the Methyl Red and Voges Proskauer (MR-VP) test. The materials needed for this test were 2 (MR-VP) broths, an inoculating loop, a bottle of methyl red, VP reagent A and reagent B, and Bacteria (B). The two test tubes were necessary because one would be used for the Methyl Red test, which tested for acids that were a result of glucose fermentation, and one for the Voges Proskauer test, which tested specifically for acetoin production. After incubation, the methyl red had been added to test tube #1 and ended with a positive result. Test tube #2 was set aside for Voges Praskauer and the reagents had been added. After letting the reagents sit for 15-30 minutes, a color change should have occurred but did not. This indicated and negative test result, that should have been positive. The reasoning for a false VP test would be not enough incubation time, or misconducted. The false VP test caused a lack of elimination for a second time and discussion with Professor Snaric revealed the needed information again. One final test was needed to verify the identity of Gram – Bacteria (B) and that was the Urea test. After allowing the Urea broth and Bacteria (B) to incubate for 2 days, the results came back negative, revealing Enterobacter Aerogenes as Bacteria (B).

 

Results:

The following tests were performed on Bacteria (A):

1. Gram Stain

2. Nitrate Test
3. Mannitol Salt Agar (MSA) Test
4. Urea Test

 

All tests had been incubated at 37 degrees Celsius for 2-5 days, depending on the test conducted. The table below shows all tests, the purpose of each, the reagents added, the observations noted, and the results.

 

Test Purpose Reagents Observations Results
Gram Stain To distinguish between Gram- (Pink/Red cells) and Gram + (Purple/Blue cells). Allows determination of cells morphology. Crystal violet, Gram’s iodine, alcohol (decolorizer), Gram’s safranin. Purple circles/balls (cocci) Gram + cocci
Nitrate Test To determine if bacterium produces enzymes nitrate reductase and nitrite reductase. Nitrate reagent A (dimethyl-naphthylamine) and Nitrate reagent B (sulfanilic acid dissolved in acetic acid) And zinc. Appearance of red color indicated reduction of nitrates to nitrites. Positive Nitrate test.
Mannitol Salt Agar (MSA) Distinguishes between organisms that are able to survive high salt concentrations and ferment mannitol. None No fermentation of mannitol, red color maintained. Negative mannitol test.
Urea Detects enzyme urease, which allows break down of urea- producing acid and causing a noticeable color change. None No color change. Negative Urea test. (*Should have been positive!)

 

The following tests were performed on Bacteria (B):

1. Gram Stain

2. Nitrate Test

3. Mannitol Salt Agar (MSA) Test

4. Simmons Citrate Test

5. Methyl-Red Test

6. Voges-Proskauer Test

7. Urea Test

 

Test Purpose Reagents Observations Results
Gram Stain To distinguish between Gram- (Pink/Red cells) and Gram + (Purple/Blue cells). Allows determination of cells morphology. Gram’s crystal violet, Gram’s iodine, alcohol (decolorizer), Gram’s safranin. Pink/Red rod shaped cells. Gram – Rods.
Nitrate Test To determine if bacterium produces enzymes nitrate reductase and nitrite reductase. Nitrate reagent A (dimethyl-naphthylamine) and Nitrate reagent B (sulfanilic acid dissolved in acetic acid) And zinc. Appearance of red color indicated reduction of nitrates to nitrites. Positive Nitrate test.
Mannitol Salt Agar (MSA) Distinguishes between organisms that are able to survive high salt concentrations and ferment mannitol. Color change of red to yellow indicated acid production and mannitol fermentation. Positive MSA test.
Simmons Citrate Differentiates organisms on ability to use citrate as primary carbon source. Color change in agar slant from green to blue. Positive Simmons Citrate test.
Methyl-Red Tests for glucose fermentation, specifically microbes that produce mixture of acids as a result of fermentation. Methyl Red. Color change from neutral to red. Positive Methyl-Red test.
Voges-Proskauer Tests for glucose fermentation, specifically organisms whose acid is converted to acetoin. V-P reagent A and V-P reagent B. No color change. Negative V-P test. (*Should have been positive!)
Urea Detects enzyme urease, which allows break down of urea- producing acid and causing a noticeable color change. No color change. Negative Urea test.

 

 




Discussion/Conclusion:

After all tests had been successfully conducted, Unknown Gram + Bacteria (A) had been determined to be Staphylococcus Epidermidis. Gram staining Bacteria (A) allowed for the elimination of all Gram – bacteria, and all rod shaped cells. Conducting the Nitrate and Mannitol (MSA) tests positively contributed to the elimination of 3 bacteria. The Urea test results however, came back negative and was incorrect. The test should have been positive, which would have eliminated the remaining 5 possible bacteria and proved that S.Epidermidis was correct. The results may have been incorrect due to a lack of growth within the Urea Broth tube, possible contamination, or simply poorly conducted. The information was reported to the Professor and the correct information verified that Staphylococcus Epidermidis was in fact correct. Unknown Gram – Bacteria (B) was more of a challenge. The Gram stain assisted with the elimination of Gram + bacteria and cocci shaped cells as with Unknown (A). All tests should have assisted with the elimination of some potential bacteria candidates but were overlapping and not concluding correctly. This resulted in more and more testing and ended with a false Voges Praskauer (VP) test. The VP test should have had a positive result, but due to lack of incubation time, the result was incorrect. Identifying with the Professor that this test was in fact incorrect, assisted with the discovery of the identity of Unknown Bacteria (B), which was Enterobacter Aerogenes.

 

 

Staphylococcus Epidermidis:

Staphylococcus Epidermidis is one of the many known species of Staphylococcus. It can be found within our mucous membranes, as a part of our skin flora, and in animals. It is also one of the most common bacterium found in laboratory tests due to contamination. Usually, Staphylococcus Epidermidis is not pathogenic, but if it comes in contact with a person who has a compromised immune system it may cause infection. These infections can be both nosocomial ( acquired in hospital) and community acquired, but pose more of a threat to hospital patients. Friedrich Julius Rosenbach found the differences between Staphylococcus Epidermidis and Staphylococcus Aureus in 1884, it is a hard microorganism consisting of non-motile Gram + cocci, arranged in grape-like clusters. S. epidermidis has the ability to form biofilms on plastic devices, causing a major virulent factor. The biofilm that grows on plastic devices generally occurs on catheters and other medical devices. These biofilms assist with the resistance to antibiotics including penicillin, amoxicillin, and methicillin.

 

 

References:

1. McDonald, Thoele, Salsgiver, Gero (Lab Manual for Gen. Microbiology STLCC Meramec)

2. Redorbit.com/education/reference_library/health_1/bacteria/2584198/staphylococcus_epidermidis/

 

 

 

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