Staphylococcus aureus or Enterococcus faecalis | How to Identify
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UNKNOWN LAB REPORT
Unknown Number 106
Bijalben Shah
INTRODUCTION
Microorganisms are everywhere. Some of these microbes are pathogenic or disease causing. Before treating patients for diseases, it is essential to identify which microorganism causes the disease and the specific drugs fight against the diseases. The techniques and procedures learned in the microbiology lab manual by McDonald et al. (1) were applied in order to identify the unknown microorganisms.
MATERIAL AND METHODS
An unknown mixture labeled 106 which contained one gram positive bacteria and one gram negative bacteria was given out by the lab instructor. To identify unknown bacteria aseptic techniques were followed per the McDonald et al. (1) lab manual.
The first step was to isolate an unknown bacterium on a solid medium to get isolated colonies. This was done by using the isolation streak method on to a nutrient agar plate as described in the McDonald et al. (1) lab manual. After that the plate were incubated at 37 degrees Celsius for two days. From that plate one isolated colony grew. Gram stains were performed on that pure culture. That process was not only to detect whether it was a gram positive or negative bacteria but, also the shape of the bacteria. The culture was determined to be gram negative rods.
Since the gram stain detected gram negative rods, the original unknown mixture was inoculated on to a mannitol salt agar plate which is selective for only gram positive bacteria. This was done by using the streak method. The MSA plate was incubated for two days and the bacterium grew. Gram stains were performed on that isolated colony using the sterile technique. This culture was determined to be gram positive cocci. After gram stains were accomplished, specific tests were performed for each gram positive and gram negative bacteria.
All of the following tests were performed on the gram negative bacteria:
- Simmon’s Citrate Test
- Methyl Red
- Voges-Proskauer
- Casein
- BCP Lactose
- BCP Maltose
All of the following tests were performed on the gram positive bacteria:
- Mannitol
- BCP Mannitol
- Nitrate
- Urea
- Catalase
RESULTS
An unknown labeled 106 was isolated by the streak method on NA plate. Only one isolated colony grew on that medium which was gram stained. The gram stain determined gram negative rods. That was inoculated onto different mediums. Table-1 lists all the biochemical tests, purpose, reagents, observations and results.
TABLE-1: GRAM NEGATIVE BACTERIA
|
TEST |
PURPOSE |
REAGENTS |
OBSERVATIONS |
RESULTS |
| Gram stain | To determine the Gram reaction of the bacterium | Crystal Violet, Iodine, Alcohol and Safranin | Pink rods | Gram negative rods |
| Citrate | To determine if bacteria produce the enzyme citrase to break down citrate | None | Color change from Green to Blue | Positive for production of citrate permease |
| Methyl Red | To determine if acid as the final end product of glucose fermentation | Methyl Red | Color change to light yellow to dark yellow | Negative for presence of a mixture of acids |
| Voges-Proskauer | To determine if organisms will produce acetoin | VP reagents A & B | After adding reagent A & B, no color change | Negative for acetoin production |
| Casein | To determine if the organism produce casease which hydrolyze milk | None | Clear zone | Positive for casease production |
| BCP Lactose | To determine the ability of bacterium to ferment a specific carbohydrate | None | No color change | Negative lactose fermenter |
| BCP Maltose | To determine the ability of bacterium to ferment a specific carbohydrate | None | No color change | Negative Maltose fermenter |
FLOW CHART FOR GRAM NEGATIVE BACTERIA
UNKNOWN # 106
Gram Stain
Gram Negative Rod
Citrate Test (Positive)
Positive
Klebsiella pneumoniae
Enterobacter aerogenes
Pseudomonas aeruginosa
Negative
Escherichia coli
Proteus vulgaris
Methyl Red Test (Negative)
Enterobacter aerogenes
Pseudomonas aeruginosa
Voges-Proskauer Test (Negative)
Pseudomonas aeruginosa
Casein Test (Positive)
Pseudomonas aeruginosa
BCP Lactose Test (Negative)
Pseudomonas aeruginosa
BCP Maltose Test (Negative)
Pseudomonas aeruginosa
Gram Negative Unknown 1 – Pseudomonas aeruginosa
Since only gram negative bacteria grew on the nutrient agar plate, an unknown was isolated and streaked on to a MSA plate which was a selective medium for gram positive bacteria. A gram stain was performed and determined to be gram positive cocci. Table-2 lists all of biochemical tests, purpose, reagents, observations and results for gram positive bacteria.
TABLE-2: GRAM POSITIVE BACTERIA
|
TEST |
PURPOSE |
REAGENT |
OBSERVATION |
RESULT |
| Gram stain | To determine the Gram reaction of the bacterium | Crystal Violet, Iodine, Alcohol and Safranin | Purple cocci | Gram positive cocci |
| Mannitol | To determine if bacteria ferment mannitol | Mannitol salt agar | Color change to yellow | Positive |
| BCP Mannitol | To determine the ability of a bacterium to ferment a specific carbohydrate | None | Color change from purple to yellow | Positive mannitol fermenter |
| Catalase | To determine if the organism release gas | Hydrogen Peroxide | Bubbling | Positive catalase test |
| Nitrate | To determine if bacteria reduce nitrate to nitrites | Nitrate broth | No color change after adding zinc | Positive nitrate test |
| Urea | To determine if bacteria produce the enzyme urease that break down urea | Urea broth | No color change | Negative urea test |
FLOW CHART FOR GRAM POSITIVE BACTERIA
UNKNOWN # 106
Gram Stain
Gram Positive Cocci
Manitol Test (Positive)
Staphylococcus aureus
Enterococcus faecalis
BCP Manitol Test (Negative)
Staphylococcus aureus
Enterococcus faecalis
Nitrate Test (Positive)
Staphylococcus aureus
Urea Test (Negative)
Staphylococcus aureus
Catalase Test (Positive)
Staphylococcus aureus
Gram Positive Unknown 2 – Staphylococcus aureus
DISCUSSION / CONCLUSION:
After performing several biochemical tests on the Gram-negative bacteria, it is established that Unknown-1 was Gram-negative rods Pseudomonas aeruginosa and Unknown-2 was Gram-positive Staphylococcus aureus. A Gram stain was performed on the Gram-negative bacteria which were negative rods. A Simmon’s Citrate test was inoculated to detect if the bacteria produce the enzyme citrase to break down citrate. The results eliminated Escherichia coli and Proteus vulgaris. A Methyl Red test was performed to determine that the microbes produce a mixture of acids as a result of glucose fermentation. Glucose was not fermented and acid was not produced, therfore that eliminated Klebsiella pneumonia. Then V-P reagent A&B was added to the inoculated medium. Acetoin was not produced which eliminated Enterobacter aerogenes. A Casein test determined that the bacteria hydrolyzed milk with the production of the enzyme casease clarifying that the unknown-1 was Pseudomonas aeruginosa.
The gram positive cocci were identified after first isolating the medium on to a Mannitol Salt Agar (MSA) plate. Then it was inoculated in BCP Mannitol broth. The color change to yellow indicated acid production by Mannitol fermentation. This indicated that the unknown was Staphylococcus aureus or Enterococcus faecalis. Then a Nitrate test was performed which had a positive reaction meaning the reduction of Nitrate to Nitrite. A Catalase test was performed which had a positive reaction for the bacterial enzyme catalase. This proved that the unknown-2 bacteria were Staphylococcus aureus.
As stated by Tortora, Funke and Case, in Microbiology textbook (2), staphylococcus aureus is Gram-positive cocci. It is found in grape like clusters, so it is known as staphylococcus. It is abbreviated as S. aureus, and the most important Stephylococcus species is Stephylococcus aureus. S. aureus is capable of secreting many exotoxins that affect wide range of infection from pimples and boils to pneumonia, food poisoning and surgical wound infections by increasing its ability to invade the body or damage tissue. The infection of S. aureus is a common problem in hospitals and its ability to develop resistance quickly against antibiotics such as penicillin. It also has rapidly acquired resistance to other antibiotic such as methichillin, which is known as MRSA, Methicillin-resistance Staphylococcus aureus. MRSA was the first healthcare-associated infection and occurs more rapidly in hospitals, nursing homes and it is a danger to patients in hospital environment.
REFERNECES:
McDonald, V., Thoele, M., Salagiver, B., & Gero, S. (2011). Lab manual for general microbiology. St. Louis, MO.
Tortora, G., Funke, B., & Case, C. (2013). Microbiology an introduction. (11 ed.). New York, NY: Pearson Education Inc.
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