UNKNOWN LAB REPORT
Unknown Number 102
May 5, 2015
Professor Jay Snaric
Microorganisms are everywhere and it is important to know, not only the type of microorganism, but what effects it can have on the environment, as well as humans. Determining which microorganism a patient is infected with is essential to determining the proper course of treatment. Improper treatment will not alleviate the infliction, and it could have disastrous short and long term effects on the patient. This study was conducted using techniques learned in microbiology class to separate and identify the gram negative and gram positive bacterium contained within a test tube.
MATERIALS AND METHODS
A test tube labeled “Unknown #102” was given by the Professor. The methods which have been learned thus far were applied to the unknown. All of the tests and procedures were applied using the methods outlined in the “Lab Manual for General Microbiology”, (3).
The unknown tube contained both a gram positive and a gram negative bacterium. The first step was to quadrant streak the unknown onto a nutrient agar plate in order to grow colonies and separate them. The plates were then incubated for at 37 degrees Celsius for 48 hours and the expected result was to grow colonies which could be visually identified as different. Due to unforeseeable circumstances, the plates were not checked after 48 hours and overgrowth occurred. The next step would have been to isolate the colonies into two separate colonies, place each colony onto a separate nutrient agar plate and allow them to incubate for another 48 hours at 37 degrees Celsius. Instead, another method, suggested by the Professor, (1), was applied. The growth on the original nutrient agar plate had a green hue around it; according to information provided by the Professor, (1), this suggested that the bacterium was gram (-) Psuedomonas aeruginosa. A new approach for isolating the gram positive bacterium was implemented at this time.
The second step was to begin tests to confirm bacterium 1 as P. aeruginosa as well as isolate the second bacterium. Since P. aeruginosa was already suspected, the tests which were chosen would each eliminate all other bacterium as possibilities. The gram stain for bacterium 1 revealed red rods.
To identify bacterium 1, a gelatin test was performed, by inoculating a gelatin tube with the unknown and incubating it at 37 degrees Celsius for 24 hours, to isolate P. aeruginosa as the only possible choice. Only P. aeruginosa is capable of breaking down gelatin, (4). Next, a maltose test was performed; by inoculating a maltose fermentation tube with the unknown and incubating it at 37 degrees Celsius for 24 hours. This test is done to determine if the microbe can ferment the carbohydrate maltose, (3). A negative result would eliminate all other bacterium as possibilities, (4). Finally, an oxidase test was performed as a final determination. The oxidase test determines if the Oxidase Cytochrome is present and functional, (3). P. aeruginosa is the only bacterium where there will be a positive result, (4); a positive reaction would confirm that the bacterium is P. aeruginosa, (4).
To isolate bacterium 2, a sample of the unknown was taken directly from the original test tube and streaked onto a Mannitol Salt Agar (MSA) plate. The MSA plate is selective for gram positive bacteria, therefore only the gram positive bacteria will grow, enabling said isolation. The plate was then incubated at 37 degrees Celsius for 48 hours. Once growth was established, a gram stain was performed to confirm a gram positive bacterium. The gram stain revealed purple cocci. Tests were then performed to eliminate other bacterium and confirm the final result.
The first test performed was a catalase test, to determine if the bacterium has catalase, a protective enzyme, which can destroy hydrogen peroxide, (3). If the hydrogen peroxide does not bubble when added to a sample of the bacterium on a slide (a negative result) Enterococcus faecalis can be eliminated, (4). A positive test will require further testing. Next, a urea test was performed by inoculating a urea broth with a sample from the MSA plate, to determine if the bacterium can use the compound urea as a source of carbon and energy for growth utilizing the enzyme urease, (3). A positive result would confirm Staphylococcus epidermidis as the bacterium, (4). Finally, a mannitol test was performed to confirm that the bacterium is S. epidermidis, (4). The test was performed by inoculating the mannitol broth tube with a sample from the MSA plate, to determine if the bacterium can ferment the carbohydrate mannitol as a carbon source, (3). A negative test will confirm S. epidermidis, (4).
All tests performed to determine bacterium 1 are outlined in Table I. The first test performed on the suspected gram negative bacterium was the gram stain to confirm gram negativity. Further tests were then performed to confirm suspected bacterium.
The tests performed on the unknown to confirm the negative bacterium were:
- Gram stain
All tests performed to determine bacterium 2 are outlined in Table II. The first test performed on the suspected gram positive bacterium was the gram stain to confirm gram positivity. Further tests were then performed to identify and confirm the bacterium. The tests performed to identify the gram positive bacterium were:
- Gram stain
Unknown # 102 had morphology on the original nutrient agar plate which suggested P. aeruginosa, (1). After determining that it was Gram negative, gelatin, maltose and oxidase tests were performed to isolate it from the other bacterium, (4). Table I, lists all of the biochemical tests, their purpose, reagents, observations, and results and interpretations. The results are also shown in a flow chart form in Figure 1.
After the gram positive bacterium was isolated on the MSA plate, and then confirmed through gram staining, other tests were performed to isolate it from the other bacterium, (4). Table II, lists all of the biochemical tests, their purpose, reagents, observations, and results and interpretations. The results are also shown in a flow chart form in Figure 2.
Table I: Tests and results to determine and confirm bacterium #1
|Nutrient Agar||To isolate colonies||None||Green tint around bacterial growth||Positive||The green tint indicates the presence of Pseudomonas aeruginosa|
|Gram Stain||1. Determine if the bacterium is gram (+) or gram (-)
2. Determine the shape of the bacterium.
|1. Crystal violet
2. Gram iodine
|Red rods when viewed under the microscope||Gram (-) rods||The bacterium is gram (-)|
|Gelatin||1. Isolate Pseudomonas aeruginosa as the only possible bacterium
2. Determine whether the microbe is capable of breaking down gelatin
|None||The gelatin liquefied in the tube
|Positive||1. Confirmation that the bacterium is Pseudomonas aeruginosa
2. Gelatinases is present and the organism is able to break down gelatin
|Maltose||1. To confirm that the bacterium is Pseudomonas aeruginosa
2. To determine if the microbe can ferment the carbohydrate maltose
|None||Color change from red to yellow||Positive||1. Further confirmation of Pseudomonas aeruginosa
2. A pH change to acidic indicating that the microbe can ferment the carbohydrate maltose as a carbon source
|Oxidase||1. Confirmation that the bacterium is Pseudomonas aeruginosa2. To determine if the Oxidase Cytochrome
is present and functional
|Oxidase||The discs turned a purple color||Positive||1. Final confirmation of Pseudomonas aeruginosa
2. Oxidase cytochrome is present and functional
Table II: Tests and results to determine and confirm bacterium #2
|MSA||1. To isolate gram (+) bacteria
2. To determine if the bacterium ferments Mannitol
|Bacteria grew and turned the agar yellow||Positive
|1. The bacterium is gram (+)
2. The bacterium is either Staphylococcus epidermidis or enterococcus faecalis
|Gram Stain||1. To determine if the bacterium is gram (+) or gram (-)2. To determine the shape of the bacterium||1. Crystal violet2. Iodine
|Round purple grape-like clusters when viewed under the microscope||Gram
|The bacterium is gram (+)|
|Catalase||1. To determine if the bacterium is Staphylococcus epidermidis or Enterococcus faecalis
2. To determine if the bacterium has catalase, a protective enzyme, which can destroy hydrogen peroxide
|Hydrogen peroxide||Bubbles formed||Positive||1. Confirmation of Staphylococcus epidermidis
2. The bacterium contains catalase
|Urea||1. To furtherconfirm that the
2. To determine if the bacterium can use the compound urea
as a source of carbon and energy for growth utilizing the enzyme urease
|None||Color changed to
2. The bacterium uses the compound
urea as a source of carbon and energy for growth utilizing the enzyme urease
|Mannitol||1. To provide final confirmation that the bacterium isStaphylococcus epidermidis
2. To determine if the bacterium can ferment the carbohydrate mannitol as a carbon source
|None||No color change||Negative||1. Final
confirmation of Staphylococcus epidermidis
2. Does not ferment mannitol as a carbon source
NOTE: Flowcharts were removed due to formatting issues.
Separating the gram negative and gram positive bacterium was an essential first step in determining the final results of unknown #102. Once the two bacteria were isolated, tests were performed to confirm the type of bacterium.
Since the original nutrient agar plate had the green hue, it was suspected that the gram negative bacteria was Pseudomonas aeruginosa, according to information provided by the Professor, (1), therefore the tests which were performed were geared to confirm that hypothesis. The gelatin, maltose and oxidase tests were performed, because each of those three tests would eliminate all other bacteria besides P. aeruginosa, (4). The gelatin and oxidase tests were both positive, eliminating all other possibilities, (4). The maltose test was negative, also eliminating all other possible bacterium, (4).
The tests performed on the gram positive bacteria were geared toward eliminating bacteria until a final result was reached. Since the gram stain revealed cocci, Bacillus cereus and Bactillus subtilis were eliminated, (4). The catalase test was positive, which eliminated Enterococcus faecalis, (4). The mannitol test was negative, leaving the only possibility as Staphylococcus epidermidis, (4). The urea test was then performed for final determination. The test was positive, therefore confirming Staphylococcus epidermidis, (4).
Staphylococcus is a genus of bacteria characterized by its round shape. This genus can be be single cells, pairs, or more frequently, clusters resembling a bunch of grapes are found. The gram stain is positive, a purple color, further resembling grapes. The name comes from the Greek terms staphyle and kokkos, meaning “bunch of grapes”, (2). Epidermidis strains, which are coagulase-negative, produce a slime that interferes with the body’s immune defenses (phagocytosis) and cause a biofilm. S. epidermidis strains are part of the body’s normal flora and do not usually cause infections, unless a person’s immune system is suppressed. It has been shown that the bacterium may actually be beneficial. S. epidermidis a nosocomial pathogen, which is quickly becoming a major issue with implanted medical devices, such as, catheters and prosthetic devices.
The danger of a S. epidermidis results from the effects on the host. The cells can be damaged as a result of membrane-altering toxins, such as alpha, beta toxins plus leucocidin, which damage cells by making holes in their membranes, (2). A major concern with regard to S. epidermidis is that it is quickly becoming resistant to many different types of antibiotics, due to the overuse and misuse of antibiotics in the past. Currently, S. epidermidis is resistant to including Methicillin, all Penicillins, Penems, Carbapanems and Cephalosporins, (5). This makes treatment for any type of staph infection much more difficult when the pathogenic form takes over. While S. Epidermidis is usually harmless, given the right conditions, it can flourish out of control and we are left with little to nothing to treat it.
- Bohra, S. (2015). Conversation with Professor. Louis
- Davis, C. (n.d.). Staph Infection: Learn About Symptoms and Treatment. Retrieved May 3, 2015, emidicinehealth. Retrieved from http://www.emedicinehealth.com/staphylococcus/article_em.html
- McDonald et al. (2011). Lab Manual for General Microbiology. St. Louis Community College – Meramec. [Lab Manual]. St. Louis
- Snaric, J. (2015). St. Louis Community College – Meramec. St. Louis.
- Snaric, J. (2015, April 21). Antibiotics. Lecture conducted from, St. Louis.