Mallory Macciomei May 13, 2011 Dr. Schnee Microbiology Determination of an Unknown Bacteria Introduction Bacteria are the cause of many illnesses in humans, and for years scientists have tried to identify unknown bacteria in order to cure and prevent our bodies from harm. A study done to identify an unknown bacterium stated “The importance of identifying these pathogens and their related epidemiology has become increasingly more important” (OPPapers 2011). There are bacteria everywhere—in your car, on your desk, even in your food. This means that we are constantly exposed to all sorts of bacteria, good and bad. So how can sickness be prevented if we are always exposed to bacteria? The first step to prevention is to identify the bacteria that are on all of the surfaces we touch. A sample of bacteria was swabbed from a laptop keyboard in order to take the first step towards identification of the unknown bacteria in our lives. Various techniques of bacteria identification were used on this unknown. It is hypothesized that the unknown bacteria from the keyboard is just a common, harmless
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Colony Morphology of 9-day old unknown bacteria Cell Morphology of 9-day old unknownbacteria
(A) (B)
Figure 1: Drawings of unknown bacteria colony and cell morphology. (A) Colony morphology of a 9-day old unknown bacteria culture grown on a nutrient agar plate. The bacteria was incubated and allowed to grow at 32 degrees Celsius for 9 days. Colonies were circular, flat, and undulate in form, elevation, and margin, respectively. Each colony was white in color. Careful observation of the nutrient agar plate helped to determine these qualities. (B) Cell morphology of unknown bacteria grown for 9 days on a nutrient agar plate at 37 degrees Celsius. Cell size and shape was determined under 1000x bright field.
Colony and cell morphology were the first observations made about the unknown
bacteria. Colony morphology was determined by carefully looking at a pure culture of the
unknown on a nutrient agar plate at all angles. Colonies were measured and recorded after
incubating at 32 degrees Celsius for nine days. The colonies were circular, flat, and undulate in
form, elevation, and margin respectively. Cell morphology was determined by observation
under 1000x bright field and phase contrast. Micrococcus luteus was used as a control in each
situation. The results were determined after using both wet mount and gram staining techniques
Figure 2: (Photo taken from “February 2009”). Contaminated nutrient agar plate. This plate shows signs of contamination because of the different colored, sized, and shaped colonies. The yellow arrow points to one colony that is white and very circular. The red arrow points to a colony that is brown and much smaller in size. These colonies are just two of perhaps many colonies on this contaminated plate.
By looking at the plate of unknown bacteria colonies, it was determined that the culture
obtained was a pure culture. In order for a culture to be pure, all colonies must be of the same
bacteria. If the culture is not pure, it is contaminated. A contaminated sample is shown in figure
2. Bacteria were found to be gram negative when observed against M. luteus and bacillus.
Because the control bacteria were gram positive, it was easy to find the unknown due to the
difference in color. The unknown bacteria exhibited a light pink color which showed that it was
indeed gram negative. All observations for gram staining, size and shape were viewed under
1000x bright field. The unknown was found to be bacilli, and size ranged from 1.5-3.0 uM in
length and .75-1.0 uM in width. The average length of the unknown was 2.0 uM and the average
width was 1.0 uM (see table 1). Size was difficult to determine because the rods were extremely
small. The unknown bacteria proved to be not motile when observed at 1000x phase contrast
against E. coli; however, the bacteria prove to be motile in later tests (see “Motility Test”
below). Brownian motion was taken into consideration when determining motility and the
bacteria seemed to just be “going with the flow” of the slide, therefore the cells did not appear
motile.
Table 1.
Unknown bacteria size range and averages
Size range Average size
Table 1: This table shows the dimensional sizes of the unknown bacteria cells. Length and width were determined by measuring in micrometers under 1000x bright field. Column one states the dimension measured. Column two shows the size range in unknown cells, and column three shows the average sizes in the unknown bacteria cells.
Figure 3Photo taken from “Kligler 2011”) Three of the possible OF test results. The first tube (far left) shows a facultative anaerobe bacteria. The bacteria grows throughout the entire tube. The second tube (middle) shows an obligate aerobe bacteria. The bacteria only grows at the top of the medium because it relies on oxygen to use energy/survive. There is no bacteria at the bottom of the tube. The third tube (far right) shows an anaerobic bacteria. The bacteria is only in the middle/bottom of the tube. There is no bacteria at the top of the medium.
An Oxidation-Fermentation (OF) test was performed on the unknown bacteria to
determine how it used energy. After the procedure and a 7 day incubation period, the bacteria
was observed. The unknown bacteria appeared to grow throughout the entire test tube, which
meant that the unknown used fermentation as a source of energy, but could tolerate the presence
of oxygen.
The unknown bacteria tested for catalase positive against nutrient agar. The nutrient agar
control helped confirm that the unknown bacteria was catalase positive because when hydrogen
peroxide was introduced to pure nutrient agar, no bubbles occurred—resulting in a negative test.
When hydrogen peroxide was introduced to the pure, unknown bacteria culture bubbles occurred
almost immediately.
When tested for cytochrome oxidase, the unknown bacteria resulted in a negative test.
The test reagent was applied to a colony on the nutrient agar plate and no color change was
observed. The control, pseudomonas, was tested and the affected area turned a dark blue within a
few minutes. Because the unknown did not show any signs of color change, it is oxidase
negative; so it does not produce the enzyme cytochrome oxidase. Refer to figure 3 for further
evidence.
Figure 4.
Oxidase Positive Results Oxidase Negative Results
(2) (B)
Figure 4: Oxidase test results—both positive and negative. Oxidase test was conducted as follows in Cappuccino et al. 1996. (A) Positive result of Oxidase test. This bacteria is oxidase positive, exhibiting a dark blue/black color on the induced area. (B) Negative result of Oxidase test. This bacteria is oxidase negative, exhibiting no color change whatsoever. In fact, to the naked eye, it does not look like a test was performed at all.
The unknown bacteria was tested for motility a second time utilizing the motility test
according to Cappuccino et al. 1996. E. coli and M. luteus were used as controls because E. coli
is motile, so the results show the bacteria throughout the test tube; while M. luteus is not motile,
so the results show the bacteria only in the area where the bacteria was first inserted with a sterile
wire loop. The unknown bacteria displayed an outcome quite like E. coli (see Fig 4). It was
obvious that the unknown was motile because there was a red color spread throughout the entire
tube (the bacteria appeared red). This contradicted the results of the wet mount motility test
which resulted in a negative test for motility.
Figure 5. Motility Test: M. luteus and Unknown
Figure 5: Motility test results: unknown bacteria (left) and M. luteus (right). Above are the results of a motility test on the unknown bacteria. The test tube on the left is the unknown, and the test tube on the right is M. luteus. The “unknown” test tube has red bacteria spread throughout the test tube, while the “M. luteus” test tube has red bacteria only in the original spot of insertion. Bacteria were inserted into the appropriate tube and allowed to incubate and grow for one week at 32 degrees Celsius.
In order to discover the unknown bacteria, motility needed to be clearer. At this point,
two motility tests had been performed (wet mount under phase contrast 1000x and the Motility
test in the test tubes) but results were mixed. When the unknown was stained for flagella, it was
observed under 1000x bright field against E. coli. E. coli is peritrichious meaning it has flagella
protruding from all areas of its body. After careful observation of the unknown after staining, it
was very difficult to determine if flagella was present. There was a faint mark at the end of each
cell that may or may not have been flagella, making the unknown monotrichious. Because the
tube motility test was positive, it is likely that the unknown do have a single flagella, but it is
very hard to see because of its size.
Figure 6. Flagella Stain: S. typhi and S. dysenteriae
Figure 6: (Photo taken from “Todar 2009”) Flagella stain of Salmonella typhi and Shigella. This flagella stain shows Salmonella bacteria which, like E. coli, is peritrichious. There are flagella protruding from all around the Salmonella cells (purple arrow). This stain also shows the bacteria Shigella which lacks flagella; there is nothing protruding from the cell (green arrow).
The unknown bacteria underwent an ultra violet (UV) ray test to determine if it could live under
those conditions or if it would die. Plate 1 (Fig 6 part A) was exposed to UV rays for 60 seconds, and
plate 2 (Fig 6 part B) was exposed for 30 seconds. After 24 hours of incubation at 32 degrees Celsius,
neither of the plates had any change whatsoever. In figure 6, it is obvious that both plates have bacteria
distributed evenly on the nutrient agar.
Figure 7. UV light test of unknown bacteria
(A) (B)
Figure 7: UV light test of unknown bacteria, each exposed to a different time limit of UV rays. Both plates have the unknown bacteria streaked thoroughly and evenly over the entire plate. (A) This plate of unknown bacteria was exposed to UV light for 60 seconds and then incubated for 24 hours at 32 degrees Celsius. There is no shape on the plate; after the 24 hour incubation period, the bacteria was still streaked thoroughly and evenly over the entire plate. (B) This plate of unknown bacteria was exposed to UV light for 30 seconds and then incubated for 24 hours at 32 degrees Celsius. There is no shape on the plate; after the 24 hour incubation period, the bacteria was still streaked thoroughly and evenly over the entire plate.
The antibiotics test performed on the unknown bacteria told us if the bacteria was
susceptible or resistant to the antibiotics. The antibiotics used were Streptomycin, Erythromycin,
and Kanamycin. Results show that the unknown bacteria was susceptible to Kanamycin and
Streptomycin. The diameter of the zones of inhibition were 18 mm and 17 mm, respectively. The
bacteria was classified as “intermediate” for resistance to Erythromycin. The diameter of the
zone of inhibition for Erythromycin was 15 mm. Figure 7 shows the nutrient agar plate with the
zones of inhibition for each antibiotic. Refer to Table 2 for a summary of the antibiotic test
results as well as all of the tests performed on the unknown bacteria.
Figure 8. Antibiotic Test Results with Zones of Inhibition
Figure 8: Antibiotic test results with zones of inhibition. This test used three different antibiotics—Kanamycin, Erythromycin, and Streptomycin. Each white circle is the antibiotic, and each antibiotic is labeled with the first letter of its name (Kanamycin is labeled “k”). The yellow arrow points to a zone of inhibition around Kanamycin. There is a distinct zone of inhibition around Streptomycin and Kanamycin, but the zone gets less defined around Erythromycin. Bacteria was incubated at 32 degrees Celsius for 24 hours after the addition of the antibiotics.
Table 2: Tests performed on unknown bacteria and results. The left lane shows the name of the test ran on the pure culture of unknown bacteria. The right lane shows the results of the tests on the unknown bacteria. Negative results are indicated by a “-“ symbol and positive results are indicated by a “+” symbol.
Discussion
The experiment was conducted to identify an unknown bacteria swabbed from a laptop
keyboard. Based on results, it was determined that the unknown belonged to the
Enterobacteriaceae family. Enterobacteriaceae are a very large family of gram negative
facultative anaerobes, and the tests performed on the unknown fit almost all of the criteria to
belong to this family. The goal of this experiment was to ideally identify the bacteria down to
the genus and even species, but more tests would have to be conducted and due to time
constraints this was not possible. The first test (gram stain) showed the first necessary
information needed to continue identifying the bacteria. Once the gram stain results were
indicated by pink colored rods under 1000x bright field, the next step was taken. An OF test
showed that the bacteria was a facultative anaerobe. This was apparent because the bacteria grew
all the way throughout the medium in the test tube. After the OF test was performed, a Catalase
Mellies, Jay. (2008). Bacterial Flagella Stain Protocol. American Society for Microbiology. < http://www.microbelibrary.org/component/resource/laboratory-test/3153-bacterial-flagella-stain-protocol>
OPPapers.com, Joining. "Identification Of Unknown Bacterium #11 - Research Paper - Organza69."
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