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Mallory MacciomeiMay 13, 2011Dr. SchneeMicrobiology

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 bacteria. Identifying this

unknown bacteria from a keyboard will allow scientists to become more knowledgeable in

common bacteria.


Methods

All methods and procedures were performed as found in Johnston 1992, unless otherwise noted.

Isolation of unknown pure culture

Sample of unknown bacteria was taken from a contaminated agar plate. The sample from a

contaminated nutrient agar plate was isolated by the method of streak plating. The sample was

allowed to grow for one week at 32 degrees Celsius in an incubator. The culture was re-streaked

until a plate with uniform colony with uniform colony morphology was obtained. Culture purity

was confirmed by examining the culture for uniform cellular morphology and gram stain.

Characterization of Cellular Morphology

Cellular morphology was determined by looking at wet mounts and gram stains. The wet mount

is a glass slide with a mix of distilled water and the bacteria of interest. By looking at the wet

mount, motility of the unknown can be determined. Cells that appear to be moving on their own

free will (against the “current” of Brownian motion) may be motile. Cells that do not appear to

move on their own (however, they may be moving due to Brownian motion) may be non-motile.

Micrococcus luteus was used as a control in the wet mount process. The slides were viewed

under 1000x phase contrast.


Characterization of Colony Morphology

Colony morphology was determined by looking at a pure culture grown on a nutrient agar plate.

The pure culture was 9 days old and grew at 32 degrees Celsius in an incubator. Colonies were

viewed in plain sight as well as under 1000x bright field. Colonies are placed into certain

categories based on appearance. See results for further description of colony morphology.

Gram Stain

A gram stain revealed cell morphology. Micrococcus luteus and bacillus were used as controls

against the unknown. Gram stains reveal the unknown bacteria to be either gram negative or

gram positive. A gram negative result leaves the unknown bacteria looking pinkish in color. A

gram positive result leaves the unknown bacteria looking purple in color. Observations were

viewed at 1000x bright field.

Oxidation-Fermentation (OF) Test

An OF test was performed to determine how the bacteria used energy. Bacteria can use either

fermentation or oxidation (or both) to use energy. .5 mL of the unknown was placed in 10mL of

medium in a test tube. .5 mL of E. coli was placed in 10 mL of medium in a different test tube as

a control. E. coli was expected to grow throughout the medium because it is a facultative

anaerobe. The caps were loosely placed on each tube and autoclaved for a total of 45 minutes.

The temperature reached 120 degrees Celsius and then was allowed to cool down to 80 degrees

Celsius before removing the tubes. Once tubes were cooled, they were allowed to incubate at 32

degrees Celsius for 7 days. After the incubation period, the tubes were analyzed. (See Figure 8).


Catalase Test

A catalase test was performed to determine if the unknown was catalase positive or negative. A

microorganism is catalase positive if it can “degrade hydrogen peroxide by producing the

enzyme catalase” (Cappuccino et al. 1996). The procedure was followed according to

Cappuccino et al. 1996. A positive test occurs if bubbles appear on the area affected by hydrogen

peroxide. If bubbles do not appear, the test is a negative test. The control used in the catalase test

was nutrient agar.

Oxidase Test

An oxidase test was performed in order to determine if the unknown bacteria was oxidase

positive or negative. A bacteria is oxidase positive if it produces the enzyme cytochrome

oxidase. When the test reagent, p-aminodimethylaniline, is introduced to the unknown bacteria

colonies on the nutrient agar plate, the colony will turn a pink color at first and eventually appear

black in color if the bacteria is oxidase positive. If the bacteria is oxidase negative, no color

change will occur. Pseudomonas was used as a control because pseudomonas results in a positive

oxidase test (color change to dark blue/black). The procedure was followed according to

Cappuccino et al. 1996.


Motility Test

A motility test was performed to further investigate the motility of the unknown bacteria. A

bacteria is motile if it is spread throughout the test tube. It is non-motile if it is only present in the

original inserted area (see Cappuccino et al. 1996 for details on procedure). Escherichia coli (E.

coli) and Micrococcus luteus (M. luteus) were used as controls.

Flagella Stain

The unknown bacteria were stained for flagella according to the methods found in Mellies 2008.

Cells were stained for ten minutes before rinsing and observing under 1000x bright field. E. coli

was used as a control. A positive flagella stain results in one or more hair-like extremities

attached to the bacteria. A negative flagella stain shows no extremities on the bacteria cells.

Figure 5 shows a positive and a negative flagella stain.

UV Light Test

The unknown bacteria were streaked onto two new nutrient agar plates with a cotton-tipped stick

so the entire plate was covered with bacteria. The plates were allowed to grow for 24 hours at 32

degrees Celsius in an incubator. After the 24 hour period, the plates were exposed to UV rays

separately. Plate 1 was exposed for 60 seconds, while plate 2 was exposed for 30 seconds. The

plates were placed upside down over a piece of paper that had a distinct picture of a star cut out

of it. This way, if the bacteria were to die because of the UV exposure, it would be visible

because there would be a star shape of absent bacteria on the plate. After appropriate time of

exposure, the plates were incubated for another 24 hours and allowed to grow at 32 degrees


Celsius. The UV light test is a positive test if the bacteria die and a shape is present on the plate.

The test is negative if no bacteria on the plate is dead.

Antibiotics Test

The unknown bacteria was tested for antibiotic resistance against three different antibiotics:

Erythromycin, Streptomycin, and Kanamycin. Each antibiotic was placed on a pure culture (on a

nutrient agar plate) with forceps and allowed to incubate for 24 hours. All three antibiotics

shared the same nutrient agar plate, but they were separated so as not to interfere with each other.

After the 24 hour incubation period, the plate was observed. If a bacteria is resistant to an

antibiotic, there will be bacteria growth around the antibiotic—no zone of inhibition. If a bacteria

is not resistant to an antibiotic, there will be no bacteria growth around the antibiotic—zone of

inhibition. Zones of inhibition were measured with a ruler in millimeters and recorded. The

Kirby-Bauer chart for zones of inhibition was used to decide how susceptible the bacteria is, if

susceptible at all. Zones of inhibition were acquired according to “Antibiotics 2011”.


Results

Figure 1.

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

(as found in Johnston 1992).


Figure 2. Contaminated Nutrient Agar Plate

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.

Length (uM) 1.5-3.0 2.0

Width (uM) .75-1.0 1.0


Figure 3. Possible OF test results

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 and ResultsTest Result

Gram Stain -Oxidase -Motility +Flagella +

UV -Antibiotic

ErythromycinStreptomycinKanamycin

Intermediate +(Susceptible) +(Susceptible)

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


test showed that the bacteria was catalase positive because of the appearance of bubbles after the

addition of hydrogen peroxide to the bacteria. The Oxidase test showed negative results because

the bacteria did not turn a dark bluish black after the chemical was added. The motility test

revealed that the bacteria was motile, but the flagella stain was very difficult to determine. A

different motility test may have been beneficial in determining the true motility. The UV test

gave negative results, which means the bacteria can tolerate UV rays. This was apparent because

there was no shape left in the bacteria after the test. The plate was still covered in bacteria.

Lastly, the antibiotic test revealed that the bacteria is susceptible to Kanamycin, Streptomycin,

and only minorly susceptible to Erythromycin. This was determined by measuring the areas

around the antibiotic (zones of inhibition). Controls also helped confirm results in all of the tests.

Based on the results of the previous mentioned tests, it was determined that the unknown bacteria

swabbed from a laptop keyboard belonged to the Enterobacteriaceae family. All determination

factors were found in Bergey’s manual (Bergey 1994). Little was determined about the genus or

species; more tests are needed to investigate further. Because this family of bacteria are typically

are part of the “natural flora” of the body, it was probably spread via careless hand washing and

transferred onto the keyboard. More bacteria should be tested and identified from common

places such as door handles and tabletops in order to become more educated in prevention!


Works Cited

"Antibiotics." Biomedical Sciences Graduate Program. Web. 10 May 2011.

<http://pathmicro.med.sc.edu/mayer/antibiot.htm>.

Bergey, D. H., and John G. Holt. Bergey's Manual of Determinative Bacteriology. Baltimore: Williams

& Wilkins, 1994. Print.

Cappuccino, James G., and Natalie Sherman. Microbiology: A Laboratory Manual. 4th ed. New York: Benjamin/Cummings, 1996. Print.

"February 2009." 4126-Spring2009. Web. 09 May 2011. <http://biol4126.blogspot.com/2009_02_01_archive.html>.

Johnston, Gail B. MELE lab manual. Iowa State University 1992, print.

"Kligler." Marietta College. Web. 12 May 2011.

<http://www.marietta.edu/~spilatrs/biol202/labresults/ftg_oxygen.html>.

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."

Free Term Papers, Research Papers, Essays, Book Reports | OPPapers.com. Web. 11 May

2011. <http://www.oppapers.com/essays/Identification-Unknown-Bacterium-11/156786>.

Todar, Kenneth. "Salmonella." Online Textbook of Bacteriology. 2009. Web. 10 May 2011.

<http://textbookofbacteriology.net/themicrobialworld/Salmonella.html>.

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mallorymacciomei.weebly.commallorymacciomei.weebly.com/.../micro_final_write_up.docx · Web viewIt is hypothesized that the unknown bacteria from the keyboard is just a common, harmless