Microbiology Unknown Report

UNKNOWN REPORT

Alyssa BredaAfternoon: TW

Dr. Debra Costa-NinoDue: 1 August 2012

Breda 2

NAME: Alyssa Breda

BACTERIA: Klebsiella pneumoniae and Staphylococcus aureus

SECTION ONE: My Steps.

One: Inoculate a back-up slant of nutrient agar from unknown broth.Purpose: Create a back-up inoculation in case of contamination or loss of media.Observation: Gas production, opaque/cream colored growth.

Two: Make a streak isolation from the unknown broth onto NAPurpose: To isolate separate and distinct colonies in hopes of isolating my different organismsObservations: Separate colonies achieved by third quadrant. One was larger and more cream colored while the other was smaller and more circular and had a more cream color with a tint of yellow in it.Conclusion/Thoughts: Since growth appeared on streaks, both organisms appear non-motile at this point. All colonies have round edges and a convex elevation.

Three: Gram stain from unknown brothPurpose: To see both organisms in one gram stain and to determine if I can observe two different organisms (Gram negative and Gram positive) on the slide.Observation: There is a Gram negative bacillus organism as well as a Gram-positive coccus organism that was arranged in long strands.Conclusion/Thoughts: I have a Gram-negative rod and a Gram-positive cocci.

Four (G-): Make a Gram stain from one of the distinct colonies present on the streak isolation plate.Purpose: To determine if the colony is pure and if so, whether it is gram negative or gram positive.How the test works: Gram stains start with transparent cells that have been aseptically transferred and heat fixed to a slide. Staining with crystal violet (positively charged) stains the cells purple since it is attracted to the negatively charged heads of the phospholipid bilayer. Next iodine creates a mordant by combining with the crystal violet, holding it stronger near the gram-positive cells with the thicker cell wall. Next the decolorizer washes the stains from gram negative while leaving the mordant on gram-positive. The counter stain of safarin then stains the gram-negative cells pink because it is positively charged and is attracted to the negative cell membrane. Thus, gram-negative cells appear pink and gram-positive cells appear purple.

Picture: streak isolation plate. The distinct colonies in toward the middle of the 3rd quadrant show more of a yellow tinge than the opaque colonies toward the edges.

Breda 3Observations: The stain is a pure Gram-negative stain. The organism is a bacillus and arrange in no specific pattern.

Conclusion/Thoughts: I have isolated a Gram-negative bacillus present in the larger opaque colonies of my streak isolation plate

Five (G-): Make a streak isolation plate on Eosin Methylene Blue Agar from the Gram-negative colony on the NA streak isolation plate.Purpose: To determine/verify if the organism is a coliform and whether it is a lactose fermenter, and if so, to what degree. Also to ensure that the organism is not inhibited by eosin and methylene blue.How the test works: Eosin Methylene Blue is a complex, selective, and differential medium. It inhibits Gram-positive growth with eosin and methylene blue. It also reacts with lactose fermenters by turning the growth either pink, dark purple, or black. Green metallic growth also occurs with Escherichia coli specifically.Observations: Good growth, growth started as pink to dark purple at 24 hours, but after 48 hours was a dark purple color. Isolation achieved by third quadrant.Conclusion/Thoughts: The dark purple growth indicates a possible coliform and the Gram-negative rod may be of the Enterobacter or Klebsiella species. The organism also ferments lactose with acid production.

Six (G-): Make an Agar Deep Stab from the EMB platePurpose: To determine the aerotolerance of the organism and to determine if it was the organism that produced gas in the original back-up slant.How the test works: Agar deep stabs are made with Tryptic Soy Agar. This is made with yeast extract to accommodate growth from a broad range of organisms. The oxygen is removed from the

Breda 4medium during its preparation but is slowly diffused back in afterwards. This means there is oxygen near the top of the agar but no oxygen at the bottom. Ergo, the medium is aerobic at the top and anaerobic at the bottom.Observation: Growth at top of agar as well as uniform throughout the medium. Gas production lifted up agar.Conclusion/Thoughts: Results show the gram negative bacillus (probable Enterobacter or Klebsiella) produces gas and is a facultative anaerobe since it grows with and without the presence of oxygen. This gas production indicates fermentative metabolic pathways.

Seven (G-): Catalase Test taken from the EMB plate.Purpose: To determine whether the gram-negative bacillus organism (Enterobacter or Klebsiella) can detoxify hydrogen peroxide into water and gaseous oxygen via the use of the catalase enzyme.How the test works: The catalase test determines a facultative anaerobe’s ability to produce catalase. Catalase is produced by an organism in order to reduce and detoxify hydrogen peroxide (formed in the electron transport chain) into water and gaseous oxygen.Observation: The organism “bubbled”, indicating a positive catalase test.Conclusion/Thoughts: The gram-negative bacillus contains the catalase enzyme, allowing it to reduce hydrogen peroxide to water and gaseous oxygen.

Eight (G-): Gram stain from EMB plate.Purpose: To ensure the culture is still pure and that there are no contaminants.Observation: Small bacilli (about 1um), still in no specific formation. Short and small bacilli.Conclusion/Thoughts: Media is still pure with no contaminants. Organism is a gram-negative bacillus (probably a Enterobacter or Klebsiella) that is approximately 1um in diameter that produces gas from fermentation.

Nine (G-): Nitrate ReductionPurpose: Nitrate reduction helps differentiate gram-negative rods from other of its kind. His test will further narrow my choices for my unknown. I will use the nitrate reduction test to determine if my gram-negative rod can reduce nitrate to nitrite.How the test works: Nitrate reduction deals with anaerobic respiration in gram-negative organisms. This occurs when electrons are passed to an electron acceptor other than oxygen (since it is anaerobic). The nitrate reduction tests for the nitrate reductase enzyme, which converts nitrate (NO3) directly to nitrite (NO2). Another way to reduce nitrate that is tested for is a multi-step process called denitrification. In this, nitrate is converted to molecular nitrogen (N2). The Durham tube in this medium tests for gas production. After incubation reagents A and B are added to the medium to determine whether reduction occurred or not. This is required since this medium contains no color indicator. The reagents will react with nitrite (if present) and will produce nitrous acid, which means that the organism was able to reduce nitrate to nitrite.

Breda 5Observation: There was no indicator of nitrate reduction after incubation and there was no gas in the Durham tube, so Reagents A and B must be added to the medium. After Reagents were added the medium turned red.Conclusion/Thoughts: The test is completed and the red color after addition of the reagents indicates that the organism reduced nitrate to nitrite. I know this because the reagents can only turn the broth red if nitrite is present, which leads to the formation of nitrous acid (HNO2).

Ten (G-): Oxidase TestPurpose: To detect the presence of cytochrome c oxidase, and therefore cytochrome c (the final electron donor in the electron transport chain). This can be useful in distinguishing between oxidase-negative Enterobacteriaceae and oxidase-positive Pseudomonadaceae.How the test works: The electron transport chain takes all of the electrons from the reduced molecules created from the Krebs cycle and glycolysis and uses them to create a proton motive force from passing the electrons down the chain until it eventually reaches the final electron acceptor. In many bacteria, the final electron donor that gives an electron is cytochrome c. The presence of cytochrome c oxidase allows the oxidation of cytochrome c, and therefore the reduction of the final electron acceptor. Cytochrome c oxidase can also catalyze the reduction of cytochrome c as well by the presence of tetramethyl-p-phenylenediamine, the chromogenic reducing agent. This is present on a Q-tip looking structure, which turns a dark color if it becomes oxidized. The oxidase test uses the Q-tip by dabbing it onto the growth of an organism. If the growth changes to a dark color, the cytochrome c oxidase is present.Observation: The culture growth on the end of the oxidase swab did not turn into a dark or black color, therefore indicating a negative test.Conclusion/Thoughts: The oxidase test was negative, meaning the organism may be within the Enterobacteriaceae family, which supports the EMB plate suggestion that the dark purple growth may prove to be an Enterobacter or Klebsiella.

Eleven (G-): Phenol Red – LactosePurpose: This test can be used to differentiate between members of Enterobacteriaceae as well as distinguish them between other gram-negative rods.How the test works: The phenol red lactose broth contains the carbohydrate lactose along with peptone, and phenol red (pH indicator). This turns yellow at a pH below 6.8 and pink above 7.4. It is red in between these pHs. This allows the broth to determine whether acid is produced from fermentation of the carbohydrate, thus lowering the pH and creating a yellow color, or if deamination of the peptone amino acids produces ammonia, therefore raising the pH and turning it pink. In addition to this, Phenol Red tubes contain a Durham tube to indicate gas production during fermentation.Observation: The Phenol Red Lactose tube turned fully yellow and gas was clearly present in the Durham tube.

Picture: Nitrate reduction test after addition of reagents A & B. Indicated the presence of nitrite, which means nitrate was reduced to nitrite by the organism.

Breda 6

Conclusion/Thoughts: The yellow broth with a bubble in the tube indicates fermentation of lactose with acid and gas end products, since the phenol red pH indicator turns yellow when pH is lowered below 6.8.

Twelve (G-): SIM MediumPurpose: This test will allow me to test for the organism’s Sulfur reduction, Indole production, and Motility. It is also part of a series of tests (Indole, Methyl red, Voges-Proskauer, and citrate) that differentiate among members of Enterobacteriaceae. How the test works: The SIM medium tests first for sulfur reduction to H2S because if H2S is produces, it reacts with iron in the form of ferrous ammonium sulfate, which forms ferric sulfide, a black precipitate. Next, Indole production is tested by the presence of tryptophan in the medium, which can be hydrolyzed by tryptophanase into pyruvate, ammonia, and indole. After incubation, Kovac’s reagent reacts with the indole and produces a pink color. Lastly, motility is tested because of the SIM’s reduced agar concentration and the single stab inoculating technique. Motile organisms spread throughout the agar, making it a cloudy appearance as opposed to growth along the single stab line.Observation: The SIM test did not produce a black precipitate, the organism showed growth strictly within the stab line, and after Kovac’s reagent was added, there was no color change.Conclusion/Thoughts: The lack of black precipitate indicates the lack of ability for the organism to reduce sulfur to H2S. The lack of color change after the addition of the Kovac’s reagent indicates that the organism does not break down tryptophan into indole and pyruvate. The lack of growth outward from the stab indicates that the organism is nonmotile.

Picture: SIM test showing negative results for Sulfur reduction to H2S (no black color), Indole production (no pink color), and no motility (no growth outward from stab line).

Breda 7

Thirteen (G-): Simmons Citrate TestPurpose: To test the ability of the organism to use citrate as its sole source of carbon. It is also part of the series of tests (Indole, Methyl red, Voges-Proskauer, and citrate) that differentiate among members of Enterobacteriaceae.How the test works: When citrate, which is used in the beginning of the Krebs Cycle in cellular respiration, is the sole source of carbon in a medium an organism must possess the citrate-permease enzyme in order to break it down and transport the molecule into the cell in order to convert it to pyruvate. Similarly, the Simmons Citrate Agar uses sodium citrate is its sole source of carbon. Ammonium phosphate is used as the source of nitrogen. Bacteria that are able to grow on the media utilize the sodium citrate and convert the ammonium phosphate into ammonia and ammonium hydroxide, which are alkaline and therefore raise the pH of the media causing the Bromthymol blue dye to turn from green to blue, which means it is a positive citrate test.Observation: The growth of the Simmons Citrate Agar was green in the slant but still blue in the butt. The organism grew well on the slant.Conclusion/Thoughts: The green color of the agar indicates a positive citrate test, meaning the citrate is utilized and the organism possesses the citrate permease enzyme. The also means ammonium phosphate was reduces to ammonia and ammonium hydroxide, increasing the pH of the medium. At this point 2 of the 4 tests that are effective at differentiating among members of Enterobacteriaceae family are completed.

Fourteen (G-): Methyl Red and Voges-Proskauer.Purpose: To further differentiate among the members of Enterobacteriaceae. This test is also the last of the series of tests (Indole, Methyl red, Voges-Proskauer, and citrate) that differentiate among members of Enterobacteriaceae. This will help eliminate many contenders for the gram-negative bacillus unknown.How the test works: The Methyl Red and Voges-Proskauer are two separate tests combined in one broth medium. The Methyl Red (MR) test indicated whether or not the organism undergoes mixed acid fermentation. Mixed acid fermentation with produce acid, lowering the pH, which will turn the medium red when Methyl Red indicator dye is added after incubation. If the MR indicator dye is added and the broth is orange or yellow, the test is a negative result. The Voges-Proskauer portion tests for an organism that ferments glucose with a quick conversion of the acid products to acetoin and 2,3-butanediol. This test is done by adding VP reagents to the medium, which oxidizes the acetoin to make diacetyl. The diacetyl then reacts with the guanidine nuclei of the peptone in the medium, making a red color to indicate a positive result.

The Citrate test is positive due to the blue color in the slant. As seen, the butt is still green, showing a good contrast between the two sections.

Breda 8

Observation: After addition of the MR indicator dye, there was no immediate color change of the broth. I added the VP reagents, and then checked at ten-minute intervals for an hour as directed by the instructions. By the 30 minute interval the solution was completely red and stayed that way through the full hour.Conclusion/Thoughts: The yellow appearance of the broth after addition of MR dye indicates a negative result for the Methyl Red mixed acid fermentation test. The red color after an hour after addition of the VP reagents indicates a positive VP test. This means the organism does not undergo mixed acid fermentation, but it does convert its acid end product of glucose fermentation to acetoin and 2,3-butanediol. This completes the series of tests that are effective in differentiating among members of Enterobacteriaceae. This will allow me to eliminate many possibilities and narrow my search for the gram-negative rod. According to the Bergey’s chart for “Family Enterobacteriaceae Lactose Positive” organisms, I now have narrowed down my choices to Klebsiella pneumoniae, Enterobacter aerogenes, Enterobacter cloacae, Enterobacter amnigenus, Erwinia carotovora, or Serratia rubidaea.

Fifteen (G-): Lysine Decarboxylase TestPurpose: This test will narrow down my choices further and hopefully lead me to the discovery of my unknown. This test again will determine if the organism has the decarboxylase enzyme necessary to decarboxylate the amino acid lysine. This will help, again, differentiate among different members of Enterobacteriaceae.How the test works: This medium contains peptone, glucose (for fermentation), bromcresol purple (pH indicator), and pyridoxal phosphate (coenzyme). The medium is purple above a 6.8 pH and yellow below a 5.2 pH. Mineral oil is used to cover the surface of the medium so fermentation occurs as a metabolic process as opposed to cellular respiration. Glucose fermentation by the organism produces acidic products, turning the medium yellow initially. This acidic environment causes organisms to produce the decarboxylase enzyme, if they are capable of doing so. The decarboxylase enzyme then breaks down lysine, and the medium will turn purple due to the production of alkaline substances. If the organism does not produce the enzyme, the medium will remain yellow, indicating a negative result.Observation: The medium was a cloudy purple color after incubation. The cloudy color indicates ample growth of the organism within the culture.Conclusion/Thoughts: The organism tested positive for the decarboxylase enzyme that broke down lysine, since the medium is purple (above a pH of 6.8). It is purple due to the alkalinity of the products produced by the breakdown of lysine. This test has narrowed down my choices to Klebsiella pneumoniae, Enterobacter aerogenes, and Serratia rubidaea. However, Serratia rubidaea has a red-pigment, which my organism does not, so that option is eliminated. My choices are between Klebsiella pneumonia and Enterobacter aerogenes of the Enterobacteriaceae family.

Breda 9

Sixteen (G-): Capsule StainPurpose: I will use this to ensure the organism has a capsule. This test will be used to determine if I am on the correct path. Both Klebsiella pneumoniae and Enterobacter aerogenes are capsulated.How the test works: The capsules that surround bacteria are mucoid polysaccharides or polypeptides that repels most stains. The is stain only colors around the cells, making the capsules visible with a white circle around the cells.Observation: The cells appear to have a white halo surrounding them when viewed under the oil immersion lens.Conclusion/Thoughts: The cells are indeed capsulated, which means I am correct in assuming that my gram-negative bacilli are either Klebsiella pneumoniae or Enterobacter aerogenes of the Enterobacteriaceae family. I believe that my organism is Klebsiella pneumonia since the SIM test proved my organism to be nonmotile, and Enterobacter aerogenes is indeed a motile organism. A final test will be sure to confirm my organism.

Seventeen (G-): Urea Hydrolysis TestPurpose: To determine if my organism can hydrolyze urea. Enterobacter aerogenes cannot hydrolyze urea, while Klebsiella pneumoniae can.How the test works: If an organism has the urease enzyme, it is capable of breaking down urea into ammonia and carbon dioxide. Urea is only produces when the decarboxylation of certain amino acids has occurred. Urea agar contains urea, peptone, potassium phosphate, glucose, and phenol red (pH indicator which is yellow/orange below 8.4 and pink above 8.4). Observation: The organism showed a slightly pink color at the top of the agar where it was inoculated after 24 hours to 48 hours.Conclusion/Thoughts: The organism has a weak urease production, and undergoes weak urea hydrolysis. This leads me to conclude that my organism is in fact Klebsiella pneumoniae since Enterobacter aerogenes does not hydrolyze urea. Enterobacter aerogenes also tests negative for the catalase test, while Klebsiella pneumoniae tests positive. My gram-negative rod has been identified as Klebsiella pneumonia.

Picture: It is difficult to see, but there is a slight pink transformation after 24 hours, indicating that the K. pneumoniae is a slow urea hydrolyzer and a weak urease producer.

Breda 10*Eighteen (G+): Make gram stain from single opaque colony on streak isolation plate from Step Two.

Purpose: To ensure this is a pure, gram-positive cocci culture as seen in the original gram strain from the unknown broth.Observation: The culture is pure. They are a gram-positive cocci but have a different formation than seen in the original gram stain (from the unknown broth). Instead of cocci arranged in thin strands, they are not arrange in clusters with clear stops in the middle of them (can be seen in the picture below).Conclusion/Thoughts: I have obtained a pure culture from the streak isolation plate that I must grow on a new media now. I have a gram-positive cocci arranged in unorganized clusters with spaces between them.

Nineteen (G+): Inoculate a Phenylethyl Alcohol Agar from the opaque colony on the streak isolate plate from Step Two.

Purpose: To encourage growth of my gram-positive organism. This test allows good growth of gram-positive Staphylococcus, Streptococcus, Enterococcus, and Lactococcus.How the test works: PEA is an undefined, selective medium. It only encourages gram-positive growth because the Phenylethyl Alcohol disrupts the gram-negative cellular membrane permeability, allowing substances to freely travel in and out of the cell. This disrupts or halts the DNA synthesis of the gram-negative cells.Observation: My gram-positive organism grew plentifully on the PEA plate and with streak isolation by the 3rd quadrant.Conclusion/Thoughts: My organism, since it grew well on the PEA, is definitely a gram-positive, and it is either a Staphylococcus, Streptococcus, Enterococcus, or Lactococcus. From here I need to find tests that help me differentiate between these types of organisms.

Twenty (G+): Make an Agar Deep Stab from an isolated colony on the PEA plate

Purpose: To determine if this organism also produced gas in the original back up slant from Step One. This will also determine the areotolerance of the organism.Observation: This organism showed growth throughout the stab as well as on the top of the agar. There was no gas production

*This is the beginning of the Gram-positive organism steps. Any tests that I have already explained the process to in the gram-negative steps are not repeated.

Breda 11Conclusion/Thoughts: This organism showed growth very similar to that of my gram-negative, however it did not produce gas. This leads me to conclude that my gram-positive is a facultative anaerobe as well.

Twenty-One (G+): Catalase TestPurpose: To determine if my organism can detoxify hydrogen peroxide into water and gaseous oxygen. This also helps differentiate between catalase-positive organisms and catalase-negative organisms. This will help narrow my search for the gram-positive.Observation: Bubbles were produced as soon as I added hydrogen peroxide to the inoculant on the slide.Conclusion/Thoughts: My gram-positive organism tested positive for the catalase enzyme that detoxifies hydrogen peroxide into water and gaseous oxygen.

Twenty-two (G+): Make a gram stain from the PEA plate.Purpose: To ensure I still have a pure culture of my gram-positive organism and to make sure there are no contaminants.Observation: My gram stain appears the same as it did in Step Eighteen.Conclusion/Thoughts: I do not have a contaminant, and I still have a pure culture.

Twenty-three (G+): Make a streak isolation on a Mannitol Salt Agar from a colony on the PEA plate.Purpose: This test will indicate whether or not my organism can grow on a high concentration of NaCl (Salt), which is able to kill most bacteria. This media is especially useful for identifying Staphylococcus aureus,How the test works: MSA plates contain Mannitol (carbohydrate fermentation), 7.5% NaCl, and phenol red (pH indicator). The media is red from a pH of 7.4 to 8.4, and pink above 8.4. Most staphylococci grow on MSA, however none except Staphylococcus aureus ferment the Mannitol and produce a yellow color.Observation: Almost the entire plate was yellow, except for a red tint in the third and fourth quadrant. The organism grew very well on this MSA plate and there was isolation by the 3rd quadrant.Conclusion/Thoughts: This organism grew in high salt concentrations, meaning it is most probably a staphylococcus. It also fermented the Mannitol which lowered the pH, turning the phenol red yellow. Usually, Staphylococcus aureus is the only bacteria to do this. I may very well have a Staphylococcus aureus as my gram-positive cocci. My gram stain supports this assumption as well, since Staphylococcus aureus appears very similar when compared to it.

Twenty-four (G+): Oxidase Test

Purpose: To determine if my gram-positive (possibly Staphylococcus aureus) has cytochrome c oxidase, and therefore cytochrome c (the final electron donor in the electron transport chain).Observation: My gram-positive inoculant did not turn to a black color on the test swabConclusion/Thoughts: My organism tested negative for cytochrome c oxidase, therefore cytochrome c. This further supports my theory that my gram-positive is Staphylococcus aureus because, since it too is oxidase-negative.

Breda 12Twenty-five (G+): Phenol Red – Glucose Test

Purpose: As a final test to determine if my organism can ferment glucose with acid end products but NO gas production (as Staphylococcus aureus does). How the test works: The phenol red lactose broth contains the carbohydrate glucose along with peptone, and phenol red (pH indicator). This turns yellow at a pH below 6.8 and pink above 7.4. It is red in between these pHs. This allows the broth to determine whether acid is produced from fermentation of the carbohydrate, thus lowering the pH and creating a yellow color, or if deamination of the peptone amino acids produces ammonia, therefore raising the pH and turning it pink. In addition to this, Phenol Red tubes contain a Durham tube to indicate gas production during fermentation.Observation: The broth turned yellow after 24 hours of incubation and there was no bubble in the Durham tube.Conclusion/Thoughts: This finalizes my search for my gram-positive cocci. Since I now have seven tests confirming that Staphylococcus aureus is my unknown gram-positive. The Staphylococcus aureus fermented the glucose with no acid production.

Breda 13

SECTION 2: Summary

For the Unknown Project, I chose a broth culture that had both a gram-negative (G-) and a gram-

positive (G+). I was able to decipher that I had a G- bacillus and a G+ coccus from the first gram stain I

took from the broth. Once I achieved isolation on my streak isolation plate, I gram stained each of the

different colonies to ensure they were pure, which they were. From here, I began testing.

Gram-negative Summary:

First, I decided to do a streak isolation on an EMB plate in order to have a pure culture of my G-.

My G- results on the EMB show that it is a possible coliform of the Enterobacter or Klebsiella species. I

then decided to make an agar deep stab in order to determine the aerotolerance of the bacteria. The

growth showed that it was a facultative anaerobe that produces gas, which lifted up the agar. To further

narrow down my choices I performed a catalase test, which came out positive. This indicated the presence

of the catalase enzyme that allows the organism to detoxify hydrogen peroxide to water and gaseous

oxygen. Next was the nitrate reduction test to determine if my G- would reduce nitrate to nitrite This

would help me narrow my choices even further between other G- rods. The result was a red color; a

positive test for nitrate reduction to nitrite. The oxidase test also narrows my choices between G- rods.

The test result was negative, meaning my bacillus does not contain the cytochrome c oxidase enzyme,

ergo cytochrome c as its final electron donor. Next I chose phenol red-lactose as a test medium. Like the

previous ones, this test will simply help me narrow my choices among the G- rods. The test showed acid

and gas production from lactose fermentation.

Each of the tests done so far has confirmed that my G- is a member of the Enterobacteriaceae

family, and as the EMB plate suggested, probably members of Enterobacter or Klebsiella. I then decided

the most rational decision would be to perform he series of tests most effective at differentiating among

members of Enterobacteriaceae: Indole, Methyl red, Voges-Proskauer, and citrate. The SIM test

determines indole production as well as sulfur reduction and motility. My G- tested negative for all three

of these.. Next, the MRVP test showed a negative result for mixed acid fermentation (methyl red), and a

Breda 14positive result for fermentation of glucose with production of acid end products that are converted to

acetoin and 2,3-butanediol (VP). Lastly, the Simmons citrate agar tested positive for the citrate permease

enzyme, and therefore the utilization of citrate. This series of tests led me to believe I have a Klebsiella

because Enterobacter is motile, which my organism tested negatively for in the SIM agar test.

The last three tests I decided to perform were to validate my assumption. These consisted of a

lysine decarboxylase test, a capsule stain, and a urea hydrolysis test. Each of these tests came out positive.

This means my G- organism produces the decarboxylase enzyme, is encapsulated, and undergoes urea

hydrolysis by use of the urease enzyme. This confirms my assumption that my G- organism is Klebsiella

pneumoniae. Enterobacter aerogenes was unable to produce urease, and tested urease negative. This I felt

was sufficient evidence to support the claim that my G- bacillus is in fact Klebsiella pneumoniae.

Gram-positive Summary:

The first step I took towards discovering my G+ coccus was to make a streak isolation from the

discrete colony on the Phenylethyl Alcohol Agar, which promotes G+ growth by disrupting G- cell

membrane permeability. This test concluded that I have either a Staphylococcus, Streptococcus,

Enterococcus, or Lactococcus for my G+ cocci. Next, I did an agar deep stab, which told me that my G+

was a facultative anaerobe since it grew in both aerobic and anaerobic conditions. A catalase test was then

performed in order to help narrow my choices of organisms. My G+ was able to successfully detoxify

hydrogen peroxide to water and gaseous oxygen. After I did a gram stain to ensure I still had a pure

culture, I made a streak isolation on a Mannitol Salt Agar (MSA). MSA plates only allow growth of

organisms that can survive in high salt concentrations (7.5% NaCl). It also indicates Mannitol

fermentation by turning yellow from a lowered pH from acid end products. My organism had good

growth on this medium as well as fermented the Mannitol, which led me to believe that there was a strong

possibly I could have Staphylococcus aureus, which is known to grow and ferment on MSA.

I then performed two final tests to ensure I was correct in assuming I had Staphylococcus aureus

as a G+ cocci. First, I did an oxidase test, which indicated a negative result for the cytochrome c oxidase.

Breda 15Lastly, I performed a phenol red-glucose test. The results of this test showed glucose fermentation with no

gas production. Both of these tests supported my theory that I had a G+ cocci classified as Staphylococcus

aureus.

SECTION 3: Dichotomous Charts

Breda 16Dichotomous Chart for Gram-negative

Gram-Negative cells?

No (Gram-positive) Yes

In the shape of rods?

No (Cocci) Yes (Bacilli)

Oxidase positive?

No (Enterobacteriaceae)

Able to ferment Lactose?

Yes (Citrobacter, Enterobacter,

Erwinia, Escherichia, Klebsiella, Serratia)

Produce Indole?

Yes (Citrobacter diversus, Escherichia coli, Erwinia chrysanthemi, Klebsiella

oxytoca)

No (The rest)

Unable to undergo mixed acid fermentation? And able to produce acetoin and 2,3-butanediol from

acid end products of glucose fermentation? MR-,

VP+)

No (E. intermedius, C. freundii, S fonticola, K.

pneumonia subsp. ozaenae)

Yes (K. pneumoniae subs pneumonia, E. aerogenes, E. cloacae, E. amnigenum, E. carotovora, S. rubidaea)

Lysine decarboxylase enzyme present?

Yes (K. pneumonia subs. pneumoniae, S.

fonticola, E. aerogenes)

Red Pigment?

No (K. pneumoniae, E. aerogenes)

Motile?

Yes (E. aerogenes)No (K. pneumoniae subsp. pneumoniae)

Yes (S. Rubidaea)

No (E. cloacae, E. amnigenus, E. carotovara)

No (Erdwardsiella, Erwinia, Morganella, Proteus,

Providencia, Salmonella, Serratia, Shigella, Yersinia)

Yes (Aeromonas, Pseudomonas,

Vibrio)

Breda 17Dichotomous Chart for Gram-positive:

SECTION 4: Summary of Pathogenicity

Gram-positive cells?

No (Gram-negative) Yes

Round-shaped cells?

No (bacilli) Yes (cocci)

Able to detoxify Hydrogen Peroxide?

Yes (Micrococcus,

Staphylococcus)

In staph arrangement?

Yes (Staphylococcus)

Can tolerate High salt concentrations?

No (Streptococci,

Lactococci)

Yes (Staphylococci)

Ferment Mannitol?

No (S. epidermis) Yes (Staphylococcus aureus)

No (Micrococcus)

No (S. pneumoniae, S mitis, S. pyogenes, Streptococcus Group B,

enterococcus, Streptococcus)

Breda 18

STAPHYLOCOCCUS AUREUS

Alexander Ogston first discovered Staphylococcus aureus in 1880. Ogston was a surgeon in

Scotland who observed pus from an abscess of a patient under a microscope. Since this type of cocci

formation was already named Staphylococci, he gave it the name of aureus due to the golden color of the

organism. Staphylococcus aureus is a rather well-known bacterial pathogen that can cause a variety of

infections, complications, and diseases. Some of these include styes, sinusitis, osteomyelitis, endocarditis,

pneumonia, meningitis, toxic shock syndrome, boils, carbuncles, furuncles, impetigo, and scalded skin

syndrome. Staphylococcus aureus is a bacteria that is commonly found on the human skin, making it

common in many infections (Todar).

Styes are usually characterized by a painful red bump on the eyelid that occurs as a result of

infection of glands or hair follicles. Staphylococcus aureus is found to be the cause of 90-95% of styes.

Styes are most commonly left to be healed on their own, however antibiotics can be prescribed (Stoppler).

Next, sinusitis is the result of bacteria causing inflammation of the sinuses. This is very common in

people with colds. When the sinuses are blocked, it is a prime location for the growth of bacteria. Acute

sinusitis can be treated with nasal decongestants, while antibiotics are more common when it comes to

chronic sinusitis. Bacteria other than Staphylococcus aureus can cause sinusitis, however Staphylococcus

aureus is indeed a bacterium known to causes this infection (“Sinusitis”).

Endocarditis is a more serious condition that involves inflammation of the cardiac tissue due to an

infection caused by bacteria or fungi. Drugs that are often used to cure this infection involve penicillin,

vancomycin, and aminoglycosides (Keys). Osteomyelitis is also a more serious infection. The bacteria

travel via the bloodstream and eventually infect the bones, causing infection. This can occur in people of

all ages, however can be treated with splinting the bones, prescribing antibiotics, or performing surgery on

the infected area (Cunha).

Pneumonia, toxic shock syndrome, and meningitis are some of the more commonly-known

infections of Staphylococcus aureus. Pneumonia is characterized by an infection of the lungs, which

Breda 19leads to cold-like symptoms followed by a more serious fever, cough accompanied by sputum, and

shortness of breath. Antibiotics are prescribed to treat pneumonia, and there are also yearly flu vaccines

available for prevention (Schiffman). Next, toxic shock syndrome in induced by a toxin produced by

Staphylococcus aureus. This can be a fatal condition, leading to cases of death in almost 50% of cases.

Treatment for TSS can involve antibiotics, IV, dialysis (if necessary), and drainage of infected area

(“Causes…”). Meningitis is another condition that can be caused by Staphylococcus aureus. It involves

inflammation of the membranes that protect both the spinal cord and the brain. It was very fatal before

antibiotics were used, however the fatality rate has dropped to about 15% with the prescription of

antibiotics as treatment (“Meningitis…”).

Boils, carbuncle, and furuncles are, like styes, due to infection of a hair follicle. They are

characterized by hurtful, red-colored sores on the skin that usually drain pus. Usually these heal on their

own but antibiotics may be used for more serious cases. Drainage may also be necessary depending on

how severe the condition is ("Folliculitis”). Similar to these is impetigo, which is also an infection of the

skin. There are two forms of this: non-bullous and bullous. Non-bullous impetigo appears as s rash on the

skin composed of small red lesions that blister and scab over within about a week or two. Bullous

impetigo separates the epidermis from the dermis, leading to a blister, which eventually burst, leaving raw

skin exposed. This also heals on its own, however both types can be treated with topical antibiotic

ointments (Mersch). The same toxin that produces impetigo also causes scalded skin syndrome. This is

most common in newborns, and also results in a large area of the skin being covered with a blistering

rash. This, too, can be treated, however is more serious in the rare event that it affects adults

(“Staphylococcal”).

Treatments for infections are usually not difficult, unless the infection is cause by a resistant strain

of Staphylococcus aureus, abbreviated MRSA or VRSA. MRSA stands for Methicillin Resistant

Staphylococcus aureus, and VRSA is the same except with vancomycin. This makes it extremely hard to

rid the patient of the infection, since the strain is resistant to most antibiotics used. HA-MRSA (Hospital-

Breda 20associated MRSA) usually occurs within patients who have lowered immune systems. The most common

of these infections include those of the bloodstream, surgical site infections, and pneumonia. A healthy

person touching an infected item or person commonly transfers this condition. CA-MRSA (community-

associated) is usually skin infections limited to boils, lesions, and abscesses. These are easier to treat,

however some specific cases have known to be fatal (Todar).

Earlier this year, there was an outbreak of a Staphylococcus aureus infection at a high school in

New Mexico. Thirteen student had skin infections thought to have been caused by the gym mats used by

both football players and cheerleaders. One cheerleader tested positive for MRSA, but luckily was being

treated with specific antibiotics. All student recovered fully, however this case of outbreak shows how

easily transmitted the infections can be (“MRSA…”).

Recently antimicrobial resistant, such as MRSA cases, are becoming a big threat to public health.

New research shows that “chemically produced antimicrobial agents” may be more effective in testing

these resistant bacteria. They are called “antimicrobial peptides” and are thought to be more effective due

to their unique effect against bacteria. Since they do not act in the same way as conventional antibiotics,

researchers think that this may work more efficiently at ridding an infected person of the resistant strain of

bacteria (“New…”).

In conclusion, Staphylococcus aureus is very common on, in, and around the human body, leading

it to cause many infections and diseases. Although many of them were once very fatal, they can now be

treated with antibiotics with a very low risk of mortality. Conditions such as styes, sinusitis, boils,

carbuncles, furuncles, impetigo, and scalded skin syndrome will eventually heal on their own, however

the more serious infections such as osteomyelitis, endocarditis, pneumonia, meningitis, and toxic shock

syndrome may need antibiotics in order for the infected person to become healthy again. MRSA

infections are much harder to treat, since this includes a resistant strain of the bacteria, and only certain

drugs are effective against it.

Breda 21KLEBSIELLA PNEUMONIAE

Klebsiella pneumoniae was first discovered in 1882 by Frielander C. Uber. It was named after

being one of the bacteria that cause pneumonia. It is a resident microbe in our digestive tract, making it a

part of the human natural flora. It can, however cause Klebsiella pneumonia, urinary tract infections,

septicemia, and meningitis. K. pneumoniae is an excellent pathogen due to the fact that it is capsulated.

(“Klebsiella Pneumoniae”).

Klebsiella pneumonia is a type of pneumonia cause by bacterial infections. It is similar to regular

bacterial pneumonia, however this is specifically cause by Klebsiella pneumoniae. Tell-tale signs include

inflammation of the lungs, cough with red-colored sputum, and fever. Klebsiella is usually a known

nosocomial infection, but can occur outside of the healthcare setting. Klebsiella pneumonia usually only

develops in already immune-compromised individuals. There are strains of this that have adapted and

become resistant, making it more difficult to treat. If untreated, Klebsiella pneumonia can cause more

serious problems. Treatment methods include antibiotics, which vary for each case depending on the

strain of the bacteria and whether it is a resistant strain or not (“Klebsiella…”).

Urinary tract infections are most commonly found in older adults, and are predominantly the result

of an E. coli infection. Klebsiella pneumoniae is the second leading cause of urinary tract infections,

however. UTI’s are also more common in healthcare settings, making it a common nosocomial infection

caused by poor hand washing of hospital staff, or infected items such as a urinary catheter. They are

usually characterized by painful urination, cloudy or bloody urine, cramping/pain in lower abdomen, or

the feeling of having to urinate. UTI’s can lead to bladder and kidney infections as well. Pain medication

and antibiotics are often used to cure the infection and relieve an discomfort (“Causes…”).

Septicemia can also be cause by Klebsiella pneumoniae. It is an infection of the blood that occurs

as a result of the bacteria traveling through the bloodstream. Fevers, decreased urination, nausea and

vomiting, diarrhea, and rapid breath are many symptoms of the infection. If untreated, sepsis may cause

Breda 22damage to vital organs. Treatment is necessary and usually consists of antibiotics accompanies by IV

fluids and oxygen supplement (“Sepsis…”).

Lastly, Klebsiella pneumoniae can cause meningitis. This can be very fatal is not treated early on.

As stated before, bacterial meningitis is a bacterial infection causing inflammation of the membrane

surrounding the brain and spinal cord (“Meningitis”). This only develops once the bacteria are already in

your body’s bloodstream. While traveling in the bloodstream, the bacteria reach the brain, and so beings

the infection. This can be spread through a simple cough, and thus is very contagious. There are

antibiotics prescribed to treat those infected, if caught early enough. Bacterial meningitis is said to be

fatal in 10% of the cases (“What…”).

Resistant strains of Klebsiella pneumoniae are become more prevalent in today’s society. A

specific strain called ESBL is becoming troublesome. In August of 2008, a man who was previously

treated for a gunshot wound to the lumbar region and who also had torn and shredded meninges was

admitted for a fever and leakage of cerebrospinal fluid. He tested positive for this resistant ESBL K.

pneumoniae and did not show progress after being treated with many antibiotics including ceftriaxone,

ciprofloxacin, teichoplanin, and pipracillin. With an alternation of many antibiotics his CSF finally tested

negative and he was discharged. This outbreak, however, shows how serious not only a  Klebsiella

pneumoniae infection of the meninges is, but also how difficult it is to treat resistant strains of bacteria

(Wadi).

In accordance with the development of new strains, researchers are attempting to determine the

genes that make Klebsiella pneumoniae resistant in order to hopefully devise a way to stop it from

happening. Thomas Russo says that the final goal of the research is to discover enough in order to

produce a vaccine for prevention or at least a better treatment to more effectively cure the infection

(“Klebsiella…”).

In conclusion, Klebsiella pneumoniae is a bacterial pathogen capable of creating life-threatening

infections if not treated effectively. Although it is a resident microbe in human bodies, if it grows out of

Breda 23control and enters the bloodstream or urinary tract it can cause serious infections. Sepsis is particularly

dangerous in the event that the bacteria travel to other parts of the body, such as the brain (cause of

meningitis). Resistant strains are also developing, making the infections harder to treat than they already

were. Research, however, is being done in hopes to find new ways to prevent and cure these infections.

Works Cited

"Causes, Incidence, and Risk Factors." Toxic Shock Syndrome. Ed. A.D.A.M. Editorial Board. U.S.

National Library of Medicine, n.d. Web. 30 July 2012.

<http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001676/>.

Breda 24

"Causes, Incidence, and Risk Factors." Urinary Tract Infection. Ed. A.D.A.M. Editorial Board. U.S.

National Library of Medicine, n.d. Web. 30 July 2012.

<http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001549/>.

Cunha, John P. "Osteomyelitis (Bone Infection) Symptoms, Treatment, Diagnosis." MedicineNet. N.p.,

n.d. Web. 30 July 2012. <http://www.medicinenet.com/osteomyelitis/article.htm>.

"Folliculitis, Furuncles, and Carbuncles." About.com Dermatology. N.p., n.d. Web. 30 July 2012.

<http://dermatology.about.com/od/infectionbacteria/a/foll_fur_carbun.htm>.

Keys, Thomas F. "Infective Endocarditis." Cleveland Clinic. N.p., n.d. Web. 30 July 2012.

<http://www.clevelandclinicmeded.com/medicalpubs/diseasemanagement/infectious-disease/

infective-endocarditis/>.

"Klebsiella Pneumoniae 'superbug' Is Being Studied." Medical Press. N.p., 17 Feb. 2012. Web. 30 July

2012. <http://medicalxpress.com/news/2012-02-klebsiella-pneumoniae-superbug.html>.

"Klebsiella Pneumoniae Urinary Tract Infection." Klebsiella Pneumoniae Urinary Tract Infection. N.p.,

n.d. Web. 30 July 2012.

<http://klebsiella-pneumoniae.org/klebsiella_pneumoniae_urinary_tract_infection.html>.

"Klebsiella Pneumoniae." Wikipedia. Wikimedia Foundation, n.d. Web. 30 July 2012.

<http://en.wikipedia.org/wiki/Klebsiella_pneumoniae>.

"Meningitis Treatments and Types." WebMD. N.p., n.d. Web. 30 July 2012.

<http://children.webmd.com/vaccines/understanding-meningitis-detection-treatment>.

"Meningitis, What Is Meningitis?" Illinois Department of Public Health. N.p., n.d. Web. 30 July 2012.

<http://www.idph.state.il.us/public/hb/hbmening.htm>.

Mersch, John. "Impetigo Pictures, Treatment, Causes, Symptoms, Prevention -

MedicineNet." MedicineNet. N.p., n.d. Web. 30 July 2012.

<http://www.medicinenet.com/impetigo/article.htm>.

"MRSA Outbreak Strikes at High School." WebWire. N.p., n.d. Web. 30 July 2012.

<http://www.webwire.com/ViewPressRel.asp?aId=151532>.

"New Class of Chemically Produced Antimicrobial Agents Could Become Future Infection Treatment

Alternative." New Class of Chemically Produced Antimicrobial Agents Could Become Future

Breda 25

Infection Treatment Alternative. N.p., 20 July 2012. Web. 30 July 2012. <http://www.news-

medical.net/news/20120720/New-class-of-chemically-produced-antimicrobial-agents-could-

become-future-infection-treatment-alternative.aspx>.

Schiffman, George. "Pneumonia Symptoms, Treatment, Vaccine - MedicineNet." MedicineNet. N.p., n.d.

Web. 30 July 2012. <http://www.medicinenet.com/pneumonia/article.htm>.

"Sepsis or Septicemia (Blood Infection) Symptoms, Causes, Treatments." WebMD. WebMD, n.d. Web.

30 July 2012. <http://www.webmd.com/a-to-z-guides/sepsis-septicemia-blood-infection?page=2>.

"Sinusitis." University of Maryland Medical Center. N.p., n.d. Web. 30 July 2012.

<http://www.umm.edu/patiented/articles/what_causes_sinusitis_000062_2.htm>.

"Staphylococcal Scalded Skin Syndrome: Diagnosis and Management." National Center for

Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 30 July 2012.

<http://www.ncbi.nlm.nih.gov/pubmed/12627992>.

"Staphylococcus Aureus." Staphylococcus Aureus. N.p., n.d. Web. 30 July 2012.

<http://textbookofbacteriology.net/staph>.

Stoppler, Marissa C. "Sty (Stye, Hordeolum) Home Remedy, Treatment, Causes, Symptoms and

Prevention by MedicineNet.com." MedicineNet. N.p., n.d. Web. 30 July 2012.

<http://www.medicinenet.com/sty/article.htm>.

Todar, Kenneth. "Staphylococcus Aureus." Staphylococcus Aureus. N.p., n.d. Web. 28 July 2012.

<http://textbookofbacteriology.net/staph_5.html>.

Wadi, Jamal A., and Fadi Selawi. "Footnotes." National Center for Biotechnology Information. U.S.

National Library of Medicine, n.d. Web. 30 July 2012.

<http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2813643/>.

"What Is Meningitis? Bacterial, Viral, and Fungal Meningitis Causes." WebMD. N.p., n.d. Web. 30 July

2012. <http://children.webmd.com/vaccines/understanding-meningitis-basics>.