Identifying Unknown Bacteria | Essay


The intent of the report is to recognize Unknown #12. The final outcome of Unknown #12's identity was reached after some checks and subcultures were conducted using aseptic techniques. Aseptic techniques are essential to avoid contaminants of the culture being examined. Dr. Burne helped in the final outcome of the identity of the unfamiliar by informing the class of the results for the Oxidase and Lipase exams. And, while both of these tests were not conducted by the students, they were still an important area of the id process.

Procedures & Tests Conducted:

Subculture: The objective of this culture approach is to have a small part of the Unidentified bacterium using aseptic techniques, and transfer it into a tube of Nutrient Broth. This process must be repeated several times (almost daily) throughout the study of the bacterias in order to keep a fresh and real culture of the Unknown. If this procedure isn't repeated, the bacterias will eventually perish from lack of nutrients and can give false excellent results.

Conventional Gram Stain: This differential stain is conducted using aseptic techniques and is utilized to determine whether the anonymous bacterium is Gram Negative or Positive. This stain can also help separate what condition the given bacterium is. Within a clinical setting, identifying whether bacterias are gram negative or positive, helps determine a quicker method of treatment. With regards to the stain itself though, gram negative and positive bacterias both stain purple primarily when the crystal violet (main stain) is applied. However, when iodine (a mordant) is applied, it combines with the crystal violet to create a crystal violet-iodine (CV-I) organic. Once shaped, "this complex is bigger than the crystal violet molecule that got into the skin cells and due to its size, it can not be washed out. " (Tortora, 69) The CV-I is thus maintained in the peptidoglycan level of the gram positive and negative bacterias until Gram's Acetone Liquor (the decolorizer) is applied. The decolorizer eats through the thin covering of peptidoglycan in gram negative bacteria's cell wall structure, allowing the CV-I to flee and triggering the bacteria to become colorless. It's for this reason it becomes necessary to use a counterstain.

The counterstain applied in this procedure is Gram's Safranin, a red/red color dye. When applied though, the counterstain does not have any effect on the gram positive bacterias because the CV-I is retained in its cell wall membrane because of its thicker coating of peptidoglycan in the cell wall. The counterstain is actually applied to give the gram negative bacterias some color. In the long run, when evaluated under a microscope, gram negative bacteria will appear green/red and gram positive can look purple.

In terms of shape, when reviewed under a microscope, gram negative bacterias typically appear as straight rods, while round/cocci shaped bacterias are generally considered to be gram positive bacterias. However, there are a few cocci formed gram negative bacteria as well. Also, it will also be considered, that while form is a feature of bacteria, some bacterias posses the capability to change condition; thus shape must not be strictly relied after for figuring out the unknown bacterias.

Streak Plate: This procedure, a culture strategy, is conducted using aseptic techniques and is utilized to obtain clean, isolated colonies. This procedure is usually performed before any biochemical tests "to be able to adequately examine and characterize. the bacterial kinds. " (Harley, 95) To get the isolated colonies, an inoculating loop can be used to copy the bacteria from a broth pipe to a plate of nutritional agar or triptic soy agar. The inoculating loop is utilized to make about five individual lines, separate in one another. Once the lines are made, the plate's incubated ugly and permitted to develop for 48 time or so and then examined for bacterial expansion. In case the isolation plate is known as to be always a success, the plate is held for use in the biochemical assessments to be run on the bacterium. If the plate is considered of no use, the task is repeated until an effective plate is obtained.

Fermentation of Sugar Tests: This series of biochemical lab tests, preformed with aseptic techniques, is used to check "the ability of microorganisms to ferment sugars. " (Harley, 130) In the event the bacteria possess the capability to ferment the carbohydrate it's examined against, the acid solution produced will lower the pH of the phenol red (pH indication) and the liquid will turn yellowish. To ascertain if the bacterium produces gas from the fermentation of the glucose, an inverted (Durham pipe) is utilized in the same pipe (of sugars). If gas development occurs, bubbles will be present in the Durham tube. For the purposes of the test, the next sugars were analyzed: Sugar, Sucrose, Lactose, Maltose, Mannitol, Sorbitol, Xylose, Dulcitol, and Arabinose.

SIM test: This biochemical test, performed with aseptic techniques, is utilized to check on for the development of Hydrogen Sulfide (H2S) and Indole, as well as Motility. To commence this test, some of the bacterias must be transferred with an inoculating needle from the broth or stable culture to a tube of SIM agar. To get this done, the needle is utilized to stab the advertising. The SIM agar, which "containsferrous ammonium sulfate, FE (NH4) SO4, as an H2S indicator, " (Harley, 151) will produce an "insoluble dark ferrous sulfide precipitate that may be seen over the line of stab inoculation" (Harley, 151) if positive. If negative, you will see no black precipitant visible. In regard to motility, if the bacterium is positive, it will grow from the stab collection; if negative, expansion will only be present in the stab brand. A positive H2S and motility test sometimes ends up with the entire tube turning dark-colored; if this is actually the case another motility test will be needed in order to determine the true motile express of the bacterias.

To check for the production of Indole, a product called Kovac's reagent must be added to the most notable of inoculated pipe. If a scarlet color quickly results after adding about five drops of the Kovac's reagent, this implies a positive consequence, while the insufficient a reddish color, means a negative result. An optimistic end result for the Indole test means that the bacterium contains the enzyme tryptophanase. The presence of this enzyme shows that the bacteria "can hydrolyze tryptophan to its metabolic products, namely indole, pyruvic acid solution, and ammonia. The bacteria use pyruvic acidity and ammonia to gratify healthy needs, " (Harley, 156) while Indole isn't used and is excatly why "it accumulates in the medium" (Harley, 156) and reacts with the Kovac's reagent when applied.

Catalase Activity Test: This biochemical test is used to see if the bacterium "contains the enzyme superoxide dismutase, which catalyses the devastation of superoxide, and either catalase or peroxidase. " (Harley, 172) Essentially this enzyme's presence helps the bacterium to defend itself "against poisonous O2 products. " (Harley, 172) To check for the enzymes existence or lack, hydrogen peroxide (H202) needs to be added to a triptic slant that's been inoculated, using aseptic techniques with the unfamiliar bacterium. Once the hydrogen peroxide's been added, bubbles will appear if positive, or no bubbles can look if negative. Bubbles developing indicate that whenever the H2O2 was added to the medium, it reacted with the superoxide dismutase enzyme and O2 gas was released.

Gelatinase Activity Test: This biochemical test investigations the bacteria's ability "to hydrolyze gelatin by secreting a proteolytic enzyme called Gelatinase. " (Harley, 167) To inoculate the pipe of nutritional gelatin, one must stab the marketing with an inoculating needle which has a portion of the Unknown using aseptic techniques. If the bacterium is positive for Gelatinase activity, the press in the inoculated tube won't be in a good state. The nutritional gelatin will become liquefied. The pipe will stay this way even after being refrigerated for around 30 minutes. However, if negative for Gelatinase activity, the nutritional gelatin that was inoculated will remain a solid. This test can even be found in a clinical setting up to determine "the pathogenicity of certain bacteriaand ability of your bacterium to break down cells collagen and spread throughout the body of a host. " (Harley, 168)

EMB & ENDO Cultures: These two biochemical tests are used to check on the bacteria's ability to ferment lactose. Both testing are performed with aseptic techniques. Together with the Eosin Methylene Blue (EMB) agar, this specific test is mainly intended to isolate and culture the bacterias but also show the bacteria's "reaction to the fermentation of lactose and/or sucrose by microorganisms. " (Electric power, 220) If non-fermenting, the bacterias will be clear or colorless. If fermenting, the bacteria will have a "characteristic green metallic sheen because of the rapid fermentation of lactose. " (Electricity 221) Together with the ENDO, when inoculated, if the blue-black color appears, it indicates that the bacterium is a lactose fermenting organism; however, like the EMB if no color change results, the bacterias is non lactose fermenting.

Phenylalanine Deaminase Test: This biochemical test bank checks the bacteria's capability to remove "amino group, NH3+, from phenylalanine. " (Harley, 199) After the tube has been inoculated using aseptic techniques, add ferric chloride to the plate of agar in track of streak. In case the test is positive, a renewable color will be obvious. If negative, no color change will be observable. A good result indicates that the bacterium "produces the enzyme phenylalanine deaminase, which deaminates phenylalanine producing phenylpyruvic acidity. " (Harley, 200) The ferric chloride (FeCl3) subsequently, combines with the phenylpyruvic acid solution and creates the green color detected.

Oxidase Test: This biochemical test, while using aseptic techniques, is conducted in order to find out whether or not the given bacteria can "produce cytochrome oxidase. " (Harley, 182) The test is conducted with the addition of an oxidase test strip to colonies which may have grown on the plate. In case the strip becomes a dark purple, it's positive. However, a light green color or no change in color shows a negative final result. A positive result signifies that the bacterias go through aerobic respiration because "cytochrome oxidase uses O2 as an electron acceptor. . . in the electron transportation system. " (Harley, 182)

Second Motility Test: This biochemical test is totally used to see if the bacteria's motile or not. To perform this test, some of the bacterias must be transferred from a broth culture to the SIM agar using aseptic techniques. In moving the bacteria, the media has to be stabbed with an inoculating needle. After some time moves, if observation suggests growth away from the stab collection, the test is positive, if there is no change or observation of growth, it's negative. This test is normally used to verify the motile point out of the bacterias observed in the SIM test.

Thioglycollate Test: This biochemical test is checking out for the bacteria's air requirements. Because of this test, the advertising used is Thioglycollate. After the tube of Thioglycollate is inoculated using aseptic techniques, if the bacterium requires oxygen, it'll grow at the top of the inoculated tube. If it requires no oxygen, it'll grow at the bottom. However, if the bacteria's a "facultative anaerobe, it'll grow either aerobically or in the absence of O2. " (Harley, 112)

Urease Activity Test: This biochemical test can be used to determine if the bacterium has the ability to "produce an enzyme called Urease that episodes nitrogen and carbon bond in urea. " (Harley, 187) To find the presence of the Urease enzyme, phenol red is used as a pH signal. After inoculating a pipe of Urea broth with the unknown, using aseptic techniques, if the test is positive, a dark pink or fuchsia color should be apparent. However, if negative an orange color or something apart from a pink color will be noticeable. The red color in a confident consequence, occurs because of "ammonia accumulating in the medium and making it alkaline" (Harley, 188) once urea has been broken down.

IMVIC Test: This biochemical test, conducted using aseptic techniques, is composed of "Indole, Methyl Red, Voges-Proskauer and Simmons-Citrate. " (Harley, 156) The Methyl Red (MR) area of the test is checking to see if the bacterium's a merged acid fermenter or a Butanediol fermenter. The Voges-Proskauer (VP) part of the test is seeking to see if the bacterium ferments glucose. The Citrate part of the test is trying to figure out if "the bacteria can use Citrate as a sole carbon source for its energy needs. " (Harley, 157) Together with the MR test, if it's positive, the red color will stay or intensify. If negative, it will lose the red color. The VP test is positive, if the color other than a watery yellow looks. The VP's negative if no color change occurs. The Citrate test is positive when a profound blue color appears. If color remains green, it's negative. On the side be aware, with the MR and VP checks, the VP test is merely about 70% exact, so if results for the MR and VP exams are negative-negative or positive-positive, only the MR test should be relied on, and the VP test should be assumed to be the opposite result of the MR.

Lipid Hydrolysis: This biochemical test is wanting to find out if the bacterias can "hydrolyze web host cell phospholipids. " (Harley, 145) After using aseptic techniques to inoculate a tube, if the bacterium is lipase positive, a blue color should be there. If negative, a lavender color will remain. A positive end result and the power of the bacteria to use web host cell phospholipids results "the discharge of fatty acids. and can contaminate food products and cause spoilage. " (Harley, 146)

Nitrate Decrease: This biochemical test is checking the ability of the bacterias to lessen Nitrate to Nitrite. If the bacteria have got the enzyme Nitrate reductase, the test is positive and a red color will be observable in the pipe that was inoculated using aseptic techniques. In the event the test consequence is negative, you will see no color change. However, there's hook glitch in this ensure that you if negative, a nitrate reagent must be put into the medium, to check on the effect for verification. The nitrate reagent is a variety of zinc dust and drinking water. Once put into the pipe of nitrate, if the red colorization results after adding the reagent, then that it is a negative test consequence. If still negative, the actual effect is positive, however the bacteria kept seeking degrading nitrate to nitrite and ended up producing ammonia.

Antibiotic Level of sensitivity: This test is merely used to check the ability of the bacterias to grow on or about certain antibiotics. The bacterium is disperse as a grass on a bowl of Mєeller-Hinton agar and the discs of antibiotics are fell into the dish in individual locations. If the antibiotics are successful in avoiding or eliminating development of the bacteria, there will be a definite space about the disk of the antibiotic. In any other case, growth on or about the antibiotic reveals it's not useful in combating development of the bacterias. The antibiotics analyzed against Unknown #12 were Penicillin, Kanamycin, Bacitracin, Doxycycline and Vancomycin.

Antiseptic Awareness: This test is similar to the antibiotic test, except it is checking the power of the bacterias to increase on or around antiseptics. With this test, the bacterium is pass on as a yard on Mєeller-Hinton agar in a dish and paper discs with the antiseptics on them are fell in split places around the plate. If the bacterium's in a position to grow in or around the disc including the antiseptic, this implies that the antiseptic is useless in preventing progress of the bacterias. However, if the antiseptic has no expansion around it, it's successful in inhibiting development of the bacteria. For this test, the antiseptics examined were Listerine (advanced) mouthwash, Hydrogen Peroxide, Germ X, Complete Easy Rub Contact Solution and Equate Antibiotic Hand Soap.

Results of Checks Conducted:

The stand below represents the consequence of each technique and biochemical test conducted.

Test Performed:

Results & Remarks:

  • Gram Stain

Gram Negative (Cocci, bacterias are round in appearance and in chains, but could be groups of direct rods as well with a slight curved appearance)

  • Glucose Fermentation

Positive for Acid; Positive for Gas Production

  • Sucrose Fermentation

Positive for Acid; Positive for Gas Production

  • Lactose Fermentation

Positive for Acid; Positive for Gas Production

  • Maltose Fermentation

Positive for Acid; Positive for Gas Production

  • Mannitol Fermentation

Positive for Acid; Positive for Gas Production

  • Dulcitol Fermentation

Negative for Acid; Negative for Gas Production

  • Xylose Fermentation

Positive for Acid; Positive for Gas Production

  • Sorbitol Fermentation

Positive for Acid; Positive for Gas Production

  • Arabinose Fermentation

Positive for Acid; Positive for Gas Production

  • SIM Test

H2S: Positive, dark-colored precipitant visible

Indole: Negative, didn't turn red with Kovac's reagent

Motile: Positive, progress throughout tube

  • Catalase Activity

Positive, bubbles shaped when hydrogen peroxide was added

  • Gelatinase Activity

Negative, still in solid state

  • EMB & ENDO Cultures

EMB:Clear & Colorless in trace of Streak.

ENDO:Clear & Colorless in trace of Streak

  • Phenylalanine Deaminase Test

Positive; turned inexperienced when citric acid was added in trace of streak

  • Oxidase Test

Negative (Dr. Burne Said all unknown's were Negative for Oxidase)

  • Second Motility Test

Positive, growth away from stab line

  • Thioglycollate Test

Facultative Aerobe. The bacterias grew at the very top, middle and lower part of the tube, but primarily at the very top.

  • Urease Activity Test

Positive, bright green color visible

  • IMVIC Test

MR: Positive, Red-Orange Color

VP: Negative, remained yellow

Citrate: Positive, converted blue

  • Lipid Hydrolysis

Negative (Dr. Burne told the class which were negative &positive).

  • Nitrate Reduction

Positive, however zinc dust with water needed to be added to the medium due to the fact the bacteria had opted too much and produced ammonia.

  • Antibiotic Sensitivity

The only successful antibiotic in preventing expansion of the Unknown was Kanamycin

  • Antiseptic Sensitivity

The only successful antiseptics in combating development of the Unknown were Hydrogen Peroxide, Equate Antibacterial Hand Soap and Listerine (Advanced) Mouthwash.


Based upon the results of the aforementioned tests and procedures that were conducted, I believe Unknown #12's individuality is Citrobacter freundii. This final result is based upon the actual fact that the Order, Enterobacteriales, are "facultatively anaerobic, gram negative rods that are motile-morphologically, the rods are upright and most are active fermenters of blood sugar and other sugars. " (Tortora, 309) This realization was further backed up by the consequence of the Oxidase test as "all members of the family Enterobacteriaceae are oxidase-negative. " (Tortora, 285) The Enterobacteriales are the only Order that matches and concurs with the results of the assessments performed and so is excatly why it's assumed to be the Order.

After deciding the Order, I knew the bacterias belonged to the Gammaproteobacteria Category because of table 11. 1 in Microbiology: an Intro. The bacterium's Family was narrowed down by the result of the Lactose, sugars fermentation test. Tortora areas that "Escherichia, Enterobacter and Citrobacter, which ferment lactose to produce acid and gas, can be distinguished from Salmonella and Shigella, which do not. " (285) Escherichia, and Enterobacter were both eliminated and Citrobacter assumed as the family following the Citrate, Urea and SIM lab tests were complete. Citrobacter is the sole family of the three which has a positive Citrate, H2S, and Urea test. Citrobacter is also the only person of the three people that has a negative Indole test result most of the time.

Furthermore, the willpower of the bacteria's Genus was able to be concluded after carefully analyzing the results of the other biochemical lab tests. Citrobacter freundii, according to Bergey's Manual has "no liquefaction by any strains, " (Breed, 339) and has a good Catalase test final result. These results and summary were further reinforced by the outcome of the Antibiotic Awareness Test. Kanamycin, which was successful in stopping expansion of the bacteria is "a bactericidal antibiotic which operates by inhibiting the synthesis of protein in prone microorganisms. Kanamycin sulfate is effective in vitro against many strains of Staphylococcus aureusand Citrobacter freundii and Citrobacter types that are frequently repellent to other antibiotics. " (Medpedia)

After reading the information of Citrobacter freundii, the studies are inconclusive with one another on the Gram Stain, almost all of the Fermentation of Sugars testing, Gelatinase, Indole, IMVIC, Citrate, SIM, Urea, Nitrate and Catalase testing. Furthermore, the results of each biochemical ensure that you procedure conducted are assumed exact, as there was no contaminants of the culture or test materials used, due to aseptic techniques being exercised with each test and technique performed.

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