Unknown Culture

The following tests according to the lab manual were performed: gram stain, fermentation tubes, methyl red, vogues proskauer, sulfur, indole, motility and growing it up on MacConkey agar. The gram stain was performed incorrectly the first time. This is because the decolorizer was not on the bacterium slide for long enough, giving a false outcome.

The first result of importance was the result of the Gram stain. The observations of the unknown bacteria from the slant culture after Gram staining showed that the unknown bacteria were Gram negative bacilli (Image 1). After determining the unknown bacteria was Gram negative, an oxidase test was conducted on a sample from the slant culture. The cotton swap with the sample of bacteria did not change color when the oxidase reagent was applied, thus providing a negative result. With a negative oxidase test, further tests were conducted to determine various characteristics of the unknown bacteria. A MR-VP broth was inoculated with a sample from a slant culture of unknown bacteria. After incubation, the methyl red reagent was added to the broth, and the broth turned red, providing a positive result (Image 2). An EMB agar streak plate was inoculated with a sample from a slant culture of the unknown bacteria, and after incubation, growth was found on the plate, providing a positive result (Image 3). A Citrate agar slant was inoculated, and after incubation, growth was found on the media, providing a positive result (Image 4). A Urea agar slant was inoculated, and after incubation, the agar had changed from a peach color to a bright pink color, providing a positive result (Image 5). Using the flowchart (Figure 1) developed from the Table of Expected Results, the lab partners started at the oxidase test. Given the negative result of the oxidase test, the flowchart is

Each of the following procedures were done separately to identify the possible organisms from both regions. The first procedure conducted was the catalase test. The catalase test was conducted by transferring ½ of the CFU to a clean microscope slide using a sterile loop. Second, a small drop of H2O2 was added to the CFU to identify a positive or negative result.

I inoculated an Eosin-Methylene Blue (EMB) agar plate and a MacConkey agar plate using Alderson’s streak plate technique (Alderson, 2015, pp. 64-65). Next, using Alderson’s procedure for aseptic techniques for inoculation (Alderson, 2015, p. 53) along with the procedures for each individual test I conducted the biochemical tests on 1 lactose Durham broth (Alderson, 2015, p. 267), 1 glucose Durham broth (Alderson, 2015, p. 267), 1 TSIA slant (Alderson, 2015, p. 267), 1 SIM agar (Alderson, 2015, p. 268), 2 MRVP broths (Alderson, 2015, p. 268) and 1 citrate agar (Alderson, 2015, p. 268). I also tested my microbe’s ability to produce oxidase (Alderson, 2015, p. 267). Finally, I tested for the presence of catalase (Aryal, 2015, para.

This test is used to see if an organism can perform fermentation. The medium for the Phenyl Red test is differential and is prepared as a base to which a carbohydrate is added (Leboffe and Pierce, 2012, p.245). The substrate is dextrose, lactose and fructose. The indicator is phenol red which turns yellow below a pH of 6.8, red to magenta above a pH of 7.4 and red in between pH 6.8 and 7.4. The enzymes include sucrase, B-glactosidase, galactokinase, hexokinase, fructokinase, phosphogluco isomerase, epimerase, phosphohexose isomerase, triosephosphate isomerase, and phosphofructokinase. Durham tubes are added to each tube to trap a portion of any gas that is produced (Leboffe and Pierce, 2012, p.245). Acid production from fermentation of the carbohydrate lowers the pH below the neutral range and turns the medium yellow. Deamination of peptone amino acids produce ammonia (NH3), which raises the pH and turns the broth a pink color (Leboffe and Pierce, 2012, p.245). A positive result will be yellow in color because acid production from fermentation of the carbohydrate lowers the pH below the neutral range of the indicator. Gas that is produced is indicated by a bubble or pocket in the Durham tube where the broth has been displaced (Leboffe and Pierce, 2012, p.245). The presence of a gas bubble will indicate that the organism can perform fermentation and is also considered a positive

After observing the results that were conducted on the unknown #222. It was determined to be Micrococcus luteus. Most of the tests observed on M. luteus had a match with the expected result, except for two tests. Those tests were the determination of glucose fermentation, and the oxidase test.

The simmon’s citrate is use to determine wether the microorganism uses citrate as the carbon source for their metabolism. If organism utilize citrate then positive result is indicated by color change from sea green to blue. However, an unknown microorganism stayed sea green color which is negative for this particular test. In h2s test, cysteine is degrade by cycsteine desulfarase enzyme into h2s and pyruvic acid. The positive result shows the black butt in the bottom of the tube for an unknown organism which indicated the production of hydrogen sulfide. whereas, negative results remains yellow. The urease test played vital role in identification of an unknown microorganism. After performing urease test on an unknown microorganism, Out

The final examination of the samples through the DNase test showed only one bacterial species to be associated with DNase production. S. Aureus had a very predominant transparent ring around the grouping of colonies. It was also found that S. Aureus turned bright yellow while growing on the plate. All other samples had no change and showed normal growth. A table of all the results can be found directly

It is also shown to not ferment lactose. It does not use the mixed acid fermentation or butylene glycol pathways during breakdown of glucose. According to the gram-negative table, this indicates that the unknown gram-negative microbe is Proteus vulgaris (5). Comparing these results to known biochemical characteristics, all results shown are consistent except for the methyl red test, which is supposed to be positive, indicating that the bacterium does use the mixed acid pathway during breakdown of glucose. This error could be due to contamination, incorrect amount of methyl red indicator added, or not being incubated for a long enough

The biochemical tests used to identify the unknown bacteria included: phenol red broth test (glucose, lactose, and sucrose), methyl red and Voges-Proskauer test, citrate test, indole test, H2S production test, urea test and motility test. The PR broths are used to differentiate members of Enterobacteriaceae and to distinguish these members from other gram-negative rods. The PR broths are useful in determining a bacterium’s fermentation abilities. If the bacterium is able to utilize the carbohydrate, then the acid production from fermentation lowers pH, which in turn turns the broth yellow. However, if the bacterium is unable to utilize the carbohydrate, but does utilize the peptone, which produces the by-product ammonia, the medium will have a rise in pH turning the broth pink.

When cultured on a Mannitol Salt Agar plate, we looked to observe if the isolate fermented mannitol. It was determined that the isolate ferments mannitol because of the yellow colonies that grew. The next test in this battery tested the presence of Catalase which is an enzyme that protects cells from damage by hydrogen peroxide by breaking it down into water and oxygen. The oxidase test which determines the presences of cytochrome oxidase determines if a bacterium can use oxygen as an energy source. The isolate tested positive for catalase but negative for oxidase. The final test to be conducted was to observe the possible lysis and degree of lysis caused by the unknown isolate. On a BPA plate, the unknown isolate lysed the surrounding red blood cells

After a couple days of incubation, the first test conducted for analysis was a gram stain. The gram stain is used to determine the cell shape and color of the bacteria cell. It can also determine whether the bacterium is positive or negative. If it is a positive bacterium, it will look purple when viewed under a microscope. If it is a negative bacterium, it will look pink. The rod shape is usually long in a cluster chain. The cocci shape is a small circle or a cluster of grapes. The next test that was conducted was the KOH test. A large inoculate of the organism was placed in the center of a slide and a drop of KOH in a corner. The two were carefully mixed together by dragging the KOH into the organism. This step was done to determine whether the test was gram-positive or gram-negative. KOH lyses the gram negative cells and results in a sticky and stringy substance while the gram- positive remain watery in appearance. Once the KOH test was complete the SIM medium test was conducted, this test was done to determine three separate tests in one test tube. We are looking for the sulfur reduction, indole production, and motility of the organism. First, a SIM media tube is prepared, and then label the SIM tube with your initials and the bacteria you’re using. Next, use a needle to pick an isolated colony from the plate. Then, inoculate the tube by making a single straight stab down the middle

cream colored colony. After determining that it was a Gram negative rod, an oxidase test was

For the Glucose/Dextrose, Lactose, and Sucrose test, we tested to see if the unknown microorganism could ferment with the specific carbohydrate that was being tested (Phenol). In doing so, we had received three test tubes with the specific carbohydrate in it, and aseptically inoculate each of the three test tubes with our specific unknown organism with a positive test showing a yellow liquid and a negative test staying red as it was in the beginning (Phenol). My results were positive for the glucose and negative for both Lactose and sucrose. The Catalase test tested for the ability of the organism to convert hydrogen peroxide to water and oxygen gas (Catalase). To test this a drop of hydrogen peroxide was placed on a slide then an isolated colony was placed in the drop, and a positive test would cause bubbles to occur, and a negative test would have no bubbles (Catalase). My result was positive with bubbles. The Oxidase test was testing the presents of Cytochrome Oxidase, which transfers electrons to oxygen, by placing a colony of the organism on a sterile swab and then placing a drop of oxidase reagent on it (Oxidase). If the colors of the swab change to a purple color, this indicated a positive test because electrons are being removed, and a negative test will have no color change (Oxidase). My results were positive with a color change to purple. The Sulfate Indole Motility tested for two things, Hydrogen Sulfide (H2S) and Indole. Some microorganisms will use indole to

Organism one demonstrated irregular, flat, wavy, medium sized and cream coloured colony morphology when viewed on agar plate, however limited conclusive data can be obtained from these observations alone. In order to provide further information for diagnosis, additional culturing was conducted on a range of selective and differential media types including Horse Blood, MacConkey, Mannitol Salt and Brilliant chromogenic UTI agar’s. This demonstrated the organism was non-haemolytic, lactose fermenting, as evident through a red colony formation on the MacConkey agar, and unable to survive in high salt concentrations, as indicated through the lack of growth on Mannitol Salt agar. The