My Unknown Tube

An unknown bacterium was handed out by Dr. Honer. The appropriate tests were prepared and applied. The first procedure that was done was the gram stain. Under a microscope, if the gram stain is purple, the bacterium is gram positive, if the stain is red, it is gram negative. The next test was the fermentation tests for glucose, sucrose and

The colonies were smooth, translucent, and had a white brownish color. The Gram stain resulted in Gram positive cocci. After the Gram stain was completed, the bacteria were streaked on a Mannitol-Salt Agar plate and a Catalase test was performed. After these test were completed a Phenol Red Dextrose Fermentation tube was inoculated, and a SIM Tube inoculated.

Preliminary studies help identify Genus species of bacteria. Two different preliminary study pathways must be used since two different pathogens were found in the sample. A dilution and a quadrant streak are the ideal methods to separate pure cultures of bacteria. MacConkey Agar and CAN (MAC) is a selective media that is used for the cultivation of gram negative bacteria. (PEA) is a selective media that is used

The purpose of this lab was to identify an unknown microorganism using lab techniques. The importance of identifying microorganisms is essential to the survival of humans, expansion of modern day medicine and improvement of quality of life. In 1884, Hans Christian Gram designed a differential staining technique to identify bacteria that would change the future of microbiology. He give rise to a staining process, known as the Gram stain to differentiate microorganisms into two groups between positive and negative gram staining microorganisms. The Gram stain is essential in a lab technique as it distinguishes the cells based on the physical properties of the individual cell walls, and is almost always the first test preformed to differentiate a microorganism. The identification of weather a microorganism is gram positive or negative can revel the bacteria’s virulence, cell wall structure, resistance to antibiotics, resistance to physical disruptions and so much more. In order to identify the unknown provided, unknown #27, the Gram stain was the first test preformed. After discovering that the unknown bacterium was indefinitely a gram positive microorganism, the vast possibilities were narrowed down. However, In order to more definitively identify the unknown, the next step was to preform biochemical tests. A biochemical test identifies metabolic

Carbohydrate testing utilizes the different ways that bacteria metabolize different sugars by inoculating different broths with the test bacteria and seeing if there is a change in acidity and/or if any gases are produced. For glucose testing, we would check to see if gas is produced and if the ph of the broth solution drops for a positive test result. For lactose, we check to see if the ph drops or becomes more acidic. For sucrose we check the same was as for lactose but use a sucrose solution instead. A negative result would mean that the solution contained a base/alkali ph.

To start off unknown #3, I picked out O17 from rack A and wrote down my name, date, class, and I first noticed the broth was a light yellow that was quite cloudy or milky. Soon later I streaked my organism onto a TSA plate and incubated it at 37oC for 24 hours, t was almost a perfect plate. There was no contamination; I could tell it there was only one species on the plate and that it was okay for me to move on. There were many isolated colonies in quadrants 3 and 4. The colonies were small and white in color, it was not translucent. I then began to make my 2 slants, inoculating a straight line on each slant, I incubated it at 37oC for 24 hours. The next day, I made sure my slants were pure and also there was a thick line of organism where I had inoculated with a yellow-white color. Now I was ready to begin making my slides and start staining. I used E. Coli and S. Aureus next to my unknown to help me

The test showed a clear zone around the colonies thus determining that the bacteria can break down starch meaning it was a positive result. With a positive starch hydrolysis result the next task was to perform a VP. The results from this test came back negative because there was no color change in the broth. My next step was to check for a swollen cell. I found that the cell was swollen looking from the spore stain slide.

The bases of this experiment was to discover the identify of the unknown from three possible specimens: Klebsiella pneumonia, Escherichia coli, and Enterobacter aerogenes. Utilizaing the T streak technique, the bacteria was isolated into pure colonies for further study. The Gram Stain method was used to identity the morhphology of the bacteria such as the shape and whether the bacteria was Gram positive or Gram negative. Biochemical test were also used to help identify the unknown bacteria. The biochemical test used was the Triple Sugar Iron Agar, Sulfur Indole Motility test, Methyl Red test, Voges-Proskauer test, Citrate test, Urease test, and the Gelatin test. After observing the morphology of the bacteria using the Gram Stain method and utilizing all the possible biochemical test, the bacteria was identified to be Enterobacter aerogenes.

The students will be able to analyze the size, margins, elevation, and color of their bacteria.

The first step in identifying my organism was to put it on a slide, stain it, and look at it under the microscope. I used the gram stain to figure out whether my organism was gram-positive or gram-negative. When I looked into the microscope it was all pink so I determined that it was gram-negative. The organism was bacillus in shape and was single and diplo. Then I determined that I had to focus on tests specific to gram-negative organisms. The tests I chose to perform were: OF glucose test, Citrate test, H2S test, Indole test, Motility test, and Urease test. The OF glucose test concluded that the organism was a fermenter. The citrate, H2S, and Urease tests were all positive. The Indole test was negative. The organism is motile. The unknown organism is Proteus mirabilis.

The bacteria that was contained within Unknown tube #12 is believed to be Pseudomonas aeruginosa, Figure 1. The bacteria tested to be Gram Stain negative, producing a pink, red color retained from the staining process. When the species of bacteria was plated on nutrient media, the cells produced an irregular and spreading configuration as shown in Figure 2. This same plating test provided the margins and elevation, lobate and hilly, respectively. The specimen was stabbed in a Fluid Thioglycollate Medium (FTM) tube using an inoculated loop of the bacteria. The results of this experimentation indicate the type of oxygen requirement of the bacteria. The test found the bacteria to be aerobic as colonies of the bacteria began to form along the top of the FTM tube (Manual 2017).

The TSIA test results after 24 hours was positive for gas production, lactose and glucose fermentation. There were several bubbles in the slant, and the slant and butt both changed to a yellow color, which indicates the lactose (slant) and glucose (butt) fermentation. Sulfur reduction was not present since the slant did not change to a black color. This test also indicates that the unknown bacteria is a facultative anaerobe due to the butt fermenting glucose.

Durham tubes differentiate a microorganism’s ability to ferment sugar (mannitol, dextrose, and lactose) that may produce a gas, but acidic byproducts will turn solution yellow. Unknown 6 was yellow in all three test tubes suggesting acidic conditions (fermentation occurred), however lactose and mannitol were cloudy, dextrose remained clear. There was no gas present in any of the Durham tube. Urease broth determines if a organism can hydrolyze urea with urease. It contains urea, nutrients, pH buffers, phenol red (indicator). Unknown 6 tested negative because it remained yellow and clear, therefore the pH did not rise, because no acid was produced from hydrolyzing urea. Sulfur Indole Motility Media (SIM deep) differentiates microorganisms that reduce sulfur, produce indole, and are motile. H2S is reduced by cysteine catabolism, or thiosulfate, and HCl, p-dimethylaminobenzaldehyde, n-amyl alcohol (Kovac’s reagent). SIM deep contains nutrients, peptone (tryptophan), iron, sodium thiosulfate. Unknown 6 tested negative to sulfur reduction and indole production, and remained yellow (after Kovac’s reagent was added), and it is non-motile since the agar remained clear. Kliger’s Iron Agar (KIA) differentiates glucose, and lactose fermentation to acids and gasses, specifically sulfur reducers. It contains small amounts of glucose (to be exhausted), phenol red (indicate acid), lactose (secondary sugar). Fissures are the result of gas byproducts. Unknown 6 fermented glucose, it failed to ferment lactose. It remained red with a yellow butt, the surface was pink. It tested negative for H2S, or gas formation.Simmons Citrate Agar tests an organism use of citrate as the only carbon source. Citrase hydrolyzes citrate into oxaloacetic and acetic acid. Oxaloacetic acid is hydrolyzed into pyruvic acid and carbon dioxide. It contains sodium citrate (carbon source), ammonium dihydrogen phosphate (nitrogen source), nutrients, and

The assigned unknown in tube #3 was classified as a gram positive bacteria shaped as Cocci Chains. Enterococcaceae is the family of the streptococcus faecalis. The gram positive bacteria included tests such as the Catalase, Coagulase, Mannitol Salt Agar (MSA), Streptococcus Faecalis Medium (SFM), and the Bile Esculin test. The purpose of these tests was to distinguish which unknowns were positive vs negative as well as to determine the morphology or shape of the unknown bacterias.

Bacteria vary greatly in terms of their characteristics and morphology. Colonies can be classified according to their colour, form, elevation, margin and size. Pure cultures of microorganisms