An Unknown Microorganism Using Biochemical Tests

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 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.

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

The purpose of this study project was to carefully isolate and identify two unknown bacteria from a mixed culture. The ability to properly evaluate biochemical test results is also necessary for the identification to be successful. The goal was to apply all of the methods and techniques that have been learned in the microbiology laboratory course for the proper identification of unknown bacteria. A certain amount of bacteria that were used throughout the course were possible bacteria that could be found in a mixed culture. The bacteria that were identified in the mixed culture were Staphylococcus Aureus and Kocuria Rhizophila.

Often scientists work with bacteria that do not come in a labeled test tube— for example, bacterial samples taken from infected human tissue or from the soil—and the scientist must then identify the unknown microorganism in order to understand what behavior to expect from the organism, for example, a certain type of infection or antibiotic resistance. However, because of the relatively few forms of bacteria compared to animals and because of the lack of bacterial fossil records due to their asexually reproductive nature, the taxonomy used to classify animals cannot be applied to bacteria (Brown 275). In order to classify unknown bacteria, a variety of physiological and metabolic tests are available to narrow a sample down from the fathomless number of possibilities into a more manageable range. Once these tests have been performed, the researcher can consult Bergey’s Manual of Determinative Bacteriology, a systematically arranged and continually updated collection of all known bacteria based on their structure, metabolism, and other attributes.

There are many reasons for knowing the identity of microorganisms. The reasons range from knowing the causative agent of a disease in a patient, so as to know how it can be treated, to knowing the correct microorganism to be used for making certain foods or antibiotics. This study was done by applying all of the methods that I have been learned so far in the microbiology laboratory class for the identification of an unknown bacterium.

There are many reasons for knowing the identity of microorganisms. The reasons range from knowing the causative agent of a disease in a patient, so as to know how it can be treated, to knowing the correct microorganism to be used for making certain foods or antibiotics. This study was done by applying all of the methods that have been learned so far in the microbiology laboratory class for the identification of unknown bacteria. The identification process can be completed with a series of deferential stains and biochemical tests. Creating a dichotomous key helps to limit the amount of biochemical tests done on an unknown organism and by observation

The Staphylococcus aureus bacteria belongs to the Staphylococcaceae family. It is small, round shaped, and non-motile. Staphylococcus aureus stains gram positive and can often be found in small clusters (Mandal, 2010). It often forms chains and is a large contributor of soft tissue infections. It is of a yellow color, hence the name ?aureus? which comes from the Latin term ?aurum? for gold (Orenstein, n.d.). Staphylococcus aureus is found in a few spots on the human body, such as the nasal passage, the skin, the oral cavity, and even the gastrointestinal tract. Staphylococci and Streptococci are two different strands of the bacteria and are very hard to distinguish from one another. In order to tell the difference between them, without a microscope, a catalase test needs to be performed. The test is undergone by adding 3% hydrogen peroxide to both samples. Since Staphylococci are catalase positive, meaning they produce catalase, they will produce O? while the Streptococci will not because Streptococci are catalase negative (Todar, n.d.).

For the Urease test, I incoluated my Urea test tube with my unkown bacteria from a TSA plate using and inoculating loop. The Urea tube was then incubated at 37 degrees Celsius for 8 days to observe for a color change. The Urea tests for the ability of a bacteria grown in urea broth produces urease. This medium contains the pH indicator phenol red. If urease is produced the pH of the media will raise thus causing the phenol red to change from yellow to a pink color.

Two smears of the unknown bacterium #5 were inoculated while the second smear was used for a back up. The unknown bacterium dried for at least forty minutes. After the smears dried, the slides were heat fixed two times to ensure the stability of the organism. The slide was placed on top of the staining rack then over the small sink.

Unknown #462. Multiple tests were performed as described in Brown’s1 manual to identify the unknown; it was determined that the unknown was Staphylococcus epidermidis. The incubation temperature of each test was 37˚C and they were incubated for 24-48 hours. The morphological test performed on the unknown #462 were all consistent with the expected results for this bacterial species such cell shape, motility, and gram stain. The bacterium was gram positive was confirmed by growth on PEA plate, inhibition of growth on EMB plate, and through gram staining. The optimal growth temperature was at 37˚C which corresponds to the optimal temperature mesophilic bacterium as stated in StrainInfo2.

Staphylococcus aureus is a gram-positive coccal bacterium, 1µm in diameter, forming grape like clusters or clumps, and is the most important pathogen amongst Staphylococci bacteria. A gram stain was performed on unknown bacteria #41, producing a purple, gram positive cocci bacteria appearing in grape like clusters or clumps under microscope. A streak plate test on nutrient agar was performed resulting in yellowish colonies on the nutrient agar. A catalase test was then performed with a positive Staphylococcus result. Mannitol Salt Agar plate was then used to determine between Staphylococcus aureus and Stapylococcus epidermidis.

The different colonies were obtained from the various surfaces and also they differed in the number. The white and cream-colored colonies obtained were identified as the coccus, foggy white colonies as Bacillus and rest colored colonies as Potentially Staphylococcus.

Correct identification of a microorganism allows for proper investigation of a particular species, and prevention or treatment of a disease if necessary. During lab, students were instructed to choose a test tube inoculated with an unknown organism and then prompted to initiate a series of appropriate lab tests to correctly identify the organism.

Oxidase Test: The oxidase results was positive as the color turned purple on the commercial rapid dry card. This means that there was a presence of cytochrome oxidase enzyme which reduces oxygen at the end of the transport chain of electrons. This test helped the identification of P. Aeruginosa along with the other tests.