Lab Report Unknown Bacteria 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 Methyl Red test is a differential test for bacterial respiration used to differentiate strains of coliform bacteria capable of performing mixed acid fermentation that will lower the pH despite the phosphate buffer (http://faculty.deanza.fhda.edu). Mixed acid fermentation is confirmed by using methyl red as an indicator. It is red ant pH 4.4 and below, yellow at pH 6.2 and above, and orange in between. Red is a positive result reported as (+), yellow is a negative result reported as (-), and orange is negative or inconclusive.

This laboratory experiment’s objective was to take a pure culture and isolate it from a mixed culture. The other part of the objective was to ascertain what species of bacteria that the pure culture was. The hypothesis made stated that so long as lab protocol was followed, the unidentified culture would be positively recognized/identified. An isolated pure colony of the unknown culture was obtained using the quadrant streak plate method. Afterward, the culture was Gram stained, and the results showed that it was Gram positive. Motility tests were done on the unknown using a filter paper bridge on a petri dish that contained TTC with agar. The unknown was revealed to not be motile, which meant that it did not possess flagella. The last test done was to learn the metabolic capabilities of the unknown bacteria. There were tests done for citrate utilization, the mixed fermentation pathway, catalase presence, carbohydrate fermentation in mannitol, lactose and glucose, urease production and the butanediol fermentation pathway in order to better identify the unknown bacteria. The results from each of the metabolic tests in conjunction with the motility and Gram staining tests were ultimately compared to results from database containing many different kinds of results from various bacteria. The unknown from the mixed culture was identified as Staphylococcus

The purpose of this lab was to identify two unknown bacteria from a mixed culture. The reason for identification of unknown bacteria was to help students recognize different bacteria through different biochemical tests and characteristics. This is important in the medical field because identification of unknown bacteria can help treat a patient by knowing the contributing source of a disease. Also knowledge of different bacteria helped others make antibiotics used today. This lab was completed by using the methods learned thus far in identification of bacteria.

The purpose of this lab was to identify two unknown bacteria cultures using various differential tests. The identification of these unknown cultures was accomplished by separating and differentiating possible

|MSA Agar |For organisms that are |Isolates for mannitol fermentation |Yellow color change in |Staphylococcus aureus |

Citrobacter Freundii is a species of bacteria that can be potentially harmful to humans. It is known to cause meningitis by protruding into the brain and replicating itself (1). The Citrobacter species has also been found as a cause of some urinary tract infections, diarrhea, and even gastrointestinal diseases and symptoms (3). C. Freundii can be located in a wide variety of soils and water (3). Lastly, it is also the cause of many nosocomial infections due to its presence in water (1).

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

I used an inoculating needle to stab the SIM test tube and then incubated it at 37 degrees Celsius for 24 hours. The SIM test was used to test whether an organism has the ability to reduce sulfur to hydrogen sulfide. Iron salts in the media reacts with the hydrogen sulfide to form a black precipitate called ferric sulfide. If sulfur can be reduced than a black color will be seen in the tube. This test also sees if an organism is and indole producer. Indole producers are bacteria that produce the enzyme trytophanase which can hydrolyze tryptophan to pyruvate, ammonia and indole. To test for indole production,

To distinguish between these four, carbohydrate fermentation tests were performed. This organism was unable to ferment any of the carbohydrates tested, including sucrose, glucose, and lactose. This was indicated by the fact that all fermentation mediums remained red, even after inoculation and incubation with Organism #2, meaning the pH remained basic, and no acid byproducts of fermentations were produced by Organism #2. P. aeruginosa is the only organism in our lab without the ability to perform any form of carbohydrate fermentation. This identification was confirmed with a gelatin test.

For the MR Test, 3 drops of Methyl Red Reagent was added to one of the test tubes and the color was observed. The color changed from yellow to red to confirm a MR positive result meaning this microbe could ferment glucose into mixed acids and lower the pH. For the VP Test, 15 drops of VP-Reagent “A” was added and mixed then 5 drops of VP-Reagent “B” was added and color was observed after 10-20 minutes, undisturbing the test tube. The analysis revealed no red top layer, a negative result, which suggests this microbe cannot ferment glucose into acetoin. For the Indole Test, 5 drops of Kovac’s Reagent was added to the green-capped test tube (Figure 5) and the top layer was to be observed. No red layer appeared, another negative result, which confirms that this microbe does not produce indole from tryptophan. The Citrate test tube appears to be positive from Figure 5 since it is blue with growth, however after confirming my results with the instructors, it is a false positive. Thus, this microbe cannot utilize citrate as a sole carbon source and the false positive could have occurred from contamination or another unknown

Determination of the bacteria being a lactose fermenter or non fermenter is done through the growth on the MacConkey agar. Knowing this allow for the student to perform the necessary tests to determine which lactose fermenter was present in the sample. The Indole test allows for the determination of whether the unknown bacteria is Escherichia coli because this genus and speices is the only lactose fermenter that will produce a positive result here. Moving onto the Methyl red test this indicates glucose fermentation, more specifically microbes that produce mixture of acids as a result of fermentation. The Voges-proskauer tests for glucose fermentation, specifically organisms whose acid is converted to acetoin. The Citrate test differentiates an organism’s ability to use citrate as its only carbon source. The urea broth culture detects the enzyme urease, which allows break down of urea producing acid, which causes a noticeable change in color. The final test is the motility test to determine if the bacteria has the capacity of movement beyond the point of

Identification of the bacterial strains:The strains were subjected to gram staining,catalase and spore formation tests. (Harrigan and McCance, 1976).All Colonies were characterized in MRS and M 17 agars.The strains that gave gram positive and catalse negative results were set aside for further identification.(Sharpe, 1979).The growth of the bacteria at different temperatures of between Growth 10-45°C for 3-6 days , resistance to 60°C for 30 min (Sherman test), growth in the presence of 2 to 6 % NaCl and different pHs (4.5 and 6.5) were used to identify the strains of LAB. Arginine and asculin hydrolysis,citrate utilistaion, acetone productionformation of gas from glucose and production of dextran from sucrose were also determined. The starins were then tested for fermentation of L-arabinose, D-xylose, galactose, D-fructose, sorbitol, lactose, melibiose, saccharose, D-raffinose, melezitose, mannose and glucose.Bacterial growth in the different temperatures were confirmed by turbidity change in MRS or M17 after incubation(after 24,48 and 72 hrs).Microbial

Gram-negative and Gram-positive bacteria can be distinguished by conducting biochemical tests. Its growth can be examined on selected mediums containing metabolic inhibitors such as antibiotics. The medias utilized in this project are: media containing the antibiotic vancomycin, Eosin Methylene Blue-lactose (EMB-lactose) agar, and Phenylethyl alcohol (PEA) agar (Holbrook and Leicht 2017). Vancomycin is most effective at inhibiting the growth of Gram-positive cocci (Rotschafer et al. 2005). EMB-lactose agar inhibits the growth of many Gram-positive bacteria and the lactose component of the medium allows for lactose-fermenting bacteria to cause color changes within the medium (Reynolds 2011). The presence of lactose fermenters in the EMB-lactose agar will cause a color change in the resulting colonies based on the strength of their acidity. A metallic green colony indicates that that active fermenters are present. A pink colony indicates that weak fermenters are present. If a bacterial species does not ferment lactose, it will appear white or colorless (Holbrook and Leicht 2017). PEA agar is a selective medium that inhibits the growth of most Gram-negative bacteria species. It does so by interrupting the lipids within the Gram-negative cell membrane, which will affect its permeability to certain molecules. This could result in the intake of materials that are supposed to be blocked and the release of potassium from within the cell, which is necessary for DNA synthesis (Lal and

To further enhance this method an indicator medium will also be used. These medium changes colour during fermentation, an indication of pH change due to the presence of acid forming species. Durham tubes will then be employed; when inverted they catch gaseous bubble formed during fermentation. The gas produced at 37C act as a strong indicator to the presence of E.coli Reasoner & Geldreich (1985).