What Is Simmon's Citrate?

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.

There are many differents ways to identify a bacterial unknown and many different situations where identification would be beneficial. One way to identify bacterial unknowns is to perform biochemical tests. In this experiment multiple biochemical tests were done, by performing these tests on the bacterial unknown received the two different bacteria were then identified. The citrate test is done to test the ability of organisms to use citrate as a carbon source. This test uses Simmons citrate agar, the agar contains sodium citrate as the only carbon source and has bromothymol blue as the pH indicator. The organisms that use citrate as a carbon source use the enzyme to transport the citrate into the cell. The cells converts ammonium dihydrogen

This experiment was centered on metabolic and biochemical testing procedures. The rationale of performing these tests was to distinguish six different microbes from one another and to compare how their metabolic and biochemical processes differ from species to species to determine the unknown sample.

I identified Citrobacter freundii, the gram negative rod, by running a series of tests. I began with the Phenol Red Lactose tests, which tests if the organism contains various enzymes that determine if it can ferment lactose. The broth turned yellow after it was incubated, indicating that the lactose was fermented to acid, and there was also gas present in the Durham tube. Since the Phenol Red Lactose Test was positive, I then ran the Phenol Red Sucrose test, which tests if the bacteria contain different enzymes that determine if sucrose can be fermented. After incubation, the broth was yellow, indicating that sugar was fermented to acid, and there was also gas present in the Durham tube. Next, I ran the Sulfide Production, Indole Formation, Motility test, but I was only testing for Hydrogen Sulfide Production to differentiate between the organisms Citrobacter freundii and Enterobacter aerogenes. This test detects if the organisms can metabolize sulfur into hydrogen sulfide, which is revealed by the formation of ferrous sulfide that causes blackening around the growth. The test also reveals if the organism can break tryptophan into indole or migrate away from initial stab area. After incubation, the agar slant was completely black, indicating that the organism produces hydrogen sulfide and is motile proving that it was Citrobacter

This test is used to detect if the bacteria contains any deoxyribonuclease activity. Because no color change was observed from blue to clear my unknown bacteria displayed a negative result.

The enzyme urease breaks urea down into NH3 and CO2. An orange broth containing urea is used for this test and needs to be inoculated with the gram negative bacteria. A pink color in the medium indicates a urease-positive organism, an orange or yellow is negative.

The filter paper was then observed to see if it changed blue or not, in order to see if the bacteria produced cytochrome c oxidase. The final test used in the experiment was an API test. To begin the API test, a solution with bacteria and 5 mL of sterile saline, had to be made with a turbidity the same as the McFarland No. 3 (BaSO4) standard. This was done by adding loopfuls of bacteria to the saline solution, mixing the solution on the vortex, and then comparing the turbidity to the McFarland No. 3 standard, until the tubes were both at the same cloudiness. This created solution was then used in the API test by adding specified amounts to each of the microtubes on the API strip. For each of the microtubes whose names were not underlined or boxed, the tubes were filled to where the microtubes met the capsule. In the microtubes whose names were underlined, the microtubes were slightly underfilled, and then the capsule was filled with mineral oil in order to create and anaerobic environment. The last of the microbes were the ones whose names were boxed. In each of these the microtube and the capsule were filled all the way up with the bacteria. The API test strip was then placed in the 37°C incubator for 20 hours. After this time, observations were made about each of the different microtubes based on a given summary of results chart for the API test. A select number of microtubes had

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,

On the first day of our investigation, we received our organism suspended in a nutrient broth. Our instructions were to perform biochemical tests and techniques to determine identity of unknown organism by consulting Bergey’s Manual. We

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.

This test should be used when trying to identify organisms that produce catalase. It is used when differentiating between Catalase positive micrococcaceae and catalase negative streptococcaceae and some variations of the catalase test are for mycobacterium.

The Winogradsky will be used as the environment to obtain the bacteria. Desulvibrio is an anaerobe organism usually found in aquatic/soil environments. This environment is presented in the Winogradsky column since it is muddy and wet. On the first day of lab a Winogradsky column was made. The procedure that was followed included the following nutrients; water, organic soil and paper towels. The Winogradsky column that was made did not have the addition of sulfur which may be a concern since the intended organism for isolating is a sulfate reducing bacteria. The exception could be obtaining Desulvibrio from an environment such like the duck pond located on campus, some different types of organisms that could also be present in that environment

With the results of the Starch Hydrolysis Test I preceded to the final test which is the Citrate Test. Using aseptic technique, I streaked the organism onto the surface of the Simmons’ citrate slant and let it incubate overnight at 37°C. The Citrate Test determines if the organism is able to

For this method, the plates containing nutrient agar was divided into 4 parts. In each part, a bacterial isolate was spotted in duplicates. The plate was incubated at 30◦C for 24 hours. Ethanolic solution of (0.02%) Sudan Black was spread over the colonies and the plate was kept undisturbed for 30 minutes. They were washed with ethanol (96%) to remove the excess stain from the colonies. The dark blue stained colonies were taken as positives. All the positive isolates were assigned a code and were sub-cultured and maintained in their respective slants at 4◦C