The Importance Of The Indole Test

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.

We set up 3 fermentation set-ups, labeling them 1, 2, and 3. Then, filled a tub with hot water and inserted the end of the plastic tubing into one of the test tubes and submerged the collection tube and plastic tubing in the tub. After that, we mixed the fermentation solutions for the other tubes, (tube 1 got 4mL of water and 3mL of corn syrup, tube 2 got 3 mL of water, 1 mL of yeast and 3 mL corn syrup, tube 3 got 1 mL water, 3 mL yeast and 3 mL of corn syrup) . We then mixed each test tube and put the rubber stoppers in the fermentation tubes. Finally, we marked the water level on each collection tube with a wax pencil to use as the baseline. Then at 5 minute intervals we measured the distance from the baseline for 20 minutes.

The Voges-Proskauer test to detect organisms that are able to ferment glucose, but convert the products to acetoin and 2,3-butanediol. This is deduced by the addition of Reagent A and Reagent B, and the observation of the color change thereafter. Reagent A is a solution of -naphthol and alcohol. Reagent A catalyzes the conversion of acetoin to diacetyl. Diacetyl thens react with guanidine-containing compounds from the peptone to form a red color in the presence of -naphthol. Reagent B is a solution of potassium hydroxide and water. It

Create a control group by testing the three reagents in distilled water. Fill three tubes one centimeter of the length with distilled water. With a permanent marker, label the test tubes according to which reagent will be used. In order to test for sugars, preheat a beaker that is three-fourths full of tap water and bring the water to a boil. In the first test tube, drop five drops of biuret reagent to test for protein, in the second, drop five drops of iodine to test for starches, and in the third, drop five drops of Benedict’s reagent to test for sugars. Using a tube grabber, place only Benedict’s reagent test tube in the boiling water for a total time of three minutes. Using the tube grabber, carefully remove the Benedict’s reagent tube from the boiling water and record the color of all 3 liquids in the test tubes. Place the tubes in the

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,

For this experiment we will be testing four different bacteria with four different tests, using glucose, lactose, and sucrose. Hopefully we will use the information from those test to be able to identify the organisms in each of the samples from the case studies. We will use the results from the four different tests along with the information of how different bacteria react to match up to the case scenario and identify the bacteria, then check to see if our guess was correct. The findings are that we were able to identify, by process of elimination, the four different test bacteria.

There was also motility because there was growth away from the point of inoculation. It looked like an upside down Christmas tree in the tube. The last thing SIM tested for was indole production. It is produced when the bacteria converts amino acid tryptophan to indole. 5 drops of dimethylaminobenzaldehyde, or Kovacs reagent, was added to the test tube. Upon addition, if it is positive for indole, it will turn a red color at the top of the medium. When the unknown was tested, it did not produce a red color. There was a slight dark red/brown color, but overall the test result was negative.

Then I did the Phenol Red Lactose test which reveals if an organism can ferment the sugar lactose. The tube was yellow after incubation which was a positive result because the acid production from fermentation lowers the pH turning the broth from red to yellow. Next I did the Urea test which is used to show a microbes ability to produce the exoenzyme urease that

: In the field of microbiology, there are times when a sample will contain more than one species of bacteria. The goal is to separate each bacterium and culture them independently from one another to identify them. This was the objective of this lab. Each stock contained two unknown bacterium, and the possible unknowns were Eserichia coli, Enterobacter aerogenes, Enterobacter cloacae, Serratia marcescens, Proteus vulgaris, Klebsiella pneumoniae, Shigella flexneri, Shigella sonnei, and Salmonella enterica. The tests available were MacConkey agar, Endo agar, Hektoen Enteric agar, Tryptone Soya Agar (TSA), carbohydrate sugar broths, Triple Sugar Iron (TSI) agar, decarboxylase broths (arginine, lysine, and ornithine), Simmon’s Citrate Agar, urease

The medias used in the experiment were the EMB agar and LT broth. LT broths are a selective/differential medium which contains sodium lauryl sulfate which is a detergent that stomach and intestinal bacteria have the ability to withstand. LT broths also contain lactose that supoorts coliform growth by fermentation. EMB agars are differential mediums that contain dyes that are inhibitors of Gram-positive organisms. They also produce dark colored colonies around

The energy sources an organism’s uses can also be determined to classify the metabolism of the unknown organism. For instance, the ability to use citrate as the sole carbon source can be assessed by growing cells in a medium with citrate as the only carbon source and a pH indicator that which changed color when the pH increases as a result of citrate metabolism (7). Similarly, the indicator methyl red can be added to a medium containing the unknown sample and various carbon sources. If the sample uses mixed acid fermentation, generating cellular energy by using more than one carbon source, then the drop in pH due to the accumulation of various acidic products will cause the indicator to turn red (7). Another such example is the use of a pH

The first method is the pyrogallic acid technique in a solid medium. This technique uses streak cultures on nutrient agar slants. A person pushes a cotton plug into the tube until it almost touches the slant. The space above the cotton gets filled with pyrogallic acid crystals and put sodium hydroxide in there as well. Insert the stopper very tightly and then invert and incubate. The chemicals absorb the oxygen which produces an anaerobic environment. The second method is the shake-culture method in a solid medium. This is a molten and cooled nutrient agar which is inoculated with a loopful of an organism. The tube gets shaken, cooled quickly, and then incubated. The position of growth in the tube makes it the index of gaseous requirements for an organism. The third method os the Paraffin plug technique in a broth medium. It is a medium that has reducing substances like cystein or ascorbic acid in it. This medium gets heated to make the oxygen go away then get quickly cooled and inoculated with a loopful of culture. After that it get immediately sealed with melted paraffin and then gets incubated. The last method is fluid thioglycollate in a broth medium. It is present in redox potential indicator like resazurin, which produces a pink color in an oxidized

This enzyme is able to produce hydrogen sulfide, this will react with the iron in the medium to form ferric sulfide. Ferric sulfide will create a black color in the medium, this indicates that cysteine desulfurase is present in the medium. The next assay that the test will determine is the indole test. The indole tests for an enzyme known as tryptophanase. This enzyme is able to detach both the amino acid group and the ring-shaped group in the medium.

On Unknown 1A, the first test required was a lactose fermentation test. I needed one tube of lactose and phenol red broth, fitted with a Durham tube. The phenol red is a Ph indicator, and would turn the broth from a red color to a yellow color if acid is produced, which would occur in lactose fermentation. The Durham tube allows us to see if any gas is produced in the fermentation process. If there is a bubble in the tube, this shows that gas was produced. Be careful when handling these tubes to ensure you do not jostle it, causing air to enter the Durham tube. I inoculated

These tests indicate metabolism characteristics that are consistent with the E. coli bacteria. The SIM agar tests for the bacteria 's ability to break down the amino acids cysteine and tryptophan. E. coli does not use cysteine as source of food, but it does use tryptophan as a food source producing indole, which is indicated by adding Kovac 's reagent. If indole is present, a reddish color will appear, if not, no change occurs. If tryptophan is broken down, a metal sulfide results which produces a black color. The SIM agar is also responsible for indicating bacterial motility, which can be observed if the agar becomes cloudy (which means the bacteria has moved outward), rather than