Lactose Fermentation Lab

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.

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.

Fermentation is a metabolic pathway that produce ATP molecules under anaerobic conditions (only undergoes glycolysis), NAD+ is used directly in glycolysis to form ATP molecules, which is not as efficient as cellular respiration because only 2ATP molecules are formed during the glycolysis. One type of fermentation is alcohol fermentation, it produces pyruvate molecules made by glycolysis and the yeast will break it down to give off carbon dioxide, the reactant is glucose and the byproducts are ethanol and carbon dioxide. In this lab, the purpose is to measure whether the changes of

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.

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.

Laboratory Tests – All laboratory tests utilize the following reference unless otherwise specified - Pagana & Pagana (2014)

Xylose was a sugar testing for fermentation for differentiation between bacteria. If the bacteria could grow and ferment xylose, then it would change the phenol red pH indicator dye to yellow and possibly black, being a positive result. The sulphur indole motility (SIM) medium contains peptone, iron, and sodium thiosulfate. The first two abilities that are tested are the sulphur reduction and the motility of the bacteria. If the bacteria can reduce sulphur, it will react with the iron present and turn the agar black resulting in a positive reaction. The motility test is determined by any movement away from the stab sight in the agar. The indole test is done lastly by adding the Kovac’s reagent to test for the presence of indole from hydrolysing tryptophan (Levell, 2007 SIM). Sulphur reduction, any movement, and indole production are all positive results. The eosin methylene blue (EMB) agar tests for lactose fermentation from gram-negative bacteria usually found in the gut. If it can ferment lactose the colonies will change to a pink/purple colour for a positive result and occasionally green if it is more aggressive (Kadam,

I examined all three slides under the microscope. After observing the slides I flushed the specimens down the drain and disinfected the area with bleach solution. After 42-72 hours I assessed the growth patterns of the tube and noted that the tube that contained the swab became yellow stained and the tube that contained the agar and tablet dissolved a little and some of the content drifted to the surface of the tube and created a film.

The Simmon’s Citrate Agar (SCA) is a media used to test the organism’s ability to use citrate as a sole carbon source. This medium contains citrate and an indicator, brom thymol blue. If the bacterium has the ability to use citrate, carbon dioxide will be produced. This will cause an elevation in pH and will turn the agar from green to blue.

A total of three gel runs were made to see if there were any fragments that indicated the presence of bacteroides. The Bacteroides products were to fall within 530 base pairs to be considered a positive result. The first gel pictured, Figure 1, on the left was the first run made to test the 1/10 dilution of Turtle Creek with sewage water, but only the ladder appeared. The second gel represented, on the right on Figure 1, a repeated test of the first gel; witch indicated the successful extraction of DNA. The last two pictures on Figure 2 are the same gel just emphasizing a bit more on the bands that were faint. This gel had a total of five DNA samples plus the controls. As indicated in the image below there was three out

1 Q-tip was then swirled around in the water in a test tube for each different type of water for 1 minute and the Q-tip was observed for any changes in color. The Q-tip was then rinsed by tap water from the sink as instructed by the kit’s instructions and properly disposed of. This was done for all the different types of water in both environments. Then for the tap water in both environments, bacteria was tested using the test kits. Using a dropper pipette provided by the kit, water was collected, and placed into a vial that contained a solution known as ‘purple reagent’ at the bottom. The vial was gently swirled and then placed on a table for 5 minutes and was undisturbed. The vial was then swirled again and a test strip that was also provided by the kit was placed into the test vial with the arrows on the test strip point down. The vial was undisturbed for 10 minutes, and the test strip was then observed for reddish lines within 1 minute after the 10 minutes. The test strip, vial, and dropper pipette were not rinsed, and were properly disposed of after

To begin with, we gathered fifteen test tubes in our test tube rack: three arabinose (A), three glucose (G), three mannitol (M), three raffinose (R), and three nitrate broth (N) tubes. We then labeled each test tube with either A, G, M, R, or N on the top of those tubes. Next we labeled these tubes with our initials, the date, and either Escherichia Coli or our unknown bacterium number. We did this so we would know which tubes to inoculate with E. Coli or our unknown and so we would know which tubes are ours. With that, we began inoculating the tubes with one loopful of E. Coli to one set of tubes in the order N to R to M to G to A. We flamed each tube before and after each inoculation in order to maintain aseptic techniques. This step was repeated two more times with each of our unknown bacteria. Finally, all of the tubes were incubated at thirty degrees Celsius. After a week on incubation, we added six drops of naphthylamine and two drops of sulfanilic acid to E. Coli N tube and our unknown tubes. This was to test for a positive

The TSIA medium test checks for hydrogen sulfide production while also fermenting glucose, lactose, or sacchorase. Acid is produced during fermentation which causes the pH indicator to change the color from red to yellow. The SIM test incorporates sulfur reduction, indole production, and motility all in one medium. If hydrogen sulfide is produced, the agar will become black. Indole production will turn a pink color during a positive reaction and motility is determined by the growth of microbes away from the stab line. The last two tests conducted were the Citrate utilization test and Urease test. Citrate utilization is used to determine if the bacterium can live with citrate as its only carbon source. Citrate utilization can be determined

Experimental results Tube Amount of 10% lactose added Enzyme present? The intensity of yellow after 30 mins Ab420 μM O-Nitrophenol produced in 30 mins μM O-Nitrophenol produced/minute A 0mL YES Yellow 0.326 81.5 2.72 B 2.0mL YES Yellow 0.032 8

Label the 3 test tubes with your initials and number each 1,2, and 3. (This allows for them to be distinguished)