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We are microbiology students of Franciscan University! Our blog is our lab report and we encourage you witness our fabulous adventure of uncovering, and discovering!

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OUR UNKNOWN IS KNOWN

After many many microbiology labs, and tirelessly preforming experiment after experiment, we finally found our bacteria!

Escherichia coli!!!!!! 

Please read our blog to witness the process of uncovering our little bacteria!!!

Immunodetective biokit

In this experiment we were testing immunology and how it applies in testing food purity. First, we had to make four wells in each section of the agar. In section one of the petri dish, we used green dye, red dye, barium chloride, and potassium sulfate. Using the dyes, we were able to see how the agar absorbed the dye. For the second portion of the dish, we used bovine albumin, goat anti-horse albumin, goat anti-bovine albumin, and goat anti-swine albumin. Also in the third, we used all of the solutions, except for the bovine albumin and replaced it with hamburger extract.

ELISA

This test helps detect antibodies in the blood to determine if someone has been exposed to a disease. ELISA is used for diverse purposes such as pregnancy tests, disease detection, detecting illegal drug use, testing indoor air quality, and determining of food is labeled properly. In this experiment, we used purified antigen, positive control, negative control, secondary control, and a substrate into a 12-well strip. We put each fluid into each well individually then waited 5 minutes to allow the antibodies the opportunity to bind to the antigen. After doing this, we would wash out the wells then go onto the next fluid. After putting in the last substrate, we waited for the results; if it turned blue then it was positive for the AIDs antigen, which our substrate was.

UV pen

In order to show us how the Uv pen worked, we took samples of all of the class's unknown bacteria and put it into a beaker of water. The shorter wave lengths of the UV pen kill bacteria!

Ahh..the wonders of a steri-pen. Our professor said that he takes his steri-pen when he travels abroad to different countries. The pen only works if there nothing causing a barrier between the bacteria and the light. For instance, the UV rays cannot pass through something as thin as paper.

Unfortunately, no one would drink the water:(

UV light

We put our bacteria on a streak plate. Covering half of the plate with an index card was supposed to stop the ultraviolet light from killing the bacteria. We put the plate under the UV light for 30 seconds in order to kill the half of the plate that was exposed. 48 hours later, we noted that the bacteria still continued to grow throughout the plate.

Yogurt

Today was a fun day in class!! We made yogurt! 

First, we boiled milk and then proceeded to let it cool until it was 37 degrees C. Next we put the cooled milk into a cup of Kefir and yogurt. Both cups were out in the incubator, and the next class we got to taste and see how much the good bacteria grew. 

To tell the truth, they did not taste good at all:(

Urea

       The purpose of this test was to determine if our bacteria could hydrolyze urea, and break it down into carbon dioxide and ammonia. 

First we inoculated a broth tube of urea with our unknown bacteria. We then incubated the sample for 48 hours before we read the results. Our results showed that the broth turned a yellow color which indicates that our bacteria is negative for hydrolyzing urea. However, if the broth turned a red/ pink color, the test would be positive. The red color is related to the ph change. Our bacterial broth had not achieved a pH change. 

Nitrate experiment

The nitrate test was performed in order to indicate if our bacteria was able to reduce nitrate ions to nitrite ions or nitrogen gasses. Nitrate reduction is usually for energy production during anaerobic conditions. To determine whether the bacteria used nitrate reduction can test the presence of nitrite ions. To detect nitrite ions, sulfanilic acid and dimethyl alpha naphthylamine are added to bacteria inoculated in nitrate broth. If the broth turns pink, nitrite is present, and the results for nitrate reduction are positive. However, having no color change does not determine a negative result. Instead, Zinc has to be added to determine if the nitrate ions were reduced to molecular nitrogen. If the color does not change after the addition of Zinc, the results are considered to be negative.  

First we inoculated a nitrate broth tube with our bacteria and let it incubate for 48 hours. Next, we added 5 drops of the two reagents listed above and proceeded to mix the contents. Right away, our broth turned red. Our bacteria is positive for nitrate reduction. We did not have to add zinc to the broth because the two reagents gave us instant results. The broth contained nitrite ions!

Citrate examination

The citrate test was performed in order to determine if citrate could act as our bacteria’s energy source. Bacteria which use citrate have membrane transporters called Citrate Permease. Citrate Permease convert the citrate to pyruvate and carbon dioxide. For this test we used Simmons citrate agar slant tube. The slant has a green agar that will turn blue if the bacteria utilizes citrate. We inoculated the slant and incubated it for 48 hours. The results turned out to be negative for the utilization of citrate as indicated by the green color of our slant agar.

Indole

       The indole test’s purpose was to determine the ability of our bacteria to split amino acids( tryptophan) into indole and pyruvic acid. The simple procedure had us first inoculate the tryptone broth with our bacteria and incubate it for 48 hours at 35 degrees C. After the incubation, we had to add 5 drops of Kovac’s reagent to broth. Right when we added the Kovac solution to the indole tube the top layer turned a redish, pink color which meant that our bacteria was positive for the presence of indole. Our bacteria successfully split the amino acid, tryptophan into indole and pyruvic acid. 

Antibiotic plate

For this experiment we wanted to test which antimicrobial agent our bacteria would be susceptible to.  We placed small samples of the antimicrobial agents onto a streaked plate of our bacteria. The next lab day we recorded the clear diameter of dead bacteria surrounding each microbial agent.

Diameter surrounding each


1.Penicillin : 1cm
2.Vancomycin: 1.2 cm
3.Novobiocin: 0 cm
4.Tetracyclic: 2 cm
5.Erythro: 1.9 cm
6.Chloramphenicol: 3 cm
7.Neomycin: 2 cm

By measuring the diameters we found that our bacteria was susceptible to Vancomycinb, Tetracyclic, Chloramphenicol, and Neomycin

Eosin methylene blue agar

       The purpose of this procedure was to isolate a gram-negative enteric bacilli. The type of agar used is called an Eosin Methylene Blue medium. The medium contains the dyes eosin, methylene, and the sugars lactose, and sucrose. The medium inhibits gram-positive bacteria growth, and only gram-negative bacteria. The primary use of this agar is to identify Enteric bacilli. 

We began by inoculating the agar with our bacteria using aseptic technique. We then let the medium incubate for 48 hours. Our results were very specific and exciting!!! The color of our bacterial streak turned to a dark purple, green metallic sheen. Our bacteria is able to ferment lactose and/or sucrose and produce acids. Our lab book stated that our results indicated that our bacteria was Escherichia coli! Exciting! This test helped us in our bacteria identification!!

MacConkey Agar

For this procedure we used a MacConkey agar in order to differentiate between gram-negative enteric bacteria and their ability to grow on the agar and ferment lactose. First we used aspetic technique to inoculate the agar plate with our bacteria. Next, we incubated the sample for 48 hours at 35 degree celsius. We examined the plate the next day and found a negative result. We concluded that because there was not a broad change of color on the agar, the bacteria was a non-lactose fermenter. 

Mannitol salt agar

The purpose of this ecperiemtn was to analyze the salt tolerance of our bacteria. Only bacteria which can tolerate 7.5% salt content will survive and grow on the medium. The salt tolerant gram positive bacteria include Staphylococcus, Micrococcus, and sometimes Enterobacteria. For this experiment we inoculated a mannitol salt agar with our bacteria and let it inoculate for 24 hours. 

Our results turned out to be negative. The bacteria is a non-fermenter of Mannitol.

Carbohydrates fermentation, TSI, MR-VP, Gelatin

Fermentation of Carbs

We performed this procedure to detect if our bacteria could ferment lactose, sucrose, or glucose by indicating the acid/gas production. The procedure began by inoculating our bacteria into three different phenol red-sugar broth, each with Durham tubes to collect the gases. Each tube was labeled with a different sugar; lactose, glucose, or sucrose. 

After letting the tubes sit for 48 hours, we recorded our results...

Lactose: pinkish/yellow, gas bubble present= acid/gas production

Sucrose: Orange/red, little gas bubble=acid/gas production

Glucose: Light Yellow, big gas bubble=acid gas production

TSI

During this lab we inoculated many tubes with our unknown bacteria. First we inoculated a triple sugar ion(TSI) slant which will indicate if the bacteria has the ability to ferment glucose, lactose, and sucrose. The triple sugar agar contains all 3 sugars and if our bacteria end up fermenting any of them, there will be a drop in pH. All we did was streak the slant in a zig zag fashion and allowed the bacteria to grow in an incubator for 24 hours. 

Unfortunately pictures are not available with the results but I will do my best to describe what happened. Our slant turned yellow. It had a yellow slant and yellow butt which means that glucose, lactose or sucrose fermented. We also noticed that there was a big gas bubble near the butt which means that it was a bacteria producing acid and gas. 

MR-VP 

The MR-VP test was to determine if the bacteria had the ability to ferment glucose via butanediol fermentation. We inoculated our bacteria into the MR-VP broth tube and incubated it for 48 hours. After 48 hours we added 1 ml of our MR-VP mixture to a smaller tube. We then added 5% alpha-napthol and 40% KOH. We proceeded to shake the smaller tube and read our results. There was no indication of color change at all because it remained yellow. If the color turned to red it would have been a positive result for the usage of butanediol fermentation. 

Next we added 10 drops of methyl red to the original tube and got a positive result for our bacteria’s ability to ferment glucose via mixed acid fermentation. The top of the broth changed to a redish color. 

Gelatin 

The gelatin test was preformed to determine the bacteria’s ability to digest gelatin. We inoculated our bacteria into the gelatin and incubated it for 48 hours. The next day we checked the liquifaction by tilting the tube. The medium remained as a liquid indicating that our bacteria is negative for hydrolyzing gelatin. 

Lipid plate

The last experiment we attempted concerning the bacteria’s ability to hydrolyze was the Lipid test. This test was to determine whether our bacteria could digest triglyceride. Some bacteria are able to grow where there are lipids present such as skin, dirt, or decomposing organisms. Most lipids are made of tryglycerides which consist of 3 fatty acids and a glycerol. In order to digest triglyceride, some bacteria need to produce lipases to break down the lipid. For this experiment we inoculated the bacteria onto a blue agar plate. If a clear zone appeared around the streak within 48 hours, the bacteria would be positive for lipases production. Our results showed that the bacteria was not positive for lipases production because there was no indication of a clear halo surrounding it. Instead, the plate turned blue-black which determined that the starch was still present. 

Skim Milk plate

  The same day we attempted the starch test was the same day we did the Casein Hydrolysis test, which tests the bacteria’s ability to digest protein in milk(casein). We first inoculated our bacteria onto a skim milk agar plate. We let the plate sit for 48 hours in an incubator and recorded the results the next lab day. Turned out that our bacteria was negative for caseinase production which means that our bacteria is unable to digest proteins. The results showed that there was no sign of a clear zone around the streak of bacteria which would have indicated a positive result. Our results showed that there was only white surrounding the bacteria, no indication of protein hydrolyzation. 

Starch agar plate

Today we attempted three different tests before going to the water plant in Steubenville Ohio. The first test was the Starch Hydrolysis test which tested the bacteria’s ability to digest starches. We inoculated our bacteria onto a starch agar plate and let the bacteria sit for 48 hours in an incubator. After 48 hours we saturated the starch plate with iodine and recorded our results. If a clear color surrounded the bacteria growth it is considered positive for amylase production. Our results showed that there was no indication of amylase production because color appeared around the growth instead of a clear color. When starch reacts with starch it causes a purplish black color to appear. Unfortunately we did not a have a camera available for the results, but just take our word for it. Our bacteria turned out to be negative for amylase production!

Culturing anaerobic bacteria:GasPak

The next experiment involved a GasPak which uses a chemical reaction to empty oxygen from a container. Our professor preformed this experiment after each group streaked a plate with their bacteria. First he cut the GasPak envelope and added water. Next he opened the indicator slip which is blue in the presence of oxygen and white when there is an absence. He sealed the container and placed the jar in an incubator for 48 hours. 

The next lab day we were surprised to find that our bacteria was facultated anaerobic bacteria. Facultated anaerobic bacteria can undergo aerobic respiration and fermentation. It can also grow under aerobic or anaerobic conditions.

Edward tried to seal the jar but he needed some help:(

Oxygen requirement

The next experiment  detected the oxygen requirements of our unknown. We used thioglycollate broth which reacts with oxygen to form water. A dye present in the broth turns the color pink or blue when oxygen is present, and remains colorless when oxygen is absent. We inoculated our bacteria into the broth and waited 48 hours for the results. 

The next lab day we were surprised to find that our bacteria was facultated anaerobic bacteria. Facultated anaerobic bacteria can undergo aerobic respiration and fermentation. It can also grow under aerobic or anaerobic conditions.