Protien-Indole (tryptophan), Citrate, Nitrate, and Urea Tests.

Uninoculated tubes:

Above (left to right) citrate tube, indole tube, nitrite tube, and urea tube

The Citrate test determines whether a bacterium can utilize citrate as its sole source of carbon and energy. If our bacteria does, it has the enzyme, citrate permease and will turn the green agar blue. Our bacteria turned slightly turned the agar blue, therefore, our bacteria does utilize citrate.

The indole test determines the ability of some bacteria to split the amino acid tryptophan into indole and pyruvic acid. If the bacteria produces indole, a red layer would form at the top of the test tube. Our bacteria did not produce a red layer, therefore, this test is negative. Our bacteria does not use the amino acid, tryptophan.

The nitrite test determines if a bacterium is able to reduce nitrate ions to either nitrite ions or to nitrogen gas. Our sample did not turn red upon adding the zinc, therefore our bacteria is positive and is capable of reducing nitrate ions.

Finally, the urea test allows us to determine whether a bacteria has the ability to hydrolyze urea. We inoculated our bacteria into the urea-containing broth and if the tube turned bright pink, the bacteria would test positive, showing that it has the enzyme urease. Our bacteria did not turn pink but remained the same color. So, our bacteria does not have urease and therefore it cannot hydrolyze bacteria.

During the second part of lab, we prepared a petri dish of our bacteria and then placed seven different antibiotic tablets in the plate: Penicillin, vancomycin, novabiocin, tetracydine, erythromycan, chloramphenicol, and neomycin. We will find out next lab whether our bacteria are sensitive to the antibiotics...

Gelatin Test, the Carbohydrates (sucrose, lactose, and glucose) Fermentation Test, TSI Agar Test, VP, Litmus Milk Test Results and Methyl Red Test

The results for our VP, gelatin, carbohydrate fermentation, TSIA, and the litmus tests are in! The following images show what we found... 

Above is the result of our VP test. The VP test determines the ability of our bacteria to use butanediol fermentation to ferment glucose. If the test is positive, the presence of oxygen will turn the liquid red upon addition of the VP reagents. As you can tell, our product has turned a reddish color (it was originally yellow) and therefore our test is positive, which means that our bacteria uses butanediol fermentation to ferment glucose.

The fermentation of carbohydrates tests (above) allow us to determine our bacteria's ability to ferment a particular carbohydrate. There are three carbohydrates that we used: sucrose, glucose, and lactose. If our bacteria uses one of these sugars for energy it will produce organic acids as waste products. If this is the case, the phenol red will turn from red to yellow in the presence of acid. As you can see from the picture, the sucrose and glucose tests were positive, meaning they use those sugars. Our bacteria does not use lactose because the medium is still red.

Above is the result of our gelatin hydrolysis test. The gelatin hydrolysis test determines if our bacteria has the enzyme, gelatinase, necessary to digest gelatin. If our bacteria does have gelatinase, it will cause the peptide bonds to cleave in the gelatin, causing the semi-solid to liquefy. If the gelatinous medium is still liquid at 4 degrees Celsius, the bacteria has gelatinase. It's hard to see in the picture, but our gelatin medium has liquefied, and so this test is positive.

The two above images are the result of the TSI (triple sugar iron) agar test. The agar in the TSI test contains lactose, sucrose, and glucose. The agar is originally red. If the bacteria uses any of these sugars, acid is a byproduct, thus causing the agar to turn from red to yellow. As you can see from the image, our slant is red and the butt is yellow. This means that our bacteria only ferments glucose and not lactose and/or sucrose. 

The purple medium in the test tube second to the left is the litmus milk test. The normally-lavender litmus milk medium allows us to differentiate the bacteria's ability to utilize lactose, protien, and litmus in litmus milk. The litmus acts as the pH indicator, turning pink in the presence of acid and blue in alkaline conditions. As you can see from the picture, our medium remained lavender in color. What you can't really see from this picture is that the medium produced a runny curd towards the surface of the medium. This means that our bacteria possesses the enzyme, rennin, to break down the casein. Our bacteria did not turn pink, meaning that it lactose fermentation did not occur.



Methyl Red Test Results: Negative



The above Methel Red test (Mixed Fermentation) shows us if the bacteria has the ability to ferment glucose via mixed-acid fermentation. 5 drops of methyl red are added to the test tube and if it turns red then the test is possitive. If there is no color change are the tube remains yellow, then the test is negative. Our test tube remained yellow so our results are negative.

Starch, Lipid, Casein, and Motility Results

At the beginning of today's lab, we retrieved our starch, lipid, and casein plates from the incubator along with our motility test slant.

Results:

Above is the result of our starch agar plate test. As you can see, not a whole lot happened here. Our test result is negative, meaning our bacteria cannot produce amylase and therefore, it does not use starch.

This is our skim milk plate that tests whether or not our bacteria uses casein. As you can see, there is a halo around where we streaked the plate. This indicates that our bacteria is able to produce protease and has used up the casein where the halo (clear spot) is. This test is positive!

This is our blue agar dish that tests whether our unknown bacteria can use lipids. Our bacteria has produced a halo around the streak, which indicates complete hydrolysis of triglycerides, positive for lipase production. The bacteria uses lipids, therefore, this test is also positive!

Although it is hard to tell from this picture, the motility slant to the right shows that our bacteria is motile because the bacteria has traveled from the original insertion line from the inoculation needle, causing the agar to appear cloudy.

Today, we also started the gelatin test, the carbohydrates (sucrose, lactose, and glucose) fermentation test, TSIA test, and the litmus milk test. We will let our bacteria grow in the incubator until next lab, when we get to determine the results.

Oxygen Requirements Lab Results and Starch, Lipid, and Casein Test

Today, we retrieved our thioglycolate broth tube from the incubator to see the oxygen requirement for our unknown sample. The result is as follows:

As you can see from the image above, our unknown bacteria thrived towards the surface of the broth and did not grow at all at the bottom. There are trace amounts of bacteria towards the center, meaning our bacteria is facultative anaerobic. We were unsure whether the bacteria in the middle is there because it grew there or whether we weren't careful and accidentally shook it when handling it. If that is the case, then our bacteria would be aerobic.

When we retrieved our petri dish from the GasPak, we discovered that our bacteria did not grow in the absence of oxygen. Therefore, our bacteria is aerobic/micro-aerophilic. This means that our bacteria needs oxygen to grow! 

Before we left lab, we inoculated three different petri plates: one was a starch agar plate, which tests whether the bacteria produces amylase to hydrolyze amylopectin and amylose into maltose, glucose, and dextrans. Another petri dish had skim milk and it discriminates bacteria that produce protease to hydrolyze the casein into clear amino acids. The last is the blue agar plate, which tests whether the bacteria produces lipase to break down lipids.

We also inoculated a solid motility medium to determine whether our bacteria has to means to move through the medium.

...the results will be determined next lab...

Oxygen Requirement Labs

Today, we got to find out whether our bacteria (environmental were aerobic or anaerobic. We performed two tests to determine if the bacteria needs oxygen and to what extent: one test we used was with thioglycollate broth and the other was with the GasPak.

With the first test using the thioglycollate broth, we used the ascetic to add our unknown bacteria to the liquid broth. We then placed the inoculated broth tube in the incubator (set at 30 degrees). There, it will grow so that next lab we can determine whether our unknown sample is aerobic or anaerobic. The results can appear as follows:

1. Obligate Aerobe

2. Facultative Anaerobe

3. Aerotolerant Anaerobe

4. Obligate Anaerobe

For the second test, we used the GasPak (shown below)

To use the GasPak, first we used the aseptic technique to transfer bacteria onto a new Petri dish. Then we placed the dishes into the air-tight chamber so that no oxygen could be present. Before we placed the lid on, we inserted a methyl-blue pad inside that would act as an indicator, turning from blue to colorless in the absence of oxygen. 

     

...And now we wait until next time to see the results...