Lab from Wednesday, Thursday, and Friday. May 22-24, 2013

May 22-23 , 2013

In the past couple of days we have learned quite a bit about our very own bacteria. Check it out!

• Our bacteria is aerobic: the new sample of bacteria in the broth culture formed near the top; therefore that means it is aerobic.
• Our bacteria can digest fat.

  

  Postive result for othe Glycoside test

• Our bacteria can excrete caseinase which is the enzyme used to digest casein molecules.



Postive result for Casein test

• Negative test for starch hydrolysis.



Before...

After Iodine.  Negative result.



• Need more time incubating to determine if there is a positive reaction for the litmus milk reaction.
• Our bacteria is motile.
• At this point, it appears as if our bacteria has a positive gelatin test, but we are going to wait a couple more days to see what happens.



Still liquid even after a 15 min. chill in the fridge indicates a positive result of the gelatin test

Test# 1: Fermentation of Carbohydrates (Burham Tube) to test for Sugars: determine ability of some bacteria to ferment a particular carbohydrate

Heterotrophic bacteria often use sugars to obtain energy by fermentation pathways.  Organic acids, alcohols, and gases accumulate as waste products.  These waste products will vary depending on the specific bacteria.  In this test, acid production is identified by a change in the color of the pH indicator, phenol red, which is included in the medium.  In the presence of acid the medium will turn yellow.  In order to collect the gases, an inverted smaller tube, called a Durham tube, is placed in the medium. 

To prepare test: A tube of phenol red containing sugar broth and a Durham tube is inoculated with our specific mystery bacteria using aseptic technique.  The test tube is then incubated for 24 hrs.  If the media turns yellow that will indicate a positive result for acid production and fermentation.  And if a gas bubble is trapped in the Burham tube, that is a positive result for gas production.  This will indicate that our bacteria is capable of fermenting digested carbohydrates. 

Results of glucose tests: positive for sucrose

Test#2:  Methyl Red Test (Mixed Fermentation): determine the ability of bacteria to ferment glucose via mixed-acid fermentation.

  The products of mixed-acid fermentation will include significant amounts of organic acids.  These acids lower the pH of medium to <5.  The pH indicator methyl red is added to the medium and will remain red as long as the pH is 4.5 and below.  This indicates a positive test.  At higher pH values or when less acid is present (indicating that the bacteria did not ferment the glucose) the color ma change to orange or yellow and therefore denote a negative result. 

To prepare the test: Our bacteria is inoculated into a tube of MR-VP broth and incubated for 48 hrs. 

Variable result for test - more pinkish color

Test#3: Voges-Proskauer Test (Butanediol Fermentation): Determine the ability of our bacteria to ferment glucose via butanediol fermentation.  If the bacteria does use butanediol fermentation, then Voges-Proskauer (VP) agents react with acetoin (a precursor for neutral alcohol 2,3 butanediol) in the presence of oxygen to form a red product.

   To prepare the test:  Inoculate a MR-VP tube with our bacteria using aseptic technique and allow to incubate for 48 hrs. 

Adding acetoin to the test tube

Test#4: Citrate Utilization Test: determine if bacteria can utilize citrate as its sole source of carbon and energy.  In order for bacteria to be able to do this it must have the membrane –associated transporter citrate permease.  These cellular enzymes convert citrate in the cell’s cytoplasm into pyruvate and carbon dioxide.  The pyruvate is used for energy and the Carbon dioxide can combine with NH4+(which is provided in the medium) to produce NaCO3, an alkaline compound.  This compound will change the pH which will be detected by the pH indicator bromothymol blue.  The color of the medium will change from green to blue and denote a positive test.

Citrate test

Test#5: Indole (Tryptophan Degradation) Test: determine the ability of bacteria to split amino acid tryptophan into indole and pyruvic acid.  Tryptophan can be used as an energy source by degrading the amino acid into pyruvate.  Indole is a byproduct not used by the bacteria.



Indole test - negative

Test#6: Nitrate Test: determine if a bacterium is able to reduce nitrate ions to either nitrite ions or to nitrogen gas. Basically there are two reactions. The first is where the nitrate ion is reduced by the enzyme nitrate reductase into nitrite ions. The second is where the nitrate is reduced completely into molecular nitrogen in the process called denitrification.

Test#7: Urease test: determine the ability of a bacterium to hydrolyze urea. Urease-producing bacteria grow in a medium containing urea, accumulates ammonia, and changes the medium to be more alkaline. The pH change is measured with phenol red which will turn to a bright pink if the pH is alkaline.

Test#8: Motility test: determine if a bacterium is motile. The presence of a flagellum are what causes the bacteria to move around. When motile bacteria is stabbed with an inoculating needle into the semisolid agar, the bacteria will swim away from the stab, showing a cloud of growth. Another way to test for motility is to prepare the hanging drop slide.

Test#9: TSI agar test: differentiate among the gram-negative enteric bacilli as to their ability to ferment glucose, lactose, and sucrose and to produce H2S. If a bacterium ferments any of the sugars present in the TSI agar (lactose, sucrose, glucose) the resulting acids cause the pH to drop.

            So far, we have performed these experiments on our bacteria and watched for the reactions to occur. The following list is incomplete but is slowly but surely directing us onward in our discovery. This has been quite an adventure for every one of us, and we each eagerly anticipate discovering just what kind of bacteria we have!

TESTS                                                                                                 RESULTS

 Glucose                                                                                               Positive

Lactose                                                                                                Negative all

Sucrose                                                                                                Positive

Mannitol                                                                                              Positive (gas)

Gelatin liquefaction                                                                             Positive

Starch                                                                                                  Negative

Casein                                                                                                 Positive

Fat                                                                                                       Negative

Indole                                                                                                  Negative

Methyl Red                                                                                         Variable (pink)

Voges-proskair                                                                                    Negative

Citrate utilization                                                                                 Positive

Nitrate reduction                                                                                 Positive

Urease                                                                                                 Negative



Bubbles!

Another Aerobic test is the Hydrogen-peroxide test.  Organisms that breath oxygen can break down Hydrogren-peroxide into Hydrogen and Water.  The visual observation is a bubbling of the liquid as the oxygen is released.  Anaerobic bacteria are unable to convernt toxic Hydrogen-peroxide and therefore die when treated with Hydrogen-peroxide, which makes this substance a good way to disinfect a live or inaminate area of anaerobic bacteria.  Our bacteria is once again an aerobe because of the bubbling result we observed.

Friday, May 24, 2013

Today, we prepared the hanging drop slide which is used to determine motility. To do this, we needed the following tools: depression slide, 5nm syringe, coverslip, vaseline, microscope, and broth culture of our bacteria. 

To begin, we put Vaseline on each of the four corners of the cover slip. Then we used the syringe to gather a very small amount of the bacteria to place onto the center of the cover slip. Then we put the cover slip onto the depression slide and placed it under the microscope with the oil immersion.

Our results were that our bacteria is motile. We were able to positively determine that because we actually saw our little bacterium swimming around.What an exhilarating discovery!

-Cecelia