Lab Monday May 20 and Tuesday May 21

Monday, May 20, 2013

Lab day: #5

At the beginning of Lab Dr. Pathakamuri checked on the bacteriaphage that he had added to a bacteria culture.  The letters he traced with the bacteriaphage, JMJ, were much clearer since the virus had had more time to destroy the bacteria.  

Letters JMJ cleared by the bacteriaphage

 

Our environmental samples grew spectacularly! We had amazing growth from our dollar bill, coin, phone, and ID card. 

Enviromental Sample Bacteria growth

We then had to prepare simple stains of them. To do this procedure we first obtained a clean slide and sterilized the inoculating loop (using the aseptic technique that we are quite proficient at now). Next, we dropped a miniscule droplet of water onto the slide and mixed bacteria into it with the now sterilized inoculating loop. After this, we waited for the bacteria and water to dry completely on the slide. To fix the bacteria on the slide, we took the dried slide and passed it over the flame of the Bunsen burner only once. We were then able to choose a stain of our choice to color the bacteria. Such fun!


Adding the stain to a fixed bacteria slide

Under the microscope, we were able to determine the shape of our bacteria. For example: we determined that the orange/pink bacteria, stained with crystal violet were bacilli-shaped. Great work team!

                                                                                    -Cecelia

View of simple stain under the microscope



Stained slide

Staining Bacteria

Gram staining is used to determine whether a bacterium is Gram-positive or Gram-negative. Begin by placing a slide with a fixed smear on a rack over a sin. Cover the smear with the dye Crystal Violet. After the duration of 20 seconds rinse the slide to remove the excess stain. Then cover the stain with Gram’s Iodine for 1 minute and rinse to remove the excess solution. Decolorize the smear by applying 95% ethanol drop by drop while holding the slide at a 45 degree angle.

Preparing Gram-stain

Once color stops running immediately rinse the slide from the decolorizing agent and cover with Safranin for 1 minute. Rinse for the final time and blot dry with bibulous paper. If the bacterium is Gram-negative it will appear a red color, and if the bacterium is Gram-positive it will have a purple-ish color. We determined that our unknown bacteria are gram-Negative because after the Gram-staining it had a pink-red color to it. -Elizabeth

View of our mystery bacteria with a gram-stain

 

Preparing a Negative stain

Negative staining is used to see more defined shapes of bacterium. Begin with a clean slide and place a small drop of Nigrosin on one end. Using a sterilized inoculating loop, transfer a small amount of bacteria onto the Nigrosin drop.  Mix well in a small diameter and be sure to flame the loop before placing it down. Touch the short end of another clean slide at a 30- 45 degree angle in the bacteria-Nigrosin drop and spread along the edge of the slide. The slide should include a thin film with a thinning trailing end. Allow the smear to dry completely before looking at it under the microscope using the immersion lens.

View of the Negative Stain

     -Elizabeth

Tuesday, May 21, 2013

Lab day: #6

Today, we conducted a capsule stain to see if our bacteria had a bacterial capsule or

Non-encapsulated bacteria

slime layer. To do this, we prepared a smear of bacteria in nigrosin as we did in the negative staining process. After this, we let the smear air dry and then covered it with safranin. We then gently washed off the excess stain and blotted the slide with the bilbulous paper. Finally, we got to examine the smear with the oil immersion lens.

We positively determined that our bacteria was non-encapsulated because we were unable to view any space between along its circumference. This, ladies and gentlemen, concluded our capsule staining- what a fascinating procedure!

The next stain we completed today was the endospore stain to determine if our bacteria contains endospores. Endospores are located in a few genera of gram-positive bacteria; therefore, we know that our bacteria will probably not contain endospores because it is gram-negative. 


To proceed with the endospore stain, we placed a slide with bacteria fixed on it on top of a beaker containing simmering water. We then had to put paper on top of the slide and keep it saturated with malachite green. This process must be kept up for five to six minutes after the malachite green began to steam, always making sure that the stain does not dry. 

Setting green dye with steam

Once the appropriate time has elapsed, we removed the slide from the heat and allowed it to cool. Next, we rinsed the cooled slide with water and covered it with safranin for about a minute. The safranin was then rinsed and we blotted the slide once again with bibulous paper. 

View of bacteria after Endospore stain

Under the microscope, we discovered that our bacteria did not contain any endospores as is visible in the pictures. Great job everyone! 

                                                                                  -Cecelia

Beginning tests to distinguish bacteria

 

At the end of lab today, we prepared some new bacteria cultures to test different properties of our mystery bacteria in order to help us come closer to identifying it.

 

Test #1 Starch Hydrolysis Test: determine bacteria’s ability to hydrolyze (digest) starch. 

Bacteria use the enzyme amylase to hydrolyze the bonds that like the glucose subunits.  Once the glucose has been broken down, the products can be used for energy and construction of other bio-molecules by the bacteria.  Determining if our mystery type of bacteria is capable of digesting starch will help us determine its identity.

To prepare the experiment we inoculated part of a starch agar plate by placing a streak of bacteria from our agar slant culture using the aseptic technique. 

 

Test #2   Casein Hydrolysis Test: determine bacteria’s ability to hydrolyze casein, the major protein of milk.  When mixed with agar, casein forms a white colloid with calcium ions.  In order to digest the casein bacteria secrete caseinase, a specific enzyme that breaks down the protins that make up casein.  The visible affirmation of a positive result of this process is a clear area around the bacterial growth.  If there is no clear area, then the bacteria is unable to break down the casein. 

To prepare the experiment we inoculated a skim milk agar plate with a streak of pure bacteria culture from our agar slant culture of mystery bacteria.

 Test #3 Gelatin Hydrolysis Test: determine the ability of bacteria to hydrolyze gelatin.  Gelatin is a processed form of collagen, an important protein of connective tissue.  Some bacteria can digest gelatin, as an animal protein, by using the enzyme gelatinase.  Gelatin is usually semi-solid at room temperature, but the enzyme breaks apart the peptide bonds in the gelatin and causes the gel to liquefy.  If the gelatin becomes liquid after cooling at 4 degrees Celsius, then the bacteria are positive for gelatinase. 

To prepare the experiment we inoculated a nutrient gelatin deep tube with a needle stab of bacteria from the agar slant culture.

 Test #4 Fat (Triglyceride) Hydrolysis Test: determine ability of bacterium to hydrolyze a triglyceride.  Some bacteria live in environments where animal or plant lipids are present, such as the bacteria that live on your skin and in the soil to digest dead plant matter.  Most lipids are composed of triglycerides.  Triglycerides are made from three fatty acids link by a single glycerol molecule.  In order to use these triglycerides for food, bacteria must break them apart into their basic compounds using enzymes called lipases.  A positive test will be indicated by a clear area around the bacteria growth. 

To prepare the experiment we inoculated a tributyrin (a type of oily triglyceride that is emulsified with melted agar) agar plate with a streak of bacteria from our agar slant tube.

Test #5   Litmus Milk Reactions: to determine the ability of our bacteria to utilize lactose, protein, and litmus in the litmus milk.  Bacteria differ in their ability to metabolize the different substrates in milk.  Litmus serves as a pH indicator within the milk.  The color will change to indicate the pH.  If the bacteria can ferment lactose the acid pH changes the color of the litmus from lavender purple to pink.  If sufficient acid is produced, the casein will become denatured and form into firm curd within the tube. 

Test#6  Urea Hydrolysis Test: to determine the ability of the bacteria to hydrolyze urea.  Urea is used by the body to excrete excess Nitrogen in urine.  Some bacteria use urease to reduce urea into carbon dioxide and ammonia.  To perform this test we inoculated a urea medium tube and let it incubate for 24 hrs.  If the color changes from yellow to bright pink then this indicates a positive test for urease as the pink indicates that the medium is more alkaline indicating that there is excess ammonia present.