Week 9

 

 

Week 9-Selective and/or Differential Media

10/22/13

 

Today in class we prepared several streak plates to test for selective and/or differential characteristics in our sample bacteria. Selective tests enable researchers to isolate a certain kind of bacteria (ex. Gram positive bacteria) by streaking bacteria on an agar plate that only that kind of bacteria can grow on. Differential tests test for a specific characteristic of the sample bacteria, and help to distinguish it from other bacterial species.

 

We prepared a blood agar streak plate to see if our bacteria was fastidious, and to test whether our bacteria was hemolytic, or had the ability to lyse red blood cells. This can occur by either B-hemolysis, in which the RBCs are completely destroyed, leaving a clear zone around the bacteria colony on the streak plate, or by a-hemolysis, in which they are only partly destroyed, leaving a green tint surrounding the bacteria. Non-hemolytic bacteria does not damage RBCs at all and causes no changes in the appearance of the plate.

 

We prepared a DNA agar plate to see if our bacteria produces the enzyme DNase, which bacteria use to hydrolyze, or digest, DNA. By flooding the DNA plate with HCl, we can see if the area surrounding the bacteria appears cloudy (due to the presence of undigested DNA molecules) or clear.

We prepared an Eosin Methylene Blue (EMB) agar streak plate. Since the dyes eosin and methylene inhibit the growth of gram-positive bacteria, EMB plates can be used to isolate gram-negative bacteria. The agar also contains lactose and sucrose. Since enteric bacteria, or bacteria of the digestive tract, are able to ferment these sugars, this plate can be used to differentiate between enteric and non-enteric bacteria. If the bacteria can ferment lactose and sucrose, acid will be produced, and the colonies will develop a greenish sheen due to the lower pH.

 

We prepared a Mannitol salt agar streak plate. Mannitol salt agar is used to isolate bacteria based on their salt tolerance, as only bacteria that can tolerate 7.5% NaCl will grow on the plate. The presence of mannitol in the agar allows us to differentiate between bacteria that can ferment mannitol and bacteria that can’t. If the sample bacteria can ferment mannitol, the acid produced will lower the pH, causing the area around the colony to turn yellow.

 

We prepared a MacConkey agar streak plate. The MacConkey plate can be used to isolate gram-negative bacteria, as the growth of gram-positive bacteria is inhibited by the crystal violet and bile salts in the agar. The lactose in the agar allows us to differentiate between lactose-fermenting and non-lactose-fermenting (enteric and non-enteric) bacteria. If the sample bacteria is able to ferment lactose, the colony will turn red due to a decrease in pH caused by acid production.

We prepared a Phenylethyl alcohol (PEA) agar streak plate. The PEA plate is used to isolate gram-positive bacteria. PEA inhibits the growth of gram-negative bacteria by interfering with DNA synthesis, while gram-positive bacteria remain unaffected.

 

We also inoculated thioglycollate broth with our sample bacteria to see if our bacteria prefers an aerobic or anaerobic medium. In our broth, an indicator turns purple in the presence of oxygen, which is mainly present near the top of the test tube.

 

10/24/13

 

These were the results of last Tuesday’s tests:

Blood Agar—our bacteria grew on the plate, showing that our bacteria is fastidious. Our bacteria partially hemolyzed the red blood cells in the plate, using a process called a-hemolysis.

EMB Agar—our bacteria grew on the plate, reaffirming our previous conclusion that our bacteria is gram-negative. Our bacteria was able to ferment the lactose and sucrose in the plate, producing an acid that lowered the pH of the plate, giving our colonies a greenish sheen. Our sample bacteria is enteric.

 

Mannitol Salt Agar—our bacteria did not grow on the plate, showing that it has a low salt tolerance (not a halophile). It was not able to ferment mannitol.

 

MacConkey Agar—our bacteria was able to grow on the plate because it is gram-negative. The bacteria was able to ferment the lactose in the plate, producing acid that turned the bacteria on the plate pink.

 

 

PEA Agar—since PEA inhibits the growth of gram-negative bacteria, our bacteria did not grow on this plate.

 

DNA Hydrolysis Test—our bacteria does not produce the enzyme DNase, so it was not able to hydrolyze the DNA in the plate.
 

 

Thioglycollate Broth—as expected, our bacteria grew best at the top of the test tube, where there had been more oxygen at the time of inoculation. Our bacteria prefers an aerobic environment.