Week 11

Week 11
11/ 12/13


Today for lab we tested several different household disinfectants on a bacterial cultures to see their effects and the strengths and weaknesses of the different varieties of disinfectants. First we took a sample from a doorknob and spread it over a nutrient agar plate for culturing. Then we divided our plate into four sections and placed samples of four different, common household disinfectants. We used hand sanitizer (60% ethanol solution), toothpaste, Clorox, and mouthwash.
We then incubated our plates at 25 degrees Celsius.

Once we were finished we made another nutrient agar spread, but this time with our unknown bacteria. We all placed our samples in an anaerobic chamber to determine if our bacteria had anaerobic properties (placing the chamber in the incubator set at 37 degree Celsius)

11/14/13

The following class we observed our disinfectant testing, and found that our bacteria was unaffected by both hand sanitizer and mouthwash (Listerine).

Then we observed the results from the anaerobic test, and our bacteria did, in fact, have  some growth (top sample). Thus showing that our bacteria is a facultative anaerobe. 

  



Week 10

11/5/13

            Today in lab, we went to the local Wastewater Treatment plant.  The tour guide showed us where the wastewater entered the building and how they began to purify the water.  The wastewater goes through many different pipes and pools while interacting with microorganisms that break down the organic material in the wastewater.  At the end of the water treatment process there are multiple samples taken of the treated wastewater to ensure treated water is being released back into the river.  The concluding part of the tour was touring the lab in the treatment plant. The tour guide showed us what the samples were tested for and why it was a vital part of the wastewater treatment plant. 

 

11/7/13

            Today in lab, we learned how to collect a nasal swab sample.  We then streaked the sample on a mannitol salt agar plate and incubated it at 37°C. 

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.

 

Week 8

For week 8, we performed several tests to determine the enzymatic properties of our bacteria. These tests included Methyl Red test (mixed fermentation), Citrate utilization test, Indole (tryptophan degradation) test, Oxidase test, Nitrate Reduction Test, and the Urea hydrolysis test.
To begin, we inoculated test tubes containing the medium of each specific test, and then incubated them until the following lab to determine the results.

The following lab period, upon retrieving our test tubes, we determined that our bacteria had a negative test with Urea, Oxidase, and Citrate, but a positive test with Nitrate, Indole, and the methyl red fermentation test.

 
For the Nitrate test, we took our inoculated nitrate broth tube, and added 5 drops of nitrate reagent A (sulfanilic acid) and 5 drops of nitrate reagent B (dimethyl-alpha-naphthylamine). Our nitrate broth immediately took on a deep red color, indicating a positive test.

For our Indole test, we took our inoculated tryptone broth tube and added 10 drops of Kovac's reagent. A red layer appeared at the tope of the tube, indicating that the test was positive.

Next, we took our methyl red broth tube and poured out half into another sterile test tube for a second fermentation test (Voges-Proskauer Test) We then added 5 drops of methyl red (pH indicator) to the first tube.

Our tube took on a reddish color, indicating a positive test.

We then took the second test tube of methyl-red broth and added 15 drops of Baritt's reagent A (alpha-naphthol) and 5 drops of Barritt's reagent B (KOH) to the tube. Our broth did not change colors, indicating a negative test.
Finally, we performed the oxidase test. Taking our inoculated broth tube for the test, we took a sample using a sterile swab. We then added the oxidase reagent to the swab. There was no change in color, indicating a negative test for oxidase.

In conclusion, through these tests, we concluded that our bacteria:
a) is able to ferment glucose via mixed-acid fermentation, but not through butanediol fermentation (methyl red)
b) does not use citrate as its sole source of carbon and energy (Citrate test)
c) has the ability to split the amino acid tryptophan into indole and pyretic acid (Indole test)
d) does not have cytochrome oxidase (oxidase test)
e) is able to reduce nitrate ions to either nitrite or to nitrogen gas (nitrate test)
f) and is unable to hydrolyze urea

Week 7

Week 7

10/10/13

This week we observed the results from all of the tests we prepared last week.

We determined that our Starch hydrolysis test was negative, thus our bacteria does not have the enzymes necessary to digest starch.

Our casein hydrolysis test (skim milk plate) also was negative.

Our gelatin test was negative 

 

 Our fat (triglyceride) hydrolysis test was negative (our bacterial sample is on the bottom)

 

And finally, our Litmus milk test results:

The white curd at the bottom detects peptonization with an alkaline reaction. Thus, our bacteria has proteolytic enzymes that digest the casein (curd) into peptides and amino acids.

We were then instructed to inoculate test tubes filled with four different mediums to test if our bacteria had enzymes to digest carbohydrates (these test tubes were filled with lactose, sucrose, glucose, and Triple Sugar Iron (TSI) mediums).

Week 6

Week 6
10/3/13

Last Tuesday we inoculated a tube of nutrient broth with our unknown bacteria sample that we received in class. We then incubated the broth solution at 37' C for the remainder of the week. 

We spent today's class streaking the same unknown bacteria over various mediums, to see which enzymes our bacteria expressed. Bacteria can be classified is by these enzymes, bringing us closer to identifying our sample.

We inoculated bacteria in litmus milk

and gelatin,


and streaked it over skim milk, starch, and spirit blue (lipid) agar plates.

We will incubate these samples at 37'C until our next class.

Week 5

Week 5 

9/24/13 - 9/26/13 

in week 5 of class we performed an acid-fast stain, an endospore stain, and determined if our bacteria was motile or not. We first started with the acid-fast staining. An acid-fast stain is used to distinguish the two groups of bacteria based on the lipid content and their cell walls: acid-fast and non-acid-fast. We first made a bacterial smear on a glass slide and then fixed it over a Bunsen burner.  We then placed the slide over a beaker of boiling water. We took a piece of bibulous paper and set it on the slide before dropping carbolfuchsin over the length of the slide. We continued to drop carbolfuchsin on the slide for 3 minutes.

We then removed the bibulous paper and retrieved the slide using forceps. We rinsed the slide using  water to remove excess stain and then a decolorizing solution, adding drop by drop until the color stopped running. We then immediately rinsed the slide to remove the decolorizing agent. We then covered the slide with methylene blue for 2 minutes.

Then we rinsed the slide with water to remove the excess coloring. After blotting the slide with pieces of bibulous paper we examined the smear under the microscope

upon examination we determined that are bacteria was a non acid-fast due to the methylene blue stain

We then were given a tube with motility test medium. Using aseptic technique, we took a sample of our bacterial culture with an inoculating needle and then stabbed it into the test medium. We then placed it in the incubator at 37' C until the next lab period.

The following lab, we worked on preparing an endospore stain. This stain is used to determine if a particular bacteria has endospores. We again fixed a smear of bacteria on a glass slide. We then placed the smear over a beaker of boiling water. Taking a piece of bibulous paper and placing it over the smear, we dropped malachite green over the smear. We let the stain set for 6 minutes while continuing to add stain as it evaporated.

 We then discarded the paper and retrieved the slide using forceps. We rinsed the slide with water to remove the excess dye. We then covered the smear with safranin for 70 seconds. We immediately rinsed the slide with water to remove the excess safranin and after blotting the slide with pieces of paper we examine the smear under the microscope.

upon examination we determine that are bacteria did not contain endospores