Week 12

Disclaimer

All content provided on this blog is representation of the blog owner and not Franciscan University of Steubenville. The information on this site is purely used for education purpose. The owner of this blog makes no representations as to the accuracy or completeness of any information on this site or found by following any link on this site. The owner will not be liable for any errors or omissions in this information nor for the availability of this information. The owner will not be liable for any losses, injuries, or damages from the display or use of this information.

Privacy

The owner of this blog does not share personal information with third-parties nor does the owner store information is collected about your visit for use other than to analyze content performance through the use of cookies, which you can turn off at anytime by modifying your Internet browser’s settings. The owner is not responsible for the republishing of the content found on this blog on other Web sites or media without permission.

Blog Comments

The owner of this blog reserves the right to edit or delete any comments submitted to this blog without notice due to;
1. Comments deemed to be spam or questionable spam
2. Comments including profanity
3. Comments containing language or concepts that could be deemed offensive
4. Comments that attack a person individually

This policy is subject to change at anytime.

In microbiology lab this week we completed an enzyme-linked immunosorbent assay (ELISA) test. This test detects antibodies in your blood to determine if you have been exposed to a disease. 

To begin we used a micropipette to add purified disease antigen to the correctly labeled wells of our microplate strip.

 We then washed the unbound antigen out of the wells and transferred the positive and negative control and the serum samples to the correctly labeled wells. 

After washing the samples out of the wells, we transferred the secondary antibody into all of the microplate wells. After waiting five minutes, we washed the unbound secondary antibody out and added an enzyme substrate into all twelve of the wells, recording our final results. 

This week in lab we also did an exercise on an antibody-antigen reaction in agar.  The purpose of this exercise was to determine if our sample hamburger extract was pure by observing a precipitin line.  First we made four wells within the agar and placed a different solution within each well. The solutions were: Bovine Albumin, Goat Anti-horse Albumin, Goat Anti-bovine Albumin, and Goat Anti-swine Albumin.  If the solution was pure there should have been a precipitin line observed between the Bovine Albumen and the Goat Anti-bovine Albumin. Unfortunately, due to the age of our solutions there was no precipitin line observed in our agar.

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

Week 4

Week 4, Day 1:
9/17/13

A New Test Tube, A New Chapter

This week, we received a test tube with a new colony of bacteria to work with. Since this bacteria was growing on an agar slant, our first step in identifying the bacteria was to analyze the growth pattern in the test tube. The bacteria we received was growing in what looked like a filiform, or even, pattern.

After observing this growth pattern, we used a sterilized inoculating loop to transfer a small amount of the new bacteria to another test tube. This tube was then placed in the incubator at 37' C to grow over the next few days. 

Using aseptic technique, we then took a loopful of bacteria from the original test tube and mixed it with a drop of distilled water on a new glass slide. We let this mixture air-dry, then passed it through the propane torch three times to heat-fix it, creating a bacterial smear.

Our first step in identifying this bacteria was to determine whether it was gram-positive or gram-negative. To do this, we performed a gram stain on our bacterial smear. We put the slide with the smear on a rack over the sink, covered the smear with crystal violet for 20 seconds, and rinsed the slide with distilled water.

We then covered the smear with Gram's iodine for 1 minute, before rinsing the slide again with distilled water.

After rinsing, we decolorized the smear by dripping 95% ethanol on the slide at a 45' angle until the color from the stain stopped running, then rinsed with distilled water.

We covered the smear with safranin for 1 minute, then rinsed the slide and blotted it with bibulous paper.

By observing the stained smear under the oil immersion lense of our microscope, we saw that our bacteria were gram-negative. Because of their thin peptidoglycan layers, the bacteria had been unable to hold the crystal violet stain, and came out safranin-pink instead.

Week 4, Day 2
9/19/13

After determining last Tuesday that our new bacteria was gram-negative, we spent Thursday's lab preparing negative and capsule stains on both our gram-positive environmental sample bacteria and the gram-negative test tube bacteria we received in class.

To prepare a negative stain on the test tube bacteria, we took two clean slides and placed a small drop of nigrosin at the end of one. Then, using aseptic technique, we used our inoculating loop to transfer a loopful of test tube bacteria to the slide. We then mixed the transferred bacteria with the nigrosin on the slide.

After transferring the bacteria, we took the second (spreader) slide and touched it to the nigrosin/bacteria drop at a 30-45' angle. Once the drop had spread across the length of the spreader slide, we quickly pushed the spreader slide across the first slide, spreading the bacteria/nigrosin mixture across the first slide.

Once this smear had air-dried completely, we examined it under the oil-immersion lense of our microscope. The negative stain allowed us to view the bacteria against a dark background, making it easy to see their shape. This method can also be used to observe bacteria that do not absorb other dyes, or bacteria that are too fragile to undergo heat-fixation when preparing a bacterial smear.

We also repeated this procedure on our environmental sample bacteria. Both bacterial strains appeared to have a spherical shape.

Once we had prepared a negative stain for our environmental and test-tube bacteria samples, we used the negative stains as a foundation for preparing a capsule stain of each type of bacteria. The capsule stain would allow us to view bacterial capsules (the outer coating of a bacteria cell) or slime layers.

To prepare a capsule stain, we took the negative stain slides for the environmental and test-tube bacteria samples, and placed them on a rack over the sink. We covered each slide with safranin for 1 minute, then rinsed off the excess stain with distilled water.

After blotting the slides with bibulous paper, we observed that our bacteria did not have any capsules. However, upon examination, we began to doubt our initial belief that our bacteria was spherical in shape.

We did another bacterial smear and determined that our bacteria was, actually, rod-shaped

Week 3

Week 3

Preparing a Gram Stain. 

9/12/13

This week the whole lab would be working with another isolated colony of their bacterial specimen and observe the colony by means of differential staining as opposed to simple staining. This week, we would focus on preparing and observing a gram stain. The preparation of this stain begins similar to the simple staining we worked on in week 2. We would again find an isolated colony of bacteria from our specimen plate:

We would then place a drop of distilled H2O (water) on a glass slide and proceed to sterilize an inoculating loop.

We used the sterile inoculating loop to capture one of the isolated, bacterial colonies and would transfer it to the drop of water on the slide. Using the inoculating loop, we thinned out the material sample and water across the whole slide.

We then proceeded to follow the directions like good students, and after the slide had air-dried, we "fixed" the smear by passing the glass through a lighted bunsen burner three times.

We then placed the fixed smear on a rack over a lab sink, and covered the smear with a crystal violet stain for 20 seconds

.

After the stain had set on the slide for 20 seconds, we rinsed the slide with distilled water to remove the excess stain. We covered the slide with Gram's iodine for 1 minute and then rinsed the slide again. Next, we held the slide at a 45 degree angle and a 95% ethanol solution (a decolorizing agent) over the slide until the run off was no longer pigmented. Once we achieved this, we rinsed the slide again and then covered it with a safranin dye for 1 minute.

 We then rinsed the slide and blotted it out with a bibulous paper. 

Once the slide was dry, we observed it under a microscope.

The purpose of this experiment was to determine if our bacterial samples were either gram-positive or gram-negative bacteria. Gram-positive bacteria have a thicker peptidogylcan layer than gram-negative bacteria, thus it (in essence) traps the violet and iodine solutions more effectively than the thinner layer found in gram negative bacteria. The 95% ethanol solution dehydrates the the peptidoglycan layer, thinning it out. The thinner layer in gram negative bacteria cannot retain the violet dye, whereas the gram positive bacteria is able to retain the stain. The safranin is added as a counter stain to then stain the now-stainless gram negative bacteria, thus providing us with a differential stain. Here we observed that our bacteria is gram positive, noted by violet stain, as well as spherical shaped. However, we also observed that there was some pinkish dyed bacteria. This could have occurred through heating up the slide too much when fixing the bacterial smear. We would repeat the procedure with a new stain to see if we could correct this error. 

The following lab period we would proceed to finish our second attempt of gram-staining our bacterial smear. We followed the same procedure and, after observation, came up with a more accurate sample. We thereby determined that our bacterial sample was, in fact, gram-positive.

Week 2

Week 2

Fun With Bacteria: Adventures in the Steubenville Port-a-Potty 

9/3/13

Last Thursday our lab group collected two bacteria samples from a urinal in a local...outhouse. We then incubated the samples in petri dishes over the weekend, so that we could check for signs of bacterial growth in class today. Our samples each grew a thin yellow film, with a few black dots (possibly mold?) near the edges of the dishes. By looking through a microscope, we were able to see the speckled, yellow-green pattern of bacterial growth that our sample produced in greater detail.

Our next task was to isolate a single strain of the bacteria, in hopes of acquiring a pure culture. Using aseptic technique, we sterilized the inoculating loop over our propane torch, waiting until the metal of the loop had turned red before removing it from heat. Once we were confident that the loop was sterile, we swiped the loop over a section of one of our bacteria samples and streaked it across the first quadrant of a new petri dish. We then took turns streaking the bacteria from the sample in the first quadrant across the second, third, and fourth quadrants (and, of course, sterilizing the inoculating loop between each streaking to prevent any contamination). Having prepared this streak plate, we placed it in the incubator at 37' C, and put the original samples into the refrigerator to prevent further growth. By Thursday's class, we hope to find isolated colonies of bacteria on the incubated streak plate, which we can then analyze as a pure bacterial culture.

Week 2

Fun With Bacteria: The Adventure Continues

9/5/13

After incubating our streak plate for two days, we started Thursday's class by looking for an isolated colony of bacteria that we could use as our pure culture. While most of our plate was covered with the same yellowish film we had last week, we also discovered a small group of dots growing on our streak plate. These "dots" of bacteria, since they grew separately from the rest of the plate, were actually pure bacterial colonies that could be used to continue with the rest of the experiment.

week 1

In week one of lab, we first learned aseptic technique. We then performed an experiment to determine how much bacteria one has on his hands before and after hand washing. Each pair of students were given an agar plate and touched the agar with a finger (once before washing ones hands and once after). The agar plate was then put in an incubator so that whatever bacteria that was on the finger, before and after, would remain at optimal temperature for growth. Our initial hypothesis was that the finger print from before the hand washing would have cultured more bacteria than the finger print after, thereby confirming the need for proper hygiene; however, our results were the opposite.
        In the next lab, to our surprise, the majority of the class experiment had developed more bacteria in the  'post-hand washing'specimen then in the specimen taken before. We concluded that one of two things may have occurred: a) we did not practice good aseptic technique; or b) something we touched after washing our hands had a large quantity of bacteria, whether it was the paper towels we used to dry our hands or turning off the faucet after rinsing our hands.
       We also made a field trip to the beautiful steubenville marina, where we collected specimen from a nearby porter john and transferred it to an agar plate for culturing. We used a sterile broth solution (which we prepared in the previous lab session) to wet the swab and collect the specimen. Upon returning to campus, we placed our specimen into incubators so that the bacteria would grow at optimum temperatures.

Disclaimer

All content provided on this blog is representation of the blog owner and not Franciscan University of Steubenville. The information on this site is purely used for education purpose. The owner of this blog makes no representations as to the accuracy or completeness of any information on this site or found by following any link on this site. The owner will not be liable for any errors or omissions in this information nor for the availability of this information. The owner will not be liable for any losses, injuries, or damages from the display or use of this information.

Privacy

The owner of this blog does not share personal information with third-parties nor does the owner store information is collected about your visit for use other than to analyze content performance through the use of cookies, which you can turn off at anytime by modifying your Internet browser’s settings. The owner is not responsible for the republishing of the content found on this blog on other Web sites or media without permission.
 

Blog Comments
 

The owner of this blog reserves the right to edit or delete any comments submitted to this blog without notice due to;
1. Comments deemed to be spam or questionable spam
2. Comments including profanity
3. Comments containing language or concepts that could be deemed offensive
4. Comments that attack a person individually

This policy is subject to change at anytime.