Day 13

Today we once again observed the EMB plate after incubating for a little while longer and confirmed again that it was E. Coli because of the green sheen.

We then discussed the different tests that hospitals run to determine different diseases, the different plates to use, colors to look for and thus we connected all the dots of things we learned this mini session. These charts below can help lay out the results for different bacterial infections and diagnosing them.

Here are a couple of points he stressed:

·      Conformation is always immunodiagnostics testing and PCR testing which is when you amplify the DNA of the bacteria. If the bacteria bind when run against unknown bacteria it is a confirmation.

·      Coliforms are lactose fermenter, which can be used to look for shigella or salmonella if a patient, has diarrhea.

·      EHEC is diarrhea with blood, while ETEC is traveler’s diarrhea.

·      With all the different differential plates you can diagnoses different diseases

·      When taking blood and urine in the hospital culturing the results is a must!

Next Dr. P showed us pictures of plates with MRSA and MSSA with their respective inhibition drugs.

Here are the results from the UV radiation of all the bacteria; the UV light killed a substantial amount of the bacteria, but not all. We can still count the colonies. Usually Dr. P said all of the bacteria would be killed and the Agar plate would be clear.

Lastly the yogurt, after the taste test the consensus was that everybody liked the taste but the texture was different than store bought yogurt.  Now we all know how to make our own yogurt, good thing because we are poor college students!!!  And with that our microbiology lab came to an end.


13 Lab days....3 girls...a whole ton of bacteria later ... is microbiology lab in a mini session!!!

Day 12

Today we started off by examining the EMB plate and saw that it showed a slight green tint meaning our mystery bacteria was E. Coli, Dr. P confirmed that the mystery was solved correctly!

Next we performed part of the Immunodetective experiment, we tested for food purity (Test 3) by creating an Agar plate with various compartments to hold: Hamburger extract, Goat Anti-Bovine Albumin, Goat Anti-Horse Albumin, and Goat Anti-Swine Albumin. We placed small amounts of each Albumin into their respective compartments and let it incubate.

We then started to Identify HIV viruses. We first took our 12 well strip and placed 50 ul of the purified antigen into all of the 9 wells we are using. We then had to wait five minutes to let the antigen bind to the plastic wells.

 We then dumped out the antigen and washed the walls of the wells with a buffer and dabbed with paper towels upside down to make sure that it is all-dry.

Next we added the serum of the patient and the positive and the negative control to the wells. We then waited to allow for the antibodies to bind to the antigens.

We washed again, so all that remains in the tube is the antibodies that have bound; we are unable to see these! We added antihuman anti-body with enzymes attached to the wells.  If color is produced the patient has antibodies. For ours, only the positive turned blue meaning the patient has antibodies for aids.

After this, Dr. P made two new Agar plates. He went around and mixed all of our bacteria broths with water. He spread the bacteria water mixture on one Agar plate. Then he used UV light to kill the bacteria in the mixture. He then prepared an Agar plate with the mixture after UV radiation. He then placed these two plates in the incubator.

Then he preformed another experiment involving yogurt. He boiled milk and let it cool. Then placed a little bit of plane Greek yogurt into each tube and incubated them. Tomorrow we will have homemade yogurt! YUM!

Day 11

Today we checked all of our plates that we made yesterday!

First off, we checked Lauren’s strep plate. Our plate showed alpha hemolysis (partial) so that means that her throat is negative for strep throat.

Then, Christina’s nose… negative for staph auereus because there was no yellow growth. 1 in 3 people do have staph auereus in their nose. 

Our plate that we spread our unknown bacteria over and placed disks of antibiotics on it showed that our bacteria is : Non sensitive to Penicillin, Intermediate to Tetracycline, Resistant to Erythromycin, Susceptible (Positive) to Chloramphenicol.

The Manitol plate of our bacteria was negative because there was no discoloration, which means there is no S. aureus.

The blood agar plate of our bacteria had partial alpha hemolysis due to the green hint of growth.

Next we evaluated all of our bacteria results to find out what unknown bacteria we had! We followed a flow chart depicted below:

We believe that we have Escherichia Coli (E Coli), but we are performing one more test to make sure! We are going to do a bacteria plate on an EMB plate to double check. 

Day 10

Day 10 

Today we performed more tests to determine our bacteria with differential media. We swabbed bacteria onto four different agar plates: McConkey, Phenol Ethanol, Blood, and Manitol.

We set those in the incubator to grow.

Next we performed antibiotic testing with our bacteria. We swabbed bacteria all over an Agar plate and then put discs of each antibiotic in a respective places. The antibiotics we used were: Penicillin, Tetracycline, Erythromycin, Neomycin, and Chloramphenicol. We also placed this in the incubator to allow it to grow.  Penicillin is used to combat the cell wall of the bacteria, Tetracycline and Erythromycin inhibits protein synthesis and Chloramphenicol inhibits the 50S Ribosome subunit. Neomycin also inhibits the protein synthesis. 

Then we got to be nurses and swab someone’s throat and someone’s nose!!! The nose swab was wet with saline solution specifically to test for MRSA. We swabbed the throat to check for strep.  

We swabbed Lauren’s throat and spread it on a blood agar plate. We then put a bacitracin disk on it and set it in the 37 degrees incubator.  

For the nasal swab we swabbed Christina’s nose and spread it on a Manitol Salt Agar plate. This also was placed in the 37 degree incubator. If it grows, the acid will change color and that will be a positive test.

Day 9

Day 9

Today we prepared a hanging drop slide in order to view our live bacteria. First we took a clean cover slide and placed petroleum jelly on all four corners. We then injected our bacteria onto the cover slip. We placed the cover slide on a depression slide and examined it under the microscope to check for motility. 

We found that our bacteria are motile!!! Check out the video below!

http://www.youtube.com/watch?v=P8lg_OTvu3s&feature=youtu.be 

We also observed our tests that we performed yesterday. 

The top one is the Oxidase tube and the bottom one is the Triple Sugar Iron Agar Slant. 

First we observed the Oxidase tube and determined our bacteria is facultative. 

For the Triple Sugar Iron Agar Slant we took a sample of the grown bacteria on a swab and added about 10 drops onto the bacteria. The bacteria did not change color, which means we had a negative test. 

Going back to the tests we performed two days ago, we had continued to incubate the sucrose, the litmus milk, and the citrate:

-There is no further growth in the citrate tube indicating a negative result.

-The sucrose still appears to be an orange color, but we will continue to incubate to further check.

-The litmus milk showed a bit of pink color and white on the bottom of the tube indicating it is an alkaline reaction. We will continue to incubate that as well to make sure it is done reacting.

Day 8

DAY 8

Today we first looked at our Skim Milk Agar and determined it was a negative.

Fermentation of Carbohydrates (Durham Tube)

·         We were looking for gas inside the Durham tube and then identify the color of our tube

·         What we found:

o   Mannitol: yellow positive, gas in the tube acid/gas (farthest right)

o   Lactose: yellow positive, gas in the tube acid/gas (2nd from right)

o   Glucose: yellow positive, gas in the tube acid/gas (2nd from left)

o   Sucrose: red negative, no gas in the tube no fermentation (far left) 
        (note: for the sucrose we are going to continue to incubate it and see if the color changes) 

Citrate utilization test

·         We found that our test tube agar slant was still green, so we are going to incubate it longer.

Triple Sugar Iron (TSI) Agar Test

·         We found that our agar slant was yellowish orange so it was an acid slant/acid butt with gas. We found that it used lactose and sucrose (still going to check). There is gas production because there are breaks in the agar. It changed color because of the pH indicator.

Urea Test:

·         Ours wasn’t a deep pink color, rather a yellow color means urea is not broken down and the enzyme is not there, therefore it is negative. However, we will incubate it longer to make sure this is true

Methyl Red Test

·         We divided our broth into 2 different tubes.  One for VP test and one for MR test. We added 15 drops of A and 5 drops of B to one tube. To the other tube we added 5 drops of methyl red.  The MR tube was positive because it was red. VP is negative because it remained yellow. tells us that it uses glucose and VP tells us that there are no end products.

Nitrate Reduction Test

·         We put on gloves and added 5 drops of reagent A and 5 drops of reagent B to the tube. We found that a red color developed meaning that the test is positive for nitrate reduction so nitrite ions are present).

Iodine (Tryptophan Degradation) test:

·         We added 10 drops of Kovac reagent to the culture. It produced the cherry red color on top so we found that it was positive.

We also looked at our litmus milk today and found that there is a little pink color so we are going to incubate it for a little longer and check it tomorrow.

We added hydrogen peroxide on top of our streak plate and found that the bacteria produced bubbles meaning that they are aerobic.

At the end of today we made a new broth culture. We inoculated our bacteria in the slant by poking and snaking up the top. Then we inoculated an oxic/anoxic test tube with our bacteria. We made sure to use aseptic technique while doing this. Lastly, we placed these tubes in the incubator.