Week 3, Day 4

May 31, 2013

Today in lab our professor brought in some MRSA (methicillin resistant Staphylococcus areus) and MSSA (methicillin sensitive Staphylococcus areus) Petri dishes for us to observe. The MRSA samples were incubated on mannitol-salt agar plates.

 In one of the samples we observed a mupirocin antibiotic disk in the agar. The bacteria in the dish was sensitive to the mupirocin and did not grow around the disk. Another dish contained a sample of bacteria that was avoiding (was sensitive to) a oxycillin antibiotic disk. Because this disk inhibited the growth of the bacteria, it indicates that the culture was MSSA and not MRSA.

                                                           

After viewing the MRSA and MSSA culture dishes we retrieved the yogurt tubes from the day before. The milk had solidified and the bacterial growth had formed yogurt in the tubes.  We were able to sample it and determine which brand of yogurt was better.

 We also observed the UV light treated bacteria water Petri dish and the untreated sample. The untreated sample had a great deal of bacteria cultures growing on, while the treated sample only had a few cultures growing on the Petri dish. Our professor told us that usually the UV treated water would have no bacterial cultures.

We also studied our food purity test from the day before. The meats that were present in the hamburger extract had reacted to the goat anti-albumins and produced precipitates, whereas the goat anti-albumins for the meats not present in the hamburger did not react with the extract at all. This test allows us to identify which meats were present in the hamburger extract, which will give us an idea of the purity of the sample.

  We finished up lab for the mini session by watching the movie 'Contagion,' and how it applied to Microbiology and our field of nursing.

  Week 3, Day 3

   May 30, 2013

Today in lab we began by checking the results of our TSIA and indole tests that we were told to re-do last session. The results were the same as before, and after checking with our professor he said there might have been a mislabeled sample giving us a different species. We definitely got the correct genus, which was Proteus. Our sample was labeled as Proteus valgaris, but tested for Proteus inconstans.

Next we moved on to the food purity testing. Our group will be testing the purity in a hamburger extract sample. We prepared a Petri dish with four different holes (one in the center with three surrounding).

Preparing the Plate

 We transferred a drop of the hamburger extract to the center well, and a drop of goat anti-bovine, goat anti-swine, and goat anti-horse albumin one each in the other three holes. We set the plate in the room temperature incubator overnight. This test will allow us to see if the hamburger extract contains beef, pork, and/or horse meats by the reaction that occurs between the hamburger extract and the anti-albumin samples (albumin is a protein found in meat).

Anti-body Albumin Samples

We also completed an HIV antibodies test. We were given a positive sample, a negative sample, and an unknown sample. We transferred 50 μL purified antigen into 9 holes of the microstrip and waited five minutes for the antigen to bind to the plastic walls.

Microstrip

 We then washed the microstrip by dumping out the purified antigen solution and filling the wells with a wash buffer.

Dumping the Wells

Wash Buffer

 We then dumped the wash buffer out and used a pipet to transfer 50 μL of the positive control into three well, the negative control into three wells, and the unknown sample into three wells.

We waited five minutes to allow the serum antibodies in the samples to bind to the antigen before washing them out again in the same manner. We then added 50 μL of secondary antibody into all nine wells and waited 5 more minutes to allow the secondary antibody to bind to the primary antibody.

 We washed the wells out twice before transferring 50 μL of enzyme substrate into all of the wells. After 5 minutes we checked the wells for a blue-ish color, which is the indicator for a positive test. The positive control turned blue while the negative and the unknown sample remained colorless. This indicates that the unknown sample was a negative test for antibodies.

Our professor also showed us how a hand-held UV light can purify bacterial filled water. We prepared a Petri dish with the before sample and after UV light treatment sample and we will check them tomorrow.

The last thing we did for the day was take cooled boiled milk and transferred them into several tubes. We then added some yogurt to each tube and set them in the 37°C incubator. Tomorrow we will observe the bacterial growth in the tubes which will form yogurt.

Week 3, Day 2

 May 29, 2013

Today in lab we started by retrieving our blood, mannitol-salt, MacConkey, and PEA samples prepared in our last lab section. Our mannitol salt agar plate tested negative, we had very little growth on the plate, and it had no yellow appearance to indicate the fermentation of mannitol. Our MacConkey agar plate was also negative, indicating a lack of ability to ferment lactose. This is consistent with our previous tests for lactose. Our blood agar plate tested for gamma (γ) hemolysis (lack of hemolysis), this means that our bacteria lacks the ability to lyse red blood cells.

Mannitol Salt Agar Plate

MacConkey Agar

Blood Agar Plate (Bottom)

 The PEA (phenylethyl alcohol) agar we will be studying tomorrow, but so far it suggests that our bacteria is gram-negative (which is consistent with our previous gram stain test). The PEA agar is a selective medium; it promotes the growth of gram-postive bacteria and slows/inhibits the growth of gram-negative bacteria.

PEA Agar (Bottom)

We also examined our throat and nasal swab samples today. Our nasal swab had been incubating at body temperature on a mannitol salt agar (a selective media). We did have some growth on our plate, indicating the presence of a halophilic bacterium, but it appeared to all be of the same type of bacterium. There was no yellow coloring of the sample, indicating that no fermentation of mannitol was taking place.

Nasal Throat Swab on Mannitol

Our throat swab had also been incubating at body temperature but had been inoculated onto a blood agar plate (differential media) with a bacitracin disk placed on the surface. We did have some different types of bacteria growing on our plate, but they were all sensitive to the bacitracin (did not grow around the disk). The bacteria observed preformed alpha (α) hemolyses (incomplete hemolysis), indicating an ability to only partially lyse red blood cells.

Throat Swab on Blood Agar with Bacitracin Disk

We also studied our Kirby-Bauer agar plate that had been inoculated with our bacterium and set with 5 different antibiotic disks - penicillin, neomycin, erythromycin, tetracycline, chloroquine. We measured the diameters of each circle surrounding the disks (the clear zones where bacteria did not grow in order to avoid the antibiotics). There were no clear zones surrounding penicillin (cell wall attacker) or erythromycin (substitute for penicillin for people with allergies; binds to 50S ribosomal subunit), indicating that the bacteria is resistant to both. The diameter of the clear zone around neomycin was ~1.78 cm. Around tetracycline was ~1.5 cm. And around chloroquine was ~1.0 cm.

Kirby-Bauer Agar Plate

We also attempted to identify our bacterium today, but some of our experimental tests were not consistent and we are going to repeat the tests for indole, oxidase, and TSIA. Tomorrow we will review our data again and identify our bacterium.

Week 3, Day 1

May 28, 2013

Today in lab we began by inoculating differential and selective agar plates. We started with the blood agar plate, which is used to isolate and support the growth of fastidious bacteria and differentiate among bacteria based on their ability to lyse red blood cells (hemolysis). We incubated the blood agar plate with a sample from our slant.

Blood Agar Plate

 The second plate we inoculated was the mannitol salt agar plate, which is used for isolating bacteria based on their salt tolerance and differentiating among these isolates for mannitol fermentation.

Mannitol Salt Agar Plate

 The third was the phenylethyl alcohol (PEA) agar, which is used to isolate gram-positive bacteria from a specimen containing a mixture of gram-positive and -negative bacteria. 

PEA Agar Plate

The final plate we inoculated was the MacConkey agar, which is used to detect and differentiate among gram-negative enteric bacilli, based on their ability to grow on the medium and to ferment lactose.

MacConkey Agar Plate

After inoculating all of the agar plates, we began work on the Kirby-Bauer technique. For this we took a Mueller-Hinton agar plate and used a swab to inoculate the plate with our bacteria.

Inoculating the Mueller-Hinton Agar Plate

 We then placed five different antibiotic disks on the surface of the agar. We did this by dipping our forceps in an ethanol solution and sterilizing it with a flame before carefully taking a disk between the forceps ends and moving it onto the surface of the agar plate. 

Alcohol Solution

Performing Aseptic Technique with Forceps

The five antibiotic disks used were penicillin, chloramphenicol, neomycin, tetracycline, and erythromycin.

 The Kirby-Bauer technique is used to determine the sensitivity of a bacterium to several antibacterial medicines. We will incubate all of our inoculated samples prepared today and examine them after the bacteria has had time to grow.

Next we moved on to the throat and nasal swab tests. For the throat swab test we took a cotton swab and ran it over the surface of the back of a lab-partners  mouth, just behind the uvula. We then swiped the cotton swab over the surface of a blood agar plate. We placed a bacitracin antibiotic disk on the surface of the blood agar plate as well, which will help in determining if the bacteria from the throat swab contains the "strep throat" bacterium, Staphylococcus progenies.

Throat Swab Test with Bacitracin Antibiotic Disk

For our nasal swab, we dipped a cotton swab in saline solution before running it over the inner surface of a lab-partners nostril. The cotton swab was then swiped over the surface of a mannitol salt agar plate. Both swab test plates were incubated at 37°C, body temperature, so the colonies would have time to grow.

Nasal Swab Plate

Nasal and Throat Swab Plates