Discovered : The Great Unknown! {Lab 13 - May 29, 2015}

     So today, on the final day of lab, while Elisabeth did ELISA testing, I sat down to discover what bacteria our elusive unknown really was. 

     I looked over our descriptive chart that we had been keeping of all our test results. I also looked at some identification flow charts that would ultimately tell us what bacteria we had been dealing with for the past few weeks. 

     After some detailed analysis of the charts, I discovered what our great unknown was! Our unknown bacteria was : Escherichia coli!

Identification flow chart

Our descriptive chart

  




















     This may be similar to the process employed by medical lab workers to identify a bacteria that is causing an illness.     

ELISA Testing {Lab 13 - May 29, 2015}

    On the last day, we worked on ELISA testing or Enzyme Linked Immune Sorbent Essay. This test is used to detect the presents of antibodies. When you get sick, your body will produce antibodies for the antigens present in the disease. Being able to detect these antibodies is useful; as in AIDS, the virus hides where it cannot be detected inside our cells. However, the antibodies are not hiding so they can be detected.

Materials: 

• ELISA kit
• piquet
• buffer
• 12-well micro plate strip
• paper towels

Procedure:  

     First, we labeled the wells. The first 3 are going to be a positive control test, the next 3 are negative control test, the next three were sample 8, and the last 3 were sample 38. 

     We add 50µl of the antigen and let it sit for 5 minutes then wash. To wash we tip the wells over on the paper towels and gently tapped, then filled the wells with the buffer and again tipped them over and tapped out the liquid on fresh paper towels. After that we added the positive control, the negative control, serum 8, and serum 38 to the appropriate wells, waited 5 minutes and washed.  Repeating the same motion we added the secondary antibody, waited and washed. The final step was to add the enzyme substrate.

Results/applications: 

     The first 2 wells and the last 3 wells turned blue right away. So one of our positive controls was not done correctly as the 3^rd well should have also turned blue. These results would also indicate that the serum 38 is positive for antibodies and serum 8 is negative.

     This test also showed us how quick and simple it is to test for antibodies to detect diseases that are able to hide in our own cells in our body.

UV lights and Yogurt {Lab 12 - May 28, 2015}

     For this lab, we sterilized our broths using a UV steri-pen and also made some delicious homemade yoghurt!

UV Light Sterilization

     

     To test how effective the UV light was, we poured our broth along with our classmates' broth into a big beaker. We inoculated three agar plates (labeled B for before) with the broth before sterilization  We then took the UV light steri-pen and went to work sterilizing the remaining broth.

Killing the bacteria
. . . We hope!

Broth filled with all
of our unknown bacteria.

     After sterilizing the broth, we inoculated it in the three agar plates (labeled A for after). The six agar plates were then allowed to incubate overnight. The next day, the results were staggering. The before plates had colonies growing everywhere. The after plates didn't have any colonies! That goes to show you just how effective the UV radiation really is. 

Before inoculation and sterilization 

Before sterilization on right;
After sterilization on left

Before - left ; After - right

Before - left ; After - right

     This was a great test to do. We were able to see just how effective the UV light is against pathogens and bacterial growth. The UV light is beginning to be used more frequently in hospitals due to its effectiveness. This is important for us to understand the capabilities of UV lights so that they can become more widespread in their usage throughout hospitals.

Yogurt Making

      To make the yoghurt, we placed some milk in a large beaker and microwaved until it was boiling. After this, we had to let it cool down before inoculating bacteria. We let it cool so that the bacteria would not be killed when added. Once the milk had cooled down, we added a spoonful of plain greek yoghurt to the milk and stirred. The inoculated milk was then placed into a 37 degree incubator overnight.

37 degrees, the perfect
temp for making yogurt 

 

Yogurt Incubator

 

     The next day, we removed the yogurt and placed it in the refrigerator until we had finished most of our lab. Then at the end of lab, we got out the yogurt and tasted what we had made. Some of us thought it was slightly too buttery tasting, so we added some salt to it. 

Final thoughts:

     I thought it was really cool that we had made yogurt even if it tasted slightly different from what we are used to eating. 

Bon Appétit! 

Further Pathogen Testing {Lab 12 - May 28, 2015}

     After the previous investigations of pathogens the class went further into testing what pathogens are resistant or susceptible to certain things.

Materials: 

• swabs
• antibiotics
• testing paper
• tweezer
• Bunsen burner
• piquet

Procedure: 

Cinnamon and Clove Mouthwash

     After preparing plates with MRSA, MRSA isolate, Staph aureus, and Staph aureus motile, we then, using the aseptic technique with the tweezer, placed a sample of penicillin (1), metecillin (2), vacomycin (3), linezolid (4), and augmentin (5) on each of the samples and placed the samples in the incubator. 

     We also prepared plates of the staph oral bacteria and tested different concentrations of salt solutions on one of the plates and on the other tried different mouth cleaning products of lemon, clove, a cinnamon and clove mouthwash, toothpaste, and chlorohexadine. 

     The last plate was our unknown which had already proven to be resistant to penicillin. So this time we treated the bacteria with penicillin on one side and an augmentin on the other.

Results/application: 

     The next day the Staph aureus proved to be resistant to the penicillin and methicillin and susceptible to the rest.

 The Staph aureus motile was sensitive to all of the antibiotics.

MRSA showed itself to be resistant to penicillin and methicillin, susceptible to vacomycin and linezolid and intermediate to augmentin.

     The MRSA isolate was penicillin, vacomycin and augmentin resistant and methicillin and linezolid susceptible. 

    

     Out of these linezolid had the greatest effect every time showing it to be the most effective antibiotic. Linezolid has recently been developed and bacteria like MRSA do not have any defenses against it.

     When checking the oral bacteria, the salt treatment seemed to have no effect on the bacteria. We suspect this is because we could not get the salt to fully dissolve in the water. However the other plate did have some results. The lemon was ineffective. The clove and cinnamon mouthwash had a moderate effect. However, it was the toothpaste and the chlorohexadine that had the greatest effect. 

     So toothpaste is a good investment in oral hygiene and if I want my mouthwash to do more then make my breath smell good I should look for some with chlorohexadine.

  

     The last plate with our unknown had results as well. Once again the penicillin had no effect. However the augment did. The augment contains clavulanic acid which destroys β-lactamase in the pathogen which prevent it from destroying the lactase ring in penicillin allowing the penicillin to do its job of breaking down the cell wall. This tells us that our bacteria has β-lactamase that works against the penicillin and it has a cell wall. 

The most curious part of our results that we did not understand was there was two rings around the augment the inside one containing no growth and the outer one containing some growth.

Movie : Contagion {May 26, 2015}

     After taking our second exam, we bought coffee and finished the movie Outbreak! This movie follows the spread of a virus via fomites (which are anything we touch and then another person touches, such as for handles). The virus was carried from Hong Kong, where it originated from bats and pigs, by a business woman who subsequently spread the virus to Chicago and Minneapolis. The movie follows the attempt to quarantine the illness and the attempts of the CDC to find a vaccine. 

Testing Antibiotics on Our Unknown {Labs 10 and 11 - May 26 and 27, 2015}

     For this test, I inoculated our unknown bacteria onto an agar plate, then placed five different antibiotics onto the plate to see if any of them would inhibit the growth of our bacteria. 

The chart to determine
susceptibility and resistance

Materials:

• Agar Plate
• Unknown bacteria
• Penicillin disk
• Tetracyclin disk
• Streptomycin disk
• Erythromycin disk
• Novobiocin disk
• Ethanol in a beaker
• Tweezers
• Gloves

Our Unknown bacteria
and the Antibiotics

Procedure:

     I began by preparing the agar plate by inoculating our bacteria. I then labeled the bottom of the agar plate with the different antibiotics that I would be testing. After labeling, I sterilized the tweezers and placed the first antibiotic disk onto the agar plate. I sterilized the tweezers between each disk. After placing all five antibiotic disks onto the plate, I incubated the plate for 24 hrs. 

 The next day, I retrieved the plate and documented the results. Our unknown bacteria is resistant to penicillin(1), Erythromycin(4), and Novobiocin(5). However, it was susceptible to both Tetracyclin(2) and Streptomycin(3). 

Testing MRSA and other Pathogens {Labs 10 and 11 - May 26 and 27, 2015}

Caution:

      For these tests we were working with pathogens meaning they can be harmful. So precautions are needed and anytime we are handling them gloves must be worn and wash hands immediately afterward.  The purpose of these tests are to see what kind of cleaning products might prevent these pathogens from growing.

Materials:

·       Gloves

 The products that we
would be putting to the test.

·       Swabs

·       dish washing foam

·       Scott cleaner (choloroxlenol)

·       Clorox bleach

·       Lysol (phenol)

·       lens cleaner (Biguaninde)

·       tincture

·       acid alcohol

·       70% isopropyl alcohol

·       H₂O₂

·       Mouthwash

·       Tweezers

·       Bunsen burner

Procedure:

      The first thing was to retrieve the pathogens from storage. Note pathogens’ plates are sealed for safety when is storage. Our group was working with MRSA and the others had VRSA, a staph bacteria that grows in the oral cavity, and a MRSA isolate. Once I had my sample I inoculate the agar plates using a swab to spread the MRSA over both plates entirely.

     Then small circular test paper must be placed in designated areas on the plates, 5 per plate. To do this tweezers are used. Before placing every testing paper the tweezers are sterilized by dipping it in 70% ethanol and then heating it with the Bunsen burner. Once the tweezer is sterilized a testing paper is picked up from its petri dish and placed in one of the designated areas. This is repeated for all 10 sections. Next a pipet is used to place 5µm of a cleaning product is placed on a testing paper. And the plates are incubated at 37⁰C for 24 hours.

Results and applications:

     The MRSA proved to be susceptible to the dish washing foam(1), the Scott’s cleaner(2), the Clorox bleach(3), the Lysol(4), and the tincture(6). 

MRSA Plates

The oral bacteria, S. aureus was susceptible to the dish washing foam (1), the Scotts cleaner (2), the bleach (3), the Lysol (4), the tincture (6), and the H₂O₂ (9). 

Staph Aureus

The VRSA was susceptible to the dish washing foam (1), the Scott cleaner (2), the bleach (3), the Lysol (4), the tincture (6), and the H₂O₂ (9). 

MRSA Isolate (middle and left)

Finally the MRSA isolate was susceptible to the dish washing foam, the bleach, the Lysol, the tincture, and the H₂O₂.

     After looking at the results it was odd to see that the MRSA was resistant to the H₂O₂ and we wonder if the 5µm were added correctly.

     The dish washing foam proved to be the most effective of all the disinfectants every time as it had the largest radius of a clean zone around it (about 30mm on the MRSA). It is good to know that the dishes at home are safe from these harmful pathogens. And a good scrub down with bleach can be effective as well. The mouthwash, which was specifically used for the oral bacteria, had no effect so that one won’t clean your mouth but it still eliminates the bad breath.

     Now we know what household cleaners can eliminate certain pathogens. So if we know what pathogen is affecting a certain family member, we know which cleaner to use so as to eliminate the pathogen.