Sample S: Identity revealed!!

All semester we have been doing procedures trying to figure our what our unknown bacteria was, and now we have come to a conclusion based on the accumulation of our results.
Here are our results compiled:

MORPHOLOGICAL CHARACTERISTICS
Cell Shape: Rods
Spore forming: yes
Gram Stain: positive (to variable)
Motility: motile
Capsules: no capsules
CULTURAL CHARACTERISTICS
Oxygen Requirements: facultative anaerobe
Optimum Temp.: 37 degrees centigrade
PHYSIOLOGICAL CHARACTERISTICS
Glucose: positive
Lactose: negative
Sucrose: negative
Mannitol: negative

Gelatin Liquefaction: negative
Starch: negative - We performed this procedure twice with the same results both times
Casein: negative
Fat: negative

Indole: negative
Methyl Red: positive
Voges-Proskauer (acetylmethylcarbinol): positive
Citrate Utilization: negative - We performed this procedure twice with the same results both times.
Nitrate Reduction: negative
Urease: negative
Catalase: positive
Oxidase: positive
DNase: negative


We had a hard time determining what our bacteria was, we were sure that it was gram positive, rods that had spores, but then we ran into some trouble with results from our starch and nitrate tests. We concluded from this that our bacteria must have at some point become contaminated by some other bacteria and this is why we were obtaining results that were not what was expected for our bacteria. From the information we had though.......we determined.......that our bacteria......is........

B. Cereus!!!! 

ELISA (Enzyme-Linked Immunosorbent Assay) Antibody Test

Today in lab we an ELISA Antibody test. The ELISA Antibody test is a test that detects antibodies in a person's blood to determine if the person has been exposed to a certain disease. 

So we first obtained all of the supplies that we needed for this test: a cup full of wash buffer, two yellow tubes containing the serum samples that will be tested for the presence of antibodies, a tube full of purified antigen (AG), a tube full of the positive control, a tube full of the negative control, a tube full of the secondary antibody (SA), and a tube full of the enzyme substrate (SUB).

After we labeled the outside wall of our 12-well strip, we began our experiment.

We first used a fresh pipet to transfer 50 microliters of the purified antigen (AG) from the green tube into all of the 12 wells and waited 5 minutes so that the antigen could bind to the plastic wells

After 5 minutes we tipped the microplate strip upside down onto the paper towels so that the sample could drain out. We then used a transfer pipet filled with the wash buffer to fill each well with the wash buffer. After that we tipped the microplate strip upside down so that the wash buffer drains out.

We then used a fresh pipet tip to transfer 50 microliters of the positive control into the three wells that were labeled "+". Afterwards we took another fresh pipet to transfer 50 microliters of the negative control into the three wells that were labeled "-"

Below is a video of Rose doing measuring 50 microliters of the negative control and carefully placing it into the three "-" wells.

We then took another fresh pipet and transferred 50 microliters of the serum sample labeled "23" and "2" and placed it into their respective wells. Then we waited 5 minutes to allow the serum antibodies in the samples to bind to the antigen. After 5 minutes we washed the samples out of the wells by using the wash buffer and repeating what we did earlier.

Afterwards we used a fresh pipet tip to transfer 50 microliters secondary antibody (SA) from the orange tube into all 12 wells of the microplate strip. Then we waited 5 minutes for the secondary antibody to bind to the primary antibody. After 5 minutes we washed the unbound secondary antibody out of the wells making sure we did it twice.

After we finished washing we used a fresh pipet tip to transfer 50 microliters of the enzyme substrate (SUB) into all 12 wells of the microplate strip.

We then waited 5 minutes and this is what came up!

As you can see our sample "23" was positive for having the antibodies. However, since there was a little bit of the blue color in the "-" labeled wells as well as in the sample "2" wells we indicated that our sample is contaminated and we would have to do the experiment again but our professor was kind enough to not have us do another experiment. 

Further interest in Antibacterial Agents

Since the mouthwash did not kill all of the bacteria that was on our hands, we decided to see how effective it is in killing the bacteria in Lindsey's mouth.

We took a swab of bacteria on Lindsey's gums and spread the bacteria on one half of a petri dish labeling it 'before mouthwash.' Then Lindsey swished a mouthful of mouthwash for 30 seconds and after she was finished we took another swab of bacteria on Lindsey's gums.

Here are the results!

We found that the mouthwash didn't really kill any of the bacteria on Lindsey's gums. We concluded that either the mouthwash was really old or maybe there needs to be a consistent use of mouthwash.

Yogurt Experiment!!!

We obtained yogurt and milk and proceeded to put a small amount of yogurt and milk in two separate cups. For one cup we heated the milk and the yogurt and then put it into the yogurt incubator for about a day. For the other cup we just put the cup into the yogurt incubator without heating the milk and the yogurt.

After a day of incubation, one of our fellow microbiology classmates took the two cups and put it in the refrigerator.

Then when we returned to the lab the following day we tasted and looked at the two yogurt cups. We found that heating the milk and yogurt not only tasted better but the consistency was a lot better than the not heated cup. The cup that we did not heat looked more liquidly than the other one and tasted really sour.

Here are some pictures of Rose tasting the yogurt and what the yogurt looked like!

Testing for Food Purity

The point of this lab was to study a technique of immunology and apply it in a test for food purity. Immunology is the study of an organism's response to a foreign organic substance or antigen.

We acquired an agar plate divided into three equal zones. And we split the plate between two other groups.

In our division of the plate we had to make four different wells in the agar. To do this we used a dropping pipet. And we first squeezed it letting out all the air, then inserted it into the agar and released the bulb to we then sucked up the small circle piece we just separated. We are arranged the four wells according to this diagram.

Once we finished this we first filled well number one with Hamburger Extract. Following the instructions we proceeded to put goat anti-horse albumin in well 2, goat anti-bovine albumin in well 3,and goat anti-swine albumin in well 4.

Once the other people put their solutions in their section of the agar plate, we placed the plate in a drawer face up to "incubate it". It had to be placed face up so that the solution that we put in the wells wouldn't pour out.

When we returned to lab in Thursday we found that our results we non-conclusive, or they did not work as they we expected to. This could have been from old solutions that we were using or some other factors.

Testing different Antibacterials..

 We decided to test how well antibacterial products actually work.
For our sample we took swabs of our hands. My sample on one half and Rose's on the other half. To obtain this sample we had to first dip the swab in a saline solution, because the sample from our hands would be dry.
Once we each smeared our half of the nutrient agar plate we each chose two different antibacterial products to test out.
I chose to test mouth wash and hand sanitizer.

While Rose tested Lysol wipes and Clorox bleach spray. 

We soaked little discs in theses multiple solutions so we could set them in the separate sections of our nutrient agar plate. 

In order to pick up the different discs we used forceps. To sterilize them we first ran them through the flame of the bunsen burner, then we dipped the tips of them in 70% ethanol alcohol. 

We then incubated our sample at 37 degrees centigrade, because that is the temperature of the human body and our hands where we obtained our sample. Next class we would see which antibacterial agents worked the best.

When we returned back to lab we found that all of the bacteria that were on our hands are able to be easily killed simply by washing their hands or by disinfecting the surface with Clorox bleach or Lysol wipes. However, if we try to wash our hands with mouthwash it is not as effective.

So wash your hands frequently and make sure to clean surfaces with disinfecting agents!!!

Antibiotics!!

As nurses we are going to have to deal with the different kinds of antibiotics all the time!
So for this lab experiment we tested different antibiotics and how the worked on our unknown bacteria.
We had already become familiar with the antibiotic Bacitracin  used for Streptococcus, or strep throat infection.

We first prepared a smear plate of our unknown bacteria using the aseptic technique.

Next using forceps, that were both sterilized in the flame of a bunsen burner and by spraying them with 70% ethanol, we removed a disc of each antibiotic and placed it is one of the five sections we divided the nutrient agar plate into.
We used: novobiocin, erythromycin, neomyosin, tetracycline, and penicillin.
We then put it in the incubator at 37 degrees centigrade, and waited to see what the results might be.



When we returned to class the next time we discovered which antibiotics worked the best.

Below are the results of our test:

As you can see all of the different antibiotics killed our bacteria. The neomyosin, however, did not work as effectively as all the other antibiotics because there was still bacteria growing on the sides of that section. The diameters for the antibacterial agents are as follows: penicillin has a diameter of 45 millimeters, tetracycline has a diameter of 45 millimeters, novobiocin has a diameter of 45 millimeters, erythromycin has a diameter of 45 millimeters, and finally neomyosin has a diameter of 30 millimeters. After determining the diameters, we can determine whether our bacteria was susceptible or resistant to the antimicrobial agents we tested. For penicillin the diameter has to be greater than 29 millimeters, for tetracycline the diameter has to be greater than 19, for novobiocin the diameter has to be greater than 22 millimeters, for erythromycin the diameter has to be greater than 23 millimeters, and finally for neomyosin the diameter has to be greater than 17 millimeters. Since all of our diameters are greater than their respective range this tells us that our bacteria is susceptible to all of the antimicrobial agents that we tested.

Therefore, we could say that if we obtain this disease we could easily kill the bacteria by virtually all the antibiotics that we tested!