Blog Post #10

Last week in lab, Hannah and I performed antibiotic sensitivity on our soil microbe in order to continue the journey of identifying our soil microbe!

Before I begin to explain our methods and results…I want to give some insight as to what antibiotics are and why they are important.  Antibiotics are powerful medicines that fight bacterial infections either by killing bacteria or keep it from reproducing. Antibiotics have no affect on viruses so they will not fight off infections such as the flu or common cold. Antibiotics are important because they can save lives and prevent infections from spreading!

Typically, if a microorganism is isolated from a patient in a hospital, they will perform antibiotic sensitivity testing on it.  The goal of this type of test is to predict the success or failure that will occur for certain types of antibiotics on that microbe. The tests are usually performed in vitro and are performed to measure the growth response of the isolated organism and its resistance or susceptibility to antibiotics.

In lab last week, we used a method called the 'disk diffusion test' to test the effectiveness of antibiotics against different species of bacteria and our unknown soil microbe.

To prepare for this procedure, we first had to start a TSB culture of our soil microbe the day before lab so that it had enough to grow.  The next day during lab we obtained four plates of antibiotic test medium.  The four plates that we chose to study were 1) unknown soil microbe, 2) E.Coli, 3) K. Pneunomonia, 4) P. Aeruginusa
Below is a snapshot of the three different species of bacteria we used and our soil microbe before we spread them across the agar plate.

The bacteria species that we chose to test and our soil microbe

We used aseptic technique and saturated a cotton swab with each of the bacterial broth cultures that we chose to use. We spread the culture uniformly across the agar medium by wiping back and forth, covering the entire agar plate. Next, we used the antibiotic disk dispenser to place antibiotic disks uniformly on the plate.  We sterilized the forceps between each dispersion of the antibiotics. Each plate was labeled with 1,2,3,4 in a clockwise direction.  The same antibiotic would go on number 1 for all plates, a different antibiotic for number 2 on all plates, and so on. Below is a snapshot of what the plates looked like after the antibiotics were dispensed on each of the plates.

Agar plates with the antibiotics for each of the bacterial species and our unknown soil microbe plates

The plates were incubated at 37 degrees celsius for 24 hours. When we returned 24 hours later, we evaluated and analyzed each of the agar plates for resistance or sensitivity to the antibiotics.

Below is a list of the organisms that were tested and the antibiotics tested and their concentrations.

Organisms Tested:  

Unknown soil sample

E. Coli

K. Pneunomonia

P. Aeruginusa           

Antibiotics and Their Concentration:

1.) Carbenicillin (100)

2.) Erythromyclin (15)

3.) Tetracycline (30)

4.) Ampicillin (10)

Below are some snapshots of the plates…if growth occurs up to the disk, the the organism is resistant to the antibiotic. If there is a clear zone of inhibition (no growth) around the disk, then the organism is sensitive to the antibiotic.

Unknown soil microbe agar plate

There is no growth around the disk for Erythromyclin and for Tetracycline, which shows that our soil microbe is sensitive to those 2 antibiotics.  Also, there is little growth around carbenicillin, but it is unclear if the organism is resistant to it or not. There is growth all the way up to the Ampicillin disk which means that our unknown soil microbe is resistant to it.

E. Coli agar plate

The E.Coli plate shows sensitivity to both the Ampicillin, Carbenicilin, and Tetracycline antibiotics.  It is resistant to the Erythromyclin antibiotic.

K. Pneunomonia agar plate

The K. Pneunomonia plate shows antibiotic sensitivity to only the Tetracycline drug. It is resistant to Carbenicillin, Erythromyclin, and Ampicillin.

P. Aeruginusa agar plate

The P. Aeruginusa agar plate showed sensitivity to only the Carbenicillin antibiotic.  It had resistance to the Carbenicillin, Erythromyclin, and Ampicillin antibiotics. 

Our soil microbe is similar to the other bacterial species in terms that E.Coli and K. Pneunomonia was also sensitive to Tetracycline.  Also, our soil microbe, K. Pneunomonia, and P. Aeruinusa were both resistant for Carbenicillin and Ampicillin.

In a previous study, we identified our soil microbe to be Gram positive. 

So far in the dichotomous key we have identified our soil microbe:

1. Gram positive

2. Morphology, Rod Shaped (Bacilli)

3. Non-acid fast organism

4. Catalase positive

5. Endospore positive

6. Motile

7. Nitrate reduction

8. Alpha hemolysis

The dichotomous key indicates that our soil microbe belongs to the genus Streptococcus!

In order to further identify our soil microbe to the particular Streptococcus species, we need to do a P/A disc test. The P disc test is an optochin susceptibility test and the A disc test is a bacitracin susceptibility test. To do this, we would need to do an experiment very similar to the one we performed in this week's lab to test antibiotic resistance/sensitivity. However, we would need to inoculate a disc that contained optochin in the center of the agar surface. The plate would need to be stored for 24 hours at 35-37 C. If the growth went all the way to the margin of the disc, then it is resistant to the chemical. If sensitive, the species that would be identified is Streptococcus pneumonia. If resistant, the species would be Streptococcus mitts.
The bacitracin test would be the same method used for the optochin test; however, the disc would contain bacitracin. If sensitive, the species that would be identified is Streptococcus progenes. If resistant, the species identified would be Streptococcus galactiae. 

References:
http://www.uphs.upenn.edu/bugdrug/antibiotic_manual/amt.html

http://www.vumicro.com/vumie/help/VUMICRO/Optochin_Susceptibility.htm

http://www.vumicro.com/vumie/help/VUMICRO/Bacitracin_Susceptibility.htm