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

Blog Post #9

blood agar plates

Last week in lab Sammy and I tested our mystery microbe's hemolytic abilities. We did this by using blood agar plates containing general nutrients and 5% sheep blood. We used the T-streak method to inoculate S. aureus, S. epiderdimis, and our unknown microbe onto three separate plates. We then incubated the plates at 37 degrees Celcius and checked them at 24 and 48 hours to check for hemolysis. 

24 hours: left: unknown, top right: S. aureus,
bottom right: S. epididermis

Unknown after 24 hours of incubation

According to our lab handout there can be divided into three groups – alpha, beta, and gamma – based upon their hemolytic activity on blood agar. Beta (β) hemolysis is defined as complete or true lysis of red blood cells. Alpha (α) hemolysis is the reduction of the red blood cell hemoglobin to methemoglobin in the medium surrounding the colony. Gamma (γ) hemolysis indicates the lack of hemolysis. After 24 hours there is already significant growth on some of our plates. Our mystery microbe grew very quickly on the blood agar and is seems to fit the description of an alpha hemolytic bacteria. As you can see in the photos there is a clear dark discoloration surrounding the colonies on the plate.

Unknown after 48 hours

Plates after 48 hours

Bacteria are able to lyse red blood cells by producing enzymes called homolysins. Thers enzyemes function by destroying the outer membrane of a red blood cell. Some create pores in the membrane, weakening it, while other hydrolyze the phospholipid components of the cell's phospholipid bilayer. Hemolytic microbes are more virulent than non-hemolytic microbes because they destory iron-containing red blood cells that fight infection. This results in a reduced number of red blood cells in the body and therefore reduced oxygen transportation to the body which can be dangerous. I would not expect a typical soil microbe to be capable of hemolysis because most of them are not pathogenic. 

We've been attempting to identify our mystery microbe this semester by using a dichotomous key. This week we determined that our microbe is capable of alpha hemolysis.

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 microbe belongs to the genus Streptococcus! 

http://en.wikipedia.org/wiki/Hemolysin

http://en.wikipedia.org/wiki/Streptococcus

Blog Post #8

Last week in lab, Hannah and I did an experiment to determine if our soil microbe reduced nitrate or not.  Nitrogen is a basic element of life and is a component of both proteins and nucleic acids.  Nitrogen is very abundant in the atmosphere; however, it is not in the accessible for that living organisms need it to be in.

In order to test if our soil microbe was a nitrate reducer or not, we used a positive control (P. Auroginosa), a negative control (E. Coli), and our soil microbe and inoculating 3 cultures (1 of each).  Each of the samples were incubated for 24-48 hours.  Our soil microbe is a fast grower, so we returned 24 hours later.

A snapshot before we returned to lab 24 hours later

The second day, we returned to observe the Durham tube in each sample.  If there were bubbles in the Durham tube, then the organism was not a fermenter.  However, our sample did not have any bubbles present so we added 8 drops of reagent A and 8 drops of reagent B and mixed well.  Once the drops were added, the sample turned red.  This means that the microbe reduced nitrate to nitrite!!  Below are snapshots of our Durham tubes during the experiment.

After reagent A & B were added, our sample turned red

Reagent A & B that were added to our soil sample

In order to further understand nitrate reduction, let me explain some information from a broader, biological context.  Most bacteria use the cellular structures called flagella for motility.  They attach to the cell surface and provide a 'swimming' movement.  There are also flagellar arrangements for bacteria that can be determined by staining and microscopic observation.  

Nitrate reduction indicates that the organism can use NO3- as an electron acceptor.  Also, nitrite may be reduced to either NO, N20, N2, and NH3 depending on the enzyme system of the organism and the atmosphere it is growing in.  Nitrate reduction often involves a shift to anaerobic metabolism.  Nitrate reduction plays a key role in the nitrogen cycle.  I think that some microbes evolved to be nitrate reducers, because nitrate reduction is one of the most fundamental biological processes that accounts for tons of inorganic nitrogen.  Simply stated, since nitrogen is a basic element for life, nitrogen reductase evolved in order to maintain the nitrogen cycle, which allows biological processes to be carried out successfully. 

I think that some microbes did not evolve to be nitrate reducers, because nitrogen was in a form that was accessible and usable to that particular organism.  Organisms that did not evolve to be nitrate reducers were able to use nitrogen in the form that it was already in, rather then converting it to a different form. 

Along our journey to identify our soil microbe, we have been keeping track by using a dichotomous key.  Below is the identification of our mysterious soil microbe thus far:

1. Gram positive

2. Morphology, Rod Shaped (Bacilli)

3. Non-acid fast organism

4. Catalase activity

5. Endospore positive

6. Motile

7. Nitrate reduction

Citations:

http://www.nature.com/scitable/knowledge/library/the-nitrogen-cycle-processes-players-and-human-15644632

http://www.microbelibrary.org/library/laboratory-test/3660-nitrate-and-nitrite-reduction-test-protocols

Blog Post #7

This past week in lab Sammy and I tested the motility of our microbe. Motility is the ability of a microbe to move on its own. By using a special, soft agar we can see motile bacteria grow throughout the medium and non-motile bacteria grow only where the inoculating needle stabbed the agar. We tested a positive control (E. coli) and a negative control (S. aureus) in the agar to compare to our mystery microbe. Unfortunately, this specific E. coli we used was actually not motile so both our controls were negative. Our mystery microbe ended up not growing within the agar or along the line of inoculation but instead on the surface of the agar. Since our triple sugar test a few weeks ago showed that our microbe can tolerate anaerobic growth it is most likely that our microbe is motile and it “swam” out of the stab and to the surface of the agar.

Bacteria need to possess flagella, threadlike organelles extending from the cells outer membrane, to be capable of motility. Different bacteria vary in their number of flagella from one to numerous. Bacteria can also move using other cellular structures including the lesser-understood gilding motility, which does not involve flagella. Another bacterial group called spirochetes move by using axial filaments, which are similar to flagella.

 

example of flagellum and axial filament structures
http://ncse.com/book/export/html/890 http://classes.midlandstech.edu/carterp/courses/bio225/chap04/lecture3.htm

Some microbes have evolved to be motile when it is evolutionarily beneficial in their environment. Some microbes need to be able to flee from a harmful environment and towards a desired environment. This type of movement is described in our textbook as chemotaxis. Although it seems evolutionarily beneficial to be motile some bacteria cannot move on their own. A possible explanation for this is that these bacteria can survive in harsher environments than motile bacteria and therefor do not need the energetically costly cellular structures necessary for motility.

So far the dichotomous key has shown us that are microbe fits the following criteria..

1. Gram positive
2. Morphology, Rod Shaped (Bacilli)
3. Non-acid fast organism
4. Catalase activity
5. Endospore positive 

The link for the key we have been using doesn't seem to be working currently but the next step, I believe, was whether our microbe was motile or non motile. According to our soft agar test we can tentatively say our microbe is motile!

6. Motile

Citations: 

http://web.biosci.utexas.edu/psaxena/MicrobiologyAnimations/Animations/BacterialMotility/PLAY_motility.html

http://inst.bact.wisc.edu/inst/index.php?module=book&type=user&func=displayarticle&aid=117

http://www.biotecharticles.com/Biology-Article/Bacterial-Locomotion-Importance-of-Bacterial-Motility-629.html