Hello dirt bloggers and welcome back to Dylan and Devin Discovering Dirt! It is with excitement that I announce this is will be our last entry into the blog! This week we ran one final experiment that will hopefully let us finally identify our soil microbe!

On our final week in the microbiology lab Devin and I conducted an experiment to test the antibiotic resistance of our microbes. To do this:

1.  We streaked 4 gel plates with 4 different microbes:
• S.aureus- control
• S.marsumes-control
• K.pneumonia- control
• Unknown-experimental

                                                       I'm glad you asked Blake!

 

        2.   We tested 4 antibiotics:

• Erythromycin- (15ug)
• Erythromycin is most effective against gram-positive bacteria such as pneumococci, streptococci, and some staphylococci. (www.infoplease.com/encyclopedia/science/erythromycin.html)
• Carbenicillin- (100ug)
• Work primarily against gram negative bacteria. (http://medical-dictionary.thefreedictionary.com/carbenicillin)
• Chloramphenicol-(30ug)
• Prescribed for Chloramphenicol treats various infections caused by susceptible strains of S. yphi, H. influenzae, E. coli, Neisseria species, Staphylococcus and Streptococcusspecies, Rickettsia, lymphogranuloma-psittacosis group of organisms, and other bacteria that cause bacteremia (bacteria in blood) and meningitis. (http://www.medicinenet.com/chloramphenicol-oral/article.htm)
• Azithromycin-(15ug)
• A derivative of Erythromycin, works in a similar manner, but it's unique chemistry allows it to be more specific for bacteria that cannot be targeted by Erythromycin. (Some mycobacteria) (http://www.nlm.nih.gov/medlineplus/druginfo/meds/a697037.html)



Specifically we used pellets with the above concentrations of each drug to administer each drug to each plate streaked with a different microbe. (seen below)

Results

Unknown

•  sensitive to Erythromycin and Azithromycin
•  resistant to Carbenicillin and Chloramphenicol.

S.aureus (g+, catalase, nitrate reduction)

• sensitive to none
• resistant to Azithromycinm, Carbenicillin, Chloramphenicol, and Erythromycin 

S. marcescens (G-,) 

• sensitive to- none
• resistant to- Azithromycinm, Carbenicillin, Chloramphenicol, and Erythromycin

K.pneumonia (g-, encapsulated, lactose fermenter, rod-shaped, facultative anaerobe)

• sensitive to Chloramphenicol
• resistant to Azithromycin, Carbenicillin, and Erythromycin

What does this mean?


These results are useful in a number of ways.

First we can use our known bacteria to gain evidence about how each drug works and make inferences on how each drug works. We may be able to use this information to add clarity to some of our earlier experiments that provided inconclusive evidence.

First, we can see that our soil microbe is sensitive to Erythromycin and Azithromycin. Literature shows that Erythromycin works best against Gram positive bacteria. This could support the idea of our microbe being Gram Positive. Our microbe is also sensitive to Azithromycin, which is commonly prescribed for anaerobic bacteria and is affective against some gram positive and gram negative bacteria. So there isn't much we an gather here, but it does not refute the idea that our unknown microbe is gram positive. It is interesting to note that both S.aureus and S.marsumes showed to resistance to all anti-biotics (which is a reason that they are dangerous pathogens). BUT if we can now assume our microbe is gram positive we can finally look at our dichotomous key and narrow down our possible names for our soil microbe!

 

The Key to naming our Microbe

Where the key leads us-


Gram +   ------------------>Bacilli/cocci
Rod Shaped---------------> Non Acid Fast/Acid Fast

Non Acid Fast------------>Catalase Negative/Catalase Positive

Catalase Negative------->Aerobic, facultative/Anaerobic

Aerobic facultative----->Endospores/ No endospores

Endospores-------------->  Clostridium , Corynebacterium

So now we know were dealing with a Clostridium spp. or Corynebacterium!
*( However, these experiments should be repeated to ensure the results are accurate, as some of the results were only accepted tentatively)

A quick trip to the google machine shows that Clostridium is very similar to our microbe as well!



Clostridium Botulinum- produces the toxin used in "Botox" facial procedures. (Endospore stain. Green=Endospore)(microbeonline.com)



Unknown bacteria slide through the
microscope lens. (Gram Stain)

So it seems our experiments a suggest we may have a Clostridium!

Not so Fast!

Further research provides that Clostridium are strict anaerobes (which differs from our dichotomous  key and our experimental results, so we may then be dealing with a corynebacterium. Unfortunately, our antibiotic resistance test did not align with his conclusion either, because corynebacterium are usually sensitive to Chloramphenicol. So, no strict conclusions can be made, but there is some support for corynebacterium (antibiotic resistance is on the rise in the United States right?).

Ideally to confirm our experimental results and conclusion further testing would need to be conducted. Specifically, testing of the aerobic/anaerobic respiration of the unknown microbe should be conducted again, and further testing could look into doing a Ponder's Stain, which stains polar granules within the microbe that are distinct to C.diphtheriae (responsible for causing diphtheria).

So, our final conclusion is that we believe we are working with a bacterium in the genus Clostridium but further testing needs to be done to verify our results.

Thank you for following our journey to discovering our soil microbe. It has been a long ride, and I hope everyone has learned something, because Devin and I sure have!

This is Dylan and Devin signing out for the last time!

Stay classy microbe enthusiast!

 

 

 

 

 

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