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!

 

 

 

 

 

So

Hello and welcome back, once again, to D⁴!! This past week Dylan and I ran an experiment that dealt with the determination of fastidious organisms.

 

Fastidious organism- is an organism that is only capable 

to growth and sustainability when their diet includes

 very specific nutrients.

Fastidious microorganism- (a more specialize term for the 

field of microbiology) describes an organism that only 

grows in a culture medium that contains that organism’s 

specific nutrient needs.

        To test to see if the controls and the unknown that we have been using throughout the duration of the semester are indeed fastidious, we acquired blood agar plates for inoculation with our microbes. Blood agar plates contain a variety of general nutrients and also contain 5% sheep blood, which gives them their vibrant RED color. When working with blood agar plates, there are some microbes that can produce hemolysins.

Hemolysins- different proteins and lipids that cause the lysis of red blood cells by the destruction of the cell membranes of the red blood cells.

        So a few questions that you may want answers to in order to 1.) better understand the project and 2.) to put it into perspective as to why we are even conducting this experiment are:

1.) How do bacteria lyse red blood cells?

• To start lysis is the compromising of cell's integrity by breaking down it's membrane.
•  In order to breakdown the cell membrane hemolysin is needed, and as previously stated hemolysin is a mix of proteins and lipids that cause the destruction of red blood cell membranes.
• This hemolysin either lyses the erythrocytes by hydrolyzing the bilayer's phospholipids or by forming pores in the phospholipid bilayer.

2.) What is the difference between alpha, beta, and gamma hemolysis?

• Alpha- is a type of hemolysis where the red blood cells are reduced, but not completely eradicated. This reduction causes a greenish brown color to appear in the medium.
• Beta- unlike alpha, is the absolute lysis of all present red blood cells. When looking at the plate after incubation, beta hemolysis can be indicated by clear zones or "windows" in the medium that surround that bacterial colonies.
• Gamma- is hemolysis that produces no hemolysis. No reaction is present after the incubation period has occurred.

3.) Would you expect hemolytic microbes to be more or less virulent than non-hemolytic microbes? why?

• I would say that hemolytic microbes are more virulent than non-hemolytic microbes because iron has been known to be a limiting factor in growth rates of numerous pathogenic bacteria.
• The reason that iron plays a large role in virulence is because RBCs are very rich in heme that contains iron, and when these RBCs lyse the iron rich heme is released into the surroundings; this heme is now free for the bacteria in the area of lysis.
• Luckily, there are numerous cases where hemolysis does not put human health at severe risk, but when the heme released from lysis teams up with other virulence factors, human lives are put at extremely high risk.
• One specific outcome that occurs due to hemolysis that makes me believe that hemolytic microbes are more virulent than non-helytic microbe is hemolytic anemia. Hemolytic anemia is the destruction of erythrocytes and then the very quick removal of those erythrocytes from the bloodstream entirely.
• This is a huge problem because the removal of these erythrocytes at such high speeds is causing a decline in RBCs because the bone marrow can't produce new erythrocytes quickly enough to meet the demands of the decline. This can lead to many issues like fatigue, overall body aches, arrhythmias (irregular heart beats), or even an enlarged heart which will eventually lead to heart failure.

4.) Would you expect a "typical" soil microbe to be capable of hemolysis?  why or why not?

• We do not think that a typical soil microbe would be capable of hemolysis because in the lab we incubated our three samples in an incubator that was set to 37 degrees Celsius.
• 37 degrees Celsius is not a temperature that these microbes normally grow at, so being incubated at this temperature definitely had an effect on the capabilities of these microbes to produce hemolytic effects.

          The procedure for this experiment had a very simple set up, and very few steps to follow in order to determine if the microbes being used were in fact fastidious. First three blood agar plates were obtained; one was labeled S. aureus, one was labeled S. epidermis, and one was labeled unknown. The three plates were T-streaked with one of the three microbes and then incubated at 37 degrees Celsius for 48 hours (maybe longer for very slow growers). After the incubation period the plates were checked on and read to determine the results of the test. In order to correctly read the plates, they must be held up to a light source, so that the light is shining through the plate from behind. This method will ensure the best reading of the blood agar plates. Below are pictures of our plates with and without the light source behind them.

Left:S. epiderimis Middle: S. aureus Right:Uknown.

Left: S. epiderimis Middle: S. aureus Right:Uknown.

       The results of our test show that S. epidermis is an alpha hemolytic species of microbe, the S. aureus is a beta hemolytic species and microbe, and that out unknown is a gamma hemolytic species. Although there is a bit of confusion with our two controls, our unknown is 100% a gamma hemolytic species. As for the S. epidermis (which we believe to be alpha) it can be seen that there is brownish coloration around the colonies as was expected, but there is also one colony on the bottom right that appears to be a beta colony. When exposed to the light source you can very easily see through the medium, but for the most part we are confident that the S. epidermis is alpha. Then for the S. aureus (which we believe to be beta) the medium has definitely become transparent as expected but the perimeter of the plate and a few spots on the middle appear to have the greenish brown characteristic of the alpha, but overall this species is clearly beta. 

      To continue on with Dylan's "What the heck is this microbe?" section, we will delve one step further into classifying WHAT THE HECK this microbe may be. As Dylan stated last week, so far we have conducted...

• Pleomorphic (between cocci and bacillus).
• Gram stain results were inconclusive (a perfect color between our + and - controls).
• Not acid fast.
• No catalase activity.
• Ferments carbohydrates via aerobic and anaerobic metabolism.
• Endospore former.
• No Motility.
• No nitrate reduction abilities.
• Hemolysis test.

      Unfortunately, it seems as though our microbe wants to stay mysterious because putting a name on this guy has been quite the struggle. Although we are starting to lean towards the genus Streptomyces, there are sources like http://terpconnect.umd.edu/~asmith/ALSACE/Discussion.html that yield different results than the ones Dylan and I have seen in lab.

      So, once again we have taken a small step forward in identifying that our microbe is a gamma hemolytic species, but until further experiments are conducted, it seems as though we are still stuck without a verdict. Streptomyces is looking promising, so hopefully our next experiment points us even further in that direction!!

Works Cited
Bhakdi S, Mackman N, Menestrina G, Gray L, Hugo F, Seeger W, Holland IB (June 1988). "The hemolysin of Escherichia coli". Eur. J. Epidemiol. 4 (2): 135–43. doi:10.1007/BF00144740. PMID 3042445.

Chalmeau J, Monina N, Shin J, Vieu C, Noireaux V (January 2011). "α-Hemolysin pore formation into a supported phospholipid bilayer using cell-free expression". Biochim. Biophys. Acta 1808 (1): 271–8. doi:10.1016/j.bbamem.2010.07.027. PMID 20692229

 Sritharan M (July 2006). "Iron and bacterial virulence". Indian J Med Microbiol24 (3): 163–4. PMID 16912433.

 "What Is Hemolytic Anemia? - NHLBI, NIH". United States National Institutes of Health. 2011-04-01. Retrieved 2012-11-24.

This past week in lab Dylan and I performed a Nitrate Reduction Test on a positive control, a negative control, and our unknown bacteria. This test has three different purposes that can be sought out through experimentation:

• The utilization of nitrate as a nitrogen source for growth, which is nitrate assimilation.
• The generation of metabolic energy by using nitrate as a terminal electron acceptor, which is nitrate respiration.
• The dissipation of excess reducing power for redox balancing, which is nitrate dissimilation.

Nitrate has proven to be significant in the fields of biochemistry, medicine, and environmental microbiology. Nitrate reduction is also very helpful when it comes to identifying different strains of bacteria; some of the more popular strains are Enterobacteriaceae, Neisseria, and Corynebacterium. We plan to use nitrate reduction to continue on in the never-ending search to  identify our UNKNOWN!

Why get irate about fixin' nitrate?

• Nitrogen is a vital element for amino acids and nucleic acids, which are vital biological processes in all organisms

• N2 gas is the major available source of nitrogen, but is highly inert due to the strength of the N2 triple bond
• Some bacteria have evolved to convert this N2 into usable forms such as NO3-Example- Rhizobium grow mutualistic with plants.Plants provide rhizobium with carbohydrates, and rhizobium give plants usable NO3- 
• These plants are then eaten by omnivores, which assimilate the Nitrogen of the eaten plant 
• These animals eventually die, and their nitrogen is utilized by other organisms and fungi

• Thus the circle of Nitrogen (and life) continues

• Information gathered via: http://www.visionlearning.com/en/library/Earth-Science/6/The-Nitrogen-Cycle/98

Nitrogen fixers vs. Non-Nitrogen

• We've previously discussed Nitrogen fixation and the benefits seen in the Nitrogen cycle, so why would it be favorable for any microbe to preform the opposite reaction and reduce nitrates?
• Microbes that reduce nitrate use it as a means of an electron acceptor in anaerobic conditions.
• This requires a low O2 concentration, allowing these microbes to be better competitors in habitats with low O2.
• These microbes are often found deeper in soil than are nitrogen fixers
• The reduction of Nitrates reaching these deep soil nitrogen reducers prevents the leeching of nitrogen into deeper soil
• The Nitrate reduction process is also used in sewage sanitation as well as industrial waste sanitation in order to remove ammonia and nitrates from waste water
• Information gathered via: https://www.boundless.com/microbiology/textbooks/boundless-microbiology-textbook/microbial-metabolism-5/anaerobic-respiration-49/nitrate-reduction-and-denitrification-314-7650/

In order to determine if our unknown bacteria reduced nitrate (NO3-) to nitrite (NO2-) the following procedure was followed:

 

Day One

• Three cultures ( a positive control, a negative control, and our unknown) were inoculated and then incubated for 24-48 hours.
  • But keep in mind that slow growing bacteria may need a longer incubation period to get the desired results!

Day Two

• Dylan and I returned back to the lab to check our cultures after 48 hours and saw that our cultures had not really reached a point for good observation, so another 24 hour incubation period was added to the 48 hours of incubation that had already occurred.

Day Three

• The Durham tubes were observed to see if any bubbles had formed inside, meaning that the controls and the unknown would produce nitrogen gas (N2).
• If there are bubbles present (which there weren’t) and the organism is not a known fermenter, then the most possible scenario is that the microbes reduce nitrate to N2.
  • If these bubbles are present then no further test need to be run, and the microbe is classified as a nitrate reducer!
• BUT…if no bubbles are present upon observation after incubation then a set of further steps must be taken in order to identify the microbes.
• First 8 drops of reagent A and 8 drops of reagent B are added to the cultures and mixed thoroughly.
  • If the sample then turns red, it is determined that the microbe does in fact reduce nitrate to nitrite.
  • BUT…if the sample does not turn red, then a small amount of zinc must be added to the culture.
    • If when the zinc is added the sample turns red, then it is determined that the microbe is not capable of nitrate reduction and the test is complete.
    • BUT…if the sample does not turn red after the addition of zinc, then it is to be decided that the microbe does reduce nitrate, but it is into something other than nitrate.

Results

• Upon completion of our Nitrate reduction test, we found:
• No bubbles-  Our microbe does not reduce NO3- to N2
• No color change after application of reagent A and B- Our microbe does not reduce NO3- to NO2-
• Color change after application of Zinc- Our microbe is unable to preform Nitrate Reduction

What the heck is this microbe?

• At this point Devin and I have had a mixed bag in the way of results for our experiments.
• Pleomorphic (between cocci and bacillus)
• Gram stain results were inconclusive (a perfect color between our + and - controls)
• Not acid fast
• No catalase activity
• Ferments carbohydrates via aerobic and anaerobic metabolism
• Endospore former
• No Motility
• No nitrate reduction abilities
• As proposed earlier our microbe looks as if it could be of the genus Streptomyces
• Unfortunately, according to http://terpconnect.umd.edu/~asmith/ALSACE/Discussion.html similar experiments showed results in Acid fast, and fermentation experiments that yielded different results.
• Maybe this week we can re-do these experiments and try to eliminate all doubt for our microbe being Streptomyces.
• Terpconnect also reports that Streptomyces are antibiotics producers, so this could be an additional experiemejnt

So, until further experiments are conducted, it seems as though we are still at a stand still with identifying our microbe. With that said we could still be on to something with Streptomyces, and will continue to look into further testing for this genus.


Tune in next week and checkout the results of our hemolysis experiment!


Is our organism fastidious? Find out next week!

 

 

     Welcome back to another riveting installment of D⁴!!! Dylan and I hope that everyone had a great holiday weekend full of family, friends, and candy, but now to the exciting stuff…

      This week in lab Dylan and I investigated the motility of our still UNKNOWN soil microbe. Unfortunately, due to time constraints in the laboratory we were unable to preform the Flagellar Stain, but did get to perform the Soft Agar Deep Test. The Soft Agar Deep Test uses a semisolid media in a tube (unlike other solid gel agar media), which allows motile bacteria to move through. The growth of motile bacteria in this type of test will produce turbidity (cloudiness) throughout the semisolid agar. This result can be compared to non-motile bacteria, which will only show growth along the agar where it was inoculated. Three different tubes were made in order to compare our unknown microbe against a positive control (E. coli) and a negative control (Staphylococcus aureus). After the tubes had been inoculated they were incubated for 24‐72 hours (depending on how slow of a grower the unknown microbe is). Based on our experiment there are a few questions that need answering!

1.) Did the microbe appear to be motile?

Left: B. megaterium, Middle: E. coli,
Right: Unknown.

      To the right is a picture of our unknown microbe next to two controls, a positive control (B. megaterium), and a negative control (E. coli). Unfortunately there was a complication with the experiment! For some reason the strain of E. coli we chose to serve as our negative control was not motile like most other strains of E. coli with peritrichous flagella…. In order to help with extrapolating our results we are using both E. coli and S. aureus as negative controls. Our unknown microbe did not resemble either the positive control or the negative control, and this could be due to a few reasons; 1.) a poor stab into the Soft Agar Deep tube, 2.) the microbe is unable to tolerate anaerobic growth, or 3.) the microbe is motile and it “swam” out of the stab and reached the surface of the agar. 

Left: E. Coli, Middle: B. Megaterium,
Right: Unknown.

• We do not think that this result is due to a poor stab, even though the stab of our negative control was not of the highest quality.
• To determine if our microbe is able to tolerate anaerobic growth we reexamined our Triple Sugar Iron. If our TSI slant shows that our microbe was unable to handle anaerobic growth then this would determine that the unknown microbe was incapable of growing in the Soft Agar Deep tube. Upon looking at our TSI slant we determined that our microbe is able to handle anaerobic growth, so this second possibility for seeing no growth is also ruled out.
• Lastly, is the possibility of the microbe “swimming” out of the site of inoculation. We are unsure as to if this is the answer that solves our problem, but it is definitely a possibility!

2.) What cellular structure(s) contribute to bacterial motility?

Peritrichous Flagella

    Bacterial cells have numerous kinds of structures for cell motility, there’s eukaryotic, prokaryotic, and archaeal; but the most common structure of locomotion is  the flagella. Flagella are rigid, filamentous organelles that are approx. 20 nm in diameter and 15-20 um long. These flagella protrude from the cell's surface, thrusting the cell through liquids or across surfaces towards more favorable environments.

Bacterial Axial Filament

   

     Certain bacteria called Spirochaetes are helical, and they have specialized locomotive structures. The specialized structure that a Spirochaete possesses is called an axial filament, and it differs from the flagella because it uses spiral motion that rotates the bacterial cell as it propels it forward.

3.) Why might some microbes have evolved to be motile?
     I would say that bacteria have evolved to be motile for numerous reasons; reproduction, food, and positive environments. Examples of this movement are positive and negative chemotaxis and phototaxis. Chemotaxis is the movement that occurs by an of an organism that is responding to a chemical stimulus. This movement is important for bacteria to find the highest concentration of food molecules in their surrounding environment or swimming away from poisons that have the potential to harm them. Phototaxis on the other hand, is the movement that occurs when an organism moves away (negative) or closer to (positive) a light stimulus. This kind of motion is very beneficial for phototrophic organisms, so they can acquire light to power photosynthesis.

Still slowly discovering our dirt... 
     As of last week we have conducted six experiments to deduce the identity of our

unknown soil microbe. We have done a Gram stain to determine if our microbe is gram 

positive or gram negative, we have tested to see if our microbe was acid or non-acid fast,

we have tested for catalase activity, we have tested to see if our microbe 

is aerobic or aerobic, we have performed an endospore stain to determine if our unknown 

is endospore forming, and most recently we used a Soft Agar Deep Test to see if our

microbe is motile or not

          We have determined so far, that our bacteria is neither gram positive or gram 

negative, but rather a bacteria called endospore-forming bacteria, which is a mix of 

bacilli and cocci shaped bacteria. We have also determined that our bacteria is non-acid

fast due to the color it appeared after staining. There was also no sign of bubble 

formation during the catalase test which means that our bacteria has no catalase activity. 

Finally, we tested to see if the bacteria was an aerobic or anaerobic bacteria, and the 

results stated that there was activity of both types of metabolism. As for the endospore

stain Dylan and I determined that our unknown microbe is endospore forming, and lastly 

our Soft Agar Deep Test has provided results that do not lead us to 100% conclusion of 

whether or not our microbe is motile or not.


http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Prepared_Slides/Bacterial_Anatomy.htm
http://www.slideshare.net/rajud521/bacterial-anatomy
http://en.wikipedia.org/wiki/Phototaxis
http://en.wikipedia.org/wiki/Chemotaxis