Hemolytic Activity of Bacterium

Last week, we continued to study our unknown soil microbe by performing a Hemolysis Test. The hemolysis test helped us determine whether our bacteria is said to be fastidious. Fastidious organisms require a rich growth medium filled with specific nutrients required to grow (Lab Handout). The medium used for this test is a Blood Agar, composed of general nutrients and 5% sheep blood. Bacteria that flourish on this blood agar produce exoenzymes called helolysins, which trigger the lysing of red blood cells. Blood agar tests are especially useful in determining streptococcal species. Based on their hemolytic activity response to the blood agar, bacteria can be categorized into three different groups:

Type	Meaning	Appearance
Beta (β) hemolysis	Complete or true lysis of red blood cells	Clear zone, almost transparent of the base medium, surrounds colonies (due to destruction of hemoglobin released from erythrocytes)
Alpha (α) hemolysis	Reduction of red blood cell hemoglobin to methemoglobin in medium surrounding colony	- Green or brown discoloration in medium (brusing color) - Cell membrane in tact
Gamma (γ) hemolysis	Lack of hemolysis	No reaction surrounding the medium

Below are photographs of our controls, Staphylococcus aureus and Staphylococcus epidermis, and our unknown bacterium after 48 hours. Although it is not fully clear which hemolytic reaction our unknown bacterium belongs to, it is most closely related to an Alpha (α) hemolysis reaction. The dark coloring surrounding the colonies is a good indicator of alpha hemolysis. After seven days, the plates were observed again to see if our prediction on alpha hemolysis was correct. After a prolonged incubation, many alpha hemolytic organisms begin to appear clearer, with still a surrounding bruised color (Lab Handout). Pictures of our unknown bacterium after a prolonged seven-day incubation are below. Our microbe did not lyse the red blood cell, and kept the cell membrane in tact.

 

S. aureus

S. epidermis

Unknown Bacterium - 48 hrs. 

Unknown Bacterium - Day 7

Hemolysins have the capability to lyse red blood cells through several mechanisms. One way this happens is through the formation of pores in phospholipid bilayers (Chalmeau et al, 2011). The pores in the phospholipid bilayer allow extracellular fluids or components, such as bacteria, to enter in the red blood cell and lyse. Many of the hemolysins are categorized as pore-forming toxins, which not only lyse red blood cells, but leukocytes, and platelets. Hemolysins also function as enzymes. They damage the membrane of the red blood cell by cleaving the phospholipid in the membrane (Honda et al, 1985). This could potentially alter the structure of the membrane, allowing swelling of the red blood cell and eventually lysing.

Virulence factors are anything that could potentially produce a disease in humans. When comparing hemolytic microbes to non-hemolytic microbes, hemolytic microbes serve as potential virulence factors when combined with other factors, and therefore more virulent than non-hemolytic microbes. The lysing of red blood cells may not be hazardous initially, but combined with other bacterial factors, the possibility of virulence increases (Woltjes and de Graff, 1983). Typical soil microbes may be hemolytic, breaking down red blood cells for nutrients to encourage growh of the bacteria. The breakdown of red blood cells is used when a physician tests for strep throat, when looking for the streptococci bacteria (Chapter 5).

           

According to the dichotomous key and the idea that our unknown soil microbe is alpha-hemolytic, our bacteria could be Streptococcus pneumoniae, which is different than our test from last week, where we believed our bacteria would be Actinomyces spp. With one week and one final test left, we hope to finally narrow down what our unknown bacterium is combining our research into discovering our culprit.

 Chalmeau, J., N. Monina, J. Shin, C. Viey, V. Noireaux. (2011) α-Hemolysin pore formation into a supported phospholipid bilayer using cell-free expression. Biochemica et Biophysica Acta (BBA) – Biomembranes. 1808(1), 271-278.

Honda, T., M. Yoh, U. Kongmuang, T. Miwatani. (1985) Enzyme-linked imunosorbent assays for detection of thermostable direct hemolysin of Vibrio parahaemolyticus. Journal of Clinical Microbiology. 22(3): 383-386.

Weeks, B. I. Edward. Alcamo. Microbes and Society. Sudbury, MA: Jones and Bartlett, 2008. Print.

Woltjes J., J. de Graaf. (1983) Virulence of beta-hemolytic and non-hemolytic Streptococcus mutans: lethal dose determinations in neonatal mice. Antonie Van Leeuwenhoek. 49(4-5): 353-360. 

To reduce or not reduce, that is the question

In searching for the identity of our unknown soil microorganism, we performed an experiment to determine whether nitrate may be reduced by our microbe. Nitrate reductase controls the reduction of nitrate to nitrite and indicates that nitrate could be used as an electron acceptor during anaerobic respiration. The experiment determines whether a microbe produces nitrogen gas, reduces nitrate to nitrite, or will reduce nitrate to another form other than nitrite. If an organism releases nitrogen gas, then bubbles will become visible at the top of the medium, as seen in the positive control. If an organism does not produce bubbles, then a solution of sulfanilic acid and alpha-naphthylamine is added to the incubated medium. Whether the medium turns red in color determines if nitrate reduces to nitrite. 

Positive Control

Negative Control

Unknown

Blank

Unknown compared to Negative control

Our unknown produced the same results as our negative control, indicating that it reduces nitrate to nitrite. Bacteria reduces nitrate for various reasons. Nitrate may be used as a source of nitrogen for cellular growth, a terminal electron acceptor in creating metabolic energy, or ridding the organism of excess reducing power in redox balancing.   

Following the dichotomous key, it appears that due to our microbes ability to reduce nitrate, our bacteria would be Actinomyces spp. Further testing must be completed in order to confirm.

The Motility Possibility

In further research to determine the elusive identity of our soil microorganism, we performed a soft agar deep test to evaluate motility. This test helps discern whether to categorize a bacteria as motile or non-motile. If a bacteria is motile, it will have the capability to move through the agar due to the nature of the semi-solid media. The positive control, E. coli, exhibits bacteria motility by expanding away from the stab inoculation line, while the negative control, S. aureus, grows solely along the inoculation line.  

E. coli 

S. aureus

Incidentally, the results of the soft agar test did not reveal a conclusive answer about the motility of our microbe. As displayed in the picture below, there is no clear growth along, or radiating from, the stab inoculation line. Though the agar appears slightly cloudy, the only definitive microbial growth occurs on the surface of the soft agar.

Unknown Microorganism

Despite not having an obvious answer regarding the motility of our unknown, we are still capable of postulating characteristics that could ultimately lead to it's identity. Last week, we determined that our unknown bacteria could be either aerobic or anaerobic. If the bacteria is aerobic, then it could not tolerate anaerobic growth. There is also the possibility that our unknown is motile and migrated to the surface of the media, instead of remaining the in the stab line.

Motile capability results from the presence of flagella on prokaryotic organisms. The momentum arises from the counterclockwise rotation of individual flagella bundled together. Bacterium often move in certain direction as a result of environmental cues, a process known as chemotaxis. They will either migrate toward or away from compounds due to the detection of concentration gradients. This process has evolved form the survival of bacterium with better receptors and flagella, and their movement to and from certain chemicals. The only downside to flagellar motility is the inability to move within small spaces. Because a microbe needs to rotate in order for flagella to create momentum, tightly compact areas prevent proper movement, inhibiting motility. 

Though motility is a helpful identifier, it does not help us narrow down our possible bacteria options using the dichotomous key. More testing, specifically on nitrate reduction, is required. Last week we stated our unknown bacterium could possibly be Lactobacillus spp., while eliminating Clostridium spp. due to the lack of endospore production. As of this week, Lactobacillus spp. and Acitonomyces spp. are contending for the identity of our unknown microorganism.

Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002. Section 34.4, A Rotary Motor Drives Bacterial Motion. 

http://www.ncbi.nlm.nih.gov/books/NBK22489/

-Palmer Miller

Endospore Testing & Verification

To further examine our bacterium, we used Endospore Testing to look at and compare our unknown bacterium to the positive and negative controls, Bacillus and E. coli, respectively. The endospore stain helps us to recognize the presence of bacterial endospores, which is only found in a very few species of bacteria. It also helps with determining the size of the bacteria, as well as morphology and the location of the endospore within the parent cell (Lab Handout). Endospores are resistant to heat, desiccation, chemicals toxic to vegetative cells, as well as UV light (Lab Handout). Endospores may serve beneficial to bacteria as they could escape certain environmental conditions in order to survive (Nicholson, 2002). They can survive severe harsh environments and are easily spread. Specifically, environmental structures they have adapted to become resistant to are stated above: heat, desiccation, chemicals, pH changes, and UV light. Other bacterium may have not formed endospores in order to have a moderation of bacteria when high numbers are present. They are resting structures and are only used when nutrient supply is depleted. Therefore, the Schaeffer-Fulton method was used to help us determine whether our unknown bacterium has or lacks endospores. The endospores we are looking for appear to be green surrounded by a pink cytoplasm.

Bacillus - Positive Control

After we stained our bacterium with Schaeffer-Fulton, we observed our slides with oil immersion and compared the three slides. Pictures of our slides are below. Our positive control, Bacillus, presented endospores. The endospores are the green colored bacteria on the picture, surrounded by the pink cytoplasm.

Our negative control, E. coli, had no endospores as we found only the pink cytoplasm and no green endospore. The image that we found is consistent with what we expected from our negative control. This control was compared to our unknown to help us determine more about our bacterium.

E. Coli - Negative Control

Our unknown bacterium appears to have a green endospore with light pink cytoplasm surrounding. However, the cytoplasm surrounding the endospore is not as prominent as what was found in the positive control. The pink bacteria surrounding the endospore are rod-shaped and only have a few bacteria present. The green center appears to trigger an idea that our unknown bacterium is endospore-positive.

Unknown Bacterium

Unknown Bacterium

Unknown Bacterium

In order to verify our results from our Endospore Stain using the Schaeffer-Fulton method, we tested each control and our unknown by observing what happened when the bacterium was “heat shocked”. As previously stated, endospores are resistant to high temperatures, desiccation, as well as radiation. We used the heat shock method to verify whether or not our bacterium did or did not have endospores. Samples were placed in an 80 °C water bath for 10 minutes and then incubated for 24 hours at room temperature. Our results are shown in pictures below. Our positive control, Bacillus, had bacteria in the medium without heat shock. After the heat shock, bacteria were still present in the tube. Serving as a positive control, we expected their to be bacteria still present after heat shock, solidifying the idea that endospores were present and could withstand the heat in the heat bath. Our negative control, E. coli, did not have bacteria present after the overnight incubation at room temperature. The medium was not clear, indicating amounts of bacteria in the tube. The heat shock for the negative control killed the bacteria, observed by a clear medium. Our unknown bacterium presented bacteria without the heat shock. The heat shock cleared up the bacteria, with no bacterial particles or murky medium shown. 

	No Heat Shock	Heat Shock	Endospore?
Bacillus – Positive Control	+	+	Endospore Positive
E. Coli – Negative Control	+	-	Endospore Negative
Unknown Bacterium	+	-	Endospore Negative

+ = Bacteria present

-  = Bacteria not present

Bacillus - Positive Control: Heat Shock

Bacillus - Positive Control: No Heat Shock

E. Coli - Negative Control: Heat Shock

E. Coli - Negative Control: No Heat Shock

Unknown Bacterium: Heat Shock

Unknown Bacterium: No Heat Shock

These results conflicted with our original findings after our initial endospore-staining test. After the original endospore staining test, we thought that our bacteria was endospore positive, like our positive control, Bacillus. However, our verification test using heat shock lead us in a different direction. The media, TSB, that the bacteria were living in, cleared up after the heat shock, showing that the bacteria were not resistant to heat. Some bacterium may not be producing endospores due to the number of already viable bacterium presented with endospores in a certain environment (Schaeffer et al., 1965).

According to the Dichotomous Key, endospore bacterium that was not produced leads us to believe that our unknown bacterium could possibly be Lactobacillus spp. We now know that since our bacterium did not produce endospores, it eliminates Clostridium spp. These observations and further testing have helped us eliminate several bacteria, but also have continued to add other bacterium to our possibility list. Next week, we will continue our search into what our bacterium exactly could be.  

Nicholson, W. L. (2002) Roles of Bacillus endospores in the environment. Cellular and Molecular Life Sciences. 59(3), 410-416.

Schaeffer, P., J. Millet, J. Aubert. (1965) Catabolic Repression of Bacterial Sporulation. Proc Natl Acad Sci USA. 54(3), 704-711.

Reactions: Catalase Test & Triple Sugar Iron Test

Earlier in the week, our microbe underwent a Catalase Test to determine whether or not there was activity or a reaction. The catalase test is used to determine whether or not there is enzyme catalase in the tested bacteria. Catalase is produced in situations of oxidative stress where it will facilitate cellular detoxification and when it correlates with pathogenicity in bacteria (lab handout). Some microbes might have evolved to have a catalase activity as a response to the oxidative damage of hydrogen peroxide to keep them from being killed off. In our catalase test of our soil microbe, we dropped 1 drop of 3% H₂O₂ onto our bacteria and waited for a reaction. Our soil microbe reacted to the H₂O₂ , leading us to believe that our bacterium resulted in a positive reaction. We compared our slides with a positive control, Staphylococcus epidermis as well as to a negative control, E. faecalis. Below is a picture of the reaction of our soil microbe in the catalase test. Our reaction was not large, however, it reacted to the H₂O₂, leading us to believe there is the catalase enzyme in our bacterium.

Catalase Reaction

We also performed a Triple Sugar Iron Test (TSI) on our soil bacterium to determine carbohydrate fermentation and hydrogen sulfide production. This test differentiates bacteria according to how they ferment lactose, glucose, and sucrose. Bacteria can metabolize carbohydrates in two ways: aerobically or fermentatively. This test will helpE. coli, B. megaterium, P. areuginosa, and P. vulgaris.

us decide according to the resulting color how our soil microbe metabolizes carbohydrates. The agar in the tube is defined as a differential medium and will select for carbohydrate fermentation and hydrogen sulfide production. Four controls were tested against our soil microbe:

Bacterium	Tube Reaction	Reaction Color
E. coli	Acid/Acid	Yellow
B. megaterium	Acid/No Change	Pink on Bottom
P. areuginosa	Alkaline/Alkaline	Dark
P. vulgaris	Acid/Acid + H2S	Yellow over Black
??? Our Soil Microbe ???

Below are pictures indicating the result of our Triple Sugar Iron Test for our controls and for our unknown soil microbe. The controls behaved as expected according to observations of the tubes with the TSI agar and from the lab handout. Our tube with our unknown bacterium in it had a pink bottom with a little yellow on the slant. The control most similar to our unknown bacterium is B. megaterium. There was no evidence of hydrogen sulfide production, as there was no observed blackening on the butt of the tube. The butt of our tube was pink, leaving us to believe that there was no yellow or black coloration and no hydrogen sulfide production. According to our initial observation after 24 hours at 37°C, our soil microbe is a glucose fermenter. Our tube reaction is alkaline over acidic (K/A), meaning that our bacteria can only metabolize glucose. Both aerobic and anaerobic metabolism can be used to produce ATP and pyruvate. Glucose is consumed by our bacterium around 18 hours and the amino acids were used as an energy source on the slant in the form or aerobic metabolism. The butt stays acidic due to the stable acid end-products of the Embden=Meyerhof-Parnas pathway that metabolizes glucose.

B. megaterium

E. coli

P. areuginosa

P. vulgaris

Unknown Soil Microbe

Unknown Soil Microbe

Unknown Soil Microbe

According to the dichotomous key, we can begin eliminating ideas and narrowing down our guesses on what our bacteria might be. Last week, our acid-fast stain left us confused in which direction to take our initial hypothesis on what our bacterium could be. This week, with further testing, we can begin to eliminate a few options and narrow our ideas. We found that our bacterium was catalase positive, and both aerobic and anaerobic. This narrows down our options to Actinomyces spp., and Peptococcus spp. However, further tests need to be done in order to solidify our observations. More tests will be needed and may lead us to reevaluate our hypothesis. But, as of right now, our tests have shown us that our bacterium could be either Actinomyces spp. or Peptococcus spp.

Tripping on Acid-Fast Staining

As we continue to characterize our soil microbe, our sights turned to determining whether our bacteria is acid-fast. This was done using Acid-fast stain, which uses the same process as a Gram stain except for the decolorizing agent used following a primary stain. The name Acid-fast derives from this variation in decolorizing agent: the combination of a mineral acid and organic solvent, such as ethanol, either together or separate.
The decolorizing agent differs from that of the Gram stain due to the physical attributes of bacteria with the acid-fast property. These bacteria all have a cell wall with a high lips content that when grown on agar medium display a wrinkled, dry surface and a pellicle in liquid medium. The lipid saturated cell wall absorbs the primary stain, so that when viewed under a microscope the microbe appears pink. A non acid-fast microbe appears blue due to the methylene blue counter stain. The Ziehl-Neelson Method is the most commonly used procedure, using carbon-fuchsin as a primary stain.
But characterizing a bacteria as acid-fast is only applicable to the genera Norcardia and Mycobacterium. Even though few species are represented, many medically pertinent bacteria display this property, such as Mycobacterium tuberculosis, the tuberculosis causing bacteria.

In order to create a point of reference from which to compare our unknown bacteria, we began by staining a known non acid-fast bacteria, Bacillus megaterium. This acted as our negative control. Below shows a microscope view of our results. The bacteria appears dark purple which indicates that the carbon-fuchsin was not absorbed into the cell wall. They also have a diplobacilus rod-shape.

Bacilus megaterium Acid-Fast Stain

After the acid-fast stain, the soil microbe appeared lighter in color compared to the B. megaterium, as seen below. But it is unclear whether the lighter hue indicates acid-fast properties or a mishap in the experimental procedure. Both cocci and bacilus bacterium are present in the culture.

Unknown Bacteria Acid-Fast Stain

To ascertain the identity of the soil microbe we can use a dichotomous key. It is clear that our microbe is prokaryotic in origin, rather than eukaryotic. We previously established that the organism is gram-positive and rod-shaped, which leads to the question of acid-fast properties. Because the results of the acid-fast stain were unclear, it will be necessary to run more tests, such as determining catalase activity. This will help determine is the bacteria is acid-fast in nature.

Positively Gram-positive: Gram-staining Results, Comparisons, & Observations

After Gram-staining our mystery bacteria last week, we initially suspect that our bacteria is Gram-positive due to many observed characteristics. Below is a photograph through a microscope of what we are basing our observations on. Our bacterium appears to be stained purple, which is a result of uptake of crystal-violet stain. The purple stain indicates a thick layer of peptidoglycan in the cell wall. The bacterium possesses a diplobacilus rod shape. 

Gram staining results

To verify our results, we used a MacConkey agar to determine whether our mystery bacterium found in the soil is Gram-negative or Gram-positive. We compared our mystery bacterium to three controls: Bacillus megaterium, which was a Gram-positive control, Pseudomonas aureoygynosa, which was a Gram-negative, non-lactose fermenting control, and Klebsiella pneumonia, a Gram-negative, lactose-fermenting control. Below is a photograph of our MacConkey agar, showing our results for growth on the agar for our controls and our bacterium. Our bacterium, according to our results on the MacConkey agar, is Gram-positive, indicating that our bacterium did not grow on the agar. 

MacConkey agar 

Gram-positive cell walls have a thick layer of peptidoglycan with teichoic acids, a polyalcohol. On the other hand, the cell wall of a bacterium that is Gram-negative will have a thin layer of peptidoglycan, with a bilayer membrane on the outside of the peptidoglycan. This bilayer membrane is composed of phospholipids, proteins, and lipopolysaccharides.

Although we are concerning ourselves here with bacterium found in soil outside, Gram-negative bacteria and Gram-positive bacteria are prevalent in our every day society. These bacterium are the cause of common infections found in almost all hospitals. Gram-negative bacterium cause many infections, such as pneumonia, bloodstream infections, wound infections, surgical site infections, and meningitis. Gram-negative bacteria exhibit multi-drug resistance to many antibiotics (CDC, 2011; Moehring & Anderson, 2014). According to the CDC, Gram-negative bacteria have built-in systems that are able to be resistant and are able to pass along genetic information that will allow other bacteria to also become drug-resistant. Antibiotics for Gram-negative bacterial infections differ from those of Gram-positive due to the risk of septic shock, morbidity, and mortality in patients. Gram-positive bacteria also pose a threat as they are becoming increasingly resistant to antibiotics used to treat infections caused by Staphylococcus aureus and Enterococcus spp. (Woodford & Livermore, 2009). More commonly known as MRSA, this Gram-positive bacteria spreads rapidly throughout hospitals. Both Gram-positive and Gram-negative exhibit multidrug resistance to antibiotics and antimicrobials aimed to control and eliminate bacterial infections.

“Gram-negative Bacteria Infections in Healthcare Settings.” Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 17 Jan. 2011. Web. 02 Mar. 2015.

http://www.cdc.gov/hai/organisms/gram-negative-bacteria.html#gi

Moehring, R., D. Anderson, “Gram-negative Bacillary Bacteremia in Adults” Gram-negative Bacillary Bacteremia in Adults. N.p., 31 Oct 2014. Web. 02 Mar. 2015.

http://www.uptodate.com/contents/gram-negative-bacillary-bacteremia-in-adults

Woodford, N., DM. Livermore, (2009) Infections caused by Gram-positive bacteria: a review of the global challenge. J Infect. 59, 60003-7.

http://www.ncbi.nlm.nih.gov/pubmed/19766888

-Heather Daniel