The Quick and Dirty: Endospore Verification in Our Unknown Bacterium

       Several types of bacteria are known to form tiny versions of themselves when the going gets tough. These structures are referred to as endospores, and allow a bacteria to preserve itself when it encounters an environment that is unfavorable for reproduction or is lacking in nutrients. These minuscule spores contain a replicated version of the cell's genetic material, several types of ribosomes, and a chemical known as dipicolinic acid, which helps the endospore remain in its dormant phase. Endospores are extremely difficult to destroy or "kill." They can survive the most extreme of conditions: extremely high or low temperatures, ultraviolet radiation, extremes in pH, and even excessive drying. What's more, endospores of some species can survive for centuries or even millennia (hopefully nowhere near my house)! The evolutionary acquisition of bacterial endospores was probably favored in species that were often exposed to nutrient-lacking or high stress environments. Some bacterial cells may not have not evolved the ability to produce endospores simply because they did not often encounter these types of environments and, thus, it would not have been energetically favorable to do so.

     In this week's experiment, we performed an endospore stain to further narrow the possible identities of our microbe. Cells lacking endospores should appear light pink in color following the stain. Cell containing endospores should contain darker blue structures much smaller than the larger pink cells when examined under a microscope. Because endospores are able to tolerate extremes in temperature, we also performed a heat shock on a sample of our unknown bacterium. If growth was observed several days following the heat shock, it would indicate that endospores were present in the sample.

     As seen in the picture to the right, growth was observed in both the heat shocked sample (right tube) and the control sample (left tube) and four days of incubation, indicating the presence of endospores in our unknown bacterium. When viewed under a microscope, we saw tiny blue/purple structures scattered across the slide among the larger bacterial cells. These were assumed to be endospores. According to the dichotomous key provided, this opens the possibility that our bacterium could be Clostridium.

http://www.biomedcentral.com/content/pdf/1471-2164-13-265.pdf
http://en.wikipedia.org/wiki/Endospore#Formation_and_destruction

Come back next week for more dirty details!
- Austin

Catalase, Slants, and Butts: A look into our soil microbe's catalase activity and carbohydrate metabolism

In lab, the week before Spring Break, Austin and I further analyzed our soil microbe using the catalase activity test and triple sugar iron test. The overall premise of the catalase activity test is to determine if the microbe is catalase positive or negative. Catalase is an antioxidant enzyme that protects cells from oxidative stress. In this case, if catalase is present, then it will neutralize the hydrogen peroxide into water and oxygen gas. This neutralization of catalase-positive samples causes bubbling after adding a drop of hydrogen peroxide, and catalase-negative remains unchanged. Some microbes have evolved to acquire catalase activity. This could be primarily due to the added protection from oxidative stress that catalase-positive microbes possess.

To determine the catalase activity of our soil microbe, we first observed the reactions of a catalase-positive and catalase-negative control. When we added hydrogen peroxide to our soil microbe, the sample bubbled indicating it was catalase-positive.

The other test we conducted was the triple sugar iron test, which is used to determine the type of carbohydrate metabolism that the microbe utilizes. This specific experiment differentiates between lactose, glucose, and sucrose fermentation and hydrogen sulfide production. We inoculated our soil microbe and 4 controls on slants containing the triple sugar iron agar. After returning to the lab after 2 days of incubation, the controls behaved as we anticipated. The controls were characterized based on the color of the slant and butt. Our soil microbe was yellow at the slant and butt. This color is characteristic of an acid over acid tube, which we also saw with the E. coli control. Metabolically, our soil microbe fermented lactose and/or sucrose. Our soil microbe and the controls can be seen in the picture below.

(FROM LEFT TO RIGHT) Our soil microbe, B. megaterium, E. coli, P. areugrinosa, P. vulgaris

The fact that our soil microbe is catalase-positive and a lactose and/or sucrose fermenter does not change any of the previous classifications of our microbe.  It is still narrowed down to either genus Myobacterium or Nocardia.

Check back next week for more information from Austin about our soil microbe's endospore stain!

-Anne

http://onlinelibrary.wiley.com/doi/10.1002/0471140856.tx0707s27/abstract

Me Dusta: A look into acid-fast staining with our mystery microbe

Acid-fast staining works similarly to gram staining, but uses an acid-alcohol mixture as the decolorizing agent instead of using just alcohol as in gram staining.  The procedure for preparing a smear is similar to the protocol we used last week for gram staining. At this point, we are basically masters at this technique. For a quick refresher, the protocol for smearing includes plating microbes on a slide with heat fixation; acid-fast staining becomes distinct from gram staining at this point in the procedure. The slide with the fixed microbe is heated over steaming water and flooded with dye.  The steam helps drive the dye into the thick cell walls of the microbes. Acid-alcohol is used to rinse the excess stain, and the slide is then flooded with a secondary dye that allows for differentiation between acid-fast and nonacid-fast microbes.

Our acid-fast soil microbe

Two genus of bacteria, Mycobacterium and Nocardia, cannot be properly stained using any technique other than acid-fast staining.  These bacteria are tough to stain because of their thick, lipid-filled cell walls that are impermeable to many dyes that are commonly used in staining.  Bacteria with acid-fast properties require higher concentrations of dyes and heat to penetrate their cell walls.  The high-lipid content of their cell walls also makes it difficult to rinse the excess dye from inside the cell.  Consequently, this characteristic is how these types of bacteria came to be known as acid-fast, since the cells hold fast to the dyes and require acid to be removed.  Acid-fast cells retain the reddish-pink dye, while nonacid-fast cells appear blue due to secondary staining.

Nonacid-fast control, M. luteus

As can be seen in the photos, we determined that our mystery microbe was, indeed, acid-fast because of its reddish-pink color. It is visibly distinct compared to the deep indigo color of the nonacid-fast control. According the dichotomous tree provided to us, the acid-fast nature of our microbe narrows its possible identity to two genus: Mycobacterium and Nocardia.

In order to help us further describe our mystery microbe, this week’s lab will focus on the organism’s catalase activity and carbohydrate metabolism. Tune in next week for more info!!!

- Anne and Austin

http://www.highlands.edu/academics/divisions/scipe/biology/labs/rome/acid_fast_stain.htm

http://www.microbelibrary.org/component/resource/laboratory-test/2870-acid-fast-stain-protocols

Dirty Deeds: A look into our filthy microbe's cell wall structure

Our next series of experiments concern the cell wall structure of our unknown bacterium. Bacterial cell walls are typically classified as gram-positive or gram-negative, a characteristic that can be determined by a process referred to as gram-staining. A stain known as crystal violet is used to dye the bacterium's cell wall. Gram-positive cell walls are extremely thick in comparison to those of gram-negative cells and consist of a single layer of a compound known as peptidoglycan. Gram-negative cell walls are bit more complex. They are composed of an inner plasma membrane, a thin peptidoglycan layer and an outer membrane that is covered by a lipopolysaccharide layer. The thick layer of peptidoglycan in gram-positive cells is able to retain the crystal violet dye, causing the cells to appear as purple following the gram-staining process. Gram-negative cells are not able to retain the dye and appear pink following this process.

So why does the structure of something that you can't even see impact your life?? The differences in cell wall structure between gram-positive and gram-negative cells affect the ways in which they are able to infect your body. The lipopolysaccharide (LPS) that covers the outside of gram-negative cells is able to act as an endotoxin that causes inflammation, high fever and even death. The outer membrane layer also allows for resistance to common antibiotics like penicillin, making them more difficult to overcome than gram-positive infections. Basically, don't try to make friends with gram-negative bacteria, they're mean. 

Last week in lab, we did our own gram-staining of the soil microbes we harvested from outside of Subway. After performing gram-stains on the gram-positive control (B.megaterium), gram-negative control (K.pneumonia), and our unknown, we viewed the cells and they looked remarkably like hot dogs. We observed their rod-like shape (bacillus) and dark color. Stains of gram-positive cells are known for their deep purple hue. Therefore, we believed our bacterium was gram-positive because of its similarity in color to our gram-positive control. In order to confirm this prediction, we plated our bacterium and the gram-positive/negative controls on MacConkey agar, which is commonly used to isolate gram-negative bacteria and differentiate between lactose-fermenting/non-fermenting bacteria. Our bacteria would not grow on this media if it were gram-positve.

As can be seen in the photo below, the only growth observed was located in the gram-negative quadrant of the plate. This result is consistent with our prediction regarding the gram-positive nature of our bacterium.

In this week's lab, we will be performing acid-fast staining on our microbe!
Check back next week for more details!

Anne and Austin

http://en.wikipedia.org/wiki/Gram-negative_bacteria#Medical_treatment
http://www.life.umd.edu/classroom/bsci424/BSCI223WebSiteFiles/GramPosvsGramNeg.htm