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

Ya Dig? A Look Into Soil Biodiversity and Why You Should Care

Here we are, one week out from our original sampling excursion outside our campus grocery store/Subway, and we are proud to report substantial microbial growth. The plated colonies have continued to grow in size (and grossness) over the past few days. The most growth remains on the plate that contains the highest concentration of soil sample (diluted 1000-fold), while the least amount of growth remains on the most diluted sample plate (diluted more than a million fold).

We now turn our focus on observations of biodiversity within our samples. After one week of incubation, we have witnessed the growth of many different types of bacterial and fungal colonies. These colonies vary in size, texture, and color. Some colonies have dark, discolored centers, while other remain uniform in color. Some are raised dramatically from the plate surface, and still others are as flat as pancakes. Some colony edges appear circular, while others stretch wildly across the plate. Every variance in appearance between colonies indicates a change in species, implying that our soil sample contained more types of organisms than could allow for peace of mind. The presence of a single fungal colony on our flourescent growth plate suggests a greater biodiversity among bacteria than fungi in the sample area. By applying some simple algebra, we estimated that there were 50,000 microbes per gram of soil at our sampling location.

You might be asking, "Why does soil biodiversity matter?" Well, it's really important - to say the least. Bacteria in the soil are responsible for the decomposition of organic matter. By breaking this matter down, they release nutrients into the soil that are vital for the production of the natural fertilizers that allow for agriculture. Basically, if we don't have any soil biodiversity, we have no food! You can find more information about the importance of soil microbes by clicking on the following link: http://www.fao.org/agriculture/crops/thematic-sitemap/theme/spi/soil-biodiversity/agriculture-and-soil-biodiversity/en/

Come back next week for updated pictures and more dirt on dirt!
- Austin and Anne

Talk Dirty To Me: A Preliminary Snapshot of Our Soil Microbiology Study

If you can call yourself a Panther, you've definitely made a few visits to the BSC C-Store/Subway. So, what's actually going on under this campus oasis? That's what we, Anne Cetto and Austin Mills, are here to investigate.

We began our study, trowel in hand, under the Subway overhang among the Common Boxwood shrubs. After practicing the proper sterilization techniques and removing the light layer of pine-straw mulch, we dug approximately one inch into the top soil layer to collect our sample. The soil in this location was cool, moist, and slightly compacted. The sampling site is located on a slope and likely comes in contact with a high amount of water runoff and pedestrian traffic.

Sampling area in front of the BSC Subway and C-store

After returning to the lab, we plated a series of diluted samples on four different laboratory bacterial growth plates. An additional plate contained a medium that was designed to grow only fungus so that we would be able to create a ratio of bacteria to fungus contained within the soil sample. These plates can be seen in the photos below.

Growth plates are pictured with the far left plate being the most concentrated sample.  The fluorescent pink plate selects for fungus as opposed to bacteria.

Growth after five days.

Growth after five days.

As seen in the above photos, the most growth occurred on the plate with the highest sample concentration. Amount of growth decreased as the sample concentration decreased, with no growth on the plate containing the least concentrated sample. As our investigation continues, we will keep you posted with the most recent information - don't forget to check back with us next week!

- Austin and Anne