Blog post #6

Last week in lab, Hannah and I verified that our soil microbe did produce endospores.  First, it is important to understand what an endospore is.  An endospore is a dormant, tough, non-reproductive structure produced by a small number of bacteria.

The function of an endospore is to ensure the survival of a bacterium through periods of environmental stress.

The benefits of an endospore is that they protect the bacteria's genome and a small amount of cytoplasm.  Endospores offer an advantage to bacteria that are able to produce them because it protects them from being destroyed.  Bacteria that does have the ability to produce endospores will have a greater chance of survival.  Bacteria that is not able to produce endospores could easily die off from harsh environmental conditions.

Some microbes have evolved to form endospores because microbes sense and adapt to changes in the environment.  They also might have evolved endospores due to response for nutrient deprivation.  Also, some microbes might not have evolved to form endospores because microbes are already adapted to conditions that are favorable for survival.  Some of these stresses could include high temperature, high UV radiation, chemical damage, etc.  A microbe may not evolve the formation of endospore because it already has nutrients that are needed and is adapted to changes in the environment.

In lab, we used aseptic technique to inoculate six 2 mL tubes of tryptic soy broth (TSB) with bacteria.  The specific cultures that we made were:
1. 2 tubes with positive Bacillus control
2. 2 tubes with negative E.coli control
3. 2 tubes with unknown soil sample
For each of the bacterium's, we labeled one tube 'HS' for heat shock.

3 tubes that contain the positive, negative, and soil microbe

Six tubes that we made using the positive control, negative control, and soil microbe before the 3 heat shocked were placed inside the water bath

After we transferred the HS tube of each bacterium, the three tubes were transferred to the 80 degree water bath.  The tubes were incubated in the water bath for 10 minutes.

Our 3 samples that were placed in the 80 degree water bath (HS samples)

After the 10 minutes, the samples were removed and all the tubes (6 total) were left on the bench to grow.

Below are some snapshots of the observations from our samples:

After 48 hours, the tubes were vortexes and observations were recorded

To interpret our results, it is important to not that all of our HS samples did have growth, which was observed by cloudiness in the tubes.  Since the E-coli served as the negative control, and did not contain endospores, the HS sample was clear because it did not survive the water bath, which is seen from the picture above.

The positive control, Bacillus served as the control that contained endospores.  This means that HS sample did survive the water bath, and thus appears cloudy.

For our soil microbe, the HS sample is exactly like the E-coli negative control.  The HS sample is clear, which means that no endospore formation was present and the soil sample did not survive the high temperatures from the water bath.

EDIT FROM LAB MARCH 31/2015.. (Repeated Experiment)
It was suggested that we repeat our endospore HS shock experiment to verify our results…
Blow is a picture that represents the results that were found

Repeated experiment on March 31, 2015

It is difficult to establish clear results from the picture, but in person it is clear that our soil sample did contain endospores.  The soil sample appeared cloudy, which means that it did contain endospores.  To verify this, our soil sample was compared to our positive control, Bacillus.

To further narrow down our soil microbe, a dichotomous key has been used: 

Below is our journey of discovery so far….
1. Gram positive
2. Morphology, Rod Shaped (Bacilli)
3. Non-acid fast organism
4. Catalase activity
5. Endospore positive (found from the Endospore stain experiment and HS experiment)

Also, we did another experiment in lab with our soil microbe and endospore stain. The stain provided us recognition if our soil sample contained a bacterial endospore.  The endospore contains a very thick wall that allows resistance to environmental conditions. We used a staining procedure called the Schaeffer-Fulton to look for endospores in bacteria.  If the sample has endospores, they will appear green in pink cytoplasm.

To do this, we prepared a smear of our soil sample, a positive sample (Bacillus) and a negative control (E.coli).   Each smear was heat fixed and then placed over a steaming beaker of water to heat the underside of the slide.  The slides were flooded with malachite green.

The positive control, negative control, and soil sample smears that were prepared

A snapshot after the malachite green was added to the slides

Each slide was heated for five minutes.  After the five minutes were up, the slides were cooled to room temperature and rinsed with water.  The slides were counterstained with aqueous safranin for 30-60 seconds and rinsed with water.  

Counterstain with aqueous safranin

After they were dry, we looked at them under a microscope at 100X using oil immersion.  Below are some snapshots of the slides under the microscope.  The positive control and negative control showed no signs of endospores.  Also, the positive control, should have contained endospores, but it was difficult to tell under the microscopes at obvious, green endospores.  The microscope was a little shaky, and was difficult to see through.  Our soil microbe did clearly contain endospores that were shown by in green towards the top of the slide.

Positive control (a species of Bacillus)

Negative control (a species of E. coli)

Our unknown soil microbe, which contained endospores shown in green (towards the top of the slide)

     

Citations:
https://micro.cornell.edu/research/epulopiscium/bacterial-endospores

http://www.sciencedaily.com/articles/e/endospore.htm

http://highered.mheducation.com/sites/0072437316/student_view0/chapter27/answers_to_text_questions.html

Blog Post #5

In our most recent lab meeting Sammy and I tested our mystery microbe for catalase activity. To accomplish this we first tested some controls that we could compare our microbe to. We used a positive (S. epideidermis) control and a negative (E. faecalis) control.  We observed bubble formation with our positive control when a drop of 3% H2O2 was added and no reaction with our negative control.

 

We then added the drop to our microbe and observed bubble formation indicating a positive result for our catalase test. A potential explanation for the evolvement of catalase activity in certain microbe is the aerobic microbes adapting to fight against elements that anaerobic microbes do not encounter such as oxygen. 

 

The other experiment we did regarding our microbe was the triple sugar iron test to determine carbohydrate metabolism. The TSI slant provides the culture with an aerobic and anaerobic environment in which to grow. This allows us to observe how different bacterium ferment and metabolize. All five of our control tubes grew as expected. I compared the information provided to us about the predicted reactions for each bacterium and confirmed the results by observing the color of the TSI agar of each control after incubation.

E. coli

P. vulgaris

P. aeruginosa

B. Megaterium 

For our unknown microbe, the media at the top of the tube was pink/red indicating an alkaline reaction. The bottom, or butt ,of the tube was yellow indicating an acidic reaction. There was no evidence of hydrogen sulfide formation. These results mean that our microbe metabolized glucose 

but not sucrose or lactose.

In the last blog post, Sammy followed the dichotomous key up until the question regarding catalase activity…

Dichotomous Key

Below, I have used a dichotomous key in order to begin identifying our microbe…Below is the steps that I followed in the dichotomous key:

1. Gram Positive

2. Morphology, Rod Shaped (Bacilli)

3. Non-acid fast organism

Now we know that our microbe is catalase (+) so we can continue!

4. Catalase positive

We know that our microbe metabolized glucose in both the aerobic and anaerobic areas of the slant so I’m unsure of the answer to the next step in the key. We might have to have further discussions to determine the answer.

Blog Post #4

Last week in lab, Hannah and I performed an acid stain test using two different stain samples:
1) a non-acid fast control (M. Luteus)
2) soil microbe

M. Luteus (non-acid fast control)

Soil Microbe

One day prior to lab, we prepared a pure culture of our soil microbe on a TSA slant.

During this acid stain procedure, we used an aseptic technique and smeared a sample of our soil microbe and the control culture onto a slide, along with a loopful of water.  We mixed the bacteria with the water and allowed it to dry. Then, we put our slide through the bunsen burner and placed the slides over a beaker with steaming water.  The slides were then flooded with carbol-fushin and cooled/rinsed with water.  The slides were depolarized with acid-alcohol until the color stopped coming off.  Lastly, the slide was flooded with methylene.

Below is a picture of the control slide & soil microbe over steaming water:

We then observed our acid fast stain slides under the microscope at 100X magnification. Sidenote:  (Oil immersion was added to the slide).

Below is a picture of the control and our soil microbe under the microscope:
The soil microbe had a blue tint with many blue rods (bacilli) distributed across the slide.

Soil Microbe

It was observed under the microscope to have very small, round, blue circles distributed evenly across the slide.

M. Luteus

Acid-Fast Stain & Background:

It is important to include some background information about an acid fast stain and what it actually is to further understand and interpret our results.  An acid fast stain is a stain that is used to identify organisms, such as members of the genus, Mycobacterium. Acid fast organisms usually have waxy cell walls that are impermeable and contain many lipids and fatty acids.  Usually to get through this cell wall, heat is involved.

It was found that our soil microbe, was non-acid fast, which means that it does not have a waxy cell wall.  It was also gram (+) in terms that a peptidoglycan wall is present. 

Dichotomous Key

Below, I have used a dichotomous key in order to begin identifying our microbe…Below is the steps that I followed in the dichotomous key:

1. Gram Positive

2. Morphology, Rod Shaped (Bacilli)

3. Non-acid fast organism

4. …The next question asks if the organism is catalase (+) or catalase (-)…Unfortunately, at this time in the experiment we do not know the answer to this question yet so we will have to look forward to next week's lab in order to further identify our mysterious microbe!

Post #3

During lab this week we Gram stained several different bacterium. First we made two control stains one Gram positive, B. megaterium, and one Gram negative, K. pneumoniae.  After viewing them under the microscope the Gram positive sample is clearly purple and the Gram negative sample is pink.

Gram positive control

Gram negative control

 We then Gram stained our mystery microbe to determine what category it fell into. When viewed under the microscope our mystery microbe was purple indicating that it is Gram positive. It also appeared rod shaped or Bacillus.

soil microbe

soil microbe under microscope

 After checking out our microbe under the microscope we streaked our soil microbe on a MacConkey agar plate along with B. megaterium, K. pneumoniae, and P. aeruginosa. We also streaked these bacterium on a Rose-Bengal plate to determine if their was any fungi present in our sample.

Our MacConkey agar confirmed our Gram staining. B. megaterium and our mystery microbe both did not grow on the agar indicating that they’re Gram positive. K. pneumoniae and P. aeruginosa successfully grew on the plate demonstrating that they are Gram negative. Also, there was no growth for our microbe on the Rose-Bengal plate. 

There are several differences between the cell wall structure of Gram positive and Gram negative bacteria. Gram positive bacteria have a cytoplasmic membrane with a thick peptidoglycan outer layer. Gram negative bacteria has a thin peptidoglycan layer sandwiched between two cytoplasmic membranes.  The outer membrane of the Gram negative  bacteria contains phospholipids, proteins, and lipopolysaccharide (LPS). LPS is an endotoxin that contains Lipid A.  When the Gram negative bacteria is killed the endotoxin is released causing negative effects in the host. This feature of the cell wall can cause issues when treating Gram negative bacterial infections. The cell wall structure also is resistant to many antibiotics making the infections difficult to treat.