Tuesday, September 24th 2013

*This blog does not reflect Franciscan University in any way*

Dear Blog and Blog Readers, 

Lab today consisted of viewing our slides that had a negative stain under the oil immersion lens, doing one last gram stain of the red pigmented soil bacteria, and making a completely new petri dish of a bacteriophage. 

First of all, we took the slides that a had negative stain and we made a capsule stain out of them by adding some crystal violet stain to the slide and then rinsing it off indirectly

Max and I staining the negative slides with crystal violet for 20 seconds. 

Rinsing off the crystal violet stain.

These next two images are the unknown (S2) negative capsule stain under the oil immersion lens.

Our conclusion is that there were no capsules. 

These next two images are our environmental sample negative capsule stain under the oil immersion lens.

Our conclusion is that we have spores but no capsules. 

Spores are often found in soil bacteria so it is not surprising that we found them in this sample. Spores allow bacteria to survive under harsh conditions such as extreme heat, light, etc. This allows the bacteria to germinate when it return to conditions that are viable for life. Swelling that is a result from endospore (a spore inside a bacterium) can shed light on the species of bacteria it is. (see bibliography)

Capsules are attached to the cell wall and very organized. They are made of polysaccharides and are used to identify a species of bacteria. Colonies with capsules are usually smooth while those without capsules are rough. This is surprising since we did not have any capsules from our unknown or our environmental sample when both colonies were smooth. (see bibliography)

Here we are making the petri dish with the bacteriophage.

 

Max sampling the bacteria "G"

Max using a wave motion to put the bacteria in a petri dish. 

Max then wrote his initials in the petri dish with a bacteriophage.

We predict that there will be no bacteria that grows where Max wrote his initials because the bacteriophage is a virus and will kill the bacteria where Max wrote his initials. 

Unfortunately our last gram stain did not work out, so we will be finishing that next class!

Bibliography:

Duben-Engelkirk, Janet, EdD, MT(ASCP). "Chapter 3: Cell Structure and Taxonomy."Burton's     Microbiology for the Health Sciences. By Paul G. Engelkirk, PhD, MT(ASCP), SM(AAM). 9th ed. Baltimore: Lippencott Williams & Wilkins, 2011. 32-34. Print.

Thursday, September 19th, 2013

*This blog does not reflect Franciscan University in any way*

Dear blog and Blog readers, 

Today in class we observed (what we thought would be) our final streak plate! To recap, this is what we discovered last class. 

On Tuesday in microbiology we obtained our streak plate from the 30 degree incubator! (this streak plate had previously started in the 30 degree incubator, was transferred the the 25 degree incubator and was then placed back into the 30 degree incubator). Here is what our streak plate looked like before this last incubator trial:

   

When we obtained the sample from the 30 degree incubator this is what we found:

Growth/cream/non-pigmentation

Since there was growth but no pigmentation and if this bacteria was the same bacteria as the red pigmented bacteria it should have become red in the 30 degree incubator we concluded that this cream/non-pigmented bacteria and the red bacteria are two different types of bacteria. 

To further prove this statement, we created one last streak plate (a brand new one) from the pure soil sample. 

    

The pure soil sample.

Our new streak plate. 

We placed this streak plate in the 30 degree incubator to see what would occur one last time. 

When we collected this streak plate today, this is what we found. 

A pure sample of the bacteria obtained from the soil near the Ohio River in Steubenville, OH. 

Dr. P advised us to prepare one more gram stain from this pure sample to see if we would still obtain the same result as we had before (which was mostly gram-negative bacteria. 

Gram Stain 3rd 30 degree streak plate soil sample:

First, we prepared a fixed smear of bacteria.

Max sterilizing the loop. 

Max putting distilled water into the sterilized loop. 

Max putting the water on the sterilized glass slide. 

Sterilizing the loop once again. 

Obtaining a pure sample of bacteria

Smearing the bacteria on the water droplet on the slide. 

Allowing the sample to air dry. 

We dropped crystal violet stain on the fixed smear for 20 seconds.

Rising the stain off with an indirect stream of DI water. 

Then we covered the smear with Gram's Iodine for one minute.

We rinsed the slide with indirect DI water. 

Next, we rinsed the slide at a 45 degree angle with 95% ethanol drop by drop until the color stopped 

running. 

Rising the stain off with an indirect stream of DI water. 

Next we covered the smear with Gram's Safranin for one minute. 

Rising the stain off with an indirect stream of DI water. 

Finally, we blotted the water from the slide with bibulous paper.

Max focusing the microscope. 

Observing the slide under the oil immersion lens. 

Interestingly enough, we observed gram-positive bacteria with some variable bacteria. There appeared to be all circles (with two colors, purple and red) but some appeared as rods. Dr. P advised us to do one more gram stain next class. 

Next, we were instructed to do a negative stain with the samples from the second, more pure test tube, "S2"(our unknown) and our pure soil bacteria sample.

S2- our test tube we created from the unknown tube Dr. P gave us last class.

The purpose of this view bacteria under a microscope with a dark background so that transparent cells are more easily seen, to make visible bacteria that is hard to stain or is changed by heat-fixation. 

First we obtained two clean slides and labeled one "S2 N" to stand for S2 negative stain.

We placed a drop of Nigrosin on one end of the slide.

We sterilized the loop.

We then obtained a sample from the S2 test tube.

We smeared the bacteria into the Nigrasin.

Max then smeared the bacteria and Nigrosin across the slide.

We waited for it to air dry!

We prepared our second slide for the negative stain and labeled it "Soil N" for the pure soil sample

We placed a drop of Nigrosin on the slide.

Then we sterilized the loop.

We obtained a little bit of the bacteria from the pure soil sample.

We then mixed it with the Nigrasin.

Max then spread the Nigrosin and bacteria on the slide with another clean slide.

We then allowed it to air dry!

Before the end of class we were not able to properly view the slides under the oil immersion lens. Check back next time for the results!

Thursday, September 12th 2013

*This blog does not reflect Franciscan University in any way*

Class date: Thursday, September 12th 2013

Dear Blog and Blog readers,

Today, we first observed our streak plates!

Previously on "Max and Sam's Adventures in Bio" . . . we placed the streak plate that had been in the 30 degree incubator (red pigment) in the 25 degree incubator and the streak plate that had been in the 25 degree incubator (cream pigment) in the 30 degree incubator. At this point we hypothesized that when they switched incubators they would switch pigments as well. We did this in an attempt to see if this was truly the same bacteria.

Here were our results:

The streak plate that had been in the 30 degree incubator and was placed in the 25 degree incubator has almost no growth, and the growth that did result had cream/no pigmentation. Keep in mind that this is what the same streak plate looked like before: 

Quite a difference!

The streak plate that had been placed in the 25 degree incubator and then transferred to the 30 degree incubator appeared the same in both cases: cream/no pigmentation. 

Our conclusion was that these were two different types of bacteria. The cream/non-pigmented bacteria is able to grow at least between the temperature range of 25 degree Celsius and 30 degree Celsius. However, the red pigmented bacteria is only able to grow at 30 degree Celsius (there may be some variation here- all we were able to determine is that it cannot grow at 25 degrees Celsius and cannot grow at 37 degrees Celsius).

To test this further, we placed the streak plate that went from the 30 degree incubator to the 25 degree incubator and placed in back in the 30 degree incubator to see what would happen. We believed that the red pigment would return, because we still had some sort of hope that this was the same type of bacteria.

                   

We were also given a agar slant test tube with a culture of (different) red bacteria and placed in the the 25 degree incubator and labeled it "S". 

Later, we took a gram stain smear of the bacterium slide from last class.

The purpose of this procedure was to determine whether the bacteria examined was gram positive or gram negative.

First, we prepared a fixed smear of bacteria.

Max sterilizing the loop. 

Max putting distilled water into the sterilized loop. 

Max putting the water on the sterilized glass slide. 

Sterilizing the loop once again. 

Obtaining a pure sample of bacteria

Smearing the bacteria on the water droplet on the slide. 

Allowing the sample to air dry. 

Heat-fixing the sample. 

We covered the smear with crystal violet for  twenty seconds.

Rinsing off the crystal violet with an indirect stream of DI water.

Then we covered the smear with Gram's Iodine for one minute.

We rinsed the slide with indirect DI water. 

Next, we rinsed the slide at a 45 degree angle with 95% ethanol drop by drop until the color stopped running. 

Following that procedure, we rinsed the slide again with DI water,

Next we covered the smear with Gram's Safranin for one minute. 

Once again, we rinsed the slide with DI water.

Finally, we blotted the water from the slide with bibulous paper.

Our slide was now ready to be used under the microscope!

We examined this slide under the oil immersion lense.

Placing the oil on the slide.

Focusing the microscope. 

Our gram-stained red pigmented bacteria from the soil around the Ohio River in Steubenville, OH under the oil immersion lens of the microscope. 

(image taken from Techniques in Microbiology: A Student Handbook by John. M. Lammert) 

*We do not own this image*

We determined that the bacteria was gram-negative bacteria. Gram-negative bacteria is in the shape of rods and has a thinner cell wall than Gram-positive bacteria. Gram-negative bacteria has a Peptidoglycan layer sandwiched between two phospholipid bilayers and has a lipopolysaccharide on it's surface which is an endotoxin. Through this we are able to tell that this bacteria is not denatured by boiling, is antigenic, does not form a toxoid, has a low potency, has a low degree of specificity, has no enzymatic activity, and is pyrogenic (fever-causing).