Would you prefer smoking, non-smoking, or oxygen free? Lab Day 4

Monday, day 4

Today we started out lab by inoculating a thioglycolate tube with our unknown bacteria.  At the top of the tube we can see that the color is red, and as you look towards the bottom of the tube, the color changes to yellow.  The red color signifies an oxygen rich environment.  The yellow part of the tube signifies an oxygen poor environment.  We did this test to see where our bacteria would grow.  If it grows in both environments, then our bacteria is facilitative, but if it only grows on the top or on the bottom then it is an obligate aerobe or obligate anaerobe.

Our next test was completed to determine if our bacteria was motile or not.  This was comprised of simply inoculating a gelatinous tube with our bacteria.  We used our pointed rod and stuck it down to the bottom of the tube.  Tomorrow we will determine if our bacteria is motile or not.  If it is motile, then the bacteria will move side-to-side and form a cloudy region around it, but if it is not motile, then not much will happen.  

The next test we performed was done to determine if our sample was an anaerobe or aerobe.  We did this by first taking a clear bin and putting all of our bacteria in it.  Next, we added a blue colored test strip that helped us indicate if oxygen was present or not.  If the strip was blue, it meant that oxygen was present.  If the strip was white, this meant that the strip was in an oxygen poor environment.  This is important because we were testing to see if our bacteria will grow in oxygen poor environments, so knowing if oxygen is present is of great importance.   After we added the strip and bacteria, we placed in the container three bags that looked and felt like hand warmers.  These bags deplete all of the oxygen in the container.  

In the last part of the lab we started by inoculating a petri dish with our unknown bacteria.  Then we added a virus to the bacteria.  In theory, when we apply the virus to our bacteria, the viral bacteriophages will inject their DNA into the bacterial cell, multiply inside the cell, and cause the lytic cycle.  This should destroy some of our bacteria, at least in the area where we applied the virus.  We’ll see tomorrow how much damage is done.

Water Colors in Microbiology - Lab day 3

Day 3 of MMBL (a hip and savvy anachronism for Medical Microbiology Lab)

Today, we performed a few bacterial stains.  We kicked it off with a good old fashioned gram stain, to determine if our unknown bacteria was gram positive or gram negative (or gram variable).  On a fixed smear of bacteria, we poured crystal violet stain for about 20 seconds, then rinsed it off.  Next, we used a decolorizing agent (95% ethanol) to rinse the color off.  Finally, we covered it with safranin stain for about a minute before rinsing that off as well, and drying the slide with bibulous paper.  

If our sample was gram positive, it would have the crystal violet stain (a bluish purple), but if if was gram negative, it would have the safranin stain (a bright red).  This is because gram positive bacteria absorb the first stain, and it is not removed by the decolorizing agent.  Thus, the end result is that it keeps the first stain.  With gram negative bacteria, however, you can easily wash the first stain out with the decolorizing agent, allowing it to absorb the second stain (in this case, the safranin).  

Examining ours under a microscope, we determined that our sample was gram negative, because it had absorbed the safranin stain.

The next stain we did was a capsule stain.  We prepared a smear of bacteria in nigrosin stain (black), making sure we spread it over most of the slide.  After that, we again added the safranin stain, then rinsed it off.  We looked at it under a microscope, searching for capsules, which should be left unstained.  The nigrosin stains the bacteria, then the safranin stains the background.  Capsules don’t take any stain, so they should be left standing out.  Unfortunately, we were not able to see very clear capsules, but apparently this is not unusual.

We also did an endospore stain.  The point of this stain is to see if you have any endospores.  Over boiling water, we stained our bacterial smear with malachite green.  If there are endospores, then the green will stain them (so long as there’s heat).  After that, we rinsed off any excess stain, then again added safranin stain for about 90 seconds, before rinsing that off as well.  Using the oil immersion lens on the microscope, we looked for endospores.  We did not see any.  If they had been there, then they would be stained with malachite green, but all we observed was the safranin stain.

The last stain we did was an acid-fast stain, to determine if our bacteria was acid-fast, or non acid-fast based on the lipid content in their cell walls.  We used the Ziehl-Neelsen method (over hot water).  First, we put a paper over the slide, and saturated it with Ziehl-Neelson carbolfuchsin stain for about 3-5 minutes.  Next we used a decolorizing agent (acid-alcohol) to remove the color.  Finally, we coverd the smear with methylene blue for about 2 minutes, then rinsed off the excess.  We did this for two samples: our unknown, and another that Dr. Pathakamuri gave us.  Examining them under the microscope, we discovered that ours was not acid-fast, while the new sample was.

MICRO - DAY 1

We kicked off the microbiology lab with a few notes on lab safety.  Generally, wear a lab coat, wash your hands, wipe your station down with 2% lysol, wash your hands, throw out hazardous material in the designated baggie, wash your hands, and generally try to avoid contracting a deadly strain of bacterial disease.

After that, we were shown how to use proper Aseptic technique, courtesy of Joseph Lister: 

(....  if those sideburns were any bigger, they'd need their own address)

Once these pleasantries were dispensed with, we got right down to the nitty gritty.  Dr. Joseph had us all begin by collecting environmental samples, in order to culture some bacteria.  Some of the other students got their samples from things like a cell phone or a door knob.  Knowing the "impeccable quality" of Steubenville's own water, we decided to take a trip down to the beautiful shores of the Steubenville River.  Since we only had ten minutes to collect the sample, we jumped into the car.  After a bumpy ride down to the Marina, we used a cotton swab to get our sample.  Using the correct technique, we rubbed the sample across the petri dish several times, rotating it as we did so.

Putting a lid on our sample, we stuck it in an incubator, which kept the sample at about 24 degrees Celsius (Stay tuned for the results).

In addition to collecting our environmental sample, we were given a specific (albeit unknown) bacteria to cultivate.  Adhering to Lister's Aseptic technique, we used a flame to disinfect our loop, then we took a dab of the unknown bacteria and inoculated one broth tube, two slants, and a petri dish (smearing it on all four sides of the petri dish in an attempt to create a pure colony).  The inoculated broth tubes, the slants, and the petri dish went into the 24 degree Celsius incubator.

<= Inoculation at work!!!  Spreadin' them germs around.

... And burning them germs =>

  Aseptic technique, ladies and      

  gentlemen.

Lab, Day 2: Microbiology's Beginning To Grow On Me

Day 2 of microbiology lab, we started off by pulling our river sample out of the incubator to see if any bacteria had been cultivated.

Our Hypothesis, considering Steubenville water's reputation, was not too far off.  We expected to see considerable bacteria growth, and we did.  Our sample had some of the most growth in the class.  We examined it in detail under a microscope to determine its characteristics.  From what we could see, the colonies were circular, with a cream/tan pigmentation, the edges were smooth (entire), and the elevation of the colony was slightly raised.

Next, we looked to our broth culture to see if there was any bacterial growth, and what kind.  At first, it appeared as though nothing had cultivated in the broth, but when we flicked the bottom of it, a flurry of particles flared up.  This meant, obviously, that they had been cultivated as a sediment, and were resting at the bottom of the tube.  

Finally, we examined the slant cultures.  Significant bacterial growth had occurred in the slants, and they had spread out from the original source of inoculation.  In both slants, the bacteria seemed to develop pointed edges, indicating an echinulate sample.

Progressing to the next experiment in the lab, we prepared a hanging-drop slide in order to better observe the motility of our bacteria samples.  The hanging-drop slide was made by putting bits of vaseline on the corners of a clear coverslip, then using a flamed inoculating loop, we transferred a sample of our broth culture onto the center of the coverslip.  We then put the coverslip onto a depression slide.  The vaseline helped it to stick, but kept it from squishing the droplet sample, allowing it to remain as a sort of "bubble."  Looking at it under a microscope, we discovered an incredible amount of movement for such a small drop of the sample.  Countless bacteria wriggled around, several trying to "swim" through the others.
















In the last section of the lab, we prepared a bacterial smear and performed a simple stain.  Smearing a sample of our agar plate culture onto a slide, we allowed it to air dry, then passed it quickly through the flame a few times to heat fix it.  We then covered it with crystal violet stain for about 30 seconds before rinsing it off.  Finally, we blotted any water from the slide using bibulous paper, and examined it under a microscope.  This time, however, we used a blue light filter on the microscope to show better contrast.  Comparatively, we got a much clearer image.