Lab Day 8 - Medical Experimentation for the lot of you!

Today, we went poking and probing at each others’ faces.  All in the name of Science, of course.  In order to test for Streptococcus pyogenes, we took a throat swab. 

Brushing the uvula with a sterile cotton swab, and careful to avoid the tongue (pushing it down with a tongue depressor), Peter took a swab of my throat.  Well, ultimately Dr. Pathakamuri took the sample, because I have a stubborn tongue. 
Regardless, we ended up with a throat swab, which we inoculated a blood agar with it.

I was happy to return the favor to Pete by taking a nasal nares swab.  I dipped the swab in saline solution first, however, because a dry swab wouldn’t work as well.  We then inoculated a mannitol salt plate with the swab in order to test for MRSA.

We finished off this medical experimentation by collecting a urine sample (which I was happy to provide).  Using a 50 microliter pipet, we inoculated an EMB plate with it.  All three of our samples went into the incubator.

Next, we performed a Kirby-Bauer test.  We did this by inoculating a nutrient agar with our unknown bacteria, and adding various antibiotics to it.  We tested Penicillin, Tetracycline, Neomycin, Cinnamon Oil, Lemon Oil, and Augmentin.  For Penicillin, Tetracycline, and Neomycin, we used sterilized forceps to place samples of these antibiotics onto our nutrient agar.  For Cinnamon Oil, Lemon Oil, and Augmentin, we placed three small paper disks onto our nutrient agar, and added 2.0 micrometers of each onto separate papers.

Lab Day 7

Thursday, Day 7

In lab today, we looked at the results of yesterday’s inoculations.  Yesterday, we inoculated tubes of tryptophan, urea, citrate, nitrate, and MR-VP, to see which amino acids our bacteria is capable of breaking down.

Starting with the tryptophan degradation test, we added 15 drops of Kovac’s reagent.  This test was to see if our bacteria had the ability to split tryptophan into indole and pyruvic acid.  If the Kovac’s reagent causes it to rapidly turn red, then it’s a positive test.  Ours showed no red, so it was a negative test.  Therefore, we know that our bacteria does not break down tryptophan.

Next we looked to our Urea Hydrolysis test, to see if our bacteria had urease, which breaks down urea.  If the urea was broken down, it would have turned bright pink, because the urea would have broken down into carbon dioxide and ammonia, causing it to turn alkaline.  This pH change is detected with phenol red, which turns bight pink in alkaline medium.  Ours was a negative test, with no color change.  Therefore, we know that our bacteria does not break down urea.

The results of the Citric Acid test were easy to observe.  If our bacteria had the enzyme citrate permease, which breaks down citrate, then the byproduct of the breakdown reaction would be CO2, which combines with NA+ in the medium to form NaCO3, an alkaline compound.  The pH indicator, bromothymol blue, turns blue in alkaline pH.  Therefore, all we need to look for is a deep blue in our Citric Acid tube.  We observed plenty of blue, so ours was positive.  Therefore, we know that our bacteria breaks down Citrate.

Next, we looked at our Nitrate Reduction test, to determine if our bacterium is able to reduce nitrate ions to either nitrite or nitrogen gas.  This reaction is anaerobic respiration, because Nitrate is accepting the electrons instead of Oxygen.  It needs Nitrate reductase enzyme to convert Nitrate to Nitrite.  So, essentially, if we’re looking to see if our bacteria has Nitrate reductase, then we’re looking for the end product, which is Nitrite.  To to this, we use two reagents: Sulfanilic acid (reagent A), and dimethyl-alpha-naphthylamine (reagent B).  If Nitrite is present, the medium will turn pink or red.  Ours was immediately positive, because it turned red.  Therefore, we know that our bacteria breaks down Nitrate.

Finally, we did our MR-VP test to see if our bacteria is able to ferment glucose via mixed acid fermentation.  The products of this fermentation are acidic, so the addition of methyl red (pH indicator) will turn the solution red.  Our methyl red test was negative, indicating that our bacteria did not ferment glucose.  

However, the results of our Voges-Proskauer (VP) test were positive, indicating that it did ferment glucose.  We added 15 drops of Barritt’s reagent A (alpha-naphthol) and 5 drops of Barritt’s reagent B (KOH).  After about five minutes, it began to turn red.  This means that our bacteria fermented the glucose and produced acid, but the acid was further converted to 2,3-butanediol.  The methyl red was initially misleading because it didn’t find any acid, because the acid had all been converted to 2,3-butanediol.  The VP, however, found the 2,3-butanediol and turned the                                                                                  medium red.  Therefore,        

                                                                           we know that our bacteria          

                          MR                                            ferments glucose.                                                   VP

Also, because we had forgotten to do it yesterday, we took a look at our now complete litmus test.  There was definite curd formation, and there was no color change.  The color didn’t change because the pH didn’t change, and the soft curd formed when cadein was altered by the bacterial enzyme renin.

Catering to a picky bacterium. - Lab Day 6

MMBL day 6.

Today, we started by checking the results of our agar plates inoculations, to see what our bacteria eats.  First, we checked the results of our starch hydrolysis test.  Starch and iodine react to make a bluish substance.  So, if the bacteria grows in starch, it will have digested the starch in our agar plate.  So, around the bacteria, there should be no starch left, and therefore nothing for the iodine to react with near the bacteria.  To determine this, we put iodine over our starch sample.  We observed no clear zones around our bacteria.  Rather, the iodine reacted with everything on the plate (our side, anyway), turning it a dark blue.  Therefore, our bacteria must not feed off of starch.

Next, we tried the casein hydrolysis test.  Similar to the starch, some bacteria can secrete enzymes that hydrolyze casein.  So, if our bacteria feeds off of casein, there will be a clear zone around the bacteria.  Once again, our bacteria showed no such clear zone.  Obviously not tempted by casein.  

We then looked to the tributyrin agar plate.  If our bacteria was partial to lipids, then it would secrete lipase and digest it, similar to the last two.  If the lipase breaks down the lipid, there will be a clear zone.  Just like the last two, ours showed no clear zone.  We have ourselves a picky little bacterium.  Three down, one more to go for the agar plates.  

For the DNA agar plate, some bacteria will secrete DNase enzyme that will hydrolyze many of the linkages between nucleotides in the DNA, leaving small DNA fragments.  To us, this should look like a clear zone around our bacteria.  Once again…  nada.  Nothing.  Negative.  

Perhaps the Gelatin?  If our bacteria hydrolyzes gelatin, then it will be liquid in our tube.  Not that we had our hopes up at this point, but this test proved negative as well.  Just to make sure, however, we decided to incubate it longer.

So…  we have failed to satisfy our bacterium with our limited menu.  Perhaps it likes Litmus milk, but we won’t know until tomorrow because we forgot to do that sample (I know, I know…  but give us a break!  There’s a big long list of similar looking tubes and agar plates to inoculate!  It seems a reasonable mistake).  So, we inoculated the Litmus milk and stuck it in the incubator.

Well, perhaps we’ll have a little more luck with the fermentation.  If our bacteria does ferment, it will produce an acid and gases.  So, we looked for growth of the bacteria, for a change in color (to yellow), and for gas bubbles.  

We tested positive for sucrose, maltose, lactose, and glucose.  So, ours is picky when it comes to food, but doesn’t mess around when it comes to fermentation.  

We then looked at the triple sugar test.  This slant contained lactose and sucrose at 1% concentration, but glucose at only 0.1%.  If the butt of the tube is yellow, then we’re positive for sucrose or lactose.  Our entire tube was yellow, so we know it fermented either sucrose or lactose.

After that, we looked at the petri dish with the virus.  Only the area where we put the virus (a little peace sign on ours) was left clear.  Everywhere else on the dish, the bacteria had grown.  When we applied the virus to our bacteria, the bacteriophages from the virus injected their DNA into the nearby bacteria, multiplied inside the cell, and caused the lytic cycle, destroying the bacteria around the virus inoculation.  

Also, because we had forgotten our Thioglycolate test two days ago, we looked at the results of the one we inoculated yesterday.  There was growth throughout the tube, showing clearly that our bacteria was facultative.  

The last result we looked at was our GasPak Anaerobic System.  Unfortunately, for the second time, the indicator strip remained blue.  So, there must have been another leak.  But, we did notice that our bacteria grew a lot, while others did not.  This is further evidence for our bacteria being facultative, because there was neither a lack of oxygen nor an abundance of it in the locked system.  

In preparation for tomorrow, we inoculated Tryptophan, Urea, Citrate, Nitrate, and MR-VP, to see which amino acids our bacteria is capable of breaking down.

Lab Day 5

Teusday, day 5

Today we did a heap of inoculation.  We were curious to find out what exactly our mystery bacteria liked to snack on.  What sort of things would it eat?  (specifically, what enzymes did it have that would allow it to break down certain substances).  So, how does one solve these sorts of questions in a microbiology class?  By inoculating everything in sight, of course. 

We tested our bacteria’s appetite with all the macromolecules.  We inoculated a nutrient gelatin tube (testing for protein hydrolysis), a starch agar plate, a skim milk agar plate (testing for Casein Hydrolysis), a tributyrin agar plate (testing for triglyceride), and a DNA agar plate (testing for nucleic acids).  We also inoculated tubes filled with various sugars: Sucrose, maltose, lactose, and glucose.

  

Essentially, we set up an all-you-can-eat buffet for our lucky bacteria.  By tomorrow, we should know a little better what exactly it feeds off of, which will help immensely in identifying it.

We did, however, do a catalase test on our bacteria, to detect the presence of catalase, an enzyme that degrades hydrogen peroxide.  Hydrogen peroxide is a reactive intermediate that forms during aerobic respiration.  So, in theory, only anaerobic bacteria will not have it.  We knew perfectly well that ours was not anaerobic, seeing as it had grown in the presence of oxygen this entire time, but we tested it anyway to see the reaction.  We dropped a dilute solution of hydrogen peroxide onto our bacteria.  The results were amusing to watch.  The catalase in our bacteria quickly broke down the H2O2 into water and O2, creating rapid formation of O2 bubbles.  Ours must have had a considerable amount of catalase.

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.