Preparing a Gram Stain to Further Identify Bacteria

Previously, we have given the procedure to make a simple stain, one that is used to identify colony and morphology of a given bacteria . 

Today, we were able to discover the colony and morphology of our unknown because our pure culture had individual colonies in it!   Here our some pictures of the unknown:

The colony was determined as predicted!  The colonies in this picture are obviously circular.  They had a cream tint to them and covered the entire surface.  They were opaque and raised off the agar medium.

Even with the colony and morphology identification, it is still impossible to know the exact identity of our bacteria without doing deeper investigation!  Our next step was to do a Gram Stain to identify the composition of our bacteria's cell wall.

To prepare a Gram Stain:

First, to create our stain, we followed the procedure used to make a simple stain: smearing, drying, and heat-fixing the bacteria onto a slide.  The full procedure for this can be found in our previous blog, "Staining Bacteria: Preparing a Smear and Preparing an Unknown Sample."

The Gram Stain, however, is more complex.  It involves the use of three stains: Crystal Violet, Gram's Iodine, and Safranin.

The initial stain used was Crystal Violet; we covered the fixed smear with this stain for 20 seconds while the slide was suspended over the sink on a slide drying rack.

After this, we rinsed the slide with water and covered the smear with dye again.  However, this time we used Gram's Iodine, keeping the smear covered for 1 minute.  After rinsing a second time, we used 95% enthanol (labeled EtOH above) to decolorize the sample.  We held the slide at a 45 degree angle until the color stopped running off the smear. It is important to rinse the slide with water immediately after the decolorizing process is complete so color doesn't continue to fade. 

After this, the last dye was used: Safranin.  This dye was left on the slide for 1 minute before rinsing for the last time and blotting the slide dry with Bibulous paper.

The results!
The Environmental Sample:

This sample is Gram-negative.  We know this because it took up the Safranin dye.  Because of the small peptidoglycan layer in a Gram-negative bacteria, the purple dyes were unable to stay fixed in the cell wall when the ethanol (decolorizing agent) was applied.  We predict that this bacteria produces endotoxins because the cell walls of Gram-negative bacteria have LPS in them.  This type of bacteria is harmful when it gets inside the body because it activates the immune system of the host.  When this cell is lysed, the LPS causes coagulation and much more.  REMEMBER: this bacteria was found on the bathroom door!  If someone were to touch this door without washing their hands and picked up enough of this bacteria, they could sick!

The Unknown "F":

This sample is Gram-positive.  This stain took up the Crystal Violet and Gram's Iodine stains because of its thick peptidoglycan layer.  The cell wall of this bacteria also has teichoic and lipoteichoic acids in it because these acids are found only in Gram-positive bacteria.  These acids stabilize the cell.

As seen in the two samples above, both of our bacteria are small, cocci bacteria.  This makes sense because as seen in the previous blog, both samples were also non-motile.  Cocci bacteria are generally non-motile.

We're getting closer and closer to identifying our bacteria!  What will they be? Harmful? Contagious?  Stay posted to find out!

The Results: Motility, Colony/Morphology, Strep, and More!

The results are in!

Were our bacteria samples motile?

Our Environmental Sample:
As seen below, there is only cloudiness evident on the length of the pin down this tube.  This proves the bacteria to be non-motile.  The bacteria grew throughout the length of the tube; however, it grew better in the bottom of the tube in anaerobic conditions (in the absence of oxygen).  Because of this growth pattern, we presume that our bacteria is an aerotolerant anaerobe bacteria.  This type of bacteria grows better in the absence of oxygen but can still grow if oxygen is present.

 Sample "F":

This sample, our unknown, is also non-motile because the cloudiness is only evident where the pin was inserted into the agar medium.  However, this bacteria grew better in aerobic conditions because the bacteria flared out on the surface of the agar.  We concluded that this was a facultative anaerobe  because they do not require oxygen for growth but utilize it when it is available.  Clearly, this bacteria utilized the oxygen present at the top of the agar medium because it grew much more rapidly there.

Left: Sample "F"  Right: Environmental Sample

This picture above is another record of our observations.  Looking at the bacteria in front of a light source helps reveal cloudiness.

Another way that we tested for motility was by observing LIVE bacteria!  We did this by using our broth samples that we prepared last class.

First, we swirled the broth tubes by flicking them with our fingers until cloudiness appeared.  After this, we applied Vasoline to the slip cover to later keep it stationed on the depression slide.  

We added a drop of the broth to the cover slip using a sterilized inoculating loop and aseptic technique.  The cover slip was then stuck to the depression slide with the drop hanging over the depression.  This is necessary to see bacteria moving!

 However, to no surprise, no bacteria was moving in our suspended drops of broth.  The only thing observable was the bacteria drifting through the broth, but not moving by its own means -- proving it to be non-motile.

What is the colony/morphology of our unknown?

This is "F":

To identify the colony/morphology of "F" we needed to view it under a microscope.  Here is "F" -- MAGNIFIED!

Sadly, our culture of "F" overgrew and we were unable to locate a single colony; however, above are some pictures of our results.  We started a new culture on a new agar plate for next class to fully be able to idnetify the colony and morphology of our unknown.  However, from what we could examine, "F" is a small cocci bacteria.  It has a flat and ridged appearance and is cream.  However, what is science without a hypothesis?  This misfortune gives us an opportunity to predict the colony and morphology of "F."  We predict that "F" will form entire, circular colonies that are opaque.

DID DR. JOSEPH HAVE STREP?

 Lysing did occur! But does that mean that he has strep?  Luckily for him, NO!  It is hard to see in the picture above, but the green residues reveals an incomplete lysis of the bacteria in the back of his throat.  The bacteria that causes strep throat is known as Streptococcus pyogenes.  In encountering the antibiotics that were present in the agar medium, this bacteria undergoes complete lysis; because complete lysis did not occur, the bacteria that causes strep is absent from the throat culture.

What happened with the T-4 Virus?

Dr. Joseph put the same bacteriophage in both of the agar plates above, but with different bacteria in them.  On the left, there is a patch of clear agar reading "JAP" (professor's initials) that indicates that lysing of bacteria did occur.  However, the second bacteria (on the right) was not affected by the T-4 bacteriophage.  This is because each bacteriophage has its own set of hosts!  Just as only certain viruses affect humans, certain bacteriophages affect certain bacteria!  This proves a point: every virus has a host range!

What will happen next week?  Plenty of surprises!  What will our new culture of "F" reveal?  Will our predictions be correct? Turn on your computer next week and check it out!

Broth Tubes, Streak Plates, Stains and More!

Today was a fun-filled day in lab!  We accomplished so many things, executing both new and old techniques.

First, we made broth tubes of both our environmental sample and our unknown sample.  To do this, we used aseptic technique.  To reiterate, this consists of sterilizing the inoculating loop between each step and sterilizing the tubes containing the broth and the sample.

 

Our bacteria will grow in the broth for next class. We placed both samples  in the proper incubator after marking them with a China Marker. (The unknown at 37 degrees Celsius and the environmental sample at room temperature.)

After this, we made a streak plate of "F," our unknown.  To do this we followed the same procedure found in "Examination of Bacteria and Creating a Pure Culture."  The purpose for this was to create a pure culture of the bacteria to further examine the details of the colony and morphology of the unknown specimen.

This is "F." We placed this sample in the 37 degree incubator as well so it can grow for our next class.

Also, we repeated the procedure to make a simple stain on a microscope slide for both samples (the unknown and the environmental sample).  The charge of the stain is very important!  There are two types of stains used in a simple staining procedure.  The first is Acidic, or anionic, and it is used to bind to proteins with a positive charge.  However, this stain won't work with bacteria because the bacteria's negative charge would repel the stain.  When staining bacteria, it is important to use a basic stain, or cationic stain, with a positive nature so the stain will reveal the mysteries of the bacteria!

We used methylene blue stain on both of the samples.  The procedure for creating a simple stain can be found in our previous blog, "Staining Bacteria: Preparing a Smear and Preparing an Unknown Sample."  However, to reiterate, "heat-fixing" the bacteria is very important.  To receive better results this time, we waited for the smear to dry thoroughly, and heat-fixed it for three quick motions.  We let the stain rest on our sample for one minute, and then we wiped it dry.  This slide reveals our wonderful bacteria!  The results will have to wait! We stored the slides in the drawer until next class.

Now it's time to dive into a new procedure, literally!  This is a new test that we find really exciting. We are now going to test the motility of our bacteria!  To do this, we used a sterilized inoculating needle to capture a small amount of bacteria.

After capturing the small amount of bacteria, we stuck the inoculating needle into a tube of 0.4% agar (this means it is semi-solid).  It was important that we stuck the needle straight down three-quarters of the way and straight back up the same hole.  After retrieving it from the incubator next class, there should be cloudiness.  Cloudiness is a result of bacterial growth.  If the cloudiness is about the whole tube, our bacteria is considered motile!  We did this for both the unknown and environmental sample.

After, we got a throat swab from Dr. Joesph!  He made a culture of his bacteria on an agar plate to grow for next class, but added something very unique to it, TWO ANTIBIOTICS!  The reason he used two, Bactitracin and Penicillin, was to discover if he had strep throat (caused by Streptococcus pyogenes).  He placed them on opposite ends of the agar plate.  Next class, by examining the growth on the plate, we will be able to diagnose our professor.  If the Penicillin kills bacteria, it doesn't necessarily mean that he as Strep.  It only means that he has bacteria in his throat, but that's a good thing, WE ALL DO!  However, if Bactitracin kills bacteria, he does have Strep and should go to the doctor because this antibiotic only kills the bacteria that causes Strep.

Next, we took a new unknown bacteria and made a pure culture of it on an agar plate.  However, to this sample we added a T-4 bacteriophage (a virus that kills bacteria).

We will not know the exact results until retrieve the dish next class. However, as a class, we hypothesized that there will be clear pockets on the plate where the T-4 bacteriophage was added to the agar.  A bacteriophage kills bacteria, so none will be present there.

So many exciting results are awaiting next class! Can our bacteria swim around?  What will our broth samples reveal?  DOES DR. JOSEPH HAVE STREP? Tune in next time!

Discovering our Simple Stains and Retrieving our Samples

In the previous entry we explained the process of creating a simple stain.  It is now time to uncover the mysteries of the bacteria found on both the bathroom door and the spout of the drinking fountain! Today will be the last time we test the sample from the drinking fountain because we were unable to obtain a pure culture with a single colony.  Here are the exciting results of our simple stains!

To view the bacteria, we had to put the compound light microscope to the highest power. A HUGE 400x!  To get enough light to clearly view the specimen, we had to use immersion oil.  Immersion oil prevents light from refracting (or bending) so that the microscope has a higher resolving power.  This resolving power is needed to see the bacteria clearly.

The bacteria found on the drinking fountain:

 Proper identification of this specimen cannot be made because there were two errors in the execution of the procedure.  First, we had too much sample, creating a slide with overlapping bacteria.  Second, and most importantly, we heated the specimen too long, altering the bacteria.  This is another reason we are dropping this sample from further exploration.

The bacteria on the bathroom door handle:

The same two mistakes were made when making this slide; however, the bacteria was identifiable to a greater extent.  It appears to be of the cocci category, and more specifically, in the staphyl (or cluster) arrangement.

After viewing our slides, we removed "F" from the incubator, finding more bacteria!

To prevent overgrowth, we moved "F" into the refrigerator for next class.  We had to create two slants because we have to use one tube for the experiments and retain one tube as a back-up.

The pure culture of the bacteria found on the door handle was also left in the refrigerator for next class for the same reason.

What fate do they await? Come back next week to see!

Staining Bacteria: Preparing a Smear and Preparing an Unknown Sample

After four days in the incubator, our single colony grew rampant. Look at the bacteria we uncovered!

Here are the results of our pure cultures:
First is the bacteria found on the outside door handle of the bathroom.  The picture on the left is the initial sample, and on the right is the pure culture of bacteria. We chose to isolate the yellow bacteria on the original plate; that is why there is a huge color difference between the two samples.

Second, we have the bacteria that was found on the spout of the drinking fountain.  The results of the pure culture can be observed in the right agar plate.  We had to make a second pure culture of the bacteria today because there were no individual colonies (single bacteria living in isolation on the plate) present.  Although at this time we do not know the exact identity of the bacteria, our professor hypothesized that there may be spores evident upon examination of this bacteria sample.

After viewing the bacteria and making a second pure culture for the latter sample, we practiced a smearing technique for the bacteria.

Preparing a Bacterial Smear:
First, we took a clean slide and put a small drop of distilled water onto the center of the slide. This was most easily done with the help of an inoculating loop.

As usual,  our next step was to light the Bunsen burner -- our main source of sterilization throughout the procedure. With the sterilized loop (which we put through the flame until it glowed red), we touched the pure culture briefly and smeared the small sample of bacteria on the slide with the water.

After the bacteria was sufficiently spread on the slide (thinly and evenly), we waited for the sample to air dry. The sample was dry when an opaque area was seen on the slide.

After our samples air-dried, we had to heat-fix the bacteria onto the slide. In order to do this we had to briefly hold the slide in the flame of the Bunsen burner for three quick motions. It is necessary to heat-fix the bacteria to the slide so the bacteria sticks.  This is needed to view the sample on a microscope.

Next, we had to do the actual staining of the slide!  Now the fun begins! Next, we added a drop of 95% Methanol to the dried smear, which was suspended over the sink on a slide rack. We had to be careful to not stain anything other than the slide with the 95% Methanol because it will permanently stain anything that it comes in contact with. We made sure to cover the entire surface of the dried smear with the 95% Methanol. After, we let it air dry for one minute.

 In order to make sure the entire smear was covered, we had to gently shift the slide in a circular motion.

In order to keep staining to a confined area, we placed the staining slide on a drying rack over the sink.

After waiting one minute, we rinsed the 95% Methanol off of the slide with distilled water. Then, to clean off excess water, we dabbed it dry with Bibulous Paper.

Then we put the slide away for further drying and examination next class after clearly marking it with a China marker.

Preparing Our Unknown Sample:

We have been given a task to identify an unknown sample of bacteria. However, before we can do any identifying we must make a pure culture of the bacteria. At this point, all we know is that our Bacteria is labeled F. "Well, F, we are going to get to know each other really well."

We had to create two slants of "F" using the aseptic technique. As a review of this process, we first sterilized the inoculating loop, and then we carefully uncapped "F" and sterilized the top of the tube. After, we carefully used the inoculating loop to get a small sample of "F" to spread in our slants.

We spread the sample of "F" in the two slants so that it could grow for further examination next class.


After our slants were created, we made sure to sterilize the equipment and put our slants away in the proper incubator. "F" required 37degrees Celsius, "oh picky 'F.'"


What new mysteries will "F" bring us? What will our newly stained slides depict? The microscopic world is unpredictable. Nobody will know until next class. Join us!