Capsule Stain

Some bacteria produce a capsule or a slime layer for protection. Bacteria that have capules or slime layers make themselves very difficult to attack and kill, or scrape off of surfaces. After preparing a capsule stain, if the bacteria have a capusule, there would be a clear area that does not pick up the stain around the bacteria. If the bacteria does not have a capsule, a clear area will not be present.

Results: No Capsule




Endospore Stain

Ever wish in stressful moments you could just curl up in a shell and weather it out? Well if you're a cell with an endospore you can! An endospore is a hard capsule that the cell shrivels up into when there is stress in the environment that makes it's habitat unlivable, such as a lack of nutrients, extreme temperatures, or lack of water. A cell can remain in this state for an indefinite time, and when favorable conditions return...POP! out comes the cell from its vegetative state and is ready to start living like nothing ever happened.  Our bacteri does have endospores and this is fairly characteristic of gram positive microbes.  The way we can tell is that there are little green spots inside which are the endospores.

Bacteria with Endospores

We lost our picture of bacteria with endospores. The above picture is a picture of Bacillis subtilis with endospores from the internet.

Acid Fast Stain

Acid-fast bacteria are gram positive and have large amounts of glycolipids, especially mycolic acids. This makes their membranes difficult to penetrate and also resistant to chemicals.

We found our Unknown E. bacteria was acid-fast positive because it turned red at the end of the staining procedure.




Acid-Fast Stain Results

 

Motility Test

Motility in a bacteria depends on the presence of flagella. We used two different techniques to check for motility in our bacteria. One was instering a straight inoculating wire into an agar tube and checking for cloudiness the next day. If the tube is cloud it means the bacteria have spread around the tube and are therefore motile. If the tube is clear, then the bacteria where not able to leave the original insertion streak and are thus not motile.

Results:
Unknown: Motile
Environmental #1 (Lauren) Motile
Encironmental #2 (Kelly): Not Motile

The second mothod involed viewing the bateria under a slide immersed in water to physically see if they were swimming or not. This is done by placing a drop of liquid agar inoculated with bateria a few days before on a cover slip. Dot the corners of the cover slip with vasaline and place a welled slide on top of the cover slip. Then slide is flipped over and this leaves us with a drop of water suspended from the cover slip and not touching anything else so the bateria are free floating in the drop of water and can be observed.

Results:
Only Environmental #1 was motile and can be seen swiming around in this video link.

These little guys are fast!

Gram Stain

Making a Gram Stain shows us if the bacteria cell wall is negative or positive which indicates the structure of the cell wall. Gram positive cell walls are thicker and have a bigger layer of peptidoglycan and teichoic acid and lopoteichoic acid molecules while gram negative cell walls are thinner due to a smaller layer of peptidoglycans, but have a complex layer of lipd macromoecules. Medically it is important to know if the cell wall of a bacteria is possitive or negative because gram negative bacteria are usually more resistant to antibiotics due to thier cell wall structure.

Two dies are used in the gram stains, Crystal Violet and Gram's Iodine. The Crystal Violet is added first, then rinsed with ethanol and then stained again with Gram's Iodine. The concept behind this is that because a gram positive cell wall is larger it will pick up the crystal violet stain while gram negative's cell wall is too small to hold the die. When we rinse the slides with ethanol, if the stain did not absorb into the cell wall it will be rinsed off allowing the next stain, gram's iodine to stain it. If the bacteria is gram positive it will already be fixed with the red crystal violet and will not have room to pick up the purple gram's iodine and will remain red. The gram negative bacterial will stain purple because they did not have room in their cell wall to pick up the crystal violet stain and it gets washed off.

Results:

Unknown bateria is Gram Positive.

Environmental #1(Lauren) is Gram Negative.

Lauren's Environmental Gram - Stain

Environmental Sample #2: Gram positive

Simple Stain

Now that we have a pure culture, we want to see what this bacteria looks like up close. To do this we need to stain the bacteria so they can be viewed under a microscope to see their structure and shape. A drop of water is placed on a slide, bacteria added and swirled in the water, fixed by letting the water air dry and running it through a flame. Then Gram’s Safranin (Red) stain and/or Crystal Violet (Purple) stain is applied, let dry, and rinsed.

Crystal Violet and Gram's Safranin Stains

Environmental Samples under low magnification:

Lauren's Env. Sample stained with Crystal Violet

                                           Kelley's Env. Sample. (Upper image under low power and lower image under oil immersion)

In order to manage a close up of the bacteria, we need to look at them using immersion oil. This oil has the same refractory index as the glass slide, and when it makes contact with the microscope lens, it prevents the light rays from bouncing or refracting out of the lens, giving a much sharper image.

Applying imersion oil to slide

Environmental Samples under high magnification using imersion oil:

Lauren's Env. Sample stained with Gram's Safrinin under high magnification using imersion oil

Taking a Pure Culture from the Environmental Samples

The object of this lab was to issolate just one type of microorganism from our environmental sample. Most of the students found many different types of bacteria or mold growing in the Petri dish. Env. Sample #1 was one of the few that had only one or two types of bacteria growing, while Env. Sample #2 had many different microbes growing including mold.  Using aseptic technique we transferred only one strain of the bacteria from our first culture to a second dish to make a pure culture.

A pure culture is made by selecting a single colony that is not touching any other colonies with an inoculating loop and using aseptic technique, transfer the bacteria to a new sterilized Petri dish.  The idea is that it only takes one bacteria to make a culture, and if the colony (the yellowish dots seen in the picture) are not touching any of the other dots, it was made from only one bacteria, and thus it contains only one type of bacteria, or a “pure culture”.

Enviromental Sample #1:
From Lauren's Env.Sample #1 there were a few larger yellow colonies of bacteria that were chosen to become the pure sample bacteria that can be seen in the picture bellow.

Environmental Sample #1 with Large Yellow colony that was selected for the Pure Culture

The colony Lauren chose was very close to all the other bacterial colonies, and therefore very difficult to obtain without being contaminated with the whitish bacteria. As a result I did not end up with a pure culture and had to redo the procedure. This is normal and it often takes many attempts before a pure culture can be obtained. If the culture is given too much time to grow the colonies can grow into one another, making it impossible to obtain a pure culture. In order to ensure a single colony of my yellow bacteria I only touched the inoculating loop to the colony to only pick up a few bacteria, then spread them in wide streaks across a new Petri dish, and only allowed the bacteria to grow in the incubator for a day. The result was much smaller colonies that were not touching. From this I was able to make a pure culture. Bellow is the final yellow Environmental Pure Culture Bacteria sample.

Env. Sample #1 Pure Culture

Env. Sample #1 Pure Culture. In this view you can see how this species forms raised colonies and has a shiny appearance.

Environmental Sample #2:
Kelley's Environmental Sample:

The colony I chose was one of the smallest colonies on my petri dish. It was the only colony of its kind growing on the agar plate and I chose it because of its unique rose color.

The above picture of is of my original environmental swab culture. As you can see, there are many different kinds of bacteria and molds that grew.

Above is my original petri dish (on left) next to my pure culture that I grew from the original (on right)

Above is a closer look at my pure culture. Notice the rose color. It is hard to tell from the picture, but the bacteria is shiny and the colonies are circular. The colony margins are entire and the elevation is convex.

Collecting an Environmental Sample of Microbes

Our first assignment was to collect a microbe from anywhere in our school environment. The purpose of this is to see that microbes live everywhere all around us in our daily lives. Lauren collected Environmental Sample #1 from the woman's room soap dispenser handle, and Kelly collected Environmental Sample #2 from a bicycle seat outside of Cosmos & Damian Hall.  Gathering a sample of microbes from any given surface is done by soaking a sterilized swab in liquid agar broth, swabing the area to be tested, and then streak or wipe the swab all over an agar plate in every direction. The plates are then put in a room temperature incubator for 24 hours to see if anything will grow.

The agar plates went from looking like this...

To an explosion of microbial growth...

Enviromental Sample #1:

Environmental Sample #1

Enviromental Sample #1 Close-up

Enviromental Sample #1 Close-up

Enviromental Sample #2:

Environmental Sample #2

Environmental Sample #2 Close-up

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