Introduction & Hypothesis

 

In unit two, we investigated and learned about Bacteria, the Aseptic technique and the Gram Staining technique.  Prior to this lab, a vast amount of information was given about bacteria, the aseptic technique and the Gram Staining technique. First, it is important to describe and explain what exactly bacteria are. Bacteria are prokaryotes that belong to the domain bacteria. Bacteria have different kind morphologies but the main groups are spherical shaped bacteria (Cocci), rod shaped bacteria (Bacilli) and spiral shaped bacteria (Spirillium).  The Aseptic technique and the Gram Staining technique are the most important procedures in microbiology because they help group bacteria and reveal beneficial or harmful bacteria.

The Aseptic technique is the technique first used in our experiment. This technique is used so that other microorganism cannot contaminate the bacteria samples. Considering this, it is extremely important to wear gloves and not to touch too many surfaces so that your hands do not pick up any additional bacteria. The second technique, Gram staining, is used to group the different bacteria.

In the technique of gram staining, there are two types of bacteria that will be found; the gram positive bacteria and the gram negative bacteria. The gram positive bacteria are the bacteria you want to be in your body aiding your body with the nutrition it needs. Gram positive bacteria have a protective cell wall made up of peptidoglycan which helps trap the crystal violet dye in the cytoplasm. Under the microscope, if a bacteria sample has a violet or purple hue to it, it is without a doubt a gram positive bacteria. Gram negative bacteria, however, are the dangerous bacteria that cause viruses and diseases in organisms. Gram negative bacteria unlike the gram positive bacteria have a thin cell wall containing a small layer of lipopolysaccharides and less peptidoglycan. When Gram negative bacteria undergo the Aseptic technique, their cell walls are rinsed of the crystal violet dye from the cytoplasm with alcohol making it appear pink.

In our experiment, four locations at The Bermuda College were chosen to extract bacteria from which was the boy’s bathroom paper towel dispenser, the coffee table in the games room, a ping pong paddle handle and a lecture hall desk. Using the Aseptic technique, we hypothesized that the ping pong paddle would have the most bacteria colonies present compared to the other locations.

 

          Material & Methods

 

• Clothes Peg                              Prepared Cultured Bacteria (E-coli & B-serous)    Inoculating Loop

• 3 x 600mL beakers                 Petri Dish containing Agar                                         Microscope 

• Bunsen Burner                     Bibulous Paper                                                           Deionized water

• Crystal Violet Stain                  Grams iodine solution                                             Ethanol in squirt bottle

• Safranin-O Stain                      Clean Glass Slides                                                    Staining Tray

 

This lab consisted of two parts using the aseptic and gram staining technique. In the first part of the procedure, cultured bacteria were transferred to an agar plate. A Bunsen burner (blue flame) was carefully lit for the sterilization of the inoculating loop to avoid contamination of the bacteria. (Before the experiment is started, ensure the petri dish is labeled on the bottom and lab gear is utilized). The inoculation loop after slowly turning it in the flame (10 secs) was used to take a bacteria sample (inoculating material) which was then transferred in different quadrants on an agar plate to ensure a separation of colonies. The separation of colonies was done by transferring less cultured bacteria into each quadrant of the agar plate. Ensure that the inoculation remains sterilized, the transfer of the bacteria cultures is done gently to avoid tearing the surface of the agar plate and the lid of the agar plate is closed as much as possible. 

 

Gram staining is the second half of the procedure. In this procedure, bacteria were transferred to a clear glass slide where it is stained and evaluated under a microscope. The tables were wiped down with ethanol and the inoculating loop was sterilized over the flame of the Bunsen burn. Label the glass slide to avoid mixing the other glass slide with each other. Held by a clothes peg the clear slide glass was topped with dH20 along with a loop of the bacteria which were then mixed together. The glass slide was then gently hovered over the flame for a short period of time (too long will kill the bacteria). On the drying rack one slide was topped with crystal violet staining solution (60 secs). Any additional staining solution was tipped off and rinsed into 3 beakers filled with water. The same procedure was performed but this time it was stained with Grams Iodine (60 secs). The smear was then stained washed with ethanol to remove any excess dye left on the bacteria. Throughout this process the beakers of water was dumped and refilled for the final staining. The smear was then stained with Safranin-o solution (30 secs) and then tipped to get rid of access solution. After all the staining was complete the slide was gently dried (dab) with bibulous paper. The slide was then viewed under a microscope using the  Oil immersion lens.

 

       Results & Analysis

Over the past weeks, our group focused on Aseptic Transfer techniques to move bacteria from one medium to another with minimal risk. In science, however, there is always a chance that errors will be made due to negligence or mistakes. Four in class transfers were conducted which involved moving E-coli from a chicken broth substance to another broth, E-coli from a chicken broth substance to a petri dish, E-coli from a petri dish to a chicken broth and finally E-coli from a petri dish to another petri dish.

After three days, we returned to the lab to check our results and found that the transfers were all successful with minimal to no unwanted substances. The E-coli in all four transfers were healthy (relatively speaking) and we found our techniques to be successful.

 

 

 

 

 

 

 

 

 

 

 

 

 

Additionally, we did another experiment in which groups travelled throughout The Bermuda College campus and did cotton swab samples on various objects. Our group selected the Lecture hall desk in Hallet Hall, the paper towel dispenser in the boys’ bathroom in Hallet Hall, the ping pong paddle in the games room, and the coffee table located in the games room. After collecting the samples, we allowed the bacteria to culture over the weekend along with the E-coli cultures. Our hypothesis states that the ping pong paddle would have the most diverse and largest amount of bacteria present.

 

Here are the results

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Unfortunately we were wrong, however it altered our perspectives. It was the lecture hall with the second most bacteria but the most diversity. Looking into the results further, another situation was brought to our attention. It appeared that in locations where food was mostly likely eaten more bacteria colonies were present. Additionally, we questioned whether or not we had taken a decent enough sample and fortunately, according to our results, there was enough evidence to produce fair observations. We found some interesting information on the colonies that we cultured. Not all of them were the same and some were extremely baffling because some contained traces of fungi.

 

Here are the Results

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Analyzing the graph, we found that there was hardly any truly noticeable diversity except for the lecture hall desk. The paper towel dispenser, Ping pong paddle and the games room desk only had small circular cultures that varied in color. The lecture hall desk, however, clearly had 3 different species growing on it; a large furry colony that covered the center of the petri dish, an elevated colony that was growing up rather  expansively and similar to the other petri dishes small circular colonies that varied in color.

 

       

 

Discussion

 

In the first part of the lab, the transfer of the bacteria (E.Coli) was successful yielding an exceptional amount of bacteria colonies. However, the aseptic technique performed could have been done more proficiently or attentively which would have resulted made the bacteria colonies easier to analyze.

 

In the second part of the lab, the bacteria were stained and view under the microscope. The staining of the bacteria was successful with no mistakes. However, if additional time was used to evaluate the shapes of the bacteria we would have had a better idea of the correct name for the bacteria structures. Overall, this lab was conducted fairly accurate with little to no mistakes. As a group we encourage other science students or anyone who is curious about the aseptic and gram staining technique and how to use them to find negative and positive bacteria.