IntroductionThe discovery and study of bacteria are on the forefront in the science community for multiple reasons. Knowing and understanding different types of bacteria leads to the ability to be able to prevent or treat the spreading of these microorganisms that could be the causation of minor to serve illnesses. One of the most commonly used techniques in determining and differentiating bacteria is called differential staining. Bacteria are transparent and are unable to be seen unless the sample is stained and viewed with the aid of a microscope.  Differential staining, better known as gram-staining, is a technique that is used in the identification of determining if the bacteria is one that contains a positive or negatively charged characteristic. This is done by staining the sample so that the bacteria can be seen through the aid of a microscope (MedNet). This staining method is used to determine the thickness of the peptidoglycan layer that covers the cell wall of the bacteria. With the cell wall being stained those that contain a thick layer of peptidoglycan are classified as being gram-positive and will stain a bluish/purple through the use of crystal violet. Those bacteria that contain a thin layer of peptidoglycan are identified as gram-negative and will stain reddish/pink through the use of safranin. (Micro). Gram staining is the most cost-effective way to obtain the initial information about the bacteria in a timely manner and simple to accomplish. This technique is done daily in hospitals, doctors offices, and laboratories across the world by obtaining samples from patients or objects that have been affected, and leads to the best treatment of the bacteria. The steps for gram staining are as follows: crystal violet, iodine, decolorizer, and then safranin in that order. The purpose of this lab is to understand and know the steps of gram staining which will lead to being able to determine the characteristics of the bacteria. The bacteria used in this experiment were Escherichia coli, Staphylococcus saprophyticus, and Bacillus subtilis all which are bacteria that are found commonly in the human body. It is hypothesized that through gram staining, one will be able to categorize bacteria as either gram-positive or gram-negative and through the aid of a light microscope will be able to identify the shape of each sample. Materials / Methods•    Crystal Violet – Primary stain•    Iodine•    Ethanol (Decolorizer)•    Safranin – Secondary stain•    Water•    Microscope and slides•    Immersion oil•    Wire loop•    Bunsen burner •    Escherichia coli•    Staphylococcus saprophyticus•    Bacillus subtilisAt the beginning of this experiment, all materials were gathered and countertops were sterilized to prevent any contamination of the bacteria. Next, the Bunsen burner was ignited to sterilize the equipment and also created an aseptic space to reduce the chance of contamination. Utilizing the wire loop, the end of the loop was placed into the flame of the burner and allowed to cool before exposing it to the bacteria samples that will be tested. Placing the hot wire into the bacteria sample before cooling would kill off all the bacteria that had been growing in the sample. It was decided to gram stain all of the bacteria samples before observing the slides under the microscope. After labeling each slide as to which bacteria was on the slide, the process of gram stain was started.Using the sterile area provided by the flame, the test tube containing the Escherichia coli was brought to the flame and the opening of the tube was sterilized by the flame for a few seconds. Next, the loop wire was placed into the tube and swirled around in the sample. Keeping the wire loop within the sterile area provided by the flame, the slide labeled Escherichia coli was brought within the sterile area and the wire loop was rubbed in a small section of the slide. The slide was then placed to the side to allow the bacteria sample to dry. Once the slide was dry, the slide was passed over the flame to adhere the sample to the slide and placed on a stand that allowed for easy applying of the stains. First, crystal violet, known as the primary stain, was poured generously over the slide and left to set for one minute. Once the crystal violet was allowed to set for the one-minute timeframe, a gentle stream of water was washed over the slide for ten seconds to remove the crystal violet that did not adhere to the bacteria. Next iodine was applied to the slide and allowed to set for one minute. The iodine acts as a mordant for the crystal violet that had been used to stain the bacteria the bluish/purple color. This step is to prepare the bacteria for the decolorizer, which would remove the crystal violet from the gram-positive bacteria if it was treated with the iodine. Once again, water was used to wash away the excess iodine for another ten seconds. Next the decolorizer, in this experiment ethanol, was applied but only allowed to stay on the slide for ten seconds. This is because the decolorizer is not only strong enough to remove the crystal violet from the gram-negative but also the gram-positive if exposed to the sample for too long. The slide is once again rinsed gently with water for ten seconds. At this point, all of the gram-positive bacteria were stained the bluish/purple, while the gram-negative bacteria were back to its original translucent state. Lastly, safranin was applied and left for one minute. Safranin was used to adhere to the gram-negative bacteria and will appear reddish/pink. The slide was rinsed one last time with water for ten seconds and then patted dry. These steps were repeated for both Staphylococcus saprophyticus and Bacillus subtilis.Results    At this point, the slides were ready to be viewed through the microscope. Bacillus subtilis was the first slide to be placed under the microscope. After focusing the image at 40x total magnification, the objective lens was increased to a total magnification of 100x and was focused only using the fine coarse adjustment knob. When the microscope was increased to 400x total magnification, it was determined that the 40x objective lens was not operating correctly. This was concluded because the image went black and nothing could be seen through the lens. Immersion oil, which is always needed when viewing a specimen at a total magnification of 1000x, was applied to the slide. The immersion oil was used because when a beam of light passes through one material index and through another, the light is refracted at a different angle (OIL). The oil lessened the refraction of the light and funneled the rays light in the direction that was needed. After numerous attempts of trying to view the slide and having to start over with the steps of adjusting the microscope, a clear image of Bacillus subtilis was never achieved.  Due to time constraints, no other slides were able to be viewed.Conclusion    Gram staining is a frequently used technique that has been used for a long time and is very effective in identifying the charge of the bacteria, makes it possible to view microorganisms, and has led to better understanding of bacteria. Gram staining provides many advantages such as quick results, cost-effective, provides directions with the proper treatment of those infected (CONNECT). With these advantages come disadvantages. Gram staining is considered easy to perform, but it also can lead to false readings of results due to the preciseness that is needed with how long a staining agent can be exposed to the bacteria. This is a technique that takes practice and even the most trained individual can very easily make a mistake in the process of staining. Overall, it is concluded that gram stain is an effective way to identify and group bacteria as gram positive or negative. Many times, individuals wait too long to seek medical attention, so it is important for healthcare professionals to be able to quickly identify the bacteria and know how to properly treat it. Through proper practice and training on gram staining, this method will continue to play an important role in providing quick preliminary information on known and unknown bacteria.


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