Simple Staining 1. Introduction: Most microorganisms, including bacteria, appear colorless when viewed through a light microscope. Therefore, in order for them to be successfully observed, bacteria need to be prepared. One of the techniques for viewing bacteria is to stain the specimen. The stain is a dye that will color the microorganism and will emphasize certain structures. However, before they are stained, the bacteria will need to be fixed to the microscope slide. Fixing the bacteria will, both, kill the microorganism and attach them to the slide. In order for a specimen to be fixed, a thin film of material containing the microorganism is spread over the surface of the slide, called a smear. (Tortora, Funke, Case, 2013). In this experiment, a stain mount of Escherichia coli , Staphylococcus aureus , and Bacillus subtilis was prepared by heat-fixing the smear of bacteria on a slide and staining it with crystal violet. The objective was to successfully stain and view each specimen under 1000x magnification. Materials: ● Slant of Escherichia coli , ● Microscope slides ● Inoculating loop ● Crystal violet (stain) ● Bunsen burner ● Microscope Staphylococcus aureus , and Bacillus subtilis 2. Procedure: ● A mount of each bacteria Escherichia coli , Staphylococcus aureus , and Bacillus subtilis was made by: ● First, the loop was sterilized in the flame and a drop of water was placed on the microscope slide. ● The loop was then resterilized and a bit of growth from the agar slant of brother was transferred to the drop of water and spread over an area of about one square inch. ● The smear was then left to air dry. ● Once the smear was dry, the bacteria was fixed to the slide by passing it through the flame. ● Crystal violet was then used to cover the smear for about 15 seconds and rinsed with water. ● The smear was left to air dry. ● Once all 3 smears were dry, they were examined under the microscope. 3. Results: Image 1: Bacillus subtilis 1000x magnification Image 2: Staphylococcus aureus Image 3: Escherichia coli 1000x magnification 1000x magnification 4. Discussion: In this experiment, 3 microorganisms were stained using crystal violet for 15 seconds and were viewed under a light microscope using oil immersion. Bacillus subtilis (image 1) was observed to be rod-shaped and some cells appear to have been forming chains. Staphylococcus aureus (image 2) appeared very populated and dense. The cells are clustered together which suggests that too much bacteria was spread over the slide which made it hard to see as many single cells such as in image 1. When viewing Staphylococcus aureus under the microscope, it was also difficult to determine the shape of the specimen. Because of this, the shape of Staphylococcus aureus was researched in order to know how it should have appeared. The cells of Staphylococcus aureus are rounded and form in grape-like clusters. (Mandal 2019). In image 2, the cells do look clustered but they are too close together to have been observed to be rounded. In order to have improved the results and observation of Staphylococcus aureus , during preparation less growth from the agar slant should have been picked up and smeared on the microscope slide. The lesser amount of growth would have made it easier to view single cells and determine the shape of the bacteria. Lastly, Escherichia coli (image 3), much like Bacillus subtilis was observed to be rod shaped as well. 5. Conclusion: A simple stain is used to highlight the whole microorganism so that the cellular shapes and basic structures are more visible. The microorganism is heat-fixed to kill the bacteria and attach them to the slide. Crystal violet was used as a simple stain on Escherichia coli , Staphylococcus aureus , and Bacillus subtilis. These microorganisms were observed under a microscope.