Lab Report Simple Staining

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Indian River State College *

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310L

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Biology

Date

Jan 9, 2024

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docx

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Uploaded by joeldube15

Simple Stain Lab Report Joel Dube March 3, 2023 3/24/23 9:00 AM Introduction (3 pts) 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. The exercise we will be performing in lab is a simple stain. 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.

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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. Most microorganisms, including bacteria, appear colorless when viewed under a light microscope. To help aid someone in the examination of microorganisms or bacteria, simple staining is used. Simple staining allows one to observe the morphology and arrangement of the bacteria cells by dying the bacteria. Using the shape of a microbe, the examiner is able to determine the type of microbe being examined. Simple staining falls under the category of basic stains; basic stains are positively charged and are attracted to a negative charge on the surface of most bacterial cells. Basic stains receive their name from the stain that they leave behind which highlights the bacterial cells, the types of stains used in a basic stain will include either methylene blue, crystal violet, or safranin. Simple staining is composed of a solvent and a colored molecule referred to as chromogen. Chromophore is the atom that gives chromogen its color and what allows simple staining to highlight the shape of the microbe being observed. The purpose of this lab will be to complete the simple staining technique and be able to view the microbes under the microscope. For the lab we will begin by placing a small drop of water on a clean slide using a sterilized inoculating loop. Aseptically add bacteria to the water. We will then mix in the bacteria and spread the drop out and then flame the loop to sterilize it. The next steps will require us to air dry the smear and then, while using a slide holder, pass the smear through the upper part of a flame two or three times. The heat fixes the preparation and coagulates cytoplasmic proteins to make the

microbes more observable. After finishing this step, we will allow the slide to cool. The next step we will take is covering the smear with a stain using a staining tray to catch the excess stain. We will then grasp the slide with a slide holder and rinse the slide with water disposing of the excess stain. The final step to preparing the stain will be to gently blot the stain in a tablet of bibulous paper or paper towels. Once this step is completed, we can observe the stain under the microscope using oil immersion. Hypothesis (2 pts) The hypothesis of this lab is that if we use simple staining techniques it is possible to highlight different bacterial species and shapes and observe them using oil immersion. Materials and Methods (2 pt) Bacterial Smear Preparation 1. Place a small drop of water on a clean slide using an inoculating loop. 2. Aseptically add bacteria to the water and mix in the bacteria and spread the drop out. Avoid spattering the emulsion as you mix and flame the loop when done 3. Allow the smear to air dry and if prepared correctly, the smear should be slightly cloudy 4. Using a slide holder, pass the smear through the upper part of a flame two or three times. The heat from the flame heat-fixes the preparation. Avoid overheating the slide as aerosols may be produced. 5. Allow the slide to cool, then continue with staining protocol Simple Stain Preparation 1. Begin with a heat-fixed emulsion 2. Cover the smear with a stain and use a staining tray to catch excess stain.

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3. Grasp the slide with a slide holder. Rinse the slide with water and dispose of the excess stain. 4. Gently blot dry the smear in a tablet of bibulous paper or paper towels. 5. Observe the smear under oil immersion. Materials:  Colony of bacteria  Microscope Slides  Slide Holder  Inoculating loop  Crystal violet (stain)  Stain Tray  Bibulous Paper  Bunsen Burner  Microscope Results (3 pts) The simple staining technique was a success as the microbes were properly stained with a positively charged dye. After oil immersion, both a rod shaped and spherical shaped bacterial cells were observed. These shapes indicated Bacilli and Diplobacilli as they are rod shaped and the spherical shaped microbes observed were most likely Cocci. The positive stain used was crystal violet so the microbes observed appeared in purple color.

Analysis (3 pts) The hypothesis presented was found to be true as we were able to observe different microbes under oil immersion as a result of using the simple staining technique. The simple staining technique used crystal violet, a positively charged dye, and stained the microbe’s purple. As the hypothesis suggested, the simple staining technique allowed us to observe the different shapes of the microbes we observed under oil immersion. This allowed us to determine that the many long rod- shaped microbes showed in the image in the results section to be Bacilli and Diplobacilli and the spherical shaped microbes to most likely be Cocci. Conclusion (2 pts) In conclusion simple staining can be used to observe the morphology and arrangement of the bacteria cell. Simple staining allowed us to visualize the bacterial cells being examined, using shape to determine the type of microbe. Various skills were required throughout the experiment to aid us in bacteria smear preparation and simple stain preparation. Some skills that would carry over from the lab to future labs would be smear preparation as well as the use of an open flame and oil immersion. In my experiment one error that occurred was I failed to properly dry the smear resulting in an unclear image under the microscope. Other possible errors that could potentially occur during this lab included over heating bacteria smear, as well as cross contamination. To avoid these errors in the future it will be important to remember to properly dry the smear after applying the stain as well as to properly sterilize the work area. Proper use of the flame during heat fixation will also be a critical part.

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