Download BIOS 242 Week 2 Assignment; Lab 4 of 14 Onsite; Simple Staining and more Assignments Health sciences in PDF only on Docsity!
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Lab 4: Simple Staining
Learning Objectives: To learn about principle of staining To learn the process of simple stain To prepare wet mount slides In light microscopy, colored dyes, called stains are used to improve contrast between the specimen and the background surrounding the specimen. Staining makes visualization of the specimen easier due to increased contrast. This is crucial for specimens that are mostly transparent. Bacteria, due to their size are difficult to visualize under light microscope without staining. Stains contain chromophores of a specific color. In addition, the stains may bind to cells based on the charges present. In simple staining, only one type of stain is used. Use of single stain allows the user to observe size, shape and arrangement of bacteria. Acidic stains are negatively charged and bind to positively-charged structures in the cells. Basic stains are positively charged and bind to negatively-charged structures, such as nucleic acids and many cell wall components. Basic stains such as Crystal violet, Safranin, and Methylene
Name: blue are some of the common stains used in a Microbiology laboratory. Differential staining uses two or more similar stains in a way that it allows observation of specific features of bacteria which often becomes basis of identification of the bacteria. In this lab, we will carry out simple staining and subsequent observation of size, shape and arrangement on bacteria and yeast. We will also make wet-mount slides of yeast and cheek cells. Exercise 1: Simple Stain Materials: Overnight cultures of S. epidermidis , B. subtilis , S. cerevisiae (yeast) , inoculating loop, glass slides, incinerators, crystal violet or any other stain chosen by instructor; tongs, staining racks/trays, immersion oil, lens paper, bibulous paper, microscope Note to students: Wear gloves and use PPE before starting the lab work. Use aseptic technique to prevent contamination. Use only 1-2 drops of stain per slide. Avoid using excess. Method:
- Obtain a clean glass slide, loop and overnight cultures. Hold the glass slides from the edges to avoid transferring grease or oil from fingers to the slide.
- Using aseptic technique, transfer a loop full of S. epidermidis culture at the middle of the glass slide. Spread it in a thin layer. a. It is important to make a thin smear. A thick smear prevents light to pass through it, may have cells too close to each other or as clumps which results in poor observation. A thin smear allows better observation of cells by allowing the light to pass through it.
- Allow the smear to air dry completely. Once the smear is completely dry, hold the glass slide using tongs and quickly pass the slide 1- times through flame of Bunsen burner to heat fix the bacteria.
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Name: Exercise 2: Wet-mount Slides Materials: Overnight cultures of S. cerevisiae (yeast), inoculating loop, sterile swabs, glass slides, cover slips, incinerators, diluted solution of crystal violet or any other stain chosen by instructor; tongs, staining racks/trays, immersion oil, lens paper, microscope Note to students: Wear gloves and use PPE before starting the lab work. Use aseptic technique to prevent contamination. Use only 1-2 drops of stain per slide. Avoid using excess. Yeast Culture:
- Obtain S. cerevisiae (yeast) culture, two glass slides, two cover slips, inoculating loop, and stain.
- Wear gloves before handling glass slides and coverslips needed to make wet-mount slides.
- Handle glass slides and cover slips by holding from sides rather than between your fingers to avoid getting the slides dirty.
- Transfer a small drop of S. cerevisiae (yeast) culture on both slides using an inoculating loop. a. Don’t forget to use aseptic techniques while handling the cultures.
- On one of the slides, add a drop of stain (in case of stained specimen), while do not use stain on the second slide (unstained specimen).
- Put a coverslips on both slides by starting with one side of coverslip touching to the liquid culture on the specimen and then dropping the coverslip at an angle. This technique is useful in preventing catching of air bubbles underneath the coverslip.
- Use wipes to carefully remove excess liquid from the edges of the coverslip.
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Lab Report:
Purpose: Describe the purpose of the lab. The purpose of the lab was to gain some knowledge on the principle of of staining and to understand the process of simple staining and learn how to prepare wet mount slides. This was a very interesting lab to me, the professor explained the lab very well. I have never done this type of lab before this was my first time doing this kind of lab. Results: Exercise 1: Simple Stain Draw the observation inside the circles provided. Label your image appropriately. Write the magnification at which the observation was made.
Name: Bacteria: S. epidermidis 40x Additional observation/notes: Bacillus/ rod shaped in chains type of cell bacteria gram postive observed at 40x size is like 1-2mm and Arrangmennt clusters
Name: Exercise 2: Wet-mount Slides Draw the observation inside the circles provided. Label your image appropriately. Write the magnification at which the observation was made. S. cerevisiae : Unstained Did not perform Additional observation/notes:
Name: S. cerevisiae : Stained Did not perform Additional observation/notes:
Name: Complete the following table: Type of Cell Shape Size Arrangement S. epidermidis Bacteria,gram postive Bacillus/ rod shaped in chains 1-2mm clusters B. Subtilis Bacteria, gram positive Bacillus, 4-10mm Chain Cheek Cells Eukaryotes Flat 5mm Random Questions: 1.Describe differences between a wet mount slide and simple staining.
In a wet mount slide, we use of water and a coverslip.Wet mount do not heat fix bacteria cells therefore, the procedure does not
damage or kill the bacteria. It retains the natural shape and arrangement. Simple staning procedure used to increase contrast on the
bacterial image, allowing one to observe cell size, morphology, and arrangement.
2.Why is air-drying important before heat fixing the specimen? It prevents spread of pathogen from the smear and it fixes the smear slide.
The air drying prevents distortion of shape of the bacteria to be staines and the smear easily accepts the stain after heat fixing and
air drying. Attempting to heat fix the slide while wet or damp will cause the cell to be denatured.
Name: 3.If you forgot to heat fix the specimen, how will it impact observation of specimen?
If this step is not done, the bacteria in the smear be washed off of the slide during the staining and decolorization steps. you would not
see anything on the slide under the microscope.
4.You observed unstained and stained yeast specimen. Which one was easier to observe? Explain the reason?
It is always better to stain the cells because performing this makes slides clearer and easier to see when viewing through a microscope
because it improves the contrast.Wet mount preparation allows you to visualize living cells under the microscope. Take a yeast
colonies from the petri plate, and use a clean toothpick to lift yeast cells from the colony. Swish the toothpick in a centrifuge tube
containing a drop of methylene blue. Live yeast cells will take up methylene blue and become stained for easy visibility.Yeast are best
viewed using the 40x objective.
