week 2 lab 4 updates

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Dec 6, 2023

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BIOS242 Week 2 Lab 4 Heat-Fixed Smear, Wet Mount, Simple Stain - Crystal Violet Name: Lab 4: Simple Staining of Heat Fixed Smears or Wet Mount Slides Learning Objectives: VIDEOS:  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 1 a) Heat Fix Bacterial Smear (Dr. Joyce Patrick) https://www.youtube.com/watch?v=MFGW3NATGsI b) The Simple Stain Technique (Secord’s Microbiology) https://www.youtube.com/watch?v=n5fXIpJUgD4 c) Saccharomyces cerevisiae Yeast Wet Mount Slide (Micro at TAMU-CC) https://www.youtube.com/watch?v=t7xzIczY5L8 d) Cheek Epithelial Cells Wet Mount Slide (Tami Guy) https://www.youtube.com/watch?v=i2x3MKSJez4

BIOS242 Week 2 Lab 4 Heat-Fixed Smear, Wet Mount, Simple Stain - Crystal Violet Name: 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 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: Heat-Fixed Bacterial Smear for 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: 1. 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. 2. 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. 2

BIOS242 Week 2 Lab 4 Heat-Fixed Smear, Wet Mount, Simple Stain - Crystal Violet Name: 3. Allow the smear to air dry completely. Once the smear is completely dry, hold the glass slide using tongs and quickly pass the slide 1-2 times through flame of Bunsen burner to heat fix the bacteria. a. Completely air drying the specimen on the slide is crucial. Complete air drying of the specimen allows the cells to attach to the glass slide without boiling of the wet smear. Boiling will cause fragmentation/shrinkage of bacteria resulting in poor specimen quality and observation. b. Once heat fixed, cells become attached to surface of the glass slide and will not get washed off during the staining process. 4. Once bacteria is heat fixed, allow the glass slide to cool to room temperature. 5. Add 1-2 drops of diluted stain and wait for 1 minute. 6. Remove the stain and gently wash it off using minimal amount of DI water. 7. Blot dry the slide and observe using 10X and then 40X lens of a compound microscope. 8. Record your observations at 40X lens in Lab report. a. Process to use Microscope is described in appendix A. b. Check with your instructor if 100X lens is needed. If 100X lens is used, don’t forget to use oil. c. Record results in the lab report. 9. Repeat the method using cultures of B. subtilis and S. cerevisiae . Record observations in Lab report and answer questions. 3

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BIOS242 Week 2 Lab 4 Heat-Fixed Smear, Wet Mount, Simple Stain - Crystal Violet Name: 4

BIOS242 Week 2 Lab 4 Heat-Fixed Smear, Wet Mount, Simple Stain - Crystal Violet Name: Exercise 2: Wet-mount Slides for Simple Stain 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: 1. Obtain S. cerevisiae (yeast) culture, two glass slides, two cover slips, inoculating loop, and stain. 2. Wear gloves before handling glass slides and coverslips needed to make wet-mount slides. 3. Handle glass slides and cover slips by holding from sides rather than between your fingers to avoid getting the slides dirty. 4. 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. 5. 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). 6. 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. 7. Use wipes to carefully remove excess liquid from the edges of the coverslip. 5

BIOS242 Week 2 Lab 4 Heat-Fixed Smear, Wet Mount, Simple Stain - Crystal Violet Name: 8. Observe slides using 10X, and 40X lenses. Record observations made using 40X lens in the Lab report. Cheek Cells: 9. Obtain glass slide, cover slip, stain, sterile swab. 10. Swab inside of your cheek using sterile swab and rub the surface of the glass slide. 11. Add 1-2 drops of diluted stain. 12. Put a coverslip at an angle by first touching one side of coverslip to the liquid on the slide and then dropping the coverslip at an angle. This technique is useful in preventing catching of air bubbles under the coverslip. 13. Use wipes to carefully remove excess liquid from the edges of the coverslip. 14. Observe using 10X and 40X lenses. Record observations made using 40X lens in the Lab report. 6

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BIOS242 Week 2 Lab 4 Heat-Fixed Smear, Wet Mount, Simple Stain - Crystal Violet Name: Lab 4 Report: Simple Stain of Heat-Fixed Smear and Wet Mount Slides Purpose: Describe the purpose of the 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. Bacteria: S. epidermidis Additional observation/notes: 7

BIOS242 Week 2 Lab 4 Heat-Fixed Smear, Wet Mount, Simple Stain - Crystal Violet Name: Bacteria: B. subtilis Additional observation/notes: 8

BIOS242 Week 2 Lab 4 Heat-Fixed Smear, Wet Mount, Simple Stain - Crystal Violet 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 Additional observation/notes: 9

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BIOS242 Week 2 Lab 4 Heat-Fixed Smear, Wet Mount, Simple Stain - Crystal Violet Name: S. cerevisiae : Stained Additional observation/notes: 10

BIOS242 Week 2 Lab 4 Heat-Fixed Smear, Wet Mount, Simple Stain - Crystal Violet Name: Cheek Cells: Additional observation/notes: 11

BIOS242 Week 2 Lab 4 Heat-Fixed Smear, Wet Mount, Simple Stain - Crystal Violet Name: Complete the following table: Type of Cell Shape Size Arrangement S. epidermidis B. Subtilis S. cerevisiae Cheek Cells Questions: 1. Describe differences between a wet mount slide and simple staining. 2. Why is air-drying important before heat fixing the specimen? 3. If you forgot to heat fix the specimen, how will it impact observation of specimen? 12

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BIOS242 Week 2 Lab 4 Heat-Fixed Smear, Wet Mount, Simple Stain - Crystal Violet Name: 4. You observed unstained and stained yeast specimen. Which one was easier to observe? Explain the reason? Grading Rubric: Activity Deliverable Points 13

BIOS242 Week 2 Lab 4 Heat-Fixed Smear, Wet Mount, Simple Stain - Crystal Violet Name: Experimental Set up and Observation Obtain/Set up the slides as directed in the lab or by instructor; Observe all assigned slides using microscope and record observation in lab report 6 Lab Report and Questions Complete lab report and answer ALL questions  Purpose (1 point)  Table (4 points)  Questions (4 points) 9 All Lab Deliverables Complete ALL lab work and lab report 15 14