Lab 4: Simple Staining

Learning Objectives:

• To learn about principle of staining
• To learn the process of simple stain
• To prepare wet mount slides

    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

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.
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.
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.
9. Repeat the method using cultures of B. subtilis and S. cerevisiae. Record observations in Lab report and answer questions.

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

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.
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.
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.

Lab Report:

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: B. subtilis

Additional observation/notes:

     Bacteria: S. epidermidis

Additional observation/notes:

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:

S. cerevisiae: Stained

Additional observation/notes:

Cheek Cells:

Additional observation/notes:

Complete the following table:

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?
4. You observed unstained and stained yeast specimen. Which one was easier to observe? Explain the reason?