Calibrating a Microscope

Procedure

1. Get an iPod with a retina display and place it under a microscope
2. Using a microscope camera, take a photo of the screen at 10x magnification, using the application ImageJ.
3. From the documentation, we know that the size of a pixel is 78 µm, corresponding to 326 pixels per inch.
4. In ImageJ, use the line tool to measure five iPod pixels, and then look at how many pixels of the camera it corresponds to.
5. Then, go to analyze, and choose set scale.
6. In set scale, choose the first box and type how many camera pixels the five iPod pixels are.
7. In the second box type how many µm the five iPod pixels are (390 µm).
8. Leave the third box alone and in the fourth box write the unit of measurement which is µm.
9. Then you can also place a scale bar by going to analyze--tools--scale bar and typing how long the scale bar should be, where it should go, and much more.

ImageJ can be found at:

http://rsb.info.nih.gov/ij/download.html

Hanging Drop Mount

Ingredients

• toothpick
• pipette
• coverslip
• well-slide
• petroleum jelly
• drop of stagnant water
• microscope

Procedure 

1. Place a coverslip on a clean, flat surface. Use a toothpick to spread a small amount of petroleum jelly around all the edges of the coverslip. Use enough to form a seal between the coverslip and the slide.
2. Using a pipette, place a drop of the  stagnant water in the center of the coverslip.
3. Invert a well slide (well side down). Center the well over the coverslip. Use just enough pressure to make sure the coverslip seals to the slide (Don't press to hard or the jelly will ooze into the specimen area).
4. Invert the assembly so the coverslip is on top. The drop should be suspended in the well from the coverslip).
5. Observe the slide under a microscope.

Observations

In the drop I found a cluster of organisms. I saw cells, long cells and some moving tiny organisms.

Long cells 100x

Cluster at  45x

More long cells 100x

Four cells 100x

Five different cells 100x

Gram Staining a Heat Fixed Smear Slide

Ingredients

• heat fixed smear mount
• paper towel
• pipettes
• distilled water
• small non corrosive glass or ceramic bowl
• ethanol
• lint-free cloth or tissue
• microscope
• hucker's crystal violet stain
• gram's iodine stain
• safranin O stain

Procedure for Gram Staining

1. Place a paper towel on a clean, flat surface, then place the heat fixed smear mount onto the towel.
2. Use a clean pipette to place a drop or two of Hucker's crystal violet stain onto the smear, then use the tip of the pipette (don't touch the smear) to spread the stain until it covers the entire smear.
3. Allow the Hucker's crystal violet stain to remain in contact with the smear for one minute.
4. Then, using a pipette filled with distilled water, hold the slide at an angle over the bowl and drip water above the stain so the slide floods with water and washes away any excess stain. 
5. Drain the slide and place it flat on the paper towel.
6. Use a clean pipette to place a drop or two of Gram's iodine stain onto the smear. Again, use the tip of the pipette to spread the stain over the entire smear.
7. Allow the Gram's iodine stain to remain in contact with the smear for one minute.
8. Fill a clean pipette with ethanol (drugstore 70% ethanol is fine). Hold the slide at an angle over the bowl and drip the ethanol above the smear so it floods the smear. Continue until the ethanol runs colorless.
9. Then repeat step 4 to rinse all of the ethanol from the slide. Drain the slide and place it flat on the paper towel.
10. Use a clean pipette to place a drop or two of safranin O stain onto the smear, and again use the tip of the pipette to spread the stain until it covers the entire smear.
11. Allow the safranin O stain to remain in contact with the smear for one minute.
12. Repeat step 4 to rinse excess safranin O stain from the slide.
13. Allow the slide to air dry. If you'r in a hurry, you can gently pat the slide dry with a lint free cloth or tissue. Do not rub the smear area.
14. Observe the slide under your microscope. You do not need a coverslip.
15. The Gram-negative bacteria should appear pink or red, and the Gram-positive bacteria should appear violet.

Observations

The bacteria stained only with
Hucker's crystal violet

After staining the smear with Hucker's crystal violet stain, I looked at it under the microscope but the bacteria was to dark to really identify anything except the blobs of bacteria.

 

The bacteria stained with Hucker's
crystal violet and Gram's iodine

After having stained the smear with Gram's iodine stain, the blobs of bacteria were considerably more transparent. I could also see that some parts of the blobs were lighter in color than the others, which could indicate that those spots were made up of Gram-negative bacteria.

After staining the smear with safranin O, I could clearly see the bacteria at 100x with the different colorations of blue and pink. I also noticed that there is more of the Gram-negative bacteria than the Gram-positive bacteria.

Simple Staining of Epithelial Cells from the Buccal Mucosa

Ingredients

• normal microscope slide and coverslip
• microscope
• toothpick
• methylene blue stain
• eosin Y stain
• paper towel

Procedure for Simple Staining

1. Transfer one drop of distilled water to the center of a slide.
2. Use a toothpick to scrape (gently) the inside of your cheek.
3. Immerse the end of the toothpick in the drop of water and stir to transfer the Epithelial cells to the water.
4. Position a coverslip over the specimen.
5. Observe the specimen under a microscope.
6. Place one drop of methylene blue stain at one edge of the coverslip.
7. Touch the corner of a paper towel to the slide at the opposite edge of the coverslip. The paper towel wicks the water from under the coverslip, drawing the drop of methylene blue stain under the coverslip.
8. Allow the stain to work for 30 seconds or so and observe the cells under a microscope.
9. Repeat steps 6 through 8 with a drop of eosin Y stain instead of methylene blue stain.
10. Observe under a microscope.

Observations

At 40x with the slide unstained I can see the cells and their nucleus without much difficulty.

At 100x with the slide unstained I can see the nucleus better and also that there are things around it.

At 450x with the slide unstained I clearly see the nucleus and what could be organelles surrounding it.

Epithelial Cells at 40x Non-stained

Epithelial Cells at 100x Non-stained

Epithelial Cell at 450x Non-stained

Due to technical difficulties with the methylene blue stain being to aqueous and made with crystals not fully dissolved, I had the slide all dirty with crystals of methylene blue stain and also the stain did not seem to work. I could not see the cells a lot and could not make any observations on them. Below is a picture of the slide with the bad stain.

The red oval indicates the
 contamination of the
methylene blue stain