III. Microscopes: Wet Mount, Bringing Specimens into Focus
by Kathy Egbert
| 6 Questions
Position of cover slip when preparing a wet mount
How to Make a Wet Mount - Need a slide, cover slip, bottle of water, and newspaper "e" specimen
1. Locate a lower case "e", font size 11 or 12, or a word with an "e" in it. Cut out the "e" or word. Your specimen should be at least 1 cm square or rectangle; about half the size of your nail on your pinkie finger. If your piece of paper is too small, it will be difficult to work with.

2. Two ways to assemble: 1) water first or 2) water last. Try both ways and see if one way is easier for you than the other. Either order is fine for grading when you demonstrate making a wet mount for a grade. Water last method: place the square/rectangle specimen "e" paper in the middle of a glass slide. Position the square so that the words are in normal reading position. In other words, don't have the "e" turned sidewards or upside-down. Using the water bottle, carefully and slowly place 1 drop of water on the paper specimen. Keep the water bottle tip about 1 cm above the slide; do not touch the water bottle tip to the paper or the paper will stick to the water bottle. (Remember the wonderful adhesive and cohesive properties of water.)

  • the rectangular piece of glass is called a slide
  • the small plastic/glass square is called a cover slip

3. Cover your specimen with a cover slip. The best way to do this is to hold the cover slip at about a 45o angle to the slide and move it over the drop. As the water touches the cover slip edge, it will start to spread evenly along the edge of the cover slip. Now, drop the cover slip. If you have done this correctly, the water will push all the air bubbles out. If you have air bubbles, redo it - it is very difficult to view a specimen slide that has lots of air bubbles trapped under the cover slip. Never press on the cover slip to try to remove the air bubbles as this may break the cover slip and/or damage your specimen.

  • cover slips can break easily; if you break one just tell the teacher and pick up the pieces and discard; be careful not to cut yourself

4. If you have extra water on the slide after you have placed the cover slip on your specimen, use a piece of paper towelling and lay it next to the edge of the cover slip. Let the paper towelling soak up the extra water before you place the slide on the microscope stage for viewing.

At some point during our microscope lab, you will need to demonstrate making a wet mount as part of your Microscope Practicum grade (5 pts).

A few last reminders and instructions...
  • Do not touch the eyepiece, lens, or objective lens with any body part (eyes, face, fingers, etc)
  • Do not use kleenex or paper towels to clean these parts of the microscope as it will scratch the lens
    • if you think one of these is dirty and needs cleaning, ask the teacher and only use lens paper to clean the lens
  • Be sure the power switch is turned on; you may need to adjust the diaphragm for the amount of light
Which objective do you use when you want the highest magnification?
A 40x high power
B 10x low power
C 4x scanning objective
Which objective allows you to see the largest area (called the field of view) of the object that you are viewing?
A 4x scanning objective
B 10x low power objective
C 40x high power objective
Describe how to increase depth of field when viewing a specimen through the microscope.
A decrease the amount of light coming in through the diaphragm
B close one eye
C increase the amount of light coming in through the diaphragm
Assume you have an amoeba clearly visible in your microscope field of view and a classmate wants to see it. What do you do?
A pick up the microscope and move it to your classmate's table so he/she can see it
B get up from your chair and allow your classmate to sit down in front of your microscope
C swing the eyepiece to the side and allow your classmate to see your specimen
D B and C are the correct choices
The 4x scanning objective is the starting point for viewing all slides.
Steps to bring your specimen into focus:
1) the 4x scanning objective is in place
2) place slide on stage and secure with stage clips
3) using the mechanical stage knobs move the slide forward/backward and left/right until the light is coming through the center of your slide
4) use the course adjustment knob to bring your specimen into focus
5) if needed use the fine adjustment knob to fine tune the focus
6) be sure your specimen is centered in the middle of your field of view prior to increasing magnification
7) turn the objectives so the 10x low power is in place
8) only use the fine adjustment knob to focus - you shouldn't have to move it much if you were already in focus at 4x
9) again, be sure your specimen is centered in the middle of your field of view prior to increasing magnification
10) turn the objectives so the 40x high power is in place
11) again, only use the fine adjustment knob to focus - if you use the course adjustment knob you will probably break the slide or scratch the lens because the lens is so close to the slide - only use the fine adjustment knob
Draw the "e" under each of the three magnifications; remember the field of view will decrease with each magnification so you will see less of the "e"
Practicum Skills
When you feel that you have mastered these skills, tell the teacher so you can be graded,

Making a Wet Mount - 5 pts
Bringing a Specimen into Focus - 5 pts
5 pts - Making a wet mount assessment. Put your name on the whiteboard to get in line to have this assessed.
5 pts - Bringing a specimen into focus. Put your name on the whiteboard to get in line for this assessment.
Clean up - everyone participates - class not dismissed until everything is cleaned up

Individual Microscope:
1. lower the stage
2. make sure the 4x objective is in the viewing position
3. remove slide from the stage and clean as instructed
4. align mechanical stage so it is flush with the right side of the stage
5. leave the diaphragm open
6. turn off the light
7. move the microscope back and away from the edge of the lab table
8. return all items to your table work tub and make sure your table is cleaned up
9. return items received from the Teacher Table (water bottle, slides, cover slips)
10. help others if needed; remember that no one leaves until all materials are cleaned up

Additional steps:
2nd and 8th class periods may need to put the covers back on the microscopes and move the work tubs to the sideboards
1st and 4th class periods may need to remove the covers from the microscopes and stack them on the sideboard and move the work tubs back to the lab table stations from the sideboards
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