MICROSCOPE USE: The arm should face you as you lift it up. Hold with one hand under base, and one hand on the arm, gently set on desk near edge, and then turn the arm away from you. Loosen the screw on the eye pieces, swivel the eye pieces towards you, then tighten the screw a little. Fold up dust cover and put it in the drawer.

PARTS OF A MICROSCOPE: The basic frame of the microscope consists of the base, stage, arm, and body tube.

Find the substage adjustment knob (left side, under stage), also called the condenser knob. What happens when you turn it? It raises and lowers the condenser. When you are done with the microscope, leave the condenser in the highest position; this is called racking up the condenser. Everyone point to this knob.

The oculars are the eyepieces. They magnify ten times and focus the image on your retina. There are two oculars. Do not touch your eyelashes to them or they will get oil on them. To clean, squirt alcohol onto lens paper and wipe, then wipe with dry lens paper.

The oculars have a diopter adjustment ring (knob with ridges), with plusses, minuses, and a zero on it. What happens when you move it? It moves the ocular up and down. The purpose of this is to allow the left eye to be focused independently of the right eye.

The coarse adjustment knob is for the dominant eye focus. Then use the diopter ring to focus for your non-dominant eye. If you still see two images, you have a convergence problem and you’ll need to keep one eye closed. Everyone has one dominant eye. You may need to close that eye when you want to use the pointer, if it is on the wrong side for you. Between the oculars is a disc with numbers on it. This determines the interpupillary distance in mm (distance between your pupils). Adjust it like binoculars to get the right width comfortable for you.

EYE DOMINANCE

Take a piece of paper, folded into a tube lengthwise. Hold the tube 12” away from your face and look at someone. Tell me what eye I am using. That’s my dominant eye. Do this now with your partner and have them tell you which eye is dominant on you. We use both of our eyes, but one more than the other.

One of your oculars has a pointer. When you are using the pointer, switch that ocular to the dominant eye. To switch the oculars, just pull them out, make sure you don’t drop them. Don’t leave them off long, or dust will get in. To turn the pointer, just turn the ocular or move the stage so the specimen meets the pointer. Always use the pointer to show me things when you have a question.

The revolving nosepiece holds the objectives. Practice turning nosepiece by the ring, not by touching the objective lenses. Listen and feel for the objective locking into place. You need to know the following about the four objectives: The scanning (red ring) objective is the smallest, and magnifies 4x. To find total magnification, multiply the ocular magnification (they come in 10x and 15x, but we only have 10x) by the magnification of the objective. What is total magnification on our microscopes with the scanning objective? Since the ocular is 10x, and the scanning objective is 4x, the total magnification is 40x. The low power (yellow ring) objective is 10x, total mag is100x. The high dry (blue ring) objective is 40x, total mag is400x. The oil immersion (white ring) objective is l00x, total mag is1000x. Always start with scanning (red) and progress to higher magnification. Don’t use the white oil immersion lens unless you are looking at bacteria, and use a drop of oil.

Focusing: Two knobs, coarse adjustment and fine adjustment. Fine adjustment is smaller and sticks out from the middle of the coarse adjustment knob. Make sure the red ring is in place, then move the coarse adjustment knob. The coarse knob moves the stage a lot, and the fine knob moves it a little. The distance between the stage and the objectives is called the “working distance”. Always start with the scanning objective (red ring), since it is the only one that can’t hit the stage and break the lens and the slide. Then make sure the condenser is racked up. Then turn the coarse knob so the stage is racked all the way up. Look at the slide, then lower the stage with the coarse knob until it comes into focus. Only after that can you switch to the next power up (yellow low power). To focus from now on, ONLY use the fine adjustment knob. You can NEVER touch the coarse knob unless the RED ring is in place. To take the slide off, switch to red ring first, then use the coarse knob to lower the stage.

PARFOCAL: This term refers to the factory adjustment which means that once you are focused with the scanning objective, you are focused with all of the objectives (except for fine adjustment for minor corrections). If you lose focus, always go back to the scanning objective.

The CONDENSER takes light from the lamp and makes the rays into a point on the slide. There is a light intensity knob on the left side of the bottom of your scope. Adjust it for comfort and visibility. Inside the condenser is a lever: the iris diaphragm lever. This opens and closes like the iris in your eye (pupil) to regulate the amount of light allowed in. When changing from low to high power, it is necessary to OPEN the iris diaphragm; increasing magnification requires MORE LIGHT.

The iris does the following four things:

a.Regulates light intensity

b.Contrast: (when iris is open, the contrast decreases)

c.Depth of field (when iris is open, only the foreground is in focus. When the iris is closed, the depth of field increases and everything is in focus).

d.Resolution (sharpness of image). Resolution is best when iris is open all the way. Different microscopes have different resolving powers. The resolving power of a microscope is a function of the numerical aperture (NA) of the lenses and the wavelength of light.

STAGE ADJUSTMENT KNOB is on the right side. It moves the stage up, down, right, left. It is also called the x-y stage knob.

BE ABLE TO MATCH THE PARTS OF A MICROSCOPE TO A PHOTO OF A MICROSCOPE FOR THE EXAM.

GETTING TO KNOW YOUR MICROSCOPE

1. What is the working distance?

Since you start with the lowest power (the shortest objective), as you increase magnification, working distance decreases because the objectives are taller as you go up in magnification. The scanning objective has the largest FIELD OF VIEW and the greatest working distance. When you are focused with one objective, you are focused with all of them. What is this called? PARFOCAL.

2. What are the functions of the parts of the microscope in italics? Know the four functions of the iris diaphragm (resolution, contrast, brightness, depth of field). Substage adjustment knob (moves condenser up and down – when down, there is poor resolution). Diopter ring (allows left eye to focus independently. Start with stage up, then bring down to focus the dominant eye.

Why does an image appear upside down and backwards? To answer this, you will look today at the letter “e” at 40x, 100x, and 400x.

FOCUS SEQUENCE

  1. Stage should be racked down.
  2. Scanning objective should be in place.
  3. Open stage clips (spring loaded).
  4. Mount slide onto stage with specimen approximately centered.
  5. Move stage up with coarse adjustment knob.
  6. Turn power on.
  7. Adjust your chair so you are not bending.
  8. Increase voltage until you see some light, but stay below maximum.
  9. For most slides you need high contrast, so open the iris to the maximum resolution. When you look at live cells, close the iris to decrease resolution.
  10. Adjust oculars for the interpupillary distance until you see just one circle of light called the FIELD. The type of microscope we have is a Brightfield.
  11. Turn coarse adjustment knob to lower the stage slowly while looking through ocular for the image to appear.
  12. Make sure the specimen is still centered.
  13. Close your left eye and use the coarse adjustment knob to focus the right eye.
  14. Close your right eye and use the diopter right to focus the left eye.
  15. Use the revolving nosepiece to move to the 10x objective (yellow).
  16. Image brightness will now decrease, so turn up the voltage control if needed.
  17. Adjust the image contrast with the iris diaphragm lever.
  18. Use the fine adjustment knob to focus. DO NOT TOUCH THE COARSE ADJUSTMENT KNOB AGAIN! If you lose sight of the specimen, go back to the scanning objective.
  19. When finished observing at that power, turn revolving nosepiece to 40x. You will see a decrease in depth of field. If you are looking at a live specimen in water, you will need to use the fine adjustment knob to follow it around.
  20. Adjust for image brightness with voltage.
  21. Adjust for contrast with iris.

You can use the x/y adjustment knobs to move the stage around if needed.

Take only one slide at a time. When done with a slide, bring it back and get another.

SLIDES TO LOOK AT NOW

  1. Letter “e”
  2. Blood smear. (look at RBC, WBC)

Practice moving the slide back and forth.

Why did the letter e look upside down and backwards?

The image gets bent as the light rays get bent through the lens, which is biconvex. Light entering top and bottom of lens gets bent (refraction). This is caused because speed of light is changing. As they travel through air, light rays go 186,000 miles per second. When they bump into some glass, they slow down and get bent. Lenses allow us to magnify the image by focusing the light rays at a point. As we move image back and forth, it allows us to focus. Then it hits mirror and is sent to oculars and is bent again with your eye lens. Wind up with a focal point. That’s why when you move slide to left, appears to go right.

Our eye lens bends things upside down and backwards; brain learns to switch the image. No brain in the microscope.

Under hi-dry look at RBC (pink) and WBC (purple nuclei). There are not as many WBCs as RBCs. Practice putting the pointer on a structure and draw and label a picture of the WBC with cell membrane, nucleus, and cytoplasm. Label the RBC with just cell membrane and cytoplasm because there is no nucleus.

BACTERIA SLIDE

Bacteria are small, so they always need 1000x to be seen.

The oil immersion lens has the smallest depth of field. Start at 40x as usual, and the bacteria will just look like little dots. As you progress to 100x, the dots are just bigger; focus again. At 400x, you will start to see shapes. The details are not seen until you get to the oil immersion lens.Why is it called an oil immersion lens? It needs a drop of synthetic immersion oil.

Move the revolving nosepiece to half way between 40x (blue) and 100x (white). Put a drop of immersion oil on the slide. When you rotate the immersion oil lens into place, it will be immersed in the oil. Now there is no air between the slide and the lens.

There are three basic shapes of bacteria:

  1. Spiral
  2. Cocci (singular is coccus)
  3. Bacilli (singular is bacillus; also known as rods)

Some slides have all three shapes. Make sure you see all three today.You don’t have to re-focus; just move the stage.

CLEANING UP OIL

Turn the nosepiece to the RED ring (not the blue). If you swivel to the blue, the oil will get in the blue objective and ruin it. Once on red, lower the stage, wipe the oil off the slide with KIMWIPES, not lens paper. Remove the oil from objective lens with LENS PAPER and alcohol, and dry it. Wipe the stage off so there is no oil there.

The oil immersion lens has a sealer around it so the oil cannot seep in, but the other objectives are not sealed, so don’t get oil near them. First, put your finger on the slide label as you lower the stage or the slide will stick. Remove the slide; clean with Kimwipe. Use lens paper to remove the oil from the objective. DO NOT RUB THE LENS OR IT WILL SCRATCH THE LENS ($150). Dab it instead on a clean spot on the paper. Dab again at a clean spot until no more oil is coming off. Take the other half of lens paper, fold it in half, spray with alcohol (there is a special oil cleaner which is a pink liquid in a clear bottle on the top of the lab desk shelves) and dab it to the lens as before. Use the alcohol soaked paper also a little on the blue (high dry) lens to be sure it is clean. Then use it to clean the stage or condenser

PUT YOUR MICROSCOPE AWAY LIKE THIS:
- stage all the way down, clean off oil and fingerprints with the bottle of pink oil cleaner on the top of the lab bench.

- condenser racked up and then rack it down just one little notch just below the slide.

- 4x (red ring lens) in position

- Loosen the screw to the eyepieces and swivel it back to face the blue side of the scope and TIGHTEN the screw a bit again.
- cord wrapped loosely behind the scope, NOT wrapped around it

- Dust cover on

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