LAB SAFETY: Open-toed shoes and shorts above the knee are not allowed since a spill can cause skin irritation. Put equipment back in tote box on the rack in the front of the class. Before leaving, make sure your area has been disinfected, left clean, and that the microscope has been put away properly. If it is found not properly put away, you will receive a note indicating what was wrong with it.

The lab counters must all be wiped down with disinfectant when you come in and when you leave. You may throw the paper towels into the regular trash afterwards.

Dispose of cover-slips, but the glass slides will be cleaned and re-used. Most fires in this lab are started from alcohol becoming ignited. Just get away from it, cover it, and let it burn out. Remember, a lab coat can be taken off and used to put out a fire if someone’s hair catches fire, etc.

There is no food, drink, gum or water bottles allowed in a micro lab.

The biohazard bag is for any and all hazardous materials, including a toothpick you put in your mouth, gloves, disposable Petri dishes, etc. Use the regular trash for everything else.

For the glassware flasks and tubes, make sure all labels are removed, and all grease marks are erased first.

If there is a spill, cover it with paper towels, saturate them with disinfectant for ten minutes, then put those paper towels in the biohazard bucket along with your gloves.

First aid kit is in the cabinet near Andrea’s door.

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

PARTS OF A MICROSCOPE: Find the substage adjustment knob (left side, under stage). 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 ocular is the eyepiece. It magnifies ten times and focuses the image on the 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 left ocular has a diopter adjustment ring (knurled knob with ridges), with plusses, minuses, and a zero on it. What happens when you move it? It moves the left ocular up and down. The purpose of this is to allow the left eye to be focused independently of the right eye (more details on this next lab). The coarse adjustment knob is for the right eye focus. 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

1 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).

The revolving nosepiece holds the objectives. Practice turning it; 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. Since the ocular is 10x, the total magnification is 40x. The low power objective is 10x, total

mag = 100x. The high dry objective is 40x, total mag = 400x. The oil immersion

objective is l00x, total mag = 1000x.

Focusing: Two knobs, coarse and fine adjustment. Fine adjustment is smaller and concentric to the coarse adjustment knob. The coarse knob moves the stage a lot, and the fine knob moves it a little. Changing the distance between the stage and the objectives to focus is called the “working distance”. Always start with the scanning objective, 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 rack up the coarse knob so the stage is 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.

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.

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. The condenser takes light from the lamp and makes the rays into a point on the slide. 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.

STORING THE MICROSCOPE: The arm should face your body, the dust cover in place, the AC (power) cord is wrapped around it. The condenser should be racked up. The toggle switch for power should be off (it is located in the front or side). The voltage regulator should be turned to zero or the lowest setting. It is located on the side near the base or below the power switch. The stage should be racked down by using the coarse adjustment knob (next to the arm). The scanning objective should be in place. Clean off the oil and other debris; wipe the ocular lens with lens paper only.

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

3 GETTING TO KNOW YOUR MICROSCOPE

1. What is the working distance? Since you start with the lowest power, as you increase magnification, working distance decreases. Scanning objective has the best depth of field 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 right 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. 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.

4 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.

WET MOUNT DEMONSTRATION Supplies needed: Lens paper Toothpicks Disposable pipettes Sterile saline in glass tubes with screw-top lids Slides Cover slips Procedure: Take the round part of the toothpick, scrape the inside of your cheek in your mouth. You will be gathering epithelium cells. Mix the toothpick well with the drop. Then take a cover slip and touch it to the drop for a few seconds so the capillary action lets the water seep up the slip. Then drop the cover slip down like a hinge. If you just drop it down quickly, bubbles will form. Throw the toothpick out in the biohazard bag.

Without staining these cells, there will not be much contrast, so this exercise it to practice adjusting the iris. The iris needs to be closed all the way at the scanning objective so you can see. Continue up to 400x and draw your picture.

5 If you see dots in your epithelium, these are normal microbes stuck to the epithelium. A pap smear is also epithelium and looks like this. The pathologist is looking for odd shaped cells, which indicate cancer. Cervical cancer can be caused from Human Papillo Virus (there’s a vaccine for that now!). Epithelium is thin, they form a sheet of tissue, they are easy to break apart form each other, and they grow back quickly. The cells you took off your cheek today will be grown back tomorrow! Now you can appreciate why we stain things and why we need to use the iris.

Cleaning the slides: Throw the coverslip into the broken glass container. Rinse the slide off in water at the sink and scrub the slide down with the cleanser. Rinse and dry and put the slides in the disinfectant tub at the sink.

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 pop 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.

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

The oil immersion lens has the narrowest 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 and 100x. Put a drop of immersion oil on the slide with the glass applicator rod. Touch the rod to the slide where the light is coming up. 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)

6 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 Remove the oil from the slide before you return it to the slide tray. You only need to clean the oil off the lens before you leave.

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; take a piece of lens paper torn in half, and use half to wipe off the oil from the slide and throw the paper in the regular trash. Take the other half of lens paper, spray it with alcohol, and wipe what’s left of the oil smear off the slide.

Take another piece of lens paper torn in half. Take one half, fold it in half, and just ouch it to the oil immersion lens. DO NOT RUB OR IT WILL SCRATCH THE LENS ($150). Dab it again at 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, 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

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