MICROSCOPY and SIMPLE STAINING

1. Watch the microscopy video and use the other resources at this site.

Microscopy Tutorial Module

Click The Virtual Scope on the Microscopy Pre-Lab Activities page (courtesy of the University of Delaware)

Microscopy video (Courtesy of the University of Leicester)

While using these resources, pay particular attention to the following parts or properties of your microscope:

ocular lenses (eyepieces)

objective lenses (low power, high dry, oil immersion)

stage and slide holder, slide movement control

condenser and iris diaphragm and their controls

arm and base (what is the correct way to lift and carry your microscope?)

lighting and its control

What is meant by the term PARFOCAL?

How does being parfocal simplify focusing?

What is the correct way to focus?

How do you clean the lenses?

Keep the head set screw tight?

A. Practice Light Control

• Turn on the light source to medium intensity
• Look through the oculars and adjust the interpupillary distance so that you see just one illuminated field. The field will tend to "dance" before your eyes. Move your head back and forth, up and down and try to determine the most comfortable viewing distance for yourself.
• While looking through the oculars, open and close the iris diaphragm. Note the effect this has on the amount of light coming through the oculars.
• While looking through the oculars, raise and lower the condenser by turning the condenser knob. What effect does this have on the light intensity?

B. Focusing and Kohler Illumination

• Place one of the prepared slides which have either the letter "e" or the colored threads mounted on them onto the stage of your microscope. Make sure that the specimen is centered over the stage aperture.
• The light source should be adjusted to medium intensity, the condenser about three-fourths of the way up and the iris should be almost closed.
• Rotate the low power objective into place over the specimen and, while looking from the side, crank the specimen up as close as it can get to the objective. (The low power objective should not be in danger of touching the specimen. However, as you gain experience with the microscope you may want to start focusing with higher power objectives. This is OK to do but be aware that the higher power objectives are longer and should not be allowed to touch the specimen.)
• Look through the oculars and start focusing by moving the specimen away from the objective by turning the coarse-adjustment knob. When the specimen comes into view, fine focus on one area of the specimen with the fine-adjustment knob. Is the whole specimen ever in focus or are some parts of the field in focus while others are not?
• Now it is time to adjust the microscope specifically for your use:
  • Close your left eye and make sure that the microscope is well focused for your right eye.
  • Now open your left eye and bring the left ocular image into focus by turning the diopter focusing ring on the left ocular.
• Now adjust your microscope for Kohler illumination:
  • With your microcope focused on the specimen as above, close the field diaphragm until it is almost closed. If you look through the oculars you should see a small circle of light.
  • Raise or lower the condenser until the edges of the circle of light come into sharp focus.
  • This represents the optimum optical adjustment of the condenser and it provides the best resolution that your microscope is capable of. Make sure that your microcope is "Kohlered" before making any critical observations.
    • (Alternative Kohler Procedure for Microscopes that don't have a field diaphragm
      Hold a sharply pointed pencil or pen over top of the light source at the bottom of your microscope.The point of your pencil should be at the center of the light source.
    • Raise or lower the condenser until the shadow of the pencil point comes into focus through your oculars.
    • As above, this represents the optimum optical adjustment of the condenser and it provides the best resolution that your microscope is capable of. Make sure that your microcope is "Kohlered" before making any critical observations. )
• Draw the letter "e" or the threads both as they appear to the naked eye and as they appear under low power of your microscope. Move the specimen using the X,Y stage control knobs. When observing the threads, focus up and down to get a sense of the depth of field.

C. Demonstrate Parfocal Property of the Microscope with the High Dry Objective

• With the letter "e" or thread slide in focus with low power, move the high dry objective into place over the specimen. Do not adjust the focus knobs before you do this. (Whenever you change objectives it is good technique to always observe the stage from the side to make sure that the objective will clear the slide. The objective lens should NEVER touch the slide!)
• Since your microscope is parfocal, the specimen should still be visible and still be in approximate focus. You may need to adjust the fine focus a bit to bring it into sharp focus however.
• Draw the letter "e" or the threads both as they appear to the naked eye and as they appear under high power of your microscope. Move the specimen using the X,Y stage control knobs. When observing the threads, focus up and down to get a sense of the depth of field.
• How does the image you see now differ from what you saw under low power? Can you make out any more details? Did you have to change the lighting?

D. Low Power Observation of Salt Crystals

• Use a bacteriological loop to transfer a small drop of 20% NaCl to the center of a microscope slide.
  • FLAME the loop and the test tube lip as if it were a bacterial culture. This fundamental procedure of aseptic technique as well the proper technique for lighting a bunsen burner will be demonstrated for you. These techniques are also demonstrated in the following film clips:
  • 
• As the slide is drying, place it on the stage and position the drying specimen over the stage aperture.
• Turn the light down low with the iris diaphragm and try to focus on the forming salt crystals with the low power lens. Use the same focusing technique you learned above. You do not need to adjust the condenser since you have already Kohlered it.
• Change the lighting with the iris and determine its effect on what you see. You can also change the condenser adjustment - but if you do you should always bring it back to Kohler illumination before moving on. Play with the rheostat and determine its effect also.
• Now move to the high dry objective and perform similar observations to what you did above.

E. Demonstrate the Parfocal Property of the Microscope with the Oil Immersion Objective

• Obtain one of the prepared tissue or bacterial slides. The tissue slides will have coverslips on them which is typical when the specimen is fragile or valuable. The bacterial smears will probably not have coverslips on them.
• Adjust the light to low with the iris diaphragm, position the specimen over the stage aperture and focus on the specimen with the low power objective. Again, play with the lighting and move the specimen around, find and identify key landmarks if you can.
• Position an interesting structure in the center of the field and then move to the high dry objective. Adjust the focus with the fine adjustment. As above, play with the lighting and move the specimen around, find and identify key landmarks if you can.
• Once again, position an interesting structure in the center of the field, rotate the high dry objective half way out of the way - enough so that you can deposit a small drop of oil onto the center of the lighted specimen. Now rotate the oil immersion objective into place. Watch from the side as you do this! The tip of the lens should pass into the oil and it should just clear the top of the cover slip (or the slide if there is no coverslip). Focus with the fine adjustment knob.
  • Your specimen should be in approximate focus but if you are over a thick area you may have to increase the lighting or move the specimen a bit to a thinner area in order to recognize anything. Conversely, if all you see is bright light, you may be over a blank area in your specimen and you may have to move your specimen slightly in order to see details. This second situation shouldn't happen if you were careful to center an object in the field you observed with the high dry lens.
• When you are done, save the smear by BLOTTING the oil off. (Don't wipe the oil off!). As was demonstrated in to microscopy video - Be sure to clean the lens with methanol at the end of the day. Be especially sure to clean off the high dry lens since it is easily contaminated with oil!

F. Wet Mounts

We have several HAY INFUSIONS available for doing wet mounts.

• It is best to sample a little of the scum floating on top with one of the wooden sticks provided.
• If going for a deeper specimen, use the plastic pipette and try to pick up a little visible debris which can act as a point of reference when focusing.
• Don't flood your slide with specimen, small is better!
• Place a coverslip over the specimen and observe under low power and high dry.
• You can move on to oil immersion but these are tricky since the oil acts as an adhesive and loosely attaches the lens to the coverslip which then slides back and forth across the fluid specimen. If you do try oil immersion on these, it is especially important that your specimen be small.
• Look at some of the identification keys available in the laboratory and see if you can identify any of the "animalicules" (as Leeuwenhoek would say) you see.

Identification Keys:

 

• Protozoa
• Flagellated Algae

G. Preparing a Bacterial Smear

Preparing a bacterial smear is a very fundamental technique that you will use many times during the course of the semester. Learn this technique by first doing it with a slant culture rather than a broth culture. All manipulations of bacterial cultures MUST BE DONE ASEPTICALLY. This means holding the cap with the "pinkie" of the hand holding the loop and always flaming the lip of the opened culture tube as demonstrated in class and in the film clip.

1. Preparing Smears from Slant Cultures

a.) Put a small drop of H2O (from the tap) onto the center of three slides. Use a bacteriological loop to pick up the water.

b.) Onto the first slide put a large amount of growth from the slant culture; onto the second slide put a pinpoint amount of growth; and onto the third slide put an invisible amount of growth. Always make sure to flame before going back into the culture tube. When applying the bacteria to the drop of water, smear the drop into the size of a dime to facilitate drying.

 c.) Once the smear is dry, HEAT FIX the smear:

Pass the bottom of the slide with the dried specimen over the flame of your bunsen burner three times. This should be done quickly and it is best to hold the slide with a clothespin so as not to burn your fingers. When pau, you should be able to place the bottom of the slide on the back of your hand and feel heat but not be burned. If the slide burns you, then the heat is intense enough to damge bacterial structure.

 2. Preparing Smears from Broth Cultures

a.) Obtain 3 microscope slides and using careful aseptic technique transfer two loopfuls from a broth culture onto the first slide. Transfer 4 loopfuls onto the second slide. Transfer 6 loopfuls onto the third slide. You will have better results if you smear each loopful out to about the size of a dime and if you allow each loopful to dry before adding the next loopful.

b.) Once the smear is dry, HEAT FIX the smear as above

H. Staining Bacterial Smears with Simple Stains

1.) Place the cooled slides on the staining rack over the sink at your lab bench.

2.) Flood the slides with methylene blue (a basic stain) and allow the stain to react for 1 minute.

3.) Wash the slides with running water from the tap. Manipulate the rubber tubing attached to the water faucet to direct the stream where you want it. With moderate water pressure you need not be concerned about washing the bacteria off of the slide. Be careful that you don't splash yourself with stain though. Pick the slides up with clothespins or forceps.

4.) Blot (don't wipe!) the slides with paper towel or Kimwipes and allow them to air dry completely.

5.) Observe as you did in "E" above.

Some Questions:

• What is meant by the term "basic dye?"

  What is a cation? Anion?

  What is the charge on a bacterial cell?

  What is a carboxyl group?

  What is meant by ionization?

copyright 2007 -- Revised Dec 2007