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Proper Use of the Microscope

illumination

  1. Condenser should always be racked up as high as it will go.
  2. BRIGHTNESS is controlled by (not all microscopes have all features):
    1. varying voltage to lamp by adjusting rheostat
    2. neutral-density filter over illuminator
    3. adjusting illuminator iris diaphragm
  3. CONTRAST is controlled by adjusting condenser iris diaphragm
  4. Centering condenser
    1. For microscope with iris diaphragm on illuminator:
      1. focus on a specimen with 10X objective
      2. close diaphragm almost completely
      3. focus spot of light by slightly lowering condenser
      4. if necessary, adjust centering screws (two knurled rods), which protrude from  condenser assembly, until the spot of light is centered in the field of view
      5. open the illuminator diaphragm until the light just in the field
    2. For microscopes with no iris diaphragm on illuminator:
      1. focus on a specimen with 10X objective
      2. remove an eyepiece (pull straight out)
      3. look down tube and close condenser iris diaphragm until a small spot of light is seen surrounded by black
      4. center bright spot as above

focusing

  1. Use coarse adjustment first, then fine adjustment (the fine adjustment may have a limited range of travel in some instruments).
  2. Oil immersion objective:
    1. If objectives are parfocal, focus at lower power, put drop of oil on slide, swing oil immersion objective into position, and adjust focus carefully with fine adjustment.
    2. If objectives are not parfocal:
      1. view objective from the side
      2. place drop of oil on specimen
      3. lower oil imm. objective with coarse adjustment until its tip just touches the slide. Note the direction the focusing knob was turning!
      4. looking into the microscope, turn the coarse focusing knob in the opposite direction slowly until the specimen comes into focus. Adjust, if necessary, with the fine-focus knob.
  3. To make objective parfocal:
    1. adjust screw on each objective (expensive models)
    2. if no adjusting screws on your instrument, use shims between each objective and turret (available from microscope supplier & usually cheap)
  4. If you find it impossible to focus on a specimen:
    1. coverslip may be too thick (usually only a problem with oil immersion)
    2. slide may be upside down
    3. oculars not matched (binocular microscopes)

tips for eyeglass wearers

  1. install rubber guards over eyepieces to prevent scratching
  2. trade in your eyepieces for "high eyepoint" ones ("exit pupil" further away from end of eyepiece)

cleaning

  1. Locating dust specks (assuming that slide is clean):
    1. if specks disappear when condenser is moved, then dust is on illuminator  bulb or filter
    2. if specks disappear while focusing, then dust is on condenser
    3. if neither of the above manipulations works, then:
      1. rotate eyepiece(s) - specks will rotate as well if dust is on them
      2. rotate objective - ditto above
  2. Removing dirt and film from lens surfaces:
    1. try using a special brush or air jet first (blower/brushes available at photo stores)
    2. wipe, using lens paper, after breathing on surface
    3. if necessary, use some alcohol - xylene is the last resort! (solvents may attack lens mounting cements)
    4. immersion oil should always be removed from objective soon after use by wiping with lens tissue - no solvents should be necessary

measuring objects under the microscope

The purchase of an ocular micrometer is highly recommended for parasitology work. It is relatively cheap and easy to install. It must be calibrated before it can be used; this procedure is simple and is described below:

 calibration of the ocular scale

Calibrating a ocular scale in a microscope is simply a matter of converting an arbitrary measure (ocular micrometer units) to a standard unit of measure (microns).

Place a stage micrometer on the microscope stage and focus on the scale using reduced illumination. Notice that the scale has large divisions which are 0.1 mm or 100 microns in length. At one end of the scale, two of the 0.1 mm divisions are each divided into 10 smaller divisions each measuring 0.01 mm (10 microns).

  1. Superimpose the ocular scale over the micrometer scale so that the zero point of each scale will coincide.
  2. Count the total number of divisions from the 0 of the ocular to one of the numbers near the end of the ocular where it exactly coincides with one of the lines on the stage. Record both numbers.
  3. Divide the stage measurements in microns by the ocular units to obtain the number of microns/ocular unit.
  4. Repeat the measurement twice to ensure that you have made no errors.
  5. Carry out the same procedure for each of the objectives on your microscope. It is not necessary to use oil with the oil immersion objective in this instance.
  6. Prepare a chart converting ocular units (at the left) to microns (at the right) for each of the microscope objectives.

 


Copyright © 2006 - University of Pennsylvania School of Veterinary Medicine, All rights reserved.
Faculty: Dr. Thomas Nolan
Students: Molly Church V'09, Diana Knight V'08, Douglas Gilson V'05, Chris Dykhouse V'04, Kimberly Mah V'00

Comments or Questions contact Dr. Tom Nolan at: