Calibration of the Ocular Micrometer on a Microscope

Microscopes are often used to measure small objects. As you can see in figure 1
below, compound microscopes have ocular micrometers, or rulers that can help you measure
items under the microscope. However, the scale on the ocular micrometer changes
with total magnification, and thus has no absolute value. Therefore, the ocular
micrometer does not have units and it needs to be calibrated prior to use.



Figure 1: Ocular micrometer superimposed over a view of red blood cells under the
microscope.

You will use a stage micrometer to calibrate the ocular micrometer. A stage
micrometer is essentially a ruler that is mounted on a microscope slide that does have units
(millimeters (mm) or micrometers (m)). When calibrating, you will line up the stage
micrometer with the ocular micrometer and count the number of divisions on the ocular
micrometer per millimeter or micrometer on the staged micrometer. The number of divisions
will change as the magnification changes.


Figure 2: Comparison of the ocular micrometer and the stage micrometer.


Example: At a total magnification of 40x, a student measured 42 ocular micrometer divisions
per millimeter. What is the distance in micrometers per ocular unit?

mm
m
x
1
1000
units ocular 42
1mm
23.8 m per ocular unit

























CALIBRATION MICROSCOPE




















I. USE OF THE OCULAR MICROMETER:
A. Procedure:
1. Place the ocular lens containing a micrometer disc on the microscope.
2. Focus on the object to be measured and determine the size in ocular units.
3. Multiply the ocular units by the calibration factor for that specific microscope, objective
and ocular micrometer. The units of the micrometer disc are arbitrary and a calibration
procedure must be done to determine the calibration factor for each different objective and
each different microscope.
B. Example:
A hypha was measured using an ocular micrometer in the eye piece of a phase contrast
scope and its 40X darkfield objective. The hypha was 3 ocular micrometer units wide. The
calibration factor for that specific micrometer used on the phase scope with the 40X darkfield
objective is 2.5 um.
3 ocular micrometer units x 2.5 um = 7.5 um ocular micrometer ocular micrometer The
hypha is 7.5 um wide.
II. CALIBRATION OF THE OCULAR MICROMETER:
Ocular micrometers are calibrated by comparing the ocular micrometer scale with a
calibrated stage micrometer. The stage micrometer is a microscope slide that has a carefully
calibrated scale which is divided into 0.1 mm and 0.01 mm units.
A. Procedure:
1. Install the 10X ocular containing the ocular micrometer disc in the microscope.
2. Place the calibrated stage micrometer slide on the stage and focus on the scale.
3. Adjust the field so that the zero line of the ocular disc scale is exactly superimposed upon
the zero line of the stage micrometer scale.
4. Without moving the stage micrometer, locate the point as far to the extreme right as
possible where any two lines are exactly superimposed upon each other.
5. Count the number of divisions (mm) on the stage micrometer between the zero line and the
superimposed line to the far right.
6. Count the number of ocular divisions between the zero line and the superimposed line to
the far right.
7. Divide the distance determined in step 5 by the number of ocular divisions in step 6 and
multiply by 1000 to give the ocular micrometer units in um.
stage micrometer divisions (mm) x 1000 um = um per ocular unit ocular micrometer
divisions mm
8. Repeat steps 3 through 7 for each objective on the microscope. If the ocular micrometer is
moved to a different scope, the calibration procedure must be repeated. If a new objective is
added to the microscope, the calibration procedure must be done for the objective.

OCULAR MICROMETER
SEC11.MAN 2
II. CALIBRATION OF THE OCULAR MICROMETER:
B. Example:
Using an ocular micrometer in the eye piece of a phase contrast scope and its 20X objective,
it is found that 45 ocular units are equal to 0.22 mm on the stage micrometer scale.
One ocular unit = 0.22 mm x 1000 um = 4.9 um 45 units mm











STAGE MICROMETER
OCULAR MICROMETER
PROCEDURE CALIBRATION MICROSCOPE
Materials
ocular micrometer
lens paper
stage micrometer
immersion oil
microscope
Procedure
1. The ocular micrometer is already in the eyepiece, so place the stage micrometer on the
stage, and center it exactly over the light source.
2. With the low power (10X) objective in position, bring the graduations of the stage
micrometer into focus using the coarse adjustment knob.
3. Adjust the amount of light and then rotate the eyepiece until the graduations of the ocular
micrometer lie parallel to the lines of the stage micrometer.
4. If the low-power objective is the objective to be calibrated, proceed to step 7.
5. If the high-dry objective is the objective to be calibrated, swing it into position and
proceed to step 7.
6. If the oil immersion lens is to be calibrated, place a drop of immersion oil on the stage
micrometer, swing the oil immersion lens into position, and bring the lines into focus; then
proceed to the next step.
7. Move the stage micrometer laterally until the lines at one end coincide. Then look for
another line on the ocular micrometer that coincides with one on the stage micrometer.
Occasionally one stage micrometer division will include an even number of ocular divisions,
as shown in illustration A. However in most cases several stage graduations will be involved.
In this instance, divide the number of stage micrometer divisions by the number of ocular
divisions that coincide. The figure you get will be that part of a stage micrometer division
that is seen in an ocular division.
Example: 4 divisions of the stage micrometer line up with 30 divisions of the ocular
organism spans over 8 ocular div
8. Replace the stage micrometer with slides of organisms and/or human chromosomes to be
measured.