[Histonet] Microscope Calibration

[Terri Braud]

A. USE EYEPIECE WITH OCULAR MICROMETER ALREADY IN PLACE. 
B. Place the stage micrometer on the stage and focus on some portion of the scale with the 10X objective.
C. Looking through the microscope, examine the ruling of the stage micrometer so that you can distinguish between
the large (0.1 mm.) and the small (0.01 mm.) divisions.
D. Adjust the field so that the 0 line on the ocular micrometer is exactly superimposed upon the 0 line on the stage
micrometer.
E. Without moving the stage micrometer find another point at the extreme right where two other lines are exactly
superimposed. The second set of superimposed lines should be as far as possible from the 0 lines, but the
distance will vary with the objective and microscope.
F. Knowing that each large division of the stage micrometer equals 0.1 mm. and each small division equals 0.01
mm., determine the total distance (in millimeters) between the two points of superimposition and the number of
small ocular units necessary to cover the same distance (Figure 1).
For example: Suppose 27 ocular units equal 2 large stage units or 0.2 mm.
G. Calculate the number of millimeters that is measured by one small ocular unit.
For example: Divide .2 mm.
27 = 0.0074
H. Since measurements of the protozoa and other small organisms or structures are usually given in microns rather
than millimeters, the above determine must be converted to microns. Since 1 millimeter equals 1,000 microns,
the millimeter determination multiplied by 1,000 will give the number of microns measured by 1 small ocular
unit.
For example: 0.0074 mm. X 1,000 = 7.4 microns (u).
I. Record the calibrations of the ocular micrometer obtained with the 10X oculars combined with each of the
three objectives. The lines of the stage micrometer will increase in magnification while those of the ocular
micrometer will remain the same. With high and oil magnifications, the thinner ocular micrometer lined must be
centered on the broader stage micrometer line for more accurate measurement.
J. The size of any microscopic object can be quickly determined by measuring it with the ocular micrometer, noting
the particular lens combination, and referring to the record of the ocular micrometer calibrations.
RECORDING RESULTS: Record results of calibration on calibration card and is kept in the cabinet behind the scope in Special
Microbiology.
REFERENCES:
Clinical Microbiology Procedures Handbook, Third edition, Vol 2. Lynne S. Garcia, ASM Press 2010.
Date Approved by

Terri L. Braud, HT(ASCP)
Anatomic Pathology Supervisor
HNL Labs, Holy Redeemer Hospital
1648 Huntingdon Pike
Meadowbrook, PA 19046
ph: 215-938-3689
fax: 215-938-3874
Care, Comfort, and Heal