Plant Physiology

Plant Physiology (Biology 327) - Dr. Stephen G. Saupe; College of St. Benedict/ St. John's University; Biology Department; Collegeville, MN 56321; (320) 363 - 2782; (320) 363 - 3202, fax; ssaupe@csbsju.edu

Using and Calibrating an Ocular Micrometer

    A precise and simple method for measuring objects in the microscope is to use an ocular micrometer. In essence, this instrument is a ruler etched in the ocular of a microscope. It consists of a series of ruled marks (Fig. 1 **not inserted**). For each objective of the microscope, the increment between each mark must be determined. Once calibrated for a given objective, the calibration need not be repeated unless the objective is switched.

    An object is measured by determining its size in ocular micrometer units. These units are then converted into actual units by multiplying by the calibration factor.

    Ocular micrometers are typically calibrated with a stage micrometer. This is a standardized glass slide with an accurately ruled scale. A typical stage micrometer is depicted in Fig. 2 (***not inserted***). The method for calibration follows:

Protocol:

Place the stage micrometer on the microscope stage and focus on the marks with low power.
Superimpose the ocular micrometer on the stage micrometer so that the left edge of the ocular micrometer ("O") lines up with the left edge of the stage micrometer.
Visually scan the two micrometers to locate a place where a line from each micrometer is perfectly superimposed.
Using the scale provided with the stage micrometer, determine the distance from the left edge of the ocular micrometer ("O") to the superimposed lines.
Divide this distance by the total number of ocular micrometer units represented by this distance to yield the calibration factor (mm omu^-1 ).
Record this value and keep it with the microscope.
Repeat step 1 - 6 with each of the other objectives.
To determine the actual size of an object, measure its size in ocular micrometer units. Then, multiply by the calibration factor (mm omu^-1 ).

Notes:
Rather than recording ocular micrometer units when taking data and then multiplying by the calibration factor, many microscopists prepare a table listing ocular micrometer units and length for each objective and then enter their data in actual units.