Micrometry

           Determination of Microbial Cell Size by Micrometry

Aim:

            To measure the dimensions of microorganisms with the help of a microscope.

 Principle: 

Microorganisms are microscopic objects that are visible only with the help of a microscope.  Sometimes it is necessary to measure its dimensions (length breadth and diameter) for its identification process. But, determination of the size of a microorganism is not an easy process. Micrometry refers to the measurement of dimensions of the desired microorganisms under a microscope which uses two micro-scales known as ‘micrometers’. At first, the diameter of the microscopic field must be established with the help these micrometers namely ocular micrometer and stage micrometer. Ocular micrometer with microscopic graduations etched on their surfaces is a circular glass disc that fits into the circular shelf inside the eyepiece of the microscope. It has 100 equally spaced divisions marked as 0 to 10. Depending on the objective being used, the distance between these graduations will vary that determines the size of the field. The stage micrometer is clipped to the stage of the microscope.  In the centre of the stage micrometer a known 1mm distance is etched into100 equally spaced divisions making each division equals 0.01 mm or 10 µm. 

Calibration:

            The calibration procedure for the ocular micrometer to requires that the graduations on both micrometers to be superimposed on each other. The number of ocular divisions that corresponds to the known distance in the stage micrometer is determined. Finally the calibration calculated as follows.

                                                              Number of divisions on stage micrometer

One division of ocular micrometer = ____________________________________ x10

                                                               Number of divisions on ocular micrometer

After calibration, the ocular micrometer measures the size of various microbes including its length, breadth, and diameter. First count the number of spaces occupied by the organism on ocular micrometer graduations. Then multiply this number by calibration factor.

Materials Required:

Stage micrometer

Ocular micrometer

Microscope

Microscopic slide

Microbial culture

Procedure

1. The eyepiece is removed from the microscope, and its top lid is unscrewed. The lid is removed. Carefully, the eye lens is removed. The ocular micrometer (a circular etched glass piece, which slips into the eye piece) is placed carefully into the eyepiece. The eye lens is placed back and the top lid is screwed to its original condition. The eyepiece is placed back in the microscope.
2. The stage micrometer is clipped to the stage and the etchings centered by moving the mechanical stage.
3. Adjust ocular and the objectives (10x, 45 x, 100x) which is to be calibrated.
4.  Superimpose the graduation of the slide with that of the ocular such that the line on the slide at one end exactly coincides with first line on the ocular. This done by rotating ocular and moving the stage micrometer.
5. Count the number ocular meter divisions coinciding between two lines of both ocular micrometer and stage micrometer.
6. The size of the bacterial cell is determined by counting the number of divisions in ocular micrometer occupies by single cell.

Result:

The size of the given microbial cell determined as ___________ (length, breadth) in 10x/40x/100x.