MICROMETRY

MICROMETRY

AIM: To measure the size of the given microorganism by using micrometry.

PRINCIPLE: Measuring the size of microorganism using a microscope is called micrometry. All measurements of length are based on a comparison of the object under scrutiny with known dimensions, or with a standardized, calibrated scale. In order to determine the length or width of a microbe, for example, a ruler or measuring tape is placed in contact with the board and the dimensions are noted by direct comparison to the graduated numerical markings on the ruler. This basic principle is applicable to the measurement of specimens observed in the microscope, is often not possible with a compound microscope to place a ruler in direct contact with the specimen. Alternative mechanisms for performing measurements at high magnifications in compound optical microscopy must be employed, and the most common of these is the application of eyepiece reticles in combination with stage micrometers. A majority of measurements made with compound microscopes fall into the size range of 0.2 micrometers to 25 millimeters. Horizontal distances below 0.2 micrometers are beneath the resolving power of the microscope, and lengths larger than the field of view of an eyepiece are usually measured with a microscope.

MATERIALS REQUIRED

Light microscope, ocular and stage micrometer, slides, bacterial cell culture

PROCEDURE

1. The ocular micrometer is placed on the circular shelf inside the eyepiece of the microscope.

2. Place the stage micrometer on the stage of a microscope and focus the graduations using low power objectives. The graduations on stage micrometer are spaced 0.01mm (10μm) apart.

3. Superimpose the two scales and record the number of ocular division coinciding exactly with the number of divisions of the stage micrometer.

4. The calibration factor or the least count of ocular micrometer is calculated

5. Now remove the stage micrometer from the stage and place the slide having cell preparation under low power magnification.

6. Position the cell being observed in such a way that the ocular micrometer is able to measure the diameter of a cell or the length/diameter of a cell component in arbitrary units.

7. Similarly for high power objective the ocular micrometer calibration has to be done again following the same procedure and then cell diameter is can be measured focusing the cell in high magnification.

8. Measure a rod-shaped bacterium, a coccus, a yeast cell, a protozoan, and a human red blood cell.  Record the results. 

Detection of calibration factor as follows:

If 13 ocular divisions coincide with 2 divisions (2X10μm=20μm) of stage micrometer

Then 1 ocular division =         20μm

-------                           = 1.54μm

13 divisions

Calculate the size as shown below:

If the diameter of a cell is occupying 5 divisions of ocular, the diameter of the cell will be:

 5 divisions X 1.54μm = 7.7μm

OBSERVATION:

The size of the given organism was measured with micrometry.

RESULT:

The size of the given organism was tabulated

S.No	Type of organism	Objective lens magnification	Calibration value	Size(mm)
1.	Bacteria/yeast/algae	10X
		40X
		100X