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Sunday, December 18, 2011

Microscope Operation & Calibration

1.0 Purpose :
The purpose of this procedure is to describe the method of operating, cleaning and Calibration of Microscope.

2.0 Scope :
This procedure applicable to compound microscope used QC department (both wet lab and microbiology department)

3.0 Responsibility :
Officer QC & Microbiologist QC.

4.0 Procedure :


4.1 Equipment and material :

4.1.1 Compound Microscope
4.1.2 Stage micrometer
4.1.3 Ocular micrometer
4.1.4 Cedar wood oil or Liquid paraffin
4.1.5 Glass slides
4.1.6 Cover slips

4.2.0 Operating procedure:
4.2.1 Place the Microscope in an upright position at a convenient height for the operation on a rigid table. Make sure all the exposed Optical surfaces are free of dirt. Plug the power cord into a Grounded outlet. Bring the light intensity regulator to lowest level and switch on the power.

4.2.2 Adjust the observation head to convenient working position.

4.2.3 Rotate the nose piece until the lowest power objective is in Viewing position. The lower the power of objective, the greater the area of the specimen surface included in the field of view. Lower power objectives also have a much greater depth of focus are generally used for initial focussing and viewing.

4.2.4 Take down the stage to a fairly low position with the help of coarse focus knob.

4.2.5 Make sure that the stage surface is free of dirt, grit or any other material that will interfere with the movement of the specimen slide and the stage between the stage fingers.

4.2.6.Position the specimen area of slide (Cover glass upside) over the centre of stage aperture. Use the stage control knobs to move specimen slide to the desired position.

4.2.7 Looking through the observation head, raise the stage by adjusting the coarse focus knob until an image appears. Focus as Sharply as possible with coarse focus knob. Adjust the fine focus to sharpen the image in the centre of the field of view.

4.2.8 Looking through the observation head, raise the stage by adjusting the coarse focus knob until an image appears. Focus as sharply as possible image. The clarity of the image depends upon the size of aperture. As the aperture becomes smaller, the contrast and depth  of focus increases but the resolving power decreases. The clearest image is produced by combination of these three factors.

4.2.9 Examine the specimen. When you find a feature you wish to observe at a higher magnification. Move the slide so that the feature gets centre in the field of view. Use the fine focus knob to sharpen the image.

4.2.10 When using objective of higher numerical aperture ( N.A.) proper focussing of the abbe condenser is important. Focus the abbe condenser by racking the condenser movement knob up and down so that the field is evenly illuminated.

4.2.11 This microscope has a sliding potentiometer coupled with electronic Circuit for regulating the light intensity slide the potentiometer from low to high as you go from low magnification to high magnification Objectives for obtaining best light in the field of view.

4.2.12 The procedure for examining a specimen using the oil immersion objectives is as follows.

4.2.13 Rotate the nose piece so that the lower power objective is in light path.

4.2.14 Place one drop of immersion oil ( Cedar wood oil ) on the lighted area of the specimen slide. Dust or air bubbles in the oil can destroy the image. If the bubbles are trapped between the objective lens and the slide, clean off the oil and start again.

4.2.15 Rotate the nose piece so that 100X oil immersion objective is in the light path.

4.2.16 With your eye at the level of the stage, use coarse focus knob to raise the stage with the specimen cover glass. When you see a flash of light at this location, the objective lens has made contact with the immersion oil and the microscope can be focussed using the fine focus knob.

4.2.17 Each time you finish using the oil immersion objective, wipe off all traces of oil from the objective and the specimen cover glass with a clean soft cloth.

4.3.0 Fine adjustment of Binocular head.

4.3.1 Rotate the Binocular head to bring it to a convenient position.

4.3.2 Adjust the interpupilary distance by bringing the eyepiece tubes closer or apart till you see one fused image.

4.3.3 If the image from both occulars does not fuse, you are required to do Dioptric adjustment on the occulars as below.

4.3.4 Bring 10X objective in position and focus the slide in the right eye with coarse and fine focus knob keeping the left eye closed.

4.3.5 Close the right eye and seeing through the left eye, focus the left Ocular up and down by rotating the focusing sleeve till the image is in sharp focus.

4.3.6 Look through both the eyes if interpupilary adjustment required.

4.3.7 fter viewing, the results should be recorded in the respective report format.

5.0 Cleaning of Microscope
5.1.1 After use clean the microscope to moisture and dust free.

5.1.2 Wipe the lens surface and objective with a cleaning soft cloth.

5.1.3 Use Xylol sparingly to remove oil from the objective.

5.1.4 Keep the Microscope in dust & moisture free wooden box.

5.15. Enter the cleaning details in the log book.

5.1.6 Frequency of cleaning: Once in a month.

6.0 Calibration of Microscope

6.1.0 Remove the eye piece and insert the ocular Micrometer.

6.1.1 The graduations in the ocular micrometer can be seen under sharp illumination.

6.1.2 The lines and distances will remain unchanged under different Objectives.

6.1.3 The stage micrometer is mounted on the microscopic field under a sharp focus.

6.1.4 This is done first with low power objectives and then under high Power objectives.

6.1.5 The scale of ocular micrometer and stage micrometer is adjusted in such a way that the lines of one should superimpose on the lines of other at one end of the microscopic field.

6.1.6 The number of divisions are counted lying in between two coinciding divisions of both ocular and stage micrometer.

6.1.7 Since we know the value of each division on stage micrometer the Value of division on the ocular can be calculated.

7.0 Frequency: Once in a month.

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