General Microbiology LaboratoryGeneral Microbiology Laboratory

Introduction to the oil immersion compound microscope

Mohammed Laqqan

• In this lab, you will become familiar with the use of the microscope (particularly oil immersion microscopy) and will compare the relative size and shape of various microorganisms.

• Most bacteria range in size between 0.5-2.0 micrometers (μm)

• There are three common shapes of bacteria: the coccus, the bacillus, and the spiral.

Mohammed Laqqan

Illustration, cocci Illustration, bacilli

                                                               

                       Illustration, spirochete

                                               

                                

Mohammed Laqqan

This is the microscope used in our class. • iris diaphragm lever: moving the

lever to the left increases the light; moving the lever to the right decreases the light.stage control "A": moves the mechanical stage holding the slide forward and backward.stage control "B": moves the mechanical stage holding the slide left and right.coarse focus: turning the knob away from you raises the stage; turning the knob towards you lowers the stage.fine focus: turning the knob away from you raises the stage; turning the knob towards you lowers the stage.

Mohammed Laqqan

Proper handling of a microscope. Both hands are used when carrying this delicate instrument.

Mohammed Laqqan

When adjusting the microscope, the technologist observes the objective carefully to prevent breaking the slide and damaging the objective lens of the microscope

Mohammed Laqqan

How to use microscope

1. Obtain a prepared slide of mixed bacteria. Mount the slide onto the stage of the microscope.

2. Start with the lowest power objective in place. Using the course adjustment knob, move the objective lens to its lowest point. Look through the ocular and focus upward with the coarse adjustment until an image comes into view. Use the fine adjustment to obtain maximum clarity.

3. Move the slide to a point of interest. Move the next objective lens into place and adjust the fine focusing knob, and adjust the iris as necessary. Repeat this step with the highest power, non-oil lens

4. Note that as the power of the objective lens increases, the distance between the objective and the specimen (working distance) decreases. Also, as magnification increases, the field of view (visible area) and depth of field/focus (visible thickness) decrease. Moving the fine adjustment up and down allows viewing of other areas along the depth of thickness of the specimen

Mohammed Laqqan

Definitions and Relationships• Resolution• Magnification• Depth of Focus• Field of Vision• Numerical Aperture

Some Concepts to Consider

Mohammed Laqqan

• Resolution is the ability to distinguish between two points.

• The closer the two points, the higher the resolution.

Resolution

Mohammed Laqqan

• Relative enlargement of the specimen

• The power of magnification is calculated by multiplying the power of the eye piece lens by the power of the objective lens.

• A specimen is magnified as light passes through the objective and ocular lens.

Magnification

Mohammed Laqqan

More Concepts…• Depth of focus - thickness of a specimen

that can be seen in focus at one time;as magnification the depth of focus .• Field of vision - the surface area ofview; the area as magnification .• Numerical aperture (N.A.) – the amountof light reaching the specimen;As N.A. resolution .

Mohammed Laqqan

Oil immersion

5. To use the oil-immersion lens, move the turret halfway between the high-power air (non-oil) lens and the oil lens. Place a drop of immersion oil directly on the slide. Move the oil-immersion lens into place and adjust the fine focusing knob. Adjust the iris as necessary. Make sure that the immersion oil does not get on the air lenses. Make note of the differences and similarities between the organisms.

Mohammed Laqqan

Resolution can be increased by using immersion oil.

Mohammed Laqqan

Cleaning

6. After using the oil lens for a specimen, wipe the lens with a piece of lens paper. Do not use anything but lens paper to clean microscope lenses. Usually, lens-cleaning fluids are not necessary unless the lens is exceptionally dirty.

Mohammed Laqqan

Care and Handling of the Microscope

1. Always use both hands to carry the microscope, one holding the arm, one under the base.

2. Before each use, examine the microscope carefully and report any unusual condition or damage.

3. Keep the oculars, objectives, and condenser lens clean. Use dry lens paper only.

4. At the end of each laboratory period in which the microscope is used, remove the slide from the stage, wipe away the oil on the oil-immersion objective, and place the low-power objective in vertical position.

5. Replace the dust cover, if available, and return the microscope to its box.

Mohammed Laqqan

END OF LECTURE