Microscope: Parts, Magnification, and Usage

• The microscope magnifies objects that cannot be seen by the naked eye.
• It uses a system of lenses with sufficient magnification and resolving power.
• Sufficient illumination helps distinguish small elements in a specimen.

Topic Learning Outcomes

• Mastering the parts of a compound microscope and their functions
• Explaining magnification and resolution principles in microscopy
• Learning to use micrometers to measure microorganisms
• Becoming familiar with the cellular structures of microorganism groups

Materials

• Compound microscopes can be monocular or binocular.
• The materials needed are, Ocular Micrometer, Stage Micrometer, immersion oil, cedarwood oil, lens paper, xylene and cotton.
• Prepared slides contain Bacteria (Bacillus, Staphylococcus, Spirillum), Protozoans (Amoeba, Euglena, Paramecium), and Fungi (Aspergillus, Penicillium, Rhizopus, Saccharomyces).

Part of a Microscope

• The ocular or eyepiece is where you look into the microscope, containing lenses to increase magnification, and inserted into the draw tube.
• The draw tube is a cylindrical part where the eyepiece is inserted.
• The body tube/barrel holds the eyepiece and objectives at the correct distance.
• The coarse adjustment knob is a large wheel to moves the body tube significantly for initial focusing, used with low power objectives.
• The fine adjustment knob is a smaller wheel that brings the specimen to its sharpest focus by moving the body tube slightly.
• The dust shield protects the objectives from dust.
• The revolving nosepiece is where objectives are attached rotates to change objectives.
• Objectives are small tubes that contain varying magnification power, attached to the nosepiece.
• The low power objective (LPO) is marked as 10x.
• Scanning Objectives can be present (4x or 5x)
• High power objectives (HPO) are typically marked 40x, 44x, or 45x.
• Advanced microscopes may also have a 100x oil immersion objective.
• The stage is the flat area where the slide is placed for examination.
• A stage aperture allows light from the mirror to pass through.
• Stage clips hold the slide in place.
• The arm supports the body tube.
• The mirror reflects light through the stage aperture, and is used to illuminate the specimen
• Concave mirrors are used when a condenser is absent.
• Flat mirrors are used if there is a condenser
• Some microscopes use a lamp as a light source.
• The condenser focuses light on the specimen.
• The pillar is an upright bar that connects the base.
• A diaphragm regulates the amount of light passing through the specimen, functioning as a rotating disk or iris.
• The inclination joint allows tilting of the microscope.
• The base is the horseshoe-shaped bottom that supports the microscope.

Microscope Objectives

• Focal length (mm) is the distance from the lens center to the point where parallel rays come to a focus.
• Numerical aperture (N.A.) measures the resolving power of an objective.
• An objective with 0.25 N.A. can distinguish 25,000 lines per inch.
• Working distance (mm) is the space between the specimen and the objective.

Magnification

• The microscope should be plugged in, and the light switch must be on.
• Place the prepared slide (Amoeba) on the stage, secured by the slide holder.
• Position the specimen area over the stage aperture.
• Looking through the eyepiece, raise the coarse knob until the image appears and focus as sharply as possible.
• If the light is too much, control iris diaphragm for best contrast
• Shift to the high-power objective (HPO). Adjust the fine knob to sharpen the image.
• If the microscopic field is dark, control the iris diaphragm to increase the light intensity.
• To retain focus, relax the eyes and keep them at a distance from the eyepiece with both eyes open.

Resolution

• Focus a prepared slide of Bacillus subtilis under low power objective (LPO), choosing a very clear area for the bacteria
• Shift to HPO and bring the bacteria into focus.
• Shift to the oil immersion objective (OIO) and adjust with the fine knob for final focusing.
• Rotate the revolving nosepiece and apply a small drop of immersion oil to the specimen above the stage aperture.
• Return the OIO to its location. The front lens should be immersed in oil and close to the slide.
• Look into the eyepiece, and adjust with fine knob for clearer focusing.
• Use lens paper moistened with xyoil to blot the oil off after use.

Measurement of the Specimen

• Use a microscope is provided with an ocular micrometer
• Using the LPO, look into the eyepiece to ensure that the grid lines are upright
• Place the stage micrometer on the stage and focus on its scale.
• Arrange the stage micrometer so that the first division coincides with a division on the stage scale.
• Look across the field for another line on the ocular micrometer coinciding with the stage scale
• Count the divisions on the ocular micrometer subtended by the divisions on the stage micrometer to compute the ocular unit.
• Repeat steps 1-5 above with HPO and OIO
• Remove the stage micrometer and replace with a prepared slide.
• Focus the specimen and line it up along the ocular micrometer.
• Determine the specimen's dimension using the formula (X)(OU) = (Y)(SU)

Formula definitions

• X = Divisions of the ocular micrometer (OM) subtended by stage micrometer (SM) divisions.
• Y = Divisions of the stage micrometer (SM) subtended by ocular micrometer (OM) divisions.
• SU = Stage units (0.01 mm)
• OU = Distance between divisions of the OM