Microscopy and Aseptic Technique

Microscopy and Aseptic Technique

        Although Robert Hooke was the first person to publish work using a light microscope, Antony van Leeuwenhoek, is “considered to have made a greater contribution to our understanding of the microscopic world” (“What is,” n.d.). Leeuwenhoek confidentially made his own microscopes and observed several specimens at microscopic levels for the first time. (Gest, 2004). He even refuted the common belief that certain organisms were formed by spontaneous generation (Gest, 2004).  Microscopy has advanced tremendously since the first observation. This includes using more than one objective lens and more advanced technology allowing greater expansion and resolution of microorganisms by using coarse and fine adjustment knobs. To focus the light source, a condenser lens is used, which also ensures greater resolution (“Light Microscopy,” n.d.).  Microscopes magnification range from 4x to 100x.  “A light microscope allows us to view objects that are as small as 0.2 micrometers” (“Light Microscopy,” n.d). This is important, because it allows us to get a better idea of how microorganisms act and observe them at different objectives.

        This experiment is an introductory measure to ensure students understand how to work the light microscope. Although this is interpreted sometimes as a simple task, it is easy to make mistakes and spend more time than necessary trying to get the optimum picture. This laboratory experiment is beneficial because light microscopy is used frequently in Microbiology and also in other courses throughout Biology.

Procedure

The first step in this experiment once you have obtained a microscope from the storage cabinet is to identify the parts of the microscope. It is important to record the color, magnification and numerical aperture of each objective lens. You should also calculate the total magnification and resolution for each objective by using the equations: Ocular magnification x Objective magnification = Total magnification & Resolution = [(λ)/(2 x NA)]. The symbol λ represents wavelength of light (0.5 µm) and NA is the Numerical Aperture, which is printed on the lens.

After obtaining a slide, place it on the stage and in the clip. The stage should be at its highest point initially and then slowly lower the stage using the fine knob until your object is in focus. Observe the effects of changing the light intensity on the object. List the name of the specimen and record what you are visualizing. Make a drawing of a few cells, minimum 2 inches in diameter, and include the total magnification of resolution. Next, observe the specimen at the 10x objective. Be sure to use the fine focus knob and move the stage to get the best visual of the specimen. Make a drawing again of what you are visualizing.  Observe the specimen at 40x objective. Even though this magnification places the lens very close to the slide, do not move the stage. Make another drawing depicting the fine details of what you are seeing. Move back to 4x magnification to wipe the slide with a kimwipe and return.