Microscope - Food Microbiology - Lab Manuals, Summaries of Microbiology

Download Microscope - Food Microbiology - Lab Manuals and more Summaries Microbiology in PDF only on Docsity! Exercise 1 The Microscope The microscope is one of the most essential tools of the microbiologist. Learning how to operate your microscope is an absolutely necessary skill to develop in order to view bacteria. Bacteria are very small and in most cases transparent. Good microscopy relies on three factors: magnification, the enlarging of the image of our specimen; resolution, the ability to distinguish individual points; and contrast, being able to distinguish the organism from its surroundings. Lenses will accomplish the magnification, light and oil will aid in resolution, and stains and dyes will improve contrast. The Merrill 29 laboratory is currently equipped with Nikon YS100 microscopes (Figure 1). These are compound light microscopes. A light source beneath the specimen creates a lit background, or bright-field. The specimen, because it blocks the light being transmitted, will appear dark against this bright-field. Because bacteria do not block much light they are generally transparent. The ability to see these organisms is improved against this bright-field by adding stains. Stains adhere to the specimen, or parts of the specimen, or to the background. The stains, being opaque, block light from being transmitted, thereby improving contrast. The Nikon YS100 is a compound microscope. There are a series of lenses used to produce and magnify the image. The first lens system, located below the stage is the condenser lens. This lens is used to focus light on the specimen. It could have easily been known as the 'light concentrator.' A lever under the left side of the stage allows us to control the positioning of the condenser lens. depending on the For our purposes, the condenser lens should be positioned all the way up so it sits just below the slide. The amount of light passing through the condenser lens can be controlled using the iris diaphragm. A lever located on the front side of the condenser permits opening and closing of the diaphragm. This allows regulation of the light intensity passing through the specimen. Often, when you close down the iris diaphragm, you can improve contrast and increase your depth of field. You can also control light intensity using the voltage control on the right side of the microscope but using the iris diaphragm is the preferred method. It should be noted Figure 1: Nikon YS100, the compound light microscope used in Bio 27 Figure 2: Under stage; iris diaphragm, condenser lens Docsity.com the condenser lens contributes significantly to the resolution of the specimen but it does not magnify the image. The next system of lenses are the objective lenses located on the rotating nosepiece. The microscope comes with four objective lenses, each with a different magnification: 4X (scanning), 10X (low power), 40X (high power), and 100X (oil immersion). Always begin your observations under the 4X objective before moving to the higher powers. Additionally, the 4X objective should be locked in position when putting your microscope away. The objective lenses magnify the specimen to produce a real image. This image is then magnified a second time by the last set of lenses, the ocular lenses, to produce a virtual image. This is what you see. The ocular lenses are located in the eye pieces you look through. These lenses will magnify the real image created by the objective lenses by 10X. This means the total magnification of the specimen in view is actually the sum of multiplying the objective lens magnification by the ocular lens magnification. For example, if using the oil immersion lens, the image will be 100X (the objective magnification) times 10X (the ocular magnification), or 1000X larger than actual size. Whenever recording the magnification of a specimen on a drawing, be sure to include the total magnification. Before moving on to discuss the other parts, and operation of, the microscope, it is important to discuss the concept of resolution. Simply defined, resolution is the ability to distinguish two objects as being separate. It refers to the fineness of detail observable in the image. The better the resolution the clearer the image. Simply magnifying the image does not necessarily mean we will have good resolution. If you magnify a fuzzing image, it is still fuzzy. Focusing light on the specimen, like the condenser lens does, can improve the resolution. Using oil with the immersion oil lens prevents the refraction of light and improves resolution further. But there is a limit to our microscopes resolving power. This limit is dictated by the wavelength of light used and the properties of the condenser lens. For most light microscopes, the best resolution achievable is about 0.2 micrometers. Turning our attention to the other parts of the microscope. The basic frame structure of most microscopes consists of the base and arm. When carrying your microscope one hand should grasp the arm and the other should support the base. Observe the horizontal platform, the stage, it is used to support the microscope slides. Notice a stage clip on the platform is used to hold the slide in place. The YS100 has what is known as a mechanical stage. The clip holds the slide in place and control knobs located on the right side of the microscope allow you to move the slide back and forth and side to side on a horizontal plane. The mechanical stage allows for smooth movements when scanning the slide. The light source is turned on and off using the switch on the backside of the microscope. The intensity of the light is controlled by the voltage control knob, or rheostat, located on the right side of the arm. The voltage control knob should be turned all the way down before turning your microscope on or off. Focus knobs are located near the base on both sides of the microscope. On the left side you will see two knobs concentrically arranged. The course focus knob is the large outer knob. This knob should only be used in conjunction with the scanning objective. The working space between your slide and the other objectives is too small, and the movements of the stage with the course knob too great, to use the course focus adjustment knob with any other objective. The fine focus adjustment knob is the inner, smaller knob of the two. You will also find a fine focus adjustment knob on the right side of the microscope. The fine focus adjustment knob can be used with all objectives when small adjustments are needed. When using the higher powered objectives you will only use the fine focus adjustment knob. Docsity.com