### Lesson 12 Microscopy

Because microorganisms and their component parts are so very small, they are measured in units that are unfamiliar to many of us in everyday life. When measuring microorganisms, we use the metric system. The standard unit of length in the metric system is the meter (m). A major advantage of the metric system is that the units are related to each other by factors of 10. Thus, Im equals 10 decimeters (dmj or 100 centimeters (em) or 1000 millimeters (mm). Units in the U.s. system of measure do not have the advantage of easy conversion by a single factor of 10. For example, we use 3 feet or 36 inches to equal I yard. Microorganisms and their structural components are sured in even smaller units, such as micrometers and nanometers.

A micrometer (j-lm) is equal to 0.000001 m (10--6 m). The prefix micro indicates that the unit following it should be divided by I million, or 106 (see the "Exponential Notation" section

in Appendix B). A nanometer (nm) is equal to 0.000000001 m (10--9 m). Angstrom (A) was previously used for 10-10 m, or 0.1 nm. Table 3. 1 presents the basic metric units of length and some of their U.S. equivalen ts. In Table 3.1, you can compare the microscopic

units of measurement with the commonly known scopic units of measurement, such as centimeters, meters, and kilometers. If you look ahead to Figure 3.2 on page 58, you will see

the rela tive sizes of various organisms on the metric scale. The simple microscope used by van Leeuwenhoek in the seventeenth century had only one lens and was similar to a magnifying glass. However, van Leeuwenhoek was the best lens grinder in the world in his day. His lenses were ground with such precision tha I a single lens could magnify a microbe 300X. His simple microscopes enabled him to be the first person to see bacteria. Contemporaries of van Leeuwenhoek, such as Robert Hooke, built compound microscopes, which have multiple lenses. In fact, a Dutch spectacle maker, Zaccharias Janssen, is credited with making the first compound microscope around 1600. However, these early compound microscopes were of poor quality and could not be used to see bacteria. It was not until about 1830 that a significantly better microscope was developed by Joseph Jackson Lister (the father of Joseph Lister). Various improvements to Lister's microscope resulted in the development of the modern compound microscope, the kind used in microbiology laboratories today. Microscopic studies of live specimens have revealed dramatic interactions between microbes Light microscopy refers to the use of any kind of microscope that uses visible light to observe specimens. Here we examine several types of light microscopy. A modern compound light microscope has a series of lenses

and uses visible ligh t as its source of illumination. With a compound light microscope, we can examine very small specimens as well as some of their fi ne detail. A series of fi nely ground lenses (Figure 3.1b) forms a clearly foc used image that is many times larger than the specimen itself. This magnification is achieved when light rays from an illuminator, the light sou rce, pass through a condenser, which has lenses that direct the light rays through the specimen. From here, light rays pass into the objective lenses, the lenses closest to the specimen . The image of the specimen is magnified again by the ocular lens, or eyepiece. We can calculate the total magnification of a specimen by multiplying the objective lens magnification (power) by the ocular lens magnification (power). Most microscopes used in microbiology have several objective lenses, including lOX (low power). 40X (h igh power), and lOOX (oil immersion, which is described shortly). Most ocular lenses magnify specimens by a factor of 10. Multiplying the magnification of a specific objective lens wi th that of the ocular, we see that the total magnifications would be lOOX for low power, 400X for high power, and lOOOX for oil immersion. Some compound light microscopes can achieve a magnification of 2000X with the oil immersion lens. Resolution (also called resolving power) is the abil ity of the lenses to distingu ish fine detail and structure. Specifically, it refers to the abili ty of the lenses to distinguish two points a specified distance aparl. For example, if a microscope has a resolving power of 0.4 nm, it can distinguish two points if they are at least 0.4 nm apart. A general principle of microscopy is that the short - er the wavelength of light used in the instrument, the greater the resolut ion. The white light used in a compound light microscope has a relatively long wavelength and cannot resolve structures smaller than about 0.2 Ilm. This fact and other practi cal. considerations limit the magnification achieved by even the best compound light microscopes to about 2000X. By comparison, van Leeuwenhoek's microscopes had a resolution of I j.Lm. The refractive index is a measure of the light-bending ability of a medium. We change the refractive index of specimens by staining them, a procedure we will discuss shortly. Light rays move in a straight line through a single medium. After the specimen is stained, when light rays pass through the two materials (the specimen and its medium) with different refractive indexes, the rays change direction (refract) from a straight path by bending or changing angle at the boundary between the materials and increase the image's contrast between the specimen and the medium. As the light rays travel away from the specimen, they spread out and enter the objective lens, and the image is thereby magnified. To achieve high magnification ( 1000X) with good resolution, the objective lens must be small. Although we want light traveling through the specimen and medium 10 refract differently, we do not want to lose light rays after they have passed through the stained specimen. To preserve the direction of light rays at the highest magnification, immersion oil is placed between the glass slide and the oil immersion objective lens (Figure 3.3). The immersion oil has the same refractive index as glass, so the oil becomes part of the optics of the glass of the microscope. Unless immersion oil is used, light rays arc refracted as they enter the air from the slide, and the objective lens would have to be increased

in diameter to capture most of them. The oil has the same effect as increasing the objective lens diameter; therefore, it improves the resolving power of the lenses. If oil is not used with an oil

immersion objective lens, the image becomes fuzzy, with poor resolution. Under usual operating conditions, the field of vision in a compound light microscope is brightly illuminated. By focusing the light, the condenser produces a brightfield illumination (Figure 3.4a). It is not always desi ra ble to stain a specimen. However, anunstained cell has little contrast with its surroundings and is therefore difficult to sec.