1. Describe the Principle of Bright Field Microscopy.

Compound light Microscopy A modern compound light microscope has a series of lenses and
uses visible light as its source of illumination.
With a compound light microscope, we can
examine very small specimens as well as some of
their fine detail. A series of finely ground lenses
forms a clearly focused image that is many times
larger than the specimen itself. This magnification
is achieved when light rays from an illuminator,
the light source, 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 (high 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 with 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 ability of the lenses to distinguish fine detail
and structure. Specifically, it refers to the ability of the lenses to distinguish two points a
specified distance apart. 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 shorter the wavelength of light used in the instrument, the greater the resolution. The white
light used in a compound light microscope has a relatively long wavelength and cannot resolve
structures smaller than about 0.2 m. This fact and other practical 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 1m.
To obtain a clear, finely detailed image under a compound light microscope, specimens must be
made to contrast sharply with their medium (substance in which they are suspended). To attain
such contrast, we must change the refractive index of specimens from that of their medium. 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 bright field illumination. It is not
always desirable to stain a specimen. However, an unstained cell has little contrast with its
surroundings and is therefore difficult to see.
G.Tortora. (2010). Microbiology: an introduction. In G. Tortora, Microbiology:an
introduction (pp. 55-60). San Francisco: Pearson Education, Inc.
Researcher: BALANGA, Lordalaiza G.