MICROSCOPY

Types of Instruments
The microscope is one of the critical tools of the clinical microbiologist. A properly functioning
microscope assists the clinical microbiologist in (1) evaluating the quality of the clinical
specimen submitted; (2) observing the presence of potential pathogens in the clinical specimen;
(3) identifying the agents of disease. The careful microscopic examination of a specimen can
allow the clinical microbiologist to suggest the presumptive identity of a potential pathogen. This
information may allow the physician to initiate appropriate therapy in an expedited manner.
Such an approach can significantly reduce patient
morbidity and mortality.

The type of microscope most frequently used in the

clinical setting is the compound bright-field
microscope. This type of microscope uses visible
light and two sets of magnifying lenses, the objective
and ocular, to enlarge an object. When properly set
up, this type of microscope is capable of enlarging
an object up to 1000 times (1000X). The light
source is usually a tungsten lamp, occasionally a
brighter halogen lamp, located below the specimen
stage. The light produced by these lamps is focused
on the specimen by means of a substage
condenser, usually an Abbe' condenser. Kölher
type illumination is used to prevent the glare of the
lamp from reaching the observer's eyes.

Unfortunately, the optics employed in the bright-field

microscope are less than perfect. The curved
surface of a lens can bend various wavelengths of
light to different degrees. This results in white light Figure 1
being broken into separate colors (prism effect).
This is known as chromic aberration. Achromatic and apochromatic lenses are corrected
for most of the effects of chromic aberration. The spherical shape of the lens causes light to
travel different path lengths depending on where the light strikes the lens. This can create
distortion where different areas of the viewing field are in focus at different points. . This is
referred to as spheric aberration. Planachromatic and planapochromatic lenses are
corrected for both spheric and chromic aberration. Lenses corrected for spheric and chromic
aberration are still imperfect. These lenses often times can not focus on the entire viewing field
at one time. Flat field lenses allow the user to focus on the entire viewing field at one time.

Resolving power is defined as the ability to distinguish two closely spaced objects as distinct
objects. Resolving power is a direct function of the wavelength (λ) of the illuminating light.
Resolving power is also directly related to the numeric aperture (NA) of the observing system.
The relationship is described by the expression: Maximum Resolution = λ /(NAobj + NAcon) where
NAobj is the numeric aperture of the objective lens and NAcon is the numeric aperature of the
condenser. The numeric aperture is a measure of the angle formed by the maximum cone of
light that can enter the objective lens. This angle (θ or α
 ) is shown in Figure 2. The
relationship between NA and θ is expressed in the equation NA = η sin θ, where η is the
refractive index of the material between the lens and the specimen. Refractive index is the

Microscopy & Staining 1

ratio of the speed of light in a vacuum to the speed of light in the material between the objective
lens and the specimen. Therefore, the resolving power of a microscope can be increased by
either increasing θ or NA. For the light microscope, θ has been optimized. The NA for most
condensers is approximately 1.25 to 1.4.

The resolving power of a microscope can

be further improved by increasing the NA
of the material between the specimen and
the objective lens. The NA of air is very
close to 1, while the NA of the specimen
and the glass of the lens and slide is
greater than 1. Consequently, as the light
moves from the specimen through the air
it is bent (refracted). Each wavelength of
light is bent a different amount so that a
significant amount of distortion is
introduced into the system. This
distortion is especially pronounced when
examining a specimen at very high
magnification. Refraction can be
minimized by placing a substance with
the same NA as glass between the
specimen and the objective lens. This is
the function of the lens immersion oil
used with the 100X oil lens. Without
immersion oil, the image observed at
1000X would be very distorted. The
effect of oil is shown in Figure 2.
Figure 2
For most observing systems, the
resolving power can be estimated by dividing the wavelength of the illuminating radiation (λ) by
2. For the typical light microscope, a blue light is used. Thus, the resolving power is equal to
400nm/2 or 0.2μm. Two objects may be as close as 0.2µm and still be resolved as two distinct
objects. Increasing the magnification of the system does not increase the resolution of the
system. Lab microscopes are optimized to yield the best resolution at 1000X total
magnification.

In addition to the bright-field microscope, there are 2-3 other types of light microscopes that are
used in the clinical microbiology laboratory.

The Fluorescence Microscope (Figure 10-18) is utilized to visualize materials that have been
stained with a fluorescent dye. Fluorescence is a property of some compounds
(fluorochromes) where light energy of one wavelength is absorbed and then immediately re-
emitted at a second wavelength. In the clinical setting, high energy ultra violet light is used to
illuminate the stained specimen. The light is absorbed by the dye and re-emitted in the visible
range. When properly stained, the specimen appears as a bright colored object against a
dark/black background. Most microscopes in the clinical setting are built for epifluorescence;
the illuminating light passes from the source (usually a mercury vapor lamp) through an exciter
filter, through the objective to the specimen. The exciter filter selects for the wavelength that
"excites" the fluorochrome to fluoresce. The re-emitted light travels back through the objectives,
past a barrier filter to the oculars. The barrier filter protects the observer's eyes from any
Microscopy & Staining 2
reflected UV light. The primary advantage of fluorescence microscopy is that, in the hands of a
trained microscopist, the amount of time required to observe a slide is dramatically reduced.
The major use of fluorescent microscopy currently is the observation of sputum for
Mycobacterium tuberculosis. The observation time per slide can be reduced from 15-30
minutes to 5 minutes. Other uses of fluorescence microscopy include the detection of certain
pathogens in clinical material through the use of fluorescent dyes coupled to specific antibody
and the rapid detection of pathogens in body fluids such as blood and spinal fluid (Figure 10-
19). These procedures will be discussed in more detail in the appropriate sections of the
course.

The Dark-field Microscope is used to improve the detection of unstained organisms in clinical
specimens. The optics of the dark-field microscope are adjusted to take advantage of the fact
that light passing through a specimen at an oblique angle will be bent (refracted). A special
condenser is used which prevents any light from directly entering the objective lens. Light
passing through a specimen will be bent slightly so that it will now enter the objective lens. The
specimen appears white against a black background. The dark-field microscope is used
primarily to detect Treponema pallidum, the causative agent of syphilis, in lesion material. It
may also be used to observe the characteristic "darting" motility of Campylobacter jejuni, an
agent of gastroenteritis, in stool specimens.

The Phase Contrast Microscope also utilizes the effect of a living, unstained specimen on the
passage of light to improve the detection of unstained specimens in clinical material. The speed
of light passing through material of differing densities is altered to varying degrees. The speed
of a ray of light passing through a microorganism will be slowed slightly in comparison to light
that misses the microorganism. The phase of the two light rays will shift with respect to each
other. When the two rays are not in phase, that is the amplitude peaks of the rays are not
aligned, interference will occur. When these two rays meet at the observer's eyes they will be
perceived as an increase or decrease in the intensity of the initial light. Imagine two waves
meeting on the surface of a pond or river. If the two wave peaks meet, the result is a single
wave the height of the two added together. If the peak of one wave meets the trough of the
other, the effect will be a flattening of the peak and trough or interference. Unfortunately, the
shifts produced when light passes through a microorganism are very small. A phase contrast
microscope uses a special condenser and objective to enhance these differences. The
condenser adjusts the light from the source so that all light rays are in phase. The objective has
a phase shift filter that further alters the phase of light rays that have passed through the
specimen. This shift increases the interference between light rays which improves the visibility
of the organism. The phase contrast microscope is sometimes used to enumerate
microorganisms directly observed in clinical material.

The Electron Microscope is being increasingly used in clinical situations, especially for the
detection of viruses from specimens. This microscope uses a beam of electrons as the
illumination source and uses a series of magnets to focus the beam. The beam is visualized by
focusing it on a phosphorescent screen similar to that used in a TV. The primary advantage of
the electron microscope is 103 fold increase in resolution which permits much higher levels of
magnification. The wavelength of the electron beam ranges between 0.4nm and 0.04 nm.
Therefore, the potential resolving power of an electron microscope is 0.2nm to 0.02nm.
Unfortunately, the cost of the instrument, the difficult staining procedures and problems with
artifacts currently limit the use of this instrument in the clinical setting.

Microscopy & Staining 3

 STAINING

While some of the instruments described above can be utilized to directly observe bacteria and
other microorganisms in clinical material, it is usually necessary to alter the specimens to
improve their contrast. This is accomplished by adding color to the structure. There are two
basic types of stains: simple stains which color all objects in the same manner and differential
stains which are used to detect differences in structure among microorganisms. Since the
surfaces of most microorganisms are negatively charged, basic (positively charged) dyes are
employed.

A simple stain is used to improve the visualization of all organisms in the specimen. Such a
stain allows for the enumeration of organisms and permits some determination of shape and
size. In order to determine more about a microorganism a variety of differential stains are
employed.

Most differential stains consist of four components. The first component is the primary stain.
The primary stain usually stains the target cell as well as most non-target cells and background
debris. The mordant is a chemical or physical process that fixes the primary stain to the target
cell. The decolorizer is a chemical that removes unfixed primary stain from non-target cells.
The final component is the counterstain that is employed in many differential staining
procedures in order to visualize non-target cells. Some differential staining protocols do not
employ a secondary or counterstain.

The most commonly employed differential stain is the Gram stain. As discussed previously,
this stain separates bacteria into two major categories based on differences in the cell wall
structure. The four components of the Gram stain are: the primary stain, crystal violet; the
mordant, Gram's iodine; the decolorizer, alcohol or acetone; and the secondary or
counterstain, safranin. Cells that are gram positive retain the primary stain after
decolorization while Gram negative cells are decolorized by the alcohol or acetone. Gram
negative cells are stained pink by the safranin counterstain. In the Gram stain the secondary
stain is additive. Gram positive cells retain both the primary and secondary stain and are
colored purple.

A second commonly used differential stain is the Acid-fast stain. This stain differentiates
members of the genus Mycobacterium and related organisms from other bacteria. Acid-fast
bacteria have unusual cell wall components that affect their staining properties. The primary
stain is carbol-fuchsin, which colors target and non-target cells red. One of two mordants is
utilized depending on the procedures, heat, or phenol. The decolorizer for either protocol is a
mixture of acid and alcohol, hence the name acid-fast for target cells. The most commonly
employed counterstain is methylene blue. When performed correctly, acid-fast organisms are
red and non acid-fast organisms are blue. A fluorescent protocol exists in which a fluorochrome
is used as the primary stain, heat is the mordant, and a non-fluorescing counterstain is used.

These are the major staining protocols for bacteria used in the clinical setting. The exact
procedures for performing each will be presented in the laboratory. Specialized staining
protocols will be introduced at the appropriate time.

Microscopy & Staining 4