Light Microscopy

RW Lovitt and CJ Wright, University of Wales, Swansea, UK

Ó 2014 Elsevier Ltd. All rights reserved.
This article is reproduced from the previous edition, volume 2, pp 1379–1388, Ó 1999, Elsevier Ltd.

Introduction
Ever since the role of microorganisms in fermentation was
realized light microscopy has become an essential technology
for the food microbiologist. Historically the techniques of light
microscopy developed from the use of single convex lens as
magnifying glasses, the invention of the first practical micro-
scope being accredited to Antony van Leeuwenhoek in 1668.
Leeuwenhoek’s microscope had a magnification ratio of 270 to
1 and consisted of a single lens that was moved up and down
by a screw mechanism. Today there is a vast variety of micro-
scopes available of different design and quality employing
a number of light microscopy techniques to study microbio-
logical structures and their interactions with the environment.
Light microscopy is routinely used in microbiological
laboratories often without consideration for the finer details of
the instrumentation. The full potential of light microscopy can
be realized when a light microscope is used by an operator
knowledgeable of the underlying principles of the instrument
and of microbiological staining methods. The specimen prep-
aration and correct staining procedures are paramount to
effective light microscopic study of food microbiological
samples. Direct examination by the experienced eye is a rapid
cost-effective diagnostic aid to estimating the quality of food,
the nature of an infection or the purity of the culture. Despite
many limitations, microscopic observation of microbes is
normally necessary for identification. There are many methods
for observing and characterizing microbes and many date back
to the last century. The primary aim is to identify and
enumerate the microorganisms present. It may also be used to
determine the strategy for further microbiological examination
and investigation.
principal focus point and thus allow the eye to focus objects at
different distances. The eye can see an object with greatest
clarity when it is positioned at the near point. A distance away
from the eye termed the least distance of distinct vision
(D)(Figure 1(c)). When using a magnifying glass the observer
moves the lens until the image is situated at the near point
(Figure 1(d)). The object is located within the focal distance.
For high magnification a lens of short focal length is required.
The fabrication of lens with focal lengths below a certain limit
is impracticable, to increase higher magnification two separate
convex lenses are used. This is the basis of a compound
microscope (Figure 1(e)). The lens close to the object is termed
an objective. The lens that is used to observe the final image is
called the eyepiece.
The power of an objective is shown by its resolution. The
resolution (R) of the objective lens is the smallest distance
between two points that can be differentiated in the generated
Principal focus
(F1)
(a)
F1 Image (I)
Object (O)
(b)
O

(c) D
Principles of Light Microscopy

A basic knowledge of the theory of light microscopy is useful to

understand how the general instrument works, optimization
procedures, instrumentation specifications and technological F1 O
(d)
innovations. A good physics textbook should be consulted if D
a more detailed explanation of image production by lenses is
required. A light microscope essentially consists of three D
components, the eyepiece, the objective and an illumination
source (condenser). The last two have greatest influence on Objective Eyepiece
image quality. Both objective and condenser are constructed I2 FE I1
from lenses. Thus their performance is governed by the effi- O Fo
ciency of their component lenses light transmission.
Convex lenses are used in light microscopy because they
converge incident light into a principal focus (Figure 1(a)).
(e)
Figure 1(b) shows how a convex lens forms an image. The
human eye contains a convex lens that produces images of Figure 1 Ray diagrams illustrating how light travels through the lenses
distant objects on the retina at the back of the eyeball. The of optical instruments: (a) and (b) convex lens; (c) human eye;
ciliary muscles change the shape of the lens to change the (d) magnifying glass; and (e) compound microscope.

684 Encyclopedia of Food Microbiology, Volume 2 http://dx.doi.org/10.1016/B978-0-12-384730-0.00213-5


image. The resolution is found to equal 0.61 l / A where l is the
wavelength of the light and A is the numerical aperture. This
equation defines the limits of light microscopy. Small values of
l improve resolution. The numerical aperture represents the
ability of the objective to gather the rays coming from each
illuminated point on the specimen and it is a function of the
refractive index of the material between the lens and the
sample. The theoretical maximum value for the numerical
aperture is equal to the refractive index of the material between
the objective and the sample. When the numerical aperture is
large then the resolution of the system is increased. Thus the
addition of immersion oil increases the magnification of
a sample, the refractive index of oil is about 1.52 compared to
1.00 that of air.
The efficiency of lenses and thus objectives and condensers
are subject to aberrations. Spherical aberration occurs with all
single lenses and results in a halo of unfocused light around the
image that is then blurred. It is due to the uneven bending of
transmitted light rays and forms images at various distances
along the axis. Chromatic aberration is due to the differential
bending of light of different wavelengths. The shorter the
wavelength the larger the bending. In this case, different
colored images are formed at different points along the prin-
cipal axis, red furthest away from the lens, violet nearest the
lens. On examination of these images they are found to have
a rainbow-like fringe due to the unfocused light of the other
colors. Spherical aberration and chromatic aberration of the
system can be corrected simultaneously by joining lenses, of
equal and opposite error, to form doublet or triplet lenses.
The choice of objective is paramount in light microscopy
and there are many types available each designed to give best
results for particular purposes. Objectives are multiple lens
systems constructed to minimize aberrations and to enhance
differentiation of sample features. To this end objective lens
systems have different numerical apertures, and different
working distances and are constructed from different lens
combinations. The working distance is an important specifi-
cation of an objective and it is the distance between the object
and the objectives front lens, when the system is correctly
focused. The objective will be redundant if the depth of the
sample and cover slip is greater than the working distance of
the objective. Achromatic objectives are economic and the
most widely used objectives, they have moderate numerical
apertures and working distances. Apochromatic objectives are
more complex lens systems than achromatic lenses and
produce very high quality images. Combinations of doublets,
triplets, specially fabricated and shaped lenses ensure that
apochromatic objectives have high numerical apertures and
perfect color rendering.
Fluorite objectives are examples of objectives that contain
lenses fabricated from a different material to glass or quartz.
Fluorite objectives characteristically increase the contrast
between objects and their surroundings; they also act to darken
some colors. Hence these lenses are useful when used in
conjunction with staining methods. Fluorite objectives have
performance close to that of apochromatic lenses. The
construction of objectives for phase contrast is discussed below.
An important consideration in light microscopy that is
often overlooked is sample illumination. It is imperative that
the plane at which the objective is focused is uniformly
MICROSCOPY j Light Microscopy 685
illuminated with light that passes through the object and forms
a cone that is large enough to fill the front of the objective. To
meet the illumination requirements of the different types of
objectives there are equally as many types of condenser.
A compromise is made with the condenser and in most
instruments a general-purpose condenser is used possibly with
the addition of a specialist unit if more advanced techniques
such as phase contrast are routine. If the specimen is mounted
on an opaque substrate then reflected light as opposed to
transmitted light can be used to view the surface. Epi-
illumination is used to direct light actually through the objec-
tive focusing light and image simultaneously.
General Design and Operation
Figure 2 shows the general design of the simple light micro-
scope used in microbiological laboratories; the following steps
describe its routine operation. A suitable slide is placed onto
the microscope stage with the lowest power lens in position.
Before viewing the sample through the eyepiece the objective
should be brought close to the slide surface using the coarse
adjustment. The sample is then focused by retracting the
objective whilst viewing through the eyepiece. This procedure
will prevent damage to the objective and the slide. The illu-
mination should then be optimized by adjusting the position
of the condenser and the size of the aperture until a homoge-
neous bright illumination is achieved without glare. Final focus
correction can be achieved by the intuitive movement of the
fine adjustment.
If required, the sample can next be viewed under a higher
magnification. Many microscopes hold parfocal objectives that
have the same focal positions. Thus only small adjustments
will be needed to focus the higher-powered lens. Care should
be taken when changing objectives as adjacent lenses may not
be parfocal and oil immersion lenses have short working
distances. In addition sample and cover slip depth can vary. If
the microscope has an achromatic oil-immersion lens, a drop
Camera/video port
Eyepiece
Revolving nosepiece
Objective
X and Y translator
Slidestage
Coarse
Substage
focusing
Substage iris
diaphragm & control Fine
Light rays focusing
Lamp house
Bulb intensity
adjustment
Figure 2 The general design of a typical microbiological laboratory light
microscope.
686 MICROSCOPY j Light Microscopy
of oil can be placed on top of the cover slip so that when the
objective is positioned a meniscus of oil forms between the
two surfaces increasing resolution. If after focusing and illu-
mination optimization procedures, the image remains poor
then the meniscus of oil may be compromised by bubbles or
lens surface grime. The solution to this problem is to raise the
objective and clean the surface oil and grime from the lens
surface before reforming the meniscus. This is modern light
microscopy in its simplest form and is often adequate when
used alongside effective staining techniques (see below). If
however staining is not a viable option or greater image
differentiation is required further light microscope techniques
should be considered.
Dark-Field Illumination
In normal light microscopy there are many objects that cannot
be seen because they are transparent. To improve the image an
increase in contrast between the object and its surroundings
is required. Dark-field illumination functions to improve
contrast. In normal light microscopy the condenser functions to
ensure the sample is uniformly illuminated and that light enters
the objective correctly. In dark-field illumination the condenser
functions to produce a hollow cone of light which is focused on
the specimen. Only light that is scattered by the specimen will
enter the objective. Thus the specimen will be seen illuminated
against a dark background. In low-power dark-field illumina-
tion the long working distance of the objective means that it is
relatively easy to reduce the entry of light into the objective. A
disc or a diaphragm with moveable leaves stops the central rays
of the cone of light. An annular cone of light can be focused on
the specimen without any direct light entering the objective.
High power dark-field illumination is used regularly alongside
oil immersion to view living organisms. However, the short
working distance of the required objectives means that a special
dark-field condenser is needed. The modern design of these
condensers is effective for objectives with numerical apertures
between 1 and 1.15, for greater apertures a further means, such
as a in-built iris diaphragm within the objective, is required to
reduce the passage of direct rays. The microbiological sample to
be viewed should be well spaced and within a single plane; thus
samples should be dilute. The spaces between the organisms
provide the dark background that contrast with the illuminated
specimen. If the objects are in multiple planes then the scat-
tering of light by all the planes will mask the dark field of the
individual plane. Both specimen and immersion oil should be
free of bubbles or dust that act on the passage of light
compromising dark-field illumination.
Phase-Contrast Microscopy
Phase-contrast microscopy is an extremely useful technique for
observing specimens that have not been stained and are in their
natural state. Objects, that under normal light microscopy
cannot be seen, are observed in sharp outline and in good
contrast to their surroundings. Light travels in the form of
waves of energy, and so has wave length and amplitude
parameters. When two waves meet, the resultant light ray will
have wave parameters that will depend on whether the two
original waves were in phase, for example peak arriving with
peak, or out of phase, valley arriving with peak. In a light
microscope the component parts ensure that direct and dif-
fracted rays meet at the eyepiece producing brightness and
shade according to the phase relationship of the incident rays.
The image observed is in reality a complex interference pattern.
The greatest contrast is achieved when the direct and diffracted
rays meet at the point of interference out of phase by about half
a wavelength. The specimen has regions of different thickness
and different refractive indices, thus rays are diffracted to
different degrees. If the specimen is transparent with a density
and thickness insufficient to create the required phase differ-
ence then contrast will be poor.
Phase-contrast objectives contain a phase plate, or a lens
face with phase rings, that retard the diffracted rays so that their
phase is altered. The phase-contrast condenser ensures that
both diffracted and direct rays are incident on the phase
changing device. The annular grooves of the phase plate allow
a differential retardation of the diffracted rays with a phase
change of about a quarter of a wavelength. This is added to the
phase change caused by the passage through the sample. The
total change is adequate to produce an image of excellent
contrast when the rays interfere at the eyepiece.
Fluorescence Microscopy
Fluorescence microscopy has become an important imaging
technique in microbiology. It is used in conjunction with
staining techniques (see below) to visualize a whole range of
intracellular structures. When certain substances are excited
by illumination of short wavelength, for example ultraviolet,
the emergent rays are converted into longer wavelength light.
Thus blue, green, yellow, or red light is emitted depending on
the composition of the substance. If this emission of long-
wavelength light only occurs while the excitation illumina-
tion is present it is called fluorescence. If the emission
continues when excitation has been removed, this is termed
phosphorescence. Many natural substances display fluores-
cence in specific colors when excited by short-wavelength
light. This is termed primary fluorescence. Many dyes
exhibit primary fluorescence and can be used in dilute
aqueous solutions to stain tissues and cells, which in turn
fluoresce. This is termed secondary fluorescence and is
exploited by fluorescence microscopy. The fluorescence stains
are used in very dilute concentrations so living cells are
exposed to minimal damage. There are many light sources
available, which excite samples not only with light within the
UV range but with other short wavelengths such as the blue
short-wave component of the visible spectrum. The light
sources are used in conjunction with filters to remove
unwanted wavelengths of light. The objectives of fluorescence
microscopy are constructed from quartz when short-wave UV
light is used, as glass is opaque to these wavelengths. When
longer UV wavelength light is used optical glass will allow the
passage of these wavelengths, negating the need for quartz
lenses and slides. A further requirement for the objectives
of fluorescence microscopy is that none of their optical
components fluoresce themselves. A further addition to the
microscope equipment is a filter that removes UV light once it
has induced the fluorescence of the specimen. This filter is
essential to protect the eyes of the operator. Advanced forms

of fluorescence microscopy use combinations of other light
microscopy techniques to improve performance.
Micrometry
Micrometry is the term used to describe the technique for
measuring detail in a microscopic sample using a light micro-
scope. For optimum results the specimen is viewed with
a superimposed grid at the highest magnification with the
greatest resolution possible. The grid is superimposed by
mounting near the focal point of the eyepiece a glass disc with
an engraved or photographically reproduced rule. This glass
disc is called an eyepiece micrometer. The micrometer must be
calibrated for each objective fitted to the microscope. To cali-
brate, a grid of known dimensions, termed a graticule, is
imaged. A typical graticule will have a 100 mm line split into
100 divisions. The number of divisions on the eyepiece
micrometer per division on the graticule is recorded. Features
in subsequent images can be measured by counting how many
divisions in the eyepiece micrometer they occupy and calcu-
lating the length using the previously recorded calibration
constant.
Another routinely used method in food microbiology is
counting cells in a known volume of solution. To do this the
cell solution is placed on a microscope slide with a chamber
of known volume. This slide is called a hemocytometer. The
chamber of known volume is normally split into smaller
sections, which simplifies counting. All micrometry methods
are aided by computer techniques. There are numerous
computer packages that can be used instead of the eyepiece
micrometer; calibration of the objective is in terms of pixels.
In addition imaging software can calculate the area of an
image that is within a contrast range, this facilitates cell
counting.
Cytological Light Microscopy
Histological Basis of Staining
The main problem with visualizing microbes under the
microscope is that organisms are virtually transparent. The
refractive index of vegetative cells is very similar to that of
water. One approach to visualizing organisms is to use phase
contrast, dark-field or fluorescence as discussed above.
However, from the earliest investigations, stains have been
used to distinguish microbes from their surroundings. The
main advantages over the more sophisticated methods are the
speed and simplicity of this approach. Thus the main objective
of staining is to increase the contrast of the cells. Although
natural dyes were used initially, artificial dyes derived from coal
tar and other aromatic materials have now replaced them.
These dyes either act directly as chromophores or become
chromogenic when they react with specific chemicals within
the cell. The chemistry of the chromaticity of these compounds
is based on the delocalization of the electronic structure as they
contain many functional groups (carbon–carbon double
bonds, carbon¼oxygen, carbon¼nitrogen, nitrogen¼nitrogen)
that cause absorption spectra in the light region. The structure
of crystal violet is shown in Figure 3 and typifies the properties
of a chromogenic compound.
MICROSCOPY j Light Microscopy 687
CH 3
Cl –
+ NCH
3
C
(CH 3) 2N N(CH 3) 2
Figure 3 Crystal violet.
For a stain to bind effectively to the cell it must be able to
react or associate with cellular materials. Dyes are normally
charged, either as cations or anions, and so bind to the many
charged sites within cellular materials (proteins, poly-
saccharides, and nucleic acids). Binding may also be aided by
the addition of a mordant that enhances the interaction or acts
as a bridging compound. Stains may be used individually,
termed simple stains, or in combinations, termed differential
staining, for example, the Gram stain or the acid fast stain.
Obtaining Samples
Appropriate sampling techniques are important if bacteria and
other microbes are to be observed. It must be remembered that
to observe a good number of bacteria in a single field at least
107 organisms per millilitre are required in a typical wet mount
under a coverslip. As a consequence sampling from air usually
requires a filtration technique or a culturing process such as
growth on an exposed agar plate. Water can be examined
directly, but in many cases the microbes in fluids are concen-
trated by filtration. Soil generally has large numbers of organ-
isms present and can be sampled by the addition of water.
There are also a number of specialized methods to observe the
growth and activity of microbes in soil. Samples from food
usually involve techniques that enrich the microbes prior to
observation.
Wet Mounts
One of the most common methods of examining microbes is
to make a wet mount of the sample. Typically this is made by
placing a small drop of the microbial suspension on a glass
slide and positioning a coverslip over the drop, the slide can
then be placed under the microscope. The hanging drop
method can also be used to ensure better conditions for
observing motility. The main advantages of wet mounts are
that the samples remain viable and it avoids drying or fixation
that could alter cell morphology. Therefore, size, shape, and
motility are normally observed using wet mounts. After
appropriate simple staining of the sample, cells may be coun-
ted directly to give total cell counts. Counts typically involve
the use of a counting chamber (see Micrometry section). Viable
counts can be made if an appropriate vital stain is used. For
example, fluorescein diacetate can be used if cells contain
688 MICROSCOPY j Light Microscopy
esterases. The enzymes will hydrolyze the fluorescein diacetate
to release fluorescence.
Permanent Mounts
For extended preservation and permanent mounting of stained
specimens, dry samples should be cleaned in xylene prior to
mounting in Canada balsam. Other mounting materials
include plastics dissolved in solvents, for example, ‘Permount,’
that may be painted directly on the stained film.
Negative Stains
Another wet mount procedure involves negative staining. This
provides the simplest and often the quickest means of gaining
information about cell shape and size, refractive inclusions,
and spores. This staining technique relies on colloidal carbon
in the form of nigrosin or Indian ink. These stains reveal
unstained bacteria against a dark sepia background. These
methods are especially useful to observe capsular layers.
Table 1 details some typical methods.
Fixation of Smears and Suspensions
For more complex differential staining methods, cells are fixed
to surfaces so that they do not wash off between the stages of
staining. Fixation also preserves structure avoiding digestion or
changes in the shape of cells. Two types of fixation methods
achieve this: heat or chemical means.
Heat Fixation
Heat fixation is the most common method used for stabi-
lizing microbial smears. Typically the smear of a bacterial
aqueous suspension is allowed to dry on a glass slide. The
slide is then passed over a flame several times taking care not
to overheat the sample. The smear is now ready for staining
procedures.
Chemical Fixation
Chemical fixation is generally better than heat fixation at
preservation of morphology. The procedures are, however,
more time consuming. Some of the most useful procedures are
fixation with aldehydes such as glutaraldehyde or formalin.
For example, a sample is mixed with formaldehyde to give
a final concentration of 1.5–2% formaldehyde. After a few
minutes the cells are smeared and dried onto a slide. Osmium
tetroxide can be used to fix wet films by exposing the wet
smear to the fumes of a 1% (w/v) solution in a closed vessel
for 2 min.
Simple Staining
Morphological studies of bacteria in culture or from natural
specimens are generally heat fixed and then stained with basic
dyes. Where a single stain is used the process is referred to as
simple staining. Slides are typically bathed in a 2% (w/v)
crystal violet/ammonium oxalate (for 10 s) or 0.8% methylene
blue in alkaline aqueous solution (for 30 s).
Other simple stains include alkaline lactophenol cotton
blue that is good at enhancing the visibility of fungal cell walls.
Nigrosin or Indian ink has also been used for negative staining
wet mounts when capsular structures are present.
Fixed Differential Staining Methods
Cells
There are a number of important differential staining methods
that are important in the characterization of microbes. Stains
for different cell wall and membrane structures and intracel-
lular bodies have been developed. Some of the most common
are discussed here.
The Gram stain is one of the most important differential
staining techniques applied to bacteria and was first developed
by Christian Gram in 1884. In theory it should be possible
to divide bacteria into two groups: Gram-positive and
Gram-negative. However, in practice it is common to observe
Gram-variable organisms. This is not surprising considering that
physiological conditions of the cell can affect wall structure.
In the Gram stain, the cells are treated with crystal violet and
then with an iodine solution that acts as a mordant binding the
dye to the cellular materials. The cells are then washed with
ethanol. Gram-positive cells retain the crystal violet whereas
Gram-negative cells do not. To make the difference obvious,
a red counter stain is used such a safranin or fuchsin. The basis
of the stain is the differential permeability to the iodine–crystal
violet complex. The complex can cross the murein layer of
Gram-negative bacteria whereas it is unable to cross the cell
wall layer of Gram-positive bacteria. A typical procedure is
outlined in Table 1.
The stain requires practice and good technique to obtain
reproducible results. For example, Gram-negative bacteria can
appear to be Gram-positive if the film is too thick or if decol-
orization washes, with ethanol, are too short. Gram-positive
organisms can appear Gram-negative if over washed with the
ethanol.
The acid-fast stain exploits the presence of waxy fatty acid
compounds in the cell wall. Certain groups of bacteria contain
long-chain fatty acids (50–90 carbons long) called mycolic
acids that give them a waxy coat which is impervious to basic
dyes such as crystal violet. Normally a detergent is required to
allow the dye to penetrate the cell. Once inside the cell the
normal acidic alcoholic solvent is unable to decolorize the cell.
The acid-fast stain is particularly useful to identify specific
groups of bacteria: Nocardia and Mycobacterium and the spores
of Cryptosporidium. The Ziehl–Neelsen staining procedure is
shown in Table 1.
Subcellular Structures
Endospores
The position of the developing spores in vegetative cells can
also be an important piece of diagnostic information. Spores
can easily be observed under phase-contrast microscope and
are strongly refractile shining brightly slightly above the true
focus. Spores can also be negatively stained using nigrosin.
Spores by their nature are very resistant to simple staining and
it requires quite harsh techniques to achieve this, however,
once stained they are also difficult to decolorize. The malachite
green stain is one such staining method and is outlined in
Table 1.
Table 1 Microscopic stains and staining procedures common in food microbiology

Type Solution Method Comments

Simple stains
Crystal violet Crystal violet staining reagent: mix 20 ml 10% (w/v)
crystal violet in ethanol with 80 ml 1% ammonium
oxalate
Methylene blue Loeffers methylene blue reagent: 30 ml of 1.6% (w/v) 1. Flood heat-fixed smear with crystal violet or Cells should take the stain to which they are exposed.
methylene blue ethanol solution is mixed with methylene blue for 10 or 30 s, respectively
100 ml of 0.01% KOH aqueous solution
2. Wash with water then blot dry
Carbol fuchsin Carbol–fuchsin stain: basic fuchsin 0.3%, phenol 5%,
ethanol 10%, water 85%
Lactophenol cotton blue Lactophenol cotton blue: a solution 0.5% cotton blue If the cells do not stain with either of the above then
in phenol (20%), lactic acid (20%), glycerol 40%, Carbol fuchsin can be used
and water (10%)
This stains colorizes fungal cell walls
Negative stain
Nigrosin Nigrosin stain: 7% nigrosin 1. Place a droplet of nigrosin on a slide These preparations reveal bacteria unstained and
standing out brightly against a sepia background
2. Mix with small sample containing microbe
3. Take another slide and smear out the mixture to
produce a gradient of film thickness
4. Allow to dry completely
Differential stains
Gram stain Crystal violet staining reagent 1. Flood an air-dried and heat-fixed smear with crystal Gram-positive organisms appear blue/black whereas
violet stain for 1 min Gram-negative organisms appear pink/red
Mix 20 ml 10% (w/v) crystal violet in ethanol with 2. Wash with water
80 ml 1% ammonium oxalate
Mordant 3. Flood the smear with mordant for 1 min

MICROSCOPY j Light Microscopy

Acid fast staining: Ziehl– Neelsen
stain
A solution of 0.33% (w/v) iodine 0.66% KI
Decolorizing reagent
Ethanol 95% solution
Counterstain: 0.25% (w/v) safranin in 10%
Ethanol water solution
Carbol–fuchsin stain: Basic fuchsin 0.3%, phenol 5%,
ethanol 10%, water 85%
Decolorizing solvent: 3 ml conc. HCl in 95% ethanol
Counter stain: 0.3% methylene blue solution
4. Wash smear with water
5. Decolorize with ethanol for 30 s
6. Wash with water
7. Flood smear with counter stain for 30 s
8. Wash smear and blot dry
1. Place an air-dried and heat-fixed smear, cover the Acid fast bacteria appear red whereas non-acid-fast
slide with basic fuchsin dye and place in steam for appear blue.
3–5 min to allow the dye to penetrate
2. After washing decolorize with the decolorizing
solvent
3. Flood smear with counter stain for 20–30 s and
wash
(Continued)

689

Table 1 Microscopic stains and staining procedures common in food microbiologydcont'd
Type
Endospores: Malachite: green
Cytoplasmic inclusions:
Poly-b-hydroxybutyrate (PHB)
Polyphosphate
Glycogen-like polysaccharides
Solution
Malachite green: 0.5% (w/v) malchite green
Counterstain: 0.25% (w/v) of safranin in 10%
ethanol water solution
Sudan Black III: 0.3% (w/v) in ethylene glycol
Washing agent
Xylene
Counterstain: 0.5% (w/v) aqueous safranin
Loeffers methylene blue reagent: 30 ml of 1.6% (w/v)
methylene blue ethanol solution is mixed with
100 ml of 0.01% KOH aqueous solution or 1%
toluidine blue
Periodic acid solution: 1% periodic acid in solution of
20 mm sodium acetate in 70% ethanol 70%
ethanol washing solution
Reducing solution: 0.05% (w/v) sodium thiosulfate,
0.1% (w/v), 20 mm HCl in 60% aqueous ethanol
Schiff reagent: 0.25% basic fuchsin in about 100 mm
HCl and 1% potassium metabisulfite
Metabisulfite washing solution: 4% potassium
metabisulfite and 1% HCl in aqueous solution
Malachite green counter stain 0.002% aqueous
solution
690
MICROSCOPY j Light Microscopy
Method Comments
1. Take a slide with an air-dried and heat-fixed smear Endospores appear bright green and vegetative cells
and flood with malachite green. appear brownish red
2. Place slide in steam for 5 min
3. Wash in water
4. Flood slide with counter stain
5. Then wash and blot dry
1. Flood heat-fixed film with Sudan Black for PHB appears as a black droplet while the cytoplasm
5–15 min appears pink.
2. Drain and air dry
3. Wash in xylene
4. Blot dry
5. Counterstain with safranin
6. Rinse with water and blot dry
1. Stain a heat-fixed smear for 10–30 s with either Under the methylene blue stain polyphosphate
Loffers methylene blue or toluidine blue appears as deep blue/ violet spheres.
2. Rinse and blot dry Using toluidine, polyphosphate appears as red
spheres while the cytoplasm appears blue
1. Flood heat-fixed slide with periodic acid reagent for Polysaccharides appear red; other cytoplasmic
5 min components appear green
2. Wash with ethanol solution
3. Flood with reducing reagent for 5 min
4. Wash with ethanol solution
5. Stain with reducing solution for 14–45 min
6. Wash several times with metabisulfite solution
7. Counterstain with malachite green for 2–3 s
8. Wash with water and blot dry
(Continued)
Type Solution Method Comments

Fat
Fluorescent stains
Acridine orange
Calcofluor white Primulin
DAPI (40 ,60 -diamidino-
2-phenylindol)
Conjugate fluorescent antibodies
Sudan black III reagent: 0.01% (w/v) Sudan III in
a solution of 50% ethanol and glycerol
Counterstain: 0.3% (w/v) methylene blue solution
Acridine orange reagent: 0.1% acridine orange in
0.2 m acetate buffer (pH 4.5)
Washing reagent: 0.2 m sodium acetate buffer pH 4.5
Calcofluor white stain: 0.1 mg l1 calcofluor, 0.1 mg l1
Primulin
DAPI staining solution DAPI 2 mg l1
Antibodies (preferable monoclonal) are raised to
specific organisms and then conjugated with
fluorescein isothiocyanate (FITC)
Flood heat-fixed Sudan black III Wash
Counterstain with methylene blue
Methanol is used to fix the smear Immerse in sodium
acetate buffer
Stain with 0.1% acridine orange 20–120 min
Add 1 drop of calcofluor white to fixed preparation
Add 1 drop of KOH Add coverslip
Mix DAPI solution with cell suspension mount the
stained suspension on the slide Apply coverslip
and view
Cover the air-dried and heat-fixed or formalin-fixed
samples with the conjugate serum for 20–30 min
taking care not to allow the preparation to dry out.
Then wet mount using (10%) glycerol solution
which is phosphate buffered (pH 7.0) under
a coverslip and examine fluorescence
Fat is stained red. This will work for bacteria, yeasts
or fungi
In yeast, cytoplasm is normally orange while the DNA
is green or green/yellow fluorescence
Fungal elements appear bright green or blue
depending on UV filter used. Primulin, yeast cell
walls fluoresce green yellow. Will allow the
observation of budding scars. In some cases can
be used to distinguish between live and dead cells,
as the dyes are not membrane permeable so if the
cytoplasm stains then membranes are damaged.
This stain is used extensively for mapping nuclei,
clear view of well spread chromosomes is possible
In yeast will stain chromosomes blue/white
These techniques of rapid identification are now being
replaced by DNA binding/probe methodologies

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Capsules
Slime layers and capsules are produced by many bacteria and
are best demonstrated by wet preparations because their highly
hydrated polymers shrink when fixed and dried. Dulguid
Indian ink is the simplest capsule stain.
Cytoplasmic Inclusions
Many bacteria will produce, as a result of metabolism, inclu-
sion bodies within the cell. These include fat droplets, poly-
hydroxybutyrate, polyphosphate and polysaccharides, sulfur
and protein crystals. Many of these can be visualized using
appropriate staining methods. Polyhydroxybutyrate is
observed using negative staining or by dyes which bind
specifically to the polymer. Polyphosphates can also be stored
by cells in the form of metachromatic granules and are detected
by treating the fixed cells with methylene blue or toluidine
blue. Glycogen-like polymers can be stained with periodic
Schiff stain. Bacterial nuclear bodies are not easy to stain
directly with basic dyes, however, fluorescence staining is
particularly useful for detecting nuclear materials.
Fluorescent Staining Procedures
There are a number of procedures that use fluorescent stains.
Their main advantages are that they allow observation at lower
magnification and detection in complex backgrounds such as
blood or animal tissues. The main disadvantage is the
requirement for a specialized microscope.
There are several popular fluorescent dyes. Acridine orange,
a fluorochrome that intercalates into nucleic acids, in both
native and denatured forms, is able to distinguish between
fungal and bacterial DNAs. Another nuclear fluorochrome is
DAPI (40 ,60 -diamidino-2-phenylindol) which is widely used
for visualization of chromosomes in eukaryotic microbes.
Calcofluor white nonspecifically binds to polysaccharides,
such as cellulose and chitin; it is thus useful in detecting fungi
and yeast. Similarly primulin will bind to chitin and, for
example, will enhance the visibility of yeast bud scars.
Antibodies conjugated with fluorochrome have also been
developed for the rapid identification of specific pathogenic
organisms such as Legionella. This is a very good method for the
detection of specific organisms. However, the modern molec-
ular biology techniques using polymerase chain reaction (PCR)
technology, DNA fingerprinting and DNA probes are beginning
to replace this technology.
Conclusions
Light microscopy is an established and vital tool for the food
microbiologist. The instrumentation continues to be improved
and exciting techniques such as confocal light microscopy are
providing new insights into the relationship between microor-
ganisms and their environment. With the advent of modern
computation methods substantial advances in image analysis are
proving extremely useful in the enumeration and morphological
measurement of microbes. The correct use of instrumentation
and optimized staining procedures will ensure that the food
microbiologist can exploit the full potential of light microscopy.
See also: Total Viable Counts: Microscopy.
Further Reading
Gerhardt, P., Murray, R.G.E., Wood, W.A., Krieg, N.R. (Eds.), 1994. Methods for
General and Molecular Bacteriology. ASM Press, Washington, DC.
Murray, P.R., Baron, E.J., Pfaller, M.A., Tenover, F.C., Yolken, R.H. (Eds.), 1995.
Manual of Clinical Microbiology, sixth ed. ASM Press, Washington DC.
Richardson, J.H., 1991. Handbook for the Light Microscope – A Users Guide. Noyes
Publications, Park Ridge, New Jersey.
Shotton, D. (Ed.), 1993. Electronic Light Microscopy – Techniques in Modern
Biomedical Microscopy. Wiley-Liss, New York.