by Joseph Jackson Lister (the father of Joseph

CHAPTER 3 Lister).
OBSERVING LIGHT MICROSCOPE
MICROORGANISMS  Light microscopy refers to the use of any
THROUGH A MICRIOSCOPE kind of microscope that uses visible light to
observe specimens
 The word microscope is derived from the
Latin word micro (small) and the Greek word Compound Light Microscope
skopos (to look at).  A modern compound light microscope (LM)
 Helicobacter pylori is a spiral-shaped has a series of lenses and uses visible light as
bacterium that was first seen in cadaver its source of illumination
stomachs in 1886  we can examine very small specimens as
 Helicobacter pylori bacteria live in the well as some of their fine detail.
human stomach and can cause ulcers  This magnification is achieved when light
rays from an illuminator, the light source, pass
through a condenser, which has lenses that
UNITS OF MEASUREMENT direct the light rays through the specimen.
 From here, light rays pass into the objective
 When measuring microorganisms, we use
lenses, the lenses closest to the specimen.
the metric system.
 The image of the specimen is magnified
 A major advantage of the metric system is
again by the ocular lens, or eyepiece.
that units relate to each other by factors of 10.
 We can calculate the total magnification of a
 Thus, 1 meter (m) equals 10 decimeters
specimen by multiplying the objective lens
(dm) or 100 centimeters (cm) or 1000
magnification (power) by the ocular lens
millimeters (mm)
magnification (power).
 Microorganisms are measured in even
 Most microscopes used in microbiology
smaller units, such as micrometers and
have several objective lenses, including 10x
nanometers.
(low power), 40x (high power), and 100x (oil
 A micrometer (μm) equals 0.000001 m (10 -6
immersion).
m).
 Most ocular lenses magnify specimens by a
 The prefix micro indicates the unit following
factor of 10
it should be divided by 1 million, or 106
 Resolution (also called resolving power) is
 A nanometer (nm) equals 0.000000001 m
the ability of the lenses to distinguish fine detail
(10-9 m).
and structure.
 Angstrom (Å) was previously used for 10 -10
 Specifically, it refers to the ability of the
m, or 0.1 nm.
lenses to distinguish two points that are a
specified distance apart.
MICROSCOPY: THE  For example, if a microscope has a
resolving power of 0.4 nm, it can distinguish
INSTRUMENTS two points if they are at least 0.4 nm apart.
 The simple microscope used by van  A general principle of microscopy is that the
Leeuwenhoek in the seventeenth century had shorter the wavelength of light used in the
only one lens and was similar to a magnifying instrument, the greater the resolution.
glass.  The white light used in a compound light
 However, van Leeuwenhoek was the best microscope has a relatively long wavelength
lens grinder in the world in his day. and cannot resolve structures smaller than
 His lenses were ground with such precision about 0.2 μm
that a single lens could magnify a microbe  To obtain a clear, finely detailed image
300x. under a compound light microscope,
 His simple microscopes enabled him to be specimens must contrast sharply with their
the first person to see bacteria medium (the substance in which they are
 Contemporaries of van Leeuwenhoek, such suspended).
as Robert Hooke, built compound microscopes,  To attain such contrast, we must change the
which have multiple lenses. refractive index of specimens from that of their
 In fact, a Dutch spectacle maker, medium.
Zaccharias Janssen, is credited with making  The refractive index is a measure of the
the first compound microscope around 1600 lightbending ability of a medium.
 It was not until about 1830 that a  We change the refractive index of
significantly better microscope was developed specimens by staining them
 After the specimen is stained, the specimen
and its medium have different refractive Phase-Contrast Microscopy
indexes.  Another way to observe microorganisms is
 When light rays pass through the two with a phase contrast microscope.
materials (the specimen and its medium), the  Phase contrast microscopy is especially
rays change direction (refract) from a straight useful because the internal structures of a cell
path by bending or changing angle at the become more sharply defined, permitting
boundary between the materials. detailed examination of living microorganisms.
 This increases the image’s contrast between  In a phasecontrast microscope, one set of
the specimen and the medium. light rays comes directly from the light source.
 As the light rays travel away from the  The other set comes from light that is
specimen, they spread out and enter the reflected or diffracted from a particular
objective lens, and the image is thereby structure in the specimen.
magnified.  Diffraction is the scattering of light rays as
 To achieve high magnification (1000x) with they “touch” a specimen’s edge.
good resolution, the objective lens must be  The diffracted rays are bent away from the
small parallel light rays that pass farther from the
 To preserve the direction of light rays at the specimen.)
highest magnification, immersion oil is placed  When the two sets of light rays—direct rays
between the glass slide and the oil immersion and reflected or diffracted rays—are brought
objective lens together, they form an image of the specimen
 The immersion oil has the same refractive on the ocular lens, containing areas that are
index as glass, so the oil becomes part of the relatively light (in phase), through shades of
optics of the glass of the microscope. gray, to black (out of phase)
 Unless immersion oil is used, light rays are
refracted as they enter the air from the slide
Differential Interference Contrast (DIC)
 The oil has the same effect as increasing
Microscopy
the objective lens diameter; therefore, it
improves the resolving power of the lenses.  Differential interference contrast (DIC)
 If oil is not used with an oil immersion microscopy is similar to phasecontrast
objective lens, the image has poor resolution microscopy in that it uses differences in
and becomes fuzzy. refractive indexes.
 By focusing the light, the condenser  However, a DIC microscope uses two
produces a brightfield illumination beams of light instead of one.
 It is not always desirable to stain a  In addition, prisms split each light beam,
specimen, but an unstained cell has little adding contrasting colors to the specimen.
contrast with its surroundings and is therefore  Therefore, the resolution of a DIC
difficult to see. microscope is higher than that of a standard
phasecontrast microscope.
Darkfield Microscopy  Also, the image is brightly colored and
 A darkfield microscope is used to examine appears nearly three-dimensional
live microorganisms that either are invisible in
Fluorescence Microscopy
the ordinary light microscope, cannot be
stained by standard methods, or are so  Fluorescence microscopy takes advantage
distorted by staining that their characteristics of fluorescence, the ability of substances to
are obscured. absorb short wavelengths of light (ultraviolet)
 A darkfield microscope uses a darkfield and give off light at a longer wavelength
condenser that contains an opaque disk. (visible).
 The disk blocks light that would enter the  Some organisms fluoresce naturally under
objective lens directly. Only light that is ultraviolet light; if the specimen to be viewed
reflected off (turned away from) the specimen does not naturally fluoresce, it is stained with
enters the objective lens. one of a group of fluorescent dyes called
 Because there is no direct background light, fluorochromes
the specimen appears light against a black  When microorganisms stained with a
background—the dark field fluorochrome are examined under a
 This technique is frequently used to examine fluorescence microscope with an ultraviolet or
unstained microorganisms suspended in liquid . nearultraviolet light source, they appear as
 One use for darkfield microscopy is the luminescent, bright objects against a dark
examination of very thin spirochetes, such as background.
Treponema pallidum the causative agent of  Fluorochromes have special attractions for
syphilis. different microorganisms.
 For example, the fluorochrome auramine O,  Because confocal microscopy uses a
which glows yellow when exposed to ultraviolet pinhole aperture, it eliminates blurring that
light, is strongly absorbed by Mycobacterium occurs with other microscopes.
tuberculosis, the bacterium that causes  As a result, exceptionally clear two-
tuberculosis dimensional images can be obtained, with
 Bacillus anthracis, the causative agent of improved resolution of up to 40% over that of
anthrax, appears apple green when stained other microscopes.
with another fluorochrome, fluorescein  Most confocal microscopes are used in
isothiocyanate (FITC). conjunction with computers to construct three-
 The principal use of fluorescence dimensional images.
microscopy is a diagnostic technique called the
fluorescent-antibody (FA) technique, or
immunofluorescence. TWO-PHOTON MICROSCOPE
 Antibodies are natural defense molecules  As in confocal microscopy, specimens are
that are produced by humans and many stained with a fluorochrome for two-photon
animals in reaction to a foreign substance, or microscopy (TPM).
antigen.  Twophoton microscopy uses long-
 Fluorescent antibodies for a particular wavelength (red) light, and therefore two
antigen are obtained as follows: an animal is photons, instead of one, are needed to excite
injected with a specific antigen, such as a the fluorochrome to emit light.
bacterium, and the animal then begins to  The longer wavelength allows imaging of
produce antibodies against that antigen. living cells in tissues up to 1 mm (1000 μm)
 After a sufficient time, the antibodies are deep
removed from the serum of the animal.  Confocal microscopy can image cells in
 a fluorochrome is chemically combined with detail only to a depth of less than 100 mm.
the antibodies. These fluorescent antibodies  Additionally, the longer wavelength is less
are then added to a microscope slide likely to generate singlet oxygen, which
containing an unknown bacterium. damages cells
 If this unknown bacterium is the same  Another advantage of TPM is that it can
bacterium that was injected into the animal, the track the activity of cells in real time.
fluorescent antibodies bind to antigens on the  For example, cells of the immune system
surface of the bacterium, causing it to have been observed responding to an antigen.
fluoresce.
 This technique can detect bacteria or other
Super-Resolution Light Microscopy
pathogenic microorganisms, even within cells,
 Until recently, the maximum resolution for
tissues, or other clinical specimens
light microscopes was 0.2 μm.
 it can be used to identify a microbe in
 However, in 2014, the Nobel Prize in
minutes.
Chemistry was awarded to Eric Betzig, Stefan
 Immunofluorescence is especially useful in
Hell, and William Moerner for the development
diagnosing syphilis and rabies
of a microscope that uses two laser beams.
Confocal Microscopy  In super-resolution light microscopy, one
wavelength stimulates fluorescent molecules to
 Confocal microscopy is a technique in light glow, and another wavelength cancels out all
microscopy used to reconstruct three- fluorescence except for that in one nanometer.
dimensional images.
 Cells can be stained with fluorescent dyes
 Like fluorescent microscopy, specimens are that are specific for certain molecules such as
stained with fluorochromes so they will emit, or DNA or protein, allowing even a single
return, light. molecule to be tracked in a cell.
 But instead of illuminating the entire field,  A computer tells the microscope to scan the
one plane of a small region of a specimen is specimen nanometer by nanometer and then
illuminated with a shortwavelength (blue) light puts the images together
which passes the returned light through an
aperture aligned with the illuminated region. Scanning Acoustic Microscopy
 Each plane corresponds to an image of a
fine slice that has been physically cut from a  Scanning acoustic microscopy (SAM)
specimen. basically consists of interpreting the action of a
sound wave sent through a specimen.
 Successive planes and regions are
illuminated until the entire specimen has been  A sound wave of a specific frequency travels
scanned. through the specimen, and a portion of it is
reflected back every time it hits an interface
within the material.
 The resolution is about 1 μm.
 SAM is used to study living cells attached to and objects are generally magnified 10,000 to
another surface, such as cancer cells, artery 10,000,000x.
plaque, and bacterial biofilms that foul  Because most microscopic specimens are
equipment so thin, the contrast between their
ultrastructure and the background is weak.
Electron Microscopy  Salts of various heavy metals, such as lead,
 Objects smaller than about 0.2 μm, such as osmium, tungsten, and uranium, are
viruses or the internal structures of cells, must commonly used as stains.
be examined with an electron microscope.  These metals can be fixed onto the
 In electron microscopy, a beam of electrons specimen (positive staining) or used to
is used instead of light. increase the electron opacity of the
 Like light, free electrons travel in waves. surrounding field (negative staining).
The resolving power of the electron microscope  Negative staining is useful for the study of
is far greater than that of the other microscopes the very smallest specimens, such as virus
described here so far. particles, bacterial flagella, and protein
 The better resolution of electron molecules.
microscopes is due to the shorter wavelengths  In addition to positive and negative staining,
of electrons; the wavelengths of electrons are a microbe can be viewed by a technique
about 100,000 times smaller than the called shadow casting.
wavelengths of visible light.  In this procedure, a heavy metal such as
 Images produced by electron microscopes platinum or gold is sprayed at an angle of
are always black and white, but they may be about 45° so that it strikes the microbe from
colored artificially to accentuate certain details. only one side.
 Instead of using glass lenses, an electron  The metal piles up on one side of the
microscope uses electromagnetic lenses to specimen, and the uncoated area on the
focus a beam of electrons onto a specimen. opposite side of the specimen leaves a clear
 There are two types: the transmission area behind it as a shadow.
electron microscope and the scanning electron  This gives a three-dimensional effect to the
microscope. specimen and provides a general idea of the
size and shape of the specimen
Transmission Electron Microscopy  Transmission electron microscopy has high
 In the transmission electron microscope resolution and is extremely valuable for
(TEM), a finely focused beam of electrons examining different layers of specimens.
from an electron gun passes through a  However, it does have certain
specially prepared, ultrathin section of the disadvantages.
specimen  Because electrons have limited penetrating
 The beam is focused on a small area of the power, only a very thin section of a specimen
specimen by an electromagnetic condenser (about 100 nm) can be studied effectively.
lens that performs roughly the same function  Thus, the specimen has no three-
as the condenser of a light microscope— dimensional aspect.
directing the beam of electrons in a straight  In addition, specimens must be fixed,
line to illuminate the specimen. dehydrated, and viewed under a high vacuum
 Instead of being placed on a glass slide, as to prevent electron scattering.
in light microscopes, the specimen is usually  These treatments not only kill the specimen,
placed on a copper mesh grid. but also cause shrinkage and distortion,
 The beam of electrons passes through the sometimes to the extent that there may
specimen and then through an appear to be additional structures in a
electromagnetic objective lens, which prepared cell.
magnifies the image.  Structures that appear as a result of the
 Finally, the electrons are focused by an preparation method are called artifacts.
electromagnetic projector lens (rather than by
Scanning Electron Microscopy
an ocular lens as in a light microscope) onto a
viewing screen and saved as a digital image.  The scanning electron microscope (SEM)
 The final image, called a transmission overcomes the sectioning problems associated
electron micrograph, appears as many light with a transmission electron microscope.
and dark areas, depending on the number of  It provides striking threedimensional views
electrons absorbed by different areas of the of specimens
specimen.  An electron gun produces a finely focused
 The transmission electron microscope can beam of electrons called the primary electron
resolve objects as close together as 10 pm, beam.
 These electrons pass through  AFM is used to image both biological
electromagnetic lenses and are directed over substances (in nearly atomic detail) and
the surface of the specimen. molecular processes (such as the assembly of
 The primary electron beam knocks electrons fibrin, a component of a blood clot).
out of the surface of the specimen, and the
secondary electrons thus produced are
transmitted to an electron collector, amplified, PREPARATION OF SPECIMENS
and used to produce an image on a viewing FOR LIGHT MICROSCOPY
screen that is saved as a digital image.
 The image is called a scanning electron  Most microorganisms appear almost
micrograph. colorless when viewed through brightfield
 This microscope is especially useful in microscopy, so we must prepare them for
studying the surface structures of intact cells observation.
and viruses.  One way is to stain (color) the specimen
 In practice, it can resolve objects as close PREPARING SMEARS FOR STAINING
together as 10 nm, and objects are generally
magnified 1000 to 500,000x.  Most initial observations of microorganisms
are made with stained preparations.
 Staining simply means coloring the
SCANNED-PROBE MICROSCOPY microorganisms with a dye that emphasizes
certain structures.
 Since the early 1980s, several new types of  Before the microorganisms can be stained,
microscopes, called scanned-probe however, they must be fixed (attached) to the
microscopes, have been developed. microscope slide.
 They use various kinds of probes to  Fixing simultaneously kills the
examine the surface of a specimen using microorganisms and fixes them to the slide. It
electric current, which does not modify the also preserves various parts of microbes in
specimen or expose it to damaging, highenergy their natural state with only minimal distortion.
radiation.  When a specimen is to be fixed, a thin film
 Such microscopes can be used to map of material containing the microorganisms is
atomic and molecular shapes, to characterize spread over the surface of the slide.
magnetic and chemical properties, and to  This film, called a smear, is allowed to air
determine temperature variations inside cells. dry.
 Among the new scannedprobe microscopes  In most staining procedures the slide is then
are the scanning tunneling microscope and the fixed by passing it through the flame of a
atomic force microscope Bunsen burner several times, smear side up, or
Scanning Tunneling Microscopy by covering the slide with methanol for 1
minute.
 Scanning tunneling microscopy (STM) uses  Stain is applied and then washed off with
a thin tungsten probe that scans a specimen water; then the slide is blotted with absorbent
and produces an image that reveals the paper.
bumps and depressions of the atoms on the  Without fixing, the stain might wash the
surface of the specimen microbes off the slide. The stained
 The resolving power of an STM is much microorganisms are now ready for microscopic
greater than that of an electron microscope; it examination.
can resolve features that are only about 1⁄100  Stains are salts composed of a positive and
the size of an atom. a negative ion, one of which is colored and is
 Moreover, special preparation of the known as the chromophore.
specimen for observation is not needed.  The color of socalled basic dyes is in the
 STMs are used to provide incredibly detailed cation; in acidic dyes, it is in the anion.
views of molecules such as DNA.  Bacteria are slightly negatively charged at
Atomic Force Microscopy pH 7. Thus, the colored cation in a basic dye is
attracted to the negatively charged bacterial
 In atomic force microscopy (AFM), a metal- cell.
anddiamond probe is gently forced down onto  Basic dyes, which include crystal violet,
a specimen. methylene blue, malachite green, and safranin,
 As the probe moves along the surface of the are more commonly used than acidic dyes.
specimen, its movements are recorded, and a  Acidic dyes are not attracted to most types
threedimensional image is produced of bacteria because the dye’s negative ions are
 As with STM, AFM does not require special repelled by the negatively charged bacterial
specimen preparation.
surface, so the stain colors the background both grampositive and gram-
instead. negative bacteria appear dark
 Preparing colorless bacteria against a violet or purple.
colored background is called negative staining. o Next, the slide is washed with
It is valuable for observing overall cell shapes, alcohol or an alcohol-acetone
sizes, and capsules because the cells are solution. This solution is a
made highly visible against a contrasting dark decolorizing agent, which
background removes the purple from the cells
 Distortions of cell size and shape are of some species but not from
minimized because fixing others.
 To apply acidic or basic dyes, o The alcohol is rinsed off, and the
microbiologists use three kinds of staining slide is then stained with safranin,
techniques: simple, differential, and special a basic red dye. The smear is
washed again, blotted dry, and
SIMPLE STAINS examined microscopically.
 A simple stain is an aqueous or alcohol  The purple dye and the iodine combine in
solution of a single basic dye. the cell wall of each bacterium and color it dark
 Although different dyes bind specifically to violet or purple.
different parts of cells, the primary purpose of  Bacteria that retain this color after the
a simple stain is to highlight the entire alcohol has attempted to decolorize them are
microorganism so that cellular shapes and classified as gram-positive bacteria;
basic structures are visible.  bacteria that lose the dark violet or purple
 The stain is applied to the fixed smear for a color after decolorization are classified as
certain length of time and then washed off. gram-negative bacteria
 The slide is dried and examined.  Because gramnegative bacteria are
 Occasionally, a chemical is added to the colorless after the alcohol wash, they are no
solution to intensify the stain; such an additive longer visible.
is called a mordant.  This is why the basic dye safranin is applied;
 One function of a mordant is to increase the it turns the gramnegative bacteria pink.
affinity of a stain for a biological specimen;  Stains such as safranin that have a
another is to coat a structure (such as a contrasting color to the primary stain are called
flagellum) to make it thicker and easier to see counterstains.
after it is stained with a dye.  Because grampositive bacteria retain the
 Some of the simple stains commonly used original purple stain, they are not affected by
in the laboratory are methylene blue, the safranin counterstain.
carbolfuchsin, crystal violet, and safranin.  different kinds of bacteria react differently to
the Gram stain because structural differences
DIFFERENTIAL STAINS in their cell walls affect the retention or escape
of a combination of crystal violet and iodine,
 Unlike simple stains, differential stains react
called the crystal violet–iodine (CV–I) complex.
differently with different kinds of bacteria and
thus can be used to distinguish them.  Among other differences, grampositive
bacteria have a thicker peptidoglycan cell wall
 The differential stains most frequently used
(disaccharides and amino acids) than gram-
for bacteria are the Gram stain and the acid-
negative bacteria.
fast stain.
 In addition, gramnegative bacteria contain a
Gram Stain layer of lipopolysaccharide (lipids and
polysaccharides) as part of their cell wall
 The Gram stain was developed in 1884 by
 When applied to both grampositive and
the Danish bacteriologist Hans Christian Gram.
gramnegative cells, crystal violet and then
 It is one of the most useful staining
iodine readily enter the cell walls.
procedures because it classifies bacteria into
 Inside the cell walls, the crystal violet and
two large groups: grampositive and gram-
iodine combine to form CV–I.
negative
 Crystal violet or iodine alone is water soluble
o A heatfixed smear is covered with
and will drain out of the cell.
a basic purple dye, usually crystal
 However, CV–I is insoluble in water, so it
violet. Because the purple stain
doesn’t easily leave the cell wall.
imparts its color to all cells, it is
referred to as a primary stain.  Consequently, grampositive cells retain the
o After a short time, the purple dye color of the crystal violet dye.
is washed off, and the smear is  In gramnegative cells, however, the alcohol
covered with iodine, a mordant. wash disrupts the outer lipopolysaccharide
When the iodine is washed off,
layer, and the CV–I complex is washed out of  The smear is then stained with a methylene
the thin layer of peptidoglycan. blue counterstain.
 As a result, gramnegative cells are  Non–acidfast cells appear blue after the
colorless until counterstained with safranin, counterstain is applied.
after which they are pink.
 In summary, grampositive cells retain the SPECIAL STAINS
dye and remain purple.  Special stains are used to color parts of
 Gramnegative cells do not retain the dye; microorganisms, such as endospores, flagella,
they are colorless until counterstained with a or capsules
red dye.
 The Gram method is one of the most
important staining techniques in medical Negative Staining for Capsules
microbiology  Many microorganisms contain a gelatinous
 The Gram reaction is most consistent when covering called a capsule.
it is used on young, growing bacteria  In medical microbiology, demonstrating the
 The Gram reaction of a bacterium can presence of a capsule is a means of
provide valuable information for the treatment determining the organism’s virulence, the
of disease. degree to which a pathogen can cause
 Grampositive bacteria tend to be killed disease.
easily by penicillins and cephalosporins.  Capsule staining is more difficult than other
 Gram negative bacteria are generally more types of staining procedures because
resistant because the antibiotics cannot capsular materials are water soluble and may
penetrate the lipopolysaccharide layer. be dislodged or removed during rigorous
washing.
 To demonstrate the presence of capsules, a
Acid-Fast Stain microbiologist can mix the bacteria in a
 Another important differential stain (one that solution containing a fine colloidal suspension
differentiates bacteria into distinctive groups) is of colored particles (usually india ink or
the acid-fast stain, which binds strongly only to nigrosin) to provide a contrasting background
bacteria that have a waxy material in their cell and then stain the bacteria with a simple
walls. stain, such as safranin
 Microbiologists use this stain to identify all  Because of their chemical composition,
bacteria in the genus Mycobacterium including capsules do not accept most biological dyes,
the two important pathogens Mycobacterium such as safranin, and thus appear as halos
tuberculosis, the causative agent of surrounding each stained bacterial cell.
tuberculosis, and Mycobacterium leprae (] the
causative agent of leprosy. Endospore (Spore) Staining
 This stain is also used to identify the  An endospore is a special resistant, dormant
pathogenic strains of the genus Nocardia structure formed within a cell that protects a
 Bacteria in the genera Mycobacterium and bacterium from adverse environmental
Nocardia are acidfast. conditions.
 In the acidfast staining procedure, the red  Although endospores are relatively
dye carbolfuchsin is applied to a fixed smear, uncommon in bacterial cells, they can be
and the slide is gently heated for several formed by a few genera of bacteria.
minutes.  Endospores cannot be stained by ordinary
 (Heating enhances penetration and retention methods, such as simple staining and Gram
of the dye.) staining, because the dyes don’t penetrate the
 Then the slide is cooled and washed with endospore’s wall.
water.  The most commonly used endospore stain
 The smear is next treated with acidalcohol, a is the Schaeffer Fulton endospore stain
decolorizer, which removes the red stain from  Malachite green, the primary stain, is
bacteria that are not acidfast. applied to a heatfixed smear and heated to
 The acidfast microorganisms retain the pink steaming for about 5 minutes.
or red color because the carbolfuchsin is more  The heat helps the stain penetrate the
soluble in the cell wall lipids than in the acid- endospore wall.
alcohol  Then the preparation is washed for about 30
 In non–acidfast bacteria, whose cell walls seconds with water to remove the malachite
lack the lipid components, the carbolfuchsin is green from all of the cells’ parts except the
rapidly removed during decolorization, leaving endospores.
the cells colorless.
 Next, safranin, a counterstain, is applied to
the smear to stain portions of the cell other
than endospores.
 In a properly prepared smear, the
endospores appear green within red or pink
cells.
 Because endospores are highly refractive,
they can be detected under the light
microscope when unstained, but without a
special stain they cannot be differentiated from
inclusions of stored material.

Flagella Staining
 Bacterial flagella (singular: flagellum) are
structures of locomotion too small to be seen
with a light microscope without staining.
 A tedious and delicate staining procedure
uses a mordant and the stain carbolfuchsin to
build up the diameters of the flagella until they
become visible under the light microscope.
 Microbiologists use the number and
arrangement of flagella as diagnostic aids