Microscopy: The technology of making very

small things visible to the naked eye.

Units of Measurement: The metric system is
Chapter 3: Observing used to measure microorganisms.
Microorganisms Through a Metric system:
Microscope u Basic unit of length: Meter.
u All units are related to each other by factors
of 10.
u Prefixes are used to indicate the relationship
of a unit to the basic unit (e.g.: meter).

Metric Units of Length and U.S. Instruments of Microscopy:

Equivalents: 1. Simple Microscopes:
Metric Relationship to U.S. uOnly have one lens, similar to a magnifying glass.
Unit basic unit (meter) Equivalent uLeeuwenhoeck’s simple microscopes allowed
kilometer (km) 1 km = 1000 m 1 mile = 1.61 km him to magnify images from 100 to 300 X.
meter (m) 1 m = 39.37 in uThey were so difficult to focus, he built a new one for
decimeter (dm) 1 dm = 0.1 m = 10-1 m 1 dm = 3.94 in each specimen, a total of 419.
centimeter (cm) 1 cm = 0.01 m = 10-2 m 2.54 cm = 1 in uHe did not share his techniques with other scientists.
millimeter (mm) 1 mm = 0.001 m = 10-3 m Even today, his source of lighting is unknown.
micrometer (um) 1 um = 0.000001 m = 10-6 m uHis daughter donated 100 of his microscopes to the
Royal Society shortly before his death in 1723.
nanometer (nm) 1 nm = 0.000000001 m = 10-9 m
picometer (pm) 1 pm = 0.000000000001 m = 10-12 m

Instruments of Microscopy: Instruments of Microscopy:

2. Compound Light (CL) Microscopy 2. Compound Light Microscopy
History of CL Microscopes: u Have several lenses:
uFirst developed by Zaccharias Janssen, Dutch 1. Light originates from an illuminator and passes
through condenser lenses, which direct light onto the
spectacle maker in 1600.
specimen.
u Poor quality
2. Light then enters the objective lenses, which magnify
u Could not see bacteria the image. These are the closest lenses to the specimen:
u Joseph Jackson Lister (Lister’s father) developed uScanning objective lens: 4 X
improved compound light microscope in 1830s. uLow power objective lens: 10 X
uHigh power objective lens: 40-45 X
u Basis for modern microscopes
uOil immersion lens: 95-100 X
uUse visible light as a source of illumination. 3. The image of the specimen is magnified once again by
the ocular lens or eyepiece (10 X).

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Instruments of Microscopy: Instruments of Microscopy:
2. Compound Light Microscopy 2. Compound Light Microscopy
u Resolution (Resolving power): Ability of
u Total magnification: Obtained by multiplying
microscope to see two items as separate and
objective lens power by ocular lens power. discrete units.
(Condenser lenses do not magnify image). u The smaller the distance between objects at which
they can be distinguished as separate, the greater the
Lens Magnification Ocular Mag. Total Mag. resolving power.
Scanning 4X 10 X = 40 X u Light must pass between two objects in order for them
to be seen as separate.
Low power 10 X 10 X = 100 X
u Depends on light wavelength. If wavelength is too
High power 45 X 10 X = 450 X long to pass between objects, they will appear as one.
uWhite light has a relatively long wavelength (550 nm), and
Oil immersion 100X 10 X = 1000 X cannot resolve structures less than 220 nm (0.2 um) apart.
Highest possible magnification with CL microscope is u Ultraviolet (UV) light has a shorter wavelength (100 to 400
nm), and can resolve distances as small as 110 nm .
about 2000 X.

Instruments of Microscopy: Instruments of Microscopy:

2. Compound Light Microscopy 3. DarkfieldMicroscopy
u Refraction: Bending of light as it passes from u Useful to examine live or unstained specimens.
one medium to another of different density.
u Light sensitive organisms
u Index of refraction: A measure of the speed at which
light passes through a material. u Specimens that lack contrast with their background.
u Can be changed by staining, which increases contrast uSpirochetes which cause syphilis.
between specimen and surrounding medium. u Darkfield condenser with opaque disc blocks
u When two substances have a different index of
refraction, the light will bend as it passes from one
light that would enter objective lens directly:
material to another. u Light reflects off specimen at an angle.
u As light passes through a glass slide, air, and the u Only light reflected by specimen enters objective lens.
objective lens, it bends each time, causing loss of light
and a blurred image.
u No direct background light.
u Immersion oil has the same index of refraction as u Image : Light specimen against dark
glass slide, preventing light loss from refraction. background.

Instruments of Microscopy: Instruments of Microscopy:

4. Phase Contrast Microscopy 5. Fluorescence Microscopy
u Useful to examine live specimens: uFluorescence: Ability of substances to absorb
short wavelengths of light (ultraviolet light) and
u Doesn’t require fixing or staining , which usually kill
emit them at a longer wavelength.
and/or distort microorganisms.
u Natural Fluorescence: Some microorganisms
u Permits detailedexamination of internal fluoresce naturally under UV light (Pseudomonas).
structures. u Fluorochrome: Fluorescent dye.
uAcridine orange: Binds to nucleic acids, colors cells orange,
u Special objective lenses and condenser with ring green, or yellow depending on light source.
shaped diaphragm accentuate small differences in u Immunofluorescence: Fluorescent antibodies can be
refractive indexes of internal structures. used to detect specific antigens. Very useful for the
rapid diagnosis of specific diseases (e.g.: syphilis).
u Image: Direct rays and reflected light rays come
together, forming an image with many shades of uImage: Luminescent bright object against a dark
gray to black. background.

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Instruments of Microscopy: Instruments of Microscopy:
Limitations of light microscopy: 6. Electron Microscopy
u Magnification: Up to 2000 X. u Electron microscopes were first developed in
1932, and became widely available in 1940s.
u Resolving Power: Up to 0.2 um. u Use a beam of electrons instead of a beam of
Because of the limits of magnification and light.
u Wavelength of electron beam is about 100,000 times
resolving power, viruses and most internal smaller than visible light.
structures of cells cannot be seen with a light u Used to examine structures too small to be resolved
microscope. with a light microscope.
u Two types of electron microscope:
A. Transmission Electron Microscope (TEM)
B. Scanning Electron Microscope (SEM)

Instruments of Microscopy: Instruments of Microscopy:

6. Electron Microscopy 6. Electron Microscopy
A. Transmission Electron Microscope (TEM) B. Scanning Electron Microscope (SEM)
u Gives excellent view ofinternal structures. u Gives excellent view ofexternal surface.
u Magnification: 100,000 X or more. u Magnification: 10,000 X or more.
u Resolving power: 20 nm or better.
u Resolving power: 2.5 nm or better.
u Three dimensional image.
u Two dimensional image.
u More recent invention than TEM. Used mainly to
u Drawbacks of TEM: observe the surfaces of cells and viruses.
u Due to limited penetrating power of electrons, can only u Specimens are covered with a layer of heavy metal
view very thin slices (70-90 nm) of specimen. (gold or palladium).
u Must slice, fix, dehydrate, and view specimen under a u A narrow beam of electrons (primary electron beam)
vacuum. Staining may be used to enhance image is swept across specimen surface.
contrast. u Electrons on the specimen surface are knocked out,
u Treatments kill specimen and may cause shrinkage and creating a secondary electron beam which is collected
and amplified to produce an image.
distortion of cells (artifacts).

7. Scanning Tunneling Microscopy and Preparation of Specimens for Light

Atomic Force Microscopy (AFM) Microscopy
u Developed in the 1980s. 1. Smear: Spread a thin film of material containing
u Used to observe structure and surface of microorganisms over slide surface. Allow to air
biological molecules and silicon computer chips. dry.
A. Scanning Tunneling Microscope (STM)
2. Fixing: Process that kills microorganisms and
Uses a thin metal probe that scans the surface of a
specimen. attaches them to a microscope slide. Fixing
B. Atomic Force Microscopy (AFM) preserves and minimizes distortion of cells.
Uses a diamond and metal probe that scans Two main methods of fixation:
surface of specimen. u Heat fixation: Pass over Bunsen burner flame several
Advantages of both microscopes: times.
u Higher resolving power than electron microscopes u Chemical fixation: Cover with methanol for 1 minute.
u No special specimen preparation required

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Preparation of Specimens for Light Preparation of Specimens for Light
Microscopy Microscopy
3. Staining: Coloring microorganisms with a dye A. Basic dyes :
that emphasizes certain structures. Before staining u Chromophor is in positive ions.
a sample, it must be fixed.
u Most commonly used dyes.
Stains are salts composed of a positive ion
u Bacteria are slightly negatively charged at pH
(cation) and a negative ion (anion).
7, therefore they stain with basic dyes.
The colored ion is called the chromophore .
u Examples:
Two types of dyes:
uCrystal violet
A. Basic dyes
u Methylene blue
B. Acidic dyes
u Saffranin

Preparation of Specimens for Light Preparation of Specimens for Microscopy

Microscopy 1. Simple Stains
B. Acidic dyes: u Aqueous or alcohol solution of a single basic
u Color is in negative ions. dye.
uStain the background: negative staining. u Primary purpose is to stain entire microorganism
u Bacteria do not stain with acidic dyes. to view cell shape and basic structures.
u Procedure:
u Used to observe cell shape, size, and capsules.
u Stain is applied for a certain time, and then washed off.
u Minimal distortion because heat fixing is not u Slide is dried and examined.
necessary an dye is not taken up by cells.
u Mordant: May be used to increase stain intensity.
u Examples: Increases affinity of stain for specimen.
u Eosin u Examples: Safranin , methylene blue, crystal
u Nigrosin violet, and carbolfuchsin.
u India ink.

Preparation of Specimens for Microscopy Preparation of Specimens for Microscopy

2. Differential Stains 2. Differential Stains
u React differently to different types of bacteria. A. Gram Stain
u Can be used to distinguish among different u Developed in 1884 by Hans Gram, a Danish
groups of bacteria. microbiologist.
u There are two important differential stains used in u The most useful staining procedure in medical
microbiology: microbiology.
A. Gram stain u Distinguishes bacteria of two large and medically
important groups:
B. Acid-Fast stain
u Gram-positive bacteria
u Gram-negative bacteria
u Provides useful information for disease treatment.

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Preparation of Specimens for Microscopy Preparation of Specimens for Microscopy
2. Differential Stains 2. Differential Stains
Steps of Gram Stain Steps of Gram Stain
3. Decolorizing: Slide is washed with alcohol,
1. Primary stain: Cover a heat fixed smear with a
which will remove stain from Gram-negative cells
basic dye (crystal violet). but not from Gram-positive cells.
u All cells, gram-positive and gram-negative, are u Gram-negative cells will be decolorized.
stained with crystal violet (appear purple). u Gram-positive cells will remain purple.
2. Mordant: After smear is rinsed with water, an 4. Counterstain: Alcohol is rinsed off. Safranin is
iodine mordant solution is applied. applied, which will stain cells that were
u Crystal violet-iodine [CV-I] complex forms
decolorized.
u Gram-negative cells are stained pink.
u Gram-positive cells remain purple.

Preparation of Specimens for Microscopy Preparation of Specimens for Microscopy

2. Differential Stain 2. Differential Stain
What accounts for the differential Applications and Limitations of the
staining between Gram-positive and Gram stain
Gram-negative cells? Chemotherapy:
uGram-positive cells with their very thick peptidoglycan cell walls,
u Gram-positive cells have very thick peptidoglycan cell
are susceptible to penicillins and cephalosporins.
walls, whereas gram-negative cells have very thin cell
uGram-negative cells with their thin cell walls and
walls. Crystal violet easily penetrates both cell types. lipopolysaccharide layer are resistant to these antibiotics.
u Because of its larger size, the crystal violet-iodine complex u Limitations :
[CV-I] is not easily removed from gram-positive cells, due u Not all bacterial cells stain well with the Gram-stain.
to their thick cell wall. The CV-I complex is readily u Gram-stain only works well on young bacterial cultures, that are
washed out of gram-negative cells with alcohol. actively growing. Therefore it is best to use cultures that are 18 to
u Counterstain only colors gram-negative cells. 24 hours old.
u Older cultures (over 24-48 hours), are often gram -variable.

Preparation of Specimens for Microscopy Preparation of Specimens for Microscopy

2. Differential Stains 2. Differential Stains
B. Acid-Fast Stain (Ziehl -Nielsen Stain) Steps of Acid-Fast Stain
u Modification of a method developed in 1882 by 1. Primary stain:
Paul Ehrlich.
u Cover a heat fixed smear with carbolfuchsin , a
u Used to detect tuberculosis and leprosy causing
red basic dye.
organisms of the genus Mycobacterium and
pathogens of the genus Nocardia. u Gently heat for several minutes to increase
u These bacteria have waxy cell walls, which penetration and retention of dye.
makes them difficult to stain. u Allow to cool and rinse with water.

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Preparation of Specimens for Microscopy Preparation of Specimens for Microscopy
2. Differential Stains 3. Special Stains
Steps of Acid-Fast Stain Used to color and isolate specific parts of
2. Decolorizing: Slide is washed with acid-alcohol. microorganisms such as:
u Non acid-fast cells will be decolorized. u Endospores
u Acid-fast cells will remain red.
u Capsules
3. Counterstain: Acid-alcohol is rinsed off.
Methylene blue is applied, which will stain cells u Flagella
that were decolorized.
u Non acid-fast cells are stained blue.
u Acid-fast cells remain red.

Preparation of Specimens for Microscopy Preparation of Specimens for Microscopy

3. Special Stains 3. Special Stains
A. Endospore Stain A. Endospore Stain
u Endospores are extremely resistant, dormant structures Steps for Schaeffer-Fulton Endospore Stain
that are formed by some gram-positive bacteria to protect 1. Primary stain: Malachite green is applied to heat
them from harsh environmental conditions: heat, drought, fixed smear and steamed for about 5 minutes.
chemicals, radiation, etc. uMalachite green will penetrate endospore.

u Ordinary staining methods cannot penetrate the thick 2. Wash: Rinse with water for 30 seconds.
endospore wall. u Removes green dye from rest of the cell, except for
endospore
u Most commonly used method is Schaeffer-Fulton
3. Counterstain: Safranin will stain rest of the cell.
endospore stain.
Appearance of cell with endospore:
Pink cell with green endospore.

Preparation of Specimens for Microscopy Preparation of Specimens for Microscopy

3. Special Stains 3. Special Stains
B. Capsule Stain C. Flagella Stain
u Capsules are gelatinous covers on top of the cell wall, u Flagella are appendages used for locomotion that are too
which are important virulence (disease) factors. thin to be seen easily with a light microscope.
u Capsules are difficult to stain because they repel most u Staining procedures are difficult. Usually involve using a
stains, are water soluble, and are easily disrupted with mordant and coating the flagellar surface with a dye or
harsh treatment. metal (e.g.: silver).
u Negative stain is used to obtain a dark background (E.g.: u The number and arrangement of flagella can be used as
India ink or nigrosin ). diagnostic aids.
u Cell is stained with a basic dye (E.g.: safranin).
Capsule appearance: Light halo around stained cell, dark
background.