SPONTANEOUS GENERATION

Learning Objectives

✓ Identify the scientists who argued in favor of spontaneous

generation.

✓ Compare and contrast the investigations of Redi, Needham,

Spallanzani, and Pasteur concerning spontaneous generation.

✓ List four steps in the scientific method of investigation.

FRANCESCO REDI
-DECAYING MEAT KEPT FROM ISOLATED FLIES, MAGGOTS NEVER DEVELOPED
-MEAT EXPOSED TO FLIES, SOON INFECTED
-WHEN THE FLASK OPENING WAS COVERED WITH GAUZE, FLIES WERE KEPT
AWAY, NO MAGGOTS APPEARED ON THE MEAT, ALTHOUGH A FEW MAGGOTS
APPEARED ON TOP OF THE GAUZE
JOHN NEEDHAM
-BOILED BEEF GRAVY AND INFUSION OF PLANT MATERIALS IN VIALS WHICH
TIGHTLY COVERED WITH CORK
-FEW DAYS AFTER, VIALS WERE CLOUDY
-AS HE EXPLAINED IT, THERE MUST BE A “LIFE FORCE” THAT CAUSES
INANIMATE MATTER TO SPONTANEOUSLY COME TO LIFE, BECAUSE HE HAD
HEATED THE VIALS SUFFICIENTLY TO KILL EVERYTHING
LAZZARO SPALLANZANI
-BOILED INFUSIONS FOR ALMOST AN HOUR AND SEALED THE VIALS BY
MELTING THEIR SLENDER NECKS CLOSED, INFUSIONS REMAINED CLEAR
-HE BROKE THE SEAL AND EXPOSED IN THE AIR, BECAME CLOUDY WITH
MICROORGANISM
-ALL LIVING THINGS ARISE FROM OTHER LIVING THINGS
LOUIS PASTEUR
-BOILED INFUSIONS AND EXAMINE IN “SWAN-NECKED FLASK”, ALLOWED AIR
TO ENTER WHILE PREVENTING INTRODUCTION OF DUST AND MICROBES
-FLASKS REMAINED FREE OF MICROBES WITHIN 18 MONTHS
-BROKE THE NECK OF SOME FLASKS, FLASKS WERE CLOUDY AFTER A DAY
-HE CONCLUDED THAT THE MICROBES IN THE LIQUID WERE THE PROGENY OF
MICROBES THAT HAD BEEN ON THE DUST PARTICLES IN THE AIR
-OBSERVED YEAST CELLS GROWING AND BUDDING IN GRAPE JUICE
-SEAL STERILE FLASKS CONTAINING GRAPE JUICE AND YEAST , OTHERS
EXPOSE TO AIR, YEASTS ARE FACULTATIVE ANAEROBES (live w/ or w/o oxygen)
-BACTERIA FERMENT GRAPE JUICE TO PRODUCE ACIDS AND THAT YEAST
CELLS FERMENT GRAPE JUICE TO PRODUCE ALCOHOL
-PASTEURIZATION, a process of heating the grape juice just enough to kill most
contaminating bacteria without changing the juice’s basic qualities, so that it could then
be inoculated with yeast to ensure that alcohol fermentation occurred.
EDUARD BUCHNER
-RESURRECTED THE CHEMICAL EXPLANATION BY SHOWING THAT
FERMENTATION DOES NOT REQUIRE LIVING CELLS.
-DEMONSTRATED THE PRESENCE OF ENZYMES, WHICH ARE CELL-PRODUCED
PROTEINS THAT PROMOTE CHEMICAL REACTIONS.
-BEGAN THE FIELD OF BIOCHEMISTRY AND THE STUDY OF METABOLISM, A
TERM THAT REFERS TO THE SUM OF ALL CHEMICAL REACTIONS WITHIN AN
ORGANISM.

WHAT CAUSE DISEASES?

Learning Objectives

✓ List at least seven contributions made by Koch to the field of

microbiology.

✓ List the four steps that must be taken to prove the cause of an
infectious disease.

✓ Describe the contribution of Gram to the field of

microbiology.

ROBERT KOCH
-DISCOVERED THE CAUSE OF ANTHRAX, WHICH TOXINS PRODUCE
ULCERATION OF THE SKIN, He observed the formation of resting stages
(endospores) within the bacterial cells and showed that the endospores always
produced anthrax when they were injected into mice. This was the first time that a
bacterium was proven to cause a disease
-KOCH’S METHOD OF ISOLATION, STANDARD TECHNIQUE IN
MICROBIOLOGICAL AND MEDICAL LABS TO THIS DAY, THOUGH A GEL CALLED
AGAR, DERIVED FROM RED SEAWEED, IS USED INSTEAD OF GELATIN OR
POTATO
-ANNOUNCES THAT CAUSE OF TUBERCULOSIS IS THE Mycobacterium
tuberculosis.
1. Simple staining techniques for bacterial cells and flagella
2. The first photomicrograph of bacteria
 3. The first photograph of bacteria in diseased tissue
4. Techniques for estimating the number of bacteria in a solution based on the
number of colonies that form after inoculation onto a solid surface
5. The use of steam to sterilize growth media
6. The use of Petri18 dishes to hold solid growth media
7. Laboratory techniques such as transferring bacteria between media using a
metal wire that had been heat-sterilized in a flame
8. Elucidation of bacteria as distinct species
-KOCH’S POSTULATE:
1. The suspected causative agent must be found in every case of the disease and be
absent from healthy hosts.
2. The agent must be isolated and grown outside the host.
3. When the agent is introduced to a healthy, susceptible host, the host must get the
disease.
4. The same agent must be found in the diseased experimental host.
HANS CHRISTIAN GRAM
-DEVELOP A STAINING TECHNIQUE, GRAM STAINING
- Results of Gram staining. Gram-positive cells (in this case Staphylococcus aureus) are
purple; Gram-negative cells (in this case Escherichia coli) are pink.

HOW CAN WE PREVENT INFECTION AND

DISEASE?
Learning Objectives

✓ Identify four health care practitioners who did pioneering research in the
areas of public health microbiology and epidemiology.

✓ Name two scientists whose work with vaccines began the field of
immunology.

✓ Describe the quest for a “magic bullet.”

FOUR HEALTH CARE PRACTITIONERS WHO WERE ESPECIALLY INSTRUMENTAL

IN CHANGING THE WAY HEALTH CARE IS DELIVERED WERE SEMMELWEIS,
LISTER, NIGHTINGALE, SNOW, JENNER, AND EHRLICH.
IGNAZ SEMMELWEIS
-
 CHAPTER 4
Microscopy,
Staining and
Classification
Clinical Case: The Salty Toddler
Lewis is an active three-year-old with a good appetite, though he is significantly smaller
than other boys his age. He loves playing with his six-year-old brother, Robert, and is
usually able to keep up with him; however, lately Lewis has been coughing a lot and has
not been running around as much as usual. He’s also had diarrhea, which seems to
come and go regardless of his diet. His mother, Dalia, has also noticed a mysterious,
distinctively salty taste on Lewis’s skin whenever she kisses him on the neck. She
wonders whether she should be concerned.
Lewis’s symptoms worsen. He starts to wheeze and cough up yellow mucus,
sometimes with a little streak of blood in it. He also develops a fever, which prompts
Dalia to take him to the hospital. There, she learns that Lewis has an unusual
respiratory infection caused by Pseudomonas, which does not typically affect healthy
children. Why, then, does Pseudomonas infect Lewis? Moreover, a lung infection
explains only some of Lewis’s symptoms. What is causing the persistent diarrhea? Why
is Lewis so small for his age?

Units of Measurement
Learning Objectives

✓ Identify the two primary metric units used to measure the diameters of
microbes.

✓ List the metric units of length in order, from meter to nanometer.

-A unit of measurement is smaller than the object being measured.
-Unit of length in metric system is meter (m)
Metric Unit Meaning of Metric U.S. equivalent Representative
prefix equivalent Microbiological
Application of
the Unit
Meter 1m 39.37 in Length of pork
tapeworm,
Taenia solium
(1.8-8.0 m)
Decimeter 1/10 0.1 m 3.94 in
Centimeter 1/100 0.01 m 0.39 in Diameter of a
mushroom cap
(12 cm)
Millimeter 1/1000 0.001 m Diameter of a
bacterial
colony (2.3
mm)
Micrometer 1/1000000 0.000001 m Diameter of a
white blood
cells (5-25 µm
Nanometer 1/1000000000 10-9 m Diameter of a
poliovirus (25
nm)

Microscopy
Learning Objective

✓ Define microscopy.

-Microscopy refers to use of light or electrons to magnify objects. The science of

microbiology began when Leeuwenhoek used primitive microscopes to observe and
report the existence of microorganisms. Since that time, scientists, and engineers have
developed a variety of light and electron microscopes.

General Principles of Microscopy

Learning Objectives

✓ Explain the relevance of electromagnetic radiation to microscopy.

✓ Define empty magnification.

✓ List and explain two factors that determine resolving power.

✓ Discuss the relationship between contrast and staining in microscopy.

-General principles involved in both light and electron microscopy include the
wavelength of radiation, the magnification of an image, the resolving power of the
instrument, and contrast in the specimen.
WAVELENGTH OF RADIATION
-Wavelength is the distance between two corresponding parts of the wave.
-Electrons are the negatively charged particles that orbit the nuclei of the atoms.
MAGNIFICATION
-Magnification is the apparent increase in size of the object. Magnification results when
a beam of radiation refracts (bends) as it passes through a lens.
-A lens refracts light because the lens is optically dense compared to the surrounding
medium (such as air); that is, light travels more slowly through the lens than through air.
-Because of its curvature, a lens refracts light rays that pass through its periphery more
than light rays that pass through its center, so that the lens focuses light rays on a focal
point.
-Microscopists could combine lenses to obtain an image magnified millions of times, but
the image would be faint and blurry. Such magnification is said to be empty
magnification
RESOLUTION
-Also called “revolving power” is the ability to distinguish objects that are close together.
-The better the resolution, the better the ability to distinguish two objects that are close
to one another.
- A principle of microscopy is that resolution distance is dependent on (1) the
wavelength of the electromagnetic radiation and (2) the numerical aperture of the lens,
which refers to the ability of a lens to gather light.
0.61 x wavelength
-resolution distance =
numerical aperture
CONTRAST
-Refers to the differences in intensity between two objects or an object and its
background.
-Most microorganisms are colorless and have very little contrast whether one uses light
or electrons. One way to increase the contrast between microorganisms and their
background is to stain them.

Light Microscopy
Learning Objectives
✓ Contrast simple and compound microscopes.

✓ Compare and contrast bright-field microscopy, dark-field microscopy, and phase

microscopy.

✓ Compare and contrast fluorescent and confocal microscopes.

-Several classes of microscopes use various types of light to examine microscopic
specimens.
-The most common microscopes are bright-field microscopes, in which the
background (or field) is illuminated.
-In dark-field microscopes, the specimen is made to appear light against a dark
background.
-Phase microscopes use the alignment or misalignment of light waves to achieve the
desired contrast between a living specimen and its background.
-Fluorescent microscopes use invisible ultraviolet light to cause specimens to radiate
visible light, a phenomenon called fluorescence.
-Microscopes that use lasers to illuminate fluorescent chemicals in a thin plane of a
specimen are called confocal microscopes.
BRIGHT-FIELD MICROSCOPES
-Simple Microscope contains a single magnifying lens.
-Compound Microscope uses series of magnifying lens.
- have four revolving nosepiece
1. Scanning objective lens (4x)
2. Low-power objective lens (10x)
3. High-power objective lens (40x)
4. Oil immersion objective lens (100x)
-Total magnification of compound microscope is determined by multiplying the
magnification of the objective lens by the magnification of the ocular lens.
-Condenser Lens directs light through the specimen.
-Photographs of a microscopic image are called light micrographs
DARK-FIELD MICROSCOPE
-Pale objects are best observed with dark-field microscopes
-Dark-field microscopes are especially useful for examining small or colorless cells
PHASE MICROSCOPE
-Used to examine living microorganisms that would be damaged or altered by attaching
them to slides or staining them.
-Light rays are said to be in phase when their crests and troughs are aligned, and out
of phase when their crests and troughs are not aligned.
-A special filter called a phase plate, which is mounted in a phase objective lens,
retards these rays another 1/4 wavelength, so that they are 1/2 wavelength out of phase
with their neighbors.
-There are two types of phase microscopes: phase-contrast and differential interference
contrast microscopes.
-Phase-contrast microscopes produce sharply defined images in which fine
structures can be seen in living cells. These microscopes are particularly useful for
observing cilia and flagella.
 -Differential Interference Contrast microscopes also called Nomarski2
microscopes create phase interference patterns. They also use prisms that split light
beams into their component wavelengths.
FLUORESCENT MICROSCOPE
-Use ultraviolet light source to fluoresce objects
-UV light increases resolution because it has a shorter wavelength than visible light.
-Pseudomonas aeruginosa and some cellular molecules such as chlorophyll in
photosynthetic organisms are naturally fluorescent. Other cells and cellular structures
can be stained with fluorescent dyes. When these dyes are bombarded with ultraviolet
light, they emit visible light and show up as bright orange, green, yellow, or other colors
against a black background.
-Some fluorescent dyes are specific for certain cells. For example, the dye fluorescein
isothiocyanate attaches to cells of Bacillus anthracis, the causative agent of anthrax,
and appears apple green when viewed in a fluorescent microscope. Another fluorescent
dye, auramine O, stains Mycobacterium tuberculosis
-Fluorescent microscopy is also used in a process called immunofluorescence
CONFOCAL MICROSCOPE
-Also use fluorescent dyes but use ultraviolet lasers to illuminate the fluorescent
chemicals in only a single plane that is no thicker than 1.0 μm.
-Visible light emitted by the dyes passes through a pinhole aperture, which helps
eliminate blurring that can occur with other types of microscopes and increases
resolution by up to 40%.
-Confocal microscopes have been particularly useful for examining the relationships
among various organisms within complex microbial communities called biofilms.

Electron Microscopy
Learning Objectives
✓ Contrast transmission electron microscopes with scanning electron microscopes in
terms of how they work, the images they produce, and the advantages of
each.Contrast simple and compound microscopes.

-Electron microscopes magnify objects 10,000 X to 100,000 X.

-Provide detailed views of the smallest bacteria, viruses, internal cellular structures, and
even molecules and large atoms.
-Cellular structures that can be seen only by using electron microscopy are referred to
as a cell’s ultrastructure
-Ultrastructural details cannot be made visible by light microscopy because they are too
small to be resolved.
-There are two general types of electron microscopes: transmission electron
microscopes and scanning electron microscopes.
TRANSMISSION ELECTRON MICROSCOPE
-generates a beam of electrons that ultimately produces an image on a fluorescent
screen.
-the path of electron is similar to the path of light in a light microscope.
-SOURCE  electrons  SPECIMEN  MAGNETIC FIELDS  focus the beam 
FLUORESCENT SCREEN  absorbs electrons
-The screen can be folded out of the way to enable the electrons to strike a
photographic film, located in the base of the microscope. Prints made from the film are
called transmission electron micrographs or TEM images
-Matter, including air, absorbs electrons, so the column of a transmission electron
microscope must be a vacuum, and the specimen must be very thin. Before thicker
specimens such as whole cells can be examined, they must be dehydrated, embedded
in plastic, and cut to a thickness of about 100 nm with a diamond or glass knife mounted
in a slicing machine called an ultramicrotome.
SCANNING ELECTRON MICROSCOPE
-uses magnetic fields within a vacuum tube to manipulate a beam of electrons, called
primary electrons.
-rapidly focuses the electrons back and forth across the specimen’s surface coated
with a metal such as platinum or gold.
-The primary electrons knock electrons off the surface of the coated specimen, and
these scattered secondary electrons pass through a detector and a photomultiplier,
producing an amplified signal that is displayed on a monitor. Typically, scanning
microscopes are used to magnify up to 10,000 X with a resolution of about 20 nm.
-Advantage of scanning microscopy over transmission microscopy is that whole
specimens can be observed, because sectioning is not required. Scanning electron
micrographs can be beautifully realistic and three-dimensional.
-Disadvantages of a scanning electron microscope are that it magnifies only the
external surface of a specimen and, like TEM, it requires a vacuum and thus can
examine only dead organisms.

Probe Microscopy
Learning Objective

✓ Describe two variations of probe microscopesLearning Objectives

-A relatively recent advance in microscopy utilizes minuscule, pointed, electronic probes

to magnify more than 100,000,000 times.
-There are two variations of probe microscopes: scanning tunneling microscopes and
atomic force microscopes.
SCANNING TUNNELING MICROSCOPE
-passes a metallic probe, sharpened to end in a single atom, back and forth across and
slightly above the surface of the specimen.
-measures the flow of electrons to and from the probe and the specimen’s surface.
-The amount of electron flow, called a tunneling current, is directly proportional to the
distance from the probe to the specimen’s surface.
-A scanning tunneling microscope can measure distances as small as 0.01 nm and
reveal details on the surface of a specimen at the atomic level.
ATOMIC FORCE MICROSCOPE
-also uses a pointed probe, but it traverses the tip of the probe lightly on the surface of
the specimen, rather than at a distance.
-deflection of a laser beam aimed at the probe’s tip measures vertical movements,
which when translated by a computer reveals the atomic topography.
-atomic force microscopes can magnify specimens that do not conduct electrons.

Staining
-increase contrast and resolution
PREPARINGSPECIMENS FOR STAINING
-Staining simply means coloring specimens with stains or dyes
-Before microbiologists stain microorganisms, they must place them on and then firmly
attach them to a microscope slide. Typically, this involves making a smear and fixing it
to the slide.
-If the organisms are growing in a liquid, a small drop is spread across the surface of
the slide.
-If the organisms are growing on a solid surface, such as an agar plate, then they are
mixed into a small drop of water on the slide.
-The thin film of organisms on the slide is called a smear.
-In heat fixation, developed more than a hundred years ago by Robert Koch, the slide
is gently heated by passing the slide, smear up, through the flame of a Bunsen burner.
-Chemical fixation involves applying a chemical such as methyl alcohol to the smear
for one minute.
-Specimens prepared for electron microscopy are also dried, because water vapor
from a wet specimen would stop the electron beam. As we have seen, transmission
electron microscopy requires that the desiccated sample also be sliced very thin,
generally before staining. Specimens for scanning electron microscopy are coated, not
stained.

Principles of Staining
Learning Objective

✓ Describe the uses of acidic and basic dyes, mentioning ionic bonding and
pH.Learning Objectives

-Dyes used as microbiological stains for light microscopy are usually salts.
-A salt is composed of a positively charged cation and a negatively charged anion.
-At least one of the two ions in the molecular makeup of dyes is colored; this colored
portion of a dye is known as the chromophore.
-Anionic chromophores are also called acidic dyes because they stain alkaline
structures and work best in acidic (low pH) environments.
-Positively charged, cationic chromophores are called basic dyes because they
combine with and stain acidic structures; further, they work best under basic (higher pH)
conditions.

Simple Stains
Learning Objective

✓ Describe the simple, Gram, acid-fast, and endospore staining

procedures.Learning Objectives

-Simple stains are composed of a single basic dye such as crystal violet, safranin, or
methylene blue.

Differential Stains
-Most stains used in microbiology are differential stains, which use more than one dye
so that different cells, chemicals, or structures can be distinguished when
microscopically examined. Common differential stains are the Gram stain, the acid-
fast stain, the endospore stain, Gomori methenamine silver stain, and
hematoxylin and eosin stain.
GRAM STAIN
-Hans Christian Gram developed the most frequently used differential stain
Six I’s