Download Ch1 bio250

Microbiology: Basic and Clinical Principles
Second Edition
Chapter 1
Introduction to Microbiology
What Is Microbiology? (1 of 6)
• Microbiology is the study of microorganisms or
microbes, which are often invisible to the naked eye.
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
What Is Microbiology? (2 of 6)
• The term microbe encompasses…
– Cellular, living microorganisms such as bacteria,
archaea, fungi, protists, and helminths
– Nonliving/noncellular entities such as viruses and
prions (infectious proteins)
– Microorganisms that are not microscopic such as
some fungi, helminths, and protists (however, part of
their life cycle is microscopic)
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
What Is Microbiology? (3 of 6)
Table 1.1 Living and Nonliving Agents Studied in Microbiology
Microbe
Cell Type
Notes
Bacteria
Prokaryotic
Unicellular;* pathogenic and nonpathogenic
Archaea
Prokaryotic
Unicellular; nonpathogenic; most live in extreme environments
Protists
Eukaryotic
Unicellular and multicellular; pathogenic and nonpathogenic
(unicellular example: amoebae; multicellular example: algae)
Fungi
Eukaryotic
Unicellular and multicellular; pathogenic and nonpathogenic
(unicellular example: yeast; multicellular example: mushrooms)
Helminths
Eukaryotic
Multicellular;* parasitic roundworms and flatworms
Viruses
Not cells; nonliving
Infect animal, plant, or bacterial cells; can have a DNA or RNA
genome
Prions
Not cells; nonliving;
infectious proteins
Not discovered until the 1980s; transmitted by transplant or
ingestion; some prion diseases are inherited
*Unicellular = one-celled organism; multicellular = organism made of many cells
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
What Is Microbiology? (4 of 6)
• At least half of Earth’s life is microbial
• Microbes inhabit almost every region of our planet
– Deep-sea trenches to glaciers
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
What Is Microbiology? (5 of 6)
• Prokaryotic cells
– Evolved about 3.5 billion years ago
– Earliest life forms
– Include unicellular bacteria and archaea
• Eukaryotic cells
– All multicellular organisms and a number of unicellular
microorganisms (e.g., amoebae and yeast)
– Endosymbiotic theory
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
What Is Microbiology? (6 of 6)
• Microbiology spans a wide variety of fields:
– Healthcare
– Agriculture
– Industry
– Environmental sciences
• Humans rely on microbes for many things:
– Food production
– Making medications
– Breaking down certain environmental hazards
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Microbes and Disease
• Pathogens are microbes that cause disease
– About 1,400 pathogens are known to infect humans
– <1% of all microbes are pathogenic
– Some “true” pathogens will always cause disease in
humans
• Opportunistic pathogens cause disease only in a
weakened host
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Great Advances Occurred in and Around
the Golden Age of Microbiology
• Golden age of microbiology (1850–1920)
– Innovations in microscopes
– Observations
– New techniques to isolate and grow microbes
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Spontaneous Generation Versus
Biogenesis (1 of 6)
• Robert Hooke (mid-1600s)
– First to publish
descriptions of cells
• Antonie van Leeuwenhoek
(1632–1723)
– Refined earlier versions
of the microscope
– First to see bacteria
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Spontaneous Generation Versus
Biogenesis (2 of 6)
With mounting observations of cells, scientists heavily
debated the origin of life…
• Spontaneous generation: life comes from nonliving
items
• Biogenesis: life emerges from existing life
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Spontaneous Generation Versus
Biogenesis (3 of 6)
“Proof” of spontaneous generation was that rotting
meat gave rise to maggots
• Francesco Redi (1626–1697)
– Meat in an uncovered jar resulted in Maggots on the
Meat
– Meat in a jar with a gauze-covered top resulted in No
Maggots on the Meat
• However, spontaneous generation theory persisted for
another 200 years!!!
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Spontaneous Generation Versus
Biogenesis (4 of 6)
• Louis Pasteur (1822–1895)
– Showed that biogenesis is responsible for the
propagation of life
– Pasteurization killed off yeast and prevented stored
wine from turning bitter
– Developed first vaccine against anthrax and rabies
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Spontaneous Generation Versus
Biogenesis (5 of 6)
• Pasteur investigated his hypothesis that air contained
contaminating microbes by performing an experiment
with a specialized S-necked flask
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Spontaneous Generation Versus
Biogenesis (6 of 6)
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Germ Theory of Disease (1 of 2)
• The germ theory of disease states that microbes cause
infectious diseases
• Robert Koch developed a technique to determine the
specific etiological agent of an infectious disease
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Germ Theory of Disease (2 of 2)
• Robert Koch (1843–1910)
– Developed staining techniques and media for the
isolation and cultivation of bacteria
– Groundbreaking work performed with anthrax
▪ Caused by bacteria (Bacillus anthracis)
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Koch’s Postulates of Disease (1 of 3)
1. Same organism must be present in every case of the
disease
2. Organism must be isolated from the diseased host and
grown as a pure culture
3. Isolated organism should cause the same disease when
inoculated into a susceptible host
4. Organism must be re-isolated from the inoculated,
diseased animal
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Koch’s Postulates of Disease (2 of 3)
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Koch’s Postulates of Disease (3 of 3)
• We’ll probably never have a complete catalog of
every infectious agent because…
– New diseases emerge
– Microbes evolve new pathogenic capabilities
– Only ~2% of bacteria can be cultured in the
laboratory
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Hand Hygiene and Aseptic Techniques (1 of 5)
• From 1800s to1900s several medical professionals
emphasized the importance of aseptic techniques in
medical settings
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Hand Hygiene and Aseptic Techniques (2 of 5)
• Ignaz Semmelweis (1818–1865)
– Developed the first aseptic techniques in the hospital
setting
– Recommended hand washing to decrease mortality
rates from childbed fever (1840s)
▪ Childbed fever (puerperal sepsis), an infection that
killed many women in childbirth before the
antibiotic’s era
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Hand Hygiene and Aseptic Techniques (3 of 5)
• Joseph Lister (1827–1912)
– Investigated processes for aseptic surgery
– Proved sterilizing instruments and sanitizing wounds
with carbolic acid prevented pus formation (1860s)
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Hand Hygiene and Aseptic Techniques (4 of 5)
• Florence Nightingale (1820–1910)
– Established aseptic techniques in nursing
– Founder of modern nursing
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Hand Hygiene and Aseptic Techniques (5 of 5)
• Aseptic processes prevent healthcare-acquired
infections or HAIs (also called nosocomial infections)
and limit the spread of diseases
• Types of aseptic techniques:
– Washing hands
– Wearing gloves
– Sterilizing instruments
– Decontaminating surfaces
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Morphology and Physiology are Central to
Bacterial Classification
• Taxonomy is the study of how organisms can be
grouped by shared features
• Early classification of bacteria included physical features
morphology (e.g., shape, size, arrangement) and
distinguishing physiological features
• Carl Linnaeus (1707–1778)
– Father of taxonomy
– Established criteria for classifying organisms
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Taxonomic Hierarchy (1 of 10)
• There are eight rankings within the taxonomic hierarchy
• Rankings range from broad overarching domains all the
way down to the precise species level
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Taxonomic Hierarchy (2 of 10)
• “Delightful King Philip came over for great spaghetti”
– Domain
– Kingdom
– Phylum
– Class
– Order
– Family
– Genus
– Species
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Taxonomic Hierarchy (3 of 10)
• Domain is the broadest grouping of organisms
• Three domains:
– Bacteria
▪ Unicellular, prokaryotic organisms
– Archaea
▪ Some live in extreme environments
▪ No known pathogens
– Eukarya
▪ Unicellular and multicellular eukaryotic organisms
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Taxonomic Hierarchy (4 of 10)
• Beneath the umbrella of domains are a variety of
kingdoms
– Number of kingdoms has fluctuated from 5 to 8
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Taxonomic Hierarchy (5 of 10)
• Older 5-kingdom classification:
– Animalia, Plantae, Fungi, Protista, Monera
– Monera includes Both Domain Archaea and Domain
Bacteria
• Newer 6-kingdom classification:
– Kingdom Monera is replaced by Kingdom Archea and
Kingdom Bacteria
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Taxonomic Hierarchy (6 of 10)
Table 1.2 Six-Kingdom Classification System
Kingdom
Archaea
Bacteria
Fungi
Plantae
Animalia
Protists*
Example
A micrograph of Sulfolobus is shown. Sulfolobus is an irregular, doughnut-shaped organelle with an uneven surface. Five such organelles have been depicted in a cluster.
Domain
Archaea
A micrograph of S. aureus is shown as a collection of densely clustered, spherical bodies.
Bacteria
A micrograph of Candida albicans is shown as multiple ovoid bodies connected in a chain. A bud from the previous ovoid allows it to remain connected to the next. Some of the ovoids
have additional spherical protrusions as well.
Eukarya
A photograph of a flowering plant shows several, long-stemmed, blue flowers. There are two layers of petals, a larger, outer layer, and a smaller, inner layer. The leaves are small in size and
two of them appear at every node.
Eukarya
A photograph of a tree frog is shown. The tree frog appears to be perched on a branch or ledge. It is lemon-green with brown speckles spread over its back and hindquarters.
Eukarya
A micrograph of Paramecium displays a single paramecium cell as observed under a bright-field microscope. The cell is shaped like the sole of a shoe. It appears to have multiple, thin cilia all
around its surface.
Eukarya
*Not a true kingdom; a catchall category for lifeforms formerly grouped in Kingdom Protista
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Taxonomic Hierarchy (7 of 10)
• Kingdom Protista adds to the elaboration…
– Miscellaneous kingdom for organisms not categorized
as plants, animals, or fungi
– Genetics now shows that protists can’t logically be
lumped into a single kingdom
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Taxonomic Hierarchy (8 of 10)
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Taxonomic Hierarchy (9 of 10)
• Eukaryotic species
– Group of similar organisms that can sexually
reproduce together
• Prokaryotic species
– Cells that share physical characteristics and have at
least 70% DNA similarity
– At least 97% identical 16S rRNA sequence similarity
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Taxonomic Hierarchy (10 of 10)
• Strain is used to recognize genetic variants of the same
species
• Mutations and gene transfer often lead to new strains
• Strain names typically include numbers and/or letters
after the species name (e.g., E. coli K-12, a strain of
Escherichia coli commonly found in laboratories)
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Classifying Microbes
• Parameters for bacterial classification are diverse
• Microbiologists have worked to unify the classification
criteria for bacteria
• Bergey’s Manual of Determinative Bacteriology is the
cornerstone reference
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Scientific Names
• Carl Linnaeus established a Binomial nomenclature
system
– Two-name system
– Genus is the first name (Capitalized)
– Species is the second name (lowercase)
– Scientific names are italicized (or underlined if
handwritten)
– For example Escherichia coli
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Microbes May Be Friends or Foes
• Microbes constitute a huge part of the Earth’s biomass
• It is suspected that there are several million species of
microbes in our world
– Over 7,000 microbes have been characterized
• Most microbes are helpful or neutral to human health
• Only a small minority are human pathogens
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Host–Microbe Interactions (1 of 2)
• A symbiotic relationship exists when two or more
organisms are closely connected
• Microbes and humans have evolved a variety of
symbiotic relationships:
– Parasitism: hurt the host
– Mutualism: help the host
– Commensalism: no perceived benefit or cost to the
host
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Host–Microbe Interactions (2 of 2)
• Pathogens are described as having a parasitic
relationship with their host
• The term parasite is commonly used to describe
helminths (worms) and protozoans
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Normal Microbiota and the Human
Microbiome (1 of 5)
• Human Microbiome Project (HMP) aims to
characterize all of the microbes in and on our bodies
– Many parts of the human body teem with microbial
life
– There are at least as many microbial cells in and on
us as there are human cells
– Our skin, nose, mouth, gut, and genital/urinary tract
harbor the most microbes
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Normal Microbiota and the Human
Microbiome (2 of 5)
• Normal microbiota (or normal flora) includes bacteria,
archaea, and eukaryotic microbes
• Functions of the normal flora:
– Train our immune system
– Produce vitamins for us
– Help us digest foods
– They may even impact our moods and brain function
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Normal Microbiota and the Human
Microbiome (3 of 5)
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Normal Microbiota and the Human
Microbiome (4 of 5)
• What makes microbiota “normal” sometimes has to do
with the location of the microbe rather than the species
itself
• Our normal microbiota often includes pathogens
– 27% percent of adults asymptomatically carry
Staphylococcus aureus on their skin
• The majority of normal microbiota is harmless
– Protects us by “crowding out” potential pathogens
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Normal Microbiota and the Human
Microbiome (5 of 5)
• Certain microbiome profiles may increase the chance of
certain chronic diseases or disorders, while others may
be protective
• There’s a long way to go in exploring potential links
between the microbiome and human physiology
• A better understanding may lead to re-tooling of normal
microbiota to treat certain diseases
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Establishing Normal Microbiota (1 of 3)
• Babies are colonized by microbes during delivery and
through early interactions with their environment and
caregivers
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Establishing Normal Microbiota (2 of 3)
• Data suggests that microbes may start to colonize us
even before birth
– Researchers found low levels of microbes in the
placenta (similar to oral bacteria)
– Oral microbiota is present in the baby’s first stool
(meconium)
– Bacteria have been isolated from umbilical cord blood
of healthy term infants
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Establishing Normal Microbiota (3 of 3)
• The developing normal microbiota of an infant is greatly
influenced by:
– Delivery (cesarean section or vaginal)
– Feeding (breast milk or formula fed)
• Normal microbiota expands, develops, and evolves
throughout the early weeks of life to adulthood
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Disruptions in Normal Microbiota (1 of 2)
• When our normal microbiota is perturbed, we are put at
risk for infections
• Normal microbiota can be disrupted with antibiotic
therapy
– Kills resident bacteria and the pathogen
– Reduction of normal microbiota allows opportunistic
pathogens to establish infections
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Disruptions in Normal Microbiota (2 of 2)
• Examples:
– Woman takes antibiotics to treat a urinary tract
infection (UTI)
▪ Candida albicans (yeast) is an opportunistic
pathogen present in the vagina
▪ Kept in check by normal vaginal microbiota
▪ Decreased vaginal microbiota leads to a vaginal
yeast infection
– Diarrhea due to antibiotic therapies
▪ Gut microbiome is affected
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Transient Microbiota
• Transient microbiota are temporary passengers that do
not persist as stable residents of our bodies
• Picked up through a handshake or contact with
environmental surfaces
• Can be removed through hygiene (e.g., proper handwashing)
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Host–Microbe Interactions Can Influence
Human Evolution
• Close ecological relationships with microbes have led
humans and microbes to coevolve
• Malaria is a mosquito-borne, tropical disease caused by
a protozoan
– kills over 600,000 people every year
• People who carry the gene for sickle cell anemia, a
blood disorder characterized by a mutation in the gene
for hemoglobin, are less likely to develop severe malaria
• Carriers of the sickle cell gene have a survival advantage
in areas where malaria is common
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Biofilms (1 of 4)
• Single, planktonic bacteria are free-floating
• Biofilms are sticky communities made up of single or
diverse microbial species
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Biofilms (2 of 4)
• Cells that seed a biofilm make adhesion factors to help
them attach to a target surface
• They secrete a sticky substance that forms a protective
matrix in which the bacteria grow
• Multiple layers tend to develop (residents of the
innermost layers are highly protected)
• Periodically, microbes in the film are released as freegrowing planktonic cells
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Biofilms (3 of 4)
• Biofilms can develop on nearly any surface, including:
– Teeth (dental plaque)
– Contact lenses
– Water filtering units
– Cutting boards
– Catheters
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Biofilms (4 of 4)
• NIH estimates that 60–80% of infectious diseases in
humans are due to biofilm-creating microbes
• Internal biofilms are not easily managed
– More resistant to antibiotics
– Protected from the immune system
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Environmental and Industrial Uses for
Microbes
• Bioremediation harnesses the power of microbes to
help clean up toxic waste
• Certain microbes can metabolize toxic substances into
harmless intermediates
• For example, hundreds of microbial species can degrade
petroleum oil spills into CO2
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
We Culture Microbes So We Can Study
Them
• First step to study a microbe is to try to grow it in the
laboratory
• Easier said than done for the majority of known species
• Microbes often require complex growth environments
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Introduction to Growth Media (1 of 2)
• Growth media (culture media) are mixtures of nutrients
that support growth in an artificial setting
• Agar is sometimes added as a solidifying agent and
allows for isolation
• Julius Richard Petri developed the petri dish
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Introduction to Growth Media (2 of 2)
• Media comes in a wide
variety of consistencies
and formulations
• Types of media:
– Broths
– Plates
– Slants
– Deeps
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Aseptic Culture Techniques (1 of 4)
• In nature, microbes do not tend to grow in single-species
groups
• Pure culture is a specific type of microbe isolated from a
diverse sample
• Aseptic culturing techniques are conditions maintained
to limit contaminants
– Sterile media
– Sterile instruments
– Decontaminating surfaces
– Gloves and other protective clothing
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Aseptic Culture Techniques (2 of 4)
• Biological safety cabinet is an
enclosed cabinet that minimizes
the chances of contaminating the
culture and protects the
researcher
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Aseptic Culture Techniques (3 of 4)
• Streak plate technique helps to isolate colonies of a
specific microbe for study
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Aseptic Culture Techniques (4 of 4)
• Colony is a grouping of cells (clones) that developed from a
single parent cell
• Mixed cultures have >1 characteristically different colonies
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Specimens Are Often Stained Before
Viewing With a Microscope (1 of 5)
• Stains, or dyes, increase contrast so the sample is easier
to see
• Most bacterial staining techniques involve:
– Making a smear of the specimen
– Fixing the specimen by exposing it to heat (or
chemical reagent)
– Staining of the specimen
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Specimens Are Often Stained Before
Viewing With a Microscope (2 of 5)
• Basic dyes are some of the most commonly used stains
– Dye is positively charged
– Attracted to the negatively charged cell surface
– Result: cell appears the color of the dye
• Examples:
– Methylene blue
– Crystal violet
– Safranin
– Malachite green
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Specimens Are Often Stained Before
Viewing With a Microscope (3 of 5)
• Acidic dyes are used in negative staining
– Dye is negatively charged
– Repelled from negatively charged cell surface
– Result: stain the background of a specimen
• Examples:
– Nigrosin
– India ink
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Specimens Are Often Stained Before
Viewing With a Microscope (4 of 5)
• Mordants are chemicals that may be required in certain
staining procedures to interact with a dye and fix, or trap,
it on or inside a treated specimen
• Examples:
– Iodine
– Alum
– Tannic acid
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Specimens Are Often Stained Before
Viewing With a Microscope (5 of 5)
• Most microbiological staining techniques are
classified as:
– Simple
– Structural
– Differential
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Simple Stains
• Simple staining techniques use one dye
– Used to determine size, shape, and/or cellular
arrangement
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Structural Stains (1 of 4)
• Flagella Staining
– Prokaryotes can have
single or multiple flagella
with diverse
arrangements
– Mordants are added to
coat the thin flagella and
then a basic dye is
applied
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Structural Stains (2 of 4)
• Capsule Staining
– Capsules are sticky
carbohydrate-based structures
that some bacteria produce
– Both a basic dye (stains the cell)
and acidic dye (stains the
background) are used
– Capsule appears as a clear halo
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Structural Stains (3 of 4)
• Bacterial Endospore Staining
– Endospores are specialized
dormant structures that certain
bacteria form in harsh
conditions
– Specimen is heated to drive the
dye (malachite green) into the
spores
– Nonsporulating cells are
stained with safranin
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Structural Stains (4 of 4)
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Differential Stains: Gram and Acid-Fast
• Differential staining highlights differences in bacterial
cell walls in order to discriminate between classes of cells
• Examples:
– Gram stain
– Acid-fast stain
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Gram Stain (1 of 6)
• Gram stain classifies bacteria as either Gram-positive or
Gram-negative
• Gram-positive cells will appear purple and Gramnegative cells will appear pink
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Gram Stain (2 of 6)
• The Gram stain technique is as follows:
– Crystal violet (primary stain) is added to a heatfixed bacterial smear
– Iodine (mordant) is added forming an insoluble
crystal violet-iodine complex (CV-I complex)
– Acetone-alcohol (decolorizing step) is used to rinse
the sample
– Safranin (counterstain) is added to the sample
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Gram Stain (3 of 6)
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Gram Stain (4 of 6)
• To understand how it works we must first discuss
cell wall composition…
– Gram-positive cell walls
▪ Contain a thick layer of peptidoglycan
▪ No outer membrane
– Gram-negative cell walls
▪ Contain a thin layer of peptidoglycan
▪ Contain an outer membrane rich in lipids
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Gram Stain (5 of 6)
Results of the acetone-alcohol treatment on…
• Gram negative:
– Dissolves the outer membrane
– Damages the thin peptidoglycan layer
– CV-I washes out
• Gram positive:
– Slightly damages the thick peptidoglycan
– Dehydration makes it less permeable
– CV-I is retained
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Gram Stain (6 of 6)
Experimental errors can happen…
• If the sample is decolorized too long
– Thick peptidoglycan layer of Gram-positive cell walls
is damaged
– CV-I complex is rinsed out of the cells
– Gram-positive cells appear Gram-negative
• To minimize Gram property errors, fresh cultures
between 24 and 48 hours old should be used
• Interpreting results can be difficult...
– Variations in cell walls
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Acid-Fast Staining (1 of 4)
• Acid-fast stain distinguishes between cells with and
without waxy cell walls
• Acid-fast bacteria
– Contain waxy cell walls rich in mycolic acid
– Retain red-colored primary dye after exposure to an
acid wash
• Non–acid-fast cells
– Red primary stain is washed away after exposure to
an acid wash
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Acid-Fast Staining (2 of 4)
• Ziehl-Neelsen method
– Carbol-fuchsin (primary dye) is added to a heatfixed smear
– Sample is steamed for several minutes to drive the
red dye into the bacteria
– Acid-alcohol (decolorizing agent) is used to rinse
the sample
– Methylene blue (counterstain) is added to the
sample
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Acid-Fast Staining (3 of 4)
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Acid-Fast Staining (4 of 4)
• Important diagnostic tool for detecting:
– Mycobacterium species
– Nocardia species
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Microscopy Is Central to Microbiology
• Van Leeuwenhoek’s microscopes reached about 300×
magnification
• Today’s microscopes allow us to see samples 20 million
times smaller than the visibility of the eye
• Micrographs, or pictures taken through a microscope,
allow us to document and share microscopy observations
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Light Microscopy
• Light microscopy uses visible light to illuminate the
specimen
• Photons in a light wave interact with the specimen and
are then channeled up to the viewer’s eyes through a
series of lenses
• The compound light microscope is the most common
type of optical microscope
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Figure 1.16 Parts of a Compound Light
Microscope
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Parts of the Compound Light
Microscope (1 of 2)
• Objective lens is near the specimen
– Come in varieties that usually include
4, 10, 40, and 100 
• Ocular lens sits at the top of the microscope near the
viewer’s eyes
• Final magnification is determined by multiplying the
magnification of the ocular and objective lenses
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Parts of the Compound Light
Microscope (2 of 2)
• Condenser lenses sharpen light into a precise cone to
illuminate the specimen
• Iris diaphragm controls amount of light aimed at the
specimen to improve contrast
• Coarse focus knob allows the viewer to roughly focus
the image by adjusting the distance between the
objective lens and specimen
• Fine focus knob allows for precision focusing
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Resolution
• Resolution is the ability to distinguish two distinct points
as separate
– The naked eye has a resolution of about 0.1 mm
(100,000 nm)
– Most compound light microscopes magnify up to
1,500  with resolution of about 200 nm
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Oil Immersion (1 of 2)
• Refractive index is the degree to which a substance
bends light
– Air has a lower refractive index than glass
– Light passes through a slide then into the air above
the slide where it scatters
– Light is not channeled through the objective lens
– To get a sharp image at 100  objective lens,
immersion oil is used
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Oil immersion (2 of 2)
• Immersion oil is formulated to have the same refractive index as glass
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Types of Light Microscopy
• Bright Field
• Dark Field
• Phase Contrast
• Differential Interference Contrast
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Electron Microscopy (1 of 4)
• Resolution improves with smaller wavelengths
– Smallest wavelength of visible light is 400 nm
– Smallest wavelength of electron beams is 1 nm
Table 1.4 Comparison of Electron Microscopy to Light Microscopy
Light Microscopes
Electron Microscopes
Use light waves to image the specimen
Use an electron beam to image the specimen
Small, portable, and affordable
Large, requires special designated space, expensive
Simple, cheap, and easy sample preparation that
requires minimal training
Lengthy and complex sample preparation requires substantial
training
Color images possible
Only black-and-white images (though color may be added
later, as an aftereffect)
Most microscopes provide a maximum of 1,000 
Can magnify over 500,000 
Resolution of 200 nm
0.2 nm or about 1,000 times better than the best compound
light microscopes
Specimens can be living or dead
Specimens are all dead
Stains often used, but certain forms can be done
without staining and can visualize live cells
Specimens often must be stained with an electron-dense
substance like osmium or gold
1,000 times
500,000 times
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Electron Microscopy (2 of 4)
• How it works…
– Shoots electrons at a specimen
– Electrons interact with the specimen and an image is
generated
– Provides high-magnification and high-resolution
images
• Very expensive
• Requires considerable training to use
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Electron Microscopy (3 of 4)
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Electron Microscopy (4 of 4)
• Two main classes of electron microscopes:
– Transmission electron microscopes (TEM)
– Scanning electron microscopes (SEM)
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Transmission Electron Microscopy
(TEM) (1 of 2)
• Most common form of electron microscopy
• 1 million times magnification and 1,000 times better
resolution
• Samples must be extensively pretreated
• Specimens cannot be thicker than 1/285th of a human hair
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Transmission Electron Microscopy
(TEM) (2 of 2)
• Electron beam passes through the specimen
• Hits a detector
• Generates 2D images of internal structures
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Scanning Electron Microscopy (SEM)
• Electron beam scans over the specimen
• Detectors sense how the electrons interact with the
surface of the specimen
• Generates a 3D image of the surface
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Using Fluorescence in Microscopy (1 of 3)
• Fluorescence occurs when a substance absorbs energy
(ultraviolet [UV] light) and then emits that energy as visible
light
• Fluorochromes are fluorescent dyes that can be used to
stain samples so they will fluoresce when illuminated by a
UV light microscope
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Using Fluorescence in Microscopy (2 of 3)
• Examples of fluorochromes
– Hoechst: binds to DNA and emits a blue glow
– Auramine-rhodamine: binds acid-fast bacteria and
emits a reddish-yellow glow
– Calcofluor-white: binds cellulose and chitin
Copyright © 2023 Pearson Education, Inc. All Rights Reserved
Using Fluorescence in Microscopy (3 of 3)
• Immunofluorescence
– Uses fluorescent dyes linked to antibodies that can
recognize a specific target
– Can be used for identification of bacteria in blood
cultures, virus identification in patient samples, and
screening for bacteria in food-processing plants
Copyright © 2023 Pearson Education, Inc. All Rights Reserved