Introduction to

Microbiology
DMB 2201 – Module 1
Microbiology
in relation to
dentistry
Microorganisms in the oral cavity

maintenance of oral health and occurrence of

disease

the oral cavity consists of a wide variety of

indigenous and supplemental microorganisms

teeth, tongue, mucosa and gingival sulci

Diseases of the oral cavity

most common lesions - dental caries

and periodontal disease
mayoclinic.org

peaceperio.com
Which microorganism causes them?

No particular microorganism has been

established to be the etiologic agent of
dental caries or periodontal disease. WHY?

Complex oral environment

Variety of microbes at different stages of
the diseases
Different host responses to
microorganisms or their products
Systemic diseases and the oral cavity

 Systemic infectious diseases often present oral

manifestations.
 dentists should identify the systemic condition that
possibly complicates the oral health of the patient or
alters the treatment of oral diseases
Systemic Disease and the Oral Cavity

 Systemic infectious diseases often present oral

manifestations.

 Knowledge on the oral manifestations of particular

systemic disease is important.
enable dentists to identify the systemic condition
that possibly complicates the oral health of the
patient or alters the treatment of oral diseases
 Infection control in dentistry

 major concerns of oral health care delivery - control of

infection and cross-infection
 proper instrument sterilization – control potential for cross-
contamination

 Chemotherapy – antibiotics
Sensitivity tests
Spectrum and dose
Antibiotic resistance → prolonged use may lead to
disturbance of the oral flora → opportunistic infections

rcdso.org
Classification of living organisms

mammothmemory.net
 Old classification (Aristotle era)
intheir time
-

they gave simple classifications

living
organisms

plants animals
I can't move to
mobile

fungi and
protozoa
bacteria
Protista kingdom
 non-nucleated and nucleated unicellular microorganisms
(Ernst Haeckel 1886)

1940 - electron microscopy revealed structural differences

among microbial cells
-

Dakita structures
Aga Kaya nagkarook
·

classifications
49

 Eukaryotes - higher protists I higher form of life

 cellular similarities to higher forms of life

 nucleated and with membrane-bound organelles
* there are more organelles

 Prokaryotes - lower protists I lower form of life

 primitive their cellular composition

 nuclear material is not enclosed by nuclear membrane and
lack organelles
↑ no
there's living organelles-oribosomes [present
5-Kingdom System (Robert Whittacker 1969)
 Based on levels of cellular
organization, nutritional
requirements, patterns of
development, and tissue
differentiation

 Kingdom Monera
(Prokaryotes) – ancestors of all
other organisms
-

 Kingdom Fungi
 Kingdom Protista
byjus.com
 Kingdom Animalia
 Kingdom Plantae
 Kingdom Fungi

form of life I do photosynthesis

higher
I

 eukaryotic, non-photosynthetic organisms that

lack tissue differentiation
 cell walls - glucans, mannans, and chitin
 cell membranes – sterol
(042)

 unicellular (yeast), multicellular (molds), and

-

macroscopic (mushrooms) species

 obtain nutrient materials by absorbing
dissolved organic matter through their cell mentalfloss.com
membranes they
-
a
absorb nutrients
 form withinthe
-gointo
rightenvironment

Molds
 Macroscopic: cottony growths on bread and other
culture media when environmental conditions are
suitable e

 Microscopic: composed of thallus from which long

filaments of linked cells called hyphae, grow
 Hyphae:
 septate - with cross-walls that separate molds into
individual, mononucleated cell-like units
 coenocytic - no septa so they appear as long,
continuous cells with many nuclei -
distinct separation
·
no

pero may nucleus

 Molds reproduce asexually by fragmentation of

hyphae or sexually and asexually by producing
exospores.
 Yeasts
 rounded or oval fungi
 macroscopically seen as white,
powdery coatings
·microscopically oval shaped
 replicate by: one yeas
 fission - divides evenly into two
cells
 budding - a protuberance forms
-

on the outer surface of a parent

-

cell that may separate from,&or

remain attached, forming a short
- -

chain of cells called

pseudohyphae
 Candida albicans are seen as yeast
when they attach to human epithelial
cells but exhibit pseudohyphae when
they invade into deep tissues
i colonizes human tissue
Dimorphism of fungi

dimorphism – fungi grow either as mold or as

-
-

yeast depending on temperature or

-
carbon dioxide

mold-like at 25°C content.

yeast-like at 37°C 3 depending

diff form
on

temperature
Fungal dimorphism

oval
Dimorphism in the fungus panaba??

Mucor indicus depends on

CO2 concentration. &

On the agar surface,

Mucor exhibits yeast-like
growth, but in the agar
where CO2 from
metabolism has
accumulated, it is mold-
like
Medically important asexual fungi
Tolerg.
He
1. Anamorphs – Epidermophyton
- dimorphic (Sporothrix and Coccidioides)
- pseudohyphae (Candida)
- Pneumocystis
2. Zygomycota – non-septate hyphae (Rhizopus and Mucur)
3. Ascomycota – dimorphic (Aspergillus, Blastomyces, and
Histoplasma)
4. Basidiomycota – septate hyphae (Cryptococcus, Malassezia)
Molds
Mucor, a common
bread mold
sciencephoto.com
Rhizopus
Rhizopus stolonifer
Aspergillus niger
Candida albicans
a b c

Characteristics of fungal hyphae. (a) Septate hyphae have cross-walls,

or septa, dividing the hyphae into cell-like units. (b) Coenocytic hyphae
lack septa. (c) Hyphae grow by elongating at the tips
 pH 5

low moisture and high salt or

Growth of sugar concentrations so
-not
moist
Of
has a
type

fungi molds – aerobic I

-
o need
survive
oxygen to

anaerobic-

yeasts - facultative
anaerobes - With or without
will liVe
oxygen, it

Both can synthesize complex

carbohydrates.
 ②
①

Kingdom Protista
 mostly unicellular and some
multicellular eukaryotic
microorganisms like protozoa, slime 3 4

molds, fungus-like water molds and

the microalgae
 generally have flagella -
movement of the

Organism
 different modes of nourishment: 6

 microalgae are photosynthetic 5

 protozoa are ingestive

 other protists are absorptive
 some overlap to the photosynthetic and
ingestive mode
on
pseudopods
 NUCIENS

- covered with rows of cilia

Paramecium -

- has specialized structures for ingestion

(cytostome), elimination of wastes (anal pore),
and the regulation of osmotic pressure
(contractile vacuoles)
- macronucleus is involved with protein synthesis
and other ongoing cellular activitie
- micronucleus functions in sexual reproduction
Slime mold
 Protozoa

unicellular microorganisms
has a unique
called
have a protective covering called pellicle
-
- >

covering - >

inhabit water and soil

exhibit a growing phase as trophozoites
that feed on bacteria and small
particulate nutrients
Giardia
trophozoites
Humalain ug
smaller bacteria
 the protozoa goes
·
pag
Protozoa outside

you
of host.
the Mamameta

 Manner of food acquisition depends on the existence of specialized

cellular structures.
 Digestion: in vacuoles and waste is eliminated through the plasma
membrane or through an anal pore
 reproduce asexually by fission, budding, or schizogony
-

no
nagfoform marami
 some may exhibit conjugation
muna

nucleus?thenmaghihiwalay

 some produce cyst parano

-
-

->

 if parasitic, enables it to survive outside a host

 classified according to their motility, cell surface, feeding structures,
and nuclear structure
· pig family organisms
of
many members
Giardia cyst

provides protection
from the environment
before it is ingested by
a new host
 Pathogenic Classes of Protozoa

Archaezoa Amoebozoa Microspora

Ciliophora Apicomplexa Euglenozoa

 a

Amoeba

Amoeba proteus

(
movement (a) To move and to engulf food,
b
engulfment amebae (such as this Amoeba proteus)
of food extend cytoplasmic structures called
*
pseudopods. Food vacuoles are
created when pseudopods surround
RBC
food and bring it into the cell. (b) -

Entamoeba histolytica. The presence of

ingested red blood cells is diagnostic for
Entamoeba

Entamoeba histolytica
Conjugation in the ciliate
protozoan Paramecium

Sexual reproduction in ciliates is by conjugation.

Each cell has two nuclei: a micronucleus and a
macronucleus.
The micronucleus is haploid and is specialized for
conjugation. One micronucleus from each cell will migrate
to the other cell during conjugation.
Both cells will then go on to produce two daughter cells.
Condensed chromosomes are visible in the micronuclei
Chilomastix Giardia trophozoites
 Kingdom Monera

 includes all prokaryotes like

bacteria
-

 Bacteria have nuclear

material without an

1.
enclosing membrane and
suspended in the
cytoplasm
membrane
pe di enclosed by a

 diverse characteristics and

requirements – difficult to
classify and identify
 Bacterial classification
--
- -

how the
cells one,
 according to the characteristics of cell walls [dusted. ec.]
-
 according cell shape, arrangement, oxygen requirements,
->

motility, and nutritional and metabolic properties

- - - - -

 Bacteria absorb nutrients from either dead or living matters

from the environment.
 Reproduction - binary fission
 Bacteria generally come in three morphological types,
(cocci, bacilli, and spirilla) although some may be star-

Copiog Food spiral,

shaped, pyramidal, or square.
shaped spring
Neisseria gonorrhoeae
 Classification based on RNA – 3 cell types

Eukaryotes - kingdoms Animalia, Plantae, Fungi,

and Protista higher form
-
s

most bacteria will fall

⑤o
Eubacteria - kingdom Monera category
under this

lower life [ Archaea – “extremophiles” prokaryotes

forms
 The 3 cell types became Domains (Carl Woese, 1978)

Eukaryotic domain - kingdoms

Animalia, Plantae, Fungi, and
Protista
Eubacteria domain - kingdom
Monera
Archaea domain - similar in
appearance with Eubacteria
 lack cell walls but if present, their cell
walls do not contain peptidoglycan

Archaea live in adverse environments such as

-

hot springs, volcanic vents, sewage

- .

domain treatment plants and ocean floors

carry out unusual metabolic

processes

physiologically classified as
extremophiles
Extremophiles

 Extreme halophiles – require salt ~ a salt

concentrations greater than 25%
 Extreme thermophiles – require temperatures
between 80°C and 121°C ·

ainitsuper!!

 Extreme acidophiles – require pH ranging from

2 to 0
 Methanogens – require anaerobic environment
and produce methane gas
 Two beliefs regarding the 3 groups…

1.
 none of the three groups is ancestral or primitive to the other
 while each domain shares certain characteristics with the others,
each has its own unique characteristics
------------
2.
 eukaryotes may have developed from a prokaryotic ancestor via
invagination of cell membrane toward the nuclear region to
produce the true nucleus of a eukaryotic cell
6-Kingdom system

 adopted to reflect the

difference between bacteria
and archaea
 Kingdom Eubacteria
Kingdom Monera
 Kingdom Archaea
 Plantae
 Fungi
Eukaryotic Domain
 Animalia
 Protista
 gadg.com

pinterest.com

businessinsider.com
 Viruses
both living and non-living
no cellular structures typical of a
living cell
made up of a nucleic acid core,
either a DNA or RNA, enclosed
by a protein coat called capsid
can replicate only within living
cells, using the synthesizing
mechanism of the cell
Viruses
 inside host cells, they the virus biologywise.com
manipulate the metabolic
- a

machinery of the host cell

 construct many copies of
themselves
 produce viral enzymes
needed for biosynthetic
activities
· 1v

 prompt the synthesis of %

specialized structures that

can transfer viral nucleic
acids to other cells in order
to amplify their infectivity
Human
immunodeficiency
viruses

news-medical.net

causative agent of AIDS

Viruses

Non-living - acellular entities

inert nucleic acids while outside a host cell

Living - when inside a host cell and viral

replication occurs, they become active units
cause infection and disease
 sphweb.bumc.bu.edu
Prions
 infectious particles that are
predominantly made up of
pure proteins
 may cause neurological
infectious diseases
 the gene for the prion protein
(PrP) found in normal host DNA
can become abnormal in form
(PrPSc)
 When introduced and
contacts a normal prion
protein, it converts normal PrP
to PrPSc that can attack other
prion proteins.
 Prion-related Diseases
PrPSc accumulates in the brain, forming plaques
spongiform encephalopathies including mad cow -

kasi
-

cal
neurologi
disease have been identified to be caused by prions
involuntary
muscle spasm

Human diseases: kuru, Creutzfeldt-Jakob disease,

- -

Gerstmann-Straussler-Scheinker syndrome and fatal

familial insomnia
may be of genetic origin but some cases have been
traced to consumption of uncooked meat of
contaminated animals

hopkinsmedicine.org
53 Importance of infection control

To prevent cross-infection via contaminated

blood and instruments

To allay patients’ fears and concerns regarding

disease transmission

Rise in emerging and re-emerging infections

-

bagontrolled na, pumalikpanaman

(8
Emerging and Re-emerging Infections
54

• caused by new agents such as the coronavirus of severe

New infections
acute respiratory syndrome (SARS-CoV2)

• known disease entities where the etiologic agents have

Old infections been recently identified through advances in technology
(e.g. Helicobacter pylori causing gastric ulcer disease)

• diseases that have returned “stronger” due to genetic and

Re-emerging structural transformations and attendant increased
infections virulence of the organism (e.g. drug-resistant
Mycobacterium tuberculosis with 'a new bag of tricks')
Reasons for emergence of infections
55
 Societal events - economic impoverishment, especially in the
developing world, war and civil conflicts, as well as mass
population migration
 Health care - new medical devices, organ/tissue transplantation,
immunosuppression, antibiotic abuse and contaminated blood
and blood products
 Human behaviour - increasing sexual promiscuity, injectable drug
abuse
 Environmental changes - deforestation, drought and floods and
global warming
 Microbial adaptation - emergence of new species from the wild
(e.g. HIV), changes in virulence and toxin production and
development of drug resistance
 Global prevalence of some emerging and re-emerging diseases. S. aureus,
Staphylococcus aureus; E. coli, Escherichia coli; vCJD, variant Creutzfeldt-Jakob
disease; SARS, severe acute respiratory syndrome; HIV, human
56
immunodeficiency virus.
58 Important terms to remember

 epidemiology: spread, distribution and prevalence of

infection in the community
 pathogenesis: the means by which microbes cause disease
in humans, an understanding of which is critical for the
successful diagnosis and management of infections
 diagnosis: detection of an infection; this depends on the
collection of the correct specimen in the most appropriate
manner, and subsequent interpretation of the laboratory
results
59 Important terms to remember

treatment: antibacterial, antifungal or antiviral

therapy combined with supportive therapy
leads to resolution of most infections
prevention (prophylaxis): immunization is the
most useful mode of preventing diseases such as
tetanus and hepatitis B; however, increasing
public awareness of diseases and their modes of
spread significantly helps to curb the spread of
infections in the community (e.g. HIV infection)
HISTORY OF MICROBIOLOGY
DMB 2201 – MODULE 2
WHO WAS THE FIRST TO OBSERVE
MICROORGANISMS?

• No one is sure but the microscope was

already available by the mid 1600s
ROBERT HOOKE (1665)

• made observations of cells in slices

of cork tissue
• recognize the smallest structural
units of life called “cells”.
• observed strands of fungi among
the specimens he viewed
• This discovery conceptualized the
cell theory: all living things are
made up of cells.
micro.magnet.fsu.edu
ANTON VAN LEEUWENHOEK

• Between 1673 and 1723, he observed small, motile

living things from suspensions and materials scraped
from his teeth through his magnifying lenses.
• called microscopic organisms “animalcules” -canimals molecules

• described protozoa, fungi, and various kinds of

bacteria
• regarded as one of the first to provide accurate
descriptions of the world of microorganisms
THE PROGRESS OF MICROBIOLOGY AFTER LEEUWENHOEK’S DEATH

• microbiology did not progress rapidly because microscopes were

rare
• interest in microorganisms was not high
• it was not until#
200 years later when definite knowledge about
microorganisms developed and influenced the founding of a
modern science, microbiology
< -

-
 TOPIC OF DEBATE DURING THAT TIME

• the origin of microbes

1st
side
• Spontaneous generation - living things, including microorganisms and larger
life-forms, arise from lifeless matter such as beef broth
-wongperiod
of time
THE DEBATE ON SPONTANEOUS GENERATION

~> di ague sa generation

opty
• Francesco Redi – in the 1600s, showed that maggots would not arise from
* -

decaying meat (as others believed) if the meat were covered to prevent the
entry of flies

~
ague
*
• John Needham - advanced the theory of spontaneous generation by
showing that microorganisms appear spontaneously in beef broth

~> disaque
*• Lazarro Spallanzani - disputed the theory by showing that boiled broth
would not give rise to microscopic forms of life
• Non-believers of spontaneous generation such as Spallanzani, Virchow and
Redi tried to disprove this theory.
• It was not until 1861 when Louis Pasteur resolved this issue.
= -

• He showed that microorganisms are indeed present in nonliving matters like

the air, solids or liquids and and grow and multiply in them but that they came
from their parents and not from the environment where they live.
• He further proved that microorganisms can be destroyed by heat and that
techniques can be used to prevent the entry of microorganisms to the
experimental medium.
• This gave way to the adoption of the concept of “Biogenesis “: that life comes
-

from life. ->

• From this work of Pasteur followed numerous breakthroughs in microbiology.

&
 Redi’s experiments to disprove
spontaneous generation

(A) Jars of decaying meat are left

-
-

-
open to the air, exposed to flies;
flies lay their eggs on the meat;
eggs hatch to maggots. Supporters
* X
of spontaneous generation
believed that the decaying meat
gives rise to the maggots.

(B) Jars covered with parchment and

- -

-
sealed; flies could not reach the
decaying meat. No maggots
appeared on the meat. Redi used
this evidence to indicate that the
maggots did not arise from the
*v
meat but from flies in the air.
 &

LOUIS PASTEUR (1800s)

• performed numerous experiments to discover why wine and dairy
products became sour

• found that bacteria were causing the souring

• called attention to the importance of microorganisms in everyday

life

• stirred scientists to think that if bacteria could make the wine “sick,”
--

then perhaps
-
they could make humans sick, too
PASTEUR AND THE SPONTANEOUS GENERATION CONTROVERSY

• Pasteur believed that microorganisms were in the air even

though they could not be seen → it was possible to
become sick by inhaling microorganisms

• many scientists continued to believe that microorganisms

arose spontaneously

• Pasteur had to disprove spontaneous generation to

maintain his own theory by performing an experiment
 (A) Nutrient- rich in a swan-necked
flask; flask of broth is heated; broth
becomes sterile; broth remains
sterile: organisms entering are
trapped in the curve of the neck →
shows that MO come from the air
rather than from the
broth

(B) Neck of the flask is removed:

& microorganisms enter: flask soon
becomes contaminated → shows
that MO exist in the air and that
they originate from the air rather
than from lifeless matter
PASTEUR AND THE GERM THEORY OF DISEASE

• Pasteur came to believe that microorganisms transmitted

by the air could be the agents of human disease.

• postulated the germ theory of disease → infectious

diseases are due to the activities of microorganisms.

• He could not prove it successfully.

ROBERT KOCH
• A German scientist who provided the proof

• cultivated the bacteria that cause anthrax apart from any other type of
organism
• injected pure cultures of anthrax bacteria into mice → showed that they
invariably caused anthrax
• proved the germ theory of disease

• procedures used by Koch came to be known as Koch’s postulates → provide

a set of principles whereby the cause of a particular disease can be identified
Koch’s postulates

(A) Blood is drawn from a sick animal

(B) brought to the laboratory
(C) sample of the blood reveal bacteria
(D) bacteria from the blood are cultivated
in a pure culture in the laboratory
(E) sample of the pure culture containing
only one kind of bacteria is injected into
a healthy animal

→ If the animal becomes sick and displays

the same symptoms as the original animal,
then evidence exists that this particular
disease is caused by this particular
organism.
GOLDEN AGE OF MICROBIOLOGY (late 1800s – early 1900s)

• scientists throughout the world seized the opportunity to

further develop the germ theory of disease ~
· fraine and cance

• many agents of different infectious diseases were

identified

• many of the etiologic agents of microbial disease trace

their discovery to that period of time
• a rapid succession of discoveries, GOLDEN AGE OF MICROBIOLOGY
influenced by Louis Pasteur and Robert
Koch
• led to the foundation of Microbiology as a
science
• discoveries of various microorganisms
that cause different infectious diseases,
fermentation, and pasteurization that
established the relationship between
microorganisms and disease
• Germ theory of disease - laboratory
techniques aimed at culturing and
identifying the specific microbial etiology microbenotes.com
of a particular disease
• scientists focused on how to prevent microorganisms from passing
infection among mankind and other organisms
• Discoveries:
• role played by immunity in arresting the disease process
• chemical activities of microorganisms
• development of vaccines and aseptic surgical techniques
listenin
• Among the scientists who ingeniously contributed
-
to these o

breakthroughs include Edward Jenner who is known as the father of

immunology and Joseph Lister, the father of aseptic surgery

vox.com
PRACTICAL APPLICATIONS OF
THE GERM THEORY OF DISEASE
• epidemics could be halted by interrupting the
- -

-spread of microorganisms
-

• public health officials began a concerted effort

to purify water, ensure that food was prepared
carefully, pasteurize milk, isolate infected
patients, employ insect control programs, and
institute other methods to interrupt the hutchregional.com

spread of disease
• Epidemics soon declined with these new
methods of infection control.
• Modern chemotherapy (1910 ) - treatment of disease by using chemical
substances
• Paul Ehrlich pioneered the quest for substances that could destroy
disease-causing microorganisms without harming the infected human.
• First observed to have therapeutic effects were synthetic substances:
• arsenic derivative salvarsan against syphilis
• quinine from the bark of a South American tree against malaria
• sulfa drugs from prontosil dye
• Alexander Fleming accidentally discovered the first antibiotic when his
- -

-
bacterial cultures became contaminated with molds.
• He saw that bacterial growth was inhibited on areas where the mold
Penicillium notatum grew.
• The molds’ active bacterial inhibitor (antibiotic ) is known as penicillin.
=

health.harvard.edu
 • New branches of microbiology: bacteriology,
mycology, parasitology, immunology, virology,
microbial genetics, molecular biology, DNA
technology or genetic engineering.
• Interest in the oral cavity: considered as the “dirtiest”
part of the body next to the colon because of the
numerous varieties of microorganisms inhabiting it
• Oral microbiology was born when concerns about
the etiology of dental caries were raised.
edelweisspublications.com • Many microbiologists tried to establish different
theories of dental caries
• the most accepted is the chemico-parasitic theory by
Miller, Black, and Williams.
Observing
Microorganisms
Through a Microscope
DMB 2201 – MODULE 3
 Microorganisms: too small to be seen with the
unaided eye; they must be observed with a
microscope.
Microorganisms Microscope: Latin word micro (small): Greek word
and skopos (to look at) something small
-

Microscopes Modern microbiologists use microscopes that

produce, with great clarity, magnifications that
range from ten to thousands of times greater than
those of van Leeuwenhoek’s single lens.
Units of Measurement
Metric units of length
Microscopy
The instsruments
van Leeuwenhoek
(17th century)
• his microscope had
only one lens and was
similar to a magnifying
glass
integratedsciencegeneral11.com

• a single lens could

magnify a microbe 300X

• enabled him to be the

first person to see
bacteria
Tortora.2016
collection.sciencemuseumgroups.com

Evolution of the
microscope
• Robert Hooke: built compound microscopes with multiple
lenses
• Zaccharias Janssen: first compound microscope around 1600
(poor quality and could not be used to see bacteria)
• Joseph Jackson Lister: about 1830 developed a significantly
better microscope
• later improvements resulted in the development of the
modern compound microscope
• microscopic studies of live specimens have revealed
dramatic interactions between microbes
Light microscopy
Any kind of microscope that uses visible light to observe
specimens
see the specimen
ruses
lightto
natural or regenerated light
 Compound light
microscopy
• compound light
microscope
• has a series of lenses
and uses visible light as
its source of
illumination
• can examine very
small specimens as well
as some of their fine
detail
 Compound light
microscope
• a series of finely ground lenses forms a clearly focused image
• many times larger than the specimen itself
• magnification is achieved when light rays from an illuminator,
the light source, pass through a condenser
• condenser: has lenses that direct the light rays through the
specimen
• light rays pass into the objective lenses, the lenses closest to the
2 where the lightcomes specimen
from

• image of the specimen is magnified again by the ocular lens, or

eyepiece
Total magnification
• total magnification = objective lens magnification
(power) X ocular lens magnification (power)
• most microscopes: 10X (low power), 40X (high
power), and 100X (oil immersion) objective lenses
·

Dolg
• most ocular lenses magnify specimens by a factor
of 10
• total magnifications: 100X for low power, 400X
for high power, and 1000X for oil immersion
Resolution (resolving power)
• ability of the lenses to distinguish fine detail and
structure
• ability of the lenses to distinguish two points that
are a specified distance apart
• example: resolving power of 0.4 nm can distinguish
two points if they are at least 0.4 nm apart
• the shorter the wavelength of light used, the
greater the resolution
• compound light microscope: cannot resolve
structures smaller than about 0.2 μm
 Soshanning electron
microscope

la
lightmicro
scope
Refractive index
• refractive index: a measure of the light-bending ability of a medium
• for a clear, finely detailed image specimens must contrast sharply with their
medium
• to attain contrast: change the refractive index of specimens from that of their
medium
• staining: changes the refractive index of specimens sAlNX1.**
bends depending on medium
the
·

light
 en.oelcheck.com

Refractive index explained

• light rays: move in a straight line through a single medium

• staining: specimen and its medium have different refractive indexes
• light rays passing through the two materials: rays change direction (refract) by
bending or changing angle at the boundary between the materials
• increases the image’s contrast between the specimen and the medium
 Oil immersion

• to preserve the direction of light rays at the highest magnification, immersion oil is
placed between the glass slide and the oil immersion objective lens
• immersion oil has the same refractive index as glass, so the oil becomes part of the
optics of the glass of the microscope
• the oil has the same effect as increasing the objective lens diameter: improves the
resolving power of the lenses
• no oil: the image has poor resolution and becomes fuzzy
Refractive indices of the
glass microscope slide
and immersion oil are the
same

light rays do not refract

when passing from one
to the other when an oil
immersion objective lens
is used
Brightfield illumination

• normally, the field of vision in a

compound light microscope is brightly
illuminated
• by focusing the light, the condenser
produces a brightfield illumination
Unstained specimens
• it is not always desirable to stain a specimen -
Ramamatay
Kasi
yung
specimen

• unstained cell has little contrast with its surroundings and is therefore difficult
to see
• unstained cells are more easily observed with the modified compound
microscopes
 Darkfield microscopy
• used to examine live microorganisms that either are:
• (1) invisible in the ordinary light microscope
• (2) cannot be stained by standard methods
• (3) distorted by staining
• uses a darkfield condenser that contains an opaque disk
• disk blocks light that would enter the objective lens directly
• only light that is reflected off the specimen enters the objective lens
• there is no direct background light: the specimen appears light against a
black background
• frequently used to examine unstained microorganisms suspended in liquid
 Phase-contrast microscopy
• internal structures of a cell become more sharply defined
• detailed examination of living microorganisms
• it is not necessary to fix or stain the specimen—procedures that could
distort or kill the microorganisms
• one set of light rays comes directly from the light source
• the other set comes from light that is reflected or diffracted from a
particular structure in the specimen
• the two sets of light rays are brought together
• image of specimen: contains areas that are relatively light (in phase),
through shades of gray, to black (out of phase)
 • similar to phase-contrast microscopy: it uses differences in
Differential refractive indexes

Interference • DIC microscope uses two beams of light instead of one

• prisms split each light beam, adding contrasting colors to the
Contrast specimen

(DIC) • the resolution of a DIC microscope is higher than that of a

standard phase-contrast microscope

Microscopy • image is brightly colored and appears nearly three-dimensional

 Fluorescence microscopy
• fluorescence: the ability of substances to absorb short
wavelengths of light (ultraviolet) and give off light at a longer
wavelength (visible)
• stain used: fluorescent dyes called fluorochromes
• microorganisms appear as luminescent, bright objects
against a dark background
• fluorochromes have different effects on microorganisms
• auramine O: Mycobacterium tuberculosis appears as bright
yellow organisms against a dark background
• fluorescein isothiocyanate (FITC): Bacillus anthracis appears sciencedirect.com
apple green
 principal use of fluorescence microscopy – diagnostic
technique

Fluorescent antibodies are produced by:

→ animal is injected with a specific antigen
Fluorescent antibody → animal produces antibodies
(FA) technique or → antibodies are removed from the serum of the animal
immunofluorescence
→ a fluorochrome is chemically combined with the
antibodies
→ fluorescent antibodies are added to a microscope slide
containing an unknown bacterium
→ fluorescent antibodies bind to antigens on the surface of
the bacterium, causing it to fluoresce
 can detect bacteria or
other pathogenic
microorganisms, even
within cells, tissues, or
other clinical specimens

The principle of
immunofluorescence

- can identify a microbe in

minutes
- especially useful in
diagnosing syphilis and
rabies
Confocal microscopy
• reconstructs three-dimensional
images: used in conjunction with
computers
• stains used: fluorochromes
• uses a pinhole aperture: it eliminates
blurring that occurs with other
microscopes
Two-Photon
Microscopy
• stains used: fluorochromes
(excited by two photons)

• uses a longer wavelength →

allows imaging of living cells in
tissues up to 1000 µm deep
(confocal microscopy: less than
100 µm)

• can track the activity of cells in

real time
Scanning acoustic
microscopy
• consists of interpreting the
action of a sound wave sent
through a specimen
• a portion of the sound wave is
reflected back every time it hits an
interface within the material
• resolution is about 1 μm
• used to study living cells
attached to another surface, such
as cancer cells, artery plaque, and
bacterial biofilms that foul
equipment
analyticalscience.wiley.com

Electron microscopy
• for objects smaller than about 0.2 μm (viruses)
• a beam of electrons is used instead of light
• much greater resolving power than other microscopes
• used to examine structures too small to be resolved with
light microscopes
• images: black and white (may be colored artificially)
• uses electromagnetic lenses to focus a beam of electrons
onto a specimen
Transmission electron microscopy

• a finely focused beam of electrons from an

electron gun passes through the specimen
• beam is focused by an electromagnetic
condenser lens
• beam of electrons passes through the specimen
• then through an electromagnetic objective lens
• electromagnetic projector lens focuses electrons
onto a fluorescent screen or photographic plate
• final image: transmission electron micrograph
• appears light and dark areas
otago.ac.nz • TEM resolution: 10 pm (objects are magnified
10,000 to 100,000X)
• stains: enhance contrast
• absorb electrons and produces a darker image
in the stained region
• commonly used stains: salts of various heavy
metals
• fixed onto the specimen (positive staining) or
used to increase the electron opacity of the
surrounding field (negative staining)
• negative staining: useful for the study of the
very smallest specimens, such as virus particles,
bacterial flagella, and protein molecules
TEM - disadvantages
• only a very thin section of a specimen (about
100 nm) can be studied effectively
• no three-dimensional aspect
• specimens must be fixed, dehydrated, and
viewed under a high vacuum to prevent electron
scattering
• preparations may kill the specimen, or cause
shrinkage and distortion, or produce artifacts
 Scanning electron microscopy
• provides striking three-dimensional views
of specimens
• electron gun produces a primary electron
beam
• electrons pass through electromagnetic
lenses and are directed over the surface of
the specimen
• primary electron beam knocks electrons
out of the surface of the specimen
• secondary electrons thus produced are
transmitted to an electron collector,
amplified, and used to produce an image on
a viewing screen or photographic plate
• image: scanning electron micrograph
• useful in studying the surface structures of
intact cells and viruses media.npr.org

• resolution: 10 nm
• objects are generally magnified 1000 to
10,000X
• images: Covid 19 TEM (top); SEM (bottom)

media.npr.org
 (left) Atomic force
microscopy:
Clostridium
Scanned probe
perfringens toxin
perfringolysin O
microscopy
• use various kinds of probes to examine the
surface of a specimen
• uses electric current, which does not
modify the specimen or expose it to
damaging, high-energy radiation
• used to map atomic and molecular shapes,
(right) Scanning tunneling
to characterize magnetic and chemical
microscopy image of RecA properties, and to determine temperature
protein from E. coli. variations inside cells
This protein is involved in
repair of DNA. • it can resolve features that are only about
1/100 the size of an atom