Introduction to

Microbiology
Terminologies
Microbiology- “mikcros”(small); “bios”(life)
“logus”(study)
study of organisms too small to be seen by the naked eye.
These are microorganisms/microbes
Cellular - prokaryotes (bacteria, cyanobacteria, and
archaeans) or eukaryotes (fungi, protozoa, and algae).
Acellular - which includes viruses.
Microbes are vitally important to all life on [Link]
play a major role in various biochemical processes such as
biodegradation, biodeterioration, climate change, food
spoilage, epidemiology and biotechnology.
 Classification
(1) Bacteriology, the study of bacteria;
(2) Virology, the study of viruses;
(3) Mycology, the study of fungi;
(4) Parasitology, the study of protozoa and parasitic
worms – (flatworms, roundworms, pinworms, etc)
(5) Phycology, the study of algae; and
(6) Immunology, the study of the immune system and the
immune response.
(7) Taxonomy – classification & naming of organisms
(8) Etiology - set of causes, or manner of causation of a
disease or condition. Epidemiology – spread of disease
(9) Pathology – manner of disease development
Why study Microbiology?
❑ Impact on the daily lives of humans. Microorganisms are
everywhere – in the air one breathes, in the environment,
and even in one’s body
❑ Essential in biotechnology and a wide range of industries
like food and beverage, pharmaceuticals, mining, genetics,
and others.
❑ Bacteria and fungi are important sources of antimicrobial
agents. Ex. penicillin was derived from the fungus
Penicillium.
❑ Microorganisms act as saprophytes or decomposers of
waste products and dead organisms essential in
maintaining a balanced ecosystem.
Why study Microbiology?

❑ Better understanding of how microorganisms produce

disease, for better disease management and control.
❑ Discovery of VACCINES that helped prevent spread
of sickness and infections.
❑ Understanding of microorganisms diverse behaviors
such as mutations, biological warfare agents, number
of pathogens developing resistance to antibiotics
❑ Research and development.
 Evolution of Microbiology
Infectious diseases have existed for thousands of years
3180 BC, an epidemic known as “plague’ broke out in
Egypt.
1122 BC, an outbreak of smallpox-like disease that
originated in China spread worldwide.
1796- smallpox
1885- rabies
1914- whooping cough (pertussis)
1945- influenza
1955- Polio
1963= Measles
 Evolution of Microbiology
1967- Mumps
1969- Rubella Virus
1952-2016 Zika virus (dengue)
1981- present –AIDS
2002-2021- SARS or MERS
2019-present – COVID-19
❑ Bacteria have been the dominant forms of life on
Earth for the past 3.5 billion years.
❑ They rapidly evolve, constantly changing their genetic
architecture through horizontal DNA transfer and
other mechanisms such as mutation
Evolution of Microbiology

Robert Hooke
▪ discovered the cell – the basic
unit of living organisms.
▪ discover microorganisms in 1665
using a compound microscope
that he built himself
▪ cell theory, which states that
living organisms are made up of
cells and that cells are the basic
unit of life.
Evolution of Microbiology

Anton von Leeuwenhoek,

▪ a Dutch merchant, created a
single-lens microscope used to
make observations of
microorganisms which he called
animalcules.
▪ He discovered both protists and
bacteria
▪ “Father of Microbiology”
 Evolution of Microbiology
▪ French chemist and
microbiologist performed
countless experiments that led to
germ theory of disease, which
states that microorganisms were
in the environment and could
cause infectious diseases.
▪ pasteurization, which kills
microorganisms in different types
of liquids, and became the basis
of aseptic techniques.
Louis Pasteur ▪ fermentation
• discovered how to make vaccines from weakened, or
attenuated, microbes. He developed the earliest vaccines
against fowl cholera, anthrax, and rabies.
 Evolution of Microbiology

▪ Koch’s postulates states that

microorganisms caused certain
diseases.
▪ Discovered specific causative
agents of deadly infectious
diseases including tuberculosis,
cholera, and anthrax,
▪ The late1800s and the first decade
Robert Koch of the 1900s came to be known as
German physician and the Golden Age of Microbiology.
microbiologist. ▪ Father of Modern Microbiology”
 Evolution of Microbiology

▪ discovered the vaccine for

smallpox”
▪ developed the concept of
vaccination and inoculation by
immunizing an eight-year-old boy
against smallpox using cowpox
fluid.
▪ Developed preventive measures
for contagious diseases
Edward Jenner
(1749-1823)
 Evolution of Microbiology

▪ Pioneer of Antiseptic Surgery

▪ promoted the idea of sterilization

in surgery

▪ Reduced the incidence of wound

sepsis and gangrene, which, in
turn, reduced the need for
Joseph Lister amputation.
(1827-1912)
 Evolution of Microbiology
▪ discovered the antibiotic
penicillin from the mold
Penicillium notatum in 1928.

▪ With the discovery of antibiotics,

the incidence of infectious
diseases like tuberculosis,
pneumonia, meningitis, and
others was significantly reduced.
Alexander Fleming
(1881-1955)
Scottish physician and
microbiologist
Microscopy
Compound Microscope

• Contains more
than one
magnifying lens
• MP up to 1000X
the original size
• Source: visible
light
Brightfield Microscope

• Utilizes visible
light
• MP 1000X-1500X
• Used to visualize
bacteria and fungi.
• Less than 0.2um
can’t be visualized
• Specimen appears
dark against the
surrounding light
• Very low contrast
Brightfield Microscope

• Utilizes visible
light
• MP 1000X-1500X
• Used to visualize
bacteria and fungi.
• Less than 0.2um
can’t be visualized
• Specimen appears
dark against the
surrounding light
• Very low contrast
Darkfield Microscope

• ideally used
to illuminate
unstained samples
causing them to
appear brightly lit
against
a dark background.
• contains a special
condenser that
scatters light and
causes it to reflect
off the specimen at
an angle.
Darkfield Microscope

• ideally used
to illuminate
unstained samples
causing them to
appear brightly lit
against
a dark background.
• contains a special
condenser that
scatters light and
causes it to reflect
off the specimen at
an angle.
Phase-contrast Microscope
• Refractive indices
and light waves
passing through
Transparent objects
assume different
phases.
• Contrast enhancing
optical technique to
produce high
contrast images
• Thin tissue slices,
culture living cells,
subcellular particles
Phase-contrast Microscope
• Refractive indices
and light waves
passing through
Transparent objects
assume different
phases.
• Contrast enhancing
optical technique to
produce high
contrast images
• Thin tissue slices,
culture living cells,
subcellular particles
Differential Interference Microscope
• Nomarski interference
contrast (NIC) or
Nomarski microscopy
• used to enhance
the contrast in
unstained, transparent
samples.
• developed by Polish
physicist Georges
Nomarski in 1952.
• Useful in examining
specimens inhibited by
standard staining
procedures.
Flourescence Microscope
• uses fluorescence and
phosphorescence
instead of scattering,
reflection, and
attenuation or
absorption
• Used to visualize
structural components
of cell
• Detect viability of cell
populations
• Visualize DNA and
RNA
Flourescence Microscope
• uses fluorescence and
phosphorescence
instead of scattering,
reflection, and
attenuation or
absorption
• Used to visualize
structural components
of cell
• Detect viability of cell
populations
• Visualize DNA and
RNA
Confocal Microscope
• Confocal microscopy
• confocal laser
scanning microscopy (CL
SM)
• laser confocal scanning mi
croscopy (LCSM), is an
optical imaging technique
for increasing optical
resolution and contrast of a
micrograph by means of
using a spatial pinhole to
block out-of-focus light in
image formation.
• Useful in the study of
cell physiology
Confocal Microscope
• Confocal microscopy
• confocal laser
scanning microscopy (CL
SM)
• laser confocal scanning mi
croscopy (LCSM), is an
optical imaging technique
for increasing optical
resolution and contrast of a
micrograph by means of
using a spatial pinhole to
block out-of-focus light in
image formation.
• Useful in the study of
cell physiology
Electron Microscope
• uses electrons to create an
image of the target. It has
much higher magnification
or resolving power than a
normal light microscope.
• Inventor: Ernst Ruska
• uses a beam of
accelerated electrons as a
source of illumination.
• Represents 3-D structure
• Image produced is black
and white
• MP: 10,000X-2M times
• Used to visualize viruses
& subcellular structures of
cell
Electron Microscope
• uses electrons to create an
image of the target. It has
much higher magnification
or resolving power than a
normal light microscope.
• Inventor: Ernst Ruska
• uses a beam of
accelerated electrons as a
source of illumination.
• Represents 3-D structure
• Image produced is black
and white
• MP: 10,000X-2M times
• Used to visualize viruses
& subcellular structures of
cell
Scanning Probe Microscope
• forms images of surfaces
using a physical probe that
scans the specimen.
• SPM was founded in 1981,
with the invention of the
scanning tunneling
microscope, an instrument
for imaging surfaces at the
atomic level.
• Used to study the
molecular & atomic
shapes of organisms
• Used to determine the
variation in temperature
inside the cell
Scanning Probe Microscope
• forms images of surfaces
using a physical probe that
scans the specimen.
• SPM was founded in 1981,
with the invention of the
scanning tunneling
microscope, an instrument
for imaging surfaces at the
atomic level.
• Used to study the
molecular & atomic
shapes of organisms
• Used to determine the
variation in temperature
inside the cell
 STAINING
a technique used to enhance contrast in samples,
generally at the microscopic level.
Stains and dyes are frequently used in histology and in
the medical fields of histopathology, hematology, and
cytopathology
focuses on the study and diagnoses of disease at a
microscopic level.
STAINING
 Simple Staining
Uses single dye aqueous or water based and or
alocohol based
Quick and easy way to visualize cell shape, size and
arrangement of bacteria
Basic dyes: Safranin, methylene blue, crystal violet
(dye is positively charged)
Most bacterial cell are negatively charged
Visiualization of bacterial cell morpohology.
Cocci in Clusters Bacilli
 Differential Staining
uses more than one chemical stain.
Using multiple stains can better differentiate between
different microorganisms or structures/cellular components
of a single organism.
The Gram stain is the most important staining procedure
in microbiology
used to differentiate between gram positive
and gram negative organisms. Hence, it is a differential
stain. Gram negative and gram positive organisms are
distinguished from each other by differences in their cell
walls.
Gram Staining
Differential Stains
COCCI – all cocci are gram positive except Neisseria,
Veilonella, & Branhamella
BACILLI – are gram negative except Corynebacterium,
Clostridium, Bacillus and Mycobacterium
Gram Staining
Acid Fast Stain
is a differential stain used to identify acid-fast organisms
such as members of the genus Mycobacterium .
Acid-fast organisms are characterized by wax-like, nearly
impermeable cell walls
contain mycolic acid and large amounts of fatty acids,
waxes, and complex lipids.
Ziehl-Neelsen Stain – hot method since it requires steam
bathing the prepared smear after addition of dye.
◦ Acid fast organisms appear red on a blue background
Kinyoun Stain – “cold method”
◦ Acid fast organisms appear red on a green background
 CULTURE MEDIA
The most ideal and specific procedure to identify the
specific organism or specimen
Used to grow microrganisms
Aqueous (water-based) solution with all the nutrients
required for growth.
Physical State:
◦ 1. liquid – (broths, milk or infusions)
● Do not solidify above freezing point
● Do not contain gelatin or agar
● Suited for propagation of huge number of organism
● For fermentation studies and other tests
 CULTURE MEDIA
Semi-solid Media
◦ Contains agar at 0.5% concentration giving its gel
consistency
◦ For culture of microaerophilic bacteria
◦ For study of bacterial motility,
Solid Media
◦ Contains solidifying agent (1.5-2%) agar giving it a firm
surface where cells can form colonies.
◦ Used for bacterial and fungal isolation
◦ For determining colony characteristics
◦ Liquefiable (reversible) & non-liquefiable
(non-reversible) solid media
 AGAR
also known as "China grass" is a jelly-like substance,
obtained from red algae. Agar is a mixture of two
components: the linear polysaccharide agarose, and a
heterogeneous mixture of smaller molecules called
agaropectin.
used in the laboratory to help feed and grow
bacteria and other microorganisms.
It acts as a culture that provides nutrients and a place
for these items to grow, but since it is indigestible to
the microorganisms, they cannot eat and destroy it.
 AGAR
used as a laxative, an appetite suppressant, a vegetarian
substitute for gelatin, a thickener for soups, in fruit
preserves, ice cream, and other desserts, as a clarifying
agent in brewing, and for sizing paper and fabrics.
 CULTURE MEDIA
Chemical Composition
◦ Synthetic Media – chemically defined substances
(inorganic and organic compounds)
◦ Non-synthetic – does not contain any chemicals
Solid Media
◦ Contains solidifying agent (1.5-2%) agar giving it a firm
surface where cells can form colonies.
◦ Used for bacterial and fungal isolation
◦ For determining colony characteristics
◦ Liquefiable (reversible) & non-liquefiable (non-reversible)
solid meadia
 CULTURE MEDIA
Functional Type
◦ General Purpose – contains nutrient mixture that support
the growth of both pathogenic and non pathogenic
organisms.
◦ Enrichment Media – organic substances like blood, serum,
or growth factors
◦ Desired to increase the number of desired microorganisms
without affecting the rest of the population
● 1. Blood Agar – blood is added to a blood agar base
● Gram positive bacteria causing RBC HEMOLYSIS
i. Beta Hemolysis – complete LYSIS
ii. Alpha hemolysis – incomplete LYSIS
iii. Gamma hemolysis – No LYSIS
CULTURE MEDIA
 CULTURE MEDIA
Selective Media
◦ is composed of specific ingredients to inhibit the growth of
certain species of microbes in a mixed culture while allowing
others to grow.
● Changing pH
● Adding substances like antibiotics, dyes, other chemicals
 CULTURE MEDIA
Mannitol Salt Agar
◦ It encourages the growth of a group of certain bacteria while
inhibiting the growth of others.
◦ Used for isolation of Staphyloccus aurius
● Changing pH
● Adding substances like antibiotics, dyes, other chemicals
 • selective and
differential medium
designed to isolate and
differentiate enterics
based on their ability to
ferment lactose.
• Bile salts and crystal
violet inhibit the growth
of Gram positive
organisms.
• Lactose provides a
source of fermentable
carbohydrate, allowing
for differentiation.
Growth of Enterobacteriaceae • Inhibits growth of gram
positive bacteria
 CULTURE MEDIA
The Löwenstein–Jensen medium, also known as
LJ medium, is a growth medium specially used for
culture of Mycobacterium tuberculosis.
M. tuberculosis appears as brown, granular colonies
(sometimes called "buff, rough and tough").
Related Online Supplemental Learning

Culture Media
Production of Agar
Gram STAINING
QUIZ MODE………..