Chapter I

Introduction to
Microbiology
Mr. Modisto Cabanesas, II, LPT
San Pedro College
 What is Microbiology?

⚫ Study of microorganisms, unseen world of living

things
 effects to living organisms
 characteristics of microbes

⚫ Microbes/Microorganisms
 either an animal, vegetable or mineral
 minute living things too small to be seen with the naked eye
 includes, bacteria, fungi (yeast and molds), protozoa, microscopic
algae, viruses
 What is Microbiology?

Microbes

Acellular
Cellular
infectious agents
microorganisms
(Prions, Viruses)

Prokaryotes Eukaryotes
(Archae, (Algae, Fungi,
Bacteria) Protozoa)
 E. coli is used in genetic engineering COVID-19 currently affects the world

Lactobacillus casei: commercially produced

Propionibacterium acnes: causes Acne Vulgaris
 History of Microbiology

⚫ Antonie van Leeuwenhoek

(1623-1723)
o referred to as the “Father of
Microbiology”
o pioneer of microscope
o observed tiny living
creatures that he called
“animalcules”
 History of Microbiology

⚫ Robert Hooke (1635-1703)

o coined the term “cell”
o published the book called
Micrographia
o did not, however, identify
bacteria
o pioneer of the Cell Theory
 History of Microbiology

⚫ Abiogenesis
o or the Theory of
Spontaneous Generation
o the idea that life can arise
spontaneously from
nonliving material

In 1668, Francesco Redi did an

experiment to disprove the
theory.
 History of Microbiology

⚫ Louis Pasteur (1822-1895)

o did another experiment to
disprove abiogenesis
o introduced the term “aerobes”
o Developed the process of
pasteurization
o Made contributions to the
germ theory
o Formulated rabies vaccine
History of Microbiology
 History of Microbiology

⚫ Robert Koch (1843-1910)

o made contributions to the
germ theory
o proved that microbes can
cause diseases through series
of steps called Koch’s
postulates
o pioneer of microbial fixing and
staining
o developed methods of
cultivating bacteria with the
help of R.J. Petri
 History of Microbiology

⚫ Koch’s Postulates

1. The microorganism must be found in abundance in all organisms suffering from the disease,
but should not be found in healthy animals.

2. The microorganism must be isolated from a diseased organism and grown in pure culture.

3. The cultured microorganism should cause disease when introduced into a healthy organism.

4. The microorganism must be re-isolated from the inoculated, diseased experimental host
and identified as being identical to the original specific causative agent
 Notable Events/Discoveries
YEAR EVENTS/DISCOVERIES PIONEER
1857 Fermentation Pasteur
1861 Disproved spontaneous generation Pasteur
1864 Pasteurization Pasteur
1867 Aseptic Surgery Lister
1876 Germ Theory Koch
1879 Discovery of Neisseria gonorrhoea Neisser
1881 Pure culture Koch
Yellow fever Finlay
 Notable Events/Discoveries
YEAR EVENTS/DISCOVERIES PIONEER
1882 Mycobacterium tuberculosis Koch
Formulation of Agar medium Hess
1883 Vibrio cholerae Koch
1884 Phagocytosis Metchnikoff
Gram-staining Gram
Escherichia coli Escherich
1887 Invention of Petri dish Petri
1889 Clostridium tetani Kitasato
1879 Discovery of Neisseria gonorrhoea Neisser
 Notable Events/Discoveries

YEAR EVENTS/DISCOVERIES PIONEER

1890 Diphteria antitoxin Von Bering
Theory of Immunity Ehrlich
1892 Sulfur Cycle Winogradsky
1898 Shigella dysenteriae Shiga
1908 Syphilis treatment Ehrlich
1928 Discovery of Penicillin Fleming
1879 Discovery of Neisseria gonorrhoea Neisser
 Specializations of Microbiology

⚫ Bacteriology – the study of the structure, functions, and activities of bacteria

⚫ Phycology - the study of the structure, functions, and activities of algae

⚫ Protozoology - the study of the structure, functions, and activities of protozoans

⚫ Mycology - the study of the structure, functions, and activities of fungi

⚫ Virology - the study of the structure, functions, and activities of viruses

 Disciplines of Microbiology

⚫ Agricultural Microbiology-beneficial and harmful roles of microbes

 soil formation and fertility
 carbon, nitrogen, phosphorus and sulfur cycles
 diseases of plants
 digestive processes of cows and other ruminants
 crops and food production

⚫ Biotechnology (Industrial Microbiology) - the exploitation of

microbes for use in industrial processes
 biopolymers
 bioremediation
 waste biotreatment
 fermentation
 Disciplines of Microbiology

⚫ Medical microbiology - study of pathogenic microbes

and the role of microbes in human illness
 epidemiology
 transmission of diseases
 treatment of infectious diseases
 disease prevention measures
 aseptic techniques
 immunology
 production of drugs
 Disciplines of Microbiology

⚫ Microbial Physiology - study of how the microbial cell

functions biochemically.
 Microbial growth
 Microbial metabolism
 Microbial cell structure

⚫ Veterinary Microbiology- role of microbes in

veterinary medicine or animal taxonomy
 Disciplines of Microbiology

⚫ Sanitary Microbiology - processing and disposal of

garbage and sewage wastes.
 inspects food processing installations and eating
establishments

⚫ Paleomicrobiology- the study of ancient microbes

 Disciplines of Microbiology

⚫ Parasitology - study of microorganisms that live on

or in another living organisms
 Parasitic protozoa
 Helminths (parasitic worms)
 Arthropods (parasitic insects)
Chapter II
Classification and
Nomenclature
Mr. Modisto Cabanesas II, LPT
Instructor
 Type of Microorganisms
Microorganisms or microbes are microscopic
organisms that exist as unicellular, multicellular,
or cell clusters. Microorganisms are widespread
in nature and are beneficial to life, but some can
cause serious harm. They can be divided into six
major types:
– bacteria,
– archaea,
– fungi,
– protozoa,
– algae, and
– viruses.
 Bacteria

Characteristics
• unicellular
• they lack a nucleus.
• they exist in four major shapes:
– bacillus (rod shape),
– coccus (spherical shape),
– spirilla (spiral shape), and
– vibrio (curved shape)

• have a peptidoglycan cell wall

• they divide by binary fission;
• and they may possess flagella for motility.
 Bacteria
Classifications
1. According to the way their cell wall structure stains
• Gram-positive
• Gram-negative

2. Based on their response to gaseous oxygen

• aerobic (living in the presence of oxygen),
• anaerobic (living without oxygen), and
• facultative anaerobes (can live in both
environments)

3. According to the way they obtain energy

• Autotrophs
• Heterotrophs
 Archaea

Characteristics
• cell wall structure lack peptidoglycans
• with avidity to extreme environmental conditions.

Based on their habitat, all Archaeans can be divided into the following
groups:
• methanogens (methane-producing organisms),
• halophiles (archaeans that live in salty environments),
• thermophiles (archaeans that live at extremely hot temperatures), and
• psychrophiles (cold-temperature Archaeans).
 Fungi

Characteristics
• eukaryotic cells (with a true nucleus)
• most are multicellular
• their cell wall is composed of chitin
• they obtain nutrients by absorbing organic
material from their environment
(decomposers), through symbiotic relationships
with plants (symbionts), or harmful
relationships with a host (parasites).
• they form characteristic filamentous tubes
called hyphae that help absorb material. The
collection of hyphae is called mycelium
• they reproduce by releasing spores
 Protozoa
Characteristics

• unicellular aerobic eukaryotes

• they have a nucleus, complex organelles, and
obtain nourishment by absorption or ingestion
through specialized structures
• they make up the largest group of organisms
in the world in terms of numbers, biomass,
and diversity
• their cell walls are made up of cellulose.
 Protozoa
Classifications

• Protozoa have been traditionally divided

based on their mode of locomotion
• Flagellates produce their own food and use
their whip-like structure to propel forward
• Ciliates have tiny hair that beat to produce
movement
• Amoeboids have false feet or pseudopodia
used for feeding and locomotion, and
• Sporozoans are non-motile. They also have
different means of nutrition, which groups
them as autotrophs or heterotrophs.
 Algae

Characteristics
• also called cyanobacteria or blue-green
algae,
• are unicellular or multicellular eukaryotes
that obtain nourishment by photosynthesis.
• they live in water, damp soil, and rocks and
produce oxygen and carbohydrates used by
other organisms.
• it is believed that cyanobacteria are the
origins of green land plants.
 Viruses

Characteristics

• are noncellular entities that consist of a

nucleic acid core (DNA or RNA)
surrounded by a protein coat
• they are not considered living organisms
• they cannot reproduce outside a host cell
and cannot metabolize on their own.
• viruses often infest prokaryotic and
eukaryotic cells causing diseases.
 Multicellular Animal Parasites

A group of eukaryotic organisms

consisting of the flatworms and
roundworms, which are
collectively referred to as the
helminths.

Since the parasitic helminths are

of clinical importance, they are
often discussed along with the
other groups of microbes.
 Taxonomy

Taxonomy is the science of naming,

describing and classifying organisms
and includes all plants, animals and
microorganisms of the world.

Carolus Linnaeus (1707-1778)

(Swedish) considered the Father of
Taxonomy developed taxonomic
system for naming plants and animals
and grouping similar organisms
together.
 Taxonomy
• Taxon, plural taxa, is any unit used in taxonomy.
• Taxa are arranged in a hierarchy from kingdom to subspecies, a given
taxon ordinarily including several taxa of lower rank.

– Kingdom (not used by most bacteriologists)

– Phylum
– Class
– Order
– Family
– Genus (plural: Genera)
– Species (both singular & plural)
Linnaean Classification
 Whittaker Classification

Based on:

• Morphology
• Metabolism (Biochemical
Activity)
• Molecular Techniques
• Fatty Acid Profiles
• Protein Differentiation
• DNA Finger Printing
 Woese Classification

Based on:

• differences in the 16S

ribosomal RNA
(rRNA) structure
• cell’s membrane lipid
structure and its
sensitivity to
antibiotics.
 Assigning Specific Names
The Binomial System of Nomenclature
• Each species is given a name that consists of two parts.
• The first part is the Genus to which the species belongs and the second part is
the species name.
• The binomial naming system was first uniformly used by Carl Linnaeus.
• Most scientific names are written in Latin or Greek or latinized words.

Rules to remember:
1. Generic part is capitalized, species is lowercase
2. Both are italicized or underlined if italics aren’t available

Staphylococcus aureus
Classifications of
Microorganisms
Prokaryotes
 Archaea

Based primarily on rRNA sequence data, domain Archaea is divided

into two phyla:
– Phylum Crenarchaeota
• originally containing thermophylic and hyperthermophilic
sulfur-metabolizing archaea

– Phylum Euryarchaeota
• contains primarily methanogenic archaea, halophilic
archaea, and thermophilic, sulfur-reducing archaea
 Selected Bacteria Phyla –
Gram-Negative
Phylum Proteobacteria
• very diverse, with all four modes
of nutrition represented
• grouped because of shared rRNA
sequence
• also has endosymbionts such as
nitrogen fixers
• of medical importance are the
chemoheterotrophs
 Selected Bacteria Phyla –
Gram-Negative
Phylum Chlamidiae
• grow only in eukaryotic host cells
• small phylum containing the genus
Chlamydia

Phylum Spirochaetes
• spiral bacteria
• characterized by flexible, helical cells
with a modified outer membrane (the
outer sheath) and modified flagella
(axial filaments) located within the
outer sheath
• important pathogenic genera include
Treponema, Borrelia, and Leptospira
 Selected Bacteria Phyla –
Gram-Negative
Phylum Bacteroidetes
• includes opportunistic pathogens
• includes genera Bacteroides,
Flavobacterium, Flexibacter, and
Cytophyga;

Phylum Cyanobacteria
• oxygenic photosynthetic bacteria
• provide an enormous amount of
food for organisms in freshwater
and marine ecosystems
 Selected Bacteria Phyla –
Gram-Positive
Phylum Firmicutes
• low G+C content;
– Bacilli include Lactobacillus,
Streptococcus, Staphylococcus,
Listeria, and Bacillus
– Clostridia includes Clostridium

Phylum Actinobacteria
• genetically, high G+C content
– Includes genera Actinomyces,
Streptomyces, Corynebacterium,
Micrococcus, Mycobacterium,
Propionibacterium
 Structure

Morphology and arrangement of bacterial cells are criteria used for

classification of bacteria into following groups:
• Cocci (singular: coccus)
• Rods / bacilli (singular: rod, bacillus)
• Vibrios (singular: vibrio)
• Spirilla (singular: spirillum)
• Spirochaetes (singular: spirochaete)
 Cocci

• Cocci in pairs are called

diplococci, for example,
meningococci and
gonococci.
• Cocci in chains are called
streptococci, for example
Streptococcus pyogenes.
• Cocci in irregular groups
are called Staphylococci,
for example,
Staphylococcus aureus.
 Bacilli

May be arranged in:

• chains, for example, Streptobacillus
species.
• branching chains, for example,
Lactobacilli .
• mass together, for example,
Mycobacterium leprae.
• remain attached at various angles
resembling Chinese letters, for
example, Corynebacterium
diphtheria.
 Spiral Bacteria

• Vibrios – rods that are slightly curved

• Spirilla – polar flagella
• Spirochaetes – axial filaments
Classifications of
Microorganisms
Eukaryotes
 Eukaryotes

Protists
• Originally, the protists were
classified under Kingdom Protista,
but has been broken up into
different kingdoms
• The term Protist is used for
eukaryotes that are not plants,
animal and fungi
 Kingdom Stramenophila
Characteristics:
normally two flagella when present,
with hairlike projections on one of them

1. Phylum Oomycota (water

molds)
2. Phylum Bacillariophyta
(diatoms)
3. Phylum Chrysophyta (golden
algae)
4. Phylum Phaeophyta (brown
algae)
 Kingdom Alveolata
Characteristics:
sac-like alveoli that form a continuous
layer just under the plasma membrane

1. Phylum Dinoflagellata
(dinoflagellates)
2. Phylum Apicomplexa
(sporozoans)
3. Phylum Ciliophora (ciliates)
 Kingdom Rhizaria
Characteristics:
produce elaborate shell-like coverings
of cells; use very slender pseudopodia
to move

1. Phylum Foraminifera (forams)

2. Phylum Radiolaria
(radiolarians)
 Kingdom Excavata
Characteristics:
possess “excavating” feeding grooves;
flagellated

1. Phylum Diplomonadida
(Giardia lamblia)
1. Phylum Parabasalia
(Trichomonas vaginalis)
 Kingdom Amoebozoa
Characteristics:
Move using relatively large lobe-
shaped pseudopodia

1. Phylum Gymnamoeba
(free-living amoebas)
2. Phylum Entamoeba
(parasitic amoebas)
3. Phylum Acrasiomycota
(cellular slime molds)
4. Phylum Myxomycota
(plasmodial slime molds)
 Opisthokonta
The opisthokonts are a broad
group of eukaryotes, including
the animal and fungus kingdoms.
The opisthokonts, are generally
recognized as a clade.

Characteristics:
flagellate cells, such as the sperm of
most animals and the spores of
the chytrid fungi, propel themselves
with a single posterior flagellum.
 Eukaryotes

Fungi
• Decomposers
• Heterotrophic; absorb nutrients. Most
have chitin in cell walls
– The feeding structures of a fungus
are threadlike filaments called
hyphae
– Hyphae branch repeatedly as they
grow, forming a mass known as a
mycelium
– Lichen is a symbiosis between
fungi and green algae
– Mycorrhiza is a symbiosis
between fungi and plant roots
 Kingdom Fungi

Phylum Chytridiomycota
(Chytrids)
• with flagellated spores; includes
parasites of other animals

Phylum Zygomycota
• have protective zygosporangia,
where zygotes produce haploid
spores by meiosis. Includes bread
molds
• may include microsporidia,
obligate intracellular parasites
 Kingdom Fungi
Phlyum Glomeromycota
• form mycorrhizae

Phylum Ascomycota
• have asci or sac (sac fungi)
• can be unicellular or
multicellular.
• includes some of the most
devastating plant pathogens,
or in lichen (e.g. morels, cup
fungi, yeasts)
 Kingdom Fungi

Phylum Basidiomycota
• club fungi with spore-producing
basidia
• excellent decomposers
• includes mushrooms and shelf
fungi
 Summary of medically
important groups
• Bacteria – prokaryotic, with peptidoglycan in cell wall, no nucleus; some
disease-causing species
– Gram – negative:
• Proteobacteria – includes coliform (e.g. E. coli), pseudomonads, and vibrios
• Chlamidiae – Chlamydia
• Spirochaetes – includes Treponema, Borrelia, and Leptospira
• Bacteroidetes – includes Bacteroidetes
– Gram – positive:
• Firmicutes – clostridia, mollicutes, bacilli (Bacillus, Lactobacillus, Streptococcus,
etc.)
• Actinobacteria – includes Streptomyces and Mycobacterium
 Summary of medically
important groups
• Eukaryotes – with nuclei
– Alveolata – includes dinoflagellates, ciliates (B. coli), and
apicomplexans (e.g. Plasmodium)
– Excavata – includes flagellated protozoans; e.g. Giardia and
Trichomonas
– Fungi – ascomycetes (e.g. Pneumocystis and Candida),
mushrooms, zygomycetes (e.g. microsporidia)
– Animals – tapeworms (e.g. Taenia), flukes (e.g.
Schistosoma), nematodes (e.g. Ascaris), arthropods (e.g.
mosquitoes, fleas, mites)
Chapter III
Prokaryotic Cells
Mr. Modisto Cabanesas, II, LPT
Instructor
 Prokaryotes Eukaryotes
Organisms Bacteria, cyanobacteria, archaea Protozoans, algae, fungi, plants, & animals

Cell size Generally 1-10μm in linear dimension Generally 5-100μm in linear dimension

Metabolism Anaerobic or aerobic Aerobic

Organelles Few or none; no nucleus Nucleus, mitochondrion, chloroplast, ER, Golgi

complex, etc. present

DNA Circular DNA in cytoplasm Linear DNA with noncoding regions in nucleus

RNA and protein RNA and protein synthesized in the same RNA in nucleus; protein in cytoplasm
compartment

Cytoplasm No cytoskeleton With cytoskeleton

Cell division Chromosomes pulled apart by membrane Uses cytoskeletal spindle apparatus
attachments

Cellular organization Mainly unicellular Mainly multicellular ,with differentiation in cell types
Generalized eukaryotic
cell structure
Generalized prokaryotic
cell structure
 Prokaryotic Cells

Cell Membrane

• similar in structure and function to the eukaryotic cell membrane

• consists of membrane proteins and phospholipids
• a flexible, semi-permeable membrane
 Prokaryotic Cells

Chromosomes
• single, long, supercoiled
• contains 450 to 8000 genes
depending on species
• Plasmid, a small, circular DNA
not part of the chromosome
may be present. It can be
transferred from one bacterium
to another. Commonly used in
genetic engineering
 Prokaryotic Cells

Cytoplasm
• About 80% water proteins
(enzymes), carbohydrates, lipids,
inorganic ions
• Major structures:
– Ribosomes
– Nucleoid - contains a single
long, continuous, and
frequently circularly arranged
thread of double-stranded DNA
called the bacterial
chromosome
 Prokaryotic Cells

Cell Wall

• With the exception of mycoplasmas, all bacteria have a

semirigid cell wall
– component: peptidoglycan, a large polymer composed of N-
acetylglucosamine and N-acetylmuramic acid
– Gram-positive bacteria have more peptidoglycan account
for their ability to retain the stain in the Gram stain procedure
– Gram-negative bacteria have more lipids in their cell wall
Bacillus Escherichia coli
Listeria Salmonella
Staphylococcus Shigella
Streptococcus Enterobacteriaceae
Enterococcus Pseudomonas
Clostridium Helicobacter
 Prokaryotic Cells

Cell Wall and Gram Staining

• Crystal violet stains both Gram-positive
and Gram-negative cells purple
– Iodine as mordant forms large complexes
that cannot escape the cell wall easily
• Alcohol dehydrates the peptidoglycan
of Gram-positive bacteria, but dissolves
the outer membrane of the Gram-
negative so they become colorless
• Safranin colors the Gram-negative cells
red or pink
 Prokaryotic Cells

Glycocalyx
• slimy, gelatinous material
produced by cell membrane and
secreted outside the cell wall
• serves as a reservoir for nutrients
and protects the organism from
changes in the environment
• when the glycocalyx is a tightly
bound structure, it is known as a
capsule and slime layer when
it is a poorly bound structure that
flows easily
 Prokaryotic Cells

Flagellum
• thread-like, protein appendages
used for locomotion
• made up of the protein flagellin.
• its shape is a 20 nanometer-thick
hollow tube
• it is helical and has a sharp bend
just outside the outer membrane;
this "hook" allows the helix to
point directly away from the cell.
 Prokaryotic Cells

Flagellum
Classification of bacterium according to
the number of flagella

a. monotrichous bacterium
b. lophotrichous bacterium
c. amphitrichous bacterium
d. peritrichous bacterium
 Prokaryotic Cells

Fimbriae
• can occur at the poles of
bacterial cells and they can be
evenly distributed over the
entire surface of the cell
• enables the cells to adhere to
surfaces, including those of the
other cells; helps the cell to
colonize mucus membranes
 Prokaryotic Cells

Pili (sing., pili)

• Hair-like structures most often
observed on Gram-negative
bacteria
• Composed of polymerized
protein called pilin
• They arise from the cytoplasm
and extends to cell membrane,
cell wall, and capsule (if
present)
• Two types: adhering pili and sex
pilus Proteus vulgaris undergoing binary fission
Schematic drawing of bacterial conjugation:

1 – Donor cell produces pilus.

2 – Pilus attaches to recipient cell, brings the
two cells together.
3 – The mobile plasmid is nicked and a single
strand of DNA is then transferred to the
recipient cell.
4 – Both cells circularize their plasmids,
synthesize second strands, and reproduce pili;
both cells are now viable donors.
 Prokaryotic Cells

Endospores

• Specialized “resting cells”

formed by some Gram-positive
bacteria such as Clostridium
and Bacillus

• Formed when nutrients are

depleted
Bacillus subtilis endospore
 Prokaryotic Cells

Cell Reproduction
• Asexual reproduction, usually by:
– Binary fission
• process results in the
reproduction of a living
prokaryotic cell by division into Binary Fission
two parts which each have the
potential to grow to the size of
the original cell
– Budding
• formation of a new organism by
the protrusion of part of another
organism
Budding
Chapter IV
Microbial Physiology
 Coverage

• Physiology and Nutrition

• Growth and Control
• Sterilization and Anti-Microbial methods
Metabolism
 Metabolism

• Two key players:

enzymes and
adenosine
triphosphate
(ATP)

• ATP is a molecule
that cells use to
manage energy
needs
 Impacts of microbial
metabolism

• Disease and food spoilage

• Nitrogen cycle
• Beverages and food
• Sewage treatment
• Drugs
 Important processes

Carbohydrate Catabolism
• Respiration – glycolysis is followed by Krebs cycle , and
electron transport chain which generates the most ATP
– 38 ATPs can be generated from 1 glucose molecule

• Fermentation – does not require Krebs cycle or ETC, and

produces end products such as lactic acid or ethanol
– 2 ATPs are produced from 1 glucose molecule
 Important processes

Lipid Catabolism
– Lipids are first broken down into component fatty acids and
glycerols by lipases
– Each component can then enter the Krebs cycle

Protein Catabolism
– Proteases and peptidases break down proteins into
component amino acids
– AAs must undergo enzymatic conversion into substances
that can enter the Krebs cycle
 Metabolic Diversity

• Phototrophs – light as energy source

• Chemotrophs – redox of in/organic compounds

• Autotrophs – self-feeders
• Heterotrophs – feed on others

Most medically important organisms are

chemoheterotrophic, because typically, infectious organisms
catabolize substances obtained from the host
Nutritional classification of
organisms
Microbial Nutrition
 Physical Requirements

Temperature
– Psychrophiles – cold-loving
– Mesophiles – moderate temperatures
– Thermophiles – heat-loving

Most bacteria grow within a limited range of temperatures

– Min and max growth temps are only 30°C apart
– Optimum temperature - temperature at which the
species can best grow
 Typical growth rates in
response to temperature
 Physical Requirements

pH
– Most bacteria grow best between pH6.5-7.5
– Few bacteria grow below pH 4

When bacteria are cultured in the lab, they often

produce acids which interfere with their growth
– Chemical buffers such as phosphate salts and
peptones are included
 Physical Requirements

Osmotic pressure
– Microbes obtain nutrients in solution from water

Adaptations
– Extreme halophiles
– Obligate halophiles
– Facultative halophiles – do not require high salt
concentrations but can grow at concentrations up to
2%
 Chemical Requirements
Carbon
– Structural backbone of living matter; needed for all organic
compounds that make up a living cell

Nitrogen
– For forming the amino group of amino acids

Sulfur
– For synthesis of sulfur-containing amino acids and vitamins
such as thiamine and biotin
 Chemical Requirements
Phosphorus
– For the synthesis of nucleic acids and phospholipids of the cell
membrane; also in ATP

Trace Elements
– Essential for some enzymes, sometimes as cofactors
– Includes iron, copper, molybdenum, and zinc

Organic growth factors

– essential compounds not synthesized which are obtained from the
environment
– include enzymes for vitamin synthesis, amino acids, purines, pyrimidines
 Chemical Requirements

Oxygen
– Obligate aerobes
– Facultative anaerobes – can use anaerobic respiration or
fermentation when oxygen is absent, e.g. E. coli and yeasts
– Obligate anaerobes - cannot use molecular oxygen for
energy-yielding reactions, e.g. Clostridium
– Aerotolerant anaerobes – e.g. lactobacilli; they can survive
convert harmful forms of oxygen to O2
– Microaerophiles – can only tolerate oxygen concentrations
lower than air
Culture Media
 Culture Media
• Nutrient material prepared for the growth of microorganisms

• Microbes that are introduced into a culture medium to initiate

growth are called inoculum

• Microbes that grow and multiply in a culture medium is called

culture

• Culture media must be initially sterile

 Agar

• A solidifying agent
• Few microbes can degrade it
• Liquefies at 100°C
• Remains liquid until temperature drops to 40°C

• NOT a nutrient
 Forms of Culture Media

Broth
– liquid

Pellicle: A mass of organisms is

floating on top of the broth.

Turbidity: The organisms appear as a

general cloudiness throughout the
broth .

Sediment: A mass of organisms

appears as a deposit at the bottom of
the tube.
 Forms of Culture Media
Slants – tubes are held at
an angle

Stab tubes / deeps –

bacteria are inoculated by
stabbing the medium

Plates – use Petri dishes;

commonly used in the
culturing, separating, and
counting of microorganisms slant deep
 Types of Culture Media
Chemically-defined media
– medium whose exact chemical composition is known
– must contain organic growth factors that serve as source of
carbon and energy
– Best for autotrophs

Complex media
– made up of nutrients including extracts from yeasts, meat,
plants or digests of proteins from cheese and other sources
– E.g. nutrient broth or nutrient agar
 Types of Culture Media
Reducing media
– contain ingredients such as sodium thioglycolate that chemically
combine with dissolved oxygen and deplete the oxygen in the
culture medium
– when the culture must be grown in Petri plates to observe
individual colonies, special anaerobic jars are used

Selective media
– designed to suppress growth of unwanted bacteria and encourage
the growth of desired microorganisms
– E.g. bismuth sulfite agar that inhibits growth of gram-positive
bacteria
 Types of Culture Media

Differential media
– make it easier to
distinguish
colonies of the
desired organism
from other
colonies growing
on the same
plate
 Mannitol Salt Agar MacConkey Agar
both differential (distinguishes mannitol Differentiates from lactose fermenters (left)
fermenters and non-fermenters) and selective and non-fermenters
(high salt con’c prevents most bacteria except
Staphylococcus spp.)
 Types of Culture Media
Enrichment culture
– selective medium and designed to increase the small numbers of
chosen microbe to observable levels
– Usually liquid and provides nutrients and environmental conditions
that favor the growth of a particular microbe but not others

Special Culture Techniques

– Mycobacterium leprae are grown in armadillos
– Treponema pallidum is grown in rabbits
– Bird eggs and cultures of living cells are used to culture and study
obligate intracellular parasites like chlamydias, rickettsias and
viruses
 Obtaining pure cultures
Most commonly used method is the streak plate method
 Preservation

• Refrigeration for short-term storage

• Deep-freezing - pure culture of microbes is placed in a

suspending liquid and quick-frozen at temperatures
ranging from -50°C to -95°C.

• Lyophilization (freeze-drying) - suspension of microbes is

quickly frozen at temperatures ranging from -54°C to -
72°C, and the water is removed by a high vacuum
 Five “I”s of Culturing Microbes

1. Inoculation: Producing a pure culture

2. Isolation: Colony on media, one kind of microbe, pure culture

3. Incubation: growing microbes under proper conditions

4. Inspection: Observation of characteristics (data)

5. Identification: use of data, correlation, to ID organism to exact species

Microbial Growth
 Microbial Growth

produces more cells thus increases microbial count and

consequently microbial growth

Binary fission is the most common mode of reproduction

Budding is another mode of reproduction

 Generation time
Generation time is the time required for a cell to divide and its
population to double

– varies considerably among organisms and with environmental

conditions such as temperature

– most bacteria have a generation time of 1-3 hours while others

require 24 hours per generation

– If binary fission continues unchecked, an enormous number of

cells will be produced
 Generation Time

Calculation:
2 # 𝑜𝑓 𝑔𝑒𝑛𝑒𝑟𝑎𝑡𝑖𝑜𝑛𝑠

Final concentration:
𝐼𝑛𝑖𝑡𝑖𝑎𝑙 # × 2# 𝑔𝑒𝑛𝑒𝑟𝑎𝑡𝑖𝑜𝑛𝑠

For example, if 5 cells were allowed to divide 9 times,

this would result to: 5 x 29 = 2560 cells
 Generation Time
How to obtain the number of generations:

log # 𝑐𝑒𝑙𝑙𝑠 (𝑒𝑛𝑑) − log # 𝑐𝑒𝑙𝑙𝑠 (𝑠𝑡𝑎𝑟𝑡)

# 𝑜𝑓 𝑔𝑒𝑛𝑒𝑟𝑎𝑡𝑖𝑜𝑛𝑠 =
0.301
*0.301 is used because it is the log of 2 (1 cell divides into two)

Generation time:
𝑚𝑖𝑛
60 𝑋 ℎ𝑟
min 𝑝𝑒𝑟 𝑔𝑒𝑛𝑒𝑟𝑎𝑡𝑖𝑜𝑛 = ℎ𝑟
# 𝑔𝑒𝑛𝑒𝑟𝑎𝑡𝑖𝑜𝑛𝑠
 Generation Time

Calculate the generation time if 100 cells growing for 5 hours

produced 1,720, 320 cells

Log1,720,320 – log 100/0.301 = 14 generations

60 mins/hour x 5 hours
__________________ = 21 minutes/generation
14 generations
 Let’s Try!

Given:
900 of the species were grown.
After 15 hours, 3,276,800 cells were produced.

Calculate the generation time.

 Growth Phases

LAG PHASE
• period of little or no cell division
• last for an hour or several days but cells aren’t dormant
• happens because cells don’t immediately reproduce in a
new medium
• microbial population during this stage is undergoing a
period of intense metabolic activity involving synthesis of
enzymes and various molecules
 Growth Phases

LOG PHASE or EXPONENTIAL GROWTH PHASE

• cells begin to divide and enter a period of growth or
logarithmic increase
• cellular reproduction is most active during this period and
generation time reaches a constant minimum
• during this period, cells are most active metabolically
• microorganisms are particularly sensitive to adverse
conditions such as antibiotics
 Growth Phases

STATIONARY PHASE
• period of equilibrium where growth rate slows
down and the number of microbial death
balances the number of new cells and the
population stabilizes
• metabolic activities of individual surviving cells
also slow down at this stage
 Growth Phases

DEATH PHASE or LOGARITHMIC DECLINE

• Number of cells dying exceeds the cells that are
newly formed
• Continues until the population diminished or few
cells remain
 Direct Measurement of
Microbial Growth

Plate counting
– Measures # of viable cells
– Takes some time, e.g. after 24hrs
– Reported as colony-forming units (CFU)
– Serial dilutions are done to ensure that colonies on the
plate are countable (30-300 colonies per plate)
– After that, pour plate or spread plate is done
 Direct Measurement of
Microbial Growth
Filtration

– used when bacterial quantity is small

– at least 100ml of water is passed through a filter;
– bacteria are filtered out and remain of filter surface
– filter is then transferred to a plate
 Direct Measurement of
Microbial Growth
Most Probable Number (MPN)
– estimates the amount of bacteria present

– statement that there is a 95% chance that the bacterial population

falls within a certain range and that the MPN is statistically the
most probable number

– most useful when microbes being counted will not grow on solid
media; or when the growth of bacteria in liquid differential media
is used to identify microbes
MPN Table
 Direct Measurement of
Microbial Growth
Direct Microscopic Count
– measured volume of a bacterial suspension is placed within a
defined area on a microscope slide, e.g. 0.01ml sample

– Once the number of bacteria has been counted in several different

fields, the average number of bacteria per viewing field can be
calculated

– the number of bacteria in the square centimeter over which the

sample was spread can also be calculated
 Indirect Measurement
Turbidity
– using a spectrophotometer to determine absorbance (used
to plot bacterial growth)
Metabolic activity
– assumes that the amount of a certain metabolic product,
such as acid or CO2 , is in direct proportion to the number
of bacteria present
Dry weight
– the fungus/bacterium is removed from the growth medium,
filtered to remove extraneous material, dried in a desiccator,
and weighed.
Sterilization and
Anti-Microbial Methods
 Methods

Sterilization - the removal or destruction of all living

microorganisms

Disinfection – control of harmful organisms

– Antisepsis (antiseptic) if directed at living tissue

Sanitization - lower microbial counts to safe public

health levels and minimize the chances of disease
transmission
 Aseptic Technique

• To protect your self from contact with bio hazards

• To protect your sample from contamination
• To protect others in the lab
 Aseptic Technique

Aseptic technique is the process of:

– Preventing contamination of a culture with
environmental microbes
– Preventing contamination of yourself or the
environment with the organism in the culture
– Remember everything is contaminated with a
variety of environmental microbes.
 Control of Growth

• Moist heat
– Best for dishes, various equipment; autoclave for media and other
items that can withstand pressure
– Kills vegetative bacterial and fungal pathogens and almost all
viruses within 10 min; less effective on endospores
– Autoclaving - at about 15 psi of pressure (121°C), all vegetative
cells and their endospores are killed in about 15 min

• Pasteurization
– Heat treatment that kills all pathogens and most non-pathogens
– Best for food
 Control of Growth

• Dry heat
– Direct – e.g. in inoculating loops
– Incineration – e.g. for paper cups, dressings
– Hot-air sterilization – empty glassware, etc.

• Filtration
– Separation of bacteria from suspending liquid
– Useful for sterilizing liquids such as vaccines that can
be destroyed by heat
 Control of Growth

• Refrigeration
– Bacteriostatic
– Best for food preservation
• Deep-freezing and freeze-drying
• High pressure
• Desiccation
• Osmotic pressure
• Radiation
 Disinfection

• Unfortunately, few chemical agents achieve

sterility; most of them merely reduce microbial
populations to safe levels or remove vegetative
forms

• No single disinfectant is appropriate for all

circumstances
Disinfection
 Disinfectants
• Phenols and phenolics
– Triclosan is a common example
– Can be put in disinfectant hand soaps
– Disrupts plasma membranes

• Biguanides (chlorhexidine) – skin disinfection, esp. for surgical

hand scrubbing

• Halogens
– Chlorine forms –HOCl which alters cellular components
 Disinfection
• Alcohols
– Protein denaturation
– Useful for thermometers and other instruments

• Heavy metals – enzyme denaturation

• Surface-active agents
– Soaps and detergents for the skin
– Acid-anionic sanitizers are used in food industry
– Cationic detergents – useful for instruments
 Disinfection
• Aldehydes – protein denaturation

• Chemical sterilization
– for sterilization of materials that may be damaged by heat
– Also for sterilizing organic medical implants

• Peroxygens and other oxygen forms

– ozone is a supplement for chlorination
– Hydrogen peroxide is a poor antiseptic but a good
disinfectant