MICROBIOLOGY

AND
IMMUNOLOGY
OPT 416
HS246
Dr MAIMUNAH MUSTAKIM
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INTRODUCTION TO
MICROBIOLOGY
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LEARNING OBJECTIVES
AT THE END OF THE LESSON, STUDENTS ARE ABLE TO
EXPLAIN OR IDENTIFY:
I. Prominent scientist in the history of microbiology.
II. Role and application of microbiology in daily life - Present
and future challenges.
III. Domain of living organisms.
IV. Nomenclature of bacteria.
V. Stains and staining methods.
VI. Morphology of Prokaryotic Cells: Shapes and Arrangements.
VII. Prokaryotic Cell Structures : External and Internal.
 Introduction
• Microbiology is the study of organisms too small to be
seen with human eye
• Includes several sub-disciplines
• Bacteriology
• Virology
• Mycology
• Parasitology
• Food microbiology
• Environmental microbiology
• Forensic microbiology
 Introduction
• Microbiology born as a science in 1674
(17th century)
• Anthony van Leeuwenhoek
• Dutch drapery merchant
• Ground lenses to view fabric
• Used lens to peer into a drop of lake water
• First glimpses of microbial world
• Called organisms “animalcules”
 The Origin of Microorganisms
• Theory of Spontaneous Generation
• Theory states
• “organisms can arise from non-living matter”
• Theory had its supporters and detractors
• Detractors included
• Francesco Redi
• Louis Pasteur
• John Tyndall
• Each contributed to disproving the theory
 Francesco Redi
• Italian biologist and physician
• Demonstrated worms found on rotting meat
came from eggs of flies landing on meat
• Proved this by placing rotting meat in jars
• Covered one jar with fine gauze
• Gauze prevented flies from depositing eggs
• No eggs – no worms
 Louis Pasteur

• Considered the father of modern microbiology

• Demonstrated that air is filled with
microorganisms
• Proved this by filtering air in cotton plug
• Identified organisms in cotton as same organisms
contaminating infusions (liquid mixtures)
Louis Pasteur
• To further show air is filled with microbes Pasteur developed swan-
necked flask
• Was able to demonstrate infusions remained sterile even if flask was
left open
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Role of Microorganisms
 Role of Microorganisms
• Microbes have enormous impact on human
existence
• Microorganisms have killed more people than
have ever been killed in war
• Without certain microorganisms life could not exist
• Organisms are responsible for the production of oxygen
and nitrogen
• Key elements for all living organisms
• Microorganisms are decomposers
• Responsible for the breakdown of a wide variety of materials
 Applications of Microbiology
• Food production
• Fermentation of milk to produce numerous
products
• Yogurt, cheese, buttermilk
• Bioremediation
• Use organisms to degrade environmental waste
• Degrade PCB’s (polychlorinated biphenyls –electrical
equipment, paints, plastics and rubber products), DDT
(Dichlorodiphenyl-trichloro-ethene - insecticides)
• Clean up oil spills
• Treat radioactive waste
 Applications of Microbiology

• Bacteria can synthesize numerous

products
• Ethanol
• Pesticides
• Antibiotics
• Dietary amino acids
 Applications of Microbiology
• Genetic engineering
• Definition: introduce genes of one organism
into an unrelated organism to confer new
properties on the organism
• Applications include engineering organisms to
produce medically important products and vaccines
• Engineered plants resist disease
• Potentially therapeutic
• Gene therapy
 Present and Future Challenges
• Infectious diseases remains a threat
• 750 million cases each year in United States
• Resulting in 200,000 deaths
• Costing tens of billions of dollars spent on health care
• Emerging diseases
• Diseases show increased occurrence with wider distribution
• Some diseases include
• Legionnaire’s disease (is a form of pneumonia caused by of Gram negative
aerobic bacterium Legionella pneumophila).
• Lyme disease (is an infectious disease caused by at least three species of
bacteria belonging to the genus Borrelia - tick bite – rash
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 Present and Future Challenges
• Severe Acute Respiratory Syndrome (SARS)
• Influenza viruses : A, B, C
• MERS-CoV (Middle East Respiratory Syndrome –
Coronavirus)
• Zika Virus
• Covid-19
• Dengue

• Factors associated with emerging disease

• Changing lifestyles
• Genetic changes in organisms
 Present and Future Challenges
• Resurgence (reappearance) of old diseases
• Diseases thought to be “defeated” increasing in
frequency
• Often more serious
• Causative agents usually resistant to treatment
• Reasons for resurgence (reappearance)
• Increase travel
• Visitors to foreign regions bring organisms from home region
• Unvaccinated individuals susceptible to infection
• Causative agents of controlled diseases still around and infect
vulnerable individuals
Ex : Tuberculosis, Ebola, Measles, Polio
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 Present and Future Challenges

• Chronic disease caused by bacteria

• Many diseases once thought caused by environmental
stressors actually caused by bacteria
• Example: gastric ulcers
• Causative agent –Helicobacter pylori
 Host-Bacterial Interactions
• These bacteria are termed normal microbiota or
normal flora
• Bacteria outnumber cells in the body 10:1
• For every one body cell, there are estimated 10 bacteria
• These bacteria compete with other organisms for
food and space
• Keep disease-causing organisms from breaching host
defenses
• Some bacteria and viruses use the human body
as a habitat for multiplication, persistence and
transmission
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The Microbial World

 The Microbial World
• All living things can be
classified in one of three
groups
• Also known as domains
• Organisms in each domain
share certain properties
• These properties distinguish
them from organisms in other
domains
• Three domains are
• Bacteria
• Archaea
• Eucarya
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 The Microbial World
• Bacteria and Archaea
• Both are single-celled
organisms
• Contain no membrane bound
nucleus
• Termed prokaryotes = pre nucleus
• Pro = pre
• karyote = nucleus
• Do not contain any other
organelles
• Cytoplasm is surrounded by
rigid cell wall
 The Microbial World
• Eucarya
• Organisms contain
membrane bound nucleus
• Termed eukaryote = true
nucleus
• Eu = true
• karyote = nucleus

• Contains internal organelles

• Making organism more complex
• Example = mitochondria

• May be single and/or

multicellular
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 Domain Bacteria
• Most common type in human infection
• Members widely diverse in shape and properties
• Most prominent features include:
• Specific shapes
• Rod-shaped, spherical and spiral
• Rigid cell walls – contain peptidoglycan
• Responsible for cell shape
• Multiply by binary fission
• One cell divides into two
• Each cell is genetically identical to the first
• Some bacteria are motile
• Move by means of flagella
 Domain Archaea
• Demonstrate a number of same features as
Bacteria
• Same shapes
• Multiply through binary fission
• Move by means of flagellum
• Archaea exhibit significant differences
• Chemical composition of cell wall differs from organisms
in other domains
• Organisms of Archaea domain found in extreme
environments
• Extreme temperatures
• Environments with high concentrations of salts
 Domain Eucarya
• All members are eukaryotic
• Microbial world composed of single-celled Eucarya
• Algae
• Fungi
• Protozoa
 Domain Eucarya
• Algae
• Diverse group
• Includes single and multicellular organisms
• All contain chlorophyll
• Pigments absorb the energy of light which is
used in photosynthesis.
• Some contain other pigments
• Usually found near surface waters
• Have rigid cell wall
• Distinct from bacterial cell walls
 Domain Eucarya

• Fungi
• Diverse single-celled and multicellular
organisms
• Single-celled = yeast
• Multi-cellular = molds
• Gain energy from organic materials
• Found wherever organic materials are present
 Domain Eucarya
• Protozoa
• Microscopic, single-celled organism
• Found in water and on land
• Complex
• Much larger than prokaryote
• Do not have a rigid cell wall
• Gain energy from organic matter
• Most are motile
• Means of motility diverse and a feature of their
classification
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Nomenclature
• Binomial naming system
• Two word naming system
• First word is genus name
• Always capitalized
• Escherichia, Salmonella, Pseudomonas
• Second word is species name
• Not capitalized
• coli, typhi, aeruginosa
• When writing full name, genus usually abbreviated
• E. coli
• Full name always italicized
• Or underlined
• Additional Symbol with numbers - strain
o O157 : H7
o K12
o E. coli O157: H7
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ØStaphylococcus aureus • Streptococcus agalactiae

ØS. epidermidis • S. pneumoniae
ØS. saprophyticus • S. pyogenes
Note: • S. viridans
Staphylococcus and
Streptococcus is not capitalized
or italicized when used in — Salmonella typhi
adjective forms, ex: — S. enteritidis
staphylococcal infection, or as
the plural (staphylococci) — S. typhimurium
— S. dublin
Note:
“sp.” (singular) or “spp.” (plural) – used when the actual specific species
name need not be specified, NOT italicized nor underlined.
Ex : Streptococcus sp. ,
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Size in the Microbial World

• Tremendous range in size
• Smallest virus approximately 1/1,000,000th size
of largest eukaryotic cell
 Microscope Techniques
• Microscopes
• Most important tool for studying
microorganisms
• Use viable light to observe objects
• Magnify images approximately 1,000x
• Electron microscope, introduced in 1931, can
magnify images in excess of 100,000x
• Scanning probe microscope, introduced in 1981,
can view individual atoms
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STAINS AND STAINING

METHODS
 Dyes and Staining
• Cells are frequently stained to observe organisms
• Stains are made of organic salts
• Dyes carry (+) or (-) charge on the molecule
• Molecule binds to certain cell structures
• Dyes divided into base or acid based on the charges
• Base dyes carry positive charge and bond to cell
structures that carry negative charge
• Commonly stain the cell
• Acidic dyes carry negative charge and are repelled by
cell structures that carry negative charge
• Commonly stain the background
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Stains and Their Characteristics

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• Basic dyes more commonly used than acidic dyes
• Common basic dyes include
• Methylene blue
• Crystal violet
• Safranin
• Malachite green
 Dyes and Staining

• Simple stain
• Uses one basic stain to stain the cell –
example: Methylene blue
• Allows for increased contrast between cell
and background
• All cells stained the same color
• No differentiation between cell types
 Dyes and Staining

• Differential Stains
• Used to distinguish one bacterial group from
another
• Uses a series of reagents
• Two most common differential stains
• Gram stain
• Acid-fast stain
 Dyes and Staining
• Gram Stain
• Most widely used procedure for staining
bacteria
• Developed over century ago
• Dr. Hans Christian Gram
• Bacteria separated into two major groups
• Gram positive
• Stained purple
• Gram negative
• Stained red or pink
 Dyes and Staining
• Gram Stain
• Involves four reagents
• Primary stain
• Crystal violet
• Stains the cell
• Mordant
• Gram’s iodine
• Holds primary dye onto cell
• Decolorizer
• Usually alcohol
• Removes primary dye from Gram
negative cell
• Counter or Secondary stain
• Safranin
• Recolors cells that lose stain through
decolorization
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Gram Stain
 GRAM STAINING
[Link]
 Dyes and Staining

• Acid-fast Stain (Ziehl–Neelsen stain)

• Used to stain organisms that resist conventional
staining
• Used to stain members of genus Mycobacterium
• High lipid concentration in cell wall prevents
uptake of dye
• Uses heat to facilitate staining
• Once stained difficult to decolorize
 Dyes and Staining
• Acid-fast Stain
• Can be used for
presumptive identification
in diagnosis of clinical
specimens
• Requires multiple steps
• Primary dye
• Carbol fuchsin
• Colors acid-fast bacteria red
• Decolorizer
• Generally acid alcohol
• Removes stains from non acid-fast
bacteria
• Counter stain
• Methylene blue
• Colors non acid-fast bacteria blue
• Color of acid-fast bacteria red
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Acid-fast Staining Method

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 Dyes and Staining

• Special Stains
• Capsule stain
• Example of negative stain
• Only stains the background
• Allows capsule to stand out around
organism
• Endospore stain
• Staining enhances endospore
• Uses heat to facilitate staining
• Flagella stain
• Staining increases diameter of flagella
• Makes more visible
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A stained preparation of the cell Bacillus subtilis showing

endospores as green and the vegetative cell as red
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Phase-bright endospores of Paenibacillus alvei imaged

with phase-contrast microscopy
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Morphology of Prokaryotic Cells:

Shapes and Arrangements
 Cells Shape
• Prokaryotes exhibit a
variety of shapes
• Most common
• Coccus

• Spherical
• Bacillus

• Rod or cylinder
shaped
• Cell shape not to be
confused with
Bacillus genus
• Prokaryotes exhibit a
variety of shapes
• Other shapes
• Coccobacillus
• Short round rod
• Vibrio

• Curved rod
• Spirillum
• Spiral shaped
• Spirochete
• Helical shape
• Pleomorphic

• Bacteria able to
vary shape
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Pleomorphic bacteria
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Corynebacterium diptheriae – pleomorphic shapes

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Cells Arrangements
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• Prokaryotic cells may — Division along a
form groupings after single plane may
cell division result in pairs or
• Cells adhere together chains of cells
after cell division for ◦ Pairs = diplococci
characteristic – Example: Neisseria
arrangements gonorrhoeae
• Arrangement depends on ◦ Chains = streptococci
plan of division – Example: species of
• Especially in the cocci Streptococcus
• Division along several random planes form clusters
• Example: species of Staphylococcus
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PROKARYOTIC CELL STRUCTURE

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EXTRACELLULAR STRUCTURES
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 Cell Wall
• Bacterial cell wall
• Rigid structure
• Surrounds cytoplasmic membrane
• Determines shape of bacteria
• Holds cell together
• Prevents cell from bursting
• Unique chemical structure
• Distinguishes Gram- positive from Gram-
negative
 Cell Wall
• Rigidity of cell wall is due to peptidoglycan
(PTG)
• Compound found only in bacteria
• Basic structure of peptidoglycan
• Alternating series of two subunits
• N-acetylglucosamine(NAG)
• N-acetylmuramic acid (NAM)
• Joined subunits form glycan chain
• Glycan chains held together by string of four amino acids
• Tetrapeptide chain
Cell Wall

• Gram-positive cell wall

• Relatively thick layer of
PTG
• As many as 30
• Regardless of thickness,
PTG is permeable to
numerous substances
• Teichoic acid component
of PTG
• Gives cell negative charge
 Cell Wall
• Gram-negative cell wall
• More complex than G+
• Only contains thin layer of
PTG
• PTG sandwiched between outer
membrane and cytoplasmic
membrane
• Region between outer membrane
and cytoplasmic membrane is
called periplasm
• Most secreted proteins contained
here
• Proteins of ABC transport system
located here
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 Cell Wall

The cytoplasmic (cell/plasma)membrane is a double- layered structure

consisting of a Phospholipid bilayer and Proteins which vary in location
and function and are able to flow laterally within it.
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INTRACELLULAR STRUCTURES
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