Lecture 1. Introduction to Medical Microbiology.pdf

Introduction to Medical
Microbiology
Mr. Mox Malama Kalumbi
March 2023

Learning Objectives
• Define Microbiology, Medical Microbiology
• Understand the History of Microbiology
• Significance of Microbiology in the Health
Field.
• Role of Microorganism in disease.
• Understand Spontaneous Generation
• Understand The Germ Theory of Disease
• Understand important early discoveries

History of Microbiology;
Spontaneous Generation
& The Germ Theory of
Disease.

• Microbiology is the study of microorganisms
usually less than 1mm in diameter which
requires some form of magnification
(Microscope) to be seen clearly
– Examples:
• Viruses
• Bacteria
• Fungi
• Algae
• Protozoa's

• Some organisms, such as bread molds (fungus),
filamentous algae, and Thiomargarita
namibiensis (bacteria), can be observed without
the use of amplification or magnification.
– These organisms are included in the discipline of
microbiology because of similarities in properties
and techniques used to study them
• Techniques necessary to isolate and culture
microorganisms include:
– Isolation
– Sterilization
– Culture in artificial media

• Microbiologists may be interested in specific
types of organisms:
– Virology - viruses
– Bacteriology - bacteria
– Phycology - algae
– Mycology - fungi
– Protozoology - protozoa

• Microbiology may have a more applied focus:
– Medical microbiology, including immunology
– Food and Dairy microbiology
– Public Health microbiology (Epidemiology)
– Industrial microbiology
– Agricultural microbiology

• Microbiologists may be interested in various
characteristics or activities of microorganisms:
– Microbial morphology
– Microbial cytology
– Microbial physiology
– Microbial ecology
– Microbial genetics and molecular biology
– Microbial taxonomy

ROBERT HOOKE
• One of the most important discoveries
of biology occurred in 1665, with the
help of a crude microscope, when
Robert Hooke stated that life’s
smallest structural units were cells.

ANTONY VAN LEEUWENHOEK
• First to observe living
microbes
• His single-lens magnified 50-
300X magnification
• Between 1674-1723 he
wrote series of papers
describing his observations
of bacteria, algae, protozoa,
and fungi (Animalcules)

SPONTANEOUS GENERATION
• Early belief that some forms of life could
arise from “vital forces” present in nonliving
or decomposing matter, abiogenesis.
• In other words, organisms can arise from
non-living matter.

LOUIS JABLOT
• In 1670 Jablot conducted an experiment in
which he divided a hay infusion that had been
boiled into two containers: a heated container
that was closed to the air and a heated
container that was freely open to the air.
• Only the open vessel developed
microorganisms.
• This further helped to disprove abiogenesis
(autogenesis, spontaneous generation).

REDI’S and JABLOT’S EXPERIMENTS

• Disproved by:
–Schwann, Friedrich Schroder and von
Dusch (1830s) – Air allowed to enter
flask but only after passing through a
heated tube or sterile wool
–John Tyndall (1820-1893) – Omission of
dust → no growth. Demonstrated heat
resistant forms of bacteria (endospores)

LOUIS PASTEUR (1822 - 1895)
• Disproved spontaneous
generation of microbes by
preventing “dust particles”
from reaching the sterile
broth
• In 1861 completes
experiments that lays to rest
spontaneous generation.
• Showed microbes caused
fermentation and spoilage

PASTEUR’S EXPERIMENT
Trapped airborne organisms in cotton; he also heated the
necks of flasks, drawing them out into long curves, sterilized
the media, and left the flasks open to the air.
In this way Pasteur disproved the theory of spontaneous
generation

IV. Role of
Microorganisms in
Disease

GOLDEN AGE OF MICROBIOLOGY
• The period from 1860 to 1900 is often
named the Golden Age of Microbiology.
During this period, rapid advances, spear-
headed by Louis Pasteur and Robert Koch,
led to the establishment of microbiology as
a science.

Robert Koch
• In 1860 developed an
elaborate technique
to isolate & identify
specific Pathogens
that cause specific
diseases.
• He isolated the
anthrax bacterium.
1843 – 1910

• Robert Koch (1843 - 1910),
– using criteria developed by his teacher, Jacob
Henle (1809-1895), established the relationship
between Bacillus anthracis and anthrax.
– His criteria became known as Koch’s Postulates
and are still used to establish the link between a
particular microorganism and a particular
disease:

History
• 1884 Koch’s Postulates of Disease
Transmission - Robert Koch

GERM THEORY OF DISEASE
• In 1876 Robert Koch proved the “Germ
theory of disease” by showing that bacteria
actually caused disease.
• Koch established a sequence of
experimental steps for directly relating a
specific microbe to a specific disease
called KOCH’S POSTULATES

➢The causative (etiological) agent must be
present in all affected organisms but
absent in healthy individuals
➢The agent must be capable of being
isolated and cultured in pure form
➢When the cultured agent is introduced to
a healthy organism, the same disease
must occur
➢The same causative agent must be isolated
again from the affected host
Koch’s Postulates

ROBERT KOCH
• Developed pure culture
methods.
• cccIdentified cause of
➢ Anthrax (Bacillus anthrax) ,
➢ TB (Mycobacterium
tuberculosis) , &
➢ Cholera (Vibrio cholera).

Development of Culture Media
• Why?
– To enable the isolation of pure cultures (only one
type of organism)
• Especially important during Koch’s period
• Gelatin not useful as solidifying agent (melts at >28 ºC
and some bacteria hydrolyze it with enzymes)
• Fannie Hesse, the wife of one of Koch’s assistants,
proposed using agar
– Not digested by most bacteria
– Melts at 100 ºC
– Used today - ~2% in solid media
• Richard Petri, another of Koch’s assistants, developed
the Petri dish

History
• Walter Hesse ( 1846-1911): Used Agar as a solidifying
agent to harden media. Agar is extracted from
seaweeds red algae.
• Rechard Petri ( 1852-1921): Used agar dish to provide
a large area to grow.
• Christian Gram ( 1853-1935): Staining method that
demonstrate bacteria and distinguish between Gram
positive and Gram negative bacteria.

History
• Raymond Sabouraud ( 1890-1910): Develop culture
media to study yeast and molds.
• Dimitri Ivanovski (1892): Tobacco mosaic virus could
pass through filters used to remove bacteria.
• Selman Waksman (1940): Discovered a number of
antibiotic such as Tetracycline and Streptomycin.

*Developed the germ
theory in 1798
*Also developed vaccine
against anthrax.
*Pasteurization technique
*Developed the germ theory
of disease
“Father of bacteriology and immunology”
Louis Pasteur 1822 – 1895

LOUIS PASTEUR
• In 1864 Pasteur established the relationship
between microbes and disease in
preventing wine from spoiling by using the
process termed pasteurization.
• This process kills bacteria in the alcohol by
heat, thus preventing the formation of acetic
acid (vinegar).

LOUIS PASTEUR
• His discovery of pasteurization, lead
Pasteur to introduce the “Germ theory of
disease” in 1864.
• Pasteur stated that diseases are caused
by the growth of microbes in the body and
not by sins, bad character, or poverty, etc.

• Oliver Holmes (1773 - 1843)
– showed that sepsis could be transmitted
by hands of medical student and may
cause disease
– M. J. Berkeley (ca. 1845)
– demonstrated that the Great Potato
Blight of Ireland was caused by a Fungus
Demonstrations that microorganisms cause
disease

• Louis Pasteur (1822 - 1895)
– showed that the pébrine disease of
silkworms was caused by a protozoan
parasite
• Edward Jenner (ca. 1798): Develop the first
Vaccine and used a vaccination procedure to
protect individuals from smallpox
• Louis Pasteur
– developed other vaccines including those for
chicken cholera, anthrax, and rabies

History
• 1885 - Vaccine against Rabies - Louis Pasteur

History
• 1796 – First vaccine (smallpox) Edward Jenner

Development of Vaccines and
Antisera
• Edward Jenner in 1796 discovered that
cowpox (vaccinia) induced protection
against human smallpox
– Called procedure vaccination

• Vaccination:
– Inoculation of healthy individuals with
weakened (or attenuated) forms of
microorganisms, that would otherwise
cause disease, to provide protection, or
active immunity from disease upon later
exposure.

• Pasteur and Roux reported that
incubating cultures longer than normal
in the lab resulted in ATTENUATED
bacteria that could no longer cause
disease.
– Working with chicken cholera (caused by
Pasteurella multocida), they noticed that
animals injected with attenuated cultures
were resistant to the disease.

• Pasteur and Chamberland developed
other vaccines:
– Attenuated anthrax vaccine
• Chemical and heat treatment (potassium
bichromate)
– Attenuated rabies vaccine
• Propagated the virus in rabbit following
injection of infected brain and spinal cord
extracts

• Passive immunization
– Work by Emil von Behring (1845-1917) and
Shibasaburo Kitasato (1852-1931)
• Antibodies raised to inactivated diphtheria
toxin by injection different host (rabbit) with the
toxin (a toxoid form)
–Antiserum recovered
»Contains antibodies specific for the toxin
»Protection from disease when injected
non -immune subject.

John Tyndall (1820 – 1893)
In 1876 discovered that there were
two different types of bacteria.
a) Heat sensitive or heat labile
forms (vegetative cells)
easily destroyed by boiling
b) Heat resistant types known as an
endospore
Tyndall demonstrated that alternate
process of heating & cooling if
repeated five times, can kill all the
endospores.
This is known as Sterilization
process or Tyndallization

FERDINAND COHN
• In 1876, a German
botanist, Ferdinand
Cohn, also discovered
“heat-resistant forms of
bacteria”.
• This bacteria are now
termed endospores
(Bacillus species and
Clostridium species)
Bacillus anthracis

History: Joseph Lister
• 1867 Antiseptic Surgery( Carbolic acid- Phenol)

• Joseph Lister (1827 - 1912)
– developed a system of surgery designed to
prevent microorganisms from entering wounds
– phenol (Carbolic Acid) sprayed in air around
surgical incision
– Decreased number of post-operative infections
in patients
– his published findings (1867) transformed the
practice of surgery

PAUL EHRLICH
➢ In the 1890’s Ehrlich proposed a theory of immunity in
which antibodies were responsible for immunity (
Antitoxin).
➢ In addition, he is known as the father of modern
chemotherapy.
➢ He speculated about some “magic bullet” that would
selectively find and destroy pathogens but not harm
the host (Selective Toxicity).
➢ He also develop a staining procedure to identify
tubercle bacilli.

History
• 1929 Discovery of Penicillin (first antibiotic) -
Alexander Fleming

ALEXANDER FLEMING
➢In 1928 Fleming observed that the growth
of the bacterium Staphylococcus aureus
was inhibited in the areas surrounding the
colony of a mold that had contaminated a
Petri plate.
➢The mold was identified as Penicillium
notatum, and its active compound was
named penicillin.

History
• Reska (1938) – First
Electron Microscope
• The electron microscope is capable of
magnifying biological specimens up to
one million times.
• These computer enhanced images of
• 1. smallpox,
• 2. herpes simplex, and
• 3. mumps are magnified, respectively,
• 150,000, 150,000 and 90,000 times.
• To study detail structures of
viruses.

WATSON and CRICK, FRANKLIN, and
WILKINS
• In 1953 Watson and Crick determined the
structure of DNA.
• They used their research, together with
the research of Franklin and Wilkins to
determine the structure of the DNA
molecule.

V. How
Microorganisms
Affect Their
Environment

• Louis Pasteur
– demonstrated that alcoholic fermentations were
the result of microbial activity,
– that some organisms could decrease alcohol yield
and sour the product, and
– that some fermentations were aerobic and some
anaerobic;
– he also developed the process of pasteurization to
preserve wine during storage

VI.
Microorganisms in
the 20th Century

• George W. Beadle and Edward L. Tatum (ca. 1941)
– studied the relationship between genes and
enzymes using the bread mold, Neurospora
– Precursor→ ornithine → citrulline → arginine
– One gene, one polypeptide hypothesis
• Salvadore Luria and Max Delbruck (ca. 1943)
– Demonstrated spontaneous gene mutations in
bacteria (not directed by the environment)
Important Early Discoveries

• Oswald T. Avery, Colin M. MacLeod, and
Maclyn McCarty (1944)
– Following initial studies by Frederick Griffith
(1928) they provided evidence that
deoxyribonucleic acid (DNA) was the genetic
material and carried genetic information during
transformation
– Worked with Streptococcus pneumoniae (rough
and smooth)

• In the 1970s new discoveries in microbiology led to
the development of :
✓ recombinant DNA technology (refers to the
joining together of DNA molecules from two different
species that are inserted into a host organism to
produce new genetic combinations that are of value to
science, medicine, agriculture, and industry) and
✓ genetic engineering (is the process of using
recombinant DNA (rDNA) technology to alter
the genetic makeup of an organism. Traditionally,
humans have manipulated genomes indirectly by
controlling breeding and selecting offspring with
desired traits).

• Medical microbiology is the study of causative
agents of infectious diseases of humans and
their reactions to such infections. In other
words it deals with etiology, pathogenesis,
laboratory diagnosis, specific treatment and
control of infection (immunization).

Modern medical microbiology
• Bacteriology – the science of bacteria, the
causative agents of a member of infectious
diseases.
• Virology – the science of viruses, non-cellular
living systems, capable of causing infectious
diseases in man.
• Immunology – the science which concerned
with mechanisms of body protection against
pathogenic microorganisms and foreign cells
and substances.
• Mycology – the study of fungi pathogenic for
man.
• Protozoology – which deals with pathogenic
unicellular animal organisms.

CLASSIFICATION OF
MICROORGANISMS
Protista Vira
DNA-viruses and RNA-viruses
• Eukaryotes Prokaryotes
• Fungi Blue-green algae
• Algae Bacteria
• Protozoa Scotobacteria Photobacteria
• Slime moulds 1. Class Bacteria
2. Class Rickettsias
3. Class Mollicutes

• Organisms can be divided into five
kingdoms:
–the Monera or Prokaryotae,
–Protista,
–Fungi,
–Animalia, and
–Plantae

• There are two types of microorganisms:
– Prokaryotes
• have a relatively simple morphology and
lack a true membrane-bound nucleus
– Eukaryotes
• are morphologically complex and have a
true, membrane-bound nucleus

Distinguishing Features of
Prokaryotic Cells:
1. DNA is:
 Not enclosed within a nuclear membrane.
 A single circular chromosome.
 Not associated with histone proteins.
2. Lack membrane-enclosed organelles like
mitochondria, chloroplasts, Golgi, etc.
3. Cell walls usually contain peptidoglycan, a
complex polysaccharide.
4. Divide by binary fission.

❖Histones are a family of basic proteins
that associate with DNA in the nucleus
and help condense it into chromatin.
❖Nuclear DNA does not appear in free
linear strands; it is highly condensed and
wrapped around histones in order to fit
inside of the nucleus and take part in the
formation of chromosomes.

Distinguishing Features of
Eukaryotic Cells:
1. DNA is:
– Enclosed within a nuclear membrane.
– Several linear chromosomes.
– Associated with histones and other proteins.
2. Have membrane-enclosed organelles like
mitochondria, chloroplasts, Golgi,
endoplasmic reticulum, etc.
3. Divide by mitosis.

Prokaryotes Eukaryotes
Cell size 0.2-2 um in diameter 10-100 um in diameter
 Nucleus Absent Present
 Membranous
Organelles Absent Present
 Cell Wall Chemically complex When present, simple
 Ribosomes Smaller (70S) Larger (80S) in cell
70S in organelles
 DNA Single circular Multiple linear
chromosome
chromosomes (histones)
 Cell Division Binary fission Mitosis

Prokaryotic and Eukaryotic cells

The Prokaryotic Cell: Size, Shape, and
Arrangement of Bacterial Cells
Cell Size:
Dimensions of most bacterial cells:
 Diameter: 0.2 to 2.0 um.
 Human red blood cell is about 7.5 -10 um in
diameter.
 Length : 2 to 8 um. Some cyanobacteria are up
to 60 um long.
Bacterial cells have large surface to volume
ratios.

Bacterial Cell Shapes & Arrangements:
• Coccus (plural: cocci): Spherical.
• May have the following arrangements:
• Diplococci : A pair of attached cocci. Remain
attached after dividing.
• Streptococci : Chainlike arrangement.
• Tetrads : Groups of four. Divide in two
planes.
• Sarcinae : Groups of eight. Divide in three
planes.
• Staphylococci : Grapelike clusters. Divide in
multiple planes.

Spherical
(cocci)
bacteria
1.Micrococci
2. Diplococci
3. Streptococci
4. Staphylococci
5. Tetracocci
6. Sarcine

The Prokaryotic Cell: Size, Shape,
and Arrangement of Bacterial Cells

The Prokaryotic Cell: Size, Shape,
& Arrangements :
 Bacterial Cell Shapes & Arrangements:
▪ Spiral Bacteria: Have one or more twists:
▪ Vibrio: A comma shaped cell. Look like curved rods.
▪ Spirilla: Helical, corkscrew shaped bacteria with rigid
bodies.
▪ Use whiplike external flagella to move.
▪ Spirochetes: Helical bacteria with flexible bodies.
▪ Use axial filaments (internal flagella) to move.

The Prokaryotic Cell: Size, Shape, and
Arrangement of Bacterial Cells
Bacterial Cell Shapes & Arrangements :
Other less common shapes:
▪ Star
▪ Flat and square
▪ Triangular
▪ Pleomorphic bacteria: Have several possible shapes.
Found in a few groups:
▪ Corynebacterium
▪ Rhizobium
▪ Most bacteria are monomorphic: Maintain a single
shape. However environmental factors may affect cell
shape.

Pleomorphic bacteria- coccobacilli
of Haemophilus influenzae

The Prokaryotic Cell Structure.
Structures External to the Cell Wall
1. Glycocalyx: “Sugar coat”.
▪ All polysaccharide containing substances found
external to the cell wall, from the thickest capsules to
the thinnest slime layers .
▪ All bacteria have at least a thin slime layer.
▪ Chemical composition varies widely with species.
▪ A glycocalyx made of sugars is called an extracellular
polysaccharide (EPS).
▪ The glycocalyx may have several functions:
▪ Attachment to host cells.
▪ Source of nutrition.
▪ Prevent dehydration.
▪ Escape host immune system.

Prokaryotic Cell Structure
1. Glycocalyx: “Sugar coat”.
A. Capsules: Organized polysaccharide substance that is firmly
attached to the cell wall.
• Not formed by all bacteria.
• Important in virulence.
▪ Anthrax bacteria only cause anthrax if have protein capsule.
▪ Only Streptococcus pneumoniae with capsule cause pneumonia.
▪ Help bacteria escape the host immune system, by preventing
destruction by phagocytosis .
▪ When bacteria lose their capsules they become less likely to cause
disease and more susceptible to destruction.

CAPSULE
Klebsiella pneumoniae
Enterobacter aerogenes

1. Glycocalyx:
B. Slime Layer: Thin polysaccharide substance that is
loosely attached to the cell wall.
▪ Not formed by all bacteria.
▪ Important for virulence.
▪ Oral bacteria stick to teeth due to slime layer and with time
produce dental plaque.
▪ Allow bacteria to adhere to objects in their environment so
they can remain near sources of nutrients or oxygen.
▪ Rock surfaces
▪ Plant roots
▪ Help bacteria trap nutrients near cell and prevent
dehydration.

2. Flagella (Sing. Flagellum):
▪ About half of all known bacteria are motile, most use
flagella.
▪ Long, thin, helical appendages.
▪ A bacterium may have one or several flagella, which
can be in the following arrangements:
▪ Monotrichous: Single polar flagellum at one end.
▪ Amphitrichous: Two polar flagella, one at each end.
▪ Lophotrichous: Two or more flagella at one or both
ends.
▪ Peritrichous : Many flagella over entire cell surface.

FLAGELLA
Electron Micrograph of Bacteria with Flagella

Flagella have three basic parts:
1. Filament: Outermost region.
▪ Contains globular protein flagellin.
▪ Not covered by a sheath like eukaryotic filaments.
2. Hook: Wider segment that anchors filament to basal
body.
3. Basal Body: Complex structure with a central rod
▪ surrounded by a set of rings.
▪ Gram negative bacteria have 2 pairs of rings.
▪ Gram positive bacteria only have one pair of rings.

• 2. Flagella (Sing. Flagellum):
▪ Bacterial flagella move by rotation from basal body.
▪ Flagellar movement may be either clockwise or
counterclockwise.
▪ Bacteria may be capable of several patterns of motility.
▪ Runs or swims: Bacterium moves in one direction.
▪ Tumbles: Bacterium changes direction. Caused by
reversal of flagellar rotation.

Vibrio cholerae has a single polar
flagellum for swimming movement.
Electron Micrograph

▪ Taxis: Movement of a cell toward or away from a
particular stimulus.
▪ Chemotaxis: Movement in response to a chemical
stimulus.
▪ Phototaxis: Movement in response to a light stimulus.
▪ Flagellar protein H antigens are used to identify
important pathogens.
▪ E. coli O157:H7: Causes bloody diarrhea associated
with food borne epidemics. Causes 200-500 deaths per
year.

3. Axial Filaments (Endoflagella):
▪ Bundles of fibers that are anchored at ends of the cell
beneath the outer sheath.
▪ Spiral around the cells.
▪ Have similar structure to flagella.
▪ Rotation of endoflagella produces a corkscrew
motion.
▪ May enable bacteria to penetrate body tissues.
▪ Found in spirochetes:
▪ Treponema pallidum: Cause of syphilis.
▪ Borrelia burgdorferi : Cause of Lyme disease.

 4. Fimbriae and Pili:
▪ Hair-like appendages that are shorter, straighter, and thinner than
flagella.
▪ Used for attachment rather than motility.
▪ Found in Gram-negative bacteria.
A. Fimbriae (Sing: fimbria)
▪ May occur at poles or over entire cell surface.
▪ Like glycocalyx, enable bacteria to adhere to surfaces. Important
for colonization of host tissue.
▪ Neisseria gonorrhoeae: Causes gonorrhea. Attach to sperm
cells and mucous membranes through fimbriae.
▪ Bacteria can attach to broth surface via fimbriae, forming a film-
like layer called pellicle.

Fimbriae of Neisseria gonorrhoeae allow
the bacterium to adhere to tissues

Fimbriae and Pili:
B. Pili (Sing: pilus): Conjugation or sex pili
▪ Only found in certain groups of bacteria.
▪ Longer than fimbriae.
▪ Cells only have one or two sex pili.
▪ Attach two cells together, and allow the transfer of
genetic material (DNA) between cells.
▪ Medically important because allow for the transfer of
antibiotic resistance genes from one cell to another.

The Cell Wall
General Characteristics:
Semi-rigid structure that lies outside the cell
membrane in almost all bacteria.
Two major functions:
1. Maintains characteristic shape of cell.
2. Prevents the cell from bursting when fluids flow
into the cell by osmosis.
Contributes to bacterial ability to cause disease.
Site of action of some antibiotics .
Very porous and does not regulate passage of
materials into the cell.

The Cell Wall Composition:
Peptidoglycan (Murein): Made up of a repeating
disaccharide attached by polypeptides to form a
lattice.
Peptidoglycan is one immense covalently linked
molecule, resembling multiple layers of chain link
fence.
Disaccharide component: Made up of two
monoscaccharides:
▪ N-acetylglucosamine (NAG)
▪ N-acetylmuramic acid (NAM)
▪ Alternating disaccharides (NAG-NAM) are linked
together in rows of 10 to 65 molecules.

The Cell Wall
Composition:
• Peptidoglycan (Murein):
▪ Adjacent disaccharide rows are linked together
by polypeptide chains which vary in
composition, but always contain tetrapeptide
side chains .
▪ Parallel tetrapeptide side chains may be
directly linked together or linked by a polypeptide
cross-bridge.
▪ Penicillin interferes with the final linking of
peptidoglycan rows by peptide cross bridges. As
a result, the cell wall is greatly weakened and
cell undergoes lysis.

The Cell Wall
Gram-Positive Cell Walls:
Consist of several layers of peptidoglycan, which
form a thick , rigid structure (20-80 nm).
Also contain teichoic acids, which are made up of an
alcohol and a phosphate group. Two types:
Lipoteichoic acids : Span cell wall, linked to cell membrane.
Wall teichoic acids: Linked to peptidoglycan layer.
Teichoic acids are negatively charged and:
▪ Bind to and regulate movement of cations into cell.
▪ Regulate cell growth and prevent cell lysis .
▪ Can be used to identify bacteria.

Gram-positive bacteria (those that retain the purple crystal
violet dye when subjected to the Gram-staining procedure)
the cell wall is a thick layer of murein.

The Cell Wall
Gram-Negative Cell Walls:
Cell wall is thinner, more complex and more
susceptible to mechanical breakage than that of Gram-
positive bacteria.
Consist of one or a few peptidoglycan layers and an
outermembrane.
Peptidoglycan is bonded to lipoproteins in:
Outer membrane
Periplasmic space: Region between outer membrane
and plasma membrane.
Periplasmic space contains degradative enzymes and
transport proteins.

Gram-negative bacteria (cells which do not retain the crystal
violet dye) the cell wall is relatively thin and is composed of a
thin layer of murein surrounded by a membranous structure
called the outer membrane.

A. Peptidoglycan Structure
B. Gram-Positive and Gram- Negative Cell Wall
Structure

Gram-negative cell wall
 Cell wall in gram-negative bacteria contains much less peptidoglycan
 Surrounded by an outer membrane
 There is much less crosslinking between the peptidoglycan.
 LPS is also present in the outer membrane and penetrates into the surrounding
environment.

Cell wall and Gram stain
We now know that the Gram
reaction is based on the structure
of the bacterial cell wall.
In Gram-positive bacteria, the
purple crystal violet stain is
trapped by the layer of
peptidoglycan which forms the
outer layer of the cell.
In Gram-negative bacteria, the
outer membrane prevents the stain
from reaching the peptidoglycan
layer in the periplasm.
The outer membrane is then
permeabilized by acetone
treatment, and the pink safranin
counterstain is trapped by the
peptidoglycan layer.

Cell wall
• In addition to conferring rigidity upon
bacteria, the cell wall protects against
osmotic damage
• Chemically, the rigid part of the cell wall is
peptidoglycan
• First described by Gram in 1884. It is' used
to study morphologic appearance of
bacteria. Gram's stain differentiates all
bacteria into two distinct groups:
• a. Gram-positive organisms
• b. Gram-negative organisms

Bacteria with deficient cell walls
• Mycoplasma: a genus of naturally occurring
bacteria which lack cell walls
• L-forms: cell-wall-deficient forms of bacteria,
usually produced in the body of patients treated
with penicillin
• Spheroplasts: derived from Gram-negative
bacteria; produced artificially by lysozyme or by
growth with penicillin or any other agent capable
of breaking down the peptidoglycan layer
• Protoplasts: derived from Gram-positive bacteria
and totally lacking cell walls; produced artificially
by lysozyme and hypertonic medium

Electron micrograph mycoplasma
cells (1) and L-forms of Proteus
mirabilis (2)
1 2

Structures Internal to the Cell Wall
1. The Plasma (Cytoplasmic) Membrane:
▪ Thin structure inside of cell wall that surrounds the
cytoplasm.
▪ Phospholipid bilayer with proteins (Fluid mosaic model).
▪ Integral membrane proteins: Penetrate membrane
completely.
▪ Peripheral membrane proteins: On inner or outer
membrane surface.
▪ Lack sterols and are less rigid than eukaryotic
membranes.
▪ Exception: Mycoplasmas

Functions of the Plasma (Cytoplasmic) Membrane:
1. Selective barrier that regulates the passage of materials in
and out of the cell.
▪ Impermeable to large proteins, ions, and most polar molecules.
▪ Permeable to water, oxygen, carbon dioxide, some simple sugars,
and small nonpolar substances.
2. Nutrient breakdown and energy (ATP) production: Site
of cellular respiration.
3. Synthesis of cell wall components

Functions of the Plasma (Cytoplasmic)
Membrane:
4. Assists with DNA replication
5. Site of photosynthesis: Photosynthetic
bacteria have membrane extensions called
thylakoids, where photosynthesis occurs.
6. Secretes proteins
7. Contains bases of flagella
8. Responds to chemical substances in the
environment

Structure of Plasma Membrane:
outer membrane

The Cell Wall
Gram-Negative Cell Walls:
2. Outer Membrane (OM):
▪ Consists of:
▪ Phospholipid bilayer
▪ Lipopolysaccharides (LPS) with two components:
▪ O polysaccharides: Antigens, used to identify bacteria.
▪ Lipid A: Endotoxin causes fever and shock.
▪ Porins: Membrane proteins that allow the passage of nucleotides,
disaccharides, peptides, amino acids, vitamins, and iron.
▪ Lipoproteins
▪ Functions of Outer Membrane: Evade phagocytosis a complement
due to strong negative charge.
▪ Barrier to antibiotics (penicillin), digestive enzymes (lysozyme),
detergents, heavy metals, dyes, and bile salts.

Movement of Materials Across Membranes:
Passive Transport Processes:
1. Simple diffusion:
Net movement of molecules or ions from an area of
high concentration to one of low concentration.
▪ Equilibrium: Net movement stops when molecules
are evenly distributed.
▪ Used by cells to transport small molecules
(oxygen, carbon dioxide) across their membranes.
▪ Example: Diffusion of perfume into the air after the
bottle is opened.

2. Facilitated diffusion:
▪ Net movement of molecules or ions from an area of high
concentration to one of low concentration.
▪ Substance to be transported combines with a Carrier
protein in plasma membrane.
▪ Extracellular enzymes may be used to break down large
substances before they can be moved into the cell by
facilitated diffusion.

3. Osmosis:
▪ Net movement of water (solvent) molecules
across a semi permeable membrane from
an area of high concentration to one of low
concentration of water.
▪ Osmotic Pressure : Pressure required to
prevent the movement of pure water into a
solution.

Osmosis: The diffusion of water across
a semipermeable membrane
3. Osmosis (Continued):
 Bacterial cells can be subjected to three different types of osmotic
solutions:
1. Isotonic: Concentration of solutes (and water) are equal on both
sides of a cell membrane (e.g.: 0.9% NaCl, 5% glucose).
Result: No net movement of water into or out of the cell.
2. Hypotonic: Solute concentration is lower outside the cell (e.g.:
pure water).
Result: Net movement of water into the cell. Most bacteria live
in hypotonic environments. Cell wall protects them from lysis.
3. Hypertonic: Solute concentration is higher outside the cell.
Result: Net movement of water out of the cell.

Active Processes:
▪ Substances are concentrated, i.e.: moved from
an area of low concentration to one of high
concentration.
▪ Require energy expenditure (ATP) by the cell.
▪ Include the following:
1. Active transport
2. Group translocation
1. Active Transport
Requires carrier proteins or pumps in plasma
membrane.

Active Transport Processes:
2. Group Translocation
▪ Similar to active transport, but substance transported is
chemically altered during process.
▪ After modification, the substance cannot leave the cell.
▪ Glucose is phosphorylated during group translocation in
bacterial cells.
▪ Note: Endocytosis (phagocytosis, pinocytosis, etc.) does
not occur in prokaryotic cells.

Structures Internal to the Cell Wall..cont’d
3. Cytoplasm
Substance inside the cell membrane.
Contains:
❖80% water
❖Proteins
❖Carbohydrates
❖Lipids
❖Inorganic ions
❖Low molecular weight compounds

4. The Nuclear Area (Nucleoid):
– Contains a single chromosome, a long circular molecule
of double stranded DNA.
– The chromosome is attached to the plasma membrane.
– May occupy up to 20% of the intracellular volume.
– Plasmids:
• Small, circular, double stranded DNA molecules.
• Found in many bacterial cells in addition to
chromosomal DNA.
• May contain from 5 to 100 genes that are usually not
essential for survival.
• Antibiotic resistance genes
• Toxins

5. Ribosomes:
▪ The site of protein synthesis (translation).
▪ Found in all eukaryotic and prokaryotic cells.
▪ Made up of protein and ribosomal RNA (rRNA).
▪ Prokaryotic ribosomes (70S) are smaller and less dense
than eukaryotic ribosomes (80S).
▪ Prokaryotic ribosomes have two subunits:
▪ Small subunit: 30S
▪ Large subunit: 50S
▪ Several antibiotics work by inhibiting protein synthesis
by prokaryotic ribosomes, without affecting eukaryotic
ribosomes.
❖ Bacteria and archaebacteria have smaller ribosomes, termed 70S
ribosomes, which are composed of a small 30S subunit and large
50S subunit. The "S" stands for svedbergs, a unit used to measure
how fast molecules move in a centrifuge.

6. Inclusions:
• Reserve deposits in the cytoplasm of cells.
• Not found in all cell types:
• 1. Metachromatic Granules:
– Contain inorganic phosphate that can be used in
the synthesis of ATP.
– Stain red with blue dyes.
– Found in bacteria, algae, protozoa, and fungi.
– Characteristic of Corynebacterium diphtheriae,
causative agent of diphtheria. Useful for
identification purposes.

Inclusions:
2. Polysaccharide Granules:
– Contain glycogen and starch.
– Stain blue or reddish brown with iodine.
3. Lipid Inclusions:
– Contain lipids, detected with fat soluble dyes.
4. Sulfur Granules:
– Contain sulfur and sulfur containing compounds.
– “Sulfur bacteria” (Thiobacillus) obtain energy by
oxidizing sulfur and its compounds.

7. Gas Vacuoles:
• Hollow cavities found in many aquatic bacteria.
• Contain individual gas vesicles, hollow cylinders covered
by protein.
• Used to regulate buoyancy so cells can
8. Endospores:
• Specialized “resting” cells formed by certain Gram-
positive bacteria.
• Genus Bacillus
• Genus Clostridium

Endospores (continued)
• Highly durable dehydrated cells with thick
cell walls and additional layers.
• Can survive extreme temperatures,
disinfectants, acids, bases, lack of water,
toxic chemicals, and radiation.
• Endospores of some thermophilic bacteria
can survive 19
• hours of boiling. Concern in food and
health industries.

Bacterial endospores.
Phase microscopy of sporulating
bacteria demonstrates the refractility
of endospores, as well as
characteristic spore shapes and
locations within the mother cell.

Endospores (continued)
➢ A bacterial structure sometimes observed as an
inclusion is actually A type of dormant cell called
an endospore.
➢ Endospores are formed by a few groups of
Bacteria as intracellular structures, but
ultimately they are released as free endospores.
Endospores exhibit no signs of life, being
described as cryptobiotic.
➢ They are highly resistant to environmental
stresses such as high temperature (some
endospores can be boiled for hours and retain
their viability), irradiation, strong acids,
disinfectants, etc. They are probably the most
durable cell produced in nature.

➢Although cryptobiotic, they retain viability
indefinitely such that under appropriate
environmental conditions, they germinate
back into vegetative cells.
➢Endospores are formed by vegetative cells in
response to environmental signals that
indicate a limiting factor for vegetative
growth, such as exhaustion of an essential
nutrient. They germinate and become
vegetative cells when the environmental stress
is relieved.
➢Hence, endospore-formation is a mechanism
of survival rather than a mechanism of
reproduction.

• Process of Sporulation: One cell produces one spore.
1. Newly replicated DNA is isolated by an in growth of the
plasma membrane called a spore septum.
2. Spore septum becomes a double-layered membrane that
surrounds chromosome and cytoplasm (forespore).
3. Peptidoglycan layer forms between membranes of
forespore.
4. Spore coat forms: Thick layer of protein around the outer
membrane. Makes endospore resistant to many harsh
chemicals.
5. Maturation: Cell wall ruptures, endospore is released.

Sporulation
 May be part of normal life cycle or triggered by adverse environmental
conditions.
 Endospores do not carry out metabolic reactions, unlike normal vegetative
cells.
 Endospores can remain dormant for thousands of years.
 Germination: Endospore returns to its vegetative state.
 Usually occurs when environmental conditions become more favorable.
 Triggered by physical or chemical damage to the spore coat.
Sporulation Germination
 Vegetative Cell ----------> Endospore ------------> Vegetative Cell
(Metabolically active), (Not metabolically active), (Metabolically active)

SPORE
Bacillus megaterium
Bacillus anthracis

Microbiological nomenclature
• In microbiology the binominal system of
nomenclature is accepted where each
species has a generic and a specific
name. The generic name is written with a
capital letter, and the specific name – with
a small letter. For example: the anthrax
bacillus – Bacillus anthracis; the tetanus
bacillus – Clostridium tetani.

The size of bacteria
• The size of bacteria is measured in
micrometer (m) or micron () (1 micron or
micrometer is one thousandth of a
millimeter) and varies from 0.1  to 16-18 .
Most pathogenic bacteria measure from 0.1
to 10 .
• The other units of measurement of
microorganisms are millimicron (m) or
nanometer (nm) (one millionth of a
millimeter) and 1 Angstrom (Å) (one tenth of
nanometer).

Morphology of Bacteria
• Bacteria are intracellular free-living
organisms having both DNA and RNA. Their
biological properties and predominant
reproduction by binary fission relates them to
prokaryotes.
• Spherical (cocci)
• Rod-shaped
(bacteria, bacilli,
and clostridia)
• Spiral-shaped
(vibriones, spirilla, spirochaetes)

Representatives of pathogenic
cocci
1.Scanning Electron Micrograph of Streptococcus pneumoniae
2.Scanning electron micrograph of a Staphylococcus aureus
1 2

Electron Micrograph of
Neisseria gonorrhoeae

Rod-shaped bacteria
• Bacteria (1) include those
microorganisms, which, as rule,
do not produce spores (E.coli,
Salmonella, Shigella).
• Bacilli (2) (B.anthracis) and
clostridia (3) (C.tetani, C.botuli-
num) include organisms the
majority of which produce spores.
• Size of rod-shaped bacteria varies
2-10 μm: small rods are 2-4 μm;
long rods are 5-10 μm.
1
2
3

ARRANGEMENT OF ROD-SHAPED
BACTERIA

Rod-shaped bacteria
1. Single Rod
2. Streptobacillus
1 2

SPIRAL FORMS
• 1. Vibrios – are cells, which resemble a
comma in appearance (curved rods).
Typical representative of this group is
Vibrio cholerae.

2. Spirilla – are coiled forms of bacteria.
Pathogenic species: Spirillum minus (1) – which
is responsible for a disease in humans
transmitted through the bite of rats – rat-bite fever
– sodoku; Helicobacter pylori (2) – causative
agent of ulcer disease of stomach.
1 2

3. Spirochaetes – are flexuous spiral forms which
include: Treponema (T.pallidum) (1), Borrelia
(B.recurrentis) (2), Leptospira (L.interrogans) (3)
1
2
3

SPIROCHAETES
• Treponema – exhibits, thin, flexible cells
with 6-14 regular twists. The size of
Treponema varies from 10-18 μ (T.pallidum).
• Leptospira – are characterized by very thin
cell structure. The leptospirae form 12-18
regular coils (primary spirals)
(L.interrogans) and C- or S- shape according
secondary twist.
• Borrelia – have large irregular spirals, the
number of which varies from 3 to 10.
(B.recurrentis, B.persica).

Morphology of viruses
• Do not possess cellular
organization
• Contain one type of nucleic acid
either RNA or DNA
• Lack enzymes necessary for
protein and nucleic acid
synthesis machinery of host
cells
• They multiply by complex
process and not by binary
fission.
• They are unaffected by
antibiotics.
• They are sensitive to interferon.

Morphology of Rickettsiae.
• They are minute organisms having properties in between
bacteria and viruses.
• It contains both DNA and RNA.
• Contains enzymes for metabolic functions.
• Multiplies by binary fission.
• It is coccobacilli 300x600 nm in size, non-motile, non-
capsulated and is Gram-negative.
• Sensitive to many antibiotics.
• Can multiply only inside living cells.

General characteristics
1. They are small, non–motile, gram negative, pleomorphic coccobacilli.
2. They are obligate intracellular parasites and can be cultured only on
guinea pigs or mice, chick embryo or tissue culture with an exception of
Rochalimea Quintana which can be grown on media containing high
concentration of hemin and serum
3. They multiply in the intestinal tract of the anthropod then excreted in the
feces or in salivary gland.
4. Rickettsial growths are enhanced by sulfonamides but para–
aminobenzoic acid (PABA) inhibits their growth.

Morphology of chlamydia.
• Chlamydiae are Gram-negative.
• They lack some important mechanisms for the
production of metabolic energy, so they are
intracellular parasites.
• Chlamydiae exist as two stages: (1) infectious
particles called elementary bodies and (2)
intracytoplasmic, reproductive forms called
reticulate bodies.
• There are 2 morphological forms of chlamydia:
• Elementary bodies
• Reticulate bodies

Developmental cycle
• The “elementary body” (the infectious particle) is so
small (0.2 to 0.4 nm) in diameter which rivals with
Mycoplasma for the smallest prokaryote.
• This EB through phagocytosis is taken by the host cell.
• This small particle is recognized into reticulate body.
• This body grows in size and divides repeatedly by binary
fission. Eventually from reticulated body, it forms an
“inclusion” in the host cell cytoplasm.
• The newly formed particle may be liberated from the host
cells to infect new cells.

Microbiology Books
1. Marjorie Kelly Cowan & Heidi Smith Microbiology
Fundamentals: A Clinical Approach: 2022 (4th Edition)
McGraw-Hill. ISBN No: 978-1260-7-0243-9
2. Jawetz, Melnick, & Adelberg (2019). Medical Microbiology
(28th Edition). McGraw-Hill. ISBN No: 978-1260-0-1202-6.
3. Warren Levinson (2014). Review of Medical Microbiology
and Immunology (13th Edition). McGraw-Hill. ISBN No: 978-
0071-8-1811-7.
4. Lansing M. (2002). Prescott Microbiology (5th Edition).
McGraw-Hill. ISBN No: 978-0072-8-2905-1
5. Tortora, Funke, & Case (2018). Microbiology – An
introduction (13th Edition). Pearson. ISBN No: 978-0134-6-
0518-0

Lecture 1. Introduction to Medical Microbiology.pdf

Lecture 1. Introduction to Medical Microbiology.pdf

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