Arsi University

College of Agriculture and Environmental Science

Department of Veterinary Science

Veterinary Microbiology

By;
Minda A (DVM, MSc)

June, 2022
 Unit 1: History of Microbiology
A. The microscope
I. Zacharias Janssen
 Developed the first compound microscope in Middleburg, Holland(1590)
 Janssen’s microscope consisted of three tubes
 One tube served as the outer casing and contained the other two tubes
 At either ends of the inner tubes were lenses used for magnification
 Janssen’s design enabled scientists to enlarge the image of a specimen 3x and 9x
times the specimen’s actual size

II. Robert Hooke

 In 1665, Robert Hooke, an English scientist, popularized the use of the
compound microscope when he placed lenses over slices of cork and viewed
little boxes that he called cells
 It was his discovery that led to the development of cell theory in the 19 th C by
Mathias Schleiden
 Theoder Schwann, and Rudolf Virchow, cell theory states that all living
things are composed of cells
III. Antony van Leeuwenhoek
 Hooke’s experiments with a crude microscope inspired Antony van
Leenwenhoek to further explore the micro world
 Van Leeuwenhock, an amateur lens grinder, improved Hooke’s microscope
by grinding lenses to achieve magnification
 His microscope required one lens
 With his improvement, van Leeuwenhock became the first person to view
living microorganisms, which he called Animalcules
 This discovery took place during the 1600s, when scientists believed that
organisms generated spontaneously and did not come from another organism
 This sounds preposterous today; however, back then scientists were just
learning that a cell was the basic component of an organism
 B. Origin of Organisms

I. Francesco Redi
 Italian physician Francesco Redi (1668) developed an experiment that
demonstrated that an organism did not spontaneously appear
 He filled jars with rotting meat
 Some jars he sealed and other he left opened
 Those that were open eventually contained maggots, which is the larval stage of
the fly
 The other jars did not contain maggots because flies could not enter the jar to lay
eggs on the rotting meat
 His critics stated that air was the ingredient required for spontaneous
generation of an organism
 Air was absent from the sealed jar and therefore, no spontaneous generation
could occur, they said Redi repeated the experiment except this time he placed a
screen over the opened jars
 This prevented flies from entering the jar
 There weren’t any maggots on the rotting meat
 Until that time scientists did not have a clue about how to fight disease

 However, Redi’s discovery gave scientists an idea

 They used Redi’s findings to conclude that killing the microorganisms that
caused a disease could prevent the disease from occurring

 A new microorganism could only be generated by another microorganism when it

underwent a reproductive process

 Kill that microorganism and you will not have new microorganisms, the theory
went – you could stop the spread of the disease

 Scientists called this the” theory of biogenesis”

 The theory of biogenesis states that a living cell is generated from another living
cell
 II. Louis Pasteur
 Although the theory of biogenesis disproved spontaneous generation, spontaneous generation was hotly
debated among the scientific community
 Louis Pasteur(1861), a French scientist, resolved the issue once and for all
 Pasteur showed that microorganisms were in the air

 He proved that sterilized medical instruments became contaminated once they were exposed to the air
 Pasteur came to this conclusion by boiling beef broth in several short-necked flasks

 Some flasks were left open to cool

 Other flasks were sealed after boiling

 The opened flasks became contaminated with microorganisms while no microorganisms appeared in the
closed flasks

 Pasteur concluded that airborne microorganisms had contaminated the opened flaks

 In a follow-up experiment, Pasteur placed beef broth in an open long-necked flask

 The neck was bent into an S-shape

 Again he boiled the beef broth and let it cool

 The S-shaped neck trapped the airborne microorganisms

 The beef broth remained uncontaminated even after months of being exposed to the air

 The very same flask containing the original beef broth exists today in Pasteur Institute in
Paris!!! and still shows no sign of contamination

 Pasteur’s experiments validated that microorganisms are not spontaneously generated

 Based on Pasteur’s findings, concerned effort was launched to improve sterilization

techniques to prevent microorganisms from reproducing

 Pasteurization, one of the best-known sterilization techniques, was developed and named for
Pasteur

 Pasteurization kills harmful microorganisms in milk, alcoholic beverages, and other foods
and drinks by heating it enough to kill most bacteria that cause spoilage
 III. John Tyndall and Ferdinand Cohn
 The work of John Tyndall and Ferdinand Cohn in the late 1800s led to one of the
most important discoveries in sterilization
 They learned that some microorganisms are resistant to certain sterilization
techniques
 Until their discovery, scientists had assumed that no microorganisms could
survive boiling water, which became a widely accepted method of sterilization
 This was wrong!!!
 Some thermophiles resisted heat and could survive a bath in boiling water
 This means that there was not one magic bullet that killed all harmfull
microorganisms
 C. Germ theory
 Until the late 1700s, not much was really known about diseases except their impact
 It seemed that anyone who came in contact with an infected person contracted the
disease
 A disease that is spread by being exposed to infection is called a contagious disease
 The unknown agent that causes the disease is called a contagion
 Today we known that a contagion is a microorganisms, but in the 1700s many found it
hard to believe something so small could cause such devastation
I. Robert Koch
 Koch made some observations on the disease caused by Bacillus anthracis called
anthrax
 Koch developed the ”germ theory” that states “disease-causing microorganisms
should be present in animals infected by the disease and not in healthy animals”
 The microorganisms can be cultivated away from the animal and used to inoculate a
healthy animal
 The healthy animal should then come down with the disease
 Samples of a microorganism taken from several infected animals are the same as the
original microorganism from the first infected animals
 Four steps used by Koch to study microorganisms are referred to as “Koch’s
Postulates”
1. The microorganism must be present in the diseased animals and not presence in
the healthy animal
2. Cultivate the microorganism away from the animal in a pure culture
3. Symptoms of the disease should appear in the healthy animal after the healthy
animal is inoculated with the culture of the microorganisms
4. Isolate the microorganism from the newly infected animal and culture it in the
laboratory
 The new culture should be the same as the microorganism that was cultivated
from the original diseased animal

 Koch‘s work with anthrax also developed techniques for growing a culture of
microorganisms
 He eventually used a gelatin surface to cultivate microorganisms
 Gelatin inhibited the movement of microorganisms

 As microorganisms reproduced, they remained together, forming a colony that

made them visible without a microscope

 The reproduction of microorganisms is called colonizing

 The gelatin was replaced with agar that is derived from seaweed and still used
today
 Richard Petri improved on Koch’s cultivating technique by placing the agar in a
specially designed disk that was later called the Petri dish which is still used
today
 D. Vaccination
 The Variola virus was one of the most feared villains in the late 1700s
 The variola virus causes smallpox
 If variola didn’t kill, it caused pus-filled blisters that left deep scars that pitted
nearly every part of your body
 Cows were also susceptible to a variation of variola called cowpox
 Milk maids who tended to infected cows contracted cowpox and exhibited
immunity to the smallpox virus
I. Edward Jenner
 Edward Jenner, an English physician, discovered something very interesting
about both smallpox and cowpox in 1796

 Those who survived smallpox never contracted smallpox again, even when they
were later exposed to someone who was infected with smallpox

 Milk maids who contracted cowpox never caught smallpox even though they
were exposed to smallpox
 Jenner had an idea
 He took scrapings from a cowpox blister found on a milkmaid and, using a
needle scratched the scrapping into the arm of James Phipps, an 8-year-old
 Phipps became slightly ill when the scratch turned bumpy
 Phipps recovered and was then exposed to smallpox
 He did not contract smallpox because his immune system developed antibodies
that could fight off variola
 Jenner’s experiment discovered how to use our body’s own defense mechanism to
prevent disease by inoculating healthy persons with a tiny amount of the disease-
causing microorganism
 Janner called this a vaccination, which is an extension of the Latin word vacca
(cow)
 The person who received the vaccination became immune to the disease-causing
microorganism
 Edward Jenner
 Also known as the “Father of Immunology”, Edward Anthony Jenner was an
English scientist and is famous for his discovery of smallpox vaccine
 II. Elie Metchnikoff
 Elie Metchnikoff, a 19th century Russian zoologist, was interested by Jenner’s
work with vaccinations
 Metchnikoff wanted to learn how our bodies react to vaccination by exploring
our body’s immune system
 He discovered that white blood cells (leukocytes) engulf and digest
microorganisms that invade the body
 He called these cells phagocytes, which means “cell eating”
 “Metchnikoff was one of the first scientists to study the new area of biology
called immunology, the study of the immune system
 E. Killing the Microorganism

I. Ignaz Semmelweis
 Great studies were made during the late 1800s in the development of antiseptic
techniques
 It began with a report by Hungarian physician Ignaz Semmelweis on a dramatic
decline in childbirth fever when physicians used antiseptic techniques when
delivering babies
 Infections become preventable through the use of antiseptic techniques

II. Joseph Lister

 Joseph Lister, an English surgeon, developed one of the most notable antiseptic
techniques
 During surgery he sprayed carbolic acid over the patient and then bandged the
patient’s wound with carbolic acid-soaked bandages
 Infection following surgery dramatically dropped when compared with surgery
performed without spraying carbolic acid
 Carbolic acid, also known as phenol was one of the first surgical antiseptic
 III. Paul Ehrlich
 Antiseptics prevented microorganisms from infecting a person, but scientists still
needed a way to kill microorganisms after they infected the body
 Scientists needed a magic bullet that cured diseases
 At the turned off the 19th C, Paul Ehrlich, a German chemist, discovered the magic
bullet
 Ehrlich blended chemical elements into a concoction that, when inserted into an
infected area, killed microorganisms without affecting the patient
 Today we call Ehrlich’s concoction a drug
 Ehrlic’s innovation has led to chemotherapy using drugs that are produced by
chemical synthesis

IV. Alexander Fleming

 Scientists from all over set out to use Ehrlich’s findings to find drugs that could make
infected patients well again

 One of the most striking breakthroughs came in 1929 when Alexander Fleming
discovered Penicillin notatum, the organism that synthesizes penicillin
 Penicillium notatum is a fungus that kills the Staphylococcus aureus
microorganism and similar microorganisms

 Fleming grew cultures of Staphyloccus aureus, a bacterium, in the laboratory

 He was also conducting experiments with Penicillium notatum, a mold

 Accidently Staphyloccous aureus was contaminated with Penicillium notatum,

causing the Staphyloccocus to stop reproducing and die

 Penicillium notatum became one of the first antibiotic

 An antibiotic is a substance that kills bacteria

 Unit 2: General Bacteriology

2.1. The Morphology and Structure of Bacteria, Fungi and Viruses

 Eukaryotic Vs Prokaryotic Cell Structure

A. The major features of eukaryotic cells. The DNA of an eukaryotic cell is

located
within the nucleus.
 Overview of Prokaryotic Cell Structure
Size, Shape, and Arrangement
 There is a remarkable amount of variation due to differences in genetics and
ecology

(a) Staphylococcus aureus.Gram stain (1,000). (b) Enterococcus faecalis. Note the
chains of cocci
 Most commonly encountered bacteria have one of two shapes:
 Cocci (s., coccus) - roughly spherical cells
 They can exist as individual cells
 Characteristic arrangements - are frequently useful in bacterial identification
 Diplococci (s., diplococcus) - arise when cocci divide and remain together to form
pairs
- Long chains of cocci - result when cells adhere after repeated divisions in one
Plane
Genera - Streptococcus, Enterococcus, and Lactococcus

- Staphylococcus divides in random planes to generate irregular grapelike clumps

 Divisions in two or three planes can produce symmetrical clusters of cocci

o Bacilli differ considerably in their length-to width ratio

o Coccobacilli - so short and wide that they resemble cocci

o The shape of the rod’s end often varies between species: may be flat, rounded, cigar-
shaped, or bifurcated
 Diversity in bacterial shape
 is often the result of life cycles that represented survival strategies in
addition to
those of maximizing growth rates
 The shape of a cell is a strategic consequence of this adaptation
 The shape of a cell is not limited in any fundamental way to a rod, coccus or
spiral
 The variety of cell shapes and groupings far exceeds those offered in early
views as rod, coccus or spiral
 There are bacteria that are amorphous, ovoid, square, stellate, filamentous
or
stalked
 They may be grouped as pairs, clumps, chains, rosettes, cuboid packets, flat
squares, networks, mycelia or fruiting bodies
→ Each type of cell morphology is uniquely appropriate for the ecological
niche of that cell
 The abundance of coccoid and rod-shaped bacteria suggests that both are very
effective growth forms and that they probably are simpler than other shapes to
generate
 Moreover, it can be argued that one or the other might have been the first
member of the Domain of Bacteria
 It is argued that, probably, the first bacterium was a Gram-positive bacillus
Diversity in Cell Size
 The conventional wisdom that prokaryotic cell diameters are typically near 1μm,
whereas eukaryotic cells are 10 μm or larger is generally true
 significant exceptions exist
 Members of the Bacteria can show enormous diversity in cell size
 Dehalococcoides ethenogenes
- a diameter of 0.4–0.5 μm and a height of 0.1–0.2 μm so that its volume is near
0.024μm3
- about one twentieth that of E. coli
 Epulopiscium fishelsonii
- found in the gastrointestinal tracts of certain fish
- a diameter as great as 80 μm and lengths up to 600 μm
 The volume of these cells is near 2 million μm3
 Bacteria vary in size as much as in shape
o The smallest are about 0.3 μm in diameter
e.g., some members of the genus Mycoplasma
- approximately the size of the largest viruses (the pox viruses)

 Nanobacteria or ultramicrobacteria appear to range from around 0.2 μm to

less than 0.05 μm in diameter
 An advantage of existing as micrometer-sized cells

- a greater surface-to-volume ratio

For example: compare spherical cells with 1μm vs 10μm diameters

it requires 1,000 of the smaller cells (ca. 0.5 μm3) to equal the larger cell (ca.500
μm3) in volume

 Moreover, the smaller cells have 10 times greater total surface area (ca.
3,140 μm2) than the larger one (ca.314μm2)

→ allowing them to present more surface to the environment for uptake of

scarce nutrients

 The smaller size apparently avoids the need for the organellar
compartmentation and internal membrane systems generally found in
eukaryotes because molecules can traverse the distances in the prokaryotic cell
more easily by diffusion
 Fine/ Ultrasructure of Bacteria
 The terms “fine structure” and “ultrastructure” refer to subcellular features that
are best observed using the electron microscope

 Scientists use a combination of procedures

- Lysis of bacterial cells

- Centrifugation to separate the various subcellular components
- Purification and biochemical analysis

 The electron microscope was used at various steps in the procedure to identify
and assess the purity of the structures
 Internal Structures
DNA
 The DNA of prokaryotes is a circular, or more rarely a linear, double-stranded
helical molecule
 The DNA appears as a fibrous material in the cytoplasm when prokaryotic cells
are viewed in thin sections
 The DNA of prokaryotes does not appear in a confined area within the cell
rather, it appears as a somewhat diffuse, dispersed fibrous material
 The region is termed as a nucleoid or nuclear area
 The typical prokaryotic DNA contains about 4 × 106 base pairs (4 mega-base
pairs, or 4 mgb)
 The DNA is many times the length of the cell and highly folded and compacted
-Ex. Escherichia coli
 Cell size: around 3–4 μm in length but contains a DNA molecule some 1400 μm
in length!
 The DNA may be associated with certain bacterial proteins, but these are not
the same as the histones found in eukaryotic chromosomes
 The cell envelope/ Cell Surface
 The interface between a bacterial cell and its surroundings is where the cell will first
have to deal with a variable environment
 It is thus the part of the bacterial cell where one might expect to find the greatest
diversity
 It is the cell surface that
- determines the nature of attachment to a substrate or to another cell
- determines what gets into and out of the cell and the nature of that transport process
 Cell Envelope : Cell wall and Cell membrane

The Cell Wall

o It is one of the most important parts of a prokaryotic cell
- Most bacteria have strong walls that give them shape and protect them from
osmotic lysis
* Exception: mycoplasmas and some Archea
 Wall shape and strength is primarily due to peptidoglycan
 Since the cytoplasm of bacteria contains high concentrations of dissolved
substances, they generally live in a hypotonic environment
i.e. one that is more dilute than their own cytoplasm
→ There is a natural tendency for water to flow into the cell, and without the cell
wall the cell would fill and burst
 you can demonstrate this by using enzymes to strip off the cell wall, leaving the
naked protoplast
 A protoplast - a cell that has had its cell wall removed
 The cell walls of many pathogens have components that contribute to their
pathogenicity
- The wall can protect a cell from toxic substances
- It is the site of action of several antibiotics
 Bacteria without cell walls survive because their cell membranes differ from
those of typical bacteria
 For example, their membranes contain sterols or other compounds that help
stabilize membrane structure in this respect they are similar to animal cell
membranes, which also lack cell walls
 Prokaryotes are classified into two groups: Archaea or Bacteria
- based on evolutionary studies of 16S rRNA
 In addition, the two domains also differ in the chemistry of their cell walls
 Almost all Bacteria have peptidoglycan, or murein in their cell walls
 Archaea do not
Bacterial Cell Wall
 Bacteria could be divided into two major groups:
-based on their response to the Gram-stain procedure
o Gram-positive bacteria – stain purple
o Gram-negative bacteria - stain pink or red
 The true structural difference between these two groups became clear with the
advent of the transmission electron microscope
 Gram-positive - cell wall consists of a single 20 to 80 nm thick homogeneous
peptidoglycan or murein layer
* lies outside the plasma membrane
 Gram-negative - cell wall is quite complex
 It has a 2 to 7 nm peptidoglycan layer
-surrounded by a 7 to 8 nm thick outer membrane
 Because of the thicker peptidoglycan layer, the walls of gram-positive cells are
stronger than those of gram-negative bacteria
Gram-Positive and Gram-Negative Cell Walls. The gram-positive envelope is
from
Bacillus licheniformis (left), and the gram-negative micrograph is of
Aquaspirillum
serpens (right). M; peptidoglycan or murein layer; OM, outer membrane; PM,
plasma membrane; P, periplasmic space; W, gram-positive peptidoglycan wall.
Periplasmic space
 A space seen between the plasma membrane and the outer membrane in
electron micrographs of gram negative bacteria
 Size of the periplasmic space in gram-negative bacteria: from 1 nm to 71 nm
 It may constitute about 20 to 40% of the total cell volume (around 30 to 70 nm)
 A similar but smaller gap may be observed between the plasma membrane and
wall in gram-positive bacteria
 Periplasm - the substance that occupies the periplasmic space

 This area, though small is important to the physiology of the cell

- The periplasm - an area of considerable enzymatic activity

- Several steps in the synthesis of the cell wall occur in this area
- Chemoreceptors involved in the chemotactic response are also located here

- The binding proteins produced by gram-negative bacteria are located in the

periplasm
 These binding proteins
- combine reversibly with substrate molecules,
- concentrate them,
- then release them to the membrane carrier proteins for transport into the
cytoplasm
 Periplasmic enzymes and other proteins – released when cell walls are disrupted
carefully or removed without disturbing the underlying plasma membrane
- may be easily studied
 The periplasmic space of gram-negative bacteria contains
- many proteins - that participate in nutrient acquisition
- hydrolytic enzymes attacking nucleic acids and phosphorylated
molecules
- binding proteins involved in transport of materials into the cell
- enzymes - involved in peptidoglycan synthesis and
- the modification of toxic compounds that could harm the cell
 Gram positive bacteria may not have a visible periplasmic space and do not
appear to have as many periplasmic proteins
 Rather, they secrete several enzymes (exoenzymes) that ordinarily would be
periplasmic in gram-negative bacteria

Peptidoglycan structure
 Peptidoglycan or murein - is an enormous polymer composed of many
identical subunits
 The polymer contains two sugar derivatives:
- N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) and
several different amino acids
 Three of the amino acids —D-glutamic acid, D-alanine, and mesodiaminopimelic
acid (DAP) —are not found in proteins
 The presence of D-amino acids – protects against attack by most peptidases
 DAP is a rare amino acid, only found in the cell walls of prokaryotes
 This is contrary to the situation in proteins and confers protection against
proteases specifically directed against L-amino acids
 The backbone of this polymer is composed of alternating N-
acetylglucosamine and N-acetylmuramic acid residues
 A peptide chain of four alternating D- and L-amino acids is connected to the
carboxyl group of N-acetylmuramic acid
 Many bacteria substitute another diaminoacid, usually L-lysine, in the third
position for meso-diaminopimelic acid
 Chains of linked peptidoglycan subunits are joined by cross links between the
peptides
 Often the carboxyl group of the terminal D-alanine is connected directly to the
amino group of diaminopimelic acid but a peptide interbridge may be used
instead
 Most gram-negative cell wall peptidoglycan lacks the peptide interbridge
 This cross-linking results in an enormous peptidoglycan sac that is actually one
dense, interconnected network
 These sacs have been isolated from gram-positive bacteria and are strong
enough to retain their shape and integrity, yet they are elastic and somewhat
stretchable
 They also must be porous, as molecules can penetrate them
 Gram Positive Cell Wall
 Normally the thick, homogeneous cell wall of gram-positive bacteria is
composed primarily of peptidoglycan
- which often contains a peptide interbridge
 In addition, gram-positive cell walls usually contain large amounts of teichoic
acids
- polymers of glycerol or ribitol joined by phosphate groups
 Amino acids such as D-alanine or sugars like glucose are attached to the
glycerol and ribitol groups
 The teichoic acids are connected to either: the peptidoglycan itself or plasma
membrane lipids; → lipoteichoic acids
 Teichoic acids - appear to extend to the surface of the peptidoglycan
 They are negatively charged
- help give the gram-positive cell wall its negative charge
 The functions of these molecules are still unclear
- they may be important in maintaining the structure of the wall
- Teichoic acids are not present in gram-negative bacteria
 Teichoic Acid Structure. The segment
The Gram-Positive Envelope of a teichoic acid made of phosphate,
glycerol, and a side chain, R. R may
represent D-alanine, glucose, or other
molecules.
 Gram Negative Cell Walls
 Gram-negative cell walls are much more complex than gram-positive walls
 The thin peptidoglycan layer next to the plasma membrane may constitute not
more than 5 to 10% of the wall weight
 In E. coli it is about 2 nm thick
-Contains only one or two layers or sheets of peptidoglycan
 The outer membrane - lies outside the thin peptidoglycan layer
 Appears similar to a cell membrane when viewed by electron microscopy
-Lipids and proteins predominate
 Polysaccharides extend into the aqueous environment
 Lipoprotein - the most abundant membrane protein
 Lipopolysaccharides (LPSs) - the most unusual constituents of the outer
membrane
 Consist of three parts:
(1) lipid A
(2) the core polysaccharide
(3) the O side chain
 The lipid A region contains two glucosamine sugar derivatives, each with
three fatty acids and phosphate or pyrophosphate attached
 It is buried in the outer membrane and the remainder of the LPS molecule
projects from the surface
 The core polysaccharide is joined to lipid A
o Somatic polysaccharides or O-polysaccharides - specific side-chain
polysaccharides
- Vary from one species of gram negative bacterium to another
 The LPS is important for several reasons other than the avoidance of host
defenses

 The core polysaccharide usually contains charged sugars and phosphate

- LPS contributes to the negative charge on the bacterial surface

 Lipid A is a major constituent of the outer membrane, and the LPS helps
stabilize membrane structure

 Furthermore, lipid A often is toxic; as a result the LPS can act as an endotoxin
- cause some of the symptoms that arise in gram-negative bacterial infections
 The Mechanism of Gram Staining
 1884 : Christian Gram - Danish
- devised a differential stain based on the ability of certain bacterial cells to retain
the dye crystal violet after decoloration with 95% ethanol
 The difference between gram-positive and gram-negative bacteria may be due to
the physical nature of their cell walls
 If the cell wall is removed from gram positive bacteria, they become gram
negative
 The peptidoglycan itself is not stained; instead it seems to act as a permeability
barrier preventing loss of crystal violet
Gram staining procedure
 The bacteria are first stained with crystal violet and next treated with iodine to
promote dye retention
 When gram-positive bacteria then are decolorized with ethanol, the alcohol is
thought to shrink the pores of the thick peptidoglycan
 Thus the dye-iodine complex is retained during the short decolorization step and
the bacteria remain purple
 In contrast, gram-negative peptidoglycan is very thin, not as highly cross-linked,
and has larger pores
 Alcohol treatment also may extract enough lipid from the gram negative wall
- increases its porosity further
 For these reasons, alcohol more readily removes the purple crystal violet-iodine
complex from gram-negative bacteria
Archaeal Cell Wall(Jumped)
 The domain Archaea consists of three major phyla:
o Crenarchaeota - contains the hyper-thermophilic organisms
o Euryarchaeota:
-the methane-producing group (methanogens) and
-the extreme halophilic group (salt-loving bacteria)
o Korarchaeota - much less well known because isolates have not yet been
obtained
 Some methanogens - have proteinaceous cell walls

 Others have cell walls with a material similar to peptidoglycan - pseudopeptidoglycan or

pseudomurein

 The amino sugar chain of the pseudomurein contains

 N-acetylglucosamine, but N-acetyltalosaminouronic acid replaces the N-acetylmuramic

acid found in bacteria

- The two amino sugars are linked in a 1,3 rather than a 1,4 configuration, and

-The amino acids of the peptide cross-links are not D stereoisomers

 The extreme halophilic and hyper-thermophilic archaea

- have glycopeptide cell walls containing a variety of sugars linked covalently to protein
 The differences in cell wall composition may explain how these species are able to
survive in their special environments
 Antibiotics and Cell Wall
 Some antibiotics exert their effect against bacteria by preventing the synthesis of
normal cell walls

 Penicillin and cephalosporin groups

- act by inhibiting the enzymes responsible for biosynthesis of peptidoglycan

 Bacteria exposed to these antibiotics produce a defective cell wall

- results in the eventual lysis, and destruction, of the bacterial cell

 Penicillin causes lysis and death of bacteria only under conditions that permit growth
and cell wall synthesis

o Thus, when antibiotics are used therapeutically, bacterial cells that are not
growing and dividing can survive and may begin growing following the antibiotic
treatment
o These cells must be eliminated by the host’s own defense activities, such as by
phagocytes
 The effect of antibiotics on the cell wall is different from that of lysozyme

 Lysozyme, as an enzyme, can continually degrade the peptidoglycan in cell

walls

 Penicillin is active only on growing cells, because it affects only the synthesis of
peptidoglycan

 Treatment of growing bacteria with penicillin in isotonic solution results in the

formation of protoplasts and spheroplasts in much the same manner as lysozyme
treatment

 The antibiotics are selectively toxic to prokaryotic organisms because

peptidoglycan is found only in Bacteria

 Thus, animals treated with these antibiotics are not affected, because animal
cells do not synthesize peptidoglycan
 Penicillin is generally much more effective against gram-positive bacteria than against
gram-negative bacteria
- the major structural component of gram-positive cell walls is peptidoglycan,
- the outer membrane of gram-negative bacteria provides a permeability barrier to antibiotics
such as penicillin G
 For use against gram negative bacteria, penicillin and other antibiotics can be modified to
permit better solubility in the lipid outer membranes
 Ampicillin, for example, is a penicillin with greater hydrophobicity that is used for gram-
negative infections
 A most important outer membrane function is to serve as a protective barrier
 It prevents or slows the entry of bile salts, antibiotics, and other toxic substances that might
kill or injure the bacterium

 Even so, the outer membrane is more permeable than the plasma membrane and permits
the passage of small molecules like glucose and other monosaccharides
 This is due to the presence of special porin proteins

 Larger molecules - vitamin B12

- must be transported across the outer membrane by specific carriers

- The outer membrane also prevents the loss of constituents like periplasmic enzymes
 Components External to the Cell Wall
 Bacteria have a variety of structures outside the cell wall that can function in
- Protection
- Attachment to objects
- Cell movement
 Capsule, slime Layers, and Surface(S) Layers
 Capsule - Some bacteria have a layer of well organized material lying outside the
cell wall
- not easily washed off
 A slime layer - a zone of diffuse, unorganized material that is removed easily
 A glycocalyx – a network of polysaccharides extending from the surface of
bacteria and other cells
 Capsules and slime layers usually are composed of polysaccharides, but they may
be constructed of other materials
 For example, Bacillus anthracis has a capsule of poly-D-glutamic acid

o Capsules are not required for bacterial growth and reproduction in laboratory
cultures

 they do confer several advantages when bacteria grow in their normal habitats
Capsule:-
i. They help bacteria resist phagocytosis by host phagocytic cells
- When it lacks a capsule, it is destroyed easily and does not cause disease,
whereas the capsulated variant quickly kills mice

ii. Capsules contain a great deal of water and can protect bacteria against
desiccation

iii. They exclude bacterial viruses and most hydrophobic toxic materials such as
detergents

 The glycocalyx - aids bacterial attachment to surfaces of solid objects in

aquatic environments or to tissue surfaces in plant and animal hosts
 Surface Layers(S-Layers)
 Many gram-positive and gram-negative bacteria have a regularly structured layer
called an S-layer on their surface

- The S layer has a pattern something like floor tiles

 They consist of one species of (glyco) protein - the S-protein, which assembles
into characteristic two-dimensional crystalline layers (lattices) at the cell surface

 The lattices can be quite porous, with pores occupying up to 70% of their surface

 S-layer proteins are among the most abundant cellular proteins - constitute
between 5-10% of the total protein content of the cell
Roles:
- It is associated with the peptidoglycan surface in gram-positive bacteria
- It may protect the cell against ion and pH fluctuations, osmotic stress, enzymes
- The S-layer also helps maintain the shape and envelope rigidity of at least some
bacterial cells

- It can promote cell adhesion to surfaces

- It functions as virulence factor and as molecular and ion traps

- The layer seems to protect some pathogens against complement attack and
phagocytosis, thus contributing to their virulence
 Pili and Fimbriae
 Fimbriae (s., fimbria) - short, fine, hairlike appendages that are thinner than
flagella and not involved in motility
 A cell may be covered with up to 1,000 fimbriae
 they are only visible in an electron microscope due to their small size
 They seem to be slender tubes composed of helically arranged protein subunits
- are about 3 to 10 nm in diameter and up to several micrometers long
 Some types of fimbriae attach bacteria to solid surfaces such as rocks in
streams and host tissues
Pili (s., pilus)
 About 1 to 10 per cell
 differ from fimbriae in the following ways:
- Pili often are larger than fimbriae (around 9 to 10 nm in diameter)
- They are genetically determined by sex factors or conjugative plasmids and are
required for bacterial mating – sex pilli
- Some bacterial viruses attach specifically to receptors on sex pili at the start of
their reproductive cycle
 A single bacterial cell may produce more than one type of fimbria or pilus

- some bacterial viruses attach to one type of fimbria or pilus

- but not to the cell surface or to other types of fimbriae or pili produced by the
same bacterial species
Importance:
o Pili are known to be important for the attachment of some bacteria
For example
-“sex pilus”- one type of pilus of Escherichia coli mediates the attachment of the
two types of mating cells during sexual conjugation
- pili produced by Neisseria gonorrhoeae are responsible for the attachment of
pathogenic strains to endothelial tissue of the genito-urinary tract in humans
 Only piliated strains cause gonorrhea
- In some Pseudomonas species, pili are involved in the extrusion of protein from
the cell
Types of pilli
-Conjugative or F (Fertility or sex) pili - are involved in the mating process
- Common or generalized pili - are also produced by numerous bacterial species
 These pili play a role in adherence of microorganisms to surfaces or to specific
receptors on eukaryotic cells
 aiding in the colonization of these ecological niches
- Type IV pili - are involved in a form of motility known as twitching
Pilli of Enterobacteriacea(Jump it)
 Members of the Enterobacteriaceae produce a wide assortment of pili or fimbriae

 In E. coli, as well as in most members of the Enterobacteriaceae, the most

prevalent class of fimbriae are of type 1

 These pili are composed of a short-tip fibrillar structure containing FimG and
the Fim H adhesin attached to a rod composed of FimA subunits

 The FimH adhesin mediates binding to mannose oligosaccharides (mannose

sensitivity)
 Flagella
 Prokaryotes that move by swimming produce flagella
- long, flexible appendages resembling “tails”
- Proteinaceous appendages
 about 10 to 20 nm in diameter and 5 to 20 µm in length
- are found in some members of the gram-positive and gram negative bacteria as
well as in some archaea
 They act like a propeller —the cell rotates the flagella and moves through the
water
 In motility, chemical energy generated from metabolism is converted into
mechanical energy
 The most common function of motility among the prokaryotes seems to be to
allow the organisms to position themselves optimally in their microenvironment
 For example: to move along a concentration gradient toward a food source or
away from a repellant
Microscopic observation
 Flagella are so thin they cannot be observed directly with a bright-field
microscope
 Must be stained with special techniques designed to increase their thickness
 The detailed structure of a flagellum can only be seen in the electron microscope

Types of flagella(Don’t Jump it)

 The number and location (pattern of distribution) of flagella on the cell surface
have been used as important features in the classification of bacteria
o Monotrichous bacteria (trichous means hair) - have one flagellum
o Polar flagella - a single polar flagellum extending from one end of the cell
o Amphitrichous bacteria (amphi means “on both sides”) have a single flagellum at
each pole
o Lophotrichous flagella (lopho means tuft) - polar tufts or bundles
o Peritrichous flagella (peri means “around”) - flagella from all locations on the
cell surface
 Ex. Proteus vulgaris
o Mixed flagella – found on a few bacteria
 For example: Some Vibrio species produce polar flagella for movement in water
but produce lateral flagella when grown on an agar surface
 The Bacterial Endospore
 Endospore - a special resistant, dormant structure formed by a number of gram-
positive bacteria
o Endospores develop within vegetative bacterial cells of several genera:
 Bacillus and Clostridium (rods), and others
 These structures are extraordinarily resistant to environmental stresses:
 heat, ultraviolet radiation, gamma radiation, chemical disinfectants, and desiccation
 Some endospores have remained viable for around 100,000 years
 Actinomycete spores have been recovered alive after burial in the mud for 7,500
year
 Spores are resistant
- Several species of endospore-forming bacteria are dangerous pathogens
→ endospores are of great practical importance in food, industrial, and medical
microbiology
 It is essential to be able to sterilize solutions and solid objects
 Endospores often survive boiling for an hour or more
→ autoclaves must be used to sterilize many materials
 Endospores are also of considerable theoretical interest
o Because bacteria manufacture these intricate entities in a very organized fashion
over a period of a few hours
 spore formation is well suited for research on the construction of complex biological
structures
 Endospores can be examined with both light and electron microscopes

 Spores are impermeable to most stains

→ they often are seen as colorless areas in bacteria treated with methylene blue and
other simple stains
→ special spore stains are used to make them clearly visible
 Spore position in the mother cell or sporangium
- differs among species, making it of considerable value in bacterial identification

 Location:
- Spores may be located
- centrally –
- close to one end (subterminally) –
- terminally
 Spore formation - sporogenesis or sporulation
 Commences when growth ceases due to lack of nutrients
 It is a complex process and may be divided into seven stages
o stage I - An axial filament of nuclear material forms
o stage II - an inward folding of the cell membrane to enclose part of the DNA
and produce the forespore septum
o stage III - the membrane continues to grow and engulfs the immature spore in a
second membrane
o stage IV - cortex is laid down in the space between the two membranes stage V
- calcium and dipicolinic acid are accumulated
o stage VI - Protein coats then are formed around the cortex, and maturation of
the spore occurs
o stage VII - lytic enzymes destroy the sporangium releasing the spore

 Sporulation requires only about 10 hours in Bacillus megaterium

 Spore Germination
 The transformation of dormant spores into active vegetative cells seems almost as
complex a process as sporogenesis
 It occurs in three stages:
(1)Activation
(2) Germination
(3) Outgrowth
 Often an endospore will not germinate successfully, even in a nutrient-rich medium,
unless it has been activated
 Activation is a reversible process that prepares spores for germination
- usually results from treatments like heating
 It is followed by germination - the breaking of the spore’s dormant state
 This process is characterized by spore swelling, rupture or absorption of the spore
coat, loss of resistance to heat and other stresses, loss of refractility, release of spore
components, and increase in metabolic activity
 Many normal metabolites or nutrients (e.g., amino acids and sugars) can trigger
germination after activation
 Germination is followed by outgrowth
 The spore protoplast makes new components, emerges from the remains of the spore
coat, and develops again into an active bacterium
 Unit 3 : Bacterial Growth
 Growth - may be defined as an increase in cellular constituents
 It leads to a rise in cell number when microorganisms reproduce by processes
like budding or binary fission
 Growth also results when cells simply become longer or larger
 It is usually not convenient to investigate the growth and reproduction of
individual microorganisms because of their small size
 Therefore, when studying growth, microbiologists normally follow changes in the
total population number
 Population growth

 Population growth is the culmination of a complex series of biochemical events

that are driven by light or chemical energy
 This energy is utilized to synthesize or assimilate monomers, and these are in
turn assembled into macromolecules
 During the course of growth and division, a bacterial cell may synthesize an
estimated:
-1,800 different proteins,
- more than 400 different RNA molecules,
- a complete copy of the genomic DNA,
- cytoplasmic membrane, and where necessary,
- sufficient cell wall to surround the newly formed cell
 The Cell Cycle in Bacteria

 The cell cycle is the period of time in which a newly formed bacterium elongates, replicates its
DNA, and divides to generate two cells
 The replication of DNA and events involved in division to form two cells is under tight regulatory
control
C phase
o In a newly formed cell there is a regulatory event that initiates the replication of the
bacterial genome
o The time required to replicate the 4.2 × 106 base pairs in the DNA of bacteria with a
generation time between 20 and 60 minutes is 40 minutes
D (delay) phase
 After completion of DNA replication (termination), there is a 20-minute period before
division occurs
 During the D phase, the replicated DNA is separated into opposite ends of the elongated
cell
 The cytoplasmic membrane is involved in this separation
 After separation of the DNA, the process of constructing cytoplasmic membrane and cell
wall begins at the midpoint of the cell
 The division into two distinct daughter cells is called transverse fission
 The Growth Curve
 Population growth is studied by analyzing the growth curve of a microbial
culture
 When microorganisms are cultivated in liquid medium, they usually are grown in
a batch culture or closed system —that is, they are incubated in a closed culture
vessel with a single batch of medium
 Because no fresh medium is provided during incubation, nutrient
concentrations decline and concentrations of wastes increase
 The growth of microorganisms reproducing by binary fission can be plotted as
the logarithm of the number of viable cells versus the incubation time
 The resulting curve – Growth curve
- has four distinct phases
Microbial Growth Curve in a Closed System
 Lag Phase

 When microorganisms are introduced into fresh culture medium, usually no

immediate increase in cell number occurs
 Cell division does not take place right away
o There is no net increase in mass
 However, the cell is synthesizing new components
 A lag phase prior to the start of cell division can be necessary for a variety of
reasons:
o The cells may be old and depleted of ATP, essential cofactors, and ribosomes;
these must be synthesized before growth can begin
o The medium may be different from the one the microorganism was growing in
previously
 Here new enzymes would be needed to use different nutrients
o Possibly the microorganisms have been injured and require time to recover
 Exponential Phase (or log phase)
 Microorganisms are growing and dividing at the maximal rate possible
 Their rate of growth is constant during the exponential phase
- i.e. the microorganisms are dividing and doubling in number at regular intervals
 The population is most uniform in terms of chemical and physiological properties
during this phase
 Exponential phase cultures are usually used in biochemical and physiological
studies
 Exponential growth is balanced growth i.e., all cellular constituents are
manufactured at constant rates relative to each other
 If nutrient levels or other environmental conditions change, unbalanced growth
results
 This is growth during which the rates of synthesis of cell components vary
relative to one another until a new balanced state is reached
- observed in a shift-up experiment in which bacteria are transferred from a
nutritionally poor medium to a richer one
 The cells first construct new ribosomes to enhance their capacity for protein
synthesis
 This is followed by increases in protein and DNA synthesis
 Finally, the expected rise in reproductive rate takes place
 Unbalanced growth also results
 When shifted to a nutritionally inadequate medium, they need time to make the
enzymes required for the biosynthesis of unavailable nutrients
 Consequently cell division and DNA replication continue after the shift-down,
but net protein and RNA synthesis slow
 The cells become smaller and reorganize themselves metabolically until they are
able to grow again
 Then balanced growth is resumed and the culture enters the exponential phase

 These shift-up and shift-down experiments demonstrate that microbial growth is

under precise, coordinated control and responds quickly to changes in
environmental conditions
The effect of changes in limiting nutrient concentration on total microbial
yield. At sufficiently high concentrations, total growth will plateau .
 Nutrient Concentration and Growth
 The rate of growth also increases with nutrient concentration, but in a
hyperbolic manner
 The shape of the curve seems to reflect the rate of nutrient uptake by microbial
transport proteins
 At sufficiently high nutrient levels the transport systems are saturated, and the
growth rate does not rise further with increasing nutrient concentration
 Stationary Phase
 Eventually population growth ceases and the growth curve becomes horizontal
 It usually is attained by bacteria at a population level of around 109 cells per ml
 Other microorganisms normally do not reach such high population densities,
protozoan and algal cultures often have maximum concentrations of about 106
cells per ml
 Final population size depends on:
 nutrient availability and other factors
 the type of microorganism being cultured
 In the stationary phase the total number of viable microorganisms remains
constant
 Implications:
 a balance between cell division and cell death
 the population may simply cease to divide though remaining metabolically active
 Microbial populations enter the stationary phase for several reasons:
 Nutrient limitation - if an essential nutrient is severely depleted, population
growth will slow
 Aerobic organisms often are limited by O2 availability
 Oxygen is not very soluble and may be depleted so quickly that only the surface of
a culture will have an O2 concentration adequate for growth
 The cells beneath the surface will not be able to grow unless the culture is shaken
or aerated in another way
 The accumulation of toxic waste products
o limits the growth of many anaerobic cultures
- Ex. Streptococci can produce so much lactic acid and other organic acids from
sugar fermentation
 Their medium becomes acidic and growth is inhibited
 Growth may cease when a critical population level is reached
 Thus entrance into the stationary phase may result from several factors operating
in concert
 Bacteria in a batch culture may enter stationary phase in response to starvation
 This probably often occurs in nature as well because many environments have quite
low nutrient levels
 They only decrease somewhat in overall size, often accompanied by protoplast
shrinkage and nucleoid condensation
 The more important changes are in gene expression and physiology
 Starving bacteria frequently produce a variety of starvation proteins
 make the cell much more resistant to damage in a variety of ways
o They increase peptidoglycan cross-linking and cell wall strength
 The Dps (DNA-binding protein from starved cells) protein protects DNA
 Chaperones prevent protein denaturation and renature damaged proteins

 As a result of these and many other mechanisms, the starved cells become
harder to kill and more resistant to starvation itself, damaging temperature
changes, oxidative and osmotic damage, and toxic chemicals such as chlorine

 These changes are so effective that some bacteria can survive starvation for
years

 These considerations are of great practical importance in medical and

industrial microbiology

 Salmonella typhimurium and some other bacterial pathogens become more

virulent when starved
 During stationary phase, some of the cells die and lyse

 These lytic products of cells can provide nutrients for other cells, and these
divide and replace the dead ones

 However, most of the population in the stationary phase survives but simply does
not proliferate
 The individual cells in this phase differ in certain biochemical components from
cells in the exponential phase
 Generally, stationary phase cells are more resistant than exponential phase cells
 to adverse physical conditions such as increased heat, radiation, or
change in pH

 They synthesize storage material such as β-hydroxybutyrate or glycogen

 Members of the genera Bacillus and Clostridium that form endospores do so
during this period
 Death Phase
 The decline in the number of viable cells
o Due to:
 Detrimental environmental changes like nutrient deprivation and the buildup of toxic
wastes
 The death of a microbial population, like its growth during the exponential phase, is
usually logarithmic
 i.e. a constant proportion of cells dies every hour
 Often the only way of deciding whether a bacterial cell is viable is by incubating it in
fresh medium
 if it does not grow and reproduce, it is assumed to be dead
 No or few new colonies are formed or
 No turbidity in broth cultures
 Although most of a microbial population usually dies in a logarithmic fashion,
 the death rate may decrease after the population has been drastically
reduced
 This is due to the extended survival of particularly resistant cells
 For this and other reasons, the death phase curve may be complex