Bacterial growth

Shimelis Teshome (BSC MLS)

02/23/2021 Shimelis Teshome (BSc MLS) 1

 Bacterial growth

Growth in microbes refers to the increase in the number

of cells rather than the increase in the cell’s size.
An increase in the number of microbes from a single
parent cell, gives rise to a single colony of cells.

Common reproduction in bacteria

Binary fission

02/23/2021 Shimelis Teshome (BSc MLS) 2

02/23/2021 Shimelis Teshome (BSc MLS) 3
 If the environment is optimum the two daughter cells may
divide into four in 20 minutes1, 2, 4, 8, 16, 32, 64... or
 20,21, 22, 23, 24, 25, …2n, exponential growth
 Mathematically this can be expressed : N=No*2n,
 =>Number at any time = Number at time zero
 times 2 to the nth power, where "n" = number of
generations

02/23/2021 Shimelis Teshome (BSc MLS) 4

 Growth Curve

Stationary

Death
FORMING

Log
COLONY

UNITS

Lag

TIME

02/23/2021 Shimelis Teshome (BSc MLS) 5

 Phases of growth
1. Lag phase
• The cells are adapting to their new niche, usually a
broth.
• Cell reproduction is not immediate.
• Enzymes are synthesized to utilize broth nutrients.
• The bacterial cells are Metabolically active.
• According to the bacteria being grown, this phase can
take from less than an hour to several days.
02/23/2021 Shimelis Teshome (BSc MLS) 6
2. log/exponential phase
• The necessary chemicals are synthesized for metabolism.
• There is a rapid replication of genetic material as well as
reproduction of new daughter cells.
• There is logarithmic growth.
• The bacteria are dividing at their greatest rate.
• Metabolic activity has peaked.
• These new cells are young, delicate and immature.
• These cells are sensitive to antibiotics and UV radiation.
• The cells can be GramShimelis
02/23/2021
stained the best during this phase.
Teshome (BSc MLS) 7
3. Stationary phase
• The growth rate slows.
• This is a phase of equilibrium, because the number of
cells being replicated equals the number of cells dying.
• Nutrients are depleted.
• Waste products are building up, causing an acidic pH
level.
• Metabolic activity is greatly reduced.

02/23/2021 Shimelis Teshome (BSc MLS) 8

4. Death/decline phase
• All nutrients are totally depleted.
• A tremendous buildup of metabolic waste products.
• Cells are dying faster than they are being replicated.
• Cell death is occurring at negative logarithmic rate.
• Gram stain reaction is Gram variable.
• The number of endospore cells exceeds vegetative cells in
G+ cells.
• Total death will occur if this culture is not transferred to a
new broth tube.
02/23/2021 Shimelis Teshome (BSc MLS) 9
 General Virology

02/23/2021 Shimelis Teshome (BSc MLS) 10

 1.1. Introduction to virus
Viruses Definition:
 Obligate intracellular parasites, with no metabolic activity

 Viruses were first described as “filterable agents.”

 The simplest viruses consist of a genome of deoxyribonucleic acid

(DNA) or ribonucleic acid (RNA) packaged in a protective shell
of protein and, for some viruses, a membrane

02/23/2021 Shimelis Teshome (BSc MLS) 11

 1.1. Introduction to virus..
• Viruses depend on cells for their replication because they lack :
 Enzyme systems that produce the basic chemical building
blocks of life: nucleotides, amino acids, carbohydrates, and
lipids

 Enzyme systems that generate useable chemical energy,

usually in the form of adenosine triphosphate (ATP),

 Ribosomes, transfer RNAs, and the associated enzymatic

machinery that directs protein synthesis

02/23/2021 Shimelis Teshome (BSc MLS) 12

1.2. Properties and Structure of virus
 Viruses are not living.
 Viruses must be infectious to continue in nature.
 Viruses must be able to use host cell processes to produce
their components (viral messenger RNA, protein, and
identical copies of the genome).
 Viruses must encode any required processes not provided
by the cell.
 Viral components must self-assemble.

02/23/2021 Shimelis Teshome (BSc MLS) 13

• Virion Structure
 The units for measurement of virion size are nanometers(nm).

 The clinically important viruses range from 18nm

(parvoviruses) to 300 nm (poxviruses)

 The virion (the virus particle) consists of a nucleic acid

genome packaged into a protein coat (capsid) or a
membrane (envelope)

02/23/2021 Shimelis Teshome (BSc MLS) 14

 Viral Structure - Overview

• Nucleic acid
• Nucleocapsid
• Capsid
• Envelope
• Viral
• protein
Membrane envelope**
protein

02/23/2021 Shimelis Teshome (BSc MLS) 15

 The structures of a naked capsid virus (top left) and enveloped viruses (bottom) with
an icosahedral (left) nucleocapsid or a helical (right) ribonucleocapsid. The helical
ribonucleocapsid is formed by viral proteins associated with an RNA genome.
02/23/2021 Shimelis Teshome (BSc MLS) 16
 Structure of virus
• Mature virus particle (virion)
– The genome (RNA or DNA)
– DNA genome
- Except for the single stranded Parvoviruses, all DNA
viruses consist of double-stranded circular or linear DNA
- Size of DNA range from 4000 bp (4 kb) corresponding to
molecular weight of 1.5 m (Parvoviruses) to more than 200
kb or molecular wt of 150 m (Poxviruses)
– RNA genome
- linear (single- or double stranded)
- May exist as a single or segmented form
- Single stranded RNA, +RNA or positive sense (the
genome can act as mRNA) or (–RNA) or negative sense (the
genome is complimentary to the mRNA)

02/23/2021 Shimelis Teshome (BSc MLS) 17

 1.2. properties and Structure of virus
• The outer layer of the virion is the capsid or envelope.

• These structures are the package, protection, and delivery vehicle

during transmission of the virus from one host to another and for
spread within the host to the target cell

• capsid and envelope mediate the interaction of the virus with the
target cell through a viral attachment protein (VAP) or
structure.
• Removal or disruption of the outer package inactivates the virus.

• Antibodies generated against the components of these structures

prevent virus infection
02/23/2021 Shimelis Teshome (BSc MLS) 18
 Structural components of viruses
• The viral capsid is assembled from individual proteins
• Individual structural proteins associate into subunits, which
associate into protomers, capsomeres and finally, a
recognizable procapsid or capsid

02/23/2021 Shimelis Teshome (BSc MLS) 19

Structural components of viruses

• The capsid is a rigid structure able to withstand harsh

environmental conditions.

• Viruses with naked capsids are generally resistant to drying,

acid, and detergents, including the acid and bile of the enteric
tract.

• Many of these viruses are transmitted by the fecal-oral route

and can endure transmission even in sewage.

02/23/2021 Shimelis Teshome (BSc MLS) 20

Structural components of viruses
• The envelope is a membrane composed of lipids, proteins,
and glycoproteins.

• The membranous structure of the envelope can be maintained

only in aqueous solutions.
• It is readily disrupted by drying, acidic conditions, detergents,
and solvents such as ether, which results in inactivation of the
virus.

• enveloped viruses must remain wet and are generally transmitted

in fluids, respiratory droplets, blood, and tissue.

• Most cannot survive the harsh conditions of the gastrointestinal

trac
02/23/2021 Shimelis Teshome (BSc MLS) 21
 1.3 Classification of medically important virus
• Viruses are divided into related groups, or families, and,
sometimes into subfamilies based on:

A. type and structure of the viral nucleic acid,

B. type of symmetry of the virus capsid (helical versus
icosahedral), and
C. presence or absence of a lipid envelope.
D. the strategy used in its replication,
E. physical and biochemical characteristics, such as size,
morphology

02/23/2021 Shimelis Teshome (BSc MLS) 22

A . type and structure of the viral nucleic acid,
• The DNA or RNA genome may be :
– ss – single stranded or
– ds – double stranded
• Genomes may be either:
– (+) sense: Positive-sense viral RNA is identical to viral
mRNA and thus can be immediately translated into protein by
the host cell.

OR
– (-) sense: Negative-sense viral RNA is complementary to
mRNA and thus must be converted to positive-sense RNA by
an RNA polymerase before translation.

• Retroviruses?

02/23/2021 Shimelis Teshome (BSc MLS) 23

 B. type of symmetry of the virus capsid (helical versus
icosahedral)
• Varies in size, shape and symmetry
• 3 types of capsid symmetry:
– Cubic (icosahedral)
• Has 20 faces, each an equilateral triangle. Eg. adenovirus
– Helical
• Protein binds around DNA/RNA in a helical fashion eg.
Coronavirus

– Complex
• Is neither cubic nor helical eg. poxvirus

02/23/2021 Shimelis Teshome (BSc MLS) 24

3. presence or absence of a lipid envelope.

02/23/2021 Shimelis Teshome (BSc MLS) 25

a. Naked virus
• Properties*
• Is environmentally stable to the following:
• Temperature, Acid, Proteases, Detergents and Drying
• Is released from cell by lysis
• Consequences*
 Can be spread easily (on fomites, from hand to hand, by dust,
by small droplets)
 Can dry out and retain infectivity
 Can survive the adverse conditions of the gut
 Can be resistant to detergents and poor sewage treatment
 Antibody may be sufficient for immunoprotection

02/23/2021 Shimelis Teshome (BSc MLS) 26

 b. Enveloped virus
• Properties*
• Is environmentally labile—disrupted by the following:
 Acid, Detergents, Drying, Heat
 Modifies cell membrane during replication
 Is released by budding and cell lysis
• Consequences
 Must stay wet
 Cannot survive the gastrointestinal tract
 Spreads in large droplets, secretions, organ transplants, and blood
transfusions
 Does not need to kill the cell to spread
 May need antibody and cell-mediated immune response for
protection and control
 Elicits hypersensitivity and inflammation to cause
immunopathogenesis
02/23/2021 Shimelis Teshome (BSc MLS) 27
02/23/2021 Shimelis Teshome (BSc MLS) 28
02/23/2021 Shimelis Teshome (BSc MLS) 29
Relative sizes of viruses and bacteria.

02/23/2021 Shimelis Teshome (BSc MLS) 30

 Replication of viruses
• Nucleic acid -- necessary genetic information
for replication
• Depend on host cell enzymes for replication
• Six phases in replication
– Adsorption or Attachment
– Penetration
– Uncoating
– Biosynthesis
– Maturation
– Release
02/23/2021 Shimelis Teshome (BSc MLS) 31
 Figure : Steps of virus replication cycle
02/23/2021 Shimelis Teshome (BSc MLS) 32
 Fig: HIV virus Replication
02/23/2021 Shimelis Teshome (BSc MLS) 33
 Adsorption
• Virion acquire contact with host cell
• Affinity between virus & host cell
• Specific receptor sites viruses can attach  host
cells
• Influenza virus -- haemagglutinin 
glycoprotein receptor sites of host cell
• HIV – gp120  CD4+ cells
• Polio virus -- lipoprotein receptor of host cells

02/23/2021 Shimelis Teshome (BSc MLS) 34

 Penetration
Penetration
 Mainly by three mechanisms
1. Whole virus enter host cell by engulfment into
intracellular vacuoles
• Endocytosis (viropexis)
2. Directly penetrate plasma membrane
3. Viral envelope fuses with plasma
membrane
• Facilitates entry of virus into host
cell
02/23/2021 Shimelis Teshome (BSc MLS) 35
 Uncoating
• Stripping of viral outer layers
• From capsid nucleic acid is released into host
cell
• Lysosomal enzymes of host cell enhance

02/23/2021 Shimelis Teshome (BSc MLS) 36

 Biosynthesis
Synthesis of nucleic acid & capsid
• Production of enzymes needed for viral
synthesis, assembly & release
• Regulator proteins are synthesized
• Regulator proteins shut down normal host cell
function
• DNA viruses usually synthesize nucleic acid in
host cell nucleus
• Exception is Pox virus synthesizes DNA in host
cell cytoplasm
02/23/2021 Shimelis Teshome (BSc MLS) 37
 Biosynthesis
• RNA viruses synthesize RNA in host cell
cytoplasm
• Viral proteins are synthesized only in cytoplasm
of the host cells
• Protein synthesis -- Transcription (DNA to
mRNA), Translation (mRNA to protein)
• Further replication cycles takes place for
synthesis of more viral structural proteins
(capsids)

02/23/2021 Shimelis Teshome (BSc MLS) 38

 Maturation
• Assembly of daughter virions into capsids -- in host cell
nucleus or cytoplasm
• Herpes and Adeno viruses -- host cell nucleus
• Picorna and Pox viruses assembly occur in cytoplasm
of host cell
• Enveloped viruses acquire envelope from host cell
plasma membrane, nuclear membrane etc. by budding

02/23/2021 Shimelis Teshome (BSc MLS) 39

 Release

• Progeny virions released from host cells by

budding from cell membrane
• Cell to cell fusion
• Rupture of host cell

02/23/2021 Shimelis Teshome (BSc MLS) 40

 Eclipse Period
• Stage from penetration till appearance of mature
daughter virions
• Viruses cannot be demonstrated inside host cell
• Temporary disappearance of infectivity
• Time required for single cycle is between 15 –
30 hours

02/23/2021 Shimelis Teshome (BSc MLS) 41

 Latent period

• Period from entry of virions up to release of

first infectious progeny virions

02/23/2021 Shimelis Teshome (BSc MLS) 42

 Viral Infections
acute infection**

virus elimination chronic infections **

Hepatitis B & C

latent infection *
immunity re-infection**
measles influenza

Slow Virus Infection * reactivation **

SSPE herpesviruses

02/23/2021 Shimelis Teshome (BSc MLS) 43

 Diagnostic methods in Virology

The laboratory methods accomplish the following results:

1. Description of virus-induced cytopathologic effects (CPEs) on
cells
2. Detection of viral particles
3. Isolation and growth of the virus
4. Detection and analysis of viral components (e.g., proteins
(antigens), enzymes, genomes)
5. Evaluation of the patient’s immune response to the virus
(serology)

02/23/2021 Shimelis Teshome (BSc MLS) 44

 Specimen & type of viral infection

 Influenza virus → nasopharyngeal swab

 Rotavirus infection, hepatitis A → feces
 Hepatitis B virus → blood specimen
 Rabies virus → saliva, brain biopsy

02/23/2021 Shimelis Teshome (BSc MLS) 45

 Direct Examination
1. Antigen Detection immunofluorescence, ELISA etc.

2. Electron Microscopy morphology of virus particles

immune electron microscopy

3. Viral Genome Detection hybridization with specific

nucleic acid probes
polymerase chain reaction (PCR)

4. Light Microscopy histological appearance

inclusion bodies

02/23/2021 Shimelis Teshome (BSc MLS) 46

 B. Indirect Examination

1. Cell Culture Cytopathic effect (CPE)

haemabsorption
immunofluorescence
2. Eggs pocks on CAM
haemagglutination
inclusion bodies
3. Animals disease or death

02/23/2021 Shimelis Teshome (BSc MLS) 47

 u
yo
k
an
Th

02/23/2021 Shimelis Teshome (BSc MLS) 48