 In Medical Microbiology, microbial cultivation is required
for the following purposes
 In Medicine

1-Diagnosis of most infectious diseases

2-Selection of drug of choice ( antibiotics ) for treatment of infection
 In other fields

3-Preparation of Vaccine

4-Research tool in molecular genetics
Microbial growth requirements
 What are the chemical growth factors required for isolation of
microbes in vitro?
 1-Carbon


Organic source
Inorganic source
Glucose
CO2
 2-Nitrogen


Organic source
Inorganic source
Protein
Atmospheric nitrogen
 Autotrophic microbes using inorganic carbon and nitrogen
 Heterotrophic microbes using organic carbon and nitrogen
 Other chemical requirements

3- Hydrogen, Oxygen, Phosphorus, Carbon, Nitrogen, Ions, and Sulfur
Physical growth factors
 1- pH and Buffer requirements
 Pathogenic bacteria grow best at neutral pH which is
typically between pH (6.8 to 7.4)
 Acidophilic bacterium is able to survive with acidic pH
 Example: Helicobacter pylori
 On the other extreme, bacteria that prefer alkaline
conditions are known as alkaliphiles

Example: Vibrio cholerae
 Fungi such as yeasts and molds; grow best at acidic
conditions ( pH 5 )
 2- Salts concentration
 A specific concentration of NaCl is required for
microbial growth in vitro
 It is equal to normal saline salts concentration (0.9%
NaCl)
 Halophilic bacteria resist high salt concentration
 3- Temperature requirements
 Mesophiles
 grow at optimum of 37 ᵒC
 human body temperature
 Pathogens
 Opportunists
 Pyschrophile
 close to freezing
 Thermophile
 close to boiling
4- Gaseous requirements and Humidity
 According to oxygen requirements, microbes can be
divided into the following groups
 1- Strict or Obligate Aerobe
 O2 must be present to grow
 Example: Mycobacterium sp.
 2- Strict or Obligate Anaerobes
 Oxygen must be absent

Killed by oxygen
 Example: Clostridium sp.
 3- Facultative anaerobes
 Adaptable organisms that use oxygen when present but
can switch to anaerobic pathways in its absence
 Survive in the presence of O2

Example: Escherichia coli
 4- Microaerophilic
 Only use low concentrations of oxygen ( around 5%)

Example : Helicobacter pylori
 Grow in low oxygen
 Killed in high oxygen
The Bacterial Growth Curve
 Bacterial growth is the division of one bacterium into two
daughter cells in a process called binary fission
 Bacterial growth curve represents the relationship between
microbial quantity and time of incubation
 During Lag phase, bacteria adapt themselves to growth
conditions (number of dividing cell is zero)
 Exponential phase ( Log phase): is a period characterized by
Rapid cell doubling
 Doubling time can be as short as twenty minutes or as long as
several days
 During stationary phase, the growth rate slows as a
result of nutrient depletion and accumulation of toxic
products
 This phase is a constant value as the rate of bacterial
growth is equal to the rate of bacterial death
 At death phase, bacteria run out of nutrients and die
Bacterial binary fission
Bacterial growth curve
Microbial Metabolism
 According to biochemical pathway used in energy
production, bacterial metabolism can be categorized
into three types:
 1-Aerobic Respiration
 Molecular oxygen serves as the final electron acceptor
 38 ATP molecules will be produced by oxidation of one
glucose molecule
 Used by obligatory aerobic bacteria for energy
production

such as: Mycobacterium sp.
 2-Anaerobic Respiration
 Inorganic sulfate or nitrate act as the final electron acceptor
 38 ATP molecules will be produced by catabolism of one
glucose molecule
 Used by obligatory anaerobic bacteria such as: Clostridium sp.
 3-Fermentation
 Lactic acid ( produced by bacteria) or ethanol ( produced by
yeast) serves as final electron acceptor
 Only 2 ATP molecules will be produced by fermentation of
one glucose molecule
 Used by facultative anaerobic bacteria such as : E. coli
Microbial Genetics
 Prokaryotic Genome
 Most prokaryotic genes are carried on the bacterial
chromosome, a single circle of DNA
 Many bacteria contain additional genes on plasmids
 Plasmid is an extra-circular supercoiled DNA that carry some
important genes such as the antibiotics resistance genes
 Both bacteria chromosome and plasmid are called replicons
 Genetics is the study of inheritance and variation
 Genetic information encoded in DNA
 Function of genetic material
 1- Replication of the genome
 2- Expression of DNA to mRNA then to protein
Definitions
 Genotype
 the complete set of genetic determinants of an
organism
 Phenotype
 expression of specific genetic material
Bacterial DNA
 2 types of DNA in bacteria
 Chromosomal
 Extra-chromosomal (plasmid)
Plasmid
 Extrachromosomal DNA
 Found in most species of bacteria.
 Govern their own replication
 Genetic exchange, amplify genes
 Transfer by conjugation
 Code for resistance to antibiotics & toxins
Bacterial Structure
Bacterial Structure
Gene Transfer
 Transfer of DNA among prokaryotes is widespread
between different strains of same bacterial species
 Mechanisms of Gene Transfer
 1- Conjugation
 2- Transduction (is a phage-mediated genetic transfer)
 3- Transformation
Bacterial conjugation
 Conjugation: is a mechanism of gene transfer by
which plasmids will be transferred from one bacterial
cell to another by a mean of Sex pili
 Hospital-dwelling bacteria resist antibiotics due to
conjugation
Conjugation
Transduction
 Transduction is the process by which DNA is
transferred from one bacterium to another by a virus
 It also refers to the process whereby foreign DNA is
introduced into another cell via a viral vector
Transduction
Bacterial transformation
 A stable genetic change brought about by the uptake
of naked DNA and competence refers to the state of
being able to take up exogenous DNA from the
environment
 There are two forms of transformation and
competence: natural and artificial
The Microbial Virulence factors
 Virulence factors are external cellular structures, enzymes,
and toxins that enhance microbial pathogenicity
 In general, the most important virulence factors are:
 1-Microbial capsule
 Microbe resist host acidic environment (stomach gastric acid)
 Microbe resist host proteolytic enzyme

(Present in Saliva, and stomach)
 Microbe resist phagocytosis
 2-Fimbriae or Pili
 Microbial adhesion to the host cell surface
 Adhesion could be also enhanced by receptor-antigen
interaction
 3-Microbial Enzymes
 Collagenase enzyme

enhances microbial invasion; due to degradation of
extracellular matrix components
 Urease
 Neutralization of acidic pH ( urine, stomach)
 Coagulase
 Catalase have different functions
 4-Bacterial Toxins
 A-Exotoxins



Well known poisonous substances.
Chemical nature
 Proteins (two polypeptide components)
Almost all are Heat-labile at 60 ˚C
 Intracellular toxin fraction could



1-Inhibit cellular protein biosynthesis
2-Cause ionic imbalance and loss of water
3-Inhibit the release of neurotransmitters
 B-Endotoxins
 Chemical nature

Lipopolysaccharide, the component of Gram’s negative bacterial
outer membrane
 Heat-Stable at 100 ˚C
 5-The microbial Hemolysin
 Degradation of RBCs, Hemoglobin and NADH will be
released
 6-The microbial Haemagglutinine and Coagulase enzyme
 Agglutination of RBCs; the microbe escapes Humoral
immunity
 7-The microbial Beta-Lactamases
 Some microbes have ability to resist antibiotics due to
production of Beta-Lactamase enzymes
 Some strains of Staphylococci can hydrolyze the Beta-
Lactam ring shown
In the absence of β-lactam antibiotics, the
bacterial cell wall plays an important role in
bacterial reproduction.
Adding β-lactam antibiotics to the cell
medium while bacteria are dividing will
cause them to shed their cell walls and fail to
divide, forming large, fragile spheroplasts.