- Understanding the physiology and genetics Chapter 1: Introduction to

of microorganisms are important to Pharmaceutical Microbiology

produce therapeutic agents
- A modern medicine must be effective,
- Knowledge of mechanisms whereby
safe, and of good quality
microorganisms are able to resist
antibiotics, colonize medical devices are
- These medicines consists of active
essential in the production of new drugs
ingredients, stable, and safe during storage
and in healthcare practices
- Analytical Chemists and Pharmacists

Chapter 2: Fundamental features of Pharmaceutical Microbiology

microbiology
- Foundation
o MICROORGANISMS
- Encompasses the subject of sterilization
- microscopic, living, single-celled
and preservation against microbial spoilage
organisms

-more versatile than mammals in breaking - Pharmacist must be responsible for the
down foods. safe and hygienic manufacturing of
medicines
Differ in:
Antibiotics
1.Shape

2.Size - Major importance in pharmacy

3.Genetic Characteristics - Naturally occurring substance that would

inhibit or kill microorganisms
4. Metabolic Characteristics

- Major groups: - Microbial metabolite

o Bacteria - Synthetic agents that are normally used

systematically to treat infection
o Fungi
- Antibiotic production began with the
o Protozoa
discovery of penicillin in the 1940s
o Viruses
- Attack and kill bacteria without harm to
the host

CLASSIFICATION OF MICROORGANISMS: Ribotyping

o Viruses - Genetic technique used to identify cross-

infection, reduce transmission and optimize
- do not have cellular structure
management of hospital-acquired infections
- Composition:
 -No nucleic acid o Nucleic acid surrounded by
proteins
-atypical form of mammalian protein
o Some posses lipid envelope
-can interact w/ normal protein molecule
associated w/ glycoprotein
and cause it to undergo conformational
change and make it into a prion and ceases o Absent:
normal function
-recognizable chromosomes
-responsible for transmissible spongiform
encephalopathies -cytoplasm

Ex. Creutzfeldt-jakob disease -cell membranes

Bovine spongiform encephalopathy -incapable of independent replication

-simplest and most recognized agents of - intracellular parasites

infectious disese
-reproduced using metabolic capabilities of
-extreme resistance to conventionall host cell
sterilizing agents (steam,gamma
-smaller than bacteria
radiation,disinfectant,etc.)
- Variation in:

o Shape (helical, linear, or

PROKARYOTIC MICROORGANISMS:
spherical)
- No true nucleus
o Size (20-400nm)
- Usually single chromosomes
o Nucleic acid composition (single
- Haploid or double-stranded,linear or
circular RNA or DNA)
- Asexual reproduction
-Viewed using electron microscope.
Examples : Bacteria, Archea

o Viroids (Virusoids)
o Archea
-simplier than viruses
-no pharmaceutical importance
-infectious particles
-capable of living in extreme environment
-single stranded RNA w/o associated
-exhibit specialized modes of metabolism proteins

Ex. Plant phatogens

o Bacteria

-unicellular o Prions
-posses prokaryotic properties -infectious agents
 - contaminate or cause spoilage of -exhibit great diversity in form, habitat,
pharmaceutical products metabolism, pathogenicity

- obtain energy by decomposition -Bacteria of interest in medicine and

of animal and vegetable materials pharmacy: Eubacteria

- described as parasites or -many bacteria would be described as

pathogens facultative anaerobes or microaerophils

-most bacteria important in medicine and

pharmacy :
Rickettsia and Chlamydia
posses cell wall
- obligate intracellular parasites
grow well at temperatures
- incapable of growing outside host between ambient and human
cell body temperature

exhibit wide variation in

requirement for oxygen
EUKARYOTIC MICROORGANISMS:
Strict aerobes = require
- true cell nucleus
atmospheric oxygen

- chromosomes separated from the Strict anaerobes = oxygen is toxic

cytoplasm by nuclear membrane
Examples:
- diploid
Eubacteria
- sexual reproduction
- Bacteria of interest in medicine
and pharmacy
Examples: fungi,protozoa,algae,etc.
- types:

o Fungi 1. Bacillus- rod shaped

2. Cocci-spherical
- structurally more complex and varied
appearance than bacteria 3. Curved or spiral cell
approximately 0.5 to 5 mm
- non-photosynthizing
- Divided into two groups acc. to
- some fungi exhibit unicellular(yeast like) Christian Grams 1884 staining procedure.
or mycelial (mould-like) upon cultivation
1. Gram positive

- Most are saprophytes w/ few pathogenic 2. Gram negative

potential

- ability to make spores that are resistant

in drying = pharmaceutical contaminants Saprophytes

Microorganisms are more versatile than humans
in breaking down food, Many can use alternative
methods in breaking down food depending on the
environment, and some can obtain energy from
carbohydrates, digestion of proteins and other non-
carbohydrate materials.
o Chemoheterotrophs
- obtain carbon from nitrogen
- gets energy from breaking down
organic compounds
- organisms of interest in pharmacy
in medicine
o Catabolic Reactions
- energy is liberated by digestion of
food materials.
o Anabolic Reactions
- use liberated energy to make
complex cellular
polymers,protein,carbohydrates,
and nucleic acids.
o Oxidation
- removal or loss of electrons
- when food materials are oxidized
energy are released.
- does not invariably involve oxygen
- when oxidizing molecules accept
electron the other molecule is
reduced; reduction and oxidation is
linked and called Redox reaction.
o Redox Potential
- indicates whether oxidation or
reduction will prevail
- Anaerobic organism = low redox
potential
Aerobes = high redox potential
- Fungus: covers both terms yeast and
moulds
Yeast
- normally unicellular
- divide by budding
- larger than bacteria
- divide by binary fission or budding
Moulds
- imprecise term to describe fungi
that doesnt form fruiting bodies to
naked eyes
- grow by extension or branching of
hyphae
- excluding toadstool and
mushrooms
- 1 to 50 um wide
- Differentiated for special
function
- consist of tangled mess or
filaments of thread
o Protozoa
- predominantly unicellular
- mostly animals than plants
- free living motile organisms
- occur in water and soil
- not normally found as contaminants
-potential to cause disease
Ex. Organism responsible for malaria and
ameobic dysentery
*for further comparison of eukaryotes and
prokaryotes check Table 2.1 page 11
NAMING OF MICROORGANISMS:
- known by two names genus and species
- written in italic or underlined
MICROBIAL METABOLISM:
 Some pathogenic bacteria can
ferment:
different sugar, acids, alcohols,
acetone, butane, etc.
o Fundamental Principle of Antibiotic
Action
- drug must exploit a difference in
metabolism between organism to be
killed and the human host.
- w/o difference it will be very
toxic to the patient.
o Primary metabolites
- metabolic products that arise
during period when microbial
culture is growing.
Ex. ethanol, organic acids
o Secondary metabolites
- diverse
- have commercial or therapeutic
importance
- produced after the cell
multiplication has stopped.
Ex. Antibiotic,enzymes,toxins,
carbohydrates
MICROBIAL CULTIVATION
CULTURE MEDIA
- Some microorganisms have different
sugar fermentation patterns
- Sugars in culture media are usually used
for identification purposes.
- NaCl may be incorporated to adjust
osmotic pressure.
o Oxidizing Agents
- molecules that can accept
electrons
o Glycolysis
- breakdown of glucose to release
energy
- metabolic pathway used by
pathogenic bacteria and mammals
- conversion of glucose to series of
reactions to pyruvic acid, oxygen is
not required.
- undertaken by both aerobic and
anaerobic
- release small amount of energy
stored in sugar molecule.
o Aerobic respiration
- Used by mammals to release more
energy in sugarcompared to
glycolysis.
- Oxygen in end of sequence of
respiratory reaction .
o Fermentation
- an anaerobic process
- alternative to respiration
- means of releasing more energy
from sugar
- a process in which in which the
final electron acceptor is inorganic
molecule
- production by yeast of ethanol and
carbon dioxide from sugar
- many organisms can be used as
apart from yeast and is not
restricted to common sugar
(sucrose).
 -Glucose: readily employed
as nutrient
CLASSIFICATION BASED ON CONSISTENCY:
1. Solid
- gelled by addition of agar (1-1.5% w/v)
- Agar: from seaweeds ; firms at 37C and
liquid at 45C.
- For anaerobic organisms contain non-
toxic
reducing agents
-low redox potential
2. Liquid
- contains no agar.
o Selective or Diagnostic Media
- restrict the growth of certain
types of microorganisms while
permitting or enhancing the growth
of others.
- use additives for identification
o Enrichment Medium
- designed to permit a particular
type of organism to grow while
others, so the one that grows
increase in relative numbers and is
enriched.
CULTIVATION METHODS
o Binary Fission
- division pattern
- cell enlarges or elongates then
forms cross wall called septum and
is followed by constriction until
finally broken and daughter cells
separate.
- For yeast and moulds have lower pH (5.5-
6.0)
- Lactic acid: used to impart lower pH
CLASSIFICATION BASED ON
COMPOSITION:
1. Truly Synthetic Media
- chemically defined
- for microorganisms that
can synthesize materials
needed for growth from
simple carbon and nitrogen.
2. Media w/ biochemicals
- used for organism that
cant synthesize
- some more commonly
used
- complex w/ precise
chemical composition
- vary slightly batch to
batch
Ex. Routine laboratory media,
General Purpose meda, Complex
Media
- aq. Soln of animals and plants
extracts that contain:
Hydrolysed protein
-inexpensive sources are meet
extracts, milk, and soya
-hydrolysed by varying degree to
give peptones or amino acids
-trypsin and other proteolytic
enzymes preffered to acids
B-group vitamins
-requirement is satisfied by
yeast extract
Carbohydrates
-in form of starch and yeast
- Several situations where number of microbial
cells in culture, sample or specimen are needed to
be measured:
Measuring levels of microbial
contamination in raw material or
manufactured medicine
Evaluating the effects of
antimicrobial chemical or
decontamination process
Using microorganisms in
manufacture of therapeutic agents
Assessing the nutrient capability of
growth medium
-In pyrogen testing and vaccine manufacture both
number of dead and living cells are required
o Total count
is a counting procedure enumerating both
living and dead cells
o Viable count
-records living cells alone
o Total Viable Count (TVC)
-used in most pharmacopoeias and by many
regulatory agencies
-mean a viable count that records all
different species or types of microorganism
that might be present in sample.
Three Traditional Methods of Viable
Counting:
Basis: Living cell will give rise to colonies when
introduced w/ suitable medium and incubated.
1.Pour Plating
- surface spread method used
2 Miles Misra Method
- Surface drop
- membrane filter methods used
ENUMERATION OF MICROORGANISMS
3. MSP (Most Probable number)
- anticipated count is very low
- In optimal conditions of laboratory
cultivation of bacteria this division
takes place every 25-30 minutes.
- growth continues until one or more
nutrient is exhausted or toxic
metabolites accumulates
o Overnight incubation in liquid media
- culture media clear but becomes
cloudyas concentration increases
- indirect means of monitoring
culture media.
o Colony
- usually arise on solid media in
petri dishes
- a collection of cells arising by
multiplication of a single original
cell or a cluster
- in microscope: hundreds to
millions
- typically 1-10 mm
- Periphery of colony; part that is
actively growing
o Petri dishes incubated in a anaerobic jar
- Where anaerobic microorganisms
grows
o Planktonic Cells
- Routinely used to testing procedures
designed used to assess the activity
- different susceptibilities of lethal
agents
- Reappraisal appropriate
 - fast ,readily automated, and eliminates
long hours of incubation and numerous petri
dishes
- not capable of reproducing colonies
Operating method principles:
o Epifluorescent Techniques
o Living cells generate ATP
o Manometer Techniques
o Resistance and capacitance or
impedance of culture media
MICROBIAL GENETICS
- Genetic material may be transferred
depending if organism is prokaryote or
eukaryote.
- Mutation is very important for resistance
of antibiotics
o Bacteria (Prokaryote)
- genes for growth and metabolism:
chromosomes of double stranded
DNA
Bacterial Chromosomes
- tightly coiled
- 1mm contain 1000-3000
genes
- additional genes for
survival advantage under
certain circumstances:
Plasmids
Plasmids
- smaller and replicate
independently
- 0.1-1% size of bacterial
chromosomes
- not essential for normal
functioning
- replicate independently
- more commonly used in water,
food, and dairy.
- poor accuracy
- last resort
*for more information about methods of viable
counting check page 18 Table 2.2 and page 19
Table 2.3
WAYS TO MEASURE BIOMASS:
1. Turbidity Measurements
- most common used in estimating the total
number of bacteria in sample
- measured using spectrophotometer or
colorimeter
- not used in fungi
2. Dry Weight Determination
-for fungi biomass
3.Direct Microscopic Counting
- for bacteria, yeast , and fungal spores
- not for moulds and indirect measure of
biomass
Limitations of traditional method of viable
counting:
Relative labour intensive
Not easy to automate
Slow due to to the need for incubation
May require relative large volumes of
culture media many petri dishes and
incubator spaces
Rapid Methods of detecting and counting
microorganisms:
- enumerate viable organisms(usually
bacteria and yeast)
- employ various means of indirect
detection of living cells
PHARMACEUTICAL IMPORTANCE OF MAJOR
CATEGORIES OF MICROORGANISMS
o Viruses
- importance is based on Pathogenic
potential
- not susceptible to antibiotics
- Hazard Category 4
- easy to destroy by heat, radiation,
or toxic chemicals
o Prions
- withstand sterilizing conditions
- ability to cause incurable and
fatal disease
o Bacteria
- important as pathogens
- ability to resist activity of
antibiotics and biocides; long
standing notoriety
- streptomycetes bacteria produce
antibiotics
- grow on diverse substrates ensure
potential as agents of spoilage
- survive well in drying, dust, and
other adverse environments
- contaminants
- can produce bacterial spores
o Fungi
- survive in drying
- produce spores
- contaminants
- can be passed to one cell
or another by various means
Genes received from other sources:
o Bacteriophages
- Genome of Cell
= Bacterial Chromosomes +
Plasmids + Bacteriophages
o Eukaryote
- nucleus that contain one or more
pairs of linear chromosomes
- DNA complexed w/ protein
- Cells may divide asexually and
undergo mitosis but many have the
potential to undergo sexual
reproduction and undergo meiosis
- based on RNA instead of DNA
- possibility of creating new gene
combination
o Genotype
- describes genetic composition
regardless if expressed or not
o Phenotypic Adaptation
- non-genetic adaptation
- bacteria adopt a phenotypic
change to counter environmental
stress.
o Genetic Adaptation
- Acquire new genes either by
mutation or conjugation
- process of selection ensure that
mutant organisms that are better
suited for new environment
becomes numerically dominant.
 organisms in order to minimize both - Less degree of resistance than
formation of damaging ice crystals bacteria
and osmotic stress that accelerate
cell death during freezing and - little threat to immunocompetent
thawing. individuals

o Protozoa
Chapter 3: Bacteria - significantly large owning to the
pathogenic potential of few species
Prokaryotes
- Smallest free living organism (bacteria - Do not poses cell wall
and archaea)
- Lack a true nuclear membrane - Do not survive drying well
* Eukaryotic cells presence of a nuclear
membrane and internal - Do not display resistance to
compartmentalization sterilization to match bacterial
Major feature: cytoplasm of membrane- spores
enclosed organelles
- More troublesome in veterinary
Differences (Bacteria and Archaea):
- Cell wall composition (major difference)
- Lipid structure making up their PRESERVATION OF MICROORGANISMS
cytoplasmic membranes
- Metabolic patterns - Manufacture of Medicines: microorganisms are
employed in variety of test and assays to
measure activity of antimicrobial chemicals.
Bacteria: Archaea:
- most are anaerobes - Aim of Culture Preservation: maintain viability of
- Vast majority of - inhabit extreme the highest possible percentage of cells and to
prokaryotes of medical environments minimize risk of selecting atypical mutants.
and pharmaceutical - greater stability under
significance extreme conditions
- no disease-causing
archaea have yet been - Gram positive bacteria tends to survive better
identified than gram negative ones.

Most common procedure for long term storage:

Bacteria 1. Freezing at -80C in refrigerators

- Represent a large diverse group of
organism that can exist as single cells or as 2. Storage in liquid nitrogen at -196C special
cell clusters vessels

3. Lyophilization or freeze-drying
- Have the ability to carry out their life
processes of growth, energy generation and o Cryoprotectant Chemicals
reproduction independently of other cells
-compounds like glycerol or
* Very different from the cells of animals dimethylsulphoxide
and plants (unable to live alone in nature,
- incorporated at concentrations
10% v/v in liquid culture of
Rarer morphological forms:
a. Actinomycetes
rigid bacteria resembling fungi that may
grow as lengthy branched filaments
b. Mycoplasmas
lack a conventional peptidoglycan
(murein) cell wall
highly pleomorphic organisms of indefinite
shape
c. Some miscellaneous bacteria stalked,
sheathed, budded, and slime producing forms
often associated with aquatic and soil
environments
Cellular components
- Simple base cell structure compared with
eukaryotic cells
Reasons to have a good knowledge of the
bacterial cell structures and functions:
- Provides an excellent route for probing the
nature of bacterial processes many of which are
shared by multicellular organisms
- Normal bacterial processes can be customized to
benefit society on a mass scale
- To know how to destroy bacterial contaminants
and disease-causing organisms (pharmaceutical and
healthcare perspective)
Cell Wall
exist only as a part of a multicellular
organism)
- Capable of growing in a range of different
environments
- Cannot only cause contamination and
spoilage but also a range of different
diseases
Bacterial Diversity and Ubiquity
- Bacteria are diverse in shape and sizes
(morphology), adaptation to environment,
survival strategies, and metabolic processes
- The presence of bacteria may be
considered ubiquitous. There is no natural
environment that is free from bacteria.
Cell Size and Shape
- Majority of bacteria are 1-5 um long and
1-2 um in diameter
* >5 um (ex: Thiomargarita namibiensis)
extremely rare
* bacterial size increased, efficient, and
rapid transport and growth rates
- Classification of bacteria is made through
morphological grounds
- Mostly unicellular and possess simple
shapes: round (cocci), cylindrical (rod,
bacillus), or ovoid
 - LPS determines the antigenicity of the
bacteria
*extremely toxic to animals
- LPS is made up of lipid A, core
polysaccharide and O-specific
polysaccharide
- The cells lose the crystal-violet iodine
complex and are rendered colorless (gram
stain)
- Appear red under the light microscope
Cytoplasmic membrane
- Fragile phospholipid bilayer with protein
distributed randomly throughout
- Involved in various transport and enzyme
functions associated with the membrane
- Transports of nutrients, energy generation
and electron transports
- Selective barrier between the cytoplasm
and the cell environment
Cytoplasm
- Consists of 80% water and contains
enzymes that generate ATP
- Compose of the ribosomes, nucleoid and
inclusion granules
Nucleoid
- Singular, covalently closed circular
molecule of double stranded DNA
Plasmids
- Relatively small circular pieces of double
stranded extrachromosomal DNA
- For autonomous replication
- Encode many auxiliary functions that are
not usually necessary for bacterial growth
(antibiotic resistance)
- Essential for the maintenance of the shape
and integrity of the bacterial cell
- An obvious target for antibiotics (Cell
Lysis)
- Provide a strong, rigid structural
component that can withstand the osmotic
pressure caused by high chemical
concentrations of inorganic ions in the cell
- Most bacterial cell walls have
peptidoglycan layer (murein/ glycopeptide)
exceptions include the Mycoplasmas,
extreme halophiles, and the archaea
- Peptidoglycan is composed of Nacetyl
muramic acid (NAM) and Nacetyl
glucosamine (NAG)
Bacteria can be divided into two large
groups (on the basis of a differential staining
technique called the Gram stain): Gram-positive,
Gram-negative
Gram-positive Cell Wall
- Consist primarily of a single type of
molecule
- Contains teichoic acids and lipoteichoic
acids (negatively charged)
- During an infection, lipoteichoic acids
molecules trigger an inflammatory response
- Retain the dye (gram stain)
- Appear purple under the light microscope
Gram-negative Cell Wall
- Multilayered structure, quite complex
- Compose of proteins, lipoproteins,
phospholipids and lipopolysaccharide that
are unique to gram negative bacteria
 - Biofilm formation begins with attaching to
surface and form cement cells to protect
the bacteria from hazardous materials
BACTERIAL SPORULATION
- Process in which the vegetative cell undergoes a
profound biochemical change to give rise to a
specialized structure called an endospore or spore
- Not part of a reproductive cycle
Spore
Highly resistant
Enables producing organism to survive in
adverse environmental conditions (lack of
moisture or essential nutrients, exposure
to toxic chemicals, radiation or high
temperatures)
All sterilization process for pharmaceutical
products have been designed to destroy
the bacterial spore
Endospore structure
Endospores
Differentiated cells that possess a
grossly different structure to that
of the parent vegetative cell in
which they are formed
Exosporium
Outermost layer
Composed of protein; within are
the spore coats (proteinaceous but
with a high cysteine content)
Cortex
Consists of loosely cross-linked
peptidoglycan
Central core
Contains the genome
Partially dehydrated (dehydration
shown to increase resistant to both
heat and chemicals)
Containing only 10-30% of the
water content of the vegetative
cells
- transfers, increasing the spread of
resistance
Ribosomes
- The site of protein synthesis
- Two subunits: 30S and 50S
Inclusion granules
- Serves as the storage material for carbon,
nitrogen, and sulphur or phosphorus
Cell surface components
Flagella
- Bacterial motility
a. Monotrichous a single polar flagellum
b. Lophotrichous two or more flagella at one pole
of the cell
c. Amphitrichous single/tuft of flagella at each
end of the cell
d. Peritrichous flagella distributed over the
entire cell
Pili and Fimbriae
a. Pili (pilus) join bacterial cell in preparation of
DNA and to environmental surfaces
involved in the genetic exchange
process of conjugation
b. Fimbriae for adherence of cells to one another
and to environmental surfaces
responsible for hemaglutination
and cell clumping in bacteria
Glycocalyx (Slime Layer and Capsule)
- General substances that surround cells
- Gelatinous polymer of polysaccharide,
polypeptide, or both
a. Slime Layer unorganized and loosely attached
to the cell wall
b. Capsule substance is organized and firmly
attached to cell wall
Biofilms
- Any surface for microbial habitat
- Usually contains more than one species of
bacteria which exist and cooperate
together
 Classified as either endotoxin
(ex. Cell wall-related,
exotoxin, products released
extracellularly as the organism
grows)
Endotoxin
Lipid A component of LPS
Possesses multiple biological
properties including ability to
induce fever, initiate the
complement and blood
cascades
Activate B lymphocytes and
stimulate production of tumor
necrosis factor
Released from lysed or
damaged cels
Depyrogenation; process where
care must be taken to
eliminate or exclude such heat-
resistant material from
parenteral products and their
delivery systems.
Exotoxin
A-B toxins; consists of a B
subunit that binds to a host
cell receptor and is also
covalently bound to the A
subunit that mediates the
enzymic activity responsible
for toxicity (ex. Diphtheria
toxin, cholera toxin)
Cytolytic toxins; do not have
seperable A and B portions but
work by enzymatically
attacking cell constituents,
causing lysis (ex. haemolysins
and phospholipases)
Superantigen toxins; lack an A-
B type structure and act by
stimulating large numbers of
immune response cells to
release cytokines, resulting in
a massive inflammatory
reaction (ex. Staphylococcus
aureus)
pH; 1 unit lower than the
cytoplasm of the vegetative cell
and contains high levels of core-
specific proteins that bind tightly
to the DNA and protect it from
potential damage
Core-specific proteins; function as
an energy source for the outgrowth
or germination of a new vegetative
cell from the endospore
Spore
Presence of dipicolinic acid and
high levels of calcium ions which
complex together
Endospore formation
Vegetative cell undergoes a complex series of
biochemical events in cellular differentiation
Sporulation; accomplished by activation of a
variety of spore-specific genes such as spo and
ssp
Leads to the production of a dry, metabolically
inert but extremely resistant endospore
Endospore germination
Reversion of endospore back to a vegetative cell
Removal of the stress inducer that initiated
sporulation
Germination loss of resistance properties; occurs
along with a loss of calcium dipicolinate and
cortex components, and degradation of the core-
specific proteins
BACTERIAL TOXINS
- Organisms, if presented with the correct set of
conditions, can cause disease
- (ex. Opportunist pathogens; Staphylococcus
epidermis,non-pathogenic environmental
organism; Ps. Aeruginosa)
Toxins
Products of bacteria that
produce immediate host cell
damage
Growth and solid surfaces
Solidified growth media are deployed to
separate different types of bacteria and
also as an aid to enumerating viable cell
numbers in the laboratory
Agar media are used in the laboratory
either poured as a thin layer into a
covered dish or contained within a small,
capped bottle
The colour, size, shape and texture of
colonies of different species of bacteria
very considerably and form a useful
diagnostic aid to identification
Growth in liquids
Growth ceases when the rate of
consumption of nutrients exceeds the rate
of supply
Bacteria (being of colloidal dimension and
sometimes highly motile, are dispersed
evenly through the fluid (nutrients are
equally available to all cells)
Liquid batch culture (closed)
Logarithmic growth phase (B); during
active growth a logarithmic plot of cell
number against time gives a straight line
Lag period (A); the inoculum adapts its
physiology to that required for growth on
the available nutrients
Late logarithmic phase; rate of growth
Stationary phase (C); eventual halt
Decline phase (D); starvation, death of
some of the cells and adaptation to a
dormant state
BACTERIAL REPRODUCTION AND GROWTH
KINETICS
- Multiplication and division cycle
Binary fission; process where the majority
of cells multiply in number
Each daughter cell will automatically
contain those materials that are dispersed
through the mother cell (mRNA, rRNA,
ribosomes, enzymes, cytochromes, etc.)
Bacterial chromosome; circular and
attached to the cytoplasmic membrane
where it is able to uncoil during DNA
replication
DNA replication if based on the number of
base pairs within it and the growth
temperature (ex. Escherichia coli;
replication of chromosomes will take
approximately 45 minutes)
Gram-negative cells; do not have rigid cell
wall, must develop a cross-wall that
divides the cell into two equal halves
Rod-shaped organisms; maintain their
diameter during the cell cycle and
increase their mass and volume by a
process of elongation
Coccal forms; increase in size by radial
expansion
Population growth
Cell numbers will increase exponentially
as a function of time
Generation time; the time interval
between one cell division and the next
A mean generation time is usually
calculated when considering a growing
culture containing thousands of cells
 F-factor (fertility factor); simplest form of
plasmid
F-factor will simply transfer a copy to a
recipient cell

ENVIRONMENTAL FACTORS THAT INFLUENCE

GROWTH AND SURVIVAL

Gram-negative bacteria tend to be aquatic

Gram-positive bacteria then to prefer
more arid conditions such as the skin
Physiochemical factors that affect
growth and survival of bacteria
Temperature
Permissive temperature; range of
temperatures under which bacteria can Growth in open culture
actively grow and multiply
Chemical and enzymic reactions within Bacteria make up >90% of the dry mass of
the cell proceed more rapidly, and growth faeces
becomes faster until an optimal rate is
achieved as temperature rises In many situations the bacteria become
Beyond such temperature, certain immobilized, as a biofilm, upon a surface
proteins may become irreversibly and extract nutrients from the bulk fluid
damaged through the thermal lysis, phase
resulting in a rapid loss of cell viability
pH
PH effects on growth are bell-shaped
Growth and genetic change
Extremes of pH can be lethal
Growth optima of 7.4 and 7.6;
Transformation
microorganisms that have medical or
pharmaceutical significance
Ability of certain types of bacteria to
May dictate the range of microorganisms
absorb small pieces of naked DNA from the
that could potentially cause its spoilage
environment that may recombine into
Water activity/ solutes
recipient chromosome
Gram-negative cell envelope cannot
Means of transferring genes between
withstand the high internal osmotic
different types of bacteria
pressures associated with rapid
Transduction
rehydration after desiccation
Bacterial DNA having moved between cells
Water activity (Aw); vapor pressure of
Temperate phage; rather than enter a
water in the space above the material
replication cycle, the viral DNA becomes
relative to the vapor pressure above pure
incorporated by recombination into the
water, can markedly affect its
chromosome of the bacterium
vulnerability to spoilage contaminants
Conjugation
Availability of oxygen
Thought to have evolved through
Oxygen acts as the terminal electron
transduction
acceptor in respiration and is essential for
Plasmids; DNA strands
growth
 concentration, do not
discriminate between living
and dead cells
Microcalorimeters; time taken
to detect such heat can be
directly related to the numbers
of viable cells present
Enrichment culture
Intended to increase the dominance of a
numerically minor component of a mixed
culture such that it can be readily
detected on an agar plate
Enrichment media; always liquid, intended
to provide conditions that are favourable
for the growth of other likely isolates
MacConkey broth; contains bile salts that
will inhibit the growth of non-enteric
bacteria and may be used to enrich for
Enterobacteriaceae
Selective media
Solidified enrichment broths,
intended to suppress the growth of
particular groups of bacteria and to
allow the growth of others
Counts of colonies obtained on
selective solid media are often
documented as presumptive counts
Identification media
(diagnostic)
Contain nutrients and reagents that
indicate, usually through some form
of colour formation, the presence
of particular organisms
Microscopy
Simple stains (such as the Gram stain)
Size, shapes, arrangement into clusters,
chain and tetrads, specific stains for the
presence of endospores, capsules, flagella
and inclusion bodies
Fermentation; carbon substrate is in
excess
Nutrition and growth
Chemolithotrops; simple inorganic forms
of elements, can utilize atmospheric
carbon dioxide and nitrogen as sources of
carbon and nitrogen
Diauxic growth; second lag phase during
the logarithmic growth period while such
adaption takes pace
DETECTION, IDENTIFICATION AND
CHARACTERIATION OF ORGANISMS OF
PHARMACEUTICAL AND MEDICAL
SIGNIFICANCE
Culture technique
- Diluting the sample to varying degrees and
inoculate the surface of a predried nutrient
agar with known volumes of those dilutions
- Enumeration media
Will only ever culture a subset
of cells towards which the
medium and incubation
conditions are directed
Simple salts media with
relatively simple sugars as
carbon sources and trace levels
of amino acids; often used to
enumerate bacteria associated
with water
Psychrophilic Gram-negative
bacteria; can be a major
source of bacterial pyrogen
Highly nutritious media are
also used as enumeration
media (ex. blood agar)
Rapid enumeration techniques
Bioluminescence
Epifluorescence
Impedance techniques
Coulter counters; used to
determine bacterial
 o Bread Biochemical testing and rapid
identification
o Enzymes
Differing ability of bacteria to ferment
sugars, glycosides and polyhydric alcohols
o Antibiotics
(widely used to differentiate the
o Recombinant proteins Enterobacteriaceae and in diagnostic
bacteriology generally)
FUNGI Results of oxidase and catalase tests
performed directly on isolated colonies
O A B T U D
O S A E S E Molecular approaches to identification
M C S L T U Have not yet become routinely adopted in
Y O I I O T the analytical or diagnostic laboratory
C M D O M E Denaturing gradient gel electrophoresis
E Y I M Y R (DGGE); isolates and amplifies 16S
T C O Y C O ribosomal DNA and, following sequencing
E E M C E M of the bases, compares this with known
S T Y E T Y sequences held in a reference library
E C T E C Gene probes carrying fluorescent dyes;
S E E S E
can be used in hybridization procedures
T S T
with the collected clinical material
E E
S S
Pharmaceutically and medically
relevant microorganisms
Broadly classified into those organisms
that are harmful or problematic, and
The Kingdom Fungi can be subdivided into six
those that can be used to our advantage
classes:

Oomycetes Refer to Table 3.3 of book; examples

of some pharmaceutically useful bacteria
-contains the mildews and water moulds

Ascomycetes

-contains the mildews, some moulds and Chapter 4: Fungi

most yeast species (including
Saccharomyces cerevisiae) Fungi
Basidiomycetes Eukaryotic organisms

-contains the mushrooms and bracket fungi Widely distributed in nature

Teliomycetes
Extremely important group of microbes in
the medical field
-contains the rust fungi (plant pathogens)
Responsible for a number of potentially
Ustomycetes
fatal diseases in humans
-contains the smuts (plant pathogens)
Great benefit in humans in terms of:
Deuteromycetes
o Production of alcoholic beverages
 Average thickness varies from 100 to 300
COMPOSITION OF FUNGAL CELL
nm WALL

S P
T R
Cell Wall R M O
G C T
U A
Glucan, the main structural component of L H E
C N
U I I
fungal cell wall, is a branched polymer of T N N
C U T
glucose. A S
A R I
N AND
L
N A N
The innermost layer is rich in glucan and chitin I
50- L
which provides rigidity to the wall and its 60% P
POLYSACCHARIDES
1-9% I
15-23%
important in regulating cell division. 25 D
% S

-contains species such as Aspergillus,

Fusarium and Penicillium

There are four distinct phyla within the fungal

kingdom; these are the Chytridiomycota,
Zygomycota, Ascomycota and Basidiomycota.
Periplasmic space

a thin region that lies directly below the

cell wall Yeast

contains secreted proteins Grow as single cells

location for a number of enzymes required Reproduce asexually by budding, although

for processing nutrients prior to entry into a minority of species reproduce by fission
the cell
Many yeast species are capable of sexual
production and formation of spores

Cell membrane / Plasmalemma Moulds

located directly below the periplasmic Grow as masses of overlapping and

space interlinking hyphal filaments

a phospholipid bilayer which contains Reproduce by producing masses of spores

phospholipids, lipids, protein and sterols.

Approximately 10 nm thick
Structure of the fungal cell

Oval in shape
Nucleus
Surrounded by a rigid cell which contains
A discrete organelle structural polysaccharides
 suppressed as a result of therapy or
disease.
The most common fungal pathogens:
o Yeasts
o Moulds
o Dermatophytes
Medically important fungal pathogens of
humans
Candida albicans
o Most frequently encountered
human fungal pathogen
o Responsible for a wide range of
superficial and systemic infections
o Opportunistic fungal pathogen
which can be present as a normal
part of the bodys microflora
o Exist in two morphologically
distinct forms: budding
blastospores or hyphae
o Plays an important role in the
degradation of the
immunoglobulins IgG and IgA
o Important in allowing yeast access
iron released from ruptured red
blood cells
o Capable of giving rise to a variety
of interconvertible phenotypes
which can be considered as
providing an extra dimension to the
existing virulence associated with
this yeast.
Aspergillus fumigatus
o a saprophytic fungus
o the dominant fungal pulmonary
pathogen of humans
Most of the cells genome is concentrated
in the nucleus
Repository of the DNA
Contains proteins in the form of histones
Mitochondrion
The powerhouse of the cell
Possesses its own DNA
Capable of producing its own proteins on
its own ribosomes, mitoribosomes
Enzymes of tricarboxylic acid cycle are
located in the matrix of the mitochondrion
Electron transport and oxidative
phosphorylation occur in the mitochondrial
inner membrane
The outer membrane contains enzymes
involved in lipid biosynthesis
Ribosomes
The site of protein biosynthesis
Mediates the export of proteins from the
cell
Vacuole
A storage space
Nutrients, hydrolytic enzymes or metabolic
intermediates are retained until required
Medical significance of fungi
A significant group of pathogens capable of
causing a range of diseases
Although majority of fungi appear to be
harmless to humans but a normally non-
pathogenic fungus can cause a clinically
relevant problem if the immune system is
 be consumed directly as a dietary
supplement
o No longer regarded as a GRAS
(generally regarded as safe) but is
now classified as a Biosafety Level
1 pathogen
o Obstructs capillaries particularly in
the brain with concomitant damage
to surrounding tissues
Penicillium marneffei
o Very rare and inconsequential
cause of disease in humans
o The most frequent cause of fungal
disease in AIDS patients who reside
in, or visited, South East Asia
o An asexual, dimorphic fungus
growing as a mycelium at 37C in
tissue and as single cells at 28C
o Reproduce by fission
Non-albicans Candida species
o Candida dubliniensis
-dominant cause of oral candidosis
of HIV-positive and negative
populations from many parts of the
world
o Candida krusei
-significant cause of disease in HIV-
positive patients, diabetics, and
Emerging fungal pathogens
cancer patients
-dominant fungal pathogen in
certain classes of diseases
o Candida glabrata
-a serious cause of disease in
neutropenic cancer patients and
o generally presents a problem in
those with pre-existing lung
disease or damage
o frequently encountered growing on
decaying vegetation and damp
surfaces
Histoplasma capsulatum
o a dimorphic fungus
o cause of histoplasmosis, the most
prevalent fungal pulmonary
infection
Cryptococcus neoformans
o an encapsulated yeast
o most frequently associated with
infection in immunocompromised
patients
o meningitis is the most common
clinical manifestation
o a facultative intracellular pathogen
that is capable of surviving and
replicating within macrophages and
withstanding the lytic activity
within these cells
Dermatophytes
o group of keratinophilic fungi which
can metabolize keratin
o the principal protein in skin, nails
and hair
Saccharomyces cerevisiae
o Better known as brewers yeast
or bakers yeast
o Used for the production of bread
and alcoholic beverages, and can
 Viral nucleic acid:
- The viral nucleic acid is composed of
either DNA or RNA
- A virus can only have a single genome
Viral capsid (protein core)
- It protects the viral nucleic acid from
detrimental, chemical and physical
conditions
- It is composed of a number of subunits
named capsomeres genetically
encoded by the viral genome
- Capsomeres give the shape of the
capsid, and provide the virus with
resistance to physical and chemical
agents
Viral envelope
- Most outer covering of a virus
- The envelope is added during the
replication process
- It can come from the host cell nuclear
membrane, or the cytoplasmic
membrane
- Enveloped viruses are considered to be
the most susceptible to chemical and
physical conditions
- They do not survive well on their own
outside the host cell, although they
can persist longer in organic soil
III. Virus-host cell interactions
Viruses can interact with the host cell in
five different ways:
1. Multiplication of the virus and
destruction of the host cell upon
release of the viral progeny
has been responsible for mortality
rates of 5-38%
-Fourth most commonly isolated
Candida species
Antibiotic production of fungi
Majority of antibiotics obtained from fungi
are produced by fermentation and most
are secondary metabolites.
Isolation of Penicillium notatum by Sir
Alexander Flemming Most important
discovery regarding the beneficial use of
fungi for humans
Antibiotic production can be maximized by
optimizing production as a result of
random mutagenesis and selection.
Chapter 5: Viruses
I. Introduction
Viruses were first discovered at the end of
19th century
They were classified as filterable agents
because they can be retained by filtration
II. General structure of viruses
Viruses are extremely diverse in size and
shape
Viruses are much smaller than bacteria
Intracellular parasites
Smallest virus: poliovirus -> 28nm in size
Largest virus: mimivirus -> 750nm in size
Viruses with envelope: enveloped
nucleocapsid
Viruses with no envelope: naked
nucleocapsid Components of a virus:
 - Envelope fuses from
the membrane
- It is a process wherein
virions can fully enter
the membrane
o Endocytosis: When an
envelope nucleocapsid fully
enters the host cell, it
undergoes endocytosis.
- Envelope fuses with the endosome
IV. Multiplication of human viruses
Uncoating - releases capsin to
free nucleic acids (DNA or RNA)
Eclipse stage: replicates and
expresses genes
Assembly or maturation -
maturation of virus cells; cells
become virions again
Release - virions are release
outside the host cell, to infect
other cells
V. Cultivation of human viruses
1. Cell culture:
May be divided into three types
according to their history:
Primary cell lines: The cell
lines are derived directly from
an intact tissue, ex: human
embryo, kidney or monkey
kidney
Secondary cell cultures:
derived from primary cultures,
usually those arising from
embryonic tissue
- The cells are more
homogenous, better
2. Multiplication of the virus and release
of the virions without the immediate
destruction of the host cell
3. Survival of the virus in a latent stage
without noticeable changes to the
infected cell
4. Survival of the infected cell in a
dramatically altered or transformed
state
5. Incorporation of the viral nucleic acid
in the host cell genome without
noticeable changes to the infected cell
Objective of replication cycle: to
ensure the multiplication of the virus
with the formation of identical viral
progeny
The multiplication cycle of human
viruses is generally slow, from 4 to
more than 40 hours
Bacterial viruses are generally faster
and can take a little as 20 minutes to
replicate within the bacterial host
There are six distinct phases in a
replication cycle:
Adsorption - attachment to the
host cell receptor
Penetration - penetrates the
virus through the cell
membrane. It has three types:
o Direct injection: type of
virus involved -> naked
nucleocapsid
- the virus did not fully
enter the host cell
o Fusion: type of virus
involved -> envelope
nucleocapsid
 - The drug used for HIV characterized, but might not
infections is called be as susceptible to viral
antiretrovirals infection as primary cell lines

- Antiretrovirals have Continuous cell lines: usually

considerably prolonged the life derived from malignant tissue,
expectancy of patients, and have the capacity to
although not without some side multiply indefinitely in vitro
effects
Cytophatic effect: a characteristic
- These drugs aim to reduce HIV morphological change in the
plasma level as much and as infected cells wherein the cells
long as possible shrink, or undergo ballooning.

They usually spread to adjacent

a Herpesvirus infections cells and will result in the
formation of a plaque that can
- Herpesviridae: a family of easily be identified following
viruses which include the staining
herpes simplex virus,
chickenpox, shingles and Plaques: used for the enumeration
cytomegalovirus of viruses

- Mild herpes simplex virus ->

treated with a topical antiviral 2. The chick embryo
drug
Fertile chicken eggs, (9-11 days old)
- Primary herpetic are used to grow a number of
gingivostomatitis -> a change human pathogenic viruses.
of diet & analgesics

- Severe infections -> systemic

antiviral is used 3. Animal inoculation

- Antiviral treatments for Animals are used to culture certain

chickenpox: recommended in viruses in order to study antiviral
patients at risk and in neonates vaccine effectiveness, and also as a
to reduce risks of severe source of cell lines for cell cultures
diseases

- Antiviral treatments for

VI. Control of viruses
herpes: is associated with a
number of side effects which 1. Antiviral chemotherapy - leads to the
may vary depending on the development of viral resistance, but is still
drug associated with a number of problems
- Antiviral treatments for a. HIV
cytomegalovirus: usually given
to immunocompromised - The role of antivirals in HIV is
patients and they tend to be to slow or halt disease
more toxic with noticeable progression
nephrotoxicity and a number of
side effect
 influenza vaccines rely on
chemically inactivated, virus
particles or components
Inactivated viruses
Use of viral components
Hepatitis B: viral DNA encoding for a
virus surface antigen expressed in
yeasts
Immunoglobulin: plays a role in the
protection of patients with a
compromised immunity against viral
infections
IM immunoglobulin used to protect
against hepa A virus
1 Viricidal effects of chemical and physical
agents on viruses
Viruses are generally transmitted
via surface and are often
associated with organic materials
In general, viruses are not
particularly resistant to chemical
or physical agents, although some
exceptions exist.
1 Control of viruses in pharmaceutical
products
2. Vaccination
Presence of certain viruses needs
to be controlled
Risk of a pharmaceutical product
being contaminated by viruses
depends on:
The origin of the product
component
The history of the donor
The amount of material used
The manufacturing process
Its capacity to remove or
destroy contaminants
a Viral hepatitis
- Treatment for acute hepatits B
-> interferons
- Treatment for chronic hepatitis
B -> antivirals
a Influenza
- Two major limitations in the
usefulness of the drug
- First: the drug needs to be
taken within a few hours of the
onset of symptoms from mild
to severe symptoms reported
- Second: the side effects have
been very severe
a Respiratory syncytial virus
- RSV is responsible for severe
bronchiolitis notably in infants
- Treatment can be a
monoclonal antibody or an
antiviral drug
the most successful measure against
microbial and viral infections
- Vaccines are preparations
containing antigents that elicit
a specific and active immunity
against an infecting agent
- Vaccines can induce the innate and the
adaptive parts of the immune system
- Viral vaccines prepare using 3 methods:
Live attenuated viruses: will
cause a strong immune
response without causing the
disease Hepatitis A and
 1 Use of bacteriophages to treat bacterial
infection
VII. Viruses as antimicrobials
Introduction of antibiotics in the
early 1940s resulted in the end of
phage therapy in the West
o Natural phages
o Non-replicating phages
o Genetically modified phages
Use of phages for surface
disinfection and antisepsis: further
work is still needed to develop
appropriate phage-based products,
especially the effect of the
different routes of administration
on phage viability and effectiveness
1 Epidemiological uses and diagnosis
Phage typing: a method that
differentiates distinct strains of the
same bacterial species on the basis
of their susceptibility to phages
VIII. Prions
Prions devoid of nucleic acid and are
extremely resistant to heating and
ultraviolet irradiation
They fail to produce an immune response
in the host
1. Bacteriophages
Bacteriophages are viruses that
infect only bacteria
They were first described at the
end of the 19th century
Size: 20-200nm, and are highly
diverse in their structure and host
range and it is likely that all
bacterial species can be infected
by a phage
Phages are extremely specific in
their host range and some will only
infect a specific bacterial strain
Most studied phages are the
complex ones, tadpole-shaped
which consists of a head that
contains the viral genome, and a
tail which function is to recognize
the host receptor, attach and
subsequently serve as a nucleic
acid injection device
Two phage replication cycles:
Lytic cycle: lysis of bacterial host
Lysogenic cycle: result of a viral
nucleic acid being integrated into
the host genome
Infection with lytic phage ->
virulent phage, results in the
replication of phage within the
susceptible bacteria and the release
of infectious phage progeny from
the host cell following cell lysis
Lysogenic cycle: viral nucleic acid
which has integrated the host
genome is called prophage
Host cell that contains the viral
genome: lysiogenic