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Chapter 1: Introduction to
Pharmaceutical Microbiology
- A modern medicine must be effective,
safe, and of good quality
- These medicines consists of activeingredients, stable, and safe during storage
- Analytical Chemists and Pharmacists
Pharmaceutical Microbiology
- Foundation
- Encompasses the subject of steriliation
and preservation against microbial spoilage
- Pharmacist must be responsible for the
safe and hygienic manufacturing of
medicines
Antibiotics
- !ajor importance in pharmacy
- "aturally occurring substance that #ould
inhibit or $ill microorganisms
- !icrobial metabolite
- %ynthetic agents that are normally used
systematically to treat infection
- Antibiotic production began #ith the
discovery of penicillin in the &'()s
- Attac$ and $ill bacteria #ithout harm to
the host
Ribotyping
- *enetic technique used to identify cross-
infection, reduce transmission and optimie
management of hospital-acquired infections
- +nderstanding the physiology and genetics
of microorganisms are important to
produce therapeutic agents
- no#ledge of mechanisms #hereby
microorganisms are able to resist
antibiotics, colonie medical devices are
essential in the production of ne# drugs
and in healthcare practices
Chapter 2: Fundamental features of
microbiology
o MICROORA!I"M"
- microscopic, living, single-celled
organisms
-more versatile than mammals in brea$ing
do#n foods
Differ in:
&%hape
.%ie
/*enetic Characteristics
( !etabolic Characteristics
- !ajor groups0
o 1acteria
o Fungi
o Protooa
o 2iruses
C#A""IFICA$IO! OF MICROORA!I"M":
o %iruses
& do not have cellular structure
& Composition0
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o "ucleic acid surrounded by
proteins
o %ome posses lipid envelope
associated #3 glycoprotein
o Absent0
-recogniable chromosomes
-cytoplasm
-cell membranes
-incapable of independent replication
- intracellular parasites
-reproduced using metabolic capabilities of
host cell
-smaller than bacteria
- 2ariation in0
o %hape 4helical, linear, or
spherical5
o %ie 4.)-())nm5
o "ucleic acid composition 4single
or double-stranded,linear or
circular 6"A or 7"A5
-2ie#ed using electron microscope
o %iroids '%irusoids(
-simplier than viruses
-infectious particles
-single stranded 6"A #3o associated
proteins
E8 Plant phatogens
o Prions
-infectious agents
-"o nucleic acid
-atypical form of mammalian protein
-can interact #3 normal protein molecule
and cause it to undergo conformational
change and ma$e it into a prion and ceases
normal function
-responsible for transmissible spongiform
encephalopathies
E8 Creutfeldt-ja$ob disease
1ovine spongiform encephalopathy
-simplest and most recognied agents of
infectious disese
-e8treme resistance to conventionall
steriliing agents 4steam,gamma
radiation,disinfectant,etc5
PRO)AR*O$IC MICROORA!I"M":
- "o true nucleus
- +sually single chromosomes
- 9aploid
- Ase8ual reproduction
E8amples 0 1acteria, Archea
o Archea
-no pharmaceutical importance
-capable of living in e8treme environment
-e8hibit specialied modes of metabolism
o +acteria
&unicellular
-posses pro$aryotic properties
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-e8hibit great diversity in form, habitat,
metabolism, pathogenicity
-1acteria of interest in medicine and
pharmacy0 Eubacteria
-many bacteria #ould be described as
facultative anaerobes or microaerophils
-most bacteria important in medicine and
pharmacy 0
✓ posses cell #all
✓ gro# #ell at temperatures
bet#een ambient and human
body temperature
✓ e8hibit #ide variation in
requirement for o8ygen
%trict aerobes : require
atmospheric o8ygen
%trict anaerobes : o8ygen is to8ic
E8amples0
,ubacteria
& 1acteria of interest in medicine
and pharmacy
& types0
& 1acillus- rod shaped
. Cocci-spherical
/ Curved or spiral cell
appro8imately ); to ; mm
- 7ivided into t#o groups acc to
Christian *ram
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- Fungus0 covers both terms yeast and
moulds
*east
- normally unicellular
- divide by budding
- larger than bacteria
- divide by binary fission or budding
Moulds
- imprecise term to describe fungi
that doesnrganism responsible for malaria and
ameobic dysentery
*for further comparison of eukaryotes and
prokaryotes check Table 2.1 page 11
!AMI! OF MICROORA!I"M":
- $no#n by t#o names genus and species
- #ritten in italic or underlined
MICRO+IA# M,$A+O#I"M:
!icroorganisms are more versatile than humans
in brea$ing do#n food, !any can use alternative
methods in brea$ing do#n 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 brea$ing do#n
organic compounds
- organisms of interest in pharmacy
in medicine
o
Catabolic Reactions- energy is liberated by digestion of
food materials
o Anabolic Reactions
& use liberated energy to ma$e
comple8 cellular
polymers,protein,carbohydrates,
and nucleic acids
o O0idation
- removal or loss of electrons
- #hen food materials are o8idied
energy are released
- does not invariably involve o8ygen
- #hen o8idiing molecules accept
electron the other molecule is
reduced? reduction and o8idation is
lin$ed and called Redo0 reaction
o Redo0 Potential
- indicates #hether o8idation or
reduction #ill prevail
- Anaerobic organism : lo# redo8
potential
Aerobes : high redo8 potential
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o O0idi/ing Agents
- molecules that can accept
electrons
o lycolysis
- brea$do#n of glucose to release
energy
- metabolic path#ay used by
pathogenic bacteria and mammals
- conversion of glucose to series of
reactions to pyruvic acid, o8ygen is
not required
- underta$en by both aerobic and
anaerobic
- release small amount of energy
stored in sugar molecule
o Aerobic respiration
- +sed by mammals to release more
energy in sugarcompared to
glycolysis
- >8ygen 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 #hich in #hich the
final electron acceptor is inorganicmolecule
- production by yeast of ethanol and
carbon dio8ide from sugar
- many organisms can be used as
apart from yeast and is not
restricted to common sugar
4sucrose5
%ome pathogenic bacteria can
ferment0
different sugar, acids, alcohols,
acetone, butane, etc
o Fundamental Principle of Antibiotic
Action
- drug must e8ploit a difference in
metabolism bet#een organism to be
$illed and the human host
- #3o difference it #ill be very
to8ic to the patient
o Primary metabolites
& metabolic products that arise
during period #hen microbial
culture is gro#ing
E8 ethanol, organic acids
o "econdary metabolites
- diverse
- have commercial or therapeutic
importance
- produced after the cell
multiplication has stopped
E8 Antibiotic,enymes,to8ins,
carbohydrates
MICRO+IA# C.#$I%A$IO!
C.#$.R, M,IA
- %ome microorganisms have different
sugar fermentation patterns
- %ugars in culture media are usually used
for identification purposes
- "aCl may be incorporated to adjust
osmotic pressure
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- For yeast and moulds have lo#er p9 4;;-
@)5
- actic acid0 used to impart lo#er p9
C#A""IFICA$IO! +A", O!
COMPO"I$IO!:
1 $ruly "ynthetic Media
- chemically defined
- for microorganisms that
can synthesie materials
needed for gro#th from
simple carbon and nitrogen
2 Media 34 biochemicals
- used for organism that
can
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& Dn optimal conditions of laboratory
cultivation of bacteria this division
ta$es place every .;-/) minutes
- gro#th continues until one or more
nutrient is e8hausted or to8ic
metabolites accumulates
o O6ernight incubation in li5uid 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 microscope0 hundreds to
millions
- typically &-&) mm
- Periphery of colony? part that is
actively gro#ing
o Petri dishes incubated in a anaerobic 8ar
- here anaerobic microorganisms
gro#s
o Plan-tonic Cells
- 6outinely used to testing procedures
designed used to assess the activity
- different susceptibilities of lethalagents
- 6eappraisal appropriate
,!.M,RA$IO! OF MICROORA!I"M"
- %everal situations #here number of microbial
cells in culture, sample or specimen are needed to
be measured0
• !easuring levels of microbial
contamination in ra# material or
manufactured medicine
• Evaluating the effects of
antimicrobial chemical or
decontamination process
• +sing microorganisms in
manufacture of therapeutic agents
• Assessing the nutrient capability of
gro#th medium
-Dn pyrogen testing and vaccine manufacture both
number of dead and living cells are required
o $otal count
is a counting procedure enumerating both
living and dead cells
o %iable count
-records living cells alone
o $otal %iable Count '$%C(
-used in most pharmacopoeias and by many
regulatory agencies
-mean a viable count that records alldifferent species or types of microorganism
that might be present in sample
$hree $raditional Methods of %iable
Counting:
Basis0 iving cell #ill give rise to colonies #hen
introduced #3 suitable medium and incubated
1Pour Plating
& surface spread method used
2 Miles Misra Method
& %urface drop
& membrane filter methods used
9 M"P 'Most Probable number(
& anticipated count is very lo#
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- more commonly used in #ater,
food, and dairy
- poor accuracy
- last resort
G for more information about methods of iable
counting check page 1" Table 2.2 and page 1#
Table 2.$
A*" $O M,A".R, +IOMA"":
1 $urbidity Measurements
& most common used in estimating the total
number of bacteria in sample
- measured using spectrophotometer or
colorimeter
- not used in fungi
2 ry eight etermination
-for fungi biomass
9irect Microscopic Counting
- for bacteria, yeast , and fungal spores
- not for moulds and indirect measure of
biomass
#imitations of traditional method of 6iable
counting:
• 6elative labour intensive
• "ot easy to automate
• %lo# due to to the need for incubation
• !ay require relative large volumes of
culture media many petri dishes and
incubator spaces
Rapid Methods of detecting and counting
microorganisms:
& enumerate viable organisms4usually
bacteria and yeast5
- employ various means of indirect
detection of living cells
- fast ,readily automated, and eliminates
long hours of incubation and numerous petri
dishes
- not capable of reproducing colonies
Operating method principles:
o Epifluorescent Techniques
o iving cells generate ATP
o !anometer Techniques
o 6esistance and capacitance or
impedance of culture media
MICRO+IA# ,!,$IC"
- *enetic material may be transferred
depending if organism is pro$aryote or
eu$aryote
- !utation is very important for resistance
of antibiotics
o +acteria 'Pro-aryote(
& genes for gro#th and metabolism0
chromosomes of double stranded
7"A
+acterial Chromosomes
& tightly coiled
- &mm contain &)))-/)))genes
- additional genes for
survival advantage under
certain circumstances0
Plasmids
Plasmids
& smaller and replicate
independently
& )&-&B sie of bacterial
chromosomes
& not essential for normal
functioning
& replicate independently
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& can be passed to one cell
or another by various means
enes recei6ed from other sources:
o +acteriophages
- %enome of !ell: 1acterial Chromosomes H
Plasmids H 1acteriophages
o ,u-aryote
- nucleus that contain one or more
pairs of linear chromosomes
- 7"A comple8ed #3 protein
- Cells may divide ase8ually and
undergo mitosis but many have thepotential to undergo se8ual
reproduction and undergo meiosis
- based on 6"A instead of 7"A
- possibility of creating ne# gene
combination
o enotype
& describes genetic composition
regardless if e8pressed or not
o Phenotypic Adaptation
- non-genetic adaptation
- bacteria adopt a phenotypic
change to counter environmental
stress
o enetic Adaptation
& Acquire ne# genes either by
mutation or conjugation
- process of selection ensure that
mutant organisms that are better
suited for ne# environment
becomes numerically dominant
P7ARMAC,.$ICA# IMPOR$A!C, OF MA;OR
CA$,ORI," OF MICROORA!I"M"
o %iruses
- importance is based on Pathogenic
potential
- not susceptible to antibiotics
- 9aard Category (
- easy to destroy by heat, radiation,
or to8ic chemicals
o Prions
& #ithstand steriliing conditions
- ability to cause incurable andfatal disease
o +acteria
- important as pathogens
- ability to resist activity of
antibiotics and biocides? long
standing notoriety
- streptomycetes bacteria produce
antibiotics
- gro# on diverse substrates ensure
potential as agents of spoilage
- survive #ell in drying, dust, and
other adverse environments
- contaminants
- can produce bacterial spores
o Fungi
& survive in drying
& produce spores
- contaminants
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- ess degree of resistance than
bacteria
- little threat to immunocompetent
individuals
o Proto/oa
- significantly large o#ning to the
pathogenic potential of fe# species
- 7o not poses cell #all
- 7o not survive drying #ell
- 7o not display resistance to
steriliation to match bacterial
spores
- !ore troublesome in veterinary
PR,",R%A$IO! OF MICROORA!I"M"
- &anufacture of &edicines: microorganisms are
employed in variety of test and assays to
measure activity of antimicrobial chemicals
- 'im of !ulture (reseration: maintain viability of
the highest possible percentage of cells and tominimie ris$ of selecting atypical mutants
- *ram positive bacteria tends to survive better
than gram negative ones
Most common procedure for long term storage:
& Freeing at -=)C in refrigerators
. %torage in liquid nitrogen at -&'@C special
vessels
/ yophiliation or freee-drying
o Cryoprotectant Chemicals
-compounds li$e glycerol or
dimethylsulpho8ide
- incorporated at concentrations
&)B v3v in liquid culture of
organisms in order to minimie both
formation of damaging ice crystals
and osmotic stress that accelerate
cell death during freeing and
tha#ing
Chapter 9: +acteria
Pro-aryotes
- %mallest free living organism 4bacteria
and archaea5
- ac$ a true nuclear membrane
G )ukaryotic cells presence of a nuclear
membrane and internal
compartmentaliation
!ajor feature0 cytoplasm of membrane-
enclosed organelles
ifferences '+acteria and Archaea(:
- Cell #all composition 4major difference5
- ipid structure ma$ing up their
cytoplasmic membranes
- !etabolic patterns
Bacteria:
- Vast majority of
prokaryotes of medical
and pharmaceutical
significance
Archaea:
- most are anaerobes
- inhabit extreme
environments- greater stability under
extreme conditions
- no disease-causing
archaea have yet beenidentified
+acteria
- 6epresent a large diverse group of
organism that can e8ist as single cells or as
cell clusters
- 9ave the ability to carry out their life
processes of gro#th, energy generation and
reproduction independently of other cells
G 2ery different from the cells of animals
and plants 4unable to live alone in nature,
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e8ist only as a part of a multicellular
organism5
- Capable of gro#ing in a range of different
environments
- Cannot only cause contamination and
spoilage but also a range of different
diseases
+acterial i6ersity and .bi5uity
- 1acteria are diverse in shape and sies
4morphology5, adaptation to environment,
survival strategies, and metabolic processes
- The presence of bacteria may be
considered ubiquitous There is no natural
environment that is free from bacteria
Cell "i/e and "hape
- !ajority of bacteria are &-; um long and
&-. um in diameter
G I; um 4e80 Thiomargarita namibiensis5
e8tremely rare
G ↓bacterial sie ↑increased, efficient, and
rapid transport and gro#th rates
- Classification of bacteria is made through
morphological grounds
- !ostly unicellular and possess simple
shapes0 round cocci+, cylindrical rod,
bacillus+, or ovoid
Rarer morphological forms:
a 'ctinomycetes
rigid bacteria resembling fungi that may
gro# as lengthy branched filaments
b &ycoplasmas
lac$ a conventional peptidoglycan
4murein5 cell #all
highly pleomorphic organisms of indefinite
shape
c ome miscellaneous bacteria stal$ed,
sheathed, budded, and slime producing forms
often associated #ith aquatic and soil
environments
Cellular components
- %imple base cell structure compared #ith
eu$aryotic cells
Reasons to ha6e a good -no3ledge of the
bacterial cell structures and functions:
- Provides an e8cellent route for probing the
nature of bacterial processes many of #hich are
shared by multicellular organisms
- "ormal bacterial processes can be customied tobenefit society on a mass scale
- To $no# ho# to destroy bacterial contaminants
and disease-causing organisms 4pharmaceutical and
healthcare perspective5
Cell all
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- Essential for the maintenance of the shape
and integrity of the bacterial cell
- An obvious target for antibiotics 4Cell
ysis5
- Provide a strong, rigid structural
component that can #ithstand the osmotic
pressure caused by high chemical
concentrations of inorganic ions in the cell
- !ost bacterial cell #alls have
peptidoglycan layer 4murein3 glycopeptide5
e8ceptions include the !ycoplasmas,
e8treme halophiles, and the archaea
- Peptidoglycan is composed of acetyl
muramic acid 4"A!5 and acetyl
glucosamine 4"A*5
+acteria can be di6ided into t3o large
groups J 4on the basis of a differential staining
technique called the %ram stain50 %ram-positie,
%ram-negatie
ram&positi6e Cell all
- Consist primarily of a single type of
molecule
- Contains teichoic acids and lipoteichoic
acids 4negatively charged5
- 7uring an infection, lipoteichoic acids
molecules trigger an inflammatory response
- 6etain the dye 4gram stain5
- Appear purple under the light microscope
ram&negati6e Cell all
- !ultilayered structure, quite comple8
- Compose of proteins, lipoproteins,
phospholipids and lipopolysaccharide that
are unique to gram negative bacteria
- P% determines the antigenicity of the
bacteria
Ge8tremely to8ic to animals
- P% is made up of lipid A, core
polysaccharide and >-specific
polysaccharide
- The cells lose the crystal-violet iodine
comple8 and are rendered colorless 4gram
stain5
- Appear red under the light microscope
Cytoplasmic membrane
- Fragile phospholipid bilayer #ith protein
distributed randomly throughout
- Dnvolved in various transport and enyme
functions associated #ith the membrane
- Transports of nutrients, energy generation
and electron transports
- %elective barrier bet#een the cytoplasm
and the cell environment
Cytoplasm
- Consists of =)B #ater and containsenymes that generate ATP
- Compose of the ribosomes, nucleoid and
inclusion granules
!ucleoid
- %ingular, covalently closed circular
molecule of double stranded 7"A
Plasmids
- 6elatively small circular pieces of doublestranded e8trachromosomal 7"A
- For autonomous replication
- Encode many au8iliary functions that are
not usually necessary for bacterial gro#th
4antibiotic resistance5
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- transfers, increasing the spread of
resistance
Ribosomes
- The site of protein synthesis
- T#o subunits0 /)% and ;)%
Inclusion granules
- %erves as the storage material for carbon,
nitrogen, and sulphur or phosphorus
Cell surface components
Flagella
- 1acterial motility
a &onotrichous a single polar flagellum
b /ophotrichous t#o or more flagella at one pole
of the cellc 'mphitrichous single3tuft of flagella at each
end of the cell
d (eritrichous flagella distributed over the
entire cell
Pili and Fimbriae
a (ili pilus+ join bacterial cell in preparation of
7"A and to environmental surfaces
involved in the genetic e8change
process of conjugation
b 0imbriae for adherence of cells to one anotherand to environmental surfaces
responsible for hemaglutination
and cell clumping in bacteria
lycocaly0 '"lime #ayer and Capsule(
- *eneral substances that surround cells
- *elatinous polymer of polysaccharide,
polypeptide, or both
a %lime ayer unorganied and loosely attached
to the cell #allb Capsule substance is organied and firmly
attached to cell #all
+iofilms
- Any surface for microbial habitat
- +sually contains more than one species of
bacteria #hich e8ist and cooperate
together
- 1iofilm formation begins #ith attaching to
surface and form cement cells to protect
the bacteria from haardous materials
+AC$,RIA# "POR.#A$IO!
- Process in #hich the vegetative cell undergoes aprofound biochemical change to give rise to a
specialied structure called an endospore or spore
- "ot part of a reproductive cycle
"pore
• 9ighly resistant
• Enables producing organism to survive in
adverse environmental conditions 4lac$ of
moisture or essential nutrients, e8posure
to to8ic chemicals, radiation or high
temperatures5• All steriliation process for pharmaceutical
products have been designed to destroy
the bacterial spore
,ndospore structure
,ndospores
• 7ifferentiated cells that possess a
grossly different structure to that
of the parent vegetative cell in
#hich they are formed
,0osporium
• >utermost layer
• Composed of protein? #ithin are
the spore coats 4proteinaceous but
#ith a high cysteine content5
Corte0
• Consists of loosely cross-lin$ed
peptidoglycan
Central core
• Contains the genome
• Partially dehydrated 4dehydration
sho#n to increase resistant to both
heat and chemicals5
• Containing only &)-/)B of the
#ater content of the vegetative
cells
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• p9? & unit lo#er than the
cytoplasm of the vegetative cell
and contains high levels of core-
specific proteins that bind tightly
to the 7"A and protect it from
potential damage
• Core-specific proteins? function as
an energy source for the outgro#th
or germination of a ne# vegetative
cell from the endospore
"pore
• Presence of dipicolinic acid and
high levels of calcium ions #hich
comple8 together
,ndospore formation
K 2egetative cell undergoes a comple8 series of
biochemical events in cellular differentiation
K %porulation? accomplished by activation of a
variety of spore-specific genes such as spo and
ssp
K eads to the production of a dry, metabolically
inert but e8tremely resistant endospore
Endospore germination
K 6eversion of endospore bac$ to a vegetative cell
K 6emoval of the stress inducer that initiatedsporulation
K *ermination loss of resistance properties? occurs
along #ith a loss of calcium dipicolinate and
corte8 components, and degradation of the core-
specific proteins
+AC$,RIA# $O
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+AC$,RIA# R,PRO.C$IO! A! RO$7
)I!,$IC"
- !ultiplication and division cycle
K Binary fission? process #here the majority
of cells multiply in number
K Each daughter cell #ill automatically
contain those materials that are dispersed
through the mother cell 4m6"A, r6"A,
ribosomes, enymes, cytochromes, etc5
K 1acterial chromosome? circular and
attached to the cytoplasmic membrane
#here it is able to uncoil during 7"A
replication
K 7"A replication if based on the number of
base pairs #ithin it and the gro#th
temperature 4e8 )scherichia coli?
replication of chromosomes #ill ta$e
appro8imately (; minutes5
K *ram-negative cells? do not have rigid cell
#all, must develop a cross-#all that
divides the cell into t#o equal halves
K 6od-shaped organisms? maintain their
diameter during the cell cycle and
increase their mass and volume by a
process of elongation
K Coccal forms? increase in sie by radial
e8pansion
Population gro#th
K Cell numbers #ill increase e8ponentially
as a function of time
K *eneration time? the time interval
bet#een one cell division and the ne8t
K A mean generation time is usually
calculated #hen considering a gro#ing
culture containing thousands of cells
ro3th and solid surfaces
K %olidified gro#th media are deployed to
separate different types of bacteria and
also as an aid to enumerating viable cell
numbers in the laboratory
K Agar media are used in the laboratory
either poured as a thin layer into a
covered dish or contained #ithin a small,
capped bottle
K The colour, sie, shape and te8ture of
colonies of different species of bacteria
very considerably and form a useful
diagnostic aid to identification
ro3th in li5uids
K *ro#th ceases #hen the rate of
consumption of nutrients e8ceeds the rate
of supply
K 1acteria 4being of colloidal dimension and
sometimes highly motile, are dispersed
evenly through the fluid 4nutrients are
equally available to all cells5
#i5uid batch culture 'closed(
K /ogarithmic groth phase 415? during
active gro#th a logarithmic plot of cell
number against time gives a straight line
K /ag period 4A5? the inoculum adapts its
physiology to that required for gro#th on
the available nutrients
K ate logarithmic phase? rate of gro#th
K tationary phase 4C5? eventual halt
K Decline phase 475? starvation, death of
some of the cells and adaptation to a
dormant state
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ro3th in open culture
K 1acteria ma$e up I')B of the dry mass of
faeces
K Dn many situations the bacteria become
immobilied, as a biofilm, upon a surface
and e8tract nutrients from the bul$ fluid
phase
ro3th and genetic change
$ransformation
K Ability of certain types of bacteria to
absorb small pieces of na$ed 7"A from the
environment that may recombine into
recipient chromosome
K !eans of transferring genes bet#een
different types of bacteria
Transduction
K 1acterial 7"A having moved bet#een cells
K Temperate phage? rather than enter a
replication cycle, the viral 7"A becomes
incorporated by recombination into the
chromosome of the bacterium
Conjugation
K Thought to have evolved through
transduction
K (lasmids? 7"A strands
K F-factor 4fertility factor5? simplest form of
plasmid
K F-factor #ill simply transfer a copy to a
recipient cell
,!%IRO!M,!$A# FAC$OR" $7A$ I!F#.,!C,
RO$7 A! ".R%I%A#
K *ram-negative bacteria tend to be aquatic
K *ram-positive bacteria then to prefer
more arid conditions such as the s$in
Physiochemical factors that affect
gro#th and survival of bacteria
Temperature
K Permissive temperature? range of
temperatures under #hich bacteria can
actively gro# and multiplyK Chemical and enymic reactions #ithin
the cell proceed more rapidly, and gro#th
becomes faster until an optimal rate is
achieved as temperature rises
K 1eyond such temperature, certain
proteins may become irreversibly
damaged through the thermal lysis,
resulting in a rapid loss of cell viability
K p9
K P9 effects on gro#th are bell-shaped
K E8tremes of p9 can be lethalK *ro#th optima of ( and @?
microorganisms that have medical or
pharmaceutical significance
K !ay dictate the range of microorganisms
that could potentially cause its spoilage
ater activity3 solutes
K *ram-negative cell envelope cannot
#ithstand the high internal osmotic
pressures associated #ith rapid
rehydration after desiccation
K ater activity 4A#5? vapor pressure of#ater in the space above the material
relative to the vapor pressure above pure
#ater, can mar$edly affect its
vulnerability to spoilage contaminants
K Availability of o8ygen
K >8ygen acts as the terminal electron
acceptor in respiration and is essential for
gro#th
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K Fermentation? carbon substrate is in
e8cess
K "utrition and gro#th
K !hemolithotrops? simple inorganic forms
of elements, can utilie atmospheric
carbon dio8ide and nitrogen as sources of
carbon and nitrogen
K Diauic groth? second lag phase during
the logarithmic gro#th period #hile such
adaption ta$es pace
,$,C$IO!= I,!$IFICA$IO! A!
C7ARAC$,RIA$IO! OF ORA!I"M" OF
P7ARMAC,.$ICA# A! M,ICA#
"I!IFICA!C,
Culture techni5ue
- 7iluting the sample to varying degrees and
inoculate the surface of a predried nutrient
agar #ith $no#n volumes of those dilutions
- Enumeration media
• ill only ever culture a subset
of cells to#ards #hich the
medium and incubation
conditions are directed
•%imple salts media #ithrelatively simple sugars as
carbon sources and trace levels
of amino acids? often used to
enumerate bacteria associated
#ith #ater
• Psychrophilic *ram-negative
bacteria? can be a major
source of bacterial pyrogen
• 9ighly nutritious media are
also used as enumeration
media 4e8 blood agar5
Rapid enumeration techni5ues
• 1ioluminescence
• Epifluorescence
• Dmpedance techniques
• Coulter counters? used to
determine bacterial
concentration, do not
discriminate bet#een living
and dead cells
• !icrocalorimeters? time ta$en
to detect such heat can be
directly related to the numbers
of viable cells present
,nrichment culture
K Dntended to increase the dominance of a
numerically minor component of a mi8ed
culture such that it can be readily
detected on an agar plate
K Enrichment media? al#ays liquid, intended
to provide conditions that are favourable
for the gro#th of other li$ely isolates
K !acCon$ey broth? contains bile salts that
#ill inhibit the gro#th of non-enteric
bacteria and may be used to enrich for
Enterobacteriaceae
"electi6e media
K %olidified enrichment broths,
intended to suppress the gro#th of
particular groups of bacteria and toallo# the gro#th of others
K Counts of colonies obtained on
selective solid media are often
documented as presumptive counts
Ddentification media
4diagnostic5
K Contain nutrients and reagents that
indicate, usually through some form
of colour formation, the presence
of particular organisms
Microscopy
K %imple stains 4such as the *ram stain5
K %ie, shapes, arrangement into clusters,
chain and tetrads, specific stains for the
presence of endospores, capsules, flagella
and inclusion bodies
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+iochemical testing and rapid
identification
K 7iffering ability of bacteria to ferment
sugars, glycosides and polyhydric alcohols
4#idely used to differentiate the
Enterobacteriaceae and in diagnostic
bacteriology generally5
K 6esults of o8idase and catalase tests
performed directly on isolated colonies
!olecular approaches to identification
K 9ave not yet become routinely adopted in
the analytical or diagnostic laboratory
K 7enaturing gradient gel electrophoresis
47**E5? isolates and amplifies &@%
ribosomal 7"A and, follo#ing sequencing
of the bases, compares this #ith $no#n
sequences held in a reference library
K *ene probes carrying fluorescent dyes?
can be used in hybridiation procedures
#ith the collected clinical material
Pharmaceutically and medically
rele6ant microorganisms
K 1roadly classified into those organisms
that are harmful or problematic, and
those that can be used to our advantage
3efer to Table $.$ of book4 eamples
of some pharmaceutically useful bacteria
Chapter >: Fungi
Fungi
• Eu$aryotic organisms
• idely distributed in nature
• E8tremely important group of microbes in
the medical field
• 6esponsible for a number of potentially
fatal diseases in humans
• *reat benefit in humans in terms of0
o Production of alcoholic beverages
o 1read
o Enymes
o Antibiotics
o 6ecombinant proteins
FUNGI
O
O
M
Y
C
E
T
E
S
A
S
C
O
M
Y
C
E
T
E
S
B
A
S
I
D
I
O
M
Y
C
ET
E
S
T
E
L
I
O
M
Y
C
E
T
ES
U
S
T
O
M
Y
C
E
T
E
S
D
E
U
T
E
R
O
M
Y
C
ET
E
S
The ingdom Fungi can be subdivided into si
classes0
Oomycetes
-contains the milde#s and #ater moulds
Ascomycetes
-contains the milde#s, some moulds and
most yeast species 4including
accharomyces cereisiae5
• +asidiomycetes
-contains the mushrooms and brac$et fungi
• $eliomycetes
-contains the rust fungi 4plant pathogens5
• .stomycetes
-contains the smuts 4plant pathogens5
• euteromycetes
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-contains species such as 'spergillus,
0usarium and (enicillium
There are four distinct phyla #ithin the fungal
$ingdom? these are the Chytridiomycota=
?ygomycota= Ascomycota and +asidiomycota
*east
• *ro# as single cells
• 6eproduce ase8ually by budding, although
a minority of species reproduce by fission
• !any yeast species are capable of se8ual
production and formation of spores
Moulds
• *ro# as masses of overlapping and
interlin$ing hyphal filaments
• 6eproduce by producing masses of spores
"tructure of the fungal cell
• >val in shape
• %urrounded by a rigid cell #hich contains
structural polysaccharides
• Average thic$ness varies from &)) to /))
nm
Cell all
•
*lucan, the main structural component offungal cell #all, is a branched polymer of
glucose
• The innermost layer is rich in glucan and chitin
#hich provides rigidity to the #all and it
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• !ost of the cell
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has been responsible for mortality
rates of ;-/=B
-Fourth most commonly isolated
Candida species
Antibiotic production of fungi
• !ajority of antibiotics obtained from fungi
are produced by fermentation and most
are secondary metabolites
• Dsolation of (enicillium notatum by %ir
Ale8ander Flemming !ost important
discovery regarding the beneficial use of
fungi for humans
• Antibiotic production can be ma8imied byoptimiing production as a result of
random mutagenesis and selection
Chapter @: %iruses
I Introduction
• 2iruses #ere first discovered at the end of
&'th century
• They #ere classified as Lfilterable agentsMbecause they can be retained by filtration
II eneral structure of 6iruses
• 2iruses are e8tremely diverse in sie and
shape
• 2iruses are much smaller than bacteria
• 56ntracellular parasites7
• %mallest virus0 polioirus -I .=nm in sie
• argest virus0 mimiirus -I ;)nm in sie
• 2iruses #ith envelope0 enveloped
nucleocapsid
• 2iruses #ith no envelope0 na$ed
nucleocapsid Components of a virus0
✓ 2iral nucleic acid0
- The viral nucleic acid is composed of
either 7"A or 6"A
- A virus can only have a single genome
✓ 2iral capsid 4protein core5
- Dt protects the viral nucleic acid from
detrimental, chemical and physical
conditions
- Dt is composed of a number of subunits
named capsomeres8 genetically
encoded by the viral genome
- !apsomeres give the shape of the
capsid, and provide the virus #ith
resistance to physical and chemical
agents
✓ 2iral envelope
- !ost outer covering of a virus
- The envelope is added during the
replication process
- Dt 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 #ell on their o#n
outside the host cell, although they
can persist longer in organic soil
III %irus&host cell interactions
• 2iruses can interact #ith the host cell in
five different #ays0
& !ultiplication of the virus and
destruction of the host cell upon
release of the viral progeny
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. !ultiplication of the virus and release
of the virions #ithout the immediate
destruction of the host cell
/ %urvival of the virus in a latent stage
#ithout noticeable changes to the
infected cell
( %urvival of the infected cell in a
dramatically altered or transformed
state
; Dncorporation of the viral nucleic acid
in the host cell genome #ithout
noticeable changes to the infected cell
I% Multiplication of human 6iruses
• >bjective of replication cycle0 toensure the multiplication of the virus
#ith the formation of identical viral
progeny
• The multiplication cycle of human
viruses is generally slo#, from ( to
more than () hours
• 1acterial viruses are generally faster
and can ta$e a little as .) minutes to
replicate #ithin the bacterial host
• There are si8 distinct phases in a
replication cycle0
✓ Adsorption - attachment to the
host cell receptor
✓ Penetration - penetrates the
virus through the cell
membrane Dt has three types0
o 7irect injection0 type of
virus involved -I na$ednucleocapsid
- the virus did not fully
enter the host cell
o Fusion0 type of virus
involved -I envelope
nucleocapsid
- Envelope fuses from
the membrane
- Dt is a process #herein
virions can fully enter
the membrane
o Endocytosis0 hen an
envelope nucleocapsid fully
enters the host cell, it
undergoes endocytosis
- Envelope fuses #ith the endosome
✓ +ncoating - releases capsin to
free nucleic acids 47"A or 6"A5
✓ Eclipse stage0 replicates and
e8presses genes
✓ Assembly or maturation -
maturation of virus cells? cells
become virions again
✓ 6elease - virions are release
outside the host cell, to infect
other cells
% Culti6ation of human 6iruses
& Cell culture0
• !ay be divided into three types
according to their history0
✓ Primary cell lines0 The cell
lines are derived directly from
an intact tissue, e: human
embryo, kidney or monkey
kidney
✓ %econdary cell cultures0
derived from primary cultures,
usually those arising from
embryonic tissue
- The cells are more
homogenous, better
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characteried, but might not
be as susceptible to viral
infection as primary cell lines
✓ Continuous cell lines0 usually
derived from malignant tissue,
and have the capacity to
multiply indefinitely in itro
• Cytophatic effect0 a characteristic
morphological change in the
infected cells #herein the cells
shrin$, or undergo ballooning.
• They usually spread to adjacent
cells and #ill result in the
formation of a pla9ue that can
easily be identified follo#ing
staining
• Plaques0 used for the enumeration
of viruses
. The chic$ embryo
K Fertile chic$en eggs, 4'-&& days old5
are used to gro# a number of
human pathogenic viruses
/ Animal inoculation
K Animals are used to culture certain
viruses in order to study antiviral
vaccine effectiveness, and also as a
source of cell lines for cell cultures
%I Control of 6iruses
& Antiviral chemotherapy - leads to the
development of viral resistance, but is still
associated #ith a number of problems
a 9D2
- The role of antivirals in 9D2 is
to slo# or halt disease
progression
- The drug used for 9D2
infections is called
antiretroirals8
- 'ntiretroirals have
considerably prolonged the life
e8pectancy of patients,
although not #ithout some sideeffects
- These drugs aim to reduce 9D2
plasma level as much and as
long as possible
a 9erpesvirus infections
- 9erpesviridae0 a family of
viruses #hich include the
herpes simple8 virus,
chic$enpo8, shingles and
cytomegalovirus
- !ild herpes simple8 virus -I
treated #ith a topical antiviral
drug
- Primary herpetic
gingivostomatitis -I a change
of diet Q analgesics
- %evere infections -I systemicantiviral is used
- Antiviral treatments for
chic$enpo80 recommended in
patients at ris$ and in neonates
to reduce ris$s of severe
diseases
- Antiviral treatments for
herpes0 is associated #ith a
number of side effects #hich
may vary depending on thedrug
- Antiviral treatments for
cytomegalovirus0 usually given
to immunocompromised
patients and they tend to be
more to8ic #ith noticeable
nephroto8icity and a number of
side effect
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a 2iral hepatitis
- Treatment for acute hepatits 1
-I interferons
- Treatment for chronic hepatitis
1 -I antivirals
a Dnfluena
- T#o major limitations in the
usefulness of the drug
- First0 the drug needs to be
ta$en #ithin a fe# hours of the
onset of symptoms from mild
to severe symptoms reported
- %econd0 the side effects have
been very severe
a 6espiratory syncytial virus
- 6%2 is responsible for severe
bronchiolitis notably in infants
- Treatment can be a
monoclonal antibody or an
antiiral drug
. 2accination
K the most successful measure against
microbial and viral infections
- 2accines are preparations
containing antigents that elicit
a specific and active immunity
against an infecting agent
- 2accines can induce the innate and the
adaptive parts of the immune system
- 2iral vaccines prepare using / methods0
✓ ive attenuated viruses0 #ill
cause a strong immune
response #ithout causing the
disease 9epatitis A and
influena vaccines rely on
chemically inactivated, virus
particles or components
✓ Dnactivated viruses
✓ +se of viral components
K 9epatitis 10 viral 7"A encoding for avirus surface antigen e8pressed in
yeasts
K Dmmunoglobulin0 plays a role in the
protection of patients #ith a
compromised immunity against viral
infections
K D! immunoglobulin used to protect
against hepa A virus
& 2iricidal effects of chemical and physical
agents on viruses
• 2iruses are generally transmitted
via surface and are often
associated #ith organic materials
• Dn general, viruses are not
particularly resistant to chemical
or physical agents, although some
e8ceptions e8ist
& Control of viruses in pharmaceutical
products
• Presence of certain viruses needs
to be controlled
• 6is$ of a pharmaceutical product
being contaminated by viruses
depends on0
✓ The origin of the product
component
✓ The history of the donor
✓ The amount of material used
✓ The manufacturing process
✓ Dts capacity to remove or
destroy contaminants
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%II %iruses as antimicrobials
& 1acteriophages
• 1acteriophages are viruses that
infect only bacteria
• They #ere first described at the
end of the &'th century
• %ie0 .)-.))nm, and are highly
diverse in their structure and host
range and it is li$ely that all
bacterial species can be infected
by a phage
• Phages are e8tremely specific in
their host range and some #ill only
infect a specific bacterial strain
• !ost studied phages are the
comple8 ones, tadpole-shaped<
#hich consists of a head that
contains the viral genome, and a
tail #hich function is to recognie
the host receptor, attach and
subsequently serve as a nucleic
acid injection device
•
T#o phage replication cycles0
✓ ytic cycle0 lysis of bacterial host
✓ ysogenic cycle0 result of a viral
nucleic acid being integrated into
the host genome
K Dnfection #ith lytic phage -I
virulent phage, results in the
replication of phage #ithin the
susceptible bacteria and the release
of infectious phage progeny from
the host cell follo#ing cell lysis
K ysogenic cycle0 viral nucleic acid
#hich has integrated the host
genome is called prophage
; 9ost cell that contains the viral
genome0 lysiogenic
& +se of bacteriophages to treat bacterial
infection
K Dntroduction of antibiotics in the
early &'()
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