micropara reviewerrrr

8/19/2019 micropara reviewerrrr

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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


16/27

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


20/27

• !ost of the cell


21/27


22/27

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


24/27

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


herpes0 is associated #ith a

number of side effects #hich

may vary depending on thedrug


cytomegalovirus0 usually given

to immunocompromised

patients and they tend to be

more to8ic #ith noticeable

nephroto8icity and a number of

side effect


25/27

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


26/27

%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 &'()


27/27

micropara reviewerrrr

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