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-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
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
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
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
1. Solid
- gelled by addition of agar (1-1.5% w/v) CLASSIFICATION BASED ON
COMPOSITION:
- Agar: from seaweeds ; firms at 37C and
liquid at 45C. 1. Truly Synthetic Media
- chemically defined
- For anaerobic organisms contain non- - for microorganisms that
toxic can synthesize materials
reducing agents needed for growth from
simple carbon and nitrogen.
-low redox potential
2. Media w/ biochemicals
2. Liquid - used for organism that
- contains no agar. cant synthesize
- some more commonly
used
o Selective or Diagnostic Media - complex w/ precise
- restrict the growth of certain chemical composition
types of microorganisms while - vary slightly batch to
permitting or enhancing the growth batch
of others.
Ex. Routine laboratory media,
- use additives for identification General Purpose meda, Complex
Media
o Enrichment Medium - aq. Soln of animals and plants
- designed to permit a particular extracts that contain:
type of organism to grow while
others, so the one that grows Hydrolysed protein
increase in relative numbers and is -inexpensive sources are meet
enriched. extracts, milk, and soya
-hydrolysed by varying degree to
give peptones or amino acids
CULTIVATION METHODS
-trypsin and other proteolytic
enzymes preffered to acids
o Binary Fission
- division pattern
B-group vitamins
-requirement is satisfied by
- cell enlarges or elongates then
yeast extract
forms cross wall called septum and
is followed by constriction until
finally broken and daughter cells Carbohydrates
separate. -in form of starch and yeast
- Several situations where number of microbial - In optimal conditions of laboratory
cells in culture, sample or specimen are needed to cultivation of bacteria this division
be measured: takes place every 25-30 minutes.
Measuring levels of microbial
contamination in raw material or - growth continues until one or more
manufactured medicine nutrient is exhausted or toxic
Evaluating the effects of metabolites accumulates
antimicrobial chemical or
decontamination process o Overnight incubation in liquid media
Using microorganisms in - culture media clear but becomes
manufacture of therapeutic agents cloudyas concentration increases
Assessing the nutrient capability of
growth medium - indirect means of monitoring
culture media.
- contaminants
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.
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: exist only as a part of a multicellular
organism)
a. Actinomycetes
rigid bacteria resembling fungi that may
grow as lengthy branched filaments
- Capable of growing in a range of different
b. Mycoplasmas environments
lack a conventional peptidoglycan
(murein) cell wall - Cannot only cause contamination and
spoilage but also a range of different
highly pleomorphic organisms of indefinite diseases
shape
- Transports of nutrients, energy generation Bacteria can be divided into two large
and electron transports groups (on the basis of a differential staining
technique called the Gram stain): Gram-positive,
- Selective barrier between the cytoplasm Gram-negative
and the cell environment
Gram-positive Cell Wall
Cytoplasm - Consist primarily of a single type of
- Consists of 80% water and contains molecule
enzymes that generate ATP
- Contains teichoic acids and lipoteichoic
- Compose of the ribosomes, nucleoid and acids (negatively charged)
inclusion granules
- During an infection, lipoteichoic acids
Nucleoid molecules trigger an inflammatory response
- Singular, covalently closed circular
molecule of double stranded DNA - Retain the dye (gram stain)
- Process in which the vegetative cell undergoes a - Two subunits: 30S and 50S
profound biochemical change to give rise to a
specialized structure called an endospore or spore Inclusion granules
- Not part of a reproductive cycle - Serves as the storage material for carbon,
nitrogen, and sulphur or phosphorus
Spore
Highly resistant Cell surface components
Enables producing organism to survive in Flagella
adverse environmental conditions (lack of - Bacterial motility
moisture or essential nutrients, exposure a. Monotrichous a single polar flagellum
to toxic chemicals, radiation or high b. Lophotrichous two or more flagella at one pole
temperatures) of the cell
All sterilization process for pharmaceutical c. Amphitrichous single/tuft of flagella at each
products have been designed to destroy end of the cell
the bacterial spore d. Peritrichous flagella distributed over the
entire cell
Endospore structure
Pili and Fimbriae
Endospores
a. Pili (pilus) join bacterial cell in preparation of
Differentiated cells that possess a
DNA and to environmental surfaces
grossly different structure to that
involved in the genetic exchange
of the parent vegetative cell in
process of conjugation
which they are formed
b. Fimbriae for adherence of cells to one another
and to environmental surfaces
Exosporium responsible for hemaglutination
Outermost layer and cell clumping in bacteria
Composed of protein; within are
the spore coats (proteinaceous but Glycocalyx (Slime Layer and Capsule)
with a high cysteine content) - General substances that surround cells
- Gelatinous polymer of polysaccharide,
Cortex polypeptide, or both
Consists of loosely cross-linked
peptidoglycan a. Slime Layer unorganized and loosely attached
to the cell wall
Central core b. Capsule substance is organized and firmly
Contains the genome attached to cell wall
Partially dehydrated (dehydration
shown to increase resistant to both Biofilms
heat and chemicals) - Any surface for microbial habitat
Containing only 10-30% of the - Usually contains more than one species of
water content of the vegetative bacteria which exist and cooperate
cells together
Classified as either endotoxin pH; 1 unit lower than the
(ex. Cell wall-related, cytoplasm of the vegetative cell
exotoxin, products released and contains high levels of core-
extracellularly as the organism specific proteins that bind tightly
grows) to the DNA and protect it from
potential damage
Endotoxin Core-specific proteins; function as
Lipid A component of LPS an energy source for the outgrowth
Possesses multiple biological or germination of a new vegetative
properties including ability to cell from the endospore
induce fever, initiate the
complement and blood Spore
cascades Presence of dipicolinic acid and
Activate B lymphocytes and high levels of calcium ions which
stimulate production of tumor complex together
necrosis factor
Released from lysed or
damaged cels Endospore formation
Depyrogenation; process where
care must be taken to Vegetative cell undergoes a complex series of
eliminate or exclude such heat- biochemical events in cellular differentiation
resistant material from Sporulation; accomplished by activation of a
parenteral products and their variety of spore-specific genes such as spo and
delivery systems. ssp
Leads to the production of a dry, metabolically
Exotoxin inert but extremely resistant endospore
A-B toxins; consists of a B Endospore germination
subunit that binds to a host Reversion of endospore back to a vegetative cell
cell receptor and is also Removal of the stress inducer that initiated
covalently bound to the A sporulation
subunit that mediates the Germination loss of resistance properties; occurs
enzymic activity responsible along with a loss of calcium dipicolinate and
for toxicity (ex. Diphtheria cortex components, and degradation of the core-
toxin, cholera toxin) specific proteins
Cytolytic toxins; do not have
seperable A and B portions but BACTERIAL TOXINS
work by enzymatically
attacking cell constituents, - Organisms, if presented with the correct set of
causing lysis (ex. haemolysins conditions, can cause disease
and phospholipases) - (ex. Opportunist pathogens; Staphylococcus
Superantigen toxins; lack an A- epidermis,non-pathogenic environmental
B type structure and act by organism; Ps. Aeruginosa)
stimulating large numbers of
immune response cells to
release cytokines, resulting in Toxins
a massive inflammatory Products of bacteria that
reaction (ex. Staphylococcus produce immediate host cell
aureus) damage
Growth and solid surfaces
MacConkey broth; contains bile salts that - Diluting the sample to varying degrees and
will inhibit the growth of non-enteric inoculate the surface of a predried nutrient
bacteria and may be used to enrich for agar with known volumes of those dilutions
Enterobacteriaceae
- Enumeration media
Will only ever culture a subset
Selective media of cells towards which the
Solidified enrichment broths, medium and incubation
intended to suppress the growth of conditions are directed
particular groups of bacteria and to Simple salts media with
allow the growth of others relatively simple sugars as
Counts of colonies obtained on carbon sources and trace levels
selective solid media are often of amino acids; often used to
documented as presumptive counts enumerate bacteria associated
Identification media with water
(diagnostic) Psychrophilic Gram-negative
Contain nutrients and reagents that
bacteria; can be a major
indicate, usually through some form
source of bacterial pyrogen
of colour formation, the presence
Highly nutritious media are
of particular organisms
also used as enumeration
media (ex. blood agar)
Microscopy
Simple stains (such as the Gram stain)
Rapid enumeration techniques
Size, shapes, arrangement into clusters,
chain and tetrads, specific stains for the
Bioluminescence
presence of endospores, capsules, flagella
Epifluorescence
and inclusion bodies
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:
Ascomycetes
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
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 Most of the cells genome is concentrated
disease. in the nucleus
o Moulds Mitochondrion
o Dermatophytes The powerhouse of the cell
- It protects the viral nucleic acid from Majority of antibiotics obtained from fungi
detrimental, chemical and physical are produced by fermentation and most
conditions are secondary metabolites.
- It is composed of a number of subunits
Isolation of Penicillium notatum by Sir
named capsomeres genetically
Alexander Flemming Most important
encoded by the viral genome
discovery regarding the beneficial use of
- Capsomeres give the shape of the fungi for humans
capsid, and provide the virus with
Antibiotic production can be maximized by
resistance to physical and chemical
agents optimizing production as a result of
random mutagenesis and selection.
Viral envelope
Chapter 5: Viruses
- Most outer covering of a virus
I. Introduction
- The envelope is added during the
Viruses were first discovered at the end of
replication process
19th century
- It can come from the host cell nuclear
membrane, or the cytoplasmic They were classified as filterable agents
membrane because they can be retained by filtration
Intracellular parasites
III. Virus-host cell interactions Smallest virus: poliovirus -> 28nm in size
Viruses can interact with the host cell in Largest virus: mimivirus -> 750nm in size
five different ways:
Viruses with envelope: enveloped
1. Multiplication of the virus and
nucleocapsid
destruction of the host cell upon
release of the viral progeny Viruses with no envelope: naked
nucleocapsid Components of a virus:
- Envelope fuses from 2. Multiplication of the virus and release
the membrane of the virions without the immediate
destruction of the host cell
- It is a process wherein
virions can fully enter 3. Survival of the virus in a latent stage
the membrane without noticeable changes to the
infected cell
o Endocytosis: When an
envelope nucleocapsid fully 4. Survival of the infected cell in a
enters the host cell, it dramatically altered or transformed
undergoes endocytosis. state