1. INTRODUCTION
2um in diameter to >5um in diameter o CELL WALL
Prokaryotes
 smallest free-living organisms (comprising
bacteria and archaea)
 lack a true nuclear membrane
Bacteria vs. Archaea
 composition of cell walls
 structure of lipids that make up their
cytoplasmic membranes
 patterns of metabolism: most archaea are
anaerobes- often found inhabiting extreme
environments
 unusual membrane structures gives archaeal
cells greater stability under extreme conditions
 no disease causing archaea
Bacteria
 large and diverse group
 can exist as single cells or cell clusters
BACTERIAL DIVERSITY AND UBIQUITY
 diversity can be seen in terms of variation in
cell size and shape (morphology)
 adaptation to environmental extremes, survival
strategies and metabolic capabilities
 allows bacteria to grow in multiplicity of
environments ranging from hot sulphur springs
(650C) to deep freezers (-200C); pH 1-13; high
(0.7m) to low osmolarity (water)
 can grow in nutritionally rich (compost) to
nutritionally poor (distilled water) situations
2. BACTERIAL ULTRASTRUCTURE
 CELL SIZE AND SHAPE
 Bacteria- smallest free-living organism
 Size measured in micrometers (microns)
 X400-1000 magnification needed
ULANGCO • GOMEZ • YANG • SERRANO • BORJA
2E PH
 0.1-0.
 Large bacteria such as Thiomargarita
namibiensis are rare
 Majority are 1-5um long and 1-2um in
diameter
 Due to its size, transport rates has most
notably increased and have been more
efficient
 16S ribosomal DNA sequencing data,
divide them into major groups on purely
morphological grounds
 Majority are unicellular and possess simple
shapes, round (cocci), cylindrical (rod;
bacillus), ovoid
 Some rods are curved (vibrios)
 spirochaetes – longer rigid curved
organism with multiple spirals
 actinomycetes – rigid bacteria resembling
fungi that may grow as lengthy branched
filaments; the mycoplasmas which lack a
conventional peptidoglycan (murein) cell
wall and are highly pleomorphic organisms
of indefinite shape; some are stalked,
sheathed, budded, and slime-producing
forms often associated with aquatic and
soil environment
 cellular arrangements- chains of rods or
cocci, paired cells (diplococci), tetrads and
clusters
 CELLULAR COMPONENTS
 Antimicrobial chemotherapy- to treat
infections antimicrobial agents are used to
inhibit the growth of bacteria
 The essence is selective toxicity,
achieved by exploiting differences
between the structure and
metabolism of bacteria and host cells;
 Most efficient when a similar target
does not exist in the host
 Essential for the maintenance of the shape
and integrity of the bacterial cell
 Obvious target for antibiotics
 Primary function is to provide a strong,
rigid, structural component that can
withstand the osmotic pressure caused by
high chemical concentrations of inorganic
ions in the cell
 Most bacterial cell walls have
peptidoglycan (murein or glycopeptide)
 Peptidoglycan- large macromolecule
containing glycan (polysaccharide)
chains that are cross-linked by short
peptide bridges
 Glycan chain- acts as a backbone to
peptidoglycan; composed of
alternating residues of N-acetyl
muramic acid (NAM) and N-acetyl
glucosamine (NAG)
 Each molecule of NAM is attached to
a tetrapeptide consisting of amino
acids: L-alanine, D-alanine, D-glutamic
acid and either lysine or
diaminopimelic acid (DAP)
 Glycan tetrapeptide repeat unit is
cross linked to adjacent glycan chains,
either through a direct peptide
linkage or a peptide interbridge
 Bacteria can be divided into 2: Gram-
positive and Gram-negative, on the basis
of a differential staining technique: Gram
stain
 Gram stain consists of treating a film
bacteria dried on a microscope slide with
a solution of crystal violet, followed by a
solution of iodine, then washed with
alcohol solution
  Gram-negative organisms lose the
crystal violet-iodine complex and are
rendered colorless
 Gram-positive cells retain the dye
 Both cell types are counter-stained
with a different colored dye;
Carbolfuchsin which is red
 Under the light microscope, gram-
negative cells appear red, while
gram-positive cells appear purple
 Gram-positive cell wall: single type of
molecule, thick (20-80 nm) 60% and 80%
peptidoglycan, extensively cross-linked in
3 dimensions to form a thick polymeric
mesh; contain acidic polysaccharide:
Teichoic acids- either ribitol phosphate or
glycerol phosphate that are connected by
phosphodiester bridges; negatively-
charged, responsible for negative charge
of the cell surface as a whole; effect
passage of metal cations through the cell
wall
 Some gram-positive bacteria glycerol-
teichoic acids are bound to membrane
lipids: Lipoteichoic acids
 During an infection, lipoteichoic acid
molecules released by killed bacteria
trigger an inflammatory response
 Gram-negative cell wall: multilayered
structure, complex; contains less
peptidoglycan (10-20% of wall); has an
outer membrane- asymmetrical,
composed of proteins, lipoproteins,
phospholipids and a component unique to
gram-negative bacteria,
lipopolysaccharide
 Proteins are also found in the outer
membrane, some of which form trimers
that transverse the whole membrane and
in so doing form water-filled channels:
ULANGCO • GOMEZ • YANG • SERRANO • BORJA
2E PH
Porins- through which small molecules can
pass
 Lipopolysaccharide- determines the
antigenicity of the gram-negative cell and
it is extremely toxic to animal cells
 Consists of 3 regions: Lipid A, core
polysaccharide and O-specific
polysaccharide
 Lipid A- composed of a disaccharide
of glucosamine phosphate bound to
fatty acids and forms the outer leaflet
of the membrane; responsible for the
toxic and pyrogenic properties
 Lipid A linked to the core
polysaccharide polysaccharide by the
unique molecule: Ketodeoxyoctonate
(KDO)
 O-polysaccharide- (O-antigen) at the
other end of the core which contains
6-carbon sugars as well as one or
more unuasual deoxy sugars such as
abequose
 Outer membrane- keep certain enzymes
that are present outside the cytoplasmic
membrane from diffusing away from the
cell; not readily penetrated by
hydrophobic compounds, resistant to
dissolution by detergents
 Periplasm- region between the outer
surface of the cytoplasmic membrane and
the inner surface of the outer membrane;
12-15nm, gel-like consistency, contains
sugars and abundance of proteins
including hydrolytic enzymes and
transport proteins
o CYTOPLASMIC MEMBRANE
 Fragile, phospholipid bilayer with proteins
distributed randomly
 Involved in transport and enzyme
functions
 Eukaryotes have sterols (e.g. cholesterol);
prokaryotes do not
 Transport nutrients, energy generation
and electron transport
 Location for regulatory and biosynthetic
proteins; acts as a semipermeable
selectivity barrier between the cytoplasm
and cell environment
 Mesosomes- invaginations of the
cytoplasmic membrane; form near the
septum of gram-positive cells serve as
organs of attachment of the bacterial
chromosome
o CYTOPLASM
 Consists 80% water and contains enzymes
that generate ATP directly by oxidizing
glucose and other carbon sources
 Contains some of the enzymes involved in
the synthesis of peptidoglycan subunits
 Ribosomes, nucleoid (DNA genome) and
inclusion granules
o NUCLEOID
 Singular, covalently closed circular
molecule of double-stranded DNA
comprising approximately 4600 kilobase
pairs
 Complexed with small amounts of
proteins and RNA
 Not associated with histones
 DNA, if linearized, about 1 mm in length
 To package this amount of material, DNA
is supercoiled into a number of domains,
which are associated with each other and
stabilized by specific proteins into an
aggregated mass or nucleoid
 Topoisomerases- enzymes that control
topological changes in DNA architecture
are different from their eukaryotic
counterparts (which act on linear
chromosomes) and therefore provide a
 unique biochemical target for antibiotic
action
o PLASMIDS
 Relatively small, circular pieces of double
stranded extrachromosomal DNA
 Autonomous replication and encode for
many auxiliary functions that are not
usually necessary for bacterial growth
 Antibiotic resistance
 May transfer readily from one organism to
another, and between species, increasing
the spread of resistance
o RIBOSOMES
 Site of protein synthesis
 70S in size, 2 subunits: 30S and 50S
 Smaller than eukaryotic cells: 80S in size
(40S and 60S)
o INCLUSION GRANULES
 Storage material; composed pf carbon,
nitrogen, sulphur or phosphorous and are
formed when these materials are replete
in the environment to act as repositories
of these nutrients when shortages occyr
 E.g. poly-β-hydroxybutyrate, glycogen,
polyphosphate
 CELL SURFACE COMPONENTS
 Portion of the organism that interacts with
the external environment most directly
 Many bacteria deploy components on
their surfaces in a variety of ways that
allow them to withstand and survive
fluctuations in the growth environment
o FLAGELLA
 Motility; long helical-shaped structures
that project from the surface of the cell
 Filament of the flagellum- built up from
multiple copies of the protein flagellin
ULANGCO • GOMEZ • YANG • SERRANO • BORJA
2E PH
 There is a hook in the flagellum, which is
attached to the cell surface by a series of
complex proteins: flagellar motor
 Flagellar motor- rotates the flagellum,
causing the bacterium to move through
the environment
 Some have a single, polar flagellum while
others are flagellate over their entire
surface (peritrichous)
o FIMBRIAE
 Not involved in motility
 Straighter, more numerous, thinner and
shorter (3um) than flagella, consist of
protein and project from the cell surface
 Act primarily as adhesins (allow
attachment to surface) and to initiate
biofilm formation
 Haemagglutination and cell clumping in
bacteria
 E.g. Type I fimbrae of enteric (intestinal)
bacteria
o PILI
 Similar to fimbriae but present in much
smaller numbers (<10) and are usually
longer
 Involved in conjugation (join bacterial cell
in prep of DNA transfer from one cell to
another)
o CAPSULES AND SLIME LAYERS
 Many bacteria excrete extracellular
polysaccharides (EPS) associated with the
exterior of the bacterial cell
 EPS- composed of c.2% carbohydrate and
98% water, provides a gummy exterior to
the cell
 2 extreme forms exist: CAPSULES- tight,
fairly rigid layer closely associated with the
cell; SLIMES- loosely associated with the
cell
 Offer protection against dessication
 Protective barrier against penetration of
biocides, disinfectants and positively
charged antibiotics
 Protect against engulfment by phagocytes
and protozoa to act as a cement binding
cells to each other and to the substratum
in biofilms
 Alginate- produced by Pseudomonas
aeruginosa
 Dextran- produced by Leuconostoc
mesenteroides
 Preferred source of alginate- seaweed
o S-LAYERS
 Most common cell wall type amongst the
archaea
 Consist of a 2-dimensional paracrystal line
array of proteins or glycoproteins which
show various ordered symmetries when
viewed under the electron microscope
 Addition to increasing the structural
robustness of the cell, S-layers can act to a
certain extent as an external permeability
barrier
3. BIOFILMS
Suface-liquid interface – allows for nutrient-rich
environment to be produced in a nutirient-poor
environment
Microbial numbers and activity – much greater on
surface than in suspension
Biofilms
– multilayered communities of bacteria, attached
to surfaces
– commonly contain more than one species of
bacteria, which exist cooperatively together as
a functional, dynamic consortium.
– Possess unique properties
 Biofilm formation
1. Begins with pioneer cells attaching to a surface
2. These cells grow and divide to produce
microcolonies, which eventually coalesce to
produce a biofilm
Key Characteristic of Biofilm
– enveloping of the attached cells in a matrix of
EPS and other macromolecules
– Help to cement cells to the surface and to each
other
– Protects the bacteria from hazardous material,
ENDOSPORE STRUCTURE
dessication, engulfment by macrophages and
phagocytes
Strands of EPS – holds the bacterial cells at a distance
from one another, enabling small watter channels to
form in the biofilm.
Water channels – act as a primitive circulatory system
carrying trapped nutrients and oxygen to the enclosed
cells and take wate products away.
In the human body, the resident cells within the biofilm
are not exposed to attach by the immune system and in
some instances can exacerbate the inflammatory
response.
Ps. aeruginosa – alginate-enclosed biofilm in the lungs of
cystic fibrosis patients.
4 . BACTERIAL SPORULATION
- vegetative cell that undergoes a profound biochemical
change that gives rise to a special structure called
Endospore or Spore
*happens in Bacillus & Clostridium
- process of sporulation is not part of a reproductive cycle
Endospore or Spore
 highly resistance cell
ULANGCO • GOMEZ • YANG • SERRANO • BORJA
2E PH
 enables it to survive in adverse environmental
conditions (lack of moisture or essential nutrients,
exposure to toxic chemicals, radiation, high
temperature)
 because of high resistance to radiation, ethylene
oxide and heat, All sterilization processes for
pharmaceutical products have been designed to
destroy the bacterial spore
 removal of environmental stress leads to
germination of the spore back to vegetative cell form
 possesses different structure from parent vegetative
cell in which they are formed
 spore is much more complex than vegetative cell (
many layers surrounding the central core)
1. EXOSPORIUM
 outermost layer
 composed of protein
2. SPORE COATS
 proteinaceous w/ high cysteine content
3. CORTEX
 loosely crossed-linked peptidoglycan
4. CENTRAL CORE
 contains genome
 core is partially dehydrated (10-30% of water
content in vegetative cell)
 Dehydration
- increases resistance to both heat and
chemicals
 pH
- 1 unit lower than the cytoplasm of veg cells
and high levels of core-specific proteins
that binds tightly to the DNA to protect
from potential damage
 Core specific proteins
-Energy source for the outgrowth or
germination of a new veg cell from the
endospore.
Characteristics
 presence of dipicolinic acid and high levels of
calcium ions
ENDOSPORE FORMATION
 vegetative cells undergoes a complex series of
biochemical events in cellular differentiation
SPORULATION
 process from vegetative cell to an endospore
 synthesis of some protein involved in
vegetative cell function cease and that specific
spore proteins are made
 activation of a variety of spore-specific genes
(spo and ssp)
 Proteins coded by the genes catalyse a series of
events leading to the PRODUCTION of a DRY,
METABOLICALLY INERT but EXTREMELY
RESISTANT SPORE
 whole process takes hours to complete under
optimal conditions
ENDOSPORE GERMINATION
- From endospore back to the vegetative cell
- endospore can lie dormant for decades but can
rapidly revert back to a vegetative cell
 activation of the process: Removal of the stress
inducer that initiated sporulation
 during germination: loss of resistance properties,
loss of calcium dipicolinate and cortex
components, degradation of core-specific proteins
 Outgrowth occurs involving water uptake,
synthesis of new RNA/proteins/DNA, until the
 vegetative cell emerges from the fractured spore
coat and begins to divide again
5 . BACTERIAL TOXINS
- Only few species of bacteria are disease-
producing/pathogenic
OPPOTUNIST PATHOGENS
- presented with correct sets of conditions, can cause
disease
Ex: Straphylococcus epidermidis
- beneficial on the skin (normal habitat) but
fatal if attached to a synthetic heart valve
Ps. Aeruginosa
- non pathogenic environmental organism but
lethal in immunocompromised patients
PATHOGENS
 cause host damage
 produce variety of molecules or factors that
promotes pathogenesis (toxins)
TOXINS
 products of bacteria
 immediate host cell damage
o ENDOTOXIN
 cell wall-related
 released from LYSED or DAMAGE CELLS
 lipid A component of the LPS
- ability to induce fever
- initiate complement blood cascades
- Activate B-lymphocytes
- Stimulate production of tumour necrosis
*Care must be taken to eliminate or exclude heat-
resistance material from parenteral products and
their delivery systems thru a process called
DEPYROGENATION*
o EXOTOXIN
- release extracellularly
 A-B TOXINS
 B subunit that binds to a host cell receptor
that is bound to the A subunit that
ULANGCO • GOMEZ • YANG • SERRANO • BORJA
2E PH
mediates enzymic activity responsible for
toxicity
Ex: diptheria toxin, cholera toxin
 CYTOLYTIC TOXINS
 haemolysins and phospholipases
 Does not have separable A and B portion
 work by enzymatically attacking cell
constituents causing lysis
SUPERANTIGEN TOXINS
 lacks A-B type structure
 stimulates large numbers of immune
response cells to release cytokines 
massive inflammatory reaction
Ex: Straphylococcus aureus
- mediated toxic shock syndrome
- cell division and the next
6. BACTERIAL REPRODUCTION AND GROWTH KINETICS
Multiplication and division cycle
 Majority of bacterial cells multiply via binary fission
(when large enough, produces 2 identical daughter
cells)
 each with at least 1 copy of bacterial
chromosome (circular, attached to the
cytoplasmic membrane, able to uncoil
during DNA replication)
 For Escherichia coli growing at 350C, replication of
chromosome will take approx. 45mins
 Gram-negative cells do not have rigid cell walls and
divide by a process of constriction then membrane
fusion
 Gram-positive cells have rigid cell walls and must
develop a cross-wall that divides into two equal
halves
 Constriction and cross wall formation take approx.
15mins to complete
 DNA replication, chromosome segregation (C-
phase) and cell division occur sequentially in slow-
growing cells with generation times of >1 hour and
are the final events of bacterial cell cycle
 It is possible for some organisms growing under
optimal conditions to divide every 15-20mins
 Rod-shaped organisms maintain their diameter
during the cell cycle and increase their mass and
volume via elongation
 Coccal forms increase in size by radial expansion
 under such circumstances, cells appear to form
chains (i.e. streptococci)
 Certain genera, e.g.
Sarcina: rotate successive division planes by 900 to
form tetrads and cubical octets
Population growth
 Generation time is the time interval between one
 1st gen: n = 1 x 2 = 21
2nd gen: n = 1 x 2 x 2 = 22
3rd gen: n = 1 x 2 x 2 x 2 = 23
xth gen: n = 1 x 2x = 2x
Growth on solid surfaces
 If microorganisms are immobilized on a solid
surface from which they can derive nutrients and
remain moist, cell division will cause the daughter
cells to form a localized colony
 Agar melts and dissolves in boiling water but will
not resolidify until the temp. is below 450C
 Agar media are used in the lab either poured as thin
layer into a covered dish (Petri dish or plate) or
contained within a small, capped bottle (slant)
 If suspensions of different species of bacteria
are spread on to the surface of a nutrient agar
plate, each individual cell will produce a single
visible colony
 These may be counted to obtain an estimate of
the original number of cells
 Transfer of single colonies from the plate to a slant
enables pure cultures of each organism to be
maintained, cultured, and identified
Growth in liquids
 Eventually, growth ceases when the rate of
consumption of nutrient exceeds the rate of
supply
 When grown in liquids, the bacteria, being of
colloidal dimensions and sometimes highly
motile, and are dispersed evenly through the
fluid
 Nutrients: equally available to cells
 Important consideration: open or closed
environment with respect to acquisition of
fresh nutrient
 Closed systems: typified by batch culture in
closed glass flasks
 waste products of metabolism are
retained and all the available
nutrients are present at the beginning
of growth
 Open systems: continual supply of fresh
nutrients and removal of waste products
Liquid batch culture (closed)
 As the increase in cell numbers is exponential
(1, 2, 4, 8, 16, etc.) then during active growth a
logarithmic plot of cell number against time
gives a straight line: logarithmic growth phase
(B)
o generation or doubling time may be
calculated from the slope of the line
 Lag period (A): inoculum adapts its physiology
to that required for growth on the available
nutrients
 Late logarithmic phase: reduced rate of growth
due to less nutrients and waste material
accumulation
 Stationary phase (C): after late log-phase
o starvation, which eventually lead to
the death of some of the cells and
adaptation to a dormant state in
others (decline phase, D)
ULANGCO • GOMEZ • YANG • SERRANO • BORJA
2E PH
 This pattern of growth occurs within
adequately preserved pharmaceutical
products, in water storage tanks and in
industrial fermentation
Growth in open culture
 Growth of bacteria in association with humans
and in our environment is subject to gradual
but continuous provision of nutrients and a
dilution of waste products
 Bacteria in our GI tracts receive a more or less
continuous supply of food and excess bacteria
are voided with the feces (bacteria >90% of the
dry mass of feces)
 In many situations, bacteria become
immobilized, as biofilm, upon a surface and
extract nutrients from the bulk fluid phase
Growth and genetic exchange
 There are three major processes of genetic
exchange that can be identified in bacteria–
transformation, transduction, conjugation
Transformation
 ability of certain types of bacteria to absorb
small pieces of naked DNA from the
environment that may recombine into the
recipient chromosome: tran
 likely to occur naturally like in septic abscesses
and in biofilms
o where high cell densities are
associated with death and lysis of
significant portions of the population
 Also exploited in molecular bio as means of
transferring genes between different types of
bacteria
Transduction
 Bacteriophages: group of viruses which have
bacterial cells as their hosts
o They inject viral DNA into the host cell
o The viral DNA is then replicated and
transcribed at the expense of host
and assembled into new viral particles
 Under normal circumstances, the host cell
becomes lysed in order to release the viral
progeny
o Under exceptional circumstances,
rather than a replication cycle, the
viral DNA becomes incorporated, by
recombination, into the chromosome
of the bacterium
o Known as temperate phage
 The viral DNA thus forms a part of the bacterial
chromosome and will be copied to all daughter
cells
 Temperate phage will become active once
again at low frequency and phasing between
temperate and lytic forms (for long-term
survival)
 Subsequent temperate infections caused by
the latter virions will result in this bacterial DNA
having moved between cells: a process of gene
movement: transduction
Conjugation
 This can be transcribed to produce singular
viral elements, which cannot assemble or lyse
the host cell
 Plasmids: DNA strands
o circular and can either be integrated
into main chromosome, in which case
they are replicated along with others
and passed to daughter cells, or they
separate from it and replicated
independently
 F-factor (fertility factor): simplest form of
plasmid
o can be transcribed at the cell
membrane to generate an F-pilus
within the cell envelope and cells
 containing an F-factor are designated o Gram-positive bacteria = arid  Proteins may become damaged through
F+ condition (skin) thermal lysis when beyond temperature,
 F-pilus: hollow appendage that is capable of resulting rapid loss of cell viability.
transferring DNA from one cell to another 7.1 Physicochemical factors that affect growth and  The optimum temp. for growth is nearer the
o through a process very similar to the maximum value than the minimum.
injection of viral DNA into a cell
during infection 3-4°C Permissive Temperature
 In its simplest form, an unassociated F-factor 10-20°C Obligate Pathogens yet
will simply transfer a copy to a recipient cell, broad
known as conjugation Temp. exceeds but lethal Organism survives but not
 Integration with, and dissociation of, F-factor temp are not achieved grow. (Gram-negative
with the chromosome occurs randomly Mesophiles - 60°C)
 When in integrated form, designated Hfr (high 105 °C and above Rapidly lethal and can
frequency of recombination), a partial or sterilize materials and
complete copy of the donor chromosome can products
also be transferred across F-pilus
 In such instances, the plasmid that is formed survival of bacteria
will transfer not only itself but also this
additional DNA into recipient cells
 The unassociated plasmid can replicate
autonomously from the chromosome to
achieve a high copy number
o It can also be transferred
simultaneously to many recipient
bacteria 7.1.2 pH

 The effect of pH is “bell-shaped” on growth

7 ENVIRONMENTAL FACTORS THAT INFLUENCE
and the its extreme can be lethal.
GROWTH AND SURVIVAL
 Microorganism that have medical or
 A microbial population growth rate depends pharmacuetical significance have pH growth of:
7.1.1 Temperature o Optima: 7.4 – 7.6
on:
o Nature o Suboptimally: 5 – 8.5
 Individual species of bacteria also have a range
o Availability of Water  The growth of lactobcilla within the vaginal
of temperature that they can actively grow and
o Nutrients multiply.
o Temperature
o pH  As the temperature rises the bacterial growth
o Solute Concentration becomes fasters until an optimal rate is
o Pressure of Oxygen achieved.
 Groups of organisms are adapted to survive
under conditions
o Gram-negative bacteria = aquatic
ULANGCO • GOMEZ • YANG • SERRANO • BORJA
2E PH
 vault reduces its pH to approx. 5.5 that
prevents growth of pathogens.
7.1.4 Availability of Oxygen
7.1.3 Water Activity/Solutes
 Gram-negative bacteria are adapted and able
to extract trace nutrients from aquatic
environment. (It has limitations)
 Gram-negative cell cannot withstand the high
internal osmotic pressure with rapid
rehydration after desiccation and unable to
grow because of high concentration of solute.
 Water Activity/ Aw – vapor pressure of water in
the space above the material relative to the
vapor pressure above pure water at the same
temperature and pressure.
Product Aw
Pure Water 1.00
Pharmaceutical Cream 0.8-0.98
Strawberry Jam 0.7
7.2 Nutrition and Growth
o Gram-negative Bacteria – cannot grow if
Aw is below 0.97
o Gram-positive Bacteria – can grow with Aw
0.8-0.98 and survive rehydration after
desiccation
o Moulds & Yeast – can grow at low Aw
ULANGCO • GOMEZ • YANG • SERRANO • BORJA
2E PH
o Phosphorus
o Potassium
o Sulfur
 Aerobic microorganism oxygen acts as terminal Trace Elements (minor requirement)
electron acceptor in respiration. (for growth)
o Magnesium
 Alternative Terminal Electron Acceptor –
o Calcium
organic molecules whose reduction leads to
o Iron
the generation of organic acids. (ex. Lactic
 Most bacteria require fixed carbon source,
Acid)
usually in the form of sugar that can be
Oxygen can be Utilized Oxygen is highly Toxic obtained from complex organic molecules:
Fermentation- carbon o Benzene
substrate is in excess Obligate Anaerobes o Paraffin Waxes
(ex. Clostridium o Proteins
Inorganic Material – iron as botulinum, Clostridium  Nitrogen can be obtained from ammonium ions
electron acceptor (ex. iron- perfringens) which is available by deamination of amino
Sulphur bacteria) acids. (carbon & nitrogen source)
 Many classes are Auxotrophic and can grow on
simple sugar with ammonium ions.
 Strongly Oxygen-dependent Bacteria – tends
to grow on the surface of liquid media where is
oxygen is most available.
 Required to grow obligate anaerobes:
o Special media
o Anaerobic Chambers
 Bacteroides and Fusobacter with strongly
aerobic streptococci are obligate anaerobes in
the mouth and gastrointestinal tract.
 Diffusion of oxygen may also be a factor
limiting the size of bacterial colonies formed on
an agar surface.
 Bacteria that is free-living environmental strain
but sometimes cause infection in
 Chemolithotrophs – independent classes of
immunocompromised people. (pseodomonads
bacteria that can utilize atmospheric Carbon
and Achromobacter)
Dioxide and Nitrogen and derive nutrients from
 Faster Rate of Growth is obtained when
simple inorganic forms of these elements:
glucose/succinate is the carbon source rather
o Carbon
than lactose/glycerol and when the source of
o Nitrogen
o Water
 nitrogen is amino acids rather than ammonium
salts.
 If the bacteria faced with a choice among
carbon and nitrogen source, it will adapt their
physiology to the preferred substrate and only
after it is depleted it will go to less preferred
substrate.
 Diauxic Growth – liquid cultures with dual
substrate that is characterized by a second lag
phase during logarithmic growth period.
 Many pathogens require complex growth
media if they are to be cultured in vitro. (ex.
Helicobacter pylori)
8. DETECTION, IDENTIFICATION, AND
CHARACTERIZATION OF ORGANISMS OF
PHARMACEUTICAL AND MEDICAL SIGNIFICANCE
Various cultural approaches are deployed in enrichment
and culture media, in order to enumerate, detect, and
identify microorganisms to make a specific diagnosis.
8.1 CULTURE TECHNIQUES
Specimens are required to be cultured to:
1. Assess the total number of specific groups of
microorganisms
2. Determine the presence or absence of
particular named species
8.1.1 Enumeration
Simplest way to enumerate the microorganisms that
contaminate an object or liquid sample:
1. Dilute the sample to varying degrees
2. Inoculate the surface of a predried nutrient
agar with known volumes of those dilutions
Rapid enumeration techniques – indirectly measure the
most probable nuber of viable cells.
8.1.1.1 Enumeration Media
ULANGCO • GOMEZ • YANG • SERRANO • BORJA
2E PH
Enumeration media – will only ever culture a subset of
cells towards which the medium and incubation
conditions are directed.
Used to enumerate bacteria:
1. Simple sugars – Carbon sources
2. Trace level of amino acids
Any description of a viable bacterial count must specify
the incubation conditions.
Psychrophilic Gram-negative bacteria
- growing in water at 10 degrees Celsius
- major source of bacterial pyrogen
Highly nutritious media
- e.g. Blood Agar
- Used as enumeration media
- Particularly used when looking for
microorganisms such as staphylococci
In the pharmaceutical industry, microbiological
monitoring will generally report the total aerobic count.
(e.g. Tryptone soya agar)
Inhibitors for bacterial growth
- e.g. Rose Bengal
- Added to select for moulds
8.1.1.2 Rapid enumeration techniques
1. Bioluminescence – can only detect those
organisms that are able to grow in the chosen
medium
2. Epifluorescence
3. Impedance techniques
Electronic particle counters
- used in the examination of pharmaceutical
waters and aqeuous pharmaceutical products
- Coulter counters – used to determine bacterial
concentration
None of the rapid techniques are able to isolate
individual organisms. They do not, therefore, aid in the
characterization or identification of the contaminants.
8.1.2 Enrichment Culture
Enrichment cultures
- intended to increase the dominance of
numerically minor component
- always liquid
- intended to provide conditions that are
favourable for the growth of the desired
organism
Enrichment culture is achieved through:
1. Manipulation of the pH and tonicity of the
medium
2. Inclusion of chemicals tat inhibit the growth of
unwanted species
MacConkey Broth – contain bile salts that will inhibit the
growth of non-enteric bacteria and used to enrich for
Enterobactriaceae
8.1.3 Selective Media
Selective media
- solidified enrichment broths
- suppress the growth of particular bacteria and
allow the growth of others
- an adjunct to characterizing the nature of
contaminants
Mannitol salts agar – favour the growth of micrococci
and staphylococci
Centrimide agar – favour the growth of Pseudomonads
Presumptive counts – Counts of colonies obtained on
selective solid media
8.1.4 Identification Media (Diagnostic)
Identification media
8.3 BIOCHEMICAL TESTING AND TAPID IDENTIFICATION
- Differing ability of bacteria to ferment sugars,
glycosides, and polyhydric alcohols is widely
used to differentiate the Enterobactriaceae
and in diagnostic bacteriology.

- contains nutrients and reagents that indicate
presence of particular organisms (usually
through color formation)
- Used only as a guide to identification
- Microscopy and biochemical or genetic
characterization are much more definitive
MacConkey Agar
- contain lactose sugar and a pH indicator that
facilitates the identification of colonies of
bacteria that can ferment lactose (Escherichia
spp., Klebsiella spp.)
- Non-fermentive coliforms (Salmonella spp.,
Shigella spp.)
Egg-yolk lecithin – gives an agar a cloudy appearance
that clears around colonies of organisms that produce
lecthinase. (e.g. a virulence factor in staphylococci)
8.2 MICROSCOPY
Multiwell microtitration plates – can be inoculated in a
single operation either with an inoculated wire or with a
suspension of pure culture.
8.4 MOLECULAR APPROACHES TO IDENTIFICATION
Denaturing gradient gel electrophoresis
– isolates and amplifies 16S ribosomal DNA
– enables phylogenetic relationships to be
derived even for those bacteria that have not
been previously identified.
Gene probes carrying fluorescent dyes
- used in hybridization procedures with the
collected clinical material
8.5 PHARMACEUTICALLY AND MEDICALLY RELEVANT
MICROORGANISMS
(See Supplementary Notes on Bacteria 2 from Ma’am Gina)

- Observation of stained and wet preparations of

clinical specimens (blood, pus, sputum) and
isolated purec ulture of bacteria
- Uses application of simple stains, such as the
Gram stain
- Observation of size, shape, arrangement
(cluster, chain, tetrads)
- Can also give an indication to the motility
status of the isolate

ULANGCO • GOMEZ • YANG • SERRANO • BORJA

2E PH