NUTRITION AND GROWTH

bacteria vary considerably in their

requirements for nutrients and in their
ability to synthesize for themselves
various vitamins and growth factors
there is a need for the provision of
carbon, nitrogen, water, phosphorus,
potassium and sulfur
minor requirement for trace elements
such as magne- sium, calcium, iron, etc
chemolithothrops - able to derive much
of their nutrition from simple inorganic
forms of these elements; can even
utilize atmospheric carbon dioxide and
nitrogen as sources of carbon and
nitrogen
many classes of bacteria are
auxotrophic and can grow on simple
sugars together with ammonium ions, a
source of potassium and trace elements
such bacteria can synthesize for
themselves all the amino acids and
ancillary factors required for growth and
division (pseudomonads and
Achromobacter)
a faster rate of growth is often obtained
when glucose or succinate is the carbon
source rather than lactose or glycerol,
and when amino acids are provided as
sources of nitrogen rather than
ammonium salts

many pathogens require complex

growth media if they are to be cultured
in vitro
possible that in the future many disease
states currently thought to have no
microbiological involvement could be
identified as being of bacterial origin
(Helicobacter pylori)

DETECTION, IDENTIFICATION AND

CHARACTERIZATION OF ORGANISMS OF
PHARMACEUTICAL AND MEDICAL
SUBSTANCES
once a microorganism has been isolated
in pure culture, usually from a single
colony grown on an agar plate, then
further characterization may be made by
the application of micros- copy together
with some relatively simple biochemical
tests
characterization of organisms has
become simplified by the introduction of
rapid identification systems
molecular approaches have enabled
identification of organisms without the
need to culture them

CULTURE TECHNIQUES
majority of samples taken for
examination contain mixtures of different
species
simple plating onto an agar surface may
fail to detect an organism that is present
at <2% of the total viable population
various enrichment culture techniques
may therefore be deployed to detect
trace numbers of particular pathogens,
prior to confirmatory identification

Diauxic growth on a mixture of glucose and

lactose.

Enumeration
simplest way in which to enumerate
the microorganisms that contaminate
an object or liquid sample is to dilute
that sample to varying degrees and
inoculate the surface of a pre-dried
nutrient agar with known volumes of
those dilutions
number of colonies formed might not
relate to the viable number of cells,
as clumps of cells will only produce a

single colony and they will only

detect a particular subset of the
viable bacteria present in the sample
that can grow under the chosen
conditions
number of techniques are currently
being developed in order speed up
the enumeration process
enumeration media will only ever
culture a subset of cells towards
which the medium and incubation
conditions are directed
simple salts media with relatively
simple sugars as carbon sources
and trace levels of amino acids are
often used to enumerate bacteria
associated with water
highly nutritious media, e.g. blood
agar, are also used as enumeration
media (Staphylococci)
Sometimes inhibitors of bacterial
growth (e.g. Rose Bengal) can be
added to a medium in order to select
for moulds
some of the rapid methods that have
been used for bacteria and other
microorganisms, e.g.
bioluminescence, epifluorescence
and impedance techniques
examination of pharmaceutical
waters and aqueous pharmaceutical
products electronic particle counters,
e.g. Coulter counters, can be used to
determine bacterial concentration
similar counters are available that
are able to analyze particles found in
air
other rapid techniques aim to detect
microbial growth rather than to
visualize individual cells and colonies
bioluminescence can only detect
those organisms that are able to
grow in the chosen medium
none of the rapid techniques are able
to isolate individual organisms; they
do not therefore aid in the
characterization or identification of
the contaminants
Enrichment Culture

Enrichment cultures are intended to

increase the dominance of a numerically
minor component of a mixed culture
such that it can be readily detected on
an agar plate
always liquid and are intended to
provide conditions that are favourable
for the growth of the desired organism
and unfavourable for the growth of other
likely isolates
McConkey broth contains bile salts that
will inhibit the growth of non-enteric
bacteria and may be used to enrich for
Enterobacteriaceae
Selective Media
selective media are solidified enrichment
broths, so again they are intended to
suppress the growth of particular groups
of bacteria and to allow the growth of
others
mannitol salts agar will favor the growth
of micrococci and staphylococci, and
cetrimide agar will favor the growth of
pseudomonads
counts of colonies obtained on selective
solid media are often documented as
presumptive counts
Identification Media (Diagnostic)
identification media contain nutrients
and reagents that indicate, usually
through some form of color formation,
the presence of particular organisms
inclusion of lactose sugar and a pH
indicator into McConkey agar facilitates
the identification of colonies of bacteria
that can ferment lactose
fermentation leads to a reduction in pH
within these colonies and can be
detected by an acid shift in the pH
indicator
the inclusion of egg-yolk lecithin into an
agar gives it a cloudy appearance that
clears around colonies of organisms that
produce lecithinase
MICROSCOPY
application of simple stains such as the
Gram stain can divide the various

genera of bacteria into two convenient

broad groups
size and shapes of individual cells and
their arrangement into clusters, chains
and tetrads will also guide identification
examination of wet preparations can
give an indication as to the motility
status of the isolate, and these
procedures all represent an important
first stage in the identification process

BIOCHEMICAL TESTING AND RAPID

IDENTIFICATION
differing ability of bacteria to ferment
sugars, glycosides and polyhydric
alcohols is widely used to differentiate
the Enterobacteriaceae and in
diagnostic bacteriology generally
identification of particular species and
genera by such processes is timeconsuming, expensive and may require
numerous media and reagents
process has become simplified in recent
years by the development of rapid
identification methods
the latter often use multiwell
microtitration plates that can be
inoculated in a single operation either
with an inoculated wire or with a
suspension of a pure culture
simple kits may perform only 815 tests,
more complex ones are capable of
performing 96 simultaneous biochemical
evaluations
large diagnostic laboratories and in
quality assurance laboratories
automated systems are deployed that
can inoculate, incubate and analyze
hundreds of individual samples at a time
MOLECULAR APPROACHES TO
IDENTIFICATION
the need to identify microorganisms
rapidly has led to the development of a
number of molecular identification and
characterization tools
these have not yet become routinely
adopted in the analytical or diagnostic
laboratory but probably will be in the
future

molecular approaches can be of

especially useful when attempting to
detect a particular species
PHARMACEUTICALLY AND MEDICALLY
RELEVANT MICROORGANISMS
microorganisms of medical and
pharmaceutical relevance can be
broadly classified into those organisms
that are harmful or problematic, and
those that can be used to our advantage
some microorganisms, depending on
the situation, can fall into both
categories
microorganisms cause some of the most
important diseases of humans and
animals and they can also be found as
major contaminants of pharmaceutical
products
many large-scale industrial processes,
e.g. antibiotic production, are based on
microorganisms, and selected species
can be used to test disinfectant efficacy
and to monitor sterilization procedures

FUNGI
yeast, such as brewers yeast, and
moulds, such as Penicillium
chrysogenum which produces the
antibiotic penicillin, are classified as
fungi
yeast cells tend to grow as single cells
which reproduce asexually in a process
known as budding
fungi are eukaryotic organisms, i.e. their
cells possess a nuclear membrane,
consequently there are many similarities
between the biochemistry of fungal cells
and human cells
medically, fungi are an extremely
important group of microbes, being
responsible for a number of potentially
fatal diseases in humans
significant number of fungi are of great
benefit to humanity in terms of the
production of alcoholic beverages,
bread, enzymes, antibiotics and
recombinant proteins
kingdom Fungi can be subdivided into
six classes
class Oomycetes contains the mildews
and water moulds

class Ascomycetes contains the

mildews, some moulds and most yeast
species
class Basidiomycetes contains the
mushrooms and bracket fungi
class Teliomycetes contains the rust
fungi
class Ustomycetes contains the smuts
class Deuteromycetes contains species
such as Aspergillus, Fusarium and
Penicillium
four distinct phyla within the fungal
kingdom; these are the Chytridiomycota,
Zygomycota, Ascomycota and
Basidiomycota

STRUCTURE OF FUNGAL CELL

typical yeast cell is oval in shape and is
surrounded by a rigid cell wall which
contains a number of structural
polysaccharides and may account for up
to 25% of the dry weight of the cell wall
the thickness of the cell wall may vary
during the life of the cell
glucan, the main structural component
of the fungal cell wall, is a branched
polymer of glucose which exists in three
forms in the cell: -1,6- glucan, -1,3glucan and -1,3,--1,6-complexed with
chitin
mannan is a polymer of the sugar
mannose and is found in the outer
layers of the cell wall
chitin, is concentrated in bud scars that
are areas of the cell from which a bud
has detached
mannoproteins form a fibrillar layer that
radiates from an internal skeletal layer

that is formed by the polysaccharide

component of the cell wall
enzymatic or mechanical removal of the
cell wall leaves an osmotically fragile
protoplast which will burst if not
maintained in an osmotically stabilized
environment
periplasmic space is a thin region that
lies directly below the cell wall
cell membrane or plasmalemma is
located directly below the periplasmic
space and is a phospholipid bilayer
which contains phospholipids, lipids,
protein and sterols
nuclear membrane contains pores to
allow communication with the rest of the
cell
nucleus is a discrete organelle and, in
addition to being the repository of the
DNA, also contains proteins in the form
of histones
mitochondrion is a semi- independent
organelle as it possesses its own DNA
and is capable of producing its own
proteins on its own ribosomes which are
referred to as mitoribosomes
fungal cell contains a vast number of
ribosomes which are usually present in
the form of polysomes lines of
ribosomes strung together by a strand of
mRNA
ribosomes are the site of protein
biosynthesis
vacuole is employed as a storage
space where nutrients, hydrolytic
enzymes or metabolic intermediates are
retained until required

MEDICAL SIGNIFICANCE OF FUNGI

yeast C. albicans is the most frequently
encountered human fungal pathogen,
being responsible for a wide range of
superficial and systemic infections
(oropharyngeal and genital conditions)
mould Aspergillus fumigatus is the
dominant fungal pulmonary pathogen of
humans and generally presents as a
problem in those with pre-existing lung
disease or damage
most commonly encountered
dermatophytic infections are athletes
foot and ringworm

ANTIFUNGAL THERAPY
choice and dose of an antifungal will
depend upon the nature of the condition,
whether there are any under- lying
diseases, the health of the patient and

whether anti- fungal resistance has been

identified as compromising therapy
ideal antifungal drug should target a
pathway or process specific to the
fungal cell, so reducing the possibility of
damaging tissue and inducing unwanted
side effects

Polyene Antifungals
polyene antifungals are characterized by
having a large macrolide ring of carbon
atoms closed by the formation of an
internal ester or lactone
principal polyenes are amphotericin B
and nystatin
amphotericin B is produced by the
bacterium Streptomyces nodosus and
its activity is due to the ability to bind
ergosterol, which is the dominant sterol
in fungal cell membranes, and
consequently increases membrane
permeability by the formation of pores
nystatin was discovered in 1950 and
exhibits the same mode of action as
amphotericin B but tends to have lower
solubility

Azole Antifungals
first generation of azole antifungals
revolutionized the treatment of mucosal
and invasive fungal infections
azoles are still the most widely used
group of anti- fungal agents
azoles in current clinical use are clotrimazole, miconazole, econazole and
ketoconazole; newer

drugs such as itraconazole, fluconazole

and voriconazole have important
applications in the treatment of systemic
infections
miconazole was the first azole used to
treat systemic fungal infections but
demonstrated a number of toxic side
effects
ketoconazole produced high serum
concentrations upon oral administration
but had poor activity against
aspergillosis
fluconazole has proved highly effective
in the treatment of infections caused by
C. albicans but shows limited activity
against Aspergillus
itraconazole became available for
clinical use in the late 1980s and was
the first azole with proven efficacy
against Aspergillus
novel azole drugs with increased ability
to inhibit the fungal 14- demethylase
are also becoming available
agents which include voriconazole,
posaconazole and ravuconazole, have a
wider spectrum of activity than
fluconazole and it has been suggested
that some of them show fungicidal
effects to some species
voriconazole is one of the newest
second-generation triazole antifungal
drugs and it shows good activity against
pulmonary aspergillosis and cere- bral
aspergillosis