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INTRODUCTION - Plant pathogen

- No known human pathogen


- Improvement of life expectancy is due to better nutrition
and sanitation PRIONS
- Development of many vaccines
- Disinfection and properties of chemicals (as antiseptics, - They contain no nucleic acid
disinfectants, and preservatives) - Atypical form of mammalian protein that can interact with a
- Benefits or use of different microorganisms in pharmacy normal protein molecule and cause it to undergo a
- Microorganisms differ in terms of: conformational change so it can become a prion and ceases
a) Shape to its normal function
b) Size - Agents responsible for transmissible spongiform
c) Appearance encephalopathies
d) Genetic and metabolic characteristics a) Creutzfeld-Jakob disease (CJD)
- All these properties are used in classifying microorganisms b) Bovine spongiform encephalopathy
into major groups: - Simplest and most recently recognized agents of infectious
a) Bacteria disease
b) Fungi - Important in pharmaceutical context owing to their extreme
c) Protozoa resistance to conventional sterilizing agents:
d) Viruses a) Steam
b) Gamma radiation
VIRUSES c) Disinfectants

- Does not have cellular structure PROKARYOTES


- Particles are composed of nucleic acid surrounded by
protein - Bacteria and archea
- Some particles possess a lipid envelope and associated - No true nucleus
glycoproteins - Possess a single chromosome not separated from the other
- recognizable chromosomes, cytoplasm and cell cell contents by the membrane
membranes are absent - Normally haploid (possess only one copy of the set of genes
- incapable of independent replication in the cell)
- does not contain the enzymes necessary to copy their own - Reproduces asexually
nucleic acid
EUKARYOTES
- all are intracellular parasite
- are reproduced using the metabolic capabilities of the host
- All other cellular microorganisms:
cell a) Fungi
- Observed in different shapes: b) Protozoa
a) Helical c) Algae
b) Linear - True cell nucleus in which the chromosomes are separated
c) Spherical
from the cytoplasm by the nuclear membrane
- Observed in different sizes: - Diploid (possess 2 copies of the set of genes in the cell)
a) 20-400nm - Reproduces sexually
- Observed in different nucleic acid compositions:
a) Single-stranded CHARACTERISTIC EUKARYOTES PROKARYOTES
b) Double-stranded Size Normally > 10µm Typically 1-5µm
c) Linear RNA/DNA
d) Circular RNA/DNA Location of Within a true In the cytoplasm
- Smaller than bacteria chromosomes nucleus separated (attached to the cell
- Cannot be seen with normal light microscope from cytoplasm (by membrane)
- Must be viewed using electron microscope nuclear membrane)
Nuclear division Mitosis and meiosis Mitosis and meiosis
VIROIDS are absent
Nucleolus Present Absent
- Also known as “virusoids” Reproduction Asexual or sexual Asexual
- Simpler than viruses Chromosome More than 1 1
number
- Infectious particles comprising, single-stranded RNA without
Mitochondria and May be present Absent
any associated protein chloroplasts
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Cell membrane Steroids present Steroids absent b) MOULD
composition - Imprecise term to describe fungi that do not
Cell wall Cell walls when Walls contain form fruiting bodies visible to the naked eye
composition present contain peptidoglycan - Excludes toadstools and mushrooms
cellulose or chitin
- Tangled mass (mycellum) of filaments of
but not
peptidoglycan threads (hyphase) which vary between 1 and
Ribosomes Cytoplasmic Ribosomes are 70S 50 µm wide
ribosomes are 80S
Flagella Structurally complex Structurally simple - Most fungi are saprophytes with relatively few having
Pili Absent Present pathogenic potential
Fimbriae Cilia Present - Their ability to form spores resistant to drying makes them
Storage compounds Poly-β- Poly-β- important as contaminants of pharmaceutical raw materials
hydroxybutyrate hydroxybutyrate (materials of vegetable origin)
absent present
PROTOZOA
BACTERIA AND ARCHAEA - Eukaryotic
- Predominantly unicellular microorganisms regarded as
A. BACTERIA
animals than plants
- Unicellular, although some species arise as sheathed
- Free-living organisms that occur in water and soil
chains of cells
- Not normally found contaminants of raw materials or
- Possess the properties listed under prokaryotes
manufactured medicines
- Exhibit great diversity of form, habitat, metabolism,
pathogenicity, etc NAMING OF MICROORGANISM
- Bacteria of interest in pharmacy is eubacteria
- Genus (plural = genera) and species
B. ARCHAEA a) GENUS – written with an upper case initial letter
- Have no pharmaceutical importance b) SPECIES - written with a lower case initial letter
- Formerly considered largely to comprise organisms - Italicized or underlined
capable of living in extreme environment: - May be abbreviated by shortening the name of the genus
a) Extreme temperature provided that the shortened form is unambiguous
b) Salnity
c) pH MICROBIAL METABOLISM
- Formerly comprised of organisms exhibiting
specialized modes of metabolism A. CHEMOHETEROTROPHS
- Obtain carbon, nitrogen and energy by breaking
C. EUBACTERIA down organic compounds
- Typically rod-shaped (bacillus), spherical (cocci),
curved or spiral cells B. CATABOLIC REACTIONS
- Approximately 0.5-5mm - Chemical reactions by which the energy is liberated
- Divided into 2 groups as either Gram-positive or by digestion of food materials
Gram-negative bacteria
C. ANABOLIC REACTIONS
FUNGI - Use of liberated energy to make complex cellular
polymers, proteins, carbohydrates, and nucleic acid
- Eukaryotes
- Structurally complex and varied in appearance than D. OXIDATION
bacteria - Removal or loss of electron
- Non-photosynthetic plants
- Covers both yeasts and moulds E. REDOX REACTION
a) YEASTS - Oxidation and reduction
- unicellular
- larger than bacteria (5-10 µm) F. GLYCOLYSIS
- divide by process of binary fission or - Conversion of glucose to pyruvic acid
budding
G. FERMENTATION
- Release more energy from sugar
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CULTURE MEDIA - Media for yeasts and moulds often have a lower pH (5.5-
6.0) than bacterial culture media (7.0-7.4)
- Liquid or solid - Lactic acid may be used to impart a low pH because it is
a) LIQUID MEDIA – gelled by addition of sugar not inhibitory to fungi at the concentrations used
b) SOLID MEDIA – designated for the growth of - Some fungal media intended for use with specimens that
anaerobic organisms that contain non-toxic may also contain bacteria may be supplemented with
reducing agents antibacterial antibiotics:
- CARBOHYDRATES are used in the form of starch or sugars a) Chloramphenicol
- GLUCOSE or dextrose is the only sugar employed as a b) Tetracyclines
nutrient
- Microorganisms differ in terms of: CULTIVATION METHODS
a) Their ability to ferment various sugars
b) Their fermentation patterns may be used as aid in - Most bacteria and some yeasts divide by a process of
identification binary fission
- Sodium chloride may be incorporated to adjust osmotic - where a cell enlarges/elongates then forms a cross-
pressure wall (septum) that separates the cell into two more or
- Buffers are added to neutralize acids that result from sugar less equal compartments each containing a copy of the
metabolism genetic material
- Routine culture media may be enriched by the addition of - Growth continues until one or more nutrients is exhausted,
materials like milk, blood or serum, and organisms that need or toxic metabolites (organic acids) accumulate and inhibit
such supplements in order to grow are described as enzyme systems
“exacting” in their nutritional requirements - Starting from a single cell many bacteria can achieve
- There are many materials commonly added to selective or concentrations of the order of 109 cells ml-1 or more
diagnostic media whose function is to restrict the growth of - At concentrations below 107 cells ml-1 culture media is clear,
certain types of microorganisms while enhancing the growth but the liquid becomes turbid as the concentration increases
of others above this value
o Examples: - Turbidity is an indirect means of monitoring culture growth
 ANTIBACTERIAL ANTIBIOTICS added to - When growing on solid media in Petri dishes, individual
fungal media to suppress bacterial bacterial cells can give rise to colonies following overnight
contaminants incubation under optimal conditions
- Colony is a collection of cells arising by multiplication of a
 BILE to suppress organisms from anatomical
single original cell or a small cluster of them (colony-forming
sites other than the GIT
unit or CFU)
- Many additives are used in media for organism
- Colony does not imply any particular number of cells
identification purposes
but usually taken to mean a number sufficiently large
- Enrichment is occasionally used in the sense of making a
to be visible by eye
medium nutritionally richer to achieve more rapid growth
- Colonies vary between bacterial spores, and their
- An enrichment medium is one designed to permit a
shapes, sizes, opacities, surface markings and
particular types of organism to grow while restricting
pigmentation may all be characteristic of the species,
others, so the one that grows increases in relative
so these properties aid in identification purposes
numbers and is “enriched” in a mixed culture
- Anaerobic organisms may be grown in Petri dishes
- Solid media designed for growth of anaerobic organisms
provided that they are incubated in an anaerobic jar
contain non-toxic reducing agents:
- Such jars are made of rigid plastic with airtight lids
a) Sodium thioglycollate
- Petri dishes are placed in them together with a low-
b) Sulphur-containing amino acids
temperature catalyst
- These compounds create redox potentials of -200 mV or
- The catalyst (consists of palladium-coated pellets or wire)
less and eliminate inhibitory effects of oxygen or
causes the oxygen in the jar to be combined with hydrogen
oxidizing molecules on anaerobic growth
that is generated by addition of water to sodium
- The inclusion of these compounds is less important in
borohydrite
liquid media (where a sufficiently low redox potential
- Usually contained in a foil sachet that is also placed in
may be achieved by boiling); this expels dissolved
the jar
oxygen, which in unstirred liquids only resaturates the
- Alternatively, oxygen may be removed by
upper few mL of liquid
combination with ascorbic acid
- Redox indicators (methylene blue or resazurin) may be
- After its removal, an anaerobic atmosphere is achieved and
incorporated in anaerobic media to confirm that a
this is monitored by redox indicator
sufficiently low redox potential has been achieved
- Resazurin is used as a solution soaking a fabric strip
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PLANKTONIC AND SESSILE (BIOFILM) GROWTH - In a surface spread technique, the sample is spread over
the surface of agar
- Freely-suspended cell - The Miles Misra (surface drop) method allows several
- Bacteria attached to a substrate in this way are described individual drops of culture to spread over discrete areas of
as sessile and exhibits the biofilm or micro-colony mode of about 1 cm diameter on the agar surface
growth - These procedures are suitable for samples expected
- Planktonic cells are routinely used for all testing to contain concentrations exceeding 100 CFU-1
procedures designed to assess the activity of antimicrobial - If there are no clear indications of the order of
chemicals and processes magnitude of the of the concentration in the sample, it
- The recognition that planktonic growth is not the natural is necessary to plate out the sample at each of two,
state for many organisms prompted investigations of the three or more dilutions
relative susceptibilities of planktonic- and biofilm-grown - Most probable number (MPN) counts may be used when
cells to antibiotics, disinfectants and decontamination or the anticipated count is relatively low
sterilization procedures - the procedure involves inoculating multiple tubes of
- Planktonic and sessile bacteria exhibit different culture medium (3-5) with 3 different volumes of
susceptibilities to these lethal agents, thus prompted a samples
reappraisals of the appropriateness of some of the - Direct microscopic counting may be an appropriate
procedures used method for bacteria, yeasts, and fungal spores but not for
moulds
ENUMERATION OF MICROORGANISMS
- Indirect measures of biomass-like assays of insoluble
- There are several situations where it is necessary to measure nitrogen, protein or nucleic acids are possible for all cell
types
the number of microbial cells in a culture, sample, or
specimen: - The traditional methods of viable counting all suffer from the
a) When measuring levels of microbial contamination same limitations:
a) Relatively labour intensive
in a raw material or manufactured medicine
b) When evaluating the effects of an antimicrobial b) Not easy to automate
chemical or decontamination process c) Slow (they require an incubation period to develop
or for liquid cultures to become turbid)
c) When using microorganisms in the manufacture of
therapeutic agents d) May require large volumes of culture media, many
d) When assessing the nutrient capability of a growth Petri dishes, and a lot of incubator splace
- Epifluorescent techniques use fluorescent dyes that:
medium
- In some cases it is necessary to know the total number of a) exhibit different colors in living and dead cells
microbial cells present in both living and dead (acridine orange)
- In vaccine manufacture, dead and living cells may both b) appear colorless outside the cell but become
produce an immune response fluorescent when absorbed and subjected to cell
metabolism (fluorescein diacetate)
- In pyrogen testing, both dead and living cells induce
fever when injected into the body - Livin cells generate ATP that can be detected by enzyme
- No fibril reaction, no pyrogen in sterile products assays (luciferin) emits light when exposed to firefly
luciferase in the presence of ATP
- Total count is a counting procedure enumerating both living
and dead cells - The resistance, capacitance or impedance of a culture
- Viable count is more common and records the living cells medium changes as a result of bacterial or yeast growth
and metabolism and these electrical properties vary in
alone
- Total viable count (TVC) is used in most pharmacopeias proportion to cell concentration
and by many regulatory agencies to mean a viable count - Manometric techniques are for monitoring the growth of
organisms that consume or produce significant quantities of
that records all different species or types of microorganisms
present in a sample gas during their metabolism (yeasts or moulds producing
- The first three traditional methods of viable counting all carbon dioxide as result of fermentation)
- Pyrogens are usually found in Gram-negative bacteria
operate on the basis that a living cell will give rise to a
visible colony when introduced into the surface of a suitable
MICROBIAL GENETICS
a suitable medium and incubated
- The procedure for pour plating involved the addition of a - The nature of the genetic material possessed by a microbial
small volume (1 mL) of sample into molten agar at 45℃ then cell and the manner in which the genetic material may be
poured into empty Petri dishes transferred to other cells depends on whether the organism
- After incubation the resultant colonies are counted and the is a prokaryote or a eukaryote
total is multiplied by the dilution factor
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- Microorganisms may adapt rapidly to new environments
and devise strategies to avoid or negate stressful or
potentially harmful circumstances
- Their ability to survive adverse conditions may result from
the organism using genes it possesses, or by acquisition of
new genetic information
- Genotype describes the genetic composition of an organism
- It is not uncommon for a microbial cell to possess a particular
gene but not to express it
- Spontaneous mutation rates are not influenced by
mutagenic chemicals or ionizing radiation

PHARMACEUTICAL IMPORTANCE OF THE MAJOR CATEGORIES


OF MICROORGANISMS

- The importance of viruses derives exclusively from their


pathogenic potential
- Because of their lack of intrinsic metabolism they are
not susceptible to antibiotics
- Viral infections are among the most dangerous and difficult
to cure
- Only viruses appear in the Hazard Category 4 (most
serious) as classified by the Advisory Committee on
Dangerous Pathogens
- Viruses are incapable of growing on manufactured
medicines or raw materials
- They do not cause product spoilage
- They have no synthetic capabilities that can be
exploited in medicines manufacture
- Easy to destroy by heat, radiation or toxic chemicals
- Pathogens are of long-standing notoriety as a result of their
ability to resist the activity of antibiotics and biocides
- The ability of bacteria to grow on diverse substrates ensures
that their potential as agents of spoilage is well recognized
- The ability of many species to survive drying means that
they survive well in dust and so become important as
contaminants of manufactured medicines
- The ability to survive in dry conditions and other adverse
environments (heat, radiation, toxic chemicals) is well
exemplified by bacterial spores
- Their pre-eminence at or near the top of the “league
table” of resistance to lethal agents has resulted in
spores acting as indicator organisms
- Fungi form spores that survive drying
- The degree of resistance presented by the spores is less
than that exhibited by bacteria
- Fungi do not represent a sterilization problem
- Fungi do not create a significant infection hazard
- Few fungal species are considered major pathogens for
animals that possess a fully functional immune system

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