PHA6114

PHARMACEUTICAL MICROBIOLOGY AND

PARASITOLOGY

FUNDAMENTAL FEATURES OF MICROBIOLOGY

ASST. PROF. RHONA P. RAMOS, MSc.
Course Facilitator
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MICROBIOLOGY

study of microscopic organisms, such as

bacteria, viruses, archaea, fungi and
protozoa

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Microorganisms differ enormously in terms of their shape,
size and appearance and in their genetic and metabolic
characteristics.
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NON-CELLULAR

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Viruses

do not have a cellular structure

particles composed of nucleic
acid surrounded by protein
(capsid)

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Viruses

incapable of independent replication

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Viruses

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Viruses

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Viruses

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 Viruses

bacteriophage H1N1 measles

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Viroids or virusoids

infectious particles comprising single-

stranded RNA without any associated protein
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Viroids or virusoids

plant pathogens

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Prions

PROTEINS - they contain NO nucleic acid

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Prions

atypical form of a mammalian protein that

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 Prions

Creutzfeldt–Jakob disease (CJD) bovine spongiform encephalopathy (BSE)

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Prions

extreme resistance to conventional sterilizing

agents like steam, gamma radiation and
disinfectants
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CELLULAR

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CELLULAR

bacteria
plants
animals

archaea
fungi
protists

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CELLULAR

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ARCHAEA

no pharmaceutical importance
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BACTERIA

unicellular; sheathed chains of cells

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BACTERIA

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BACTERIA

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BACTERIA

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BACTERIA
harmless or positively beneficial
saprophytes
parasites or pathogens e.g. Rickettsia and
chlamydia

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BACTERIA

possess cell walls - resistant to osmotic stress

grow well at temperatures between ambient
and human body temperature

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BACTERIA
exhibit wide variations in their requirement for,
or tolerance of, oxygen
strict aerobes
strict anaerobes
facultative anaerobes
microaerophils
M. tuberculosis Clostridium tetani E. coli H. pylori

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FUNGI

structurally more complex and varied in

appearance than bacteria
non-photosynthesizing plants
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FUNGI

YEAST
unicellular organisms that are larger than
bacteria (typically 5–10µm)
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FUNGI

YEAST

division through binary fission

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FUNGI

YEAST

division through budding

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FUNGI

MOULD
term used to describe fungi that do not form
fruiting bodies visible to the naked eye

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FUNGI

MOULD
consist of a tangled mass (mycelium) of
filaments or threads (hyphae) which vary
between 1 and over 50 µm wide
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FUNGI

they have the ability to form spores that are resistant to

drying makes them important as contaminants of
pharmaceutical raw materials, particularly materials of
vegetable origin

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PROTOZOA

free-living motile organisms that occur in

water and soil

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PROTOZOA

Plasmodium malaria Entamoeba histolytica

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PROTOZOA

not normally found as contaminants of raw materials or

manufactured medicines

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 NAMING

Escherichia coli staphylococci

genus species

in italics or underlined

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METABOLISM
CHEMOHETEROTROPHS

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METABOLISM

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METABOLISM

REDOX POTENTIAL
indicates whether oxidizing or reducing
conditions prevail in a particular situation

Anaerobic - thrive in low redox potentials environments

Aerobic - thrive in high redox potential environments
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 METABOLISM

GLYCOLYSIS
conversion of glucose to pyretic
acid in which oxygen is not
required

Aerobic - oxygen is the final electron acceptor

Anaerobic - nitrate or fumarate are the final electron acceptor

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METABOLISM
FERMENTATION
a process in which the final
electron acceptor is an
organic molecule
starting materials (sugars and
other organic material)
metabolic products (acids,
alcohols, and other solvents

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METABOLISM

materials as food
mineral salts and sugar
carbohydrates
proteins
non-carbohydrate foods

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METABOLISM

switching to different metabolic pathways

explains why none of the major antibiotics work by
interfering with the chemical reactions microorganisms use
to metabolize their food
antibiotic action must exploit a difference in metabolism
between the organism to be killed and the human host

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METABOLIC PRODUCTS
primary metabolites
products that arise during the period when a microbial
culture is actually growing
ethanol from yeasts
organic acids that decreases pH during microbial
growth further metabolized to increase pH after
growth

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METABOLIC PRODUCTS

secondary metabolites
produced after cell multiplication has slowed or
stopped, i.e. in the ‘stationary phase’
many of them have commercial or therapeutic
importance
antibiotics, enzymes, toxins, carbohydrates

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CULTIVATION

culture media

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CULTIVATION

culture media
PROTEIN
meat extracts, milk, soya
Trypsin

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CULTIVATION

culture media
PROTEIN
meat extracts, milk, soya
B-GROUP VITAMINS
from yeast extract

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CULTIVATION

culture media
PROTEIN
meat extracts, milk, soya
B-GROUP VITAMINS
from yeast extract
CARBOHYDRATES
starch or sugars (glucose/dextrose)

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CULTIVATION

culture media
OTHER SUGARS for diagnostic purposes
NaCl - to adjust osmotic pressure
BUFFERS - to neutralize acids from
metabolism

routine culture media may be enriched

with milk, blood or serum
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CULTIVATION

culture media
fluid

solid

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CULTIVATION
sets at 40°C
culture media do not re-liquefy until 90 °C
forms a firm gel at 37 °C

when used as a liquid at 45°C

sufficiently low temperature
to avoid killing
agar (1 - 1.5%) microorganisms

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CULTIVATION

culture media
to restrict growth of others
selective or diagnostic media
antibacterial antibiotics
added to fungal media to suppress bacterial
contaminants
bile
to suppress organisms from anatomical sites
other than the gastrointestinal tract
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CULTIVATION

culture media

enrichment media
used in the sense of making a medium nutritionally richer
to achieve more rapid or profuse growth
designed to permit a particular type of organism to grow
while restricting others

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CULTIVATION

culture media

reducing agents, e.g. sodium thioglycollate or sulphur-containing

amino acids for anaerobic organisms
create redox potentials of −200 mV or less
to diminish or eliminate the inhibitory effects
of oxygen or oxidizing molecules on
anaerobic growth

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CULTIVATION

culture media

pH (5.5–6.0)
for yeasts and moulds
pH (7.0–7.4)
for bacterial culture

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CULTIVATION

methods
binary fission

may take place every 25–30

minutes under optimal conditions
growth at infection sites much
slower

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CULTIVATION

methods
binary fission
overnight incubation of a single cell can achieve 109
cells/mL or more
107 cells/mL - culture media is clear
liquid becomes progressively more cloudy (turbid) as
the concentration increases above this value

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CULTIVATION

methods
binary fission

turbidity is an indirect means of

monitoring culture growth

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CULTIVATION

methods

budding extension and branching

yeast mould
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CULTIVATION

methods
colony
a collection of cells arising by multiplication of a single
original cell or a small cluster of them (called a colony-
forming unit or CFU)

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CULTIVATION

methods
colony

vary between bacterial species, and their shapes, sizes,

opacities, surface markings and pigmentation
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CULTIVATION

methods
anaerobic organisms

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CULTIVATION

methods
colony

mould colony consists of a mycelium that may be loosely or

densely entangled depending on the species, often with
the central area showing pigmentation associated with
spore production

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CULTIVATION

planktonic and sessile (biofilm) growth

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ENUMERATION OF MICROORGANISMS

it is necessary to measure the number of microbial cells in a

culture, sample or specimen
when measuring the levels of microbial contamination in a raw material
or manufactured medicine
when evaluating the effects of an antimicrobial chemical or
decontamination process
when using microorganisms in the manufacture of therapeutic agents
when assessing the nutrient capability of a growth medium

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 ENUMERATION OF MICROORGANISMS

total count counting procedure enumerating

both living and dead cells

viable count records the living cells alone

total viable count viable count that records all the

different species or types of
microorganism that might be present
in a sample
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ENUMERATION OF MICROORGANISMS

counting methods
1. Pour plate
2. Surface spread or surface drop
3. Membrane filter methods
4. Most probable number (MPN)

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ENUMERATION OF MICROORGANISMS
counting methods

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ENUMERATION OF MICROORGANISMS
counting methods

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ENUMERATION OF MICROORGANISMS
counting methods

Miles Misra
several individual drops of culture are allowed to spread
over discrete areas of about 1 cm diameter on the agar
surface
for concentrations exceeding 100 CFU/ml
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ENUMERATION OF MICROORGANISMS
counting methods

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ENUMERATION OF MICROORGANISMS
counting methods
Most probable number (MPN)
from <1 up to 100
microorganisms/ml
more commonly used in the
water, food and dairy industries
poor accuracy and precision

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ENUMERATION OF MICROORGANISMS
counting methods

turbidity measurement using

spectrophotometer or colorimeter

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ENUMERATION OF MICROORGANISMS
counting methods
relatively labor intensive
not easy to automate
slow, because they require an incubation period for
colonies to develop or liquid cultures to become turbid
may require relatively large volumes of culture media,
many Petri dishes and a lot of incubator space

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ENUMERATION OF MICROORGANISMS
rapid counting methods
VIABLE NON-VIABLE

epifluorescent techniques -
use of fluorescent dyes

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ENUMERATION OF MICROORGANISMS
rapid counting methods

enzyme assays - detects ATP

generated by living cells

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ENUMERATION OF MICROORGANISMS
rapid counting methods

The changes in (a) resistance and (b)

capacitance are displayed as a function of
yeast (A12O) growth over time.
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ENUMERATION OF MICROORGANISMS
rapid counting methods

manometric techniques -
measurement of consumed or
produced gas during metabolism

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genetics

bacteria

chromosome - ccc ds DNA

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genetics

bacteria
single chromosome - ccc ds DNA
1mm or more in length, tightly coiled
contain about 1000–3000 genes

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genetics

bacteria

plasmids

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genetics

bacteria
plasmids - resemble chromosomes except that they are
approximately 0.1–1.0% of the size of a bacterial
chromosome
may code for a property that affords a survival advantage
in certain environmental conditions
antibiotic resistance

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genetics

bacteria
plasmids
replicate independently
may also be passed from one cell to another by various
means
some may cross between different species within a
genus or between different genera
conjugative (self- transmissible) plasmids
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genetics

eukaryotes
nucleus - linear
chromosomes, ds DNA +
protein

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genetics

eukaryotes
asexual - mitosis

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genetics

eukaryotes
sexual - meiosis

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genetics
genetic variation and gene expression
genotype - genetic composition of an organism whether
they are expressed or not

phenotypic adaptation genetic adaptation

possessing a acquire new genes either by
particular gene but mutation or by conjugation
not expressing it to
to be better suited to the
avoid wasting energy
new environment

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genetics
genetic variation and gene expression
mutation
important mechanism by which resistance to antibiotics and
other antimicrobial chemicals is achieved

mutation rates
a mutant arises once in every 100 000 to every 10 million
cell divisions

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PHARMACEUTICAL importance

virus
lacks intrinsic metabolism that they are not susceptible to antibiotics
most dangerous and difficult to cure
incapable of growing on manufactured medicines or raw materials,
so they do not cause product spoilage
no synthetic capabilities that can be exploited in medicines
manufacture
easy to destroy by heat, radiation or toxic chemicals

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PHARMACEUTICAL importance
prions

questionable categorization as microorganism

ability to cause, as yet incurable, fatal disease,
extreme resistance to lethal agents
easily withstand sterilizing conditions

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PHARMACEUTICAL importance
bacteria
pathogens
ability to resist the activity of antibiotics and biocides
(disinfectants, antiseptics and preservatives)
meticillin-resistant Staphylococcus aureus (MRSA),
vancomycin-resistant enterococci (VSE) and multiply
resistant Mycobacterium tuberculosis

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PHARMACEUTICAL importance

bacteria
clinically important antibiotics are produced by species of bacteria,
streptomycetes
used in the manufacture of steroids, enzymes and carbohydrates
agents of spoilage in manufactured medicines and raw materials
survive not only in dry conditions but in other adverse environments
(heat, radiation, toxic chemicals), e.g. bacterial spores

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PHARMACEUTICAL importance

fungi
form spores that survive drying, as contaminants of manufactured
medicines
do not create a significant infection hazard
few fungal species are considered major pathogens
can be opportunist pathogens

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PHARMACEUTICAL importance

protozoa
pathogens
do not possess cell walls they do not survive drying well
do not display resistance to sterilizing processes
more troublesome in veterinary medicine and in the tropics

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PRESERVATION

long-term
freezing at −80°C (or lower) in refrigerators
liquid nitrogen at −196 °C in special vessels
freeze-drying

cryoprotectant chemicals—compounds like glycerol or

dimethylsulphoxide - to minimize both the formation of damaging
ice crystals and osmotic stresses

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PRESERVATION

Reference culture
well-defined biosynthetic capabilities or resistance properties
obtained in a freeze-dried form from internationally accessible
culture collections
American Type Culture Collection (ATCC)
UK National Collection of Industrial and Marine Bacteria (NICMB)

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