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LECTURE 4: MICROBIAL NUTRITION AND
METABOLISM

Introduction: Microorganisms require about ten
elements in large quantities because they are used to
construct carbohydrates, lipids, proteins, and nucleic
acids. Several other elements are needed in very small
amounts and are parts of enzymes and cofactors.

Biochemical Components of Cells
- Water: 80% of wet weight
- Dry Weight
o Protein: 40-70%
o Nucleic Acid: 13-34%
o Lipid: 9-15%
o Polysaccharide: 5.0%
o Lipopolysaccharide: 3.4%
o DNA: 3.1%
o RNA: 20.5%
o Also monomers, intermediates, and
inorganic ions

Macronutrients cells make proteins, nucleic acids
and lipids.
- Macromolecules, metabolism
- Dry weight percentage: C (50%), O (17%), N
(13%), H (8.2%), P (2.5%), S (1.8%), Se
(<0.01%)
- Other elements: K, Mg, Fe
- Sources: organic compounds and inorganic
salts

Micronutrients elements needed in trace quantities
- Refer to Table 4.1 (Trace Metals) in Lecture 4,
Slide 7
o Co: Vitamin B
12
; transcarboxylase
(only in propionic acid bacteria)
o Cu: In respiration, cytochrome c
oxidase; in photosynthesis,
plastocyanin, some superoxide
dismutases
o Mn: Activator of many enzymes;
component of certain superoxide
dismutases and of the water-splitting
enzyme in oxygenic phototrophs
(photosystem II)
o Zn: Carbonic anhydrase; alcohol
dehydrogenase; RNA and DNA
polymerases; and many DNA-binding
proteins
o V: Vanadium nitrogenase;
bromoperoxidase
- Enzymes and Tap water
- Refer to Lecture 4, Slide 8
o Essential to all: H, C, N, O, P, S, Se
o Essential cations and anions for most:
Na, Mg, Cl, K, Ca
o Trace metals: Y, Mn, Fe, Co, Ni, Cu,
Zn, Mo, W
o For special functions: B, F, Si, As, Sr,
Cd, Ba
o Unessential but metabolized: the rest
o Unessential, unmetabolized: Noble
gases, Zr, Nb, Hf, Ta, Fe, Ds, Ir, At,
Rs

Growth Factors
- Biotin: carboxylation (Leuconostoc)
- Cyanocobalaman or Vit B
12
: molecular
rearrangements (Euglena)
- Folic Acid: one-carbon metabolism
(Enterococcus)
- Pantothenic Acid: fatty acid mechanism
(Proteus)
- Pyridoxine or Vit B
6
: transamination
(Lactobacillus)
- Niacin: precursor of NAD and NADP
(Brucella)
- Riboflavin or Vit B
2
: precursor of FAD and
FMN (Caulobacter)
- Thiamine or Vit B
1
: aldehyde group transfer
(Bacillus anthracis)

Energy Sources
Phototrophs Light
Chemotrophs Oxidation of organic or
inorganic compound
Hydrogen and Electron Sources
Lithotrophs Reduced inorganic
molecules
Organotrophs Organic molecules
Carbon Sources
Autotrophs CO
2
sole or principal
biosynthetic carbon
source
Heterotrophs Reduced, preformed,
organic molecules from
other organisms

Major Nutritional
Types
Sources of Energy,
Hydrogen/Electron
s or Carbon
Examples
Photolithotrophic
Autotrophy
Light energy
Inorganic
hydrogen/electron
donor
CO2 carbon source
Algae;
Purple and
green sulphur
bacteria;
Blue-green
algae
(cyanobacteria
)
Photoorganotrophi
c Heterotrophy
Light energy
Organic materials
Purple and
green non-
sulfur

Major Nutritional
Types
Sources of Energy,
Hydrogen/Electron
s or Carbon
Examples
Chemolithotrophic
Autotrophy
Chemical energy
source (inorganic)
Inorganic
hydrogen/electron
donor
CO2 carbon source
Sulfur-
oxidizing
bacteria;
Hydrogen
bacteria;
Nitrifying
bacteria;
Iron bacteria
Chemoorganotrophi
c Heterotrophy
Chemical energy
source (organic)
Organic materials
Protozoa;
Fungi;
Most non-
photosyntheti
c bacteria


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Chemoorganotrophs are by definition heterotrophs.

Most chemolithotrophs and phototrophs are
autotrophs.

Autotrophs are sometimes called primary
producers. They synthesize new organic matter from
CO
2
.

Virtually all organic matter on Earth has been
synthesized by primary producers, in particular, the
phototrophs.

MICROBIAL PHOTOSYNTHETIC SYSTEMS:
Property Cyanobacteria
Green
and
Purple
Bacteria
Purple
Nonsulfur
Bacteria
Photo
pigment
Chlorophyll
Bacterio-
chlorophyll
Bacterio-
chlorophyll
O
2
Prod. Yes No No
e
-
donors H
2
O H
2
, H
2
S, S H
2
, H
2
S, S
Carbon
source
CO
2
CO
2

Organic /
CO
2

Primary
products
of
energy
cnvrsn
ATP + NADPH ATP ATP

CHEMOAUTOTROPH
Bacteria
Electron
Donor
Electron
Acceptor
Products
Alcaligens
and
Pseudomonas
sp.
H2 O2 H2O
Nitrobacter NO2
-
O2 NO3
-
, H2O
Nitrosomonas NH4
+
O2

NO2
-
, H2O
Desulfovibrio H2 SO4
2-
H2O, H2S
Thiobacillus
denitrificans
S
0
, H2S NO3
-
SO4
2-
, N2
Thiobacillus
ferroxidans
Fe
2+
O2 Fe
3+
, H2O

UPTAKE OF NURIENTS nutrient molecules
frequently cannot cross selectively permeable plasma
membranes through passive diffusion and must be
transported by one of three major mechanisms
involving the use of membrane carrier proteins.
1. Phagocytosis protozoa
2. Permeability absorption most organisms
a. Simple transport
i. Passive or simple diffusion
process in which molecules
move from a region of higher
conc. to one of lower conc. as
a result of random thermal
agitation. A few substances,
such as glycerol, can cross
the plasma membrane by this
process.



ii. Facilitated diffusion (against or
with the conc. gradient) rate
of diffused increases by the
use of carrier proteins
(permeases), which are
embedded in the plasma
membrane. The rate of
facilitated diffusion increses
with the conc. gradient much
more rapidly and at lower
conc. of the diffusion molecule
than that of passive diffusion.

Note: The membrane carrier can change conformation
after binding an external molecule and subsequently
release the molecule on the cell interior. It then returns
to the outward oriented position and is ready to bind
another solute molecule. (No energy input)

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iii. Symport and Antiport



iv. Active Transport is the
transport of solute molecules
to higher conc., or against,
with the use of metabolic
energy input.



b. Group translocation (against the
conc. gradient) the best-known group
translocation system is the
phosphoenolpyruvate: sugar
phophotransferase system (PTS),
which transports a variety of sugars
into prokaryotic cells while
simultaneously phosphorylating them
using phosphoenolpyruvate (PEP) as
the phosphate donor.

()
()



c. ATP-Binding Cassette (ABC)
transporter (against conc. gradient)
for the uptake of organic compounds
like sugars and amino acids; and
inorganic compounds such as
sulphate and phosphate; and trace
metals. And this process includes the
following proteins:
i. Periplasmic binding protein
high affinity to substrate even
at low conc. (less than 1 M).
ii. Membrane spanning
transporter forms the
transport channel
iii. ATP hydrolysing protein
supply energy



SIMPLE COMPARISON OF TRANSPORT SYSTEMS

Items
Passive
Diff.
Facilitated
Diffusion
Group
Trans-
location
ABC
Trans.
Carrier
Proteins
None Yes Yes Yes
Transport
Speed
Slow Rapid Rapid Rapid
Against
Gradient
No Yes / No Yes Yes
Transport
Molecule
No
spec.^
Spec.^* Spec.^ Spec.^
Metabolic
Energy
No need Need* Need Need
Solute
Molecules
Not
changed
Changed* Changed
Not
Changed
^specificity
*may or may not be

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CULTURE MEDIA are the nutrient solutions used to
grow microorganisms (mostly chemotrophs) in the
laboratory. Note: Most microorganisms have yet to be
cultured.

MAJOR CLASSES OF MEDIA
1. Defined precise amounts of highly purified
inorganic or organic chemicals; exact
composition (qualitative and quantitative
sense)
2. Complex employ digests of microbial,
animal or plant products, such as casein (milk
protein), beef (beef extract), soybeans (tryptic
soy broth), yeast cells (yeast extract), or any of
a number of other highly nutritious yet impure
substances.
3. According to Use
a. Enriched enhances growth (e.g.
Chocolate and Blood Agar)
b. Selective inhibits growth of other
bacteria (e.g. EMBA, BGLB)
c. Differential differentiates two types
of microorganisms in a single medium
(e.g. EMBA, BGLB)
d. General Purpose all-purpose
medium (e.g. Nutrient A/B)
4. Fluidity
a. Solid contain 1.5-1.7% agar
b. Semi Solid contain 0.5-0.7% agar;
for studying motile bacteria
c. Liquid no agar

DEVELOPMENT OF SOLID MEDIUM
- Before Agar liquid medium
- Potato Slices Robern Koch (1881) used
boiled potato slices but not all bacteria grew
well
- Gelatin Frederick Loeffler; meat extract
medium + gelatin; liquid at room temperature
- Agar Fannie Hesse (1882); agar is used for
jams and jellies; generally not metabolized by
microbes; liquefies at 100
o
C and solidifies at
40
o
C.

ANAEROBIC CONDITION FOR GROWTH
- Reducing Media contain chemicals
(thioglycollate or oxyrase) that combine O
2
;
heated to drive off or use up O
2
.
- Anaerobic Jar production of an anaerobic
environment; used in culturing bacteria that die
or fail to grow in the presence of oxygen.



METABOLISM total of all chemical reactions
occurring in the cell.
- Anabolism nutrients from the environment
or those generated from catabolic reactions
are converted to cell components.
- Catabolism energy sources from the
environment are converted to waste products.
o Fermentation anaerobic
catabolism; organic compound is both
an electron donor and acceptor; ATP
is produced by substrate-level
phospholyration; since many microbial
habitats lack O
2
, this is the only option
for energy conservation by
chemoorganotrophs.
o Respiration catabolism in which a
compound is oxidized; O
2
(or
substitute) as the terminal electron
acceptor; usually accompanied by
ATP production by oxidative
phospholyration; more ATP produced
than in fermentation (preferred).


A simple view of cell metabolism depicts how catabolic
degradative reactions supply energy needed for cell
functions and how anabolic reactions bring about the
synthesis of cell components from nutrients

ENZYMES biological catalysts; lowers activation
energy of a reaction; generally much larger than the
substrate(s).
- Active Site portion of the enzyme to which
substrate binds

GLYCOLYSIS (Embden-Meyerhof-Parnas Pathway)
- Stage I: Preparatory reactions
o Not redox reactions and do not
release energy

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o Production of key intermediate of
pathway.
- Stage II: Production of NADH, ATP, and
Pyruvate
o Redox reactions
o Energy is conserved in the form of
ATP, and two molecules of pyruvate
are formed
o Redox balance has not yet been
achieved
- Stage III: Consumption of NADH and
Production of Fermentation Products
o Redox reactions occur once again
o Fermentation products formed

Note: Refer to Slides 42 and 43 in Lecture 4 for
flowchart of the Glycosis pathway and products formed


KREB CYCLE (Citric Acid Cycle)


RESPIRATION AND ELECTRON TRANSPORT
- the biochemical pathways involved in the
transformation of organic carbon to CO
2

- the way electrons are transferred from the
organic compound to the terminal electron
acceptor, driving ATP synthesis at the
expense of the proton motive force

ELECTRON TRANSPORT composed of membrane
associated electron carriers.
- To accept electrons from an electron donor
and transfer them to an acceptor
- To conserve some of the energy released
during electron transfer for synthesis of ATP
- Redox Enzymes involved in Electron
Transport:
o NADH dehydrogenase
o Riboflavin-containing electron carriers,
generally called flavoproteins
o Iron-sulfur proteins
o Cytochromes
o Non-protein carriers, lipid-soluble
quinones

ATP AND CELL YIELD the amount of ATP
produced by an organism has a direct effect on cell
yield
- Cell yield is directly proportional to the amount
of ATP produced
- Energy costs for assembly of macromolecules
are much the same for all microorganisms

ENERGY STORAGE most microorganisms produce
insoluble polymers that can later be oxidized for the
production of ATP
- Polymer formation: potential energy stored in
stable form; little effect on the internal osmotic
pressure of cells; do not interfere with other
cellular processes; readily accessible

PROTON MOTIVE FORCE
- Carriers in the Electron Transport Chain (ETC)
o Arranged in increasing positive
reduction potential
o Oriented in such a way that as
electrons are transported, protons are
separated
o The final donating the electrons plus
protons to a terminal electron acceptor
such as O
2

- H
+
are extruded to the other surface of the
membrane
- From two sources:
o NADH nicotinamide adenine
dinucleotide
o The dissociation of water into
hydronium ion and hydroxide
- The extrusion of hydronium ion to the
environment results in the accumulation of
hydroxide on the inside of the membrane
- As a result, the two sides of the membrane
differ in both charge and pH
- Formation of an electrochemical potential
across the membrane = Proton Motive Force

ATP SYNTHASE (ATPase) the use of PMF to
general ATP
- Catalysed a reversible reaction between ATP
and ADP + P
1

- Two components:

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o F
1
complex carries out the chemical
functions;
3

3

o F
0
complex ion-translocating
functions; a, b
2
, c
12



CATABOLIC DIVERSITY Refer to Slide 54 in
Lecture 4


ANABOLISM: Biosynthesis
- Sugar


- Amino Acids



- Fatty acids



REGULATION OF THE ACTIVITY OF ENZYMES
- Feedback Inhibition reaction shut off
because of an excess of the end product


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- Allosteric Inhibition allosteric enzymes has
two binding sites: the active site (where
substrate binds) and the allosteric site, where
the end product of the pathway binds

- Covalent Modification when cells are
grown with excess ammonia, glutamine
synthetase (GS) is covalently modified by
adenylylation; as many as 12 AMP groups can
be added.

LECTURE 5: MICROBIAL REPRODUCTION AND
GROWTH

GROWTH increase in cellular constituents; leads to
a rise in cell number
- Budding: is a form of asexual reproduction in
which a new organism develops from an
outgrowth or bud due to cell division at one
particular site.
- Binary Fission: meaning "division in half",
refers to a method of asexual reproduction. It
is the most common form of reproduction in
prokaryotes and occurs in some single-celled
eukaryotes.



- Coenocytic organisms (multinucleate):
growth results in increased cell size not in
number

GROWTH CURVE population growth is studied by
analysing the growth curve of microorganisms
- Batch Culture: microorganisms are cultivated
in a liquid medium; closed system; incubated
in a closed culture vessel with a single batch
of medium; no fresh medium provided during
incubation; nutrient concentration decline and
concentrations of waste increase during
incubation period
- Plotted as the logarithm of cell number
versus incubation time



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LAG PHASE no immediate increase in cell mass or
number (synthesizing new components)
- Necessity of a lag phase:
o Cells may be old and ATP, essential
cofactors and ribosomes depleted
(synthesized first before growth can
begin)
o Medium may be different from the one
the microorganism was growing
previously (new enzymes would be
needed to use different nutrients)
o Microorganisms have been injured
and require time to recovery
- Cells retool, replicate their DNA, begin to
increase in mass and finally divide
- Long Lag Phase: inoculum from old culture;
from refrigerated source; into a chemically-
different medium
- Short Lag Phase (or even absent): young,
vigorously growing exponential phase culture
is transferred to fresh medium of same
composition

EXPONENTIAL PHASE/LOG PHASE
microorganisms are growing and dividing at the
maximal rate possible even their genetic potential,
nature of medium and conditions under which they are
growing
- Rate of growth is constant
- Microorganism doubling at regular intervals
- Population is most uniform in terms of
chemical and physiological properties
- Smooth curve because each individual divides
at a slightly different moment

STATIONARY PHASE population growth ceases
and the growth curve becomes horizontal (around 10
9

cells on the average)
- Due to: nutrient limitation (slow growth);
oxygen limitation; accumulation of toxic waste
products

DEATH PHASE detrimental environmental changes
like nutrient depletion and build-up of toxic wastes lead
to the decline in the number of viable cells
- Usually logarithmic (constant every hour)
- Death: no growth and reproduction upon
transfer to new medium
- Death rate may decrease after the population
has been drastically reduced due to resistant
cells

MATHEMATICS OF GROWTH
- Generation Time: time required for a
microbial population to double in number
- Mean Growth Rate Constant (k): rate of
microbial population growth expressed in
terms of the number of generations per unit
time
- Mean Generation Time (g):

Example: Given an initial density of 4 x 10
4
. After 2
hours, the cell density became 1 x 10
6
. Compute for
the generation time.




(

) (

GENERATION OF SOME COMMON MICROBES

Microorganisms Temperature(
o
C)
Generation
Time (hr)
Escherichia coli 40 0.35
Bacillus subtilis 40 0.43
Mycobacterium
tuberculosis
37 12
Euglena gracilis 25 10.9
Giardia lamblia 37 18
Sacharomyces
cerevisiae
30 2

MEASUREMENT OF CELL NUMBERS AND MASS
- Direct
o Total Cell Count or Direct
Microscopic Counts

o Viable Cell Count



o Membrane Filtration Method: used
to test large volumes of sample; size
exclusion principle; usually used for
coliform detection

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- Indirect
o Biomass Determination: Biomass is
biological material derived from living,
or recently living organisms.
o Turbidity Measurements



o Most Probable Number: count
positive tubes and compare to
statistical MPN table



CONTINUOUS CULTURE SYSTEM a culture
system with constant environmental conditions
maintained through continual provision of nutrients and
removal of wastes.
- Chemostat: feeds medium into the culture
vessel at the same rate as medium containing
microorganisms is removed; contains one
essential nutrient in limiting quantities

o Dilution Rate = Nutrient Exchange:

o Rate if nutrient exchange
Expressed as the dilution rate
(D)
Rate at which medium flows
through the culture vessel to
the vessel volume
D = f/V; where f is the flow
rate (mL/hr) and V is the
vessel volume (mL)
- Turbidostat: equipped with a photocell that
adjusts the flow of medium through the culture
vessel so as to maintain a constant cell
density or turbidity
o Difference from Chemostat: dilution
rate in a turbidostat varies rather than
remaining constant and its culture
medium lacks nutrients
o Operates best at high dilution rates
(chemostat most stable stable and
effective at low dilution rates)

ENVIRONMENTAL GROWTH FACTORS
- Solutes and Water Activity
o Water Activity (a
w
= P
sol
/P
water
; P =
vapour pressure): degree of water
availability; inverse proportional to
osmotic pressure; 1/100 the relative of
the solution when expressed as
percent; example: osmotolerant
yeasts and bacteria
o Compatible Solutes: solutes that are
compatible with metabolism and
growth when at high intracellular
concentrations; example:
Halobacterium (require high salt
concentration for normal activity)
o Types of Microbes According to
Solutes and Water Activity:
Osmophilic, osmophobic,
osmotolerant (osmotic
pressure)
Halophilic, halophobic,
halotolerant (salt content)
Xenophilic, xenophobic,
xenotolerant (dry
environment)

Water
Activity
Source Bacteria Fungi Algae
1.00 (pure
water)
Blood
Most
Gram (-)
and non-
halophile
None None
0.90 Ham
Most
cocci
and
Bacillus
Fusarium,
Mucor,
Rhizopus

0.60 chocolate None S. rourii None
0.55 (DNA
disordered)



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- Osmotic Pressure
o Hypertonic environments, increase
salt or sugar, cause plasmolysis (loss
of water of the cell)
o Extreme or obligate halophiles require
high pressure
o Facultative halophiles tolerate high
osmotic pressure
- pH most bacteria grow between 6.5 & 7.5
o Molds and Yeasts grow between 5&6
o Types: acidophiles, alkalophiles,
neutrophils
- Temperature cardinal temperature:
minimum, optimum, maximum
o Dependence on enzyme activity: as
the temp. rises, chemical and
enzymatic reactions in the cell
proceed at more rapid rates and
growth becomes faster
o Above a certain temp., proteins,
nucleic acids, and other cellular
components may irreversibly
damaged
o Dependence on lipid content
Psychrophily: high content of
unsaturated fatty acids; help
maintain a semi-fluid
membrane state at low temp.
Thermophily: proteins or
enzymes = increased number
of salt bridges (resist unfolding
in the aqueous milieu;
membranes = rich in saturated
fatty acids (stable at high
temp.)
o Temperature Range
Stenothermal Microbes:
small range; e.g. Neisseria
gonorrhoea
Eurythermal Microbes: wide
range; e.g. Enterococcus
faecalis
o Spoilage: microbes grow between
0
o
C and 20-30
o
C, causing food
spoilage




- Oxygen Concentration
o Obligate aerobes: top
o Faultative anaerobes: mostly on top
o Obligate anaerobes: bottom
o Aerotolerant anaerobes: mostly
bottom
o Microaerophiles: middle



o Toxic Forms of Oxygen
Single Oxygen: O
2
boosted to
a higher energy state


Peroxide anion: O
2
2-


Hydroxyl radical: OH

- Pressure: 1 atm (atmosphere)
o Barotolerant: increased pressure
does adversely affect them but not as
much as it does non-tolerant bacteria
o Barophilic: grow more rapidly at high
pressures

TRIVIA: One barophile has been recovered from the
Mariana Trench near the Philippines (10,500 m depth).
It can only grow at pressure greater than 400-500 atm
(at 2
o
C).

ADDITIONAL NOTES ON TEMPERATURE




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LECTURE 6: MICROBIAL CONTROL

HISTORY Bubonic Plague or the Black Death
- Epidemic swept thru Europe in the Middle
Ages (13
th
and 14
th
centuries)
- 40 million people were killed (about 1/3 of the
population of the continent)
- Etiological agent: Yersinia pestis Gram (-) rod
- 2 vectors (carriers): flea and rat
- Infection:
o Flea bite with Yersinia pestis
o Bacteria multiply in the bloodstream
(Bacteremia)
o Bacteria localize in lymph nodes,
especially axillary and groin areas
o Hemorrhaging occurs in lymph nodes,
resulting in reddish rash that will
eventually become black and blue
swellings or Buboes (hence the
name, Black Death or Bubonic
Plague)
o If untreated, about 50% mortality rate
o If bacteria spread to the lungs, it
becomes Pneumonic Plague and is
now highly contagious (almost 99%
mortality rate)
- In relation to the song, a pouch of sweet
smelling herbs or posies (other versions use
flowers) was carried due to the belief that the
disease was transmitted by bad smells (or
sneezes).






- Other versions use Ashes! Ashes! since the
corpses of the infected were incinerated to kill
the microbe.

HUMANS VS. MICROBES most of history, microbes
have been winning the battle; in the last 100 years or
so the battle swung in our favour because of our
increasing knowledge of how to control Microbial
Growth (e.g. Smallpox (Variola virus) eradicated in
1977 (Somalia))

DEFINITION OF TERMS

STERILIZATION destroying all forms of life
DISINFECTION destroying pathogens or unwanted
organisms

DISINFECTANT antimicrobial agent used on
inanimate object
ANTISEPTIC antimicrobial agent used on living
tissue

BACTERICIDAL kills bacteria
BACTERISTATIC inhibits bacterial growth

ASEPTIC TECHNIQUE use of specific methods to
exclude contaminating microorganisms from an
environment
DECONTAMINATION treatment used to reduce the
number of disease-causing microbes to a level that is
considered safe to handle
DEGERM treatment used to decrease the number of
microbes in an area
PASTEURIZATION a brief heat treatment used to
reduce the number of spoilage organisms and to kill
disease-causing microorganisms
PRESERVATION process of inhibiting the growth of
microorganisms in products to delay spoilage
ANITIZE reduce the number of microorganisms to a
level that meets public health standards implies
cleanliness as well
TERILANT chemical used to destroy all
microorganisms and viruses in a product, rendering it
sterile
STERILE completely free of all microorganisms and
viruses; an absolute term
VIRICIDE inactivates viruses
GERMICIDE kills microorganisms and inactivates
viruses

COMMERICIAL STERILIZATION: killing C. botulinim
endospores

MICROBIAL DEATH: a microbe is defined dead if it
does not grow when inoculated into culture medium
that would normally support its growth; defined as the
inability of the organisms to form a visible colony



Bacterial poulations die at a constant logarithmic
rate. It takes more time to kill a large population of
bacteria than it does to kill a small population, because
only a fraction of organisms die during a given time
interval.

CONDITIONS INFLUENCING EFFECTIVENESS OF
ANTIMICROBIAL AGENT ACTIVITY
- Population Size: larger population requires a
longer time to die
- Population Composition: microorganisms
vary markedly on susceptibility; vegetative
versus spores; young versus mature cells
- Concentration or Intensity of an
Antimicrobial Agent: the more concentrated
an agent the more rapidly microbes can be
destroyed; but sometimes an agent may be

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more effective at lower concentrations (e.g.
70% alcohol)



- Duration of Exposure: the longer the
exposure to an agent the more they will be
killed
- Temperature: an increase in temperature at
which chemical acts often enhances its activity
(e.g. acids used in high temp. = more
effective)
- Local environment: pH, organic matter, etc.;
controls or protects the pathogen



TARGETS OF ANTIMICROBIAL AGENTS
- Cell membrane
- Enzymes & Proteins
- DNA & RNA

METHODS OF MICROBIAL CONTROL
- Physical: heat, filtration, radiation
- Chemical

MICROBIAL CONTROL: HEAT
- Thermal Death Point (TDP): lowest
temperature at which a microbial suspension
is killed in 10 minutes
- Thermal Death Time (TDT): shortest time
needed to kill all organisms in a microbial
suspension at a specific temperature and
under defined conditions
- Decimal Reduction Time or D value: time
require to kill 90% of the microorganisms or
spores in a sample test at a specified
temperature; time required for the line to drop
by one log cycle or tenfold; used to estimate
the relative resistance of a microbe to different
temperatures

Note: However, such a destruction is logarithmic and it
is theoretically not possible to completely destroy
microbes in a sample.

- Z value: increase in temperature required to
reduce D to 1/10 of its value or to reduce it by
one log cycle
- F value: time in minutes at a specific
temperature needed to kill a population of cells
or spores; usually at 121
o
C



PHYSICAL METHODS: MOIST HEAT
- Calculations using D and z value
o Given: For Clostridium botulinum
spores suspended in phosphate
buffer, D
121
= 0.204 min
o How long would it take to reduce a
population of C. botulinum spores in
phosphate buffer from 10
12
spores to
10
0
spores (1 spore) at 121
o
C?
o Answer: Since 10
12
to 10
0
is 12
decimal reductions, then the time
required is 12 x 0.204 min = 2.45 min
o Given the D value at one temperature
and the z value, we can derive an
equation to predict D value at a
different temperature:

(

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o Given: For Clostridium botulinum
spores suspended in phophsate
buffer, D
121
= 0.204 min and z = 10
o
C
o How long would it take to reduce a
population of C. botulinum spores in
phosphate buffer from 10
12
spores to
10
0
spores (1 spore) at 111
o
C?
o Answer: To answer the question we
need to know D
111
, which we can
calculate from the formula:
(

()
(

)
o Given: For Staph. aureus in turkey
stuffing, D
60
= 15.4 min and z = 6.8
o
C
o How long would it take to reproduce a
population of Staph. aureus in turkey
stuffing from 10
5
cells to 10
0
cells at
55
o
C, 60
o
C, and 65
o
C?
o Answers: Work it out for yourself.
55
o
C: 419 min
60
o
C: 77 min
65
o
C: 14.2 min

APPLICATION: FOOD INDUSTRY
- If the z value for Clostridium spores is 10
o
C, it
takes a 10
o
C change in temp. to alter the D
value tenfold
- Thus, if the cans are to be processed at 111
o
C
rather than 121
o
C, the D value would increase
by tenfold t 2.04 minutes; 12D value = 24.5
minutes

MOIST HEAT STERILIZATION
- Horizontal and vertical autoclaves have the
same efficiency, according to Maam Suyom.
-


HEAT flaming; incineration; hot-air sterilization

Hot-air Autoclave
Equivalent
treatments
170
o
C, 2 hours 121
o
C, 15 min

HOW DOES HEAT KILL MICROBES?
- Moist Heat: kill effectively by degrading
nucleic acids and by denaturing enzymes and
other essential proteins; may also disrupt cell
membranes
- Dry Heat: microbial death results from the
oxidation of cell constituents and denaturation
of proteins

CHEMICAL CONTROL
- Chemical Agents

Chemical
Agent
Effectiveness Against
Endospores Mycobacteria
Phenolics Poor Good
Quats None None
Chlorines Fair Fair
Alcohols Poor Good
Glutaraldehyde Fair Good

o Phenolics: first widely used
antisepctic and disinfectant; Joseph
Lister (1867) reduced the risk of
infection during operations; act by
denaturing proteins and disrupting cell
membranes; Lysol and bisphenols:
hexachlorophene, Triclosan



Advantages: effective in the
presence of organic material
and remain active on surfaces
long after application
Disadvantages: disagreeable
odor and can cause skin
irritation and in some
instances brain damage
(hexachlorophene)
o Alcohols: ethanol, Isopropanol (70-
80% concentration); act by denaturing
proteins and possibly by dissolving
membrane lipids; 10-15 soaking in
alcohol is sufficient to disinfect
thermometers and small instruments




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o Halogens
Iodine: kills by oxidizing cell
constituents and iodinating
cell proteins; kills spores at
high conc.; disadvantage: a
stain may be left (answer =
iodophor)
Chlorine: usually for water
supply; kills by oxidization of
cellular materials and
destruction of vegetative
bacteria and fungi; will not kill
spores; death within 30 mins
o Heavy Metals: Hg, As, Zn, Cu
used as germicides; heavy
metals combine with proteins,
often with their sulhydryl group
and inactivate them; may also
precipitate cell proteins
o Quartnary Ammonium Compounds
(Detergents): amphiphatic (both polar
and non-polar ends); kill by disrupting
microbial membranes and denature
proteins
Advantage: stable, non-toxic
Disadvantage: inactivated by
hard water

Soap
Acid-anionic
Degerming
Detergents Sanitizing
Quats
Cationic detergents
Bactericidal, denature
proteins, disrupt plasma
membrane

o Aldehydes: formaldehyde; very
reactive molecules that combine with
proteins and inactivate them;
sporicidal and can be used as
sterilants
o Sterilizing Gases

EVALUATION OF ANTIMICROBIAL AGENT
EFFECTIVENESS
- Phenol Coefficient Test
o Best-known disinfectant screening
test
o Potency of a disinfectant is compared
with that of phenol
o The highest dilution that killed
bacteria after a 10 minute exposure
are used to calculate phenol
coefficient
o The higher the phenol coefficient
value, the more effective the
disinfectant under these conditions;
directly proportional
o The reciprocal of the appropriate
test disinfectant dilution is divided
by that for phenol to obtain the
coefficient
o Example: phenol dilution = 1/90 and
the maximum effective dilution for
disinfectant X = 1/450
o Phenol coefficient = 5
- Use Dilution Test
o Metal rings dipped in test bacteria are
dried
o Dried cultures placed in disinfectant
for 10 min at 20
o
C
o Rings transferred to culture media to
determine whether bacteria survived
treatment
- Disk Diffusion



CHEMOTHERAPHY: Chemotherapeutic Agent
- Antibiotic: a product produced by a
microorganism or a similar substance
produced wholly or partially by chemical
synthesis, which in low conc., inhibits the
growth of other microorganisms
o Antibiotics are medicines used to treat
infections caused by bacteria only
o Infections are usually caused by
bacteria or viruses
o Antibiotics, therefore, do not cure all
infections
o Many infections like the common cold,
flu, mild sore throat, or diarrhea are
caused by viruses
- Broth Dilution Minimum Inhibitory
Concentration (MIC) Test





- Agar Dilution MIC Test

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- Agar Diffusion Kirby-Bauer Disk Diffusion



- Diffusion depends on:
o Concentration
o Molecular weight
o Water solubility
o pH and ionization
o Binding to agar
- Zones of Inhibition (~antimicrobial activity)
depend on:
o pH of environment
o Media components (agar depth,
nutrients)
o Stability of drug
o Size of inoculum
o Length of incubation
o Metabolic activity of organisms



When bacteria are exposed to an antibiotic, they either
die or adapt.

Those that survive carry genes that protect them
against the antibiotic and pass those genes to other
bacteria.

Since bacteria multiply very quickly and can be easily
spread among people, resistant bacteria can easily
occur in places like hospitals and nursing homes,
where a lot of people are gathered and antibiotic use is
high.
EMERGENCE OF ANTIMICROBIAL RESISTANCE



SELECTION FOR ANTIMICROBIAL RESISTANT
STAINS



RESISTANCE: Physiological Mechanism
- Lack of entry tet, fosfomycin
- Greater exit efflux pumps, tet (R factors)
- Enzymatic inactivation bla (penase),
hydrolysis, CAT chloramphenicol acetyl
transferase, aminoglycosides & transferases
- Altered target
o RIF altered RNA polymerase
(mutants)
o NAL altered DNA gyrase
o STR altered ribosomal proteins
o ERY methylation of 23S rRNA
- Synthesis of resistant pathway TMP
r
plasmid has gene for DHF reductase;
insensitive to TMP

CELL WALL SYNTHESIS INHIBITORS
- Resistance to -Lactams Gram (+)




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- Resistance to -Lactams Gram (-) [more
resistant than (+)]



MECHANISM OF RESISTANCE




CONSEQUENCE OF RESISTANCE: Ecology of
Pathogenesis
- Bacteria grow fast high population densities
great competition for resources
- Pathogens = normal bacteria that has gained
access to a new resource through new genes
competitive advantage

Antibiotics revolutionized medicine.

The first antibiotic, penicillin, was discovered by
Alexander Fleming in 1929. It took less than 20 years
for bacteria to show signs of resistance.

Staphylococcus aureus, which causes blood poisoning
and pneumonia, started to show resistance in 1950s.
Today, there are different strains of S. aureus resitant
to every form of antibiotic in use (MRSA = Multiple
Resistance S. aureus).
WHERE DO WE GET ANTIOBIOTIC RESISTANT
BACTERIA
- If a patient taking a course of antibiotic
treatment does not complete it or forgets to
take the doses regularly, then resistant
strains get a chance to build up.
- When antibiotics are used on a person, the
numbers of antibiotic resistant bacteria
increase in other members of the family.
- In places where antibiotics are used
extensively e.g. hospitals and farms,
antibiotic resistant bacteria increase in
number.

MYTHS AND FACTS ABOUT ANTIBIOTICS AND
RESPIRATORY ILLNESS

Myth: Taking antibiotic means I or my child can return
to work or childcare sooner.
Fact: Antibiotics do not shorten the duration of viral
illnesses.

Myth: Cold and flu symptoms will feel better or get
better faster on antibiotics.
Fact: Antibiotics cannot ease the symptoms of viral
illnesses; these infections resolve on their own.

Myth: Illnesses with the same symptoms require
antibiotics.
Fact: Illnesses with similar symptoms can be caused
by different germs.

Myth: If I take an antibiotic, I wont spread my illness
to others.
Fact: Viral illnesses (cold, flu, etc.) usually spread from
person to person before the onset of symptoms; before
a person becomes ill.

Antibiotics cannot stop the spread of viral illnesses.

HOW TO STOP ANTIBIOTIC MISUSE?
- Dont ask for antibiotics let your doctor
decided if you need them.
- Always take antibiotics exactly as prescribed.
- Finish the whole prescription do not stop
even when you feel better.
- Never save antibiotics for a future illness or
share with others.

ANTIMICROBIAL RESISTANCE: Key Prevention
Strategies





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12 STEPS TO PREVENT ANTIMICROBIAL
RESISTANCE: HOSPITALIZED ADULTS

- Prevent Infection
1. Vaccinate
2. Get the catheters out

- Diagnose and Treat Infection Effectively
3. Target the pathogen
4. Access the experts

- Use Antimicrobials Wisely
5. Practice antimicrobial control
6. Use local data
7. Treat infection; not contamination
8. Treat infection, not colonization
9. Know when to say no to vanco
10. Stop treatment hen infection is cured or
unlikely

- Prevent Transmission
11. Isolate the pathogen
12. Contain the contagion






ADDITIONAL FIGURES AND FLOW CHARTS

GLYCOLYSIS (In reference to Page 5)



STAGES I-III OF GLYCOLYSIS

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PRODUCTS OF PYRUVATE

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FERMENTATION VS. RESPIRATION