Control of Microorganisms

Unit 4
Dr. Neetu Kumra Taneja
Lecture 1-3
 Unit 1
 Introduction :Scope and history of microbiology

Unit 2:
Microbial Ultra Structure and Functions:General structure of
 prokaryotic and eukaryotic cell, cell wall, plasma membrane,
protoplasm, endoplasmic reticulum, lysosome, golgi apparatus,
centriole, cilia, flagellum, storage bodies and ribosomes,
chloroplasts, mitochondria and nucleus.


Unit 3
Microbial Morphology, Metabolism and Growth: General
morphological , cultural characteristics and reproduction of
bacteria (bacterial metabolism – nutrition and respiration,
bacterial reproduction, growth curve) fungi, algae, protozoa and
viruses (bacterial viruses, animal viruses)

 Unit 4
Cultivation and preservation of micro-organisms : Importance,
methods of isolation and cultivation, pure culture technique,
enumeration methods for micro-organisms, preservation of
microorganism
Unit 5
Control of microorganisms: Physical and Chemical agents,
antibiotics and other chemotherapeutic agents
Unit 6
Bacterial genetics :Structure of DNA, difference between DNA
and RNA, bacterial recombination (transformation, transduction,
conjugation), bacterial mutations (spontaneous and induced),
plasmids – properties, types, applications
Unit 7
Principles of immunology: Innate and acquired immunity,
difference between active and passive immunity , antigen and
antibody reactions – neutralization, precipitation and agglutination
Unit 8
Applied and environmental microbiology: Definitions and
scope of industrial and environmental microbiology, microbiology
of water and waste water, air, soil and industrial microbiology.
Industrial uses of bacteria, yeasts and molds.
 Control of Microorganisms
• Control: refers to reduction in
numbers/activity of total microbial flora.
• Reason:
– To prevent disease transmission and infection
– to prevent contamination by or growth of
undesirable m/o
– To prevent deterioration and spoilage of materials
by m/o
 Terms for Microbial Control
• Sterilization
• Disinfection
• Sanitization
• Degermation

Microbial agents
• Bactericide
• Sporocide
• Fungicide
• Viricide
• Microbistasis
• Bacteriostatic
• Fungistatic
 Definitions
• Sterilization: A treatment that kills or removes
all living cells, including viruses and spores,
from a substance or object
• Disinfection: A treatment that reduces the
total number of microbes on an object or
surface, but does not necessarily remove or
kill all of the microbes
 Definitions
• Sanitation: Reduction of the microbial
population to levels considered safe by public
health standards

• Antiseptic: A mild disinfectant agent suitable

for use on skin surfaces
 Definitions
• -cidal: A suffix meaning that “the agent kills”.
For example, a bacteriocidal agent kills
bacteria

• -static: A suffix that means “the agent inhibits

growth.” For example, a fungistatic agent
inhibits the growth of fungi, but doesn’t
necessarily kill it.
CHEMICAL AGENTS
 Conditions Influencing Antimicrobial
Activity
• Under most circumstances, a microbial
population is not killed instantly by an agent
but instead over a period of time
• The death of the population over time is
exponential, similar to the growth during log
phase
Factors Influencing Antimicrobial Activity

• Several critical factors play key roles in determining

the effectiveness of an antimicrobial agent, including:
– Population size
– Types of organisms
– Concentration of the antimicrobial agent
– Duration of exposure
– Temperature
– pH
– Organic matter
– Biofilm formation
Factors altering effectiveness
Primary Targets of
Control
 Cellular Targets of Control

• Attack lipids and

proteins in the
cytoplasmic membrane

• Inhibit or denature
enzymes
 Physical and Chemical Control of
Microorganisms
Controlling Microorganisms
• Reduce or destroy undesirable microbes in a
given area
• Physical
• Chemical
• Mechanical
Methods of Microbial Control
 Physical Methods
• Moist Heat
• Dry Heat
• Low Temperatures
• Filtration
• Radiation
Physical Methods: High
Temperatures/Heat
 Physical Methods: Moist Heat
• Mechanism of killing is a combinantion of
protein/nucleic acid denaturation and
membrane disruption
• Effectiveness Heavily dependent on type of
cells present as well as environmental
conditions (type of medium or substrate)
• Bacterial spores much more difficult to kill
than vegetative cells
 Physical Methods: Moist Heat
• Measurements of killing by moist heat
– Thermal death point (TDP): Lowest temperature at
which a microbial suspension is killed in 10 minutes;
misleading because it implies immediate lethality
despite substrate conditions
– Thermal death time (TDT): Shortest time needed to kill
all organisms in a suspension at a specified
temperature under specific conditions; misleading
because it does not account for the logarithmic nature
of the death curve (theoretically not possible to get
down to zero)
 Physical Methods: Moist Heat
• Measurements of killing by moist heat (cont.)
– Decimal reduction time (D value): The time required to
reduced a population of microbes by 90% (a 10-fold, or
one decimal, reduction) at a specified temperature and
specified conditions
– z value: The change in temperature, in ºC, necessary to
cause a tenfold change in the D value of an organism
under specified conditions
– F value: The time in minutes at a specific temperature
(usually 121.1°C or 250 °F) needed to kill a population
of cells or spores
 Physical Methods: Moist Heat
• Calculations using D and z values
– Given: For Clostridium botulinum spores
suspended in phosphate buffer, D121 = 0.204 min
– How long would it take to reduce a population of
C. botulinum spores in phosphate buffer from 1012
spores to 100 spores (1 spore) at 121°C?
Answer: Since 1012 to 100 is 12 decimal reductions,
then the time required is 12 x 0.204 min = 2.45
min
 Physical Methods: Moist Heat
• Calculations using D and z values (cont.)
– Given the D value at one temperature and the z
value, we can derive an equation to predict the D
value at a different temperature:
D T  Tb
log( b )  a
Da z

Db
 10Ta Tb /z 
Da
 Physical Methods: Moist Heat
• Calculations using D and z values (cont.)
» First, write an equation for this line. Since the y axis is
on a log scale, then y = log (D). The slope of the line is
-1/z; we’ll let the y intercept be equal to c. Therefore:
T
log(D)  c
z
» At a given temperature Ta, D = Da, so we can eliminate
the “c” term
 Ta
log(D a )  c
z
T
c  log(D a )  a
z
T T
log(D)   log(D a )  a
z z
 Physical Methods: Moist Heat
• Calculations using D and z values (cont.)
» We can explicitly refer to the second temperature and D
value as Tb and Db, so:
 Tb T
log(D b )   log(D a )  a
z z
T  Tb
log(D b )  log(D a )  a
z
D T  Tb
log( b )  a
Da z
Db
 10Ta Tb / z 
Da
 Physical Methods: Moist Heat
• Calculations using D and z values (cont.)
– Given: For Clostridium botulinum spores suspended in
phosphate buffer, D121 = 0.204 min and z = 10°C
– How long would it take to reduce a population of C. botulinum
spores in phosphate buffer from 1012 spores to 100 spores (1
spore) at 111°C?
Answer: To answer the question we need to know D111, which
we can calculate from the formula:
log(D111/0.204) = (121-111) /10
D111 = 0.204(10) = 2.04 min
12D111= 24.5 min
 Physical Methods: Moist Heat
• Calculations using D and z values (cont.)
– Given: For Staph. aureus in turkey stuffing,
D60 = 15.4 min and z = 6.8°C
– How long would it take to reduce a population of
Staph. aureus in turkey stuffing from 105 cells to 100
cells at 55°C, 60°C, and 65°C?
Answers: Work it out for yourself. Here are the
answers.
At 55°C: 419 min
At 60°C: 77 min
At 65°C: 14.2 min
 Physical Methods: Moist Heat
• Methods of Moist Heat
– Boiling at 100°C
• Effective against most vegetative cells; ineffective against
spores; unsuitable for heat sensitive chemicals & many foods
– Autoclaving/pressure canning
• Temperatures above 100°C achieved by steam pressure
• Most procedures use 121.1°C, achieved at approx. 15 psi
pressure, with 15 - 30 min autoclave time to ensure
sterilization
• Sterilization in autoclave in biomedical or clinical laboratory
must by periodically validated by testing with spores of
Clostridium or Bacillus stearothermophilus
 Physical Methods: Moist Heat
• Methods of Moist Heat
– Pasteurization
• Used to reduce microbial numbers in milk and other
beverages while retaining flavor and food quality of the
beverage
• Retards spoilage but does not sterilize
• Traditional treatment of milk, 63°C for 30 min
• Flash pasteurization (high-temperature short term
pasteurization); quick heating to about 72°C for 15 sec,
then rapid cooling
 Physical Methods: Moist Heat
• Methods of Moist Heat
– Ultrahigh-temperature (UHT) sterilization
• Milk and similar products heated to 140 - 150°C for
1 - 3 sec
• Very quickly sterilizes the milk while keeping its flavor &
quality
• Used to produce the packaged “shelf milk” that does
not require refrigeration
 Physical Methods: Dry Heat
• Incineration
– Burner flames
– Electric loop incinerators
– Air incinerators used with ferementers; generally operated
at 500°C
• Oven sterilization
– Used for dry glassware & heat-resistant metal equipment
– Typically 2 hr at 160°C is required to kill bacterial spores by
dry heat: this does not include the time for the glass to
reach the required temp (penetration time) nor does it
include the cooling time
 Physical Methods:
Low Temperatures
• Refrigerator:
– around 4°C
– inhibits growth of mesophiles or thermophiles;
psychrophiles will grow
• Freezer:
– “ordinary” freezer around -10 to -20°C
– “ultracold” laboratory freezer typically -80°C
– Generally inhibits all growth; many bacteria and
other microbes may survive freezing temperatures
 Physical Methods: Filtration
• Used for physically removing microbes and dust
particles from solutions and gasses; often used to
sterilize heat-sensitive solutions or to provide a
sterilized air flow
• Depth filters: eg. Diatomaceous earth, unglazed
porcelean
• Membrane filters: eg. Nitrocellulose, nylon,
polyvinylidene difluoride
• HEPA filters: High efficiency particulate air filters
used in laminar flow biological safety cabinets
Physical Methods: Filtration
Physical methods: radiation
Text, Fig. 11.8
 Physical Methods: Radiation
• Ultraviolet Radiation
– DNA absorbs ultraviolet radiation at 260 nm
wavelength
– This causes damage to DNA in the form of
thymine dimer mutations
– Useful for continuous disinfection of work
surfaces, e.g. in biological safety cabinets
Text, Fig. 11.10
 Physical Methods: Radiation
• Ionizing Radiation
– Gamma radiation produced by Cobalt-60 source
– Powerful sterilizing agent; penetrates and damages
both DNA and protein; effective against both
vegetative cells and spores
– Often used for sterilizing disposable plastic
labware, e.g. petri dishes; as well as antibiotics,
hormones, sutures, and other heat-sensitive
materials
– Also can be used for sterilization of food; has been
approved but has not been widely adopted by the
food industry
Methods of Microbial Control
CHEMICAL AGENTS
 CHEMICAL AGENTS
• ‘-cide’ meaning ‘to kill’ to define an antimicrobial agent
aimed at destroying a certain group of microorganisms.
• Eg: Microbicide
• Bactericide is an agent that destroys bacteria except those
in the endospore stage
• Fungicide is a chemical that kill fungal spores, hyphae and
yeasts.
• Virucide is a chemical known to inactivate viruses
especially on living tissue.
• Sporicide is an agent capable of destroying bacterial
endospores.
 MICROBISTATIC
• Suffix ‘-static’ condition in which: they denote
a condition in which microbes are prevented
from multiplying/inhibted but are not killed
outrightly.
• Eg. Microbistatics:
• Bacteriostatic
• Fungistatic
 CHEMICAL AGENTS
• Germicide, also called a microbicide, is any chemical
agent that kills microbes.
• Any physical or chemical agent that ‘kills germs’ is said
to have germicidal properties

• Disinfection, refers to a physical process or a chemical

agent (disinfectant) to destroy vegetative pathogens but
not bacterial endospores.

• It is important are normally used only on inanimate

objects because in the concentrations required to be
effective, they can be toxic to human and animal tissue.
 Chemical Agents
• Phenolics
• Alcohols
• Halogens
• Heavy metals
• Quaternary Ammonium Compounds
• Aldehydes
• Sterilizing Gases
 Chemical Agents: Phenolics
• Aromatic organic compounds with attached -OH
• MOA: Denature protein & disrupt membranes
• Eg: Phenol, orthocresol, orthophenylphenol,
hexachlorophene
• Commonly used as disinfectants (e.g. “Lysol”); are
tuberculocidal, effective in presence of organic matter,
remain on surfaces long after application
• Disadv: Disagreeable odor & skin irritation;
hexachlorophene once used as an antiseptic but its use
is limited as it causes brain damage
 Phenols
phenol p-cresol (lysol)

OH OH

Phenolics affect
CH3
protein function
and/or disrupt
Hexachlorophene (phisohex)
membranes
OH OH
Cl CH2
Cl

Cl Cl
Cl Cl
 Chemical Agents: Alcohols
• Eg: Ethanol; isopropanol; used at
concentrations between 70 – 95%
• MOA: Denature proteins; disrupt membranes
• Kills vegetative cells of bacteria & fungi but not
spores
• Uses: Used in disinfecting surfaces;
thermometers; “ethanol-flaming” technique
used to sterilize glass plate spreaders or
dissecting instruments at the lab bench
 Chemical Agents: Halogens
• MOA: Act as oxidizing agents; oxidize proteins &
other cellular components
• Eg.1: Chlorine compounds
– Used in disinfecting municipal water supplies (as
sodium hypochlorite, calcium hypochlorite, or
chlorine gas)
– Sodium Hypochlorite (Chlorine Bleach) used at 10 -
20% dilution as benchtop disinfectant
– Halazone tablets (parasulfone dichloroamidobenzoic
acid) used by campers to disinfect water for drinking
 Chemical Agents: Halogens
• Eg.2: Iodine Compounds
– Tincture of iodine (iodine solution in alcohol)
– Potassium iodide in aqueous solution
– Iodophors: Iodine complexed to an organic carrier;
e.g. Wescodyne, Betadyne
– Used as antiseptics for cleansing skin surfaces and
wounds
 Chemical Agents: Heavy Metals
• Eg: Mercury, silver, zinc, arsenic, copper ions
• MOA: Form precipitates with cell proteins
• At one time were frequently used medically as
antiseptics but much of their use has been replaced
by less toxic alternatives
• Examples: 1% silver nitrate was used as opthalmic
drops in newborn infants to prevent gonorrhea; has
been replaced by erythromycin or other antibiotics;
copper sulfate used as algicide in swimming pools
 Chemical Agents: Quaternary
Ammonium Compounds
• Quaternary ammonium compounds are cationic
detergents
• Amphipathic molecules that act as emulsifying
agents
• MOA: Denature proteins and disrupt
membranes
• Used as disinfectants and skin antiseptics
• Examples: cetylpyridinium chloride,
benzalkonium chloride
 Chemical Agents: Aldehydes
• Eg: Formaldehyde and glutaraldehyde
• MOA: React chemically with nucleic acid and
protein, inactivating them
• Aqueous solutions can be used as
disinfectants
Chemical Agents: Sterilizing Gases
• Eg: Ethylene oxide (EtO)
– Used to sterilize heat-sensitive equipment and plasticware
– Disadv: Explosive; supplied as a 10 – 20% mixture with
either CO2 or dichlorofluoromethane
– Disadv: Its use requires a special EtO sterilizer to carefully
control sterilization conditions as well as extensive
ventilation after sterilization because of toxicity of EtO
– Much of the commercial use of EtO (for example, plastic
petri dishes) has in recent years been replaced by gamma
irradiation
Chemical Agents: Sterilizing Gases
• Eg: Betapropiolactone (BPL)
– In its liquid form has been used to sterilize vaccines
and sera
– Decomposes after several hours and is not as difficult
to eliminate as EtO, but it doesn’t penetrate as well as
EtO and may also be carcinogenic
– Has not been used as extensively as EtO
• Eg: Vapor-phase hydrogen peroxide
– Has been used recently to decontaminate biological
safety cabinets
 CHEMOTHERAPEUTIC AGENTS
• Chemotherapeutic Agents: chemical agents used to
treat disease.

• Chemotherapeutic agents destroy pathogenic

microorganisms or inhibit their growth at low
concentrations (no damage to host cells).
• Selective toxicity: Harm pathogen not the host

– Antibiotics
• produced by microbes
– Antimicrobial chemotherapeutic chemicals
• Produced by chemists
 Definitions of Antibiotics

• An antibiotic is a chemical substance produced by

various species of microorganisms that is capable
in small concentrations of inhibiting the growth of
other microorganisms
 History
Paul Ehrlich
“Magic Bullet”
– Chemicals with selective toxicity

ORIGIN: Selective Stains

DRUG: Arsphenamine (1910)
“606” Salvarsan

NOBEL: 1908
 History
(cont’d)
Alexander Fleming
– Microbes make antibiotics

ORIGIN: moldy culture plate

DRUG: Penicillin (1928)

NOBEL: 1945
 History
(cont’d)

Selman Waksman
– Soil Streptomyces make antibiotics
– comes up with definition of antibiotic

ORIGIN: Penicillin development

DRUG: Streptomycin (1943)

NOBEL: 1952
Impact of Modern Healthcare on Life
Expectancy
 The Ideal Drug*
1. Selective toxicity: against target pathogen but not against
host
2. Bactericidal vs. bacteriostatic
3. Favorable pharmacokinetics: reach target site in body
with effective concentration
4. Spectrum of activity: broad vs. narrow
5. Lack of “side effects”
6. Little resistance development

* There is no perfect
drug.
How do antimicrobials work?
 Terms related to antimicrobials
• Spectrum of activity
– Broad spectrum acts on many different types (eg.
Tetracyclines act against G+ve and G-ve bacteria)
– Narrow spectrum acts on only a few types (eg. Penicillins act
against G+ve bacteria)

• Mode of action
– Inhibit cell wall synthesis (Eg. Penicillins, Cephalosporins)
– Inhibit protein synthesis (eg. Streptomycin, Tetracycline)
– Inhibit Nucleic acid synthesis (eg. Rifampicins)
– Attack the cytoplasmic membrane (eg. Polymyxins, Polyenes)
ANTIMICROBIAL SPECTRUM OF ACTION FOR
SELECTED CHEMOTHERAPEUTIC AGENTS
 Mechanism of Action
1. ANTIMETABOLITE ACTION
 Sulfonamides
 an analog of PABA, works by competitive inhibition

 Trimethoprim-sulfamethoxazole
 a synergistic combination; useful against UTIs
Mechanism of Action
ANTIMETABOLITE ACTION
(cont’d)

tetrahydrofolic acid
 Mechanism of Action
(cont’d)

2. ALTERATION OF CELL MEMBRANES

 Polymyxins and colistin
 destroys membranes
 active against gram negative bacilli
 serious side effects
 used mostly for skin & eye infections
Mechanism of Action
ALTERATION OF CELL MEMBRANES
(cont’d)
 Mechanism of Action
(cont’d)

3. INHIBITION OF PROTEIN SYNTHESIS:

Steps in synthesis:
1. Initiation
2. Elongation
3. Translocation
4. Termination

• Prokaryotes and eukaryotes (80S) have a different

structure to ribosomes so can use antibiotics for
selective toxicity against ribosomes of prokaryotes
(70S)
 Mechanism of Action
INHIBITION OF PROTEIN SYNTHESIS

(cont’d)

• Aminoglycosides
– bind to bacterial ribosome on 30S subunit; and
blocks formation of initiation complex. Both
actions lead to mis-incorporation of amino acids
– Examples:
Gentamicin Tobramycin
Amikacin Streptomycin
Kanamycin Spectinomycin
Neomycin
 Mechanism of Action
INHIBITION OF PROTEIN SYNTHESIS
(cont’d)

• Aminoglycosides (cont’d)
– broad spectrum
• Gram negative rods
• P. aeruginosa
• Drug-resistant gram negative rods
• Plague, Tularemia, Gonorrhea
• Pre-op (bowel)
• External (skin)
– toxic at some level to eighth cranial nerve
 Mechanism of Action
INHIBITION OF PROTEIN SYNTHESIS

(cont’d)

• Macrolides: chloramphenicol & erythromycin

– bind to 50S subunit and blocks the translocation step
Anaerobes
Typhoid
Meningitis
 Chloramphenicol: broad spectrum

 Erythromycin:
Mycoplasma
Legionella
S. pyogenes
 Mechanism of Action
INHIBITION OF PROTEIN SYNTHESIS

(cont’d)
• Clindamycin
– binds to 50S subunit and interferes with binding
of the amino acid – acyl-tRNA complex and so
inhibits peptidyl transferase
– works best against
• Staphylococcus
• Bacteroides & anaerobic gram neg rods
– Penicillin allergic people
 Mechanism of Action
INHIBITION OF PROTEIN SYNTHESIS

(cont’d)
• Tetracyclines
– bind to 30S subunit and interferes with the
attachment of the tRNA carrying amino acids to
the ribosome
– effective against:
• Chlamydia
• Rickettsia
• Mycoplasma
• Brucella
 Mechanism of Action
(cont’d)

4. INHIBITION OF DNA/RNA SYNTHESIS

 Rifampin
 binds to RNA polymerase
 active against gram positive cocci
 bactericidal for Mycobacterium
 used for treatment and prevention of
meningococcus
 Mechanism of Action
INHIBITION OF DNA/RNA SYNTHESIS
(cont’d)

 Metronidazole
 breaks down into intermediate that causes
breakage of DNA
 active against:
– protozoan infections
– anaerobic gram negative infections

 Quinolones and fluoroquinolones

 effect DNA gyrase
 broad spectrum
Mechanism of Action
INHIBITION OF DNA/RNA SYNTHESIS
(cont’d)
 Mechanism of Action
(cont’d)
5. CELL WALL SYNTHESIS INHIBITORS
Steps in synthesis:
1. NAM-peptide made in cytoplasm
2. attached to bactoprenol in cell membrane
3. NAG is added
4. whole piece is added to growing cell wall
5. crosslinks added

• the β-Lactams
• the non β-Lactams
 Mechanism of Action
(cont’d)

5. CELL WALL SYNTHESIS INHIBITORS

β-Lactam Antibiotics
– Penicillins
– Cephalosporins
– Carbapenems
– Monobactams
 Mechanism of Action
CELL WALL SYNTHESIS INHIBITORS
(cont’d)

β-Lactam ring structure

 Mechanism of Action
CELL WALL SYNTHESIS INHIBITORS
(cont’d)

Action of β-Lactam antibiotics

1. Bactericidal; growing cells only
2. Drug links covalently to regulatory enzymes called PBPs (penicillin-binding
proteins)
3. Blocks cross-linkage of peptidoglycan
 Mechanism of Action
CELL WALL SYNTHESIS INHIBITORS
(cont’d)

Action of β-Lactam antibiotics

For E. coli
> MIC

 wall damage
 autolysins
 spheroplasting
 cell lysis
< MIC

 no septa
 filaments
 Mechanism of Action
CELL WALL SYNTHESIS INHIBITORS

(cont’d)
Resistance to β-Lactams – Gram pos.
 Mechanism of Action
CELL WALL SYNTHESIS INHIBITORS

(cont’d)

Resistance to β-Lactams – Gram neg.

 Mechanism of Action
CELL WALL SYNTHESIS INHIBITORS

(cont’d)
Non - β-Lactams

 Vancomycin
active against gram positive cocci, but not
gram negative because too large to pass
through outer membrane
 interferes with PG elongation

 Cycloserine, ethionamide and isoniazid

inhibits enzymes that catalyze cell wall
synthesis
for Mycobacterial infections
 Mechanisms of Antibiotic resistance
• Detoxify antibiotic
• Modify target so
antimicrobial won’t
bind
• Pump antimicrobial out
of the cell
• Modify metabolic
pathway
How do we test effectiveness of
Antimicrobial Agents?

Agar diffusion assay