o b i a l

Mic r
n t r o l
C o

Dr. R. Tan
 Primary target of microbial control
Microorganisms capable of causing infection or
spoilage that are constantly present in the external
environment and on the human body

Relative Resistance of Microbial Forms

Highest Moderate Least resistance
resistance
bacterial endospore resistance
protozoan cyst most bacterial vegetative
(Bacillus & some fungal spores cells
Clostridium) some naked virus ordinary fungal spores &
vegetative bacteria hypae
that enveloped virus
have higher Yeasts
resistance Trophozoites
( M. tuberculosis,
S.aureus,
Pseudomonas)
 Definition of Frequently Used
Sterilization Terms
- process of destruction or removal of all viable
microorganisms from an object or from a particular
environment
- total inactivation of all forms of microbial life in terms
of the organism’s ability to reproduce

Disinfection
- refers to the use of physical process or chemical agent
(disinfectants) that promotes killing, inhibition, or
removal of pathogenic microorganisms (usually on
inanimate objects) but not bacterial endospore

Sanitization
- is any cleansing technique that mechanically removes microorg.
to reduce the level of contaminants / microbial population to a
safe level as determined by public health standards
Sepsis – growth of microorganisms or the presence of
microbial toxins in the blood and other tissues

Asepsis - refers to any practice that prevents the entry of

infectious agents into sterile tissues and thus prevents
infection

Antisepsis – aseptic technique practiced in health care,

range from sterile methods that uses chemical agents
(antiseptics) which are applied directly to exposed body
surfaces, wounds, and surgical incision to destroy or inhibit
vegetative pathogens
Disinfectant - is an agent used to reduce the viability of
a microbial population below a threshold
level that causes infection
- Disinfectants are used on inanimate objects

Antiseptic - chemical agents that opposes sepsis or putrefaction

either by killing microorganisms or by preventing
their growth
- Term commonly used for agents that are applied
topically to living tissues

This distinguishes them from antiseptics that serve the

same purpose but are used on living tissues without
causing toxicity.
suffix “cide” – meaninig to kill
- added when a killing action is implied
- a suffix indicating that the antimicrobial agent will kill or
destroy a certain group of microorganism
viricide – destroys virus
fungicide – destroys fungi
bactericide – destroys bacteria
germicide – (or disinfectant) – agent that kills
microorg. capable of producing an
infection

Suffix “static/stasis” – meaning to stand still

- a suffix indicating that the agent will prevent the growth or
multiplication of the type of organism but are not killed
outright
bacteriostatic - prevents the growth of bacteria
fungistatic – prevents the growth of fungi
 Dynamics of Sterilization &
Disinfection
microorganisms are not killed instantly when
exposed to a lethal agent
The kinetics of death of a microbial population is
exponential: the number of survivors decreases
with time
(exponential killing)
because of the exponential form of survivor-time
curve, the larger the initial number of cells to
be killed, the more intense or prolonged is the
treatment required for sterilization
The rate of disinfection varies with the
concentration of the disinfectant
 Factors affecting disinfectant
potency
1. Number of microorganisms
- larger the initial number of cells to be killed, the more
intense or prolonged is the treatment required for
sterilization

2. Nature of microorganism
- the efficacy of a chemical agent depends on these
properties: specie , growth phase of culture, and
presence of special structure (such as spores and
capsules), and the number of the organisms in the
test system

3. Temperature
- the killing of bacteria by chemical agents increases
with an increase in temperature
- for each 10°C increase in temperature, there is
doubling of the death rate
4. pH
- Hydrogen ion concentration influences bactericidal action
by affecting both the organism and the chemical
agent
- Some disinfectants are more effective at alkaline pH
(glutaraldehyde) while others are more effective at acid pH
(e.g. phenols)

5. Time
- when bacteria are exposed to a specific concentration of a
bacterial agent, even in excess, not all organisms die at the
same time; rather, there is a gradual decrease in the
number of living cells

6. Mode of action of the agents

 damage cell membrane function
 denatures protein
 induce extensive nucleic acid damage
7. Concentration of the agent
- the concentration required to produce a given effect
varies with the disinfectant, the organism and the
method of testing
- high conc. → lethal to bacteria
- low conc. → may stimulate, retard, or even kill the
organism
- An effective concentration must be used

8. Presence of exogenous materials

- presence of organic matter (serum, blood, pus) alter
disinfectant activity
- alter disinfectant activity by:
 surface absorption of the disinfectant by protein
colloids
 formation of a chemically inert or less active compound
 binding of the disinfectant by active groups of foreign
protein
 Evaluation of
Disinfectants
Association of Official Analytical Chemists (AOAC)
methods
Approved protocol for evaluation purposes
Phenol Coefficient Test
Phenol → reference standard agent
official method used to test disinfectant potency
designed to determine the ratio of the highest
dilution of the germicide that will kill the test
organism within a specified time to the greatest
dilution of phenol showing the same result
 Methods of
Microbial Control
Physical Agents
Chemical Agents
Chemotherapeutic
Agents
• Heat
• Freezing
• Radiation
• Filtration
• Ultrasonic and
SonicVibration
 HEAT
- most reliable and universally applied method of
sterilization
- whenever possible, should be the method of choice
- 2 kinds of heat : 1. Dry
2. Moist

Thermal Death Time

- refers to minimum time required to kill all microbes at a
specified temperature in a specified environment
- time required inversely related to temperature of
exposure
- sterilization time is directly related to the number of
organisms in a suspension

Thermal Death Point

- lowest temperature required to kill all microbes when
DRY HEAT
Sterilization that requires higher temperature and
longer period of heating
denotes air with a low moisture content that has been
heated by flame or electric heating coil
temp ranges from 160°C – several thousand °C
MOA :
 denaturation of proteins
 oxidative damage
 toxicity effects of elevated levels of electrolytes
Its use is limited primarily to sterilization of glasswares
and such materials as oils, jellies, and powders that
are impervious to steam
Forms:
1. Direct flaming
- bacteriological loop, needle, glass rods through
the flame of a Bunsen burner

2. Incineration / cremation
- burn to ashes
- useful for decontaminating infected lab. Animals

3. Hot air oven

- most widely used type of dry heat
- oven heat set 1800C for 2-4 hours
- temp. enough to kill all type of organism,
including the sporeformer
MOIST HEAT
 kills bacteria faster than dry heat
 preferred because of its more rapid killing
 temp ranges from 60-135°C
 MOA:
 denaturation and coagulation of proteins
 production of single-stranded breaks of DNA
 loss of functional integrity of membrane and
leakage of small molecules
microorganism Time required for
sterilization
Most mesophilic nonsporeformers 60°C for 30min
S. Aureus & S. faecalis 60°C for 60min
Vegetative form of all bacteria, fungi & 80°C for 5-10min
yeast
Heat resistant sporeformers 120°C for 4min
100°C for 5.5min is
required
Forms:
1. Boiling
- can be relied only for disinfection and not for
sterilization
- 30 minutes boiling 1000C adequate to kill
vegetative cell → Bacteria, Fungi, Protozoa
- sporeformer 5½ hours

2. Free – Flowing steam

- live steam → Arnold sterilizer
- same sterilizing action with boiling
 3. Steam Under Pressure
- steam sterilization is carried out in an autoclave
using pressured steam
- the principal requirement : the whole of the material
to be sterilized remain in contact with saturated steam
at the required temperature for the necessary
period of time
- 1210C 15 – 20 minutes (15lbs steam pressure per
sq. inch)
- most dependable moist heat method of sterilization
for hospital clinical lab. & research
facilities
- very effective against vegetative cells and most
bacterial endospores
- commonly used for sterilizing :
1. Hospital instruments ( surgical & medical )
4. Tyndallization
- Fractional /Intermittent sterilization
- free flowing steam 80-1000C 30 minutes for 3
consecutive days
involves exposing the material to elevated temp
(killing the vegetative cell)

incubate at 37°C
(to allow spores to germinate to form new vegetative cell)

expose to elevated temp again

(to kill the newly germinated vegetative cells)

- for sterilization of certain liquid or semisolid materials that are

easily destroyed by heat
- it is used to sterilize heat-sensitive culture media containing
such materials ac carbohydrates, egg or serum
- kills both sporeformer & non-sporeformer bacteria
5. Pasteurization
- applied to fresh beverages such as milk, milk
products, beer and wine
- heat is applied to these liquids to kill potential agents
of infection and spoilage at the same time retaining
the liquid`s flavor and food value
- 63-66°C for 30 minutes followed by rapid cooling
- the temperature does not sterilize the milk, but it does
kill all disease producing bacteria commonly
transmitted by milk
- primary target: non spore forming pathogens
(Salmonella sp., Campylobacter jejuni, Listeria
monocytogenes, Brucella sp., Coxiella sp. and
Mycobacterium sp.)
FREEZING
Is not a reliable method of sterilization
Primarily used in the preservation of bacterial cultures
In freezing, the formation of ice crystals outside the
cell causes the withdrawal of water from the cell
interior, resulting in an increased intracellular
electrolyte concentration and denaturation of proteins
The cell membrane is damaged, and a leakage of
intracellular organic compounds ensues

Lyophilization ( Freeze-drying )
 A process used for preserving biological material, by
removing the water from the sample, which involves
first freezing the sample and then drying it, under a
vacuum, at very low temperatures
RADIATION
- defined as energy emitted from atomic activities and
dispensed at high velocity through matter or space
-Sunlight possesses appreciable bactericidal activity and
plays an important role in the spontaneous
sterilization that occurs under natural conditions
-2 types
Ionizing radiation: Radiation that have sufficient energy to
remove an electron completely from an atom and
produce an electrical charge (ionization)
Nonionizing radiation: Energy absorbed by the molecule
cannot remove an electron completely, the
excitation produced often leads to
photochemical changes
Ionizing radiation
- Ex: electromagnetic rays: X-ray, alpha, beta & gamma rays
- uses short wavelength
- much higher energy content than UV rays
- has greater power penetration
- can penetrate a solid barrier, bombard a cell, enter it, and
dislodge electrons from molecules

breakage of DNA creates massive mutations

greater capacity to produce lethal effects
- used for sterilization of cutgut , nylon sutures, plastic,
syringe, catheter, prosthesis & plastic tubings
Non-ionizing radiation
- Ex. Ultraviolet light
- effectiveness of UV light as a lethal and mutagenic agent is
closely
related to its wavelength (240-280nm); optimum at about
260nm which corresponds with the absorption maximum of
DNA
- Lethal effect on bacteria is attributed to absorption and
resultant
damage of DNA formation of abnormal bonds
→ (damage) killing of the organism
- Energy of UV radiation is low
- penetrating ability is very poor
- primarily used to control airborne infections, where it is used
for the disinfection of enclosed areas such as nurseries,
hospital wards & OR
FILTRATION
-an effective method to remove microbes from air and liquids
- process of separating microorganism from contaminated
solution
- filtration sterilization is used to prepare liquids that cannot
withstand heat (heat-labile), including serum and other
blood products, vaccines, drugs, IV fluids, enzymes and
culture media
- useful for trapping microorganism only
- types filters
1. Seitz – asbestose – cellulose
2. Sintered glass – glass filaments
3. Chamberland – unglazed porcelain
4. Berkefeld – diatomaceous earth
5. Membrane filter – cellulose ester
-High efficiency particulate air (HEPA) filters are used to
ULTRASONIC AND SONIC VIBRATIONS
- Sound vibration at high frequency, in an upper audible and
ultrasonic range (20-1000 kc), provide a useful
technique for disruption & disintegration of the cell
-The passage of sound thru a liquid produces alternating
pressure changes, which, if the sound intensity is
sufficiently great, causes cavities to form in the liquid
- the cavities grow in size until they collapse violently
leading to cell disintegration
- No practical value in sterilization & disinfection
- uses 1. research laboratories
2. treating sewage H2O
 destroys structural integrity of cell membrane (protein &
lipids)
 interferes w/ normal membrane function
 the net effect is the release of small metabolites from
the cell and interfere with the active transport and
energy metabolism
 Agents:
 Surface active agents
• Cationic – most effective
• Anionic
• Non-ionic – not effective
• Amphoteric
 Phenolic compound
 Alcohol
I. Surface active agents
- Substances that alter the energy relationship at
interfaces producing a reduction of surface or
interfacial tension
- Compounds that possess both water-attracting
(hydrophilic) and water-repelling (hydrophobic)
groups
- They disrupt the integrity of cell membrane
This results in the loss of  molecules from the
cytoplasm and affects the proton motive force which
provides energy for solute transport
- The interface between the lipid-containing membrane of
a bacterial cell and the surrounding aqueous medium
provides a susceptible target site for agents of this
type
Cationic Agents
Quarternary ammonium compound
- QAC are any of a group of ammonium salts in which organic
radicals have been substituted for all four hydrogens of the
original ammonium cation
- This is the most important antibacterial surface-active
agent
- Bactericidal for a wide range of organisms, gram (+)
species are more susceptible
- Ex. Benzalkonium chloride ( Zephiran )
 used primarily in hand or face washes
 must not be applied to areas which have not been
fully rinsed as it is inactivated by organic
compounds
 Benzalkonium application many include disinfecting
instruments and preserving drugs in low
Anionic Agents
- Among the anionic detergents are soaps and fatty acids that
dissociate to yield a negatively charged ion
- agents are most active in an acid pH
- Causes gross disruption of the lipoprotein framework of the
cell wall
- By combining an anionic agent with acid, a very effective
acid-anion surfactant sanitizer
- display very rapid bactericidal action (within 30sec)

- Effective against gram (+) organisms but are relatively

ineffective among gram (-) species because of their
lipopolysaccharide outer membrane
 The activity of all four Surface active agents may be
enhanced by incorporating other disinfectants such as the
diguanide

 Diguanides
- have antimicrobial activity against vegetative bacteria,
yeasts, protozoans and enveloped viruses but not against
spores, protozoan cysts and mycobacteria
- They function by disrupting cytoplasmic membrane to cause
cell leakage and may enter cells to cause coagulation of the
cell cytoplasm
- It may be used together with surface active disinfectants at
a concentration of 1 – 4%
- Ex. chlorhexidine which is more effective at pH 7 – 8
 used as a safe antiseptic to apply to prevent body infection
and in oral rinses for treating sore gums and mouth ulcers
and preventing plaque on teeth
II Phenolic compound
- at low concentration, these compounds are rapidly
bactericidal causing leakage of cell contents and irreversible
inactivation of membrane-bound oxidases and
dehydrogenases
- Parent compound : Carbolic acid ( phenol )
- excellent for disinfecting feces, blood, pus, sputum &
other proteinaceous material
- primarily use for testing new bactericidal agent
- It had been replaced as a practical disinfectant by less
caustic and less toxic phenol derivative
- Cresols
- Xylenols
- Diphenyl compound
- Phenolics are effective against bacteria, fungi and virus
Phenol derivatives:
• Cresols
- the simplest of the alkyl phenols
- Ortho-, meta-, paracresols – are applicably more active than
phenol
- usually employed as a mixtureCresols obtained industrially
by the distillation of coal tar -- usual source of these products
- Cresols are used to dissolve other chemicals, as disinfectants
and deodorizers, and to make specific chemicals that kill Insect
pests
- sold under the trade names: Lysol and Creolin

• Xylenols
- Dimethylphenols
- important class of phenolics with great industrial importance
- are used as pesticides and in the manufacture of antioxidants
 Kills
99.9
%
of
germ
30 se s in • A general purpose Disinfectant
cond
s • Kills most bacteria on surface
• Destroys odors, cleans and
disinfects against germs and
against staph
• Good septic tank
disinfectant

• Hospital disinfectant-deodorant is highly

effective against TB, MRSA, and HIV-1
• Tuberculocidal, virucidal, fungicidal, bactericidal
• Minimizes concern over the spread of germs
in public facilities
• Prevents odors and growth of mold and mildew
• Diphenyl compound
- the halogenated diphenyl compounds exhibit unique
antibacterial properties
- effective against gram + bacteria ( staph & strep );
offers no protection against gram-negative infections
-The most impt, is the chlorinated derivative,
Hexachlorophene
• very useful as a topical anti-infective, anti-bacterial agent,
often used in soaps, toothpaste and antiperspirant
• also used in agriculture as a soil fungicide, plant bactericide
• bacteriostatic skin cleanser
• used as a preservative in cosmetic products
• pHisoHex, was widely used as a very effective antibacterial skin
cleanser in the treatment of acne
III Alcohol
-Alcohols provide an insight into the interaction of organic
solvents with lipid membranes
- they disorganize lipid structure by penetrating into the
hydrocarbon region
-Alcohols are normally used at concentrations between 50%-90%
and have optimal activity at 70% - 75% as the presence of water
helps to denature membranes, dissolve cell membrane lipids
causing cell lysis
- In addition to their effect on the cell membrane, alcohols
also denature proteins
- effective against vegetative bacteria, fungi and viruses
- have little sporicidal activity
- usually used to disinfect work surfaces and, as antiseptics on skin
- Antiseptic activity may be enhanced by incorporating other
antimicrobial compounds: chlorhexidine (0.5%)
iodine (1% to 2%)
Ethanol / Ethyl alcohol
- Used as skin disinfectant because of their bactericidal
action and ability to remove lipids from the skin surfaces
- their action as disinfectant is restricted by their inability at
normal temperatures to kill spores not relied for
sterilization of
instruments
- Uses: - to sterilize skin before cutaneous injections
- to disinfect thermometers
- Most effective at 50-70%
- effective against gram (+) , gram (-) , AF bacteria
Isopropyl alcohol
- Most effective at 50-70%
- most effective type of alcohol
- bactericidal activity is slightly greater than ethanol
- less volatile
- for this reason, it had been recommended as replacement
for the sterilization of thermometers
-Toxic effect is greater and long lasting
-Necrosis may result from absorption of vapors through the
lungs during alcohol sponge bath
- In its native state, each protein possesses a characteristic
conformation that is required for its proper function
- Agents that alter the conformation of the proteins by
denaturation cause an unfolding of the polypeptide chain
so that the chain becomes randomly and irregularly
looped or coiled
- Agents:
1. Acid & alkalies
- exert their antibacterial activity through their free H+ and OH-
ions, through undissociated molecules or by altering the
pH of organism’s environment
Benzoic acid
Lactic acid used as food preservative
Acetic acid
Propionic acid
2. Alcohol
3. Acetone
- The catalytic site of an enzyme contains specific
functional groups that bind the substrate and initiate the
catalytic events
- Inhibition of enzyme activity results in one or more of
these functional groups is altered or destroyed
- Important functional groups of the cell wall, membrane
and nucleic acids are also susceptible to inactivation
-Agents:
1. Heavy metals
2. Oxidizing agents
3. Dyes
4. Alkylating agents
Heavy Metals
– soluble salts of mercury, silver , arsenic and other heavy
metals poison enzyme activity by forming mercaptides with
the sulfhydryl groups of cysteine residue
• Mercurials
• Mercuric chloride – very toxic disinfectant, not used
today
• Metaphen, Merthiolate, Mercuchrome – less toxic,
unreliable as skin disinfectant
• Silver compounds
• widely used as antiseptic ; either as soluble silver salts
or as colloidal preparations
• inorganic silver salts - efficient bactericidal agents but
has irritant and caustic
effects
• Silver nitrate – highly bactericdal for gonococci and
routinely used as prophylaxis against
opthalmia neonatorum in neonates
Oxidizing agents
• most useful antimicrobial agents: halogens (iodine, chlorine)
hydrogen peroxide

• Halogens
- chlorine and iodine are among the most useful disinfectants
- Almost exclusively bactericidal and effective against
sporulating bacteria, virus, fungi and protozoa
(1) iodine
- exist principally in the form of I2 at pH value below 6
where maximal bactericidal action is manifested
- iodine tinctures came into wide use as microbicides
- Iodine quickly kills a broad range of microorganisms
- destroys many microorganisms and viruses within 3-5 minutes
- used in cleansing skin or disinfecting small wounds but
presented with some problems
- strong smell and can stain skin and clothing.
- react to metals and are rather unstable
- skin irritant
 Betadine Solution
- various mixtures and solutions of
iodine, called Iodaphores, have
been formulated that greatly lessen such
disadvantages and dangers
- enhance stability and microbe killing
properties.

-Contains 10% povidone-iodine

- broad spectrum topical iodophor microbicide
- available in "Swab Aid" pads, Swab Sticks and as a
Surgical Scrub
- It is a fast-acting, broad-spectrum antiseptic that kills
bacteria (including antibiotic resistant organisms), as well as
fungi/yeasts, viruses and protozoa
- It is indicated for degerming skin, wounds and mucous
membranes
- widely use in hospitals, sanitation and water purification,
(2) chlorine
- 3 types of Chlorine compound: hypochlorite,
inorganic & inorganic
chloramines
- The active moiety of hypochlorites and chlorine is
hypochlorous acid which is stable at a low pH, which is a
strong oxidizing agent and an effective disinfectant

- Water disinfectant
- hypochlorites - most useful of the chlorine compound
- widely used for sanitizing dairy products
and food processing equipment
- employed as sanitizers in most households,
hospitals, and public buildngs
- marketed as: Chlorox, Zonrox, Purex
• Hydrogen peroxide
- antibacterial action is secondary to its oxidizing
ability as well as formation of a more toxic free
hydroxyl radical from the peroxide in an iron-
dependent reaction
- It is a weak acid
- In a 3% solution, it is harmless but very weak
antiseptic whose primary clinical use is in the
cleansing of wounds
- It has strong oxidizing properties and is therefore a
powerful bleaching agent that is mostly used for bleaching
paper
Dyes
- some of the coal-tar dyes not only stain bacteria but are
inhibitory at very high dilutions
- within the usual pH range, the basic dyes are the most
effective
- their current medical use is limited primarily to the
treatment of dermatologic lesions
• Triphenyl methane dyes
• aniline dye derivatives especially Crystal violet, Malachite
green, Brilliant green
• Highly selective for gram (+) organisms
• Used in the laboratory in the formulation of selective
culture media
• Acidine dyes
• Often referred as Flavines because of their yellow color
• exert a bactericidal and bacteriastatic effect upon a
number of organisms
• Compounds of clinical use : proflavine, acriflavine
Alkalyting agent
- The lethal effect results from their alkylating action on
proteins
- Glutaraldehydes and formaldehydes are the most commonly
used aldehydes
-They are active against bacteria and their spores, viruses, fungi
and protozoa
-Antimicrobial activity occurs as a result of cross-linking proteins
and nucleic acids in fungi, protozoa and bacteria and capsid
nucleic acid complexes in viruses
-These effects are mediated predominantly via amine, sulfurhydryl
and carboxyl groups on microbial surface proteins
Formaldehyde
- One of the least agents acting on proteins
- commercially available in:
• aqueous solution containing 37% formaldehyde (Formalin)
• paraformaldehyde a poymer (contain 91-99% formaldehde)
- Formalin - used for preserving fresh tissues
- major component of embalming fluids
- when used in high concentration, it destroys all
organisms, including spore
- Used to inactivate virus in the preparation of vaccines
- As a gas – used to decontaminate rooms, buildings, fabrics,
and instruments
Glutaraldehyde
- used as “cold sterilant” for surgical instruments
- 10x more effective than formaldehyde as a bactericidal
and sporocidal agent and less toxic

Ethylene oxide
- Employed in gaseous sterilization, especially on
materials that would be damaged by heat
(polyethylene tubings, electric & medical instruments,b
iologicals and drugs)
- Active against all types of bacteria, including spores &
TB bacilli