MEDICAL MICROBIOLOGY

Course for medical students

Associate Professor L. Yocheva, Ph.D.

Department of Biology, Medical Genetics and

Microbiology
Medical Faculty
SU “St. Kliment Ohridski”

Email: [email protected]
 Lecture 4
Influence of Physical and Chemical Factors on
Microorganisms.
Sterilization and Disinfection

1. Influence of physical factors on microorganisms:

heat, drying, lyophilization, etc.
2. Influence of chemical factors on microorganisms
3. Sterilization: definition, methods
4. Disinfection: disinfectants, antiseptics,
preservatives - groups, mechanism of action,
representatives
 Key words

• Sterilization
• Hot air sterilizer
• Autoclave
• Plasma gas sterilization
• Disinfection
• Disinfectants
• Antiseptics
• Preservatives
Influence of Physical and
Chemical Factors on
Microorganisms
 Influence of Physical Factors on
Microorganisms
• Temperature
• Desiccation
• рН of medium
• Pressure
˗ Osmotic
˗ Hydrostatic
• Sunlight
• Irradiation
˗ Ultraviolet rays
˗ Ionizing radiation
• Sonic energy
 Temperature
• Bacteria do not have thermoregulatory mechanisms
• They accept the temperature of the environment they live in
• Temperature affects:
˗ the growth rate of bacteria
˗ the rate of cellular reactions
˗ the nature of cellular metabolism
˗ the cell’s requirements for nutrients
• Temperature range is outlined by:
˗ minimum temperature
˗ optimal temperature
˗ maximum temperature
• Application
˗ transport of clinical specimen - 22 ℃ /37 ℃ for bacteria and
4 ℃ for viruses
˗ cultivation – at optimal temperature
˗ preservation – anabiosis; at 4 ℃, -20 ℃, -70 ℃, -196 ℃
˗ destruction – 160-170 ℃
 Desiccation
• Water content in the microbial cell - 73-90%
• All life functions take place in an aquatic environment
• Only free water in the environment is available
• When the microbial cells are dried, they fall into anabiosis
• Bacteria are most sensitive to drying, followed by yeasts
and molds
˗ vegetative forms are much more sensitive to desiccation
than spores
˗ sensitive to desiccation – meningococci, gonococci,
influenza and AIDS viruses
˗ Mycobacterium tuberculosis can survive in dried
secretions for 1 year
• Application
˗ cultivation - agar content adjustment in solid media
˗ storage – lyophilization
˗ destruction - drying of non-sterilized products (meat, fish,
fruits, vegetables)
 рН
• An indicator of the hydrogen ions concentration in the
medium
• It affects the chemical processes in the microbial cell:
˗ directly - through the immediate impact of H+
˗ indirectly on:
▪ the ionic state and accessibility of inorganic ions
and metabolites
▪ the stability of macromolecules
▪ the balance of electric charges on the surface of the
cell
• Microbial enzymes are active in the pH range of 4.0 - 9.0
• Most pathogenic microorganisms thrive at neutral pH
• Maintained by buffer solutions
• It depends on the temperature
 Osmotic Pressure
• Normal osmotic pressure in the bacterial cell – 3-30 atm
• Isotonic medium - with a defined salt concentration (0.5%
to 3% NaCl)
• Plasmoptysis - rupture of microbial cells during their rapid
transfer to an environment with low osmotic pressure
(hypotonic solution) due to the entry of water into them
• Plasmolysis - when transferred to hypertonic solution, the
cytoplasm and membrane shrink
• Osmophilic microbes - live in an environment with high
osmotic pressure; found in seawater or in concentrated
sugar solutions
• Halophiles - grow at 13-29% NaCl (Staphylococcus aureus
– 15%)
• In the food industry - for preservation of meat, fish,
vegetables and fruits
 Hydrostatic Pressure

• Hydrostatic pressure over 1000 atm causes

irreversible changes in bacteria:
˗ change in viscosity of protoplasm
˗ denaturation of cellular proteins
• Barophilic - marine bacteria; sulfate-reducing
bacteria isolated from petroleum
• Application
˗ sterilization by autoclaving
 Irradiation
• Short-wavelength radiation - with biological activity
˗ mechanism of action - cause photochemical reactions in
microbial cells
• Infrared rays - with a physiological effect on
microorganisms
• Visible light - a source of energy for photosynthetic
bacteria to assimilate CO2
• Gram-negative bacteria - more resistant to sunlight
 Ultraviolet Rays
• Biological activity - 200-295 nm (max 250-260 nm)
˗ mutagenic effect – thymine dimers (point mutations) in
one strand of DNA
˗ lethal effect – thymine dimers in both complementary
strands; replication stops
• Repair enzyme systems – activated by light
(photoreactivation) and remove damaged DNA regions
• The most sensitive are the young, multiplying cells in log-
phase
• The bactericidal effect is determined by:
˗ radiation intensity
˗ absorption coefficient of the rays by the microbes
˗ exposure time
• Application
˗ air disinfection in operating rooms
˗ sterilization of drugs, safety cabinets, plastic products
 Ionizing Radiation
• Corpuscular (particulate):
˗ β/ E-beam (high-speed electrons)
• Electromagnetic:
˗ γ- and Rö (X)-rays
▪ penetrate deep into the cell
▪ cause ionization of atoms and destruction of molecules
• Mechanism of action:
˗ target theory - enzymes or DNA directly absorb photons
˗ free radical theory - water, absorbing energy, splits into
free H+ and OH- radicals, which can cause destructive
oxidation-reduction reactions
• Pigmented bacteria and spores are more resistant
• Sterilization of plastic items; surgical sutures; bandages;
biological preparations (vitamins, hormones); antibiotics;
food
 Sonic Energy
• Bacteria are only sensitive to high frequency ultrasound:
2.104-2.108Hz
• Cavitation – in a liquid medium, ultrasound waves cause
the protoplasm to contract and relax, resulting in the
formation of bubbles of dissolved gases. They lead to the
destruction of the microbial cell by the applied pressure
or by the dissociation of water molecules with the
formation of toxic radicals
• Application
˗ mechanical cleaning of medical, dental and optical
instruments
˗ extraction of microbial antigens, enzymes, endotoxins
 Chemical Factors
• Chemical factors - those of the chemical substances that
have a toxic effect or cause the death of the bacterial cell
• Types: natural; synthetic; substances resulting from the
metabolic activity of different organisms
• The effect on the microorganism depends on:
˗ the chemical nature, structure and concentration of the
substance
˗ the species, physiological state and number of affected
microorganisms
˗ exposure time
˗ the temperature at which the effect occurs
˗ the presence of another chemical factor
synergism (additive or potentiated action)
antagonism (weakens the effect of the first factor)
˗ the presence of organic substances (blood, serum, pus)
Sterilization
History

Surgical clothing,
19th century
Semmelweis and Lister – Founders of Antiseptics

• Ignaz Semmelweis (1818 –1865)

˗ Known as an early pioneer of antiseptic procedures
˗ Described as the "Savior of mothers“
˗ Discovered that the incidence of puerperal fever could be
drastically cut by the use of hand disinfection instead
hand washing in obstetrical clinics
˗ In 1847, using the practice of hands disinfection with
chlorinated lime solutions puerperal fever fall from 18%
to 2,5%
• Joseph Lister (1827-1912)
˗ British surgeon and a pioneer of antiseptic surgery
˗ In 1867 Lister successfully introduced carbolic acid (1-2
% phenol) to sterilize surgical instruments and to clean
wounds
˗ This led to a reduction in post-operative infections and
made surgery safer for patients
 Sterilization

• Sterilization: destruction of all forms of

microbial life including bacteria, spores,
viruses and fungi

• Performed mostly by physical means

• Possible even with chemical or physio-

chemical means
 Methods of Sterilization
• Physical Methods
˗ Heat
▪ Dry heat – Red heat, Flaming, Incineration, Hot Air
Sterilizer
▪ Moist Heat – Tyndallization, Pasteurization, Boiling,
Fractional Sterilization with Steam
▪ Steam Under Pressure - Autoclaving
- Cold
▪ Filtration – of liquids and gases (air)
▪ Irradiation
➢ Non-ioning Radiation - Ultra violet rays
➢ Ioning Radiation - Gamma-rays, E-beam
• Chemical – solutions, gases, aerosols of substances with
sporicidal activity
• Physio-chemical - a combination of a physical factor and
a chemical substance (Plasma-gas Sterilization)
Methods of Sterilization
Physical Methods of Sterilization
 Heat Sterilization

• Heat: the most reliable and universally

applicable method of sterilization
• Mechanism of action:
˗ Dry heat denatures proteins
˗ Moist heat denatures and coagulates
proteins
• Dry heat is less efficient and requires high
temperature and longer heating period
compared to moist heat
 Heat Sterilization - Methods
• Flaming and Red heat - performed in the flame of a
Bunsen burner/spirit lamp
˗ short time – 5-7 seconds
˗ small and fast heating metal tools: inoculating wires,
loops and points of forceps are sterilized by holding
them until they are red hot
˗ scissors, scalpels, spatulas, necks of flasks and test
tubes are sterilized by passing through flame – by
flaming
˗ vegetative and spore forms are killed
• Incineration - an efficient method of sterilization and
disposal of infectious materials, including bodies of
animals that died from anthrax
 Hot Air Sterilizer (Oven)
• It is carried out in a Hot air sterilizer (oven)
• Vegetative and spore forms are killed
• It is essential that hot air circulates between the
objects being sterilized
• These must be loosely packed with an
adequate space to ensure best heat transfer
• It is done by applying 160 ℃ for 2 hours
• Usage for sterilization of glassware, oils,
greases, powders; metal instruments
• For materials which can withstand high
temperatures
 Moist Heat for Sterilization
• Boiling
˗ done by applying 100 ℃ for 30 minutes
˗ performed in a water bath
˗ glass, metal, rubber and plastic objects; fabrics and clothes
˗ not a reliable method of sterilization - only vegetative forms are
killed
• Tyndallization (Fractional Sterilization)
˗ performed in a water bath
˗ done by applying 56-58 ℃ for 1-2 h /5-7 consecutive days
˗ medicines, serums and foods with thermolabile ingredients
• Pasteurization (it is not sterilization)
˗ exposure of liquids to a temperature below the boiling point in order
to remove all vegetative bacteria of significance for human diseases
˗ avoids changing the molecular structure of the liquids
˗ 80 ℃ /15 min or 62 ℃/30 min followed by rapid cooling to 13 ℃
˗ food products and beverages: milk, wine, beer, ketchup, juices; for
the preparation of bacterial vaccines
˗ UHT pasteurization of milk - 138 ℃ /4 seconds – shelf life 6 months
 Steam Sterilization

Fractional Sterilization
• in Koch’s steamer
• steaming of the material is done at 100 ℃ for 1h
on three consecutive days
• spores which survived the heating process
would germinate before the next thermal
exposure and then would be killed
• for sterilization of nutrient media with
components that do not withstand heating
above 100 ℃ (carbohydrates, amino acids,
vitamins, ammonium salts)
 Autoclaving - Steam Under Pressure
• The most effective form of moist heat sterilization
• Autoclave – 121 ℃ at 1 atm for 15-20 min
• Vegetative cells and endospores are killed
• N.B. Very effective way of sterilization, quicker than hot
air oven, excellent for killing the spores
• Based on the principle that when water is boiled at increased
pressure, hot saturated steam will be formed and will
penetrate and give up its latent heat when it condenses on
cooler objects
• Hot saturated steam in autoclaving acts as an excellent agent
for sterilization because of: high temperature; high latent
heat; ability to form water of condensation; contraction in
volume that occurs during condensation
• Nutrient media, rubber, metal, plastic and composite
products, filters, gowns, medical and surgical equipment
 Methods of Controlling Sterilization
• Recording of temperature and time of each
sterilizing cycle

• By chemical indicators used in:

– autoclaving
– dry heat sterilizer
▪ heat-sensitive tape is fixed to the outside
of each pack
▪ a change in the color of the paper strips/
tape indicates complete sterilization
• By biological indicator
 Lyophilization (Freeze-drying)

• A method for creation and long-term storage of

stable biological products
• Includes:
˗ rapid freezing
˗ subsequent dehydration of the frozen product
under high vacuum by sublimation
• Use:
˗ preservation of microbial cultures
˗ preservation of vaccines
▪ Live vaccines are lyophilized
 Filtration
• Filtration is useful for removing bacteria and fungi from:
˗ air – with high-efficiency particulate air filters – HEPA
˗ solutions – through membrane filters

• Filter pores – 14–0.023 µm

– for viruses
– for bacteria - membrane filters made of polymer materials;
pores 0.2-0.45 µm
• Use:
˗ Sterilization of thermolabile solutions (amino acids,
sugars, vitamins, easily oxidizable mineral ingredients),
serums, plasma, vaccines, infusion solutions, drugs,
nutrient media
˗ Microbial evaluation of water purity
˗ Viable counting procedures
Membrane Filters
 Ioning Radiation
• Ioning radiation includes χ rays, γ rays and β rays (E
beam)
• Gamma rays are generated by the decay of the
radioisotope cobalt 60 and β rays are generated by
electron accelerators
• The resulting photons are of high energy and good
penetrating power
• An effective method of sterilization at low temperatures
• They are used for sterilization of a number of medical
products (tissues for transplantation, drugs, medical
devices)
• Sterilization of plastic items in microbiology
• Food sterilization
 Ultraviolet Radiation
• Has less quantum energy with low penetrating power than
ionic radiation
• Bacterial spores are more resistant to ionic and ultra violet
radiation than bacteria and viruses
• UV rays are generated using a high-pressure mercury vapor
lamp
• Longer exposure
• Suitable for disinfection of surfaces and air in hospital rooms
(operating and delivery rooms, isolators) and in laminar boxes
• Sterilization of surgical sutures, catheters, petri dishes
• Disinfection of drinking water, titanium implants and contact
lenses
• Culture media while dispensing and pharmaceutical products
like hormones, enzymes and antibiotics
• Damages human tissue and many plastics
• Strong natural sunlight has microbicidal activity
Chemical Sterilization
 Gas Vapor Sterilization with Ethylene Oxide
• Mechanism of action: alkylates sulfhydryl-, amino-,
carboxyl- and hydroxyl groups.
• Use: highly effective hemisterillant capable of killing spores
rapidly.
• It has good penetration and is well adsorbed by porous
materials.
• Sterilization of:
˗ heat-labile products (bedding; textiles, rubber or plastic
goods - syringes, petri dishes)
˗ complex devices - equipment for ventilation, for
maintaining cardiac activity; in dentistry.
• Flammable; sterilization is carried out in a hermetically
sealed chamber with moisture and CO2.
• Very toxic (residual toxic radicals), carcinogenic,
mutagenic, irritating to eyes, skin, mucous membranes.
 Plasma-gas Sterilization
• A new plasma-based sterilization technology
• H2O2 in vacuum at low temperatures (highly ionized gas)
• The by-products are water vapor and O2
• Mode of action: Combined action of H2O2 gas and
generated free radicals from H2O2 vapor during a plasma
phase of the cycle. Radicals interact with cellular
components and disrupt the metabolism of
microorganisms
• Microbicidal activity – including bacterial spores
• It is used for the sterilization of materials and devices
that cannot withstand high temperatures and humidity,
such as plastics, electrical and electronic devices and
corrosion-sensitive metal alloys.
Disinfection
 Disinfection

• Use a chemical agents (disinfectants) to

destroy most microbial forms
• Spores and other resistant organisms (e.g.
Mycobacteria, viruses, and fungi) may
remain viable
• Disinfectants destroy microbes without
showing any form of selectivity
• Disinfectants are used very often for
inanimate surfaces
 Effects of Chemical Agents on
Microorganisms
• Germicide agents – chemical agent capable of killing
microbes; spores may survive
• Sporicidal agents: germicide capable of killing
bacterial spores
• Bactericidal effect – chemical agent kills the bacterial
cell
• Bacteriostatic effect – chemical agent inhibits
(suppresses) bacterial cell growth; when the
substance is removed, reproduction continues
• Fungicidal effect – chemical agent kills the fungal cell
• Fungistatic effect – chemical agent inhibits the growth
of the fungal cell
• Virucidal effect – chemical agent inactivates viral
particles
 Types of Disinfectants

• Disinfectants - chemicals used in disinfection

- equally toxic to microbes and human cells
- not applied to humans for internal use (oral and
parenteral)
- applied to inanimate objects
• Antiseptics
- less toxic
- applied locally on skin and mucosa
• Preservatives
- prevent the growth of microorganisms in
sera, vaccines, pharmaceuticals, foodstuffs and
cosmetics
 Antisepsis and Asepsis
• Antisepsis - use of chemical agents on skin or other
living tissue to inhibit or eliminate microbes; no
sporicidal action is implied
˗ Requirements for antiseptics – never be toxic to
cells; never be corrosive; should never change
nature of skin
˗ Examples
▪ Alcohol (ethyl, isopropy) - 70-90%
▪ Aqueous Iodine, Iodine Tincture, Iodophors
▪ Chlorhexidine

• Asepsis – the employment of techniques (such as

gloves, air filters, UV rays) to achieve a microorganism
-free environment
 Categories of Disinfectants
• High level of disinfectants
˗ They kill vegetative cells, mycobacteria, fungi, viruses and bacterial
spores
˗ Sporicidal agents – H2O2, glutaraldehyde, formaldehyde, chlorine
compounds
˗ Certain types of endoscopes and surgical instruments with plastic
components in invasive procedures
• Intermediate level disinfectants
˗ They have activity against vegetative cells, mycobacteria, fungi, viruses;
have no effect on bacterial spores
˗ Clinical contact surfaces and semi-critical instruments with or without
visible contamination with body fluids (flexible fiber optic endoscopes,
laryngoscopes, vaginal speculums, anesthesia breathing devices).
˗ Examples: alcohols, halogens, phenolic compounds
• Low-level (“hospital”) disinfectants
• They kill vegetative cells, fungi and some viruses
• Clinical contact surfaces and non-critical instruments not visibly
contaminated with body fluids (blood pressure cuffs, electrocardiogram
electrodes, stethoscopes)
• Examples: quaternary ammonium compounds
 Mechanism of Action of Disinfectants

• Damage to lipids and/or proteins of the semipermeable

cytoplasmic membrane or cell wall, leading to disruption
of their integrity
• Denaturation of the proteins in bacterial protoplasm and
microbial enzymes, which leads to disruption or blocking
of metabolic processes
• Inhibition of enzyme activity by modification (oxidation or
alkylation) of essential sulfhydryl-, amino-, carboxy- and
hydroxyl groups of enzymes
• Nucleic acid damage that results in mutations or
cessation of replication
 Surface Active Agents

• Destroy the membrane

• Soluble in water
• Dissolve fats - cleaning agents
▪ Soaps – NaOH or KOH and fats
– alkaline, remove bacteria mechanically
▪ Detergents – synthetic surface active agents
– Cationic (+ charged) – Quaternary
ammonium compounds – have limited
antibacterial activity; low level disinfectants
– Anionic (- charged)
 Phenolics and Derivatives of Phenol
• They destroy the membrane, precipitate proteins and
inactivate enzymes
• Disinfectants in high concentrations, antiseptics - in low
concentrations
• Broad-spectrum of activity
• In the past - have been widely used as general-purpose
environmental disinfectants in hospitals
• Neurotoxic - not used today
• Examples
– Lysol
– Hexachlorophene - the halogenated bis-phenol is a
disinfectant and antiseptic with bacteriostatic activity;
used as a topical anti-infective and antibacterial agent;
used in toothpaste, soaps, creams and other
preparations for skin problems
– Chlorocresol - in pharmacy
 Biguanide Compounds
• Effective against Gram-positive and Gram-negative bacteria,
but poor activity against viruses
• Effectiveness is enhanced and it is more rapid in onset when
diluted with alcohol and very often used in these combination
• Inactivated by soap and anionic detergent
• Chlorhexidine
˗ At physiologic pH, chlorhexidine dissociate and release the
positively charged chlorhexidine cation. The bactericidal
effect is a result of the binding of this cationic molecule to
negatively charged bacterial cell walls
˗ At low concentrations of chlorhexidine, this results in a
bacteriostatic effect; at high concentrations, membrane
disruption results in a cell death
˗ Disinfection of skin, mucosa, hands
˗ In cosmetics and pharmaceutical products (preservative in
eye drops, active substance in wound dressings and
antiseptic mouthwashes)
 Alcohols

• Alcohols dehydrate cells, disorganize cell

membrane lipid structure, and cause protein
coagulation
• They have a bactericidal effect against
mycobacteria and complex viruses
• They do not kill spores
• 70% aqueous solution – the best effect (better
penetration in the microbial cell)
• Ethyl alcohol, isopropyl alcohol
˗ Antiseptics
˗ Disinfects clinical thermometers and surfaces
 Oxidizing Agents - Halogens
• Mode of action – oxidizing agents modifying sulfhydryl groups
of enzymes
• Chlorine-containing agents
– Chlorine reacts with water to form hypochlorous acid with a
microbicidal effect
– Chlorine: water disinfectant; inactivated by organic materials
– Hypochlorite: sanitizing dairy and food processing
industries, households and hospitals
– Organic or inorganic chloramine: effective water disinfectant
acting by liberating chlorine

• Iodine-containing agents
– Aqueous iodine
– Iodine tincture: 2% iodine and 70% ethanol; antiseptic
– Idiophores (e.g. Betadine – Iodine and organic component):
Less toxic and less active than aqueous iodine and iodine
tincture; for pre- and post-operative skin disinfection
 Oxidizing Agents - Hydrogen Peroxide
• Н2О2 releases free hydroxyl radicals, damaging cellular
proteins and DNA
• 3% Н2О2 solution
˗ Antiseptic
˗ Deodorizing wounds and ulcers – it kills anaerobic
bacteria
• 6% Н2О2 solution
˗ Disinfection of surgical instruments, ventilators and
contact lenses
• Strong solutions are sporicidal
• Degraded by catalase; decomposed by light; organic
matter on instruments reduces activity of Н2О2
 Alkylating Agents
• Formaldehyde, Glutaraldehyde, Ethylene Oxide
• They add amino, hydroxyl and carboxyl functional
groups to biological molecules
• Used as sterilants and high level disinfectants
• Expose of skin and mucous membrane to them
may be toxic
• Formaldehyde – inactivates viruses and toxins;
for production of toxoids and killed vaccines
• Glutaraldehyde – cold sterling of surgical
instruments; endoscopes (cystoscopes,
bronchoscopes); anesthetic equipment's
• Ethylene Oxide – for chemical gaseous
sterilization of products not resistant to heat
 Dyes
• Acridine dyes
˗ Intercalate between base pairs in DNA. They are
more effective against Gram-positive bacteria.
They are bactericidal and germicidal in action.
˗ Rivanol – wounds
• Aniline dyes
˗ Form pseudobases that are liposoluble and easily
enter the cell
˗ Inhibit the synthesis of peptidoglycan
˗ Active only on Gram-positive bacteria
˗ Malachite green, brilliant green, crystal violet
▪ for treatment of dermatological lesions
▪ for formulation of selective culture media