Control of

microbes






Dr. Jos L. Navarro
Clinical Microbiologist, (Madrid, Spain)


http://www.4shared.com/dir/HZVz_Hvv/MICROBIOLOGY_PHARMACY.html
Introduction.
Surgery during 19
th
century
Surgery was very dangerous.
Very high risk of infection.
The operating room,
the surgeon's hands, and
the surgical instruments were full of microbes.
Wearing their street clothes,
Without washing their hands.
Ordinary sewing thread to suture wounds

Introduction.
Ignaz Semmelweis (1818 1865)
Assistant obstetrician at the
Vienna General Hospital.
Childbed fever,
which we now know is caused
primarily by group A
streptococci,
600 to 800 maternal deaths
per year at that time
He noticed that the death
rate in one of the two
divisions of the hospital was
10 times that in the other.
Introduction.
Ignaz Semmelweis (1818 1865)
First clinic
was attended by students of medicine after autopsies.
Second clinic
was attended by student midwives
who had no contact with corpses.
With a much lower mortality rate.
Cadaverous particles" on their hands.
In 1847,
he instituted a policy of handwashing something similar to
bleach between autopsy and examination.
Mortality rate in the First Clinic dropped 90%
Introduction.
Joseph Lister (1827 1912)
British surgeon at the
Glasgow Royal Infirmary.
He promoted the idea of
sterile surgery.
In 1865,
he introduced carbolic acid
(now known as phenol)
to sterilize surgical
instruments
and to clean wounds,
Reduction in post-operative
infections and made
surgery safer for patients.
Introduction.
Robert Koch (1843 1910)
The germ theory of disease
1884, Kochs
postulates
Sequence of
experimental steps
for directly relating a
specific microbe to
a specific disease.
Basic concepts
What is death?
Microbial death:
The permanent loss of reproductive capability,
even under optimum growth conditions.
Basic concepts
Sterilization:
Use of a physical process or a chemical agent to
destroy all forms of microbial life, including
viruses.
Any material that has been subjected to this
process is said to be sterile.
Absolute concept: YES or NO
it is either sterile or not sterile.
Generally reserved for inanimate objects
Basic concepts
Disinfection:
Use of a physical process or a chemical agent (a
disinfectant) to destroy vegetative pathogens
but NOT bacterial endospores.
Normally used only on inanimate objects.
Basic concepts
Antisepsis:
Disinfection, using a chemical agent (antiseptic),
applied to living tissues.
Thus, antiseptics are disinfectant agents that can
be used on body surfaces
such as the skin or mucosa
lower toxicity than disinfectants used in the
environment.
Basic concepts
Sanitization:
Less precise term
between disinfection and cleanliness.
Process made to decrease the number of microbes on
eating, drinking and home stuff.
In housekeeping and food preparation.
Basic concepts
Asepsis:
Practice that prevents the entry of infectious
agents into sterile tissues.
To prevent infection.
Thus, asepsis applies sterilization, disinfection
and antisepsis to create a protective environment.
Examples:
Preparing the skin before surgical incisions with
iodine compounds,
swabbing a wound with hydrogen peroxide.

Physical methods.
Heat
Heat kills microorganisms
by denaturing their enzymes
changes to the three-dimensional shapes of these
proteins.
Physical methods.
Heat
The two physical states of heat used in microbial
control are
Moist
Dry
Physical methods.
Heat. Moist heat
Sterilization with steam under pressure
At sea level, normal atmospheric pressure is
1 atmosphere.
At this pressure, water will boil at 100C,
and the resultant steam will remain at exactly that 100C
But it is too low to kill all microbes.
In order to increase the temperature of steam,
the pressure at which it is generated must be increased.
If pressure is increased, the temperature at which water
boils and the temperature of the steam produced both
increase.
Physical methods.
Heat. Moist heat
So, how can we get higher pressures?
Special device, closed
Autoclave
A sealed heating device that uses steam under
pressure to kill microorganisms
121C, 10 15 minutes, and 1 atm over normal (2
atm).
The duration of the process is adjusted according to
the bulkiness (weight and volume) of the items in the
load.
Physical methods.
Heat. Moist heat
Non-pressurized Steam
Some substances that cannot resist the high
temperature of the autoclave can be subjected to
intermittent sterilization, also called tyndallization.
100C steam for 30 to 60 minutes.
Not sufficient to kill spores,
so a single exposure will not be enough.
After that, items are incubated at appropriate
temperatures for 23 to 24 hours,
and then again steam treatment is applied
This cycle is repeated for 3 days.
Because the temperature never gets above 100C,
highly resistant spores could survive even after 3 days
of this treatment
Physical methods.
Heat. Moist heat
Boiling Water
Disinfection
NOT sterilization
Because 100C will not kill all resistant cells.
Dont kill bacterial spores
Boiling water for 30 minutes will kill most non-
spore-forming pathogens.
But the items can be easily contaminated again
when removed from the water.
Physical methods.
Heat. Moist heat
Pasteurization
Milk, fruit juices, beer, and wine are easily contaminated
during collection and processing.
Heat is applied to liquids to kill potential agents of infection
and spoilage,
retaining the liquids flavor and food value.
A widely used pasteurization technique is the flash
method,
that exposes the liquid to heat at
71.6C for 15 seconds.
The milk is then rapidly cooled.
Another method: Ultra High Temperature (UHT):
134C for 2 to 5 seconds.

Physical methods.
Heat. Dry heat
Incineration
The most rigorous of all heat treatments.
The flame of a Bunsen burner reaches 1,870C at its
hottest point, and incinerators operate at temperatures of
800C to 6,500C.
Everything becomes ashes and gas.
Very common practice in the microbiology laboratory.
Fast and effective,
but it is also limited to metals and heat-resistant glass
materials.
Also to burn hospital wastes
Physical methods.
Heat. Dry heat
Hot-air oven
Provides another means of dry-heat sterilization.
The dry oven is usually electric (occasionally gas)
heat within an enclosed compartment.
A cycle takes from 12 minutes to 4 hours to
complete and temperatures of 150C to 180C.

Physical methods.
Radiation
Irradiation
Energy emitted from atomic activities and
dispersed at high velocity.
Alteration of DNA.
Types:
Ionizing radiations.
Gamma rays,
X rays,
Produces sterilization.
Non-ionizing radiations.
Ultraviolet radiation.
Produces disinfection.
Physical methods.
Radiation
Physical methods.
Radiation
Physical methods.
Radiation
Physical methods.
Irradiation
Physical methods.
Irradiation
Physical methods.
Others
Filtration:
For heat-sensitive liquids and gases.
Disinfection of gases and sterilization of liquids.
The liquid or gas is passed through a filter,
a device with pores too small for the passage of
microorganisms,
but large enough to allow the passage of the liquid or
gas

Chemical methods.
Phenolics
Phenol was the first widely used antiseptic
and disinfectant.
Used as disinfectants in laboratories and
hospitals.
Phenolics act by
denaturing proteins and
disrupting cell membranes.
However, they have a disagreeable odor and
can cause skin irritation.
Chemical methods.
Alcohols
Protein denaturants
Need to be mixed with water.
70% to 95% alcohol.
Solutions of 100% alcohol dehydrate organisms rapidly but
fail to kill.
They are inactive against bacterial spores and many
viruses.
They need 10 15 minutes to begin their effect.
Ethanol (70-90%) and isopropyl alcohol (90-95%)
are widely used as skin decontaminants
before simple invasive procedures such as venipuncture.
Isopropyl alcohol has largely replaced ethanol in hospital
use because it is somewhat more active.
Chemical methods.
Halogens. Iodine
Effective disinfectant that acts by interfering
with the disulfide bonds of proteins.
Its original use was as a tincture of
2% iodine in 50% alcohol, which kills more rapidly
and effectively than alcohol alone.
Now, preparations iodine is combined with
carriers (povidone) or nonionic detergents.
Chemical methods.
Halogens. Chlorine
Chlorine is a highly effective oxidizing agent.
Chlorine is lethal within seconds to most vegetative
bacteria, and it inactivates most viruses.
This efficacy accounts for its use in water treatment.
But its activity is lost quickly in the presence of
organic material.
This property, combined with its toxicity, renders it
ineffective on body surfaces.
It is usually applied as a 5% solution called
hypochlorite. Bleach
Chemical methods.
Hydrogen peroxide
Powerful oxidizing agent that attacks
membrane lipids and other cell components.
Rapidly
Slower with bacteria that produce catalase and
spores less rapidly.
Useful in disinfecting items such as contact
lenses.
Chemical methods.
Detergents and soaps
Surfactants. Compounds with hydrophobic and
hydrophilic groups that attach to and solubilize
various compounds or alter their properties.
Anionic detergents
Soaps
highly effective cleansers, but have little direct antibacterial
effect.
remove dirt, oil, and contaminants as well as some resident
microbes.
Cationic detergents
Quaternary ammonium compounds ("quats") such as
benzalkonium chloride.
Chemical methods.
Detergents and soaps. Quats
Their name is derived from the fact that they
are modifications of the four-valence
ammonium ion, NH
4
.
are highly bactericidal in the absence of
contaminating organic matter.
Alter the membrane's surface properties and
its permeability.
They do not kill endospores.
Chemical methods.
Detergents and soaps. Quats
Chemical methods.
Detergents and soaps.
Washing hands with
water and soap (or quat)
is the most important
method to avoid the
spread of infections in
the hospital.
Chemical methods.
Chlorhexidine
Containing chlorine and two phenolic rings.
Acts both cell membranes and denaturation
of proteins.
Inactive against spores.
Low toxic, and rapid action.
Chemical methods.
Aldehydes
Glutaraldehyde and Formaldehyde
Combine with nucleic acids and proteins, and
inactivate them, probably by cross-linking and
alkylating molecules.
Sporicidal.
Formaldehyde gas is an excellent disinfectant.
It is more commonly available as formalin
37% aqueous solution of formaldehyde gas.
Used extensively to preserve biological specimens and
inactivate bacteria and viruses in vaccines.
Chemical methods.
Aldehydes
Glutaraldehyde
Less irritating and more effective than
formaldehyde.
Used to disinfect hospital instruments, including
endoscopes and respiratory therapy equipment.
Can be considered a sterilizing agent.
Chemical methods.
Aldehydes
Chemical methods.
Gas ethylene oxide
Inflammable and explosive gas.
It is an alkylating agent, altering DNA.
Ethylene oxide sterilizers resemble autoclaves
10% ethylene oxide in CO
2
at 50 to 60C under controlled
conditions of humidity.
4 to 6 hours
Followed by a prolonged period of aeration
Aeration is essential, because absorbed gas can cause
damage to tissues or skin.
Ethylene oxide is a mutagen.
Effective sterilizing agent for heat-labile devices such as artificial
heart valves that cannot be treated at the temperature of the
autoclave.
Chemical methods.
Gas ethylene oxide
Mechanism of action Sporicidal Adverse effects Uses Others
Phenolics Denaturalization of proteins.
Disruption of cell membrane.
No Corrosive, irritant.
Can be absorbed by skin
causing CNS
damage.
Antiseptic at low doses.
Disinfectant at high doses.
Alcohols Denaturalization of proteins. No Irritant, dries skin. Antiseptic of skin. Needs water to act.
Iodine Interfering with the disulfide
bonds of proteins.
Yes (slowly) Corrosive, irritant, staining. Antiseptic of skin. Allergies in some
people
Chlorine Oxidizing agent. Yes (slowly) Corrosive, irritant, toxic. Disinfectant.
Treatment of drinking water.
Inactivation in the
presence of
organic material.
H
2
O
2
Oxidizing agent. Yes Toxic. Antiseptic.
Disinfectant.
Soaps Hydrophobic and hydrophilic
groups that attach to and
solubilize various
compounds or alter their
properties.
No Cleanser. Good cleansers but
little direct
antibacterial
effect.
Quats Similar. No Irritant to mucous
membranes.
Antiseptic of skin.
Disinfectant.
Much more
bactericidal than
soaps.
Chlorhexidine Denaturalization of proteins.
Disruption of cell membrane.
No Antiseptic of skin.
Aldehydes Cross-linking and alkylating. Yes Toxic, irritant. Disinfectants, preservators.
Glutaraldehyde can be
considered sterilizing
agent.
Gas ethylene
oxide
Alkylating agent Yes Toxic, explosive.
Mutagen.
Sterilization.
Summary.
Microbial control methods