Islamic Islamic University University of Gaza of Gaza Faculty of Faculty of Medicine Medicine Medical Medical Microbiology Microbiology 2008-2009 2008-2009 Prepared by: Prepared by: Sohaib M. El-Hams Sohaib M. El-Hams PRESENTED TO: PRESENTED TO: Dr.Abdelraouf Ali El Manamma Dr.Abdelraouf Ali El Manamma
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Islamic University of Gaza Faculty of Medicine Medical Microbiology 2008-2009 Prepared by: Sohaib M. El-Hams PRESENTED TO: Dr.Abdelraouf Ali El Manamma.
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Prepared by:Prepared by:Sohaib M. El-HamsSohaib M. El-Hams
PRESENTED TO:PRESENTED TO: Dr.Abdelraouf Ali El Manamma Dr.Abdelraouf Ali El Manamma
Sterilization & DisinfectionSterilization & Disinfection Sterilization:Sterilization: is the killing or removal of all is the killing or removal of all
microorganisms, including bacterial spores, which are microorganisms, including bacterial spores, which are
highly resistant.highly resistant.
SterilizationSterilization is usually carried out by autoclaving, is usually carried out by autoclaving,
which consists of exposure to steam at which consists of exposure to steam at 121°C121°C under a under a
pressure of pressure of 15 lb/in 215 lb/in 2 for for 15 minutes15 minutes..
Surgical instruments that can be damaged by moist heat Surgical instruments that can be damaged by moist heat
are usually sterilized by exposure to ethylene oxide gas, are usually sterilized by exposure to ethylene oxide gas,
and most intravenous solutions are sterilized by and most intravenous solutions are sterilized by
filtration.filtration.
Sterilization & DisinfectionSterilization & Disinfection Disinfection:Disinfection: is the killing of many, but not all, is the killing of many, but not all,
microorganisms. microorganisms.
For adequate disinfection, pathogens must be killed, but For adequate disinfection, pathogens must be killed, but
some organisms and bacterial spores may survive. some organisms and bacterial spores may survive.
Disinfectants vary in their tissue damaging properties Disinfectants vary in their tissue damaging properties
from the corrosive phenol-containing compounds, which from the corrosive phenol-containing compounds, which
should be used only on inanimate objects, to less toxic should be used only on inanimate objects, to less toxic
materials such as ethanol and iodine, which can be used materials such as ethanol and iodine, which can be used
on skin surfaces. on skin surfaces.
Chemicals used to kill microorganisms on the surface of Chemicals used to kill microorganisms on the surface of
skin and mucous membranes are called antiseptics. skin and mucous membranes are called antiseptics.
RATE OF KILLING OF RATE OF KILLING OF MICROORGANISMSMICROORGANISMS
Death of microorganisms occurs at a certain rate dependent Death of microorganisms occurs at a certain rate dependent
primarily upon two variables: primarily upon two variables:
(1) The concentration of the killing agent.(1) The concentration of the killing agent.
(2) The length of time agent is applied.(2) The length of time agent is applied.
The rate of killing is defined by the relationship:The rate of killing is defined by the relationship:
N ∞1/CT N ∞1/CT
which shows that the number of survivors, N, is inversely which shows that the number of survivors, N, is inversely
proportionate to the concentration of the agent, C, and to the proportionate to the concentration of the agent, C, and to the
time of application of the agent. Collectively, CT is often time of application of the agent. Collectively, CT is often
referred to as the dose. referred to as the dose.
Stated alternatively, the number of microorganisms killed Stated alternatively, the number of microorganisms killed
is directly proportionate to CT. The relationship is usually is directly proportionate to CT. The relationship is usually
stated in terms of survivors, because they are easily stated in terms of survivors, because they are easily
measured by colony formation.measured by colony formation.
Death is defined as the inability to reproduce. In certain Death is defined as the inability to reproduce. In certain
circumstances, the physical remains of dead bacteria can circumstances, the physical remains of dead bacteria can
still cause problems.still cause problems.
CHEMICAL AGENTSCHEMICAL AGENTS
Chemicals vary greatly in their ability to kill Chemicals vary greatly in their ability to kill
microorganisms.microorganisms.
A quantitative measure of this variation is expressed as A quantitative measure of this variation is expressed as
the phenol coefficient, which is the ratio of the the phenol coefficient, which is the ratio of the
concentration of phenol to the concentration of the concentration of phenol to the concentration of the
agent required to cause the same amount of killing agent required to cause the same amount of killing
under the standard conditions of the test.under the standard conditions of the test.
Chemical agents act primarily by one of three Chemical agents act primarily by one of three
mechanisms: mechanisms:
(1) Disruption of the lipid-containing cell membrane. (1) Disruption of the lipid-containing cell membrane.
(2) Modification of proteins. (2) Modification of proteins.
(3) Modification of DNA.(3) Modification of DNA.
Each of the following chemical agents has been Each of the following chemical agents has been
classified into one of the three categories, but some of classified into one of the three categories, but some of
the chemicals act by more than one mechanism. the chemicals act by more than one mechanism.
DISRUPTION OF CELL DISRUPTION OF CELL MEMBRANESMEMBRANES
1.1. AlcoholAlcohol
2.2. DetergentsDetergents
3.3. PhenolsPhenols
1.1. Alcohol:-Alcohol:-
* Ethanol is widely used to clean the skin * Ethanol is widely used to clean the skin
before immunization or venipuncture. before immunization or venipuncture.
* It acts mainly by disorganizing the lipid * It acts mainly by disorganizing the lipid
structure in membranes, but it denatures structure in membranes, but it denatures
proteins as well. proteins as well.
* Ethanol requires the presence of water * Ethanol requires the presence of water
for maximal activity; i.e., it is far more for maximal activity; i.e., it is far more
effective at effective at 70%70% than at than at 100%.100%.
* Seventy percent ethanol is often used as an antiseptic * Seventy percent ethanol is often used as an antiseptic to clean the skin prior to venipuncture.to clean the skin prior to venipuncture.
* However, because it is not as effective as iodine-* However, because it is not as effective as iodine-
containing com- pounds, the latter should be used prior containing com- pounds, the latter should be used prior
to obtaining a blood culture and installing intravenous to obtaining a blood culture and installing intravenous
catheters.catheters.
2.2. DetergentsDetergents:-:-
* Detergents are "surface-active" agents composed of a * Detergents are "surface-active" agents composed of a
long-chain, lipid-soluble, hydrophobic portion and a long-chain, lipid-soluble, hydrophobic portion and a
polar hydrophilic group, which can be a cation, an polar hydrophilic group, which can be a cation, an
anion, or a nonionic group.anion, or a nonionic group.
* These surfactants interact with the lipid in the cell * These surfactants interact with the lipid in the cell
membrane through their hydrophobic chain and with membrane through their hydrophobic chain and with
the surrounding water through their polar group and the surrounding water through their polar group and
thus disrupt the membrane.thus disrupt the membrane.
* Formaldehyde, which is available as a * Formaldehyde, which is available as a 37%37% solution solution
in water (in water (FormalinFormalin), denatures proteins and nucleic ), denatures proteins and nucleic
acids. acids.
* Both proteins and nucleic acids contain essential -* Both proteins and nucleic acids contain essential -
NH 2NH 2 and- and-OHOH groups, which are the main sites of groups, which are the main sites of
alkylation by the hydroxymethyl group of formaldehyde.alkylation by the hydroxymethyl group of formaldehyde.
* Glutaraldehyde, which has two reactive aldehyde * Glutaraldehyde, which has two reactive aldehyde
groups, is 10 times more effective than formaldehyde groups, is 10 times more effective than formaldehyde
and is less toxic. and is less toxic.
* In hospitals, it is used to sterilize respiratory therapy * In hospitals, it is used to sterilize respiratory therapy
equipment. equipment.
6. 6. Ethylene Oxide:Ethylene Oxide:
* Ethylene oxide gas is used extensively in hospitals for * Ethylene oxide gas is used extensively in hospitals for
the sterilization of heat-sensitive materials such as the sterilization of heat-sensitive materials such as
surgical instruments and plastics.surgical instruments and plastics.
* It kills by alkylating both proteins and nucleic acids; * It kills by alkylating both proteins and nucleic acids;
i.e., the hydroxyethyl group attacks the reactive hydrogen i.e., the hydroxyethyl group attacks the reactive hydrogen
atoms on essential amino and hydroxyl groups.atoms on essential amino and hydroxyl groups.
7.7. Acids & Alkalis:Acids & Alkalis:
* Strong acids and alkalis kill by denaturing proteins. * Strong acids and alkalis kill by denaturing proteins.
Although most bacteria are susceptible, it is important to Although most bacteria are susceptible, it is important to
note that Mycobacteriurn tuberculosis and other note that Mycobacteriurn tuberculosis and other
mycobacteria are relatively resistant to mycobacteria are relatively resistant to 2% NaOH2% NaOH, which , which
is used in the clinical laboratory to liquefy sputum prior to is used in the clinical laboratory to liquefy sputum prior to
culturing the organism.culturing the organism.
* Weak acids, such as benzoic, propionic, and citric * Weak acids, such as benzoic, propionic, and citric
acids, are frequency used as food preservatives because acids, are frequency used as food preservatives because
they are bacteriostatic. they are bacteriostatic.
* The action of these acids is partially a function of the * The action of these acids is partially a function of the
organic moiety, e.g., benzoate, as well as the low organic moiety, e.g., benzoate, as well as the low pHpH..
MODIFICATION OF NUCLEIC MODIFICATION OF NUCLEIC ACIDSACIDS
A variety of dyes not only stain microorganisms but also inhibit their A variety of dyes not only stain microorganisms but also inhibit their
growth.growth.
One of these is crystal violet (gentian violet), which is used as a skin One of these is crystal violet (gentian violet), which is used as a skin
antiseptic.antiseptic.
Its action is based on binding of the positively charged dye molecule Its action is based on binding of the positively charged dye molecule
to the negatively charged phosphate groups of the nucleic acids. to the negatively charged phosphate groups of the nucleic acids.
Malachite green, a triphenylamine dye like crystal violet, is a Malachite green, a triphenylamine dye like crystal violet, is a
component of Lowenstein- Jensen's medium, which is used to grow component of Lowenstein- Jensen's medium, which is used to grow
M. tuberculosis.M. tuberculosis.
The dye inhibits the growth of unwanted organisms in the sputum The dye inhibits the growth of unwanted organisms in the sputum
during the 6-week incubation period. during the 6-week incubation period.
PHYSICAL AGENTSPHYSICAL AGENTS
The physical agents act either by imparting The physical agents act either by imparting
energy in the form of heat or radiation or by energy in the form of heat or radiation or by
removing organisms through filtration.removing organisms through filtration.
(1) Heat(1) Heat
(2) Radiation(2) Radiation
(3) Filtration(3) Filtration
PHYSICAL AGENTSPHYSICAL AGENTS (1)(1) Heat:Heat: Heat energy can be applied in three ways: in the form ofHeat energy can be applied in three ways: in the form of
moist moist heat (heat (either boiling or autoclavingeither boiling or autoclaving) or ) or dry heatdry heat or or
by by pasteurizationpasteurization..
In general, heat kills by denaturing proteins, but In general, heat kills by denaturing proteins, but
membrane damage and enzymatic cleavage of DNA may membrane damage and enzymatic cleavage of DNA may
also be involved. also be involved.
Moist heatMoist heat sterilizes at a lower temperature than dry sterilizes at a lower temperature than dry
heat, because water aids in the disruption of non covalent heat, because water aids in the disruption of non covalent
bonds, e.g., hydrogen bonds, which hold protein chains bonds, e.g., hydrogen bonds, which hold protein chains
together in their secondary and tertiary structures. together in their secondary and tertiary structures.
Moist-heat sterilization, usually autoclaving, is the most Moist-heat sterilization, usually autoclaving, is the most
frequently used method of sterilization. frequently used method of sterilization.
Because bacterial spores are resistant to boiling (100°C Because bacterial spores are resistant to boiling (100°C
at sea level), they must be exposed to a higher at sea level), they must be exposed to a higher
temperature; this cannot be achieved unless the temperature; this cannot be achieved unless the
pressure is increased. pressure is increased.
For this purpose, an autoclave chamber is used in which For this purpose, an autoclave chamber is used in which
steam, at a pressure of 15 lb/in 2, reaches a steam, at a pressure of 15 lb/in 2, reaches a
temperature of 121°C and is held for 15-20 minutes.temperature of 121°C and is held for 15-20 minutes.
This kills even the highly heat-resistant spores of This kills even the highly heat-resistant spores of
Clostridium botulinum, the cause of botulism, with a Clostridium botulinum, the cause of botulism, with a
margin of safety. margin of safety.
Sterilization by Sterilization by dry heatdry heat, on the other hand, requires , on the other hand, requires
temperatures in the range of temperatures in the range of 180°C180°C for for 2 hours2 hours..
This process is used primarily for glassware and is used This process is used primarily for glassware and is used
less frequently than autoclaving.less frequently than autoclaving.
Pasteurization,Pasteurization, which is used primarily for milk, which is used primarily for milk,
consists of heating the milk to consists of heating the milk to 62°C62°C for for 30 minutes30 minutes
followed by rapid cooling. followed by rapid cooling. ("Flash" pasteurization at ("Flash" pasteurization at
72°C for 15 seconds is often used72°C for 15 seconds is often used) This is sufficient to ) This is sufficient to
kill the vegetative cells of the milk-borne pathogens, e.g., kill the vegetative cells of the milk-borne pathogens, e.g.,