Download Microbial control agents

General Microbiology Laboratory
Microbial control agents
Controlling Microorganisms
 Many microorganisms are beneficial and necessary for human
well-being.
 However, microbial activities may have undesirable
consequences, such as food spoilage and disease.
 It is essential to be able to kill microorganisms or inhibit their
growth to minimize their destructive effects.
 Physical, chemical, and mechanical methods to destroy or
reduce undesirable microbes in a given area
 Primary targets are microorganisms capable of causing
infection or spoilage:
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vegetative bacterial cells and endospores
fungal hyphae and spores, yeast
protozoan trophozoites and cysts
worms
viruses
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Terminology of Microbial Control
Sterilization: Killing or removing all forms of
microbial life (including endospores) in a
material or an object.
Commercial sterilization: sufficient heat to
kill Clostridium botulinum endospores (some
non-pathogenic thermophilic bacteria may
survive)
Disinfection: destruction of vegetative
pathogens on inert substances
Antisepsis: destruction of vegetative pathogens
on living tissue
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Biocide or germicide: kills microorganisms.
Fungicide: kills fungi.
Bacteriostatic agent: stops growth of bacteria.
Aseptic technique minimizes contamination.
Degerming: mechanical removal of microbes
from limited area.
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Microbial Death
Microbes die at a constant rate
Factors affecting how long it takes to kill
bacteria
 number of microbes
 environment (slowed by organic materials,
biofilms - hastened by prior cleaning, heat).
 time of exposure
 characteristics of microbes: most resistant are
• spores
• thick lipid coats
• protozoan cysts
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Actions of Microbial Control Agents
Cell wall.
Cell membrane.
Nucleic acid synthesis.
Protein synthesis.
Protein function.
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Cell wall
Bacteria and fungi.
 Block synthesis.
 Degrade cellular components.
 Destroy or reduce stability.
Agent: Penicillin, detergents, alcohols
Cell membrane
All microbes and enveloped viruses.
 Bind and penetrate lipids.
 Lose selective permeability (leakage).
Agent: Polymyxin B
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Nucleic acid synthesis
Irreversible bind to DNA.
 Stop transcription and translation.
 Mutations.
Agent:
 Chemical agent – formaldehyde
 Physical agent – radiation
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Protein synthesis
Binds to ribosome's.
 Stops translation.
 Prevents peptide bonds.
Agent: chloramphenicol
Protein function
Block protein active sites.
Prevent binding to substrate.
Denature protein.
Agent
 Physical – Heat, pH change
 Chemical – alcohols, acids, phenolics, metallic ions
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Physical Methods of Microbial Control
1.
2.
3.
4.
Heat – moist and dry
Cold temperatures
Radiation
Filtration
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Mode of Action and Relative Effectiveness of Heat
Moist heat: lower temperatures and shorter
exposure time; coagulation and denaturation of
proteins
Dry heat: moderate to high temperatures;
dehydration, alters protein structure.
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Pasteurization
Pasteurization: heat is applied to kill potential agents
of infection and spoilage without destroying the food
value.
63°C - 66°C for 30 minutes.
 71.6°C for 15 seconds.
Not sterilization - kills non-spore-forming pathogens
and lowers overall microbe count; does not kill
endospores or many nonpathogenic microbes.
Dry heat: using higher temperatures than moist heat
Dry ovens – 150-180oC- coagulate proteins
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Radiation
• Ionizing radiation:
deep penetrating
power that has
sufficient energy to
cause electrons to
leave their orbit,
breaks DNA,
 gamma rays, X-rays,
cathode rays
 used to sterilize
medical supplies and
food products
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Radiation
• Nonionizing
radiation: little
penetrating power
must be directly
exposed
• UV light creates
thymine dimers,
which interfere
with replication.
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USING
ANTIMICROBIAL CHEMOTHERAPY
TO
CONTROL MICROORGANISMS
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Antimicrobial Drugs
Chemotherapy
Antibiotics
Antimicrobial
chemotherapeutic
chemicals
Selective toxicity
The use of drugs to treat a
disease
produced by a microbe that
inhibits another microbe
chemicals synthesized in the
laboratory which can be used
therapeutically on microorganisms.
kills harmful microbes
without damaging the host
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 In fact, only 3 major groups of microorganisms have
yielded useful antibiotics: the actinomycetes
(filamentous, branching soil bacteria such as
Streptomyces), bacteria of the genus Bacillus, and the
saprophytic molds Penicillium and Cephalosporium.
To produce antibiotics, manufacturers inoculate large
quantities of medium with carefully selected strains
of the appropriate species of antibiotic-producing
microorganism. After incubation, the drug is
extracted from the medium and purified. Its activity is
standardized and it is put into a form suitable for
administration.
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Some antimicrobial agents are:
 cidal in action: they kill microorganisms
(e.g., penicillins, cephalosporins, streptomycin,
neomycin).
 Others are static in action: they inhibit
microbial growth long enough for the body's
own defenses to remove the organisms (e.g.,
tetracyclines, erythromycin, sulfonamides).
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Antimicrobial agents also vary in their spectrum.
 Drugs that are effective against a variety of both
gram-positive and gram-negative bacteria are said to
be broad-spectrum (e.g., tetracycline, streptomycin,
cephalosporins, ampicillin, sulfonamides).
 Those effective against just gram-positive bacteria,
just gram negative bacteria, or only a few species are
termed narrow-spectrum (e.g., penicillin G,
erythromycin, clindamycin, gentamicin).
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Antimicrobial Drugs
Antibiotic Resistance: bacteria gain ability to
grow.
Antiretroviral: act specifically against viruses
 Combination of drugs:
• Synergism: action of two antibiotics greater
• Antagonism: action of drug is reduced;
less effective
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Five Modes of Antimicrobial Activity
1. Injury to Plasma Membrane
• polymixin B
2. Inhibition of Cell Wall Synthesis
• penicillins, bacitracin, vancomycin
3. Inhibition of Protein Synthesis (translation)
4. Inhibition of Nucleic Acid replication &
transcription
5. Inhibition of essential metabolites
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For some microorganisms, susceptibility to
chemotherapeutic agents is predictable. However, for
many microorganisms (Pseudomonas,
Staphylococcus aureus, and gram-negative enteric
bacilli such as Escherichia coli, Serratia, Proteus,
etc.) there is no reliable way of predicting which
antimicrobial agent will be effective in a given case.
This is especially true with the emergence of many
antibiotic-resistant strains of bacteria.
Because of this, antibiotic susceptibility testing is
often essential in order to determine which
antimicrobial agent to use against a specific strain of
bacterium.
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Antibiotic Susceptibility Testing
 Several tests may be used to tell a physician
which antimicrobial agent is most likely to
combat a specific pathogen:
1. Tube dilution tests
2. The agar diffusion test (Bauer-Kirby test)
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Tube dilution tests
 In this test, a series of culture tubes are prepared, each
containing a liquid medium and a different concentration of a
chemotherapeutic agent. The tubes are then inoculated with the
test organism and incubated for 16-20 hours at 35C. After
incubation, the tubes are examined for turbidity (growth). The
lowest concentration of chemotherapeutic agent capable of
preventing growth of the test organism is the minimum
inhibitory concentration (MIC).
 Sub culturing of tubes showing no turbidity into tubes
containing medium but no chemotherapeutic agent can
determine the minimum bactericidal concentration (MBC).
MBC is the lowest concentration of the chemotherapeutic
agent that results in no growth (turbidity) of the subcultures.
These tests, however, are rather time consuming and expensive
to perform.
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The agar diffusion test (Bauer-Kirby test)
 A procedure commonly used in clinical labs to determine
antimicrobial susceptibility is the Bauer-Kirby disc diffusion
method. In this test, the in vitro response of bacteria to a
standardized antibiotic-containing disc has been correlated with
the clinical response of patients given that drug.
 In the development of this method, a single high-potency disc of
each chosen chemotherapeutic agent was used. Zones of growth
inhibition surrounding each type of disc were correlated with the
minimum inhibitory concentrations of each antimicrobial agent
(as determined by the tube dilution test).
 The MIC for each agent was then compared to the usuallyattained blood level in the patient with adequate dosage.
Categories of "Resistant," "Intermediate," and "Susceptible"
were then established.
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End of lecture
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