General Microbiology LaboratoryGeneral Microbiology Laboratory

Microbial control agentsMicrobial control agents

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Controlling MicroorganismsControlling 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: vegetative bacterial cells and endospores fungal hyphae and spores, yeast protozoan trophozoites and cysts worms viruses prions

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Terminology of Microbial ControlTerminology 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

Degerming: mechanical removal of microbes from limited area

Sanitization: lowering microbial counts on eating and drinking utensils to safe levels

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Biocide or germicide: kills microorganismsFungicide: kills fungiVirocide: inactivates viruses

E.g. microbiocides for HIV used in spermacides

Bacteriostatic agent: stops growth of bacteriaSepsis: bacterial contaminationAsepsis: absence of significant contaminationAseptic technique minimizes contamination

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Microbial DeathMicrobial Death

Microbes die at a constant rateFactors affecting how long it takes to kill bacteria

number of microbes environment

• slowed by organic materials, biofilms (e.g., feces, sewage)

• hastened by prior cleaning, heat

Factors affecting how long it takes to kill bacteria time of exposure characteristics of microbes: most resistant are

• spores

• thick lipid coats

• protozoan cysts

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Actions of Microbial Control AgentsActions of Microbial Control Agents

Cell wallCell membraneNucleic acid synthesisProtein synthesisProtein function

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Cell wall Bacteria and fungi

Block synthesis Degrade cellular components Destroy or reduce stability

Agent Penicillin, detergents, alcohols

Cell membraneAll microbes and enveloped viruses

Bind and penetrate lipids Lose selective permeability (leakage)

Agent Surfactants

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Nucleic acid synthesisNucleic acid synthesis

Irreversible bind to DNA Stop transcription and translation mutations

Agent Chemical agent – formaldehyde Physical agent – radiation

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Protein synthesisBinds to ribosomes

Stops translation Prevents peptide bonds

Agent chloramphenicol

Protein functionBlock protein active sitesPrevent binding to substrateDenature proteinAgent

Physical – Heat, pH change Chemical – alcohols, acids, phenolics, metallic ions

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Physical Methods of Microbial ControlPhysical Methods of Microbial Control

1. Heat – moist and dry

2. Cold temperatures

3. Desiccation

4. Radiation

5. Filtration

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Mode of Action and Relative Effectiveness of HeatMode 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; incineration

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Heat Resistance and Thermal DeathHeat Resistance and Thermal Death

Bacterial endospores most resistant – usually require temperatures above boiling

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Moist Heat MethodsMoist Heat Methods

Steam under pressure – sterilizationAutoclave 15 psi/121oC/10-40minSteam must reach surface of item being

sterilizedItem must not be heat or moisture sensitiveMode of action – denaturation of proteins,

destruction of membranes and DNA

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Boiling WaterBoiling Water

Boiling at 100oC for 30 minutes to destroy non-spore-forming pathogens

Disinfection

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PasteurizationPasteurization

Pasteurization – heat is applied to kill potential agents of infection and spoilage without destroying the food flavor or value

63°C - 66°C for 30 minutes (batch method)71.6°C for 15 seconds (flash method)Not sterilization - kills non-spore-forming

pathogens and lowers overall microbe count; does not kill endospores or many nonpathogenic microbes

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Dry HeatDry Heat

Dry heat using higher temperatures than moist heat

Incineration – flame or electric heating coil ignites and reduces microbes and other

substances

Dry ovens – 150-180oC- coagulate proteins

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ColdCold

Microbiostatic – slows the growth of microbesRefrigeration 0-15oC and freezing <0oCUsed to preserve food, media and cultures

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DesiccationDesiccation

Gradual removal of water from cells, leads to metabolic inhibition

Not effective microbial control – many cells retain ability to grow when water is reintroduced

Lyophilization – freeze drying; preservation

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RadiationRadiation

• 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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RadiationRadiation

• Nonionizing radiation – little penetrating power – must be directly exposed

• UV light creates thymine dimers, which interfere with replication.

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FiltrationFiltration

Physical removal of microbes by passing a gas or liquid through filter

Used to sterilize heat sensitive liquids and air in hospital isolation units and industrial clean rooms

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USING

ANTIMICROBIAL CHEMOTHERAPY

TOCONTROL MICROORGANISMS

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Antimicrobial DrugsAntimicrobial Drugs

Chemotherapy

Antibiotics

Antimicrobial chemotherapeutic chemicals

Selective toxicity

The use of drugs to treat adisease

produced by a microbe that inhibits another microbe

kills harmful microbeswithout damaging the host

chemicals synthesized in the laboratory which can be used therapeutically on microorganisms.

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Most of the major groups of antibiotics were discovered prior to 1955, and most antibiotic advances since then have come about by modifying the older forms.

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 DrugsAntimicrobial Drugs

Antibiotic Resistance - bacteria gain ability to grow

no longer sensitive to drug 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 ActivityFive 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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Antibiotic Susceptibility Testing Antibiotic Susceptibility Testing

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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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)

3. Automated tests

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Tube dilution testsTube 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).

Subculturing 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)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 usually-attained blood level in the patient with adequate dosage. Categories of "Resistant," "Intermediate," and "Susceptible" were then established.

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ProcedureProcedure

a. Prepare a standard turbidity inoculum of the test bacterium so that a certain density of bacteria will be put on the plate.

Select 3-5 isolated colonies of the bacterium that is being tested.

If the organism is a Staphylococcus or is fastidious and grows unpredictably in broth like the streptococci, suspend the colonies is saline, Mueller Hinton broth or trypticase soy broth. If the organism grows rapidly in broth, place the colonies in Mueller Hinton broth or trypticase soy broth and incubate 2-8 hours.

Match the turbidity of the test suspension or culture with a 0.5 McFarland standard. (McFarland standards are tubes containing either latex particles or barium sulfate and adjusted to a standard turbidity.) If the bacterial suspension is too turbid, add more saline or broth. If the bacterial suspension is too light, pick off more colonies and

suspend them in the broth or incubate longer.

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b. Inoculate a 150mm Mueller-Hinton agar plate with the standardized inoculum so as to cover the entire agar surface with bacteria.

Dip a sterile swab into the previously standardized tube of the bacterium being tested. Squeeze the swab against the inner wall of the tube to remove excess liquid.

Swab the entire plate from top to bottom, edge-to-edge leaving no gaps.

Rotate the plate approximately 60 degrees and using the same swab, again swab the entire plate from top to bottom.

Rotate the plate approximately 60 degrees and using the same swab, and swab the entire plate from top to bottom a third time.

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c. Place standardized antibiotic-containing discs on the plate.

d. Incubate the plate agar side upat 35°C for 16-18 hours.

e. Measure the diameter of any resulting zones of inhibition in millimeters (mm)

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f. Determine if the bacterium is susceptible, moderately susceptible, intermediate, or resistant to each antimicrobial agent using a standardized table

If there is a double zone of inhibition, measure the diameter of the innermost zone.

If there is a zone containing colonies, measure the diameter of the colony free zone.

If there is a feathered zone, measure the diameter of the point where there is an obvious demarcation between growth and no

growth. When testing swarming Proteus mirabilis, ignore the

swaming. When testing Staphylococcus aureus, the haze around an oxacillin should not be ignored. Measure the diameter of the

zone free of growth or haze.

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The term intermediate generally means that the result is inconclusive for that drug-organism combination.

The term moderately susceptible is usually applied to those situations where a drug may be used for infections in a particular body site, e.g., cystitis because the drug becomes highly concentrated in the urine.

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Automated tests Automated tests

Computerized automated tests have been developed for antimicrobial susceptibility testing. These tests measure the inhibitory effect of the antimicrobial agents in a liquid medium by using light scattering to determine growth of the test organism. Results can be obtained within a few hours.

Labs performing very large numbers of susceptibility tests frequently use the automated methods but the equipment is quite expensive.

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End of lecture